Patent Application: US-37022103-A

Abstract:
cross - linkable , phosphonate containing supramoleuclar aggregates are disclosed , which may be used to advantage , for example , as drug delivery vehicles for the treatment of bone - related disorders .

Description:
the supramolecular aggregate of the invention may comprise various phosphonate compounds , which are derivatives of phosphonic acid having the formula r — po ( oh ) 2 , r represents a straight or branched chain hydrocarbon , which has at least one cross - linkable moiety , e . g . unsaturation bonds , and which may have a hydrocarbon chain length of c 6 to c 22 . in a preferred embodiment the hydrocarbon chain length is between c 8 - c 16 . alkylphosphronic acids derivatives of the foregoing formula can be prepared according to the following reaction scheme , which is provided by way of example and not limitation . these long chain phosphonates self - assemble into aggregate form when exposed to an aqueous environment under conditions described by schulz et al . ( 21 ). there are certain advantages in using polymerizable phosphonates to prepare the targeted delivery vehicles of the invention . chapman and ringsdorf first used photopolymerizable phospholipids in the early 1980s as a means of preparing aggregates that were stable towards changes in their local environment . ( ringsdorf , h ., et al . j . angew . chem . ( 1988 ) 100 : 117 ); johnston et al ., biochem . biophys . acta ( 1980 ) 602 : 57 . incorporating the ability to polymerize the aggregates following extrusion enables the size distribution of the vesicles to be “ locked - in ”. the morphology of the polymerized vesicles should thus remain stable to environmental changes such as ph or metal addition . moreover , the polymerized aggregates could be directly targeted to bone tissue solely through their terminal phosphonic acid head groups , thus obviating the stabilizing metal layer and the exterior bisphosphonic targeting component , which are included in the “ complexes of alkylphosphonic acids ”, which is the subject of international patent application no . pct / us00 / 32057 , commonly owned . the introduction of polymeric character into the vesicle structure can also be used to potentially control the release characteristics of the aggregates . one way this can be accomplished is through variation in the number of unsaturated moieties incorporated into the monomers . by introducing a higher degree of unsaturation , the number of potential cross - linking polymer bonds can be increased and thus the ability of therapeutic or diagnostic agents trapped within this hydrophobic cure region to escape would be reduced . another way of controlling the release of active agents is by modifying the hydrophobic tail group of at least one of the phosphonate compounds to include a functional group , which is utilized to covalently bind the therapeutic or diagnostic agent to the aggregate . the covalently bound therapeutic or diagnostic agent may be either hydrophilic or hydrophobic . the chemical nature of the agent will dictate the nature of the functional group ( s ) utilized for covalent binding thereof . preferably the bonds thus formed are hydrolyzable . following polymerization , water molecules near or penetrating the outer region of the hydrocarbon core of the vesicle could slowly degrade these bonds to release the contents of the aggregate . for example , if the therapeutic agent is a compound having a free amine group (— nh 2 ), the alkylphosphonic acid may be modified to include a free carboxyl group (— cooh ), preferably at the terminus of the hydrophobic tail , thereby allowing for covalent binding of the therapeutic agent to the aggregate via an amide bond formed between the amine group and the carboxyl group . alternatively , the aggregates may be formed using one or more non - polymerizable phosphonate monomers , thereby to create tiny channels in the aggregates through which trapped therapeutic or diagnostic agent could escape . see , for example , u . s . pat . no . 5 , 366 , 881 to singh . in a preferred embodiment of the invention , the composition further comprises a suitable physiologically compatible buffer . buffers suitable for this purpose are known in the art and include , but are not limited to , phosphate buffered saline ( pbs ), 3 -[ n - morpholino ] propanesulfonic acid ( mops ) and 3 -[ n - morpholino ] ethanesulfonic acid ( mes ). the compositions of the invention may be administered both systemically as well as injected directly at a disease site . the means of incorporating therapeutic or diagnostic agents into the aggregates may vary , depending on the relative hydiophobicity of the active agent . hydrophobic agents can readily be incorporated into the core hydrophobic regions of the aggregate . this provides the most direct and general ( i . e . a host of hydrophobic drugs , e . g ., paclitaxel , could be used ) way to incorporate therapeutic or diagnostic substances into the aggregate delivery system . another means of incorporating a drug or diagnostic agent into the aggregates involves the use of a modified monomer phosphonic acid in which the drug is covalently attached to the tail of the amphiphile , as briefly mentioned above . this second approach has been used successfully to attach the hydrophobic anti - cancer drug adriamyacin ( adr ) to the interior of several block copolymer micelles ( 8 ). for example , the tail of the hydrophobic portion of the alkylphosphonic acid monomer may be converted to a carboxylic acid , which allows for the formation of an amide linkage with the amine on adr . upon the breakdown of this linkage , the drug is released from the hydrophobic interior and can diffuse from the aggregate to enter the body . this monomer can be used in various ratios with unmodified phosphonic acid monomers to prepare aggregates . the supramolecular aggregates described above may have biologically compatible metal ions bound to the exterior thereof in accordance with the present invention . the metal component of the aggregate can optimally be further functionalized with a targeting component for the purpose of enhancing site - specific delivery of the therapeutic or diagnostic agent . the biologically compatible metal ions are preferably selected from the group consisting of fe ( iii ), co ( ii ), zn ( ii ), zr ( iv ), mg ( ii ) and ca ( ii ). of course other biologically compatible metals may be used , if desired . the metal ions are bound to the exterior of the above - described aggregate by direct injection of salt solution into the aggregate preparation , followed by dialysis of excess or unbound ions through dialysis tubing . solutions were dialyzed for approximately one week . the targeting component of the composition may include but is not limited to antibody , fragment of an antibody , protein ligand , polysaccharide , polynucleotide , polypeptide , low molecular mass organic molecule and the like . such targeting group can be linked covalently to the phosphonate , or can be non - covalently incorporated in the compositions , e . g ., through hydrophobic , electrostatic interactions or hydrogen bonds . preferably the targeting component is α , ω bisphosphonic acid , such as decyl ( bis ) phosphonic acid ( dbpa ), geminal bisphosphonic acid ( the latter being characterized by a p — c — p bond ) or tetrakisphosphonic acid ( the latter containing two p — c — p bisphosphonate moieties connected by a carbon chain ). because dbpa is soluble in water , the addition of this component to the aggregates can be carried out at room temperature ( i . e . similar conditions to those used for preparing self - assembled multilayers with dbpa ) ( 20 ). size and molecular weight of the resulting delivery vehicle may be determined by static and dynamic light scattering and tem acquired by negatively staining the samples . due to the large negatively charged acid head groups on the exterior of the resulting delivery vehicle system , it should have a long circulation time within the bloodstream and not be taken up by other organs in the body ( 16 , 17 ). in order to enhance release from these metallated materials , optionally , one may add small amounts of impurities , such as chemically distinct surfactant , e . g ., one with a sulfonate head group , which is unable to bind metal ions . this would reduce the number of sites available for metal binding on the exterior of the aggregate and should increase the fluidity of the outer shell and promote release . suitable surfactants for this purpose are described in liposomes : a practical approach , irl press , oxford , 1997 . the targeting component may be chemically modified to allow for binding of the therapeutic or diagnostic agent thereto . the diagnostic agents that may be used in the practice of this invention include those having utility in imaging . suitable therapeutic agents are those capable of acting on a cell , organ or organism to create a change in the functioning of the cell , organ or organism , including but not limited to pharmaceutical agents or drugs . such agents include a wide variety of substances that are used in therapy , immunization or otherwise are applied to combat human and animal disease . such agents include but are not limited to analgesic agents , anti - inflamatory agents , antibacterial agents , antiviral agents , antifungal agents , antiparasitic agents , tumoricidal or anti - cancer agents , proteins , toxins , enzymes , hormones , neurotransmitters , glycoproteins , immunoglobulins , immunomodulators , dyes , radiolabels , radio - opaque compounds , fluorescent compounds , polysaccharides , cell receptor binding molecules , anti - inflammatories , anti - glaucomic agents , mydriatic compounds and local anesthetics . exemplary non - steroidal anti - inflamatories include , but are not limited to , indomethacin , salicylic acid acetate , ibuprofen , sulindac , piroxicam , and naproxen , antiglaucomic agents such as timolol or pilocarpine , neurotransmitters such as acetylcholine , anesthetics such as dibucaine , neuroleptics such as the phenothiazines ( e . g ., compazine , thorazine , promazine , chlorpromazine , acepromazine , aminopromazine , perazine , prochlorperazine , trifluoperazine , and thioproperazine ), rauwolfia alkaloids ( e . g ., resperine and deserpine ), thioxanthenes ( e . g ., chlorprothixene and tiotixene ), butyrophenones ( e . g ., haloperidol , moperone , trifluoperidol , timiperone , and droperidol ), diphenylbutylpiperidines ( e . g ., pimozde ), and benzamides ( e . g ., sulpiride and tiapride ); tranquilizers such as glycerol derivatives ( e . g ., mephenesin and methocarbamol ), propanediols ( e . g ., meprobamate ), diphenylmethane derivatives ( e . g ., orphenadrine , benzotrapine , and hydroxyzine ), and benzodiazepines ( e . g ., chlordiazepoxide and diazepam ); hypnotics ( e . g ., zolpdem and butoctamide ); beta - blockers ( e . g ., propranolol , acebutonol , metoprolol , and pindolol ); antidepressants such as dibenzazepines ( e . g ., imipramine ), dibenzocycloheptenes ( e . g ., amtiriptyline ), and the tetracyclics ( e . g ., mianserine ); mao inhibitors ( e . g ., phenelzine , iproniazid , and selegeline ); psychostimulants such as phenylehtylamine derivatives ( e . g ., amphetamines , dexamphetamines , fenproporex , phentermine , amfeprramone , and pemoline ) and dimethylaminoethanols ( e . g ., clofenciclan , cyprodenate , aminorex , and mazindol ); gaba - mimetics ( e . g ., progabide ); alkaloids ( e . g ., codergocrine , dihydroergocristine , and vincamine ); anti - parkinsonism agents ( e . g ., l - dopamine and selegeline ); agents utilized in the treatment of altzheimer &# 39 ; s disease , cholinergics ( e . g ., citicoline and physostigmine ); vasodilators ( e . g ., pentoxifyline ); and cerebro active agents ( e . g ., pyritinol and meclofenoxate ). anti - neoplastic agents can also be used advantageously as biological agents in the compositions of the invention . representative examples include , but are not limited to paclitaxel , daunorubicin , doxorubicin , carminomycin , 4 ′- epiadriamycin , 4 - demethoxy - daunomycin , 11 - deoxydaunorubicin , 13 - deoxydaunorubicin , adriamycin - 14 - benzoate , adriamycin - 14 - actanoate , adriamycin - 14 - naphthaleneacetate , vinblastine , vincristine , mitomycin c , n - methyl mitomycin c , bleomycin a 2 , dideazatetrahydrofolic acid , aminopterin , methotrexate , cholchicine and cisplatin . representative antibacterial agents are the aminoglycosides including gentamicin . representative antiviral compounds are rifampicin , 3 ′- azido - 3 ′- deoxythymidine ( azt ), and acylovir . representative antifungal agents are the azoles , including fluconazole , macrolides such as amphotericin b , and candicidin . representative anti - parastic compounds are the antimonials . suitable biological agents also include , without limitation vinca alkaloids , such as vincristine and vinblastine , mitomycin - type antibiotics , such as mitomycin c and n - methyl mitomycin , bleomycin - type antibiotics such as bleomycin a 2 , antifolates such as methotrexate , aminopterin , and dideaza - tetrahydrofolic acid , taxanes , anthracycline antibiotics and others . the compositions of this invention also can utilize a variety of polypeptides , such as antibodies , toxins , such as diphtheria toxin , peptide hormones , such as colony stimulating factor , and tumor necrosis factors , neuropeptides , growth hormone , erythropoietin , and thyroid hormone , lipoproteins such as μ - lipoprotein , proteoglycans such as hyaluronic acid , glycoproteins such as gonadotropin hormone , immunomodulators or cytokines such as the interferons or interleukins , as well as hormone receptors such as the estrogen receptor . the compositions also can comprise enzyme inhibiting agents such as reverse transcriptase inhibitors , protease inhibitors , angiotensin converting enzymes , 5μ - reductase , and the like . typical of these agents are peptide and nonpeptide structures such as finasteride , quinapril , ramipril , lisinopril , saquinavir , ritonavir , indinavir , nelfinavir , zidovudine , zalcitabine , allophenylnorstatine , kynostatin , delaviridine , bis - tetrahydrofuran ligands ( see , for example ghosh et al ., j . med . chem . 1996 , 39 : 3278 ), and didanosine . such agents can be administered alone or in combination therapy ; e . g ., a combination therapy utilizing saquinavir , zalcitabine , and didanosine , zalcitabine , and zidovudine . see , for example , collier et al ., antiviral res . 1996 , 29 : 99 . the compositions described herein may also comprise nucleotides , such as thymine , nucleic acids , such as dna or rna , or synthetic oligonucleotides , which may be derivatized by covalently modifying the 5 ′ or the 3 ′ end of the polynucleic acid molecule with hydrophobic substituents to facilitate entry into cells ( see for example , kabanov et al ., febs lett . 1990 , 259 , 327 ; kabanov and alakhov , j . contr . rel . 1990 , 28 : 15 ). additionally , the phosphate backbone of the polynucleotides may be modified to remove the negative charge ( see , for example , agris et al ., biochemistry 1968 , 25 : 6268 , cazenave and helene in antisense nucleic acids and proteins : fundamentals and applications , mol and van der krol , eds ., p . 47 et seq ., marcel dekker , new york , 1991 ), or the purine or pyrimidine bases may been modified , for example , to incorporate photo - induced crosslinking groups , alkylating groups , organometallic groups , intercalating groups , biotin , fluorescent and radioactive groups ( see , for example , antisense nucleic acids and proteins : fundamentals and applications , mol and van der krol , eds ., p . 47 et seq ., marcel dekker , new york , 1991 ; milligan et al ., in gene therapy for neoplastic diseases , huber and laso , eds . p . 228 et seq ., new york academy of sciences , new york , 1994 ). such nucleic acid molecules can be , among other things , antisense nucleic acid molecules , phosphodiester , oligonucleotide α - anomers , ethylphospotriester analogs , phosphorothioates , phosphorodithioates , phosphoroethyletriesters , methylphosphonates , and the like ( see , e . g ., crooke , anti - cancer drug design 1991 , 6 : 609 ; de mesmaeker et al , acc . chem . res . 1995 , 28 : 366 ). the compositions of the invention may also include antigene , ribozyme and aptamer nucleic acid drugs ( see , for example , stull and szoka , pharm . res . 1995 , 12 : 465 ). other suitable biologically active agents include oxygen transporters ( e . g . porphines , porphirines and their complexes with metal ions ), coenzymes and vitamins ( e . g . nad / nadh , vitamins b12 , chlorophylls ), and the like . suitable biologically active agents further include those used in diagnostic visualization methods , such as magnetic resonance imaging ( e . g ., gadolinium ( iii ) diethylenetriamine pentaacetic acid ), and may be a chelating group ( e . g ., diethylenetriamine pentaacetic acid , triethylenetriamine pentaacetic acid , ethylenediamine - tetraacetic acid , 1 , 2 - diaminocyclo - hexane - n , n , n ′, n ′- tetraaceticacid , n , n ′- di ( 2 - hydroxybenzyl ) ethylene diamine ), n -( 2 - hydroxyethyl ) ethylene diamine triacetic acid and the like ). such agents may further include an alpha -, beta -, or gamma - emitting radionuclide ( e . g ., galliun 67 , indium 111 , technetium 99 ). iodine - containing radiopaque molecules are also suitable diagnostic agents . the diagnostic agent may also include a paramagnetic or superparamagnetic element , or combination of paramagnetic element and radionuclide . the paramagnetic elements include but are not limited to gadolinium ( iii ), dysporsium ( iii ), holmium ( iii ) europium ( iii ) iron ( iii ) or manganese ( ii ). the compositions of the present invention allow diverse routes of administration , including but not limited to parenteral ( such as intramuscular , subcutaneous , intraperitoneal , and intraveneous ), oral , otic , topical , vaginal , pulmonary , and ocular . these compositions can take the form of aqueous solutions , suspensions , micelles , vesicles , emulsions and microemulsions . conventional pharmaceutical formulations may be employed . when aqueous suspensions are required for oral use , the composition can be combined with emulsifying and suspending agents . for parenteral administration , sterile solutions of the composition are usually prepared , and the ph of the solutions are suitably adjusted and buffered . for intravenous use , the total concentration of solutes should be controlled to render the preparation isotonic . for ocular administration , ointments or droppable liquids may be delivered by well - known ocular delivery systems such as applicators or eyedroppers . such compositions can include mucomimetics such as hyaluronic acid , chondroitin sulfate , hydroxypropyl methylcellulose or polyvinyl alcohol , preservatives such as sorbic acid , edta or benzylchronium chloride , and the usual quantities of diluents and / or carriers . for pulmonary administration , diluents and / or carriers will be selected to be appropriate to allow for formation of an aerosol . it is known that phosphonic acid moieties bind specifically to bone tissue . for about the past two decades geminal bisphosphonic acids have been increasingly studied for use as therapeutic agents for osteoporosis ( 16 , 17 ). osteoporosis is a condition in which the natural process of bone breakdown by osteoclastic cells ( resorption ) is dramatically increased without subsequent reformation of new tissue ( 16 ). rapid breakdown of the bone matrix dramatically increases the amount of exposed ca ++ at the bone surface , and the strongly binding phosphonic acids are readily targeted to the affected area . although the exact mechanism is still unknown , it is believed that the negatively charged acid headgroups bind tightly to exposed calcium ions thus blocking osteoclastic activity ( 15 , 17 ). this binding “ locks ” the calcium in place strengthening the weakened tissue , while providing a protective coat over the surface to prevent further resorption ( 15 , 17 ). the compositions of the invention are , therefore , unique in that they enable targeted delivery of an encapsulated therapeutic or diagnostic agent to one specific site . when a cancer cell metastasizes to the bone , it also triggers the release of chemical stimulators which increase bone breakdown ( 15 ). using the knowledge that phosphonic acids will specifically target areas where resorption is occurring rapidly , a phosphonic acid derivative is used to target drug delivery to areas where cancer has metastasized . the resulting functionalized aggregate has free acids extended out from the main delivery vehicle assembly which serve to target drug delivery . the following examples are provided to illustrate certain embodiments of the present invention . these examples set forth the best mode presently contemplated for carrying out the invention described herein ; they are not intended to limit the invention in any way . the materials used in this example , including 1 - chloro - 5 - hexyne , 1 - hexyne , copper ( i ) chloride , hydroxylamine hydrochloride , bromotrimethylsilane , butyl lithium ( 2 . 5 m in hexanes ), reagent grade sodium hydroxide , and ethyl amine were used as received from aldrich chemical company . triethylphosphite ( 98 %) was purchased from aldrich chemical company and distilled from sodium metal under nitrogen prior to use . diethyl ether was purchased from fisher chemical and was distilled from sodium prior to use . 1 h and 13 c nmr spectra were acquired using a varian unity - inova 300 spectrometer at room temperature . infrared spectra were recorded using a nicolet magna 550 ft - ir with omnic software . uv - vis spectra were acquired on a hp 8453 uv - vis spectrometer attached to a hp kayak xa running chemstation ( rev a . 06 . 01 ) software . the melting points of the compounds were determined using a mel - temp ii capillary melting point apparatus equipped with a thermocouple . differential scanning calorimetry experiments were conducted using a ta instruments 2920 modulated dsc . 1 - iodo - 1 - hexyne ( 1 ). ether ( 30 ml ) was transferred via cannula to a clean , flame dried , 500 ml 3 neck round bottom flask with stir bar that was fitted with a rubber septum , a glass stopper , and an equalizing addition funnel . 1 - hexyne ( 10 . 04 g , 0 . 121 moles ) was added to the flask and stirred while being cooled in a dry ice acetone bath to − 50 ° c . after reaching temperature , 2 . 5 m butyl lithium ( 46 ml , 0 . 115 moles ) was added dropwise to the stirring reaction mixture . immediately following the addition of this solution , iodine ( 20 . 36 g 0 . 16 moles ) dissolved in ether ( 60 ml ) was added dropwise through the addition funnel . following the addition of this solution the reaction was transferred to an ice bath and brought to oc . the reaction was quenched through the slow addition of water to the reaction mixture with vigorous stirring . the biphasic mixture was transferred to a separation funnel and the water layer was removed . the ether layer was washed two more times with water discarding the water layer after each wash . the ether layer was washed once with an aqueous sodium thiosulfate solution followed by one more water wash . the ether layer was then dried over sodium sulfate and the solvent removed using rotary evaporation . no further purification was attempted as 1 h nmr showed only a slight trace of starting material . 16 . 56 g ( 66 %) of i was isolated as an orange oil . 1 h nmr ( cdcl 3 ): δ 0 . 91 ( t , 3h ), 1 . 46 ( m , 4h ), and 2 . 37 ( t , 2h ). 1 - iodo - 5 - hexyne ( 2 ). sodium iodide ( 50 . 1 g , 0 . 88 moles ) and 1 - chloro - 5 - hexyne ( 13 . 13 g , 0 . 111 moles ) was dissolved in 250 ml of reagent grade acetone and the reaction mixture refluxed for 12 hours . the reaction was cooled and the white precipitate removed using filtration . the acetone solution was reduced in volume using rotary evaporation and 100 ml of water was added . this solution was extracted with ether and the resulting ether layer washed three times with water before drying over sodium sulfate . the ether was removed under vacuum . the resulting product was distilled ( 55 ° c . @ 2 mm hg ) to yield 20 . 92 g ( 90 %) of ii as a yellow oil . 1 h nmr ( cdcl 3 ): δ1 . 65 ( q , 2h ), 1 . 96 ( m , 3h ), 2 . 24 ( td , 2h ), and 3 . 22 ( t , 2h ). ir : 3295 , 2940 , 2861 , 2836 , 2117 , 1430 , and 1211 cm − 1 . 1 - hexynyl phosphonic acid ( 3 ) in a three neck flask equipped with a stir bar and set up for simple distillation , molecule 2 ( 9 . 13 g , 0 . 044 moles ) was added . into this triethylphosphite ( 7 . 65 g , 0 . 066 moles ) was cannulated under nitrogen and the reaction was heated to 140 ° c . for 4 hours . the reaction was then cooled to room temperature and distilled under vacuum ( 94 ° c . @ 2 mm hg ) to collect 4 . 13 g ( 43 %) of a colorless oil . following distillation , 3 . 14 g ( 0 . 0144 moles ) of the oil was transferred to a 50 ml round bottom flask fitted with a small stir bar and rubber septum along with 20 ml of dichloromethane . the mixture was stirred to ensure complete dissolution of the phosphonate before bromotrimethylsilane ( 3 . 79 ml , 0 . 0248 moles ) of was added dropwise via gas tight syringe . the reaction was allowed to stir over a period of 12 hours . 3 m aqueous sodium hydroxide was added slowly until the ph of the solution was 10 in order to quench the reaction . this reaction mixture was extracted into chloroform three times . the chloroform was dried briefly over sodium sulfate before being removed by rotary evaporation to yield an off white crystalline product . the water layers from the earlier extraction were also evaporated to dryness and the remaining solid dissolved in acetone . filtration of this solution to remove sodium chloride also yielded a tiny amount of product that was combined with that from the chloroform extraction . recrystallization of this material from a 10 : 90 ethyl acetate : petroleum ether solvent mixture yielded 0 . 095 g ( 4 . 1 %) of white crystals that were pure by 1h nmr spectroscopy . melting point 114 . 4 - 115 . 7 ° c . 1 h nmr ( cdcl 3 ): δ1 . 77 ( m , 6h ), 1 . 97 ( t , 2h ), 2 . 23 ( td , 2h ), and 6 . 04 ( s , 2h ). 31 p nmr ( cdcl 3 ) δ 37 . 180 . ir : 3290 , 2946 , 2117 , 1450 , 1292 , 1101 , and 995 cm − 1 . dodeca - 5 , 7 - diynoyl phosphonic acid ( 4 ). in a clean 3 neck round bottom flask with stir bar and fitted with a rubber septum , an equalizing addition funnel , and a glass stopper , sodium hydroxide ( 0 . 026 g , 6 . 48 × 10 − 4 moles ) was dissolved in ethanol / water ( 3 ml , 1 : 1 ). to this solution 3 ( 0 . 095 g , 5 . 89 × 10 - 4 moles ) was added and stirred to dissolve . in a separate flask ethylamine ( 0 . 5 ml , 30 % aqueous ) was combined with copper ( i ) chloride ( 0 . 014 g , 1 . 47 × 10 − 4 moles ). following the complete dissolution of the solid copper , this mixture was transferred via pipette to the stirring phosphonic acid mixture . to this deep blue solution a few crystals of hydroxylamine hydrochloride were added until the reaction turned a yellow green color . to the stirring reaction mixture 1 ( 0 . 122 g , 5 × 10 − 4 moles ) was added dropwise from a gas tight syringe . aqueous hydroxylamine hydrochloride ( 10 %) was added through the addition funnel as needed to keep the copper catalyst active ( indicated by the yellow green color of the reaction ). following the addition of 1 , the solution was allowed to stir for 1 hour before 10 % hcl was added to quench the reaction . the mixture was transferred to a separatory funnel and extracted three times with ether . the ether was dried over sodium sulfate and dried using rotary evaporation to yield white crystals . the crystals were recrystallized from ethyl acetate / petroleum ether ( 10 : 90 ) to yield 0 . 023 g ( 14 . 5 %) of white crystals that were pure by nmr , product 4 . melting point 135 . 4 - 137 . 0 ° c . 1 h nmr ( cdcl 3 ): δ 0 . 91 ( t , 3h ), 1 . 46 ( m , 6h ), 1 . 71 ( m , 4h ), 2 . 28 ( m , 4h ), and 7 . 52 ( s , 2h ) 31 p nmr ( cdcl 3 ): δ 37 . 05 . mass spectrometry ( m + ) 243 . 1 hz . twice the value of the cmc determined for n - dodecanephosphonic acid ( 2 . 94 × 10 − 4 m ) 4 was used to assure aggregate formation in these experiments due to the limited amount of the final product . all samples were prepared in the manner described by schulz , supra , using solutions buffered to a ph of 5 . 5 with 5 mm mes . samples prepared in the above manner were imaged using tem by negatively staining the samples with 1 % uranyl acetate . fig2 is a picture of unpolymerized vesicles of dodeca - 5 , 7 - diynoyl phosphonic acid within four days of their initial preparation . it appears from this micrograph that the aggregates are small and relatively spherical compared to the fully saturated aggregates of n - dodecanephosphonic acid prepared as described hereinbelow . the average size of these vesicles is roughly 50 nm in diameter with the sizes of individual aggregates ranging from 4 nm to over 400 nm . interestingly , it appears that some of these vesicles tend to roll into cylinders . this behavior was only observed with freshly prepared aggregates prior to their exposure to light . one explanation for this behavior is that it represents an artifact of drying the sample in the presence of the metal stain used to visualize it . [ 0053 ] fig3 is a micrograph of the same sample after one week . it is noteworthy that the vesicles are still spherical though they now appear to be smaller and have a more irregular exterior surface . the average size of the aggregates is still close to 50 nm in diameter , but the size dispersity has dramatically decreased with no evidence of vesicles larger than 100 nm present in the sample . moreover , there was no evidence of the rolled cylinders previously imaged in the fresh aggregate solutions . a fresh solution of the vesicles of dodeca - 5 , 7 - diynoyl phosphonic acid was prepared and aged three days before polymerization using an immersion uv light operating at 450 w . the polymerization of the aggregates was followed using uv - vis spectroscopy and the results are shown in fig4 . fig4 a is the spectrum of the unpolymerized vesicle . as the polymerization proceeds there is a red shift in the spectrum as the conjugation of the system increases . see fig4 b . within the first hour of polymerization the peaks at 239 nm and 254 nm completely disappear while the peak centered at 226 nm grows dramatically . this peak slowly coalesces with the peak at 209 nm to form a broad featureless peak after 510 minutes of polymerization . extending the polymerization time past this point produced no further changes in the uv - vis spectrum of this sample . the dodeca - 5 , 7 - diynoyl phosphonic acid obtained in this example readily dissolves in buffered aqueous solutions to form vesicles . using tem to analyze these phosphonic acid vesicles it appears that the age of the solution affects the size dispersity and possibly the stability of the aggregates . aggregates that had aged one week appeared to be smaller and more monodipserse compared to solutions less than five days old . this may be due to polymerization of small amounts of the phosphonic acid in the aggregate solutions upon exposure to ambient light . an aggregate was prepared using n - dodecanephosphonic acid . specifically , a 3 × 10 − 4 molar ( on average ) solution of n - dodecanephosphonic acid was used , which was prepared by adding 17 mg dodecanephosphonic acid to 250 ml of a 5 mm solution of morpholinoethanesulfonic acid ( mes ) buffer solution . this solution was heated in a boiling water bath for 40 - 60 minutes . the solution was then cooled and allowed to sit undisturbed for 3 days ( following shulz &# 39 ; procedure ( 21 )). this preparation was used to produce the desired micelles and the reported critical micelle concentration ( cmc ) was independently confirmed through measurement of the surface tension by means of a wilhelmy plate balance ( 22 ). it was found that the aggregates formed through this preparation are sperical in shape with an average diameter of 20 - 120 nm as visualized through tem . it has also been determined that these aggregates are stable over the ph range of 4 - 6 , with minimal structural changes . the choice of biologically compatible metal ion used to bind to the micelle exterior directly influences the size and the shape of the resulting aggregates . iron ( iii ) salts are preferred because they are relatively inexpensive , readily available in several simple forms and known to be biologically compatible . however , other metal ions may be used if desired , including , without limitation , zr ( iv ), mg ( ii ), ca ( ii ), co ( ii ) and zn ( ii ) salts . the tem images in fig3 represent three aggregate samples from the same starting micelle solution that have been metallated with different iron salt sources ; fig3 a : fe ( clo 4 ) 3 ; fig3 b : fe ( no 3 ) 3 ; fig3 c : fecl 3 . the aggregates in each of these samples are similar , ranging in size from 25 nm to 275 nm in diameter with an average size of 110 nm . aggregates of a relatively uniform size distribution can be obtained by multiple extrusions through a nuclepore filter ® ( whatman ) prior to metallation . specifically this entails multiple extrusions through a membrane filter to yeild aggregates that are more monodisperse in size , i . e ., on the order of 25 - 75 nm . the diversity of sizes of the metallated aggregates and the change in shape relative to the metal - free aggregates can only be explained if some type of reorganization is occurring upon addition of the metal . for the large spheres observed upon metallation with fe ( clo 4 ) 31 the size of these aggregates indicates reorganization to form larger vesicular species . this type of reorganization phenomenon has been previously reported in vesicle systems with the addition of group ii elements such as magnesium and calcium ( 23 , 24 ). in these systems it was found that the addition of metal ions to the exterior of the vesicle negated the surface charge of the exposed headgroups . this in turn allowed the aggregates to approach each other closely and fuse together to form larger more stable “ super vesicles ” ( 23 , 24 ). 1 ) “ the challenge of liposome targeting in vivo ” poste , g . ; 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papahadjopoulos , d . proc . natl . acad . sci . u . s . a . 1976 , 73 , 1603 . 24 ) papahadjopoulos , d . ; vail , w . j . ; newton , c . ; nir , s . ; jacobson , k . ; poste , c . ; lazo , r . biochim . biophys . acta 1977 , 465 , 579 . 25 ) “ formation of unilamellar vesicles ” lasic , d . d . j . colliod interface sci . 1988 , 124 , 428 . 26 ) “ characterization of vesicles by classical light scattering ” van zanten , j . h . ; monbouquette , h . g . j . colloid inter . sci . 1991 , 146 , 330 . while certain preferred embodiments of the present invention have been described and specifically exemplified above , it is not intended that the invention be limited to such embodiments . various modifications may be made thereto without departing from the scope and spirit of the present invention , as set forth in the following claims .