Nanoparticles as microcarriers for anticancer drugs

Advanced Drug Delivery Reviews, 5 (1990) 209-230

209

Elsevier A D R 00069

Nanoparticles as microcarriers for anticancer drugs P. Couvreur a, L. Roblot-TreupeF, M.F. Poupon b, F. Brasseur c and F. Puisieux ~

aLaboratoire de Physico-Chimie des Surfaces et Innovation en Pharmacotechnie, CNRS URA 1218, Universit6 Paris-Sud, Chfitenay-Malabry, France, bLaboratoire de Biologie des M6tastases, CNRS UPR 6, Institut de Recherches Scientifiques sur le Cancer, Villejuif, France, and CLaboratoire de Canc6rologie Experimentale, Universit6 Catholique de Louvain, Bruxelles, Belgium (Received January 26, 1990) (Accepted May 9, 1990)

Key words: Dactinomycin; Doxorubicin; Drug targeting; Experimental cancer; Multidrug resistance; Nanoparticle; Tissue distribution; Toxicity

Contents Summary .................................................................................................................

210

I. Introduction ...................................................................................................

210

II. Binding of antineoplastic drugs to nanoparticles: physicochemical characterization ....... 1. Preparation and characterization of nanoparticles loaded with anticancer drugs ...... 2. Drug-release from nanoparticles ...................................................................

211 211 213

III. Nanoparticles loaded with anticancer drugs: cell culture experiments ......................... 1. Nanoparticles increase the concentration of dactinomycin in macrophages ............. 2. Doxorubicin-loaded nanoparticles bypass tumor cell resistance ...........................

214 214 216

IV. Tissue distribution of anticancer drugs associated with nanoparticles ..........................

218

V. Doxorubicin-loaded nanoparticles: toxicological data ..............................................

221

Abbreviations: AUC, area under the concentration-time curve; Dact, dactinomycin; DL, free doxorubicin; DN, doxorubicin associated with polyisobutylcyanoacrylate nanoparticle; Dox, doxorubicin; GPC, gel permeation chromatography; HT, healthy hepatic tissue; ILS, increased life span; i.v., intravenous; LTS, long-term survivor; NP-Dox, doxorubicin-loaded nanoparticle; PACA, polyalkylcyanoacrylate; PBCA, polybutylcyanoacrylate; PECA, polyethylcyanoacrylate; PHCA, polyhexylcyanoacrylate; PIBCA, polyisobutylcyanoacrylate; PIHCA, polyisohexylcyanoacrylate; PMCA, polymethylcyanoacrylate; SEM, scanning electron microscopy; T r , tumoral hepatic tissue. Correspondence: L. Roblot-Treupel, Laboratoire de Physico-Chimie des Surfaces et Innovation en Pharmacotechnie, CNRS URA 1218, Universit6 Paris-Sud, Chfitenay-Malabry, France.

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VI. Doxorubicin-loaded nanoparticles: increased efficiencyagainst experimental cancers .... 1. L-1210leukemia ........................................................................................ 2. M-5076hepatic metastases...........................................................................

223 223 225

VII. Conclusion.....................................................................................................

228

References...............................................................................................................

228

Summary This paper surveys the current status of anticancer drug delivery by means of polyalkylcyanoacrylate nanoparticles. In spite of the development of numerous antineoplastic drugs, no ideal agent has been found yet. One reason is the poor selectivity of these drugs for tumor cells, thus leading to toxicity towards proliferating normal cells. The potential usefulness of polyalkylcyanoacrylate nanoparticles as carriers for anticancer drugs has attracted considerable interest. These biodegradable ultrafine particles are able to carry anticancer compounds and to modify their distribution profile in vivo considerably. Both reduced toxicity and increased efficiency were reported in experimental animal models. Moreover, encouraging results were obtained concerning the possibility to overcome multidrug resistance with these nanoparticles.

I. Introduction For the treatment of cancer approaches which kill tumor cells while leaving nontarget tissues unaffected are highly desirable. Although success has been achieved in controlling several neoplastic diseases, failures result from inadequacies in obtaining therapeutically effective drug levels at the tumor site. Classically, an intravenously administered anticancer drug is distributed throughout the body as a function of the physicochemical properties of the molecule. A pharmacologically active concentration is reached in the tumor tissue at the expense of massive contamination of the rest of the body. For cytostatic compounds, this poor specificity raises a toxicological problem which presents a serious obstacle to an effective therapy. A number of efforts made to develop more rational approaches to specific cancer therapy are based on the concept of drug targeting. Phospholipid vesicles (liposomes) were proposed as microcarriers for application in tumor diagnosis [1,2] or treatment [3,4]. However, problems with the use of phospholipids include: poor stability during storage, exchange of phospholipids with certain blood components and, to a lesser extent, difficulties in obtaining a reproducible preparation on a large scale. Polyacrylamide [5] or polymethylmetacrylate [6] nanoparticles were then developed as an alternative to targeting with liposomes. Although more stable, these ultrafine polymeric systems were not observed to degrade in vivo. Thus, the risk of polymeric tissue or cellular overloading restricted their possible clinical use. With these considerations in mind, biodegradable nanoparticles made by poly-

NANOPARTICLESAS CARRIERSFOR ANTICANCERDRUGS

211

merization of various alkylcyanoacrylate monomers were proposed [7]. These polymers were chosen because of their biodegradability [8]. Their extensive use in surgery was, from a toxicological point of view, an advantage [9]. The objective of this review is to evaluate the current status of polyalkylcyanoacrylate (PACA) nanoparticles for the delivery of anticancer drugs. The possible increase of their therapeutic indices will be discussed in light of the alteration of the tissue distribution profile of these agents when attached to nanoparticles. Furthermore, the hope that multidrug resistance might be overcome by using drug-loaded nanoparticles will also be considered.

II. Binding of antineoplastic drugs to nanoparticles: physico-chemical characterization

H.1. Preparation and characterization of nanoparticles loaded with anticancer drugs PACA nanoparticles can be prepared by emulsion polymerization, in which droplets of water-insoluble monomers are emulsified in an aqueous phase [7]. Anionic polymerization (Fig. 1) takes place in micelles after diffusion of monomer molecules through the water phase and is initiated by negatively charged compounds. The polymerization of the alkylcyanoacrylate monomers occurs spontaneously at room temperature with stirring, leading to the formation of 180 nm (-+ 20 nm) colloidal particles. Anticancer drugs can be combined with nanoparticles after dissolution in the polymerization medium, either before the introduction of the monomer or after its polymerization [10]. The carrier capacity of nanoparticles varies greatly, depending upon whether they were loaded by an adsorption or an incorporation process. Table I shows the percentage of adsorption to PACA nanoparticles for different anticancer compounds. Maximal drug-loading capacity per weight of nanoparticles was obtained at a ratio 1:10; i.e., using doxorubicin as a typical example, 10 mg of the active drug was found entrapped inside 100 mg of nanoparticles. Concerning morphological characterization of PACA nanoparticles, scanning electron microscopy (SEM) shows generally spherical particles with a diameter of about 200 nm [11]. After freeze-fracturing, it was observed that these particles were solid; their inner structure was highly porous [12]. The nanoparticles do not appear as a hollow vesicle, but rather as spheres with a dense polymeric network. From a total of 250 measured distribution profiles, the radius distribution ranged from 25 to 340 nm. The specific surface area of polybutylcyanoacrylate (PBCA) nanoparticles (average size 51 nm) was calculated by Kreuter to be 106 m2/g [12]. Using gel permeation chromatography (GPC), it was shown that PACA nanoparticles are built by an entanglement of numerous small oligomeric subunits rather than by the rolling up of one or a few long polymer chains [13]. Chromatographic profiles suggested a uniform distribution of molecules with unexpectedly low molecular weights ranging between 500 and 1000. Furthermore, no monomeric residues were found. The low molecular weight of oligomeric subunits is consistent with observations concerning excretion and metabolism of nanoparticles (see below).

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ing of doxorubicin (Fig. 2). This could be due to the intervention of doxorubicin as initiator (by its free amine function) in the anionic polymerization of the cyanoacrylic monomer. The drug might also be covalently linked to the beginning of the polymer chain. After freeze-drying, both doxorobucin and dactinomycin-loaded nanoparticles were easily resuspended in water. Comparative particle size measurements and chromatographic determinations did neither show significant modification of carrier dimensions nor changes in the molecular weight distribution profile of the polymers [14]. Likewise, the extent of drug binding to nanoparticles was not changed by the freeze-drying process for both doxorubicin and dactinomycin.

H.2. Drug-releasefrom nanoparticles A characteristic of cyanoacrylate polymers is that their rate of degradation is dependent upon the length of their alkyl chains [8, 15]. Bioerosion is prolonged with increasing alkyl chain length. Furthermore, using dactinomycin as model drug, it was demonstrated [16] that drug release could be a consequence of polymer degradation, which probably occurs through an enzymatic pathway consisting of ester hydrolysis of the side chain with production of a water-soluble polycyanoacrylic acid. Since the biodegradability of polyalkylcyanoacrylates depends upon the nature of the alkyl chain, it is possible to choose a monomer whose polymerized form has a biodegradability corresponding to the requested program for release of the biologically active compound. This has been shown for dactinomycin; the release rate from various cyanoacrylic copolymers was found to correlate with the rate of polymer bioerosion [10] (Fig. 3).

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IlI. Nanoparticles loaded with anticancer drugs: cell culture experiments Over the past several years, many papers have been published concerning the use of carriers, mainly liposomes, for introducing a molecule into cells. However, the mechanism of vesicle-cell interactions is often not clear. Indeed, various mechanisms can occur simultaneously in a cell suspension: adsorption, endocytosis, fusion and lipid transfer [17]. With regard to polymeric nanoparticles, the main mechanism reported is endocytosis leading to an intralysosomal localization of the carrier [18,19]. Polyalkylcyanoacrylate nanoparticles have been studied with various anticancer drugs with the aim of introducing such molecules into cancer cells.

HI. 1. Nanoparticles increase the concentration of dactinomycin in macrophages Polybutylcyanoacrylate nanoparticles have been investigated with [3H]-dactinomycin as the anticancer agent, in order to evaluate some parameters involved in cell-particle interactions [20]. Mouse peritoneal macrophages in culture were chosen as the cell model. In these experiments, the adherent macrophages were incubated in the presence of particles for various time intervals and at different temperatures with or without serum. Dactinomycin uptake was found to be three times greater when this compound was associated with nanoparticles and incubated with cells in the presence of serum compared to the uptake of free dactinomycin. By constrast, in the absence of serum no increase of uptake was observed (Fig. 4). The n u m b e r of nanoparticles per cell was time-dependent. It is interesting to

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o b s e r v e that a plateau was reached w h e n there w e r e about 120 nanoparticles per cell. M o r e o v e r , the intracellular concentration of d a c t i n o m y c i n was found to increase with the t e m p e r a t u r e up to 37°C, suggesting that the association was cellenergy d e p e n d e n t (Fig. 5). 0.35

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111.2. Doxorubicin-loaded nanoparticles bypass tumor cell resistance Doxorubicin (Dox) is one of the most active chemotherapeutic agents for the treatment of human cancer. Nevertheless, the ability of tumor cells to develop simultaneous resistance to multiple lipophilic compounds represents a major problem in cancer chemotherapy. Cellular resistance to anthracyclines has been attributed to an active drug effiux from resistant cells [21-23]. Kartner et al. [24] were the first to show that multidrug resistance was associated with decreased intracellular drug accumulation, and to identify, in resistant cells, the presence of transmembrane Pglycoprotein, which was not detectable in the parental drug-sensitive cell line. Drugs, such as doxorubicin, appear to enter the cell by passive diffusion through the lipid bilayer. Upon entering the cell, these drugs bind to P-glycoprotein which forms transmembrane channels and uses the energy of ATP hydrolysis to pump these compounds out of the cell [25]. To solve this problem, many authors have proposed the use of competitive Pglycoprotein inhibitors, such as verapamil, which are able to bind to P-glycoprotein, and to overcome pleiotropic resistance. The results obtained demonstrated that drug effiux can be inhibited by this calcium channel blocker. However, as the adverse effects of verapamil are serious, its clinical use to overcome multidrug resistance is limited. During the past few years, many studies have been devoted to evaluate the antitumor potential of carrier-drug complexes (e.g., Ref. 26). However, only a few reports exist on the ability of carriers to overcome drug transport resistance and they concern mainly liposomes. Poste and Papahadjopoulos [27] have demonstrated that dactinomycin-loaded liposomes enabled a 120-fold reduction in the dose of dactinomycin required to produce a 50% inhibition of cell growth. We evaluated the effect of nanoparticles loaded with doxorubicin, whose mechanism of pleiotropic resistance is known to be related to the presence of P-glycoprotein. We compared the cytotoxicity of free-Dox, Dox-loaded polyisohexylcyanoacrylate (PIHCA) nanoparticles (NP-Dox) (mean diameter 300 nm) and nanoparticles without drug (NP), against sensitive (MCF7) and pleiotropic resistant (Dox R MCFT) human breast cancer cell lines. This cytotoxic effect was evaluated after 6 h of incubation at appropriate Dox concentrations [28]. MCF7 cells were more sensitive to free-Dox (170 ng/ml gave 50% surviving cells, TD 50) than Dox R MCF7 cells which required about 26 ~g/ml, i.e., 150-fold more (Figs. 6 and 7). Drug-free nanoparticles were relatively non-toxic up to at least 1 ~g of polymer per ml of cell culture for the two breast cancer cell lines (90% survival). No significant difference was observed in the survival rate of MCF7 treated with free-Dox or NP-Dox (Fig. 6). In contrast, for Dox R MCFT, the required concentration of Dox leaving 50% viable cells was decreased 130-fold when NP-Dox were used instead of free-Dox (Fig. 7) [28]. These results indicated that nanoparticles provided an effective carrier for introducing cytotoxic doses of doxorubicin into the pleiotropic resistant human cancer cell line Dox R MCF7. Since then, additional experiments, conducted with other sensitive and resistant cell lines, have confirmed the above-mentioned efficiency of nanoparticles (Table

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218

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TABLE II SENSITIVITY OF DIFFERENT CANCER CELL LINES TO DOXORUBICIN (Dox) AND DOXORUBICIN-LOADED NANOPARTICLES (NP-Dox) IN VITRO (REFS. 28 AND 29) n.d., not determined Cancer cell lines TD 50 (ng/ml) MC7 Dox-R-MC7

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IV. Tissue distribution of anticancer drugs associated with nanoparticles Various studies have c o m p a r e d the tissue distributions of an anticancer drug in free form with this drug associated with several different P A C A nanoparticles. These experiments generally showed that drug-loaded nanoparticles are rapidly r e m o v e d from the bloodstream by the reticuloendothelial system, whose cells are abundant in the liver and the spleen. Thus, tritiated vinblastine or dactinomycin coupled to polymethyl ( P M C A ) or polyethylcyanoacrylate ( P E C A ) nanoparticles showed an increased uptake of radioactivity in liver, spleen and lungs [30]. An even higher accumulation in the spleen and liver was found with dactinomycin-loaded polybutylcyanoacrylate (PBCA) nanoparticles [11] (Fig. 8). Indeed, 44- and 64-fold higher drug concentrations were found in the spleen and liver, respectively, when [3H]-dactinomycin was associated with P B C A nanoparticles and injected intravenously into rats. Similar patterns were observed when 5-fluorouracil-containing P B C A nanoparticles were administered to Swiss albino mice [31]. The results of Kreuter et al. [32] were surprising as they showed a low hepatic accumulation of [75Se]norcholestenol containing P B C A nanoparticles when given intravenously to rabbits. The pharmacokinetic parameters were compared in mice after i.v. injection of dactinomycin alone or associated with nanoparticles [33] (Fig. 9). A double-label-

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ing technique enabled the simultaneous measurement of the change in the blood concentration with a 14C-labeled carrier and a 3H-labeled cytostatic agent. When dactinomycin was associated with nanoparticles, a slowdown of its blood clearance was observed. The pharmacokinetic changes were more pronounced when the cytostatic drug was bound to polyhexylcyanoacrylate(PHCA) nanoparticles which, compared in vitro with polyisobutylcyanoacrylate (PIBCA) nanoparticles, were more resistant to bioerosion. The tissue distribution of PACA nanoparticles was also investigated in tumorbearing mice. Whole-body autoradiography was performed on mice with subcutaneously implanted Lewis lung carcinoma and injected with [~4C]PIBCA nanoparticles [34]. Intratumoral accumulation of nanoparticles reached a maximum 4 h postinjection (2% of injected dose), when the carrier had been totally removed from the bloodstream. This observation suggests a possible passage of submicroscopic particles through the vascular endothelium, which could be explained by a change in the structure of the capillary wall due to the inflammatory process at the tumor site. However, the most interesting observation was the intense pulmonary capture of the carrier in tumor-bearing animals. No carrier accumulation in the lung was observed in healthy animals. Because histological studies have shown the presence of numerous metastases in the lungs, this phenomenon could be a valid reason for using PACA nanoparticles to target anticancer drugs to lung metastases. In contrast, no tumor accumulation was seen 4 days after i.v. administration of PHCA nanoparticles to CBA mice bearing a human osteosarcoma [35]. To collect further information on nanoparticle distributions in tumor bearing animals, Gipps et al. [36] studied the tissue distribution of PHCA nanoparticles in nude mice bearing a human osteogenic sarcoma. In this experiment, the highest levels of radioactivity were found on day 7 and the concentration of the polymer in the tumor was found to be 40 times higher than that in muscle tissue. However, the fraction of radioactivity detected in the tumor was generally less than 1% of the injected dose. Furthermore, the concentration of the radioactivity in the tumor varied. Higher levels of radioactivity were correlated with low amounts of tumor necrosis, indicating the importance of viable tumor tissue for accumulation of the radiolabel in this particular animal model. Taken together, studies conducted on the distribution patterns of cytostatic drugs associated with PACA nanoparticles provided evidence that nanoparticles can modify markedly the drug distribution profile in tissues. However, the predominant capture by the liver and the spleen reduces the probability of the particles penetrating the capillaries of the tumor. Furthermore, the direct interaction of nanoparticles with the target cells requires that the particles cross the mechanical barrier presented by the endothelial cells comprising the capillary wall. Although this could be due to the presence of inflammatory sites, nanoparticle entry into tumor interstitial spaces might occur as a consequence of the presence of endothelial gaps which are known to exist in some malignancies [37]. Another important observation is that nanoparticles can accumulate in metastatic tissue.

NANOPARTICLES AS CARRIERS FOR ANTICANCER DRUGS

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V. Doxorubicin-loaded nanoparticles: toxicological data The toxicity and side effects of certain compounds may be considerably reduced after linkage to nanoparticles because of the modified tissue distribution o f the agent. This was shown with doxorubicin which has extensive side effects involving the heart (acute and chronic cardiomyopathy). It was clearly demonstrated that doxorubicin associated with PIBCA nanoparticles was less toxic than the free drug after i.v. administration to mice [38]. Fig. 10 shows the survival of the mice injected on three consecutive days. Regardless of whether doxorubicin was free or bound, no deaths were observed at doses of 5.0 and 7.5 mg doxorubicin per kg per day (not shown in the figure), whereas 15 mg doxorubicin per kg per day was clearly an overdose. At the intermediate dose level (10 and 12.5 mg doxorubicin per kg per day), a longer survival of the nanoparticle-treated mice was observed. Other evidence of the lower overall toxicity of the bound drug form compared to the free drug is the more favorable body weight evolution of the mice treated with doxorubicin loaded nanoparticles (Fig. 11). At the 5 mg doxorubicin per kg per day dose, for instance, the maximal body weight loss (as compared to body weight on day zero) of the free doxorubicin group was twice as much as it was for the nanoparticle group. In fact, the weight loss of the free doxorubicin group was significantly higher than that of the nanoparticle group (Student's t-test; P>0.975) at all three doses tested (5, 7.5 and 10 mg doxorubicin per kg per day). Moreover, at the 10 mg doxorubicin per kg per day dose, the weight difference between the two test groups was most likely underestimated. No mice died in the doxorubicin nanoparticle group, while those mice in the free doxorubicin group which died were those which had lost most weight; they were subsequently excluded from the weight loss computation.

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In the next step of this toxicity study, intravenously administered free doxorubicin and nanoparticles with doxorubicin were compared for their effects on body weight [39]. The decrease in total body weight of the doxorubicin-treated mice (as compared to the weight of the control mice) was less pronounced in the doxorubicin nanoparticle group ( - 1 2 % ) than in the free doxorubicin group ( - 2 3 % ) , 4 days after i.v. injection of 7.5 mg of drug per kilogram of body weight per day during 3 consecutive days. The same pattern was found in each organ weighed, with marked differences in organ weight loss (between free and nanoparticle-associated doxorubicin) being especially marked in the heart (29%), liver (28%) and kidneys (16%). This reduced toxicity towards the heart could be correlated to a decreased cardiac capture of the drug when administered in the form of nanoparticles. Indeed, using an autofluorographic histological technique, a lack of doxorubicin fluorescence was observed in the cardiac muscle of mice given different doxorubicinloaded nanoparticle formulations [38]; after administration of free doxorubicin fluorescence was observed in cardiac tissue samples. This discrepancy was quantitatively confirmed using HPLC: drug associated to PIBCA nanoparticles was eliminated from the plasma while the cardiac level of the bound drug was drastically reduced in comparison with that of the free drug [14] (Fig. 12). This decrease in cardiac uptake could be attributed to the low endocytotic activity of cardiac cells, and indirectly, reflects the high stability of the carrier-drug complex. Histopathological findings confirmed the reduction of doxorubicin's toxicity when the drug was attached to PACA nanoparticles [39]. Regarding intestinal mucosa thickness, there was a smaller decrease (as compared to the thickness of mucosa in the control group) in the nanoparticle group ( - 1 % ) than in the free doxorubicin group ( - 5 2 % ) in the colon, while the same

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was true for the ileum ( - 1 5 and - 2 2 % for nanoparticle and free doxorubicin, respectively); further experiments are needed to explain these observations. Taking the megakaryocyte count of the control group as 100%, those for the free doxorubicin and doxorubicin-nanoparticle groups were 21 and 58%, respectively, four days after the last injection. Nine days later, the megakaryocyte count of the free doxorubicin group was higher than that of the control group (117%), while that of the nanoparticle group remained unchanged (58%). The other histopathological findings supported the more quantitative observations described above. In general, symptoms of organ, tissue and cell atrophy were prevalent in the free doxorubicin group, with concomitant disturbance of structure, perturbation of cell renewal or growth, signs of altered regeneration, and a few manifestations of degeneration. These alterations were particularly striking in the pancreas, liver, kidneys, testes, thymus and bone marrow. VI. Doxorubicin-ioaded cancers

nanoparticles: increased efficiency against experimental

VI.1. L-1210 leukemia The antitumor efficacy of doxorubicin-loaded nanoparticles was first tested using the lymphoid leukemia L-1210 as the tumor model. In this study, one injection of doxorubicin-loaded P I B C A nanoparticles was found to be more effective against L-1210 leukemia than when the drug was administered in its free form following the same dosing schedule [40]. Indeed, the increased life span (ILS %) of mice injected with doxorubicin-loaded PIBCA nanoparticles was twice as high as the ILS for free doxorubicin (Fig. 13). However, there were no long-term survivors (LTSs). An interesting observation was that this enhanced effectiveness of doxorubicin nanoparticles compared to free doxorubicin became more apparent after increasing the number of drug administrations. Thus, 30% more LTS were rec-

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Fig. 13. Increased life span (ILS %) of L-1210 leukemia-bearing mice treated intravenously with different doses of free doxorubicin (DL) or doxorubicin associated with polyisobutylcyanoacrylate nanoparticles (DN). Details can be found in Ref. 40.

orded for some administration schedules involving doxorubicin associated with PIBCA nanoparticles, whereas no improvement in the therapeutic efficiency was noted with the free drug treatment. In control experiments it was confirmed that unloaded nanoparticles exerted no anticancer activity. Furthermore, similar efficiencies were obtained after intravenous administration of free doxorubicin and injection of a single mixture of free doxorubicin and unloaded nanoparticles. These findings strongly suggested that the enhanced efficiency was correlated with the modified tissue distribution profile of the drug when associated with nanoparticles. The effectiveness of doxorubicin-loaded polyisohexylcyanoacrylate (PIHCA) nanoparticles against L-1210 leukemia was even more pronounced than that of doxorubicin loaded PIBCA nanoparticles. The drug's toxicity was markedly decreased when it was bound to PIHCA nanoparticles, so that impressive results were obtained with that formulation at doses for which the therapeutic efficiency of free doxorubicin was completely masked by the overpowering toxicity of the drug [40]. Nearly 70% LTS were recorded after administration of 17 mg/kg doxoru-

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bicin associated with PIHCA nanoparticles. Finally, preliminary experiments suggested that one i.v. bolus injection of doxorubicin-loaded nanoparticles was more active on L-1210 bearing mice, than the perfusion of the free drug for 24 h. VL2. M-5076 hepatic metastases The superiority of doxorubicin targeted with the aid of PACA nanoparticles was then confirmed on murine hepatic metastases model (M-5076 reticulosarcoma) [41]. Irrespective of the dose and the administration schedule, the reduction in the number of metastases was much greater with doxorubicin-loaded nanoparticles than with free doxorubicin. For instance, when the drug (10 mg/kg) was administered i.v. twice, on days 11 and 13 after tumor inoculation, no difference in terms of metastasis counts was observed between free doxorubicin (124+-18) and controls (138 +- 25). By contrast, the number of metastases was dramatically reduced to 11---8 with doxorubicin-loaded nanoparticles (Fig. 14). The improved efficacy of the targeted drug was clearly confirmed by histological examinations showing that both the number and the size of the tumor cores were lower when doxorubicin was administered in its nanoparticulate form [41]. Furthermore, liver biopsies of animals treated with the nanoparticle-targeted drug showed a lower cancer cell density inside tumor tissue• Necrosis was often less widespread with the nanoparticle-associated drug than in the control group and the group treated with free doxorubicin. Studies performed on total homogenates of livers from both healthy and metastases-bearing mice have shown extensive capture of nanoparticulate doxorubicin by the liver; no difference in hepatic concentrations was noted between healthy and

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T A B L E III AREAS UNDER THE CURVES OVER DIFFERENT TIME INTERVALS DERIVED FROM T H E P H A R M A C O K I N E T I C P R O F I L E S O F D O X O R U B I C I N G I V E N IN ITS F R E E ( D O X ) A N D N A N O P A R T I C U L A T E (NP-DOX) F O R M , IN H E A L T H Y (HT) A N D N E O P L A S T I C (TT) HEPATIC TISSUES Time intervals

0-6 h 0--48 h

Areas under the curves ~g • h • (g tissue) Dox-HT

Dox-TI"

NP-Dox-HT

NP-Dox-TT

16 98

52 164

289 946

74 414

tumor-bearing animals [41]. In order to elucidate the mechanism behind the enhanced efficiency of doxorubicin-loaded nanoparticles, doxorubicin measurements were made in both metastatic cores and neighboring healthy hepatic tissue. This provided quantitative information concerning the drug distribution over these tissues [42]. The area under the concentration-time curve (AUC) was calculated for different time intervals (0-6 h, 0-48 h) by the trapezoidal rule (Table III). For each dosage form (either free or nanoparticle-bound doxorubicin), the tumor/liver A U C ratio was used, for a given time interval, as an indicator of the relative exposure of these tissues to the drug. After administration of the free drug, the tumor/liver ratio was 3.2 for the 0-6 h interval and 1.7 for the 0-48 h interval [42]. These high ratios may be attributed to the high affinity of free doxorubicin for cellular DNA, presumably abundant in rapidly dividing tissue. After administration of the nanoparticle-associated drug, the tumor/liver ratio increased from 0.26, for the 0-6 h interval, to 0.43, for the 0--48 h interval [42]. Interestingly, doxorubicin concentrations observed in neoplastic tissue for the 6-24 h interval reached the levels recorded in healthy hepatic tissue (Fig. 15). Since the drug carrier could not move from one tissue to another, it was clear from this result that free doxorubicin became available in healthy tissue for diffusion into tumoral tissue. Drug clearance from both tissues was low, and this redistribution led to an increased doxorubicin concentration in neoplastic cells. When administered in its nanoparticulate form, 18-fold more doxorubicin concentrated in the liver during the 0-6 h interval than when the free form was administered (Table III)] This finding was caused by the rapid uptake of the colloidal carrier by Kupffer cells. Administering doxorubicin in its nanoparticulate form resulted only in a 1.4-fold increase in drug accumulation in the neoplastic tissue, during the 0-6 h interval. This indicates clearly that nanoparticles have no special affinity for tumor cells compared to free doxorubicin. However, during the 0-48 h interval, metastatic tissue was exposed to 2.5-times more doxorubicin in the case of doxorubicin nanoparticles injection than in the case of free doxorubicin. Moreover, the drug concentration in the tumor tissue was clearly higher after 24 h and 48 h for the nanoparticulate formulation than for the free compound (11.1 ~g/g wet tissue

NANOPARTICLES AS CARRIERS FOR ANTICANCER DRUGS

227

~.100

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Time (h) Fig. 15. Doxorubicin concentration vs. time curves in healthy (HT) and tumoral ('IT) hepatic tissues after i.v. administration of doxorubicin (10 mg/kg corresponding to 133 mg NP-Dox (total weight)/kg of polymer) in its free (Dox) or nanoparticle-bound (NP-Dox) forms in mice. n=4. Details can be found in Ref. 42.

vs. 2.2 I~g/g wet tissue at 24 h and 3.1 I~g/g wet tissue vs. 0.7 Ixg/g wet tissue at 48

h). Electron microscopic examinations have shown that Kupffer cells were filled with numerous spherical nanoparticles, transparent to the electron beam [42]. They were found as early as 15 min after intravenous administration. Nanoparticles were also taken up, but to a lesser extent, by hepatocytes and endocytes. Phagocytosis of nanoparticles by circulating polynuclear cells in the sinuses was also observed. Some spherical vesicles could be seen in tumor cells occasionally, but full proof identification as nanoparticles was not possible yet. Liver examination at later times (18 h and 36 h after administration) already showed partly degraded nanoparticles remaining in lysosomal vesicles of Kupffer cells. Hepatocytes, particularly those close to Kupffer cells, looked different from those seen earlier; they showed enhanced activity with enlarged endoplasmic reticula and numerous transparent vesicles. The mechanism by which nanoparticle association of doxorubicin increases the therapeutic index of the drug is still not fully elucidated. Direct uptake of nanoparticles by neoplastic tissue is unlikely. The 2.5-fold increase in the doxorubicin concentration in tumor tissue (Table III) probably resulted from doxorubicin released from healthy tissue, in particular Kupffer cells. Hepatic tissue could play the role of drug reservoir from which prolonged diffusion of the free drug (from nanoparticles entrapped in Kupffer cell lysosomes) towards the neighboring malignant cells

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occurs. Similar results were obtained with doxorubicin-containing liposomes [43,44]. This hypothesis raises questions concerning the long-term effect of an 18-fold increase of doxorubicin concentration in the liver. Although toxicological data have shown that doxorubicin-loaded nanoparticles were not significantly or unexpectedly toxic to the liver [39], this possibility should nonetheless be explored in detail in the near future. VII. Conclusion We have focused on the rationale of using nanoparticles as drug carriers in cancer therapy. Direct targeting to tumors is unrealistic because there are no differences between the behaviour of tumor cells and normal cells as far as nanoparticles are concerned. In addition, tumors are composed of multiple subpopulations of phenotypically distinct cell subsets. Finally, intravenously injected nanoparticles are a priori too large to pass efficiently across epithelial and endothelial cell layers, so that availability to most tumors would be limited anyway. In spite of these limitations it was found that modification of the tissue distribution of a nanoparticulate anticancer agent combination and its release from a 'storage' organ can decrease drug side effects, especially when this organ consists of tissues that are not sensitive to the toxic effects of the compound. This concept was illustrated with doxorubicin, whose reduced toxicity could be correlated to a decreased capture of the drug by the cardiac tissue. Accumulation of biodegradable nanoparticles loaded with doxorubicin in liver Kupffer cells presumably created a drug concentration gradient and, therefore, prolonged diffusion of the free drug towards the neighboring neoplastic tissue. The results obtained in vitro with multidrug-resistant cancer cells were very impressive and indicate that nanoparticles may indeed be able to circumvent this resistance. Although they need to be confirmed in in vivo experimental models, these results open new perspectives in rendering multidrug-resistant cancers vulnerable to a 'carrier-mediated chemotherapy'. References 1 Gregoriadis, G. (1976) The carrier potential of liposomes in biology and medicine, New Eng. J. Med. 295,704-710. 2 Caride, V.J., Taylor, W., Cramer, J.A. and Gottchalk, A. (1976) Evaluation of liposome entrapped radioactive tracers as scanning agents. Part I. Organ distribution of liposome (99Tc-DTPA)in mice, J. Nucl. Med. 17, 1067-1072. 3 Gabizon, A., Dagau, A., Goreu, D., Barenholz, Y. and Fuks, Z. (1984) Liposomes as in vivo carriers of adriamycin: reduced cardiac uptake and preserved antitumor activity in mice, Cancer Res. 42, 4734-4739. 4 Forssen, E.A. and T6kes, Z.A. (1983) Improved therapeutic benefits of doxorubicin by entrapment in anionic liposomes, Cancer Res. 43,546--550. 5 Birrenbach, G. and Speiser, P. (1976) Polymerized miceiles and their use as adjuvants in immunology, J. Pharm. Sci. 65, 1763-1766.

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6 Kreuter, J. and Speiser, P. (1976) In vitro studies of poly(methylmethacrylate) adjuvants, J. Pharm. Sci. 65, 1624-1627. 7 Couvreur, P., Kante, B., Roland, M., Guiot, P., Baudhuin, P. and Speiser, P. (1979) Polycyanoacrylates nanocapsules as potential lysosomotropic carriers: preparation, morphological and sorptive properties, J. Pharm. Pharmacol. 31,331-332. 8 Vezin, W. and Florence, A. (1978) In vitro degradation rates of biodegradable poly-N-alkylcyanoacrylates, J. Pharm. Pharmacol. 30, Suppl. 5P. 9 Leonard, F., Kulkarni, R., Nelson, J. and Brandes, G. (1967) Tissue adhesives and hemostasis-inducing compounds: the alkylcyanoacrylates, J. Biomed. Mat. Res. 1, 3-6. 10 Couvreur, P., Kante, B., Roland, M. and Speiser, P. (1979) Adsorption of antineoplastic drugs to polyalkylcyanoacrylate nanoparticles and their release characteristics in a calf serum medium, J. Pharm. Sci. 68, 1521-1524. 11 Kante, B., Couvreur, P., Lenaerts, V., Scailteur, V., Roland M. and Speiser, P. (1980) Tissue distribution of [3H]actinomycin D adsorbed on polybutylcyanoacrylate nanoparticles, Int. J. Pharm. 7, 45-53. 12 Kreuter, J. (1983) Physicochemical characterization of polyacrylic nanoparticles, Int. J. Pharm. 14, 43-58. 13 Van Snick, L., Couvreur, P., Christiaens-Leyh, D. and Roland M. (1985) Molecular weights of free and drug-loaded nanoparticles, Pharm. Res. 1, 36-41. 14 Verdun, C., Couvreur, P., Vranckx, H., Lenaerts, V. and Roland M. (1986) Development of a nanoparticle controlled release formulation for human use, J. Controlled Rel. 3,205-211. 15 Leonard, F., Kulkarni, R., Brandes, G., Nelson, J. and Cameron J. (1966) Synthesis and degradation of poly(alkylcyanoacrylates), J. Appl. Polym. Sci. 10,259-272. 16 Lenaerts, V., Couvreur, P., Christiaens-Leyh, D., Joiris, E., Roland, M., Rollman, B. and Speiser, P. (1984) Biodegradation of polyisobutylcyanoacrylate nanoparticles, Biomaterials 5, 65-68. 17 Pagano, R.E. and Weinstein, J.N. (1978) Interactions of liposomes with mammalian cells, Annu. Rev. Biophys. 7,435-468. 18 Couvreurp., Tulkens, P., Roland, M., Trouet, A. and Speiser P. (1977) Nanocapsules: a new type of lysosomotropic carrier, FEBS Lett. 84,323-326. 19 Guise, V., Jaffray, P., Delattre, J., Puisieux, F., Adolphe, M. and Couvreur, P. (1987) Comparative cell uptake of propidium iodide associated with liposomes or nanoparticles, Cell. Mol. Biol. 33 (3), 397-405. 20 Guiot, P. and Couvreur, P. (1983) Quantitative study of the interaction between polybutylcyanoacrylate nanoparticles and mouse peritoneal macrophages in culture, J. Pharm. Belg. 38 (3), 130-134. 21 Fry, D.W. and Jackson, R.C. (1986) Membrane transport alterations as a mechanism of resistance to anticancer agents, Cancer Surv. 5 (1), 47-79. 22 Kaye, S. and Merry, S. (1985) Tumour cell resistance to anthracyclines - a review, Cancer Chemother. Pharmacol. 14, 96-103. 23 Moscow, J.A. and Cowan, K.H. (1988) Multidrug resistance, J. Natl. Cancer Inst. 80 (1), 14-20. 24 Kartner, N., Evernden-Porelle, D., Bradley, G. and Ling, V. (1985) Detection of P-glycoprotein in multidrug-resistant cell lines by monoclonal antibodies, Nature 316,820-823. 25 Kartner, N. and Ling, V. (1989) Multidrug resitance in cancer, Sci. Am., March 1989, 44-51. 26 Brasseur, F., Couvreur, P., Kante, B., Deckers-Passau, L., Roland, M., Deckers, C. and Speiser, P.P. (1986) Actinomycin D adsorbed on polymethylcyanoacrylate nanoparticles: increased efficiency against an experimental tumor, Eur, J. Cancer 16, 1441-1445. 27 Poste, G. and Papahadjopoulos, D. (1976) Drug-containing lipid vesicles render drug-resistant tumour cells sensitive to actinomycin D, Nature 261,699-701. 28 Roblot-Treupel, L., Cuvier, C., Poupon, M.F., Couvreur, P. and Puisieux, F. (1990) Doxorubicin loaded nanospheres bypass tumor cell multidrug resistance, Biochem. Pharmacol., in press. 29 Kubiac, C., Couvreur, P., Manil, L. and Clausse, B. (1989) Increased cytotoxicity of nanoparticlecarried adriamycin in vitro and potentiation by verapamil and amiodarone, Biomaterials 10, 553556. 30 Couvreur, P., Kante, B., Lenaerts, V., Scailteur, V., Roland M. and Speiser, P. (1980) Tissue distribution of antitumor drugs associated to polyalkylcyanoacrylate nanoparticles, J. Pharm. Sci. 69,199202.

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31 Kreuter, J. and Hartmann, H. (1983) Comparative study on the cytostatic effects and the tissue distribution of 5-fluorouracil in a free form and bound to polybutylcyanoacrylate nanoparticles in sarcoma 180-bearing mice, Oncology 40,363-366. 32 Kreuter, J., Mills, S.N., Davis, S.S. and Wilson, C.G. (1983) Polybutylcyanoacrylate nanoparticles for the delivery of [75Se]norcholestenol, Int. J. Pharm. 16, 105-113. 33 Grislain, L., Couvreur, P. and Roland, M. (1985) Modification de la pharmacocin6tique de mol6cules associ6es aux nanoparticules, Stp. Pharma 1, 1038-1042. 34 Grislain, L., Couvreur, P., Lenaerts, V., Roland, M., Deprez-Decampeneere, D. and Speiser, P. (1983) Pharmacokinetics and distribution of a biodegradable drug-carrier, Int. J. Pharm. 15,335345. 35 Ilium, L., Jones, P.D.E., Baldwin, R.W. and Davis, S.S. (1984) Tissue distribution of poly(hexyl-2cyanoacrylate) nanoparticles coated with monoclonal antibodies in mice bearing human tumor xenographs, Pharm. Exp. Ther. 230,733-736. 36 Gipps, M.E., Arshady, R., Kreuter, J., Groseurth, P. and Speiser, P. (1986) Distribution of polyhexylcyanoacrylate nanoparticles in nude mice bearing human osteosarcoma, J. Pharm. Sci. 75, 256-258. 37 Peterson, H.I. (1979) Vascular and extravascular spaces in tumor: tumor vascular permeability. In: H.. Peterson (Ed.), Tumor blood circulation, CRC Press, Boca Raton, FL, pp. 77-86. 38 Couvreur, P., Kante, B., Grislain, L., Roland, M. and Speiser P. (1982) Toxicity of polyalkylcyanoacrylate nanoparticles. II. Doxorubicin-loaded nanoparticles, J. Pharm. Sci. 71,790-792. 39 Couvreur, P., Grislain, L., Lenaerts, V., Brasseur, F., Guiot P., Biornacki, A. Biodegradable polymeric nanoparticles as drug carrier for antitumor agents. In: P. Guiot and P. Couvreur (Eds.), Polymeric nanoparticles and microspheres, CRC Press, Boca Raton, FL, pp. 27-94, 1986. 40 Brasseur, F., Verdun, C., Couvreur, P., Deckers, C., Roland, M. (1986) Evaluation exp6rimentale de I'efficacit6 th6rapeutique de la doxorubicine associ6e aux nanoparticules de polyalkylcyanoacrylate. Proceedings of the 4th International Conference on Pharmaceutical Technology, Vol. 5, 177186. 41 Chiannilkulchai, N., Driouich, Z., Benoit, J.P., Parodi, A.L., Couvreur, P. (1989) Doxorubicinloaded nanoparticles: increased efficiency in murine hepatic metastases, Select. Cancer Ther. 5, 111. 42 Chiannilkulchai, N., Ammoury, N., Caillou, B., Devissaguet J.Ph. and Couvreur, P. (1990) Hepatic tissue distribution of doxorubicin-loaded nanoparticles after, I.V. administration in reticulosarcoma M 5076 mestastases-bearing mice, Cancer Chemother. Pharmacol. 26, 122-126. 43 Storm, G., Gessel, H.J.G.M., Steerenberg, P.A., Speth, P.A.J., Roerdink, F.H., Regts, J., Van Veen, M. and De Jong, W.H. Investigation of the role of mononuclear phagocytes in the transportation of doxorubicin-containing liposomes into a solid tumor, Cancer Drug Deliv. 4 (2), 89-104, 1987. 44 Mayhew. E., Rustum, Y. and Vail, W.J:(1983) Inhibition of liver metastases of M 5076 tumor by liposome-entrapped adriamycin, Cancer Drug Deliv. 1, 43-57.