Proteolytically activated anti-bacterial hydrogel microspheres

Abstract

Hydrogels are finding increased clinical utility as advances continue to exploit their favorable material properties. Hydrogels can be adapted for many applications, including surface coatings and drug delivery. Anti-infectious surfaces and delivery systems that actively destroy invading organisms are alternative ways to exploit the favorable material properties offered by hydrogels. Sterilization techniques are commonly employed to ensure the materials are non-infectious upon placement, but sterilization is not absolute and infections are still expected. Natural, anti-bacterial proteins have been discovered which have the potential to act as anti-infectious agents; however, the proteins are toxic and need localized release to have therapeutic efficacy without toxicity. In these studies, we explore the use of the glutathione s-transferase (GST) to anchor the bactericidal peptide, melittin, to the surface of poly(ethylene glycol) diacrylate (PEGDA) hydrogel microspheres. We show that therapeutic levels of protein can be anchored to the surface of the microspheres using the GST anchor. We compared the therapeutic efficacy of recombinant melittin released from PEGDA microspheres to melittin. We found that, when released by an activating enzyme, thrombin, recombinant melittin efficiently inhibits growth of the pathogenic bacterium Streptococcus pyogenes as effectively as melittin created by solid phase peptide synthesis. We conclude that a GST protein anchor can be used to immobilize functional protein to PEGDA microspheres and the protein will remain immobilized under physiological conditions until the protein is enzymatically released.

Keywords: Glutathione; Glutathione s-transferase; Hydrogel; Microparticles; Recombinant protein; Thrombin.

Fig. 1. Schematic of PEGDA-GSH microspheres and predicted structure of GST-GFP and GST-melittin

(A) Schematic showing half of a GST-GFP (green/top) and half of a GST-melittin (orange/bottom) microsphere with a magnified region of the microspheres suggesting the surface and internal structure. In the magnified region, black entanglements indicate the crosslinked (grey circles) PEGDA mesh with pendant glutathione (GSH; gold spheres). GST (blue)-GFP (green) and GST-melittin (pink) fusion protein are shown binding to GSH. Thrombin (brown) is shown cleaving melittin or GFP from GST fusion partners by acting on a thrombin cleavage site in linker fragment. (B) Schematic of the chemical structure of the PEGDA-GSH hydrogel. (C) Predicted three-dimensional structures of GST (blue)-GFP (green) fusion protein and GST (blue)-melittin (red) fusion protein monomers [18] used to estimate the size of the fusion proteins. Distances are approximations of the longest three dimensions of the fusion proteins.

Fig. 2. Binding of GST-GFP to PEGDA-GSH microspheres

Brightfield (upper left) and pseudo-color composite micrograph (lower right) of GST-GFP (green/upper right) bound to GST-PEGDA microspheres counterstained with trypan blue (red/lower left). The scale bar is 10 µm.

Fig. 3. Size Distribution of PEGDA-GSH and PEGDA-cys microspheres

Representative histogram showing the diameters of PEGDA-GSH (blue) and PEGDA-cys (red) microspheres from a single production batch. Three batches were used to calculate average microsphere diameter and are presented in Table 2.

Fig. 4. Release of GST-GFP in the presence of biologic fluids

(A) Release of GST-GFP in the presence of 10 mM (), 1 mM (), 0.1 mM (), 0.01 mM (), and 0 mM () GSH containing phosphate buffered saline. Each point represents the mean plus or minus the standard deviation of three independent samples. (B) Human plasma mediated release of GFP from GST-GFP loaded PEGDA-GSH microspheres. Microspheres were incubated with human plasma for 6 days at 37°C prior to measuring GFP in the supernatant by fluorescence. Each point represents the mean plus or minus the standard error of the mean of three independent samples in plasma from 3 human donors. (C) Gross photographs of microspheres under UV illumination after 6 days of incubation in human plasma or PBS at 37°C. The scale bar is 1 mm.

Fig. 5. Thrombin cleavage of GST-GFP from with PEGDA microspheres

(A) Release of GFP from GST-GFP PEGD-GSH microspheres in the presence of () 0, 11 (), 22 (), 44 (), or 110 () nM thrombin as detected by fluorescent GFP signal. Each point represents the mean plus or minus the standard deviation of three independent samples. (B) Representative brightfield and epifluorescent micrographs depicting GST-GFP (green) associated with microspheres before (top row) or after (bottom row) release in response to thrombin (1.8 µM). (C) Rate (v) of GFP liberation from GST-GFP (unbound; ) and GST-GFP (bound; ) bound to PEGDA-GSH microspheres for the linear portion of the rate curve, typically the first 1 hour. Each point represents the mean plus or minus the standard deviation of three independent samples.

Fig. 6. GST-melittin microspheres inhibit growth of S. pyogenes

Growth inhibition of S. pyogenes cells exposed for 10 hours to chemically defined growth medium (CDM), CDM with synthetic melittin (syn. Melittin), or CDM mixed with the releasate from PEGDA-GSH microspheres with thrombin, PEGDA-GSH microspheres bound with GST-melittin, or PEGDA-GSH microspheres bound with GST-melittin and exposed to thrombin (1.8 µM) for 2 hours. The data is presented as the absorbance at 600 nm following 10 hours of growth divided by the absorbance at 600 nm following 1 hour of growth. Each bar represents the mean plus or minus the standard error of the mean of three to four technical replicates.