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Background and Objectives: The industrial development of polymeric nanoparticle suspensions, and drug delivery system, is limited due to the problems in maintaining stability of suspensions. In this work, amphotericin B (AmB) nanoparticles prepared by means of a spray-dried technique.
Material and Methods: The dried nanocapsule and nanosphere suspensions prepared by nanoprecipitation. Total AmB was measured by High performance liquid chromatography, after dissolution of the nanoparticles in DMSO. Then AmB-loaded nanoparticles efficiency of each formulation was assessed. The minimal inhibitory concentrations (MICs) of these nanoparticles to Candida. albicans was determined and compared to those of respective free AmB using micro dilution method. Furthermore, the in vivo antifungal activity was evaluated.
Results: The MICs of all AmB nanoparticles against C. albicans tested were reduced compared to that of free antibiotic. Drug entrapment efficacy for nanospheres were significantly (Pâ‰¤0.05) higher (65% Â± 0.10) than those of nanocapsules (52% Â± 0.14), respectively. The AmB-loaded nanosphere and nanocapsule found to be 6.1 and 5.9 times less toxic than free AmB. Incubation experimental findings indicate that infected animals treated with AmB nanoparticles significantly reduced CFU values, whereas infected animals treated with free drug showed insignificant reduction in CFU. A marked increase in the percent survival was observed in the case of animals treated with AmB-loaded nanospheres.
Conclusion: The results suggest that AmB-loaded nanoparticles prepared by spray-drying technique may be an appropriate delivery system for AmB to be used in the treatment of fungal infections.
Keywords: Nanocapsule, Candida albican,
Received: 29 December 2009
Address communications to: Mr Mohsen Mirzaee, Department of Laboratory sciences, Medical school, Islamic Azad University, Boroujerd Branch, Boroujerd, Iran
Amphotericin B (AmB) is a polyene macrolide antifungal agent and the drug of choice for systemic fungal infection. Unfortunately, it is poorly absorbed from the gastro-intestinal tract due to its low aqueous solubility (1). Thus, it must be given parenterally to treat systemic fungal infections. Currently, two types of drug formulations for AmB are available. The first one is a micellar solution of AmB with deoxycholate as surfactant which may show serious nephrotoxicity. The second ones are lipid-based nanoparticulate formulations. These formulations have been found to reduce nephrotoxicity, but are quite expensive (2). Thus, much effort has been spent to develop cheaper delivery systems with reduced amphotericin B toxicity. Polymeric nanoparticle delivery systems are one approach that has been extensively investigated (3-5). Nanoparticles, polymeric submicron carriers with nanometric size, are a general name to describe nanocapsules and nanospheres (6). According to the literature, the nanocapsules correspond to a polymeric wall enveloping an oil core, whereas the nanosphere consists of a polymeric matrix (7;8). Nanoparticle suspensions have been developed as drug targeting delivery system, using polyesters, poly (alklylcyanoacrylate), poly (alkyl methacrylate-co-acrylic acid) and other polymers (9;10). The industrial development of these systems is limited due to the problems in maintaining stability of suspensions for a prolonged time period (11). The colloidal suspension, in general, dose not tend to separate just after preparation because submicron particles sediment slowly and aggregation effect is counteracted by mixing tendencies of diffusion and convection (8). However, after several months of storage, aggregation can occur. According to the literature, freeze-drying is good method to dry nanospheres in order to increase the stability of these colloidal systems. The aims of the present study were to apply the spray-dried technique for drying AmB-loaded nanocapsule and nanosphere suspensions, using poly (Ñ”-caprolactone) or poly [()-lactic acid] as polymers and mineral oil as core in the case of nanocapsules, as well as to study the stability and comparative hemolytic and therapeutic efficacy of each formulations by in vitro and in vivo experiments.
Material and Methods:
Poly [()-lactic acid] (PLA) and AmB obtained from Sigma (St. Louis, MO, USA). Mineral oil, sorbitan monostearate and polysorbate 80 were supplied by Delaware (Porto Alegre, Brazil). Colloidal silicon dioxide (Aerosil 200Â®) was obtained from Degussa (São Paulo, Brazil). All other chemicals and solvents used were of pharmaceutical grade. All reagents were used as received.
In this work, HPLC apparatus (Thermo Separation Products, CA, USA) equipped with a 15 cm Ã- 4.6mm (i.d.) reversed-phase C18 column with a 5 Âµm particle size (Gemini Phenomenex, CA, USA) was applied. Also a Mini-spray-dryer Büchi MSD 190 (Flawil, Switzerland) and Rotary evaporator (RV 05 ST, IKAÂ®, VWR International Eurolab, Barcelona, Spain) were used.
Preparation and characterization of colloidal suspensions
All samples were prepared by nanoprecipitation, of preformed polymers was based on the method of Pohlmann et al. (12), with some modification by Espuleas et al. method (13). For nanocapsules, the organic phase was constituted by the PLA (l g), AmB (0.150 g) the sorbitan monostearate (0.766 g) and the mineral oil (3.3 ml) dissolved in acetone (270 ml) and acidified by 0.1 N HCl to solubilize amphotericin B. This organic phase was added with moderate magnetic stirring into an aqueous solution (530 ml) containing the polysorbate 80 (0.766 g). After nanoprecipitation, the acetone and some water were removed by evaporation under reduced pressure and the final volume adjusted with water to 100 ml (1.5 mg / ml of AmB).
For nanocapsules, formulations were prepared by nanoprecipitation of PLA, AmB, sorbitan monostearate and poly-sorbate 80 at the same concentration described above, omitting the oil. All formulations were made triplicate.
Preparation and characterization of spray-dried powders
5 ml Aerosil 200Â® solution [3.0% (w/v) in water] was added to the 10 ml colloidal suspensions of nanocapsules or nanospheres and the mixture was fed into a mini-spray-dryer Büchi MSD 190 (Flawil, Switzerland) with two component nozzle and co-current flow. The inlet temperature at the drying chamber was maintained around 804 °C. The outlet temperature was 504 °C. A dispersion of 3% (w/v) Aerosil 200Â® in water was spray-dried in the same conditions for comparison. Moisture content of each spray-dried product was determined with the Mettler LP16 (Greisensee, Switzerland) infrared dryer and LJ16 moisture analyzer. Empty nanoparticles were also prepared for appropriate comparison.
In vitro dissolution studies
AmB possesses very poor aqueous solubility. In order to increase its solubility, 1% (v/v) Tween 80 in 10mM HEPES buffer, pH 7.4, was used as a dissolution medium. The drug release profile of AmB nanoparticles was studied at for three months at 37 Â± 0.5 °C. Nanoparticles containing 30 Âµg of AmB were placed into 20 mL of the dissolution medium and shaked in the dark place. An aliquot (1 mL) was taken at predetermined time intervals of 1, 2 and 3 months. The samples were filtered through a 0.2 Âµm membrane and the amount of AmB released was determined using HPLC method which was modified from Hosotsubo and Hosotsubo (14). One hundred microliters of filtered sample, 500 ÂµL of internal standard (0.5 Âµg/mL p-nitroaniline), and 400 ÂµL of mobile phase were mixed. Fifty microliters of the mixture was then injected into the column. An HPLC apparatus (Thermo Separation Products, CA, USA) equipped with a 15 cm Ã- 4.6mm (i.d.) reversed-phase C18 column with a 5 Âµm particle size (Gemini Phenomenex, CA, USA) and UV-visible detector was used. The HPLC assay conditions were as follow: mobile phase: 10mM acetate buffer, pH 7.2, and acetonitrile (63:37); flow rate: 1.2 ml/min; detection wavelength: 408 nm. Dissolution studies were performed in triplicate. AmB content was determined by calculating the peak-height ratio of AmB to an internal standard.
Determination of Entrapment Efficiency
The content of AmB in loaded nanoparticles was determined by HPLC has been described previously (15). Twelve milligrams of spray-dried nanoparticles were weighed and then dissolved in 0.5 mL of DMSO and then centrifuged at 12,000Ã-g for 20 min. Twenty microliters of the supernatant were mixed with 0.98 mL of methanol: water (1:1) solution. The mixture was ultrafiltered and fifty microliters of that injected into the column. The percentage of drug entrapped was then calculated from:
Amount of AmB in particle Ã-Volume tested Ã-100%
Total sample volume Ã- Initial amount of AmB
Percent drug entrapment =
In vitro toxicity
In vitro toxicity of free, nanosphere and nanocapsule forms of AmB was determinate by Chakraborty et al., method (16).
In brief, blood samples were drawn from rabbit in the presence of heparin (50-units/ ml) and washed 3 times with 0.15 M PBS (pH 7.4). A series of free AmB samples were prepared for haemolysis experiment by further diluting stock solution of AmB in PBS (pH 7.4) to get varying concentration ranged 0 - 250 Î¼g AmB / ml, whereas dose levels of AmB used in the in-vitro analysis assay was ranged from 0 - 250 Î¼g AmB /ml for each of nanoparticles. Subsequently, 0.8 ml of 0.1% red blood cells (vol. /vol.) were mixed with 0.2 ml of buffer containing varying amounts of free, nanosphere and nanocapsule forms of AmB. The dose levels of AmB used in the in vitro haemolysis assay was ranged from 0 to 250 Î¼g AmB/ml. The mixture was then incubated at 37°C for 1 hr and centrifuged at 1000Ã-g for 2 min. The amount of hemoglobin released in the presence of 0.3% Triton X-100 was taken as measure of complete (100%) lysis.
Antifungal susceptibility testing
Susceptibility testing was performed in triplicate according to Clinical and Laboratory Standards Institute (formerly NCCLS) M38-A microdilution methodology (17). Briefly, conidial suspensions of ~1 Ã- 106 conidia/ml were diluted 1:50 in RPMI growth medium (buffered to pH 7.0 with 0.165 M 4-morpholinepropanesulfonic acid) and dispensed (100 Î¼l) into a microtiter tray containing serial twofold dilutions of AMB. The tray was then incubated for 48 h at 37Â°C, and the MIC was read at 48 h as the lowest drug concentration that showed complete growth inhibition. Two separate experiments in triplicate were performed for each formulation.
Rabbit model of Candidiasis
Sixty Male New Zealand White (NZW) rabbits (2.5-3 kg body weight obtained from the National Institute of Pasture, Iran) were infected with 0.25 ml Candida albicans cell suspension (7Ã-106 cells ml-1) in normal saline via the caudal vein. The infected rabbit were divided into 5 groups. Group 1 received free AmB (0.5 mg Kg-1, I.V.); groups 2 & 3 received AmB-nanocapsules or AmB-nanospheres (0.5 mg kg-1, I.V.); groups received 4 empty nanoparticles and group 5 received physiological saline (1ml kg-1). Each group (excepting 4 & 5) was administered with their respective amphotericin B preparation on an alternate days starting from the 3rd day of infection for 13 days, similarly group 5 was treated with saline as control. The progress of infection and mortality of rabbit were monitored for 13 - 16 days. Survival rate was recorded with the last dose of drugs. The rabbit were killed 2 days after the last dose and the fungal load was determined, in terms of colony forming units (Cfu) in lung, liver, kidney, spleen and brain.
The in vivo antifungal activity was evaluated on the basis of survival rate of animals and number of Cfus in homogenates of lungs, liver, kidney, spleen and brain. The organs were excised aseptically washed with physiological saline and then homogenized in saline. A 25-fold serial dilution was placed in Sabouraud dextrose agar plates and then counting for Cfu after 48 h incubation at 37 °C (18).
The Cfu data were statistically evaluated by analysis of variance of one-way classification with unequal frequencies (19). The heterogeneity of means for the various organs was tested by the F ratio of treatment variance to the experimental error variance. The survival data were analyzed using Chi-squared with Yates correction and by Fisher's exact test (20).
Determination of entrapment efficacy
The entrapment efficacy was done by dissolving AmB nanoparticles in DMSO and measured the amount of AmB by HPLC. The results showed that entrapment rate of nanosphere forms of AmB (65% Â± 0.10), higher than rate of nanocapsule forms of AmB (52% Â± 0.14), respectively.
In vitro dissolution rate
The percentage of cumulative AmB released from nanoparticles at varying time intervals in the dissolution medium, consisting of 1% (v/v) Tween 80 in HEPES buffer pH 7.4, at 37 °C was examined. Dissolution rate for nanospheres and nanocapsules were 8% Â± 0.09 and 2% Â± 0.3%, respectively. AmB recoveries after 3 months were shown in Fig. 1.
Fig 1: AmB recoveries from nanocapsules and nanospheres prepared with PLA after 3 months of storage at 37 °C.
In vitro toxicity study
Fig. 2. Show the hemolytic ability of AmB-loaded nanoparticles prepared by spray-dried method showed in Fig. 2. The dose level of AmB ranged from 0 to 250 Î¼g AmB /ml. In all cases, the degree of haemolysis increased as the dose increased. However, at any dose of AmB, the hemolytic ability of the different nanoparticles showed considerable differences. For instance, AmB-loaded nanospheres showed comparative mild toxicity (15% lysis of erythrocytes) at a concentration of 38Î¼g/ml, whereas at the same concentration of free AmB complete lysis (100%) of erythrocytes was observed. The corresponding hemolytic value (100% lysis) for AmB-loaded nanocapsules and nanospheres was estimated to be 226 and 234 Î¼g / ml, respectively.
Fig 2: Erythrocyte lysis caused by amphotericin B when delivered through different nanoparticulate preparations. Values are expressed as mean of % lysis of three separate experiments + standard error of means.
The MIC values of AmB in either free or nanoparticulate forms for Candida albicans shown in Table 1. The MICs of nanospheres and nanocapsules forms were than those free AmB agents Candida albicans decreased 3- and -2 folds than respectively. Empty nanoparticles have no effect on fungal growth. The combination of empty nanoparticles and free AmB has an antifungal activity similar to that of respective free antibiotic.
Table1. In vitro antifungal activities of AmB in either free or nanoparticulate forms for Candida albicans (ATCC 90028)
Minimum inhibitory concentration (Âµg/ml)a
Candida albicans(ATCC 90028)
Standard microdilution method modified from the macrodilution method of the National Committee for Clinical Laboratory Standards. Two-fold serial dilutions of free amphotricn-B (F), AmB-loaded nanospheres (AmB-nanosphere) and AmB-loaded nanocapsule (AmB-nanocapsule) + free amikacin was prepared in a broth medium and was mixed with fungi suspension to achieve a final inoculums 1Ã-105 cfu/ml. The round-bottom 96-well plate was incubated for 72 h at 30° C and then read. The MIC was recorded to be the lowest concentration of the drug that prevented visible growth and expressed in Âµg/ml.
Therapeutic efficacy of various nanoparticulate AmB preparations on candidiasis rabbit model
Candida albicans-infected rabbit treated with AmB-loaded nanospheres and nanocapsules showed significant reduction in Cfu values in kidney and spleen compared with control animals (Table 2). The single dose (0.5 mg kg-1 I.V.) was administered to the in vivo situation considering the degree of haemolysis (100 %) elicited by free AmB at a concentration of 38 Î¼g/ml. The Cfu reduction effect of AmB-loaded nanospheres was higher than the AmB-loaded nanocapsules. It was also observed that AmB nanoparticles treated rabbit showed a complete absence of Cfu in the spleen and kidney. Treatment with free AmB showed a significant reduction in Cfu values in the lung only when compared with nontreated control rabbit. It was observed that control animals infected with Candida albicans elicited more than 50 and 90% mortality after 9 and 15 days respectively. Treatment with free drug, AmB-loaded nanocapsules and nanospheres increased the survival rate by 20, 50 and 60% respectively.
Table 2: Colony-forming units (Cfu) of Candida albicans in different organs and percent survival of infected rabbit and effect of chronic treatment with free and AmB-loaded nanospheres and nanocapsules.
Percentage of survival 15 day after the therapy (n=12)
Control without drug
4.312 Â± 0.5
4.901 Â± 0.4
4.723 Â± 1.1
4.842 Â± 1.3
4.221 Â± 1.6
(0.5 mg Kg-1, I.V.)
4.310 Â± 0.3
4.943 Â± 0.7
4.701 Â± 0.8
4.898 Â± 0.9
4.250 Â± 1.1
(0.5 mg Kg-1, I.V.)
4.017 Â± 0.03
4.616 Â± 0.13
4.436 Â± 0.19
4.680 Â± 1.1
3.510 Â± 0.20
(0.5 mg Kg-1, I.V.)
3.131 Â± 0.09
2.751 Â± 0.03
1.161 Â± 0.05 *
(0.5 mg Kg-1, I.V.)
2.433 Â± 0.04
2.019 Â± 0.03
1.116 Â± 0.03 *
The values are expressed as mean Â± S.E. 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 and ** p <0.001.
A new approach of reducing the toxicity of amphotericin B, but maintaining its antifungal activity, would represent a significant clinical advance in the management of fungal infections. Superiority of liposome-encapsulated AmB in vitro and in vivo studies has been clearly demonstrated. Many nanoparticulate AmB formulations displayed reduced toxicity to mammalian cells (erythrocytes, macrophages, renal tubular cells), while maintaining activity against yeast (21). It has been documented that mammalian cell (RBC) toxicity arises from the formation of conducting pores in the cell membrane, when AmB binds to cholesterol, a major sterol in mammalian membranes (22-25).
Results showed the spray-dried nanoparticles containing AmB displayed a distinctly decreased hemolytic activity of AmB as compared to that of free AmB. Our data was agreement with the observations of Fukui et al. reported that LNS-AmB (a lipid nanosphere incorporating AmB), have less toxicity to erythrocytes as compared to that of free AmB (26). In vitro antifungal-activity experiments, AmB-loaded nanoparticles maintained the potent activity of AmB against fungal cells even though the AmB was entrapped into spray-dried polyester nanoparticles. In vivo, AmB-loaded nanoparticles showed a stronger protective effect against candidiasis than did free AmB. This difference reflects the difference in the MICs of the formulations: in this experiment, we found, the MICs for AmB-loaded nanoparticles were lower than those for free form of AmB. This phenomenon indicates that the effective therapeutic dose of prepared nanoparticles like LNS-AmB used for the treatment of mycosis, including candidiasis, has a low level of toxicity (27). The mean sizes of both nanoparticle types were characterized at two different times: before they were spray-dried and after they were redissolved from the spray-dried powders. Both nanoparticle types remained in the nano-range size after spray-drying also the mean nanoparticle sizes were increased after spray-drying, though this increase was statistically significant only for the nanospheres. In vitro dissolution studies in the presence of Tween 80 showed that this nanoparticle system provided very slow release of AmB suggesting that AmB was in its majority incorporated into nanoparticles, but don't likely adsorbed onto the particle surface. Tiyaboonchai et al. (1) reported that nanoparticles prepared with chitosan-dextran have very low stability and AmB released from particles with in 5min. Nevertheless, AmB is insoluble in water; therefore, the release rate of AmB in vivo should be lower than that in vitro studied which may lead to slowly increasing AmB concentration in the plasma to avoid toxic effects (28).
In the present study, we compared the in vitro and in vivo potencies of free AmB and prepared nanospheres and nanocapsules against C. albicans (ATCC 90028). This strain was more susceptible to nanospheres that to nanocapsules and free AmB; i.e., a smaller dose of nanospheres was required to inhibit fungal growth. Our study agreement with Pahls et al. (29), who reported among three AmB formulations NS-718, was found to be the most efficacious against Cryptococcus neoformans isolates in vitro and NS-718 was also found to be more effective than liposomal AmB against clinical isolates of Candida albicans. Our results in this study indicate that dryied-spray AmB nanoparticles were efficacious in the treatment of rabbit infected with C. albicans, which correlates with the results of the in vitro study. Prepared AmB nanoparticles at a dose of 0.5 mg/kg inhibited the growth of candidia in the spleen and kidney, but the antifungal activity of free AmB was weaker than that of spray-dried AmB nanoparticles. We investigated that all rabbits died rapidly after therapy with a high dose of free AmB (3.0 mg/kg/day) because of the acute toxicity of AmB, but equivalent doses of spray-dried AmB nanoparticles were well tolerated, indicating a reduction in toxicity with the nanoparticulate formulations. One of the reasons for the differences in the antifungal potencies and toxicities among AmB formulations was suggested by Espuelas et al. (13). Their formulation, which is an AmB-poly (Îµ-caprolacton) nanosphere, was less toxic than free form of AmB. Their results indicated that their nanoparticles containing AmB were more potent in vitro against C. albicans than free AmB. They mentioned that if the high degree of stability of AmB formulations can explain its lower level of toxicity, it is also responsible for the increased efficacy reported by several investigators (30;31).
The results obtained in the present study suggest that prepared, spray-dried AmB nanoparticles may represent a more appropriate choice for patients with mycosis because of the good balance between efficacy and toxicity. Because spray-dried AmB nanoparticles were more efficacious than free AmB, the prepared AmB-loaded nanoparticles, probably in future serve as an effective novel antifungal drug delivery system for the treatment of patients with fungal infections.