Chromatography and Anti-Biofilm Activity of Chiral Carbon Nanoparticles

Abstract

Biofilms have evolved to be resistant to natural and man-made antibiotics. The biochemical composition and barrier function of the extracellular polymeric substance prevent penetration of traditional antimicrobials reduce proliferation of bacterial cells embedded in it. Design of the agents capable of dispersing the biofilm matrix is obscured by its heterogeneity and variability. Here we seek to explore chiral nanoparticles as means to enantioselectively disperse amyloid rich biofilms. The first scope of this work is synthesize and characterize chiral nanoparticles, namely chiral carbon nanoparticles as a new class of anti-biofilm agents. Chiral carbon nanoparticles (CNPs) represent a rapidly evolving area of research for optical and biomedical applications. Similar to small molecules, further development of chiral chemistry of CNPs and fundamental understanding of the structural origin of their optical activity would greatly benefit from their enantioselective separations using established methods of chromatography with chiral stationary phases. However, this technique remains in its infancy for chiral CNPs and the possibility of such separations using high performance liquid chromatography (HPLC) remains an open question. In Chapter 2, we report a detailed methodology of HPLC for successful separation of chiral CNPs and establish a path for its future optimization. A mobile phase of water/acetonitrile was able to achieve chiral separation of CNPs derived from L- and D-cysteine denoted as L-CNPs and D-CNPs. Molecular dynamics simulations show that the teicoplanin-based stationary phase has a higher affinity for L-CNPs than for D-CNPs, in agreement with experiments. The experimental and computational findings jointly indicate that chiral centers of chiral CNPs are present at their surface, which is essential for the multiple applications of these chiral nanostructures and equally essential for interactions with biomolecules and circularly polarized photons. In Chapter 3, we show that cysteine-derived chiral carbon nanoparticles (C3NPs) are capable to disperse established amyloid-rich Staphylococcus (S. aureus) biofilms and reveal three modes of function-relevant biological interaction. We found that C3NPs from ¬D-cysteine (D-C3NPs) are much more efficient in biofilm dispersal than those from ¬L-cysteine (L-C3NPs). Unlike small molecules, the cooperativity of the intermolecular interactions leads to strong binding between C3NPs and phenol soluble modulins (PSM), the monomeric form of amyloid fibers within the biofilm. First, the formation of PSMα1 nanofibers known as the essential structural component of the extracellular polymeric substance was prevented by both D- and L-C3NPs due the strong change in their assembly patterns leading to formation of rigid nanotubes disrupting the co-assembly with other biofilm components. Second, D- but not L-C3NPs distort conformation and thus prevent fibrillation of PSMα3 peptide known for nucleating assembly of other nanofibers. Molecular dynamics simulations confirm chirality-dependent interactions of C3NPs with amino acid residues essential for PSM self-assembly. Third, agglutination of both types of PSMs promoted by NPs prevented their dispersal as virulence factors as confirmed by cytotoxicity data that including hemolysis rate and cytokine production. Subsequent engineering of molecular and nanoscale chirality of C3NPs will enable their further adaptation to the biomedical needs and clinical requirements.