Combining Inducible Lectin Expression and Magnetic Glyconanoparticles for the Selective Isolation of Bacteria from Mixed Populations.

The selective isolation of bacteria from mixed populations has been investigated in varied applications ranging from differential pathogen identification in medical diagnostics and food safety to the monitoring of microbial stress dynamics in industrial bioreactors. Selective isolation techniques are generally limited to the confinement of small populations in defined locations, may be unable to target specific bacteria, or rely on immunomagnetic separation, which is not universally applicable. In this proof-of-concept work, we describe a novel strategy combining inducible bacterial lectin expression with magnetic glyconanoparticles (MGNPs) as a platform technology to enable selective bacterial isolation from cocultures. An inducible mutant of the type 1 fimbriae, displaying the mannose-specific lectin FimH, was constructed in Escherichia coli allowing for "on-demand" glycan-binding protein presentation following external chemical stimulation. Binding to glycopolymers was only observed upon fimbrial induction and was specific for mannosylated materials. A library of MGNPs was produced via the grafting of well-defined catechol-terminal glycopolymers prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization to magnetic nanoparticles. Thermal analysis revealed high functionalization (≥85% polymer by weight). Delivery of MGNPs to cocultures of fluorescently labeled bacteria followed by magnetic extraction resulted in efficient depletion of type 1 fimbriated target cells from wild-type or afimbriate E. coli. Extraction efficiency was found to be dependent on the molecular weight of the glycopolymers utilized to engineer the nanoparticles, with MGNPs decorated with shorter Dopa-(ManAA)50 mannosylated glycopolymers found to perform better than those assembled from a longer Dopa-(ManAA)200 analogue. The extraction efficiency of fimbriated E. coli was also improved when the counterpart strain did not harbor the genetic apparatus for the expression of the type 1 fimbriae. Overall, this work suggests that the modulation of the genetic apparatus encoding bacterial surface-associated lectins coupled with capture through MGNPs could be a versatile tool for the extraction of bacteria from mixed populations.

The In Vitro, Ex Vivo, and In Vivo Effect of Polymer Hydrophobicity on Charge-Reversible Vectors for Self-Amplifying RNA.

RNA technology has the potential to revolutionize vaccination. However, the lack of clear structure-property relationships in relevant biological models mean there is no clear consensus on the chemical motifs necessary to improve RNA delivery. In this work, we describe the synthesis of a series of copolymers based on the self-hydrolyzing charge-reversible polycation poly(dimethylaminoethyl acrylate) (pDMAEA), varying the lipophilicity of the additional co-monomers. All copolymers formed stable polyplexes, showing efficient complexation with model nucleic acids from nitrogen/phosphate (N/P) ratios of N/P = 5, with more hydrophobic complexes exhibiting slower charge reversal and disassembly compared to hydrophilic analogues. The more hydrophobic copolymers outperformed hydrophilic versions, homopolymer controls and the reference standard polymer (polyethylenimine), in transfection assays on 2D cell monolayers, albeit with significantly higher toxicities. Similarly, hydrophobic derivatives displayed up to a 4-fold higher efficacy in terms of the numbers of cells expressing green fluorescent protein (GFP+) cells in ex vivo human skin (10%) compared to free RNA (2%), attributed to transfection enrichment in epithelial cells. In contrast, in a mouse model, we observed the reverse trend in terms of RNA transfection, with no observable protein production in more hydrophobic analogues, whereas hydrophilic copolymers induced the highest transfection in vivo. Overall, our results suggest an important relationship between the vector lipophilicity and RNA transfection in vaccine settings, with polymer biocompatibility potentially a key parameter in effective in vivo protein production.