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Abstract

Natamycin (NAT) is the drug of choice for the treatment of fungal keratitis (FK). However, its inherent shortcomings, such as poor solubility, high dosing frequency, and long treatment cycle, need to be urgently addressed by designing a new delivery to widen its clinical utility. Growing research has confirmed that clotrimazole (CLZ) plays a significant role in fungal growth inhibition. Hence, coaxial electrospray (CO-ES) technology is used herein to prepare nano-systems with an average hydrodynamic particle size of 309-406 nm for the co-delivery of NAT and CLZ in chitosan (CTS) and poly(lactic-co-glycolic acid) (PLGA). The resulting NAT/CLZ@CTS/PLGA formulations were characterized by a transmission electron microscope (TEM) and in vitro release test. The results show that the formulations had obvious core-shell structures, uniform particle distribution, and also can sustain the release of drugs over 36 h. Furthermore, in vitro hemolysis, in vivo corneal irritation test, local allergenic test, and antifungal activity analyses are performed to evaluate the safety and efficiency of the formulations. Thus, good biosafety along with a significant anti-candidiasis effect are found in the NAT/CLZ@CTS/PLGA nanoparticles (NPs). Taken together, the results suggest that this design may provide a promising drug delivery system and a new option for the treatment of FK.

Preparation of Electro-Sprayed NPs

Various NP formulations were produced, including the single drug-loaded formulations (NAT@CTS/PLGA and CLZ@CTS/PLGA) and the co-loaded formulation NAT/CLZ@CTS/PLGA.

In vitro Drug Release

The in vitro release of NAT and CLZ from electro-sprayed NPs was performed using a dialysis bag (3,500 molecular weight cutoff, membrane-cell, Chicago, IL, United States) (). First, the samples were centrifuged at 12,000 rpm for 10 min and then the supernatant was discarded and the recovered NPs were rinsed with distilled water to exclude the free drugs. Subsequently, 2.0-ml samples were added into a dialysis bag, which was then immersed in PBS (50.0 ml) and gently swung at 37°C and 100 rpm. The release medium was sampled (1.0-ml) at predetermined time and immediately replaced with 1 ml fresh release medium (phosphate buffered saline, PBS, pH 7.4) after each sampling. The cumulative dose of NAT and CLZ was measured by HPLC method as described above.

Abstract

Natamycin (NAT) is the drug of choice for the treatment of fungal keratitis (FK). However, its inherent shortcomings, such as poor solubility, high dosing frequency, and long treatment cycle, need to be urgently addressed by designing a new delivery to widen its clinical utility. Growing research has confirmed that clotrimazole (CLZ) plays a significant role in fungal growth inhibition. Hence, coaxial electrospray (CO-ES) technology is used herein to prepare nano-systems with an average hydrodynamic particle size of 309-406 nm for the co-delivery of NAT and CLZ in chitosan (CTS) and poly(lactic-co-glycolic acid) (PLGA). The resulting NAT/CLZ@CTS/PLGA formulations were characterized by a transmission electron microscope (TEM) and in vitro release test. The results show that the formulations had obvious core-shell structures, uniform particle distribution, and also can sustain the release of drugs over 36 h. Furthermore, in vitro hemolysis, in vivo corneal irritation test, local allergenic test, and antifungal activity analyses are performed to evaluate the safety and efficiency of the formulations. Thus, good biosafety along with a significant anti-candidiasis effect are found in the NAT/CLZ@CTS/PLGA nanoparticles (NPs). Taken together, the results suggest that this design may provide a promising drug delivery system and a new option for the treatment of FK.

Preparation of Electro-Sprayed NPs

Various NP formulations were produced, including the single drug-loaded formulations (NAT@CTS/PLGA and CLZ@CTS/PLGA) and the co-loaded formulation NAT/CLZ@CTS/PLGA.

Zone of Inhibition Tests

The antifungal activity of electro-sprayed NPs was assessed against C. albicans by the zone of inhibition (ZOI) test (), which determined the level of antifungal activity that inhibits the growth of C. albicans. The NAT/CLZ@CTS/PLGA NPs were tested at a concentration of drugs equivalent to that of the NAT@CTS/PLGA NPs and CLZ@CTS/PLGA NPs. First, the C. albicans were co-cultured with sterilized SDB slants at 37°C and diluted with fresh medium to a concentration of 106 CFU/ml after 24 h of co-culture. Meanwhile, the sterilized SGA medium was poured into a disposable Nest culture plate and allowed to coagulate at room temperature (25 ± 1°C). The C. albicans (0.5 ml) were then evenly swabbed onto each plate and a 6-mm-diameter sterilized filter paper was then placed in the center of each culture plate. Then, an 8-μl sample of the NAT/CLZ@CTS/PLGA NPs was added to a fresh, sterilized 6-mm-diameter filter paper in a separate plate, and PBS (0.02 M, pH 7.4) was added as the negative control. To allow sufficient time for the fungi to grow and the formulations to diffuse, all the plates were placed in a low-temperature incubator (Shanghai Jinghong Experimental Equipment Co. LTD, SHP-150, China) at 37°C for 48 h. The potential antifungal activity was then presented as the diameter (mm) of ZOI.