Vascular magnetic imaging method and agent comprising biodegradeable superparamagnetic metal oxides

The preparation and isolation of biodegradable superparamagnetic MR imaging contrast agents for the vascular compartment is described. These aggregates are comprised of individual biodegradable superparamagnetic metal oxide crystals which aggregates have an overall mean diameter less than about 4000 angstroms. The preferred vascular imaging contrast agent is comprised of aggregates of iron oxide crystals having an overall mean diameter less than about 500 angstroms. These contrast agents may be associated with a macromolecular species, which assist, among other things, in the preparation of these extremely small materials, and may be dispersed or dissolved in a physiologically acceptable medium. Preferred media also stabilize the materials against further aggregation even under harsh sterilization conditions. The autoclaved biodegradable superparamagnetic iron oxides of the invention are ideally suited for a pharmaceutical preparation and enjoy several advantages over prior intravascular imaging contrast media including low osmolality, low effective dose requirements, high relaxivities, long blood lifetimes, rapid biodegradability, and versatility with respect to a wide range of applicable MR data acquisition parameters.

1. INTRODUCTION 
This invention relates to methods for enhancing magnetic resonance (MR) 
images of the vascular compartment of animal or human subjects. The 
methods of the present invention involve the use of biodegradable 
superparamagnetic contrast agents which enhance MR images of the vascular 
compartment. These methods, in turn, allow one to image organ or tissue 
perfusion, as well as blood flow. The use of these contrast agents, 
preferably administered as a superparamagnetic fluid, offers significant 
advantages over existing methodologies including inter alia high proton 
relaxivities, rapid biodegradability, control of blood lifetimes, 
versatility in choice of pulse sequence weighting schemes and other MR 
experimental parameters, low dosage requirements, low osmolality, and 
little or no toxicity. 
2. BACKGROUND OF THE INVENTION 
Magnetic resonance (MR) imaging is widely regarded as a powerful technique 
for probing, discovering, and diagnosing the presence and progress of a 
pathological condition or disease. The MR method of imaging is also 
regarded as the least invasive of the imaging techniques presently 
available and does not expose the patient or subject to potentially 
harmful high-energy radiation, such as X-rays, or radioactive isotopes, 
such as technetium-99m. Technological developments in both instrumentation 
and computer software continue to improve the availability and quality of 
the images produced. Researchers discovered quickly, however, that the 
relative differences between the chemical and magnetic environments of 
water molecules, whose proton nuclei provide by far the largest source of 
measurable signal intensity within the body, whether these molecules be 
located in organs, tissues, tumors, or in the vascular compartment, are 
often quite small and, consequently, the resulting images are poorly 
resolved. Fortunately, this inherent limitation can be overcome by the use 
of proton relaxation agents, also known as contrast agents, which are 
absorbed selectively by different types of tissues and/or sets of organs, 
and thus create a temporary condition in which the magnetic environments 
of neighboring water molecules are measurably dissimilar. 
According to their magnetic properties, there are three general types of MR 
contrast agents: paramagnetic, ferromagnetic, and superparamagnetic. The 
weak magnetism of paramagnetic substances arises from the individual 
unpaired electrons while the stronger magnetism of ferromagnetic and 
superparamagnetic materials results from the coupling of unpaired 
electrons made possible by their presence in crystalline lattices. 
Ferromagnetic materials retain their magnetism in the absence of an 
applied magnetic field while superparamagnetic materials lose their 
magnetism when the applied magnetic field is removed. With respect to 
effects on proton relaxation, paramagnetic agents have been termed T.sub.1 
type agents because of their ability to enhance spin-lattice or 
longitudinal relaxation of proton nuclei. Ferromagnetic agents have been 
termed T.sub.2 type agents because of their specific effects on T.sub.2, 
sometimes called the spin-spin or transverse relaxation. 
There are three major disadvantages to the use of paramagnetic chelates as 
vascular MR contrast agents. The first is that many low molecular weight, 
ionic materials which are commonly used as paramagnetic chelates are 
hypertonic. The use of hypertonic solutions often results in adverse 
reactions upon injection. The second disadvantage is that paramagnetic 
chelates have short blood lifetimes whereas a vascular MR contrast agent 
should remain confined to the vascular compartment for long periods of 
time. Third, removal of paramagnetic chelates from the vascular 
compartment can result in release of the paramagnetic ion from the 
chelate. Paramagnetic ions of iron, manganese, and gadolinium, for 
example, are toxic in their free ionic form. Vascular MR contrast agents 
should have a benign metabolic fate after removal from the vascular 
compartment. These three disadvantages are explained further below. 
Paramagnetic materials that have been used as T.sub.1 contrast agents 
generally include organic free radicals as well as transition metal salts 
and chelates. These compounds can be quite soluble and, in the case of 
most transition metal complexes, are highly charged, ionic species. Due to 
their relatively low relaxivities (their ability to increase the 
relaxation rates of protons as a function of dose), high concentrations of 
transition metal chelates are needed to effect useful alterations in the 
relaxation times of blood. In addition, the ionic nature of many 
transition metal ion salts and chelates contributes to the high osmotic 
pressure of the injected diagnostic solutions. The end result is that 
solutions of paramagnetic materials, whether they are used as MR agents or 
not, tend to be hyperosmotic relative to blood. The administration of 
hyperosmotic solutions into the subject is widely believed to be a major 
cause of adverse reactions to radiographic and MR contrast media (See, 
McLennan, B. L. Diagnostic Imaging Supplement 1987, 16-18 (December); 
"Contrast Media: Biological Effects and Clinical Application," Vol. I, 
Parvez, Z., Moncada, R., and Sovak, M. (Eds.), CRC Press, Boca Raton, Fla. 
(1987)). 
The usual approach to the development of MR contrast agents confined to the 
vascular compartment for long periods of time is to increase the molecular 
weight of paramagnetic chelates by attaching the chelates to high 
molecular weight polymers. After injection, high molecular weight forms of 
the chelates cannot be excreted by glomerular filtration, and consequently 
have longer residence times within the vascular compartment. High 
molecular weight forms of chelates can be made by covalently attaching 
chelators to macromolecules such as human serum albumin (Schmiedl et al. 
Radiology 1987, 162, 205-210)). With this approach to the design of 
vascular MR contrast agents, the fate of the gadolinium after degradation 
of the agent presents serious problems. The long term retention of 
gadolinium not eliminated by glomerular filtration, and the potential for 
delayed toxicity from that element, pose major obstacles to the 
administration of high molecular weight gadolinium chelates to humans. 
A major disadvantage of present ferromagnetic contrast agents is that such 
materials are relatively large and, frankly particulate in character. 
Frankly particulate materials, those generally having overall dimensions 
between about 0.5-10 microns, are quickly removed from the blood by the 
phagocytic action of the cells of the reticuloendothelial system, limiting 
the duration of their effects on the spin-spin and spin-lattice relaxation 
times of blood. Hence their usefulness as vascular MR contrast agents is 
limited. All particulate agents suffer a similar limitation. 
3. DEFINITIONS 
Unless otherwise noted, the term "sterilized" describes a sample or a 
preparation which has been subjected to any method known in the art which 
completely destroys all bacteria and other infectious agents which may be 
present in the sample or preparation. Nonlimiting examples of such methods 
include autoclaving, ultraviolet or gamma irradiation, cold membrane 
filtration, or chemical treatment. The resulting preparation is then 
suitable for in vivo and/or clinical use with or without further 
treatment. The term "biodegradable" describes a property of a compound or 
complex which allows the compound or complex to be broken down into 
smaller innocuous components and be excreted from or utilized within the 
body. The term "polyfunctional" applies to a molecule which contains a 
plurality of identical functional groups. A "macromolecular species" may 
include any molecule, natural or synthetic, which has a molecular weight 
in excess of 1 kilodalton. 
Abbreviations used in the text are defined below. 
T.sub.1 =spin-lattice relaxation time 
T.sub.2 =spin-spin relaxation time 
EM=transmission electron microscopy 
MR=magnetic resonance or nuclear magnetic resonance (NMR) 
RES=reticuloendothelial system 
MW=molecular weight 
kD=kilodalton 
4. SUMMARY OF THE INVENTION 
This relates to a method for enhancing an MR image of the vascular 
compartment of an animal or human subject. The enhanced MR image is 
realized by administering to such an animal or human subject an effective 
amount of a contrast agent prepared from aggregates of individual 
biodegradable superparamagnetic metal oxide crystals. The aggregates, 
whose metal oxide crystals may be associated with a macromolecular 
species, are dispersed in a physiologically acceptable medium, preferably 
an aqueous citrate buffer, which stabilizes the aggregates of the 
resulting fluid against further aggregation even under the sometimes 
extreme conditions of sterilization. The preparation of biodegradable 
superparamagnetic aggregates are described whose properties and 
characteristics, such as overall sizes and blood circulation lifetimes, 
for example, may be varied. 
One of the objects of the present invention is to provide a pharmaceutical 
preparation of a biodegradable superparamagnetic contrast agent suitable 
for human clinical use in magnetic resonance experiments. In particular, 
preferred embodiments of the invention have great utility in obtaining 
enhanced images of the vascular system, of the blood pool, or of organ or 
tissue perfusion. By practicing the methods of the invention, blood 
vessels, arteries, veins, capillaries, as well as lymph vessels may be 
imaged successfully. In particular capillary beds of various organs or 
tissues such as the brain, the kidney, the lung, and the heart can be 
visualized. Such methods can thus provide valuable information regarding 
the condition of these vessels, organs, or tissues including the presence 
of microocclusions, embolisms, aneurysm, restricted blood flow, and the 
onset or recession of arterial disease. 
It is also an object of this invention to provide a contrast agent which 
can serve as a brightening or a darkening agent, or both, and which is 
effective at low doses. This invention also seeks to provide a contrast 
agent which is rapidly biodegraded, being either excreted from or utilized 
within the body of the subject, as evidenced by a return of the proton 
relaxation rates of affected tissue to pre-administration levels within 
about one week of the initial administration. Yet another object of the 
present invention is to provide sterile solutions of a contrast agent 
which have a low osmotic pressure, low enough, in fact, that added salt 
may be included in the formulation if so desired. Hypotonic solutions of 
contrast media may thus be prepared, if so desired. It is also an object 
of the present invention to provide a contrast agent whose blood lifetime 
may be adjusted to accommodate the variable needs of the MR imaging 
experiment. 
It is thus an object of the instant invention to provide an effective 
contrast agent with all the advantages enumerated above yet is 
substantially free of the limitations and objectionable aspects associated 
with paramagnetic and ferromagnetic materials. 
After a thorough consideration of the foregoing disclosures and those that 
follow, other objects of the present invention become readily apparent to 
those readers skilled in the art.

6. DETAILED DESCRIPTION OF THE INVENTION 
Superparamagnetic metal oxides are a unique class of magnetic materials 
because of their large effects on both the T.sub.1 and T.sub.2 relaxation 
times of magnetically active nuclei. We have described in detail the 
utility of biodegradable superparamagnetic materials as MR contrast agents 
in prior co-pending U.S. application Ser. Nos. 882,044 and 067,586, filed 
July 3, 1986 and June 26, 1987, respectively. The disclosures in both 
Applications are incorporated herein by reference. Others have described 
the use of superparamagnetic contrast agents since (See, for example, U.S. 
Pat. No. 4,675,173 issued to Widder; Hemmingson et al. Acta. Radiologica 
1987, 28(6), 703; Saini et. al Radiology 1987, 162, 211). When present as 
a component of a superparamagnetic lattice, transition metals, such as 
iron, are more potent enhancers of proton relaxation than the same metal 
as the central ion of a simple paramagnetic chelate. This increased 
"potency" of the superparamagnetic iron is reflected in, for example, the 
spin lattice relaxivity value measured for a biodegradable 
superparamagnetic fluid, comprising the biodegradable superparamagnetic 
iron oxide and a citrate buffer, which value is 50 times greater than that 
found for a chelated form of iron. Indeed, as the sizes of the aggregates 
of iron oxide crystals decrease, their effects on T.sub.1 increase 
relative to their effects on T.sub.2. As disclosed in the present 
invention, superparamagnetic fluids comprised of very small metal oxide 
aggregates are excellent intravascular MR contrast agents and can be used 
in a variety of modes including T.sub.1 -weighted (brightening agents), 
T.sub.2 -weighted (darkening agents), or combinations of these and other 
pulse techniques. The present invention is directed to agents and to a 
method for the enhancement of MR images of the vascular compartment. The 
enhancement of the MR image is made possible by the use of a sterile 
biodegradable superparamagnetic contrast agent whose magnetic component is 
comprised of three-dimensional aggregate of individual biodegradable 
superparamagnetic metal oxide crystals. 
An important prerequisite for the successful use of biodegradable 
superparamagnetic materials as intravascular agents in humans is the 
ability to control the blood lifetimes of the materials. In other words, 
an effective intravascular agent must have a useful lifetime in the 
circulatory system so that the MR experiment may be completed before a 
significant proportion of the administered contrast agent is removed from 
circulation by organs or tissues of, for example, the reticuloendothelial 
system (RES). The injection of higher concentrations of any contrast agent 
will, of course, increase its longevity in the vascular compartment and 
prolong its effect on the nuclear relaxation times of the blood. Such a 
strategy, while useful in demonstrating the efficacy of an MR contrast 
agent in animal models of human pathology, is not ideal for the 
formulation of a pharmaceutical, clinical form of the contrast agent. 
Safety considerations preclude the use of overly large doses of a 
pharmaceutical or diagnostic agent. Imaging techniques, such as those 
described herein, which are able to enhance the blood pool while 
maintaining the minimum effective dose are highly desirable. Moreover, the 
ability of the practitioner to manipulate or control the blood lifetime of 
the biodegradable superparamagnetic contrast agents of the invention 
permits an unsurpassed flexibility previously unavailable in the design 
and operation of the MR experiment. A variety of pharmacokinetic patterns 
might be used, for example. Hence, for an experiment designed to delineate 
tissue blood pool volume, an agent with a long blood lifetime would be 
used having all the advantages outlined above for biodegradable 
superparamagnetic materials. In other applications, an agent which is 
rapidly removed from the vascular compartment may be used to "wash in" to 
damaged tissue and an image taken very soon thereafter. Areas where the 
blood brain barrier have broken down due to the presence of tumors may be 
successfully examined using these methods. Moreover, because discrete size 
ranges of the contrast agent can be obtained, for example, by size 
exclusion chromatography, the practitioner can tailor and control the 
amount of time (e.g., blood halflives) that these agents spend circulating 
within the vascular compartment. Generally, the blood lifetime is 
inversely proportional to the overall size of the contrast agent. Some 
agents of the present invention, because of their extremely small 
dimensions, are able to circulate within the vascular compartment for 
relatively long periods of time. The crystalline nature of these agents, 
which agents characteristically provide solutions of low osmolality 
compared with paramagnetic agents, in combination with the above-mentioned 
properties and characteristics make these biodegradable superparamagnetic 
metal oxides uniquely suitable as intravascular contrast agents. 
The biodegradable superparamagnetic metal oxides can be prepared as a 
superparamagnetic dispersion or as a superparamagnetic fluid by general 
methods described previously in applicants, prior co-pending U.S. 
Applications identified above. These procedures include precipitating the 
metal oxide crystals, in the presence or absence of a macromolecular or 
polymeric species, from a solution of the trivalent and divalent ions of a 
metal by the addition of a sufficient quantity of base. Preferred metal 
ions include those of chromium, cobalt, copper, iron, manganese, 
molybdenum, nickel, and tungsten. A solution of hydrated ferric and 
ferrous salts is particularly preferred. The alkaline reaction may be 
carried out in the presence of a macromolecular species which aids in 
dispersing the resultant superparamagnetic metal oxide crystals. In 
general, the aggregates that form initially, in the absence of the 
polymeric substance, are larger. Some suitable macromolecular species 
include proteins, polypeptides, carbohydrates, mono-, oligo-, or 
polysaccharides. Preferred macromolecules include human or bovine serum 
albumin, polyglutamate, polylysine, heparin, hydroxyethyl starch, gelatin, 
or dextran, with the lattermost being particularly preferred. The dark 
slurry which forms after the addition of base is then sonicated to further 
reduce the size of the aggregates. The sonication also serves to oxygenate 
the mixture and assures the full oxidation of the divalent metal ion to 
the trivalent oxidation state. Larger particulate aggregates that may form 
during the procedure may be removed by centrifugation. The resulting 
supernatant contains only nonparticulate-sized aggregates. 
The next stage of the preparation involves the dilution of the supernatant 
with distilled water and subsequent ultrafiltration through a hollow fiber 
dialysis apparatus. The diluted supernatant is dialysed against an aqueous 
solution or buffer of a polyfunctional organic molecule. Molecules 
containing positively charged groups may be utilized. These molecules may 
include but are not limited to polylysine, polyornithine, and 
polyarginine. However, the functional groups of the organic molecule are 
preferably ionizable to give negatively charged groups such as phosphates, 
phosphinates, phosphonates, sulfinates, sulfonates, carboxylates, and the 
like. Polycarboxylate compounds are particularly effective, with salts of 
citrate ion being most preferred. Besides being commonly used and widely 
accepted as safe in clinical preparations, the aqueous citrate buffer 
serves to stabilize the aggregates of the resulting superparamagnetic 
fluid to further clustering or aggregation. The inventors have found, 
quite unexpectedly, that the resulting fluid is stable even under the 
extreme conditions of prolonged heating in an autoclave. In addition, the 
dialysis step also removes most of the macromolecular species, e.g. 
dextran. Also, dialysis against citrate scavengers isolated free metal 
ions probably through coordination with the anionic groups of the 
polyfunctional molecule. If so desired, greater than ninety percent of the 
dextran used initially can be removed in this manner. 
The resulting dialysed superparamagnetic fluid is then diluted further and 
sterilized by any means well known in the art. Sterilization may be 
accomplished by autoclaving, irradiation, sterile filtration, chemical 
treatment, or other means. Sealing the samples in appropriate containers 
and heating them in an autoclave is most convenient. It is understood 
that, depending upon the particular application at hand, substantially all 
the added macromolecular species may be removed or another introduced by 
any appropriate means well known in the art including exhaustive dialysis. 
The size distribution and architecture of the metal oxide crystal 
aggregates can be examined by light scattering methods and by electron 
microscopy (EM). The EM studies are particularly revealing and show that 
the aggregates are indeed comprised of individual metal oxide crystals 
which are interconnected to form irregularly shaped three-dimensional 
structures (See, for example, FIG. 3). The dimensions of the aggregates 
depend on the number of component crystals present and, for one specific 
example, fall in the range of 10-125 nm. The majority of the aggregates 
have dimensions smaller than about 50 nm. The results of light scattering 
methods give similar, but slightly larger, values. This slight difference 
is probably due to the fact that larger particles will tend to scatter 
more light and thus skew the value slightly toward the higher end. Light 
scattering methods give a value of about 75 nm for the overall mean 
diameter of the sample described above. 
In a specially modified procedure, extremely small superparamagnetic 
materials particles are obtained at the outset. In this modification, a 
solution of ferric and ferrous salts is adjusted first to a pH of about 
2.3 before the addition of dextran. The resulting mixture is stirred and 
heated to about 60.degree.-70.degree. C. for several minutes and then 
allowed to cool to a temperature of about 40.degree.-45.degree. C. To this 
solution is added a sufficient amount of ammonium hydroxide to bring the 
pH to about 10. The resulting suspension is then heated to about 
100.degree. C. until a black suspension forms. After an ultrafiltration 
step to remove most of the unbound dextran, light scattering experiments 
showed that dextranized particles of this preparation have an overall mean 
diameter of about 40 nm. 
The superparamagnetic fluid prepared according to the general methods can 
be separated into fractions of decreasing aggregate diameter by size 
exclusion gel chromatography with a suitable buffer as eluent. The 
collected fractions are analyzed by UV-vis spectroscopy for metal oxide 
content and by light scattering methods for an estimate of their crystal 
aggregate dimensions. Predictably, the larger aggregates elute first 
followed by the smaller ones. The fractions can be divided into four 
groups, for the sake of convenience, and tested separately. A standard 
solution comprised of dextran blue (2,000 kD), ferritin (443 kD), and 
bovine serum albumin (65 kD) was fractionated under the same conditions as 
the superparamagnetic fluid. The fraction number or elution volume at 
which the known proteins emerged served as points of reference from which 
the average "molecular weights" of the crystal aggregates of the contrast 
agent fractions could be related. The results are summarized in Section 
7.5. 
Proton relaxation experiments are carried out in vivo by intravascularly 
injecting mice with solutions of fractionated as well as unfractionated 
contrast agents. The effect on proton relaxation, T.sub.1 for example, may 
be monitored as a function of time for each of the samples, and the 
results show that the smaller the aggregate size the longer the blood 
serum lifetime of the contrast agent. The unfractionated superparamagnetic 
contrast agent shows a moderate serum blood halflife, Blood t.sub.1/2, of 
about 13.5 minutes while the smallest aggregates have a Blood t.sub.1/2 
value of 49.6 minutes. 
These superparamagnetic metal oxide preparations serve as excellent 
contrast agents for MR imaging. They may be successfully used as 
brightening agents, for example, as can be demonstrated by a relative 
comparison of the Brightness/Intensity effects of the materials of the 
invention versus samples of pure water and aqueous solutions of typical 
paramagnetic T.sub.1 relaxation agents such as manganese(II) chloride or 
ferric chloride. All the superparamagnetic metal oxide preparations 
described above are able to brighten water signals relative to pure water 
and in one case, the brightness effects even exceeds that of manganese(II) 
chloride, heretofore recognized as a very effective T.sub.1 contrast agent 
(See FIG. 4). 
7. EXAMPLES 7.1. PREATION OF STERILIZED SUPERAMAGNETIC FLUIDS 
To a vigorously stirred aqueous 16% ammonium hydroxide solution (5 liters) 
of dextran (2.500 kg, 10-15 kilodalton) is added gradually, and over a 5 
minute period, an aqueous solution (5 liters) of ferric chloride 
hexahydrate (FeCl.sub.3.6H.sub.2 O, 0.755 kg) and ferrous chloride 
tetrahydrate (FeCl.sub.2.4H.sub.2 O, 0.320 kg). The black magnetic slurry 
which forms is then pumped at a rate of about 0.4 liters per minute 
through a continuous flow sonicating apparatus. It is sometimes 
advantageous, although not necessary, to heat the mixture through a 
sonicating apparatus which comprises a sonicator connected in series to a 
100.degree. C. heating coil unit followed by a cooling coil unit. The 
resulting dispersed mixture is next centrifuged at 2,000.times.g for 20 
minutes to separate the larger aggregates which are discarded. 
The supernatant is diluted with deionized, sterile water to a volume of 
about 20 liters. Ultrafiltration of the diluted supernatant against water 
and citrate buffer is then carried out in a noncontinuous fashion using a 
large hollow fiber dialysis/concentrator, Model DC 10 (Amicon Corp., 
Danvers, Mass.), equipped with a 100 kilodalton molecular weight cutoff 
dialysis cartridge. In this manner, a substantial amount of the dextran 
used initially is removed along with any free metal ions. 
After ultrafiltration, a solution of 0.20M Fe and 0.025M citrate is 
obtained by the addition of 1M citrate and adjusting to a pH of about 8 
with 1N NaOH. The solution is then autoclaved for 30 minutes (121.degree. 
C.). Autoclaving is the preferred technique for sterilization since the 
bottle or container need not be sterile prior to fill. An alternative to 
sterilization is filtration, but superparamagnetic fluids at high 
concentrations filter poorly. Dilute superparamagnetic fluids can be 
filter sterilized, but the added water must then be removed under sterile 
conditions. 
7.2 REMOVAL OF DEXTRAN FROM STERILIZED SUPERAMAGNETIC FLUIDS 
The dextran remaining after ultrafiltration is largely dissociated from the 
superparamagnetic iron oxide-dextran by autoclaving. 
To determine what part of the dextran is attached to superparamagnetic iron 
oxide after autoclaving, dialysis is used to separate dextran from 
superparamagnetic iron oxide-dextran complexes. The autoclaved 0.20M Fe 
solution from above is diluted into 25 mM citrate buffer (pH 8) to an iron 
concentration of about 2 mM and the solution incubated overnight at 
37.degree. C. to release any dextran weakly adsorbed to the 
superparamagnetic iron oxide. The solution is then applied to a centrifuge 
micropartition system equipped with a cellulose acetate membrane (Amicon, 
Danvers, Mass.), where the filtrate is forced through the membrane by 
centrifugation. The dextran employed initially has a molecular weight of 
10-15 kD and passes through the membrane while superparamagnetic iron 
oxide does not. 
Prior to autoclaving (but after ultrafiltration) there were 8.45 mg/mL of 
dextran present in a solution containing 11.2 mg/mL of Fe (0.2M Fe=11.2 mg 
Fe/mL). The concentration of dextran passing through the micropartition 
system membrane was 7.49 mg/mL, indicating that most of the dextran had 
detached from the superparamagnetic iron oxide by the autoclaving step. 
Iron is measured by atomic absorption spectrophotometry after dissolving 
the iron oxide in 0.01N HCl. Dextran is measured by a phenol-sulfuric acid 
method for total carbohydrates (See, C. E. Meloan and Y. Pomeranz, "Food 
Analysis Laboratory Experiments," The Avi Publishing Co., Westport Conn., 
pp. 85-86 (1973)). 
The small aggregates of superparamagnetic iron oxide crystals seen in EM 
studies are largely uncoated after autoclaving and are stabilized through 
their interactions with polyvalent anions such as citrate. The majority of 
the dextran is present as a dialyzable species free in solution. 
7.3 MODIFIED PROCEDURE FOR THE PREATION OF EXTREMELY SMALL BIODEGRADABLE 
SUPERAMAGNETIC AGGREGATES 
To an aqueous solution (250 mL) of FeCl.sub.3.6H.sub.2 O (35 g) and 
FeCl.sub.2.4H.sub.2 O (16 g) is added a sufficient amount of aqueous 10% 
sodium carbonate to bring the pH of the solution to a value of about 2.3. 
Solid dextran (150 g) is then added. The solution is stirred and heated to 
about 60.degree.-70.degree. C. for about 15 min and then allowed to cool 
to 40.degree.-45.degree. C. To the reddish solution is added aqueous 7.5% 
NH.sub.4 OH to a final pH between 9.5 and 10.0. A greenish suspension is 
produced which is subsequently heated to 95.degree.-100.degree. C. for 15 
min. The resulting black suspension is then subjected to an 
ultrafiltration step using an Amicon RA 2000 hollow fiber dialysis unit 
equipped with a cartridge having a nominal cutoff of 100 kilodaltons. 
Light scattering measurements reveal that the dextranized particle has an 
overall mean diameter of about 40 nm. 
7.4 GEL EXCLUSION CHROMATOGRAPHY OF SUPERAMAGNETIC MATERIALS 
The superparamagnetic fluid described in Section 7.1 can be applied to the 
top of a chromatography column (2.5 cm.times.100 cm) packed with Sepharose 
4B. The sample is eluted with 20 mM sodium citrate buffer (pH=8). 
Fractions are collected and analyzed for their optical density at 340 nm. 
A separate sample containing a mixture of known proteins is treated and 
fractionated under the same chromatography conditions. A plot of the 
optical density (OD) versus the fraction number and elution volume yields 
a chromatogram which is illustrated in FIG. 1. The reference proteins 
elute at the fraction numbers indicated at the bottom of the plot. The 
overall mean diameter in nanometers may be determined for a given set of 
superparamagnetic fluid fractions by light scattering methods. These 
results are listed across the top of FIG. 1. As is readily evident from 
the chromatogram and plot, the overall size decreases with increasing 
fraction number and elution time. Likewise, the higher molecular weight 
proteins elute faster than the smaller ones. The eluted fractions of 
superparamagnetic materials can be combined as follows: Sample A, 
fractions 29-31; Sample B, fractions 34-37; Sample C, fractions 43-49; and 
Sample D, fractions 55-62. 
7.5 DETERMINATION OF BLOOD LIFETIMES OF SUPERAMAGNETIC MATERIALS AS A 
FUNCTION OF SIZE 
The samples B, C, and D, described above, are intravenously injected 
separately into a rat at the same dose of 2 mg of iron per kg of rat. The 
spin-lattice relaxation rate, 1/T.sub.1, can then be measured as a 
function of time and reflects the blood concentration. FIG. 2 is a 
graphical representation of the results for samples B, C, D, and 
unfractionated superparamagnetic fluid. An examination of the plot in FIG. 
2 shows that the blood lifetimes of the different fractions increase with 
decreasing overall size. Table I summarizes the results of the above 
experiments. The blood half-life (Blood t.sub.1/2) is determined from a 
nonlinear least squares fit to a single exponential decay process. The 
molecular weight (MW) values of samples A, B, C, and D are those estimated 
from extrapolated values derived from the standard proteins. 
TABLE I 
______________________________________ 
PROPERTIES OF NATIVE AND FRACTIONATED 
SUPERAMAGNETIC FLUID 
Sample Fraction # 
OMD.sup.a 
MW Blood t.sub.1/2 
Dose 
______________________________________ 
Native.sup.b 
All 75 nm -kD 13.5 min 
1 mg/kg 
A 29-31 97 1,500 -- 2 
B 34-37 46 500 4.05 2 
C 43-49 18 170 16.6 2 
D 55-62 &lt;15 60 49.6 2 
______________________________________ 
.sup.a Overall mean diameter as measured by light scattering methods. 
.sup.b Unfractionated fluid prepared by the general methods. 
7.6 TRANSMISSION ELECTRON MICROSCOPY STUDIES 
An electron micrograph of the sterilized superparamagnetic fluid, prepared 
according to 7.1 is shown in FIG. 3. The EM study shows the individual 
particle to be aggregates of iron oxide, with individual iron oxide 
crystals having a diameter of about 5-7 nm. The crystals of each particle 
are in direct contact with each other and are not separated by, or 
embedded in, a matrix or polymer of any kind. EM studies (beyond those 
shown in FIG. 3) have indicated the presence of aggregates from as small 
as 10 nm to as large as 300 nm. Both EM and gel chromatography (see 
Section 7.4) indicate that the superparamagnetic fluid is a heterogeneous 
mixture of different-sized particles. EM studies reveal the presence of a 
large number of small aggregates which comprise a very small percentage of 
the volume of iron mass. Consequently the average volume of an aggregate 
by EM studies is only about 20 nm. (Average volume =the total volume of 
all aggregates divided by the total number of aggregates.) 
For EM studies it is important to prevent changes in size distribution of 
particles during sample preparation. The preparation of the sample for EM 
studies is as follows. A solution of 5% molten agar is added to the 
sterile superparamagnetic fluid to give a final agar concentration of 1% 
and the agar allowed to harden. The agar is minced into pieces of about a 
millimeter and dehydrated by addition of ethanol/water mixtures of 
decreasing water content until water is replaced by 100% ethanol. The 
ethanol is then replaced by propylene oxide in an analogous fashion. The 
propylene resin finally yielding a 100% epoxy resin which is hardened by 
curing overnight at 60.degree. C. The hardened resin is then sliced with 
an ultramicrotone (0.5 micron thick sections) and placed on a 400 mesh 
copper grid. 
A Phillips model 410 LS transmission electron microscope is used to take 
micrographs at 100 kV. 
7.7 SUPERAMAGNETIC FLUIDS AS BRIGHTENING AGENTS 
Superparamagnetic materials are used as MR contrast agents due to their 
ability to promote the relaxation of magnetically active nuclei. As the 
particle size of iron oxide cluster in superparamagnetic fluids becomes 
smaller and smaller, they become more powerful as brightening agents in 
T.sub.1 -weighted MR pulse sequences. FIG. 4 shows the effects of 
different concentrations of four MR contrast agents on the signal 
intensity (S) of an MR image using a T.sub.1 -weighted pulse sequence of 
TE=200 msec and TR=15 msec. The imager was a GE CSI 2 Tesla imager. The 
signal intensity of distilled water is set at 1.0. 
Various concentrations of agents were added to distilled water and the 
molar concentration of metal is given on the x-axis. Agents that brighten 
increase the value of S while those that darken decrease the value of S. A 
standard T.sub.1 type MR brightening agent is MnCl.sub.2 and has a strong 
image brightening effect over a wide range of concentrations. A standard, 
more purely T.sub.2 type MR agent is provided by a large silanized cluster 
of superparamagnetic materials made according to the teachings of U.S. 
Pat. No. 4,695,392 which is incorporated herein by reference. On a molar 
basis the smallest fraction obtained from the chromatogram of FIG. 1 (D of 
Table I) is a more potent brightening agent than MnCl.sub.2, while the 
larger parent material (Native of Table I) is considerably less potent as 
a brightening agent. As shown in FIG. 4, the large silanized cluster is 
the least potent T.sub.1 type agent. The strong effects of sterile 
superparamagnetic fluids on MR image brightening, particularly as particle 
size decreases, allows such materials to be used as vascular MR imaging 
with T.sub.1 - or T.sub.2 - weighted pulse sequences. When T.sub.1 
-weighted sequences are used, such materials perform as contrast agents in 
a manner analogous to paramagnetic brightening agents such as Gd/DTPA or 
MnCl.sub.2. 
It should be apparent that other modifications and embodiments can be 
contemplated without departing significantly from the scope and spirit of 
the present invention. The invention should, therefore, not be limited by 
the foregoing examples and descriptions thereof but only as enumerated in 
the following claims.