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Development of PLGA–PEG encapsulated miltefosine based drug delivery system against visceral leishmaniasis
Materials Science and Engineering C 59 (2016) 748–753
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Development of PLGA–PEG encapsulated miltefosine based drug delivery system against visceral leishmaniasis Rishikesh Kumar a, Ganesh Chandra Sahoo a,⁎, Krishna Pandey b,⁎, V.N.R. Das b, Roshan K. Topno b, Md Yousuf Ansari c, Sindhuprava Rana a, Pradeep Das d a
Biomedical and Nanomedicine Department, Rajendra Memorial Research Institute Medical Science (ICMR), India Clinical Medicine Department, Rajendra Memorial Research Institute Medical Science (ICMR), India Pharmacoinformatics Department, National Institute Pharmaceutica Research and Education Industrial Area, Hajipur, India d Molecular Biology Department, Rajendra Memorial Research Institute Medical Science (ICMR), India b c
a r t i c l e
i n f o
Article history: Received 9 July 2015 Received in revised form 28 September 2015 Accepted 26 October 2015 Available online 29 October 2015 Keywords: Miltefosine Transmission electron microscope Nanoscale Amastigotes Amphoterecin B
a b s t r a c t Targeted drug delivery systems are ideal technology to increase the maximum mechanism of action with smaller dose, we have developed miltefosine encapsulated PLGA–PEG nanoparticles (PPEM) to target macrophage of infected tissues against Leishmania donovani. The structural characterization of PLGA–PEG by transmission electron microscopy (TEM) has shown a size range of 10 to 15 nm. Synthesis and drug encapsulation confirmed by dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR) and confirmed NP encapsulation. The dose of nano encapsulated miltefosine decreased by fifty percent as compared to that of a conventional miltefosine and Amphoterecin B. The inhibition of amastigotes in the splenic tissue with nano encapsulated miltefosine (23.21 ± 23) was significantly more than the conventional miltefosine (89.22 ± 52.7) and Amphoterecin B (94.12 ± 55.1). This study signifies that there is an increased contact surface area of the nano encapsulated drug and significant reduction in size, improved the efficacy in both in vitro and in vivo study than that of the conventional miltefosine, Amphoterecin B. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Leishmaniasis is cause by Leishmania donovani, and is presently a major cause concern of a severe health issue in the developing world. Leishmaniasis parasitic infections found to be a number of clinical symptoms, of which, visceral leishmaniasis (VL) is the most prominent in the eastern part of India. Approximately 500,000 new cases of VL with over 90% of cases has been seen every year in India, Bangladesh, Nepal, Sudan, and Brazil. VL has become a frequent coinfection in HIV-positive individuals in endemic areas and is associated with enhanced onset of AIDS-related illness and increased VL treatment failure [1]. The treatment options for leishmaniasis caused by L. donovani are limited and unsatisfactory. Parental drugs are mostly available for treatment; these drugs have higher side effects and toxicity. This problem is mostly confined in the eastern part of India, where widespread resistance to pentavalent antimonial has been used [2–4].The treatment of VL patients such as sodium stibogluconate (Pentostan), N-methylglucamine (Glucantime), amphotericin B (AmpB) and pentamidine have been involved [5]. Resistance of drugs and antimonial-resistant parasites has been a major limiting factor in infected regions [6,7]. Pentamidines have been used as drugs of second choice in visceral leishmaniasis and
⁎ Corresponding authors.
http://dx.doi.org/10.1016/j.msec.2015.10.083 0928-4931/© 2015 Elsevier B.V. All rights reserved.
are effective against kala-azar by inhibiting amino acylation and translation of the replicating parasite [8–13]. AmpB is very successful and widely used against visceral leishmaniasis but its higher toxicity and solubility are very low [14–16]. Formulation of deoxycholate complexed AmB micelles (Fungizone) has been used but caused high toxicity to patients [17,18]. In the early 1990s, miltefosine used as an anticancer drug later on was found to have antileishmania activity in animal models. Miltefosine is a membrane active alkyl phospholipid found to activate orally and previous studies suggest that the optimal dose to balance efficacy and tolerance was 100 mg/day for 28 days [19–21]. Animal studies have shown that it has some reproductive toxicity and thus it is contraindicated in pregnancy, and needs to be used with caution in women of reproductive age. Gastrointestinal side effects appear to be common but rarely severe enough to warrant stopping treatment [22]. To avoid toxicity, premature degradation of drug, drug failure, insolubility and development of resistance, to reduce target effect of antileishmanial drug, supreme technique is required to overcome the problem hence PLGA(poly (lactide-co-glycolide)) PLGA–PEG(poly ethylene glycol) nano carrier has been developed to deliver miltefosine to target the macrophage of infected cells. In this study, we have developed PLGA–PEG encapsulated miltefosine (PPEM) nanoparticles to target the macrophage of infected tissues against L. donovani. Different characterization techniques have been used to confirm the synthesis and size of PLGA–PEG encapsulated miltefosine. Nanoparticle syntheses
R. Kumar et al. / Materials Science and Engineering C 59 (2016) 748–753
and sizes of the particles were confirmed by transmission electron microscope (TEM), dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR). Furthermore in vitro and in vivo study was carried out to evaluate efficacy during promastigotes stage of VL found to two fold increase and in the case parasite inhibition in splenic tissue of animal model hamster were shown 93.69% inhibition. 2. Methods
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(10 min, 10,000 g) was used to purify NPs. Similarly the PLGA–b–PEG NPs were again disolved, washed with water, and collected [24]. 2.7. Characterization of nanoparticle The characterization of nano-encapsulated antileishmanial compound was carried out by transmission electron microscope (TEM), dynamic light scattering (DLS) and Fourier transform infrared (FTIR).
2.1. Chemical 2.8. Transmission electron microscope Chemical like PLGA, COOH–PEG–NH2, methlyne chloride, Nhydoxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)-carbodiimide (EDC) were purchased from Sigma-Aldrich, USA and Milli-Q water purification system from Millipore, Billerica, MA, USA. 2.2. Parasites Indian L. donovani promastigotes strain MHOM/IN/83/AG83 was obtained from the culture bank of Rajendra Memorial Research Institute of Medical Sciences (ICMR), Patna, India. The cryo-cells were revived and grown in RPMI1640 medium (Sigma-Aldrich) supplemented with 10% Fetal Calf Serum (FCS: Sigma-Aldrich) in BOD incubator at 22 °C. 2.3. Drug preparation The experimental drugs have been obtained from Sigma-Aldrich, USA and stored at −20 °C. The stock solutions were diluted in methanol medium, to require concentrations, before drug sensitivity assay.
Transmission electron microscope (TEM) measurements were performed using Philips CM 200 TEM in SAIF department of Indian Institute of Technology, Bombay (IITB). 2.9. Fourier transform infrared Fourier transform infrared (FTIR) measurements were performed using Perkin Elmer in Metallurgical Engineering and Materials Science department of IITB. 2.10. Dynamic light scattering The average size distribution of the nanoparticle was determined by dynamic light scattering (Beckman Coulter instrument) from IIT, Bombay.
2.4. Formulation of miltefosine drug-loaded PLGA
2.11. In vitro drug sensitivity assay
Copolymer PLGA–b–PEG has been synthesized by the mix of COOH– PEG–NH2 to PLGA–COOH. PLGA–COOH (1 g) in methylene chloride (2 ml), it converted into PLGA–NHS with excess N-hydroxysuccinimide (27 mg) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 46 mg). PLGA–NHS was precipitated with ethyl ether in 01 ml, andrepeatedly washed in an ice-cold mixture of ethyl ether and methanol to remove residual NHS. PLGA–NHS (1 g) was dissolved in chloroform (4 ml) followed by addition of NH2–PEG–COOH (250 mg) and N, N-diisopropylethylamine (28 mg) after vacuum drying. The co-polymers were precipitated with cold methanol after 12 h and again washed with the same solvent (3 × 5 ml) to remove untreated PEG. The resulting PLGA–PEG block co-polymers were dried under vacuum [23].
L. donovani promastigotes were subjected to in vitro drug sensitivity assays. Each test was carried out in duplicate and the mean IC50 was determined based on the two values.
2.5. Chemical carboamide cross linking of antibody to CD14 to polymeric surface PLGA–b–PEG NPs (10 μg/ml) were incubated with EDC (400 mM) and NHS (200 mM) for 20 min followed by repeatedly washed NPs in DNase-, RNase-free water (30 ml).The NPs activated with NHS were reacted with amine terminal of CD14 (1 μg/ml). The resulting NPCD14 bio encapsulates were washed with ultrapure water (15 ml) by ultra filtration, and the surface-bound CD14 was denatured at 90 °C and allowed to assume binding conformation during snap-cooling on ice. The NP suspensions were kept at 4 °C until use [24]. 2.6. Formulation of miltefosine drug-loaded PLGA For the formation of drug encapsulated carboxylated PLGA–b–PEG NPs nanoprecipitation method was conducted. In various organic solvents which are miscible with water miltefosine was dissolved. Similarly, the drug polymer was also dissolved and mixed. The drug polymer solution was added to water to form Nps. For 6 h at room temperature the NP solution was allowed to stir uncovered. The centrifugation
2.12. Assay on promastigotes In vitro drug sensitivity assay: To compare the efficacy of nano-AmpB formulation with AmpB (Hyclone) on L. donovani parasites, 1 × 106 parasites/ml in M199 medium supplemented with 10% FBS were seeded in 96-well plate in triplicate and treated with different concentrations (0–10 μg/ml) of either test compound or AmpB for 24 h. The viable cells after 24 h drug treatment were determined using Cell proliferation reagent WST-1 (Roche), according to manufacturer's recommendations and use for deducing 50% inhibitory concentration (IC50) values for both test compound and AmpB. The absorbance values were converted to % viability of parasites assuming parasites without drug treatment as corresponding to 100%. The experiment was repeated thrice and identical results were observed. 2.13. Hamster studies Hamsters were infected, via the tail vein, with 2 × 107 amastigotes of Ag83 L. donovani. The mean values of infected and treated hamsters were taken for further analysis and the results of both experiment sets were merged. Groups of five mice (50–60 g), received miltefosine (2.5 mg/kg) and PPEM (NPs) also received (2.5 mg/kg) days; smear liver impression were made 24 days post infection and before and after the treatment groups of mice were weighed, and the change in weight percent was record. The number of amastigotes per liver cell in treated and untreated mice activity was determined by microscopically, counting. After autopsy the weight of the spleen was measured immediately. The Giemsa-stained imprints were monitored and visceral infection microscopically in which parasite burdens was measured [24].
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R. Kumar et al. / Materials Science and Engineering C 59 (2016) 748–753
Fig. 1. FTIR image showing PLGA–PEG–CD14 encapsulation of miltefosine.
Fig. 2. a. TEM images of PLGA–PEG nanoparticles. b. TEM images of PLGA–PEG nanoparticles.
R. Kumar et al. / Materials Science and Engineering C 59 (2016) 748–753
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The following formulae has been used to calculate the percentage of inhibition
respectively (Fig. 1). Finally, stable amide bond formed as a result of the reaction of carboxyl groups in PLGA–PEG with CD14 [24].
PI ¼ ðPA−NAÞ 100 PA:
3.3. Determination of particle size of synthesized nanoparticles
where PI is the percentage of inhibition, PA the number of amastigotes per 500 nuclei in spleen before treatment, NA the number of amastigotes per 500 nuclei after treatment. 2.14. Statistical analysis Inhibition of amastigotes in vivo values were calculated by using Graph Pad Prism 5 version (La Jolla, CA). 3. Result 3.1. Nanoparticle synthesis By direct encapsulation of PLGA–COOH with NH2–PEG–COOH, copolymers PLGA–PEG were synthesized, to generate PLGA–b–PEG– COOH both having fixed block length. At the terminal end of the hydrophilic PEG block the carboxyl group in the copolymer is located; therefore, upon NP formulation, making them available for surface chemistry the PEG should facilitate the presentation of the carboxyl groups on the NP surface. After preparing the nanoparticle, the size of this nanoparticle was characterized by TEM (transmission electron microscope). 3.2. Chemical carboamide cross-linking of antibody to CD14 to the polymeric surface FT-IR spectroscopic analysis of PLGA–PEG NPs CD14 conjugate demonstrates that CD14 was indeed covalently linked to the PEG via amide bond free carboxyl group on the surface of PLGA nanoparticles through carbodiimide chemistry (Fig. 1).The PLGA–g–PEG composite preserved the characteristic peaks of each component can be seen in FTIR spectra of the pure PLGA–PEG. The peak around 1748 cm−1 was characteristics to the stretching of carbonyl groups (C_O) from the polymer. In PLGA– PEG bonding, the peak at 1084 cm−1 belonged to the stretch band of the C–O bond. Peaks of 1650 cm− l and 1530 cm− l assigned to C_O stretching vibration and –NH-bending vibration of amide appeared
The particle size distributions of the synthesized nanoparticles of PLGA–PEG and PPEM nanoparticle were measured by transmission electron microscope (Fig. 2a and b). TEM micrograph (the inset shows the particle size distribution) exhibited nearer to spherical nature of PLGA–PEG nanoparticles and the particle size ranges from 10 nm to 20 nm with an average size of about 15 nm (Fig. 2a) and PPEM nanoparticles showing that they are spherical in shape and the particle size distribution was 10 to 25 nm (Fig. 2b). According to TEM analysis, with the attachment of miltefosine, the diameter of the PPEM nanoparticles (than PLGA–PEG NPs alone) exhibited marked increase in diameter. Encapsulation of miltefosine further supported by DLS analysis, reveals that DLS measurements exhibit that the nanoparticle is highly mono-disperse in isopropanol medium. The mean hydrodynamic diameters of the PLGA–PEG encapsulated miltefosine (Fig. 3) clearly indicate that there is an increase in size seen in PLGA–PEG encapsulated miltefosine [24]. 3.4. In vitro drug sensitivity assay The in vitro drug sensitivity assay was performed on L. donovani parasites to compare the efficacy of PLGA–PEG encapsulated miltefosine against miltefosine and AmpB (Fig. 4a–c). An IC50 value of ~0.1 μg/ml, ~ 0.2 μg/ml and ~ 1 μg/ml was observed for PLGA–PEG encapsulated miltefosine, miltefosine and AmpB, respectively, which shows that PPEM formulation displays 50% better efficacy than conventional miltefosine formulation and 100 times against L. donovani parasites under in vitro culture conditions. 3.5. In vivo study Hamster studies of L. donovani infections indicated that PPEM nanoparticles were significantly more active than miltefosine or amphotericin B alone as measured by load of parasites liver and mean values were taken for further analysis (Table 1). The mean value taken was found to be respectively lower for PPEM (23.21 ± 23) compared with miltefosine (89.12 ± 52.7)and Amphoterecin B (94.12 ± 55.1). There was a highly significant reduction in the total parasite burden in
Fig. 3. DLS showing average size distribution of PPEM nanoparticles with marked increase in diameter size.
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Fig. 4. (a–c). The in vitro drug sensitivity assay was performed on L. donovani parasites to compare the efficacy of PLGA–PEG encapsulated miltefosine (PPEM) formulation against conventional miltefosine and AmpB.
spleen in the treated groups, 93.67% parasite inhibition has been seen with PLGA–PEG encapsulated miltefosine which is showing very high % as compared to miltefosine with 75.22% and AmpB with 74.41% parasite inhibition. 4. Discussion VL remains a challenging disease to treat. The main drugs available for treatment of VL are the systemic agents like antimony, amphotericin, paromomycin and now the oral drug miltefosine. We have reviewed the efficacy and safety of antimony. Newer drugs such as liposomal amphotericin are promising, especially in the single day treatment regimen but need to be tested in different leishmania endemic settings. The experience with paromomycin, where a drug effective in India was not effective in East Africa shows that there are multiple factors to be considered in the development of an effective leishmania treatment program [25]. The high cost, specific toxicities, parenteral administration (technical efficiency required) and spread of drug resistance, and relapses in HIV–Leishmania co-infected patients are several limitations in the treatment of visceral leishmaniasis. Synthetic and natural polymers of different types with biodegradable and biocompatible characteristics have been explored. N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer has shown a good result in the delivery of an antileishmanial activity of poly (HPMA)–amphotericin B [26]. Polymeric nanoparticles generally described as nanospheres and nanocapsules, have been proposed for use as passive drug delivery to macrophages because of their long circulation time in the body and rapid clearance from the plasma by the mononuclear phagocyte system (MPS) [27]. The polymer drug PLGA–PEG Amphoterecin B nanoparticle was synthesized from a polymeric precursor by aminolysis followed by substitution at the terminal amino group of the antileishmanial drug and during encapsulation the target moiety CD14 was introduced, it showed better result from conventional antileishmanial drug by reducing drug dose and enhancing the inhibition of parasite both in vitro and in vivo [24] therefore we have developed PLGA–PEG nanoparticle (NP) conjugated with antibody to CD14 (targeting macrophage) with antileishmanial compound miltefosine to overcome these drawbacks. The poorly soluble, poorly absorbed substances to biologically active substances convert to promising deliverable drugs by the nano encapsulate system. Due to the
particulate form of polymer and biodegradable nature, the macrophages engulfed the drugs, which results in drug delivery. The PLGA– PEG encapsulated miltefosine nanoparticles are considerably smaller (15–25 nm) and effectiveness of PLGA–PEG encapsulated miltefosine B was two fold increase as compare to conventional AmpB and recent development nano form of Amphoterecin B deoxycholate whereas it was 1.4 fold [28] better during promastigotes or less than 1.4 fold [29, 30]. Moreover these particles didn't lose their original characters, the macrophage phagocyte system leads to engulfment of these particles, this results in enhancement as nanosizing adhesion properties from cells to tissues [31,32]. PLGA–PEG nano encapsulates regain their antileishmanial activities against amastigotes when evaluated in vivo studies showing higher parasite load, it may be due to the phagolysosome acidic pH accelerating the degradation of PLGA, promoting specific release of the drug in the vicinity of the amastigotes and likely to be taken up by the macrophage phagocytic system, thereby considerably reducing the systemic side effects of miltefosine and Amphoterecin B akin to that seen with liposomal Amphoterecin B [24, 33,34]. This study signifies that there is an increased contact surface area of the drug and significant reduction in size, improved efficacy than that of miltefosine, Amphoterecin B and it is well targeted to macrophage. Thus, smaller doses of PPEM nanoparticle are required to achieve the better rate of success in treatment in the future. Hence, PPEM nanoparticles are likely to be taken up by the macrophage phagocytic system, thereby considerably reducing the systemic side effects of miltefosine and Amphoterecin B akin to that seen with liposomal Amphoterecin B. 5. Conclusion Improved antileishmanial efficacy and bioavailability of bioactive drug miltefosine in PLGA–PEG were synthesized and it was observed that nano encapsulation and nanoparticle were in a size range of 10 to 20 nm with an increased localization in macrophages predominantly infested with leishmaniasis parasite. In vitro and in vitro antileishmanial activities of PPEM were found to be more effective than that of free miltefosine in terms of therapeutic efficacy. It will be very useful in those cases where VL patients confected with other hepatic disorders and most importantly, it is applicable to those infected persons, where lower dose is preferred to eliminate the toxicity. If the cost of the
Table 1 In vivo activity of PLGA–PEG encapsulated miltefosine (PPEM), miltefosine and amphotericin B (group of hamster). Parameters
Before treatment
PLGA–PEG encapsulated miltefosine Group A(5)
Miltefosine Group B(5)
Amphotericin B(AMB) Group B(5)
Control Group C(5)
Spleen weight (g) Amastigotes/500 nuclei ± SD Percentage inhibition + SD
1.052 367.82 ± 31.11
0.4780 23.21 ± 23 93.69
0.6712 89.12 ± 52.7 75.22
0.6823 94.12 ± 55.1 74.41
1.5582 2397.59 ± 1459
P-value
0.005 0.0045
R. Kumar et al. / Materials Science and Engineering C 59 (2016) 748–753
production of PPEM nanoparticle works out to be considerably less than that for liposomal Amphoterecin B, it will be worthwhile to instigate a large-scale in vitro and in vivo study to look at the efficacy and toxicity of PPEM nanoparticle. Further studies are ongoing for the development of both the antileishmanial drug AmpB and miltefosine conjugate and encapsulate into PLGA–PEG for combinational therapy to check the efficacy and to overcome the drug resistance property of leishmaniasis parasite. Declaration of interest The authors are thankful to ICMR (Grant No. BMS/NTF/6/10-11), for providing financial support for carrying out nanomedicine related research work in our institute. The authors report no conflicts of interest. References [1] J. Alvar, S. Yactayo, C. Bern, Leishmaniasis and poverty, Trends Parasitol. 22 (12) (2006) 552–557. [2] S. Sundar, et al., Failure of pentavalent antimony in visceral leishmaniasis in India: report from the center of the Indian epidemic, Clin. Infect. Dis. 31 (4) (2000) 1104–1107. [3] P.F.d. Prado, et al., Epidemiological aspects of human and canine visceral leishmaniasis in Montes Claros, State of Minas Gerais, Brazil, between 2007 And 2009, Rev. Soc. Bras. Med. Trop. 44 (5) (2011) 561–566. [4] A. Awasthi, R.K. Mathur, B. Saha, Immune response to leishmania infection, Ind. J. Med. Res. 119 (2004) 238–258. [5] H.W. Murray, Clinical and experimental advances in treatment of visceral leishmaniasis, Antimicrob. Agents Chemother. 45 (8) (2001) 2185–2197. [6] M. Grogl, T.N. Thomason, E.D. Franke, Drug resistance in leishmaniasis: its implication in systemic chemotherapy of cutaneous and mucocutaneous disease, Am.J.Trop. Med. Hyg. 47 (1) (1992) 117–126. [7] L.F. Oliveira, et al., Systematic review of the adverse effects of cutaneous leishmaniasis treatment in the new world, Acta Trop. 118 (2) (2011) 87–96. [8] L. Monzote, Current treatment of leishmaniasis: a review, Open Antimicrob. Agents J. 1 (2009) 9–19. [9] C. Thakur, Epidemiological, clinical and therapeutic features of Bihar kala-azar (including post kala-azar dermal leishmaniasis), Trans. R. Soc. Trop. Med. Hyg. 78 (3) (1984) 391–398. [10] M. Paul, et al., Physicochemical characteristics of pentamidine-loaded polymethacrylate nanoparticles: implication in the intracellular drug release in Leishmania major infected mice, J. Drug Target. 5 (6) (1998) 481–490. [11] P. Chakraborty, M.K. Basu, Leishmania phagolysosome: drug trafficking and protein sorting across the compartment, Crit. Rev. Microbiol. 23 (3) (1997) 253–268. [12] J. Berman, R. Dietze, Treatment of visceral leishmaniasis with amphotericin B colloidal dispersion, Chemotherapy 45 (Suppl. 1) (1999) 54–66.
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Development of PLGA–PEG encapsulated miltefosine based drug delivery system against visceral leishmaniasis Rishikesh Kumar a, Ganesh Chandra Sahoo a,⁎, Krishna Pandey b,⁎, V.N.R. Das b, Roshan K. Topno b, Md Yousuf Ansari c, Sindhuprava Rana a, Pradeep Das d a
Biomedical and Nanomedicine Department, Rajendra Memorial Research Institute Medical Science (ICMR), India Clinical Medicine Department, Rajendra Memorial Research Institute Medical Science (ICMR), India Pharmacoinformatics Department, National Institute Pharmaceutica Research and Education Industrial Area, Hajipur, India d Molecular Biology Department, Rajendra Memorial Research Institute Medical Science (ICMR), India b c
a r t i c l e
i n f o
Article history: Received 9 July 2015 Received in revised form 28 September 2015 Accepted 26 October 2015 Available online 29 October 2015 Keywords: Miltefosine Transmission electron microscope Nanoscale Amastigotes Amphoterecin B
a b s t r a c t Targeted drug delivery systems are ideal technology to increase the maximum mechanism of action with smaller dose, we have developed miltefosine encapsulated PLGA–PEG nanoparticles (PPEM) to target macrophage of infected tissues against Leishmania donovani. The structural characterization of PLGA–PEG by transmission electron microscopy (TEM) has shown a size range of 10 to 15 nm. Synthesis and drug encapsulation confirmed by dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR) and confirmed NP encapsulation. The dose of nano encapsulated miltefosine decreased by fifty percent as compared to that of a conventional miltefosine and Amphoterecin B. The inhibition of amastigotes in the splenic tissue with nano encapsulated miltefosine (23.21 ± 23) was significantly more than the conventional miltefosine (89.22 ± 52.7) and Amphoterecin B (94.12 ± 55.1). This study signifies that there is an increased contact surface area of the nano encapsulated drug and significant reduction in size, improved the efficacy in both in vitro and in vivo study than that of the conventional miltefosine, Amphoterecin B. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Leishmaniasis is cause by Leishmania donovani, and is presently a major cause concern of a severe health issue in the developing world. Leishmaniasis parasitic infections found to be a number of clinical symptoms, of which, visceral leishmaniasis (VL) is the most prominent in the eastern part of India. Approximately 500,000 new cases of VL with over 90% of cases has been seen every year in India, Bangladesh, Nepal, Sudan, and Brazil. VL has become a frequent coinfection in HIV-positive individuals in endemic areas and is associated with enhanced onset of AIDS-related illness and increased VL treatment failure [1]. The treatment options for leishmaniasis caused by L. donovani are limited and unsatisfactory. Parental drugs are mostly available for treatment; these drugs have higher side effects and toxicity. This problem is mostly confined in the eastern part of India, where widespread resistance to pentavalent antimonial has been used [2–4].The treatment of VL patients such as sodium stibogluconate (Pentostan), N-methylglucamine (Glucantime), amphotericin B (AmpB) and pentamidine have been involved [5]. Resistance of drugs and antimonial-resistant parasites has been a major limiting factor in infected regions [6,7]. Pentamidines have been used as drugs of second choice in visceral leishmaniasis and
⁎ Corresponding authors.
http://dx.doi.org/10.1016/j.msec.2015.10.083 0928-4931/© 2015 Elsevier B.V. All rights reserved.
are effective against kala-azar by inhibiting amino acylation and translation of the replicating parasite [8–13]. AmpB is very successful and widely used against visceral leishmaniasis but its higher toxicity and solubility are very low [14–16]. Formulation of deoxycholate complexed AmB micelles (Fungizone) has been used but caused high toxicity to patients [17,18]. In the early 1990s, miltefosine used as an anticancer drug later on was found to have antileishmania activity in animal models. Miltefosine is a membrane active alkyl phospholipid found to activate orally and previous studies suggest that the optimal dose to balance efficacy and tolerance was 100 mg/day for 28 days [19–21]. Animal studies have shown that it has some reproductive toxicity and thus it is contraindicated in pregnancy, and needs to be used with caution in women of reproductive age. Gastrointestinal side effects appear to be common but rarely severe enough to warrant stopping treatment [22]. To avoid toxicity, premature degradation of drug, drug failure, insolubility and development of resistance, to reduce target effect of antileishmanial drug, supreme technique is required to overcome the problem hence PLGA(poly (lactide-co-glycolide)) PLGA–PEG(poly ethylene glycol) nano carrier has been developed to deliver miltefosine to target the macrophage of infected cells. In this study, we have developed PLGA–PEG encapsulated miltefosine (PPEM) nanoparticles to target the macrophage of infected tissues against L. donovani. Different characterization techniques have been used to confirm the synthesis and size of PLGA–PEG encapsulated miltefosine. Nanoparticle syntheses
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and sizes of the particles were confirmed by transmission electron microscope (TEM), dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR). Furthermore in vitro and in vivo study was carried out to evaluate efficacy during promastigotes stage of VL found to two fold increase and in the case parasite inhibition in splenic tissue of animal model hamster were shown 93.69% inhibition. 2. Methods
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(10 min, 10,000 g) was used to purify NPs. Similarly the PLGA–b–PEG NPs were again disolved, washed with water, and collected [24]. 2.7. Characterization of nanoparticle The characterization of nano-encapsulated antileishmanial compound was carried out by transmission electron microscope (TEM), dynamic light scattering (DLS) and Fourier transform infrared (FTIR).
2.1. Chemical 2.8. Transmission electron microscope Chemical like PLGA, COOH–PEG–NH2, methlyne chloride, Nhydoxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)-carbodiimide (EDC) were purchased from Sigma-Aldrich, USA and Milli-Q water purification system from Millipore, Billerica, MA, USA. 2.2. Parasites Indian L. donovani promastigotes strain MHOM/IN/83/AG83 was obtained from the culture bank of Rajendra Memorial Research Institute of Medical Sciences (ICMR), Patna, India. The cryo-cells were revived and grown in RPMI1640 medium (Sigma-Aldrich) supplemented with 10% Fetal Calf Serum (FCS: Sigma-Aldrich) in BOD incubator at 22 °C. 2.3. Drug preparation The experimental drugs have been obtained from Sigma-Aldrich, USA and stored at −20 °C. The stock solutions were diluted in methanol medium, to require concentrations, before drug sensitivity assay.
Transmission electron microscope (TEM) measurements were performed using Philips CM 200 TEM in SAIF department of Indian Institute of Technology, Bombay (IITB). 2.9. Fourier transform infrared Fourier transform infrared (FTIR) measurements were performed using Perkin Elmer in Metallurgical Engineering and Materials Science department of IITB. 2.10. Dynamic light scattering The average size distribution of the nanoparticle was determined by dynamic light scattering (Beckman Coulter instrument) from IIT, Bombay.
2.4. Formulation of miltefosine drug-loaded PLGA
2.11. In vitro drug sensitivity assay
Copolymer PLGA–b–PEG has been synthesized by the mix of COOH– PEG–NH2 to PLGA–COOH. PLGA–COOH (1 g) in methylene chloride (2 ml), it converted into PLGA–NHS with excess N-hydroxysuccinimide (27 mg) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 46 mg). PLGA–NHS was precipitated with ethyl ether in 01 ml, andrepeatedly washed in an ice-cold mixture of ethyl ether and methanol to remove residual NHS. PLGA–NHS (1 g) was dissolved in chloroform (4 ml) followed by addition of NH2–PEG–COOH (250 mg) and N, N-diisopropylethylamine (28 mg) after vacuum drying. The co-polymers were precipitated with cold methanol after 12 h and again washed with the same solvent (3 × 5 ml) to remove untreated PEG. The resulting PLGA–PEG block co-polymers were dried under vacuum [23].
L. donovani promastigotes were subjected to in vitro drug sensitivity assays. Each test was carried out in duplicate and the mean IC50 was determined based on the two values.
2.5. Chemical carboamide cross linking of antibody to CD14 to polymeric surface PLGA–b–PEG NPs (10 μg/ml) were incubated with EDC (400 mM) and NHS (200 mM) for 20 min followed by repeatedly washed NPs in DNase-, RNase-free water (30 ml).The NPs activated with NHS were reacted with amine terminal of CD14 (1 μg/ml). The resulting NPCD14 bio encapsulates were washed with ultrapure water (15 ml) by ultra filtration, and the surface-bound CD14 was denatured at 90 °C and allowed to assume binding conformation during snap-cooling on ice. The NP suspensions were kept at 4 °C until use [24]. 2.6. Formulation of miltefosine drug-loaded PLGA For the formation of drug encapsulated carboxylated PLGA–b–PEG NPs nanoprecipitation method was conducted. In various organic solvents which are miscible with water miltefosine was dissolved. Similarly, the drug polymer was also dissolved and mixed. The drug polymer solution was added to water to form Nps. For 6 h at room temperature the NP solution was allowed to stir uncovered. The centrifugation
2.12. Assay on promastigotes In vitro drug sensitivity assay: To compare the efficacy of nano-AmpB formulation with AmpB (Hyclone) on L. donovani parasites, 1 × 106 parasites/ml in M199 medium supplemented with 10% FBS were seeded in 96-well plate in triplicate and treated with different concentrations (0–10 μg/ml) of either test compound or AmpB for 24 h. The viable cells after 24 h drug treatment were determined using Cell proliferation reagent WST-1 (Roche), according to manufacturer's recommendations and use for deducing 50% inhibitory concentration (IC50) values for both test compound and AmpB. The absorbance values were converted to % viability of parasites assuming parasites without drug treatment as corresponding to 100%. The experiment was repeated thrice and identical results were observed. 2.13. Hamster studies Hamsters were infected, via the tail vein, with 2 × 107 amastigotes of Ag83 L. donovani. The mean values of infected and treated hamsters were taken for further analysis and the results of both experiment sets were merged. Groups of five mice (50–60 g), received miltefosine (2.5 mg/kg) and PPEM (NPs) also received (2.5 mg/kg) days; smear liver impression were made 24 days post infection and before and after the treatment groups of mice were weighed, and the change in weight percent was record. The number of amastigotes per liver cell in treated and untreated mice activity was determined by microscopically, counting. After autopsy the weight of the spleen was measured immediately. The Giemsa-stained imprints were monitored and visceral infection microscopically in which parasite burdens was measured [24].
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Fig. 1. FTIR image showing PLGA–PEG–CD14 encapsulation of miltefosine.
Fig. 2. a. TEM images of PLGA–PEG nanoparticles. b. TEM images of PLGA–PEG nanoparticles.
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The following formulae has been used to calculate the percentage of inhibition
respectively (Fig. 1). Finally, stable amide bond formed as a result of the reaction of carboxyl groups in PLGA–PEG with CD14 [24].
PI ¼ ðPA−NAÞ 100 PA:
3.3. Determination of particle size of synthesized nanoparticles
where PI is the percentage of inhibition, PA the number of amastigotes per 500 nuclei in spleen before treatment, NA the number of amastigotes per 500 nuclei after treatment. 2.14. Statistical analysis Inhibition of amastigotes in vivo values were calculated by using Graph Pad Prism 5 version (La Jolla, CA). 3. Result 3.1. Nanoparticle synthesis By direct encapsulation of PLGA–COOH with NH2–PEG–COOH, copolymers PLGA–PEG were synthesized, to generate PLGA–b–PEG– COOH both having fixed block length. At the terminal end of the hydrophilic PEG block the carboxyl group in the copolymer is located; therefore, upon NP formulation, making them available for surface chemistry the PEG should facilitate the presentation of the carboxyl groups on the NP surface. After preparing the nanoparticle, the size of this nanoparticle was characterized by TEM (transmission electron microscope). 3.2. Chemical carboamide cross-linking of antibody to CD14 to the polymeric surface FT-IR spectroscopic analysis of PLGA–PEG NPs CD14 conjugate demonstrates that CD14 was indeed covalently linked to the PEG via amide bond free carboxyl group on the surface of PLGA nanoparticles through carbodiimide chemistry (Fig. 1).The PLGA–g–PEG composite preserved the characteristic peaks of each component can be seen in FTIR spectra of the pure PLGA–PEG. The peak around 1748 cm−1 was characteristics to the stretching of carbonyl groups (C_O) from the polymer. In PLGA– PEG bonding, the peak at 1084 cm−1 belonged to the stretch band of the C–O bond. Peaks of 1650 cm− l and 1530 cm− l assigned to C_O stretching vibration and –NH-bending vibration of amide appeared
The particle size distributions of the synthesized nanoparticles of PLGA–PEG and PPEM nanoparticle were measured by transmission electron microscope (Fig. 2a and b). TEM micrograph (the inset shows the particle size distribution) exhibited nearer to spherical nature of PLGA–PEG nanoparticles and the particle size ranges from 10 nm to 20 nm with an average size of about 15 nm (Fig. 2a) and PPEM nanoparticles showing that they are spherical in shape and the particle size distribution was 10 to 25 nm (Fig. 2b). According to TEM analysis, with the attachment of miltefosine, the diameter of the PPEM nanoparticles (than PLGA–PEG NPs alone) exhibited marked increase in diameter. Encapsulation of miltefosine further supported by DLS analysis, reveals that DLS measurements exhibit that the nanoparticle is highly mono-disperse in isopropanol medium. The mean hydrodynamic diameters of the PLGA–PEG encapsulated miltefosine (Fig. 3) clearly indicate that there is an increase in size seen in PLGA–PEG encapsulated miltefosine [24]. 3.4. In vitro drug sensitivity assay The in vitro drug sensitivity assay was performed on L. donovani parasites to compare the efficacy of PLGA–PEG encapsulated miltefosine against miltefosine and AmpB (Fig. 4a–c). An IC50 value of ~0.1 μg/ml, ~ 0.2 μg/ml and ~ 1 μg/ml was observed for PLGA–PEG encapsulated miltefosine, miltefosine and AmpB, respectively, which shows that PPEM formulation displays 50% better efficacy than conventional miltefosine formulation and 100 times against L. donovani parasites under in vitro culture conditions. 3.5. In vivo study Hamster studies of L. donovani infections indicated that PPEM nanoparticles were significantly more active than miltefosine or amphotericin B alone as measured by load of parasites liver and mean values were taken for further analysis (Table 1). The mean value taken was found to be respectively lower for PPEM (23.21 ± 23) compared with miltefosine (89.12 ± 52.7)and Amphoterecin B (94.12 ± 55.1). There was a highly significant reduction in the total parasite burden in
Fig. 3. DLS showing average size distribution of PPEM nanoparticles with marked increase in diameter size.
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Fig. 4. (a–c). The in vitro drug sensitivity assay was performed on L. donovani parasites to compare the efficacy of PLGA–PEG encapsulated miltefosine (PPEM) formulation against conventional miltefosine and AmpB.
spleen in the treated groups, 93.67% parasite inhibition has been seen with PLGA–PEG encapsulated miltefosine which is showing very high % as compared to miltefosine with 75.22% and AmpB with 74.41% parasite inhibition. 4. Discussion VL remains a challenging disease to treat. The main drugs available for treatment of VL are the systemic agents like antimony, amphotericin, paromomycin and now the oral drug miltefosine. We have reviewed the efficacy and safety of antimony. Newer drugs such as liposomal amphotericin are promising, especially in the single day treatment regimen but need to be tested in different leishmania endemic settings. The experience with paromomycin, where a drug effective in India was not effective in East Africa shows that there are multiple factors to be considered in the development of an effective leishmania treatment program [25]. The high cost, specific toxicities, parenteral administration (technical efficiency required) and spread of drug resistance, and relapses in HIV–Leishmania co-infected patients are several limitations in the treatment of visceral leishmaniasis. Synthetic and natural polymers of different types with biodegradable and biocompatible characteristics have been explored. N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer has shown a good result in the delivery of an antileishmanial activity of poly (HPMA)–amphotericin B [26]. Polymeric nanoparticles generally described as nanospheres and nanocapsules, have been proposed for use as passive drug delivery to macrophages because of their long circulation time in the body and rapid clearance from the plasma by the mononuclear phagocyte system (MPS) [27]. The polymer drug PLGA–PEG Amphoterecin B nanoparticle was synthesized from a polymeric precursor by aminolysis followed by substitution at the terminal amino group of the antileishmanial drug and during encapsulation the target moiety CD14 was introduced, it showed better result from conventional antileishmanial drug by reducing drug dose and enhancing the inhibition of parasite both in vitro and in vivo [24] therefore we have developed PLGA–PEG nanoparticle (NP) conjugated with antibody to CD14 (targeting macrophage) with antileishmanial compound miltefosine to overcome these drawbacks. The poorly soluble, poorly absorbed substances to biologically active substances convert to promising deliverable drugs by the nano encapsulate system. Due to the
particulate form of polymer and biodegradable nature, the macrophages engulfed the drugs, which results in drug delivery. The PLGA– PEG encapsulated miltefosine nanoparticles are considerably smaller (15–25 nm) and effectiveness of PLGA–PEG encapsulated miltefosine B was two fold increase as compare to conventional AmpB and recent development nano form of Amphoterecin B deoxycholate whereas it was 1.4 fold [28] better during promastigotes or less than 1.4 fold [29, 30]. Moreover these particles didn't lose their original characters, the macrophage phagocyte system leads to engulfment of these particles, this results in enhancement as nanosizing adhesion properties from cells to tissues [31,32]. PLGA–PEG nano encapsulates regain their antileishmanial activities against amastigotes when evaluated in vivo studies showing higher parasite load, it may be due to the phagolysosome acidic pH accelerating the degradation of PLGA, promoting specific release of the drug in the vicinity of the amastigotes and likely to be taken up by the macrophage phagocytic system, thereby considerably reducing the systemic side effects of miltefosine and Amphoterecin B akin to that seen with liposomal Amphoterecin B [24, 33,34]. This study signifies that there is an increased contact surface area of the drug and significant reduction in size, improved efficacy than that of miltefosine, Amphoterecin B and it is well targeted to macrophage. Thus, smaller doses of PPEM nanoparticle are required to achieve the better rate of success in treatment in the future. Hence, PPEM nanoparticles are likely to be taken up by the macrophage phagocytic system, thereby considerably reducing the systemic side effects of miltefosine and Amphoterecin B akin to that seen with liposomal Amphoterecin B. 5. Conclusion Improved antileishmanial efficacy and bioavailability of bioactive drug miltefosine in PLGA–PEG were synthesized and it was observed that nano encapsulation and nanoparticle were in a size range of 10 to 20 nm with an increased localization in macrophages predominantly infested with leishmaniasis parasite. In vitro and in vitro antileishmanial activities of PPEM were found to be more effective than that of free miltefosine in terms of therapeutic efficacy. It will be very useful in those cases where VL patients confected with other hepatic disorders and most importantly, it is applicable to those infected persons, where lower dose is preferred to eliminate the toxicity. If the cost of the
Table 1 In vivo activity of PLGA–PEG encapsulated miltefosine (PPEM), miltefosine and amphotericin B (group of hamster). Parameters
Before treatment
PLGA–PEG encapsulated miltefosine Group A(5)
Miltefosine Group B(5)
Amphotericin B(AMB) Group B(5)
Control Group C(5)
Spleen weight (g) Amastigotes/500 nuclei ± SD Percentage inhibition + SD
1.052 367.82 ± 31.11
0.4780 23.21 ± 23 93.69
0.6712 89.12 ± 52.7 75.22
0.6823 94.12 ± 55.1 74.41
1.5582 2397.59 ± 1459
P-value
0.005 0.0045
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