Glutathione-Conjugated Hydrogels: Flexible Vehicles for Personalized Treatment of Bacterial Infections

Abstract

Purpose: Skin and soft tissue infections are increasingly prevalent and often complicated by potentially fatal therapeutic hurdles, such as poor drug perfusion and antibiotic resistance. Delivery vehicles capable of versatile loading may improve local bioavailability and minimize systemic toxicities yet such vehicles are not clinically available. Therefore, we aimed to expand upon the use of glutathione-conjugated poly(ethylene glycol) GSH-PEG hydrogels beyond protein delivery and evaluate the ability to deliver traditional therapeutic molecules.

Methods: PEG and GSH-PEG hydrogels were prepared using ultraviolet light (UV)-polymerization. Hydrogel loading and release of selected drug candidates was examined using UV-visible spectrometry. Therapeutic molecules and GST-fusion protein loading was examined using UV-visible and fluorescent spectrometry. Efficacy of released meropenem was assessed against meropenem-sensitive and -resistant P. aeruginosa in an agar diffusion bioassay.

Results: For all tested agents, GSH-PEG hydrogels demonstrated time-dependent loading whereas PEG hydrogels did not. GSH-PEG hydrogels released meropenem over 24 h. Co-loading of biologic and traditional therapeutics into a single vehicle was successfully demonstrated. Meropenem-loaded GSH-PEG hydrogels inhibited the growth of meropenem-sensitive and resistant P. aeruginosa isolates.

Conclusion: GSH ligands within GSH-PEG hydrogels allow loading and effective delivery of charged therapeutic agents, in addition to biologic therapeutics.

Keywords: antibiotic delivery; biomaterials; glutathione; hydrogel; infection; protein delivery.

Figure 1.. Swelling behavior of PEG and GSH-PEG hydrogels in water.

Equilibrium swelling ratios (A) and mass change (%) over time from the relaxed state (B) for PEG (●) and GSH-PEG (■) hydrogels in deionized water. Points and bars represent the mean plus or minus (±) the standard deviation of three independent experiments, where ** indicates statistical difference, p-value < 0.01, between the groups as determined by unpaired Student’s t-test.

Figure 2.. Vancomycin, meropenem, and pravastatin loading into PEG and GSH-PEG hydrogels over time.

PEG (●) and GSH-PEG (■) hydrogels were incubated within vancomycin (A), meropenem (B), and pravastatin (C) solutions [1 mM all solutions] for the time indicated and measured. Charcoal (♦) at an equal mass to GSH-PEG served as a positive loading control demonstrating full loading of the active therapeutic. Loading curves were obtained using a one-phase association exponential fit. Points represent the mean plus or minus (±) the standard deviation of three independent experiments.

Figure 3.. Meropenem loading and release from hydrogels.

Hydrogels were incubated for 1 hour in 160 μg/mL (A,B), 1,600 μg/mL (C,D), and 16,000 μg/mL (E,F) meropenem solutions. Meropenem loading (A,C,E) and cumulative release over time (B,D,F) are displayed for PEG (●) and GSH-PEG (■) hydrogels. Points and bars represent the mean plus or minus (±) the standard deviation of three independent experiments, where ** and *** indicates statistical difference, p-value < 0.01 and p-value < 0.001 respectively, between the groups as determined by unpaired Student’s t-test.

Figure 4.. Quantity of meropenem permeated per hydrogel unit area over time.

Flux of meropenem across PEG (●) and GSH-PEG (■) hydrogels. Points represent the mean plus or minus (±) the standard deviation of three independent experiments.

Figure 5.. Hydrogel swelling in response to pH and ionic content.

Lyophilized PEG (●) and GSH-PEG (■) hydrogels were equilibrated in PBS solutions of various pH (A, constant I=0.15) or ionic strength (B, constant pH 7.4). Points represent the mean plus or minus (±) the standard deviation of three independent experiments.

Figure 6.. Ionic strength influence on meropenem loading into hydrogels.

PEG (●) and GSH-PEG (■) hydrogels unequilibrated (A), equilibrated with PBS solutions of varying ionic strength (B), or equilibrated with deionized water (C). Incubation occurred over the course of 1 hour within meropenem [160 μg/mL] solutions of corresponding ionic strength. Points represent the mean plus or minus (±) the standard deviation of three independent experiments.

Figure 7.. Influence of meropenem on GST-GFP protein loading of GSH-PEG.

Hydrogels were loaded in the presence (+) or absence (−) of GST-GFP protein and/or meropenem [160 μg/mL]. Loading occurred independently (−/+), concurrently (+/+) or in a step-wise fashion (^1,2). Bars represent the mean plus or minus (±) the standard deviation of three independent experiments, where ** and *** indicates statistical difference, p-value < 0.01 and p-value < 0.001 respectively, between the specified groups as determined by the Dunnett post-hoc test.

Figure 8.. Pseudomonas aeruginosa growth inhibition with different GSH-PEG meropenem loading approaches.

Meropenem loaded within GSH-PEG hydrogels assessed by bacterial inhibition, as measured by zone of inhibition of susceptible (A; MIC 0.5 μg/mL) and resistant (B; MIC 8 μg/mL) P. aeruginosa. Hydrogels were loaded in the presence (+) or absence (−) of GST-GFP protein and/or meropenem [160 μg/mL], either concurrently or independently in a step-wise fashion (^1,2). Points and bars represent the mean plus or minus (±) the standard deviation of three independent experiments, where **** indicates statistical difference, p-value < 0.0001 between the specified groups as determined by the Dunnett post-hoc test.

Scheme 1.

General schema of charged species transition within a thiol conjugated GSH tripeptide as a function of pH.

Scheme 2.. Proposed parallel (A) and anti-parallel (B) orientations of conjugated GSH tripeptides with accompanying interactions at pH of approximately 7.4.

This figure applies for pH values between approximately 3.6 and 9.6 which correspond to two glutathione pKas as described in Scheme 1. Orange arrows depict potential electrostatic repulsions and light blue ellipses indicate potential hydrogen bonding.