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Title

SUSTAINED RELEASE OF PHOSPHATE-BASED THERAPEUTICS FOR ATTENUATION OF PATHOGEN-INDUCED PROTEOLYTIC MATRIX DEGRADATION

Creator

Bittencourt Pimentel, Marja

Date

2019

Description

Loss of the normal intestinal microbiome community structure and its replacement by pathogenic microbes contributes to severe persistent...
Show moreLoss of the normal intestinal microbiome community structure and its replacement by pathogenic microbes contributes to severe persistent inflammation in diseases such as ulcerative colitis and inflammatory bowel disease. While host-derived proteases are known to contribute to this pathogenesis, the role of increased production of microbial-secreted proteases due to virulent phenotypes remains unclear. Following surgical removal of diseased intestinal tract, increased bacterial protease expression is a key phenotype involved in intestinal healing impairment. Antibiotic administration is ineffective for treating these complications as it inadvertently eliminates normal flora while allowing pathogenic bacteria to acquire antibiotic resistance. Prior research has shown that intestinal phosphate depletion in the surgically stressed host triggers bacterial virulence which is suppressed under phosphate abundant conditions. To address this issue our previous work has demonstrated that the use of free monophosphate (-Pi) and polyphosphate (-PPi), as well as post-loaded PPi nanoparticles (NP-PPi) attenuate collagenase production of gram-negative (Pseudomonas aeruginosa and Serratia marcescens) but not gram-positive (Enterococcus faecalis) pathogens expressing high collagenolytic activity. Due to the variation in phosphate metabolism among microbial species we investigated the in vitro efficacy of a combination treatment of phosphates delivered in a sustained release format using NP-PPi and NP-Pi on collagenase and biofilm attenuation across gram-positive and gram-negative test pathogens.Collagenase screening was assessed using two in vitro models. The first in vitro assay involved culturing pathogens in the presence and absence of NP-Pi and/or NP-PPi treatment using two-dimensional (2D) commercially available fluorogenic protease-sensitive peptide substrates. Although these substrates are among the most commonly used for screening protease activity and inhibition in vitro, their application does not translate to three-dimensional (3D) matrix degradation. Additionally, the addition of drug-loaded nanoparticles directly in bacterial culture does not recapitulate the in vivo sustained release of phosphates due to nanoparticles embedded within tissue. Thus, the second model involved the development of a novel cell culture platform which utilized a proteolytically degradable hydrogel scaffold and a non-degradable nanocomposite hydrogel scaffold. In this assay NP-Pi and NP-PPi were entrapped in a non-degradable poly(ethylene) glycol (PEG) hydrogel to form of a nanocomposite matrix which served as a reservoir for sustained release of phosphates. Bacteria producing high levels of proteases were cultured in the presence of the nanocomposite phosphate releasing reservoir and the proteolytically degradable PEG hydrogel scaffold to determine the efficacy of sustained release of phosphates in attenuating proteolytic hydrogel degradation. To correlate matrix degradation with bacterial enzymes secreted in the culture medium, we also developed a method to efficiently measure hydrogel degradation rate until complete material degradation with a greater degree of accuracy compared to the commonly employed method utilizing gravimetric measurements in gel wet weight. Combined, the in vitro platform and our proposed degradation assay provide a novel approach for screening the effect of therapeutics for attenuation of bacterial protease-induced matrix degradation.The 2D in vitro study demonstrated that the combination treatment (NP-PPi + NP-Pi) confers broad spectrum efficacy for suppression of collagenase and biofilm production across test pathogens. Conversely, the 3D in vitro model demonstrated that the combination treatment (NP-PPi + NP-Pi) attenuated protease production for gram-negative pathogens, while the gram-positive test pathogen exhibited significant decreases in protease levels only in the presence of NP-Pi. Finally, our novel Sirius red absorbance assay for quantifying hydrogel degradation was found to provide greater accuracy when compared to gravimetric measurements in gel wet weight. It also enabled real-time monitoring of 3D matrix degradation kinetics as well as the time required for complete material dissolution in the presence of bacterial proteases and active human MMP-9 enzyme solutions. These findings highlight the importance of designing relevant in vitro platforms for screening therapeutic efficacy in the presence of cells and nanomaterials.
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