Patent Publication Number: US-5527524-A

Title: Dense star polymer conjugates

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation-in-part of our application Ser. No. 654,851, filed Feb. 13, 1991 now U.S. Pat. No. 5,338,532, which is a continuation-in-part of Ser. No. 386,049, filed Jul. 26, 1989, now abandoned, which is a continuation-in-part of Ser. No. 087,266, filed Aug. 18, 1987, now abandon, which is a continuation-in-part application of Ser. No. 897,455, filed Aug. 18, 1986, now abandon. All of these prior application documents are hereby incorporated by reference. 
    
    
     FIELD OF THE INVENTION 
     The present invention concerns the use of dense star polymers as carriers for agricultural, pharmaceutical, and other materials. 
     BACKGROUND OF THE INVENTION 
     In recent years polymers referred to as dense star polymers or STARBURST™ polymers (a trademark of Dendritech Inc.) have been developed. It has been found that the size, shape and properties of these dense star polymers or STARBURST™ polymers can be molecularly tailored to meet specialized end uses. STARBURST™ polymers have significant advantages which can provide a means for the delivery of high concentrations of carried material per unit of polymer, controlled delivery, targeted delivery and/or multiple species delivery or use. 
     SUMMARY OF THE INVENTION 
     In its broadest aspect, the present invention is directed to polymer conjugate materials comprising dense star polymers or STARBURST™ polymers associated with desired materials (hereinafter these polymer conjugates will frequently be referred to as &#34;STARBURST™ conjugates&#34; or &#34;dense star polymer conjugates&#34; or &#34;conjugates&#34;), process for preparing these conjugates, compositions containing the conjugates, and methods of using the conjugates and compositions. 
     The conjugates of the present invention are suitable for use in a variety of applications where specific delivery is desired, and are particularly suited for the delivery of biologically active agents. In a preferred embodiment of the present invention, the STARBURST™ conjugates are comprised of one or more STARBURST™ polymers associated with one or more bioactive agents. 
     The STARBURST™ conjugates offer significant benefits over other carriers known in the art due to the advantageous properties of the STARBURST™ polymers. STARBURST™ polymers exhibit molecular architecture characterized by regular dendritic branching with radial symmetry. These radially symmetrical molecules are referred to as possessing &#34;STARBURST™ topology&#34;. These polymers are made in a manner which can provide concentric dendritic tiers around an initiator core. The STARBURST™ topology is achieved by the ordered assembly of organic repeating units in concentric, dendritic tiers around an initiator core; this is accomplished by introducing multiplicity and self-replication (within each tier) in a geometrically progressive fashion through a number of molecular generations. The resulting highly functionalized molecules have been termed &#34;dendrimers&#34; in deference to their branched (tree-like) structure as well as their oligomeric nature. Thus, the terms STARBURST™ oligomer and STARBURST™ dendrimer are encompassed within the term STARBURST™ polymer or dense star polymer. Topological polymers, with size and shape controlled domains, are dendrimers that are covalently bridged through their reactive terminal groups, which are referred to as STARBURST™ &#34;bridged dendrimers.&#34; The term bridged dendrimer is also encompassed within the term &#34;STARBURST™ polymer&#34;. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The following description of the figures aid in understanding the present invention. 
     FIG. 1 depicts various generations of STARBURST™ dendrimers. 
     FIG. 2 depicts a dendrimer having unsymmetrical (unequal) branch junctures, and also depicts a dendrimer having symmetrical (equal) branch junctures. 
     FIG. 3 depicts dendrimer sizes relative to antibody dimensions. 
     FIG. 4 shows carbon-13 spin lattice relaxation times (T 1 ) for aspirin incorporated into various dendrimer generations, Example 1. 
     FIG. 5 shows the results of the dynamic analysis of Example 2. 
     FIG. 6 shows the influence of generation 6.5 dendrimer on the dialysis rate of pseudoephedrine at pH 9.5 from Example 2. 
     FIG. 7 shows the effect of dendrimer hydrolysis on the permeability of pseudoephedrine of Example 3. 
     Example 8 shows the comparison of percent salicylic acid released into the receptor compartment in the presence of STARBURST™ polymer (Gen=4.0) at pH 5.0 and 6.65 with salicylic acid control, Example 4. 
     FIG. 9 shows the comparison of percent salicylic acid lost from donor compartment with STARBURST™ polymer (Gen=4.0) in receptor compartment at pH 8.0 to salicylic acid content, Example 4. 
     FIG. 10 shows the comparison of percent salicylic acid lost from donor compartment in presence of STARBURST™ polymer (Gen=4.5) to salicylic acid control, Example 4. 
     FIG. 11 shows carbon-13 spin lattice relaxation times (T 1 ) for 2,4-D incorporated into various dendrimer generations, Example 15. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The STARBURST™ polymers are illustrated by FIG. 1 wherein the circled I [herein &#34;core (I)&#34;]  represents an initiator core (in this figure a tri-functional initiator core shown by the far left drawing,); Z represents a terminal group, shown in the first instance by the second drawing from the left, referred to as a starbranched oligomer; A, B, C, D, and E represent particular molecular generations of STARBURST™ dendrimers; and (A) n , (B) n , (C) n , (D) n , and (E) n  represent STARBURST™ bridged dendrimers. 
     The STARBURST™ dendrimers are unimolecular assemblages that possess three distinguishing architectural features, namely, (a) an initiator core, (b) interior layers (generations, G or Gen) composed of repeating units, radially attached to the initiator core, and (c) an exterior surface of terminal functionality (i.e., terminal functional groups) attached to the outermost generation. The size and shape of the STARBURST™ dendrimer molecule and the functional groups present in the dendrimer molecule can be controlled by the choice of the initiator core, the number of generations (i.e., tiers) employed in creating the dendrimer, and the choice of the repeating units employed at each generation. Since the dendrimers can be isolated at any particular generation, a means is provided for obtaining dendrimers having desired properties. 
     The choice of the STARBURST™ dendrimer components affects the properties of the dendrimers. The initiator core type can affect the dendrimer shape, producing (depending on the choice of initiator core), for example, spheroid-shaped dendrimers, cylindrical or rod-shaped dendrimers, ellipsoid-shaped dendrimers, or mushroom-shaped dendrimers. Sequential building of generations (i.e., generation number and the size and nature of the repeating units) determines the dimensions of the dendrimers and the nature of their interior. 
     Because STARBURST™ dendrimers are branched polymers containing dendritic branches having functional groups distributed on the periphery of the branches, they can be prepared with a variety of properties. For example, the macromolecules depicted in FIG. 2A (such as Denkewalter, U.S. Pat. No. 4,289,872), and the present STARBURST™ dendrimers, such as those depicted in FIG. 2B have distinctly different properties due to the branch length. The dendrimer type shown in FIG. 2A possesses unsymmetrical (unequal segment) branch junctures, exterior (i.e., surface) groups (represented by Z), interior moieties (represented by Z&#39;) but much less internal void space. The preferred dendrimer type shown in FIG. 2B possesses symmetrical (equal segment) branch junctures with surface groups (represented by Z), two different interior moieties (represented respectively by X and Z&#39;) with interior void space which varies as a function of the generation (G). The dendrimers such as those depicted in FIG. 2B can be advanced through enough generations to totally enclose and contain void space, to give an entity with a predominantly hollow interior and a highly congested surface. 
     It is the tiered structure that is the essence of the STARBURST™ dendrimers rather than the elemental composition. Therefore, the repeating units may be composed of a combination of any elements, so long as these units possess the properties of multiplicity and are assembled into the tiered structure as described herein. These repeat units may be composed entirely of elements that are commonly seen in polymeric structures, such as carbon, hydrogen, oxygen, sulfur, and nitrogen, or may be composed of less traditional elements, provided that these repeat units allow a stable branched structure to be constructed. For example, metalloids and transition metals are well known in the art to form stable covalent compounds and complexes with organic moieties. These stable covalent compounds and complexes with organic moieties can exist as branched materials such as, for example, boranes, borates, germanes, stannanes, and plumbanes, or non-branched linkages such as, for example, dialkyl zincs or mercuries. The use of appropriate ligands can make a transition metal, such as cobalt, function as a branching unit (by connecting three separate ligands) or a non-branched linkage (by connecting two separate ligands). Therefore, branched structures fitting the patterns described herein and incorporating any element are within the scope of the present invention. 
     Also, STARBURST™ dendrimers, when advanced through sufficient generations exhibit &#34;STARBURST™ dense packing&#34; where the surface of the dendrimer contains sufficient terminal moieties such that the dendrimer surface becomes congested and encloses void spaces within the interior of the dendrimer. This congestion can provide a molecular level barrier which can be used to control diffusion of materials into or out of the interior of the dendrimer. 
     Surface chemistry of the dendrimers can be controlled in a predetermined fashion by selecting a repeating unit which contains the desired chemical functionality or by chemically modifying all or a portion of the surface functionalities to create new surface functionalities. These surfaces may either be targeted toward specific sites or made to resist uptake by particular organs or cells, e.g. by reticuloendothelial cells. 
     In an alternative use of the STARBURST™ dendrimers, the dendrimers can themselves be linked together to create polydendritic moieties (STARBURST™ &#34;bridged dendrimers&#34;) which are also suitable as carriers. 
     In addition, the dendrimers can be prepared so as to have deviations from uniform branching in particular generations, thus providing a means of adding discontinuities (i.e., deviations from uniform branching at particular locations within the dendrimer) and different properties to the dendrimer. 
     The STARBURST™ polymers employed in the STARBURST™ conjugates of the present invention can be prepared according to methods known in the art, for example, U.S. Pat. No. 4,587,329, the disclosure of which is hereby incorporated by reference. Polyamine dendrimers may be prepared by reacting ammonia or an amine having a plurality of primary amine groups or secondary amine groups with N-substituted aziridine, such as N-tosyl or N-mesyl aziridine, to form a protected first generation polysulfonamide. The first generation polysulfonamide is then activated with acid, such as sulfuric, hydrochloric, trifluoroacetic, fluorosulfonic or chlorosulfonic acid, to form the first generation polyamine salt. Preferably, the desulfonylation is carried out using a strong acid which is volatile enough to allow removal by distillation, such as hydrochloric acid. The first generation polyamine salt can then be reacted further with N-protected aziridine to form the protected second generation polysulfonamide. The sequence can be repeated to produce higher generation polyamines. 
     Polyamidoamines can be prepared by first reacting ammonia (or an amine having a puralitity of primary and/or secondary amine groups) with methyl acrylate under conditions sufficient to cause the Michael addition of one molecule of the ammonia to three molecules of the methyl acrylate to form the core adduct. Following removal of unreacted methyl acrylate, this compound is reacted with excess ethylenediamine under conditions such that one amine group of the ethylenediamine molecule reacts with the methyl carboxylate groups of the core adduct to form a first generation adduct having three amidoamine moieties. Following removal of unreacted ethylenediamine, this first generation adduct is then reacted with excess methyl acrylate under Michael&#39;s addition conditions to form a second generation adduct having terminal methyl ester moieties. The second generation adduct is then reacted with excess ethylenediamine under amide forming conditions to produce the desired polyamidoamine dendrimer having ordered, second generation dendritic branches with terminal amine moieties. Similar dendrimers containing amidoamine moieties can be made by using organic amines as the core compound, e.g., ethylenediamine which produces a tetra-branched dendrimer or diethylenetriamine which produces a penta-branched dendrimer. 
     To prepare anhydrous STARBURST™ polyethyleneimines, after acid cleavage of the sulfonamide bonds, a solvent which will form an azeotrope with water, such as benzene, toluene, xylene or mesitylene, preferably toluene, can be added and the resulting water/solvent azeotrope removed by azeotropic distillation, such as by heating the mixture to reflux with water removal carried out by a Dean-Stark trap. Alternatively, chlorinated solvents in which anhydrous polyethyleneimine is soluble, such as chloroform, can be used in the drying step. The addition of a chlorinated solvent or solvent which forms an azeotrope with water, avoids the necessity of having to heat the polymer at temperatures which char or degrade the polymer. Anhydrous polyethyleneimines are particularly useful as carriers for antigenic materials. 
     Dendrimers can be prepared having highly uniform size and shape and most importantly allow for a greater number of functional groups per unit of surface area of the dendrimer, and can have a greater number of functional groups per unit of molecular volume as compared to other polymers which have the same molecular weight, same core and monomeric components and same number of core branches as the STARBURST™ polymers. The increased functional group density of the dense STARBURST™ polymers may allow a greater quantity of material to be carried per dendrimer. Since the number of functional groups on the dendrimers can be controlled on the surface and within the interior, it also provides a means for controlling, for example, the amount of bioactive agent to be delivered per dendrimer. In a particularly preferred embodiment of the present invention, the STARBURST™ polymers, particularly the STARBURST™ dendrimers, are targeted carriers of bioactive agents capable of delivering the bioactive agents to a particular target organ or to a particular determinant or locus in a target organism. 
     An analogy can be made between early generation STARBURST™ dendrimers (i.e. generation=1-7) and classical spherical micelles. The dendrimer-micelles analogy was derived by comparing features which they had in common such as shape, size and surface. 
     
                       TABLE I                                                     
______________________________________                                    
             Regular Classical                                            
Parameter    Micelles     Starburst Dendrimers                            
______________________________________                                    
Shape        Spherical    Spherical                                       
Size         20-60Å   17-67Å                                      
(diameter)                                                                
Surface      4-202        Z = 6-192                                       
aggregation number        (Z is the number of                             
                          surface groups)                                 
                          (generation = 2-7)                              
area/surface group                                                        
             130-80Å.sup.2                                            
                          127-75Å.sup.2                               
(Å.sup.2)                                                             
______________________________________                                    
 (1Å = 10.sup.-1 nm; 1Å.sup.2 = 10.sup.-2 nm.sup.2)               
 
    
     In Table I, the shape was verified by scanning transmission electron microscopy (STEM) and intrinsic viscosity [η] measurements. The size was verified by intrinsic viscosity [η] and size exclusion chromatography (SEC) measurements. The surface aggregation numbers were verified by titrimetry and high field NMR. The area/surface group was calculated from SEC hydrodynamic measurements. 
     The first five generations of STARBURST™ polyamidoamine (PAMAM) dendrimers are microdomains which very closely mimic classical spherical micelles in nearly every respect (i.e., shape, size, number of surface groups, and area/surface groups). A major difference, however, is that they are covalently fixed and robust compared to the dynamic equilibrating nature of micelles. This difference is a significant advantage when using these microdomains as encapsulation devices. 
     As further concentric generations are added beyond five, congestion of the surface occurs. This congestion can lead to increased barrier characteristics at the surface and manifests itself as a smaller surface area per head (surface) group as shown in Table II. 
     
                                           TABLE II                                
__________________________________________________________________________
PAMAM Dendrimer Features vs. Generation                                   
Generations                                                               
          1    2    3    4     5   6    7    8    9                       
__________________________________________________________________________
# of surface                                                              
          3    6    12   24    48  96   192  384  768                     
groups, Z                                                                 
Molecular wt.                                                             
          275  875  2411 5147  10,619                                     
                                   21,563                                 
                                        43,541                            
                                             87,227                       
                                                  174,779                 
Diameter* 10.4Å                                                       
               15.8Å                                                  
                    22Å                                               
                         31Å                                          
                               40Å                                    
                                   53Å                                
                                        67Å                           
                                             76Å                      
                                                  88Å                 
measured SEC                                                              
Surface area per                                                          
          366Å.sup.2                                                  
               783Å.sup.2                                             
                    1519Å.sup.2                                       
                         3018Å.sup.2                                  
                               5024Å.sup.2                            
                                   8,820Å.sup.2                       
                                        14,096Å.sup.2                 
                                             18,136Å.sup.2            
                                                  24,328Å.sup.2       
dendrimer                                                                 
Surface area per Z                                                        
          122Å.sup.2                                                  
               131Å.sup.2                                             
                    127Å.sup.2                                        
                         126Å.sup.2                                   
                               104Å.sup.2                             
                                   92Å.sup.2                          
                                        73Å.sup.2                     
                                             47Å.sup.2                
                                                  32Å.sup.2           
group                                                                     
Distance between                                                          
          12.4Å                                                       
               12.8Å                                                  
                    12.7Å                                             
                         12.6Å                                        
                               11.5Å                                  
                                   10.8Å                              
                                        9.8Å                          
                                             7.75Å                    
                                                  6.28Å               
Z groups                                                                  
Void Volume                                                               
          311.6Å.sup.3                                                
               1,470.2Å.sup.3                                         
                    4,737.9Å.sup.3                                    
                         11,427.0Å.sup.3                              
                               --  --   --   --   --                      
__________________________________________________________________________
 *Hydrodynamic diameters determined by size exclusion chromatogaphy       
 measurements calibrated against monodisperse (Mw/Mn = 1.02)              
 polyethyleneoxide standards.                                             
 1Å = 10.sup.-1 nm; 1Å.sup.2 = 10.sup.-2 nm.sup.2 ; 1Å.sup.3 =
 10.sup.-3 nm.sup.3.                                                      
 
    
     For example, amine terminated generations 5.0, 6.0, 7.0, 8.0 and 9.0 have decreased surface areas of 104, 92, 73, 47 and 32Å 2  per Z group, respectively. This characteristic corresponds to a transition from a less congested micelle-like surface to a more congested bi-layer/monolayer barrier-like surface normally associated with vesicles (liposomes) or Langmuir-Blodgett type membranes. 
     If this surface congestion is occurring, the change in physical characteristics and morphology should be observed as the generations increase from the intermediate generations (6-8) to the more advanced generations (9 or 10). The scanning transmission electron micrographs (STEM) for generations=7.0, 8.0 and 9.0 were obtained after removing the methanol solvent from each of the samples to provide colorless, light yellow solid films and staining with osmium tetraoxide. The morphological change predicted occurred at the generation (G)=9.0 stage. The hollow interior at G=9.0 measures about 63Å in diameter and is surrounded by a darkened rim which is about 22Å thick. Apparently methanolic solvent has been entrapped within the 22Å outer membrane-like barrier to provide the colorless interior. Thus, at G=9.0, the STARBURST™ PAMAM is behaving topologically like a vesicle (liposome). However, this STARBURST™ is an order of magnitude smaller and very monodispersed compared to a liposome and is much more physically stable than a liposome. Consequently, the present dendrimers can be used to molecularly encapsulate solvent filled void spaces of as much diameter as about 63Å (volume about 131,000Å 3 ) or more. These micelle sized prototypes appear to behave like a covalently fixed liposome in this advanced generation stage. This behavior enables these prototypes to have additional capability as carriers for, for example, non-chelating radionuclides in STARBURST™ antibody conjugates for the treatment of various mammalian diseases. 
     Since the number of functional groups on the dendrimers can be controlled on the surface and within the interior, it also provides a means for controlling the amount of carried material to be delivered per dendrimer. In one embodiment, the dendrimers are targeted carriers of agents, capable of delivering the carried material, for example, a bioactive agent, to, for example, a plant or pest or a particular determinant or locus in a target organism. 
     Dendrimers suitable for use in the conjugates of the present invention include the dense star polymers or STARBURST™ polymers described in U.S. Pat. Nos. 4,507,466, 4,558,120, 4,568,737 and 4,587,329, which are hereby incorporated by reference. 
     In particular, the present invention concerns a STARBURST™ conjugate which comprises at least one STARBURST™ polymer associated with at least one carried agricultural, pharmaceutical, or other material. STARBURST™ conjugates included within the scope of the present invention include those represented by the formula: 
     
         (P).sub.x *(M).sub.y                                       (I) 
    
     wherein: 
     each P represents a dendrimer; 
     x represents an integer of 1 or greater; 
     each M represents a unit (for example, a molecule, atom, ion, and/or other basic unit) of a carried material, said carried material can be the same carried material or a different carried material, preferably the carried material is a bioactive agent; 
     y represents an integer of 1 or greater; and 
     * indicates that the carried material is associated with the dendrimer; and 
     with the proviso that the carried material maintains its effectiveness. 
     Preferred STARBURST™ conjugates of formula (I) are those in which M is a drug, pesticide, radionuclide, chelant, chelated metal, toxin, antibody, antibody fragment, antigen, signal generator, for example, fluorescing entities, signal reflector, for example, paramagnetic entities, or signal absorber, for example, electron beam opacifiers, fragrance, pheromones, or dyes. The terms &#34;signal generator&#34; and &#34;signal absorber&#34; are well known to one skilled in the art. A signal generator may be defined as an entity which emits a detectable amount of energy in the form of electromagnetic radiation (such as X-rays, UV radiation, IR radiation and the like) and include phosphorescent and fluorescent entities, and gamma and X-ray emitters, or matter (such as neutrons, positrons, β-particles, α-particles, and the like) and include radionuclides, positron emitters and the like. A signal absorber may be defined as an entity which absorbs a detectable amount of energy in the form of electromagnetic radiation or matter. Some examples are dyes, contrast agents, and boron (which absorbs neutrons). A given entity can be both a signal absorber and a signal generator, i.e., fluorescent or phosphorescent substances can absorb light and emit light; boron absorbs neutrons and emits radiation, and many other such examples. The term pheromone is a well known to those skilled in the art and is defined and exemplified, for example, in the McGraw-Hill Encyclopedia of Science &amp; Technology, 7th ed., Vol. 13, pg 360-361; and &#34;Insect Pheromone Technology:Chemistry and Applications&#34;, ACS Symposium Series 190 (1982). Examples of suitable fragrances, such as perfumes and flavors, are well known to those skilled in the art, and are illustrated and defined further in Perfume and Flavor Chemicals, Vols. I and II, by Steffen Arctander, pub. Montclair, N.J. (1969). Dyes and dye moieties are well known to those skilled in the art, and are illustrated and defined in, for example, Webster&#39;s Third New International Dictionary, pp 706-710, pub. G. C. Merriam Company (1981); Kirk-Othmer Encyclopedia of Chemical Technology, Vol. 8, pp 151-406, 3rd ed., pub. John Wiely &amp; Sons (1979). It is particularly preferred that x=1 and y=2 or more. 
     Also included are STARBURST™ conjugates of formula (I) wherein the STARBURST™ dendrimers are covalently linked together, STARBURST™ bridged dendrimers, optionally via linking groups, so as to form polydendritic assemblages (i.e., where x&gt;1). Uses of these STARBURST™ bridged dendrimers include topical controlled release agents, radiation synovectomy, and others. 
     As used herein, &#34;associated with&#34; means that the carried material(s) can be physically encapsulated or entrapped within the core of the dendrimer, dispersed partially or fully throughout the dendrimer, or attached or linked to the dendrimer or any combination thereof, whereby the attachment or linkage is by means of covalent bonding, hydrogen bonding, adsorption, absorption, metallic bonding, van der Waals forces or ionic bonding, or any combination thereof. The association of the carried material(s) and the dendrimer(s) may optionally employ connectors and/or spacers to facilitate the preparation or use of the STARBURST™ conjugates. Suitable connecting groups are groups which link a targeting director (i.e., T) to the dendrimer (i.e., P) without significantly impairing the effectiveness of the director or the effectiveness of any other carried material(s) (i.e., M) present in the STARBURST™ conjugate. These connecting groups may be cleavable or non-cleavable and are typically used in order to avoid steric hindrance between the target director and the dendrimer, preferably the connecting groups are stable (i.e., non-cleavable). Since the size, shape and functional group density of the STARBURST™ dendrimers can be rigorously controlled, there are many ways in which the carried material can be associated with the dendrimer. For example, (a) there can be covalent, coulombic, hydrophobic, or chelation type association between the carried material(s) and entities, typically functional groups, located at or near the surface of the dendrimer; (b) there can be covalent, coulombic, hydrophobic, or chelation type association between the carried material(s) and moieties located within the interior of the dendrimer; (c) the dendrimer can be prepared to have an interior which is predominantly hollow allowing for physical entrapment of the carried materials within the interior (void volume), (e.g. magnetic or paramagnetic cores or domains created by the chelation and reduction of metal ions to the zero valence state within the dendrimer), these dendrimers containing magnetic interiors can be used for harvesting various bioactive entities that can be complexed with various dendrimer surfaces by use of magnets and the like, wherein the release of the carried material can optionally be controlled by congesting the surface of the dendrimer with diffusion controlling moieties; or (d) various combinations of the aforementioned phenomena can be employed. 
     Dendrimers, herein represented by &#34;P&#34;, include the dense star polymers described in U.S. Pat. No. 4,507,466, 4,558,120, 4,568,737 or 4,587,329. 
     In a preferred embodiment, the carried materials, herein represented by &#34;M&#34;, are pharmaceutical materials. Such materials which are suitable for use in the STARBURST™ conjugates include any materials for in vivo or in vitro use for diagnostic or therapeutic treatment of mammals which can be associated with the dense star dendrimer without appreciably disturbing the physical integrity of the dendrimer, for example, but not limited to: 
     drugs, such as, but not limited to, antibiotics, analgesics, antihypertensives, cardiotonics, and the like; examples are acetaminophen, acyclovir, alkeran, amikacin, ampicillin, aspirin, bisantrene, bleomycin, neocardiostatin, chloroambucil, chloramphenicol, cytarabine, daunomycin, doxorubicin, fluorouracil, gentamycin, ibuprofen, kanamycin, meprobamate, methotrexate, novantrone, nystatin, oncovin, phenobarbital, polymyxin, probucol, procarbazine, rifampin, streptomycin, spectinomycin, symmetrel, thioguanine, tobramycin, trimethoprim, and valban; 
     toxins, such as, but not limited to, diphtheria toxin, gelonin, exotoxin A, abrin, modeccin, ricin, or toxic fragments thereof; 
     metal ions, such as, but not limited to, the metals in the Periodic Table Groups VIIIA (Fe, Co, Ni, Ru, Rh, Pd, Os, Ir, Pt), IVB (Pb, Sn, Ge), IIIA (Sc, Y, lanthanides and actinides), IIIB (B, Al, Ga, In, Tl), IA alkali metals (Li, Na, K, Rb, Cs, Fr), and IIA alkaline-earth metals (Be, Mg, Ca, Sr, Ba, Ra) and transition metals; 
     radionuclides, such as, but not limited to, those generated from actinides or lanthanides or other similar transition elements or from other elements, such as  47  Sc,  67  Cu,  67  Ga,  82  Rb,  89  Sr,  88  Y,  90  Y,  99m  Tc,  105  Rh,  109  Pd,  111  In,  115m  In,  125  I,  131  I,  140  Ba,  140  La,  149  Pm,  153  Sm,  159  Gd,  166  Ho,  175  Yb,  177  Lu,  186  Re,  188  Re,  194  Ir, and  199  Au, preferably  88  Y,  90  Y,  99m  Tc,  125  I,  131  I,  153  Sm,  166  Ho,  177  Lu,  186  Re,  67  Ga,  111  In,  115m  In, and  140  La; 
     signal generators, which includes anything that results in a detectable and measurable perturbation of the system due to its presence. A signal generator may be defined as an entity which emits a detectable amount of energy in the form of electromagnetic radiation (such as X-rays, ultraviolet (UV) radiation, infrared (IR) radiation and the like) and include phosphorescent and fluorescent entities, and gamma and X-ray emitters, or matter (such as neutrons, positrons, β-particles, α-particles, and the like) and include radionuclides, positron emitters and the like; such as, but not limited to, fluorescing entities, phosphorescence entities and radiation, such as radionuclides, particles and radiation sources; 
     signal reflectors, such as, but not limited to, paramagnetic or magnetic entities, for example, Fe, Gd, or Mn, nitroxyl radicals, NMR shift reagents such as Eu or Pr salts; 
     chelated metal, such as, but not limited to, any of the metals or their ions given above, whether or not they are radioactive, i.e. contrast agents, paramagnetic or magnetic entities, metals ions, when associated with a chelant; 
     signal absorbers may be defined as an entity which absorbs a detectable amount of energy in the form of electromagnetic radiation or matter. Some examples are dyes, contrast agents, electron beam opacifiers, aromatic UV absorber, and boron (which absorbs neutrons). A given entity can be both a signal absorber and a signal generator, i.e., fluorescent or phosphorescent substances can absorb light and emit light; boron absorbs neutrons and emits radiation, and many other such examples; i.e. such as, but not limited to, contrast agents, for example, Gd, Mn or Fe, and electron beam opacifiers such as Pb or Fe; 
     antibodies, including monoclonal antibodies and anti-idiotype antibodies; 
     antibody fragments; 
     hormones; 
     biological response modifiers such as, but not limited to, interleukins, interferons, viruses and viral fragments; 
     diagnostic opacifiers, such as a signal absorbers above, biological stains and the like; 
     fluorescent moieties; and 
     scavenging agents such as chelants, antigens, antibodies or any moieties capable of selectively scavenging therapeutic or diagnostic agents. 
     In another embodiment, the carried materials, herein represented by &#34;M&#34;, are agricultural materials. Such materials which are suitable for use in the STARBURST™ conjugates include any materials for in vivo or in vitro treatment, diagnosis, or application to plants or non-mammals (including microorganisms) which can be associated with the STARBURST™ dendrimer without appreciably disturbing the physical integrity of the dendrimer. For example, the carried materials can be 
     toxins, such as, but not limited to, diphtheria toxin, gelonin, exotoxin A, abrin, modeccin, ricin, or toxic fragments thereof; 
     metal ions, such as described above for pharmaceutical carried materials; 
     radionuclides, such as those generated from actinides or lanthanides or other similar transition elements or from other elements, such as  47  Sc,  67  Cu,  67  Ga,  82  Rb,  89  Sr,  88  Y,  90  Y,  99m  Tc,  105  Rh,  109  Pd,  111  In,  115m  In,  125  I,  131  I,  140  Ba,  140  La,  149  Pm,  153  Sm,  159  Gd,  166  Ho,  175  Yb,  177  Lu,  186  Re,  188  Re,  194  Ir, and  199  Au, or those as described above for pharmaceutical carried materials; 
     signal generators, which includes anything that results in a detectable and measurable perturbation of the system due to its presence, such as fluorescing entities, phosphorescence entities and radiation, or those as described above for pharmaceutical carried materials; 
     signal reflectors, such as paramagnetic entities, for example, Fe, Gd, or Mn, or those as described above for pharmaceutical carried materials; 
     signal absorbers, such contrast agents and as electron beam opacifiers, for example, Fe, Gd, or Mn or those as described above for pharmaceutical carried materials; 
     pheromones or hormones; 
     biological response modifiers, such as interleukins, interferons, viruses and viral fragments; pesticides, including antimicrobials, algicides, arithelmetics, acaricides, insecticides, attractants, repellants, herbicides and/or fungicides, such as acephate, acifluorfen, alachlor, atrazine, benomyl, bentazon, captan, carbofuran, chloropicrin, chlorpyrifos, chlorsulfuron, cyanazine, cyhexatin, cypermithrin, 2,4-dichlorophenoxyacetic acid, dalapon, dicamba, diclofop methyl, diflubenzuron, dinoseb, endothall, ferbam, fluazifop, glyphosate, haloxyfop, malathion, naptalam, pendimethalin, permethrin, picloram, propachlor, propanil, sethoxydin, temephos, terbufos, trifluralin, triforine, zineb, and the like; and 
     scavenging agents such as chelants, chelated metal (whether or not they are radioactive) or any moieties capable of selectively scavenging therapeutic or diagnostic agents. 
     In another embodiment, the carried material, herein represented by &#34;M&#34;, are immuno-potentiating agents. Such materials which are suitable for use in the STARBURST™ conjugates include any antigen, hapten, organic moiety or organic or inorganic compounds which will raise an immuno-response which can be associated with the STARBURST™ dendrimers without appreciably disturbing the physical integrity of the dendrimers. For example, the carried materials can be synthetic peptides used for production of vaccines against malaria (U.S. Pat. No. 4,735,799), cholera (U.S. Pat. No. 4,751,064) and urinary tract infections (U.S. Pat. No. 4,740,585), bacterial polysaccharides for producing antibacterial vaccines (U.S. Pat. No. 4,695,624) and viral proteins or viral particles for production of antiviral vaccines for the prevention of diseases such as AIDS and hepatitis. 
     The use of STARBURST™ conjugates as carriers for immuno-potentiating agents avoids the disadvantages of ambiguity in capacity and structure associated with conventionally known or synthetic polymer conjugates used to give a macromolecular structure to the antigen-carrier. Use of the STARBURST™ dendrimers as carriers for immuno-potentiating agents, allows for control of the size, shape and surface composition of the conjugate. These options allow optimization of antigen presentation to an organism, thus resulting in antibodies having greater selectivity and higher affinity than the use of conventional adjuvants. It may also be desirable to connect multiple antigenic peptides or groups to the STARBURST™ dendrimer, such as attachment of both T- and B-cell epitopes. Such a design would lead to improved vaccines. 
     It may also be desirable to conjugate pesticides or pollutants capable of eliciting an immune response, such as those containing carbamate, triazine or organophosphate constituents, to a STARBURST™ dendrimer. Antibodies produced to the desired pesticide or pollutant can be purified by standard procedures, immobilized on a suitable support and be used for subsequent detection of the pesticide or pollutant in the environment or in an organism. 
     In a further embodiment, the carried materials, herein represented by &#34;M&#34;, which are suitable for use in the STARBURST™ conjugates include any materials other than agricultural or pharmaceutical materials which can be associated with the STARBURST™ dendrimer without appreciably disturbing the physical integrity of the dendrimer, for example: 
     metal ions, such as the alkali and alkaline-earth metals, magnetic interiors, or as for the pharmaceutical carried material as defined before; 
     signal generators, which includes anything that results in a detectable and measurable perturbation of the system due to its presence, such as fluorescing entities, phosphorescence entities and radiation sources, or as for the pharmaceutical carried material as defined before; 
     signal reflectors, such as paramagnetic entities, for example, Fe, Gd, or Mn, or as for the pharmaceutical carried material as defined before; 
     signal absorbers, such as contrast agents and an electron beam opacifiers, for example, Fe, Gd, or Mn; pheromone moieties, or as for the pharmaceutical carried material as defined before; 
     fragrance moieties; 
     dye moieties; and the like; and 
     scavenging agents such as chelants or any moieties capable of selectively scavenging a variety of agents. 
     Preferably the carried materials are bioactive agents. As used herein, &#34;bioactive&#34; refers to an active entity such as a molecule, atom, ion and/or other entity which is capable of detecting, identifying, inhibiting, treating, catalyzing, controlling, killing, enhancing or modifying a targeted entity such as a protein, glycoprotein, lipoprotein, lipid, a targeted cell, a targeted organ, a targeted organism [for example, a microorganism, plant or animal (including mammals such as humans)] or other targeted moiety. 
     The STARBURST™ conjugates of formula (I) are prepared by reacting P with M, usually in a suitable solvent, at a temperature which facilitates the association of the carried material (M) with the STARBURST™ dendrimer (P). 
     Suitable solvents are solvents in which P and M are at least partially miscible and inert to the formation of the conjugate. If P and M are at least partially miscible with each other, no solvent may be required. When desired, mixtures of suitable solvents can be utilized. Examples of such suitable solvents are water, methanol, ethanol, chloroform, acetonitrile, toluene, dimethylsulfoxide and dimethylformamide. 
     The reaction conditions for the formation of the STARBURST™ conjugate of formula (I) depend upon the particular dendrimer (P), the carried material (M), and the nature of the bond (*) formed. For example, if P is the PEI (polyethyleneimine) STARBURST™ dendrimer with a methylene carboxylate surface, M is a radionuclide, e.g. yttrium, then the reaction is conducted at room temperature in water. However, if P is an ester terminated polyamidoamine (PAMAM) STARBURST™ dendrimer, M is aspirin, then the reaction is conducted at room temperature in chloroform. Typically, the temperature can range from room temperature to reflux. The selection of the particular solvent and temperature will be apparent to one skilled in the art. 
     The ratio of M:P will depend on the size of the dendrimer and the amount of carried material. For example, the molar ratio (ratio of moles) of any ionic M to P usually is 0.1-1,000:1, preferably 1-50:1, and more preferably 2-6:1. The weight ratio of any drug, pesticide, organic or toxin M to P usually is 0.1-5:1, and preferably 0.5-3:1. 
     When M is a radionuclide, there are three ways the STARBURST™ conjugate can be prepared, namely: (1) P can be used as a chelant. For example, a methylene-carboxylate surface PEI or PAMAM will chelate a metal such as yttrium or indium. (2) A chelate can be covalently bonded to P. For example, an amine terminated PEI STARBURST™ dendrimer can be reacted with 1-(p-isothiocyanatobenzyl)diethylenetriaminepentaacetic acid and then chelated, or a complex such as rhodium chloride chelated with isothiocyanatobenzyl-2,3,2-tet can be reacted. (3) A prechelated radionuclide can be associated with P by hydrophobic or ionic interaction. 
     Other STARBURST™ conjugates, which are particularly preferred for use with pharmaceutical materials, are those conjugates which contain a target director (herein designated as &#34;T&#34;) and which are represented by the formula: 
     
         (T).sub.e *(P).sub.x *(M).sub.y                            (II) 
    
     wherein: 
     each T represents a target director; 
     e represents an integer of 1 or greater; and 
     P, x, *, M, and y are as previously defined herein; and 
     with the proviso that M maintains its effectiveness. 
     Preferred among the STARBURST™ conjugates of formula (II) are those in which M is a drug, pesticide, radionuclide, chelator, chelated metal, toxin, signal generator, signal reflector, or signal absorber. Also preferred conjugates are those conjugates in which e=1 or 2; and those in which x=1 and y=2 or more. Particularly preferred conjugates are those in which x=1, e=1, y=2 or more and M and T are associated with the polymer via the same or different connectors. 
     The STARBURST™ conjugates of formula (II) are prepared either by forming T*P and then adding M or by forming P*M and then adding T. Either reaction scheme is conducted at temperatures which are not detrimental to the particular conjugate component and in the presence of a suitable solvent when required. To control pH, buffers or addition of suitable acid or base is used. The reaction conditions are dependent on the type of association formed (*), the STARBURST™ dendrimer used (P), the carried material (M), and the target director (T). For example, when T is a monoclonal antibody and M is a radionuclide, the T*P association is done through a functional group such as an isothiocyanate in water or in water with an organic modifier such as acetonitrile or dimethylformamide. Usually, the conjugation is done in a buffer at pH 7-10, preferably pH 8.5-9.5. The formed conjugate is then chelated with a radionuclide such as yttrium acetate, preferably at room temperature. Alternatively, P and M can be chelated, usually in water, before conjugation to T. The conjugation with T is carried out in a suitable buffer. 
     The ratio of T:P is preferably 1:1, especially when T is an antibody or fragment. The ratio of M:P will be as before. 
     Target directors capable of targeting the STARBURST™ conjugates are entities which when used in the STARBURST™ conjugates of the present invention result in at least a portion of the STARBURST™ conjugates being delivered to a desired target (for example, a protein, glycoprotein, lipoprotein, lipid, a targeted cell, a targeted organ, a targeted organism or other targeted moiety) and include antibodies, preferably monoclonal antibodies, antibody fragments such as Fab, Fab&#39;, F(ab&#39;) 2  fragments or any other antibody fragments having the requisite target specificity, hormones, biological response modifiers; epitopes; chemical functionalities exhibiting target specificity; and the like. 
     The antibodies or antibody fragments which may be used in preferred STARBURST™ conjugates described herein can be prepared by techniques well known in the art. Highly specific monoclonal antibodies can be produced by hybridization techniques well known in the art, see, for example, Kohler and Milstein (1975, Nature 256:495-497; and 1976, Eur. J. Immunol. 6:511-519). Such antibodies normally have a highly specific reactivity. 
     In the antibody targeted STARBURST™ conjugates, antibodies directed against any antigen or hapten may be used. Although conventional polyclonal antibodies may be used, monoclonal antibodies offer several advantages. Each monoclonal antibody is highly specific for a single epitope. In addition, large amounts of each monoclonal antibody can be produced. Antibodies used in the present invention may be directed against, for example, tumors, bacteria, fungi, viruses, parasites, mycoplasma, differentiation and other cell membrane antigens, nucleic acids such as DNA or RNA, pathogen surface antigens, toxins, enzymes, allergens, drugs and any biologically active molecules. For a more complete list of antigens see U.S. Pat. No. 4,193,983. 
     It may be desirable to connect more antibodies or fragments to the dendrimer, and in particular instances to connect antibodies of different specificities. For example, a bifunctional conjugate which has the ability to localize and bind to a tumor and then scavenge circulating cytotoxic, diagnostic, or biostatic compounds can be designed. 
     In the absence of a target director (or in the presence of a target director if desired), due to the number of functional groups which can be located at or near the surface of the dendrimer, all or a substantial portion of such functional groups can be made anionic, cationic, hydrophobic or hydrophilic to effectively aid delivery of the STARBURST™ conjugate to a desired target of the opposite charge or to a hydrophobic or hydrophilic compatible target. 
     Preparation of the conjugates of formula (II) using a P with a protected handle (S) is also intended as a process to prepare the conjugates of formula (II). The reaction scheme is shown below: ##STR1## where S*P represents the protected dendrimer; 
     S*P*M represents the protected dendrimer conjugated with M; 
     P*M represents the dendrimer conjugated with M (STARBURST™ conjugate); 
     T*P*M represents the STARBURST™ conjugate linked to the target director. 
     Suitable solvents can be employed which do not effect P*M. For example when S is t-butoxycarbonyl, S can be removed by aqueous acid. 
     Also preferred when the carried materials are pharmaceutical materials are STARBURST™ conjugates in which the polymer is associated directly, or via connectors; these STARBURST™ conjugates are represented by the formula: 
     
         [(T).sub.e --(C&#39;).sub.f ].sub.g *(P).sub.x *[(C&#34;).sub.h --(M).sub.y ].sub.k(III) 
    
     wherein: 
     each C&#39; represents the same or different connecting group; 
     each C&#34; represents the same or different connecting group; 
     g and k each individually represent an integer of 1 or greater; 
     f and h each individually represent an integer of 0 or greater; 
     -- indicates a covalent bond in instances where a connecting group is present; and 
     P, x, *, M, y, T, and e are as previously defined herein; and 
     with the proviso that M maintains its effectiveness. 
     Preferred among the STARBURST™ conjugates of formula (III) are those in which M is a radionuclide, drug, toxin, signal generator, signal reflector or signal absorber. Also preferred are those conjugates in which x=1. Particularly preferred conjugates are those in which x, e, f, h, and y are each 1, and g is 1 or more and k is 2 or more. Most preferred are those conjugates in which x, e, f, h, y and g are each 1, and k is 2 or more. Also particularly preferred are those STARBURST™ conjugates in which M represents a bioactive agent such as a radionuclide, drug, or toxin. 
     Suitable connecting groups which are represented by C&#34; are groups which link the carried pharmaceutical material to the dendrimer without significantly impairing the effectiveness of the carried pharmaceutical material or the effectiveness of the target director(s) present in the STARBURST™ conjugate. These connectors must be stable (i.e., non-cleavable) or cleavable depending upon the mode of activity of the carried pharmaceutical material and are typically used in order to avoid steric hindrance between the carried pharmaceutical material and the polymer. 
     Most preferred are conjugates in which the dendrimer is associated directly, or via connecting group(s), to one antibody or antibody fragment. The polymer in these preferred conjugates may, in addition, be optionally associated either directly, or via connecting group(s), to one or more other carried materials, preferably a radioisotope. Such STARBURST™ conjugates are represented by the formula: 
     
         [(Antibody).sub.e --(C&#39;).sub.f ].sub.g *(P).sub.x *[(C&#34;).sub.h --(M).sub.y ].sub.k                                                   (IV) 
    
     wherein 
     each Antibody represents an antibody or antibody fragment capable of interacting with a desired epitope; 
     -- indicates a covalent or coulombic bond in instances where a connecting group is present; and 
     P, x, *, M, T, e, y, C&#39;, C&#34;, g, k, f, and h are as previously defined herein; and 
     with the proviso that M maintains its effectiveness. 
     For the above synthesis of STARBURST™ dendrimers (P) which have a functional group available for linking (C&#39; or C&#34;) with a targeting director (T), the preferred process requires that the reactive functionality be protected as a synthetic precursor. This protection is preferred because it enables the synthesis of dendrimer or conjugates of very high quality. This process allows for the chemical binding of a unit of carried pharmaceutical material (M) to the terminal functional groups of the STARBURST™ dendrimer (P) in ways which would otherwise result also in reaction with a linking functional group, thus making it impossible to attach to the targeting director (T). Subsequent deprotection or synthetic conversion into the desired linking functional group thus enables the STARBURST™ conjugate to be linked to the targeting director. 
     One of the preferred &#34;functional groups for linking&#34; (hereafter referred to as a &#34;handle&#34;) is an aniline moiety. This group is preferred because it can be used directly for linking to the targeting director, or it can be readily modified to other functional groups suitable for reaction with the targeting director, e.g. isothiocyanate, isocyanate, semithiocarbazide, semicarbazide, bromoacetamide, iodoacetamide, and maleimide. The aniline moiety is also preferred as a handle for linking with the targeting directors because it can be readily protected for use in STARBURST™ dendrimer synthesis, or the nitro group can be used as a precursor which can be converted into the desired amino function at the end of the synthesis. 
     There are a number of protecting groups which are suitable for protecting the anilino amino functionality during STARBURST™ dendrimer synthesis. (See Theodora W. Green, Protective Groups In Organic Synthesis., Pub. John Wiley &amp; Son, New York, 1981). A preferred class of protecting groups are the carbamates shown below, wherein R&#39; represents a dendrimer. ##STR2## Many carbamates have been used for protection of amines. The most preferred carbamates for STARBURST™ dendrimer synthesis is the t-butoxycarbamate, R=--C(CH 3 ) 3 . Deprotection is achieved by mild acid hydrolysis. Also preferred is the benzylcarbamate protecting group, ##STR3## which is preferred when the dendrimer is susceptible to acid hydrolysis. Deprotection is achieved by catalytic hydrogenation. 
     Other protecting groups which can be used for protecting the reactive moieties at the desired generation level include 9-fluorenylmethylcarbamate ##STR4## and the phthalimide protecting group, ##STR5## 
     Other protecting groups used for amines which are well known in the literature could also be used in this synthetic scheme. The above preferences are given as illustrative examples only but are not the only protecting groups which can be used. Any protecting group which is stable under the reaction conditions and can be removed without altering the integrity of the STARBURST™ dendrimer can be employed. 
     An alternate process involves the reaction of an activated aryl halide, e.g. 4-nitrofluorobenzene, 2,4-dinitrofluorobenzene, with an amino-function on the agent for conjugation, e.g. STARBURST™ polyethyleneimines (PEI), and subsequent catalytic hydrogenation of the nitro group to the aniline functionality for subsequent conjugation. It is particularly useful for agents, e.g. polyamines, which need further modification prior to use, due to the relative chemical inertness of the nitrophenyl functionality to all non-reducing reaction conditions. Various coupling reagents suitable for the conjugation reaction are well known in the art, such as those discussed in European Published Application 0430863, published Jun. 5, 1991. The more common bifunctional linking agents, e.g. active esters or diisocyanates, which are reactive under a large number of reaction conditions and which would render them usable for conjugation, include: ##STR6## 
     The invention also includes the use of nitro-substituted arylsulphonyl halides to give sulphonamides, ##STR7## 
     The advantage of this process over known processes of introducing an aminophenyl group for conjugation is that it takes place at a late stage of the synthesis. Gansow et al., U.S. Pat. No. 4,472,509, in his process introduced the nitrophenyl group at the first step of a long synthetic procedure, thereby having limitations on the chemistry available. 
     This process also introduces a handle which is clearly differentiable from the remainder of the molecule. Manabe et al., disclosed that the ring opening of succinic anhydride by residual amines gave a coupling group through which conjugation to an antibody was possible. This method however gave no means of differentiating between any unchelated sites on the polymer, since the chelating groups were the same as the linking group. 
     The above process can introduce an aminophenyl functionality into any agent containing an amino group which is then conjugated with some bioactive agent, e.g. monoclonal antibody or enzyme. The agent can be conjugated by oxidative coupling to carbohydrates on the bioactive agent, e.g. antibody. The aminophenyl group also can be converted into an isothiocyanate or isocyanate for subsequent reaction with the pendant amino groups of lysine residues on the bioactive agent. 
     The present process also provides for direct chelation of lanthanides with STARBURST™ dendrimers, preferably by PEI acetate dendrimer. In contrast, Denkewalter, U.S. Pat. No. 4,289,872, states that just putting acetates on the surface works. However, the present reaction shows that PEI acetate, works much better than PAMAM, i.e., surface of iminodiacetates is only part of the story, the nature of the backbone, and branching is very important as well. The PEI acetate has better chelating properties than the PAMAM acetate. ##STR8## 
     Preferred among the STARBURST™ conjugates of formula (IV) are those in which M is a radionuclide, drug, toxin, signal generator, signal reflector or signal absorber. Also preferred are those conjugates in which x=1. Particularly preferred are those conjugates in which x, e, f, h, and y are each 1, and g is 1 or more and k is each individually 2 or more. Most preferred are those conjugates in which x, e, f, h, y, and g are each 1, and k is 2 or more. Also particularly preferred are those STARBURST™ conjugates in which &#34;Antibody&#34; represents a monoclonal antibody or an epitope binding fragment thereof; and especially preferred are those in which M represents a bioactive agent such as a radionuclide, drug, or toxin. 
     The STARBURST™ conjugates can be used for a variety of in vitro or in vivo diagnostic applications such as radioimmunoassays, electron microscopy, enzyme linked immunosorbent assays, nuclear magnetic resonance spectroscopy, contrast imaging, and immunoscintography, in analytical applications, in therapeutic applications as a carrier of antibiotics, radionuclides, drugs or other agents suitable for use in the treatment of disease states such as cancer, autoimmune diseases, genetic defects, central nervous system disorders, infectious diseases, and cardiac disorders; in biological control applications as a means of delivering pesticides such as herbicides, fungicides, repellants, attractants, antimicrobials or other toxins; or used as starting materials for making other useful agents. 
     The present invention is also directed to STARBURST™ conjugate compositions in which the STARBURST™ conjugates are formulated with other suitable vehicles. The STARBURST™ conjugate compositions may optionally contain other active ingredients, additives and/or diluents. 
     In the agricultural materials embodiment of the invention, the STARBURST™ conjugates can be formulated with suitable vehicles useful in agriculture such as on crops, fallow land, or as pesticides, or in treatment of in vivo or in vitro testing of non-mammals. An agriculturally acceptable carrier or diluent which may also be present with one or more STARBURST™ conjugates of the present invention includes those carriers or diluents customarily used in granular formulations, emulsifiable concentrates, solutions, or suspensions such as, for example, toluene, xylene, benzene, phenol, water, methane, hydrocarbons, naphthalene and others. 
     The preferred STARBURST™ polymer for use in the STARBURST™ conjugates of the present invention is a polymer that can be described as a STARBURST™ polymer having at least one branch (hereinafter called a core branch), preferably two or more branches, emanating from a core, said branch having at least one terminal group provided that (1) the ratio of terminal groups to the core branches is more than one, preferably two or greater, (2) the density of terminal groups per unit volume in the polymer is at least 1.5 times that of an extended conventional star polymer having similar core and monomeric moieties and a comparable molecular weight and number of core branches, each of such branches of the extended conventional star polymer bearing only one terminal group, and (3) a molecular volume that is no more than about 80 percent of the molecular volume of said extended conventional star polymer as determined by dimensional studies using scaled Corey-Pauling-Koltun (CPK) molecular models. As used herein, the term &#34;dense&#34; as it modifies &#34;star polymer&#34; or &#34;dendrimer&#34; means that it has a smaller molecular volume than an extended conventional star polymer having the same molecular weight. The extended conventional star polymer which is used as the base for comparison with the dense star polymer is one that has the same molecular weight, same core and monomeric components and same number of core branches as the dense star polymer. By &#34;extended&#34; it is meant that the individual branches of the conventional star polymer are extended or stretched to their maximum length. In addition while the number of terminal groups is greater for the dense star polymer molecule than in the conventional star polymer molecule, the chemical structure of the terminal groups is the same. 
     Dendrimers used in the conjugates of the present invention can be prepared by processes known in the art. The above dendrimers, the various coreactants and core compounds, and process for their preparation can be as defined in U.S. Pat. No. 4,587,329. For example, core compounds may be anything on which a further X group may be attached. 
     The dendrimers, for use in the conjugates of the present invention, can have terminal groups which are sufficiently reactive to undergo addition or substitution reactions. Examples of such terminal groups include amino, hydroxy, mercapto, carboxy, alkenyl, nitrile, allyl, vinyl, amido, halo, urea, oxiranyl, aziridinyl, oxazolinyl, imidazolinyl, sulfonato, phosphonato, crown esters, bipyridines, chloromethylphenyl, isocyanato and isothiocyanato. The terminal groups can be modified to make them biologically inert, for example, to make them non-immunogenic or to avoid non-specific uptake in the liver, spleen or other organ. The dendrimers differ from conventional star or star-branched polymers in that the dendrimers have a greater concentration of terminal groups per unit of molecular volume than do conventional extended star polymers having an equivalent number of core branches and an equivalent core branch length. Thus, the density of terminal groups per unit volume in the dendrimer usually is at least 1.5 times the density of terminal groups in the conventional extended star polymer, preferably at least 5 times, more preferably at least 10 times, most preferably from 15 to 50 times. The ratio of terminal groups per core branch in the dense polymer is preferably at least 2, more preferably at least 3, most preferably from 4 to 1024. Preferably, for a given polymer molecular weight, the molecular volume of the dense star polymer is less than 70 volume percent, more preferably from 16 to 60, most preferably from 7 to 50 volume percent of the molecular volume of the conventional extended star polymer. 
     Preferred dendrimers for use in the conjugates of the present invention are characterized as having a univalent or polyvalent core that is covalently bonded to dendritic branches. Such ordered branching can be illustrated by the following sequence wherein G indicates the number of generations: ##STR9## 
     Mathematically, the precise sequencing involved in the preparation of dendrimers leads to precise, predictable stoichiometries within each dendrimer molecule. For example, the number of repeat units within a dendrimer depends on the number of generations (G), the multiplicity of the repeat unit (N r ), and the multiplicity of the core molecule or core atom (N c ). In a homopolymeric dendrimer, where the same repeat unit is employed throughout the molecule, the total number of repeat units within a dendrimer, its degree of polymerization (DP), is given by: ##EQU1## 
     The total valency or number of terminal moieties on the dendrimer (Y) is given by: 
     
         Y=N.sub.c N.sub.r.sup.G. 
    
     Often it is more convenient or intuitive to consider the number of terminal groups on the dendrimer (Z) because each group may contain a multiplicity of valencies or a number of valencies that varies in subsequent chemical modifications. Therefore, the number of terminal groups is defined by: ##EQU2## 
     This may be illustrated by the amine terminal group, which has a multiplicity of 2 in polyamine synthesis. In this case, the ##EQU3## 
     For subsequent modifications of dendrimers, this number is more convient because the valency of the amine group depends upon the chemistry employed. For example, a primary amine end group will react readily with only one molecule of ester to form an amide with two molecules of alkylating agent (under mild conditions, e.g. Eschweiler-Clarke conditions) to form a tertiary amine, or with three molecules of alkylating agent (under more rigorous conditions, e.g. reaction with alkylsulfates) to form a quaternary amine salt. 
     Accordingly, the dendrimers of this invention can be represented in its component parts as follows: ##EQU4## wherein the Core (I) is an initiator molecule or atom; Terminal Moiety is the atom or molecule which occupies the binding sites of the Repeat Unit, e.g. hydrogen or methoxy group of an ester; G and N c  are as defined before; and the Repeat Unit has a valency or functionality of N r  +1 wherein N r  is as defined before. 
     A copolymeric dendrimer which is a preferred dendrimer for the purposes of this invention is a unique compound constructed of polyfunctional monomer units in a highly branched (dendritic) array. The dendrimer molecule is prepared from a polyfunctional initiator unit (core compound, core (I), polyfunctional repeating units and terminal units which may be the same or different from the repeating units. The core compound is represented by the formula (I) (Z c ) Nc  wherein core (I) represents the core, Z c  represents the binding sites available on the core (I) and N c  represents the core functionality or number of binding sites, which is preferably 2 or more, most preferably 3 or more. Thus, the dendrimer molecule comprises a polyfunctional core, (I), bonded to a number (N c ) of functional groups, Z c , each of which is connected to the monofunctional tail of a repeating unit, X 1  Y 1  (Z 1 ) N .spsb.1, of the first generation and each of the Z groups of the repeating unit of one generation is bonded to a monofunctional tail of a repeating unit of the next generation until the terminal tier or layer is reached. 
     In the dendrimer molecule, the repeating units are the same within a single generation i, but may differ from generation to generation. In the repeating unit, X 1  Y 1  (Z 1 ) N .spsb.1, X 1  represents the monofunctional tail, of the first generation repeating unit, Y 1  represents the moiety constituting the first generation, Z 1  represents a binding site of the polyfunctional head of the repeating unit of the first generation and may be the same as or different from the binding sites of the core compound, (I) (Z c ) Nc , or other generations; and N 1  is a number of 2 or more, most preferably 2, 3 or 4, which represents the multiplicity of the polyfunctional head of the repeating unit in the first generation. Generically, the repeating unit is represented by the formula X i  Y i  (Z i ) N .spsb.i wherein &#34;i&#34; represents the particular generation from the first to the t-1 generation. Thus, in the preferred dendrimer molecule, each Z 1  of the first generation repeating unit is connected to an X 2  of a repeating unit of the second generation and so on through the generations such that each Z i  group for a repeating unit X i  Y i  (Z i ) N .spsb.i in generation number &#34;i&#34; is connected to the tail (X i+1 ) of the repeating unit of the generation number &#34;i+1&#34;. The final or terminal tier of a preferred dendrimer molecule comprises terminal units, X t  Y t  (Z t ) N .spsb.t wherein t represents a terminal unit and X t , Y t , Z t  and N t  and may be the same as or different from X i , Y i , Z i  and N i  except that there is no succeeding generation connected to the Z t  groups and N t  may be less than two, e.g., zero or one and all these terminal units, X t  Y t  (Z t ) N .spsb.t, need not be identical to each other. Therefore, the preferred dendrimer has a molecular formula represented by ##EQU5## wherein i is 1 to t-1; the symbols are as previously defined. The II function is the product of all the values between its defined limits. Thus ##EQU6## which is the number of repeat units, X i  Y i  (Z i ) N .spsb.i, comprising the ith generation of one dendritic branch 
     and when i is 1, then ##EQU7## 
     In copolymeric dendrimers, the repeat unit for one generation differs from the repeat unit in at least one other generation. The preferred dendrimers are very symmetrical as illustrated in structural formulas described hereinafter. Preferred dendrimers may be converted to functionalized dendrimers by contact with another reagent. For example, conversion of hydroxyl in the terminal layer or tier to ester by reaction with an acid chloride gives an ester terminally functionalized dendrimer. This functionalization need not be carried out to the theoretical maximum as defined by the number of available functional groups and, thus, a functionalized dendrimer may not have high symmetry or a precisely defined molecular formula as is the case with the preferred dendrimer. 
     In a homopolymeric dendrimer, all of the repeat units, X i  Y i  (Z i ) N .spsb.i, are identical. Since the values of all N i  are equal (defined as N r ), the product function representing the number of repeat units reduces to a simple exponential form. Therefore, the molecular formula may be expressed in simpler form as: ##EQU8## where i=1 to t-1 
     This form still shows the distinction between the different generations i, which each consist of N c  N r .sup.(i-1) repeating units, X i  Y i  (Z i ) N .spsb.i. Combining the generations into one term gives: ##EQU9## wherein X r  Y r  (Z r )N r  is the repeating unit which is used in all generations i. This formula is essentially the same as that on page 46, line 10 with G=t-1. 
     Consequently, if a polymer compound will fit into these above formulae, then the polymer is a STARBURST™ polymer. Conversely, if a polymer compound will not fit into these above formulae, then the polymer is not a STARBURST™ polymer. Also, to determine whether a polymer is a STARBURST™ polymer, it is not necessary to know the process by which it was prepared, but only whether it fits the formulae. The formulae also demonstrate the generations (G) or tiering of dendrimers. 
     Clearly, there are several ways to determine the ratio of agent (M) to dendrimer (P) which depend upon how and where the association of P*M occurs. When there is interior encapsulation, the weight ratio of M:P usually is 10:1, preferably 8:1, more preferably 5:1, most preferably 3:1. The ratio can be as low as 0.5:1 to 0.1:1. When interior stoichiometry is used, the weight ratio of M:P is the same as for interior encapsulation. When exterior stoichiometry is determined, the mole/mole ratio of M:P is given by the following formulae: 
     
         ______________________________________                                    
M                   :     P                                               
______________________________________                                    
(A) 5 N.sub.c N.sub.t N.sub.r.sup.t-1                                     
                      1                                                   
(B) 3 N.sub.c N.sub.t N.sub.r.sup.t-1                                     
                      1                                                   
(C) 1 N.sub.c N.sub.t N.sub.r.sup.t-1                                     
                      1                                                   
______________________________________                                    
 
    
     where N c  means the core multiplicity, N t  means the average terminal group multiplicity, and N r  means branch juncture multiplicity. The N c  N t  N r   t-1  term will result in the number of terminal moieties. Thus, for example, (A) above may result when proteins, enzymes or highly charged molecules are on the surface; (B) above when it is aspirin, 2,4-D or octanoic acid; (C) above when converting surface ester groups to carboxylate ions or groups. 
     Of course other structures of various dimensions can be readily prepared by one skilled in the art by appropriately varying the dendrimer components and number of generations employed. A roughly scaled comparison of three different dendrimer series relative to an IgG antibody is seen in FIG. 3. The series of drawings indicated by FIG. 3(B) I shows the STARBURST™ polyamidoamines (PAMAM); by II shows the STARBURST™ polyethers (PE); and by III shows the STARBURST™ polyethyleneimines (PEI). In a manner similar to that of FIG. 1, all three series (I, II and III) have their far left drawing showing the initiator core, the next drawing from the left showing the starbranch oligomer, and the remaining drawings showing the STARBURST™ oligomers, and respective STARBURST™ bridged dendrimers. It can be seen that in these series of scale drawings that the dendrimer dimensions are close to and in fact smaller than those noted for the whole IgG antibody FIG. 3(A). The IgG antibody is shown to the far left in FIG. 3. The scale is 1 mm=3.5Å. In FIG. 3(A) the variable region is shown by (A); the constant region by (B); and the carbohydrate attachment sites by (C). Approximate measurements shown on FIG. 3 are (1) is 35-40Å; (2) is 70Å; and (3) is 60Å. These dimensional properties are preferred for instance where targeting involves exiting from the vascular system. Therefore dendrimer diameters of 125 Angstrom units or less are particularly preferred in that they may allow exiting from the vascular system to targeted organs serviced by continuous or fenestrated capillaries. These dimensions are significant in that they are small compared to the size of a potential targeting component such as an antibody (see FIG. 3). A linear polymer of comparable molecular weight would have a radius of gyration, (in its fully extended form), that would be much larger than the same molecular weight dendrimer. A linear polymer of this type would be expected to adversely affect the molecular recognition properties of many accepted targeting components. It is also desirable that the conjugates be of minimum molecular volume so as not to discourage extravascularsation, e.g., by coupling Fab, Fab&#39;, Fab&#39; 2  or single chain or portions thereof, or other appropriate antibody fragment to low molecular volume dendrimers. 
     Dendrimers are desirable for the delivery of radionuclides or strongly paramagnetic metal ions to tumor sites because of their ability to chelate a number of metal ions in a small volume of space. Coupling to antibodies or antibody fragments which are specific for tumors may deliver a number of metals per antibody, with only a single modification to the antibody. 
     Linking target directors to dendrimers is another aspect of the present invention. In preferred embodiments of the present invention, particularly where it is desired to use an antibody as a target director, a reactive functional group such as a carboxyl, sulfhydryl, reactive aldehyde, reactive olefinic derivative, isothiocyanato, isocyanato, amino, reactive aryl halide, or reactive alkyl halide can conveniently be employed on the dendrimer. The reactive functional groups can be introduced to the dendrimer using known techniques, for example: 
     (1) Use of a heterofunctional initiator (as a starting material for synthesizing the dendrimer) which has incorporated into it functional groups of different reactivity. In such heterofunctional initiator at least one of the functional groups will serve as an initiation site for dendrimer formation and at least one of the other functional groups will be available for linking to a target director but unable to initiate dendrimer synthesis. For example, use of protected aniline allows further modification of NH 2  groups within the molecule, without reacting the aniline NH 2 . 
     The functional group which will be available for linking to a target director may be part of the initiator molecule in any one of three forms; namely: 
     (a) In the form in which it will be used for linking with the target director. This is possible when none of the synthetic steps involved in the dendrimer synthesis can result in reaction at this center. 
     (b) When the functional group used for linking to the targeting director is reactive in the synthetic steps involved in the dendrimer synthesis, it can be protected by use of a protecting group, which renders the group unreactive to the synthetic procedures involved, but can itself be readily removed in a manner which does not alter the integrity of the remainder of the macromolecule. 
     (c) In the event that no simple protecting group can be formed for the reactive functionality to be used for linking with the targeting director, a synthetic precursor can be used which is unreactive in all the synthetic procedures used in the dendrimer synthesis. On completion of the synthesis, this functional group must be readily convertible into the desired linking group in a manner which does not alter the integrity of the remainder of the macromolecule. 
     (2) Coupling (covalently) the desired reactive functional group onto a preformed dendrimer, the reagent used must contain a functionality which is readily reacted with the terminal functional groups of the dendrimer. The functional group to be ultimately used to link with the targeting agent can be in its final form, as a protected functionality, or as a synthetic precursor. The form in which this linking functionality is used depends on its integrity during the synthetic procedure to be utilized, and the ability of the final macromolecule to withstand any conditions necessary to make this group available for linking. Examples of suitable linking agents may be found in the art, for example European Published Application 0430863, published Jun. 5, 1991. For example, the preferred route for PEI uses ##STR10## 
     Examples of heterofunctional initiators for use in (1) above, include the following illustrative examples: ##STR11## 
     There are several chemistries of particular importance: 
     1) STARBURST™ Polyamidoamines (&#34;PAMAM&#34;) Chemistry; 
     2) STARBURST™ Polyethyleneimines (&#34;PEI&#34;) Chemistry; 
     3) STARBURST™ PEI compound with a surface of PAMAM; 
     4) STARBURST™ polyether (&#34;PE&#34;) chemistry. 
     Modifications of the dendrimer surface functionalities may provide other useful functional groups such as the following: 
     --OPO 3  H 2 , --PO 3  H 2 , --PO 3  H.sup.(-1), --PO 3 .sup.(-2), --CO 2 .sup.(-1), --SO 2  H, --SO 2 .sup.(-1), --SO 3  H, --SO 3 .sup.(-1), --NR 1  R 2 , --R 5 , --OH, --OR 1 , --NH 2 , polyethers, perfluorinated alkyl ##STR12## wherein: R represents alkyl, aryl or hydrogen; 
     R 1  represents alkyl, aryl, hydrogen, or ##STR13## R 2  represents alkyl, aryl, or ##STR14## R 3  represents --OH, --SH, --CO 2  H, --SO 2  H, or --SO 3  H; 
     R 4  represents alkyl, aryl, alkoxy, hydroxyl, mercapto, carboxyl, nitro, hydrogen, bromo, chloro, iodo, or fluoro; 
     R 5  represents alkyl; 
     X represents NR, O or S; and 
     n represents the integer 1, 2 or 3; 
     polyethers; or other immuno insensitive moieties wherein for all the above, alkyl is a linear or branched C 1  -C 18  hydrocarbon and aryl is a benzyl or napthyl which may be substituted with one or more C 1  -C 4  alkyl, bromo, chloro, iodo, fluoro, or trifluoromethyl moieties. 
     The choice of functional group depends upon the particular end use for which the dendrimer is designed. For example, a dendrimer having 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-tris-(carbomethoxymethyl)-1,4,7,10-tetraazacyclodocecane (DO3A methyl ester) as the functional group can be used as carriers for magnetic resonance imaging or as nuclear magnetic resonance reagents. 
     These functionalized dendrimers are prepared, for example, by reacting the functionalizing group, such as DO3A methyl ester, with an epoxide or epoxide substituted with a C 1  -C 18  alkyl group to form a hydroxyethylated product. The hydroxyethylated product is reacted with a coupling agent, such as dicyclohexyl carbodiimide (DCC), and then reacted with a PAMAM STARBURST™ polymer. A dendrimer having DOTA as a functional group can be prepared by using an isothiocyanate derivativized DOTA, such as, 1-[1-carboxy-3(4&#39;-isothiocyanatophenyl)propyl]-4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane, with a PAMAM STARBURST™ polymer. Other suitable chelates can be added by known synthetic techniques. When desired, a lanthanide ion or pseudolanthanide ion can be incorporated by conventional methods. 
     Linking of antibodies to dendrimers is another aspect of the present invention. Typically, the antibodies or antibody fragments are linked to the dendrimer by techniques well known in the art such as attachment between a functional group on the dendrimer and moieties such as carbohydrate, amino, carboxyl, or sulfhydryl functionalities present in the antibody or antibody fragment. In some instances connecting groups may be used as connectors or spacers between the dendrimer and antibody or antibody fragment. The attachment of the dendrimer to the antibody or antibody fragment should be performed in a manner which does not significantly interfere with the immunoreactivity of the antibody or antibody fragment, that is, by binding the antibody or antibody fragment via a functionality in the antibody or antibody fragment which is not a part of the antigen recognition and binding site. 
     The following examples further illustrate the invention but are not to be construed as a limitation on the scope of the invention. The lettered examples concern the preparation of starting materials; the numbered examples concern the preparation of products. 
     Example A: Preparation of 2-Carboxamido-3-(4&#39;-nitrophenyl)propanamide 
     p-Nitrobenzyl malonate diethylester (2.4 grams (g), 8.13 mmole) was dissolved in 35 mL of methanol. The solution was heated to 50°-55° C. with stirring and a stream of anhydrous ammonia was bubbled through the solution for 64 hours. The solution was cooled and the white, flocculant product was filtered and recrystallized from 225 mL of boiling methanol to afford 1.85 g (7.80 mmole) of bisamide in 96% yield (mp=235.6° C.(d)). 
     The structure was confirmed by MS,  1  H and  13  C NMR spectroscopy. 
     
         ______________________________________                                    
Anal: Calc. for C.sub.10 H.sub.11 O.sub.4 N.sub.3                         
        C           H      N                                              
______________________________________                                    
Theo:     50.63         4.69   17.72                                      
Found:    50.75         4.81   17.94                                      
______________________________________                                    
 
    
     Example B: Preparation of 1-Amino-2-(aminomethyl)-3-(4&#39;-nitrophenyl)propane 
     2-Carboxamido-3-(4&#39;-nitrophenyl)propanamide (2.0 g, 8.43 mmole) was slurried in 35 mL of dry tetrahydrofuran under a nitrogen atmosphere with stirring. To this mixture was added borane/tetrahydrofuran complex (106 mL, 106 mmole) via syringe. The reaction mixture was then heated to reflux for 48 hours during which time the suspended amide dissolved. The solution was cooled and the tetrahydrofuran was removed in vacuo using a rotary evaporator. The crude product and borane residue was dissolved in 50 mL of ethanol and this solution was purged with anhydrous hydrogen chloride gas. The solution was refluxed for 1 hour and the solvent removed in vacuo. The crude hydrochloride salt was dissolved in 15 mL of deionized water and extracted with two 50 mL portions of methylene chloride. The aqueous layer was cooled in an ice bath under an argon blanket and 50% sodium hydroxide was slowly added until basic pH=11.7. The basic aqueous layer was extracted with four 25 mL portions of methylene chloride and these combined extracts were evaporated using a rotary evaporator to give 1.45 g of amber colored oil. This oil was triturated with diethyl ether (50 mL) and filtered under pressure through a short silica gel (grade 62 Aldrich) column. The column was washed with 100 mL of ether and the combined filtrates were vacuum evaporated giving 1.05 g (5.02 mmole) of the titled diamine as a clear oil (mp=275°-278° C. (d) bis HCl salt). 
     The structure was confirmed by MS,  1  H and  13  C NMR spectroscopy. 
     
         ______________________________________                                    
Anal: Calc. for C.sub.10 H.sub.17 N.sub.3 O.sub.2 Cl.sub.2                
        C           H      N                                              
______________________________________                                    
Theo:     42.57         6.07   14.89                                      
Found:    43.00         6.14   15.31                                      
______________________________________                                    
 
    
     Example C: Preparation of 1-Amino-2-(aminomethyl)-3-(4&#39;-aminophenyl)propane 
     Borane/tetrahydrofuran solution (70 mL, 70 mmole) was added under nitrogen via a cannula needle to a flask containing 4-aminobenzyl malonamide (1.5 g, 7.24 mmole) with stirring. The solution was brought to reflux for 40 hours. The colorless solution was cooled and excess tetrahydrofuran was removed by rotary evaporation leaving a clear gelatinous oil. Methanol (50 mL) was cautiously added to the oil with notable gas evolution. Dry hydrogen chloride was bubbled through the suspension to effect dissolution and the solution was then refluxed for 1 minute. The methanol/HCl solution was removed by rotary evaporation and the resulting hydrochloride salt was carried through the same dissolution/reflux procedure again. The hydrochloride salt obtained was dissolved in 10 mL of water and cooled in an ice bath under argon. Concentrated sodium hydroxide (50%) was added slowly with stirring to pH=11. The aqueous portion was then extracted with 2×100 mL portions of chloroform which were combined and filtered through a short silica gel plug without drying. The solvent was removed in vacuo (rotary evaporator) affording the title compound (0.90 g, 5.02 mmole) in 70% yield (R f  =0.65--CHCl 3  /MeOH/NH 4  OH conc--2/2/1). The structure was confirmed by  1  H and  13  C NMR and used without further purification. 
     Example D: Preparation of 6-(4-Aminobenzyl)-1,4,8,11-tetraaza-5,7-dioxoundecane 
     4-Aminobenzyl malonate dimethylester (2.03 g, 8.43 mmole) was dissolved in 10 mL of methanol. This solution was added dropwise to a stirred solution of freshly distilled ethylenediamine (6.00 g, 103.4 mmole) in 10 mL of methanol under nitrogen over a 2 hour period. The clear solution was stirred for 4 days and Thin Layer Chromotography (TLC) analysis indicated total conversion of diester (R f  =0.91) to the bis amide (R f  =0.42--20% conc NH 4  OH/80% ethanol). This material was strongly ninhydrin positive. The methanol and excess diamine were removed on a rotary evaporator and the resulting white solid was vacuum dried (10 -1  mm, 50° C.) overnight to afford crude product (2.45 g, 8.36 mmole) in 99% yield. An analytical sample was recrystallized from chloroform/hexane, MP=160°-161° C. The mass spectral,  1  H and  13  C NMR data were consistent with the proposed structure. 
     Example E: Reaction of Mesyl Aziridine with 1-Amino-2-(aminomethyl)-3-(4-nitrophenyl)propane 
     1-Amino-2-(aminomethyl)-3-(4-nitrophenyl)propane (400 mg, 1.91 mmole, &gt;96% pure) was dissolved in 10.5 mL of absolute ethanol under nitrogen. Mesyl aziridine (950 mg, 7.85 mmole) was added to the stirred diamine solution as a solid. The reaction was stirred at 25° C. for 14 hours using a magnetic stirrer and during this period a white, gummy residue formed on the sides of the flask. The ethanol was decanted and the residue was triturated with another 15 mL portion of ethanol to remove any unreacted aziridine. The gummy product was vacuum dried (10 1  mm, 25° C.) to afford the tetrakis methyl sulfonamide (1.0 g, 1.44 mmole) in 75% yield (R f  =0.74--NH 4  OH/ethanol--20/80). The structure was confirmed by  1  H and  13  C nuclear magnetic resonance (NMR) spectroscopy. 
     Example F: Preparation of 2-(4-Nitrobenzyl)-1,3-(bis-N,N-2-aminoethyl)diaminopropane 
     The crude methylsulfonamide from Example E (650 mg, 0.94 mmole) was dissolved in 5 mL of nitrogen purged, concentrated sulfuric acid (98%). This solution was maintained under nitrogen and heated to 143°-146° C. for 27 minutes with vigorous stirring. A slight darkening was noted and the cooled solution was poured into a stirred solution of ether (60 mL). The precipitated white salt cake was filtered and immediately dissolved in 10 mL of deionized water. The pH of the solution was adjusted to pH=11 with 50% NaOH under argon with cooling. The resulting solution was mixed with 90 mL of ethanol and the precipitated inorganic salts were filtered. The solvent was removed from the crude amine under reduced pressure and to the resulting light brown oil was added 190 mL of toluene under nitrogen. The mixture was stirred vigorously and water was removed through azeotropic distillation (Dean-Stark trap) until the remaining toluene acquired a light yellow color (30-40 mL remaining in pot). The toluene was cooled and decanted from the dark, intractable residues and salt. This solution was stripped of solvent in vacuo and the resulting light yellow oil was vacuum dried (0.2 mm, 35° C.) overnight affording 210 mg of the product (60%) which was characterized by MS,  1  H and  13  C NMR. 
     Example G: Preparation of a STARBURST™ Polymer (containing an aniline derivative) of One Half Generation Represented by the Following Scheme: ##STR15## 
     Methyl acrylate (2.09 g, 24 mmole) was dissolved in methanol (15 mL). The compound 6-(4-aminobenzyl)-1,4,8,11-tetraaza-5,7-dioxoundecane (1.1 g, 3.8 mmole) (i.e., Compound #1, the preparation of this compound is shown in Example D) was dissolved in methanol (10 mL) and was added slowly over 2 hours with rigorous stirring to the methyl acrylate solution. The reaction mixture was stirred for 48 hours at ambient temperatures. The solvent was removed on the rotary evaporator maintaining the temperature below 40° C. The ester (Compound #2) was obtained as a yellow oil (2.6 g). No carboxyethylation of the aniline function was observed. 
     Example H: Preparation of a STARBURST™ Polymer (containing an aniline moiety) of One Generation; Represented by the Following Scheme: ##STR16## 
     The ester (Compound #2) (2.6 g, 3.7 mmole) was dissolved in methanol (100 mL). This was carefully added to a vigorously stirring solution of ethylenediamine (250 g, 4.18 mole) and methanol (100 mL) at such a rate that the temperature did not rise above 40° C. After complete addition the reaction mixture was stirred for 28 hours at 35°-40° C. (heating mantle). After 28 hours no ester groups were detectable by infrared spectroscopy. The solvent was removed on the rotary evaporator at 60° C. The excess ethylenediamine was removed using a ternary azeotrope of toluene-methanolethylenediamine. Finally all remaining toluene was azeotroped with methanol. Removal of all the methanol yielded 3.01 g of the product (Compound #3) as an orange glassy solid. 
     Example I: Preparation of a STARBURST™ Polymer (containing an aniline moiety) of One And One Half Generations Represented by the Following Scheme: ##STR17## 
     The amine (Compound #3) (2.7 g, 3.6 mmole) was dissolved in methanol (7 mL) and was added slowly over one hour to a stirred solution of methyl acrylate (3.8 g, 44 mmole) in methanol (15 mL) at ambient temperatures. A slight warming of the solution was observed during the addition. The solution was allowed to stir at ambient temperatures for 16 hours. The solvent was removed on a rotary evaporator at 40° C. After removal of all the solvent and excess methyl acrylate the ester (Compound #4) was obtained in 4.7 g yield as an orange oil. 
     Example J: Preparation of a STARBURST™ Polymer (containing an aniline moiety) of One Half Generation Represented by the Following Scheme: ##STR18## 
     The triamine (Compound #5, the preparation of this compound is shown in Example C) (0.42 g, 2.3 mmole) was dissolved in methanol (10 mL) and was added dropwise over one hour to methyl acrylate (1.98 g, 23 mmole) in methanol (10 mL). The mixture was allowed to stir at ambient temperatures for 48 hours. The solvent was removed on a rotary evaporator, maintaining the temperature at no higher than 40° C. The excess methyl acrylate was removed by repeated azeotroping with methanol. The ester (Compound #6) was isolated as an orange oil (1.24 g). 
     Example K: Preparation of a STARBURST™ Polymer (containing an aniline moiety) of One Generation; Represented by the Following Scheme: ##STR19## 
     The ester (Compound #6) (1.24 g, 2.3 mmole) was dissolved in methanol (50 mL) and was added dropwise over two hours to ethylenediamine (73.4 g, 1.22 mole) in methanol (100 mL). A small exotherm was noted, vigorous stirring was maintained. The solution was left to stir at ambient temperatures for 72 hours. The solvent was removed on a rotary evaporator at 60° C. The excess ethylenediamine was removed using a ternary azeotrope of toluene-methanol-ethylenediamine. Finally all remaining toluene was removed with methanol and then pumping down with a vacuum pump for 48 hours gave the amine (Compound #7) (1.86 g) as a yellow/orange oil. 
     Example L: Preparation of a STARBURST™ Polymer (containing an aniline moiety) of One And One Half Generations; Represent by the Following Scheme: ##STR20## 
     The amine (Compound #7) (1.45 g, trace of methanol remained) was dissolved in methanol (100 mL) and was added slowly over 11/2 hours to a stirred solution of methyl acrylate (5.80 g) in methanol (20 mL). The solution was allowed to stir for 24 hours at room temperature. Removal of the solvent followed by repeated azeotroping with methanol enabled the removal of all the excess methyl acrylate. After pumping down on a vacuum pump for 48 hours the ester (Compound #8) was isolated as an orange oil (2.50 g, 1.8 mmole). 
     Example M: Hydrolysis of 4.5 Generation Dendrimer and Preparation of Calcium Salt 
     4.5 Generation PAMAM (ester terminated, initiated from NH 3 ) (2.11 g, 10.92 meq) was dissolved in 25 mL of methanol and to it was added 10% NaOH (4.37 mL, 10.92 meq) (pH=11.5-12). After 24 hours at room temperature, the pH was about 9.5. After an additional 20 hours, the solution was removed using a rotary evaporator, 50 mL of toluene added, and evaporated again. 
     The resulting oil was dissolved in 25 mL of methanol and precipitated as a white gum upon addition of 75 mL of diethyl ether. The liquid was decanted, and the gum was rotary evaporated to give a very fine off-white powder which upon further drying gives 2.16 g of product (98% yield). No ester groups were found upon NMR and infrared analysis. 
     The sodium salt of 4.5 Generation PAMAM (ester terminated, initiated from NH 3 ) was replaced by the calcium salt via dialysis. The sodium salt (1.03 g) was dissolved in 100 mL of water and passed through hollow fiber dialysis tubing (cut off=5000) at 3 mL/minute. The exterior of the tubing was bathed in 5% CaCl 2  solution. This procedure was then repeated. 
     The resulting solution was again dialyzed, this time against water, then repeated two additional times. 
     Evaporation provided 0.6 g of wet solid, which was taken up in methanol (not totally soluble) and is dried to give 0.45 g of off-white crystals. 
     
         ______________________________________                                    
C.sub.369 H.sub.592 O.sub.141 N.sub.91 Ca.sub.24 M Wt. = 9526.3           
         C     H          N       Ca                                      
______________________________________                                    
Theo:      46.5,   6.32,      13.38,                                      
                                    10.10                                 
Found:     47.34,  7.00,      13.55,                                      
                                     8.83                                 
______________________________________                                    
 
    
     Example N: Preparation of Dendrimers with Terminal Carboxylate Groups 
     Half-generation STARBURST™ polyamidoamines were hydrolyzed to convert their terminal methyl ester groups to carboxylates. This generated spheroidal molecules with negative charges dispersed on the periphery. The dendrimers hydrolyzed ranged from 0.5 generation (three carboxylates) to 6.5 generation (192 carboxylates). 
     The products could be generated as Na + , K + , Cs +   or Rb +   salts. 
     Example O: N-t-butoxycarbonyl-4-aminobenzyl Malonate Dimethylester 
     4-Aminobenzyl malonate dimethylester (11.62 g, 49 mmol) was dissolved in 50 mL of t-butanol:water (60:40 with stirring. Di-t-butoxydicarbonate (19.79 g, 90 mmol) was added and the reaction mixture stirred overnight. The butanol was removed on a rotary evaporator, resulting in a yellow suspension of the product in water. Extraction into methylene chloride, drying (MgSO 4 ) and evaporation gave a yellow oil (21.05 g, contaminated by di-t-butoxydicarbonate). Recrystallization from 2-propanol:water (75:25) yield pale yellow crystals (11.1 g, 33 mmol, 67%). The structure was confirmed by  13  C NMR and purity checked by HPLC analysis (Spherisorb™ ODS-1, 0.05M H 3  PO 4  pH 3:CH 3  CN 55:45). The material was used without further purification. 
     Example P: N-t-butoxycarbonyl-6-(4-aminobenzyl)-1,4,8,11-tetraaza-5,7-dioxoundecane 
     N-t-butoxycarbonyl-4-aminobenzyl malonate dimethylester (8.82 g 26 mmol), prepared in Example O, was dissolved in 50 mL of methanol, This solution was added dropwise (2 hours) to a solution of freshly distilled ethylenediamine (188 g 3.13 mole) and 20 mL of methanol, under a nitrogen atmosphere. The solution was allowed to stir for 24 hours. The ethylenediamine/methanol solution was removed on a rotary evaporator. The product was dissolved in methanol and toluene added. Solvent removal on the rotary evaporator gave the crude product as a white solid (10.70 g contaminated with ethylenediamine). The sample was divided into two samples for purification. Azeotropic removal of ethylenediamine with toluene, using a soxhlet extractor with sulphonated ion exchange beads in the thimble to trap the ethylenediamine, resulted in partial decomposition of the product, giving a brown oil. The remaining product was isolated as a white solid from the toluene on cooling (2.3 g approximately 50 percent). Analysis of a 10 percent solution in methanol by gas chromatography (Column, Tenax 60/80) showed no ethylenediamine detectable in the sample (&lt;0.1 percent). The second fraction was dissolved in methanol to give a 10 percent solution (by weight) and purified from the ethylenediamine by reverse osmosis, using methanol as the solvent. (The membrane used was a Filmtec™ FT-30, in an Amicon™ TC1R thin channel separator, the ethylenediamine crossing the membrane.) The product was isolated as a white solid (2.7 g), in which no detectable amounts of ethylenediamine could be found by gas chromatography. The  13  C NMR data and HPLC analysis (Spherisorb ODS-1, 0.05M H 3  PO 4  pH 3:CH 3  CN 55:45) were consistent with the proposed structure. The product was used with no further purification. 
     Example Q: Preparation of a STARBURST™ Dendrimer of One Half Generation from N-t-butoxycarbonyl-6-(4-aminobenzyl)-1,4,8,11-tetraaza-5,7-dioxoundecane 
     N-t-butoxycarbonyl-6-(4-aminobenzyl)-1,4,8,11-tetraaza-5,7-dioxoundecane (5.0 g 13 mmol), prepared in Example P, was dissolved in 100 mL of methanol. Methyl acrylate (6.12 g, 68 mmol) was added and the solution stirred at ambient temperatures for 72 hours. The reaction was monitored by HPLC (Spherisorb™ ODS1, acetonitrile:0.04M ammonium acetate 40:60) to optimize conversion to the desired product. The solution was concentrated to 30 percent solids, and methyl acrylate (3.0 g, 32 mmol) was added. The reaction mixture was stirred at ambient temperatures until no partially alkylated products were detectable by HPLC (24 hours). Removal of the solvent at 30° C. by using a rotary evaporator, and pumping down at 1 mm Hg for 24 hours gave the product as yellow viscous oil, yield 7.81 g. The  13  C NMR data was consistent with the proposed structure. The product was used without further purification. 
     Example R: Preparation of a STARBURST™ Dendrimer of One Full Generation from N-t-butoxycarbonyl- 6-(4-aminobenzyl)-1,4,8,11-tetraaza-5,7-dioxoundecane 
     The half generation product (Example Q) (7.70 g, 10.45 mmol) was dissolved in 75 mL of methanol and added dropwise over 2 hours to a stirred solution of ethylenediamine (400 mL, 7.41 mol) and methanol (50 mL). The reaction mixture was stirred at ambient temperatures for 48 hours. The ethylenediamine and methanol were removed by rotary evaporation to give a yellow oil (11.8 g contaminated with ethylenediamine). The product was dissolved in 90 mL of methanol, and purified from the ethylenediamine by reverse osmosis (Filmtec™ FT-30 membrane and Amicon™ TC1R thin channel separator, methanol as solvent). After 48 hours, no ethylenediamine could be detected by gas chromatography (Column, Tenax™ 60/80). Removal of the solvent on a rotary evaporator, followed by pumping down on a vacuum line for 24 hours gave the product as a yellow glassy solid (6.72 g). Analysis by HPLC, PLRP-S column, acetonitrile:0.015M NaOH, 10-20 percent gradient in 20 min.) and  13  C NMR analysis was consistent with the proposed structure. 
     Example S: Preparation of a STARBURST™ Polymer of One and One Half Generation from N-t-butoxycarbonyl-6-(4-aminobenzyl)-1,4,8,11-tetraaza-5,7-dioxoundecane 
     The one generation product (Example R) (2.14 g, 25 mmol) was dissolved in 12.5 mL of methanol, and methyl acrylate (3.5 g, 39 mmol) in 5 mL of methanol was added. The solution was stirred at ambient temperatures for 48 hours, monitoring the progress of the reaction by HPLC (Spherisorb ODS-1, acetonitrile:0.04M ammonium acetate, 60:40). A second aliquot of methyl acrylate was added (3.5 g 39 mmol) and the reaction mixture stirred at ambient temperatures for a further 72 hours. Removal of the solvent on the rotary evaporator gave the product as a yellow oil (3.9 g) after pumping down overnight with a vacuum pump. The product was used with no further purification. 
     Example T: Preparation of a STARBURST™ Polymer of Two Full Generations from N-t-butoxycarbonyl-6-(4-aminobenzyl)-1,4,8,11-tetraaza-5,7-dioxoundecane 
     The one and one half generation product (Example S) (3.9 g, 2.5 mmol) was dissolved in 50 mL of methanol, and was added dropwise over 2 hours to a stirred solution of ethylenediamine (600 g, 10 mol) and methanol (50 mL). The solution was stirred at ambient temperatures under an atmosphere of nitrogen for 96 hours. The ethylenediamine/methanol was removed on the rotary evaporator to give a yellow glassy solid (4.4 g contaminated with ethylenediamine). A 10 percent solution of the product was made in methanol, and purified from the ethylenediamine by reverse osmosis (membrane used as a Filmtec™ FT-30, in an Amicon™ TC1R thin channel separator) until no ethylenediamine could be detected by gas chromatography (Column, Tenax 60/80. Removal of the solvent gave the product as a yellow glassy solid (3.52 g). The  13  C NMR data and HPLC analysis (PLRP-S column, acetonitrile:0.015M NaOH, 10-20 percent gradient in 20 minutes) were consistent with the proposed structure. 
     Example U: Reaction of the Two Generation STARBURST™ with Bromoacetic Acid to Give a Methylene Carboxylate Terminated STARBURST™ Dendrimer 
     The second generation product (Example T) (0.22 g, 0.13 mmol) was dissolved in 15 mL of deionized water and the temperature equilibrated at 40.5° C. Bromoacetic acid (0.48 g, 3.5 mmol) and lithium hydroxide (0.13 g, 3.3 mmol) were dissolved in 5 mL of deionized watery and added to the reaction mixture. The reaction pH was carefully maintained at 9, with the use of a pH stat (titrating with 0.1N NaOH), at 40.5° C. overnight. Monitoring by reverse phase HPLC, (Spherisorb ODS-1 column, eluent 0.25M H 3  PO 4  pH 3 [NaOH]; acetonitrile 85:15) confirmed the synthesis of predominantly a single component. 
     Example V: Preparation of Isothiocyanato Functionalized Second Generation Methylene-carboxylate Terminated STARBURST™ Dendrimer 
     Five mL of a 2.8 mM solution of the second generation methylenecarboxylate terminated STARBURST™ dendrimer (Example U) was diluted with 20 mL water and the pH adjusted to 0.5 with concentrated hydrochloric acid. After one hour at room temperature the mixture was analyzed by HPLC to verify the removal of the butoxycarbonyl group and then treated with 50 percent sodium hydroxide to bring the pH to 7. A pH stat (titrating with 0.1N NaOH) was used to maintain the pH at 7 and 225 μL thiophosgene was added. After 15 minutes at room temperature the pH of the mixture was adjusted to 5 with 1N HCl. The mixture washed with chloroform (20 mL×2) then concentrated on a rotary evaporator at reduced pressure. The residue recovered, 0.91 g, is a mixture of the isothiocyanate and salts. 
     Example W: Preparation of Second Generation STARBURST™ Polyethyleneimine-methane Sulfonamide 
     To a solution of 125 g N-methanesulfonylaziridine in 50 mL ethanol was added 25.0 g tris(2-aminoethyl)amine. The solution was stirred at room temperature for 4 days. Water was added to the reaction mixture as needed to maintain the homogeneity of the solution. The solvent was removed by distillation in vacuo to give the 2nd generation STARBURST™ PEI-methane sulfonamide as a yellow glass (161 g). 
     Example X: Cleavage of Methane Sulfonamides to Form Second Generation STARBURST™ Polyethyleneimine 
     A solution of 5.0 g of second generation STARBURST™ PEI-methane sulfonamide, from Example W in 20 mL of 38 percent HCL was sealed in a glass ampoule. The ampoule was heated at 160° C. for 16 hours, then cooled in an ice bath and opened. The solvent was removed by distillation in vacuo and the residue dissolved in water. After adjusting the pH of the solution to greater than or equal to 10 with 50 percent NaOH, the solvent was removed by distillation in vacuo. Toluene (150 mL) was added to the residue and the mixture heated at reflux under a Dean-Stark trap until no more water could be removed. The solution was filtered to remove salts and the filtrate concentrated in vacuo to give 1.9 g second generation STARBURST™ PEI as a yellow oil. 
     Example Y: Preparation of Third Generation STARBURST™ Polyethyleneimine-methane Sulfonamide 
     To a solution of 10.1 g second generation STARBURST™ PEI, from Example X, in 100 mL ethanol was added 36.6 g N-methanesulfonylaziridine. The solution was stirred at room temperature for 1 week. Water was added as needed to maintain the homogeneity of the solution. The solvent was removed by distillation in vacuo to give third generation STARBURST™ PEI-methane sulfonamide as a yellow glass (45.3 g). 
     Example Z: Cleavage of Methane Sulfonamides to Form 3rd Generation STARBURST™ Polyethyleneimine 
     The methane sulfonamide groups of third generation STARBURST™ PEI-methane sulfonamide (5.0 g), from Example Y, were removed by the same procedure as described for the second generation material in Example X to give 2.3 g third generation STARBURST™ PEI as a yellow oil. 
     Example AA: Reaction of a Third Generation STARBURST™ Polyethyleneimine with (4-Fluoro)nitrobenzene 
     The third generation STARBURST™ polyethyleneimine (Example Z) (1.06 g, 1.2 mmol) was dissolved in 12 mL of absolute ethanol. (4-Fluoro)nitrobenzene (120 μL, 1.2 mmol) was added and the reaction mixture refluxed overnight. The solvent was removed on the rotary evaporator, and the bright yellow oil dissolved in water. The aqueous solution was washed with chloroform to remove any unreacted (4-fluoro)nitrobenzene. Removal of the water gave the product as a deep yellow oil (0.80 g). The  13  C NMR spectrum was consistent with the proposed structure. (No attempt was made to determine the nature of the statistical distribution). The product was used without further purification. 
     Example BB: Reaction of the Nitrophenyl Derivative of the Third Generation STARBURST™ Polyethyleneimine with Glycolonitrile 
     The nitrophenyl derivative of the third generation STARBURST™ polyethyleneimine (Example AA) (0.80 g) was dissolved in 20 mL of deionized water. Sodium hydroxide (2.80 g, 50 percent w/w) was added to the stirred solution, and the solution purged with nitrogen, venting through a sodium hydroxide scrubber. Glycolonitrile (2.85 mL of a 70 percent aqueous solution) was added at ambient temperatures. A yellow precipitate was observed to form after a few minutes. After two hours, the temperature was slowly raised to a reflux, and the solution maintained at a reflux with a nitrogen purge for 24 hours. Removal of the water gave the product as a yellow solid contaminated with glycolic acid and sodium hydroxide. The  13  C NMR spectrum was consistent with the proposed structure. The product was used without further purification. 
     Example CC: Hydrogenation of the Nitrophenyl Derivative to the Aminophenyl Methylenecarboxylate Terminated Third Generation STARBURST™ Polyethyleneimine 
     The yellow solid from Example BB (1.70 g) was dissolved in 10 mL of deionized water, the resulting pH of the solution was approximately 11. Palladium on charcoal (200 mg of 5 percent Pd/C) was added to the reaction mixture in a glass Parr shaker bottle. The reaction mixture was placed under a pressure of 40 psi (275 kPa) of hydrogen, and shaken at ambient temperature in a Parr hydrogenation apparatus, for 6 hours. The reaction mixture was then filtered through a 0.5 μm Millipore™ filter to remove the Pd/C and the solvent removed in vacuo and was gel filtered through a Biogel P2 resin (25 g swollen with water). Acidification with HCl resulted in an orange brown solution, which was purged with nitrogen overnight. Removal of the solvent in vacuo gave the product as the hydrochloride salt which was a pale brown solid (3.98 g, contaminated with NaCl and glycolic acid, maximum theoretical amount of product 1.15 g). The product was used with no further purification. 
     Example DD: Preparation of 4-Isothiocyanatophenyl Methylenecarboxylate Terminated Third Generation STARBURST™ Polyethyleneimine 
     The product from Example CC (3.98 g) was dissolved in 15 mL of deionized water and an aliquot (2.5 mL) of this solution was diluted with 10 mL water. The pH of the solution was adjusted to 7 with sodium hydroxide. A pH stat (titrating with 1N NaOH) was used to maintain the pH and 200 μL thiophosgene was added. After 10 minutes the pH of the mixture was adjusted to 4 with hydrochloric acid. Water was removed on a rotary evaporator at reduced pressure (a small amount of n-butanol was added to prevent foaming). The residue was washed with methylene chloride and then dried. The crude product (0.95 g) a mixture of isothiocyanate (0.14 g) and salts was used without further purification. 
     Example EE: Preparation of a Methylenecarboxylate-terminated Second Generation STARBURST™ Polyamidoamine (initiated from ammonia) 
     The second generation STARBURST™ polyamidoamine (2.71 g, 2.6 mmol) and bromoacetic acid (4.39 g, 31.6 mmol) were dissolved in 30 mL of deionized water and the pH adjusted to 9.7 with 5N NaOH using a pH stat. The reaction was maintained at this pH for a half hour, and the temperature was slowly raised to 60° C. and was maintained at 60° C. for three hours at constant pH. The pH was raised to 10.3, and the reaction mixture remained under control of the pH stat at ambient temperatures overnight. The reaction mixture was refluxed for a further four hours prior to work up. Removal of the solvent, and azeotroping the final traces of water with methanol gave the product as a pale yellow powder (8.7 g, contaminated with sodium bromide). The  13  C/NMR spectrum was consistent with the propose structure (with some contamination due to a small amount of defected material as a result of some monoalkylation). 
     Example FF: Preparation of a Methylenecarboxylate Terminated Second Generation STARBURST™ Polyethyleneimine (initiated from ammonia) 
     The second generation STARBURST™ polyethyleneimine (2.73 g, 6.7 mmol), from Example X, and bromoacetic acid (11.29 g 81 mmol) were dissolved in 30 mL of deionized water. The pH was slowly raised to pH 9.5 maintaining the temperature below 30° C. The temperature was raised slowly to 55° C., and the reaction pH maintained at 9.5 for 6 hours with the aid of a pH stat (titrating with 5N NaOH). The pH was raised to 10.2, and maintained at that pH overnight. Removal of the solvent on a rotary evaporator, and azeotroping the final traces of water using methanol, gave the product as a yellow powder (17.9 g, contaminated with sodium bromide). The  13  C NMR spectrum was consistent with the proposed structure (with some contamination due to a small amount of defected material as a result of some monoalkylation). 
     Example GG: Preparation of 3.5, 4.5, 5.5 and 6.5 Generation STARBURST™ PAMAM 
     To a 10 weight percent methanolic solution of 2.46 g 3 generation PAMAM STARBURST™ was added 2.32 g of methyl acrylate. This mixture was allowed to sit at room temperature for 64 hours. After solvent and excess methyl acrylate removal, 4.82 g of product was recovered (105% of theoretical). 
     Preparation of higher half generation STARBURST™ PAMAM&#39;s 
     Generations 4.5, 5.5 and 6.5 were prepared as described above with no significant differences in reactant concentrations, reactant mole ratios or reaction times. 
     Example HH: Preparation of 4, 5 and 6 Generation STARBURST™ PAMAM 
     To 2000 g of predistilled ethylenediamine was added 5.4 g of 4.5 generation STARBURST™ PAMAM as a 15 weight percent solution in methanol. This was allowed to sit at room temperature for 48 hours. The methanol and most of the excess ethylenediamine were removed by rotary evaporation under water aspirator vacuum at temperature less then 60° C. The total weight of product recovered was 8.07 g. Gas chromatography indicated that the product still contained 34 weight percent ethylenediamine at this point. A 5.94 g portion of this product was dissolved in 100 mL methanol and ultrafiltered to remove the residual ethylenediamine. The filtration was run using an Amicon™ TC1R thin channel recirculating separator equipped with an Amicon™ YM2 membrane. An in-line pressure relief valve was used to maintain 55 psig (380 kPa) pressure across the membrane. The 100 mL was first concentrated to 15 mL by forcing solvent flow exclusively through the membrane. After this initial concentration, the flow was converted to a constant volume retentate recycle mode for 18 hours. After this time, 60 mL of methanol was passed over the membrane to recover product still in the module and associated tubing. The product was stripped of solvent and 2.53 g of 5 generation STARBURST™ PAMAM was recovered. Analysis by gas chromatography indicated 0.3 percent residual ethylenediamine remained in the product. 
     Preparation of generation 4 and 6 proceeded as above with the only difference being the weight ratio of ethylenediamine to starting material. To prepare 4th generation this ratio was 200:1 and for 6th generation this ratio was 730:1. 
     Example II: Modification of Polyamidoamine Dendrimers by Reaction with Epoxyoctane 
     To a solution of 0.50 g of sixth generation PAMAM in 5 mL of methanol was added 0.56 g of epoxyoctane. After 6 days at room temperature, the solvent was evaporated in vacuo to give 0.80 g of colorless oil. The material was soluble in chloroform, toluene or methanol, but not soluble in water. The  13  C-NMR spectrum was in accord with a dendrimer with C-8 alkyl groups attached to its terminal amines. 
     Example JJ: Modification of Polyamidoamine Dendrimers by Reaction with t-butyl Glycidyl Ether 
     To a solution of 0.50 g sixth generation PAMAM in 5 mL methanol was added 0.57 g of t-butyl glycidyl ether. After 6 days at room temperature, the solvent was evaporated in vacuo to give 1.0 g of colorless oil. The material was soluble in chloroform, toluene or methanol, but not soluble in water. The  13  C-NMR spectrum was in accord with a dendrimer with 3-(t-butoxy)- 1-propan-2-ol groups attached to its terminal amines. 
     Example KK: Modification of Polyamidoamine Dendrimers by Reaction with Epoxyoctadecane 
     To a solution of 0.50 g of sixth generation PAMAM in 25 mL of methanol was added 1.1 g epoxyoctadecane. The solution was heated at reflux for 5 days. During the reflux time, a colorless viscous oil precipitated from the solution. The solvent was evaporated in vacuo to give 1.6 g of white foam. The material was soluble in chloroform or toluene, but not soluble in water or methanol. The  13  C-NMR spectrum was in accord with a dendrimer with C-18 alkyl groups attached to its terminal amines. 
     Example LL: Reaction of STARBURST™ PEI with a Hydrophobic Epoxide 
     Into a flask equipped with a magnetic stirrer was added 0.397 g (2×10 -4  moles) of fourth generation PEI (NH 3  core; MW 1,955) and 2.1 g (9.6×10 -3  moles) of methyl 10,11-oxoundecanoate (MW 214.3); in 5 mL of methanol. The reaction mixture was stirred at room temperature for 1 day and then warmed at 80° C. for 8 hours to give a brown viscous syrup. Complete ring opening reaction to produce the carbomethoxy terminated, hydrophobic dendrimer was confirmed by NMR analysis and comparison to model systems. 
     The above hydrophobic dendrimer was converted into a water soluble form by simply combining an equivalent weight of this product with sodium hydroxide in water and heating for 30 to 60 minutes. A homogeneous solution, which showed no detectable carbomethoxy groups, was obtained. Addition of excesses of NaOH or NaCl caused the sodium carboxylate salt to become cloudy and phase out as an oil. 
     The preparation of the conjugates of this invention uses the starting materials described above, or starting materials that are prepared in an analogous manner, or starting materials described in the art, or starting materials that are available. 
     Example 1: Incorporation of 2-(Acetyloxy)benzoic Acid (aspirin) into STARBURST™ Dendrimers 
     A widely accepted method for ascertaining whether a &#34;probe molecule&#34; is included in the interior of a micelle is to compare its carbon-13-spin lattice relaxation times (T 1 ) in a non-micellized versus micellized medium. A substantial decrease in T 1  for the micellized medium is indicative of &#34;probe molecule&#34; inclusion in the micelle. Since STARBURST™ dendrimers are &#34;covalently fixed&#34; analogs of micelles, this T 1  relaxation time technique was used to ascertain the degree/extent to which various pharmaceutical type molecules were associated with STARBURST™ polyamidoamines. In the following examples, T 1  values for (acetyloxy)benzoic acid (I) (aspirin) were determined in solvent (CDCl 3 ) and then compared to T 1  values in CDCl 3  at various [I:dendrimer] molar ratios. 
     Inclusion of aspirin (I) into various STARBURST™ polyamidoamine dendrimers as a function of generation. 
     Various half generation (ester terminated, initiated from NH 3 ) STARBURST™ polyamidoamine dendrimers (G=0.5→5.5) were combined with 2-(acetyloxy)benzoic acid in CDCl 3  to give acid:tertiary amine ratios of =1.0. A plot of T 1  values for 2-(acetyloxy)benzoic acid versus generation of STARBURST™ dendrimer added (see FIG. 4 where represent C-4, □ represent C-6, and ∘ represent C-5) shows that T 1  reaches a minimum over the generation range of 2.5→5.5 for carbons 4, 5 and 6 in 2-(acetyloxy)benzoic acid. This demonstrates interior association of 2-(acetyloxy)benzoic acid in the dendrimers (G=2.5→5.5) and further confirms that polyamidoamine dendrimers (Gen=2.5 or greater) can function as carrier molecules. 
     Example 2: Release of Pseudoephedrine from STARBURST™ Dendrimer-PAMAM 
     Pseudoephedrine (0.83 mg/mL) and STARBURST™ PAMAM dendrimer [1.0 mg/mL; G=6.5; terminal group (Z)=192 (methyl ester)] were dissolved in deionized distilled water and the pH of the donor phase was adjusted to 9.5, with sodium hydroxide solution, and stored at room temperature for about 12 hours. Solution of pseudoephedrine alone was treated in the same way (control). The drug dendrimer solution was stored at 40° C. for 8 hours after the first experiment and dynamic dialysis performed. Dialysis membrane used was a SpectraPor™ 7, MWCO 1,000, 28.6 mm in diameter in spectrum separation cells (half cell volume 5 and 10 mL, cell dimensions: 38 mm diameter for both the cells and the cell depth of 10 and 20 mm for 5 and 10 mL cells, respectively). 
     Samples were analyzed by an HPLC procedure developed for pseudoephedrine conditions for which are as follows: 
     Column: μBondapak™ C-18 
     Mobile phase: pH 3.2 phosphate buffer plus acetonitrile (80:20) 
     Flow rate: 0.3 mL/min 
     Detection: UV at 210 nm 
     Retention time: 13.3 min 
     The dialysis membrane was washed with deionized water and was kept soaking in the receptor phase for at least 12 hours prior to use. The dialysis membrane was placed in between the donor and the receptor compartment, and the donor compartment was stirred with a small magnetic spin bar. Known volumes of donor and receptor solutions were introduced into the respective compartments and transfer of pseudoephedrine to the receptor compartment was followed as a function of time. To maintain sink conditions the entire receptor phase was removed periodically (every 30 minutes) and replaced with fresh receptor phase. The amount of pseudoephedrine was assayed in the sampled receptor phase. Experiments were conducted at room temperature (22° C.). The receptor phase was plain deionized distilled water. 
     The results of dynamic analysis are shown in FIG. 5. In FIG. 5, the represents pseudoephedrine only (control), the 
     represents pseudoephedrine plus the dendrimer, and the 
     
         ○ 
    
     represents pseudoephedrine plus the dendrimer held at 40° C. for 8 hours prior to dialysis. It is apparent that in presence of G 6.5 dendrimer in the donor compartment the rate of dialysis of pseudoephedrine is reduced. Storing the donor solution at 40° C., appears to further reduce the rate of dialysis. 
     The experiment was repeated at lower concentrations (the ratio of number of drug molecules to the number of terminal groups was kept the same). G 6.5 dendrimer 120 μg/mL pseudoephedrine 100 μg/mL (122 μg/mL salt). 
     Dynamic dialysis of pseudoephedrine (alone) at this lower concentration was almost identical to that at higher concentration. FIG. 6 summarizes the results of this experiment where   represents pseudoephedrine only (control), and ∘ represents pseudoephedrine plus dendrimer. 
     Example 3 
     The procedure of Example 2 was repeated using the modifications given below. 
     Receptor phase: pH 7.4 phosphate buffer 
     Donor phase: pH 7.4 phosphate buffer plus drug and dendrimer in the following ratios: 
     1. G 6.5:Drug :: 1:192 
     2. G 5.5:Drug :: 1:96 
     3. G 4.5:Drug :: 1:48 
     4. G 6.5H:Drug :: 1:192 
     5. G 5.5H:Drug :: 1:96 
     6. G 4.5H:Drug :: 1:48 
     The above donor phase compositions plus pseudoephedrine alone were subjected to dynamic dialysis. The letter &#34;H&#34; after the dendrimer generation number stands for hydrolyzed dendrimer. Hydrolysis was accomplished by the procedure described in Examples M and N. 
     The results of these experiments are summarized in FIG. 7 where the donor and receptor compartment contained pH 7.4 phosphate buffer. For pseudoephedrine alone (P) the mean curve of three experiments is plotted (shown by the solid line), and one typical run from the other experiments are shown. In FIG. 7, the following symbols represent the dendrimer of the indicated generation. 
     
                       TABLE III                                                   
______________________________________                                    
Symbol      Dendrimer Generation                                          
______________________________________                                    
            5.5                                                           
            6.5                                                           
            4.5                                                           
θ     5.5 H                                                         
◯                                                             
            6.5 H                                                         
            4.5 H                                                         
______________________________________                                    
 
    
     Pseudoephedrine appears not to associate with the dendrimer (hydrolized) at pH 7.4. Hydrolysis of the terminal functional groups into carboxylate form, has a dramatic effect on the dialysis rate (reduction). The generation number appears not to influence the dialysis rate. 
     Example 4: Interaction Studies of Salicylic Acid with PAMAM STARBURST™ Dendrimers 
     This example evaluated interaction characteristics of salicylic acid with PAMAM STARBURST™ dendrimers. These dendrimers consisted of an ammonia initiated core with repeating units derived from N-(2-aminoethyl) acrylamide. Both full (amine terminated functions) and half (ester terminal groups) generation polymers were included in the studies. The ratio of salicyclic acid to STARBURST™ dendrimers utilized in the experiments resulted in approximately one salicyclic acid molecule to one terminal amine functional group for full generation polymers. In the half-generation polymer study, the same ratio was employed with adjustments made for the higher molecular weight polymer. 
     The experiments were conducted at room temperature using an equilibrium static cell dialysis methodology. Spectrum static dialysis cells (half cell volume, 10 mL) separated by SpectraPor 6 membranes (molecular weight cutoff=1000) were utilized for all experiments. Transport of salicylic acid was monitored as a function of time by removing aliquots from appropriate cell compartments and assayed by HPLC analysis using a U.V. detector at 296 nm (Bondapak C-18 Column, eluting mobile phase of acetonitrile/0.1M phosphate buffer (pH=3.2) at a ratio of 20:80 (V/V), set at a flow rate of 30 mL/hour). 
     Ten mL of a solution containing 1 mg/mL salicyclic acid and 2.5 mg/mL STARBURST™ polymer (Gen=4.0) adjusted to pH 6.65 and 5.0 with HCL solution were placed in the donor compartment of the dialysis cell and an equal volume of purified water adjusted to the same pH&#39;s placed in the receptor compartment. Transport of salicyclic acid into the receptor compartment was monitored. The results are given in FIG. 8. In FIG. 8, the free acid is represented by  , the acid plus generation 4.0 dendrimer, pH 6.65 is represented by 0, and the acid plus generation 4.0 dendrimer, pH 5.00 is represented by □. 
     Due to the lower percent ionization of the amine groups on the polymer at pH 6, a greater extend of interaction with salicylic may be expected at pH 5, resulting in less compound transported at the lower pH. The results given in FIG. 8 indicate a much lower percentage of salicylic acid transported in the presence of polymer at both pH&#39;s studied compared to the salicyclic acid control study. It is also observed that more salicyclic acid is transported at pH 6.65 than at pH 5.0 as predicted. The data demonstrates an interaction of the STARBURST™ polymer with salicylic acid that can be controlled by pH. Sustained release characteristics are also implied by these data since the salicyclic acid levels in the presence of polymer continue to rise past the approximate 12-hour equilibrium point observed in the control study. 
     To further investigate the interaction characteristics of salicylic acid with STARBURST™ polymers (Gen=4.0) an experiment was designed at pH 8.0. The design of the study differed from that previously described in that only the salicylic acid solution (1 mg/mL), adjusted to pH 8.0, was placed in the donor compartment and the polymer solution (2.5 mg/mL) placed in the receptor compartment. Loss of salicylic acid from the donor compartment was monitored as previously described. The results of the experiment are given in FIG. 9. In FIG. 9, the free acid is represented by - -, and the acid plus generation 4.0 dendrimer at pH 8.0 is represented by ---Δ---. 
     As indicated in FIG. 9, the equilibrium characteristics of salicylic acid in the donor compartment with STARBURST™ polymers in the receptor compartment differs from the salicylic acid control study. Based on the ionization characteristics of the molecules at pH 8, approximately 6-7% interaction is expected. The observed extent of interaction is indicated to be on the order of 4-5%. The lower association observed may be due to experimental variability or to an ionization constant of less than one. 
     This experiment indicates an uptake or removal of free salicylic acid from the continuous phase of the system by the polymer. This type of action could result in suppression of reactivity of molecules suggesting a possible chelating or molecular association type of property associated with the polymers. 
     The interaction characteristics of salicylic acid at pH 6.65 with a half generation STARBURST™ polymer (Gen=4.5) having ester terminated functional groups were evaluated. Salicylic acid (1 mg/mL) was combined with STARBURST™ polymer (Gen=4.5) 3.6 mg/mL at pH 6.65. Ten mL of the solution was placed in the donor compartment and transport from the donor compartment was monitored as previously described. The results are given in FIG. 10. In FIG. 10, the free acid is represented by - -, and the acid plus polymer is represented by ---∘---. 
     Under these experimental conditions no charge interaction is predicted to occur since the tertiary amine groups are non-ionized at pH 6.65. As is indicated in FIG. 10, the loss of salicylic acid in the presence of polymer (Gen 4.5) is virtually identical during the first 10 hours of dialysis to that of the salicylic acid control study. 
     The following observations are made from the data presented in this example: 
     (1) Full generation PAMAM STARBURST™ polymers function as a carrier for salicylic acid. 
     (2) Full generation PAMAM STARBURST™ polymers have sustained release functionality for salicylic acid. 
     (3) Salicylic acid carrier properties of full generation PAMAM STARBURST™ polymers can be controlled by pH. 
     Example 5: Demonstration of Multiple Chelation of Iron by a Sodium Propionate Terminated Sixth Generation STARBURST™ Polyamidoamine 
     The sodium propionate terminated sixth generation polyamidoamine (initiated from ammonia), prepared as in Example M and N, (97.1 mg, 2.45 mol.) was dissolved in 1.5 mL of deionized water. Addition of 0.5 mL of 0.5N HCl reduced the pH to 6.3. Ferric chloride was added (0.5 mL of 0.1.2M solution, 0.051 mmol) producing a light brown gelatinous precipitate. On heating at 60° C. for 0.5 hours, the gelatinous precipitate became soluble, resulting in a homogeneous orange solution. The solution was filtered through Biogel P2 acrylamide gel (10 g, twice) isolating the orange band (free of halide contamination). Removal of the solvent in vacuo gave the product as an orange film (30 mg). Analysis was consistent with chelation of approximately 20 moles of ferric ions per mole of STARBURST™ dendrimer. 
     
                       TABLE IV                                                    
______________________________________                                    
       Theoretical                                                        
Found    Na.sub.4 Fe.sub.20 H.sub.128 SB                                  
                     Na.sub.5 Fe.sub.20 H.sub.127 SB                      
                                 Na.sub.6 Fe.sub.20 H.sub.126 SB          
______________________________________                                    
Na 0.39, 0.24                                                             
         0.25        0.31        0.38                                     
(0.31 0.1%)                                                               
Fe 3.14, 3.11                                                             
         3.05        3.05        3.04                                     
(3.12 0.02%)                                                              
C 47.11  49.87       49.84       49.81                                    
H 7.33   7.31        7.30        7.29                                     
N 14.81  14.49       14.48       14.47                                    
O --     25.03       25.02       25.01                                    
Mwt.     36632.23    36654.21    36676.18                                 
______________________________________                                    
 SB = C.sub.1521 H.sub.2467 N.sub.379 O.sub.573                           
 
    
     These results confirm chelation of 20 ±2 moles of ferric ions per mole of STARBURST™ dendrimer. 
     Example 6: Preparation of a Product Containing More Than One Rhodium Atom Per STARBURST™ Polymer 
     2.5 Gen PAMAM (ester terminated, initiated from NH 3 ), prepared as in Example GG, (0.18 g, 0.087 mmole) and RhCl 3 .3H 2  O (0.09 g, 0.3 mmole) were mixed in dimethylformamide (DMF) (15 mL) and heated for 4 hours at 70° C. The solution turned crimson and most of the rhodium was taken up. The unreacted rhodium was removed by filtration and the solvent removed on the rotary evaporator. The oil formed was chloroform soluble. This was washed with water and dried (MgSO 4 ) before removal of solvent to yield a red oil (0.18 g). The NMR spectrum was recorded in CDCl 3  only minor differences were noted between the chelated and unchelated STARBURST™. Dilution of some of this CDCl 3  solution with ethanol followed by NaBH 4  addition resulted in rhodium precipitation. RhCl 3 .3H 2  O is insoluble in chloroform and in chloroform STARBURST™ solution thus confirming chelation. 
     Example 7: Preparation of a Product Containing Pd Chelated to a STARBURST™ Polymer 
     3.5 Generation PAMAM (ester terminated, initiated from NH 3 ), prepared as in Example GG, (1.1 g, 0.24 mmole) was dissolved with stirring into acetonitrile (50 mL). Palladium chloride (0.24 g, 1.4 mmole) was added and the solution was heated at 70°-75° C. (water bath) overnight. The PdCl 2  was taken up into the STARBURST™. After removal of the solvent, the NMR in CDCl 3  confirmed that chelation had occurred. Dilution of the CDCl 3  solution with ethanol and addition of NaBH 4   resulted in precipitation of the palladium. The chelated product (1.23 g) was isolated as a brown oil. 
     Example 8: Demonstration of Multiple Chelation of Yttrium by a Methylene Carboxylate Terminated Second Generation STARBURST™ Polyethyleneimine by Trans Chelation from Yttrium Acetate 
     The STARBURST™ polyethyleneimine methylene carboxylate terminated material (0.46 g, 52.5 percent active, remainder sodium bromide, 0.18 mmol active STARBURST™ dendrimer), from Example FF, was dissolved in 4.5 mL of deuterium oxide. The resultant pH was 11.5-12. A solution of yttrium acetate was prepared by dissolving yttrium chloride (0.15 g, 0.5 mmol) and sodium acetate (0.41 g, 0.5 mmol) in 1.5 mL of deuterium oxide (2.9 moles of yttrium per mole of dendrimer). Aliquots of 0.5 mL of the yttrium acetate solution were added to the dendrimer solution and the  13  C NMR spectra recorded at 75.5 MHz. 
     The  13  C NMR spectrum of yttrium acetate shows two resonances, 184.7 ppm for the carboxyl carbon and 23.7 ppm for the methyl carbon, compared with 182.1 and 24.1 ppm for sodium acetate, and 177.7 and 20.7 ppm for acetic acid (Sadtler  13  C NMR Standard Spectra). Monitoring the positions of these bands indicates degree of chelation with the STARBURST™ dendrimer. The most informative signal for the STARBURST™ dendrimer which is indicative of chelation is the α-CH 2  (of the methylene carboxylate group involved in chelation), which appears at 58.4 ppm in the unchelated dendrimer, and 63.8 ppm in the chelated dendrimer. Upon chelation with yttrium, the spin lattice relaxation times of the time α-CH 2  shortens as expected from 0.24 ±0.01s to 0.14 ±0.01s, indicative of chelation. 
     Following the addition of 0.5 mL of the yttrium acetate solution to the STARBURST™ dendrimer, all the yttrium appeared to be chelated by the dendrimer, confirmed by the signals for the acetate being that of sodium acetate. The same observation was noted for the addition of a second 0.5 mL aliquot of the yttrium acetate solution. Upon addition of the third aliquot of yttrium acetate, not all of the yttrium was observed to be taken up as the STARBURST™ chelate, the acetate carboxyl resonance was observed to shift to 183.8 ppm indicating that some of the yttrium was associated with the acetate. The integrated area of the chelated --CH 2  groups on the dendrimer increased, indicating that some of the third mole equivalent of yttrium added was indeed chelated with the dendrimer. These results indicate that the dendrimer can chelate from 2-3 yttrium ions per dendrimer molecule (Generation=2.0). 
     Example 9: Demonstration of Multiple Chelation of Yttrium by a Methylene Carboxylate Terminated Second Generation STARBURST™ Polyamidoamine by Trans-chelation from Yttrium Acetate 
     The same experimental methods were used for this study as were used for Example 8. The STARBURST™ polyamidoamine methylene-carboxylate terminated material (0.40 g 62.5% active, remainder sodium bromide, 0.12 mmol) was dissolved in 4-5 mL of deuterium oxide. The resultant pH was 11.5-12, which was lowered to 9.4 with 6N HCl prior to the experiment. A solution of yttrium acetate was prepared by dissolving yttrium chloride (0.1125 g, 0.37 mmol) and sodium acetate (0.0915 g, 1.1 mmol) in 1.5 mL of deuterium oxide, thus every 0.5 mL of solution contains one mole equivalent of metal. 
     The first two mole equivalents of yttrium acetate added were fully chelated by the STARBURST™ polyamidoamine. On addition of a third mole equivalent of yttrium, precipitation of the product occurred and as such no NMR data could be obtained. The signals which gave the most information about chelation by the STARBURST™ dendrimer were those of the two carbons adjacent to the chelating nitrogen. The chemical shifts of these carbons in the unchelated dendrimer occurred at 59.1 ppm for the α-CH 2 , and 53.7 ppm for the first methylene carbon of the backbone. Upon chelation these two resonances were observed to shift downfield to 60.8 and 55.1 ppm respectively. The trans chelation shows that two metal ions can be readily chelated per dendrimer molecule, however upon chelation of some unknown fraction of a third mole equivalent, the product precipitates out of solution. 
     Example 10: Demonstration of Multiple Chelation of  90  Y by a Methylenecarboxylate Terminated Second Generation STARBURST™ Polyethyleneimine 
     Standard solution of yttrium chloride (3×10 -2  M, spiked with non-carrier added  90  Y) and methylenecarboxylate terminated second generation STARBURST™ polyethyleneimine of Example FF (6×10 -2  M) were prepared. These were reacted together at various metal:STARBURST™ ratios in HEPES buffer. The complex yield was determined by ion exchange chromatography using Sephadex G50 ion exchange beads, eluting with 10% NaCl:NH 4  OH, 4:1 at pH 10. Noncomplexed metal is removed on the column, complexed metal elutes. Yields were obtained by comparing the radioactivity eluted with that on the column, using a well counter. 
     
                       TABLE V                                                     
______________________________________                                    
Chelation of 2.5 Gen. PEI Acetate with .sup.90 Y                          
        Vol.   Vol                                                        
Vol. Y + 3                                                                
        PEI    HEPES    M:L Theor.                                        
                                % Complex                                 
                                        M:L Act.                          
______________________________________                                    
 5      30     370      0.1     110     0.1                               
 10     30     360      0.2     101     0.2                               
 20     30     350      0.4     95      0.4                               
 30     35     340      0.5     97      0.5                               
 30     30     340      0.5     102     0.5                               
 60     30     310      1.0     99      1.0                               
120     30     250      2.0     100     2.0                               
180     30     180      3.0     94      2.8                               
250     30     120      4.1     80      3.3                               
300     20      80      7.5     44      3.3                               
300     20      70      5.0     40      2.0                               
300     20      70      5.0     41      2.0                               
______________________________________                                    
 
    
     All volumes in Table V are in microliters 
     Within the accuracy of the experiments, these results indicate that the 2.5 Gen. STARBURST™ PEI acetate can chelate between 2 and 3 metals per dendrimer giving a soluble complex. 
     Example 11: Conjugation of 4-Isothiocyanatophenyl Methylenecarboxylate Terminated Third Generation STARBURST™ Polyethyleneimine with IgG Monoclonal Antibody 
     The isothiocyanate, 10 mg (50 μmoles), from Example DD was dissolved in 500 μL of 3 mM indium chloride which had been spiked with radioactive indium-111 chloride and the pH was adjusted to 9 with 660 μL of NaOH. Aliquots of whole monoclonal antibody IgG CC-46 were then mixed with aliquots of the chelated STARBURST™. The mixtures were shaken then left for 18 hours. The mixtures were then analyzed by HPLC (column Dupont Zorbax Biosphere GF-250; eluent 0.25M sodium acetate, pH 6) and a UV detector at 254 nm and a radioactivity detector. Results are shown in Table VI. 
     
                       TABLE VI                                                    
______________________________________                                    
Starburst-IgG Conjugates                                                  
             1    2         3     4                                       
______________________________________                                    
IgG solution (μL)                                                      
               20     20        20  20                                    
Chelated Starburst                                                        
               5      20        50  100                                   
solution (μL)                                                          
% Radioactivity                                                           
               6      5          5   3                                    
on IgG                                                                    
% IgG conjugated                                                          
               2      7         17  22                                    
______________________________________                                    
 
    
     Example 12: Conjugation of 4-Isothiocyanatophenyl Methylenecarboxylate Terminated Third Generation STARBURST™ Polyethyleneimine with IgG Monoclonal Antibody 
     The isothiocyanate from Example DD, 4 mg (20 μmoles) was mixed with 200 μL of 3 mM indium chloride (60 μmoles). A 20 μL aliquot of the solution was then spiked with radioactive indium-111 chloride and the pH adjusted to 9 by the addition of 30 μL 1N NaOH and 10 μL of 0.1N HCL. The indium chelate was mixed with 150 μL of CC-49 whole antibody IgG, 10 mg/mL in 50 mM HEPES buffer at pH 9.5. After 18 hours at room temperature the antibody was recovered by preparative HPLC (column Dupont Zorbax Biosphere GF 250; eluent 0.25M sodium acetate, pH 6); and a UV detector at 254 nm and a radioactivity detector. The recovered antibody was concentrated on an Amicon membrane and exchanged into PBS buffer (phosphate buffered saline pH 7.4 which contains 0.12M NaCl, 2.7 mM KCl and 10.0 mM phosphate) at pH 7.4. The recovered antibody had specific activity of approximately 0.5 μCi/100 μg. 
     Example 13: In vivo Localization of  111  In Labeled STARBURST™ Antibody Conjugate 
     The usefulness of the labeled STARBURST™ antibody conjugate prepared in Example 12 was demonstrated by measuring the uptake of the material by a human tumor xenograft in an athymic mouse. Female athymic mice were innoculated subcutaneously with the human colon carcinoma cell line, L-174T (approximately 4×10 6  cells/animal). Approximately two weeks after innoculation, each animal was injected via the tail vein. The mice were sacrificed after 17 and 48 hours (five animals at each time point), the tumor and selected tissues were excised and weighed, and radioactivity was measured in a gamma counter. After 17 hours 13.5 percent of the injected dose per gram of tissue had localized at the tumor. After 48 hours 21.6 percent of the injected dose per gram of tissue had localized at the tumor. 
     Example 14: Attachment of Herbicidal Molecules (2,4-D) to the Surface of STARBURST™ Dendrimers 
     Third generation PAMAM (initiator core=NH 3 ) (2.0 g, 0.8 mmole) was dissolved in H 2  O (10 mL) and combined with toluene (20 mL). The two-phase system was then stirred and cooled with an ice bath at which time the acid chloride of 2,4-D [2,4-dichlorophenoxy)-acetic acid] (2.4 g, 12 equiv) dissolved in toluene (10 mL) was added dropwise over 30 minutes. When the addition was nearly complete, NaOH (0.5 g, 12.5 mmole, 50% w/w solution) was added and the solution stirred for an additional two hours. The reaction mixture was then evaporated to dryness and the resulting solid residue repeatedly taken up in CHCl 3  /MeOH (1:1) and filtered. The tan solid was not totally soluble in CHCl 3  and appeared to be insoluble in water; however, the addition of acetone facilitated dissolution. The tan solid was stirred in CHCl 3  for 24 hours and the solution filtered (a sticky tan solid was obtained). After drying over MgSO 4 , the filtrate was concentrated to give a viscous orange oil which solidified on standing. The  13  C NMR indicated partial amidation at the surface by 2,4-D and is consistent with the association of the 2,4-D to STARBURST™ polymer. 
     Example 15: Inclusion of 2,4-Dichlorophenoxyacetic Acid (2,4-D) into STARBURST™ Dendrimers 
     A widely accepted method for ascertaining whether a &#34;probe molecule&#34; is included in the interior of a micelle is to compare its carbon-13-spin lattice relaxation times (T 1 ) in a non-micellized versus micellized medium. A substantial decrease in T 1  for the micellized medium is indicative of &#34;probe molecule&#34; inclusion in the micelle. Since STARBURST™ dendrimers are &#34;covalently fixed&#34; analogs of micelles, this T 1  relaxation time technique was used to ascertain the degree/extent to which various herbicide type molecules were associated with STARBURST™ polyamidoamines. In the following examples, T 1  values for 2,4-dichlorophenoxy-acetic acid (I) (2,4-D) were determined in solvent (CDCl 3 ) and then compared to T 1  values in CDCl 3  at various [I:dendrimer] molar ratios. 
     Inclusion of 2,4-D into various STARBURST™ polyamidoamine dendrimers as a function of generation. 
     Various half generation (ester terminated, initiated off NH 3 ) STARBURST™ polyamidoamine dendrimers (Generation (Gen)=0.5, 1.5, 2.5, 3.5, 4.5 and 5.5) were combined with 2,4-dichlorophenoxyacetic acid (I) in CDCl 3  to give an acid:tertiary amine ratio of 1:3.5 and molar ratios of acid:dendrimer of 1:86 as shown in Table VII. The relaxation times (T 1 ) obtained for the various carbon atoms in 2,4-dichlorophenoxyacetic acid and a generation=3.5 STARBURST™ PAMAM dendrimers are shown in Table VIII, both for 1:1 acid/amine ratios and for saturated solutions of 2,4-D. 
     
                       TABLE VII                                                   
______________________________________                                    
                                    (D)                                   
                            (C)     Molar Ratio                           
      (A)        (B)        Acid/Total                                    
                                    (Acid/Star-                           
Gen   Acid/Amine Acid/Amine Nitrogen                                      
                                    burst)                                
______________________________________                                    
0.5   1          --         1       1                                     
1.5   1          1.33       0.57    6                                     
2.5   1 (3.5)*   1.11 (3.8)*                                              
                            0.53 (1.8)*                                   
                                     9 (34)*                              
3.5   1 (3.0)*   1.05 (3.2)*                                              
                            0.51 (1.6)*                                   
                                    20 (67)*                              
4.5   1          1.02       0.51    42                                    
5.5   1          1.01       0.50    86                                    
______________________________________                                    
 *represents examples of 2,4D inclusion into the interior of the dendrimer
 in amounts greater than stoichiometric.                                  
 
    
     
                       TABLE VIII                                                  
______________________________________                                    
T.sub.1 &#39;s for 2,4-D/G = 3.5 PAMAM STARBURST ™                         
Inclusion complex: Concentration Effects                                  
                   (B)                                                    
(A)                Saturated with                                         
1:1 Acid/Amine     2,4-D                                                  
Carbon T.sub.1   .sup.13 C**                                              
                           T.sub.1   .sup.13 C**                          
______________________________________                                    
1      3.19 ± .12                                                      
                 (152.73)  3.08 ± .09                                  
                                     (152.30)                             
3      0.34 ± .01                                                      
                 (128.64)  0.29 ± .01                                  
                                     (129.62)                             
5      0.38 ± .01                                                      
                 (127.41)  0.32 ± .01                                  
                                     (127.34)                             
2      3.28 ± .08                                                      
                 (125.79)  2.72 ± .68                                  
                                     (125.99)                             
4      4.58 ± .16                                                      
                 (123.27)  3.95 ± .07                                  
                                     (123.16)                             
6      0.31 ± .01                                                      
                 (114.66)  0.28 ± .01                                  
                                     (114.48)                             
CH.sub.2                                                                  
       0.16 ± .01                                                      
                  (67.29)  0.146 ± .003                                
                                      (66.79)                             
C═O                                                                   
       1.24 ± .07                                                      
                 (170.12)  --        --                                   
______________________________________                                    
 **represents .sup.13 C chemical shifts referenced to chloroform at 76.9  
 ppm.                                                                     
 
    
     These data show that larger than stoichiometric amounts of 2,4-dichlorophenoxyacetic acid (i.e., [(I):Gen=3.5 dendrimer)]=67) can be used without increasing the T 1  in any case in the saturated state (see Columns (A) and (B) in Table VIII). In fact, the relaxation times T 1  in Column (B) are decreased slightly, thus indicating that larger than stoichiometric amounts of 2,4-dichlorophenoxyacetic acid can be included into the interior of the dendrimer. For example, a molar ratio of [(I):Gen=2.5 dendrimer]=34 whereas [(I):Gen=3.5 dendrimer]=67, (see Column D in Table VII). 
     FIG. 11 is a plot of T 1  values for carbons-3, 5 and 6 in 2,4-dichlorophenoxyacetic acid as a function of dendrimer generation (i.e., 0.5→5.5). The lower dashed line (---) is for carbons-3 and 6 and the upper solid line is for carbon 5. A minimum in T 1  is reached in all cases for generation 2.5→5.5, thus indicating incorporation in that dendrimer generation range is occurring. FIG. 11 also includes T 1  values for 2,4-D in the presence of triethylamine [N(Et) 3  ] (points B1 and B2) and N(Et) 3  +N-methylacetamide (points A1 and A2). It can be seen that these values are much larger than for dendrimers G=1.5→5.5, thus further supporting molecular incorporation into the dendrimer molecule. 
     Example 16: Preparation of a Product Containing Fluorescein with a STARBURST™ Polymer 
     A sample of 5-carboxyfluorescein (0.996 g) and STARBURST™ polyethyleneimine (Gen=2.0; amine terminated, initiated off NH 3 ) (0.202 g) were mixed in 10 mL of methylene chloride and 5 mL of methanol and allowed to reflux for 10 minutes. Upon filtering, an insoluble red powder (0.37 g) was obtained (mostly unreacted 5-carboxy fluorescein). From the filterate was isolated 0.4 g of a brilliant-red solid which exhibited a softening point of 98°-103° C. and foamed to a brilliant red melt at 175°-180° C.; NMR spectra (D 2  O) of this product were consistent with dendrimer having fluorescein bound to the surface. 
     Example 17 
     In a procedure similar to that described in Example 16, STARBURST™ polyethyleneimine (Gen=2.0; amine terminated, initiated off NH 3 ) was reacted with fluorescein isothiocyanate to give a brilliant-red iridescent solid which was suitable for use as a fluorescent labelling reagent. 
     Example 18: Encapsulation of R(+)--Limonene in Polyamidoamine STARBURST™ Dendrimers 
     A 5-50 weight percent solids solution in methanol of STARBURST™--PAMAM dendrimer (M.W. about 175,000; generation=9.0) is added dropwise to R(+) limonene in methanol until saturated. The solution is stirred at room temperature (about 25° C.) for several hours and then devolatized on a Buchi rotovap at room temperature to give a solid product. Warming at temperatures greater than 80° C. gives solvent insoluble products which retain substantial amounts of R(+) limonene in an encapsulated form. These products are excellent prototypes for slow release of (R+)-limonene as a fragrance and deodorizer product. 
     Example 19: Encapsulation of Heavy Metal Salts in Polyamidoamine STARBURST™ Dendrimers 
     A 5-50 weight percent solids solution in water of STARBURST™ PAMAM dendrimer (M.W. about 350,000; generation=10.0) is stirred as a saturated solution of lead acetate [Pb(C 2  H 3  O 2 ) 2  ] is added dropwise. The solution is stirred at room temperature (about 25° C.) for several hours and then devolatilized on a Buchi rotovap to give solid products. Scanning transmission electron-micrograph of these products showed that these heavy metal salts are encapsulated in the interior of the dendrimers. These films containing heavy metal salts are useful as shields for absorbing electromagnetic radiation. 
     Example 20: Encapsulation of Fluorescein (water soluble) Dye in Polyamidoamine STARBURST™ Dendrimers 
     A 5-50 weight percent solids solution (H 2  O/CH 3  OH) of STARBURST™-PAMAM dendrimer (M.W. about 175,000; generation=9.0) is stirred as fluorescein, disodium salt (Acid Yellow 73, Cl. 45350; Uranine; available from Aldrich (Chemical Co., Milwaukee, Wis.) is added until saturated. The solution is stirred at room temperature (about 25° C.) for several hours and then devolatilized at room temperature to give a colored solid product. These dye encapsulated dendrimers are excellent reference probes for calibrating ultrafiltration membranes. 
     Example 21: Preparation of Dendrimers with Terminal Fluorescent Groups 
     A. Reaction of Amine Terminated Dendrimer with N-Dansyl Aziridine 
     A sample (1.5 g, 1.6×10 -3  mole) of STARBURST™ polyethyleneimine (PEI), G=3.0, terminal groups (Z)= 12, M.W.=920) is dissolved in 20 mL of methanol. The solution is stirred and 0.884 g (3.84×10 -2  mole) of a solution of N-dansyl aziridine (ICN Biomedicals, Costa Mesa, Calif.) is added dropwise over a period of 20 minutes. The reaction mixture is allowed to stir at room temperature overnight. Removal of solvent under vacuum gives a solid product. NMR and infrared analysis indicate that the product possessed covalently bonded dansyl groups on the surface of the dendrimer. 
     B. Reaction of Amine Terminated Dendrimers with Dansyl Chloride 
     A solution of STARBURST™ polyamidoamine (1.0 g, 1.9×10 -4  mole) (initiated from ammonia, G=4.0, terminal groups (Z)=24, M.W.=5,147) in 30 mL of water is stirred in a 3-neck flask with 80 mL of toluene while a solution of dansyl chloride (1.23 g, 4.5×10 -3  mole) (5-dimethyl-amino-1-naphthalenesulfonyl chloride, from Aldrich Chemical Co., Milwaukee Wis.) in 40 mL of toluene is added dropwise while cooling with ice. Concurrently, a solution of 10% NaOH (13.3 mole, 10% excess) is added to the reaction mixture to give an oily ball. The product is washed with water, dissolved in methanol, and precipitated with diethyl ether to give a solid product. NMR and infrared analyses are consistent with covalently bonded dansyl groups on the dendrimer surface. 
     Example 22: Preparation of Manganese PAMAM Dense Star Polymer 
     Following the procedure set forth in Example 1 of U.S. Pat. No. 4,587,329, a third generation ester-terminated polyamidoamine dense star polymer is prepared. The ester moieties are hydrolyzed to sodium salt form by dissolving the dense star polymer in 0.1N NaOH. A 131-mg (0.496 mmol) portion of the resulting crude sodium salt of the dense star polymer is dissolved in water (1 mL, pH 11.4) and neutralized to a pH of 7.0 by adding 0.5M HCl. A 26-mg (2 equiv) portion of MnCl 2 .4H 2  O is added to the neutralized solution and the solution is heated at 55 C for 2 days. The resultant light yellow solution is purified using a Bio-Gell P2 column (15.0 g, 150 mm length×25 mm diameter) eluting with watery and isolating the colored band. The product was confirmed to be chloride free (e.g. NaCl, MgCl 2 ) by silver nitrate test. The eluate is stripped to dryness to produce a clear gel which is dried under high vacuum to yield 45 mg of the desired dense star polymer complex which is determined to have the following elemental constituency, [NaMn 2  H 7  ][C 69  H 105  N 19  O 33  ]. 
     
         ______________________________________                                    
        Na     Mn     C         H    N                                    
______________________________________                                    
Calculated:                                                               
          1.23;    5.88;  44.35;  5.66;                                   
                                       14.24                              
Found:    1.42;    4.80;  43.33;  7.91;                                   
                                       14.72.                             
______________________________________                                    
 
    
     Following a similar procedure using a seventh generation sodium carboxylate-terminated polyamidoamine dense star polymer, an additional manganese dense star polymer complexis prepared which is determined to have the following elemental constituency, [Na 30  H 354  ][Mn 10  C 3057  H 4965  N 763  O 1149  ], 
     
         ______________________________________                                    
         Mn    C          H      N                                        
______________________________________                                    
Calculated:                                                               
           0.759;  50.72;     7.41;                                       
                                   14.76;                                 
Found:     0.72;   46.34;     7.91;                                       
                                   14.72.                                 
______________________________________                                    
 
    
     The relaxivities for the foregoing complexes are measured in water and the results are reported in Table IX. For the purposes of comparison, the relaxivities of complexes of manganese with two known complexing agents are calculated from the corresponding relaxation times measured by nuclear magnetic resonance. The results are reported in Table IX. 
     
                       TABLE IX                                                    
______________________________________                                    
                RELAXIVITY*                                               
COMPOUND          R.sub.1   R.sub.2                                       
______________________________________                                    
MnCl.sub.2        5.2       &gt;20                                           
MnEDTA            2.9       --                                            
MnDTPA            1.3       --                                            
Mn.sub.2 [PAMAM-A(2.5 G)]                                                 
                  3.63 ± 0.02                                          
                            16.35 ± 0.78                               
______________________________________                                    
 *(mM · sec).sup.-1, 37° C., 90 MHz. Values per metal atom
                                                                          
 
    
     Example 23 
     Following the procedure of Example 22, dense star polymer complexes of iron are prepared. In one embodiment, the sodium salt of the acid form of a sixth generation polyamidoamine is prepared by dissolving the ester form of the polyamidoamine made in accordance with the excess reactant method of U.S. Pat. No. 4,587,329 in 1N NaOH. The sodium salt (97 mg, 2.4×10 -3  mmol) is then dissolved in 2 mL of water and the solution (pH of 11.3) is neutralized to a pH of 7 with 0.5M HCl. To this neutralized solution is added an aqueous solution of FeCl 3  (0.102M, pH 1.4, 0.5 mL, 0.051 mmol, 21.6 equiv). The resulting deep orange solution (pH 4.9) is heated at 55° C. for 23 hr. The solution is cooled to ambient temperature and filtered by gel filtration chromatography (Biogel P2 column, 175×25 mm). The orange eluate is stripped to dryness under vacuum at 50° C. to yield 45 mg of an orange solid having the following molecular formula: [Na 5  H 127  ][Fe 20  (PAMAM-P(6.5G))] wherein PAMAM-P represents the dense star polyamidoamine in the propionate terminated form. This orange solid is determined to be a complex of iron and the polyamidoamine dense star polymer represented by the formula Na 5  H 127  Fe 20  (C 1521  H 2469  N 379  O 573 ), 
     
         ______________________________________                                    
        Na     Fe     C         H    N                                    
______________________________________                                    
Calculated:                                                               
          0.31;    3.05;  49.84;  7.30;                                   
                                       14.48                              
Found:    0.31;    3.12;  47.11;  7.33;                                   
                                       14.81.                             
______________________________________                                    
 
    
     Following the above procedure, similar complexes of iron and acetate-terminated polyamidoamine dense star polymers or acetate-terminated polyethylenimines dense star polymers having from 2 to 6 generations are prepared. The relaxation times for these complexes are measured using nuclear magnetic resonance and the corresponding relaxivities are calculated and reported in Table X. 
     
                       TABLE X                                                     
______________________________________                                    
                 RELAXIVITY*                                              
COMPOUND           R.sub.1    R.sub.2                                     
______________________________________                                    
FeCl.sub.3         8.4 ± 0.1                                           
                              --                                          
FeEDTA             1.7        --                                          
FeDTPA             0.73       0.85                                        
FeEHPG             0.95       1.1                                         
Fe.sub.2 [PEI-A(2.5G)]                                                    
                   0.40 ± 0.02                                         
                              0.88 ± 0.03                              
Fe.sub.2 [PAMAM-A(2.5G)]                                                  
                   0.77 ± 0.01                                         
                              --                                          
Fe.sub.2 [PAMAM-P(2.5G)]                                                  
                   0.135 ± 0.004                                       
                              0.42 ± 0.02                              
Fe.sub.4 [PAMAM-P(4.5G)]                                                  
                   0.038 ± 0.006                                       
                              1.01 ± 0.05                              
Fe.sub.20 [PAMAM-P(6.5G)]                                                 
                   0.047 ± 0.007                                       
                              0.50 ± 0.07                              
Fe.sub.50-60 [PAMAM-P(7.5G)]                                              
                   about 0.3**                                            
                              about 62**                                  
{SPEI-[DTPA][Fe(DTPA)].sub.2 }                                            
                   1.094 ± 0.050                                       
                              1.70 ± 0.02                              
______________________________________                                    
 *(mM · sec).sup.-1, 37° C., 90 MHz. Values per metal atom
 **Values per complex.                                                    
 
    
     Example 24 
     Using the method of Example 22 complexes of gadolinium and the polyamidoamine and polyethyleneimine dense star polymers described in Example 23 are prepared and their relaxation times are measured and relaxivities calculated with the result being shown in Table XI. As an illustration of the preparation of the gadolinium complex, 0.40 g (0.16 mmol) of the sodium acetate form of a polyethyleneimine dense star polymer (second generation) prepared as in Example 6 of U.S. Pat. No. 4,587,329 and followed by reaction with an excess of bromoacetic acid at pH=9, 40° C., is dissolved in 5 mL of water. The pH of the resulting solution is lowered to 9.2 with 0.5N HCl and 3.2 mL of an aqueous solution of GdCl 3  (0.31 mmol) is added. The pH of the resulting solution is increased from 4.1 to 8.3 with 1N NaOH and the solution is allowed to stand for 24 hrs. The volatiles are then removed from the solution in vacuo and the residue twice chromatographed in chelex resin. The recovered light yellow solid was found to have an atomic ratio of Gd to N of 2:1. 
     Found (%): C, 14.5; H, 2.1; N, 3.6; Gd, 7.9; Br, 28.7. 
     Following the above procedure, similar complexes of gadolinium and acetate-terminated polyamidoamine dense star polymers or acetate-terminated polyethylenimines dense star polymers having from 2 to 6 generations are prepared. The relaxation times for these complexes are measured using nuclear magnetic resonance and the corresponding relaxivities are calculated and reported in Table XI. 
     
                       TABLE XI                                                    
______________________________________                                    
                 RELAXIVITY*                                              
COMPOUND         R.sub.1                                                  
______________________________________                                    
Gd(NO.sub.3).sub.3                                                        
                 8.6 ± 0.3                                             
GdCl.sub.3       8.7 ± 0.1                                             
GdCl.sub.3 (0.15M saline)                                                 
                 10.43 ± 0.36                                          
GdEDTA           6.5                                                      
GdDTPA           3.9                                                      
GdDOTA           about 4                                                  
Gd(PAMAM-A)      9.90 ± 0.43                                           
Gd.sub.2 (PAMAM-A)                                                        
                 13.00 ± 0.33                                          
Gd(PEI-A)        8.85 ± 0.19                                           
Gd.sub.2 (PEI-A) 15.35 ± 0.17                                          
Gd.sub.2 (PEI-A) + 3PEI-A                                                 
                  8.0 ± 0.19                                           
Gd(DTPMP)        10.8 ± 0.4                                            
Gd.sub.2 (PEI-A) + 2EDTA                                                  
                 about 6                                                  
______________________________________                                    
 *(mM · sec).sup.-1, 37° C., 90 MHz. Values per metal atom
                                                                          
 
    
     Example 25: Preparation of Polyamidoamine STARBURST™ Conjugate with Bradykinin C 
     Bradykinin Potentiator C, 4.7 mg (Sigma Chemical Company), was taken up in 90 μL of N-methylpyrrolidinone (NMP) containing 7.1 mg/mL N-hydroxybenzotriazole (HOBT) and mixed with 33 μL of a freshly prepared solution of dicyclohexylcarbodiimide (DCC) (28 mg/mL NMP). After four hours at room temperature, 60 μL of fifth generation (5G) PAMAM in NMP (13.4 mg/mL) was added to the Bradykinin mixture. After 72 hours at room temperature, 1.5 mL of 17 mM acetic acid was added. The mixture was centrifuged and the solution decanted from the solid and the solid then washed with 1.5 mL of dilute acetic acid. The solution and washings were combined and filtered through a 25 mm PTFE 0.45μ syringe filter. Unreacted peptide, HOBT and solvent were separated from the conjugate by ultra-filtration using two Centricon-10 Microconcentrators. The conjugate was washed with 2 mL of 17 mM acetic acid, then 2 mL PBS buffer. The conjugate was recovered in 450 μL of retentate and diluted to 750 μL with fresh PBS buffer. 
     Four aliquots of the conjugate were hydrolyzed with concentrated HCl at 115° C. for 20 hours and analyzed for glutamic acid. The glutamic acid was quantified as the o-phthalaldehyde/2-mercaptoethanol derivative using reverse phase HPLC with a florescence detector (column: Whatman Partisil 5CCS/C8 (10 cm); Gilson Spectra/Glo detector; tyrosine as internal standard; eluent 50 mM sodium acetate and methanol; gradient: 12% to 30% methanol in 5 min., held for 1 min. at 30%, then increased to 80% methanol over 14 min. and held at 80% for 5 min.). The o-phthalaldehyde derivative was prepared by mixing 25 μL of the amino acid solution with 50 μL fluoraldehyde™ o-phthalaldehyde reagent solution (Pierce), mixing for one minute and then diluting with 0.05 mL of PBS buffer (phosphate buffered saline; 120 millimolar sodium chloride, 2.7 millimolar potassium chloride, 10 millimolar phosphate buffer salts, pH 7.4). For a 25 μL aliquot of the conjugate, 58 nmoles of glutamic acids were found. This corresponds to 1.74 μmoles Bradykinin C on the conjugate. 
     Example 26: Preparation of Polyamidoamine STARBURST™ Conjugate with BOCPheLeuPheLeuPhe 
     N-t-butoxy-carbonyl-L-Phenylalanyl-D-Leucyl-L-Phenylalanyl-D-Leucyl-L-Phenylalanine (BOCPheLeuPheLeuPhe), 4.1 mg (Sigma Chemical Company), was taken up in 100 μL NMP which contained 7.1 mg/mL HOBT and mixed with 30 μL of a freshly prepared solution of DCC (36 mg/mL) in NMP. After 90 minutes at room temperature, 60 μL of a solution of 5G PAMAM in NMP (13.4 mg/mL) was added. After 72 hours at room temperature, the conjugate was isolated following the procedure in Example 24. The conjugate was recovered in 750 μL of retentate. 
     Four aliquots of the conjugate were hydrolyzed with concentrated HCl at 115° C. for 20 hours and analyzed for phenylalanine. The phenylalanine was quantified as the o-phthalaldehyde/2-mercaptoethanol derivative using reverse phase HPLC with a fluorescence detector (HPLC conditions as Example 24 except the eluent gradient was 12% to 80% methanol over 20 min. and then held at 80% for 5 min.). The o-phthalaldehyde derivative was prepared as given in Example 24. For a 25 μL aliquot of the conjugate, 26 nmoles phenylalanine were found. This corresponds to 0.26 μmoles BOCPheLeuPheLeuPhe on the conjugate. 
     Example 27 
     To a methanol solution of 1,4,7-tris-(carbomethoxymethyl)-1,4,7,10-tetraazacyclododecane (DO3A methyl ester) is added 1.0 equivalent of freshly prepared anhydrous sodium methoxide and the solution is allowed to stir for several hours. To this solution is added one equivalent of 10,11-epoxyundecanoic acid and the solution is brought to reflux overnight. The solvent is removed in vacuo to give a crude hydroxyethylated product, 1,4,7-tris-(carbomethoxymethyl)-10-[2&#39;-hydroxy-10&#39;-carboxy(decyl)]-1,4,7,10-tetraazacyclododecane. Two to five equivalents of this product are dissolved in dimethylformamide (DMF) and corresponding equivalents of dicyclohexyl-carbodiimide (DCC) and N-hydroxysuccinimide are added with stirring overnight. To this solution is added one equivalent of generation 2 PAMAM and stirring is continued another 24 hours. The corresponding DO3A methyl ester conjugate is selectively hydrolyzed by treatment with aqueous sodium hydroxide in methanol. Treatment of the DO3A conjugate with excess Gadolinium(III) acetate (Gd(OAc) 3 ) (buffered to pH=6) followed by incubation with excess diethylenetriaminepentaacetic acid (DTPA) and purification of the conjugate using preparative reverse phase HPLC gives a hydroxyethyl DO3A PAMAM STARBURST™ conjugate product which contains two to five equivalents of gadolinium which can not be removed by further challenges with excess DTPA. 
     Example 28 
     To an aqueous solution of generation 2 PAMAM, which is buffered to pH=9.0, is added two to five equivalents of 1-[1-carboxy-3(4&#39;-isothiocyanatophenyl)propyl]-4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane prepared by the method of described in EP published application 0353450, published Feb. 7, 1990. The solution is stirred overnight at room temperature. Treatment of the solution with excess Gd(OAc) 3  at pH=6.0 results in the formation of a thiourea linked DOTA PAMAM STARBURST™ conjugate which contains two to five equivalents of inertly bound gadolinium which can not be removed with a DTPA challenge. 
     Example 29: Preparation of IgG/dendrimer-FITC I Conjugate 
     A: Preparation of PAMAM dendrimer conjugated to fluorescein isothiocyanate 
     A 2.2 mL methanolic solution of generation 8.0 PAMAM dendrimer was dried under vacuum on a rotary evaporator. Approximately 300 mg (7.4 μmol) of the dried dendrimer was quickly removed and dissolved in 1 mL of deionized water to form the stock dendrimer solution. A 60 mg/mL solution was prepared by mixing 200 μL of the stock dendrimer solution and adding it to 800 μL of deionized water. In order to achieve a final dendrimer concentration of 10 mg/mL (final dendrimer solution), 5 mL of 0.1M phosphate buffer (pH=9.0) was added to the 60 mg/mL solution. The final dendrimer solution, 3 mL, was transfered to a 2 dram vial (30 mg, 0.13 μmol). 
     Fluorescein isothiocyanate (FITC I, Molecular Probes), 5 mg, was dissolved in 1 mL of dimethyl sulfoxide in the dark. The fluorescein isothiocyanate solution (200 μL, 2.57 μmol) was added to the stirring dendrimer solution. The resulting reaction mixture was protected from light and stirred at room temperature for 15 to 20 hours. The solution remained clear and amber in appearance. 
     After stirring, the reaction mixture was placed into two Centricon™ microconcentrator tubes (molecular weight cutoff: 10,000 Daltons), and was ultrafiltered to remove any unreacted FITC I. During the ultrafiltration, ten, 2 mL additions of 0.1M phosphate buffer of pH 9.0 were made to facilitate the purification. The ultra filtrations were monitored for purity by thin-layer chromatography in order to insure the complete removal of any unreacted FITC I. The thin-layer chromatography was run using silica gel 60 plates (Merck) using an ethyl acetate (100%) solvent system. While the labelled dendrimer remained at the origin, the remaining FITC I eluted near an R f  =0.7. Approximately 2 mL of conjugated 8.0 generation PAMAM dendrimer-FITC I was recovered from the purification. 
     B: Preparation of PAMAM dendrimer conjugated to fluorescein isothiocyanate and IgG 
     Rabbit immunoglobulin (IgG, Sigma Chemical Co.), 9.8 mg, was dissolved in 1 mL of 30 mM of sodium acetate and 150 mM of sodium chloride buffer (pH=5.5). To the IgG solution was added 1.5 mg of sodium periodate with gentle stirring until dissolved. The mixture was protected from light and gently stirred at room temperature for 15-18 hours. After stirring, the reaction mixture was placed into two Centricon™ microconcentrator tubes (molecular weight cutoff: 10,000 Daltons), and was ultrafiltered into 20 mM phosphate buffer of pH=6.0 (4×2 mL). Approximately 1-2 mL of the IgG dialdehyde solution was recovered from the purification. 
     An aliquot of the labelled dendrimer-FITC I solution from Step A (0.88 mL, 17.6 mg, 0.075 μmol) was added dropwise to the IgG dialdehyde solution. (This represents about 1.15:1 ratio of labelled dendrimer to antibody.) Upon the addition of the first drop of the labelled dendrimer solution, the reaction mixture precipitated. An additional 3 mL of 20 mM phosphate buffer (pH=6.0) was added, but it did not solubilize the solids. 
     The IgG/dendrimer-FITC I conjugate was protected from the light and allowed to stir at room temperature for 1 hour. After that hour, 1.7 mg of sodium cyanoborohydride was added and the reaction was stirred overnight (15-20 hours) at room temperature. The solution was a fluorescent orange with some precipitate. The reaction product, including the precipitate, was placed into into two Centricon™ microconcentrator tubes (molecular weight cutoff: 100,000 Daltons), and was ultrafiltered. Six additional 2 mL volumes of the 20 mM phosphate buffer (pH=6.0) were added during the process to remove any unbound dendrimer-FITC I. The IgG/dendrimer-FITC I conjugate was recovered and transferred to an amber glass vial for storage at -10° C. 
     Example 30: Preparation of IgG/dendrimer-dansyl Conjugate 
     A: Preparation of 8.0 Generation PAMAM dendrimer-dansyl Conjugate 
     Using 200 μL of the stock dendrimer solution prepared in Example 29A and adding 800 μL of deionized water, the 60 mg/mL solution was prepared. To achieve the final dendrimer concentration of 10 mg/mL, 5 mL of 0.1M phosphate buffer (pH=9.0) was added to the 60 mg/mL solution. 
     Dansyl chloride (13.9 mg, 0.052 mmol, Aldrich Chemical Co.) was added to 5 mL of acetone and shaken for 5-10 minutes. (This solution was a deep yellow color and had traces of solid present.) The dansyl chloride solution was added to the dendrimer solution over a 30 second period and the reaction flask was placed in a water bath at 40° C. for 90 minutes. During the heating, the reaction mixture was shaken intermittently. The reaction mixture was slightly hazy in appearance and the color faded from a deep yellow to a faint yellow. After 90 minutes, the reaction flask was removed from the heat and allowed to cool to room temperature. The reaction mixture was then placed on a rotary evaporator, under vacuum at 30°-35° C. to remove the acetone. The reaction product was placed into two Centricon™ microconcentrator tubes (molecular weight cutoff: 100,000 Daltons), and was ultrafiltered to remove any unbound dansyl chloride. Ten, 2 mL additions of O.1M phosphate buffer (pH=9.0) were made during the ultrafiltration. 
     The ultrafiltrations were monitored for purity by thin-layer chromatography in order to insure the complete removal of any unreacted dansyl chloride. The thin-layer chromatography was run using silica gel 60 plates (Merck) using an ethyl acetate (100%) solvent system (visualization by UV light). While the labelled dendrimer remained at the origin, the remaining dansyl chloride eluted near an R f  =0.3. Approximately 1-2 mL of conjugated 8.0 generation PAMAM dendrimer-dansyl was recovered from the purification. 
     B: Preparation of PAMAM dendrimer conjugated to dansyl and IgG 
     Rabbit immunoglobulin (IgG, Sigma Chemical Co.), 9.8 mg, was dissolved in 1 mL of 30 mM of sodium acetate and 150 mM of sodium chloride buffer (pH=5.5). To the IgG solution was added 1.5 mg of sodium periodate with gentle stirring until dissolved. The mixture was protected from light and gently stirred at room temperature for 15-18 hours. After stirring, the reaction mixture was placed into two Centricon™ microconcentrator tubes (molecular weight cutoff: 100,000 Daltons), and was ultrafiltered into 20 mM phosphate buffer of pH=6.0 (4×2 mL). Approximately 1.9 mL of the IgG dialdehyde solution was recovered was recovered from the purification. 
     An aliquot, 1.25 mL, of the labelled dendrimer-dansyl solution from Step A (14.1 mg/mL) was added dropwise to the IgG dialdehyde solution. (This represents about 1.15:1 ratio of labelled dendrimer to antibody.) Upon the addition of the first drop of the labelled dendrimer solution, the reaction mixture precipitated. 
     The IgG/dendrimer-dansyl conjugate was protected from the light and allowed to stir at room temperature for 1 hour. After that hour, 1.7 mg of sodium cyanoborohydride was added and the reaction was stirred overnight (15-20 hours) at room temperature. The reaction product, including the precipitate, was placed into into two Centricon™ microconcentrator tubes (molecular weight cutoff: 100,000 Daltons), and was ultrafiltered. Six additional 2 mL volumes of the 20 mM phosphate buffer (pH=6.0) were added during the process to remove any unbound dendrimer-dansyl. The IgG/dendrimer-dansyl conjugate was recovered and transferred to an amber glass vial for storage at -10° C. 
     Example 31: Preparation of PAMAM Dendrimer Conjugated to HRP and IgG 
     A: Preparation of PAMAM dendrimer conjugated to IgG 
     Rabbit immunoglobulin (IgG, Sigma Chemical Co.), 9.6 mg, was dissolved in 1 mL of 30 mM of sodium acetate and 150 mM of sodium chloride buffer (pH=5.5). To the IgG solution was added 1.5 mg of sodium periodate with gentle stirring until dissolved. The mixture was protected from light and gently stirred at room temperature for 15-18 hours. After stirring, the reaction mixture was placed into two Centricon™ microconcentrator tubes (molecular weight cutoff: 100,000 Daltons), and was ultrafiltered into 20 mM phosphate buffer of pH=6.0 (4×2 mL). Approximately 1.5 mL of the IgG dialdehyde solution was recovered was recovered from the purification. 
     An aliquot, 100 μL, dendrimer stock solution from Step A of Example 29 was added to 100 μL of deionized water to yield 150 mg/mL solution. A final concentration of 15 mg/mL was attained by further dilution of 100 μL of the dendrimer solution with 900 μL of deionized water. The final dendrimer solution, 1 mL, was added dropwise to the IgG dialdehyde solution and the mixture stirred for 1 hour at room temperature. A precipitate was formed. After that hour, 1.7 mg of sodium cyanoborohydride was added and the reaction was stirred overnight (15-20 hours) at room temperature. The reaction product, including the precipitate, was placed into into two Centricon™ microconcentrator tubes (molecular weight cutoff: 100,000 Daltons), and was ultrafiltered. Four additional 2 mL volumes of the 20 mM phosphate buffer (pH=6.0) were added during the process to remove any unbound dendrimer-IgG. The IgG/dendrimer conjugate was recovered and transferred to a 2 dram vial. 
     B: Preparation of PAMAM dendrimer/IgG-HRP Conjugate 
     Horse-radish peroxidase (HRP, 2.6 mg, Pierce) was dissolved in 0.5 mL of deionized water to form the HRP solution. Sodium periodiate, 10.7 mg, was dissolved in 5 mL of 10 mM sodium phosphate (pH=7.0). Approximately 1.6 mL of the periodiate solution was added to the HRP solution at room temperature. The enzyme reaction mixture was protected from light and gently stirred at room temperature for 30 minutes. The oxidized HRP product was placed into into two Centricon™ microconcentrator tubes (molecular weight cutoff: 10,000 Daltons), and was ultrafiltered. Four additional 2 mL volumes of the 30 mM sodium acetate/150 mM sodium chloride buffer (pH=5.5) were added during the process. 
     The oxidized enzyme solution was added to the PAMAM dendrimer-IgG conjugate mixture with stirring. Although the percipitate remained in the reaction, the mixture was allowed to stir for 2 hours at room temperature. After the 2 hours, 2.1 mg of sodium borohydride was dissolved in 1 mL of deionized water and 100 μL of the borohydride solution added to the PAMAM dendrimer-IgG-HRP mixture. The reaction was stirred overnight (15-20 hours) at 2°-8° C. 
     The reaction product, including the precipitate, was placed into two Centricon™ microconcentrator tubes (molecular weight cutoff: 100,000 Daltons), and was ultrafiltered. Four additional 2 mL volumes of the 20 mM phosphate buffer (pH=6.0) were added during the process. The IgG/dendrimer/HRP conjugate was recovered and transferred to a 2 dram vial for storage at -10° C. 
     Example 92: Preparation of IgG/Dansylated PAMAM Dendrimer/HRP Conjugate 
     A: Preparation of HRP Solution 
     Horse-radish peroxidase (HRP, 2.6 mg, Pierce) was dissolved in 0.5 mL of deionized water to form the HRP solution. Sodium periodate, 10.7 mg, was dissolved in 5 mL of 10 mM sodium phosphate (pH=7.0). Approximately 1.6 mL of the periodiate solution was added to the HRP solution at room temperature. The enzyme reaction mixture was protected from light and gently stirred at room temperature for 30 minutes. The oxidized HRP product was placed into into two Centricon™ microconcentrator tubes (molecular weight cutoff: 10,000 Daltons), and was ultrafiltered. Four additional 2 mL volumes of the 30 mM sodium acetate/150 mM sodium chloride buffer (pH=5.5) were added during the process. The oxidized enzyme solution was recovered and placed into a 2 dram glass vial. 
     B: Preparation of Dansyl Generation 3.0 PAMAM Dendrimer Solution 
     A 5 mL methanolic solution of 12.7% (w/w) 3.0 generation PAMAM dendrimer (NH 3  core) solution was prepared and dried under vacuum on a rotary evaporator. Approximately 40 mg (7.4 μmol) of the dried dendrimer was removed and dissolved in 5 mL of 0.1M phosphonate buffer (pH=9.0). 
     A 10 mg (0.037 mmol) quantity of dansyl chloride was added to 5 mL of acetone and shaken for 5-10 minutes. The solution was a deep yellow and had traces of solid present. The dansyl chloride solution was added to the dendrimer solution over a 30 second period and the reaction flask was placed in a water bath at 40° C. for 90 minutes. During the heating, the reaction mixture was shaken intermittently. The color of the solution was a faint yellow. After the 90 minutes, the reaction flask was removed from the heat and allowed to cool to room temperature. The reaction mixture was slightly hazy in appearance. 
     The reaction solution was evaporated to remove the acetone using a rotary evaporator under vacuum at 30°-35° C. The solution was clarified by this process. The reaction mixture was then transferred into 2 centrifuge tubes and centrifuged at 5000 rpm for 30 minutes. The dendrimer solution was then placed into two Centricon™ microconcentrator tubes (molecular weight cutoff: 3,000 Daltons), and was ultrafiltered four times from a volume of 2 mL to remove any unbound dansyl chloride. Approximately 1-2 mL of the dansylated dendrimer product solution was recovered. 
     C: Preparation of IgG/Dansyl Generation 3.0 PAMAM Dendrimer Solution 
     A 6.9 mg quantity of rabbit immunoglobulin (IgG) was dissolved in 1 mL of 50 mM phosphate buffer) pH=7.2). A 14.8 mg quantity of sodium periodate was added and the mixture was swirled until the solids were dissolved. The mixture was protected from light and stirred for an additional 30 minutes at room temperature. The solution was then placed into two Centricon™ microconcentrator tubes (molecular weight cutoff: 3,000 Daltons), and was ultrafiltered four times from a volume of 2 mL each time using 50 mM phosphate buffer (pH=7.2) to ensure the removal of any unreacted sodium periodate. Approximately 1 mL of the dialdehyde solution was recovered and transferred to a vial in preparation for coupling with the dansylated dendrimer. 
     An aliquot of the labelled dansyl dendrimer from Part B was removed from stock and diluted to a final dendrimer concentration of 3.0 mg/mL. A 100 μL volume of the diluted dansylated dendrimer was added to the IgG dialdehyde solution and dispersed. This represents about 1.2:1 ratio of the labelled dendrimer to IgG. 
     The IgG/dansylated dendrimer conjugate was protected from the light and allowed to stir at room temperature for 1 hour. After that time, 1.4 mg of sodium cyanoborohydride was added and the reaction stirred overnight (15-20 hours) at room temperature. The solution appeared clear. An electrophoresis sample, 200 μL, was removed and the rest of the solution was then placed into two Centricon™ microconcentrator tubes (molecular weight cutoff: 100,000 Daltons), and was ultrafiltered. Four additional 2 mL volumes of the 50 mM phosphate buffer (pH=7.2) was added during the process to remove any unbound dansyl dendrimer. The desired conjugate was recovered. 
     D: Preparation of 3.0 PAMAM dendrimer/dansyl/IgG-HRP Conjugate 
     To 650 μL (10.1 mg) of IgG/Dansyl Generation 3.0 PAMAM dendrimer solution (prepared in Part C) was added 2.6 mg of the oxidized HRP. The IgG/dansyl dendrimer/HRP conjugate was protected from light and allowed to stir at room temperature for 2 hours. After the 1 hour, 2.1 mg of sodium borohydride was dissolved in 1 mL of deionized water and 100 μL of the borohydride solution added to the PAMAM dendrimer-IgG-HRP mixture. The reaction was stirred overnight (15-20 hours) at 2°-8° C. The mixture was a clear amber liquid. 
     The reaction product was placed into two Centricon™ microconcentrator tubes (molecular weight cutoff: 100,000 Daltons), and was ultrafiltered. Four additional 2 mL volumes of the 20 mM phosphate buffer (pH=6.0) were added during the process. The IgG/dansyl/dendrimer/HRP conjugate was recovered and transferred to a amber glass vial for storage at -10° C. 
     D: Characterization of 3.0 PAMAM dendrimer/dansyl/IgG-HRP Conjugate 
     Aliquates of the IgG/Dendrimer/HRP conjugates were evaluated for the presence of HRP enzyme using 3,3&#39;,5,5&#39;-tetramethylbenzidine (TMB). HRP catalyzes the hydrogen peroxide oxidation of substrates, such as TMB, by transferring electrons from the TMB to the peroxide to yield a colored product. Therefore, the loss of one electron yields a blue color and the loss of two electrons produces a yellow color. 
     The following dendrimer/dansyl/IgG-HRP conjugates were prepared to confirm the presence of HRP enzyme: 
     (1) 0.1 mg/mL IgG/dansyl Generation 3.0 PAMAM dendrimer 
     (2) 0.1 mg/mL IgG/dansyl Generation 3.0 PAMAM dendrimer/HRP 
     (3) 0.1 mg/mL IgG/Generation 8.0 PAMAM dendrimer/HRP 
     (4) Ultrafiltrate from IgG/Generation 8.0 PAMAM dendrimer/HRP, containing unbound HRP 
     The TMB substrate solution was prepared using TMB-dihydrochloride. Each of the above diluted samples, 200 μL, were added to 200 μL of TMB substrate solution. The two HRP conjugates (#2 &amp; #3) immediately turned green, then yellow. The ultrafiltrate (#4) turned bright yellow. The IgG/dendrimer (#1) remained colorless. All four samples were incubated at room temperature for 30 minutes, then quenched with 200 μL of 1N sulfuric acid. The yellow color of the enzyme conjugates (#2 &amp; #3) became more intense. The IgG/dendrimer (#1) was slightly green and showed fluorescence under UV light. 
     Aliquots of all the conjugates were analyzed by polyacrylamide gel electrophoresis. Sample concentrations of approximately 1 mg/mL were prepared for the electrophoresis, which corresponds to about 10 μg of electrophoresis sample. An Amicon Gradipore™ (Hylinx) 5-50% T gel was used in combination with 0.05M sodium acetate buffer (0.025% in sodium azide) of pH=4.0. The gel was run at a constant voltage of 200 V for 2 hours. Conjugates containing precipitate were centrifuged prior to electrophoresis analysis. The supernant was removed and used for the electrophoresis. The following samples were run: 
     (1) Generation 8.0 PAMAM dendrimer 
     (2) Dansylated Generation 8.0 PAMAM dendrimer 
     (3) Generation 8.0 PAMAM dendrimer-FIT I 
     (4) IgG (control) 
     (5) IgG/Dansyl Generation 8.0 PAMAM dendrimer 
     (6) IgG/Generation 8.0 PAMAM dendrimer-FIT I 
     (7) IgG/Generation 8.0 PAMAM dendrimer/HRP 
     (8) HRP (control) 
     (9) IgG/Dansyl Generation 3.0 PAMAM dendrimer/HRP 
     (10) Open 
     (11) Repeat of #7 
     (12) Repeat of #9 
     Prior to staining, the gel was placed under UV light to identify any fluorescence. Only Samples #2, 3 and 6 showed fluorescence. 
     Visualization of the gel following TMB, but prior to staining showed Samples #7, 8, 9, 11, and 12 present and confirmed the presence of enzyme at the higher molecular weights on the gel. 
     The gel was subsequently stained with 0.025% Coomassie Blue Stain overnight in 5% acetic acid and 7% methanol. Sample #10 was the only unstained lane on the gel. Samples #4 and 8 were at the lower molecular weights. All other Samples showed higher molecular weights without any lower molecular weights present. 
     Example 33: Preparation of Generation 5.0 PAMAM Dendrimer/Dichlorotriazinyl Fluorescein 
     Generation 5.0 PAMAM dendrimer, 300 μL (21.5% solids, NH 3  core) was added to 700 μL of deionized water and dispersed (64.5 mg, 2.24 μmol). The dendrimer was further diluted with 4 mL of deionized water to a concentration of 12.9 mg/mL. 
     Dichlorotriazinyl fluorescein hydrochloride DTAF I, Abbott Laboratories), 6.0 mg (11.3 μmol) was dissolved in 0.5 mL of methanol in the dark, and 2 drops of triethylamine added. The DTAF I solution was added to the stirring dendrimer solution dropwise over 30 seconds. The reaction mixture was protected from the light and stirred at room temperature for 15-20 hours. The solution remained clear and orange throughout the reaction. After stirring, the reaction mixture was placed into two Centricon™ microconcentrator tubes (molecular weight cutoff: 10,000 Daltons), and was ultrafiltered to remove any unreacted DTAF I. Fifteen, 2 mL volumes of 0.1M phosphate buffer (pH=9.0) were added during the process. Approximately 3 mL of conjugated 5.0 generation PAMAM dendrimer/DTAF I were recovered. 
     Example 34: Preparation of Generation 4.5 PAMAM Dendrimer/Aminomethyl Fluorescein 
     Generation 4.5 PAMAM dendrimer, 1 mL solution (4.2% solids, 42 mg, 1.7 μmol) was added to a 2 dram vial. 
     In a separate vial, 6.6 mg (1.7 μmol) of 4&#39;-aminomethyl fluorescein (AMF) was dissolved in 0.5 mL of deionized water. The pH was adjusted to 9 by added 2 drops of triethylamine. 
     In a third vial, 63.8 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) was dissolved in 1 mL of deionized water. 
     The AMF solution was added to the stirring dendrimer solution over 1-2 minutes at room temperature. The pH of the dendrimer-AMF solution was adjusted to 4.5-5.5 using 1N hydrochloric acid. The solution remained clear. With continued stirring, to the mixture was added the EDAC solution, in 200-300 μL increments, at 15-20 minute intervals over 1 hour. The pH of the reaction mixture was maintained between 4.5-5.5 using 1N hydrochloric acid. The reaction mixture was protected from light and stirred at room temperature for 15-20 hours. The solution was clear and orange. 
     After stirring, the reaction mixture was placed into two Centricon™ microconcentrator tubes (molecular weight cutoff: 10,000 Daltons), and was ultrafiltered to remove any unreacted AMF. Fifteen, 2 mL volumes of 0.1M phosphate buffer (pH=9.0) were added during the process. Approximately 2 mL of conjugated 4.5 generation PAMAM dendrimer/AMF were recovered. 
     Example 35: Preparation of 3.0 Generation PAMAM/Isatoic Anhydride Conjugate 
     Generation 3.0 PAMAM, 2 g, was dissolved in methanol and cooled in an ice bath. To this solution was added 0.01 g of isatoic anhydride. The mixture was stirred for 2 hours, the bath allowed to warm to room temperature, and stirring continued for several days. The methanol was then removed by rotary evaporation and the product taken up in water. The solution was then dialyzed using a Spectrapor #1 membrane. The solution was then concentrated to 38 mL and its fluorescence spectrum run by using irradiation at 340 nm and detection at 420 nm. The fluorescence was still detectable after a 10,000 fold dilution, and the response was linear for dilutions of 100 and 1,000. 
     Example 36: Preparation of 4.0 Generation PAMAM Dendrimer/DTPA/Gd Conjugate 
     A: Preparation of a 4.0 Generation PAMAM dendrimer, methyl ester terminated 
     A 3.5 generation PAMAM dendrimer, methyl ester terminated, was prepared by sequential reaction with methyl acrylate and ethylenediamine. Then to 1,000 g of predistilled ethylenediamine was added about 5 g of 3.0 generation PAMAM dendrimer, methyl ester terminated, as a 15 wt % solution in methanol. The solution was allowed to stand at room temperature for 48 hours. The methanol and most of the excess ethylenediamine were removed by rotary evaporation under vacuum at a temperature of less than 60° C. A total of about 7.5 g of product was recovered. To remove entrapped ethylenediamine, the product was dissolved in 100 mL of methanol and ultrafiltered two Centricon™ microconcentrator tubes (molecular weight cutoff: 2,000 Daltons). When most of the solution had passed through the membrane, several additional portions of methanol were added and the ultrafiltration process was repeated. The retentate was transferred to a round-bottomed flask and the filter cup was rinsed repeatedly with methanol and combined with the retentate. Volatile solvents were removed from the retentate solution by rotary evaporation under vacuum. About 6  g of 4.0 generation PAMAM (MW 5,147 Daltons, 24 terminal amino groups) was recovered. 
     B: Preparation of 4.0 Generation PAMAM dendrimer/DTPA Anhyride Conjugate 
     A solution of PAMAM (4 g, 7.6×10 -4  mol) in 300 mL of water was stirred in a three-neck flask. Over the next hour, 29 g (72×10 -3  mol) of the solid, N 3  -(2,6-dioxomorpholinoethyl)-N 6  -ethoxycarbonylmethyl-3,6-diazaoctane diacid (DTPA anhydride) was added by portions. During the addition the pH was maintained at 8.8-9.2 by the addition of 1N aqueous sodium hydroxide solution. When the addition was completed, the solution was stirred for an additional 30 minutes. The solution pH was adjusted to a pH of 7 by the addition of an ion exchange resin (H +   form), and the resulting slurry was filtered by suction to remove the ion exchange resin. The filtrate was transferred to two Centricon™ microconcentrator tubes (molecular weight cutoff: 5,000 Daltons) and ultrafiltered. The filtrate was collected and freeze-dried. The yield was about 12 g of a colorless flaky power. The product was a polyamidoamide (24 terminal amino groups) having each terminus linked via an amide bond to one of the carboxyl groups of DTPA. 
     C: Preparation of 4.0 Generation PAMAM dendrimer/DTPA/Gd Conjugate 
     About 10 g of the chelating agent described above was dissolved in 600 mL of water and mixed with 2.8 g of Gd 2  O 3 . The slurry was stirred for about 90 minutes at 80° C. After cooling to room temperature, the solution pH was adjusted to 7 by the addition of an ion exchange resin. The resulting slurry was filtered by suction to remove the ion exchange resin. The filtrate was transferred to two Centricon™ microconcentrator tubes (molecular weight cutoff: 5,000 Daltons) and ultrafiltered. The filtrate was collected and freeze-dried. The yield was about 12 g of colorless, flaky lyphilizate, known to contain Gd by atomic absorption spectroscopic analysis. 
     Example 37: Preparation of a 3.0 Generation, Amino Terminated Dendrimer/DTPA/Gd Conjugate 
     A: Preparation of a 3.0 Generation, Hydroxy terminated dendrimer 
     A 3.0 generation, hydroxy terminated dendrimer was prepared from pentaerythrityltetrabromide and 4-hydroxymethyl-2,6,7-trioxabicyclo[2.2.2]-octane as described by Tomalia et al. in U.S. Pat. No. 4,587,329, Example 11. Following the procedure of Step C of Example 11 of U.S. Pat. No. 4,587,329, the second generation hydroxy-terminated polyether dendrimer was converted to the corresponding perbromide. Then, following the procedures of Steps A and B of that Example, the brominated derivative of Step C was allowed to react first with a 4-hydroxymethyl-2,6,7-trioxabicyclo[2.2.2]-octane and then demasked with water to form the desired third generation dendrimer. 
     B: Preparation of a 3.0 Generation, Chloride terminated dendrimer 
     A 500 mL, three-neck flask equipped with a stirrer, condenser and addition funnel is charged with a 0.1 mole of the third generation dendrimer from Part A and 15 mole of freshly distilled thionyl chloride is added at a rate to maintain the temperature below 45° C. Upon completion of the addition, the solution is warmed to about 50° C. and maintained at this temperature for about 4 hours. The excess thionyl chloride is removed under vacuum. By this process the hydroxy groups are converted to chloride moieties. 
     C: Preparation of a 3.0 Generation, Amino terminated dendrimer 
     The chloride terminated dendrimer from Part B was transferred to a large polyolefin bottle and a large excess of ammonium hydroxide solution was added. The bottle was sealed and the contents were stored at about 55° C. for several days. The solution formed a residual oil which was redissolved in a small volume of water, the pH adjusted to 8 by addition of sodium bicarbonate, and the desired amine was extracted with chloroform. The chloroform extracts were dried over anhydrous magnesium sulfate, filtered and concentrated to a residual oil. By this process the chloride terminated dendrimer was converted to an amino terminated dendrimer. 
     D: Preparation of a 3.0 Generation, Amino terminated dendrimer 
     Alternatively, the hydroxy terminated dendrimer from Part A was dissolved in dimethylformamide and allowed to react with an excess of methanesulfonyl chloride. The resulting tosylate ester was isolated by dilution of the dimethylformamide with water and extraction with chloroform. The chloroform extracts were washed with dilute sodium bicarbonate solution and with water, then dried over anhydrous magnesium sulfate. After filteration to remove any solids, the filtrate was concentrated to dryness. The residual oil, the desired polymethanesulfonate ester, was used without further purification, was transferred to a large polyolefin bottle and a large excess of ammonium hydroxide solution was added. The bottle was sealed and the contents were stored at about 55° C. for several days. The amino-terminated dendrimer was isolated as described above in Part C. 
     E: Preparation of a 3.0 Generation, Amino terminated dendrimer/DTPA Conjugate 
     A mixed anhydride of DTPA was prepared by reaction of diethylenetriaminepentaacetic acid (DTPA) and isobutyl chloroformate. The anhydride (1 mole) was dissolved in dimethylformamide, and the solution was added to a solution of amine-terminated dendrimer (from either Part C or D) in the same solvent. To the solution was added 2,6-lutidine as necessary to maintain a basic environment. By this means, covalent bonds were formed between the amino terminus of the dendrimer and the carboxyl group of DTPA. 
     F: Preparation of a 3.0 Generation, Amino terminated dendrimer/DTPA Conjugate 
     Alternatively, 1 mmole of an amine-terminated dendrimer (from either Part C or D) was dissolved in 300 mL of water. Within 2 hours, 150 mmol of the solid form of N 3  -(2,6-dioxomorpholinoethyl)-N 6  -ethoxycarbonylmethyl-3,6-diazaoctane diacid was added by portions to the solution. The pH was maintained at about 9.0 by the addition of 1N sodium hydroxide. The solution was stirred for an additional 30 minutes, the pH adjusted to 7.0 with an ion exchange resin (H + ), and then filtered to remove the ion exchange resin. The solution was placed into two Centricon™ microconcentrator tubes (molecular weight cutoff: 5,000 Daltons) and ultrafiltered. The filtrate was collected and freeze-dried. The DTPA-conjugated dendrimer was obtained as a colorless flaky powder. 
     G: Preparation of a 3.0 Generation, Amino terminated dendrimer/DTPA/Gd Conjugate 
     About 10 g of the DTPA-conjugated dendrimer was dissolved in 600 mL of water for injection and mixed with about 3 g of Gd 2  O 3 . The slurry was stirred for about 90 minutes at 80° C. After cooling, the solution pH was adjusted to 7 by the addition of an ion exchange resin. The solution was filtered to remove the resin and then ultrafiltered as described above. The filtrate was collected and freeze-dried. The Gd/DTPA-conjugated dendrimer was obtained as a colorless, flaky lyophilizate containing Gd by AAs analysis. 
     Example 38: Zero Valent Nickel in Dendrimers 
     To 10 g of 1% NiCl 2  (aqueous) was added 177 mg generation 0.5 diethylenetriamine (DETA) Core STARBURST™ dendrimer (Na-salt form). The solution turned pale blue. To this solution was added 0.5 mL of 28% aqueous ammonium hydroxide, causing the solution to turn a darker blue. Addition of 12 g of 2.78% aqueous sodium hypophosphite to the solution and then heating to boiling gave a precipitate of nickel metal with a supernatant blue solution. The supernatant was concentrated in vacuo to give the metallized dendrimer as a mixture with excess phosphites (0.6 g). Purification was carried out by ultrafiltration on a hollow fiber filtration unit (The Dow Chemical Company). (A control reaction, carried out the same as above except that no dendrimer was added, gave a colorless solution with complete precipitation of nickel metal after boiling.) 
     Example 39: Zero Valent Nickel in Dendrimers 
     To 10 g of 1% aqueous NiCl 2  was added 270 mg of generation 1.5 linear polyethyleneimine (PEI) core STARBURST™ dendrimer (sodium carboxylate form). The solution turned a darker green than the control mixture (no STARBURST™ added). After addition of 0.5 mL of concentrated ammonium hydroxide, the control was blue and the STARBURST™ containing solution was still green. Addition of 12 g of 2.78% aqueous sodium hypophosphite and heating to boiling caused, in the control, precipitation of black nickel metal and gave a colorless solution. The STARBURST™ containing solution gave a pale green suspension under the same conditions. The pale green suspension was centrifuged to remove a small amount of solids (50 mg) and concentrated in vacuo to give 500 mg of pale green powder. Ultrafiltration of an aqueous solution of this mixture of metallized dendrimer and phosphites using an Amicon™ YM2 membrane was continued until no more green color was seen in the filtrate. Concentration in vacuo of the green retentate solution gave approximately 100 mg of a dark green glass. 
     Example 40: Preparation of Epoxyoctane Modified Generation 5.0 PAMAM Dendrimer 
     Generation 5.0 PAMAM dendrimer, 0.30 g (2.7 meq) was dissolved in 10 mL of methanol. Epoxyoctane, 0.5 g, (Aldrich, 3.9 mmol) was added to the solution. The solution was then heated at 40° C. in an oil bath for 3 days. The solvent was removed by distillation in vacuo, then the residue was devolatized at room temperature under high vacuum to give 0.60 g (93% of theory) of the modified dendrimer as a colorless oil. The  13  C and  1  H NMR spectra indicated attachment at the surface by epoxyoctane and is consistent with the association of the epoxyoctane dendrimer. 
     When the above procedure was repeated using generation 2.0, 3.0, 4.0, 6.0 or 7.0 PAMAM dendrimer, the corresponding conjugate was formed. 
     Example 41: Preparation of Abscisic Acid Modified Generation 4.5 PAMAM Dendrimer 
     An aqueous solution of a generation 4.5 PAMAM dendrimer was hydrolyzed by NaOH/MeOH (1 mM, 10 mL) and the pH adjusted to 11 by NaOH/HCl. To the solution was added a large excess of solid abscisic acid. The solution was stirred vigorously at room temperature. After 1, 2, 6 and 24 hours, 500 μL of the solution was removed into an Eppendorf tube (1.5 mL microcentrifuge tube) and any undissolved abscisic acid removed by centrifugation at 15,000 rpm for 1 minute. The abscisic acid concentration was calculated from the absorbance of the supernatant (λ=260 nm, ε=19400). 
     When the procedure was repeated using different generations of PAMAM dendrimers, the higher the generation, the more abscisic acid was dissolved. The generation 5 and 6 dendrimers dissolved more of the acid than generation 4. At pH=7, only 0.004M of the abscisic acid dissolved in water and β-CD solution, while the dendrimer solutions dissolved 0.02-0.08M. The half-generations of dendrimers dissolved the same amounts of acid as the whole generations. Thus the abscisic acid is believed to be taken into the dendrimer interior. 
     When the above procedure was used with nifedipine (ε=8300 in chloroform), the nifedipine took a long time to dissolve in water, but once dissolved, it was taken into the dendrimer very rapidly. 
     Other embodiments of the invention will be apparent to those skilled in the art from a consideration of this specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.