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Therapeutic efficacy of artemisinin-loaded nanoparticles in experimental visceral leishmaniasis
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Therapeutic efficacy of artemisinin-loaded nanoparticles in experimental visceral leishmaniasis Muzamil Yaqub Want a , Mohammad Islamuddin a , Garima Chouhan a , Hani A. Ozbak b , Hassan A. Hemeg b , Anjan Kumar Dasgupta c , Asoke Prasun Chattopadhyay d , Farhat Afrin a,b,∗ a
Parasite Immunology Laboratory, Department of Biotechnology, Jamia Hamdard (Hamdard University), New Delhi 110062, India Department of Medical Laboratories Technology, Faculty of Applied Medical Sciences, Taibah University, P.O. Box 344, Universities Road, Medina 30001, Saudi Arabia c Department of Biophysics and Biochemistry, Ballygunge Science College, University of Calcutta, Kolkata 700019, India d Department of Chemistry, University of Kalyani, West Bengal 741235, India b
a r t i c l e
i n f o
Article history: Received 9 January 2015 Received in revised form 4 April 2015 Accepted 6 April 2015 Available online xxx Keywords: Visceral leishmaniasis Leishmania donovani Nanoparticles Antileishmanial Drug delivery
a b s t r a c t Visceral leishmaniasis (VL) is a fatal vector-borne parasitic syndrome attributable to the protozoa of the Leishmania donovani complex. The available chemotherapeutic options are not ideal due to their potential toxicity, high cost and prolonged treatment schedule. In the present study, we conjectured the use of nano drug delivery systems for plant-derived secondary metabolite; artemisinin as an alternative strategy for the treatment of experimental VL. Artemisinin-loaded poly lactic co-glycolic acid (ALPLGA) nanoparticles prepared were spherical in shape with a particle size of 220.0 ± 15.0 nm, 29.2 ± 2.0% drug loading and 69.0 ± 3.3% encapsulation efficiency. ALPLGA nanoparticles administered at doses of 10 and 20 mg/kg body weight showed superior antileishmanial efficacy compared with free artemisinin in BALB/c model of VL. There was a significant reduction in hepatosplenomegaly as well as in parasite load in the liver (85.0 ± 5.4%) and spleen (82.0 ± 2.4%) with ALPLGA nanoparticles treatment at 20 mg/kg body weight compared to free artemisinin (70.3 ± 0.6% in liver and 62.7 ± 3.7% in spleen). In addition, ALPLGA nanoparticle treatment restored the defective host immune response in mice with established VL infection. The protection was associated with a Th1-biased immune response as evident from a positive delayed-type hypersensitivity reaction, escalated IgG2a levels, augmented lymphoproliferation and enhancement in proinflammatory cytokines (IFN-␥ and IL-2) with significant suppression of Th2 cytokines (IL-10 and IL-4) after in vitro recall, compared to infected control and free artemisinin treatment. In conclusion, our results advocate superior efficacy of ALPLGA nanoparticles over free artemisinin, which was coupled with restoration of suppressed cell-mediated immunity in animal models of VL. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Visceral leishmaniasis (VL) or kala-azar is a debilitating disease caused by protozoa of the Leishmania donovani complex (L. donovani and L. infantum) that imperils 200 million people in 62 countries with an estimated 500,000 new cases and 60,000 deaths annually [1]. VL leads to substantial health problems and death with more than 90% cases occurring in India, Nepal, Bangladesh, Sudan and Brazil [2]. Leishmania parasites are digenetic having
∗ Corresponding author at: Department of Medical Laboratories Technology, Faculty of Applied Medical Sciences, Taibah University, P.O. Box 344, Universities Road, Medina 30001, Saudi Arabia. Tel.: +966 4 8460008; fax: +966 4 8461407; mobile: +966 509862875. E-mail addresses: afrin [email protected], [email protected] (F. Afrin).
two morphological forms: promastigotes in the digestive organs of the sand fly vector and amastigotes, a clinically relevant form in the mammalian host [3]. The parasite infects cells of the reticuloendothelial system and results in hepatosplenomegaly, anemia, hypergammaglobulinemia and chronic immunosuppression. Successful treatment is associated with restoration of Th1-biased response and suppression of Th2 cytokines that aid in parasite clearance [4,5]. Due to unavailability of vaccines, chemotherapy remains the only option for the treatment of VL. The present regimen for VL includes pentavalent antimonials, amphotericin B (AmB), liposomal amphotericin B, miltefosine and paromomycin that are unsatisfactory owing to high cost, toxicity and resistance in some cases. Artemisinin is a secondary metabolite of the herb Artemisia annua, which has shown tremendous potential in the treatment
http://dx.doi.org/10.1016/j.colsurfb.2015.04.013 0927-7765/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: M.Y. Want, et al., Therapeutic efficacy of artemisinin-loaded nanoparticles in experimental visceral leishmaniasis, Colloids Surf. B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.04.013
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of experimental cutaneous and visceral leishmaniasis; however, its efficacy is compromised due to serious drawbacks such as low bioavailability and short half-life [6–8]. These limitations can be partly overcome by encapsulating in colloidal nanoparticles that can potentiate the therapeutic effectiveness of artemisinin. Earlier, we have shown enhanced efficacy of artemisinin in colloidal nanoparticles as delivery system against L. donovani amastigotes ex vivo with reduced toxicity on mammalian macrophages [9]. The present study is aimed at investigating the immunomodulatory and therapeutic efficacy of these nanoparticles in experimental VL. 2. Materials and methods 2.1. Materials Artemisinin was procured from Baoji Herbest Bio-Tech Ltd, China; poly (D,L-lactide-co-glycolide) (PLGA Resomer® 503H, lactide:glycolide 50:50), carboxyfluorescein succinimidyl ester (CFSE), AmB, RPMI-1640 medium, M-199 medium, penicillin, streptomycin, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), anti-mouse IgG and isotype antibodies, o-phenylenediamine dihydrochloride (OPD) were procured from Sigma-Aldrich (St Louis, MO, USA). Fluorochrome-conjugated antibodies such as anti-mouse CD4-phycoerythin, anti-mouse CD8fluorescein isothiocyanate, anti-mouse CD80-allophycocyanin, anti-mouse CD86-phycoerythin cyanine dye 7 and anti-mouse CD40-fluorescein isothiocyanate, isotype controls and cytokine bead array kit (CBA) were procured from BD Pharmingen, USA. Aspartate aminotransaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), creatinine and urea kits were obtained from Span Diagnostics Ltd (Surat, Gujarat, India). All the other reagents were of analytical grade. 2.2. Methods 2.2.1. Preparation of artemisinin-loaded nanoparticles ALPLGA nanoparticles were prepared by solvent displacement method, as described earlier [9]. In brief, solution of 12.8 mg PLGA (MW 24,000–38,000) and 1.69 mg artemisinin (in 2 ml acetone) was added drop wise to a specified volume of polyvinyl alcohol (0.47% w/v in 10 ml water). The nanoparticles were then precipitated and washed three times to purify the particles from the excess of polyvinyl alcohol and non-encapsulated artemisinin by centrifugation (45,000 × g, 20 min, 10 ◦ C) (Hermle Labor Technik, Germany). Empty nanoparticles were prepared in the same manner, except that the drug was not added. These nanoparticles were lyophilized (Martin Christ, Germany) and stored at 4 ◦ C until use to the prevent nanoparticles from degradation. 2.3. Characterization of nanoparticles The morphology, shape and size distribution of nanoparticles were assessed by transmission electron microscopy (FEI TechnaiTM TF20, USA) at 200 kV. Briefly, 1 mg of nanoparticles was reconstituted in Milli Q water and diluted to approximately 5–20 g/ml, such that the sample was transparent. A drop of the diluted sample was placed on a 300-mesh carbon-coated grid (Applied Biosystems, India). The sample was dried under vacuum and analyzed microscopically without staining. At least three samples were assessed for the determination of morphology and size of nanoparticles.
2.5. Efficacy of artemisinin-loaded nanoparticles in experimental VL 2.5.1. Animals Female BALB/c mice (25–30 g) maintained at the Central Animal House Facility of Jamia Hamdard were used for all experiments. The animal studies were approved by the Jamia Hamdard Animal Ethics Committee (JHAEC, Approval Number 458), and experiments were performed in accordance with the guidelines of Council for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. 2.5.2. Parasite culture The WHO strain of L. donovani (MHOM/IN/1983/AG83) was maintained by serial passage in BALB/c mice and hamsters [10]. These parasites were subcultured every 5 days at 22 ◦ C in Medium 199 supplemented with 20% heat-inactivated fetal bovine serum, 25 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin G and 100 g/ml streptomycin. 2.5.3. Infection and treatment of BALB/c mice for antileishmanial activity in vivo Female BALB/c mice at 6–8 weeks of age were infected intravenously (i.v.) with 2.5 × 107 late log or stationary phase promastigotes/mouse in PBS. At 8 weeks, at least three animals were euthanized, and the infection was validated in giemsa-stained tissue imprints of spleen and liver by counting the number of amastigotes per 500 macrophages under oil immersion (100×) using a microscope (Nikon Eclipse 80i, Japan). The infected mice were then assigned into seven groups. Group I comprised normal mice and Group II infected control. Animals of Groups III–VIII were infected and subsequently treated for 10 days: artemisinin daily intraperitoneally (i.p.), 10 mg/kg bw (Group III) or 20 mg/kg bw (Group IV), ALPLGA nanoparticles were administered alternately (i.p.), 10 mg/kg bw (Group V) or 20 mg/kg bw (Group VI) to reduce the dosing frequency, empty nanoparticles alternately (i.p.), equivalent to the highest concentration in nanoparticles (Group VII) and AmB alternately (i.v.) at 5 mg/kg bw (Group VIII). 2.5.4. Assessment of hepatosplenomegaly and parasite burden after treatment Infected mice were administered artemisinin daily, whereas ALPLGA nanoparticles and AmB were given alternatively for a period of 10 days. One week post treatment, animals were euthanized and weights of spleen and liver taken. Splenic and hepatic impression smears were made, amastigotes counted and parasite burden as Leishman Donovan Units (LDUs) and percentage inhibition of parasites were calculated in accordance with the formula [11]. LDU = Number of amastigotes per 500 nuclei × organ weight (mg) Percentage inhibition (PI) =
LDU(Infected control) − LDU(Treated group) LDU(Infected control) × 100
2.6. Preparation of antigen 2.4. Drug loading and encapsulation efficiency The lyophilized nanoparticles were used for determining the amount of drug loading and encapsulation efficiency, as described earlier [9].
Leishmanial antigen was prepared as reported previously with modifications [12]. In brief, stationary phase L. donovani promastigotes at third or fourth passage in liquid culture were harvested and washed four times with cold PBS. For freeze thawed (FT) antigen,
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the promastigotes were subjected to six sequential cycles of freezing (−70 ◦ C and 30 min) and thawing (37 ◦ C and 15 min). Soluble leishmanial antigen (SLA) was prepared similarly with two additional freeze–thaw cycles, followed by ultrasonication for 3 min at 4 ◦ C (Sonics and Materials Inc., VC 505, USA) and centrifugation at 5250 × g, 10 min, 4 ◦ C (Allegra® X-15R, Beckman Coulter, USA) and the supernatant (SLA) harvested. The protein content was determined by the Lowry method [13], and the antigens were stored at −70 ◦ C until use. 2.7. Delayed type hypersensitivity (DTH) To ascertain whether the ALPLGA nanoparticle treatment helped to regain cell-mediated immunity, all animals 1 week post treatment with higher dose of nanoparticles were intradermally injected with 50 l (800 g/ml) of FT antigen in the right hind footpad. In the control left footpad, an equivalent volume of PBS was injected. After 24 h, the swelling in the footpad was measured using vernier calipers, and the difference in the thickness (mm) between the right and left footpads was calculated [10]. 2.8. T-cell proliferation and cytokine analysis Proliferative response of in vitro restimulated splenocytes was measured by CFSE dilution [14]. Briefly, splenocytes from the uninfected, untreated and treated animals were stained with CFSE (1 M) for 15 min in dark. The cells were washed three times and dispensed at a density of 5 × 106 cells/well in triplicates in 96-well tissue culture plates. The splenocytes were pulsed with SLA (10 g/ml) or Con A (2.5 g/ml) as positive control or left unstimulated (null) at 37 ◦ C and 5% CO2 . The proliferative response of splenocytes was measured after 72 h using flow cytometer (BD LSR II, BD Biosciences, San Diego, CA, USA). In parallel, the levels of Th1 (IL-2 and IFN-␥) and Th2 (IL-4 and IL-10) cytokines were analyzed in the 72 h culture supernatant of unlabeled splenocytes, stimulated or not, using a cytokine bead array (CBA) kit [15]. 2.9. Serum antibody analysis Parasite-specific antibody isotypes, IgG1 and IgG2a, indirect surrogate markers of Th2 and Th1 response, respectively, were measured 1 week post-treatment in the sera of untreated and treated animals by conventional ELISA [16]. 2.10. Expression of co-stimulatory molecules
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the retro-orbital plexus under mild anesthesia. Sera were separated and analyzed for the estimation of hepatic and renal toxicity markers using commercially available kits (Span Diagnostics Ltd, Gujarat, India). 2.12. Statistical analysis Data were expressed as mean ± SE and were representative of two independent experiments performed. In each experiment, three to five mice were used per group, and the results were analyzed by one-way ANOVA using Graph Pad Prism 5.03 software and differences at P < 0.05 were considered statistically significant. 3. Results 3.1. Preparation and characterization of artemisinin-loaded nanoparticles ALPLGA nanoparticles were spherical in shape with a particle diameter of 220.0 ± 15.0 nm. The drug loading and encapsulation efficiency was found to be 29.2 ± 2.0% and 69.0 ± 3.3%, respectively (Fig. 1). The empty nanoparticles were also spherical in shape with a particle size of 195.0 ± 12.0 nm (data not shown). 3.2. Liver and spleen weight Animals treated with ALPLGA nanoparticles (10 and 20 mg/kg bw) showed a significant decline in the liver weight compared to infected control group (P < 0.01 and P < 0.001), whereas administration of artemisinin even at higher doses resulted in mild reduction (P < 0.05). However, AmB ensued a dramatic fall in hepatomegaly compared with infected control (P < 0.001) and was not statistically significant compared with ALPLGA nanoparticle treatment (Supplementary Material, Fig. S1a). Moreover, ALPLGA nanoparticles (20 mg/kg bw) as well as AmB led to a decline in size and weight of spleen comparable to that of normal animals, and the difference was significant compared to the treatment with higher dose of artemisinin (P < 0.05). In addition, it was evident from the results that there was no significant pathological effect in the liver and spleen of infected animals treated with empty nanoparticles compared with infected control (Supplementary Material, Fig. S1b). 3.3. Parasite burden In the liver, reduction of parasite burden or percentage inhibition of L. donovani infection one week post-treatment with
Post treatment, macrophages were aseptically isolated from the peritoneal cavity of uninfected, untreated and treated mice. The cells were washed with RPMI-1640, cell density adjusted to 2 × 106 cells/ml and stained with anti-mouse antibodies – FITC-conjugated anti-CD40, PE-CY7-conjugated anti-CD86 and APC-labeled anti-CD80 or appropriate isotype controls for 15 min on ice in dark. This was followed by three washes to remove the unbound antibody, acquisition of cells (after resuspending in FACS buffer) on a BD LSR II flow cytometer and analysis using FACS Diva software [17]. 2.11. In vivo hepatic and renal toxicity of artemisinin-loaded nanoparticles Toxicity study was carried out in healthy mice at a higher therapeutic dose by estimating the serum levels of hepatic markers; AST, ALT, ALP and renal toxicity markers; creatinine and urea. At 24 h post-administration of the last dose, blood was collected from
Fig. 1. TEM image of optimized nanoparticles at a scale of 200 nm depicting spherical morphology of nanoparticles.
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Fig. 2. Percentage inhibition of parasites in (a) liver and (b) spleen 1-week post-treatment determined, as described in Section 2. Data are expressed as mean ± SE and are from one of the two independent experiments having five animals per group.
ALPLGA nanoparticles at higher doses was comparable to the levels with the standard drug, AmB (85.4± 5.4% versus 98.6 ± 0.2%), and was significantly different (P < 0.05) when compared with free artemisinin at both lower and higher doses (68.1 ± 2.8% versus 53.0 ± 2.0% and 85.4 ± 5.4% versus 70.3 ± 0.6%), respectively (Fig. 2a). Similarly, percentage of parasite inhibition in the spleen of animals treated with ALPLGA nanoparticles was 66.3 ± 2.4% and 82.0 ± 2.4% at lower and higher doses, respectively, whereas free artemisinin exerted moderate inhibition of parasite replication (Fig. 2b). 3.4. Evaluation of cell-mediated immunity after treatment 3.4.1. Delayed type hypersensitivity reaction A positive DTH response was observed in animals treated with ALPLGA nanoparticles and AmB, thus aiding to regain cellmediated immunity, which was otherwise quenched during active VL. DTH reaction observed in animals treated with ALPLGA nanoparticles and AmB was significantly different compared with free artemisinin (P < 0.01 and P < 0.001), respectively, whereas no
significant difference was found between the free artemisinin, empty nanoparticles treated and infected control groups (P > 0.05) (Fig. 3a).
3.4.2. Lymphoproliferation and cytokine production Mice treated with ALPLGA nanoparticles and AmB showed a higher proliferation of splenocytes compared with free artemisinin (P < 0.05 and P < 0.001), whereas splenocytes from infected control and empty nanoparticles-treated animals failed to respond to in vitro stimulation with SLA (Fig. 3b and Supplementary Material, Fig. S2). In the culture supernatant of infected control splenocytes, IFN␥ and IL-2 levels diminished significantly, whereas on the contrary IL-4 and IL-10 accrued compared to untreated uninfected control. Animals treated with artemisinin-loaded nanoparticles (20 mg/kg bw) and AmB led to restitution of both IFN-␥ and IL-2, whereas the levels of IL-4 and IL-10 diminished upon treatment, normalizing their levels analogous to the uninfected group. Treatment with empty nanoparticles did not depict any noticeable alteration on
Fig. 3. (a) DTH response to leishmanial antigen in age-matched untreated and differently treated animals at higher dose 1-week post-treatment. Data are represented as mean ± SE of five animals in each group, representative from one of two independent experiments. (b) Proliferation of splenocytes after 72 h of stimulation with and without SLA measured by labeling with CFSE using a flow cytometer. Data are shown for four animals per group and are representative of one of the two independent experiments.
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Table 1 Effect of artemisinin-loaded nanoparticles on the induction of Th1 and Th2 cytokines in the culture supernatant of mice splenocytes. Group
IFN- ␥ (pg/ml)
Naïve Infected control Artemisinin ALPLGA nanoparticles Empty nanoparticles AmB
465.4 286.9 331.2 501.8 279.1 523.3
± ± ± ± ± ±
IL-2 (pg/ml)
32.9* 24.7 22.5 36.1*,‡ 11.03 17.9*,‡
6.4 2.9 3.8 5.2 2.5 5.9
± ± ± ± ± ±
0.5** 0.4 0.1 0.3**,‡ 0.1 0.6**,‡
IL-10 (pg/ml) 107.0 482.5 448.3 245.2 470.9 143.7
± ± ± ± ± ±
1.9*** 14.3 19.4 9.7***,‡‡‡ 9.4 15.0***,‡‡‡
IL-4 (pg/ml) 0.4 1.0 0.8 0.5 1.0 0.4
± ± ± ± ± ±
0.0*** 0.1 0.1 0.0**,‡ 0.0 0.0***,‡‡‡
Data represent the mean ± SE of four animals per group and are representative from one of two independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 versus infected control and empty nanoparticles and ‡ P < 0.05 and ‡‡‡ P < 0.001 versus artemisinin assessed by one-way ANOVA followed by the Tukey–Kramer multiple comparison test.
the production of these cytokines compared to uninfected group, affirming its immunological inertness (Table 1). 3.4.3. Humoral immune response post-treatment Increase in Th1 or Th2 cytokines was also indirectly supported by the levels of IgG2a and IgG1, surrogate markers of Th1 and Th2 response, respectively, in the sera of mice with and without treatment. Our results demonstrate that IgG1 levels were high as compared to IgG2a in the infected control and empty nanoparticlestreated animals. The animals treated with ALPLGA nanoparticles and artemisinin at the higher dose demonstrated a reverse trend in the ratio of IgG1 and IgG2a. Elevated levels of IgG2a over IgG1 were found in the ALPLGA nanoparticles-treated mice compared to free artemisinin, as also observed with AmB (Fig. 4). 3.4.4. Expression of CD40 and B7 co-stimulatory molecules on the peritoneal macrophages post-treatment L. donovani infection impairs the activation of macrophages by triggering down regulation of CD40 molecules. Treatment with ALPLGA nanoparticles significantly rescued the levels of CD40 molecules on the macrophages of infected animals compared with free artemisinin (P < 0.001) (Fig. 5 and Supplementary Material, Fig. S3). Expression of CD86 (B7.2) co-stimulatory molecules on the macrophages of untreated and treated animals was not significantly different, compared with the basal levels of expression (data not shown). On the contrary, there was a comprehensive refurbishment of CD80 (B7.1) molecules on the BALB/c derived-macrophages 1 week post-treatment with the higher dose of ALPLGA nanoparticles and partial restoration in artemisinin-treated animals. The standard antileishmanial drug, AmB, exhibited higher levels of
Fig. 5. Differential expression of CD80 and CD40 on mouse macrophages in agematched controls and animals treated with artemisinin, ALPLGA nanoparticles, empty nanoparticles and AmB 1-week post-treatment. Data are shown as mean ± SE for four animals per group and are representative of one of the two independent experiments.
expression of CD80 molecules on the macrophages, following treatment comparable to the levels in the uninfected group (Fig. 5 and Supplementary Material, Fig. S4). 3.4.5. Evaluation of toxicity of artemisinin-loaded nanoparticles in vivo Quantitative estimation of hepato- and renal toxicity markers studied 24 h post-administration of artemisinin and artemisininloaded nanoparticles (20 mg/kg bw) in healthy animals revealed no significant enhancement in the levels of these enzymes. There was a drastic elevation in the serum levels of creatinine and urea upon administration of AmB (5 mg/kg bw), indicating its renal toxicity (Supplementary Material, Table S1). 4. Discussion
Fig. 4. Leishmania-specific antibody levels 1-week post-treatment with artemisinin, ALPLGA nanoparticles, empty nanoparticles and AmB at higher doses measured by ELISA. Bars represent mean ± SE of five animals per group.
Nano-mediated drug delivery of plant-derived products is one of the most important strategies for the treatment of VL in the absence of adequate antileishmanial drugs. Artemisinin, an endoperoxide sesquiterpene lactone from A. annua, has been traditionally used for the treatment of malaria and has been reported to exhibit antileishmanial and antitumor activities [6,18]. Earlier, we have shown that formulation of artemisinin-loaded nanoparticles improved its activity against L. donovani amastigotes ex vivo. The present study demonstrates therapeutic efficacy of artemisinin-loaded nanoparticles in L. donovani-infected BALB/c mice, an experimental model for VL. Our study depicts that encapsulation of artemisinin in
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nanoparticles enhances its curative effect as noticed by the significant decrease in the hepatic and splenic parasite burden and organ weights after treatment. On the contrary, free artemisinin caused a marginal reduction in organ weight, reflecting the partial clearance of parasites from these organs. The reason behind encouraging results may be due to the small size of the nanoparticles that are easily taken up by the macrophage phagocytic system as evidenced by other drug delivery systems against the Leishmania infection, hence increasing the therapeutic accessibility of the drug to the amastigote-infected macrophages, thereby clearing the parasites [19,20]. However, AmB reduced the organ weight to normal levels and showed the highest activity almost eliminating the parasites in these organs, and the protection was significantly different compared to the other groups [21]. In the experimental VL, profound impairment of the host immune system is a grave concern requiring involvement of Tcells and effective cell-mediated immunity pivotal to successful chemotherapy [22]. In this study, we investigated the DTH response to leishmanial antigen (FT), an index of classical cell-mediated immunity. We found contemptible levels of Leishmania-specific DTH response in the control mice that adds to the failure of cell-mediated response corresponding to disease aggravation. The highest DTH was observed in the group treated with artemisininloaded nanoparticles and was comparable to AmB treatment, which was associated with a fall in liver and splenic parasite load. The positive DTH triggered by AmB is well corroborated by earlier studies, highlighting induction of cell-mediated immunity [23]. In the animals treated with artemisinin, minimal and insignificant gain in the DTH was detected, correlating with a partial reduction in splenic and liver parasite burden [24]. Active VL is characterized by T-cell unresponsiveness as well as production of Th2 cytokines to leishmanial antigen upon in vitro recall, whereas successful therapy is associated with restoration of host effective response with Th1-biased response [24,25]. Free artemisinin-treated group showed a marginal increase in the number of splenocytes, whereas no such change was depicted in the splenocytes of the group treated with empty nanoparticles. On the contrary, successful therapy with artemisinin nanoparticles as well as AmB manifested a significant increase in the lymphoproliferative response compared to the untreated infected group, revelatory of antileishmanial activity, hence almost completely restoring cell-mediated immunity instrumental for the elimination of the intracellular pathogen [26]. Lymphoproliferative response in artemisinin-loaded nanoparticles-treated animals was comparable to the levels induced upon AmB treatment, thus attributing its improved antileishmanial efficacy via modulation of the host immune system. Disease progression devolves on the switch of proinflammatory cytokines to anti-inflammatory cytokines, distorting the normal immune response by dampening the host-protective Th1 cytokines such as IFN-␥ and IL-2 and enhancing the secretion of Th2 cytokines such as IL-4 and IL-10 that cause the proliferation of B cells and production of non-specific Leishmania-specific IgG1 antibodies [27]. We attempted to evaluate the polarity of T-cell subsets toward protective Th1 or disease-exacerbating Th2 response in BALB/c mice models with established L. donovani infection. We found enhanced levels of IFN-␥ and IL-2 and diminishing levels of IL-4 and IL-10 in the artemisinin-loaded nanoparticles and AmB-treated animals compared to the infected untreated group. Our findings are in line from earlier studies leading to a plausible transition of host immune response from a disease-encouraging pattern to a diseaseresolution form after therapy [28]. Hypergammaglobulinemia and non-specific polyclonal antibody production are characteristic features associated with infection and are reinstated upon successful therapy [29]. Induction in the levels of parasite-specific antibodies such as IgG2a after
treatment is dependent on IFN-␥, whereas IgG1 correlates with IL-4. IgG2a and IgG1 are therefore used as surrogate markers of Th1 and Th2 response, respectively [28]. In the present study, we observed elevated levels of IgG2a parasite-specific antibodies after treatment with artemisinin-loaded nanoparticles and AmB, connotative of Th1 bias whereas low levels of IgG2a and higher IgG1 were observed in the infected control and empty nanoparticle-treated groups, suggestive of a dominance of Th2 response, indicating disease progression [30]. Artemisinin-treated group demonstrated a slight increase in the IgG2a parasite-specific antibody, reflecting an elusive shift toward Th1 immunity [31]. Apart from antigen T-cell receptor complex, a second stimulatory signal important for T-cell proliferation and optimal cytokine production is CD80. There is ample evidence to support that up-regulation of this molecule contributes to the restoration of cell-mediated immune response in experimental as well as clinical VL [32]. We thus investigated the status of B7 costimulatory molecules, CD80 (B7-1) on the macrophages derived from untreated uninfected, infected and treated BALB/c mice. The extent of CD80 expression mainly depends on the parasite load as has been reported earlier with lower level of expression on the infected macrophages or the antigen presenting cells, in vivo as well as in vitro [33]. In the present study, artemisinin-loaded nanoparticles triggered restoration of CD80 molecules, whereas partial restoration was observed on the macrophages derived from artemisinin-treated group, indicating enhanced therapeutic efficacy of the former. CD80 expression in the macrophages of AmB-treated animals was the highest and significantly different compared to other groups, indicating that AmB activates the antigen-presenting cells of the host and thereby modulates the immune system toward therapeutic cure. Thus, we believe that apart from killing the parasites directly, artemisinin-loaded nanoparticles also refurbish the expression of CD80 molecules needed for restoration of appropriate effector T-cell response. It has also been reported that Leishmania infection alters the expression of CD40 molecules on the antigen-presenting cells of murine model translating to disease susceptibility [33]. Restoration of CD40 expression on macrophages induces leishmanicidal activity and has been implicated in driving the immune response toward Th1 type, resulting in the clearance of parasites from the liver and spleen of infected animals. Our study clearly demonstrates that in the animals treated with artemisinin-loaded nanoparticles, expression of CD40 on murine macrophages is rescued which may be due to increased contact of drug pertaining to the large surface-to-volume ratio of nanoparticles leading to enhanced killing of the intracellular parasites [34]. Similarly, AmB treatment reverted CD40 levels toward normal levels and was significantly different compared to artemisinin-loaded nanoparticles treated and other groups. Immunomodulatory activity of nanoparticles or herbal extract loaded nanoparticles has recently been reported [35–37]. However, immunomodulation by nanoparticles loaded with a plant secondary metabolite, artemisinin, has not been reported yet. Thus, this study was the first to report immunopotentiation by artemisinin-loaded nanoparticles in experimental VL, augmenting its antileishmanial activity. Immunostimulation by artemisininloaded nanoparticles may be due to increase in the concentration of the drug in the vicinity of parasites that results in parasite killing and presentation of Leishmania antigens to the immune cells resulting in the enhancement of immune cell-related activities including lymphoproliferation, antibody and cytokine production. Artemisinin is usually considered as a safe drug without increased evidence of toxicity in vivo [38]. In this study, no overt signs of toxicity were observed in the animals administered with artemisinin-loaded nanoparticles or empty nanoparticles or free artemisinin, compared to normal control. However, AmB, the standard antileishmanial drug used for in vivo studies, resulted
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in elevated levels of creatinine and urea in the animals confirming its renal toxicity [39]. Thus, the present study suggests that artemisinin-loaded nanoparticles at the highest therapeutic dose administered to BALB/c mice are safe and do not cause any statistically significant variation in the levels of toxicity markers of liver and kidney. In conclusion, the findings of this study serve to emphasize that the leishmanicidal activity of artemisinin in experimental VL was accelerated by encapsulating them in PLGA nanoparticles. However, the curative effect was not as efficient as the standard antileishmanial drug, AmB. The enhanced parasite killing may have been mediated by inducing the delivery of artemisinin to the liver and spleen, hence restoring the cell-mediated immune response in immunosuppressed animals, leading to cure. Thus, the nano-encapsulation of artemisinin or conventional drugs or a combination of both may be a better strategy for extirpating the non-healing VL as well as other chronic intracellular infections. Conflict of interest None to declare. Author contributions F.A., M.Y.W., A.P.C. and A.K.D. conceived and designed the experiments; M.Y.W. and F.A. performed the experiments; M.I., G.C. and F.A. assisted in in vivo experiments; M.Y.W. and F.A. analyzed the data; M.Y.W. and F.A. wrote the paper and F.A., M.Y.W., H.A.H. and H.A.O. reviewed the manuscript. Acknowledgements The authors are highly thankful to Dr Nahid Ali for providing the Indian strain of L. donovani and BD FACSTM Jamia Hamdard for providing the facility of flow cytometer. This work was supported by research grants from the Department of Biotechnology (DBT, BT/PR9715/PBD/17/534/2007, BT/PR10876/NNT/28/137/2008), Department of Science and Technology (SR/FT/L-102/2006) and Central Council for Research in Unani Medicine (F. No. 3-77/2005-CCRUM/Tech), Government of India. M.Y.W. deeply acknowledges DBT and University Grants Commission, Government of India for providing the research fellowship.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]
[21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37]
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.04. 013
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[38] [39]
P. Desjeux, Comp. Immunol. Microbiol. Infect. Dis. 27 (2004) 305–318. C.R. Davies, P. Kaye, S.L. Croft, S. Sundar, Br. Med. J. 326 (2003) 377–382. R.J. Burchmore, M.P. Barrett, Int. J. Parasitol. 31 (2001) 1311–1320. A.K. Haldar, S. Banerjee, K. Naskar, D. Kalita, N.S. Islam, S. Roy, Exp. Parasitol. 122 (2009) 145–154. S. Kaur, K. Chauhan, H. Sachdeva, J. Med. Microbiol. 63 (2014) 1328–1338. R. Sen, S. Ganguly, P. Saha, M. Chatterjee, Int. J. Antimicrob. Agents 36 (2010) 43–49. Y. Chen, X. Lin, H. Park, R. Greever, Nanomedicine 5 (2009) 316–322. D.M. Yang, F.Y. Liew, Parasitology 106 (1993) 7–11. M.Y. Want, M. Islamuddin, G. Chouhan, A.K. Dasgupta, A.P. Chattopadhyay, F. Afrin, J. Colloid Interface Sci. 432 (2014) 258–269. F. Afrin, N. Ali, Infect. Immun. 65 (1997) 2371–2377. L.A. Stauber, Rice Institute Pamphlet 45 (1958) 80–96. K. Maasho, H.O. Akuffo, Scand. J. Immunol. 11 (1992) 179–184. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951) 265–275. D. Fulcher, S. Wong, Immunol. Cell Biol. 77 (1999) 559–564. T.M. McHugh, Methods Cell Biol. 42 (1994) 575–595. R. Banduwardene, A.B. Mullen, K.C. Carter, Int. J. Immunopharmacol. 19 (1997) 195–203. C. De Trez, M. Brait, O. Leo, T. Aebischer, F.A. Torrentera, Y. Carlier, E. Muraille, Infect. Immun. 72 (2004) 824–832. M.P. Crespo-Ortiz, M.Q. Wei, Biomed. J. Biotechnol. 2012 (2012) 247597. G.K. Gupta, S. Kansal, P. Misra, A. Dube, P.R. Mishra, AAPS PharmSciTech 10 (2009) 1343–1347. H. Van de Ven, M. Vermeersch, R.E. Vandenbroucke, A. Matheeussen, S. Apers, W. Weyenberg, S.C. De Smedt, P. Cos, L. Maes, A. Ludwig, J. Drug Target 20 (2012) 142–154. M. Mishra, U.K. Biswas, A.M. Jha, A.B. Khan, Lancet 344 (1994) 1599–1600. L. Das, N. Datta, S. Bandyopadhyay, P.K. Das, J. Immunol. 166 (2001) 4020–4028. E. Cenci, A. Mencacci, G. Del Sero, F. Bistoni, L. Romani, J. Infect. Dis. 176 (1997) 217–226. G.S. Nabors, J.P. Farrell, J. Infect. Dis. 173 (1996) 979–986. J.P. Haldar, S. Ghose, K.C. Saha, A.C. Ghose, Infect. Immun. 42 (1983) 702–707. E.M. Carvalho, O. Bacellar, C. Brownell, T. Regis, R.L. Coffman, S.G. Reed, J. Immunol. 152 (1994) 5949–5956. A.P. Taylor, H.W. Murray, J. Exp. Med. 185 (1997) 1231–1239. G.D. Miralles, M.Y. Stoeckle, D.F. McDermott, F.D. Finkelman, H.W. Murray, Infect. Immun. 62 (1994) 1058–1063. E.M. Carvalho, R.S. Teixeira, W.D. Johnson, Infect. Immun. 33 (1981) 498–500. J. Joshi, N. Malla, S. Kaur, Parasitol. Int. 63 (2014) 612–620. J. Roychoudhury, R. Sinha, N. Ali, PLoS ONE 6 (2011) 1–12. B. Saha, G. Das, H. Vohra, N.K. Ganguly, G.C. Mishra, Eur. J. Immunol. 25 (1995) 2492–2498. P.M. Kaye, N.J. Rogers, A.J. Curry, J.C. Scott, Eur. J. Immunol. 24 (1994) 2850–2854. H.W. Murray, C.M. Lu, E.B. Brooks, R.E. Fichtl, J.L. DeVecchio, F.P. Heinzel, Infect. Immun. 71 (2003) 6453–6462. B.C. Schanen, S. Das, C.M. Reilly, W.L. Warren, W.T. Self, S. Seal, D.R. Drake, PLoS ONE 8 (2013) 1–12. Y.C. Seo, W.Y. Choi, C.G. Lee, S.W. Cha, Y.O. Kim, J.C. Kim, G.P. Drummen, H.Y. Lee, Int. J. Mol. Sci. 12 (2011) 9031–9056. A. Dube, J.L. Reynolds, W.C. Law, C.C. Maponga, P.N. Prasad, G.D. Morse, Nanomedicine 10 (2014) 831–838. B. Angus, Expert. Rev. Clin. Pharmacol. 7 (2014) 299–316. R. Dietze, S.M. Fagundes, E.F. Brito, E.P. Milan, T.F. Feitosa, F.A. Suassuna, G. Fonschiffrey, G. Ksionski, J. Dember, Trans. R. Soc. Trop. Med. Hyg. 89 (1995) 309–311.
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Therapeutic efficacy of artemisinin-loaded nanoparticles in experimental visceral leishmaniasis Muzamil Yaqub Want a , Mohammad Islamuddin a , Garima Chouhan a , Hani A. Ozbak b , Hassan A. Hemeg b , Anjan Kumar Dasgupta c , Asoke Prasun Chattopadhyay d , Farhat Afrin a,b,∗ a
Parasite Immunology Laboratory, Department of Biotechnology, Jamia Hamdard (Hamdard University), New Delhi 110062, India Department of Medical Laboratories Technology, Faculty of Applied Medical Sciences, Taibah University, P.O. Box 344, Universities Road, Medina 30001, Saudi Arabia c Department of Biophysics and Biochemistry, Ballygunge Science College, University of Calcutta, Kolkata 700019, India d Department of Chemistry, University of Kalyani, West Bengal 741235, India b
a r t i c l e
i n f o
Article history: Received 9 January 2015 Received in revised form 4 April 2015 Accepted 6 April 2015 Available online xxx Keywords: Visceral leishmaniasis Leishmania donovani Nanoparticles Antileishmanial Drug delivery
a b s t r a c t Visceral leishmaniasis (VL) is a fatal vector-borne parasitic syndrome attributable to the protozoa of the Leishmania donovani complex. The available chemotherapeutic options are not ideal due to their potential toxicity, high cost and prolonged treatment schedule. In the present study, we conjectured the use of nano drug delivery systems for plant-derived secondary metabolite; artemisinin as an alternative strategy for the treatment of experimental VL. Artemisinin-loaded poly lactic co-glycolic acid (ALPLGA) nanoparticles prepared were spherical in shape with a particle size of 220.0 ± 15.0 nm, 29.2 ± 2.0% drug loading and 69.0 ± 3.3% encapsulation efficiency. ALPLGA nanoparticles administered at doses of 10 and 20 mg/kg body weight showed superior antileishmanial efficacy compared with free artemisinin in BALB/c model of VL. There was a significant reduction in hepatosplenomegaly as well as in parasite load in the liver (85.0 ± 5.4%) and spleen (82.0 ± 2.4%) with ALPLGA nanoparticles treatment at 20 mg/kg body weight compared to free artemisinin (70.3 ± 0.6% in liver and 62.7 ± 3.7% in spleen). In addition, ALPLGA nanoparticle treatment restored the defective host immune response in mice with established VL infection. The protection was associated with a Th1-biased immune response as evident from a positive delayed-type hypersensitivity reaction, escalated IgG2a levels, augmented lymphoproliferation and enhancement in proinflammatory cytokines (IFN-␥ and IL-2) with significant suppression of Th2 cytokines (IL-10 and IL-4) after in vitro recall, compared to infected control and free artemisinin treatment. In conclusion, our results advocate superior efficacy of ALPLGA nanoparticles over free artemisinin, which was coupled with restoration of suppressed cell-mediated immunity in animal models of VL. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Visceral leishmaniasis (VL) or kala-azar is a debilitating disease caused by protozoa of the Leishmania donovani complex (L. donovani and L. infantum) that imperils 200 million people in 62 countries with an estimated 500,000 new cases and 60,000 deaths annually [1]. VL leads to substantial health problems and death with more than 90% cases occurring in India, Nepal, Bangladesh, Sudan and Brazil [2]. Leishmania parasites are digenetic having
∗ Corresponding author at: Department of Medical Laboratories Technology, Faculty of Applied Medical Sciences, Taibah University, P.O. Box 344, Universities Road, Medina 30001, Saudi Arabia. Tel.: +966 4 8460008; fax: +966 4 8461407; mobile: +966 509862875. E-mail addresses: afrin [email protected], [email protected] (F. Afrin).
two morphological forms: promastigotes in the digestive organs of the sand fly vector and amastigotes, a clinically relevant form in the mammalian host [3]. The parasite infects cells of the reticuloendothelial system and results in hepatosplenomegaly, anemia, hypergammaglobulinemia and chronic immunosuppression. Successful treatment is associated with restoration of Th1-biased response and suppression of Th2 cytokines that aid in parasite clearance [4,5]. Due to unavailability of vaccines, chemotherapy remains the only option for the treatment of VL. The present regimen for VL includes pentavalent antimonials, amphotericin B (AmB), liposomal amphotericin B, miltefosine and paromomycin that are unsatisfactory owing to high cost, toxicity and resistance in some cases. Artemisinin is a secondary metabolite of the herb Artemisia annua, which has shown tremendous potential in the treatment
http://dx.doi.org/10.1016/j.colsurfb.2015.04.013 0927-7765/© 2015 Elsevier B.V. All rights reserved.
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of experimental cutaneous and visceral leishmaniasis; however, its efficacy is compromised due to serious drawbacks such as low bioavailability and short half-life [6–8]. These limitations can be partly overcome by encapsulating in colloidal nanoparticles that can potentiate the therapeutic effectiveness of artemisinin. Earlier, we have shown enhanced efficacy of artemisinin in colloidal nanoparticles as delivery system against L. donovani amastigotes ex vivo with reduced toxicity on mammalian macrophages [9]. The present study is aimed at investigating the immunomodulatory and therapeutic efficacy of these nanoparticles in experimental VL. 2. Materials and methods 2.1. Materials Artemisinin was procured from Baoji Herbest Bio-Tech Ltd, China; poly (D,L-lactide-co-glycolide) (PLGA Resomer® 503H, lactide:glycolide 50:50), carboxyfluorescein succinimidyl ester (CFSE), AmB, RPMI-1640 medium, M-199 medium, penicillin, streptomycin, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), anti-mouse IgG and isotype antibodies, o-phenylenediamine dihydrochloride (OPD) were procured from Sigma-Aldrich (St Louis, MO, USA). Fluorochrome-conjugated antibodies such as anti-mouse CD4-phycoerythin, anti-mouse CD8fluorescein isothiocyanate, anti-mouse CD80-allophycocyanin, anti-mouse CD86-phycoerythin cyanine dye 7 and anti-mouse CD40-fluorescein isothiocyanate, isotype controls and cytokine bead array kit (CBA) were procured from BD Pharmingen, USA. Aspartate aminotransaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), creatinine and urea kits were obtained from Span Diagnostics Ltd (Surat, Gujarat, India). All the other reagents were of analytical grade. 2.2. Methods 2.2.1. Preparation of artemisinin-loaded nanoparticles ALPLGA nanoparticles were prepared by solvent displacement method, as described earlier [9]. In brief, solution of 12.8 mg PLGA (MW 24,000–38,000) and 1.69 mg artemisinin (in 2 ml acetone) was added drop wise to a specified volume of polyvinyl alcohol (0.47% w/v in 10 ml water). The nanoparticles were then precipitated and washed three times to purify the particles from the excess of polyvinyl alcohol and non-encapsulated artemisinin by centrifugation (45,000 × g, 20 min, 10 ◦ C) (Hermle Labor Technik, Germany). Empty nanoparticles were prepared in the same manner, except that the drug was not added. These nanoparticles were lyophilized (Martin Christ, Germany) and stored at 4 ◦ C until use to the prevent nanoparticles from degradation. 2.3. Characterization of nanoparticles The morphology, shape and size distribution of nanoparticles were assessed by transmission electron microscopy (FEI TechnaiTM TF20, USA) at 200 kV. Briefly, 1 mg of nanoparticles was reconstituted in Milli Q water and diluted to approximately 5–20 g/ml, such that the sample was transparent. A drop of the diluted sample was placed on a 300-mesh carbon-coated grid (Applied Biosystems, India). The sample was dried under vacuum and analyzed microscopically without staining. At least three samples were assessed for the determination of morphology and size of nanoparticles.
2.5. Efficacy of artemisinin-loaded nanoparticles in experimental VL 2.5.1. Animals Female BALB/c mice (25–30 g) maintained at the Central Animal House Facility of Jamia Hamdard were used for all experiments. The animal studies were approved by the Jamia Hamdard Animal Ethics Committee (JHAEC, Approval Number 458), and experiments were performed in accordance with the guidelines of Council for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. 2.5.2. Parasite culture The WHO strain of L. donovani (MHOM/IN/1983/AG83) was maintained by serial passage in BALB/c mice and hamsters [10]. These parasites were subcultured every 5 days at 22 ◦ C in Medium 199 supplemented with 20% heat-inactivated fetal bovine serum, 25 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin G and 100 g/ml streptomycin. 2.5.3. Infection and treatment of BALB/c mice for antileishmanial activity in vivo Female BALB/c mice at 6–8 weeks of age were infected intravenously (i.v.) with 2.5 × 107 late log or stationary phase promastigotes/mouse in PBS. At 8 weeks, at least three animals were euthanized, and the infection was validated in giemsa-stained tissue imprints of spleen and liver by counting the number of amastigotes per 500 macrophages under oil immersion (100×) using a microscope (Nikon Eclipse 80i, Japan). The infected mice were then assigned into seven groups. Group I comprised normal mice and Group II infected control. Animals of Groups III–VIII were infected and subsequently treated for 10 days: artemisinin daily intraperitoneally (i.p.), 10 mg/kg bw (Group III) or 20 mg/kg bw (Group IV), ALPLGA nanoparticles were administered alternately (i.p.), 10 mg/kg bw (Group V) or 20 mg/kg bw (Group VI) to reduce the dosing frequency, empty nanoparticles alternately (i.p.), equivalent to the highest concentration in nanoparticles (Group VII) and AmB alternately (i.v.) at 5 mg/kg bw (Group VIII). 2.5.4. Assessment of hepatosplenomegaly and parasite burden after treatment Infected mice were administered artemisinin daily, whereas ALPLGA nanoparticles and AmB were given alternatively for a period of 10 days. One week post treatment, animals were euthanized and weights of spleen and liver taken. Splenic and hepatic impression smears were made, amastigotes counted and parasite burden as Leishman Donovan Units (LDUs) and percentage inhibition of parasites were calculated in accordance with the formula [11]. LDU = Number of amastigotes per 500 nuclei × organ weight (mg) Percentage inhibition (PI) =
LDU(Infected control) − LDU(Treated group) LDU(Infected control) × 100
2.6. Preparation of antigen 2.4. Drug loading and encapsulation efficiency The lyophilized nanoparticles were used for determining the amount of drug loading and encapsulation efficiency, as described earlier [9].
Leishmanial antigen was prepared as reported previously with modifications [12]. In brief, stationary phase L. donovani promastigotes at third or fourth passage in liquid culture were harvested and washed four times with cold PBS. For freeze thawed (FT) antigen,
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the promastigotes were subjected to six sequential cycles of freezing (−70 ◦ C and 30 min) and thawing (37 ◦ C and 15 min). Soluble leishmanial antigen (SLA) was prepared similarly with two additional freeze–thaw cycles, followed by ultrasonication for 3 min at 4 ◦ C (Sonics and Materials Inc., VC 505, USA) and centrifugation at 5250 × g, 10 min, 4 ◦ C (Allegra® X-15R, Beckman Coulter, USA) and the supernatant (SLA) harvested. The protein content was determined by the Lowry method [13], and the antigens were stored at −70 ◦ C until use. 2.7. Delayed type hypersensitivity (DTH) To ascertain whether the ALPLGA nanoparticle treatment helped to regain cell-mediated immunity, all animals 1 week post treatment with higher dose of nanoparticles were intradermally injected with 50 l (800 g/ml) of FT antigen in the right hind footpad. In the control left footpad, an equivalent volume of PBS was injected. After 24 h, the swelling in the footpad was measured using vernier calipers, and the difference in the thickness (mm) between the right and left footpads was calculated [10]. 2.8. T-cell proliferation and cytokine analysis Proliferative response of in vitro restimulated splenocytes was measured by CFSE dilution [14]. Briefly, splenocytes from the uninfected, untreated and treated animals were stained with CFSE (1 M) for 15 min in dark. The cells were washed three times and dispensed at a density of 5 × 106 cells/well in triplicates in 96-well tissue culture plates. The splenocytes were pulsed with SLA (10 g/ml) or Con A (2.5 g/ml) as positive control or left unstimulated (null) at 37 ◦ C and 5% CO2 . The proliferative response of splenocytes was measured after 72 h using flow cytometer (BD LSR II, BD Biosciences, San Diego, CA, USA). In parallel, the levels of Th1 (IL-2 and IFN-␥) and Th2 (IL-4 and IL-10) cytokines were analyzed in the 72 h culture supernatant of unlabeled splenocytes, stimulated or not, using a cytokine bead array (CBA) kit [15]. 2.9. Serum antibody analysis Parasite-specific antibody isotypes, IgG1 and IgG2a, indirect surrogate markers of Th2 and Th1 response, respectively, were measured 1 week post-treatment in the sera of untreated and treated animals by conventional ELISA [16]. 2.10. Expression of co-stimulatory molecules
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the retro-orbital plexus under mild anesthesia. Sera were separated and analyzed for the estimation of hepatic and renal toxicity markers using commercially available kits (Span Diagnostics Ltd, Gujarat, India). 2.12. Statistical analysis Data were expressed as mean ± SE and were representative of two independent experiments performed. In each experiment, three to five mice were used per group, and the results were analyzed by one-way ANOVA using Graph Pad Prism 5.03 software and differences at P < 0.05 were considered statistically significant. 3. Results 3.1. Preparation and characterization of artemisinin-loaded nanoparticles ALPLGA nanoparticles were spherical in shape with a particle diameter of 220.0 ± 15.0 nm. The drug loading and encapsulation efficiency was found to be 29.2 ± 2.0% and 69.0 ± 3.3%, respectively (Fig. 1). The empty nanoparticles were also spherical in shape with a particle size of 195.0 ± 12.0 nm (data not shown). 3.2. Liver and spleen weight Animals treated with ALPLGA nanoparticles (10 and 20 mg/kg bw) showed a significant decline in the liver weight compared to infected control group (P < 0.01 and P < 0.001), whereas administration of artemisinin even at higher doses resulted in mild reduction (P < 0.05). However, AmB ensued a dramatic fall in hepatomegaly compared with infected control (P < 0.001) and was not statistically significant compared with ALPLGA nanoparticle treatment (Supplementary Material, Fig. S1a). Moreover, ALPLGA nanoparticles (20 mg/kg bw) as well as AmB led to a decline in size and weight of spleen comparable to that of normal animals, and the difference was significant compared to the treatment with higher dose of artemisinin (P < 0.05). In addition, it was evident from the results that there was no significant pathological effect in the liver and spleen of infected animals treated with empty nanoparticles compared with infected control (Supplementary Material, Fig. S1b). 3.3. Parasite burden In the liver, reduction of parasite burden or percentage inhibition of L. donovani infection one week post-treatment with
Post treatment, macrophages were aseptically isolated from the peritoneal cavity of uninfected, untreated and treated mice. The cells were washed with RPMI-1640, cell density adjusted to 2 × 106 cells/ml and stained with anti-mouse antibodies – FITC-conjugated anti-CD40, PE-CY7-conjugated anti-CD86 and APC-labeled anti-CD80 or appropriate isotype controls for 15 min on ice in dark. This was followed by three washes to remove the unbound antibody, acquisition of cells (after resuspending in FACS buffer) on a BD LSR II flow cytometer and analysis using FACS Diva software [17]. 2.11. In vivo hepatic and renal toxicity of artemisinin-loaded nanoparticles Toxicity study was carried out in healthy mice at a higher therapeutic dose by estimating the serum levels of hepatic markers; AST, ALT, ALP and renal toxicity markers; creatinine and urea. At 24 h post-administration of the last dose, blood was collected from
Fig. 1. TEM image of optimized nanoparticles at a scale of 200 nm depicting spherical morphology of nanoparticles.
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Fig. 2. Percentage inhibition of parasites in (a) liver and (b) spleen 1-week post-treatment determined, as described in Section 2. Data are expressed as mean ± SE and are from one of the two independent experiments having five animals per group.
ALPLGA nanoparticles at higher doses was comparable to the levels with the standard drug, AmB (85.4± 5.4% versus 98.6 ± 0.2%), and was significantly different (P < 0.05) when compared with free artemisinin at both lower and higher doses (68.1 ± 2.8% versus 53.0 ± 2.0% and 85.4 ± 5.4% versus 70.3 ± 0.6%), respectively (Fig. 2a). Similarly, percentage of parasite inhibition in the spleen of animals treated with ALPLGA nanoparticles was 66.3 ± 2.4% and 82.0 ± 2.4% at lower and higher doses, respectively, whereas free artemisinin exerted moderate inhibition of parasite replication (Fig. 2b). 3.4. Evaluation of cell-mediated immunity after treatment 3.4.1. Delayed type hypersensitivity reaction A positive DTH response was observed in animals treated with ALPLGA nanoparticles and AmB, thus aiding to regain cellmediated immunity, which was otherwise quenched during active VL. DTH reaction observed in animals treated with ALPLGA nanoparticles and AmB was significantly different compared with free artemisinin (P < 0.01 and P < 0.001), respectively, whereas no
significant difference was found between the free artemisinin, empty nanoparticles treated and infected control groups (P > 0.05) (Fig. 3a).
3.4.2. Lymphoproliferation and cytokine production Mice treated with ALPLGA nanoparticles and AmB showed a higher proliferation of splenocytes compared with free artemisinin (P < 0.05 and P < 0.001), whereas splenocytes from infected control and empty nanoparticles-treated animals failed to respond to in vitro stimulation with SLA (Fig. 3b and Supplementary Material, Fig. S2). In the culture supernatant of infected control splenocytes, IFN␥ and IL-2 levels diminished significantly, whereas on the contrary IL-4 and IL-10 accrued compared to untreated uninfected control. Animals treated with artemisinin-loaded nanoparticles (20 mg/kg bw) and AmB led to restitution of both IFN-␥ and IL-2, whereas the levels of IL-4 and IL-10 diminished upon treatment, normalizing their levels analogous to the uninfected group. Treatment with empty nanoparticles did not depict any noticeable alteration on
Fig. 3. (a) DTH response to leishmanial antigen in age-matched untreated and differently treated animals at higher dose 1-week post-treatment. Data are represented as mean ± SE of five animals in each group, representative from one of two independent experiments. (b) Proliferation of splenocytes after 72 h of stimulation with and without SLA measured by labeling with CFSE using a flow cytometer. Data are shown for four animals per group and are representative of one of the two independent experiments.
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Table 1 Effect of artemisinin-loaded nanoparticles on the induction of Th1 and Th2 cytokines in the culture supernatant of mice splenocytes. Group
IFN- ␥ (pg/ml)
Naïve Infected control Artemisinin ALPLGA nanoparticles Empty nanoparticles AmB
465.4 286.9 331.2 501.8 279.1 523.3
± ± ± ± ± ±
IL-2 (pg/ml)
32.9* 24.7 22.5 36.1*,‡ 11.03 17.9*,‡
6.4 2.9 3.8 5.2 2.5 5.9
± ± ± ± ± ±
0.5** 0.4 0.1 0.3**,‡ 0.1 0.6**,‡
IL-10 (pg/ml) 107.0 482.5 448.3 245.2 470.9 143.7
± ± ± ± ± ±
1.9*** 14.3 19.4 9.7***,‡‡‡ 9.4 15.0***,‡‡‡
IL-4 (pg/ml) 0.4 1.0 0.8 0.5 1.0 0.4
± ± ± ± ± ±
0.0*** 0.1 0.1 0.0**,‡ 0.0 0.0***,‡‡‡
Data represent the mean ± SE of four animals per group and are representative from one of two independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 versus infected control and empty nanoparticles and ‡ P < 0.05 and ‡‡‡ P < 0.001 versus artemisinin assessed by one-way ANOVA followed by the Tukey–Kramer multiple comparison test.
the production of these cytokines compared to uninfected group, affirming its immunological inertness (Table 1). 3.4.3. Humoral immune response post-treatment Increase in Th1 or Th2 cytokines was also indirectly supported by the levels of IgG2a and IgG1, surrogate markers of Th1 and Th2 response, respectively, in the sera of mice with and without treatment. Our results demonstrate that IgG1 levels were high as compared to IgG2a in the infected control and empty nanoparticlestreated animals. The animals treated with ALPLGA nanoparticles and artemisinin at the higher dose demonstrated a reverse trend in the ratio of IgG1 and IgG2a. Elevated levels of IgG2a over IgG1 were found in the ALPLGA nanoparticles-treated mice compared to free artemisinin, as also observed with AmB (Fig. 4). 3.4.4. Expression of CD40 and B7 co-stimulatory molecules on the peritoneal macrophages post-treatment L. donovani infection impairs the activation of macrophages by triggering down regulation of CD40 molecules. Treatment with ALPLGA nanoparticles significantly rescued the levels of CD40 molecules on the macrophages of infected animals compared with free artemisinin (P < 0.001) (Fig. 5 and Supplementary Material, Fig. S3). Expression of CD86 (B7.2) co-stimulatory molecules on the macrophages of untreated and treated animals was not significantly different, compared with the basal levels of expression (data not shown). On the contrary, there was a comprehensive refurbishment of CD80 (B7.1) molecules on the BALB/c derived-macrophages 1 week post-treatment with the higher dose of ALPLGA nanoparticles and partial restoration in artemisinin-treated animals. The standard antileishmanial drug, AmB, exhibited higher levels of
Fig. 5. Differential expression of CD80 and CD40 on mouse macrophages in agematched controls and animals treated with artemisinin, ALPLGA nanoparticles, empty nanoparticles and AmB 1-week post-treatment. Data are shown as mean ± SE for four animals per group and are representative of one of the two independent experiments.
expression of CD80 molecules on the macrophages, following treatment comparable to the levels in the uninfected group (Fig. 5 and Supplementary Material, Fig. S4). 3.4.5. Evaluation of toxicity of artemisinin-loaded nanoparticles in vivo Quantitative estimation of hepato- and renal toxicity markers studied 24 h post-administration of artemisinin and artemisininloaded nanoparticles (20 mg/kg bw) in healthy animals revealed no significant enhancement in the levels of these enzymes. There was a drastic elevation in the serum levels of creatinine and urea upon administration of AmB (5 mg/kg bw), indicating its renal toxicity (Supplementary Material, Table S1). 4. Discussion
Fig. 4. Leishmania-specific antibody levels 1-week post-treatment with artemisinin, ALPLGA nanoparticles, empty nanoparticles and AmB at higher doses measured by ELISA. Bars represent mean ± SE of five animals per group.
Nano-mediated drug delivery of plant-derived products is one of the most important strategies for the treatment of VL in the absence of adequate antileishmanial drugs. Artemisinin, an endoperoxide sesquiterpene lactone from A. annua, has been traditionally used for the treatment of malaria and has been reported to exhibit antileishmanial and antitumor activities [6,18]. Earlier, we have shown that formulation of artemisinin-loaded nanoparticles improved its activity against L. donovani amastigotes ex vivo. The present study demonstrates therapeutic efficacy of artemisinin-loaded nanoparticles in L. donovani-infected BALB/c mice, an experimental model for VL. Our study depicts that encapsulation of artemisinin in
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nanoparticles enhances its curative effect as noticed by the significant decrease in the hepatic and splenic parasite burden and organ weights after treatment. On the contrary, free artemisinin caused a marginal reduction in organ weight, reflecting the partial clearance of parasites from these organs. The reason behind encouraging results may be due to the small size of the nanoparticles that are easily taken up by the macrophage phagocytic system as evidenced by other drug delivery systems against the Leishmania infection, hence increasing the therapeutic accessibility of the drug to the amastigote-infected macrophages, thereby clearing the parasites [19,20]. However, AmB reduced the organ weight to normal levels and showed the highest activity almost eliminating the parasites in these organs, and the protection was significantly different compared to the other groups [21]. In the experimental VL, profound impairment of the host immune system is a grave concern requiring involvement of Tcells and effective cell-mediated immunity pivotal to successful chemotherapy [22]. In this study, we investigated the DTH response to leishmanial antigen (FT), an index of classical cell-mediated immunity. We found contemptible levels of Leishmania-specific DTH response in the control mice that adds to the failure of cell-mediated response corresponding to disease aggravation. The highest DTH was observed in the group treated with artemisininloaded nanoparticles and was comparable to AmB treatment, which was associated with a fall in liver and splenic parasite load. The positive DTH triggered by AmB is well corroborated by earlier studies, highlighting induction of cell-mediated immunity [23]. In the animals treated with artemisinin, minimal and insignificant gain in the DTH was detected, correlating with a partial reduction in splenic and liver parasite burden [24]. Active VL is characterized by T-cell unresponsiveness as well as production of Th2 cytokines to leishmanial antigen upon in vitro recall, whereas successful therapy is associated with restoration of host effective response with Th1-biased response [24,25]. Free artemisinin-treated group showed a marginal increase in the number of splenocytes, whereas no such change was depicted in the splenocytes of the group treated with empty nanoparticles. On the contrary, successful therapy with artemisinin nanoparticles as well as AmB manifested a significant increase in the lymphoproliferative response compared to the untreated infected group, revelatory of antileishmanial activity, hence almost completely restoring cell-mediated immunity instrumental for the elimination of the intracellular pathogen [26]. Lymphoproliferative response in artemisinin-loaded nanoparticles-treated animals was comparable to the levels induced upon AmB treatment, thus attributing its improved antileishmanial efficacy via modulation of the host immune system. Disease progression devolves on the switch of proinflammatory cytokines to anti-inflammatory cytokines, distorting the normal immune response by dampening the host-protective Th1 cytokines such as IFN-␥ and IL-2 and enhancing the secretion of Th2 cytokines such as IL-4 and IL-10 that cause the proliferation of B cells and production of non-specific Leishmania-specific IgG1 antibodies [27]. We attempted to evaluate the polarity of T-cell subsets toward protective Th1 or disease-exacerbating Th2 response in BALB/c mice models with established L. donovani infection. We found enhanced levels of IFN-␥ and IL-2 and diminishing levels of IL-4 and IL-10 in the artemisinin-loaded nanoparticles and AmB-treated animals compared to the infected untreated group. Our findings are in line from earlier studies leading to a plausible transition of host immune response from a disease-encouraging pattern to a diseaseresolution form after therapy [28]. Hypergammaglobulinemia and non-specific polyclonal antibody production are characteristic features associated with infection and are reinstated upon successful therapy [29]. Induction in the levels of parasite-specific antibodies such as IgG2a after
treatment is dependent on IFN-␥, whereas IgG1 correlates with IL-4. IgG2a and IgG1 are therefore used as surrogate markers of Th1 and Th2 response, respectively [28]. In the present study, we observed elevated levels of IgG2a parasite-specific antibodies after treatment with artemisinin-loaded nanoparticles and AmB, connotative of Th1 bias whereas low levels of IgG2a and higher IgG1 were observed in the infected control and empty nanoparticle-treated groups, suggestive of a dominance of Th2 response, indicating disease progression [30]. Artemisinin-treated group demonstrated a slight increase in the IgG2a parasite-specific antibody, reflecting an elusive shift toward Th1 immunity [31]. Apart from antigen T-cell receptor complex, a second stimulatory signal important for T-cell proliferation and optimal cytokine production is CD80. There is ample evidence to support that up-regulation of this molecule contributes to the restoration of cell-mediated immune response in experimental as well as clinical VL [32]. We thus investigated the status of B7 costimulatory molecules, CD80 (B7-1) on the macrophages derived from untreated uninfected, infected and treated BALB/c mice. The extent of CD80 expression mainly depends on the parasite load as has been reported earlier with lower level of expression on the infected macrophages or the antigen presenting cells, in vivo as well as in vitro [33]. In the present study, artemisinin-loaded nanoparticles triggered restoration of CD80 molecules, whereas partial restoration was observed on the macrophages derived from artemisinin-treated group, indicating enhanced therapeutic efficacy of the former. CD80 expression in the macrophages of AmB-treated animals was the highest and significantly different compared to other groups, indicating that AmB activates the antigen-presenting cells of the host and thereby modulates the immune system toward therapeutic cure. Thus, we believe that apart from killing the parasites directly, artemisinin-loaded nanoparticles also refurbish the expression of CD80 molecules needed for restoration of appropriate effector T-cell response. It has also been reported that Leishmania infection alters the expression of CD40 molecules on the antigen-presenting cells of murine model translating to disease susceptibility [33]. Restoration of CD40 expression on macrophages induces leishmanicidal activity and has been implicated in driving the immune response toward Th1 type, resulting in the clearance of parasites from the liver and spleen of infected animals. Our study clearly demonstrates that in the animals treated with artemisinin-loaded nanoparticles, expression of CD40 on murine macrophages is rescued which may be due to increased contact of drug pertaining to the large surface-to-volume ratio of nanoparticles leading to enhanced killing of the intracellular parasites [34]. Similarly, AmB treatment reverted CD40 levels toward normal levels and was significantly different compared to artemisinin-loaded nanoparticles treated and other groups. Immunomodulatory activity of nanoparticles or herbal extract loaded nanoparticles has recently been reported [35–37]. However, immunomodulation by nanoparticles loaded with a plant secondary metabolite, artemisinin, has not been reported yet. Thus, this study was the first to report immunopotentiation by artemisinin-loaded nanoparticles in experimental VL, augmenting its antileishmanial activity. Immunostimulation by artemisininloaded nanoparticles may be due to increase in the concentration of the drug in the vicinity of parasites that results in parasite killing and presentation of Leishmania antigens to the immune cells resulting in the enhancement of immune cell-related activities including lymphoproliferation, antibody and cytokine production. Artemisinin is usually considered as a safe drug without increased evidence of toxicity in vivo [38]. In this study, no overt signs of toxicity were observed in the animals administered with artemisinin-loaded nanoparticles or empty nanoparticles or free artemisinin, compared to normal control. However, AmB, the standard antileishmanial drug used for in vivo studies, resulted
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in elevated levels of creatinine and urea in the animals confirming its renal toxicity [39]. Thus, the present study suggests that artemisinin-loaded nanoparticles at the highest therapeutic dose administered to BALB/c mice are safe and do not cause any statistically significant variation in the levels of toxicity markers of liver and kidney. In conclusion, the findings of this study serve to emphasize that the leishmanicidal activity of artemisinin in experimental VL was accelerated by encapsulating them in PLGA nanoparticles. However, the curative effect was not as efficient as the standard antileishmanial drug, AmB. The enhanced parasite killing may have been mediated by inducing the delivery of artemisinin to the liver and spleen, hence restoring the cell-mediated immune response in immunosuppressed animals, leading to cure. Thus, the nano-encapsulation of artemisinin or conventional drugs or a combination of both may be a better strategy for extirpating the non-healing VL as well as other chronic intracellular infections. Conflict of interest None to declare. Author contributions F.A., M.Y.W., A.P.C. and A.K.D. conceived and designed the experiments; M.Y.W. and F.A. performed the experiments; M.I., G.C. and F.A. assisted in in vivo experiments; M.Y.W. and F.A. analyzed the data; M.Y.W. and F.A. wrote the paper and F.A., M.Y.W., H.A.H. and H.A.O. reviewed the manuscript. Acknowledgements The authors are highly thankful to Dr Nahid Ali for providing the Indian strain of L. donovani and BD FACSTM Jamia Hamdard for providing the facility of flow cytometer. This work was supported by research grants from the Department of Biotechnology (DBT, BT/PR9715/PBD/17/534/2007, BT/PR10876/NNT/28/137/2008), Department of Science and Technology (SR/FT/L-102/2006) and Central Council for Research in Unani Medicine (F. No. 3-77/2005-CCRUM/Tech), Government of India. M.Y.W. deeply acknowledges DBT and University Grants Commission, Government of India for providing the research fellowship.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]
[21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37]
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.04. 013
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[38] [39]
P. Desjeux, Comp. Immunol. Microbiol. Infect. Dis. 27 (2004) 305–318. C.R. Davies, P. Kaye, S.L. Croft, S. Sundar, Br. Med. J. 326 (2003) 377–382. R.J. Burchmore, M.P. Barrett, Int. J. Parasitol. 31 (2001) 1311–1320. A.K. Haldar, S. Banerjee, K. Naskar, D. Kalita, N.S. Islam, S. Roy, Exp. Parasitol. 122 (2009) 145–154. S. Kaur, K. Chauhan, H. Sachdeva, J. Med. Microbiol. 63 (2014) 1328–1338. R. Sen, S. Ganguly, P. Saha, M. Chatterjee, Int. J. Antimicrob. Agents 36 (2010) 43–49. Y. Chen, X. Lin, H. Park, R. Greever, Nanomedicine 5 (2009) 316–322. D.M. Yang, F.Y. Liew, Parasitology 106 (1993) 7–11. M.Y. Want, M. Islamuddin, G. Chouhan, A.K. Dasgupta, A.P. Chattopadhyay, F. Afrin, J. Colloid Interface Sci. 432 (2014) 258–269. F. Afrin, N. Ali, Infect. Immun. 65 (1997) 2371–2377. L.A. Stauber, Rice Institute Pamphlet 45 (1958) 80–96. K. Maasho, H.O. Akuffo, Scand. J. Immunol. 11 (1992) 179–184. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951) 265–275. D. Fulcher, S. Wong, Immunol. Cell Biol. 77 (1999) 559–564. T.M. McHugh, Methods Cell Biol. 42 (1994) 575–595. R. Banduwardene, A.B. Mullen, K.C. Carter, Int. J. Immunopharmacol. 19 (1997) 195–203. C. De Trez, M. Brait, O. Leo, T. Aebischer, F.A. Torrentera, Y. Carlier, E. Muraille, Infect. Immun. 72 (2004) 824–832. M.P. Crespo-Ortiz, M.Q. Wei, Biomed. J. Biotechnol. 2012 (2012) 247597. G.K. Gupta, S. Kansal, P. Misra, A. Dube, P.R. Mishra, AAPS PharmSciTech 10 (2009) 1343–1347. H. Van de Ven, M. Vermeersch, R.E. Vandenbroucke, A. Matheeussen, S. Apers, W. Weyenberg, S.C. De Smedt, P. Cos, L. Maes, A. Ludwig, J. Drug Target 20 (2012) 142–154. M. Mishra, U.K. Biswas, A.M. Jha, A.B. Khan, Lancet 344 (1994) 1599–1600. L. Das, N. Datta, S. Bandyopadhyay, P.K. Das, J. Immunol. 166 (2001) 4020–4028. E. Cenci, A. Mencacci, G. Del Sero, F. Bistoni, L. Romani, J. Infect. Dis. 176 (1997) 217–226. G.S. Nabors, J.P. Farrell, J. Infect. Dis. 173 (1996) 979–986. J.P. Haldar, S. Ghose, K.C. Saha, A.C. Ghose, Infect. Immun. 42 (1983) 702–707. E.M. Carvalho, O. Bacellar, C. Brownell, T. Regis, R.L. Coffman, S.G. Reed, J. Immunol. 152 (1994) 5949–5956. A.P. Taylor, H.W. Murray, J. Exp. Med. 185 (1997) 1231–1239. G.D. Miralles, M.Y. Stoeckle, D.F. McDermott, F.D. Finkelman, H.W. Murray, Infect. Immun. 62 (1994) 1058–1063. E.M. Carvalho, R.S. Teixeira, W.D. Johnson, Infect. Immun. 33 (1981) 498–500. J. Joshi, N. Malla, S. Kaur, Parasitol. Int. 63 (2014) 612–620. J. Roychoudhury, R. Sinha, N. Ali, PLoS ONE 6 (2011) 1–12. B. Saha, G. Das, H. Vohra, N.K. Ganguly, G.C. Mishra, Eur. J. Immunol. 25 (1995) 2492–2498. P.M. Kaye, N.J. Rogers, A.J. Curry, J.C. Scott, Eur. J. Immunol. 24 (1994) 2850–2854. H.W. Murray, C.M. Lu, E.B. Brooks, R.E. Fichtl, J.L. DeVecchio, F.P. Heinzel, Infect. Immun. 71 (2003) 6453–6462. B.C. Schanen, S. Das, C.M. Reilly, W.L. Warren, W.T. Self, S. Seal, D.R. Drake, PLoS ONE 8 (2013) 1–12. Y.C. Seo, W.Y. Choi, C.G. Lee, S.W. Cha, Y.O. Kim, J.C. Kim, G.P. Drummen, H.Y. Lee, Int. J. Mol. Sci. 12 (2011) 9031–9056. A. Dube, J.L. Reynolds, W.C. Law, C.C. Maponga, P.N. Prasad, G.D. Morse, Nanomedicine 10 (2014) 831–838. B. Angus, Expert. Rev. Clin. Pharmacol. 7 (2014) 299–316. R. Dietze, S.M. Fagundes, E.F. Brito, E.P. Milan, T.F. Feitosa, F.A. Suassuna, G. Fonschiffrey, G. Ksionski, J. Dember, Trans. R. Soc. Trop. Med. Hyg. 89 (1995) 309–311.
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