Int J Clin Exp Med 2015;8(4):5150-5162 www.ijcem.com /ISSN:1940-5901/IJCEM0005923 Original Article Improved antifungal activity of amphotericin B-loaded TPGS-b-(PCL-ran-PGA) nanoparticles Xiaolong Tang1,2*, Ronghong Jiao3*, Chunmei Xie4, Lifa Xu1, Zhen Huo1, Jingjing Dai1, Yunyun Qian1, Weiwen Xu4, Wei Hou2, Jiang Wang1, Yong Liang5 1Stem Cell Engineering Research Center, School of Medical, Anhui University of Science & Technology, Huainan 232001, Anhui, P.R. China; 2State Key Laboratory of Virology/Institute of Medical Virology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071, P.R. China; 3Department of Clinical Medical Laboratory, Shanghai Pudong New Area People’s Hospital, Huai’an, Shanghai 201299, P.R. China; 4School of Bioteclmology, Southern Medical University, Guangzhou 510515, China; 5Clinical Laboratory, The Affiliated Huai’an Hospital of Xuzhou Medical College, Huai’an, Jiangsu 223002, P.R. China. *Equal contributors. Received January 14, 2015; Accepted March 19, 2015; Epub April 15, 2015; Published April 30, 2015 Abstract: To develop amphotericin B-loaded biodegradable TPGS-b-(PCL-ran-PGA) nanoparticles (PLGA-TPGS-AMB NPs) for fungal infection treatment, PLGA-TPGS NPs and PLGA NPs were synthesized by a modified double emulsion method and characterized in terms of size and size distribution, morphology and zeta potential. Drug encapsulation efficiency, in vitro drug release, and in vitro/vivo tests against Candida glabrata were completed. The data showed that both of the two AMB-loaded NPs (PLGA-AMB NPs, PLGA-TPGS-AMB NPs) achieved significantly higher level of antifungal effects than water suspended AMB. In comparison with PLGA-AMB NPs, PLGA-TPGS-AMB NPs had a stronger protective effect against candidiasis and gained an advantage of prolonged antifungal efficacy. In conclu- sion, PLGA-TPGS-AMB NPs system significantly improves AMB bioavailability by increasing the aqueous dispersibility and improving the antifungal activity. And this would be an excellent choice for the antifungal treatment of the en- trapped drug because of its low toxicity and higher effectiveness. Keywords: Antifungal, Candida glabrata, amphotericin B, nanoparticle, TPGS-b-(PCL-ran-PGA) Introduction tides and amino acids leak, which in turn leads to cellular dysfunction and eventually to cell Amphotericin B (AMB) is a lipophilic polyene death [4, 5]. This is mainly valid to AMB for antifungal agent, which was initially secluded Candida, Cryptococcus, Histoplasma, yeast, from a strain of Streptomyces nodosus. It is an dermatitis blastomycosis and Coccidioides. amphoteric compound composed of a hydro- Broad antibacterial spectrum is in favor of the philic polyhydroxyl chain along one side and a treatment of fungal infections via oral/intrave- lipophilic polyene hydrocarbon chain on the nous administration. However, AMB has low bio- other [1]. AMB is sparingly soluble in water [2], availability via oral administration because it is and has a poor oral absorption. It has antibac- a highly hydrophobic weak base with a low terial activity against almost all fungi at the aqueous solubility of approximately 1 ng/ml at minimum inhibitory concentration (MIC) of pH 7 [1, 2]. 0.02-1 mg/L. AMB inhibits membrane enzymes like proton ATPase in fungal cells [2, 3] and Recently, drug delivery system (DDS) has Na+/K+-ATPase in mammalian cells and this received substantial attention in the field of inhibitory activity depletes cellular energy drug development. In DDS, pharmacological reserves and inhibits cell proliferative ability techniques are used to control pharmacokinet- [4]. Moreover, the interaction of AMB with mem- ic properties (absorption, distribution, metabo- brane sterol changes the fungal cell membrane lism, and excretion) and to improve the efficacy permeability and results in an essential cellular and safety of a drug. Copolymer formulations, substances such as potassium ions, nucleo- such as nanoparticles and emulsion based car- PLGA-TPGS-AMB NPs improved antifungal activity Figure 1. Schematic description of the synthesis of PLGA-TPGS random copolymer. riers, are highly predictable and are now ual degradation in the body fluid, and variable explored in numerous directions, and several routes of administration [8]. D-α-tocopheryl products have already been made commercial- polyethylene glycol 1000 succinate-b-poly ly available [6, 7]. Nanoparticle drug delivery (ε-caprolactone-ran-glycolide) [TPGS-b-(PCL- systems such as biodegradable nanoparticles ran-PGA), PLGA-TPGS] was a recently devel- of poly (D, L-lactide-co-glycolide) (PLGA) have oped diblock copolymer. It demonstrated good the advantages of increasing drug bioavailabil- biocompatibility and biodegradability [8]. ity, decreasing the fluctuation of drug concen- Moreover, PLGA-TPGS NPs have higher drug tration in blood, continuous drug release, grad- encapsulation and cellular uptake, longer half 5151 Int J Clin Exp Med 2015;8(4):5150-5162 PLGA-TPGS-AMB NPs improved antifungal activity Figure 2. The physical properties of the star-shaped NPs. A, B: FESEM image of the PLGA and PLGA-TPGS NPs. life and higher therapeutic effects of the formu- ly 23,000) copolymers were obtained from the lated drug than PLGA NPs [8, 9]. Graduate School at Shenzhen, Tsinghua Uni- versity. Acetonitrile and methanol were pur- Fungal infections have significantly contributed chased from EM Science (Chrom AR, HPLC to the increasing morbidity and mortality of grade, Mallinckrodt Baker, USA). All other patients, especially immunocompromised pa- agents were of analytical grade or higher quali- tients [8, 9]. Therefore, this research aims to ty commercially available. Millipore water was investigate the fungistatic and fungicidal prepared by a Milli-Q Plus System (Millipore effects of the AMB-loaded PLGA-TPGS NPs/ Corporation, Breford, USA). C. Glabrata (ATCC PLGA NPs. Since Candida spp. represent one of 90030) was obtained from American Type the most common pathogenic yeasts blamed Culture Collection (ATCC; Rockville, MD, USA). for life-threatening fungal infections [10], we chose Candida glabrata as (C. glabrata) a Animals and fungal inoculation model fungus for the antimycotic capability test 20-22 g BALB/c mice of 6-8 weeks old were of drug-loaded NPs. purchased from the animal house of Anhui Materials and methods Medical University (China). They had free access to food and water. The local Institute’s Chemicals Animal Ethics Committee approved all of the animal studies. The investigations conformed D-α-tocopheryl polyethylene glycol 1000 succi- to the Guide for the Care and Use of Laboratory nate (TPGS, C O H (CH CH O) ), D, L-lactide Animals published by the US National Institute 33 5 54 2 2 23 (3,6-dimethyl-1,4-dioxane-2,5-dione, C H O ) of Health (NIH Published No. 85-23, revised 6 8 4 with a purity greater than 99% and glycolide 1996). Three days before the challenge, (1,4-Dioxane-2,5-dione, C H O ) (purity > 99%) Candida glabrata ATCC 90030 was subcul- 4 4 4 and poly (lactide-co-glycolide) (PLGA, Mw tured daily in Sabouraud’s dextrose broth. The approximately 25,000), Amphotericin B (AMB), subculture was pelleted and rinsed twice in Stannous octoate (Sn(OOCC H ) )and couma- sterile phosphate buffered saline (PBS) on the 7 152 rin-6 were purchased from Sigma-Aldrich (St. day of challenge. The final pellet was resus- Louis, MO, USA). PLGA-TPGS (Mw approximate- pended in PBS. The concentration of blasto- 5152 Int J Clin Exp Med 2015;8(4):5150-5162 PLGA-TPGS-AMB NPs improved antifungal activity Table 1. Characterization of AMB-loaded nanoparticles (n = 3) surement. Zeta potential of the nanoparticles was determined Polymer Particle size (nm) PDI ZP (mV) LC (%) EE (%) by Zeta Plus zeta potential ana- PLGA-AMB 112.2 ± 4.3 0.265 -23.6 ± 0.5 8.11 76.01 lyzer (Brookhaven Corporation). PLGA-TPGS-AMB 122.7 ± 3.9 0.199 -19.5 ± 0.6 9.45 84.37 10 μL suspended NPs were Note: PDI = polydispersity index, ZP = zeta potential, LC = loading content, EE = dropped onto platinum-coated entrapment efficiency. copper grids and stained with 2% wt/vol phosphotungstic acid. spores was counted with a hemacytometer and The particles were then coated with a platinum prepared for the following experiments. layer using a JFC-1300 automatic fine platinum coater (JEOL) for 30 seconds in a vacuum. The Preparation of PLGA NPs and PLGA-TPGS NPs surface morphologies of the NPs were observed by a field emission scanning electron micros- PLGA-TPGS random copolymers were synthe- copy using a JEOL JSM-6700F system (JEOL, sized and characterized by a previously des- Tokyo, Japan) operated at a 5.0 kV accelerating cribed method [8, 9]. 50 mg PLGA-TPGS ran- voltage. dom copolymer and 10 mg AMB were fully dis- solved in 10 mL dichloromethane (DCM). The To determine the contents of drug loading (LC) formed solution was poured into 50 mL 3% and entrapment efficiency (EE) of the AMB- (w/v) polyvinyl alcohol solution under stirring. loaded nanoparticles, a predetermined amount The mixture was sonicated for 10 min with puls- of nanoparticles was dissolved in 1 mL methy- es of 10 s on and 10 s off at 600 W output to lene dichloride under vigorous vortexing. The form oil/water (O/W) emulsion. The organic sol- solution was transferred to 5 mL of mobile vent was then allowed to evaporate overnight phase consisting of acetonitrile and deionized under continuous magnetic stirring at 500 rpm. water (50:50, v/v). A nitrogen stream was intro- The reaction synthesis is schematically des- duced to evaporate the methylene dichloride cribed in Figure 1. The hydroxyl end of TPGS for approximately 20 min, and then a clear solu- served as initiator that selectively cleaves acyl tion was obtained for HPLC analysis (LC 1200, oxygen chain of lactide or glycolide. The forma- Agilent Technologies, Santa Clara, CA, USA). A tion of the NPs and the encapsulation of AMB reverse-phase C18 column (250 × 4.6 mm, 5 within the polymeric matrix were accomplished μm, Agilent Technologies, Santa Clara, CA, USA) during the DCM evaporation process. The sus- was used at 25°C. The flow rate of the mobile pension was then dialyzed in a Regenerated phase was 0.5 mL/min. The column effluent Cellulose Dialysis Membrane (Spectra/Por 6, was detected using a UV detector at λmax of MWCO = 1000; Spectrum, Houston, Texas) bag 297 nm. The measurement was performed in with a molecular weight cut-off of 100 kDa to triplicate. The LC and EE of the AMB-loaded remove the emulsifier and unencapsulated nanoparticles were calculated by the following AMB. The bag was put into a 3 L tank with nano- equations, respectively: pure water, at a ratio of 100:1 nanopure water to NPs, under gentle stirring for 8 h with the LC(%) = (Weight of AMB in the nanoparticles)/ water changed every 4 h. The resulted suspen- (Weight of the nanoparticles) × 100%; sion was freeze-dried at -50°C at a reduced pressure of 0.13 Pa for 48 h. The resulted EE(%) = (Weight of AMB in the nanoparticles)/ PLGA-TPGS-AMB NPs were stored at -8°C for (Weight of the feeding AMB) × 100%. further analysis. PLGA-AMB NPs were synthe- In vitro release of AMB from NPs sized following the same procedure. Nanoparticles characterization and drug con- In vitro AMB release from nanoparticle formula- tent and entrapment efficiency tions was performed as described previously [8, 9]. 10 mg of accurately weighted lyophilized The average size and size distribution of the nanoparticles was put into a centrifuge tube prepared NPs were measured by Dynamic Light and redispersed in 15 mL PBS (containing 0.1% Scattering (Brookhaven Instruments Corpo- w/v Tween 80, pH 7.4). The tube was put into an ration, Holtsville, New York). 2 mg dried NPs orbital shaker water bath and vibrated at 150 were suspended in 200 μl ddH O before mea- rpm at 37°C. At certain time intervals, the tube 2 5153 Int J Clin Exp Med 2015;8(4):5150-5162 PLGA-TPGS-AMB NPs improved antifungal activity incubation for 12 h, the C. gla- brata cells were harvested and washed twice with PBS (pH 7.4). The cell wall was incubated with a lysing enzyme mixture (1.2 U of chi- tosanase, 1.3 U of chitinase, 1.5 U of lyticase, and 10 mg/ ml lysing enzyme [Sigma]) for 5 h at 30°C. It was then digested and analyzed for the presence of apoptotic mark- ers with Annexin V/PI; stains show the presence of apop- totic cells (green fluores- cence) and necrotic cells (red fluorescence), as described by Madeo and Shirazi and Kontoyiannis [11]. The apop- totic and necrosis features in PLGA-TPGS-AMB NPs-treated C. glabrata cells were evalu- ated using annexin V-FITC and PI staining, respectively. Figure 3. Cumulative release of AMB from PLGA-TPGS-AMB nanoparticles Annexin V-FITC (Annexin V and PLGA-AMB nanoparticles (mean ± S.E.M., n = 5). Apoptosis Detection Kit; BD Pharmingen) was used to was taken out and centrifuged at 25,000 rpm detect phosphatidylserine that was translocat- for 15 min. The supernatant was then trans- ed to the plasma membrane surface at the ferred to a glass test tube for HPLC analysis. onset of apoptosis in C. glabrata. Five microli- The pellet was resuspended in 8 mL fresh PBS ters of annexin V-FITC (50 μg/ml) and 5 μl of and placed back into the shaker bath for subse- propidium iodide (PI) (200 μg/ml) were added quent determination. The accumulative release to 1 ml of cell suspension, and the mixture was of AMB from nanoparticles was plotted against incubated for 20 min in the dark at RT [12]. time. Antifungal activity in vitro Cellular uptake of nanoparticles Antifungal activity in vivo was tested by paper- In this research, coumarin 6 served as a model plate technique. 10 µl of conidial suspension C. fluorescent molecule, which can be entrapped glabrata (1 × 105 spores/ml) was spread over in the PLGA-TPGS nanoparticles for qualitative the glucose agar plate. 5 mm diameter filter and quantitative studies on fungal cellular paper was made with a sterile borer and steril- uptake. The cells were incubated with 250 μg/ ized. Three pieces of sterile filter paper were mL PLGA-TPGS-coumarin 6 nanoparticles at placed on the solidified agar layer. Since AMB 37°C for 24 hrs, rinsed with cold PBS solution 3 minimum inhibitory concentration (MIC) for C. glabrata was 0.2-1.0 μg/ml [13], 2.5/5/ times, and then fixed in methanol for 25 min 10/20/40 µl of PLGA-AMB NPs and PLGA- before analysis by fluorescence microscopy. TPGS-AMB NPs at the concentration of 12.5 Detection of apoptosis markers in C. glabrata μg/ml and 10.6 μg/ml (equivalent to 1.0 μg/ml of AMB) respectively, and free AMB at 1.0 µg/ C. glabrata spores (1 × 106 spores/ml) were mL emulsion were separately dropped onto the inoculated in RPMI 1640 medium at 37°C for 5 sterile filter paper. Petri plates were kept for 4 h with and without antifungal agents (PLGA- hours at room temperature and then were incu- TPGS-AMB NPs, 10.0 μg/ml) and incubated for bated at 28°C for 9 days. Digital photos of the 12 h at 37°C with shaking (180 rpm). After cultures were taken after treatments were initi- 5154 Int J Clin Exp Med 2015;8(4):5150-5162 PLGA-TPGS-AMB NPs improved antifungal activity Table 2. Effect of free AMB and AMB-loaded sue) were expressed as mean ± S.E.M. Di- nanoparticles on MIC against C. Glabrata fferences between paired groups were ana- lyzed by Student’s unpaired t-test. Probability MIC (μg/ml, n = 3) values of P < 0.05 were considered to be statis- Free AMB 0.87 tically significant. PLGA-AMB NPs 0.96 PLGA-TPGS-AMB NPs 0.89 Results Empty PLGA nanoparticles > 10.0 Empty PLGA-TPGS nanoparticles > 10.0 NP characterization: morphology, size, zeta potential, encapsulation efficiency and release Abbreviations: AMB, amphotericin B; MIC, minimum inhibi- tory concentration; PLGA, poly (d,l-lactide-co-glycolide); TPGS, d-α-tocopheryl polyethylene glycol 1000 succinate. Particle size and surface properties of the nanoparticles play a crucial role in drug release kinetics, cellular uptake behavior as well as in ated and the antifungal activity was evaluated vivo pharmacokinetics and tissue distribution every day. PLGA-TPGS-AMB NPs were com- [14]. The physical properties of the PLGA-TPGS- pared against free AMB and PLGA-AMB NPs. AMB NPs and PLGA-AMB NPs were displayed in Figure 2 and Table 1. The average hydrodynam- Animal studies ic size of the PLGA-TPGS NPs and PLGA-TPGS- In vivo therapeutic efficacy of the prepared AMB NPs were approximately 110~120 nm and nanoparticles was tested by a described meth- 120~130 nm in diameter respectively. These od [8, 9]. 60 BALB/c mice were challenged are within the size range for accumulating intravenously via the tail vein with 0.1 ml of C. readily in tumor vasculature due to enhanced glabrata cell suspension (1 × 107 cells/ml) in permeation and retention effects [15]. The par- normal saline. All the treatments were intraper- ticle size and size distribution of the PLGA- itoneally carried out 24 h after the challenge for TPGS-AMB NPs were detected by dynamic light the next 3 days and then randomly divided into scattering (DLS) equipment, and the data were 6 groups. All groups received the drug intrave- displayed in Table 1. According to PDI, ZP (mV), nously as follows: Groups I received PBS (1.0 Particle Size (nm), LC (%) and EE (%) parame- mg/kg, pH = 7.4); group II received free AMB ters, PLGA-TPGS Copolymer nanoparticles dis- (1.0 mg/kg); group III received empty PLGA NPs play perfect advantage for an efficient drug (12.5 mg/kg, equivalent PLGA-AMB NPs 12.5 delivery vehicle. Zeta potential is an important mg/kg); group IV received PLGA-AMB NPs (12.5 predictor of dispersion stability of nanoparti- mg/kg, equivalent AMB 1.0 mg/kg); group V cles. A high absolute value of the zeta potential received empty PLGA NPs (10.6 mg/kg, equiva- means high surface charge of the nanoparti- lent PLGA-AMB NPs 10.6 mg/kg); group VI cles. As displayed in Table 1 and Figure 1, the received PLGA-AMB NPs (10.6 mg/kg, equiva- zeta potential of the PLGA-TPGS-AMB NPs was lent AMB 1.0 mg/kg). All groups were treated determined to be -19.5 mV, which was slightly as described above for 10 days starting from higher than that of the PLGA-AMB NPs of zeta the 3rd day of infection. At the end of the exper- potential of about -23.6 mV. The negative sur- iment, the mice that survived were anesthe- face charge of the nanoparticles may be due to tized and sacrificed. Then fungal colony forming the presence of ionized carboxyl groups of PLA units (CFU) in liver, kidney, spleen and right lung and PGA segments [8, 9, 15]. were determined, and the left lung tissue was It could also be deduced from Table 1 that the embedded in paraffin and sectioned at 5 μm. contents of drug loading and entrapment effi- Haematoxylin & eosin (HE) stain or Crystal vio- ciency of the PLGA-TPGS NPs were higher than let stain and methylene blue stain were then those of the PLGA NPs, indicating the higher performed. binding affinity between the PLGA-TPGS and Statistical analysis hydrophobic AMB. Moreover, the drug loading content of AMB in the PLGA-TPGS NPs could Qualitative data such as AMB content loaded in reach approximately 9.5%, which is ideal for an PLGA-TPGS/PLGA NPs, biochemical data of efficient drug delivery vehicle. After redisper- PLGA-TPGS/PLGA NPs, and fungal burden in sion in PBS, the mean size and size distribution the mice lung tissue (CFU per gram of lung tis- of the AMB-loaded nanoparticles were almost 5155 Int J Clin Exp Med 2015;8(4):5150-5162 PLGA-TPGS-AMB NPs improved antifungal activity Figure 4. Representative photomicrographs of AMB-treated C. Glabrata cells using a fluorescence microscope. A: Bright-field images of C. Glabrata. B: C. Glabrata cellular uptake and fluorescence image of the coumarin 6-loaded nanoparticles after 24 h incubation with the PLGA-TPGS-coumarin 6 nanoparticles. The cellular uptake was visual- ized by overlaying images obtained by EGFP filter. C: Effects of 24 h treatment with AMB on the activity of C. Glabrata as confirmed using Annexin V/PI. D: Effects of 48 h treatment with AMB on the activity of C. Glabrata as confirmed using Annexin V/PI. Annexin V/PI stains show the presence of apoptotic cells (green fluorescence) and necrotic cells (red fluorescence). The experiments were performed in triplicate and repeated three times. Magnification × 400. unchanged during the 3 months of follow-up. solubility of AMB in the PBS and to prevent the This suggested that the AMB-loaded nanopar- adhesion of AMB onto the tube wall [16]. The ticles had good stability and redispersion AMB release from the PLGA-TPGS NPs and ability. PLGA NPs displayed an initial burst of 47.38% and 33.35% respectively in the first 5 days. It In vitro drug release was followed by a second slow-release phase sustained for up to 22 days, which was predom- The in vitro drug release profiles of the freshly inantly attributed to the diffusion of the drug. prepared AMB-loaded nanoparticles in PBS After 22 days, the accumulative AMB release of (containing 0.1% w/v Tween 80, pH 7.4) in the nanoparticles reached 45%~65%. The accu- first 22 days was studied and displayed in mulative AMB release in the first 22 days Figure 3. Tween 80 was applied to improve the was found in the following order: PLGA-TPGS 5156 Int J Clin Exp Med 2015;8(4):5150-5162 PLGA-TPGS-AMB NPs improved antifungal activity Table 3. The ratio of survived mice and colony-forming units (CFU) in different organs of infected-mice Log CFU gram tissue (n = 3) Survival ratioa (%, n = 20) Lung Liver Kidney Spleen Control 3.51 ± 0.53 3.13 ± 0.36 3.91 ± 0.46 2.22 ± 0.65 0.0 PBS 3.38 ± 0.46 3.10 ± 0.42 3.89 ± 0.52 2.11 ± 0.48 0.0 Free AMB 2.31 ± 0.03* 1.11 ± 0.13* 1.12 ± 0.29* 1.20 ± 0.24* 30.0 PLGA 3.40 ± 0.32 3.15 ± 0.71 3.90 ± 0.81 2.21 ± 0.46 0.0 PLGA-AMB 1.08 ± 0.04* 1.05 ± 0.03* Nil** Nil** 45.0 PLGA-TPGS 3.44 ± 0.47 3.11 ± 0.75 3.87 ± 0.88 2.19 ± 0.91 0.0 PLGA-TPGS-AMB 1.02 ± 0.03* 1.01 ± 0.02* Nil** Nil** 85.0 The values are expressed as means ± S.E.M. from three separate experiments. Analysis of variance of one-way classification between the treatment means was heterogeneous, and the t-test values (two-tailed) were significant. *P < 0.05, **P < 0.001. a: Percentage of survival 10 days after the therapy (n = 20). nanoparticles > PLGA nanoparticles, indicating showed the images of C. glabrata cells in light that the PLA-TPGS copolymer was capable of and fluorescence microscopy after 24 h incuba- displaying faster drug release than the PLGA tion with the PLGA-TPGS-coumarin six nanopar- nanoparticles when the copolymers had the ticles at the concentration of 250 μg/mL. It same molecular weight. could be seen from this figure (Figure 4B) that the PLGA-TPGS-coumarin 6 nanoparticles In vitro antifungal activity (green) were closely located around the fungi, indicating that the nanoparticles show high To evaluate the antifungal activity of the AMB- affinity to C. glabrata cells. loaded nanoparticles, the minimum inhibitory concentration of free AMB, AMB-loaded To investigate whether the drug can actually nanoparticles, empty PLGA and PLGA-TPGS induce apoptosis and necrosis in C. glabrata copolymer nanoparticles were tested with C. cells, the apoptotic and necrosis features in glabrata. As shown in Table 2, the minimum PLGA-TPGS-AMB NPs-treated C. glabrata cells inhibitory concentration of the AMB-loaded using annexin V-FITC and PI double-staining nanoparticles was similar to that of free AMB, method were evaluated. Apoptotic cells are while the empty polymeric nanoparticles had a stained with annexin V-FITC, whereas PI accu- much higher minimum inhibitory concentration mulates in the nuclei of necrotic cells via mem- value, indicating that AMB-loaded nanoparti- brane permeabilization [18]. Following PLGA- cles have an antifungal potential similar to that TPGS-AMB NPs (5.0 μg/ml, equivalent AMB 0.5 of free AMB. However, the PLGA, PLGA-TPGS μg/ml) exposure at 37°C for 24 h, about 96% of copolymer did not affect the antifungal activity the cells were stained with annexin V-FITC, of the polymeric nanoparticles. while only 25% to 43% of the cells were stained with PI. However, when PLGA-TPGS-AMB NPs Cellular uptake of fluorescent PLGA-TPGS treatment was extended to 48 h, almost of all nanoparticles and antifungal activity the cells were stained with annexin V-FITC, and only 85% to 95% of the cells were PI positive as The therapeutic effects of the drug-loaded poly- shown in Figure 4C and 4D. These results sug- meric nanoparticles were dependent on inter- gested that PLGA-TPGS-AMB induced apopto- nalization and sustained retention of the sis in C. glabrata cells. nanoparticles by fungal cells [17]. The in vitro studies were capable of providing some circum- Therapeutic efficacy of AMB-loaded nano- stantial evidence to show the advantages of spheres the nanoparticle formulation compared with the free drug. Coumarin-6 served as a fluores- The treatment of C. glabrata-infected mice with cent probe in an attempt to represent the drug AMB-loaded nanospheres as compared with in the nanoparticles for visualization and quan- control animals showed a significant reduction titative analysis of cellular uptake of the nano- in CFU values in evaluated organs especially in particles [8, 9]. Figure4A and 4B respectively kidney and spleen (Table 3). It was discovered 5157 Int J Clin Exp Med 2015;8(4):5150-5162 PLGA-TPGS-AMB NPs improved antifungal activity 5158 Int J Clin Exp Med 2015;8(4):5150-5162 PLGA-TPGS-AMB NPs improved antifungal activity Figure 5. Photomicrographs of infected lung tissue section of C. glabrata. A: Normal healthy lung section stained with H&E. B, C: Infected lung tissue section of C. glabrata was stained with H&E and Crystal violet showing pulmo- nary interstitial edema, with numerous intracellular and extracellular circular to oval, elongated, thin-walled yeast- like moniliform hyphal organisms. D, F: Infected lung tissue treated with free AMB (1.0 μg/mg) and PLGA-AMB NPs (12.5 μg/mg, equivalent AMB 1.0 μg/mg), showing that pulmonary interstitial edema significantly reduced pulmo- nary interstitial edema, and fungal cells proliferation were significantly inhibited. E, G: Infected lung tissue treated with empty PLGA NPs and PLGA-TPGS NPs vector showing that changes in the lung tissue were the same with that of the infected-control group. H: Infected lung section treated with PLGA-TPGS-AMB NPs (10.3 μg/mg, equivalent AMB 1.0 μg/mg), it shows that lung tissue was free from edema and damage with much lower level of fungal spores after the PLGA-TPGS-AMB treatment. Magnification × 400. that the mortality of C. glabrata-infected mice fungal cells infiltrated the lung tissue (Figure used as control (without AMB administrated) 5E and 5G). Nevertheless, the histological was 100% after 10 days, whereas mice treated results of the free AMB group and PLGA-AMB with free AMB and AMB-loaded nanospheres group generally improved relative to those of showed increase in survival rate of 40.0 and the C. glabrata infected models, although lung 85.0%, respectively. One of the reasons for the tissues in the two groups showed histological differences in the antifungal potencies and tox- changes with occasional inflammatory cell infil- icities among free AMB and prepared AMB for- tration similar to that seen in the normal group. mulations could be due to high stability of AMB- They also showed significantly reduced pulmo- loaded nanoparticles [8, 9]. nary interstitial edema (Figure 5D and 5F). Interestingly, data of the fungal burden of the Discussion different organs analyzed showed that free AMB would favorably reduce the amount of Pulmonary fungal infections are serious life- bacteria in the lungs, kidneys and livers. threatening diseases. To improve the treatment However, its therapeutic effect was significantly of pulmonary infections, several antifungal weaker than that of PLGA-AMB and PLGA-TPGS- agents including formulations of echinocan- AMB (Table 3). The kidneys and spleens of dins, pyrimidines and polyenes were used. PLGA-AMB and PLGA-TPGS-AMB groups had no Amphotericin B (AMB) is a kind of systemic anti- fungal growths. Also the fungal amount in the fungal polyene holding broad-anti-fungal spec- lungs and livers were effectively suppressed trum, and has a good antibacterial effect for a compared with the control group. variety of deep fungal infections such as Cryptococcus neoformans, Candida albicans, Lung histological analysis Coccidioides immitis, Histoplasma capsulatum fungus, dermatitis blastomycosis, Schenck As shown in Figure 5, lung tissue structures in Sporothrix, Aspergillus, and Mucor [2, 6, 19]. the normal group were well defined without dis- Having the broad antifungal spectrum, AMB cernible damage or edema, and the trachea has become the most important antifungal and blood vessels were not infiltrated peripher- agents [1, 20]. However, safety remains an ally by inflammatory cells (Figure 5A). However, important consideration. AMB has common lung tissue and bronchial structures in the C. adverse reactions of the digestive system, seri- glabrata-infected model mice were disturbed ous toxicity to the kidneys, and even causes by internal hemorrhage and edema; the bron- leukopenia and liver damage [15, 21]. The goal chial and vascular walls were thickened and of this study is to determine whether a novel infiltrated by a considerable number of inflam- nanoparticle delivery platform for AMB is less matory cells (Figure 5B and 5C). Lung tissue disruptive to lung epithelium than existing for- was free from edema and damage after 10 mulations of AMB without affecting the efficacy days of the PLGA-TPGS-AMB treatment (Figure of AMB. We hypothesized that AMB would 5H). In the PLGA NPs and PLGA-TPGS NPs treat- cause minimal damage to the lung when formu- ment groups, there was marked irregular archi- lated within the nanoparticles (NPs). tecture and inflammatory cell infiltration. As fungal infected control groups, there was a The stability of nanocarriers that will minimize notable histological changes; the bronchial and the release of drug in the blood circulatory sys- vascular walls were thickened, and numerous tem plays a major role in determining the rate 5159 Int J Clin Exp Med 2015;8(4):5150-5162