Free Nystatin Research Articles

Design of transfersomal nanocarriers of nystatin for combating vulvovaginal candidiasis; A different prospective

The objective of this study was to prepare and evaluate Nystatin (NYS) loaded transfersomes to achieve better treatment of vulvovaginal candidiasis. Nystatin transferosomes were formulated utilizing thin film hydration method. A 32 full factorial design was employed to evaluate the effect of different formulation variables. Two independent variables were chosen; the ratio between lecithin surfactant (X1) was set at three levels (10–40), and the type of surfactants (X2) was set at three levels (Span 60, Span 85 and Pluronic F-127). The dependent responses were; entrapment efficiency (Y1: EE %), vesicles size (Y2: VS) and release rate (Y3: RR). Design Expert® software was utilized to statistically optimize formulation variables. The vesicles revealed high NYS encapsulation efficiency ranging from 97.35 ± 0.03 to 98.01 ± 0.20% whereas vesicle size ranged from 194.8 ± 20.42 to 400.8 ± 42.09 nm. High negative zeta potential values indicated good stability of the prepared formulations. NYS release from transfersomes was biphasic and the release pattern followed Higuchi’s model. The optimized formulation (F7) exhibited spherical morphology under transmission electron microscopy (TEM). In-vitro and in-vivo antifungal efficiency studies revealed that the optimized formula F7 exhibited significant eradication of candida infestation in comparison to free NYS. The results revealed that the developed NYS transfersomes could be a promising drug delivery system to enhance antifungal efficacy of NYS.

Mucoadhesive assessment of different antifungal nanoformulations

Oral candidiasis is an important opportunistic fungal infection and polyenes and azoles are still the most used antifungal agents. However, the oral absorption resulting from most available treatments is generally poor and, consequently, a very high frequency of administrations of antifungal agents is strongly required. Therefore, the major challenge is to improve the retention of the antifungal agents in buccal mucosa, and the encapsulation into mucoadhesive systems may be considered as a possible strategy to achieve this objective. Three types of mucoadhesive polymeric nanoparticles (polylactic acid (PLA), polylactic-co-glycolic acid (PLGA) and alginate) were prepared using nystatin as model drug. The drug-loaded nanoparticles were then included in toothpaste, oral gel and oral films, respectively. The results demonstrated that the loaded nanoparticles were successfully produced, presenting a mean size between 300–900 nm and with a negative surface charge. Also, the determination of the encapsulation efficiency of all nanoparticles showed values above 70%. In terms of the in vitro mucoadhesion, the best formulation was the oral film loaded with the PLGA nanoparticles followed by the oral gel with PLA nanoparticles and thirdly the toothpaste with alginate nanoparticles. This was confirmed in an in vitro rinsing model with mucus producing HT29-MTX cells, where the percentage of nystatin retained to the cells after 40 min of simulated saliva flow was between 10–27% when formulations were used and only 4% for free nystatin. Further studies will include in vivo testing using animal models.

Effects of Archaeal Tetraether Lipids on Membrane Partitioning of the Antifungal Drug Nystatin

Nystatin is a polyene antibiotic that is frequently used in the treatment of cutaneous, vaginal, and oral fungal infections. It interacts with ergosterol in the fungal cell plasma membrane, causing membrane disruption and leakage, and leading to cell death. Liposomal nystatin has good antifungal activity but with significantly less systemic toxicity compared to free nystatin. In this study, we attempt to use archaeal tetraether lipids to increase the stability and drug loading of liposomal nystatin. Using a fluorometric method, we have determined the partition coefficient of nystatin into liposomes composed of cholesterol and POPC (80 mol%) in the absence and presence of archaeal tetraether lipids. We found that nystatin partition into liposomes increases significantly (by a factor of ∼4-10) with the addition of 0.5 mol% archaeal tetraether lipids PLFE to replace 0.5 mol% cholesterol. PLFE is the polar lipid fraction E isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius. This dramatic change in nystatin partitioning engendered by PLFE was surprising and may result from the presence of interfacial regions between diester and tetraether lipid domains. This result plus our previous findings in PLFE-enhanced membrane stability suggest that tetraether lipids can be a useful additional component in liposomal nystatin formulations.

Niosomes as a potential drug delivery system for increasing the efficacy and safety of nystatin

Nonionic surfactant (NIS) vesicles (niosomes) formed from self-assembly of hydrated synthetic NIS monomers are capable of entrapping a variety of drugs and have been evaluated as an alternative to liposomes. Nystatin (NYS) is a polyene antifungal drug that has been used in the treatment of cutaneous, vaginal and oral fungal infections since the 1950s. The aim of this work is to encapsulate NYS in niosomes to obtain a safe and effective formula administered parenterally for neutropenic patients. NYS niosomes were prepared by the thin-film hydration method using Span 60 or Span 40 and cholesterol (CHOL). Stearylamine and dicetyl phosphate were added as the positive and negative charge-inducing agents (CIA), respectively. Two molar ratios were used, namely NIS/CHOL/CIA (1:1:0.1 and 2:1:0.25). Neutral and positively charged niosomes gave the highest encapsulation efficiencies. NYS niosomes were characterized using transmission electron microscopy, differential scanning calorimetry and dynamic light scattering. The release of neutral and negatively charged NYS niosomes was estimated, and it showed a slow sustained release profile. A 25-kGy γ-irradiation dose was sufficient to sterilize the investigated vesicles. NYS niosomes exerted less nephrotoxicity and hepatotoxicity in vivo, showed higher level of drug in vital organs and revealed pronounced efficacy in elimination of the fungal burden in experimental animals infected with Candida albicans compared with those treated with free NYS. Niosomal encapsulation thus provided means for parenteral administration of NYS, reducing its toxicity and making it a more active antifungal agent.

Inhibition of Candida albicans Biofilm Formation by Antimycotics Released from Modified Polydimethyl Siloxane

Unlike various disinfectants, antifungals have not been commonly incorporated so far in medical devices, such as catheters or prostheses, to prevent biofilm formation by Candida spp. In the present study, five antimycotics were added to polydimethyl siloxane (PDMS) disks via admixture (nystatin) or impregnation (trimethylsilyl-nystatin (TMS-nystatin), miconazole, tea tree oil (TTO), zinc pyrithione). Nystatin-medicated PDMS disks exhibited a concentration-dependent inhibitory effect on biofilm formation in a microtiter plate (MTP) but not in a Modified Robbins Device (MRD). This observation, together with HPLC data and agar diffusion tests, indicates that a small fraction of free nystatin is released, which kills Candida albicans cells in the limited volume of a MTP well. In contrast, biofilm inhibition amounted to more than one log unit in the MRD on disks impregnated with miconazole, TTO, and zinc pyrithione. It is hypothesized that the reduction in biofilm formation by these compounds in a flow system occurs through a contact-dependent effect.

Safety, efficacy and pharmacokinetics of tuftsin-loaded nystatin liposomes in murine model

Present study was performed to evaluate the efficacy, toxicity and pharmacokinetics of antifungal drug nystatin incorporated in immunomodulator tuftsin-bearing liposomes. In vitro toxicity of free nystatin and nystatin incorporated in tuftsin-free or tuftsin-loaded liposomes was assessed by incubation of nystatin formulations with human erythrocytes. The toxicity profile of free nystatin and liposomal formulations of nystatin with or without tuftsin was also analyzed by monitoring the level of blood urea nitrogen (BUN) and serum creatinine in the treated BALB/c mice. The results of the present work showed that tuftsin-loaded nystatin liposomes like conventional nystatin liposomes exerted less toxicity to human erythrocytes as compared with free nystatin. Moreover, mice treated with tuftsin-loaded nystatin liposomes showed insignificant elevation in the biochemical values of serum creatinine and blood urea. The stability of nystatin liposomes upon incorporation of tuftsin was evaluated by monitoring the leakage of the entrapped drug in human serum. Tuftsin-loaded liposomes held nystatin for longer duration in the presence of serum than identical nystatin liposomes without tuftsin. Pharmacokinetics of the both tuftsin-free or tuftsin-loaded liposomal formulations nystatin was analyzed by determining the level of nystatin in the systemic circulation of mice at different time points. Mice injected with tuftsin-loaded nystatin liposomes showed higher level of the drug in the systemic circulation compared with those treated with conventional nystatin liposomes. The efficacy of tuftsin-loaded nystatin liposomes against A. fumigatus was evaluated by assessing the fungal burden in the lungs of treated mice. Treatment with tuftsin-loaded nystatin liposomes was most effective in eliminating fungal burden from lung tissues of infected mice compared to those treated with free nystatin or nystatin liposomes without tuftsin. The immunopotentiating activity, increased stability and less toxicity of tuftsin-incorporated nystatin liposomes, supports the idea for its prophylactic and therapeutic use in the clinical setting.

New nystatin polymeric complexes and their in vitro antifungal evaluation in a model study with Fusarium oxysporum.

Six water-soluble nystatin-polyvilnylpyrrolidone complexes with respective MW of 10 kDa (NC1), 25 kDa (NC2), 30 kDa (NC3), 40 kda (NC4), 90 kDa (NC5), 360 kDa (NC6) were synthesized. The activity of the complexes was compared with that of nystatin against growth and spore germination of Fusarium oxysporum f.sp. radiciscucumerinum. The ED50 value (effective dose) of free nystatin in aqueous solution on growth inhibition on solid medium was determined at 35.7 ppm. The ED50 of the complexes NC3, NC4, NC5, and NC6 ranged from 2.2 to 4 times lower than that of nystatin. The NC6 complex exhibited the highest activity, followed by NC5, NC4, and NC3. The activities of NC1 and NC2 were about 3 and 1.7 times higher than nystatin respectively in the same in vitro model. The complexes NC6. NC1 and NC4 were 25.4, 13.6 and 6.9 times more active respectively than nystatin against spore germination of E oxysporum. The activity of the nystatin complexes was dependent on the molecular weight of the polymeric carrier.

Species differences in the proportion of plasma lipoprotein lipid carried by high-density lipoproteins influence the distribution of free and liposomal nystatin in human, dog, and rat plasma.

The objective of this study was an interspecies comparison of free nystatin (NYS) and liposomal NYS (Nyotran) distribution in plasma. NYS and liposomal NYS at concentrations of 5, 10, and 20 microg of NYS/ml were incubated in human, dog, and rat plasma for 5, 60, and 180 min at 37 degrees C. Following these incubations, plasma samples were separated into their high-density lipoprotein (HDL), triglyceride-rich lipoprotein, low-density lipoprotein, and lipoprotein-deficient plasma (LPDP) fractions by density-gradient ultracentrifugation, and each fraction was assayed for NYS by high-pressure liquid chromatography. Total plasma and lipoprotein cholesterol, triglyceride, and protein concentrations in each human, dog, or rat plasma sample were determined by enzymatic assays. When NYS and liposomal NYS were incubated in human, dog, or rat plasma, the majority of the NYS was recovered in the LPDP fraction. For the 5- and 60-min incubation times for all plasmas measured, a significantly greater percentage of NYS was recovered in the lipoprotein fraction (primarily HDL) following the incubation of liposomal NYS than following the incubation of NYS. There was a significant correlation between the lipoprotein lipid and protein profiles in human, dog, and rat plasmas and the distribution of NYS and liposomal NYS in plasma. In particular, differences in the proportion of plasma lipoprotein cholesterol, triglyceride, and apolar lipids (cholesteryl ester and triglycerides) carried by HDL influenced the distribution of NYS and liposomal NYS within plasmas of different species. These findings suggest that the distribution of NYS among plasma lipoproteins of different species is defined by the proportion of lipid carried by HDL, and this is possibly an important consideration when evaluating the pharmacokinetics, toxicities, and activities of these compounds following administration to different animal species.

Plasma Lipoprotein Distribution of Liposomal Nystatin Is Influenced by Protein Content of High-Density Lipoproteins

The plasma lipoprotein distribution of free nystatin (Nys) and liposomal nystatin (L-Nys) in human plasma samples with various lipoprotein lipid and protein concentrations and compositions was investigated. To assess the lipoprotein distributions of Nys and L-Nys, human plasma was incubated with Nys and L-Nys (equivalent to 20 microg/ml) for 5 min at 37 degreesC. The plasma was subsequently partitioned into its lipoprotein and lipoprotein-deficient plasma fractions by step-gradient ultracentrifugation, and each fraction was analyzed for Nys content by high-pressure liquid chromatography. The lipid and protein contents and compositions of each fraction were determined with enzymatic kits. Following the incubation of Nys and L-Nys in human plasma the majority of Nys recovered within the lipoprotein fractions was recovered from the high-density lipoprotein (HDL) fraction. Incorporation of Nys into liposomes consisting of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol significantly increased the percentage of drug recovered within the HDL fraction. Furthermore, it was observed that as the amount of HDL protein decreased the amounts of Nys and L-Nys recovered within this fraction decreased. These findings suggest that the preferential distribution of Nys and L-Nys into plasma HDL may be a function of the HDL protein concentration.

Activity of liposomal nystatin against disseminated Aspergillus fumigatus infection in neutropenic mice.

The purpose of this study was to examine the activity of liposomal nystatin against a disseminated Aspergillus fumigatus infection in neutropenic mice. Mice were made neutropenic with 5-fluorouracil and were administered the antifungal drug intravenously for 5 consecutive days beginning 24 h following infection. Liposomal nystatin, at doses as low as 2 mg/kg of body weight/day, protected neutropenic mice against Aspergillus-induced death in a statistically significant manner at the 50-day time point compared to either the no-treatment, the saline, or the empty-liposome group. This protection was approximately the same as that for free nystatin, a positive control. Histopathological results showed that liposomal nystatin cleared the lungs, spleen, pancreas, kidney, and liver of Aspergillus and that there was no organ damage at the day 5 time point, which was after only three doses of liposomal nystatin. Based on these results in mice, it is probable that liposomal nystatin will be effective against Aspergillus infection in humans.

Physical characteristics and lipoprotein distribution of liposomal nystatin in human plasma.

The physical characteristics and lipoprotein distribution of free nystatin (NYS) and liposomal NYS (L-NYS) in human plasma were investigated. To determine the percentage of NYS that was lipid associated following incubation in human plasma, C18 reverse-phase extraction columns were used. To assess plasma drug distribution, NYS and L-NYS (20 microg/ml) were incubated in human plasma for 5, 60, and 120 min at 37 degrees C. After each interval, plasma was removed and separated into its lipoprotein and lipoprotein-deficient plasma (LPDP) fractions by ultracentrifugation and assayed for NYS by high-pressure liquid chromatography. Further studies evaluated the liposome structure of L-NYS by filtering through a 0.14-microm-pore-size microfilter before and after the addition of human plasma. When reconstituted L-NYS (mean particle diameter +/- standard deviation, 321 +/- 192 nm) was applied to a C18 column, 67% +/- 4% of the initial NYS concentration was associated with the lipid. When plasma samples containing L-NYS that had been incubated for 5 to 120 min at 37 degrees C were applied to C18 columns, 66 to 76% of the NYS was lipid associated. Incubation of NYS in human plasma for 5 min at 37 degrees C resulted in 3% +/- 1% of the initial NYS concentration incubated in the low-density lipoprotein (LDL) fraction, 23% +/- 4% of that in the high-density lipoprotein (HDL) fraction, and 66% +/- 10% of that in the LPDP fraction. In contrast, the distribution of NYS following incubation of L-NYS in human plasma for 5 min was 13% +/- 2% in the LDL fraction, 44% +/- 5% in the HDL fraction, and 42% +/- 5% in the LPDP fraction. Similar results were observed following 60 and 120 min of incubation. In addition, the liposome structure of L-NYS was quickly lost when mixed with plasma. These findings suggest that rapid disruption of the L-NYS structure upon incubation in human plasma is consistent with its rapid distribution in plasma. The preferential distribution of NYS into the HDL fraction upon incubation of L-NYS may be a function of its phospholipid composition.

Formulation, toxicity, and antifungal activity in vitro of liposome-encapsulated nystatin as therapeutic agent for systemic candidiasis.

Multilamellar vesicles containing nystatin (NYS) were compared with vesicles containing the free drug for toxicity to erythrocytes and for antifungal activity in vitro. Liposomal nystatin was as active as free NYS was against a wide variety of yeasts and fungi. The antifungal activity against Candida albicans was maintained with different liposome compositions and without sterols. Liposome encapsulation also protected the erythrocytes from the toxicity of free NYS.

Toxicity and therapeutic effects in mice of liposome-encapsulated nystatin for systemic fungal infections.

The therapeutic activity of nystatin (NYS) incorporated in multilamellar liposomes (L-NYS) was studied in vivo. Hale-Stoner mice injected intravenously with various doses of L-NYS and free NYS showed a significant reduction in toxicity of NYS after the NYS was incorporated into liposomes (maximal tolerated doses, 16 and 4 mg/kg of body weight, respectively). The maximal tolerated dose of free NYS had no effect in the treatment of mice infected with Candida albicans, whereas L-NYS at an equivalent dose improved the survival of mice. A marked increase in survival was observed when L-NYS was administered in higher and multiple doses (total doses up to 80 mg/kg). Liposome encapsulation thus provided a means for intravenous administration of NYS, reducing its toxicity and making it an active systemic antifungal agent.