This study aimed at preparing chitosan nanoparticles (C.N.P.s) from fungal Chitosan for the purpose of producing nano-drug delivery systems for antifungal drugs. Chitosan was extracted from mycelia of Aspergillus niger using modified methods of George et al., (2011) and (Naghdi et al., 2014). For confirmation, the FTIR spectrum of the isolated Chitosan was compared with standard. A nanoformulation for Voriconazole antifungal drug was synthesized via ionic gelation method and characterized for their particle size, polydispersity index, surface charge, functional group composition using and Malvern Zeta Sizer ZS-90, P.C.S., FTIR, S.E.M. The prepared nanoformulation was also checked for its antifungal activity using Aspergillus niger and Aspergillus fumigatus as an indicator organism. Voriconazole showed maximum growth with no mycelial growth while CNPS showed minimum growth of about 70 percent, and Voriconazole loaded CNPS showed inhibitory growth effects on the mycelial growth of A. niger and A. fumigatus. Our results indicated that Chitosan Nanoparticles have huge potential against fungal diseases caused by Aspergillus species.
Research Article
Extraction and characterization of fungal chitosan and voriconazole loaded chitosan antifungal nanoformulations
https://doi.org/10.21203/rs.3.rs-957555/v1
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Chitosan
nanoparticles
Aspergillus niger
PCS
FTIR
SEM. Voriconazole
antifungal
Fungi are the prominent kingdom in the phylogenetic tree of the eukaryotic domain. There are around one million species of fungi, of which only ten percent (100,000) are present to date. The fungal field contains yeast, a single-celled fungus (Saccharomyces cerevisiae), in addition to molds and mushrooms, which are multicellular. Mostly the fungi life cycle stays in a haploid state. During sexual reproduction, they formed diploid cells. Like the cells of other organisms, and the fungi have cell walls. However, fungi are distinct in their cell walls because they consist of chitin, unlike others that contain cellulose. Chitin is a unique and environmentally friendly polymer of which the exoskeleton of specific organisms is made up (Webster et al., 2012).
Most fungi are eukaryotic; there are membrane-bound components and a well-defined nucleus. When there was less research on fungi, they thought to put them under kingdom Plantae. However, on examination, it was found that fungi do not undergo photosynthesis because they lack chlorophyll. They are also distinguished structural and physiological features from plants because of their cell wall components and cell membrane. Fungi have no standard features with that of other living organisms, including animals. They are heterotrophic because of their mode of growth and nutrition. Fungi grow from hyphae that make the cascade of hyphae and make a mycelial cluster, and they absorb organic matter before digesting it as a source of food (Vernon Ahmadjian et al).
People are openly exposed to many contagious spores consistently, generally they have self-influenced consequence on their wellbeing. In the Respiratory System of patients that need adequate pneumonic insusceptible precautions, Aspergillus. fumigatus spores can develop and grow the branch into contagious hyphae. The Infection would spread to different organs all throughout the body. Patients with Strong insusceptible immune system can fight its contagious spores by mucociliary clearance, macrophages, and other basically aspiratory guard instruments. C. albicans, in actuality, is a commensal life form dwelling in the gastrointestinal lot and oral, and vaginal mucosa of most solid and healthy individuals, where it commonly doesn't hurt healthy people. In any case, Candida unreasonable development can become indicative causing mucosal layer contaminations, the most well-known being thrush and vaginal candidiasis. Extreme fundamental Candida diseases (Candidemia) can spread to all through circulation system and cause serious contamination to immunocompromised patients. Current methodologies for identifying IFIs depend on clinical signs and manifestations and microbial examines. For instance, pneumonic contagious diseases are regularly inspected by means of CT examine, circled back to Broncho alveolar lavage (BAL) and biopsy. Fundamental yeast diseases, for example, candidiasis can be analyzed by the blood culture. Nonetheless, current symptomatic apparatuses as a rule don't recognize fungal pathogens at initial phases, and hence, antifungal treatment in many cases is non effective(Vega & Kalkum, 2012).
Invasive fungal infections (IFIs) are rapidly identified as a major concerning factor in severely immunocompromised adult and paediatric patients, with significant morbidity and mortality rate. They can be broadly classified into coarse of its action and can be life threatening in some individuals (Gullo, n.d.). Despite recent breakthroughs in the etiology and treatment of invasive fungal infections (IFIs), morbidity and mortality from these infections remain very high, particularly in the absence of early diagnosis and prompt antifungal treatment initiation, two factors that are the most important predictors of survival from these infections. IFIs caused by Aspergillus spp. and Candida spp., account for about 95% of cases of IFI (Jenks et al., 2020).
Fungal infections are considered a global crisis, significantly effective with high morbidity and mortality ratios. Many effective antifungal agents exist, while their uses are limited due to toxicity and other physicochemical factors. Nanoformulations have a significant advantage to bypass such limitations due to the small size, multifunctional, target-specific, and biocompatible, which is the most crucial objective for any formulation. Lipid-based Nano-drug delivery modes are the most extensively researched therapy as nanoformulations compared to others such as Polymeric systems (Soliman, 2017).
Nanoparticles synthesized by the assembly of biologically active polymers have extensive research in drug delivery, precision medicine. Polymeric systems show excellent in improving the therapeutics while diminishing its withdrawal effects. Many Polymeric-based nanoformulations of cytotoxic drugs are already in clinical studies, while many others are in the developmental stage. By the method of their synthesis, they are categorized in nanosphere, nanocapsules, and polymeric micelles. On voriconazole-based polymeric systems, Chitosan-coated poly lactic-co-glycolic acid nanoparticles of voriconazole were synthesized to improve efficacy for extended periods and provide sustained drug release localized therapy for recurrent lung-fungal infection (Paul et al., 2018; Soliman, 2017).
To improve the pharmaceutical and pharmacological properties of antifungal drugs nanotechnology can be explored. Nanoformulations of antifungal drugs can potentially improve their solubility and bioavailability at the site of infection. Moreover, nanoformulations can help us develop more convenient dosage forms of drugs which cannot be administered orally by enhancing their GI compatibility and safety. This research intended to develop fungal chitosan based nanoformulation of Voriconazole and assess its pharmaceutical and pharmacological properties.
Microorganism used
Aspergillus niger was utilized during this research for the production of Chitosan. Aspergillus niger was isolated from stale bread.
Cultivation medium
Sabouraud dextrose broth (Oxoid) and Sabouraud Dextrose Agar was used for the cultivation of fungi.
Culture Conditions
The culture media was prepared and kept for sterilization at 121 oC for 20 minutes. After sterilization, the antibacterial agent (ciprofloxacin) was added to the culture to prevent bacterial contamination. The fungal strains were inoculated on medium, and they were allowed to grow for 2-3 weeks at 30 oC.
Isolation of Aspergillus Niger
Aspergillus niger was isolated and purified from a stale bread Sample. An inoculum was taken from stale bread and cultivated into sabouraud dextrose agar, allowed to grow at 30 oC for two weeks. Then samples were transferred to a fresh broth. In fresh broth, the growth of fungi is visible in 2 days. After that, this method is adopted for mass culture of Aspergillus niger after microscopic identification. The inoculum was prepared and transferred to Sabouraud Dextrose Broth (OXOID). They were kept for their growth for 2 to 3 weeks at 30o C.
Extraction of chitosan from fungal mycelia
Aspergillus niger fungal strain, purified the strain from stale bread sample and cultured on Sabouraud Dextrose Agar and then transferred the inoculum to Sabouraud Dextrose Broth to undergo the fermentation process, which was kept for incubation at 30o C for 2-3 weeks.
Collection of Alkali Insoluble Material (AIM)
For collecting Alkali Insoluble Material, Aspergillus niger was allowed to grow for 2-3 weeks at 30o C. The fungal mycelium (mat-like Structure black and white) was collected using an aseptic technique and was weighed and grounded with pestle and mortar. It was treated with 1N NaOH (50 ml/gram) and 2N NaOH (30ml/gram) respectively, the solution containing grounded mycelium was subjected to autoclaving at 121 oC for 20 mins to obtain Alkali insoluble material. A.I.M. will be Collected by Centrifugation at 9000 RPM for 20 mins. Then A.I.M. is subjected to washing with distilled water for the normalization of pH 7, A.I.M. is Kept for oven drying at 40 oC overnight.
Acid treatment of AIM
After drying, the A.I.M. were weighed, now ready for further extraction. Both samples (Chitosan-AA and Chitan L1) were treated with 2 % Acetic Acid and 0.1 N Lactic Acid (30ml/gram). They are now kept for reflux conditions (Continuous Stirring at room temperature) to dissolve Chitosan from A.I.M. Now they are subjected to centrifugation at 9000 Rpm for 20 mins.
Precipitation of Chitosan
The supernatant containing Chitosan solution was harvested, the pH was adjusted to 10-13 by 2N Sodium Hydroxide to precipitate fungal Chitosan. The alkali containing Chitosan solution then recentrifuged to collect the Chitosan pellet at 9000 Rpm for 20 mins. The extracted Chitosan was washed 4-5 times with distilled water to normalize the pH, and finally, it was rinsed with 95 % ethanol and air-dried.
Characterization of Chitosan
Fourier transformed infrared Spectroscopy (FT IR IRTracer-100, UET CASE) was used to identify the chitosan using KBr pellets.
Synthesis of Chitosan Nanoparticles
Voriconazole matrixed Chitosan nanoparticles were prepared by the Ionic gelation method which was developed by (Zhang et al., 2020) improved protocol. The synthesis involved the combination of sodium T.P.P. (Glentham life Sciences, U.K.) (10 mL, 1 mg /mL) in droplets form in the solution of extracted Chitosan (20 mL, 1mg/ml) with and without voriconazole (20 mg/mL; dissolved in DMSO). 1 % Acetic acid (v/v) was used to make the solution of Chitosan via continuous stirring at room temperature overnight at 500 Rpm. The free ions concentration was adjusted to 4.6 pH using 0.1 N sodium Hydroxide, they were subjected to undergo 8-20 hours of continuous stirring using a magnetic stirrer at room temperature, the synthesized Chitosan Nanoparticles and Nanoformulations (V.R. 5, V.R. 10, and V.R. 20, VR 100) were subjected to sonication for 20 minutes afterward subjected centrifugation at 9500 Rpm for 20 mins. The resultant pellets were washed thrice with distilled water to bring normal pH and before storage, it was washed with 10 % Ethanol, the Nanoformulation were air-dried and refrigerated at 4o C for further use.
Loading and Encapsulation Efficiency of Voriconazole
The Voriconazole Nanoformulations were dispersed in phosphate-buffered saline (PBS,6 ml) for the identification of free drug concentration using calibration curve as standard operation. Few milligrams of voriconazole-based nanoformulations were centrifuged at 9000 rpm for 30 min in PBS. The superficial liquid was collected to measure free drug concentration with UV Vis spectral absorption band at 286 nm. Free drug conc will be calculated by the following equation.
Invitro Drug Release Studies
The release of nanoformulation in simulated fluid was calculated by making a clear suspension of 15 mg of each sample in 100 ml Phosphate buffer Saline (PBS) neutral pH at 37o C and Kept it in a shaking incubator at 120 rpm. Over 30 hours at predetermined time intervals, take out 5 ml aliquots and measure concentration using U.V. Spectrophotometer (Multiskan Go, Thermo scientific). A fresh 5ml medium replaced the dissolution medium to maintain the volume.
Invitro Antifungal activity
The antifungal activity of voriconazole pure and synthesized nanoformulations were checked against Aspergillus niger & Aspergillus fumigatus. Nutrient agar media was prepared, autoclaved, and poured into a sterile test tube to prepared slant, incubated at 37oC for 24 hours to check sterility. After sterility check, the inoculation of the fungal strains was done using a spirit lamp to maintain sterile conditions, to obtained seven days old culture of fungal species as per the requirement of standard procedure. Sabouraud dextrose agar media was prepared in test tubes and autoclaved. After autoclave, when the media temperature decreases up to 50 oC, the test solution of 82.98 µl was added to separate the test tube from the stock solution of pure voriconazole chitosan nanoparticles (100 ugs/mL). Test tubes were placed in a slanting position, and then the fungal strains were inoculated in the test tube and incubated for one week at 30 ºC. After one week, the results were noted by checked the liner growth inhibition of fungal species. The percent growth inhibition was obtained through the following formula.
Characterization of Voriconazole loaded Chitosan Nanoparticles
UV-Vis spectral analysis
UV-Vis Spectroscopy, performed by (Multiskan GO, Thermo Scientific) by using a quartz cuvette and a range of 200-500 nm, respectively. Each sample was repeated three times to maintain the quality of the research.
FT-IR characterization
IR-Tracer 100 (Shimadzu Instrument at UET CASE) FTIR was used to analyze Chitosan nanoparticles, and VR Loaded CNPS by KBr pellet technique. The Nanoformulations samples were crushed with KBr to form a transparent body and run on a spectral range of 4000 cm to 400 cm.
Zeta Potential Analysis
The charge on nanoformulations will be identified by using a zeta potential analysis (Malvern Zetasizer Nano ZS90 UOP). The study will be conducted at a dispersing angle of 90° at the temperature of 25 °C with the nanoformulation dispersed into Milli-Q water. The niche of the zeta charge studies is based on the speed regulation of the particles under the pressure of pre-determined electrical impulses.
Photon Correlation Spectroscopy
The atomic size and polydispersity index (PDI) of Voriconazole-based nanoformulation were determined by photon correlation spectroscopy (PCS) with a ZetasizerZS-90 (Malvern, UOP). Before Sample analysis, all nanoformulations of Voriconazole was dispersed into Milli Q water. All experiments were run thrice to keep the quality of the research.
Scanning Electron Microscopic Analysis (SEM)
The morphological study of Chitosan Nanoparticles and Voriconazole-based nanoformulations will be measured through Scanning Electron Microscope (Jeol JSM-IT-100, NCEG, U.O.P.). The sample was stuck by carbon tape to hold the samples, and it is followed by Gold or Carbon coating as materials are non-conductive.
Extraction of Chitosan from Fungal Mycelia
The growth of Aspergillus niger was observed for 2-3 weeks. For the Identification of Species, microscopic analyses were performed. All fungi identification was performed with isolated plates of the most grown fungi present in each sample Plate. Fungal identification was completed using gross and microscopic morphology of the fungi. Lactophenol methylene blue and Lugol’s iodine with 0.25 % glycerol were used to stain the fungal samples for easier microscopic identification. The yield of chitosan from Aspergillus niger was 50mg/g from wet weight using lactic acid extraction, while the product was little from acetic acid was 35mg/g of the wet weight of alkali-insoluble material.
Table 1 Microscopic Characteristics of Aspergillus niger
|
Aspergillus Species |
Size |
Stipes Color |
Surface |
Vesicle Serration |
Metula Covering |
Shape |
Conidia Surface |
|
A. niger |
400-3000 |
Slightly brown |
Smooth |
Biseriate large size |
Entirely |
globose |
Very rough Irregular |
Table 2 Yield of Chitosan from Aspergillus niger using two acids with slight modification
|
Fungi used |
Growth Condition |
pH |
Yield of Chitosan (mg/g) |
|
Aspergillus niger was isolated from stale bread and further identified by Leica DM 500 microscope. |
SDA for 2 3 weeks at 30o C Antibiotic was added to prevent any bacterial contamination |
5.6 0.2 |
35 mg/ g via 2 % acetic acid and 50 mg/g via 0.1 N lactic acid w/c involves phosphate removal step via 0.1N H2SO4 |
|
1 N NaOH and 2 N NaOH were used as Deproteinization. |
For extraction of Chitosan pH was adjusted to 10-13 |
Chitosan was confirmed by FT IR Identification (UET CASE Peshawar) |
FT IR Identification of extracted chitosan
The standard chitosan (Glentham Lifesciences) and fungal chitosan extracted from mycelia of Aspergillus niger with a modified method of acetic acid as mentioned by (George et al., 2011; Sebastian et al., 2019) and lactic acid method mentioned by (Naghdi et al., 2014) respectively, both methods shared similarities with that of standard. There was a strong peak between 3200-3500 cm-1 which indicates the hydroxyl group (OH). while the spectral peaks at 1650,1590, and 1380 indicate the expression of glucose(glucosamine) units. The absorption band at 1150 cm-1 is a feature of saccharide structure (Valencia et al., 2021). These spectral bands indicate the presence chitosan structure. which was confirmed by Fourier Transform Infrared Spectroscopy (I.R. tracer 100, Shimadzu Instruments) at the facility of UET CASE Peshawar.
Synthesis of Chitosan and Voriconazole Loaded Chitosan Nanoparticles
Chitosan nanoparticles and Voriconazole-based nanoformulations were successfully synthesized by ionic gelation method using chitosan as polymer, Sodium tripolyphosphate (NaTPP) as the cross-linking agent, and voriconazole as an encapsulant. The ionic gelation method is used because very easy to operate as it does not require high-class equipment. It takes is a less time-consuming and very economical way to synthesized polymeric nanoparticles. Chitosan nanoparticles were formed with aggregation. Therefore, it was concluded that while Standard method for nanoparticle formation remain unchanged, particle size differed in most of experiments. Results indicated that particle size was influenced by the concentration of cchitosan Tripolyphosphate, the concentration higher than 0.5 will lead to quite a large particle size of 100-1000 nm so to form low molecular size nm the minimum concentration should be 0.5-1. so, it is concluded that chitosan TPP conc should be 1:1 or 2:1. to need more uniform size particles then more optimization is required. The P.D.I. value emphasizes the distribution molecular size; Experimental formulations with a wide range of nanoparticles size have much greater P.D.I. values, whereas precisely distributed nanoparticles have decreased P.D.I. concentrations (Masarudin et al., 2015).
UV Vis Spectral Analysis
The UV-Vis Spectroscopic measurements are shown in Figure 7. The peaks of chitosan N.P.s, Voriconazole, and VR-CNPs were in between at 284, 286, and 292 nm, respectively.
FT IR Characterization
The FTIR spectrum was analyzed for measuring the peaks at different bandwidth positions of voriconazole-based nanoformulations and the bandwidth of each biomolecule individually (Voriconazole (ref), Chitosan, Chitosan Nanoparticles) which are depicted below. A surge in bandwidth at 3500 to 3200 cm−1 was reported for the activated group of Chitosan and is due to the hydroxyl stretching. The emergence of bands at 1630, 1531 cm−1 is because of the factor which stimulates amino group vibration of positively charged amines and Carbon and hydrogen group are because of flexion in vibration or inclusion of alkyl functional group. The spectral peaks at 1059 and 886 cm−1 are identified by glucopyranose ring in chitosan stacks. There is a result of TPP interaction with ammonia group in chitosan ring and several hydrogen bonds in Chitosan TPP matrix which in resulted in lowering of absorption peaks (Saharan et al., 2013). The C.N.P.s peak changed the course of its spectra to 1576 cm because of twitching of the ammonia NH2 bond, and the significantly crucial surge of the peak at 1412 cm-1 is because of the Carbon-Hydrogen bonds flexion of vibration in the alkyl group. The ionic interaction with two biomolecules indicates the conversion of chitosan polymer in the nano form, forming a cross-link with the treated molecules.
The voriconazole spectrum indicates the presence of hydroxyl group elevating at 3200–3046 cm, Carbon-Nitrogen stretching at 1510.28–1451.28 cm−1, and C-F bandwidth at 1587.44–1451.28 cm. further, it is indicated that the Physical mixture of Chitosan, T.P.P., and Voriconazole showed no significant shifting of any functional peaks between the spectra of polymer and drug and their combined effect; hence it is observed that there is no interaction between Chitosan and voriconazole.
Particle Size, Zeta Potential and Poly Dispersity Index
The Voriconazole-based nanoformulations were measured using Malvern Zeta sizer ZS90 at the Department of Pharmacy University of Peshawar. The particle size of the formulation of Chitosan Nanoparticle was found to be 156 nm and Voriconazole-based chitosan nanoparticles were found to be 188 nm, respectively. The average size of the nanoformulation is based on the Chitosan T.P.P. concentration used during the formulating process. The results obtained clearly state that the zeta potential of the synthesized Voriconazole-based nanoformulation was found to be +26 mV. In context with dispersity index, the nanoformulations are highly polydisperse with multiple particle size communities. The polydispersion index was 0.745 for Chitosan nanoparticles and 0.803 for Voriconazole-based nanoformulation, respectively. higher charge cationic strength indicates that the nanoformulation was highly stable.
Table 3 Particle size, PDI, and Zeta potential analysis of synthesized VR CNPS
|
NPS |
Particle Size (nm) |
PDI |
Zeta Potential |
|
CNPS |
156.4 10.8 |
1 |
0.745 |
|
5 ug/ml VRCNP |
220.2 |
0.282 |
0.948 |
|
10 ug/ml VRCNP |
211 |
0.62 |
0.902 |
|
20 ug/ml VR CNP |
198.11 |
0.645 0.4 |
0.950 |
|
100 ug/ml VRCNP |
188.4 10.2 |
0.803 |
26.6 4.85 |
Scanning Electron Microscope
S.E.M. images indicate the size and morphology of the combined N.P.s. The dimensions of chitosan N.P.s integrated with Voriconazole-based nanoformulations were found to be 156.8 nm and 188.4 nm, respectively, while the nanoparticles were formed circularly. The emergence of the particles is because of the strong interaction of the voriconazole in the presence of Chitosan N.P.s. The above results clearly show that C.N.P.s show a remarkably potent location due to compounding symptoms. The powerful, porous, and agglomeration capabilities of the C.N.P. provide them with benefits as a sensitive bio-nano pesticide based on Chitosan nanoparticles. Accumulation is considered a key component of C.N.P. novels applications in the pharmaceutical and Biotechnology domain. The same idea holds for agriculture. Thus, the dynamic environment can trap extinguished molecules to promote harmful chemicals in the soil. (Rizeq et al., 2019; Saharan et al., 2013)
Loading and Encapsulation Efficiency
The entrapment of Voriconazole-based nanoformulations is measured at four different concentrations (5, 10, 20, and 100 μg /ml). The loading and entrapment potency is measured on the concentration of 100 μg /ml values of 19.75% and 82.98%.
Table 4 Loading and encapsulation efficiency of VR CNPS
|
Formulation |
Drug loading |
Encapsulation Efficiency |
|
5 ug /ml |
3.48 |
25.2 1 |
|
10 ug /ml |
3.95 0.5 |
27.02 2 |
|
20 ug/ml |
5.77 0.6 |
24.9 0.8 |
|
100 ug/ml |
19.75 2 |
82.98 2 |
Invitro Drug Release Profile
The most characteristics of a polymeric drug delivery vehicle are its potential to deliver a drug that binds to the specific target cells. The release profile of Voriconazole-based antifungal nanoformulation was initiated from zero hours to thirty hours (0-30 hrs). For this purpose, PBS at pH 7.4 was used to study Voriconazole Release from nanoformulation in simulated pH media. Voriconazole effects on C.N.P.s reached 63.08% after six h due to the presence of control release. The tangible attraction between polymer and drugs has played an essential role in regulating drug release. The cumulative production of V.R. from CNPS was 11.01%, 17.34%, 23.33%, and 30.08% after two h, respectively. The C.N.P.s showed the release of control after the initial drug release.
Invitro Antifungal Activity
Antifungal activity of voriconazole (82.98 ugs/ml), chitosan nanoparticles, and VR-CNPS (82.98 ugs/ml) was checked in S.D.A. slants. In the conducted study, voriconazole showed 99.99%, while CNPS showed 70 %, and Voriconazole-based nanoformulation showed 85 % inhibition against controlled Aspergillus niger compared with Aspergillus fumigatus there was a slight difference among minimum inhibition.
Table 5 : Antifungal activity of Voriconazole, CNPS, and VR CNPS
|
|
Percent inhibition |
||||
|
Positive control |
Negative control |
CNPS |
VR-CNPS (100ug/ml) |
VR |
|
|
Aspergillus niger |
100 |
0 |
70 |
85 |
100 |
|
Aspergillus fumigatus |
100 |
0 |
65 |
80 |
98 |
Aspergillus spp from a stale bread sample during this study to obtain chitosan from a biological source. Chitosan nanoparticles were synthesized by ionic gelation technique containing voriconazole in assessing the potential changes in the potency of the antifungal agent. However, the antifungal activity of fungal Chitosan piled voriconazole was not reported previously. The concentration of Chitosan and T.P.P. used was 2:1, which resulted in the aggregation of particles, thus making them polydisperse. Results showed that molecule size was not controlled by the grouping of Chitosan or T.P.P. utilized. However, it is instead dictated by different variables.
P.D.I. has been used as a sign of nanoparticle sensitivity and developmental harmony. P.D.I. mirrors showing nanoparticle size distribution; tests with a wide range of molecular sizes have higher P.D.I. perceptions, while tests containing equally measured particles have lower P.D.I. values than randomly distributed particles. In our study, low sensitivity exhibits multiple C.N.P.s and more significant molecular sensitivity, while higher values indicate lower cell viability and the presence of integrated Chitosan-based nanomaterials. The entrapment efficiency was measured at 286 nm utilizing the standard curve for voriconazole. We dissolved our drug in 100% DMSO (20mg/ml). As mentioned by (al Mohaimeed et al., 2018), The absorption spectra in DMSO and D.M.F. show broadband, but this broadband shifted to a higher wavelength in dioxane. The broadening and shifting of the absorption peaks at lower energy (longer wavelength) are due to the coordination of solvent with VR-CNPs. Our research demonstrated that voriconazole-loaded chitosan nanoformulations have significant antifungal activity against Aspergillus spp, including A. niger and A. fumigatus.
This research opens up a whole new world, searching for an effective combination of Chitosan-based products and other early varieties to ensure broader antimicrobial, strong anti-leishmanial, and antibacterial resistance significant problem for future generations.
Chitosan-based nanomaterials are among the most highly recommended complex carbohydrate biomaterials for various functions due to their specific properties, environmentally friendly, safety, and antimicrobial properties. Current research is centered around improving, biocompatibility with other active molecules and a combination of novel C.N.P.s to upgrade their potency in topical nano-drug delivery systems and other bio-based applications. Likewise, a few C.N.P.s and nanocomposites with polymeric frameworks include polyglycolic corrosive, polylactic corrosive, metal and metal oxides, and carbon nanostructures created to beat a couple of constraints of unblemished C.N.P.s, including aggregation, solvency, and antimicrobial activity. (Rizeq et al., 2019; Saharan et al., 2013).
Fungal chitosan based antifungal nanoformulations were successfully formulated via ionic gelation method. Voriconazole based antifungal nanoformulations were effective as chitosan can increase their drug loading and entrapment efficiency. It increases the chitosan activity against Aspergillus spp. It can be concluded that chitosan based antifungal nanoformulation can effective in treating fungal infections caused by Aspergillus spp.
Future Prospects
More studies are required in order to increase of chitosan from fungal mycelia which holds very bright future. While the use of nanomaterials offers great advantages in the biomedical field, current research on the safety of various NPs is not enough for their application in the biomedical field. Commonly, Chitosan was found to be relatively safe due to its biodegradable and biocompatible properties. However, several studies showed the cytotoxicity of CNPs in vitro and in vivo. Thus, the present knowledge on chitosan-based nanomaterials is not developed enough, and extensive research on the fabrication of CNPs and their biological properties is urgently needed.(Rizeq et al., 2019; Saharan et al., 2013)
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Acknowledgements
I would like to thank CECOS University for their support
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