Amphotericin B and Curcumin Co-Loaded Porous Microparticles as a Sustained Release System against Candida albicans.

Amphotericin B (AMB) is an antifungal drug used for serious fungal infections. However, AMB has adverse reactions such as nephrotoxicity, which limit the clinical application of AMB alone or in combination with other antifungal drugs. Nano or micro drug delivery systems (DDS) have been proven to be effective in reducing the toxic and side effects of drugs. Further, the combination of AMB with other compounds with antifungal activity, such as curcumin (CM), may enhance the synergistic effects. Herein, AMB and CM were co-loaded into porous poly (lactic-co-glycolic acid) (PLGA) microparticles (MPs) to prepare AMB/CM-PLGA MPs. The AMB/CM-PLGA MPs showed a remarkably reduced hemolysis (62.2 ± 0.6%) compared to AMB (80.9 ± 1.1%). The nephrotoxicity of AMB/CM-PLGA MPs is significantly lower than that of AMB. In vitro, AMB/CM-PLGA MPs had better inhibitory effects on the adhesion and biofilm formation of Candida albicans compared with AMB. Experiments on mice infected with C. albicans showed that AMB/CM-PLGA MPs have a better therapeutic effect than AMB in vivo. In summary, AMB/CM-PLGA MPs may be a novel and promising therapeutic candidate for fungal infection.

Fungus-mediated green synthesis of nano-silver using Aspergillus sydowii and its antifungal/antiproliferative activities.

Due to the increasing demand for eco-friendly, cost-effective and safe technologies, biosynthetic metal nanoparticles have attracted worldwide attention. In this study, silver nanoparticles (AgNPs) were extracellularly biosynthesized using the culture supernatants of Aspergillus sydowii. During synthesis, color change was preliminarily judge of the generation of AgNPs, and the UV absorption peak at 420 nm further confirms the production of AgNPs. Transmission electron microscopy and X-ray diffraction were also used to identify the AgNPs. The results shows that AgNPs has crystalline cubic feature and is a polydisperse spherical particle with size between 1 and 24 nm. Three main synthesis factors (temperature, pH and substrate concentration) were optimized, the best synthesis conditions were as follows 50 °C, 8.0 and 1.5 mM. In the biological application of AgNPs, it shows effective antifungal activity against many clinical pathogenic fungi and antiproliferative activity to HeLa cells and MCF-7 cells in vitro. Our research finds a new path to biosynthesis of AgNPs in an eco-friendly manner, and bring opportunity for biomedical applications in clinic.

Micronized curcumin fabricated by supercritical CO2 to improve antibacterial activity against Pseudomonas aeruginosa.

Curcumin (CM) is a natural polyphenolic compound with multiple biomedical functions. However, clinical applications face more challenges due to its low dissolution rate and poor bioavailability. Micronization is an effective strategy to overcome these drawbacks. Herein, CM nanoparticles (CM NPs, ∼300 nm) were fabricated using solution enhanced dispersion by supercritical CO2 (SEDS). The solubility of CM NPs was remarkably enhanced. Aim to study the effects of micronization on the biological functions of CM, we investigated the antibacterial activity of original CM and CM NPs upon Pseudomonas aeruginosa. In vitro, the minimal inhibitory concentrations (MIC) assay, solid-medium spot assay, growth kinetics assay and morphologic observation using atomic force microscopy (AFM) confirmed that the anti-P. aeruginosa activity of CM NPs was enhanced compared to original CM. Moreover, CM NPs also showed stronger inhibition for adhesion and biofilm formation of P. aeruginosa compared to original CM. Experiments on mice infected with P. aeruginosa showed that CM NPs have a better therapeutic effect than the original CM in vivo. In summary, CM NPs may be a novel and promising therapeutic candidate for bacterial infection.

Curcumin-Silk Fibroin Nanoparticles for Enhanced Anti-Candida albicans Activity In Vitro and In Vivo.

Curcumin (CM) has multiple pharmacological activities including anti-fungal activity, but its clinical application is limited due to low solubility in aqueous media, poor bioavailability and extensive first pass metabolism. We aimed to resolve the limitation and enhance antifungal activity of CM using nanotechnology. Using silk fibroin (SF) as a carrier, we fabricated curcumin-silk fibroin nanoparticles (CM-SF NPs) by solution enhanced dispersion of supercritical CO2 (SEDS) technique. The characterization of CM-SF NPs was analyzed using scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FTIR), differential scanning calorimeter (DSC) and thermo gravimetric apparatus (TGA). Following characterization of the NPs, we evaluated the antifungal activity of CM-SF NPs against Candida albicans in vitro and in vivo. A SF-based drug delivery system (CM-SF NPs, 85 ± 15 nm) was established by SEDS. Compared to original CM, water solubility of CM-SF NPs was improved, and its antifungal activity was enhanced. The natural compound-loaded SF nanoparticles may be a promising therapeutic candidate for fungal infection.

Reducing the toxicity of amphotericin B by encapsulation using methoxy poly(ethylene glycol)-b-poly(l-glutamic acid-co-l-phenylalanine).

Amphotericin B (AmB) is an antifungal drug used for serious fungal infections and leishmaniosis. However, its clinical application is limited because of its high toxicity. To resolve this problem, herein we loaded AmB into methoxy poly(ethylene glycol)-b-poly(l-glutamic acid-co-l-phenylalanine) (mPEG-b-P(Glu-co-Phe)) nanoparticles (l-AmB) via electrostatic, hydrophobic and π-π interactions. The l-AmB has excellent stability both in PBS and in plasma and shows a remarkably reduced hemolysis (17.1 ± 1.5%, 6 h) compared to the free AmB (94.2 ± 5.3%, 6 h). The nephrotoxicity of l-AmB is significantly lower than that of free AmB. The maximum tolerance dose (MTD) of l-AmB is 3.0 mg kg-1, which is 3.75 fold that of free AmB (MTD = 0.8 mg kg-1). The antimicrobial activity of the conjugate was retained in vivo, with l-AmB proving to be a more protective treatment for Aspergillus fumigatus infections in mice than AmB alone. These indicate that l-AmB is a formulation of AmB with low side effects.

Deletion of afpab1 Causes Increased Sensitivity to Oxidative Stress and Hypovirulence in Aspergillus fumigatus.

Aspergillus fumigatus AFPAB1 is the ortholog of the Aspergillus oryzae cytoplasmic messenger ribonucleoprotein granules AOPAB1 that function to depress the initiation of translation during stress. A. fumigatus can regulate its cellular physiology in response to environmental stresses, but there has been no research on Pab1 in A. fumigatus. The associated gene afpab1 was replaced with a hygromycin-selectable marker to generate the strain Δafpab1. Phenotypic analysis showed that the Δafpab1 grew more weakly than the wild-type strain. Also the germination rate of Δafpab1 was decreased when exposed to oxidative stress. The morphology of Δafpab1 spores also showed great changes. The killing rate of Δafpab1 by RAW264.7 murine macrophage cells was increased, and the reactive oxygen species (ROS) scavenging ability of Δafpab1 was decreased. Pathogenicity testing showed that the deletion strain had decreased virulence. Therefore, we conclude that afpab1 activity is correlated with susceptibility to oxidative stress, and deletion of afpab1 from A. fumigatus possibly leads to observed hypovirulence in an immunosuppressed mouse model.

Biosynthesis of silver nanoparticles by the fungus Arthroderma fulvum and its antifungal activity against genera of Candida, Aspergillus and Fusarium.

The objective of this study was to find one or more fungal strains that could be utilized to biosynthesize antifungal silver nanoparticles (AgNPs). Using morphological and molecular methods, Arthroderma fulvum was identified as the most effective fungal strain for synthesizing AgNPs. The UV-visible range showed a single peak at 420 nm, which corresponded to the surface plasmon absorbance of AgNPs. X-ray diffraction and transmission electron microscopy demonstrated that the biosynthesized AgNPs were crystalline in nature with an average diameter of 15.5±2.5 nm. Numerous factors could potentially affect the process of biosynthesis, and the main factors are discussed here. Optimization results showed that substrate concentration of 1.5 mM, alkaline pH, reaction temperature of 55°C, and reaction time of 10 hours were the optimum conditions for AgNP biosynthesis. Biosynthesized AgNPs showed considerable activity against the tested fungal strains, including Candida spp., Aspergillus spp., and Fusarium spp., especially Candida spp.

Identification and characterization of an anti-oxidative stress-associated mutant of Aspergillus fumigatus transformed by Agrobacterium tumefaciens.

Aspergillus fumigatus is one of the most common opportunistic pathogenic fungi, surviving in various environmental conditions. Maintenance of the redox homeostasis of the fungus relies upon the well­organized regulation between reactive oxygen species generated by immune cells or its own organelles, and the activated anti­oxidative stress mechanism. To investigate such a mechanism, the present study obtained a number of randomly­inserted mutants of A. fumigatus, mediated by Agrobacterium tumefaciens. In addition, a high throughput hydrogen peroxide screening system was established to examine ~1,000 mutants. A total of 100 mutants exhibited changes in hydrogen peroxide sensitivity, among which a significant increase in sensitivity was observed in the AFM2658 mutant. Further investigations of the mutant were also performed, in which the sequence of this mutant was characterized using thermal asymmetric interlaced­polymerase chain reaction. This revealed that the insertion site was located on chromosome 2 afu1_92, and the 96 bp sequence was knocked out, which partially comprised a sequence localized between the integral membrane protein coding region and the helix­loop­helix transcription factor coding region. A decrease in the levels of anti­oxidative stress­associated mRNAs were observed, and an increase in reactive oxygen species were detected using fluorescence. The results of the present study demonstrated that this sequence may have a protective role in A. fumigatus in the presence of oxidative stress.