Chitosan-based delivery system enhances antimicrobial activity of chlorhexidine

Infected chronic skin wounds and other skin infections are increasingly putting pressure on the health care providers and patients. The pressure is especially concerning due to the rise of antimicrobial resistance and biofilm-producing bacteria that further impair treatment success. Therefore, innovative strategies for wound healing and bacterial eradication are urgently needed; utilization of materials with inherent biological properties could offer a potential solution. Chitosan is one of the most frequently used polymers in delivery systems. This bioactive polymer is often regarded as an attractive constituent in delivery systems due to its inherent antimicrobial, anti-inflammatory, anti-oxidative, and wound healing properties.

However, lipid-based vesicles and liposomes are generally considered more suitable as delivery systems for skin due to their ability to interact with the skin structure and provide prolonged release, protect the antimicrobial compound, and allow high local concentrations at the infected site. To take advantage of the beneficial attributes of the lipid-based vesicles and chitosan, these components can be combined into chitosan-containing liposomes or chitosomes and chitosan-coated liposomes. These systems have previously been investigated for use in wound therapy; however, their potential in infected wounds is not fully investigated.

In this study, we aimed to investigate whether both the chitosan-containing and chitosan-coated liposomes tailored for infected wounds could improve the antimicrobial activity of the membrane-active antimicrobial chlorhexidine, while assuring both the anti-inflammatory activity and cell compatibility. Chlorhexidine was incorporated into three different vesicles, namely plain (chitosan-free), chitosan-containing and chitosan-coated liposomes that were optimized for skin wounds. Their release profile, antimicrobial activities, anti-inflammatory properties, and cell compatibility were assessed in vitro.

The vesicles comprising chitosan demonstrated slower release rate of chlorhexidine and high cell compatibility. Additionally, the inflammatory responses in murine macrophages treated with these vesicles were reduced by about 60% compared to non-treated cells. Finally, liposomes containing both chitosan and chlorhexidine demonstrated the strongest antibacterial effect against Staphylococcus aureus. Both chitosan-containing and chitosan-coated liposomes comprising chlorhexidine could serve as excellent platforms for the delivery of membrane-active antimicrobials to infected wounds as confirmed by improved antimicrobial performance of chlorhexidine.

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Materials

Chitopharm™ S-Chitosan with low M_w (50–1000 kDa), degree of deacetylation >70% was kindly provided by Chitinor (Tromsø, Norway). Lipoid S100 was kindly provided by Lipoid GmbH (Ludwigshafen, Germany). Methanol ≥99.9%, HiPerSolv CHROMANORM® for LC–MS, phosphate buffered-saline (PBS, pH 7.4) tablets and acetic acid glacial were procured from VWR International (Fontenay-sous-Bois, France). Chlorhexidine ≥99.5%, glycerol solution (86–89%), glycine hydrochloride ≥99% (HPLC), sodium chloride, hydrochloric acid, Cell Counting Kit-8 (CCK-8), and Kollisolv® polyethylene glycol (PEG) E 400 were obtained from Sigma-Aldrich (St. Louis, MO, United States). Cibacron Brilliant Red 3BA was a product from Santa Cruz Biotechnology (Dallas, TX, United States). Ortho-phosphoric acid ≥85% was purchased from Kebo Lab Ab (Oslo, Norway). Penicillin–streptomycin and Roswell Park Memorial Institute (RPMI) medium 1640 were purchased from Sigma-Aldrich (Steinheim, Germany). Lipopolysaccharide (LPS, from Escherichia coli 055:B5), sulfanilamide ≥98% and N-(1-Naphthyl)ethylenediamine dihydrochloride ≥98% were obtained from Sigma Life Science Norway AS (Oslo, Norway). Dulbecco’s Modified Eagle Medium high glucose w/ l-glutamine (DMEM-hg) and sodium pyruvate and fetal bovine serum (FBS) were purchased from Biowest (Nuaillé, France). Blood agar plates, saline solution, and Mueller–Hinton broth were supplied by University Hospital of North Norway (Tromsø, Norway). Murine macrophage RAW 264.7 cells were ordered from ATCC (Manassas, VA, United States). Human Dermal Fibroblasts, Neonatal (NHDF-neo) were obtained from Lonza (Basel, Switzerland), and HaCaT cell line (immortalized human keratinocytes) from CLS Cell Lines Service GmbH (Eppelheim, Germany). Staphylococcus aureus (ATCC® BAA-1721™) MSSA476 was ordered from LGC standards AB (Borås, Sweden).

Hemmingsen LM, Panchai P, Julin K, Basnet P, Nystad M, Johannessen M and Škalko-Basnet N (2022) Chitosan-based delivery system enhances antimicrobial activity of chlorhexidine. Front. Microbiol. 13:1023083.
doi: 10.3389/fmicb.2022.1023083