Tailored anti-biofilm activity – Liposomal delivery for mimic of small antimicrobial peptide

The eradication of bacteria embedded in biofilms is among the most challenging obstacles in the management of chronic wounds. These biofilms are found in most chronic wounds; moreover, the biofilm-embedded bacteria are considerably less susceptible to conventional antimicrobial treatment than the planktonic bacteria. Antimicrobial peptides and their mimics are considered attractive candidates in the pursuit of novel therapeutic options for the treatment of chronic wounds and general bacterial eradication. However, some limitations linked to these membrane-active antimicrobials are making their clinical use challenging. Novel innovative delivery systems addressing these limitations represent a smart solution. We hypothesized that incorporation of a novel synthetic mimic of an antimicrobial peptide in liposomes could improve its anti-biofilm effect as well as the anti-inflammatory activity.

Highlights

• A novel mimic of an antimicrobial peptide was incorporated into liposomes.

• The novel system significantly reduced inflammatory response in macrophages.

• The novel system inhibited S. aureus, E. coli and P. aeruginosa biofilm formation.

• The system improved eradication of S. aureus, E. coli, and P. aeruginosa biofilms relative to the mimic alone.

The small synthetic mimic of an antimicrobial peptide, 7e-SMAMP, was incorporated into liposomes (~280 nm) tailored for skin wounds and evaluated for its potential activity against both biofilm formation and eradication of pre-formed biofilms. The 7e-SMAMP-liposomes significantly lowered inflammatory response in murine macrophages (~30 % reduction) without affecting the viability of macrophages or keratinocytes. Importantly, the 7e-SMAMP-liposomes completely eradicated biofilms produced by Staphylococcus aureus and Escherichia coli above concentrations of 6.25 μg/mL, whereas in Pseudomonas aeruginosa the eradication reached 75 % at the same concentration. Incorporation of 7e-SMAMP in liposomes improved both the inhibition of biofilm formation as well as biofilm eradication in vitro, as compared to non-formulated antimicrobial, therefore confirming its potential as a novel therapeutic option for bacteria-infected chronic wounds.

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Materials

Lipoid S100 (phosphatidylcholine content >94 %) was kindly provided by Lipoid GmbH (Ludwigshafen, Germany). Methanol (≥99.9 %), HiPerSolv CHROMANORM® for LC-MS, acetonitrile (≥99.9 %), HiPerSolv CHROMANORM®, gradient grade for HPLC and acetic acid (>99.9 %) were purchased from VWR International (Fontenay-sous-Bois, France). Phosphoric acid (>85 %) was acquired from Kebo lab (Oslo, Norway). Albunorm (200 mg/mL human serum albumin) was acquired from Octapharma AG (Lachen, Switzerland). Sepharose™ CL-4B was purchased from GE Healthcare (Uppsala, Sweden). 1,2-Dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DOPG), and 1-palmitoyl-2-(6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl)-sn-glycero-3-[phospho-rac-(1-glycerol)] (ammonium salt) (C6-NBD-PG) were obtained from Avanti Polar Lipids (Alabaster, AL, USA). 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was obtained from Lipoid GmbH (Ludwigshafen, Germany). Trifluoroacetic acid (TFA) for HPLC (≥99.0 %), chloroform (99.0–99.4 %, GC), glycerol solution (86–89 %), sodium phosphate dibasic dihydrate, potassium phosphate monobasic, sodium chloride, EDTA sodium, sodium hydrogen carbonate, potassium chloride, calcium chloride dehydrate, cardiolipin sodium salt from bovine heart (≥97 %), N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), sodium dithionite, fluorescein isothiocyanate–dextran average molecular weight (Mw) 3000–5000 Da (FITC–dextran 4400), fluorescein isothiocyanate–dextran average Mw 20,400 Da (FITC–dextran 20,400), Kollisolv® PEG E 400, Phospholipid Assay Kit for colorimetric or fluorometric tests, N-(−1-Naphthyl)ethylenediamine dihydrochloride, sulphanilamide, Triton™ X-100, and Cell Counting Kit-8 (CCK-8) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Penicillin-streptomycin (10,000 units/mL and 10 mg/mL, respectively), lipopolysaccharides (LPS) from Escherichia coli O55:B5, RPMI-1640 medium (with l-glutamine and sodium bicarbonate) and fetal bovine serum (FBS) were obtained from Sigma-Aldrich (Steinheim, Germany). Dulbecco’s Modified Eagle Medium high glucose (DMEM-hg) w/l-glutamine and sodium pyruvate was purchased from Biowest (Nuaillé, France). Nutrient Broth (NB) was supplied by Becton Dickinson and Company (Sparks, MD, USA). Murine macrophage, RAW 264.7 cell line was obtained from ATCC (Manassas, VA, USA). HaCaT cell line (immortalized human keratinocytes) was purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany). Staphylococcus aureus ATCC29213, Escherichia coli ATCC11105, Pseudomonas aeruginosa ATCC10145 were purchased from American Tissue and Cell Culture Corp. (Manhasset, Virginia, USA). Staphylococcus aureus SO2, SO83, SO86, and SO88 are clinical isolates (Ospedale Sant’Orsola-Malpighi, Bologna, Italy).

Lisa Myrseth Hemmingsen, Barbara Giordani, Marianne H. Paulsen, Željka Vanić, Gøril Eide Flaten, Beatrice Vitali, Purusotam Basnet, Annette Bayer, Morten B. Strøm, Nataša Škalko-Basnet, Tailored anti-biofilm activity – Liposomal delivery for mimic of small antimicrobial peptide, Biomaterials Advances, Volume 145, 2023, 213238, ISSN 2772-9508, https://doi.org/10.1016/j.bioadv.2022.213238.