INTRODUCTION:

The microsponge drug delivery system (MDS) has become highly emphasis among researchers for their cost effective, less side effects and its enhanced efficacy in the treatment of diseases. Since the microsponges prepared from synthetic polymers, it will protect the entrapped drug from any kind of degradation. These kinds of encapsulated drugs within microsponge can overcome all the above requirements with targeted drug delivery^1,2. Metal nanoparticles such as gold, silver and copper are reported as highly toxic to micro-organisms^3,4. In recent years, it has been extensively used for the production of medical products like wound dressing because of its strong cytotoxicity^4-6. For Example, copper nanoparticles embedded hydrogels are a promising candidate for skin tissue regeneration and potentially valuable for clinical applications⁷.

Impaired wound healing continues to be a major health issues that leads to infections, long term morbidity and mortality^8-10, particularly in high risk patients suffer from diabetes- induced skin ulcers and burning¹¹.

Many researchers are being tried to find ideal clinical wound healing biomaterials or agents with low cost and enhanced healing¹¹. Meanwhile, in traditional medicine, some plant extracts or even parts of the plant are used for the treatment of cuts, wounds and burns to accelerate the healing process¹². Therefore, there is a new interest of developing a low cost drug delivery system that will be used for a better wound healing activity with less side effects and without any amputation of a patient.

Hence, this current study involves an evaluation of green synthesized copper nanoparticles loaded microsponges towards its in-vitro drug release study and its kinetics, cytotoxicity effect and in-vitro wound scratch assay(Fig 1). The in vitro scratch assay is a straightforward and economical method to study cell migration in vitro¹³. The basic steps involve creating a “scratch” in a cell monolayer, capturing the images at the beginning and at regular intervals during cell migration to close the scratch and comparing the images to quantify the migration rate of the cells¹⁴.

MATERIALS AND METHODS:

Green synthesis of copper nanoparticles:

The copper nanoparticles[CuNps(B)] were synthesized from the Leaf extract of Hibiscus rosa-sinensis.the leaf extract was mixed with 0.05M copper sulphate solution in the ratio of 1:1. The black precipitate of CuNps(B) were lyophilized and characterized with UV-Vis, FT-IR, SEM-EDX, XRD and evaluated its antimicrobial and antioxidant activity¹⁵.

Fig 1: Outline of the proposed work

Synthesis of copper nanoparticles loaded microsponges:

Copper nanoparticles loaded Microsponges were formulated by Quasi-Emulsion Solvent Diffusion method. Five batches of microsponges with varying proportions of Ethyl Cellulose (EC) and Polyvinyl alcohol (PVA) were taken. The Dispersed Phase consists of Copper Nanoparticles (B -CuNps) and required amount of EC dissolved in 20mL of Dichloromethane (DCM). It was slowly added to PVA in 150mL of aqueous continuous phase. Then it was stirred at 1000 rpm under magnetic stirrer for 3 hours. The microsponges formed were filtered and dried in oven at 40–50ºC for 24 hours. Then the dried microsponges were stored in vacuum dessicator for further use. It was characterized by HRSEM, PSA, Drug content and entrapment efficiency. The best CuNps loaded microsponge (NS₄Cu_B) was selected by the better entrapment efficiency value which was subjected to evaluate the in-vitro antimicrobial and antioxidant activity¹⁶.

Characterization:

The chosen copper nanoparticles loaded microsponge was evaluated for its in-vitro drug release study and its kinetics, cytotoxicity and wound healing activity.

In-vitro drug release study:

The in-vitro release of copper nanoparticles loaded microsponge was studied through cellophane membrane using an open end cylinder. The medium used was freshly prepared phosphate buffer pH 7.4. Cellophane membrane, previously soaked overnight in the dissolution medium, was tied to one end of a specifically designed glass cylinder (open at both ends and of 5 cm diameter). The formulation was accurately weighed and placed in a membrane and attached to this assembly. The cylinder was attached to the metallic driveshaft and suspended in 50mL of dissolution medium maintained at 37± 1°C so that the membrane just touched the receptor medium surface. The medium was stirred at 50 rpm using magnetic stirrer. Aliquots, each of 1mL volume, were withdrawn at different time intervals and replaced by an equal volume of the receptor medium. The aliquots were diluted with the receptor medium and analyzed by UV-Vis spectrophotometer at 650nm^17,18.

In-vitro drug release kinetics:

The kinetic modelling was done by fitting the results obtained from the in-vitro drug release into the various mathematical models given below².

· Zero order rate kinetics - Cumulative percent drug released Vs. Time

· First order kinetics - Log Cumulative percent drug retained Vs. Time

· Higuchi matrix - Cumulative percent released Vs. a square root of Time

· Korsmeyer Peppas - Log cumulative percent drug released Vs. Log Time

The plots were plotted for all the kinetic models and find out the regression coefficient (R²). This value was taken as criteria for choosing the most appropriate model.

Cytotoxicity test (MTT assay):

The cytotoxic activity of samples on VERO cells were determined by the MTT assay. Cells (1 × 10⁵/well) were plated in 0.2mL of medium/well in 96-well plates. Incubate at 5% CO₂ incubator for 72 hours. Then, added various concentrations of the sample (NS₄Cu_B) in 0.1% DMSO for 24 hrs at 5% CO₂incubator. View the images under Inverted microscope 40X and take the photos. After removal of the sample solution and 20µl/well MTT reagent was added. Viable cells were determined by the absorbance at 540nm. Measurements were carried out in triplicate. 50% inhibition of cell viability (IC₅₀) value was determined graphically¹⁹. Cell viability in control medium without any treatment was represented as 100%. The effect of the samples on the proliferation of VERO cells was expressed as the % cell viability²⁰, using the following formula:

% Cell viability = A540 of treated cells/A540 of control cells × 100%

In-vitro scratch wound healing assay:

VERO cells were seeded into 96 well plates and After 72 hours, the cell monolayer was scraped with a sterile 200μL micropipette tip to create a wound, examined under inverted microscope. Thereafter, the cells were treated with sample (NS₄Cu_B) of 125µg and 62.5µg. After 24 hours treatment period, the plates were view under microscope and the images were captured at 10× magnification. The wound healing process was determined by the % cell migration that described about the distance traversed by cells migrating into the denuded area^13,14,²⁰.

RESULTS AND DISCUSSION:

In-vitro drug release study:

The drug release profile was investigated on copper nanoparticles loaded microsponge (NS₄Cu_B) at different time intervals of 3 hrs, 6 hrs and 9 hrs. The profile shows a cumulative release of copper nanoparticles from the beginning (Fig 2). By nearing 9^th hour, almost 100% of the CuNps from the microsponge system got released. It shows an efficient drug release profile of this microsponge system.

Fig 2: In-vitro drug release profile of copper nanoparticles loaded microsponge (NS₄Cu_B)

In-vitro drug release kinetics:

To gain a good understanding into the mechanism of the copper nanoparticles release from the microsponge, kinetic modeling was done. All the data obtained from the release profile was applied to the plot of zero order, first order, Higuchi and korsemeyer-peppas kinetic models (Fig 3-6). These models will explain the release rate. The best fit model was chosen by the high correlation coefficient (R²) value from the plots (Table 1). Here, the selected sample copper nanoparticles loaded microsponge NS₄Cu_Bhas highest value of R² value 0.9990 in the peppas model. Hence, it follows korsemeyer-peppas kinetics in which the value of release exponent n is found to be 0.618 which follows non-fickian diffusion.

Table:1 R²value of the kinetic models

S. No.	Kinetic model	R²value
1.	Zero order kinetics	0.9573
2.	First order kinetics	0.9819
3.	Higachi-release kinetics	0.9945
4.	Korsemeyer -peppas kinetics	0.9990

Cytotoxicity test (MTT assay):

The CuNps loaded microsponge was evaluated for its cytotoxicity using MTT assay in which different concentrations of sample has been checked for the cell viability (Fig 7). The % cell viability of the cells of the sample (NS₄Cu_B) was 92.3% at 31.2µg/mL and its toxicity was within the acceptable range for human use. The IC50 value was found to be 125µg/mL which was determined graphically. This value depicted the less toxicity of the sample.

In-vitro scratch wound healing assay:

The sample (NS₄Cu_B) was subjected to scratch assay in order to find out the wound healing process (Fig 8). The IC50 value of the sample concentration was analysed. The % cell migration was calculated using the imagej software. Here, the distance traversed by the cells from the edges of the wound created was measured. At 24^th hour, the NS₄Cu_B of 125 µg/mL showed 35.8% and 62.5 µg/mL showed 59.6% cell migration which depicted the effectiveness of the wound healing process.

Fig 3: kinetic release profile of NS₄Cu_B- Zero Order

Fig 4: kinetic release profile of NS₄Cu_B- First Order

Fig 5: kinetic release profile of NS₄Cu_B- Higuchi plot

Fig 6: kinetic release profile of NS₄Cu_B- Peppas plot

Fig 7: Cytotoxic activity of CuNps Loaded microsponge (NS₄Cu_B)

Fig 8: Wound healing assay of CuNps Loaded microsponge (NS₄Cu_B)

CONCLUSION:

The effectiveness of the copper nanoparticles loaded microsponge was checked for its in-vitro drug release study followed by cytotoxic and wound healing activity. The loaded microsponge system has proven antimicrobial and antioxidant activity^15,16. Besides, it has been checked for its effective wound healing property. The in-vitro drug release study depicted the cumulative release which releases copper nanoparticles from microsponge nearly 100 % at 9^th hour. This data was best fitted into the korsemeyer-peppas kinetic model which showed non- fickian diffusion mechanism of drug release. The sample was checked for the in- vitro cytotoxicity using MTT assay in which it showed % cell viability of 92.3% and the IC50 value was found to be 125 µg/mL. It proves that by decreasing the sample concentration, the % cell viability may lead to 100%. This will be very much useful for the applications in the medical field. The in-vitro scratch wound healing assay was also evaluated for the copper nanoparticles loaded microsponge in which it showed nearly 60% of cell migration that ensures the potential wound healing process. In future, it can be used for the in-vivo wound healing study followed by the clinical trials. Hence, the present work concludes that the green synthesised copper nanoparticles loaded microsponge act as a potential wound healing agent with high antimicrobial and antioxidant activity with less side effect and better results. This is the first kind of work as far as we know, hence it can be exclaimed to the next level of research like producing biomaterials of this microsponge system.

CONFLICT OF INTEREST:

Nil.

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Received on 22.10.2019 Modified on 19.12.2019

Research J. Pharm. and Tech 2020; 13(9):4357-4360.

DOI: 10.5958/0974-360X.2020.00770.2