The development of electrospun chloramphenicol containing wound dressing

Wound healing is among the most complex processes in the human body that is greatly coordinated with a focus to rebuild tissue integrity and restore the skin´s protective barrier. The normal healing process is disrupted in chronic wounds, leading to delayed wound healing due to several underlying factors such as aging, obesity, and diabetes. Among chronic wounds 50 % are infected and up to 78 % of these wounds have presence of biofilm. The management of these wounds is complicated, even with antibiotics. Antibiotic resistance is a growing issue worldwide. Thus, there is a need for innovative treatments that are capable of accelerating wound healing process, and at the same time, prevent and fight bacterial contamination and growth. Electrospinning is a favorable method for making nanofiber dressings. These nanofibers can be added several active ingredients to obtain multifunctional wound dressings. This project aims as developing multifunctional nanofibers, containing the active ingredient soluble beta-1,3/1,6-glucan (SBG) with an immune stimulating effect, and chitosan (CHI) applied for its antimicrobial effect. In addition, we incorporated chloramphenicol (CAM) as the antimicrobial ingredient. Hydroxypropyl methylcellulose (HPMC) and polyethylene oxide (PEO) were used as co-polymers, and water, ethanol, and acetic acid were used as solvents. The focus of this master project was to process and characterize these multifunctional nanofiber formulations together with appropriate control formulations. The nanofibers were produced using the needle-free NanospiderTM technology. To assess the properties of fabricated nanofibers, suitable methods for characterization of both polymer solutions without and with CAM and final nanofibers were applied. Conductivity, pH, surface tension and viscosity were examined to characterize the polymer solutions. The produced nanofibers were evaluated for absorption capacity, tensile properties, morphologies, and diameter. Fabricated nanofibers with CAM were evaluated for its content and in vitro release. In addition, all nanofibers were assessed for in vitro cell toxicity. Polymer solutions containing CAM and -glucan (G) did not affect solution properties. CHI containing polymer solutions had increased conductivity, pH, and viscosity. The electrospinning process was not influenced by these increased properties. The temperature and relative humidity were successfully controlled during the electrospinning process for all nanofibers. All nanofibers were uniform and had diameter in the range from 129.5 to 200.9 nm. Nanofibers containing CHI did hold their structure in simulated wound fluid (SWF) and was possible to be investigated for absorption capacity. These nanofibers had absorption capacity up to 1055 % and was judged suitable for treatment of moderate to high exudative wounds. The method used for absorption capacity was not able to be performed on nanofibers without CHI since these nanofibers dissolved fast in SWF. The tensile strength of all produced nanofibers was in the skin range and proved to have good strength. However, the elongation at break for all nanofibers was poor and suggested to be less elasticity compared to the native skin. CHI and G nanofibers did not affect the in vitro CAM release, since all nanofibers had burst release of CAM. The high recovery of CAM from the in vitro CAM release, from 85 to 99 %, indicates that CAM is stable in all nanofibers and tolerate the electrospinning process well. Lastly, all nanofibers did not show cytotoxicity, indicating good cytocompatibility on human immortalized keratinocytes (HaCaT) cell lines. Keywords: Chitosan; SBG; -glucan; Chloramphenicol; Nanofiber; Needle-free electrospinning; NanospiderTM; Wound dressing; Wound healing; Chronic wound

Electrospinning of chloramphenicol-containing nanofibrous dressings for treatment of chronic wounds

Background An efficient treatment is crucial to overcome the delayed healing process and high infection rate in chronic wounds. Nanofibrous dressings, having a nano‐sized fiber structure, can enhance cell ingrowth and support the healing process. In addition, nanofibers can deliver active ingredients e.g. antibiotics into the wound to treat wound infection. By incorporating broad‐spectrum antibiotics like chloramphenicol into a nanofibrous dressing, wound infection can be treated whereas systemic side effects are avoided. Active nanofiberforming polymers can be added to achieve more functionalities. Examples are the β‐glucan‐ and chitosan polymers, with their immunostimulating and antimicrobial properties, respectively. Combining these polymers in one dressing might provide a multifunctional and more efficient wound dressing. Goals The goal of this project is to electrospin a multifunctional dressing comprising the active polymers β‐glucan and chitosan. For this, we investigated the effect of the different active ingredients on nanofiber characteristics such as morphology, diameter, swelling index and cytotoxicity. Methods Nanofibers were spun from preformed polymer‐solutions using the needle‐free NanospiderTM technology. Nanofiber morphology and diameter were determined by SEM and the image‐processing program ImageJ. The thickness was measured using a micrometer and swelling index was determined by submerging the nanofibers into artificial wound fluid. Cytotoxicity of the nanofibers was tested using the Cell Counting Kit‐8 (Sigma‐Aldrich). Results and Conclusion Nanofibers comprising 20% chitosan and 20% βG together with the copolymers polyethylene oxide and hydroxypropylmethylcellulose and 1% chloramphenicol were successfully fabricated. All fibers had a randomly distributed fiber structure with a diameter around 100 nm. Nanofibers containing chitosan had a reduced thickness with values from 0.03 mm to 0.05 mm, compared to fibers without chitosan that had a thickness from 0.06 mm to 0.08 mm, but an improved stability upon contact with water. In addition, chitosan containing nanofibers showed a high swelling index, ranging from 700 to 1200 %. Fibers without chitosan disintegrated upon contact with water, the swelling index could therefore not be measured. All fibers showed no cytotoxicity compared to medium as control when tested on human keratinocytes. The incorporation of chloramphenicol neither influenced the fiber morphology nor the swelling index or cytotoxicity, proving that the design of nanofibers containing both active polymers together with chloramphenicol was successful

Electrospinning of chloramphenicol-containing nanofibrous dressings for treatment of chronic wounds

Background An efficient treatment is crucial to overcome the delayed healing process and high infection rate in chronic wounds. Nanofibrous dressings, having a nano‐sized fiber structure, can enhance cell ingrowth and support the healing process. In addition, nanofibers can deliver active ingredients e.g. antibiotics into the wound to treat wound infection. By incorporating broad‐spectrum antibiotics like chloramphenicol into a nanofibrous dressing, wound infection can be treated whereas systemic side effects are avoided. Active nanofiberforming polymers can be added to achieve more functionalities. Examples are the β‐glucan‐ and chitosan polymers, with their immunostimulating and antimicrobial properties, respectively. Combining these polymers in one dressing might provide a multifunctional and more efficient wound dressing. Goals The goal of this project is to electrospin a multifunctional dressing comprising the active polymers β‐glucan and chitosan. For this, we investigated the effect of the different active ingredients on nanofiber characteristics such as morphology, diameter, swelling index and cytotoxicity. Methods Nanofibers were spun from preformed polymer‐solutions using the needle‐free NanospiderTM technology. Nanofiber morphology and diameter were determined by SEM and the image‐processing program ImageJ. The thickness was measured using a micrometer and swelling index was determined by submerging the nanofibers into artificial wound fluid. Cytotoxicity of the nanofibers was tested using the Cell Counting Kit‐8 (Sigma‐Aldrich). Results and Conclusion Nanofibers comprising 20% chitosan and 20% βG together with the copolymers polyethylene oxide and hydroxypropylmethylcellulose and 1% chloramphenicol were successfully fabricated. All fibers had a randomly distributed fiber structure with a diameter around 100 nm. Nanofibers containing chitosan had a reduced thickness with values from 0.03 mm to 0.05 mm, compared to fibers without chitosan that had a thickness from 0.06 mm to 0.08 mm, but an improved stability upon contact with water. In addition, chitosan containing nanofibers showed a high swelling index, ranging from 700 to 1200 %. Fibers without chitosan disintegrated upon contact with water, the swelling index could therefore not be measured. All fibers showed no cytotoxicity compared to medium as control when tested on human keratinocytes. The incorporation of chloramphenicol neither influenced the fiber morphology nor the swelling index or cytotoxicity, proving that the design of nanofibers containing both active polymers together with chloramphenicol was successful

Multifunctional Nanofibrous Dressing with Antimicrobial and Anti-Inflammatory Properties Prepared by Needle-Free Electrospinning

An active wound dressing should address the main goals in wound treatment, which are improved wound healing and reduced infection rates. We developed novel multifunctional nanofibrous wound dressings with three active ingredients: chloramphenicol (CAM), beta-glucan (βG) and chitosan (CHI), of which βG and CHI are active nanofiber-forming biopolymers isolated from the cell walls of Saccharomyces cerevisiae and from shrimp shells, respectively. To evaluate the effect of each active ingredient on the nanofibers’ morphological features and bioactivity, nanofibers with both βG and CHI, only βG, only CHI and only copolymers, polyethylene oxide (PEO) and hydroxypropylmethylcellulose (HPMC) were fabricated. All four nanofiber formulations were also prepared with 1% CAM. The needle-free NanospiderTM technique allowed for the successful production of defect-free nanofibers containing all three active ingredients. The CAM-containing nanofibers had a burst CAM-release and a high absorption capacity. Nanofibers with all active ingredients (βG, CHI and CAM) showed a concentration-dependent anti-inflammatory activity, while maintaining the antimicrobial activity of CAM. The promising anti-inflammatory properties, together with the high absorption capacity and antimicrobial effect, make these multifunctional nanofibers promising as dressings in local treatment of infected and exuding wounds, such as burn wound