Amphotericin B, poly-lactide co-glycolide (PLGA) and polyvinyl alcohol (PVA) were purchased from Sigma–Aldrich, USA. All other chemicals used in this study were of highest purity.

Pathogen free Swiss albino female mice with an average weight of 18–22g were used throughout the study. The animals were fed rodent feed and filtered water ad libitum. The animals were housed in propylene cages on wood powder bedding under standard atmospheric conditions (22±1°C temperature; 12h light/12 dark photoperiod and 50–60% humidity). All experiments were performed according to the Guide for Experimental Animal Care Review Board, College of Pharmacy, King Saud University.

C. neoformans (ATCC 24067) strain was procured from American Type Culture Collection, USA. The strain was recovered from glycerol stocks and maintained in yeast extract peptone dextrose (YPD) media while shaking at 37°C for 48h. The cell suspension was centrifuged at 5000×g for 15min at 4°C and washed thrice with sterile normal saline. After quantifying viable cell count the mice were infected intravenously with C. neoformans (1×106cfu/mouse).

AMB was diluted firstly in 5% glucose and subsequently in phosphate buffered saline (PBS) and was given intraperitoneally (ip).

For isolation of plasma, blood was collected through retro-orbital puncture from healthy mice with a heparinized capillary. Blood in heparin was centrifuged at 400×g at 4°C and plasma (supernatant) was isolated. An aliquot of plasma (250μL) was immediately used for preparation of plasma beads whereas rest of the plasma was stored at −20°C for further use. Plasma beads were prepared according to the published protocol.21

Preparation of PLGA was based on water in-oil-in-water (W/O/W) system. Microspheres were prepared by solvent evaporation technique according to the published protocol.22 For the preparation of AMB encapsulated PLGA emulsion: 5mg of AMB in 5% glucose was mixed with 190mg PLGA dissolved in dichloromethane, 10% PVA was added to primary emulsion formed by sonication. After homogenization the resulting O/W emulsion was stirred for 18h after which the formulation of AMB-PLGA microsphere was centrifuged and washed. The final purified formulation was kept in dessication after lyophilyzation at 4°C.

The fibrin microsphere based dual delivery system was prepared by encapsulating AMB in PLGA microsphere and further entrapped in fibrin beads according to the published protocol.4 Plasma (250μL) was mixed with the suspension of AMB encapsulated PLGA microsphere in PBS. To the mixture, calcium chloride (40mM) was also added. Aliquots (3μL) of the mixture were transferred as droplets over a parafilm covered glass slide which were then incubated at 37°C for 40min. The beads after collecting from the parafilm were washed with normal saline and then with PBS.

Healthy Swiss albino mice were divided into six groups of five mice each. One group served as control and received 0.25mL PBS, ip. The remaining groups were injected with different amount of AMB. After 24h, the mortality rate of each group was determined. The 0.5mg/kg b.w. dose of free drug resulted in almost 50% survival of the treated mice. Therefore, this dose was used in present experiment. The selected dose is similar to the already reported dose.18

Antifungal treatment was begun one day post-infection for a period of seven days. Treatment was given once in the 1st, 3rd, and 5th day post-infection. The pathogen infected mice were divided into five groups comprising of 10 animals each. The mice of group I were used as untreated control and received no treatment. Group II mice were injected with free AMB at a single dose of 0.5mg/kg b.w. ip. Group III mice were injected ip with 500μL of AMB-plasma beads. In Group IV, the mice were treated with AMB-PLGA microsphere at a dose of 0.5mg/kg b.w. in 500μL; ip. Group V mice were injected ip with 500μL of AMB-fibrin microsphere.

The efficacy of AMB-fibrin microsphere against severity of C. neoformans was confirmed by assessing the cfu count in various vital organs, namely lung, brain and blood. The mice from experimental groups were euthanized in the 7th, 14th and 21st day post-infection and organs were taken out aseptically. The organs were homogenized separately in 5mL PBS and an aliquot of the homogenate was plated on YPD agar plates after appropriate dilutions. The number of colonies in each group were noted after 48h of incubation at 37°C. The fungal load in each organ was calculated by multiplying with the respective dilution factor.

The efficacy of the formulation was further confirmed by monitoring the survival of remaining mice from experimental groups. For survival studies, mortality of the mice was observed twice each day during 60 days of observation.

The data were analyzed by one-way analysis of variance (ANOVA) following Student's t-test. p values of <0.05 were considered statistically significant. All analyses were performed with SPSS v20.0 software.

Fig. 1a shows the fibrin microsphere beads formed with 3μL calcium-enriched plasma. The beads were spherical in shape with diameter between 1.0 and 1.5mm. Fig. 1b shows the size and surface morphology of AMB-PLGA microspheres as revealed by SEM. PLGA microspheres were spherical in structure and their size varied in the range of 300–800nm (Fig. 1b). The size of the AMB-PLGA microsphere was further confirmed with the particle size analyser and it was in good agreement with their diameter (Fig. 1c).

Fig. 1.

Image depicting AMB-fibrin microsphere. (A) A picture of fibrin beads bearing AMB loaded PLGA microspheres (AMB-fibrin microsphere), (B) scanning electron micrograph of AMB loaded PLGA microspheres, AMB loaded PLGA microspheres visualized by the nanophox particle size analyzer.

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The entrapment efficiency of AMB-PLGA microsphere and AMB-fibrin microsphere was obtained to be 65.6±2.5 and 78.31±4.7, respectively. The zeta-potential stability of AMB-fibrin microsphere was found to be −35±2.0mV.

The AMB-fibrin microsphere was found to withstand both normal and alkaline conditions for a period of 48h (Fig. 2). For first two days, the formulation at pH 7 showed sustained release pattern, where only ∼10 to 30% of AMB was released from the total amount loaded in the fibrin microsphere. The rest of the 89% of AMB was released from formulation in one week. However, the whole AMB leaked out in the 4th and 5th days at pH 4 and 9, respectively.

Fig. 2.

Percent release of AMB from fibrin microsphere. Fibrin microsphere containing entrapped AMB was incubated in different pH at 25°C and release kinetics of the drug was determined spectrophotometrically. Each value represents mean±SD of three determinations.

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The effects of AMB-fibrin microsphere were investigated against C. neoformans in murine model. In the first set of experiments, the fungal burden in tissues was taken into consideration. All the mice of treatment groups were infected with 1×106cfu/mouse of C. neoformans. The antifungal therapy was started one day post-infection on alternate days. The mice were euthanized in the 7th day of therapy for determining early tissue burden and in the 14th and 21st days for determining late tissue burden. The effect of therapy on reduction in fungal burden in blood, brain, and lung is shown in Figs. 3–5, respectively. In the blood, AMB-fibrin microsphere therapy was more effective than free AMB (Fig. 3). The AMB-fibrin microsphere therapy showed significant (p<0.05) reduction in the fungal load in blood from 4.12±0.019 mean cfu on day 7 to 1.68±0.018 mean cfu on day 21 as compared to the fungal burden in untreated control (4.91±0.001 mean cfu on day 7). Free AMB was able to reduce fungal burden till day 7 only (4.00±0.026 mean cfu). In contrast, AMB-PLGA microsphere was also able to reduce the total load of C. neoformans present in blood from 3.18±0.019 mean cfu on day 7 to 2.68±0.018 mean cfu on day 21.

Fig. 3.

Effect of AMB-fibrin microsphere therapy on fungal burden in blood of mice infected with C. neoformans. Antifungal efficacy was recorded as reduction in fungal load at days 7, 14, and 21 post-infection. The data represent the mean cfu/organ of three different experiments±SD.

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Fig. 4.

Effect of AMB-fibrin microsphere therapy on fungal burden in brain of mice infected with C. neoformans. Antifungal efficacy was recorded as reduction in fungal load at days 7, 14, and 21 post-infection. The data represent the mean cfu/organ of three different experiments±SD.

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Fig. 5.

Effect of AMB-fibrin microsphere therapy on fungal burden in lung of mice infected with C. neoformans. Antifungal efficacy was recorded as reduction in fungal load at days 7, 14, and 21 post-infection. The data represent the mean cfu/organ of three different experiments±SD.

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AMB-fibrin microsphere was superior at reducing fungal burden in brain (Fig. 4). The mean cfu values 4.01±0.021 (day 7), 3.23±0.021 (day 14), and 2.19±0.023 (day 21) demonstrates a significant (p<0.05) reduction in fungal burden in brain upon treatment with the formulation as compared to mean cfu value of 4.91±0.005 (day 7) and 6.34±0.003 (day 14) of untreated controls. Free AMB was unable to invoke significant antifungal response.

In the lung, the AMB-fibrin microsphere therapy was also found more effective than free AMB (Fig. 5). For C. neoformans, the fibrin microsphere as well as AMB-PLGA microsphere with the exception of free AMB and AMB-fibrin beads were significantly effective at reducing fungal burden relative to untreated controls (p<0.05). The AMB-fibrin microsphere therapy was significantly more effective (mean cfu 4.10±0.003, day7; 3.01±0.014, day 14; 1.65±0.015, day 21) than untreated controls (mean cfu 4.89±0.005, day 7; 6.87±0.007, day 14) at reducing fungal burden in the lung.

For survival studies, the mice were observed throughout 60 days, and deaths were recorded twice daily. The survival curves of all treated groups are presented in Fig. 6. For C. neoformans infection, AMB-fibrin microsphere was more effective in prolonging survival than AMB-PLGA microsphere, AMB-fibrin beads or free AMB. However, the survival of mice treated with the formulation and AMB-PLGA microsphere were significantly different relative to the untreated controls (p<0.05). AMB-fibrin microsphere based dual therapy increased the survival time of treated mice where almost 80% of the animals survived day 60 post-infection. Treatment with free AMB resulted in survival of 40% of the animals till day 60. AMB-PLGA microsphere and AMB-fibrin beads therapy did also improved the survival rate by 60% and 50%, respectively, as compared to the untreated controls (p<0.05) at day 60 post-infection. In the untreated control group, infection with C. neoformans resulted in 100% mortality by day 20 post-infection.

Fig. 6.

Prophylactic potential of AMB-fibrin microsphere in terms of survival of mice that were infected with C. neoformans. The mice were monitored for survival till day 60 post-infection. Data represents mean±SD of three different experiments.

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