- Nano Commentary
- Open Access
Enhanced Antifungal Activity by Ab-Modified Amphotericin B-Loaded Nanoparticles Using a pH-Responsive Block Copolymer
- Xiaolong Tang†1, 2,
- Jingjing Dai†1,
- Jun Xie†2,
- Yongqiang Zhu1,
- Ming Zhu2,
- Zhi Wang2,
- Chunmei Xie3,
- Aixia Yao1,
- Tingting Liu1,
- Xiaoyu Wang1,
- Li Chen1,
- Qinglin Jiang1,
- Shulei Wang4,
- Yong Liang5Email author and
- Congjing Xu2Email author
© Tang et al. 2015
- Received: 9 April 2015
- Accepted: 2 June 2015
- Published: 10 June 2015
Fungal infections are an important cause of morbidity and mortality in immunocompromised patients. Amphotericin B (AMB), with broad-spectrum antifungal activity, has long been recognized as a powerful fungicidal drug, but its clinical toxicities mainly nephrotoxicity and poor solubility limit its wide application in clinical practice. The fungal metabolism along with the host immune response usually generates acidity at sites of infection, resulting in loss of AMB activity in a pH-dependent manner. Herein, we developed pH-responsive AMB-loaded and surface charge-switching poly(d,l-lactic-co-glycolic acid)-b-poly(l-histidine)-b-poly(ethylene glycol) (PLGA-PLH-PEG) nanoparticles for resolving the localized acidity problem and enhance the antifungal efficacy of AMB. Moreover, we modified AMB-encapsulated PLGA-PLH-PEG nanoparticles with anti-Candida albicans antibody (CDA) (CDA-AMB-NPs) to increase the targetability. Then, CDA-AMB-NPs were characterized in terms of physical characteristics, in vitro drug release, stability, drug encapsulation efficiency, and toxicity. Finally, the targetability and antifungal activity of CDA-AMB-NPs were investigated in vitro/in vivo. The result demonstrated that CDA-AMB-NPs significantly improve the targetability and bioavailability of AMB and thus improve its antifungal activity and reduce its toxicity. These NPs may become a good drug carrier for antifungal treatment.
- Amphotericin B
- C. albicans
Amphotericin B (AMB), which has been considered to associate with ergosterol in fungal cell membranes to mediate ion-permeable pores, is still one of the most effective agents for the treatment against systemic fungal infections [1–3]. However, the association leading to the elevation of the intracellular Ca2+ concentration can result in renal tubular cell injury . Recently, new lipid formulations of AMB have been developed to reduce the nephrotoxicity and improve the therapeutic index [4, 5], while new lipid formulations still remain limited to be used mainly due to the high cost.
In recent years, advances in gaining drug potency or in vivo properties of nanoparticles (NPs) for drug delivery applications by increasing targetability and environmental sensing leading to NP property switching and sustained drug release have been improving the bioavailability of AMB with few side effects for the treatment against systemic fungal infections. The fungal metabolism and the host immune response often cause localized acidity, which results loss of drug activity in a pH-dependent manner [6, 7]. Fungi can adjust to local conditions such as ambient pH, temperature, and nutrient supply through shared metabolic systems including the Pal/PacC pathway in Aspergillus sp. and the RIM101 pathway in Candida sp. to infect tissue [8–10].
Developing systemically available nanodrug carriers that can target and improve antibiotic properties in the setting of localized acidity may therefore be a method to improve the treatment of these and potentially other infections [9, 10]. Herein, we developed pH-responsive, drug-encapsulated, surface charge-switching poly(d,l-lactic-co-glycolic acid)-b-poly(l-histidine)-b-poly(ethylene glycol) (PLGA-PLH-PEG) nanoparticles for treating fungal infections. The PLH segment would become positively charged under acidic conditions, imparting the PLGA-PLH-PEG NP surface an overall positive zeta potential, facilitating interactions with the negatively charged elements of the fungal cell wall leading to strong multivalent electrostatic-mediated binding. At the same time, in order to further increase the targeting efficacy for fungal infections, we prepared anti-Candida albicans antibody (CDA)-modified AMB-loaded PLGA-PLH-PEG nanoparticles (CDA-AMB-NPs). As CDA reacts with antigens presenting at the cell wall of C. albicans, these CDA-AMB-NPs delivering AMB efficiently are designed to target to and bind avidly to C. albicans in acidity [11, 12], reducing the loss of antifungal activity in acidity.
The result demonstrates that CDA-AMB-NPs switched their surface charge from a negative zeta potential (−3.82 ± 0.53 mV) at pH 7.35 to a positive one (2.81 ± 0.34 mV) at pH 6.8, yielding a significant increase in targetability and antifungal efficacy at pH 6.8 compared to pH 7.35 with reduced hemolytic activity and cytotoxicity on human renal tubular epithelial cells.
Herein, the CDA-AMB-NPs might step up the development of systemically administered and targeted drug carriers that can potentially treat systemic candidiasis infection associated with acidity.
All chemical reagents used in this study were of HPLC grade and were purchased from Sigma-Aldrich, St. Louis, MO, USA, unless otherwise noted. l-Histidine (His), AMB, and coumarin 6 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Thionyl chloride and PLGA-N-hydroxysuccinimide (NHS) were obtained from the Graduate School at Shenzhen, Tsinghua University. l-His was derivatized by introducing a carbobenzoxy (CBZ) group to the α-amino group; the amino group in the imidazole ring of N α-CBZ-l-His was protected with a dinitrophenyl (DNP) group. N α-CBZ-N im -DNP-l-His was then transformed to N-carboxy anhydride (NCA) form by thionyl chloride. Acetonitrile and methanol were purchased from EM Science (Chrom AR, HPLC grade, Mallinckrodt Baker, USA). All the other chemicals and reagents used were of analytical grade and commercially available. Millipore water was prepared by the Milli-Q Plus System (Millipore Corporation, Bedford, MA, USA). C. albicans (ATCC 90029) were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). mPEG-OPSS polymer (MW 5000) and mPEG-NH2 (MW 5000) were obtained from Laysan Bio (Arab, AL, USA). Phosphate-buffered solution (PBS) and fetal bovine serum (FBS) were purchased from Gibco Co. (Uxbridge, UK).
Fabrication of CDA-AMB-NPs and Characterization
Modified emulsion/solvent evaporation techniques were utilized to form AMB-loaded NPs . In brief, a pre-weighed amount of AMB powder and 10.0 mg of PLGA-PLH-PEG copolymer were dissolved in a volume of 5.0 mL of 15:85 v/v DMSO/ethyl acetate solution by vortexing and sonication. The resulted nanoparticle suspension was stirred at room temperature overnight and centrifuged at 25,000 rpm for 20 min. The AMB-loaded NPs were then hardened by allowing slow organic solvent evaporation for 24 h in the hood. AMB-loaded NPs were purified by triple filtration using Amicon Ultra-4 100,000 NMWL centrifugal filter units (Millipore, Billerica, MA, USA) using sterile water and freeze-dried using 10 % w/v of sucrose as a cryoprotectant.
To form CDA-modified NPs, AMB-loaded PLGA-PLH-PEG copolymer was coupled to CDA using EDC/NHS chemistry and purified by precipitation and drying in vacuo . After the conjugation reaction, the CDA-AMB-NPs were purified by centrifugation at 14,000 rpm × 30 min and redispersed in phosphate-buffered solution. Finally, CDA-AMB-NPs were lyophilized for 48 h for further use. As a model control molecule, coumarin 6 can be entrapped in PLGA-PLH-PEG NPs for qualitative and quantitative analyses of cellular uptake by candidiasis cells. CDA-coumarin 6-NPs were fabricated in a similar manner.
As a fungal infection usually generates weak localized acidity as far as pH = 6.8 [8–10], the particle size and zeta potential of the PLGA-PLH-PEG NPs in 6.8 and 7.35 pH-adjusted PBS were determined by using a Zeta PALS dynamic light scattering detector (15-mW laser, incident beam 676 nm, Brookhaven Instrument Corporation). PLGA-PLH-PEG NPs were resuspended at 2.5 mg/mL in a 1 % w/v uranyl acetate solution (pH ~5.5), deposited onto carbon-supported copper TEM grids and dried, and then detected by a JEOL 200 CX TEM (MIT CMSE). And the content of AMB loaded in the micelle was quantified by reverse-phase HPLC. Drug release was conducted by suspending NPs in 4 mL of pH-adjusted PBS, and the drug release rate was measured by spectrophotometry. The released drug was quantified at each time point in triplicate. Drug delivery efficiency in vitro was also detected. 1 × 105 C. albicans cells were incubated with 10 mL of CDA-coumarin 6-NP solution (equivalent to 1.0 μg/mL coumarin 6) for 12 h at different pH (pH = 6.8 and pH = 7.35) and finally subjected to flow cytometry analysis to determine the intracellular drug delivery efficiency of CDA-drug-NPs.
Biological Effect of CDA-AMB-NPs
The hemolytic activity of CDA-AMB-NPs was evaluated using human red blood cells (RBCs) . Erythrocytes were washed twice with saline solution and then suspended in saline at 2 % of hematocrit. Different concentrations of AMB solution and AMB-incorporated PLGA-PLH-PEG micelle solution were incubated with properly diluted RBCs at 37 °C for 1 h. After centrifugation at 800 g for 15 min, the supernatant was analyzed for hemoglobin at 545 nm using a UV spectrophotometer. The percentage of hemolysis was determined as follows: Hemolysis (%) = [(ODs − ODmin)/(ODmax − ODmin)] × 100, where ODs is the absorbance of the supernatant from samples incubated with the drugs. Diluted RBCs incubated with PBS and with distilled water were used as minimal (ODmin) and maximal (ODmax) hemolytic controls, respectively .
Determination of Cytotoxicity on HKC
Immortalized human renal tubular epithelial cells (HKC) were cultured at 37 °C with humidified atmosphere of 5 % CO2 in DMEM/F12 supplemented with 10 % fetal bovine serum (FBS) and 2 ng/mL mouse epidermal growth factor and were seeded into 96-well culture plates (Corning, Schiphol-Rijk, The Netherlands) for 24 h. Subconfluent cells (culture surface covering nearly 70 %) were then exposed to various AMB or AMB formulation concentrations (1.0–10.0 μM). As kidney injury molecule-1 (KIM-1) and type I transmembrane glycoprotein neutrophil gelatinase-associated lipocalin (NGAL) have been reported to correlate with proximal tubular injury [17, 18], the cytotoxicity was measured periodically by measuring KIM-1 and NGAL using immunofluorescence, WB, and RT-PCR methods to determine early damage caused by exposure to AMB and AMB formulations. The prime set for NGAL was forward, 5′-TCA CCT CCG TCC TGT TTA G-3′ and reverse, 5′-CTC CTT GGT TCT CCC GTA-3′, and that for KIM-1 was forward, 5′-ACTCCTGCAGACTGGAATGG-3′ and reverse, 5′-CAAAGCTCAGAGAGCCCATC-3′. For normalization, the expression of β-actin was examined: forward, 5′-TGC TTC TAG GCG GAC TAT GA-3′ and reverse, 5′-CTC GGC CAC ATT GTG AAC TT-3′ .
In Vivo Toxicity Analysis
The toxicities of AMB and AMB formulations were assessed with BALB/cA mice (7–9 weeks old). The Committee of Laboratory Animal Welfare and Ethics, Anhui University of Science and Technology has approved all of the animal studies. Five healthy mice were chosen for each route of administration. AMB or AMB formulation was delivered through an intravenous (2.0 mg/kg/day for 3 days, pH = 7.4) route. All animals were monitored through 1 week to determine mortality. Biochemical analyses of blood urea nitrogen (BUN) and creatinine (Cr) levels were determined to assess the nephrotoxicity of CDA-AMB-NPs . And major organs were used for light microscopic evaluation with hematoxylin and eosin (HE) or IHC to evaluate cytological and histological alterations including injury, degeneration, apoptosis, or even necrosis.
In Vitro Antimicrobiological Analysis
Susceptibility test for C. albicans to different drugs was performed by the agar-based E-test method (bioMérieux, Sweden) with 8.4 g/L RPMI 1640 (RPMI, Sigma Chemical Co., St. Louis, MO, USA), 1.5 % agar, and 1.5 % glucose, poured in 110-mm-diameter plates. The plates were inoculated by dipping a sterile swab into the inoculum suspension adjusted to the turbidity of a 0.5 McFarland standard (106 cells/mL) and streaking it across the surface of the agar in four directions. The plates were dried at ambient temperature for 20 min before applying the E-test strips. The minimum inhibition concentration (MIC) endpoints were determined after 24 h of incubation at 35 °C. The MIC was read for different amphotericin B deforms, and null NPs were used as a blank . Experiments were performed in duplicate.
Detection of Apoptosis Markers in C. albicans
C. albicans spores (1 × 105 cells/mL) were inoculated in RPMI 1640 medium at 37 °C for 3 h with different antifungal agents (free AMB, AMB-NPs, CDA-AMB-NPs, 10.0 μg/mL) and were incubated for 24 h at 36 °C with shaking (150 rpm). After incubation for 12 h, C. albicans cells were harvested and then digested with a lysing enzyme mixture (2.0 U of lyticase, 2.5 U of chitinase, 2.0 U of chitosanase, and 15 mg/mL of lysing enzyme [Sigma]) for 10 h at 35 °C. And the presence of apoptotic markers was analyzed with annexin V-FITC/PI stains. Then, the apoptotic and necrotic features in the drug-treated C. albicans cells were evaluated using fluorescence microscopy and flow cytometry instrument detection, respectively.
In Vivo Antifungal Activity
The in vivo therapeutic efficacy of the prepared nanoparticles was tested by a described method . Sixty 20–22-g BALB/c mice (6–8 weeks old) were infected by inhalation with a volume of 20 μL of PBS containing 5.0 × 105 contagious C. albicans cells. After 3 days, all infected mice were randomly divided into four groups. All groups received the drug intravenously as follows: Group A received PBS (10.0 mg/kg/day for 4 days, pH = 7.35); group B received free AMB (10.0 mg/kg/day for 4 days); group C received AMB-NPs (equivalent AMB 10.0 mg/kg/day for 4 days); group D received CDA-AMB-NPs (equivalent AMB 10.0 mg/kg/day for 4 days).
After 2 weeks, the survived mice were anesthetized and sacrificed. Then, the histopathologic changes and fungal colony-forming units (CFU) in the liver, kidney, heart, and right lung were determined.
Qualitative data was expressed as means ± SEM. Differences between groups were assessed using one-way ANOVA. Differences between paired groups were analysed by Student’s unpaired t test. A significance level of p < 0.05 was used for all comparisons.
Polymer Characterization: Morphology, Size, Zeta Potential, Encapsulation Efficiency, and Release
Characterization of AMB-loaded nanoparticles (n = 3)
Particle size (nm)
109.2 ± 4.1
121.1 ± 3.5
To evaluate the zeta potential (ZP; mV) property which was key to tailoring NP-fungal interactions, CDA-AMB-NPs were resuspended in PBS with appropriate pH (6.8, 7.0, and 7.35). The results showed that the CDA-AMB-NPs switched their surface charge from a negative zeta potential at pH 7.35 (ζ = −3.82 ± 0.53 mV, N = 3) to a positive one at pH 6.8 (ζ = 2.81 ± 0.34 mV, N = 3) (Fig. 2c). This change is due to the increasing presence of positive charges from the imidazole group of the PLH with reductions in pH . Polydispersion (PDI), particle size (nm), and EE (%) parameters are displayed in Table 1.
Intracellular Drug Delivery and Drug Release Efficiency Analysis
The in vitro drug release profiles of the freshly prepared CDA-AMB-NPs in PBS (containing 0.1 % w/v Tween 80) in the first 10 days are displayed in Fig. 3b. Tween 80 was applied to improve the solubility of AMB in the PBS and to avoid the adhesion of AMB onto the tube wall [21, 22]. The AMB release from the CDA-AMB-NPs displayed an initial burst of 58.3 % in the first 6 days at pH = 6.8, which was much higher than that at pH = 7.35. A second slow-release phase sustained for up to 18 days, which was predominantly attributed to the diffusion of the drug. After 18 days, the accumulative AMB release of nanoparticles reached to 81.4 % at pH = 6.8, which indicates that the PLGA-PLH-PEG copolymer was capable of displaying sound drug release at pH = 6.8 (Fig. 3b).
In Vitro Antifungal Activity
Effect of free AMB and AMB-loaded NPs against C. albicans
MIC (μg/mL, pH = 7.35)
MIC (μg/mL, pH = 6.8)
1.0 ± 0.1
1.0 ± 0.2
10.0 ± 0.2
11.0 ± 0.2
11.0 ± 0.2
11.0 ± 0.2
In Vitro Hemolytic Effects of CDA-AMB-NPs
Hemolytic activity of CDA-AMB-NPs and free AMB against RBCs
Cytotoxicity on HKC
In Vivo Nephrotoxicity Study
Therapeutic Efficacy of CDA-AMB-NPs
The ratio of survived mice and CFU in different organs of infected mice
Log CFU gram tissue (n = 3)
Survival ratioa (%, n = 20)
4.35 ± 0.27
3.08 ± 0.25
4.78 ± 0.33
3.41 ± 0.34
4.29 ± 0.35
3.14 ± 0.21
4.75 ± 0.29
3.34 ± 0.33
2.51 ± 0.11*
1.12 ± 0.09*
1.21 ± 0.26*
1.87 ± 0.25*
1.01 ± 0.06*#
In in vivo studies, the lesions demonstrated diffuse exudative change accompanied by necrosis and granulomatous formation in the control group. Many hyphae could be found in the lesions. The same pathological changes also existed in the free AMB treatment group. But the therapeutic efficacy of CDA-AMB-NPs was detected in murine candida pneumonia. CDA-AMB-NPs significantly increased the survival time and were more effective in reducing the CFU counts in the lungs than free AMB (Fig. 7).
AMB has been reported to cause hemolysis and cytotoxicity [1, 24]. In order to reduce the toxicity of AMB, we used a nanomaterial which has a slow-release effect to wrap the drug. The result presented that CDA-AMB-NPs increased drug targeting, delayed drug release, prolonged plasma circulation, altered the physical properties of the drug, and reduced hemolytic toxicity (Table 3). The reduction of hemolysis of the free AMB loaded in PLGA-PLH-PEG may be due to the sustained targeting release of the drug in monomeric mode, which reduces the concentration of AMB in the blood, compared with free AMB . CDA-AMB-NPs were also found to be significantly less nephrotoxic, as CDA-AMB-NPs significantly reduced the expression levels of KIM-1 and NGAL which mark the damage of the kidney cells  and the BUN and Cr levels in serum. These data demonstrated that CDA-AMB-NPs have therapeutic superiority to free AMB.
To investigate the CDA-AMB-NPs’ targetability, we incubated CDA-coumarin 6-NPs and C. albicans together. The results showed that the green fluorescence on the fungal cell wall increased over time (Fig. 3a). In acidic conditions, the release speed of AMB from the nanodrug is more efficient, as CDA-AMB-NPs can more dramatically induce C. albicans apoptosis and necrosis at pH 6.8 than free AMB (Fig. 4).
At the same time, the treatment of experimental infection mice with CDA-AMB-NPs resulted in 95 % survival in contrast to 0 % survival in the control group and only 55 % survival in the free AMB group (Table 4). What is more, CDA-AMB-NP treatment also significantly reduced the quantity of pathogens in various organs of the infected mice. And the living bacterium could even be completely eliminated in parts of tissues and organs (Fig. 7 and Table 4). For mice fungal infection of the lungs, both PAS and target blue staining results showed that after the treatment of CDA-AMB-NPs, the quantity of fungal spores in the infected lungs of mice significantly reduced, the alveolar structure returned to normal, and the edema subsided. These results showed that CDA-AMB-NPs can effectively kill fungus, improve lung inflammation and seepage, and promote lung tissue lesion to return to normal physiological status and function. All these efficacy data demonstrated a therapeutic superiority of CDA-AMB-NPs over free AMB.
The developed CDA-AMB-NPs gave appropriate particle size, extended release, and zeta potential. Compared with AMB, the in vitro/in vivo data of the present study against C. albicans showed that CDA-AMB-NPs had less toxicity and significantly higher antifungal activity, which indicated that the developed CDA-AMB-NP delivery system may serve as an effective novel low-dose, less side effect antifungal therapeutic system for AMB.
Further experiment on CDA-AMB-NPs including their specific molecular mechanism, targetability, and release mechanism and the efficiency of the loaded drug in fungi-infected lesions will be conducted to reveal more of their characteristics. Moreover, a detailed in vivo study is required before this therapy moves to the bedside. Nevertheless, these results raise interesting possibilities for future studies.
The authors are grateful to the Open Research Fund Program of the State Key Laboratory of Virology of China (2014KF004), the National High Technology Research and Development Program (863 Program) (2011AA02A111), and the 2011 Infectious Disease Prevention and Control Technology major project (2012ZX10004903).
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