Peptide-Polysaccharide Conjugates: a Click Approach to Functional Wound Dressings

This thesis investigates the synthesis of bioactive materials capable of enhancing the wound healing process by preventing bacterial infections or accelerating wound closure. This was achieved by combining natural polysaccharides with peptides using a click chemistry approach. The polysaccharides cotton, chitosan, and hyaluronic acid were selected due to their nontoxicity, biocompatibility, and intrinsic bioactivities. Peptides were used as active molecules due to their versatile biological properties, which include antimicrobial, antiviral, anti-inflammatory, and collagen-stimulating activities. PMAP-36(12-24), Pal-HAaH, 3.1 and (RW)3 were selected for their broad-spectrum antimicrobial activity, while PP4 and Ac-SDKP were chosen for their potential skin regenerative properties. This work reports on (1) three chemoselective strategies for conjugating peptides to cotton; (2) a dual chemoselective ligation approach for conjugating peptides 3.1 and peptide PP4 to chitosan; (3) the use of thiazolidine conjugation to bind peptides to hyaluronic acid and the electrospinning of the peptide-hyaluronic acid conjugates to form textiles made of nanometric fibers. Cotton is the most widely used material for wound bandages but lacks intrinsic bioactivity. Therefore, aldehydes were introduced to its backbone through TEMPO/laccase/O₂ catalyzed oxidation, enabling thiazolidine and oxime ligations with active peptides. The reaction conditions for the two types of chemoselective ligation were studied using model peptides. Consecutive thiazolidine bonds of PMAP-36(12-24) and Pal-HAaH or PP4 were successfully carried out, producing double functionalized peptide-cotton conjugates capable of inhibiting the growth of Acinetobacter baumannii. PMAP-36(12-24) was also successfully bound to norbornene-functionalized cotton through a photoinitiated thiol-ene reaction. The resulting peptide-cotton conjugates demonstrated comparable peptide loading and antibacterial activity to that of the conjugates obtained via thiazolidine bond. The intrinsic antibacterial and hemostatic properties of chitosan make it a promising wound-healing material, and the presence of an hydroxyl and amine group in its repeating unit permit a double functionalization of the polysaccharide. Aldehydes and azides were introduced onto chitosan to enable binding of peptide cys3.1 via thiazolidine chemistry and peptide PraPP4 via azide–alkyne cycloaddition. The binding of both peptides to modified chitosan was confirmed by FT-IR, XPS and amino acid analyses. Antibacterial tests against Staphylococcus aureus and Pseudomonas aeruginosa demonstrated the ability of the 3.1–chitosan conjugate to strongly inhibit the growth of both bacterial strains. However, the tests also revealed the dual conjugate 3.1–chitosan–PP4 to be less effective. Hyaluronic acid is a key component in the wound healing process. Conjugating hyaluronic acid with active peptides and electrospinning the resulting conjugate enables the creation of nanostructured textiles with enhanced healing properties. NaIO4 and TEMPO/laccase/O₂ oxidation were compared as methods to introduce the aldehyde onto hyaluronic acid. TEMPO/laccase/O₂ was chosen because it has a better environmental impact and preserves the integrity of hyaluronic acid better. Electrospun nanofibers were successfully fabricated from aldehyde-functionalized hyaluronic acid/polyethylene oxide blends, and the same electrospinning setup was used for the successful electrospinning of peptide-hyaluronic acid conjugates with analogues of Ac-SDKP and (RW)3. The conjugation and electrospinning processes did not impair the ability of (RW)3 to inhibit the growth of Staphylococcus aureus.