Drug Targeting with Polyalkylcyanoacrylate nanoparticles: in vitro activity of primaquine-loaded nanoparticles agaisnt intracellular Leishmania donovani

Annals of Tropical Medicine and Parasitology, Vol. 86, No. I, 41-49 (1992) ,._——é Drug targeting with polyalkylcyanoacrylate nanoparticles: in vitro activity of primaquine-loaded nanoparticles against intracellular Leishmania donovani BY ROGERIO GA_SPAR*, FRED R. OPPERDOEST, VERONIQUE PREAT* AND MICHEL ROLAND* ”“Laboratoire de Pharmaeie Galénique, Universite’ Catholique ale Louvain, Av. Emmanuel Mounier 73.20, 1200 Bruxelles, Belgium and TReseareh Unit for Tropical Diseases, International Institute of Cellular and /l/Ioleeular Pathology, /17;. Hippoerate 74.39, 1200 Brussels, Belgium Reeeivecl 4 February 1991, Revised 29 November I 99], Accepted 2 Deeember 1991 The efficacy of primaquine-loaded polyisohexylcyanoacrylate (PIHCA) nanoparticles was evaluated using J77-4G8 macrophage-like cells infected with Leislzmania zlonovani: as an in vitro model of visceral leishmaniasis. The in oitro antileishmanial activity of primaquine—loaded nanoparticles showed a 21-fold increase in ED50 compared with free primaquine. Although unloaded PIHCA nanoparticles also exhibited a significant anti- leishmanial eifect, the loaded nanoparticles showed a synergistic effect compared with a mixture of unloaded nanoparticles and free primaquine at equivalent concentrations. Primaquine release and isohexanol production were evaluated in a lysosomal fraction; the correlation of both with protein concentration and the rapid drug release indicate the processes are associated with an enzymatic degradation. The results indicate that PIHCA and other polyalkylcyanoacrylates may be useful for targeting drugs at intracellular Leisltmania, and that the unloaded carrier itself could be of interest in experimental chemotherapy of leishmaniasis. The leishmaniases are widespread parasitic dis- eases, occurring in most tropical and subtropical countries and affecting the lives of more than 12 million people. The haemoflagellates which cause these diseases invade macrophages, surviving and multiplying in phagolysosomal compartments of the host cells. Treatment is limited to a few drugs, of which sodium stibogluconate, an antimonial, is the most important. However, the treatment re- quires repeated daily injections and causes serious side—efl"ects, and therefore new and better drugs are urgently required. Drug targeting, using colloidal systems, has led both to a reduction in toxicity and increased 0003-4983/92/01004-1+ 09 $03.00/() efficacy of antileishmanial drugs. Colloidal car- riers, such as liposomes, have been shown to be able to increase the therapeutic index of pentava- lent antimonials in animal models (Alving et al., 1978), while experiments with several carriers, including lipoproteins (Hart, 1987), rescaled erythrocytic ‘ghosts’ (Berman et al., 1986), nio- somes (Baillie et al., 1986), microparticles (Stjarnkvist et al., 1987) and nanoparticles (Fouarge et al., 1989), have all demonstrated the potential value of drug targeting in improving the chemotherapy of visceral leishmaniasis. Among polymeric synthetic colloidal sys- tems, nanoparticles of polyalkylcyanoacrylate © 1992 Liverpool School of Tropical Medicine 42 GASPAR ET AL. (PACA) have been well characterized with re- spect to size, molecular weight, biodegradabi- lity, toxicity and biodistribution (Kante et al., 1982; Lenaerts et al., l984a,b; Couvreur, 1988). These nanoparticles can be easily prepared on an industrial scale (Verdun et al., 1986), while their ability to be phagocytosed by macrophages and their proven localization in both Kiippfer cells and spleen macrophages (Grislain et al., 1983; Lenaerts, 1984) render them good candi- dates for the targeting of drugs against visceral leishmaniasis. Our overall aim was to improve the chemo- therapy of visceral leishmaniasis by reducing drug toxicity and/or increasing therapeutic efficacy through the use of PACA nanoparticles as drug carriers. Primaquine, widely used as a gametocidal drug in the clinical treatment of malaria (Webster, 1985), also possesses some activity against Visceral leishmaniasis (Peters et al., 1980; Berman, 1985; Neal, 1987) and we have therefore developed and characterized primaquine-loaded nanoparticles of PIHCA to test them for antileishmanial activity (Gaspar et al., 1991). We present the results of preliminary exper- iments designed to evaluate the ability of poly- meric nanoparticles to reduce intracellular infection with Leisbmania donovani in vitro. MATERIALS AND METHODS Polymeric Nanoparticles Unloaded nanoparticles of polyisohexylcyano- acrylate (PIHCA) were prepared by anionic polymerization of 12 mg ml” isohexylcyano— acrylate (Sopar Pharma, Belgium) in a medium containing 5% (w/v) glucose (Merck, Germany) and 1% (w/V) dextran 40 (‘Rheomacrodex’; Pharmacia, Sweden), adjusted to pH 3-0 with 0001 M citric acid (Merck). Primaquine (janssen Chimica, Belgium) was adsorbed to some of the PIHCA (Gaspar et al., 1991) and the loaded and unloaded nanoparticles were freeze-dried in a LyoFreeze 111 GT 15 (Leybold Heraeus) and stored at — 30°C. They were redispersed in 0'02M NaHCO3 when needed. The primaquine-loaded nanoparticles had a unimodal size distribution between 200 and 300nm (Gaspar et al., 1991). The complete preparation of the nanoparticles was performed in a sterile room according to standard procedures (Verdun et al., 1986). Free primaquine in 5% glucose and 1% dex- tran 40 was freeze-dried under sterile con- ditions as described above,and redispersed like the nanoparticles. Drug Release by Incubation with a Lysosomal Fraction Primaquine-loaded nanoparticles were incu- bated with a crude lysosomal (ML) fraction obtained by differential centrifugation, first at 1600g and then at 15 000g, of homogenized Wistar rat livers in sucrose-imidazole buffer (De Duve, 1975). The ML fraction was used after adjustment of the protein concentration to give 5 mg ml” protein and 1 mg mr‘ PIHCA in the citrate-phosphate assay buffer, pH 5-0, which contained 01% (v/v) Triton X-100. Protein concentrations were measured by standard procedures (Lowry et al., 1951). Isohexanol production was evaluated by gas chromatographic determinations of the super- natants produced by centrifugation of the incu- bated nanoparticle suspensions at 20 000g for two hours. The IGC-120FB chromatograph (Intersmat, Belgium) was fitted with a carbowax 20M column and a flame-ionisation detector. n—Butanol (25 ug ml”) was used as internal standard. Primaquine release was determined in the same supernatants using high-performance liquid chromatography (Laakso et al., 1987), with 8-aminoquinoline as internal standard. Macrophage—1ike Cells The macrophage-like cell line ]774G8, derived from a BALB/c murine reticulum cell sarcoma (Chang, 1980; Murray, 1981; Hart et al., 1989) was used in in vitro infection experiments. The cells were cultivated in Dutch-modified RPM1-1640 medium (Gibco, U.K.) with 10% (v/v) heat-inactivated foetal calf serum (HIFCS) (Gibco), supplemented with L-glutamine (Flow Laboratories, U.K.) just prior to use. Incubation was at 37°C in an atmosphere containing 5% co, USE OF NANOPARTICLES AGAINST LEISH/l/IANIA 43 The attached cells were resuspended and counted in a Biircker cell counter. For the assays, the cells were cultured in flat-bottomed 24-well plates (Flow), with wells of 17-8>< 16 mm each containing 3 X 10° cells ml”. Parasites Leisbmania donovani infantum (MHOM/67/ MA(BE)/ITMAP 263) was isolated by Pro- fessor D. Le Ray in 1967 from a Moroccan child admitted to Hospital Brugman, Brussels, and was stabilized after the second passage through hamsters. Promastigote cultures obtained in vitro were maintained in SDM-79 medium (Brun and Schonenburger, 1979) at 28°C in concentrations of 5 X 107-1 >< 10° cells ml_1, and subcultured weekly. These cultures were used to infect cultured ]774G8 cells. Cytotoxicity Assay ]774G8 cells were incubated with different con- centrations of unloaded PIHCA nanoparticles, and their viabilities were determined by the trypan blue exclusion test after two, four, 24 and 48 hours incubation. In V itro Antileishmanial Assay ]774G8 cells were infected with La’. infantum by incubating them at a ratio of five promasti- gotes per cell in Dutch-modified RMPI-1640 with 10% HIFCS at 37°C and in 5% CO2. After 48 hours all promastigotes were completely transformed to intracellular amastigotes. The culture medium was then refreshed and the antileishmanial agents added. These agents consisted of free primaquine and primaquine- loaded and unloaded PIHCA nanoparticles at various concentrations. To evaluate the agents’ antileishmanial activity the infected cells were incubated for further 24 or 48 hours after the agent was added, and 200 ul aliquots of each suspension were spun down onto microscope slides in a Cytospin centrifuge (Shannon Ltd.) at 1000 g for three minutes. The percentage of infected ]774G8 cells and the average number of amastigotes per cell were determined by examination of these slides at 1000 >< magnification after May—Grunwald—Giemsa staining. The results given are means and standard deviations for five experiments. Values for ED50 were determined from plots of experimental data. RESULTS Incubation of primaquine-loaded nonoparticles with a lysosomal fraction showed that all primaquine was released within one hour (at a protein concentration of 5 mg mg” PIHCA) and that PIHCA degradation occurred in paral- lel with isohexanol release. The degradation of the polymer, based on isohexanol production, was faster for primaquine-loaded nanoparticles than for unloaded nonoparticles (Fig. 1). Primaquine release and isohexanol production were directly correlated with protein concen- tration (data not shown), suggesting that both were a result of enzymatic degradation. To determine the highest possible polymer concentration and the optimal incubation time, a preliminary cytotoxicity assay on _l774G8 cells was carried out (Fig. 2). Only concentrations of PIHCA greater than 48 ug ml” showed sig- nificant toxicity up to 48 hours of incubation. Therefore, in all further experiments, no con- centrations greater than 48 ug ml” were used in the assays of in vitro antileishmanial activity. The doses of polymer required to kill 50% of the ]774G8 cells, the LD50 values, were 173 pg ml‘1 over 24 hours and 86 ug ml” over 48 hours. In the control group of cells the level of infec- tion, both in terms of number of parasites per cell and percentage of cells infected, increased significantly between 72 and 96 hours after infection, whereas it fell significantly in all treated cells over the same period, which was 24-48 hours after treatment (Table). All further data are expressed in terms of the percentage reduction in parasite burden, i.e. the reduction in number of amastigotes per cell, compared with the control Value 24 hours after infection. Figure 3 is a plot of percentage reduction in parasite burden v. the concentration of the vari- ous antileishmanial agents used. The ED50 values for the agents were 3-4 ug primaquine ml” for free primaquine, 016 ug primaquine ml” and 1-92 pg PIHCA ml” for primaquine-loaded nanoparticles and 6-0 ug PIHCA ml” for 44 GASPAR ET AL. '4 -D— Unloaded nanoparticles (,u.mol isohexanol/lumol IHCA) x I00 ,2 _ -I- Primaquine nanoparticles I00 80 60 °/0 Of total primaquine 40 - E 20 _ / -£}— Primaquine (free) ‘"/E —I- Primaquine-nanoparticles T I I 1 I 1 1 I I I I I O 10 20 3O 40 50 60 Time (minute) Fig. 1. Release of products from primaquine-loaded and unloaded nanoparticles by incubation with a lysosomal fraction: (A) isohexanol production; (B) primaquine release; (N = 5). PIHCA nanoparticles. Thus primaquine-loaded nanoparticles showed a 21-fold increase in antileishmanial activity compared with free primaquine, and the carrier itself showed a significant antileishmanial effect. The primaquine-loaded nanoparticles had a synergistic effect on activity compared with a mixture of free primaquine and unloaded PIHCA nanoparticles at equivalent concen- trations; the ED50 increased from 1-92 pg ml” to 3-76 pg mr‘ ofPIHCA and 0-16 pg m1-1 to 0-31 pg ml” of primaquine (Fig. 4). DISCUSSION The purpose of this preliminary investigation was to study the potential of PIHCA nano- particles as drug carriers in parasitic diseases in which macrophages are infected. A Leis/Lmcznm— infected macr0phage—like cell line, ]774G8, was therefore chosen as a model system. The _I774G8 cell line has previously been used as an in vitro model for infection with the causative agents of both cutaneous (Chang, 1980; Hart et 51]., 1989) and visceral leishmaniasis (Murray, 1981), as it supports the intracellular growth of amastigotes Without stimulation of significant microbicidal activity. The 8—aminoquinolines, and especially pri- maquine, are well known for their antimalarial action on the exoerythrocytic or liver stages of Plasmodium (Webster, 1985). This group, of f compounds also exhibits significant antileish-.4 manial activity (Peters et 41]., 1980; Neal, 1987), USE OF NANOPARTICLES AGAINST LEISHMANI/1 45 100 80- 4O— Ratio (dead cells/total cells) x 100 Incubation time (hour) Fig. 2. Cytotoxicity of unloaded nanoparticles of PIHCA, expressed as percentage of dead cells evaluated by the trypan blue exclusion test (N: 6). Macrophages were a ]774G8 cell line (3 x 105 cells ml“), incubated in 24-well microplates with different concentrations of unloaded nanoparticles (concentrations expressed as equivalent weight of monomer/ml). (Cl), Control; (Q), 24 pg ml”; (E), 48 pg ml'l; (E), 96 pg ml”; (I), 192 pg ml”. probably through metabolization of the 8- aminoquinolines to more active compounds by the host cell (Berman, 1985). Primaquine is known to be concentrated to a large extent in the liver, but significant concentrations have been found also in the lungs (McChesney er al., 1987), and the drug has a significant toxicity towards the red blood cell (Tarlov at a/., 1962). Therefore, concentrating primaquine inside carriers such as polymeric nanoparticles would be an interesting way to reduce its toxicity and at the same time increase its antileishmanial activity by targeting the drug directly to the infected phagocytizing host cell. Primaquine- loaded nanoparticles of PIHCA have been developed and characterized in terms of their physico-chemical properties and their size dis- tribution. Such particles exhibit a significant reduction in acute toxicity of primaquine compared with the free drug after intravenous administration in NMRI mice (Gaspar er al., 1991). In the present study, incubation of primaquine-loaded nanoparticles in a lysosomal fraction showed their biodegradability and the complete release of entrapped primaquine, and supported earlier results which indicated that PACA were biodegraded in the lysosomes by an enzymatic procedure involving hydrolysis of its ester (Lenaerts er al., 198441). Our present data show that the in vitro antileishmanial activity of primaquine increased 21-fold when the drug was loaded in PIHCA nanoparticles, which suggests that use of these drug-loaded nanoparticles may lead to a better intracellular delivery of the drug. This is corro- borated by the fact that the loaded nanoparticles were more effective than a comparable mixture of unloaded nanoparticles and free primaquine. The unloaded nanoparticles are known to exert activity against Leishmania donovani infec- tion in rats (Fouarge et al., 1987) and against bloodstream trypanosomes (Lherm et al., 1986). In the present study this potential carrier also showed significant antileishmanial activity in vitro. This could be explained by the release of degradation products, since PACA are known to release cyanoacetate and the corresponding alkyl side-chain alcohols together with small quantities of formaldehyde (Kante et al., 1982; Lenaerts et al., l984a,l9) or by macrophage acti- vation following phagocytosis of the polymeric particles (Artursson at al., 1987). Activation 46 GASPAR ET AL. Iwfim iwva .:.HE fiwwmm -._.w3w EMS an at iwvov 21:15 was? 3+E.M i_+~.§ §+w.~ m: X 3+ 2; 3. +39 oopi EH9: E flam pop? ma 2 She: 5 his m.3..~.m o.mH.-at 2_.trl..~m gap 3 M1 c ifs? 331% 2 H S m+HN.ov Shmi. m.o.fE 2. M 5:5 iflém M..?:rm.$ EH2. N.m..TZ~ M-NH mm. Sam... 2: Ala 2_sSe_s_ 9: 3H~.~.m EH.-% EH 2 m.._,.fl-Q «TIL-£ 2r:£__ 2: efieaoaa. 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