Characterizing the Molecular Architecture of Hydrogels and Crosslinked Polymer Networks beyond Flory-Rehner. II: Experiments.

We previously [Borges, F. T. P. Biomacromolecules 2020, 21(12), 5104-5118] introduced a novel methodology for the characterization of the dimensions and architecture of hydrogel networks that provides more detailed information than the classical Flory-Rehner theory [Canal, T.; Peppas, N. A. J. Biomed. Mater. Res. 1989, 23, 1183-1193]. In this article, we illustrate our methodology by applying it to the phototerpolymerization of N-vinyl-2-pyrrolidone (NVP), ethylene glycol methyl ether acrylate (EGA), and poly(ethylene glycol) diacrylate (PEGDA). The experimental design includes 120 formulations using different fractions of the three monomers. Experimental measurements determined the mass swelling ratio and were coupled with theory to compute the internal dimensions of the network. Results demonstrate how the use of a macromeric crosslinker leads to unique network architectures not predicted by classical F-R theory, e.g., the figure shows that the mass between crosslinks predicted by F-R is actually distributed between branches and the backbone. The methodology presented offers a path toward optimizing/customizing hydrogel properties to suit the size and shape of the specific therapeutic targeted for drug delivery.

Characterizing the Molecular Architecture of Hydrogels and Crosslinked Polymer Networks beyond Flory-Rehner-I. Theory.

In the early 1940s, Paul Flory and John Rehner published a series of papers on the properties of swellable polymeric networks. Originally intended for vulcanized rubber, their development has since been extensively used and extended to much more complex systems, such as hydrogels, and used to estimate the mesh size of such networks. In this article, we take a look at the development of the Flory-Rehner equation and highlight several issues that arise when using such a theory for the described hydrogel networks. We then propose a new approach and equations to accurately calculate the backbone molecular weight in-between crosslinks while explicitly accounting for the molecular mass of the crosslinker and branch segments. The approach also provides more applicable mesh dimensions, for complex networks with macromeric crosslinkers and/or a high degree of branching, as is the case of biocompatible hydrogels. The approach is finally illustrated by a case study comparing the values obtained with our proposed approach to those using the state-of-the-art approach.

An in vitro tissue model for screening sustained release of phosphate-based therapeutic attenuation of pathogen-induced proteolytic matrix degradation.

Tissue response to intestinal injury or disease releases pro-inflammatory host stress signals triggering microbial shift to pathogenic phenotypes. One such phenotype is increased protease production resulting in collagen degradation and activation of host matrix metalloproteinases contributing to tissue breakdown. We have shown that surgical injury depletes local intestinal phosphate concentration triggering bacterial virulence and that polyphosphate replenishment attenuates virulence and collagenolytic activity. Mechanistic studies of bacterial and host protease expression contributing to tissue breakdown are difficult to achieve in vivo necessitating the development of novel in vitro tissue models. Common techniques for screening in vitro protease activity, including gelatin zymography or fluorogenic protease-sensitive substrate kits, do not readily translate to 3D matrix degradation. Here, we report the application of an in vitro assay in which collagenolytic pathogens are cultured in the presence of a proteolytically degradable poly(ethylene) glycol scaffold and a non-degradable phosphate and/or polyphosphate nanocomposite hydrogel matrix. This in vitro platform enables quantification of pathogen-induced matrix degradation and screening of sustained release of phosphate-based therapeutic efficacy in attenuating protease expression. To evaluate matrix degradation as a function of bacterial enzyme levels secreted, we also present a novel method to quantify hydrogel degradation. This method involves staining protease-sensitive hydrogels with Sirius red dye to correlate absorbance of the degraded gel solution with hydrogel weight. This assay enables continuous monitoring and greater accuracy of hydrogel degradation kinetics compared to gravimetric measurements. Combined, the proposed in vitro platform and the presented degradation assay provide a novel strategy for screening efficacy of therapeutics in attenuating bacterial protease-induced matrix degradation.

Sustained Release of Phosphates From Hydrogel Nanoparticles Suppresses Bacterial Collagenase and Biofilm Formation in vitro.

Intestinal disease or surgical intervention results in local changes in tissue and host-derived factors triggering bacterial virulence. A key phenotype involved in impaired tissue healing is increased bacterial collagenase expression which degrades intestinal collagen. Antibiotic administration is ineffective in addressing this issue as it inadvertently eliminates normal flora while allowing pathogenic bacteria to "bloom" and acquire antibiotic resistance. Compounds that could attenuate collagenase production while allowing commensal bacteria to proliferate normally would offer major advantages without the risk of the emergence of resistance. We have previously shown that intestinal phosphate depletion in the surgically stressed host is a major cue that triggers P. aeruginosa virulence which is suppressed under phosphate abundant conditions. Recent findings indicate that orally administered polyphosphate, hexametaphosphate, (PPi) suppresses collagenase, and biofilm production of P. aeruginosa and S. marcescens in animal models of intestinal injury but does not attenuate E. faecalis induced collagenolytic activity (Hyoju et al., 2017). Systemic administration of phosphates, however, is susceptible to rapid clearance. Given the diversity of collagenase producing bacteria and the variation of phosphate metabolism among microbial species, a combination therapy involving different phosphate compounds may be required to attenuate pathogenic phenotypes. To address these barriers, we present a drug delivery approach for sustained release of phosphates from poly(ethylene) glycol (PEG) hydrogel nanoparticles. The efficacy of monophosphate (Pi)- and PPi-loaded NPs (NP-Pi and NP-PPi, respectively) and a combination treatment (NP-Pi + NP-PPi) in mitigating collagenase and biofilm production of gram-positive and gram-negative pathogens expressing high collagenolytic activity was investigated. NP-PPi was found to significantly decrease collagenase and biofilm production of S. marcescens and P. aeruginosa. Treatment with either NP-Pi or NP-Pi + NP-PPi resulted in more prominent decreases in E. faecalis collagenase compared to NP-PPi alone. The combination treatment was also found to significantly reduce P. aeruginosa collagenase production. Finally, significant attenuation in biofilm dispersal was observed with NP-PPi or NP-Pi + NP-PPi treatment across all test pathogens. These findings suggest that sustained release of different forms of phosphate confers protection against gram-positive and gram-negative pathogens, thereby providing a promising treatment to attenuate expression of tissue-disruptive bacterial phenotypes without eradicating protective flora over the course of intestinal healing.

Synthesis of Polyphosphate-Loaded Nanoparticles Using Inverse Miniemulsion Polymerization for Sustained Delivery to the Gastrointestinal Tract.

Polyphosphate salts, such as sodium hexametaphosphate (PPi), are effective in the attenuation of collagenase and biofilm production and prevention of anastomotic leak in mice models. However, systemic administration of polyphosphate solutions to the gut presents a series of difficulties such as uncontrolled delivery to target and off-site tissues. In this article a process to produce PPi-loaded poly(ethylene glycol) diacrylate (PEGDA) hydrogel nanoparticles through miniemulsion polymerization is developed. The effects of using a polyphosphate salt, as compared to a monophosphate salt, is investigated through cloud point measurements, which is then translated to a change in the required HLB of the miniemulsion system. A parametric study is developed and yields a way to control particle swelling ratio and mean diameter based on the surfactant and/or initiator concentration, among other parameters. Finally, release kinetics of two different crosslink density particles shows a sustained and tunable release of the encapsulated polyphosphate.