Evaluation of Antibacterial Activity and Drug Release Behavior of Chitosan-Based Nanofibers (In Vitro Study)

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1 UK Journal of Pharmaceutical and Biosciences Vol. 2(3), 01-05, 2014 RESEARCH ARTICLE UK Journal of Pharmaceutical and Biosciences Available at Evaluation of Antibacterial Activity and Drug Release Behavior of Chitosan-Based Nanofibers (In Vitro Study) Hassan Nageh 1, Metwally Ezzat 1, Mohammed Ghanim 1, Ahmed Hassanin 2, Ahmed Abd El-Moneim 1* 1 Materials Science and Engineering Department, Egypt-Japan University of Science and Technology, New Borg El-Arab, Alexandria 21934, Egypt. 2 Faculty of Engineering-Alexandria University-Egypt. Article Information Received 01 May 2014 Received in revised form 25 June 2014 Accepted 29 June 2014 Keywords: Chitosan Poly (caprolactone) Poly (vinyl alcohol) Ciprofloxacin.HCl Drug release * Corresponding Author: ahmed.abdelmoneim@ejust.edu.eg Tel.: Abstract Spinning of chitosan-poly (vinyl alcohol); Cs/PVA and chitosan- poly (caprolactone); Cs/PCL polymer blends were successfully achieved using electrospinning technique. The nanofiber mats of Cs/PVA and Cs/PCL were used as carriers for examining the release profile of Ciprofloxacin.HCl as broad-spectrum antibiotic. The shape, size, structure and composition of the nanofibers were characterized using FT-IR Spectroscopy and Scanning Electron Microscope (SEM). The release behavior was studied in Phosphate Buffer Solution (ph=7.4) using a UVspectrophotometer. The antimicrobial activity of the produced nanofiber mats was studied using different strains of human pathogenic bacteria and Fungi. Compared to release profile from Cs/PVA/Ciprofloxacin.HCl nanofibers show faster drug release than that of Cs/PCL/Ciprofloxacin.HCl system,indicating that these nanofiber mats are promising active wound dressing materials for wound dressing applications. 1 Introduction Electrospinng is a unique fiber formation technique in which nanofibers are formed via utilization of electrical forces with high voltage to produce fibers with different diameters ranging from subnano to several micrometers 1. There are many parameters which affect the fiber formation process by electrospinning including; the applied voltage, tip-to-collector distance, feed rate of polymer solution, polymer concentration and solvent type 2-5. These nanofibers have many properties such as high porosity, small pore size, large surface area and gas exchange ability; making them promising materials for different applications like tissue engineering scaffolds, nanocatalysis, filtration, biomedical, pharmaceutical and many other applications 6. Chitosan a natural polysaccharide, obtained from marine crustaceans such as lobster, crab and shrimp and it is the second most abundant natural polymer after cellulose. Chitosan is nontoxic polymer, biocompatible and biodegradable 7. In addition, it has several unique properties making it very useful in pharmaceutical applications (i.e. antimicrobial activity, haemostatic properties and wound healing ability) 8,9. These properties arise from the presence of active amino and hydroxyl groups on chitosan backbone. Chitosan is insoluble in water but soluble in slightly acidic medium. It is difficult to electrospin pure chitosan because of inefficient entanglement of chitosan chains thus chitosan is often blended with synthetic polymers such as poly (vinyl alcohol), poly (caprolactone) and other polymers to enhance the spinnability of chitosan polymer and to increase the mechanical properties of the resulted nanofibers Controlled release is an efficient process for delivering drugs in medical applications in which active ingredients are released and absorbed by the body 13. Nanofibers exhibit many advantages for sustained release applications where the drug can be loaded with the nanofiber easily via electrospinning process In the present investigation, chitosan-pva (hydrophilic nanofiber system) and chitosan-pcl (hydrophobic nanofiber system) are used for the first time as nonwoven nanofiber matrices for examining the release profile of water soluble ciprofloxacin.hcl drug for wound dressing applications.

2 2 Materials and Method 2.1 Materials Chitosan with an average molecular weight of KDa and degree of deacetylation (DD) of 90% prepared in our lab. poly (caprolactone) with Mv of KDa purchased from Sigma Aldrich, Ciprofloxacin.HCl is a gift from Pharco Corporation (Egypt), Acetic acid ( 99.98%) purchased from Sigma Aldrich, poly (vinyl alcohol) Mv of 72 KDa purchased from (Applichem,Germany),trifluoroacetic acid (TFA) purchased from (SDSCL,India), Luria Bertani (LB) agar medium (Amresco, USA), Klebsiella pneumonia ATCC , Escherichia coli BAL2296 and candida albicans ATCC Preparation for Electrospinning For the preparation of Cs/PCL nanofibers system: 2% chitosan (w/v) and 10% PCL (w/v) were dissolved in TFA in the ratio of 30:70 (wt./wt.) Cs/PCL for 24 hours at room temperature. Then the blend was loaded with 10% of Ciprofloxacin.HCl drug. For the preparation of CS/PVA 30:70 (wt/wt) nanofibers system: 2% chitosan was dissolved in 90% acetic acid (w/v) and 10% PVA dissolved in distilled water and stirred at 80 o C for 5 hours then loaded with 10% of Ciprofloxacin.HCl. Electrospinning was performed at applied voltage of 28 KV, feed rate of 0.9 ml/hr. and the distance between nozzle and collector was 12 cm to produce good nanofiber. 2.3 Antibacterial activity of Cs/PVA and Cs/PCLnanofibers loaded with Ciprofloxacin.HCl The antibacterial activity of nanofiber mats of Cs/PVA/Ciprofloxacin.HCl and Cs/PCL/Ciprofloxacin.HCl were examined against some human pathogenic bacteria and fungi. The antimicrobial study was performed using the disc diffusion method as follow 17 : Agar media were prepared according to the manufacturer instructions, sterilized at 15 psi and 121 o C for 15 min then poured into sterile Petri plates. One hundred micro liters of overnight broth cultures of the human pathogens was spread over the prepared LB plates. Sterile filter paper discs were loaded with Cs/PVA/Ciprofloxacin.HCl and Cs/PCL/Ciprofloxacin.HCl separately and added to the plate surfaces. The plates were then incubated at 30 o C for 24 h; the formed clear zones were measured and recorded. 2.4 Characterization The nanofiber mats were characterized by SEM (JEOL JSM- 6010LV) for surface morphology, FT-IR (Vertex 70) for chemical structure, UV-Spectrophotometer (Hitachi U-3900 spectrophotometer) for studying the behavior of the drug release profile. 3 Results and Discussion 3.1 FT-IR and SEM Results FT-IR spectra were measured in the range of cm -1 using KBr pellets to study the possible interaction sites between the polymer and the drug. Figure 1 (a, b) illustrates FT-IR spectra of Cs/PVA/Ciprofloxacin.HCl and Cs/PCL/Ciprofloxacin.HCl. The spectra of pure chitosan, chitosan/pva and chitosan/pcl were used for comparison. The observed peaks are indicated in table (1) As shown in the spectra of Cs/PVA/Ciprofloxacin.HCl and Cs/PCL/Ciprofloxacin.HCl, very slightly unnoticeable change in their spectra indicating that the bonding between the drug and nanofibers are very weak. Figure 2 shows the SEM images of both (a) Cs/PVA/Ciprofloxacin.HCl nanofibers (30:70) and (b) Cs/PCL/Ciprofloxacin.HCl nanofibers (30:70) the Ciprofloxacin.HCl content was 10%. It s obvious from this figure that the average nanofiber diameters are 103 nm and 265 nm, respectively, which indicates the formation of good nanofibers under optimized spinning conditions. Table 1: The observed peaks from FT-IR spectra 3400:3200 cm -1 O-H group and N-H stretching 3400:3200 cm -1 C-H stretching 1630 cm -1 COO cm -1 C-O 3.2 Drug release studies The difference in drug release behavior between PVA/Cs and PCL/Cs polymeric systems has be studied in PBS (ph=7.4) using UV-Vis spectrophotometer as observed in figure 3. It shows that systems which have PVA as hydrophilic polymer release about 99% of Ciprofloxacin.HCl drug as burst release within the first two hours, while systems that contain PCL as hydrophobic polymer release about 97% of the drug loaded amount within the first five hours which is more efficient than Cs/PVA nanofibers in wound dressing applications and as antibacterial agents. 3.3 Antimicrobial studies The antimicrobial activity of Cs/PVA/Ciprofloxacin.HCl and Cs/PCL/Ciprofloxacin.HCl was investigated as in table (2) and figure 4. It has been found that both nanofiber systems have a great antimicrobial activity against some human pathogenic bacteria and fungi. In general, these nanofibers showed very strong antibacterial activity against the tested human pathogenic bacteria and fungi. It could be concluded that both nanofiber systems loaded with UK J Pharm & Biosci, 2014: 2(3); 2

3 T % (a.u.) T % (a.u.) Ciprofloxacin.HCl drug are recommended to be used in the wound dressing applications and as antibacterial agents especially those that can cause skin infections. Table 2: Diameter of clear zones recorded for Cs/PVA/Ciprofloxacin.HCl and Cs/PCL/Ciprofloxacin.HCl nanofibers against human pathogenic bacteria and fungi Microbial Gram Diameter of Clear Zones (mm) Strain Reaction Cs /PVA Cs/PCL 4 Conclusions Two types of nanofiber mats were fabricated using the electrospinning technique for wound dressing applications and loaded with broad spectrum antibiotic (Ciprofloxacin.HCl) to enhance the wound healing ability. The release profile of both nanofibers indicated that hydrophobic polymers have more sustained release than hydrophilic polymers. The prepared nanofiber mats shows very strong antimicrobial activity against different skin pathogens which are promising wound dressing nanofibers. 5 References Escherichia coli Klebsiella pneumonia Gram Negative Gram Negative New N, Furuike T, Tamura H. The Mechanical and Biological Properties of Chitosan Scaffolds for Tissue Regeneration Templates Are Significantly Enhanced by Chitosan from Gongronellabutleri. Materials. 2009; 2(2): Candida albicans Uni-Cellular Fungi (a) (b) Wavenumber (Cm-1) Cs Cs/PCL Cs/PCL/Cipro Fig 1: FT-IR spectra of (a) Cs/PCL /Ciprofloxacin.HCl nanofibers and (b) Cs/ PVA / Ciprofloxacin.HCl nanofibers Wavenumber (Cm-1) Cs Cs/PVA Cs/PVA/Cipro (a) (b) Fig 2: SEM images of (a) Cs/PVA nanofibers (30:70) and (b) Cs/PCL nanofibers (30:70), the Ciprofloxacin.HCl content was 10% UK J Pharm & Biosci, 2014: 2(3); 3

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