Synthesis and Characterization of Peptide–Chitosan Conjugates (PepChis) with Lipid Bilayer Affinity and Antibacterial Activity

Abstract

Antimicrobial peptides appear among innovative biopolymers with potential therapeutic interest. Nevertheless, issues concerning efficiency, production costs, and toxicity persist. Herein, we show that conjugation of peptides with chitosans can represent an alternative in the search for these needs. To increase solubility, deacetylated and degraded chitosans were prepared. Then, they were functionalized via N-succinimidyl-S-acetylthiopropionate or via glutathione (GSH), an endogenous peptide linker. To the best of our knowledge, it is the first time that GSH is used as a thiolating agent for the conjugation of peptides. Next, thiolated chitosans were conjugated through a disulfide bond with designed shortchain peptides, one of them derived from the antimicrobial peptide Jelleine-I. Conjugates and respective reaction intermediates were characterized by absorciometry, attenuated total reflectance−Fourier transform infrared, and 1 H NMR. Zeta potential measurements showed the cationic nature of these biomacromolecules and their preferential partitioning to Gram-positive bacterial-like model membranes. In vitro investigation using representative Gram-positive and -negative bacteria (Staphylococcus aureus and Escherichia coli, respectively) showed that the conjugation strategies lead to enhanced activity in relation to the unconjugated peptide and to the unconjugated chitosan. The obtained products showed selectivity toward S. aureus at low cytotoxicity as determined in NIH/3T3 cells. Overall, our study demonstrates that an appropriate choice of antimicrobial peptide and chitosan characteristics leads to increased antimicrobial activity of the conjugated product and represents a strategy to modulate the activity and selectivity of antimicrobials resorting to low-cost chemicals. The present proposal starts from less expensive raw materials (chitosan and short-chain peptide), is based on aqueous solvents, and minimizes the use of reactants with a higher environmental impact. The final biopolymer contains the backbone of chitosan, just 3−6% peptide derived from royal jelly and GSH, all of them considered safe for human use or as a physiological molecule.

Towards Robust Delivery of Antimicrobial Peptides to Combat Bacterial Resistance

“…Being a linear polysaccharide made up of glucosamine and N -acetylglucosamine subunits [ 133 ], chitosan is a biocompatible, non-toxic and biodegradable polysaccharide that also displays bioadhesive and wound healing properties [ 79 ]. Furthermore, it itself displays mild antibacterial properties against a broad spectrum of microorganisms, which can be tuned by molecular weight and chemical modification via amine groups (e.g., deacetylation) [ 79 ]; as such, it has gained some attention as a promising delivery vehicle for AMPs. Much work has been published detailing the utilization of chitosan to improve the biocompatibility of peptides while maintaining or enhancing their antimicrobial activity [ 79 , 133 , 134 , 135 , 136 ].…”

“…Furthermore, it itself displays mild antibacterial properties against a broad spectrum of microorganisms, which can be tuned by molecular weight and chemical modification via amine groups (e.g., deacetylation) [ 79 ]; as such, it has gained some attention as a promising delivery vehicle for AMPs. Much work has been published detailing the utilization of chitosan to improve the biocompatibility of peptides while maintaining or enhancing their antimicrobial activity [ 79 , 133 , 134 , 135 , 136 ]. For example, Hou et al developed self-assembled short chain chitosan-polylysine AMP nanoparticles that displayed strong broad-spectrum activity both in vitro and in vivo with very low hemolytic activity and improved selectivity [ 136 ].…”

Cationic chitosan derivatives as potential antifungals: A review of structural optimization and applications

“…In the process, 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are often used to activate the carboxyl groups of amino acids in the reaction process (Zhou et al, 2019). To avoid side reactions and reduce reaction steric hindrance, some spacer molecules are often used in the coupling process (Table 3) (Denora et al, 2016;Petrin et al, 2019). In some cases, to prevent amino acids from self-polymerization in the presence of EDC, the primary amino groups of amino acids will be protected in advance (Muhsin et al, 2014).…”

Antimicrobial Chitosan Conjugates: Current Synthetic Strategies and Potential Applications

“…Although the presence of hydroxyl and amino groups in the chitosan molecule can allow structural modification to be easily carried out, in terms of conjugates that result in chitosan conjugates with more structural diversity, it is very necessary to explore a functional group conversion strategy to introduce new reactive groups to the chitosan backbone. It has been demonstrated that the amino group of chitosan can be converted into azide group [87], substituted carboxyl group [88], substituted mercapto group [89], etc., and the hydroxyl group can be azidated [90], aminated [91,92], oxidized to an aldehyde [93] or carbonyl group [94], or further oxidized to a carboxyl group [95]. Figure 4 lists some common functional group conversion methods used in the preparation of common chitosan conjugates.…”