As society draws closer to the post-antibiotic era and the pipeline for alternatives dries, there is an urgent need for the development of novel antimicrobial therapies that do not promote bacterial resistance, particularly for immunocompromised chronic wound patients. Antimicrobial peptides (AMPs), including human-derived LL37, show considerable promise as broad spectrum alternatives that also have wound healing properties; however, few have been clinically implemented as novel antimicrobials due to their cytotoxicity stemming from a poor understanding of their mechanisms and low stability in vivo. It has been suggested that tethering, or attaching AMPs onto surfaces, is a viable strategy of delivering bioactive AMPs to surfaces while reducing cytotoxicity and improving stability. Thus, we designed new chimeric versions of LL37 with collagen-binding domains (CBD), derived from collagenase (cCBD-LL37) and fibronectin (fCBD-LL37) for non-covalent tethering onto collagen, a prevalent biopolymer in commercially available wound dressings and scaffolds. Our overall hypothesis was that CBDs would mediate stable tethering of broadly active, non-cytotoxic CBD-LL37 onto collagen-based scaffolds. We first studied the loading, release and bioactivities (e.g. antimicrobial activity and cytotoxicity) of each CBD-LL37 on commercially available 100% collagen type I PURACOL® wound scaffolds. We found that both cCBD-LL37 and fCBD-LL37 bound highly to collagen, were active against relevant wound pathogens, demonstrated stable activity after 14 days of release, and were not cytotoxic to human fibroblasts. The addition of different CBDs onto LL37 also markedly altered their soluble bioactivities. Using similar methods, we then studied the loading, release and bioactivity of each CBD-LL37 on a commercially available FIBRACOL® wound scaffolds, comprised of 90% collagen type I and 10% calcium alginate biopolymers. We found that both cCBD-LL37 and fCBD-LL37 also bound highly to and retained on collagen for 14 days, but were only active against Gram-negative P. aeruginosa. This suggested that the presence other biopolymers in addition to collagen, which is common among commercial wound dressings, could cause significant differences in binding, retention and bioactivities of CBD-LL37. To better understand how CBD modification affected CBD-LL37 structure leading to different bioactivities, we studied the CBD sequence-, peptide structure-, concentration-, time-, and bilayer composition-dependent interactions of soluble CBD-LL37 and compared these findings with the properties of unmodified LL37. Using Molecular Dynamics (MD) simulations, circular dichroism (CD) spectroscopy, quartz crystal microbalance with dissipation (QCM-D), and fluorescent bilayer imaging we determined the structural basis behind CBD alterations in bioactivities. MD and CD, in addition to other intrinsic CBD properties (helicity, amphiphilicity, charge) we hypothesized that cCBD-LL37 utilized similar mechanisms as unmodified LL37 while fCBD-LL37 demonstrated based primarily on surface adsorption. We used QCM-D and Voigt-Kelvin viscoelastic modeling to determine the time- and concentration-dependent interactions of unmodified LL37 with model mammalian lipid bilayers, the mechanisms of which are still controversial in literature despite being widely studied. These results were used to propose a model for the interaction mechanism of LL37 with zwitterionic bilayers that aligned with its bioactive concentrations. LL37 adsorbed at concentrations where it is immunomodulatory until reaching a threshold which corresponded with its antimicrobial concentrations. The threshold was correlated to lipid bilayer saturation, after which LL37 formed transmembrane pores. We observed collapse of the bilayer into a rigid proteolipid film at concentrations higher than the reported cytotoxic threshold of LL37. The mechanistic and structural information for each CBD-LL37 and unmodified LL37 provided a baseline for QCM-D and Voigt-Kelvin viscoelastic modeling to further elucidate the time-, concentration-, lipid composition- and CBD sequence-dependent basis behind the observed bioactivities of cCBD-LL37 and fCBD-LL37. We found that similar to LL37, cCBD-LL37 demonstrated pore formation mechanisms likely due to their similar charges, structural content and amphiphilicity. fCBD-LL37 demonstrated time-dependent, adsorption-based mechanism likely due to its anchoring aromatic residues, low charge, and low amphiphilicity. Knowledge gained from this study allowed mechanistic predictions of two newly designed hypothetical CBD-LL37 peptides. Results from this study contribute to a better understanding of a new class of antimicrobial, non-cytotoxic therapies based on collagen-tethered CBD-LL37, bringing it closer to clinical implementation in chronic wound applications and demonstrate the viability of biopolymer tethering as a platform toward using AMPs to quench the resistance crisis.

As a direct consequence of the misuse of antibiotics since its first discovery, bacteria have developed extensive resistance mechanisms making them once again potential lethal threats. This eventuality has triggered a vast research effort from scientists worldwide to find solutions to mitigate antimicrobial resistance. One such option is the identification of new potential antimicrobial agents, like for example antimicrobial peptides (AMPs). Various methods have been applied to evaluate the properties and determine the complex mechanism of AMPs. However, the details of the mechanism remain unknown and hence the work in this project seeks to examine the suitability of using TIR Raman, a vibrational spectroscopy technique which is sufficiently surface sensitive to study the interaction of AMPs in contact with lipid bilayers, which are just a few nanometres thick. In order to evaluate the information that could be extracted from TIR Raman, measurement of solid supported lipid bilayers in the absence of peptides were first carried out. In particular, the lipid DMPC with a phase transition close to room temperature, was measured at various temperatures to determine spectral changes associated with the transition. For the peptide-membrane interactions, the AMP LL37 was put into contact with solid supported lipid bilayers modelling the cell membranes of bacteria (DOPE, DOPG) or humans (DOPC) respectively. The data clearly indicates that the membrane composition, and specifically the lipid head group charge, play an important role in the peptide-membrane interactions. In the bilayers mimicking bacteria cell membranes, the peptide either absorbed onto or inserted into the bilayer. In contrast, for the bilayer modelling a human cell membrane, no significant variations were detected, indicating no interaction with LL37. The findings presented in this work generally coincide with similar research of LL37 using other techniques. At hand of the herein presented data, TIR Raman has proven suitable and effective in detecting effects of antimicrobial peptides in contact with model lipid bilayers, and hence can be recommended for further studies. / Som en direkt följd av missbruket av antibiotika sedan det först upptäcktes, har bakterier utvecklat omfattande resistensmekanismer vilket föranlett dem att återigen utgöra potentiellt dödlig hot. Denna situation har manat fram en väsentlig forskningsinsats från forskare världen över att hitta lösningar för att minska antimikrobiell resistens. Ett sådant alternativ har varit identifieringen av nya potentiella antimikrobiella substanser, så som till exempel antimikrobiella peptider. Ett flertal metoder har använts för att både evaluera peptiders egenskaper och fastställa deras komplexa mekanism. Detta till trots förblir de exakta detaljerna i mekanismen okända, vilket föranlett arbetet i detta projekt att undersöka lämpligheten i att använda TIR Raman, en vibrational-spektroskopisk metod som är tillräckligt ytkänslig för att studera interaktionen hos antimikrobiella peptider i kontakt med lipidmembran, vilka endast är några få nanometer tjocka. För att evaluera informationen som kan utvinnas med TIR Raman, utfördes först mätningar av lipidmembran, skapade på ett solitt underlag, utan tillägg av peptider. Mer noggrant, har lipiden DMPC med en fasövergång nära vid rumstemperatur, mätts vid olika temperaturer för att fastställa spektrala variationer associerade till övergången. För peptid-membran interaktionerna, sattes den antimikrobiella peptiden LL37 i kontakt med lipidmembran som modellerar cellmembranet hos bakterier (DOPE, DOPG) respektive människor (DOPC). Mätdatan indikerar tydligt att membrankompositionen, och där specifikt laddningen av lipidens huvudgrupp, spelar en viktig roll i membran-peptid interaktionerna. För lipidmembranen som imiterar bakteriella cellmembran, adsorberade peptiden till membranet eller integrerades in i det. Till skillnad från detta, kunde inga signifikanta variationer detekteras för lipidmembranet som modellerade ett mänskligt membran vilket indikerar att det inte interagerar med peptiden LL37. Upptäckterna som presenteras i detta arbete sammanfaller generellt med andra, liknande studier där andra instrument använts för att undersöka LL37. Det kan ur materialet som presenterats här utläsas att TIR Raman visat sig lämpligt och effektivt i detekteringen av antimikrobiella peptider i kontakt med modeller av lipidmembran, och kan därav rekommenderas för fortsatta studier.