Formulation and Evaluation of Nano-fiber-based Transdermal patch of Cephalexin

 

Sidra Choudhary¹, Sanket Dharashivkar¹, Chetan Mahajan², Madhuri Gaikwad³

¹Department of Pharmaceutics, Dr. L. H Hiranandani College of Pharmacy, Ulhasnagar,

²Scientist-B, Wool Research Association, Thane,

³Department of Pharmaceutics, AIKTC School of Pharmacy, Panvel

 

ABSTRACT:

In the present work polymeric nano-fiber patches were developed for the effective treatment of skin infection using cephalexin as model drugs. The nano-fibers were prepared by electro spinning technique by using polyvinyl alcohol (PVA) as a polymer and fibers were characterized on the basis of entrapment efficiency, fiber diameter, morphology, x-ray diffraction analysis, drug release behavior and in vitro antimicrobial test. The entrapment efficiency of the optimized formulation was 95%. The % cumulative release of drug from nano-fiber patch across the dialysis membrane was 96% at the end of 24h. The ex-vivo release of drug through nano-fiber patch across the goat skin was 81% at the end of 24 h.

 

 

INTRODUCTION:

A recent approach to transdermal drug delivery system is to deliver the drug via nano-fiber patch into systemic circulation at predetermined rate using skin as a site of application. Nano-fibers are a nanomaterial in a nanometric range. Electro spinning process is use to form a fine filament of the polymer solution. The benefits of electro spinning technology are; a) high rate of nano-fiber can be produces; b) simple set up and production costs is low^1,2. Small size, high surface area, lightweight, and flexibility in surface functionalities are some of the characteristics that make the nano-fibers appropriate candidates for transdermal drug delivery. Nano-fibers have large specific surface area with small pore size and can be used in management of wound¹.

 

The skin provides a good barrier against bacterial infections. Although many bacteria come in contact with the skin, when bacterial skin infection occurs, it can range in size from a tiny spot to the entire body surface. They can cause serious skin infection³. Skin infections come in many forms. Most commonly skin infection is Cellulitis. It is an acute, usually non-contagious, inflammation of the connective tissue of the skin, resulting from bacterial infection and characterized by localized warmth, erythema, pain and swelling to the affected area. Infections of the skin and soft tissues are common and usually uncomplicated. Skin infections can be caused by many things. Skin infections can be either bacterial, viral, fungal, or caused by parasites. Skin infections can cause swelling, itching and redness³.

 

Cephalexin (CEX) is an antibiotic drug which is used in various diseases like skin and tissue infection, urinary tract infection, bone infection, diabetic foot infection. There are no topical formulations available in the market so the transdermal patch is beneficial to avoid the side effect associated with oral dosage form like throat swelling, wheezing, difficulty in breathing, diarrhea, abdominal pain, nausea and vomiting, dizziness, tiredness, headache, stomach upset, mouth ulcer and improve the patience compliance³. CEX belonging to the BCS class-III i.e. high solubility and low permeability so to increase the permeability of the drug it can be loaded into the nano-fibre patch.

 

MATERIAL AND METHODS:

MATERIAL:

Cephalexin was purchased from local market. Polyvinyl alcohol and methanol was purchased from molychem Mumbai. All other chemicals and solvents used during the experiments were of analytical grade and procured from SD Fine Chemicals, Mumbai, India.

 

Fabrication of the electrospun nano-fibers patch:

PVA nano-fibers were produced by an electrospinning technique. PVA (10% W/V) solution was prepared in distilled water. CEX (1%W/V) was added to the homogeneous polymeric solution with constant stirring. The final solution was subjected to electro spinning using rotating cylindrical drum as a collector placed at a distance of 20cm from the needle under an applied voltage of 20 KV and flow rate of 0.3 ml/h. The nano-fiber sheets were cut into of 1cm² patches and used for further characterization⁴.

 

Optimization of formulation using 2³ factorial design:

2 level 3 factor was designed to optimize the formulation using design expert software version 11. Polymer concentration (X1), voltage (X2), electrode distance (X3) was selected as independent variables and entrapment efficiency of formulation was selected as dependent variable⁵. The different levels for the independent variables for the 2³ factorial design are given in Table 1. Table 2 shows 2³ factorial design for optimization of formulation.

 

Table 1. Factors and factor levels investigated in 2³ factorial design

 

Table 2. 2³ factorial design for optimization of developed formulation

 

Characterization of nano-fiber patches:

Nano-fiber patches were characterized on the basis of various parameters such as surface morphology, entrapment efficiency, x-ray diffraction analysis, in vitro drug release, ex-vivo drug release and in vitro antimicrobial study.

 

Entrapment efficiency:

Electrospun nano-fibers are expected to possess high drug entrapment efficiency (EE) due to presence of high surface area. EE describes the efficiency of the preparation technique to incorporate drug into carrier system⁶. The drug loaded nano-fibre patch were weighed and dissolved in phosphate buffer pH 7.4. The solution was assayed  in triplicate for entrapped drug concentration by UV spectrophotometer at 261nm.

 

The percentage drug EE was calculated as follows:

               

      Entrapped drug

EE %  =  ––––––––––––––––  × 100

               Total amount of drug

 

Surface morphology and fiber diameter:

Surface characteristics such as uniformity in diameter, smoothness of surface are important parameters to evaluate the quality of the developed nano-fibers sheet. Surface morphology and fiber diameter of the optimized nano-fibers was analyzed by using scanning electron microscope (SEM)⁶.

 

In vitro drug release:

The in-vitro diffusion study was carried out using Franz diffusion cell. The receptor compartment was filled with 20 ml of phosphate buffer pH 7.4 and maintained at 37⁰C±0.5⁰C. Optimized nanofiber sample was kept in donor compartment over a dialysis membrane. The aliquot of 1ml was taken in suitable interval of time and replaced with fresh buffer solution maintained at same temperature.  Sample was analyzed using UV spectrophotometer in triplicate at 261nm^7,8.

 

X-ray diffraction (XRD):

X-ray diffraction (XRD) analysis were performed to illustrate the crystalline structures of CEX powders and drug present in optimized nano-fiber patch using a Philips X’Pert-Pro MPD with a 3 KW ceramic tube as the X-ray source (Cu-K_) and an X’Celerator detector. Cu-K radiation was used with a diffraction angle range of 20–50⁰ at 45 kV and 40 mA at a scanning rate of 10⁰/min⁸.

 

Ex-vivo drug release:

In the present study, hairless goat skin was used. Hairs were removed with animal hair clipper. Skin was washed with phosphate buffer pH 7.4 and used immediately. Ex-vivo release studies on optimized nano-fiber patch were performed using Franz-diffusion cell. The goat skin was mounted between the donor and receptor compartment. Same procedure was followed as discussed under in vitro drug release study⁷.

Microbial study:

The microbial study of the formulation was carried out by zone inhibition method by using sterilized agar medium on three different organism namely Stephylococcus aureus, Klebsiella aerogenes, Escherichia coli⁹. Bacterial suspension was streaked aseptically over the plate containing agar medium and was spread uniformly. A blank polymeric nano-fiber and drug solution was used as a positive and negative control respectively. Blank nano-fibers and drug loaded optimized nano-fibers were gently placed at the center of the solidified agar gel in different petri dishes. Aqueous drug solution (1%) was poured in the well formed using 12 mm borer.  The plates were incubated at 37°C for 24 h. The bacterial growth was compared with the controls^8,10. The experiment was carried out in triplicate.

 

RESULTS AND DISCUSSION:

Optimization of formulation using 2³ factorial design:

Excipient selection was made based on information available in literature, physicochemical properties of drug and compatibility studies. Nanofiber patch was prepared by electrospinning method. Table 3 shows the EE of experimental batches.

 

 

Table 3. 2³ factorial design showing results for response of entrapment efficiency

 

Selection of the optimized batch:

A 2³ full factorial design was employed to evaluate the individual and combined effects of three formulation variables on nano-fiber patch performance and characteristics. In this design, three factors were evaluated and experimental trials were performed at all eight possible combinations. The effects of selected independent variables namely polymer concentration (X1), voltage (X2), electrode distance (X3) was observed on EE as a dependent variable. The optimized batch was selected based on highest EE in Table 3. The high value (0.94) of correlation coefficient (R2) indicates a good fit. The model F value of (14.08) implies that the model is significant. The values of Prob was <0.05.

 

Software provides 8 new batches (Table 4), batch with highest entrapment value was selected and performed experimentally. Contour plots (Fig.1) were further applied to explore the effects of the independent factors on the responses.

 

Table 4. Software provided batches

 

Fig 1. Contour plot

Entrapment efficiency:

The entrapment efficiency of optimized batch was found to be 95%. This slight decrease in the entrapment efficiency compared to software predicted batch (Table 4) could be due to the decreasing potential drainage capacity of collector as the fiber get deposited on the surface of the collector leading to distraction of fibers from the collector site.

 

Surface morphology:

Diameter and shape of the nano-fibers was determined using SEM. The diameter of optimized nano-fibers was found to be in the range of 200–600 nm. Fig. 2 shows the SEM images of optimized formulation which confirms the presence of smooth round shaped fibers.

 

Fig 2. SEM images of cephalexin nanofiber

 

In vitro drug release:

In vitro release studies showed that the developed nano-fibers are capable of sustained release drug delivery up to 24 h. Fig. 3 shows release of 96% at the end of 24h.

 

Fig 3. in vitro drug release

 

XRD analysis:

XRD patterns with distinctive crystalline peaks of CEX are shown in Fig. 4. As seen in figure the XRD spectrum of CEX displayed sharp and intense peaks of crystallinity, which suggested a highly crystalline nature. The XRD spectra of the nanofiber patch containing CEX showed a reduction of peak intensity, as compared to the CEX, which indicated decreased crystallinity or conversion into an amorphous phase of the drug.

 

Fig 4. XRD of drug and formulation

 

Ex-vivo drug release:

Ex-vivo release studies showed that the developed nano-fibers are capable of sustained release drug delivery up to 24 h. Fig. 5 shows release of 81% at the end of 24h. Decrease in the release compared to in vivo study can be due to difficulty that drug might have faced while penetrating to thick skin of goat.

 

Fig 5. EX-Vivo release study

 

Microbial study:

The microbial study on the organism shows a zone of inhibition as mentioned in Table 5. Blank nano-fiber patch did not show any zone of inhibition. Fig. 6 shows the images of zone of inhibition. Increased zone of inhibition with drug solution might be because of ease of release of drug through solution compared to nano-fibers.

 

 

Table 5. Zone of inhibition

Microrganisms	Zone of inhibition (mm)
	Formulation	Drug solution
Staphylococcus aureus	12	17
Klebsiella aerogenes	13	18
Escherichia coli	13	16

 

Fig 6. Microbial study

 

CONCLUSION:

In the present work biodegradable polymeric nano-fibers of CEX were successfully developed by using electrospinning technique. The microscopic study indicated that the nano-fibers were uniform in diameter with smooth surface. Entrapment efficiency achieved was very high (95% ) with good zone of inhibition against different microbes. Hence, the nano-fibers patch of CEX can be effectively utilized for the treatment various skin infections.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

ACKNOWLEDGEMENT:

I am thankful to Indian Institute of Technology, Mumbai for providing facility to perform SEM analysis. I am also thankful to SAIF Chandigarh for providing facility to perform XRD analysis.

 

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Accepted on 21.01.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(6): 2787-2791.