{"id":56503,"date":"2024-03-20T11:38:17","date_gmt":"2024-03-20T11:38:17","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=56503"},"modified":"2024-04-01T19:06:07","modified_gmt":"2024-04-01T19:06:07","slug":"evaluation-of-the-antifungal-activity-of-fluconazole-nanovesicles-against-aspergillus-fumigatus","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no1\/evaluation-of-the-antifungal-activity-of-fluconazole-nanovesicles-against-aspergillus-fumigatus\/","title":{"rendered":"Evaluation of the Antifungal Activity of Fluconazole Nanovesicles Against Aspergillus fumigatus"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Rhinosinusitis occurs when the mucosa of the nasal and paranasal sinuses is inflamed 1<\/sup>. It may be infectious or non-infectious due to immunological or non-allergic causes. Infectious rhinosinusitis is caused by fungal pathogens such as Bipolaris, Curvularia, and Aspergillus species2<\/sup>. The most prevalent causative agent appears to be Aspergillus. The diagnosis is histological, based on allergic mucin exhibiting fungal components in Gomori’s methenamine silver staining method. The diagnosed Aspergillus fumigatus is tested by various methods to identify the minimum inhibitory concentration (MIC) of fluconazole3<\/sup>. One of the most common fungal isolates causing sinusitis is Aspergillus fumigatus. Surgical debridement followed by prolonged oral antifungal therapy results in chances of attaining renal impairment. <\/p>\n\n\n\n

Fungal rhinosinusitis demands immediate attention, as its urgency\nnecessitates swift initiation of both aggressive antifungal therapy and prompt\nsurgical intervention to effectively address and mitigate the potentially\nsevere consequences of this condition4<\/sup>.<\/p>\n\n\n\n

Fluconazole is\na triazole antifungal medication employed for the treatment of various local\nand systemic fungal infections5<\/sup>. It functions as an inhibitor of\nergosterol, disrupting the integrity and fluidity of the fungal cell membrane,\nthereby impeding fungal growth 6<\/sup>. It exhibits limited solubility in\nwater and boasts a remarkable 90% absolute oral bioavailability. The\nrecommended oral dosage typically ranges from 50 to 400 mg, dependent on the\nspecific pathological conditions7<\/sup>.<\/p>\n\n\n\n

Nanovesicles\nare commonly utilized as carriers or targeting vehicles for active substances\nwithin various body organs and tissues 8<\/sup>. These nanovesicles\nprepared in different forms, including micelles, cubosomes, noisomes, liposomes,\ntransferosomes, and ethosomes 9-11<\/sup>. All serve as a versatile platform\nfor enhancing the drug characteristics such as stability, solubility, and\nrelease characteristics, and overall bioavailability 12<\/sup>. The\nnanovesicle has been fine-tuned to be compatible with multiple administration\nroutes, include ocular, transdermal, oral, parenteral and nasal routes.\nFurthermore, specialized adaptations have been made to create vector oriented\nnanovesicle designed to target drugs to specific locations such as the colon,\nbrain, liver, or tumors13<\/sup>.<\/p>\n\n\n\n

This study was\naiming to determine the appropriate range of nanovesicles loaded with\nfluconazole to be directly exposed to the ATCC strains of Aspergillus\nfumigatus. This is to estimate the MIC of the antifungal agent to determine the\nminimal dosage of the drug to be administered to the infected individual\nrescuing them from the renal impairments that could occur.<\/p>\n\n\n\n

Material and\nMethods<\/strong><\/p>\n\n\n\n

Materials<\/strong><\/p>\n\n\n\n

Fluconazole was provided as a gift from Julphar\n(Gulf Pharmaceutical Industries), United Arab Emirates. Cetyl alcohol and tween\n80 was purchased form Sigma\u2013Aldrich, Co. (St. Lois, United States). Other\nchemical materials of the analytical grade used were not purified further.<\/p>\n\n\n\n

Fluconazole Nanovesicles Preparation <\/strong><\/p>\n\n\n\n

A modified thin film hydration process was used to prepare the\nnanovesicles 14<\/sup>. In a nutshell, fluconazole, Tween 80, in addition\nto cetyl alcohol were precisely measured, dissolved in 2\/1, v\/v mixture of\nmethanol and chloroform 10 mL, and placed in 250 mL flask round bottom. The\nrotary evaporator machine (Heidolph, Rotavapor, VV2000) was then employed under\nvacuum conditions, evaporating the organic solvent mixture at 60 \u00baC and 150 rpm\nfor 20 minutes. The resulting thin film on the wall was hydrated with a 10 mL of\nthe low molecular weight chitosan solution in 0.1 M acetic acid under normal\npressure. To prevent aggregation, the prepared nano-vesicles underwent\nsonication in an ultrasonic bath from (SH 15O-41, PCl Analytic Pvt, Ltd) for a\nperiod of 1 minute 15<\/sup>.<\/p>\n\n\n\n

Statistical Designing <\/strong><\/p>\n\n\n\n

The research employed central composite design with\nDesign Expert\u00ae 7 software (Version 7. Stat_Ease Inc, MN) to investigate the\nimpact of formulation variables on the characteristics of nanovesicles. Two\nindependent factors, namely Tween\/Cetyl with ratio (\u2716l) and chitosan ratio (\u27162), were examined. The response tracked included\nparticle size (PS, Yl), polydispersity index (PDl, Y2), zeta potential (ZP.,\nY3), and encapsulation efficiency (EE., Y4). Additionally, desirability value was\ncalculated to aid in selecting the optimized formula 16<\/sup>.<\/p>\n\n\n\n

Characterization of the prepared fluconazole nanovesicle<\/strong><\/p>\n\n\n\n

Analysis of particle polydispersity index, zeta potential and the size<\/strong><\/p>\n\n\n\n

The Zeta sizer (Nano ZS, Malvern Instrument, Malvern,\nUnited Kingdom) employed dynamic scattering of light for analyzing the polydispersity\nindex (PDI), zeta potential (ZP), and particle size (PS) of the nano vesicular\nformulations. Prior to analysis, dilution of samples from each formulation were\nperformed to the point of haziness.<\/p>\n\n\n\n

Assessment of the encapsulation effectiveness of the formulated fluconazole nanovesicle<\/strong><\/p>\n\n\n\n

Nano-vesicles loaded with\nfluconazole underwent centrifugation at 20,000 revolutions per minute for 1\nhour at 40\u00b0C using a high-speed cool centrifuge (Andres Hetich Gmb.H and Co.\nKG, Tutlingen, Germany) to separate them from the un-encapsulated drug. The\nfluconazole concentration in the supernatant was determined by analyzing it\nwith a UV-spectrophotometer (Shimadzu, Tokyo, Japan) according to previously\nestablished curve of calibration.<\/p>\n\n\n\n

Drug release from nanovesicular formulations in an in-vitro setting<\/strong><\/p>\n\n\n\n

The release of the drug from\nthe optimized nano vesicular formulation in addition to the drug suspensions were\nassessed utilizing the reverse dialysis method using a USP II dissolution machine\n(Pharm Test, Hainburg, Germany)17<\/sup>. A phosphate buffer (pH 6.6) of\n900 mL served as the dissolution medium. Dialysis bags having a cut-off\nmolecular weight of 12-14 kDa were filled with 3 mL of dissolution media. The speed\nof the rotation was set to 50 revolutions per minute, at 37 \u00b1 1 o<\/sup>C.\nSamples were collected at specified time intervals up to 240 minutes, and the concentration\nof the drug was analyzed using spectrophotometer at the predetermined \u03bb max. To\nstatistically compare the release profiles of fluconazole from the nano-vesicular\nformulation and the drug suspension, the similarity factor (f2) was calculated.<\/p>\n\n\n\n

Microbiology Test<\/strong><\/p>\n\n\n\n

Test organism<\/strong><\/p>\n\n\n\n

Aspergillus fumigatus <\/em>ATCC 1022 reference strain were used. <\/p>\n\n\n\n

Inoculum preparation<\/strong><\/p>\n\n\n\n

The test organisms’ inoculants were prepared in accordance with the instructions of the National Committee for Clinical Laboratory Standards (NCCLS) document M38-A 18<\/sup>. These cultures were cultivated on Potato Dextrose Agar (PDA) slants at a temperature of 35\u00b0C for a period of 7 days. For preparation of the conidial inoculants, the cultures were flooded with a sterile solution consisting of 0.85% normal saline and 0.025% Tween 20 (obtained from Sigma company), and slowly agitated using a tip of the pipette. Following this, the resulting suspension was subjected to vortexing, and the heavier particles were allowed to settle for a duration of 3 to 5 minutes. Subsequently, the supernatant was adjusted to achieve a transmission reading of 80 \u2013 82 percent spectrophotometrically, with a wavelength set at 530 nm.<\/p>\n\n\n\n

Broth Microdilution Method<\/strong><\/p>\n\n\n\n

Out of nine fluconazole nanovesicle prepared, one (F2) is tested\nagainst aqueous suspension of the drug. Both the aqueous suspension and\nnanovesicle forms of fluconazole were mixed with RPMI 1640 medium containing\nL-glutamine lacking bicarbonate. The pH of the mixture was adjusted to 7.0 by using\n(0.165 M.MOPS solution by Sigma Company). Serial dilution of the fluconazole\nwere then prepared in nine microtiter plate wells, ranging from 1.0 to 0.2\nmg\/ml. Each well received 100 \u00b5l of the diluted fungal suspensions along with\n100 \u00b5l of the fluconazole solution, resulting in a serial of diluted\nfluconazole concentrations. In order to ensure the accuracy of the experiment, each\nset was subjected to growth and sterile controls as part of the experimental\nprocedure.<\/p>\n\n\n\n

The microtiter plate was subsequently incubated at 35\u00b0C, and after\n48 hours, they were examined to determine the Minimum Inhibitory Concentration\n(MIC). The MICs interpretation was the drug concentration at which 50% inhibition\nof growth was observed. Microscopic examination was used to determine the MICs,\nspecifically identifying the lowest fluconazole concentration that led to morphological\nabnormality of the fungal hyphae characterized by short, numerous branching 19<\/sup>.<\/p>\n\n\n\n

Agar dilution technique<\/strong><\/p>\n\n\n\n

Fluconazole was prepared in serial dilution in a molten medium that\nhad been equilibrated to a temperature of 50\u00b0C. This medium consisted of RPMI\n1640 with 2% glucose (sourced from Sigma Company) and 1.5% Agar. The purpose of\nthis dilution was to create a series of drug concentrations. Subsequently, one\nmilliliter of this mixture was dispensed into each well of a 12-well cell\nculture plate with a flat bottom and kept for solidification. In the middle of\neach well, 10 \u00b5l of the conidial suspension was introduced. To serve as a\ncontrol, organism-free wells were included.<\/p>\n\n\n\n

Minimal Inhibitory Concentrations (MICs) for mold fungi were examined\nafter incubation for 48 h at a temperature of 35\u00b0C. The MICs were determined as\nthe lowest drug concentration that effectively inhibits the fungal growth on\nthe solid agar medium 20.<\/p>\n\n\n\n

Galactomannan\n(GM) antigen release method <\/strong><\/p>\n\n\n\n

Alexander Imhof devised a Galactomannan antigen release method to assess the growth of the fungi independently of colony characteristics or growth density21<\/sup>. This approach was selected because literature had indicated that Galactomannan is released in the growing media in quantities that correlate with the fungal burden21<\/sup>. The microtiter plate was prepared using antifungal substances and fungal samples following the same procedure as the broth microdilution method described earlier. After incubating for 24 hours at a temperature of 35\u00b0C. From each well, 5 \u00b5l of the liquid was added to 5 ml of saline making overall dilution 1:1,000 ratio. The released GM was quantified using an enzyme immunoassay, specifically the GM Test Fungiopert Aspergillus Galactomannan ELISA Detection Kit, following the instructions of the manufacturer.<\/p>\n\n\n\n

Next, 50 \u00b5l of these dilutions were transfer to microtiter plate wells that had been sensitized with EB-A2 monoclonal antibody, which targets Aspergillus Galactomannan. The microtiter plate was then placed in the incubator at 37\u00b0C for 90 minutes. Following this incubation, the microtiter plates washed, then 100 \u00b5l of a buffer solution contain ortho-phenylenediamine dihydro-chloride was added. The microtiter plates were placed in the incubator for an additional 30 minutes in the absence of light at room temperature, before 100 \u00b5l of a 1.5 M. sulfuric acid solution was added to halt the reaction. The reading of the optical density (OD) was taken at 450 nm.<\/p>\n\n\n\n

The optical density index was determined\nby dividing the optical density of each sample by the optical density of a\ncontrol sample that contain 1 ng of Galactomannan \/ml. The rate between the Galactomannan\nindices in the samples and those in the controls were computed, and the concentration\nof the drug at which this ratio approached 0.5 is determined as a Minimum\nInhibitory Concentration (MIC).<\/p>\n\n\n\n

Results and\nDiscussion:<\/strong><\/p>\n\n\n\n

Effect of formulations factors on particle size (PS) <\/strong><\/p>\n\n\n\n

The particle size measurements of the\nfluconazole nanovesicles prepared varied between 450 and 753. The equation for calculating\nthe particle size analysis was:<\/p>\n\n\n\n

particle size (PS) = 633.38 \u2212 114.83 X1<\/sub>\n+ 1.67 X2<\/sub>                                        (1)<\/p>\n\n\n\n

Figure 1A shows that only tween\/cetyl ratio (X1) significantly affected the PS values of the prepared nanovesicular formulations. Where the PS values were significantly decreased with increasing the tween\/cetyl ratio. This could be attributed to the effect of tween 80 in reducing the interfacial tension between the nanovesicular surface and the surrounding aqueous medium. Additionally, the prepared nanovesicles were stabilized and protected from aggregation by the steric hindrance of the used surfactant. These findings are consistent with that published by Elsayed et al., who investigated the effect of tween 80 concentration on the vesicle size of rosuvastatin calcium elastic nanovesicles 23<\/sup>.<\/p>\n\n\n\n

The values of the particle size (PS), polydispersity index (PDI), zeta potential (ZP), and encapsulation efficiency (EE) of the prepared nanovesicular formulations is published in table 1 (part 1 of this project) by Ahmed et al 28<\/sup>. <\/sub><\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: Response surface plots for the effects of tween\/cetyl alcohol (T\/C) ratio (\u27161) and chitosan % (\u27162) on the particle size (a), polydispersity index (b), zeta potential (c), and encapsulation efficiency % (d) of fluconazole nanovesicular formulations.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Effect of\nformulations factors on polydispersity index (PDI) <\/strong><\/p>\n\n\n\n

The polydispersity index (PDI) measurement of the prepared fluconazole\nnanovesicle varied from 0.152 to 1. As illustrated by Ahmed et al 28<\/sup>.\nThe derived equation used for the PDI analysis was:<\/p>\n\n\n\n

PDI = 0.66\n\u2212 0.33 X1 <\/sub>\u2212 0.043 X2<\/sub>                                                        (2)<\/p>\n\n\n\n

Fig. 1B illustrates that only tween\/cetyl\nratio (X1) had a significant impact on the PDI values of the prepared\nnanovesicular formulations. Where the PDI values were significantly decreased\nwith increasing the tween\/cetyl ratio. This could be attributed to the\nefficiency of Tween 80 as a surfactant in preventing the aggregation of the\nprepared nanovesicles. Zambaux et al., and Ruiz et al., also assured that the increase\nof the surfactant concentration resulted in a considerable decrease in the PDI values\n24, 25<\/sup>.<\/p>\n\n\n\n

The PDI is used to make sure that\nthere is no variation in particle size. The formula that has less variation in\nparticle size will has less PDI.<\/p>\n\n\n\n

Effect of formulations factors on zeta potential (ZP) <\/strong><\/p>\n\n\n\n

ZP for the prepared fluconazole\nnanovesicle varied from 7.25 to -2.46. As illustrated by Ahmed et al 27<\/sup>.\n<\/sub>The calculated equation for the zeta potential analysis was:<\/p>\n\n\n\n

ZP = 0.6492 \u2013 1.76 X1<\/sub> \u2013 1.49 X2<\/sub>                                                        (3)<\/p>\n\n\n\n

Figure 1C illustrates that both the\nindependent variables X1 and X2 had no
\nsignificant impact on the zeta potential of all\nthe prepared nanovesicular formulations. <\/p>\n\n\n\n

In general, the zeta potential indicates\nthe potential stability of the colloidal system in solution.<\/p>\n\n\n\n

Effect of\nformulations factors on encapsulation efficiency (EE) <\/strong><\/p>\n\n\n\n

EE % for the prepared fluconazole\nnanovesicles ranged between 92.00 and 96.56 %. As illustrated by Ahmed et al 28<\/sup>.\n<\/sub>The calculated equation for the EE% analysis was:<\/p>\n\n\n\n

EE = 92.54 \u2013 0.753 X1<\/sub> \u2013 0.837 X2<\/sub> + 0.164 X1<\/sub> X2<\/sub> + 1.57 X1<\/sub>2<\/sup> + 0.476 X2<\/sub>2<\/sup>                (4)<\/p>\n\n\n\n

Figure 1D illustrates that both the\nindependent variables X1 and X2 had a significant impact on the EE%\nof all the prepared nanovesicular formulations. The EE% was decreased with increasing\nthe tween\/cetyl ratio.  This\ncould be accredited to that increasing tween\nconcentration led to decreasing the vesicle sizes, hence decreasing the EE% due\nto the small inner space of the prepared vesicles. These findings are in consistency\nwith that stated by Duong et al., who found that the EE% of the formulated\nvesicles were significantly decreased with increasing the surfactant\nconcentration25<\/sup>.  Additionally,\nthe chitosan % (X2) significantly impacted the EE% of the nanovesicles. Where\nthe EE% was significantly decreased with increasing the chitosan %. Increasing\nthe chitosan concentrations could result in increasing the solution viscosity\nwhich could hinder the drug entrapment. This agrees with the findings stated by\nValente et al., who found that the drug EE was significantly decreased with\nincreasing the chitosan % 27<\/sup>. <\/p>\n\n\n\n

Statistical\nanalysis of central composite design <\/strong><\/p>\n\n\n\n

The effect of the studied formulation\nfactors on the characteristics of the prepared nanovesicles was investigated using\ncentral composite <\/strong>design.\nEach response was investigated individually and fabricated using different\norder models. As displayed in Table 1, the predicted R2<\/sup> values of\nall the examined responses were in harmony with the adjusted R2<\/sup>. A\nprecision value higher than 4 was attained in all responses, assuring the\nsuitability of the designed model to navigate the design space.<\/p>\n\n\n\n

Selection\nof the optimized Fluconazole nanovesicular formulation <\/strong><\/p>\n\n\n\n

To identify the optimized nanovesicular formula, it was practically\nhard to provide all the needed responses at the same time., as the optimal\ncondition met for one response may have a detrimental effect on another.\nHowever, the desirability function aggregated all desired responses in one\nvariable to determine the optimum level of the studied factors. Figure 2 shows the\nhighest desirability value was 0.681 for the optimized fluconazole\nnanovesicular formulation (F7) containing tween\/cetyl ratio of <\/strong>15:1 and chitosan concentration of 0.1%. This optimized formulation\ncollectively showed the maximal PS and PDI and maximal ZP and\nEE%. According to the comparison of the observed and predicted values, a\nnotable similarity was observed, as indicated in Table 1. Consequently, the\nnanovesicular formulation that was optimized has been selected for further\ninvestigation.<\/p>\n\n\n\n\n\n
\"\"<\/a><\/td>\n\n

Figure 2: Responses surface plot for the effect of tween\/cetyl (T\/C) ratio (Xl) and chitosan % (X2) on the desirability of fluconazole nanovesicular formulation.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table 1: Output data of the central composite responses surface design and predicted and observed values of the optimized nanovesicular formula.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Responses<\/p>\n<\/td>\n

\n

PS (nm)<\/p>\n<\/td>\n

\n

PDI<\/p>\n<\/td>\n

\n

ZP (mV)<\/p>\n<\/td>\n

\n

EE (%)<\/p>\n<\/td>\n<\/tr>\n

\n

Minimum<\/p>\n<\/td>\n

\n

450.08 \u00b1 7.66<\/p>\n<\/td>\n

\n

0.152 \u00b1 0.01<\/p>\n<\/td>\n

\n

-2.46 \u00b1 0.07<\/p>\n<\/td>\n

\n

92.00 \u00b1 1.02<\/p>\n<\/td>\n<\/tr>\n

\n

Maximum<\/p>\n<\/td>\n

\n

753.15 \u00b1 6.24<\/p>\n<\/td>\n

\n

1 \u00b1 0.00<\/p>\n<\/td>\n

\n

7.25 \u00b1 0.68<\/p>\n<\/td>\n

\n

96.55 \u00b1 2.64<\/p>\n<\/td>\n<\/tr>\n

\n

Model<\/p>\n<\/td>\n

\n

Linear<\/p>\n<\/td>\n

\n

Linear<\/p>\n<\/td>\n

\n

Linear<\/p>\n<\/td>\n

\n

Quadratic<\/p>\n<\/td>\n<\/tr>\n

\n

P-value<\/p>\n<\/td>\n

\n

0.0003<\/p>\n<\/td>\n

\n

< 0.0001<\/p>\n<\/td>\n

\n

0.0697<\/p>\n<\/td>\n

\n

0.0001<\/p>\n<\/td>\n<\/tr>\n

\n

Adequate precision<\/p>\n<\/td>\n

\n

10.97<\/p>\n<\/td>\n

\n

17.33<\/p>\n<\/td>\n

\n

6.35<\/p>\n<\/td>\n

\n

18.35<\/p>\n<\/td>\n<\/tr>\n

\n

Adjusted R2<\/sup><\/p>\n<\/td>\n

\n

0.762<\/p>\n<\/td>\n

\n

0.870<\/p>\n<\/td>\n

\n

0.295<\/p>\n<\/td>\n

\n

0.929<\/p>\n<\/td>\n<\/tr>\n

\n

Predicted R2<\/sup><\/p>\n<\/td>\n

\n

0.604<\/p>\n<\/td>\n

\n

0.795<\/p>\n<\/td>\n

\n

-0.402<\/p>\n<\/td>\n

\n

0.802<\/p>\n<\/td>\n<\/tr>\n

\n

R2<\/sup><\/p>\n<\/td>\n

\n

0.802<\/p>\n<\/td>\n

\n

0.892<\/p>\n<\/td>\n

\n

0.412<\/p>\n<\/td>\n

\n

0.959<\/p>\n<\/td>\n<\/tr>\n

\n

Significant factors<\/p>\n<\/td>\n

\n

X1<\/sub><\/p>\n<\/td>\n

\n

X1 <\/sub><\/p>\n<\/td>\n

\n

None<\/p>\n<\/td>\n

\n

X1<\/sub>, X2<\/sub>, X1<\/sub>2<\/sup>, X2<\/sub>2<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

Observed values of optimal formulation<\/p>\n<\/td>\n

\n

480.35<\/p>\n<\/td>\n

\n

0.495<\/p>\n<\/td>\n

\n

2.256<\/p>\n<\/td>\n

\n

94.4262<\/p>\n<\/td>\n<\/tr>\n

\n

Predicted values of optimal formulation<\/p>\n<\/td>\n

\n

516.885<\/p>\n<\/td>\n

\n

0.376<\/p>\n<\/td>\n

\n

2.380<\/p>\n<\/td>\n

\n

94.505<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Presented data mean \u00b1 SD (n = 3).<\/p>\n\n\n

In vitro drug release from the optimized nano vesicular formulation<\/strong><\/p>\n\n\n\n

The optimized nano vesicular\nformulation significantly increased the Fluconazole release in comparison to\nthe drug suspension. After 240 minutes, 57.14 % of the drug was released from\nthe optimized formulation, while just 17.94 % of the drug was released from the\ndrug suspension, as shown in Figure 3, with a similarity factor (f2<\/em>) of\n33. This considerable increase in fluconazole release from the optimized\nnanovesicular formulation could be due to the large surface area of the prepared nanovesicles\nand the incorporation of tween 80 that enhanced fluconazole diffusion from the\nprepared vesicles to the medium 23<\/sup>.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 3: Fluconazole release profiles from the optimized lipotomal formula compared to the drug suspension.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Microbiology test<\/strong><\/p>\n\n\n\n

Fluconazole suspension and fluconazole nanovesicles MICs were\ndetermined by agar dilution, broth microdilution and GM antigen release against\nAspergillus fumigatus <\/em>ATCC 1022. Aspergillus fumigatus <\/em>showed resistance\nto all concentrations ofregular\nfluconazole by using the three methods.<\/p>\n\n\n\n

The MIC obtained by broth microdilution were similar to the GM antigen release (0.5 mg\/ml) as shown Figure 6. On the other hand, MIC obtained by agar dilution method show little higher (0.6 mg\/ml) compared to the other two methods Figure 4.<\/p>\n\n\n\n

The result of the broth microdilution method was confirmed by examining the microtiter plate under microscope x40 HPF, sign of growth was observed (hyphal conidia and conidiophores) in wells containing 0.2, 0.3, and 0.4 mg\/ml Figure 5.<\/p>\n\n\n\n\n\n
\"\"<\/td>\nFigure 4: Agar dilution technique. Well No.1 control negative (No organism), wells from 2 to 10 fluconazole serial dilution concentration 1 to 0.2 mg\/ml. <\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 5: Broth microdilution method. Upper row shows Aspergillus resistance to normal fluconazole in all concentrations (from 1 to 0.2 mg\/ml). <\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 6: Range of aqueous and fluconazole nanovesicles MIC using different methods, organism is resistant to normal fluconazole, or the MIS is >1 mg\/ml (range used 1 – 0.2).<\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

 Conclusions<\/strong><\/p>\n\n\n\n

In this study, the optimized\nfluconazole nanovesicular formulation and the appropriate concentration\nof fluconazole nanovesicles was identified to be directly exposed to the ATCC\nstrains of Aspergillus fumigatus. This is to determine the minimal dosage of\nthe drug to be administered by the infected individuals rescuing them from the\nrenal impairments that could occur. Finally, fluconazole nanovesicles had a better\neffect and lower MIC when compared to the aqueous fluconazole suspension. Considering these results obtained, the enhanced\nantifungal effect of fluconazole can be attributed to the promising\nnanocarriers provided by the optimized nanovesicular formulation.<\/p>\n\n\n\n

Acknowledgement<\/strong><\/p>\n\n\n\n

None to declare<\/p>\n\n\n\n

Conflict\nof Interest<\/strong><\/p>\n\n\n\n

All authors declared that there is no\nconflict of interest in this research.<\/p>\n\n\n\n

Funding\nSource<\/strong><\/p>\n\n\n\n

This research is funded by Gulf Medical University, Ajman, UAE, grant\nnumber is GMU\/COHS\/GR\/2019-10\/003<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

  1. Greiner, A. N., Hellings, P. W., Rotiroti, G., & Scadding, G. K. J. T. L. Allergic rhinitis., 2011; 378<\/em>(9809), 2112-2122.
    CrossRef<\/a><\/li>
  2. Ciecko, S. C., Scher, R. J. E. E., Nose, & Journal, T. Invasive fungal rhinitis caused by Paecilomyces lilacinus infection: Report of a case and a novel treatment., 2010; 89<\/em>(12). <\/li>
  3. Wallace, D. V., Dykewicz, M. S., Bernstein, D. I., Blessing-Moore, J., Cox, L., Khan, D. A.,  immunology, c. The diagnosis and management of rhinitis: an updated practice parameter., 2008; 122<\/em>(2), S1-S84.
    CrossRef <\/a><\/li>
  4. Piromchai P, Thanaviratananich S. Impact of treatment time on the survival of patients suffering from invasive fungal rhinosinusitis. Clin Med Insights Ear Nose Throat. 2014; 9;7:31-4. doi: 10.4137\/CMENT.S18875. PMID: 25288891; PMCID: PMC4167318.
     CrossRef <\/a><\/li>
  5. Soliman, O. A. E.-A., Mohamed, E. A., Khatera, N. A. A. J. P. D., & Technology. Enhanced ocular bioavailability of fluconazole from niosomal gels and microemulsions: Formulation, optimization, and in vitro\u2013in vivo evaluation., 2019; 24<\/em>(1), 48-62.
    CrossRef <\/a><\/li>
  6. Cardoso NN, Alviano CS, Blank AF, Romanos MT, Fonseca BB, Rozental S, Rodrigues IA, Alviano DS. Synergism Effect of the Essential Oil from Ocimum basilicum var. Maria Bonita and Its Major Components with Fluconazole and Its Influence on Ergosterol Biosynthesis. Evid Based Complement Alternat Med. 2016;2016:5647182.
    CrossRef <\/a><\/li>
  7. Moffat, A. C., Osselton, M. D., Widdop, B., & Watts, J. Clarke’s analysis of drugs and poisons<\/em>., 2011 (Vol. 3): Pharmaceutical press London.<\/li>
  8. Elsaied, E. H., Dawaba, H. M., Ibrahim, E., & Afouna, M. I. J. U. J. o. P. R. Effect of pegylated edge activator on Span 60 based nanovesicles: comparison between Myrj 52 and Myrj., 2019; 59. 4<\/em>(4), 1-8.
    CrossRef <\/a><\/li>
  9. Costa, R., & Santos, L. J. P. T. Delivery systems for cosmetics-From manufacturing to the skin of natural antioxidants., 2017; 322<\/em>, 402-416.
     CrossRef <\/a><\/li>
  10. Javadzadeh, Y., & Bahari, L. A. Therapeutic nanostructures for dermal and transdermal drug delivery. In Nano-and Microscale Drug Delivery Systems<\/em>., 2017;<\/em> (pp. 131-146): Elsevier.
     CrossRef <\/a><\/li>
  11. Mathur, M., & Devi, V. K. J. J. o. D. T. Potential of novel drug delivery systems in the management of topical candidiasis., 2017; 25<\/em>(8), 685-703.
    CrossRef <\/a><\/li>
  12. Indulkar, A. S., Mo, H., Gao, Y., Raina, S. A., Zhang, G. G., & Taylor, L. S. J. P. r. Impact of micellar surfactant on supersaturation and insight into solubilization mechanisms in supersaturated solutions of atazanavir., 2017; 34<\/em>(6), 1276-1295.
    CrossRef <\/a><\/li>
  13. Hsu, C.-Y., Chen, C.-H., Aljuffali, I. A., Dai, Y.-S., & Fang, J.-Y. J. N. Nanovesicle delivery to the liver via retinol binding protein and platelet-derived growth factor receptors: how targeting ligands affect biodistribution., 2017; 12<\/em>(4), 317-331.
    CrossRef <\/a><\/li>
  14. Abdel-Hafez SM, Hathout RM, Sammour OA. Curcumin-loaded ultradeformable nanovesicles as a potential delivery system for breast cancer therapy. Colloids Surfaces B Biointerfaces. 2018;167:63\u201372.
    CrossRef <\/a><\/li>
  15. Xu Y, Zhang X, Zhang Y, Ye J, Wang H-L, Xia X, et al. Mechanisms of deformable nanovesicles based on insulin-phospholipid complex for enhancing buccal delivery of insulin. Int J Nanomedicine. 2018;13:7319.
     CrossRef <\/a><\/li>
  16. Elsayed I, El-Dahmy RM, Elshafeey AH, El Gawad A, Abdelaziz N, Gazayerly E, et al. Tripling the Bioavailability of Rosuvastatin Calcium Through Development and Optimization of an In-Situ Forming Nanovesicular System. Pharmaceutics. 2019;11(6):275.
     CrossRef <\/a><\/li>
  17. Abdel-Messih HA, Ishak RAH, Geneidi AS, Mansour S. Tailoring novel soft nano-vesicles \u2018Flexosomes\u2019 for enhanced transdermal drug delivery: Optimization, characterization and comprehensive ex vivo\u2013in vivo evaluation. Int J Pharm. 2019;560:101\u2013115.
     CrossRef <\/a><\/li>
  18. Wayne, P. J. C. d. M.-A. Reference method for broth dilution antifungal susceptibility testing of yeasts, approved standard., 2022 <\/li>
  19. Arikan, S., Lozano-Chiu, M., Paetznick, V., Rex, J. H. J. A. A., & Chemotherapy. In vitro susceptibility testing methods for caspofungin against Aspergillus and Fusarium isolates., 2001; 45<\/em>(1), 327-330.
    CrossRef <\/a><\/li>
  20. Therese, K., Bagyalakshmi, R., Madhavan, H., & Deepa, P. J. I. j. o. m. m. In-vitro susceptibility testing by agar dilution method to determine the minimum inhibitory concentrations of amphotericin B, fluconazole and ketoconazole against ocular fungal isolates., 2006; 24<\/em>(4), 273-279.
    CrossRef <\/a><\/li>
  21. Alexander Imhof, S. Arunmozhi Balajee, and Kieren A. Marr, New Methods To Assess Susceptibilities of Aspergillus Isolates to Caspofungin, Journal Of Clinical Microbiology., 2003; p. 5683\u20135688.
     CrossRef <\/a><\/li>
  22. R. M. Winn, A. Warris, T. G. Abrahmsen, and P. Gaustad, Abstr. 6th Congr. Eur. Confed. Med. Mycol. Soc., abstr. 2000; P3-016.<\/li>
  23. Elsayed I, El-Dahmy RM, El-Emam SZ, Elshafeey AH, Abd El Gawad NA, El-Gazayerly ON.<\/em> Response surface optimization of biocompatible elastic nanovesicles loaded with rosuvastatin calcium: enhanced bioavailability and anticancer efficacy. Drug Deliv Transl Res.<\/em> 2020;10:1459\u20131475. doi.org\/10.1007\/s13346-020-00761-0.
     CrossRef <\/a><\/li>
  24. Zambaux MF, Bonneaux F, Gref R, Maincent P, Dellacherie E,
    Alonso MJ, et al. Influence of experimental parameters on the
    characteristics of poly (lactic acid) nanoparticles prepared by a
    double emulsion method. J Control Release Elsevier. 1998;50:
    31\u201340<\/li>
  25. Ruiz CC, Hierrezuelo JM, Peula-Garc\u00eda JM, Aguiar J. Interaction
    between n-octyl-b-D-thioglucopyranoside and bovine serum albumin. Open Macromol J. 2008; 2: 6\u201318.<\/li>
  26. Duong V-A, Nguyen T-T-L, Maeng H-J. Preparation of Solid Lipid Nanoparticles and Nanostructured Lipid Carriers for Drug Delivery and the Effects of Preparation Parameters of Solvent Injection Method. Molecules<\/em>. 2020; 25(20):4781. https:\/\/doi.org\/10.3390\/molecules25204781.
     CrossRef <\/a><\/li>
  27. Valente JFA, Pereira P, Sousa A, Queiroz JA, Sousa F. Effect of Plasmid DNA Size on Chitosan or Polyethyleneimine Polyplexes Formulation. Polymers<\/em>. 2021; 13(5):793. https:\/\/doi.org\/10.3390\/polym13050793.
    CrossRef <\/a><\/li>
  28. Ahmed Luay Osman1, Salah Eldin Omar Hussein, Iqra Nizam, Deepa Dilip, Mariam Mahamadou, Jood Al Herafi, Sana Gulroz, Ibrahim Elsayed, Abd Elgadir Elamin Eltom, Devapriya Finney, Praveen Kumar Kandakurti. An In Vitro Evaluation of Anti-fungal Activity of Different Nano forms of Fluconazole Against Candida albicans, Biomedical & Pharmacology Journal., 2023; 16(3), 1421-1430.
     CrossRef <\/a><\/li><\/ol>\n","protected":false},"excerpt":{"rendered":"

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