Mognefk’ Resonance Printed in the USA.
l
Imagmg. All rights
Vol. 9. pp. 215-283, reserved.
1591 Copyright
0730-725X/91 $3.04 + .oO 0 1991 Pergamon Press plc
Original Contribution IRON OXIDE NANOPARTICLES FOR USE AS AN MRI CONTRAST PHARMACOKINETICS AND METABOLISM
AGENT:
D. POULIQUEN, J.J. LE JEUNE, R. PERDRISOT, A. ERMIAS, AND P. JALLET Laboratoire de Biophysique, Faculte de Medecine, 1 Rue Haute de ReculCe,49000 Angers, France The pharmacokinetics and metabolism of a new preparation of superparamagnetic iron oxide nanoparticles were evaluated by 59Fe radiotracer studies and histologic examination of mice liver and spleen tissues (light and transmission electron microscopy). In the first 30 min following IV injection of the product half of the dose injected remains in the blood, the other part being sequestered mainly by the mononuclear phagocyte system (MPS). In the first five days following IV administration of the nanoparticles, early metabolization of the iron oxide cores occurs, revealed by modification of their aspect in the lysosomes of Kupffer cells and macrophages of the splenic red pulp. The incorporation of s9Fe is then observed in RBC of the mice. These results are discussed in relation with the physicochemical properties of this new preparation of nanoparticles, and compared with current pharmacokinetic data concerning injectable particle systems. Keywords: Contrast agent; Superparamagnetic iron oxide; Pharmacokinetics; Metabolism.
which presents differences in degree in relaxometry and biodistribution as compared to previously described onesIS and a considerable safety margin.
INTRODUCTION
The use of ferromagnetic or superparamagnetic iron oxide particles to enhance the contrast of liver and spleen MR images is now well established. Since the last two years the field of investigation of these new contrast agents has been extended to gastrointestina1’s2 and cardiovascular imaging.3*4 Although all these materials are well known to be very efficient contrast agents, particle preparations differ greatly upon their physicochemical and biological properties. These characteristics are related mainly to the dimension and charge parameters of the particles and to the structural properties of the coating polymer. To avoid aggregation of the magnetic particles, proteins,5T6 lipids,’ and either synthetic’-” or natural polymers have been successively used.‘2-‘4 Whatever the coating material used, most publications described rapid liver sequestration of the particles, when intravenously injected into animals. We report herein the pharmacokinetic and metabolic study of a new superparamagnetic iron oxide nanoparticle preparation developed in our laboratory
Superparamagnetic iron oxide nanoparticles (MD) were prepared by modification of Molday’s method16: 80 ml of 50% (w/w) Dextran T-40 (Pharmacia, Uppsala, Sweden) were mixed with 40 ml of an aqueous solution containing 1.8 g of anhydrous FeC13 and 1.2 g FeC12.4H20. While stirring and heating to 65°C in a water bath, the mixture was titrated to pH lo-11 by the dropwise addition of NaOH N. The superparamagnetic nanoparticles were separated from unbound dextran by gel filtration (Sephacryl S 300, Pharmacia, Uppsala, Sweden) chromatography. The nanoparticles collected were finally dialyzed overnight using distilled water at 4°C and then filtered. The preparation is a very stable aqueous colloid. Nanoparticle range of size distribution was analyzed with an N4 nanosizer (Coultronics, France) and iron
RECEIVED6/15/90; ACCEPTED10/22/90. Acknowledgments-The authors thank Mrs. M. Moreau, G. Tanguy, and P. Legras for their excellent technical assistance and Mr. R. Filmon for electron microscopy.
This work was supported by research grant 87-9008 from the Institut National de la SantC et de la Recherche Medicale (INSERM) and the Association pour la Recherche contre le Cancer (ARC).
MATERIAL AND METHODS Iron Oxide Nanoparticles
215
276
Magnetic Resonance Imaging 0 Volume 9, Number 3, 1991
core dimension distribution was examined by transmission electron microscopy (TEM JEOL IOOB). Zeta potential was determined from measurements of particle electrophoretic mobility in suspension with a DELSA 440 Coulter (Coultronics, France). Dextran and iron concentrations were measured by a spectrophotometric method (phenol/sulfuric acid”) and a plasma torch absorption spectrophotometer, respectively. Rl and R2 relaxivities were determined on a,PC 20 spectrometer (Bruker, Rheinstetten, Germany) at 0.47 T, 20 MHz, and 4°C. Animals
Wistar rats were anesthetized via intramuscular administration of 100 mg/kg Ketamine (Vetoquinol S.A, Lure, France) and 35 mg/kg Thiopental (Specia, Paris, France). Twenty-one Wistar male rats (mean weight: 250 g) were used for radiotracer studies; Other earlier and later pharmacokinetics data were obtained on Swiss female mice (mean weight: 25-30 g). Experimental cirrhosis in rats was produced by a combined carbon tetrachloride-phenobarbitone regimen, using the Courtoy procedure. ‘s Hepatocarcinoma nodules were chemically induced in rats by adding of diethylnitrosoamine (DENA) (Sigma Chem. Corp., St. Louis, USA) into drinking water during three months. l9 Relaxation Time Measurements MR relaxation times of liver, spleen and plasma were determined to measure the effect of MD nanoparticles on each tissue. The particles were injected at a dose of 1.5 mg Fe/kg to normal Swiss mice. Animals were sacrificed by exsanguination 20 min, 1, 2, 18, 72 and 144 hr (6 days) after injection and organs were removed immediately, T2 values were measured at 4°C and 20 MHz within 1 hr after sacrifice using an NMR spectrometer (Bruker PC 20). T2 was obtained from 10 data points generated with a Carr-PurcellMeiboom-Gill pulse sequence (7 = 2 to 4 msec for liver tissue). l/T2 values were plotted as a function of time after injection. 5gFe Radiotracer Studies “Fe oxide nanoparticles were synthesized by incorporating 59FeC13during the synthesis. All the 59Fe was associated with the iron oxide particles, as verified by radioactivity measurements after 48 hr dialysis against distilled water. Organ biodistribution was determined by injection of 59Fe oxide nanoparticles into Swiss mice and Wistar rats at a dose of 7.5 mg and 2 mg Fe/kg, respectively (0.75 and 0.2 &i/kg). For the early pharmacokinetic study the mice were sacrificed 5, 10, 20, 30, 40, 50 and 60 min after injection (five animals at each time
point). The radioactivity of all organs was measured together with a 59Fe standard in a gamma counter (Intertechnique CC 4000-Kontron, France). The percentage of the injected dose in blood was determined by measuring the radioactivity of the blood collected from sacrificed animals. In the later pharmacokinetic evaluation the fraction of the dose injected in blood, liver, spleen and carcass was measured 1, 2, 4, 6, and 8 wk after injection of 59Fe nanoparticles (7.5 mg Fe/kg) into normal Swiss mice. Incorporation of 59Fe into red blood cells was investigated after separation from plasma. Complementary pharmacokinetic data were obtained in Wistar rats at 20 min, 2, 6, 24, 48 hr and 5 days postinjection. Metabolism of MD Nanoparticles The distribution and metabolism of MD nanopartitles in liver and spleen were examined, following administration of 100 to 1000 mg iron/kg (10’6-10’9 particles) to normal male Swiss mice. Animals were sacrificed 10 min, 2 hr, and 1, 5, 28 days after injection. Tissue intended for electron microscopy was prefixed in vivo by IV injection of a PBS buffered 4% glutaraldehyde solution adjusted to pH 7.4, and fixed ex vivo by immersion in a cacodylate buffered 5% glutaraldehyde solution, during 1 hr at 4°C. Liver and spleen samples were washed in PBS and then postfixed during 18 hr at 4°C in cold 0.2 M cacodylate buffered 1% osmium tetroxide before being dehydrated first in ethanol, then in propylene oxide and finally embedded in epon. Liver and spleen histological specimens were stained with many1 acetate and lead citrate and examined on a TEM JEOL 100 B. Biodistribution of sgFe MD Nanoparticles in Liver Diseases Biodistribution of 59Fe MD nanoparticles in diseased liver (cirrhosis, hepatocarcinoma) was determined 20 min after injection of 2 mg Fe/kg (0.75 $X/kg = 28 kBq/kg) to Wistar rats. We have defined an index i, for cirrhosis, hepatocarcinoma and normal liver tissue: % of the injected dose per gram of liver ’ = 9’0 of the injected dose per gram of blood *
RESULTS Physicochemical Properties and Morphometry of MD Nanoparticles Iron oxide (MD) nanoparticles’3 consist of an iron oxide core and a Dextran coating. The stability of the product in aqueous solution is related to the low range
Iron oxide nanoparticles for use as an MRI contrast agent 0 D.
Table 1. Main physicochemical properties of iron oxide nanoparticles
% iron (w/w) 30
Mean diameter (nm)
R;
48.9
368.4
R;
R,/R2
Zeta potential (mv)
8.4
0
(S-l mM_‘) 43.6
*Measurements in NaCl 9%1~at 4°C.
of nanoparticle size distribution: lo-70 nm (Table 1 and Fig. 1) and to the uniformity of the Dextran coating which leads to a neutral material (zeta potential = 0 mV) (Table 1). The range of size of the iron oxide core, analyzed by TEM, is about 5 to 20 nm (Fig. 2). T2 Measurements Spin-spin relaxation time measurements showed an early increase (20 min) in l/T’ of plasma, spleen, and liver (Fig. 3). Thereafter l/T, decreased consistently. No persistent effect is observed in the plasma after 2 hr. At 72 and 144 hr no difference in l/T, values are observed compared to tissues without injection of contrast agent: the half-time of the tissue l/7” effect (2 hr for liver) is shorter than the organ clearance given by 59Fe phamacokinetics data (6-8 wk for liver).
100
Size
Mean 48.9 SD 1.2 cv 26%
NM
Amount
Sue
NM
AmOUnt
10.0
0%
147
0%
14.7 21.5 31 .6
0%
215 316
0% 0%
11%
46.4 66.1 100
65% 23% .5%
464 681 1000
0% 0% 0%
1%
Fig. 1. Laser light scattering determination tribution of (MD) iron oxide nanoparticles.
of the size dis-
ET AL.
277
Pharmacokinetics The results show that in the first 30 min postinjection, about 47% of the product is still in the blood: uptake of 59Fe oxide (MD) nanoparticles is very rapid during the first 10 min and slows down during the following 20 min. Although uptake begins to rise again after 30 min, about 20% of the product remains in the blood after 1 hr (Fig. 4). Uptake by Tissue Measurement of radioactivity in the liver shows an early uptake of 20% in the first 5 min, followed by a 15min period with no significant change. Uptake then increases again between 20 min and 50-60 min (Fig. 4), and then remains stable until day 5 (Fig. 5). The radioactivity of liver then begins to decrease 2 wk after injection (Fig. 6). In the other organs (kidneys, heart, lung, spleen, intestine) a relatively small fraction of the injected doses was found. Metabolism s9Fe oxide (MD) nanoparticle metabolism was determined by measuring the incorporation of “Fe into RBC hemoglobin after separation of the plasma. The localization of iron oxide (MD) nanoparticles in tissue in the first 5 days was analyzed by light and transmission electron microscopy, after massive IV injection of nanoparticles (100 mg to 1 g/kg). l
Partide Diameter (NM)
POULIQUEN
between day 5 (Fig. 5) and week 6 (Fig. 6) radioactivity in the RBC regularly increased. In the last two weeks of the investigation, the percentage of the injected dose in the RBC rose more slowly. These results correlate well with the decrease in radioactivity in the RES tissue: spleen, liver and carcass (Fig. 6). iron oxide (MD) nanoparticles were localized in the Kupffer cells of the liver and in macrophage cells in the splenic red pulp by both light and transmission electron microscopy (TEM). the light photomicrograph of liver tissue demonstrates an important uptake in the Kupffer cells: the nanoparticles were directly visualized in a large number of lysosomes (Fig. 7), by their brown color (arrows) the TE micrographs of Kupffer cells (Fig. 8) show the internal structure of the lysosomes which are filled with iron oxide (MD) nanoparticles. Magnification of the lysosome shows two different aspects of particles (Fig. 9) in the central part of the lysosome (arrows), we can observe the same ultrastructural aspect of iron oxide nanoparticles (MD) as in solution (Fig. 2) round to the center, the particles are regular in shape and smaller than in Fig. 2. The aspect and di-
278
Magnetic Resonance Imaging 0 Volume 9, Number 3, 1991
Fig. 2. TEM photograph
of MD nanoparticles
iron oxide cores (x270.000,
mension of such particles look like ferritin cores. So, the discrepancy observed between 59Fe radiotracer and T2 relaxation times pharmacokinetic data can be explained by the metabolism of the
the scale bar represents
10 nm).
product in liver and spleen: the iron oxide crystals from the particles being probably transformed into an oxyhydroxide form of iron, which is considerably less efficient on relaxation times than magnetite or maghemite. We may also mention that the return to normal Tz values of the tissues occurs here more rapidly for MD nanoparticles, as compared to other preparations.”
lfrZ(S-1) ar
Biodistribution of 59Fe MD Nanoparticles in Liver Diseases The value of the index i, defined above, decreases
--.
by about 30%-35% for cirrhosis (15 i Z@Io)or HC (17 f 3070/o), as compared with normal liver tissue (26 + 4%) (Fig. 10). These results are related to the important modifications in RES functions which occur in these liver diseases.
Spleen
10
,
0
0
2
18
72
' 144
Plasma
DISCUSSION I
TIME (hrs) Fig. 3. Changes in T2 relaxation rate for plasma liver and spleen after injection of 1.5 mg Fe/kg of MD nanoparticles into mice.
The MR contrast agents currently in use consist of low molecular weight chelates: DTPA,” DOTA,” which bind the paramagnetic Gd3+ ion. Although these products have shown high stability in solution and good physicochemical properties, their use as MR
Iron oxide nanoparticles for use as an MRI contrast agent 0 D. POULIQUEN
c 20
40
50
60
TtMEAFERINEClTON(MIN)
Fig. 4. Early pharmacokinetic data in organs of the RES system after injection of 7.5 mg Fe/kg of 59Fe MD nanoparticles into mice.
contrast agents is limited by their lack of specificity and their diffusion in the extravascular space. A new approach consists of preparing superparamagnetic iron oxide nanoparticles as a magnetically active labe1.22 These new contrast agents are of great interest,
ET AL.
219
because of their high efficacy and their mainly cellular localization. All iron oxide nanoparticle preparations in current use are taken up massively by the liver within 5 min of IV injection. As for other foreign particles introduced intravenously,‘3 about 90% are extracted by liver, 5% by the spleen and a few percent enter the bone marrow.” This phenomenon is directly related to the function of the hepatic part of the mononuclear phagocyte system (MPS), but the MPS not only includes the Kupffer stellate cells lining the wall of the hepatic sinusoids, but also the phagocytically active reticulum cells of the reticular connective tissue, the Hortegal cells (microglia) of the central nervous system, the histiocytes (i.e., tissue macrophages) and the blood macrophages or monocytes. The dominance of the liver in extracting foreign particles from the blood does not therefore mean that the macrophage cells are concentrated within the liver, but that they are simply more easily accessible to the blood that passes through the liver. We conducted a study on the pharmacokinetic and metabolic behavior of biodegradable iron oxide nanoparticles prepared by a new technique. Our preparation (MD) is not exclusively sequestered by blood-sinuslining macrophages: half of the injected dose remains in the blood and only 25% is taken up by liver in the first 30 min following an IV injection. These results differ greatly from previous published data with other iron oxide preparations, showing 82.6% uptake by
%
Fig. 5. Pharmacokinetics of 5gFe MD nanoparticles after injection of 2 mg Fe/kg into rats.
Magnetic Resonance Imaging 0 Volume 9, Number 3, 1991
280
Y ..#--.._
_____~_________-.-----~ ‘~-o________________.~......-----
1
.
1
4
6
8
TIME AFIER INJEClTON WEEKS)
Fig. 6. Later pharmacokinetic nanoparticles into mice.
liver 1 hr after injectionI Although the mean size higher than MD a clear iron oxide core dimension
data (incorporation
of 59Fe into red blood cells) after injection
and 6 min blood half-life.24 of AM1 25 is only 2 times discrepancy exists between (about 5 nm) determined by
Fig. 7. Light microscopy photograph
of 7.5 mg Fe/kg of MD
TEM and the total mean size of the particles (80-120 nm) measured by laser light scattering, suggesting that aggregation of the crystals occurs in solution.25 Some authors23*26 have reviewed the different factors re-
of Kuppfer cells filled with MD nanoparticles
(arrows).
Iron oxide nanoparticles for use as an MRI contrast agent 0 D.
Fig. 8. TEM photograph 140 nm).
of Kuppfer
of Kuppfer Fig. 9. TEM photograph core :s) (x 50.000-scale bar represents
cell (arrows
= lysosomes
cell Iysosomes: 55 nm).
POULIQUEN ET AL.
filled with MD nanoparticles)
magnification
(arrows
= nonmetabolized
(x 20.000-scale
281
bar repre :sents
MD nanoparticles
iron
Magnetic Resonance Imaging 0 Volume 9, Number 3, 1991
282 INDEX
0.0 NORMAL LIVER
CIRRHOSIS
HEPATOCARCINOMA
Fig. 10. Values of the index i for normal and diseased liver rat tissue.
lated to the clearance of the particulate systems from blood: size, dose, surface charge, nature of the particle matrix, particle stability and physiological state of the animal species. Within them the relationship between size parameters and liver sequestration have already been described: the higher the particle size, the higher the liver uptake. Consequently, the biodistribution of a monodispersed particle preparation (MD) is expected to be very different as compared to a polydispersed one (AMI 2.9, with a liver sequestration much lower for the former as compared to the latter. The small size of the particles (MD) means they can reach sites such as the spaces of Disse,27 the fenestrae of the liver sinusoidal wall and of the endothelial capillaries” which are inaccessible to the other particulate systems (microspheres and microcapsules). Particles must be less than 100 nm in diameter in the first case and less than 30 nm in the second one. However, the mean diameter of 50 nm of the MD particles is not the only explanation for their low concentration level in the liver. A number of authors have already described in detail the interactions between particles and biological macromolecules.29 The sign and importance of the surface charge of a particulate system is directly related to its chemical components as well as to the technique used for preparation. The surface charge is neutralized in part by the free ions in the biological environment. On injection, the phenomena of deposit and adhesion observed in these particles are linked to the surface adsorption of a number of biological macromolecules. Since the nanoparticles were coated in Dextran, which is a neutral material, they were not affected by interaction energy which would cause them to adhere to biological surfaces. Dextran-coated nanoparticle surfaces may, however, adsorb macromolecules, provoking minor interactions such as hydrogen bonds and Van der Waals links.
A phenomenon of reversible blockage of the Kupffer cell-phagocyte function after IV injection of Dextran sulfate has already been described.30 In vivo, certain plasma proteins aggregate and are then filtered by the liver. A number of authors have shown that the reaction lasted 4 days in the mouse3* and that the Kupffer cell function returned to normal through bone marrow phagocyte cell lines.32 Although the Dextran we used does not have sulfate functional groups, the choice of different physicochemical conditions for the preparation of the particles may lead to a biochemical activation of plasma proteins and then to a reversible blockage of liver phagocyte function.26*30 Further biochemical studies are needed to analyze in detail the mechanisms that cause iron oxide nanoparticles (MD) to remain in the blood. The structureactivity relationship of this new type of contrast agent may also contribute to a fuller understanding of the distribution of nanoparticle systems. Also, the detailed metabolic process involved for such particles must be clarified, especially the relations between lysosome behavior and ferritin core formation. REFERENCES 1. Hahn, P.F.; Stark, D.D.; Saini, S.; Lewis, J.M.; Wittenberg, J.; Ferrucci, J.T. Ferrite particles for bowel contrast in MR imaging: Design issues and feasibility studies. Radiology 164:37-41; 1987. 2. Lonnemark, M.; Hemmigsson, A.; Carlsten, J. Superparamagnetic particles as a MRI contrast agent for the gastrointestinal tract. Acta Radiol. 29~599-602; 1988. 3. Majumdar, S.; Zoghbi, S.S.; Gore, J.C. Regional differences in rat brain displayed by fast MRI with superparamagnetic contrast agents. Magn. Reson. Imaging 6:611-615;
1988.
4. Rozenman, Y.; Zou, X. Cardiovascular MRI with ferrite particles: The utility of a superparamagnetic contrast agent. Abstract SMRM Amsterdam. 1:353; 1989. 5. Renshaw, P.; Owen, C.S.; Mac Laughlin, A.; Frey, T.G.; Leigh, J.S.J. Ferromagnetic contrast agents: A new approach. Magn. Reson. Med. 3:217-225; 1986. 6. Widder, D. J.; Edelman, R.R.; Grief, W.L.; Monda, L. Magnetite albumin suspension: A superparamagnetic oral MR contrast agent. AJR 149:839-843; 1987. 7. Morimoto, Y.; Sugibayashi, K.; Akimoto, M. Magnetic guidance of ferro-colloid-entrapped emulsion for sitespecific drug delivery. Chem. Pharm. Bull. 31(1):279285; 1983.
8. Kronick, P.L.; Campbell, G.L.; Joseph, K. Magnetic microspheres prepared by redox polymerization used in a cell separation based on gangliosides. Science 200: 1074-1076; 1978. 9. Ibrahim, A.; Couvreur, P.; Roland, M.; Speiser, P. New magnetic drug carrier. J. Pharm. Pharmacol. 3559-61; 1983.
Iron oxide nanoparticles for use as an MRI contrast agent 0 D. POCJLIQUEN ET AL. 10. Patton, W.F.; Kim, J.; Jacobson, B.S. Rapid, high-yield purification of cell surface membrane using colloidal magnetite coated with polyvinylamine: Sedimentation versus magnetic isolation. Biochim. Biophys. Acta. 816: 83-92; 1985. Il. Kemshead, J.T.; Treleaven, J.G.; Gibson, EM.; Ugelstad, J.; Rembaum, A.; Philip, T. Removal of malignant cells from bone marrow using magnetic microspheres and monoclonal antibodies. Prog. Exp. Tumor. Res. 29:249-255; 1985. 12. Saini, S.; Stark, D.D.; Hahn, P.F.; Wittenberg, J.; Brady, J.T.; Ferrucci, J.T. Ferrite particles: A superparamagnetic MR contrast agent for the reticuloendothelial system. Radiology 162:211-216; 1987. 13. Pouliquen, D.; Perdrisot, R.; Ermias, R.; Akoka, S.; JaIlet, P.; Le Jeune, J.J. Superparamagnetic iron oxide nanoparticles as a liver MRI contrast agent: Contribution of microencapsulation to improved biodistribution. Magn. Reson. Imaging 7(6):619-627; 1989. 14. Fahlvik, A.K.; Holtz, E.; Leander, P.; Schroder, U.; Klaveness, J. Magnetic starch microspheres, efficacy and elimination, a new organ-specific contrast agent for magnetic resonance imaging. Invest. RadioI. 25(2): 113120; 1990. 15. WeissIeder, R.; Stark, D.D.; Engelstad, B.L.; Bacon, B.R.; Compton, C.C.; White, D.L.; Jacobs, P.; Lewis, J. Superparamagnetic iron oxide: Pharmacokinetics and toxicity. AJR 152:167-173; 1989. 16. Molday, R.S.; MacKenzie, D. Immunospecific ferromagnetic iron-Dextran reagents for the labeling and magnetic separation of cells. J. Zmmunol. Methods 52: 353-367; 1982. 17. Dubois, M.; Gilles, K.A.; Hamilton, J.K.; Rebers, P.A.; Smith, F. Calorimetric method for determination of sugars and related substances. Anal. Chem. 28(3): 350; 1956. 18. Courtoy, P.J.; Feldman, G.; Rogier, E.; Moguilevsky, N. Plasma protein synthesis in experimental cirrhosis; morphologic demonstration and function correlations. Lab. Invest. 45(1):67; 1981. 19. Lacassagne, A.; Buu-Hoi, N.P.; Giao, N.B.; Hurot, L.; Fernando, R. Comparaison des actions hepatocancCrogenes de la diCthyInitrosamine et du p-dim&hylaminoazobenzkne. Znt. J. Cancer 21425; 1967. 20. Weinmann, H.J. Chelated contrast agents. In: Contrast agents in magnetic resonance imaging. Proceedings of
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an international workshop. San Diego, Excerpta Medica, 19-23; 1986. 21. Desreux, J.F.; BarthClCmy, P.P. Highly stable lanthanide macrocyclic complexes: In search of new contrast agents for NMR imaging. Nucl. Med. Biol. 15(1):9; 1988. 22. Weissleder, R.; Hahn, P.; Stark, D.D.; Rummeny, E.; Saini, S.; Wittenberg, J.; Ferrucci, J.T. MR imaging of splenic metastases: Ferrite-enhanced detection in rats.
AJR 149:723-726; 1987. 23. Tomlinson, E. Theory and practice of site-specific drug delivery: X-Intravascular targets. Adv. Drug Deliv. Rev. 1:131; 1987. 24. Weissleder, R.; Elizondo, G.; Wittenberg, J.; Rabito, C.A.; Bengele, H.H.; Josephson, L. Ultrasmall superparamagnetic iron oxide: Characterization of a new class of contrast agents for MR imaging. Radiology 175:
489-493; 1990. 25. Josephson, L.; Lewis, J.; Jacobs, P.; Hahn, P.F.; Stark, D.D. The effects
of iron oxides on proton
relaxivity.
Magn. Reson. Imaging 6:647-653; 1988. 26. Miiller, R.H. Colloidal
27.
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29.
30.
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32.
carriers for controlled drug delivery: Modification, characterization and in vivo distribution. Ph.D. Thesis, University of Kiel, Germany; 1989. Wisse, E. An electron microscopic study of the fenestrated endothelial lining of rat liver sinusoids. J. Mr. Res. 31:125; 1970. Tomlisson, E. Theory and practice of site-specific drug delivery: XI - Extravasation. Adv. Drug Deliv. Rev. 1: 147; 1987. Norde, W. Physicochemical aspects of the behaviour of biological components at solid/liquid interfaces. In: S.S. Davis, L. Illum, J.G. McVie and E. Tomlinson (Eds.), Microspheres and Drug Therapy. Pharmaceutical, Immunological and Medical Aspects. New York: Elsevier Science Publishers B.V., 1984:~~. 39-59. Bradfield, J.W.B. A new look at reticuloendothelial blockade. Br. J. Exp. Path. 61:617; 1980. Souhami, R.L.; Bradfield, J.W.B.; Parker, N.E. The effect of cytotoxic drugs on the formation of mononuclear phagocytes. In: Perspectives in Inflammation. Lancaster: MTP; 1977:~. 29. Souhami, R.L.; Bradfield, J.W.B. The recovery of hepatic phagocytosis after blockade of Kiipffer cells. J.
Reticuloendoth.
Sot. 16:75: 1914.
l
Imagmg. All rights
Vol. 9. pp. 215-283, reserved.
1591 Copyright
0730-725X/91 $3.04 + .oO 0 1991 Pergamon Press plc
Original Contribution IRON OXIDE NANOPARTICLES FOR USE AS AN MRI CONTRAST PHARMACOKINETICS AND METABOLISM
AGENT:
D. POULIQUEN, J.J. LE JEUNE, R. PERDRISOT, A. ERMIAS, AND P. JALLET Laboratoire de Biophysique, Faculte de Medecine, 1 Rue Haute de ReculCe,49000 Angers, France The pharmacokinetics and metabolism of a new preparation of superparamagnetic iron oxide nanoparticles were evaluated by 59Fe radiotracer studies and histologic examination of mice liver and spleen tissues (light and transmission electron microscopy). In the first 30 min following IV injection of the product half of the dose injected remains in the blood, the other part being sequestered mainly by the mononuclear phagocyte system (MPS). In the first five days following IV administration of the nanoparticles, early metabolization of the iron oxide cores occurs, revealed by modification of their aspect in the lysosomes of Kupffer cells and macrophages of the splenic red pulp. The incorporation of s9Fe is then observed in RBC of the mice. These results are discussed in relation with the physicochemical properties of this new preparation of nanoparticles, and compared with current pharmacokinetic data concerning injectable particle systems. Keywords: Contrast agent; Superparamagnetic iron oxide; Pharmacokinetics; Metabolism.
which presents differences in degree in relaxometry and biodistribution as compared to previously described onesIS and a considerable safety margin.
INTRODUCTION
The use of ferromagnetic or superparamagnetic iron oxide particles to enhance the contrast of liver and spleen MR images is now well established. Since the last two years the field of investigation of these new contrast agents has been extended to gastrointestina1’s2 and cardiovascular imaging.3*4 Although all these materials are well known to be very efficient contrast agents, particle preparations differ greatly upon their physicochemical and biological properties. These characteristics are related mainly to the dimension and charge parameters of the particles and to the structural properties of the coating polymer. To avoid aggregation of the magnetic particles, proteins,5T6 lipids,’ and either synthetic’-” or natural polymers have been successively used.‘2-‘4 Whatever the coating material used, most publications described rapid liver sequestration of the particles, when intravenously injected into animals. We report herein the pharmacokinetic and metabolic study of a new superparamagnetic iron oxide nanoparticle preparation developed in our laboratory
Superparamagnetic iron oxide nanoparticles (MD) were prepared by modification of Molday’s method16: 80 ml of 50% (w/w) Dextran T-40 (Pharmacia, Uppsala, Sweden) were mixed with 40 ml of an aqueous solution containing 1.8 g of anhydrous FeC13 and 1.2 g FeC12.4H20. While stirring and heating to 65°C in a water bath, the mixture was titrated to pH lo-11 by the dropwise addition of NaOH N. The superparamagnetic nanoparticles were separated from unbound dextran by gel filtration (Sephacryl S 300, Pharmacia, Uppsala, Sweden) chromatography. The nanoparticles collected were finally dialyzed overnight using distilled water at 4°C and then filtered. The preparation is a very stable aqueous colloid. Nanoparticle range of size distribution was analyzed with an N4 nanosizer (Coultronics, France) and iron
RECEIVED6/15/90; ACCEPTED10/22/90. Acknowledgments-The authors thank Mrs. M. Moreau, G. Tanguy, and P. Legras for their excellent technical assistance and Mr. R. Filmon for electron microscopy.
This work was supported by research grant 87-9008 from the Institut National de la SantC et de la Recherche Medicale (INSERM) and the Association pour la Recherche contre le Cancer (ARC).
MATERIAL AND METHODS Iron Oxide Nanoparticles
215
276
Magnetic Resonance Imaging 0 Volume 9, Number 3, 1991
core dimension distribution was examined by transmission electron microscopy (TEM JEOL IOOB). Zeta potential was determined from measurements of particle electrophoretic mobility in suspension with a DELSA 440 Coulter (Coultronics, France). Dextran and iron concentrations were measured by a spectrophotometric method (phenol/sulfuric acid”) and a plasma torch absorption spectrophotometer, respectively. Rl and R2 relaxivities were determined on a,PC 20 spectrometer (Bruker, Rheinstetten, Germany) at 0.47 T, 20 MHz, and 4°C. Animals
Wistar rats were anesthetized via intramuscular administration of 100 mg/kg Ketamine (Vetoquinol S.A, Lure, France) and 35 mg/kg Thiopental (Specia, Paris, France). Twenty-one Wistar male rats (mean weight: 250 g) were used for radiotracer studies; Other earlier and later pharmacokinetics data were obtained on Swiss female mice (mean weight: 25-30 g). Experimental cirrhosis in rats was produced by a combined carbon tetrachloride-phenobarbitone regimen, using the Courtoy procedure. ‘s Hepatocarcinoma nodules were chemically induced in rats by adding of diethylnitrosoamine (DENA) (Sigma Chem. Corp., St. Louis, USA) into drinking water during three months. l9 Relaxation Time Measurements MR relaxation times of liver, spleen and plasma were determined to measure the effect of MD nanoparticles on each tissue. The particles were injected at a dose of 1.5 mg Fe/kg to normal Swiss mice. Animals were sacrificed by exsanguination 20 min, 1, 2, 18, 72 and 144 hr (6 days) after injection and organs were removed immediately, T2 values were measured at 4°C and 20 MHz within 1 hr after sacrifice using an NMR spectrometer (Bruker PC 20). T2 was obtained from 10 data points generated with a Carr-PurcellMeiboom-Gill pulse sequence (7 = 2 to 4 msec for liver tissue). l/T2 values were plotted as a function of time after injection. 5gFe Radiotracer Studies “Fe oxide nanoparticles were synthesized by incorporating 59FeC13during the synthesis. All the 59Fe was associated with the iron oxide particles, as verified by radioactivity measurements after 48 hr dialysis against distilled water. Organ biodistribution was determined by injection of 59Fe oxide nanoparticles into Swiss mice and Wistar rats at a dose of 7.5 mg and 2 mg Fe/kg, respectively (0.75 and 0.2 &i/kg). For the early pharmacokinetic study the mice were sacrificed 5, 10, 20, 30, 40, 50 and 60 min after injection (five animals at each time
point). The radioactivity of all organs was measured together with a 59Fe standard in a gamma counter (Intertechnique CC 4000-Kontron, France). The percentage of the injected dose in blood was determined by measuring the radioactivity of the blood collected from sacrificed animals. In the later pharmacokinetic evaluation the fraction of the dose injected in blood, liver, spleen and carcass was measured 1, 2, 4, 6, and 8 wk after injection of 59Fe nanoparticles (7.5 mg Fe/kg) into normal Swiss mice. Incorporation of 59Fe into red blood cells was investigated after separation from plasma. Complementary pharmacokinetic data were obtained in Wistar rats at 20 min, 2, 6, 24, 48 hr and 5 days postinjection. Metabolism of MD Nanoparticles The distribution and metabolism of MD nanopartitles in liver and spleen were examined, following administration of 100 to 1000 mg iron/kg (10’6-10’9 particles) to normal male Swiss mice. Animals were sacrificed 10 min, 2 hr, and 1, 5, 28 days after injection. Tissue intended for electron microscopy was prefixed in vivo by IV injection of a PBS buffered 4% glutaraldehyde solution adjusted to pH 7.4, and fixed ex vivo by immersion in a cacodylate buffered 5% glutaraldehyde solution, during 1 hr at 4°C. Liver and spleen samples were washed in PBS and then postfixed during 18 hr at 4°C in cold 0.2 M cacodylate buffered 1% osmium tetroxide before being dehydrated first in ethanol, then in propylene oxide and finally embedded in epon. Liver and spleen histological specimens were stained with many1 acetate and lead citrate and examined on a TEM JEOL 100 B. Biodistribution of sgFe MD Nanoparticles in Liver Diseases Biodistribution of 59Fe MD nanoparticles in diseased liver (cirrhosis, hepatocarcinoma) was determined 20 min after injection of 2 mg Fe/kg (0.75 $X/kg = 28 kBq/kg) to Wistar rats. We have defined an index i, for cirrhosis, hepatocarcinoma and normal liver tissue: % of the injected dose per gram of liver ’ = 9’0 of the injected dose per gram of blood *
RESULTS Physicochemical Properties and Morphometry of MD Nanoparticles Iron oxide (MD) nanoparticles’3 consist of an iron oxide core and a Dextran coating. The stability of the product in aqueous solution is related to the low range
Iron oxide nanoparticles for use as an MRI contrast agent 0 D.
Table 1. Main physicochemical properties of iron oxide nanoparticles
% iron (w/w) 30
Mean diameter (nm)
R;
48.9
368.4
R;
R,/R2
Zeta potential (mv)
8.4
0
(S-l mM_‘) 43.6
*Measurements in NaCl 9%1~at 4°C.
of nanoparticle size distribution: lo-70 nm (Table 1 and Fig. 1) and to the uniformity of the Dextran coating which leads to a neutral material (zeta potential = 0 mV) (Table 1). The range of size of the iron oxide core, analyzed by TEM, is about 5 to 20 nm (Fig. 2). T2 Measurements Spin-spin relaxation time measurements showed an early increase (20 min) in l/T’ of plasma, spleen, and liver (Fig. 3). Thereafter l/T, decreased consistently. No persistent effect is observed in the plasma after 2 hr. At 72 and 144 hr no difference in l/T, values are observed compared to tissues without injection of contrast agent: the half-time of the tissue l/7” effect (2 hr for liver) is shorter than the organ clearance given by 59Fe phamacokinetics data (6-8 wk for liver).
100
Size
Mean 48.9 SD 1.2 cv 26%
NM
Amount
Sue
NM
AmOUnt
10.0
0%
147
0%
14.7 21.5 31 .6
0%
215 316
0% 0%
11%
46.4 66.1 100
65% 23% .5%
464 681 1000
0% 0% 0%
1%
Fig. 1. Laser light scattering determination tribution of (MD) iron oxide nanoparticles.
of the size dis-
ET AL.
277
Pharmacokinetics The results show that in the first 30 min postinjection, about 47% of the product is still in the blood: uptake of 59Fe oxide (MD) nanoparticles is very rapid during the first 10 min and slows down during the following 20 min. Although uptake begins to rise again after 30 min, about 20% of the product remains in the blood after 1 hr (Fig. 4). Uptake by Tissue Measurement of radioactivity in the liver shows an early uptake of 20% in the first 5 min, followed by a 15min period with no significant change. Uptake then increases again between 20 min and 50-60 min (Fig. 4), and then remains stable until day 5 (Fig. 5). The radioactivity of liver then begins to decrease 2 wk after injection (Fig. 6). In the other organs (kidneys, heart, lung, spleen, intestine) a relatively small fraction of the injected doses was found. Metabolism s9Fe oxide (MD) nanoparticle metabolism was determined by measuring the incorporation of “Fe into RBC hemoglobin after separation of the plasma. The localization of iron oxide (MD) nanoparticles in tissue in the first 5 days was analyzed by light and transmission electron microscopy, after massive IV injection of nanoparticles (100 mg to 1 g/kg). l
Partide Diameter (NM)
POULIQUEN
between day 5 (Fig. 5) and week 6 (Fig. 6) radioactivity in the RBC regularly increased. In the last two weeks of the investigation, the percentage of the injected dose in the RBC rose more slowly. These results correlate well with the decrease in radioactivity in the RES tissue: spleen, liver and carcass (Fig. 6). iron oxide (MD) nanoparticles were localized in the Kupffer cells of the liver and in macrophage cells in the splenic red pulp by both light and transmission electron microscopy (TEM). the light photomicrograph of liver tissue demonstrates an important uptake in the Kupffer cells: the nanoparticles were directly visualized in a large number of lysosomes (Fig. 7), by their brown color (arrows) the TE micrographs of Kupffer cells (Fig. 8) show the internal structure of the lysosomes which are filled with iron oxide (MD) nanoparticles. Magnification of the lysosome shows two different aspects of particles (Fig. 9) in the central part of the lysosome (arrows), we can observe the same ultrastructural aspect of iron oxide nanoparticles (MD) as in solution (Fig. 2) round to the center, the particles are regular in shape and smaller than in Fig. 2. The aspect and di-
278
Magnetic Resonance Imaging 0 Volume 9, Number 3, 1991
Fig. 2. TEM photograph
of MD nanoparticles
iron oxide cores (x270.000,
mension of such particles look like ferritin cores. So, the discrepancy observed between 59Fe radiotracer and T2 relaxation times pharmacokinetic data can be explained by the metabolism of the
the scale bar represents
10 nm).
product in liver and spleen: the iron oxide crystals from the particles being probably transformed into an oxyhydroxide form of iron, which is considerably less efficient on relaxation times than magnetite or maghemite. We may also mention that the return to normal Tz values of the tissues occurs here more rapidly for MD nanoparticles, as compared to other preparations.”
lfrZ(S-1) ar
Biodistribution of 59Fe MD Nanoparticles in Liver Diseases The value of the index i, defined above, decreases
--.
by about 30%-35% for cirrhosis (15 i Z@Io)or HC (17 f 3070/o), as compared with normal liver tissue (26 + 4%) (Fig. 10). These results are related to the important modifications in RES functions which occur in these liver diseases.
Spleen
10
,
0
0
2
18
72
' 144
Plasma
DISCUSSION I
TIME (hrs) Fig. 3. Changes in T2 relaxation rate for plasma liver and spleen after injection of 1.5 mg Fe/kg of MD nanoparticles into mice.
The MR contrast agents currently in use consist of low molecular weight chelates: DTPA,” DOTA,” which bind the paramagnetic Gd3+ ion. Although these products have shown high stability in solution and good physicochemical properties, their use as MR
Iron oxide nanoparticles for use as an MRI contrast agent 0 D. POULIQUEN
c 20
40
50
60
TtMEAFERINEClTON(MIN)
Fig. 4. Early pharmacokinetic data in organs of the RES system after injection of 7.5 mg Fe/kg of 59Fe MD nanoparticles into mice.
contrast agents is limited by their lack of specificity and their diffusion in the extravascular space. A new approach consists of preparing superparamagnetic iron oxide nanoparticles as a magnetically active labe1.22 These new contrast agents are of great interest,
ET AL.
219
because of their high efficacy and their mainly cellular localization. All iron oxide nanoparticle preparations in current use are taken up massively by the liver within 5 min of IV injection. As for other foreign particles introduced intravenously,‘3 about 90% are extracted by liver, 5% by the spleen and a few percent enter the bone marrow.” This phenomenon is directly related to the function of the hepatic part of the mononuclear phagocyte system (MPS), but the MPS not only includes the Kupffer stellate cells lining the wall of the hepatic sinusoids, but also the phagocytically active reticulum cells of the reticular connective tissue, the Hortegal cells (microglia) of the central nervous system, the histiocytes (i.e., tissue macrophages) and the blood macrophages or monocytes. The dominance of the liver in extracting foreign particles from the blood does not therefore mean that the macrophage cells are concentrated within the liver, but that they are simply more easily accessible to the blood that passes through the liver. We conducted a study on the pharmacokinetic and metabolic behavior of biodegradable iron oxide nanoparticles prepared by a new technique. Our preparation (MD) is not exclusively sequestered by blood-sinuslining macrophages: half of the injected dose remains in the blood and only 25% is taken up by liver in the first 30 min following an IV injection. These results differ greatly from previous published data with other iron oxide preparations, showing 82.6% uptake by
%
Fig. 5. Pharmacokinetics of 5gFe MD nanoparticles after injection of 2 mg Fe/kg into rats.
Magnetic Resonance Imaging 0 Volume 9, Number 3, 1991
280
Y ..#--.._
_____~_________-.-----~ ‘~-o________________.~......-----
1
.
1
4
6
8
TIME AFIER INJEClTON WEEKS)
Fig. 6. Later pharmacokinetic nanoparticles into mice.
liver 1 hr after injectionI Although the mean size higher than MD a clear iron oxide core dimension
data (incorporation
of 59Fe into red blood cells) after injection
and 6 min blood half-life.24 of AM1 25 is only 2 times discrepancy exists between (about 5 nm) determined by
Fig. 7. Light microscopy photograph
of 7.5 mg Fe/kg of MD
TEM and the total mean size of the particles (80-120 nm) measured by laser light scattering, suggesting that aggregation of the crystals occurs in solution.25 Some authors23*26 have reviewed the different factors re-
of Kuppfer cells filled with MD nanoparticles
(arrows).
Iron oxide nanoparticles for use as an MRI contrast agent 0 D.
Fig. 8. TEM photograph 140 nm).
of Kuppfer
of Kuppfer Fig. 9. TEM photograph core :s) (x 50.000-scale bar represents
cell (arrows
= lysosomes
cell Iysosomes: 55 nm).
POULIQUEN ET AL.
filled with MD nanoparticles)
magnification
(arrows
= nonmetabolized
(x 20.000-scale
281
bar repre :sents
MD nanoparticles
iron
Magnetic Resonance Imaging 0 Volume 9, Number 3, 1991
282 INDEX
0.0 NORMAL LIVER
CIRRHOSIS
HEPATOCARCINOMA
Fig. 10. Values of the index i for normal and diseased liver rat tissue.
lated to the clearance of the particulate systems from blood: size, dose, surface charge, nature of the particle matrix, particle stability and physiological state of the animal species. Within them the relationship between size parameters and liver sequestration have already been described: the higher the particle size, the higher the liver uptake. Consequently, the biodistribution of a monodispersed particle preparation (MD) is expected to be very different as compared to a polydispersed one (AMI 2.9, with a liver sequestration much lower for the former as compared to the latter. The small size of the particles (MD) means they can reach sites such as the spaces of Disse,27 the fenestrae of the liver sinusoidal wall and of the endothelial capillaries” which are inaccessible to the other particulate systems (microspheres and microcapsules). Particles must be less than 100 nm in diameter in the first case and less than 30 nm in the second one. However, the mean diameter of 50 nm of the MD particles is not the only explanation for their low concentration level in the liver. A number of authors have already described in detail the interactions between particles and biological macromolecules.29 The sign and importance of the surface charge of a particulate system is directly related to its chemical components as well as to the technique used for preparation. The surface charge is neutralized in part by the free ions in the biological environment. On injection, the phenomena of deposit and adhesion observed in these particles are linked to the surface adsorption of a number of biological macromolecules. Since the nanoparticles were coated in Dextran, which is a neutral material, they were not affected by interaction energy which would cause them to adhere to biological surfaces. Dextran-coated nanoparticle surfaces may, however, adsorb macromolecules, provoking minor interactions such as hydrogen bonds and Van der Waals links.
A phenomenon of reversible blockage of the Kupffer cell-phagocyte function after IV injection of Dextran sulfate has already been described.30 In vivo, certain plasma proteins aggregate and are then filtered by the liver. A number of authors have shown that the reaction lasted 4 days in the mouse3* and that the Kupffer cell function returned to normal through bone marrow phagocyte cell lines.32 Although the Dextran we used does not have sulfate functional groups, the choice of different physicochemical conditions for the preparation of the particles may lead to a biochemical activation of plasma proteins and then to a reversible blockage of liver phagocyte function.26*30 Further biochemical studies are needed to analyze in detail the mechanisms that cause iron oxide nanoparticles (MD) to remain in the blood. The structureactivity relationship of this new type of contrast agent may also contribute to a fuller understanding of the distribution of nanoparticle systems. Also, the detailed metabolic process involved for such particles must be clarified, especially the relations between lysosome behavior and ferritin core formation. REFERENCES 1. Hahn, P.F.; Stark, D.D.; Saini, S.; Lewis, J.M.; Wittenberg, J.; Ferrucci, J.T. Ferrite particles for bowel contrast in MR imaging: Design issues and feasibility studies. Radiology 164:37-41; 1987. 2. Lonnemark, M.; Hemmigsson, A.; Carlsten, J. Superparamagnetic particles as a MRI contrast agent for the gastrointestinal tract. Acta Radiol. 29~599-602; 1988. 3. Majumdar, S.; Zoghbi, S.S.; Gore, J.C. Regional differences in rat brain displayed by fast MRI with superparamagnetic contrast agents. Magn. Reson. Imaging 6:611-615;
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