基于聚乳酸羟乙酸共聚物的青蒿琥酯纳米颗粒的制备，及其抗疟活性和毒性评价

Poly( D,L -lactic- co -glycolic acid)-based artesunate nanoparticles: formulation, antimalarial and toxicity assessments 基于聚乳酸羟乙酸共聚物的青蒿琥酯纳米颗粒的制备，及其抗疟活性和毒性评价 1 first-author 1 1 1 2 3 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zootund@yahoo.comoyeyemio@babcock.edu.ng †E-mail:zootund@yahoo.comoyeyemio@babcock.edu.ng zootund@yahoo.com 3 3 3 Department of Zoology, University of Ibadan, Ibadan 200284, Nigeria Department of Zoology, University of Ibadan, Ibadan 200284, Nigeria Department of Basic Sciences (Biology Programme), Babcock University, Ilishan-Remo 121103, Nigeria Department of Basic Sciences (Biology Programme), Babcock University, Ilishan-Remo 121103, Nigeria Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India Objective: 2 The aim of this study was to formulate polymer-based artesunate nanoparticles for malaria treatment. Methods: 2 Artesunate was loaded with poly( D,L -lactic- co -glycolic acid) (PLGA) by solvent evaporation from an oil-in-water single emulsion. Nanoparticles were characterized by X-ray diffraction and differential scanning calorimetry analyses. In vivo antimalarial studies at 4 mg/kg were performed on Swiss male albino mice infected with Plasmodium berghei . Hematological and hepatic toxicity assays were performed. In vitro cytotoxicity of free and encapsulated artesunate (Art-PLGA) to cell line RAW 264.7 was determined at concentrations of 7.8–1000 µg/ml. Results: 2 The particle size of the formulated drug was (329.3±21.7) nm and the entrapment efficiency was (38.4±10.1)%. Art-PLGA nanoparticles showed higher parasite suppression (62.6%) compared to free artesunate (58.2%, P < 0.05). Platelet counts were significantly higher in controls (305 000.00±148 492.40) than in mice treated with free artesunate (139 500.00±20 506.10) or Art-PLGA (163 500.00±3535.53) ( P < 0.05). There was no sign of hepatic toxicity following use of the tested drugs. The half maximal inhibitory concentration (IC 50 ) of Art-PLGA (468.0 µg/ml) was significantly higher ( P < 0.05) than that of free artesunate (7.3 µg/ml) in the in vitro cytotoxicity assay. Conclusions: 2 A simple treatment of PLGA-entrapped artesunate nanoparticles with dual advantages of low toxicity and better antiplasmodial efficacy has been developed. 目的 2 为疟疾治疗制定基于聚合物的青蒿琥酯纳米粒。 创新点 2 以聚乳酸羟乙酸共聚物（PLGA）为载体，制备青蒿琥酯纳米颗粒。并以小鼠为模型，评估其抗疟疗效和安全性。 方法 2 以PLGA为载体，采用从单一的水包油乳剂中进行溶剂蒸发的方法制备青蒿琥酯纳米颗粒。借助X射线衍射和差示扫描量热分析对纳米颗粒进行表征。以4 mg/kg的剂量对感染疟原虫的雄性瑞士白化小鼠进行体内抗疟活性的研究，测定血液和肝毒性的相关指标。体外实验以小鼠腹腔巨噬细胞细胞系RAW 264.7为模型，在7.8~1000 µg/ml浓度范围内，测定游离型和包裹型青蒿琥酯的细胞毒性。 结论 2 实验结果表明，纳米颗粒的粒径为(329.3±21.7) nm，包封率为(38.4±10.1)%。与游离青蒿琥酯（58.2%）相比，基于PLGA的青蒿琥酯纳米颗粒（Art-PLGA）具有较高的抑虫率（62.6%）， P < 0.05。就血小板计数结果而言，对照组（305 000.00±148 492.40）明显地高于游离青蒿琥酯组（139 500.00±20 506.10）和Art-PLGA组（163 500.00±3535.53）， P < 0.05。因此，药物的使用没有导致肝毒性的产生。体外细胞毒性试验结果表明，Art-PLGA的半数抑制浓度（IC 50 ，468.0 µg/ml）显著高于游离青蒿琥酯（7.3 µg/ml）， P < 0.05。基于PLGA的青蒿琥酯纳米颗粒是一种有效安全的抗疟治疗方法。 Poly( D,L -lactic- co -glycolic acid) (PLGA) Artesunate-PLGA delivery system Antiplasmodial Toxicity 青蒿琥酯-PLGA缓释系统 抗疟原虫 毒性 Introduction 1 Malaria is a life threatening disease, which has attracted significant attention in terms of developing or improving drug regimens. The problems of toxicity and poor bio-distribution of most of the mainstay drugs for most parasitic diseases have necessitated the search for better alternatives. Artesunate, a partially synthetic derivative of artemisinin, has been approved for the treatment of malaria and has also been extensively documented for its anticancer properties (Woerdenbag et al., 1993 1993 ; Efferth, 2007 2007 ; Liu et al., 2011 2011 ). Despite the therapeutic uses of artesunate, it has the disadvantage of a short half-life, therefore requiring frequent administration (Chadha et al., 2012 2012 ; Nguyen et al., 2015 2015 ). Its instability often causes easy degradation, resulting in poor pharmacokinetics and low bioavailability and pharmacological activity (Agnihotri et al., 2013 2013 ; Meng et al., 2014 2014 ). Therefore, development of a carrier that can maintain a sustained release profile and avoid rapid degradation of the drug is essential for its effective therapeutic usage. The high bioavailability, good encapsulation, controlled release, and low toxicity of biodegradable nanoparticles have resulted in their frequent use as drug delivery vehicles (Nguyen et al., 2015 2015 ). Poly( D,L -lactic- co -glycolic acid) (PLGA) is one of the most successful biodegradable polymers used for the development of nanoparticle treatments. Its hydrolysis within the body produces glycolic acid, which is a non-toxic biodegradable metabolite monomer (Kumari et al., 2010 2010 ). Along with approval for use in humans by the US Food and Drug Administration (Jain, 2000 2000 ), this polymer is a good candidate for preparation of drug delivery systems for diseases like cancer, schistosomiasis, malaria, and other infectious agents (Mainardes and Evangelista, 2005 2005 ; Acharya and Sahoo, 2011 2011 ; Pradhan et al., 2013 2013 ; Nguyen et al., 2015 2015 ). While polymeric particle delivery of artesunate has undeniably enjoyed wide therapeutic application, its antimalarial potency and toxicological effects have not been studied. Therefore, the aim of this study was to develop a polymeric drug delivery system for artesunate that has proper physical properties for improved malaria therapy and low toxicity. Materials and methods 1 Materials 2 Artesunate (from Artemisia annua L. (Asteraceae)), polyvinyl alcohol (PVA; M W =30–70 kDa), D -mannitol, dimethyl sulfoxide (DMSO), and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). PLGA (intrinsic viscosity η =0.41 dl/g, copolymer ratio 50:50, 45 kDa) was purchased from Purac Biochem, the Netherlands. Dichloromethane and acetone were procured from Merck Serono Ltd., Middlesex, UK. Water was purified using a Milli-Q plus system from Millipore (MQ water, Ontario, Canada). Culture medium RPMI-1640, fetal calf serum (FCS), and antibiotic-antimycotic were obtained from GIBCO Invitrogen (Grand Island, NY, USA). All other chemicals were of analytical grade. Preparation of nanoparticles 2 Artesunate-loaded PLGA (Art-PLGA) nanoparticles were formulated by solvent evaporation from an oil-in-water single emulsion (Chittasupho et al., 2009 2009 ). Briefly, 5 mg of artesunate was added to the organic phase containing 50 mg of polymer dissolved in 3.5 ml of dichloromethane and 0.5 ml of acetone to constitute a 1:10 (drug to polymer, w/w) ratio. The organic phase was then added drop-wise to 16 ml aqueous solution (2% PVA as emulsifier) with sonication using a sonicator (Qsonica sonicators, Newtown, USA) at 30 W with a 40% duty cycle for 3 min in ice-cold water. The emulsion was continuously stirred by magnetic stirrer (Thermo Scientific, Massachusetts, USA) at 300 r/min for 6 h to evaporate the solvent, leaving behind a colloidal suspension of the drug-encapsulated nanoparticles in aqueous phase. The formulation was centrifuged at 16 000 r/min for 15 min using an ultracentrifuge (Beckman Coulter, Atlanta, USA) and then washed three times with MQ water. Dry powders were obtained by lyophilization of frozen samples in a freeze dryer (Labconco FreeZone 2.5, Kansas City, USA) in the presence of 5% mannitol as a cryoprotectant. Measurement of particle size and zeta potential 2 The particle diameter, zeta potential, and polydispersity index (PDI) of the Art-PLGA-entrapped nanoparticulate drug were measured by a dynamic light scattering method using a Zetasizer Nano-ZS system (Malvern Instruments, UK). A homogenous solution obtained from dispersal of an appropriate amount of the formulated particle in MQ water was transferred to a clear disposable sizing cuvette for size and PDI measurements. A clear zeta cell was used for zeta potential analysis. Readings were taken in triplicate and results were expressed as mean±standard deviation (SD). X-ray diffraction analysis and differential scanning calorimetry 2 X-ray diffraction (XRD) analysis was conducted on lyophilized Art-PLGA nanoparticles using an X’Pert-Pro multipurpose X-ray diffractometer (PANalytical, the Netherlands). The CuKα radiation was generated at 45 kV and 40 mA in the diffraction angle (2 θ ) range of 5°–40°. Differential scanning calorimetry (DSC) was conducted on Art-PLGA nanoparticles to characterize the physical state of the drug. Thermograms were obtained using a Pyris 1 differential scanning calorimeter (PerkinElmer, USA). Dry nitrogen gas was used to purge the DSC cell at a flow rate of 40 ml/min. About 6–8 mg of the formulation was sealed in a standard aluminum pan with a lid and heated at a rate of 5 °C/min from 50 °C to 300 °C. Drug entrapment and encapsulation efficiency 2 Drug entrapment and encapsulation efficiency were determined using a modified form of the method described by Anitha et al. ( 2011 2011 ). Lyophilized drug-loaded nanoparticles (10 mg) were dissolved in 1 ml acetonitrile (a common solvent for polymeric particles and drugs). The solution was thoroughly mixed and subjected to solvent evaporation for 9–10 h at 50 °C using a heater (CH-100, Biosan Ltd., Cambridge, UK). The residue was resuspended in 500 µl methanol, vortexed and centrifuged at 13 000 r/min for 20 min. The supernatant (500 µl) was collected and stored. The process was repeated with 500 µl of acetonitrile. A stock solution (1000 µg/ml) of artesunate was prepared by dissolving 5 mg of the drug in methanol (5 ml). The drug concentration of non-entrapped artesunate was determined using an ultraviolet-visible (UV-vis) spectrophotometer (Ultrospec 2100 pro, Amersham Biosciences, USA) at λ max of 222 nm. Calibration plots of UV-vis spectrophotometer analysis of non-entrapped drug were obtained at concentrations ranging from 5 to 70 µg/ml. The supernatant containing encapsulated drug was analyzed by the same method. Then the drug content and encapsulation efficiency of the formulation were estimated. Murine model and parasite strain 2 In vivo assays adhered to the Principles of Laboratory Animal Care (National Research Council, 2010 2010 ). Swiss male albino mice were obtained from the Animal House Centre of the Department of Pharmacology, University of Ibadan, Nigeria. The rodent malaria line used was Plasmodium berghei NK-65 obtained from the Institute of Advanced Medical Research and Training (IAMRAT), University College Hospital, University of Ibadan, Nigeria. Animals and conditions 2 A total of 15 Swiss male albino mice ((20.0±2.0) g) of 5–6 weeks old were used in a Peters’ 4-d suppressive test on three groups with five mice each group. Animals were maintained in standard pathogen-free conditions and fed ad libitum. Antiplasmodial evaluation using the Peters’ 4-d suppressive test 2 On Day 0, P . berghei -infected erythrocytes (pEry) were obtained from an infected donor Swiss male mouse and diluted in physiological saline to 1×10 7 pEry/ml. Mice were infected intraperitoneally (ip) with an aliquot of 0.2 ml of the parasite suspension. The lyophilized Art-PLGA-entrapped nanoparticles were reconstituted in distilled water containing 10% Tween 80. At 2 h post-infection, the mice were treated orally with 0.1 ml of the nanoparticulate drug suspension with an equivalent artesunate composition of 4 mg/kg of body weight (Chinaeke et al., 2015 2015 ). The positive control group was treated orally with free artesunate at a dose of 4 mg/kg. The negative control group received 0.2 ml of the vehicle. On Days 1–3 post-infection, the Day 0 treatments were repeated in the experimental groups of mice. On Day 4 post-infection, blood smears prepared from the tail vein were Giemsa-stained and examined microscopically (Cheesbrough, 1998 1998 ). The percentage parasitemia was determined microscopically by counting the number of infected erythrocytes among total erythrocytes in four fields of view. The percentage suppression of P . berghei was evaluated on the 4th day after treatment using the formula: ( A − B )/ A ×100%, where A is the mean parasitemia in the untreated group (negative control) and B is the mean parasitemia in each treated group (Tona et al., 2001 2001 ). Animals were monitored daily for clinical signs and weight loss. Rectal temperature and changes in weight 2 The rectal temperature and body weight of the mice and their baseline mean parasitemia before treatment were measured. The same measurements were repeated 4 d post treatment with free artesunate or the PLGA-encapsulated artesunate nanoparticulate drug. Hematological and hepatic toxicity assays 2 Acclimatized albino rats (weight 94–105 g) were administered with drugs at similar concentrations to those used for the antiplasmodial study (i.e. 4 mg/kg for free and nano-formulated artesunate) for 4 d. Blood samples (2 ml) were collected from the rats through a retro-orbital puncture, into ethylene diamine tetraacetic acid (EDTA) tubes. Hematological parameters including packed cell volume (PCV), red blood cell (RBC) count, white blood cell (WBC) count, neutrophils, eosinophils and hemoglobin (Hb) levels were evaluated in accordance with standard procedures. The cyanomethemoglobin method was used for the measurement of Hb levels; a hemocytometer was used to estimate the RBC and WBC counts, while PCV was measured by the conventional method of filling capillary tubes with blood and centrifuging using a microhematocrit centrifuge. The concentrations of plasma enzymes like aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphate (ALP) were determined using Randox diagnostic kits. Cell lines and culture 2 Murine RAW 264.7 macrophages were obtained from Product Development Cell-1 (National Institute of Immunology, New Delhi, India). The equilibrated cells were incubated in RPMI-1640 (Sigma, St. Louis, MO, USA) containing 10% FCS and 1% penicillin G/streptomycin/amphotericin B (PSA) at 37 °C and 5% CO 2 in an air humidified incubator. Cells were harvested when confluence was reached, and were washed with RPMI medium. The cells were diluted to 5×10 4 cells/ml with 200 µl of the cell suspension seeded per well in sterile 96-well plates and incubated for 24 h to allow cell attachment. In vitro cell viability 2 The method of Zhang and Feng ( 2006 2006 ) was adopted with some modifications. Briefly, the seeded RAW 264.7 cells (density, 5×10 4 viable cells/well) were incubated with free drug or the drug-loaded PLGA nanoparticle suspension at concentrations ranging from 7.8 to 1000 μg/ml for 24 h. At designated time intervals, the medium was removed and 100 µl of culture medium and 10 µl of MTT (5 mg/ml in phosphate-buffered saline (PBS)) were added to the wells. After incubation for 3–4 h, the culture solution was removed, leaving the precipitate of formazan crystals. DMSO (100 µl) was then added to each well before the plate was analyzed by a microplate reader at 570 nm. Cell viability was calculated using the following equation: Cell viability (%)=Abs s /Abs control ×100%, where Abs s is the absorbance of the cells incubated with the PLGA-loaded nanoparticle suspension, and Abs control is the absorbance of the control containing cells incubated with the culture medium only. IC 50 is the concentration at which the free drug or nanoparticle-entrapped drug inhibits cell growth by 50%, and is calculated using GraphPad Prism 6 statistical software (GraphPad Software Inc., La Jolla, CA, USA). Statistical analysis 2 Data were entered in an MS-Excel spreadsheet and transferred to GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA, USA) for analysis. Two-way analysis of variance (ANOVA) and Tukey’s multiple comparison tests were used to test for significant differences. A P -value of < 0.05 was considered to be statistically significant. Cytotoxicity was expressed as the mean inhibition relative to the unexposed control±SD for three parallel readings. Results 1 Characterization of nanoparticles 2 The size, PDI, and zeta potential of Art-PLGA-entrapped nanoparticles were (329.3±21.7) nm, 0.355±0.04, and (−17.4±7.1) mV, respectively. The drug entrapment efficiency (%EE) was (38.4±10.1)%. The effects of surfactants and drug/polymer ratios on nanoparticle characteristics are presented in Table 1 1 . Data are expressed as mean±SD ( n =3); OP: organic phase; EAP: external aqueous phase; PVA: polyvinyl alcohol; SLS: sodium lauryl sulfate; PDI: polydispersity index  Surfactant OP:EAP Drug:PLGA Particle size (nm) * * Zeta potential (mV) * * PDI * *  1% PVA 1:5 (4 ml:20 ml) 1:5 (10 mg:50 mg) 3330.00±1.88 −13.3±3.3 0.869±0.094  2% SLS 1:4 (4 ml:16 ml) 1:10 (2.5 mg:25 mg) 340.60±5.15 −14.1±3.9 0.322±0.124  2% PVA 1:5 (4 ml:20 ml) 1:5 (10 mg:50 mg) 7300.00±1.70 −24.5±12.4 1.000±0.000  2% PVA 1:4 (4 ml:16 ml) 1:10 (5 mg:50 mg) 329.30±21.70 −17.4±7.1 0.355±0.040  1% SLS 1:4 (4 ml:16 ml) 1:10 (5 mg:50 mg) 3700.00±0.60 −16.6±22.5 0.653±0.122 The crystalline contents of the formulated nanoparticles were determined by powder X-ray diffraction (P-XRD) analysis. The P-XRD diffractograms for Art-PLGA showed two peaks representing 2 θ of 42.1° and 43.7°, respectively (Fig. 1 1 ). The DSC thermogram of PLGA-entrapped artesunate showed no intensive endothermic peak for artesunate. However, a sharp PLGA relaxation peak at 47 °C was observed along with a diminished PLGA glass transition peak from 55 °C to 65 °C (Fig. 2 2 ). A very weak thermal event was present between 140 °C and 150 °C. Intensive endothermic events occurred from 260 °C to 300 °C. Parasite suppression 2 The percentage parasite suppression was lower (58.2%) in the free artesunate group than in the group treated with the nano-synthesized form (62.6%) at 4 d post-treatment. No parasite reduction was observed in the control group (Table 2 2 ). Similar superscript letters in the same column denote no significant difference while different letters denote a significant difference. Parasitemia denotes mean of parasite counts in 5 mice representing each experimental group. Data are expressed as mean±SD ( n =5) 2 Group 2 Parasitemia * * 2 Parasite suppression (%) 3 Weight (g) * * 3 Temperature (°C) * * Before After P Before After P Art-free 1.9±0.7 a 58.2 28.0±3.2 26.0±1.3 >0.05 34.5±1.0 36.8±1.0 <0.05 Art-PLGA 1.7±0.1 a 62.6 28.2±2.7 29.2±1.7 >0.05 33.7±0.4 36.2±0.9 <0.05 Control 4.5±0.6 b 24.2±3.2 23.2±1.2 >0.05 36.1±0.5 36.3±1.0 >0.05 Variation in body weight and rectal temperature 2 The body weight of mice decreased from (28.0±3.2) g on Day 0 to (26.0±1.3) g on Day 4 after treatment with free artesunate, and from (24.2±3.2) g to (23.2±1.2) g in the control group during the same period ( P > 0.05). However, there was a weight gain from (28.2±2.7) g to (29.2±1.7) g in mice treated with the Art-PLGA nanoparticulate drug ( P > 0.05) (Table 2 2 ). There was a significant temperature gain from (34.5±1.0) °C to (36.8±1.0) °C in mice treated with free artesunate and from (33.7±0.4) °C to (36.2±0.9) °C in mice treated with Art-PLGA nanoparticulate drugs ( P < 0.05) (Table 2 2 ). No temperature gain was observed in the control group. Variation in hematological and liver function parameters 2 There was no significant variation in most of the observed hematological parameters in free or nano-formulated artesunate exposed groups compared with the control group ( P > 0.05). However, the platelet counts (305 000.00±148 492.40) observed in the control group were significantly higher than those in the free (139 500.00±20 506.10) and nano-formulated drug (163 500.00±3535.53) treatment groups (Table 3 3 ). There were no significant differences in AST or ALT concentrations in all experimented groups, whereas the ALP concentration was significantly lower in the free artesunate group ((70.5±19.1) U/L) than in the control ((120.5±6.4) U/L) and the nano-synthesised groups ((110.5±4.9) U/L) ( P < 0.05; Fig. 3 3 ). AST: aminotransferase; ALT: alanine aminotransferase; ALP: alkaline phosphate; Art: artesunate; Art-PLGA: artesunate entrapped PLGA nanoparticles. Data are expressed as mean±SD ( n =5) Similar superscript letters in the same column denote no significant difference while different letters denote a significant difference. PCV: packed cell volume; Hb: hemoglobin; RBC: red blood cell; WBC: white blood cell. Data are expressed as mean±SD ( n =5) Group PCV (%) Hb (g/dl) RBC (×10 6 /cm 3 ) WBC (×10 3 /cm 3 ) Platelet (×10 3 /cm 3 ) Lymphocyte (%) Neutrophil (%) Monocyte (%) Artesunate 37.00±2.83 a 12.20±0.92 a 5.80±0.61 a 6.10±1.17 a 139.50±20.51 a 64.00±2.83 a 31.50±3.54 a 2.50±0.71 a Art-PLGA 37.50±2.12 a 12.00±0.57 a 6.20±0.08 a 8.80±2.47 a 163.50±3.54 a 70.00±0.00 a 26.50±0.71 a 1.50±0.71 a Control 37.00±2.83 a 12.10±0.78 a 5.70±0.59 a 12.00±8.94 a 305.00±148.49 b 63.50±2.12 a 32.00±1.41 a 2.00±1.41 a In vitro cell viability 2 Art-PLGA nanoparticles showed lower toxicity than the free drug. Cell viability data following treatment with free artesunate or Art-PLGA-entrapped nanoparticles are presented in Fig. 4 4 . Cell viability varied in a concentration-dependent manner ( P < 0.001). Generally, cells became less viable at higher doses. The IC 50 of Art-PLGA (468.0 µg/ml) was significantly higher than that of free artesunate (7.3 µg/ml). IC 50 : half maximal inhibitory concentration; Art: artesunate; Art-PLGA: artesunate-entrapped PLGA nanoparticles. Data are expressed as mean±SD ( n =3). *** P < 0.001, vs. free Art Discussion 1 The method employed in the formulation yielded particles which appeared soluble and finely dispersed in water when compared to the free non-entrapped drugs. Dry lyophilized nanoparticles have been shown to have good physical and chemical stability and can be stored at room temperature for over 6 months without decomposition or aggregation (Bhawana et al., 2011 2011 ). The influence of the larger surface area of nanoparticles in promoting dissolution has been emphasized (McNeil, 2005 2005 ). The negative zeta potential denotes non-toxicity and the values for all formulations fall within the acceptable range for easy charge neutralization, thus favoring less interference in in vivo studies (Nordström, 2011 2011 ). The PDI of our formulation, which was less than 0.4, will enhance moderate distribution (Nobbmann, 2014 2014 ). The nanoparticles could, however, be further optimised to give a narrow range of monodispersed PDI for better drug distribution. The mean Art-PLGA nanoparticle size ((329.3±21.7) nm) observed in our study was larger than that found in a related study ((289.6±4.6) nm) (Nguyen et al., 2015 2015 ). Our formulation also showed lower drug entrapment efficiency ((38.4±10.1)%) than that shown in that study ((42.36±0.73)%). The PLGA concentration, volume of the aqueous phase, and type of surfactants and stability agents used are known to affect particle size, PDI, and encapsulation efficiency (Wohlfart et al., 2011 2011 ; Cooper and Harirforoosh, 2014 2014 ). An increase in aqueous phase volume produces small nanoemulsion droplets, which decrease the size and PDI of the particles while an increase in PLGA concentration increases the viscosity of the polymer solution, resulting in poorer dispersibility of the organic phase in the aqueous phase (Nguyen et al., 2015 2015 ). The diffractogram patterns showing peaks at 42.1° and 43.7° 2 θ suggest the amorphous nature of Art-PLGA (Panda et al., 2016 2016 ). The absence of a decomposition exotherm indicates the increased physical stability of the drug in the formulation (Chadha et al., 2012 2012 ). The thermogram of the Art-PLGA nanoparticles, which did not show any intensive endothermic peaks, indicates that chemical interactions which could alter the drug or the polymer properties did not occur during particle formulation (Panda et al., 2016 2016 ). However, the presence of endothermic events at 260–300 °C could be due to residual excipients or the presence of impurities in the drug or polymer. It is also possible that polymers are corrosive in nature and their melting and interaction with the metal pan in previous samples may have resulted in the generation of peaks in non-specific regions. The very weak thermal event between 140 °C and 150 °C could have been due to the melting of artesunate. However, in the Art-PLGA formulation, the amount of artesunate was small compared to that in PLGA, and hence no significant endotherm was visible. The increase in suppression of P . berghei in Art-PLGA-treated mice compared with those in the free drug and control groups was consistent with the findings of a report on the antimalarial properties of artesunate-loaded solid lipid microparticles, in which improved activity was linked to the drug’s sustained release action (Chinaeke et al., 2015 2015 ). There was no parasite suppression in the control group since no drug was administered. The P . berghei murine model has been widely adopted for systematic screening of potential antimalarial drugs at the early stages of drug discovery. The screening results, however, do not always translate to the occurrence of Plasmodium falciparum malaria in man. Thus, there is a need to perform similar studies in a P . falciparum animal model (Ibrahim et al., 2014 2014 ). The efficacy of Art-PLGA nanoparticles may be related to their ability to penetrate mucus and microvilli barriers. Nanoparticles as large as 500 nm encapsulated with a muco-inert polymer have been observed to traverse physiological human mucus rapidly, with diffusivities only 4-fold lower than their rates in pure water (Lai et al., 2007 2007 ). The capacity of the polymeric nanoparticles to protect the encapsulated drug and its stability in the digestive tract have been reported to facilitate improved cellular uptake and controlled drug release at the target (des Rieux et al., 2006 2006 ). The negative association between P . berghei parasitemia and rectal temperature as observed in free artesunate and Art-PLGA-treated groups was consistent with earlier reports (Dikasso et al., 2006 2006 ; Chinaeke et al., 2015 2015 ). While weight loss in the control group could be associated with parasite burden, the loss of weight in the group treated with free artesunate could also result from toxic effects of the drug. There were no significant differences in most of the observed hematological parameters between mice administered with free artesunate or Art-PLGA nanoparticles and the control groups. However, the significantly lower platelet counts in the treated groups compared with the control group were consistent with the claim that the best side effect of artemisinin-based compounds is that they lower reticulocyte counts (Clark, 2012 2012 ). Artesunate’s interaction with erythrocyte plasma membranes initiates a reaction between the drug and ferrous iron in the heme prosthetic group of hemoglobin, thus activating the drug to form toxic free radicals (Clark, 2012 2012 ). The mechanism of action of artesunate and its ability to accumulate in the erythrocytes are associated with hemotoxicity (Bigoniya et al., 2015 2015 ). The controlled release of artesunate from PLGA, however, could have resulted in the higher reticulocyte counts compared with the free drug. The lack of significant differences in transaminases, the important markers of hepatocellular toxicity and damage (Faber et al., 1981 1981 ), suggests that free and nanoparticulate drugs are not toxic to the liver at the tested concentrations. 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The drug encapsulation efficiency, in vitro drug release, cellular uptake and cytotoxicity of paclitaxel-loaded poly(lactide)-tocopheryl polyethylene glycol succinate nanoparticles Oyetunde OYEYEMI acknowledges the Centre for Science and Technology of the Non-Aligned and Other Developing Countries (NAM S&T Centre) in collaboration with the Department of Science & Technology (DST), the Government of India for “Research Training Fellowship for Developing Country Scientists (RTF-DCS)” award, undertaken at the National Institute of Immunology, New Delhi, India. Compliance with ethics guidelines Kabiru DAUDA, Zulaikha BUSARI, Olajumoke MORENIKEJI, Funmilayo AFOLAYAN, Oyetunde OYEYEMI, Jairam MEENA, Debasis SAHU, and Amulya PANDA declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed. 10.1631/jzus.B1600389 2016-09-05 2016-12-14 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany 2017 2017 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 11 18