Nano-sized chitosan/VS2 nanocomposite like flowers for potential pharmaceutical applications

A nano-sized chitosan/VS2 nanocomposite for use in various pharmaceutical applications. The nano-sized chitosan/VS2 nanocomposite can be formed as a flower nanocomposite.

BACKGROUND

The present disclosure relates to a nano-sized chitosan/VS2nanocomposite for use in various pharmaceutical applications.

2. Description of the Related Art

Recently, nanotechnology has begun to be investigated for use in obtaining nanoscale materials with antibacterial properties in order to create novel therapeutic goods and efficient prophylactic and treatment methods for infections. Naturally occurring polysaccharides have been utilized as powerful templates for stabilizing a variety of metal ions, metal sulfides, and metal oxides because of their low toxicity, low cost, and excellent biological properties. For this purpose, numerous polymer-supported metal or metal oxide nanocomposite materials have been designed in an attempt to find materials providing unique properties and fit many applications not achievable separately by each component. Currently, a growing interest among researchers is the design and growth of metal oxide polymer complexes that can be successfully utilized in many applications like water treatment, nano catalysis, and as biologically active agents, among many other applications.

Chitosan (CS), the partial deacetylated form of chitin, is prepared via alkaline deacetylation under certain conditions. During this process, acetamide groups are converted into primary amino groups, as shown below. Chitosan has been intensively utilized due to its superior biodegradability, biocompatibility, low toxicity, and film forming properties. Chitosan and its derivatives have a wide range of biological activities that have been widely explored, particularly in biomedical science. Chitosan and its derivatives, more so than most other polysaccharides, are considered as powerful templates for the preparation of metal oxide nanoparticles owing to their unique ability to combine with metal ions via the hydroxyl and amino groups.

So far, numerous effective techniques such as chemical vapor deposition, electro-Fenton processing, electrodeposition, liquid exfoliation, solvothermal methods, and microwave heating processes, have been carried out to synthesize transition metal dichalcogenide (TMDC) materials like VS2. These techniques frequently have drawbacks because they take an extended period of time, require difficult conditions, and/or utilize risky and expensive organic solvents.

Accordingly, compositions and methods solving the aforementioned problems are desired.

SUMMARY

The present subject matter relates to the use of a combination of hydrothermal and calcination synthesis methods as the most advantageous for the economy and the environment. Such methods result in a novel chitosan/VS2nanocomposite which has never previously been achieved and having various pharmaceutical applications.

Accordingly, the present subject matter relates to a chitosan/VS2nano composite having the formula:

In one embodiment, the present subject matter relates to a method for making the chitosan/VS2nano composite described herein, the method comprising: stirring chitosan in an acetic acid solution to produce a chitosan solution; adjusting pH of the chitosan solution to a pH of about 6 to about 7 to obtain a pH adjusted chitosan solution; adding a VS2nanoflower to the pH adjusted chitosan solution while stirring to obtain a mixture; drying the mixture to remove any remaining acetic acid; and obtaining the chitosan/VS2nano composite.

In an embodiment, the present subject matter relates to a method for treating a microbial infection in a patient, the method comprising administering a microbial treating effective amount of the chitosan/VS2nano composite as described herein to a patient in need thereof.

In another embodiment, the present subject matter relates to a method for treating cancer in a patient, the method comprising administering a cancer treating effective amount of the chitosan/VS2nano composite as described herein to a patient in need thereof.

In a further embodiment, the present subject matter relates to a method for promoting an antioxidant effect in a patient, the method comprising administering an antioxidant amount of the chitosan/VS2nano composite as described herein to a patient in need thereof.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions

It should be understood that the drawings described above or below are for illustration purposes only. The drawings are not necessarily to scale, with emphasis generally being placed upon illustrating the principles of the present teachings. The drawings are not intended to limit the scope of the present teachings in any way.

The term “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, “optionally substituted alkyl” means either “alkyl” or “substituted alkyl,” as defined herein.

“Subject” as used herein refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, and pet companion animals such as household pets and other domesticated animals such as, but not limited to, cattle, sheep, ferrets, swine, horses, poultry, rabbits, goats, dogs, cats and the like.

“Patient” as used herein refers to a subject in need of treatment of a condition, disorder, or disease, such as by way of non-limiting example a microbial infection, cancer, or an antioxidant needing condition.

The present subject matter relates to the use of a combination of hydrothermal and calcination synthesis methods as the most advantageous for the economy and the environment. Such methods result in a novel chitosan/VS2nanocomposite which has never previously been achieved and having various pharmaceutical applications.

Accordingly, the present subject matter relates to a chitosan/VS2nano composite having the formula:

In certain embodiments, the chitosan/VS2nano composite can be formed as flower-like particles.

In additional embodiments, the VS2can be immobilized on the chitosan.

In further embodiments, the chitosan/VS2nano composite can have antibacterial, antifungal, and anti-cancer properties.

In one embodiment, the present subject matter relates to a method for making the chitosan/VS2nano composite described herein, the method comprising: stirring chitosan in an acetic acid solution to produce a chitosan solution; adjusting pH of the chitosan solution to a pH of about 6 to about 7 to obtain a pH adjusted chitosan solution; adding a VS2nanoflower to the pH adjusted chitosan solution while stirring to obtain a mixture; drying the mixture to remove any remaining acetic acid; and obtaining the chitosan/VS2nano composite.

In an embodiment of the present production methods for making the chitosan/VS2nano composite, the chitosan can be stirred in the acetic acid solution for about 5 hours.

In another embodiment of the present production methods for making the chitosan/VS2nano composite, the chitosan solution can have a concentration of chitosan of about 2% w/v.

In a further embodiment of the present production methods for making the chitosan/VS2nano composite, the pH of the chitosan solution can be adjusted by adding a 0.5 M NaOH solution.

In an additional embodiment of the present production methods for making the chitosan/VS2nano composite, the mixture can be dried for about 4 hours at about 60° C.

In one embodiment of the present production methods for making the chitosan/VS2nano composite, the dried mixture can be dehydrated.

In an embodiment, the present subject matter relates to a method for treating a microbial infection in a patient, the method comprising administering a microbial treating effective amount of the chitosan/VS2nano composite as described herein to a patient in need thereof.

In one embodiment, the microbial infection is caused by bacteria. In one embodiment in this regard, the microbial infection can be caused byPseudomonasbacteria. In another embodiment in this regard, the microbial infection can be caused by a fungus.

In another embodiment, the present subject matter relates to a method for treating cancer in a patient, the method comprising administering a cancer treating effective amount of the chitosan/VS2nano composite as described herein to a patient in need thereof. In an embodiment in this regard, the cancer can be colon cancer.

In a further embodiment, the present subject matter relates to a method for promoting an antioxidant effect in a patient, the method comprising administering an antioxidant amount of the chitosan/VS2nano composite as described herein to a patient in need thereof.

The present teachings are illustrated by the following examples.

EXAMPLES

Preparation of the Chitosan-VS2Flower Like Nanocomposite

A calculated amount of chitosan (1 g) was swirled for 5 h at room temperature with a magnetic agitator in a 1% (v/v) acetic-acid solution to produce a chitosan solution of 2% (w/v). The pH of the resultant chitosan solution was brought up to the pH range of 6-7 by gradually adding a calculated amount of 0.5 M NaOH solution while continuously stirring. The anticipated quantity of VS24%) nano-flower was then gradually added to the Chitosan solution, portion by portion, while the mixture was continuously stirred. The chitosan-VS2nanocomposite was obtained by firstly cast of the above solution into a 100 mm Petri dish, drying there for 4 hrs at 60° C. to get rid of any remaining acetic acid, and then removed. The chitosan-VS2nanocomposite was dehydrated at 60° C. and rinsed with distilled water before fully dried and further used for characterization.

Antimicrobial Activity

To evaluate antimicrobial activity, the bacterial strain (Pseudomonas(-ve)), and fungal (Aspergillus flavus), were used. These strains cultured in nutrient agar and Muller-Hinton medium. Ofloxacin and Fluconazole compounds were used as standard drug for comparison.

The susceptibilities of such growth rate of microorganisms were measured in vitro by agar well diffusion method. The tested nano complex was dissolved in dimethylsulfoxide at different concentrations (10 and 20 mg/ml). 1 cm3of a 24 h broth culture containing 106CFU/cm3was placed in sterile Petri-dishes. Molten nutrient agar (15 cm3) maintained at 45° C. was then poured into the Petri-dishes and allowed to solidify. Then holes of 6 mm diameter were formed in the agar using a sterile cork borer and these holes were completely filled with the test solutions. The plates were incubated for 24 h at 37° C. After the incubation period, the zone of inhibition of each well was determined by measuring the zones of growth inhibition (mm) against the test microorganisms with zone reader (Hi Antibiotic zone scale). In order to clarify the effect of solvent (DMSO) on the biological screening, DMSO alone was used as control, and it showed no activity against microbial strains. The measurements were made in triplicate for each compound and their average values are reported.

Antifungal activities of the prepare nano-complex were studied against three fungal cultures using the well diffusion method. The tested fungi were inoculated in Sabouraud dextrose broth medium (Hi-Media Mumbai) and incubated at 35° C. for 72 h and subsequently a suspension of about 1.60×104-6.00×104c.f.u/ml was introduced agar plates and a sterile glass spreader was used for even distribution of the inoculum. The discs measuring 6 mm in diameter were prepared from Whatman No. 1 filter paper and sterilized by dry heat at 140° C. for 1 h. The sterile discs previously soaked in known concentration of the tested compounds were placed in Sabouraud dextrose Agar (SDA) plates. The plates were inverted and incubated at 35° C. for 7 days. The susceptibility was assessed on the basis of diameter of inhibition againstalbicansand non-albicansstrain of fungi.

Minimum inhibitory concentrations (MICs) are defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. MICs are used by diagnostic laboratories mainly to confirm resistance of microorganism to antimicrobial agents and also to monitor the activity of new antimicrobial agents. MIC was determined in vitro in liquid medium by serial broth dilution method. The MIC values keep up a correspondence to the most minuscule concentrations that did not allow for the recognition of any visible growth.

Anticancer Activity

The anticancer activity was made at the National Cancer Institute, Cancer Biology Department, Pharmacology Department, Cairo University. The absorbance or optical density (O.D.) of each well was measured spectrophotometrically at 564 (nm) with an “ELIZA” micro plate reader (Meter tech. Σ 960, “USA”). Evaluation of the cytotoxic activity of the prepared nano-complex was carried out against Breast cancer cells line. The evaluation process was carried out in vitro using the Sulfo-Rhodamine-B-stain (SRB). Cells were placed in 96-multiwell plate (104cells/well) for 24 hrs before processing with the complexes to allow attachment of cell to the wall of the plate. Various concentrations of the compounds under test in DMSO (0, 1, 2.5, 5 and 10 μM) were added to the cell monolayer. Monolayer cells were incubated with the complexes for 48 hrs at 37° C. and in atmosphere of 5% CO2. After 48 hrs, cells were fixed, rinsed, and stained with Sulfo-Rhodamine-B-stain. Excess stain was washed with acetic acid and attached stain was treated with Tris EDTA buffer. Color intensity was measured in an ELISA reader. IC50was evaluated and potency was calculated with regard to percentage of change of (vistabline standard). The relation between surviving fraction and compound concentration is plotted to get the survival curve of each tumor cell line after the specified compound. The experiment was carried out once and each concentration repeated 3 times.

The inhibitory concentration percent (IC %) was estimated according to the equation for Inhibition concentration:
(IC) %=(Control O.D.−Ligand O.D.)×100/Control O.D

The prepared chitosan-VS2like flower nanocomposite show super anticancer activity with IC501.25 μg/μl against a colon cancer cell line compared with the vinblastine standard drug (IC50=4.75 μg/μl)

Antioxidant Activity

In vitro antioxidant activity of the newly nano-complex was evaluated using scavenging the stable DPPH radical modified method. The model of scavenging the stable DPPH radical is a method that is widely used to evaluate antioxidant activities in a relatively short time compared with other methods. DPPH′ radical scavenging test relies on the absorbance change of the radical when deactivated by antioxidants, which easily observable with naked eye as color changes from purple to yellow. Stock solutions of the investigated compounds were dissolved in methanol-DMSO (4:1) was diluted to final concentration of 10, 25, 50, 100 and 150 M. Methanolic DPPH (2,2-diphenyl-1-picrylhydrazyl) solution (1 mL, 0.3 mmol) was added to 3.0 mL of the synthesized compounds as well as standard compound (Ascorbic acid). The tube was protected from light by covering with aluminum foil and the absorbance was measured at 517 nm after 30 min. using methanol as a blank. All the tests were made in triplicates. Vitamin C was used as standard or positive control, parallel to the test compound and in the absence of the test compound/standard used as the negative control. The reduction in the absorbance of DPPH was calculated relative to the measured absorbance of the control. Lower absorbance values of reaction mixture indicated higher free-radical-scavenging activity.

The percentage of DPPH radical scavenging activity was calculated using the below equation:

%⁢DPPH⁢scavenging⁢activity=AC-ASAC
where ACis the absorbance of the L-ascorbic acid (Standard) and ASis the absorbance of different compounds. The methanolic DPPH solution (1 mL, 0.3 mM) was used as control. The effective concentration of sample required to scavenge DPPH radical by 50% (IC50 value) was obtained by linear regression analysis of dose-response curve plotting between % inhibition and concentrations.

The prepared chitosan-VS2like flower nanocomposite shows super Anti-oxidant activity with IC50of 5.5 μg/μl compared with l-ascorbic acid standard antioxidant (IC50=50.8 μg/μl).