PREPARATION AND CHARACTERIZATION OF HYDROGEL WOUND DRESSING LOADED PLGA/ CIPROFLOXACIN HYDROCHLORIDE NANOSPHERES FOR USED AS PRESSURE ULCER TREATMENT

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1 PREPARATION AND CHARACTERIZATION OF HYDROGEL WOUND DRESSING LOADED PLGA/ CIPROFLOXACIN HYDROCHLORIDE NANOSPHERES FOR USED AS PRESSURE ULCER TREATMENT Chasuda Choipang a, Orawan Suwantong b, Piyachat Chuysinuan c, Pongpol Ekabutr a, Pitt Supaphol a a The Petroleum and Petrochemical College, Chulalongkorn University b School of science, Mae Fah Luang University c Chulabhorn Research Institute Keywords : Hydrogel, Pressure ulcer, PLGA nanoparticles, Ciprofloxacin Hydrochloride, Wound dressing ABSTRACT Pressure ulcers are areas of damage to skin and underlying tissue caused by continuous pressure or friction on part of the body which are the most common problems that found in chronic wound. In this case, a hydrogel dressing loaded with poly (lactic-co-glycolic acid) or PLGA nanospheres containing ciprofloxacin hydrochloride (fluoroquinolone antibiotic) prepared by water in oil technique, can be used for a treatment for this type of wound since not only the drug can inhibit the bacteria in the wound site but also the flexible hydrogel can absorb a pressure force generated from a body that could make the wound more severe. In this research, morphology, size, entrapment efficiency, kinetics of release and stabilities of nanospheres are systematically studied. Afterwards, PLGA/Ciprofloxacin hydrochloride nanospheres with various ratios were blended in hydrogel matrixes which were optimized with the suitable physical properties. In addition, the antibacterial activity of hydrogel against E.coli and S.aureus are determined as a percentage of bacterial reduction at 24 hours. Also, a cytotoxicity test of material is performed by using mouse fibroblast (L929) cell as a model to verify that developed dressing can be safely used on human and animal skins. *pitt.s@chula.ac.th INTRODUCTION One of the most common problems that found in chronic wounds is pressure ulcer or pressure sore. This problem has the negative effects on the physical and psychological health. It causes the pain, depresses the mood, limits social activities, and significantly impacts on the quality of life of patients. Furthermore, it also effects on the cost of healthcare systems (Ciliberti et al., 2014). Pressure ulcer or pressure sore is the localized injury of the skin or the subcutaneous tissue, normally over a bony prominence, that caused by pressure, friction, shearing, and moisture induced necrosis of soft tissues (Hunter et al., 2014). The treatments of pressure ulcer are always complicated by the presence of bacteria in a wound, may result in contamination, colonization, or infection (Ip, 2011). The infected pressure ulcers always include P. aeruginosa, E. coli, and S. aureus in a wound (Livesley et al., 2002; Ciliberti et al., 2014). It has been reported that the bacterial infections are estimated to be the fifth-leading cause of death in the hospital (Jovanović et al., 2010). Thus, the prevention and reduction of these bacterial loads are necessary for the treatments. There are several techniques have been developed to treat pressure ulcer and also promote wound healing. One of the most popular techniques is using of wound dressings, especially hydrogels. Hydrogels are threedimensional, hydrophilic, polymeric networks capable of absorbing large amounts of water or biological fluids (Caló et al., 2015). These hydrogels are used to provide a moist wound Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 1

2 environment, protect the wound from external environment, absorb wound exudate, induce autolytic debridement, promote tissue regeneration, and facilitate wound healing. Besides the above mentioned, the other benefits of using hydrogel-based dressings are easy to remove without further trauma to the wound and short the recovery period (Moura et al., 2013; Archana et al., 2015; Caló et al., 2015). Nanoparticles are solid and spherical structures prepared from natural or synthetic polymers. A wide variety of drugs can be delivered using nanoparticles such as hydrophilic small drugs, hydrophobic small drugs, vaccines and biological macromolecules. Nanoparticles also allow a targeted administration to specific organs or cells or controlled drug delivery (Hans and Lowman, 2002). PLGA polymers is one of the most used biodegradable polymer Because these two monomers are endogenous and easily metabolized by the body via the Krebs cycle, a minimal systemic toxicity is associated with the use of PLGA for drug delivery or biomaterial applications (Kumari et al., 2010),widely available and approved by regulatory agencies such as FDA. PLGA particles may decrease toxicity, protectively, enhance, and prolong active agent presentation (Silva et al., 2016). Ciprofloxacin hydrochloride (CPFX) is one of the most widely available the monohydrochloride monohydrate and fluoroquinolone. Ciprofloxacin is used to treatment of wide range of infectious and chronic diseases because broad spectrum antimicrobial activity and the frequency of spontaneous resistance to ciprofloxacin is very low. The purpose of this research is to prepare and characterize the hydrogel which has the properties that can fulfill the goal of pressure ulcer treatment. The effects of PLGA/ Ciprofloxacin hydrochloride on morphology, size distribution, zeta-potential, capsulate efficiency, antibacterial activities and cytotoxicity by in-vitro cytotoxicity was assessed via MTT assay will also be evaluated. EXPERIMENTAL A. Materials All chemicals were used without further purification. PLGA 50:50 and PVA (MW 88,000 and 125,000) were purchased from Sigma Aldrich. Ciprofloxacin hydrochloride (CPFX) were purchased from SRL chemical. Dichloromethane were obtained from Lab scan. Dialysis membrane with molecular weight cut-off (MWCO) size of 12,000 Da were obtained from Sigma Aldrich. Phosphate buffer saline (PBS) ph 7.4 was used as releases medium. B. Preparation of PLGA nanoparticles 1.) PLGA 50mg dissolve in DCM 1ml following by vortex in high setting until the solution mix well to obtain the organic phase. 2.) Drop wise the W/O mixture into 2% PVA 5 ml and stirred at 500 rpm 10 minutes. 3.) After that find the optimum processing by various the probe sonicated at 50, 80, and 100% amplitude for 5, 12, and 20 minutes, respectively. 4.) Bring it to centrifuge 11,000 rpm for 25 min. Finally, wash 3 times by DI water and kept in freezer 4 until used. C. Preparation of PLGA nanoparticles loaded ciprofloxacin hydrochloride by Solvent Evaporation Method 1.) PLGA 50mg dissolve in DCM 1ml following by vortex in high setting until the solution mix well 2.) CPFX with various ratio in 2, 4, and 6 fold from the MIC value of CPFX (2.5 mg/ml) and added into PLGA solution and then sonicated in sonication bath 60s to obtain W/O. 3.) Drop wise the W/O mixture into 2% PVA 5 ml and stirred at 500 rpm 10 minutes. After that probe sonicated at 100% amplitude for 12 minutes and bring it to centrifuge 11,000 rpm for 25 min. Finally, wash 3 times by DI water and kept in freezer 4 until used. Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 2

3 D. Characterization of PLGA/ ciprofloxacin hydrochloride nanoparticles 1.) PVA solution was prepared at a concentration of 15% (w/v) in distilled water under mechanical stirring until a homogeneous solution was formed. 2.) PLGA nanoparticle was added to the solution in various concentrations and then the solutions were poured into the nylon bags, sealed, and then exposed to gamma radiation at dose of 25 kgy in order to obtain crosslinking and sterilization simultaneously. E. Characterization of PLGA nanoparticles with and without ciprofloxacin hydrochloride The particle size, size distribution, and zeta potential of nanoparticles was determined by Photon Correlation Spectroscopy with a Zetasizer 3000 (Malvern Instruments). The morphology was determined by transmission electroscopy (TEM) which a drop of nanoparticle suspension was placed on a TEM copper grid coated with carbon film and dried at room temperature. FTIR spectra were measured in transmission mode with a Thermo Nicolet Nexus 670 Fourier transform infrared spectrometer using 32 scans at a resolution of 4 cm -1 from 4000 to 400 cm -1. UV-Vis absorption spectra were recorded over wavelengths ranging from 200 to 400 nm by a Shimadzu UV-1800 spectrophotometer (Japan). F. Indirect Cytotoxicity Each of hydrogel was determined for their indirect cytotoxicity and TCPS was used as a control. The extraction medium was prepared by immersing the sample in 24-well plate containing a serum-free medium (SFM) which was kept at 37ºC under 5% CO 2 for 1, 3 and 7 days. 40,000 L929 cells/well were separately cultured in another 24-well plate for 1 day to allow cells to attach to the plate. The cells were then starved with SFM for another 1 day. After the starvation, the culture medium was replaced with the prepared extraction medium. The cells were cultured in the extraction medium for 24 h before the MTT assay was performed to determine the number of viable cells. RUSULTS AND DISCUSSION A. The morphology of PLGA nanoparticles. First of all in this study, the PLGA nanoparticle was fabricated using ultrasonicator by solvent evaporation method to generated nanoparticles. The morphology of the PLGA nanoparticle is shown in Table 1 shows TEM observation of PLGA nanoparticle with various %amplitude and time. The result shown that the spherical nanoparticles was observed. B. Particle size and surface charge (zeta potential) The average and standard deviation values of the PLGA nanoparticle which various % amplitude and time were calculated as shown in Table 1. From Table 1, the particles size of the PLGA nanoparticle with 50% amplitude with 5, 12, and 20 minutes was ± 79.93, ± 92.26, and 385.6± 145.2, respectively. At 80% amplitude with 5, 12, and 20 minutes was ± 41.83, ± 18.05, and ± respectively. At 100% amplitude with 5, 12, and 20 minutes was ± 58.91, 52.9 ± 4.23, ± and 95.2 ±29.19, respectively. From these results, the mean particles size decreased with increasing % amplitude because it induced it dispersed to a small size but the particles size increased with increasing time because the particle aggregated again due to the ultrasonic generated energy lead to high temperature. Furthermore, a zeta potential values obtained with PLGA nanoparticle without CPFX ( Table 1) was calculated to be ± 4.44, -32 ± 5.63, and ± 4.89 mv for 50% amplitude with 5, 12, and 20 minutes, respectively ± 4.44, -32 ± 5.63, and ± 4.89 mv for 80% amplitude with 5, 12, and 20 minutes, respectively ± 9.13, ± 7.19, and ± 5.18 mv for 100% amplitude with 5, 12, and 20 minutes, respectively. Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 3

4 Table 1 Selected TEM images, particle sizes, and zeta potential of the PLGA nanoparticle without ciprofloxacin hydrochloride. Condition 50 %amplitude 50 %amplitude 50 %amplitude Particle size ± ± ± Zeta potential ± ± ± 4.89 Condition 80 %amplitude 80 %amplitude 80 %amplitude Particle size ± ± ± Zeta potential ± ± ± 5.18 Condition 100 %amplitude 100 %amplitude 100 %amplitude Particle size ± ± ± , 95.2 ±29.19 Zeta potential ± ± ± 6.48 Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 4

5 CONCLUSIONS The PLGA nanoparticle were successfully fabricated using solvent evaporation method. The effect of % amplitude and time on the morphology, particle sizes, and zeta potential was investigated. From TEM images, the PLGA nanoparticle had the spherical. The particles size decreased with increasing % amplitude because it induced it dispersed to a small size but the particles size increased with increasing time. Thus, the suitable processing condition was 100% amplitude with 12 minutes. ACKNOWLEDGEMENTS The author would like to thank for the scholarship and funding of the thesis work provided by the Petroleum and Petrochemical College, Chulalongkorn University, Thailand. Center of Excellence on Petrochemical and Materials Technology. A fund received from The Research Pyramid. REFERENCES Jain, D., & Banerjee, R. (2008). Comparison of ciprofloxacin hydrochloride loaded protein, lipid, and chitosan nanoparticles for drug delivery. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 86(1), Dorati, R., Genta, I., Montanari, L., Cilurzo, F., Buttafava, A., Faucitano, A., & Conti, B. (2005). The effect of γ-irradiation on PLGA/PEG microspheres containing ovalbumin. Journal of controlled release, 107(1), Kumari, A., Yadav, S. K., & Yadav, S. C. (2010). Biodegradable polymeric nanoparticles based drug delivery systems. Colloids and Surfaces B: Biointerfaces, 75(1), Dillen, K., Vandervoort, J., Van den Mooter, G., Verheyden, L., & Ludwig, A. (2004). Factorial design, physicochemical characterisation and activity of ciprofloxacin-plga nanoparticles. International journal of pharmaceutics, 275(1), Dillen, K., Vandervoort, J., Van den Mooter, G., & Ludwig, A. (2006). Evaluation of ciprofloxacin-loaded Eudragit RS100 or RL100/PLGA nanoparticles. International journal of pharmaceutics, 314(1), Mobarak, D. H., Salah, S., & Elkheshen, S. A. (2014). Formulation of ciprofloxacin hydrochloride loaded biodegradable nanoparticles: optimization of technique and process variables. Pharmaceutical development and technology, 19(7), Petrochemical and Materials Technology Tuesday May 23, 2017, Pathumwan Princess Hotel, Bangkok, Thailand Page 5