Thiol–Norbornene Photoclick Chemistry for Grafting Antimicrobial Peptides onto Chitosan to Create Antibacterial Biomaterials

Abstract

Covalent conjugation of antimicrobial peptides (AMP) is considered an effective approach to overcome their potential degradation in vivo. As the high cost of AMP-based therapies is one of the major hurdles toward their clinical application, it is urgently needed to explore high yield reactions. Herein, the highly efficient thiol–norbornene photoclick chemistry (TNPC) was explored for AMP grafting onto chitosan. Norbornenes were introduced onto chitosan (NorChit) in an aqueous/organic system, followed by UV-triggered conjugation of N- and C-terminus cysteine-modified Dhvar5 (NorChit-Dhvar5). Up to 0.38 norbornene groups per chitosan repeating unit and up to 43% conjugation yield in NorChit-Nt-Dhvar5 (80 μmol of Dhvar5/g) were achieved, while in NorChit-Ct-Dhvar5 conjugation yield was 30% (55 μmol of Dhvar5/g). Finally, NorChit-Dhvar5 ultrathin films showed up to a 35% reduction of total adhered Gram-positive Staphylococcus epidermidis and increased the adhesion and killing of Gram-negative Pseudomonas aeruginosa compared to unmodified chitosan. Moreover, NorChit-Dhvar5 was noncytotoxic to human neonatal dermal fibroblasts, according to ISO 10993-1. Overall, our findings indicate TNPC as a high yield strategy for AMP grafting onto norbornene-functionalized biopolymers toward the fabrication of antibacterial biomaterials.

Hydrophobic PEGylation of Chitosan: A Graft Copolymer Approach toward Developing Nontoxic Antimicrobial Chitosan

“…Chitosan is a well-known polysaccharide, which can be easily prepared via the deacetylation of Chitin, the second most abundant natural polysaccharide. Antimicrobial derivatives of chitosans were reported (Figure ), following the earlier discussed principles of structure–activity relation of tuning hydrophobicity and cationic balance. − While there is a definite advantage of chitosan, such as the presence of cationic amine groups in the backbone, biodegradability, and biocompatibility, the major drawback lies in the lower water solubility of chitosan, especially hydrophobically modified ones . This hinders their antimicrobial efficacy, especially at ∼ pH 7, and results in higher toxicity and lesser selectivity, as revealed by their lower MIC/HC values .…”

One-Pot Microfluidics to Engineer Chitosan Nanoparticles Conjugated with Antimicrobial Peptides Using “Photoclick” Chemistry: Validation Using the Gastric Bacterium Helicobacter pylori

“…AMP (MSI-78A-SH: GIGKFLKKAKKFAKAFVKILKK-Ahx-C) was synthesized with a flexible 6-aminohexanoic acid (Ahx, Novabiochem) spacer and a cysteine (Novabiochem) at the Cterminus, as previously described. 14,19 Norbornene-modified chitosan (NorChit; Figure 1A) was prepared by us according to Alves et al 21 Briefly, purified squid chitosan (France Chitine) with a degree of acetylation (DA) of 2−3% ( 1 H NMR and FTIR chitosan spectra and respective DA calculations are in S1 and S2) and molecular weight of 363 ± 28 kDa was solubilized in 0.1 M acetic acid (AppliChem) with a final concentration of 0.4% w/v. To introduce the norbornene groups, chitosan in solution reacted with carbic anhydride in N,N-dimethylformamide (DMF; 0.15 M, Merck), which was added every 30 min, three times.…”

“…This click chemistry has been extensively studied for the functionalization of biomaterials (Table 1) and, more recently, for the grafting of an AMP (Dhvar5) onto chitosan hydrogels to prepare thin coatings against Staphylococcus epidermidis (ATCC 35984) and Pseudomonas aeruginosa (ATCC 27853). 21,28,37,39 The optimal pH for TNPC is between 4 and 7. 45 At pH > 9, the reaction yield decreases drastically due to the predominance of thiolate forms, which do not react with norbornene groups.…”

Photocrosslinkable natural polymers in tissue engineering

“…Norbornene is incorporated into natural polymers through amide reactions (Devaraj et al, 2008). Natural polymers incorporated with norbornene exhibit low solubility under acidic aqueous solvent conditions (Mũnoz et al, 2014;Alves et al, 2022). This is because the overall hydrophobicity was increased by the norbornene moiety, and the positive elective charge was reduced due to the conversion of the amine into the amide (Michel et al, 2019).…”