Ciprofloxacin and Fluconazole Containing Fibrin Nanoparticles

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Ciprofloxacin and Fluconazole Containing Fibrin Nanoparticles Incorporated Chitosan Bandages for the Treatment of Polymicrobial Wound Infections Nimal Thattaruparambil Raveendran, Annapoorna Mohandas, Riju Ramachandran Menon, Arun Somasekharan Menon, Raja Biswas, and Rangasamy Jayakumar ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00585 • Publication Date (Web): 21 Dec 2018 Downloaded from http://pubs.acs.org on December 22, 2018

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ACS Applied Bio Materials

Ciprofloxacin and Fluconazole Containing Fibrin Nanoparticles Incorporated Chitosan Bandages for the Treatment of Polymicrobial Wound Infections Nimal Thattaruparambil Raveendran# †, Annapoorna Mohandas# †, Riju Ramachandran Menon§, Arun Somasekharan Menon¥, Raja Biswas*†, Rangasamy Jayakumar*† †Centre for Nanosciences and Molecular Medicine, §Department

of General Surgery, ¥Department of Endocrinology, Amrita Institute of Medical

Sciences and Research Centre, Amrita Vishwa Vidyapeetham, Kochi-682041, India

______________________________________ Author Contributions#. T.R.N and A.M contributed equally to the study *Corresponding Author: *Email: [email protected] & [email protected] Tel. No: +91 484 2801234 Fax. No: +91 484 2802020 *E-mail: [email protected] & [email protected] Tel: +91-484-2801234 Fax: 91-484-2802030

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ABSTRACT: Polymicrobial wound infections often requires high dosage of antibiotics and fungicides. However, prolonged antimicrobial therapies are associated with potential systemic side effects and increased risk for the development of drug resistant microbes. With this focus, we aimed at developing chitosan bandages loaded with anti-microbial (ciprofloxacin and fluconazole) drug loaded nanoparticles for a sustained slow release of drugs. The particle sizes of the prepared ciprofloxacin and flucanazole loaded fibrin nanoparticles were observed to be 132 ± 16 and 175 ± 17 nm respectively. The chitosan bandages with drug containing nanoparticles were flexible with adequate tensile strength and porosity of 80-85%, which would favour excess exudates absorption in an infectious wound. The in vitro toxicity of the bandages studied against human dermal fibroblast (HDF) cell line proved its cytocompatibility. Ciprofloxacin and fluconazole from bandages released upto 14 days in a sustained manner. The antimicrobial drugs loaded bandages showed significant antimicrobial activity towards polymicrobial cultures of C. albicans, E. coli & S. aureus in vitro and ex vivo. In vivo studies were conducted on polymicrobial infected rat wound model. A significant reduction in microbial load was obtained upon application of antimicrobial drug loaded chitosan bandages in vivo.

KEY

WORDS:

Polymicrobial

infection,

Antimicrobial

nanocomposite chitosan bandage.


agents,

fibrin

nanoparticles,

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1. INTRODUCTION Infections can be either acute or chronic and ranges from superficial paronychia to deep tissue infections, which include cellulitis, myositis, abscesses, necrotizing fasciitis, tendinitis, and osteomyelitis.1 Most of the chronic wound infections are polymicrobial in nature. In a polymicrobial infection, various microorganisms share their niche and survive in a dependent manner.2 Gram negative, Pseudomonas and Escherichia sps and Gram positive bacteria: Staphylococcus sps and fungi, Candida sps generally predominates the polymicrobial population.3 The infectious wound site produces free radicals and lytic enzymes like matrix metalloproteinase that has detrimental effect on the cellular events in wound healing. The exposure of wound bed with these proteases can lead to chronic inflammation.4 According to the American Diabetes Association, 25% of diabetic patients have a delayed wound healing, reduced neuronal sensations and decreased blood flow to the wound area. Current first aid care available for polymicrobial wound infections are cleansing the wound with antiseptics and oral or parenteral administration of various antibiotics. Ciprofloxacin and fluconazole are the commonly used antibiotic and antifungal drugs for wound infections.5 Intravenous injection and oral delivery are the commonly chosen practice of drug administration. However, the availability is lesser in patients suffering from peripheral arterial diseases.6 Topical application of antimicrobial drugs on superficial wound is a considered as a superior drug delivery system for its minimal systemic side effects.7-10 Novel therapeutic drugs not useful as systemic drug can be better used topically. Incorporation of antimicrobial drugs in wound dressing material can be used as topical antimicrobial drug delivery system for infectious wounds.11,12 Bandages protects wounds from injury, maintains a moist environment, absorbs wound exudates, helps to prevent maceration of surrounding tissue, allow gas and



moisture vapour exchange, helps in autolytic debridement and infection control.

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13Antibiotics

loaded polymeric wound dressing material has the potential to deliver drugs in a sustained manner for longer duration than the traditionally available dressings.14 Polysaccharides like chitin, chitosan, alginate and heparin, proteoglycans and proteins like fibrin, collagen, gelatin, silk fibroin and keratin are excellent materials for wound management because of their biodegradability, biocompatibility and resemblance to the extracellular matrices.15 Chitosan based material is used as an efficient drug delivery system.16-21 Chitosan, a polysaccharide made of β-1, 4-linked 2-amino-2-deoxy-β-D-glucose (glucosamine) and N-acetyl glucosamine can induce natural blood clotting and their degradation products can stimulate fibroblast proliferation and collagen deposition.22,23 The degradation products, N-acetyl glucosamine was studied to increase the wound healing rate and decrease scar formation.24,25 The functionality of chitosan wound dressings can be enhanced by incorporating metallic nanoparticles such as silver, gold and copper or antimicrobial drug loaded nanoparticles like liposomes, poly(lactic-co-glycolic acid), hyaluronic acid, gelatin and fibrin.26,27,28,29 The cytotoxicity of metallic nanoparticles and decreased bioactivity of synthetic polymeric nano careers makes natural polymers based nano careers a promising drug delivery system for wound healing and tissue engineering applications. Fibrin is an insoluble coagulation protein, which forms a crosslinked network during haemostasis of any wound healing process and it plays significant role in the subsequent phases of wound healing. Fibrin enhances the migration of fibroblasts and keratinocytes towards the wound bed and is also known to improve vascularisation.26,27 In addition to that , fibrinogen does not possess any immunogenic reactions within the same species.31 The objective of this research is to develop chitosan bandage containing ciprofloxacin and fluconazole loaded fibrin nanoparticles


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separately which would provide sustained release of drugs and therefore control the polymicrobial load on infectious wounds.

2. MATERIALS AND METHODS 2.1. Materials. Chitosan (Molecular weight: 100-150 kDa, Degree of deacetylation: 85%) was purchased from Koyo Chemical Industry Ltd, Japan. Bovine fibrinogen, Chloramphenicol, Luria-Bertani (LB) broth, Agar-agar, Mannitol salt agar, Mac Conkey agar and Sabouraud dextrose agar were purchased from Himedia, India. Ciprofloxacin and fluconazole were obtained from Micro Labs, India. Tigecycline was obtained from Glen Mark Pharmaceuticals Ltd, India. Cell culture consumables such as Fetal Bovine Serum (FBS), Dulbecco’s Minimal Essential Medium (DMEM), Alamar blue, trypsin etc were procured from Gibco, India. Hematoxylin, eosin, silicone sheet (0.5-mm-thick), Triton X-100 and ultra-centrifugal filter units were purchased from Sigma-aldrich, India. Thrombin from bovine plasma was obtained from Merck, India. Filter top cages were purchased from Vishnu traders, India. PTAH stain was obtained from Abcam. Sodium hydroxide, Formaldehyde and glacial acetic acid were purchased from Thermo Fisher Scientific India Private Limited, India. 2.2. Methods 2.2.1. Preparation of Ciprofloxacin and Fluconazole Loaded Nanoparticles (cFNPs and fFNPs). The drug loaded fibrin nanoparticles were prepared by emulsification method.32, 33Briefly, 5 ml of 40 mg/ml fibrinogen solution containing 1 mg ciprofloxacin or fluconazole and 5 ml of 12 U/ml of thrombin were added simultaneously to preheated coconut oil at 55±5 ºC. The solution was stirred at 500 rpm for 8 hrs. The emulsion was further centrifuged (HERMLE Labortechnik, Germany) at 10,000 rpm for 15 mins and the pellet was washed thrice using deionized water



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(18.2 MΩ cm, Millipore). The pellets were further lyophilized (Christ Alpha 1-2 LD) and stored at -20 ºC for further studies. 1 mg of cFNPs and fFNPs were suspended in 10 ml of deionized water. The suspensions were probe sonicated at 35% amplitude for 20 mins (Sonics vibra cell CV18). The fibrins in the sonicated suspensions were separated to minimize its interference in UV absorbance measurement by filter centrifugation at 5000 rpm for 30 mins using amicon ultra centrifugal filter units having molecular weight cut-off of 10 kDa. The filtrates were collected and the presence of ciprofloxacin and fluconazole were quantified by measuring the UV absorbances of each drug (UV-1700 Pharma Spec, Shimadzu) at 275 and 210 nm respectively. The concentrations corresponding to the UV absorbance’s were determined from concurrently plotted standard graphs. The obtained concentrations (µg/ml) were converted to amount (µg) and calculated the entrapment efficiency (EE) and loading efficacy (LE) using following equations.

(1) (2) Where, Wt = Total amount of ciprofloxacin/fluconazole added for the nanoparticle preparation. Wf = Amount of ciprofloxacin/fluconazole entrapped in the nanoparticles. Y = Total dry weight of the nanoparticles. 2.2.2. Characterization of cFNPs and fFNPs. The size, polydispersity index (PDI) and stability of the nanosuspensions was measured using Malvern Zetasizer Nano Series. The particle sizes were further confirmed using Transmission Electron Microscopy (TEM). PTAH staining was performed to confirm the formation of fibrin nanoparticles.34 Briefly, 5 µl of PTAH stain was


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added to 5 ml of 0.5 mg/ml nanosupension and incubated at 60ºC for 15 mins. The stained nanoparticles were imaged under microscope (Olympus-BX-51). 2.2.3. Preparation of cFNPs and fFNPs Loaded Chitosan Bandages. Chitosan hydrogel was prepared by previously established method.35 The chitosan hydrogel was washed thrice and collected by centrifugation (5000 rpm for 10 mins). To the prepared hydrogel, cFNPs pooled from 3 batches (corresponding to 4.5 mg of ciprofloxacin) and fFNPs pooled from 5 batches (corresponding to 4.2 mg of flucanazole) were resupended in 3 ml of water and added to 10 gm chitosan hydrogel and mixed thoroughly using overhead stirrers for 15 mins. The resulting slurry was then poured in a suitable polytetrafluoroethylene mould and was frozen at -20°C for 24 hours. The frozen gel was lyophilized for 24 hrs to obtain cFNPs and fFNPs loaded chitosan bandages (cFNPs + fFNPs-CH). After lyophilisation, the total weight of hydrogel gets reduced by 4 fold. The control bandages used for the studies were chitosan bandage (CH), chitosan bandage loaded with fibrin nanoparticles without drug (FNPs-CH) and chitosan bandages containing fibrin nanoparticles loaded with fluconazole (fFNPs-CH) and ciprofloxacin (cFNPsCH). The steps involved in the fabrication of cFNPs + fFNPs-CH is summarized in Figure 1.

Figure 1. Schematic representation is showing the cFNPs + fFNPs-CH bandage preparation. 7 ACS Paragon Plus Environment


2.2.4. Characterization of cFNPs+fFNPs-CH Bandages. To examine the porous structure of the cFNPs/fFNPs-CH bandages, the bandages were sectioned; sputter coated with gold and visualized using Scanning Electron Microscopy (SEM JEOL JSM-6490LA Analytical SEM). The chemical characterization was done using Fourier Transform Infrared spectroscopy-FTIR (Shimadzu IRAffinity-1S). Samples were ground, mixed with KBr and pelletised prior to FTIR analysis. 2.2.5. Porosity and Swelling Ratio Evaluation of Bandages. The porosity of CH, FNPs-CH, cFNPs-CH, fFNPs-CH and c-FNPs+fFNPs-CH bandages were quantified using alcohol displacement method.36 1 X 1 cm2 bandages of uniform thickness (0.5 cm) were soaked in absolute ethanol for 5 mins. The bandages were weighed and porosity was determined using the following equation:

Where, Wa- Weight of the bandage after immersing in ethanol Wi- Weight of the bandage before immersing in ethanol V- Volume of ethanol used for the study ρ- Density of ethanol The swelling of CH, FNPs-CH, cFNPs-CH, fFNPs-CH and cFNPs + fFNPs-CH bandages in 100 mM Tris buffer (pH 8) were performed. Briefly, 1 X 1 cm2 bandages of uniform thickness (0.5 cm) were incubated with sterile 100 mM Tris buffer (pH 8) at 37 ºC for 1 to 4 days. The weights of the swollen bandages were measured every day and the degree of swelling (DS) was calculated using the following formula.


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(4) Where, Ww - Wet weight of the bandages Wd - Dry weight of the bandages. 2.2.6. Mechanical Properties of Bandages. Universal Testing Machine (UTM) was used to analyse the tensile strength and elongation percentage of 1 X 1 cm2 bandages of uniform thickness (0.5 cm). The load cell was 250 N with crosshead speed and gauge length of 25 mm/h and 5 cm (Tinius Olsen) respectively.37 2.2.7. In Vitro Drug Release and its Kinetic Studies. The release pattern of ciprofloxacin and fluconazole from cFNPs+fFNPs-CH bandages were studied in 5 ml 100 mM Tris buffer (pH 8). The buffer having pH 8 was selected so as to mimic the alkaline pH of chronic wounds.38 1 X 1 cm2 bandages of uniform thickness (0.5 cm) were immersed in 20 ml autoclaved 5 ml 100 mM Tris buffer of pH 8 and incubated in 37ºC shaking incubator for 30 days. The release buffer were withdrawn every day and replaced with fresh buffer. The ciprofloxacin and fluconazole released in the withdrawn buffers were quantified by measuring the UV absorbances at 275 and 210 nm respectively using a spectrophotometer (SHIMADZU UV-2600). The amounts of individual drugs released were calculated from the absorbance values with the help of concurrently plotted standard graphs. The percentage of drug release was calculated by the formula:

The release of ciprofloxacin and fluconazole from the cFNPs+fFNPs-CH bandages were compared to the release profile of ciprofloxacin and fluconazole loaded chitosan bandages (c+fCH). Further, the mechanism of ciprofloxacin and fluconazole release from cFNPs+fFNPs-CH 9 ACS Paragon Plus Environment


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bandages were studied using important kinetic models like zero order, first order, Higuchi and Korsmeyer-Peppas.39 The obtained drug release data were fitted into the release models. The linearity and slope of each model were determined to study the release mechanism. 2.2.8. Cell Culture. The Human Dermal Fibroblast cell line (HDF; Promocell, Germany) was cultured and maintained in DMEM supplemented with 10% (v/v) FBS (high glucose) at 37ºC in 5% carbon dioxide (CO2) incubator. 2.2.9. Cytotoxicity Assay. The cytotoxicity of CH, FNPs-CH, cFNPs-CH, fFNPs-CH and cFNPs+fFNPs-CH bandages were tested against HDF cell line using alamar blue assay.40 EtO sterilised 1X1 cm2 bandages of uniform thickness (0.5 cm) were placed in non-tissue culture treated 24 well plates (1 bandages per well) 20,000 HDF cells were seeded on top of the bandages. The cell seeded bandages was further placed in 37ºC-5% CO2 incubator for 2 h. After incubation, 500 µl of culture media was carefully added to every well and incubated at 37ºC in 5% CO2 incubator. After particular time intervals, the media was replaced with 10 % Alamar containing basal media. The colour change in the Alamar blue solution after 4 hrs incubation was quantified by measuring the absorbance at 570 nm, using 600 nm as reference wavelength. 0.1% (v/v) Triton X-100 treated cells were used as positive control. The cell viability was calculated using the following formula:

(6) 2.2.10. Microbial Strains and Culture Conditions. S. aureus strain, SA113 (ATCC35556) and E. coli (ATCC25922) were cultured in sterile LB broth. C. albicans (ATCC2091) was cultured in sterile Sabouraud Dextrose (SD) broth. The liquid cultures were incubated in 37ºC shaking incubator at 120 rpm. 10 ACS Paragon Plus Environment

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2.2.11. In Vitro Antimicrobial Activity. 2.2.11.1. Broth Dilution Assay. The antimicrobial activities of the developed bandages were evaluated using broth dilution assay.41 1 X 1 cm2 bandages of uniform thickness (0.5 cm) were incubated in 10 ml of LB broth containing 106 CFU/ml of microorganisms at 37ºC in shaking incubator (120 rpm) for 12 hrs. The different microbial cell densities (CFU/ml) used in this study were obtained from a standard microbial suspension which had an absorbance at 600 nm similar to that of McFarland Standard No. 0.5 (1.5X108 CFU/ml).

The cultures were serially diluted

and were spread plated on LB agar and incubated in 37ºC incubator. The number of colonies formed after the incubation were quantified for microbial colonies. In addition, the antimicrobial activity of the prepared bandages against the mixed cultures of S. aureus, E. coli and C. albicans (106: 106: 107 CFU/ml) was studied using the above-mentioned method. 2.2.11.2. In Vitro Antimicrobial Evaluation by SEM. The 100 µl LB broth containing 106 CFU of S. aureus, 106 CFU of E. coli and 107 CFU of C. albicans and a mixture of these organisms were added on 1 X 1 cm2 bandages of uniform thickness (0.5 cm) and incubated for 12 hrs at 37ºC incubator with relative humidity of 95% without disturbing the bandages. Bandages were then fixed using 2% (v/v) glutaraldehyde for 15 mins, followed by a dehydration using gradient ethanol (50%, 75%, 90% and 100%). The improved sustained drug releasing property of cFNPs+fFNPs-CH bandages over control bandages were confirmed using long term antimicrobial activity assays. The sterile bandages were cut into 1 X 1 cm2 bandages of uniform thickness (0.5 cm) and incubated in 10 ml LB broth at 37ºC in shaking incubator (120 rpm) for 14 days. The culture broth was collected every day and replaced with fresh LB broth. Long term antimicrobial activity was studied by incubation of mixed culture of 106 CFU/ml of S. aureus, 106 CFU/ml E. coli and 107 CFU/ml C.



albicans in the 14 days sample elute. After 12 hrs of incubation with the organisms, the samples were serially diluted followed by plating on agar plates. The number of colonies formed after 12 hrs incubation at 37ºC was enumerated. 2.2.12. Ex-Vivo Antimicrobial Activity. Ex-vivo antimicrobial activity of the bandages was studied using pig skin. Mixed culture of 106 CFU of S. aureus, 106 CFU E. coli and 107 CFU C. albicans were inoculated on 1 X 1 cm2 tissue samples in 12 well plate and incubated at 37ºC in 5% CO2 incubator. 1 X 1 cm2 bandages of uniform thickness (0.5 cm) FNPs-CH and cFNPs+fFNPs-CH bandages were kept over the infected tissue and incubated further for 12 hrs. After the incubation period, the tissue samples were homogenised (IKA T20 basic ULTRATURRA) for the microbial enumeration. The homogenate was serially diluted and spread plated on the agar medium. 2.2.13. Animal ethical statement. All in vivo experiments were approved by the Institutional Animal Ethical Committee (IAEC), at Amrita Institute of Medical Sciences and Research Center, Kochi, India. 2.2.14. In Vivo Evaluation of c-FNPs+fFNPs-CH Bandages on Polymicrobial Infected Wounds. Female SD rats (average weight 275 ± 50 g) were randomly grouped into three, animals with cotton gauze, commercial control Biatain® Ag and test bandages c-FNPs+fFNPs-CH. All the rats were anesthetized by intraperitoneal injection of 35 mg/kg ketamine and 5 mg/kg xylazine. The dorsal skins of the rats were shaved and disinfected using 70% (v/v) ethanol. 1.5 X 1.5 cm2 full thickness excisional wounds were created on the depilated area using surgical blade and scissors.26 Silicon splints of thickness 0.5 mm were glued around the wounds to reduce the chances of skin contraction. 106 CFU of S. aureus, 106 CFU of E. coli and 107 CFU C. albicans in 200 µl sterile PBS were inoculated over the wound bed. After 24 hrs, 2 X 2 cm2 bandages


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were kept on the wound surface and wrapped using conventional cotton gauze as secondary dressing. The steps involved in the optimization of polymicrobial rat wound model are represented in Figure 2. Rats after recovering from anaesthesia were housed individually in filter top cages at 25 ± 3 ºC for 3 weeks. The dressing materials were changed on 7th and 14th postoperative days. Three rats from each group were euthanized every week for microbial enumeration. Wound surface was swabbed ten times using a cotton swab and the swab was vortexed in 5 ml PBS for 5 mins. The tissues were weighed and homogenised in 5 ml sterile PBS. The homogenate of wound tissue and vortexed swab solution were serially diluted and plated on mannitol salt agar. The bacterial and fungal growth on the solid medium was confirmed based on the colony morphology and enumerated.

Figure 2. The steps involved in the creation of polymicrobial infected wounds on SD rats. 2.2.15. In Vivo Drug Release. The amount of drug released from the bandages during animal studies was examined on 3rd and 7th day after dressing. The bandages were chopped into small pieces and vortexed vigorously in 10 ml of water: ethanol (1: 1) solution for 30 mins. The UV



absorbances of ciprofloxacin and fluconazole in the solution were measured by taking the absorbances of the releasate at 275 and 210 nm respectively using UV spectrophotometer. 2.2.16. Statistics. All the experiments were performed in triplicate and the values are represented as average ± standard deviation. The results were analysed statistically using student’s t test. The probability values were represented with * (p

Ciprofloxacin and Fluconazole Containing Fibrin Nanoparticles

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