Molecularly imprinted polymers (MIPs) with selective affinity for protein biomarkers could find extensive utility as environmentally robust, cost-efficient biomaterials for diagnostic and therapeutic applications. In order to develop recognitive, synthetic biomaterials for prohibitively expensive protein biomarkers, we have developed a molecular imprinting technique that utilizes structurally similar, analogue proteins. Hydrogel microparticles synthesized by molecular imprinting with trypsin, lysozyme, and cytochrome c possessed an increased affinity for alternate high isoelectric point biomarkers both in isolation and plasma-mimicking adsorption conditions. Imprinted and non-imprinted P(MAA-co-AAm-co-DEAEMA) microgels containing PMAO-PEGMA functionalized polycaprolactone nanoparticles were net-anionic, polydisperse, and irregularly shaped. MIPs and control non-imprinted polymers (NIPs) exhibited regions of Freundlich and BET isotherm adsorption behavior in a range of non-competitive protein solutions, where MIPs exhibited enhanced adsorption capacity in the Freundlich isotherm regions. In a competitive condition, imprinting with analogue templates (trypsin, lysozyme) increased the adsorption capacity of microgels for cytochrome c by 162% and 219%, respectively, as compared to a 122% increase provided by traditional bulk imprinting with cytochrome c. Our results suggest that molecular imprinting with analogue protein templates is a viable synthetic strategy for enhancing hydrogel-biomarker affinity and promoting specific protein adsorption behavior in biological fluids.
Tuning the composition
of antimicrobial nanogels can significantly
alter both nanogel cytotoxicity and antibacterial activity. This project
investigated the extent to which PEGylation of cationic, hydrophobic
nanogels altered their cytotoxicity and bactericidal activity. These
biodegradable, cationic nanogels were synthesized by activators regenerated
by electron transfer atom transfer radical polymerization (ARGET ATRP)
emulsion polymerization with up to 13.9 wt % PEG (MW = 2000) MA, as
verified by 1H NMR. Nanogel bactericidal activity was assessed
against Gram-negative E. coli and P. aeruginosa and Gram-positive S.
mutans and S. aureus by measuring membrane lysis with a LIVE/DEAD assay. E. coli and S. mutans viability was further validated by measuring metabolic activity
with a PrestoBlue assay and imaging bacteria stained with a LIVE/DEAD
probe. All tested nanogels decreased the membrane integrity (0.5 mg/mL
dose) for Gram-negative E. coli and P. aeruginosa, irrespective of the extent of PEGylation.
PEGylation (13.9 wt %) increased the cytocompatibility of cationic
nanogels toward RAW 264.7 murine macrophages and L929 murine fibroblasts
by over 100-fold, relative to control nanogels. PEGylation (42.8 wt
%) reduced nanogel uptake by 43% for macrophages and 63% for fibroblasts.
Therefore, PEGylation reduced nanogel toxicity to mammalian cells
without significantly compromising their bactericidal activity. These
results facilitate future nanogel design for perturbing the growth
of Gram-negative bacteria.
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