Journal of Controlled Release 171 (2013) 288–295

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Journal of Controlled Release

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Proteolytically activated anti-bacterial hydrogel microspheres

Jason S. Buhrman a, Laura C. Cook b, Jamie E. Rayahin a, Michael J. Federle b, Richard A. Gemeinhart a,c,d,⁎a Department of Biopharmaceutical Sciences, University of Illinois, Chicago, IL 60612-7231, USAb Center for Pharmaceutical Biotechnology, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago, IL 60607-7173, USAc Department of Bioengineering, University of Illinois, Chicago 60607-7052, USAd Department of Ophthalmology and Visual Sciences, University of Illinois, Chicago, IL 60612-4319, USA

⁎ Corresponding author at: 833 South Wood Street (MCUSA.

E-mail address: rag@uic.edu (R.A. Gemeinhart).

0168-3659/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.jconrel.2013.06.023

a b s t r a c t

a r t i c l e i n f o

Article history:Received 31 March 2013Accepted 18 June 2013Available online 28 June 2013

Keywords:Recombinant proteinGlutathione s-transferaseGlutathioneThrombinHydrogelMicroparticles

Hydrogels arefinding increased clinical utility as advances continue to exploit their favorablematerial properties.Hydrogels can be adapted for many applications, including surface coatings and drug delivery. Anti-infectioussurfaces and delivery systems that actively destroy invading organisms are alternative ways to exploit the favor-able material properties offered by hydrogels. Sterilization techniques are commonly employed to ensure thematerials 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. Inthese 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 ther-apeutic levels of protein can be anchored to the surface of themicrospheres using the GST anchor.We comparedthe 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 path-ogenic bacterium Streptococcus pyogenes as effectively as melittin created by solid phase peptide synthesis. Weconclude that a GST protein anchor can be used to immobilize functional protein to PEGDA microspheres andthe protein will remain immobilized under physiological conditions until the protein is enzymatically released.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Most modern advances in medical technology put sterility as a firstpoint in design principles. Conventionally, these systems are passivelysterilized to prevent infection before they are used. Being an incompleteprocess, materials implanted or inserted into the body are expected tohave an incidence of infection with even the best methods of steriliza-tion. Nosocomial infections frompassively sterilizedmaterials, e.g. cath-eters, artificial joints, pacemakers and drug pumps, cause more than1.5 million deaths each year [1–3]. Therefore, methods beyond passivesterilization are necessary. Active anti-infectious materials are thoughtto be superior to the currently used, passively sterilized, noninfectiousmaterials in maintaining sterility in non-sterile environments.

Recent reports of actively anti-infectious systems are emerging.These systems include the use of charged polymers that interactwith membranes of bacteria [4], nitric oxide coatings [5], antibioticcoatings [6], silver coatings [7], and antibody coatings [8] (reviewedin [9]). These systems have been shown to actively inhibit the growthof invasive bacteria and reduce the risk of infection. Local delivery of

865), Chicago, IL 60612-7231,

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anti-bacterial therapy has been achieved by polymeric microspheres[10–12]. Further improvements are necessary to limit local infections.

Hydrogels are becoming more prevalent in medical use as deliverysystems [13,14], implants [14], drug excipients [14,15], and coatings[16]. Hydrogels are highly adaptable and have the potential as a plat-form for modifying other materials. In this report, we describe amicrosphere-based, active antibacterial hydrogel delivery system. Thehydrogel microsphere system is, to our knowledge, the first activatedby the local immune response to bacterial infection into releasing ananti-bacterial compound. The system utilizes the tripeptide glutathione(GSH), a highly versatile molecule readily attachable to most surfaces.GSH was immobilized onto poly(ethylene glycol) diacrylate (PEGDA)hydrogelmicrospheres to allow for amodular systemof anchoring ther-apeutic glutathione s-transferase (GST) fusion proteins. The GSH–GSTinteraction has long found utility in recombinant protein technology[17] but not in drug delivery to date.

Here, we present data that GST fusion proteins may be bound toGSH-laden microspheres. To deliver specific proteins during infection,thrombin-cleavable sites were engineered in the junction betweenGST and the fused protein to serve as an activation site. Upon thrombincleavage, the target protein dissociates from the immobilized GST. Mi-crospheres with GST attached to green fluorescent protein (GFP) weremade as a control to demonstrate proper formation of the complexand proper release by thrombin (Fig. 1A).

Fig. 1. Schematic of PEGDA–GSH microspheres and predicted structure of GST–GFP andGST–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 microspheressuggesting the surface and internal structure. In the magnified region, black entangle-ments indicate the crosslinked (gray circles) PEGDA mesh with pendant glutathione(GSH; gold spheres). GST (blue)–GFP (green) and GST–melittin (pink) fusion proteinsare shown binding to GSH. Thrombin (brown) is shown cleaving melittin or GFP fromGST fusionpartners by acting on a thrombin cleavage site in linker fragment. (B) Schemat-ic of the chemical structure of the PEGDA–GSH hydrogel. (C) Predicted three-dimensionalstructures of GST (blue)–GFP (green) fusion protein and GST (blue)–melittin (red) fusionprotein monomers [18] used to estimate the size of the fusion proteins. Distances are ap-proximations of the longest three dimensions of the fusion proteins.

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As a model anti-bacterial therapeutic, melittin was attached to mi-crospheres to act as a lytic peptide against bacterial pathogens [19–21]following cleavage. Thrombin cleavagewas chosen due to the fact that

thrombin is activated by the host immune system at sites of inflamma-tion caused by bacterial pathogens, such as the Gram-positive bacteri-um Streptococcus pyogenes, among others [22]. Both the therapeuticmolecule and protease are models and can be readily altered in thisplatform. GSH-laden PEGDA hydrogel microspheres, thus, can interactwith GST-fused proteins and act as a delivery system for antibacterialproteins or peptides, such asmelittin. It is reasoned that proteolysis byhost-derived thrombin at sites of infection will facilitate release ofmelittin and subsequently, the killing of pathogenic bacteria.

2. Materials and methods

2.1. Expression and purification of recombinant proteins

Proteinswere expressed and purified bymethods previously reported[23]. In brief, GFP was cloned from the pWiz-GFP plasmid (Aldevron,Madison, WI) into the pGex-6p-1 plasmid (GE Healthcare, Waukesha,WI) by primer extension PCR. A hexa-histidine tag and thrombin cleavagesite (LVPRGS)were added to theN-terminal ofGFPprior to insertion, gen-erating pJB-HTS-GFP [24]. Melittin (GIGAVLKVLTTGLPALISWIKRKRQ)[19,20] was codon optimized using JCAT [25]: (AGC GGA TCC GGT ATCGGT GCT GTT CTG AAA GTT CTG ACC ACC GGT CTG CCG GCT CTG ATCTCT TGG ATC AAA CGT AAA CGT CAG TAG GAA TTC TCA CG) and clonedinto the same vector replacing GFP after removal by BamHI and EcoRI(NEB, Cambridge, MA) generating pJB-HTS-melittin [23].

Proteins were expressed in BL21 and Rosetta (Novagen/EMDMillipore, Billerica, MA) Escherichia coli cells for GST–GFP and GST–melittin, respectively. In both cases, bacterial cells were grown toan absorbance at 600 nm (A600) of 0.4 at 37 °C before addition of0.1 mM IPTG [24] to induce protein production. Cells were removedto 25 °C and incubated for 16 h to produce the fusion proteins. Bac-terial cells were centrifuged before re-suspension in lysis buffer(50 mM NaHPO4, 300 mM NaCl, buffered to pH 8.0). Cells werelysed by three freeze–thaw cycles on dry ice and 4 °C before three15-second sonication (Misonix model XL2015) rounds at 40% inten-sity with 15-second incubation on ice between sonication rounds.The lysate was centrifuged at 12,000 RPM and 4 °C for 30 min to pel-let the insoluble material. GST–GFP was predominantly in the solu-ble fraction and was purified by Ni-NTA chromatography (Qiagen,Germantown, MA) according to the manufacturer's recommenda-tion. GST–melittin was predominantly in the insoluble fraction andwas extracted by 1% Tween-20. The extract was then purified byNi-NTA chromatography yielding a column elution product deter-mined to be approximately 50% GST and 50% GST–melittin.

2.2. PEGDA microsphere formation, protein loading, and characterization

All chemicals are fromThermo Fisher Scientific (Waltham,MA) unlessotherwise noted. PEGDA microspheres were created through a modifiedprocess of reverse phase emulsion polymerization [24,26]. PEGDA(300 μL; MW = 575 g/mol, Sigma-Aldrich, St. Louis, MO) was dilutedin 300 μL PBS containing 30 mg reduced glutathione (GSH) or 12 mg(equimolar with GSH) reduced cysteine (Sigma-Aldrich, St. Louis, MO).The microspheres made in the presence of glutathione and cysteine arereferred to as PEGDA–GSH and PEGDA–cys, respectively. Mineral oil(2 mL) was added to a borosilicate culture tube (1.5 mm diameter,10 mm length). Monomer solution (100 μL) was added to the culturetube while vortexing for 10 s at full speed. While still vortexing, 100 μLof 20% ammonium persulfate (in PBS) was added followed by 50 μL N,N,N′,N′-tetramethylethylenediamine. The mixture was vortexed for anadditionalminute. Deionizedwater (2 mL)was addedprior to centrifuga-tion at 4 °C, 4000 RPM for 1 min to recover the microspheres. Micro-spheres were washed over the course of a week at 4 °C with greaterthan a 10-fold volume of PBS and with 5 to 10 changes of PBS per day.Microspheres were loaded over the course of 3 h with 50 to 100 μg ofprotein in PBS. Loading occurred on a rotator wheel, with gentle rotation

Table 1Calculated dimensions of GST and GST fusion proteins.

Protein x × y × z (nm × nm × nm)a

Monomer Dimer

GSTb 4 × 4.5 × 9 5 × 9 × 9GST–GFPc 4 × 6 × 10 8 × 10 × 10GST–melittinc 4 × 5 × 7 8 × 10 × 7

a x, y, and z lengths of the protein using arbitrary coordinates similar to those repre-sented in Fig. 1.

b Based upon published crystal structures for the dimer [38,39] and MacPyMol [42]measurement of a single monomer from the dimer crystal structure.

c Measured in MacPyMol [42] from Raptor X structure prediction and alignment ofthe GST–GST dimer [18].

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at 20 RPM. After loading, microspheres were washed one time with10-fold excess volume of PBS.

Microscopy was carried out on an Olympus IX70 inverted micro-scope with an epifluorescent illuminator. Images were captured by aQ-imaging Retiga 1300 CCD and analyzed onQ-capture suite. The diam-eter of at least 130 particles was measured from at least three differentparticle preparations for each formulation. Fluorescent images arepseudocolor micrographs using the fluorescent properties of GFP (exci-tation and emission: 488 and 519 nm) or trypan blue (excitation andemission: 595 and 660 nm) [27–29]. To determine particle density insolution PEGDA microspheres were counted using hemocytometerafter staining with trypan blue (1% in PBS).

Mesh size of macroscopic hydrogels formed using identical condi-tions without emulsification was measured as previously described[30–32]. Briefly, after polymerization in an 8 mm × 2 mm cylindricalmold, polymers were suspended in 1-butanol and the mass of butanoldisplaced by the suspended polymer was noted. Hydrogels were swol-len over 24 h in deionized water, and their masses recorded. Thehydrogels were returned to water and masses recorded at three-hourintervals until there was a difference between subsequent masses ofless than 5%, deemed equilibrium swelling. At this point, the swollenhydrogels were re-suspended in butanol and the mass of butanoldisplaced by the suspended polymer was determined. Hydrogels werethen freeze–dried for 24 h, and their dry weight recorded. Mesh sizewere estimated based upon the Flory–Rehner swelling theory [33].

2.3. Protein identity, quantitation, and kinetics

Total protein was determined by the Bradford Assay (Pierce) [34]according to the manufacturer recommendations and measured witha Beckman model DU640 spectrophotometer. Acrylamide gels wereprepared in house by conventional protocols [35]. All gels were be-tween 12% and 15% acrylamide and crosslinked at a ratio of 37.5 to 1acrylamide to bis-acrylamide. Gels were stained by Coomassie bril-liant blue. Images were captured on a light table with a Sony DSC-H9cybershot CCD.

Reaction kinetics were measured by semi-quantitative densitome-try of SDS–PAGE gels and fluorometry. Reactions with equivalent con-centrations of GST–GFP substrate and varying concentrations ofthrombin (0.1, 0.33, 0.75, 1.5, and 3 μM) were incubated at 37 °C forvarying amounts of time. In groups where cleavage from microspheresis reported, approximately 10,000 microspheres were used. Reactionsfor densitometry were stopped by addition of Laemmli sample buffer[36] (containing SDS) and stored at 4 °C until run on SDS–PAGE.Reactions for fluorometry were stopped by addition of 1 mMphenylmethylsulfonyl fluoride (PMSF) and immediately read on aTurner Quantech Fluorometer (Cole Parmer, Vernon Hills, IL). Theexcitation filter was a band pass 490 nm and the emission filterwas long pass 550 nm. All gels were run with known concentrationsof BSA standards. Density measurements were made with NIHimageJ software with gel backgrounds normalized. Each thrombinconcentration was run one time, except for 0.73 μM, which was runin biological triplicate.

2.4. Protein release

GSH mediated release studies were carried out over 6 days. Proteinrelease was measured from 10,000 to 20,000 microspheres into 500 μLPBS in the presence or absence of glutathione. Release was measuredwith three independent replicates per experimental group. Preliminarystudies (not shown) indicated that in this system, 50% of reduced gluta-thione becomes oxidized within 24 h. Therefore, at each 24-hour timepoint, glutathione containing buffers was completely replaced. Re-moved buffer was frozen for later analysis. At the completion of the ex-periment, all frozen samples were thawed and 50 μL of each samplewas diluted in PBS and read on a Quantech fluorometer.

Anti-coagulated (citrate) human plasma (Innovative Research, Novi,MI) from three different donors was incubated with microspheres at37 °C for six days. Plasma (100 μL) was incubated in a 96-well plate inthe presence of PEGDA–GSH microspheres anchoring GST–GFP. At theend of six days, photographs were taken of the wells, and fluorescencewas measured in the plasma supernatant. The University of Illinois atChicago Institutional Review Board approved the study protocol foruse of human plasma.

2.5. S. pyogenes growth inhibition

In a volume of 500 μL, approximately two million PEGDA–GSH mi-crospheres were loadedwith GST–melittin over a period of 3 h. Themi-crospheres were washed with 10 times volume of PBS one time, anddivided into two tubes. Thrombin (2U) was added to one tubecontaining one-million GST–melittin loadedmicrospheres, and two ad-ditional units of thrombin were loaded to a third tube containingone-million PEGDA–GSH microspheres that were not loaded withGST–melittin. Sampleswere incubated at 37 °C for 2 h before the super-natant was removed.

S. pyogenes cells were grown in Todd Hewitt broth (BectonDickenson) supplemented with 0.5% yeast extract. Following over-night growth, cells were diluted 1:100 into chemically defined medi-um [37] to a final concentration of ~107 colony forming units/mL.Supernatant (50 μL) of washed spheres was added to 400 μL ofcells. Each sample (50 μL) was added, in triplicate wells, to a96-well optical bottom plates (Greiner BioOne). This plate was incu-bated at 37 °C for 12 h while shaking. At ten-minute intervals, theabsorbance at a wavelength of 600 nm (A600) was recorded (BioTekSynergy 2 spectrophotometer; Winooski, VT).

2.6. Statistical analysis

ANOVA was used to test all groups, and post-hoc Tukey analysiswas utilized for pairwise comparison if ANOVA suggested significantdifferences between the groups. In all cases α less than or equal to0.05 was considered significant.

3. Results

GST is encoded as a monomer, but forms a dimer in the native statein solution [38,39]. The dimer has been crystallized in its dimeric form[40] as has the GFP protein [41]. Using this information and structurepredictions [18], the size of fusion proteins (Fig. 1B) in the monomericand dimeric forms (Table 1). To predict if proteins of these dimensionswould enter the hydrogels, we measured the mesh size (ξ) of thePEGDA–GST hydrogels to be 4.6 ± 0.2 nm. The addition of 5 to 1molar ratio of PEGDA to GSH did not significantly affect mesh sizefrom the PEGDA hydrogels without GSH, which had a mesh size of4.5 ± 0.1 nm. Based upon estimates of fusion protein size and themesh size, GSTmonomers or dimers would be unlikely to enter the hy-drogel mesh even without being fused to another protein. To further

Fig. 3. Size distribution of PEGDA–GSH and PEGDA–cysmicrospheres. Representative his-togram showing the diameters of PEGDA–GSH (blue) and PEGDA–cys (red)microspheresfrom a single production batch. Three batcheswere used to calculate averagemicrospherediameter and are presented in Table 2.

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support this, PEGDA–GSHmicrosphereswere loadedwithGST–GFP andnonspecifically stainedwith trypan blue to allow localization of the pro-tein. Microspheres visualized by epifluorescent microscopy exhibitedGFP (green) predominantly on their surface while trypan blue (red)was observed throughout the microspheres in a diffuse pattern(Fig. 2). We concluded that the interaction between GST–GFP andPEGDA–GSH microspheres was occurring predominantly at surface ofthe microspheres.

Assuming that the entire surface of a PEGDA microsphere wasavailable for interaction with GST–GFP, we calculated the numberof GST–GFP that could bind to the surface assuming perfect, mono-layer coverage. Hydrogel microspheres had an average diameter of46 ± 16 μm(Fig. 3, Table 2). Using individual microsphere diameter,the average surface area of an average sphere was calculated to be6647 ± 804 μm2 (Table 2) after synthesis, washing, and loadingwith GST–GFP. Measurements on control PEGDA–cysteine (cys) micro-spheres showed similar size and surface area distributions (Fig. 3).Assuming an ellipsoid projection of the dimeric GST–GFP or GST–melittin with binding in the pocket on the x–y plane (Fig. 1B, left), wereasoned that the binding area of a GST fusion protein molecule, eitherGST–GFP or GST–melittin, to be 1.4 × 10−5 μm2 (Fig. 1B, right). Giventhese values and the assumption that all GST–GFP loading is on the sur-face, we predicted that monolayer coverage of an average PEGDA–GSHmicrosphere would consist of 4.7 × 108 molecules of GST–GFP or GST–melittin.

With the estimate of the amount of protein loaded on the micro-spheres, the ability to release the protein in a specific manner wasassessed. PEGDA–GSH and PEGDA–cys microspheres were first load-ed for 3 h with GST–GFP. PEGDA–cys microspheres are used, in thiscase, as a control for non-specific binding and release of protein.When placed in buffer, PEGDA–cys microspheres released 0.98 ±0.4 μg of GFP in 6 days, but PEGDA–GSH microspheres released nomeasurable GFP in this period. Although the washing of PEGDA–cysmicrospheres was not complete and some surface binding of the fu-sion protein was detected (1.82 ± 1.1 × 108 molecules per micro-sphere), this was rapidly released from microspheres without needfor protease or glutathione. For the PEGDA–GSH microspheres, thatdid not elute any detectable GFP in 6 days, thrombin was able tocleave 9.9 ± 2.5 × 108 molecules of GFP per PEGDA–GSH micro-sphere. Based upon this result, GST–GFP bound to PEGDA–GSH

Fig. 2. Binding of GST–GFP to PEGDA–GSH microspheres. Brightfield (upper left) andpseudo-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.

microspheres represents significantly more than monolayer cover-age. And it is clear that the interaction between GST fusion proteinsand the PEGDA–GSH microspheres is specific.

To further confirm the specificity of the interactions of GST–GFPwith the surface and to give a preliminary understanding of theexpected response of the microspheres following injection into extra-cellular fluids, we examined the release of protein from microspheresin the presence of free GSH. GSH is available at low levels (0.1 mM) inextracellular compartments of the body [43]. Extracellular GSH willelute proteins immobilized by GST–GSH interactions, but based uponthe knowledge of the association constant, Km, between GSH and GST(2.2 × 10−2 mM) [44], the elution of GST–GFP protein anchored toPEGDA–GSH microspheres was expected to be low. Over a period oftime, GST–GFP elution was measured in buffer containing various GSHconcentrations. GSH at intracellular levels (10 mM and 1 mM) eluteda significant portion of GST–GFP within 48 h. Extracellular levels ofGSH, i.e. concentrations below 1 mM GSH had a much less prominentdisruption of the microsphere GST–GFP interaction with more than80% of the protein remaining associated with the particles after 6 days(Fig. 4A).

To further understand the strength of the interaction in complexbiological fluids, we challenged PEGDA–GSH microspheres anchor-ing GST–GFP with anti-coagulated human plasma for six days at37 °C. We found that after six days almost no GFP was released intothe plasma (Fig. 4B), and the microspheres remained brightly fluo-rescent suggesting that the bound protein was still active (Fig. 4C).Protein is clearly not released from the microspheres without thepresence of proteases in the fluid. Although plasma does not containmany blood proteases in the active form due to calcium depletion,the small molecules, ions, and active proteases in citrated plasmado not release proteins significantly. Because many plasma proteases,

Table 2Summary of physical parameters of PEGDA–GSH microspheres.

Parameter Calculated value Observed value

Particle Diameter (μm) – 46 ± 16Surface area (μm2) – 6647 ± 804Mesh size (nm) – 4.6 ± 0.2

GST–GFP Cross sectional areaa

(μm2/molecule)1.4 × 10−5 –

Capacitybc (molecule/microsphere)

4.7 × 108 9.9 ± 2.5 × 108

a Estimated based upon the crystal structure of a GST dimer and predicted structureof the fusion proteins.

b Estimated from the observed microsphere diameter and estimated cross sectionalarea of GST–GFP.

c Observed value was determined from thrombin release from microspheres.

Fig. 4. Release of GST–GFP in the presence of biologic fluids. (A) Release of GST–GFP in thepresence of 10 mM ( ), 1 mM ( ), 0.1 mM ( ), 0.01 mM ( ), and 0 mM ( ) GSHcontaining phosphate buffered saline. Each point represents the mean plus or minus thestandard deviation of three independent samples. (B) Human plasma mediated releaseof GFP from GST–GFP loaded PEGDA–GSH microspheres. Microspheres were incubatedwith human plasma for 6 days at 37 °C prior tomeasuring GFP in the supernatant by fluo-rescence. Each point represents the mean plus orminus the standard error of the mean ofthree independent samples in plasma from3human donors. (C) Gross photographs ofmi-crospheres under UV illumination after 6 days of incubation in human plasma or PBS at37 °C. The scale bar is 1 mm.

Fig. 5. Thrombin cleavage of GST–GFP fromwith PEGDAmicrospheres. (A) Release of GFPfrom GST–GFP PEGD–GSHmicrospheres in the presence of ( ) 0, 11 ( ), 22 ( ), 44 ( ),or 110 ( ) nM thrombin as detected by fluorescent GFP signal. Each point represents themean plus or minus the standard deviation of three independent samples. (B) Represen-tative brightfield and epifluorescent micrographs depicting GST–GFP (green) associatedwith microspheres before (top row) or after (bottom row) release in response to throm-bin (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 h. Each point represents the mean plus or minus the standard devia-tion of three independent samples.

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including prothrombin, become activated by blood draw, addition ofcalcium can be used to achieve active proteolytic plasma, but this acti-vated plasma has significantly higher protease activity than plasma be-fore the blood draw [45–47].

With an understanding that the association is specific, we sought todetermine the release in the presence of physiologic thrombin concen-trations. Comparedwith the absence of thrombin (Fig. 4A), significantlyfaster GFP release was observed when thrombin mediated the release(Fig. 5A). Protein was released on a time scale of minutes compared todays when no enzymatic response was present. Microscopy clearlydemonstrates the release of GFP fromGST–GFP loaded PEGDA–GSHmi-crospheres following thrombin exposure (Fig. 5B). By comparing the

rate of GST–GFP cleavage between free GST–GFP and PEGDA–GSH asso-ciated GST–GFP, the rate of cleavage was slightly but significantlyhigher for free (unbound) GST–GPF compared to bound GST–GPF atall concentrations of thrombin (Fig 5C). This would be expected sincethe orientation and mobility of the substrate would be restrictedresulting in diminished binding of thrombin and possibly restrictedcleavage due to the orientation of the molecules.

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To determine the effectiveness of the GSH–GST interaction as athrombin mediated, anti-bacterial delivery system, we generated GST–melittin and loaded this protein on PEGDA–GSHmicrospheres in aman-ner equivalent to the GST–GFP protein. GST–melittin binds to PEGDA–GSH microspheres similarly to GST–GFP with an estimated 7.8 ± 1.5molecules of melittin per microsphere. This is not significantly different(p-value greater than 0.05) from the number of GFP molecules estimat-ed to be present on the surface of PEGDA–GSH microspheres, 9.9 ± 1.3molecules per microsphere. Following exposure of these GST–melittinloaded microspheres to thrombin, the released protein, or appropriatecontrols, was incubated with S. pyogenes cells. GST–melittin loaded mi-crospheres that had been activated with thrombin inhibited S. pyogenesgrowth similarly to 10 μM synthetic melittin over a period of 12 h. Pro-tein released frommicrosphereswithout thrombin treatment had a lim-ited growth inhibition (Fig. 6).

4. Discussion

We chose to conduct these studies with PEGDA as the base for thehydrogels because PEGDA is an FDA approvable material already inuse in clinics [48]. Hydrogels can be surface-modified [49] or layeredonto various surfaces with the potential for controlling cellular interac-tions and for drug delivery [50–54]. We also chose PEGDA as the poly-mer due to the thiol–ene addition of GSH to PEGDA in a single step[55]. Although the system is developed with PEGDA, this approach iseasily amendable to nearly any hydrogel system, and possibly beyondhydrogel systems. Hydrogel microspheres were utilized because of theincreased surface to volume ratio allowed high binding capacity the po-tential for clinical application as locally injected systems [56–58].

One property of hydrogels that can bemodulated is themesh size. Inour current system, themesh size of the hydrogel microspheres limitedproteins from entering the microspheres. Based upon our past experi-ence with PEGDA hydrogels [13,30,59], we designed the hydrogel tohave a mesh that would exclude the GST fusion proteins from the inte-rior of the microspheres. The calculated mesh size agreed with this de-sign prediction. From themeasurements of the size of themicrospheres,a monolayer of dimeric GST–GFP would consist of approximately4.7 × 108 molecules. The specific interactions between GST and GSHappear to account for more than monolayer coverage of the micro-spheres based upon the number of GFP molecules that are released.

Fig. 6. GST–melittin microspheres inhibit growth of S. pyogenes. Growth inhibition ofS. pyogenes cells exposed for 10 h to chemically defined growth medium (CDM), CDMwith synthetic melittin (syn. melittin), or CDM mixed with the releasate fromPEGDA–GSH microspheres with thrombin, PEGDA–GSH microspheres bound withGST–melittin, or PEGDA–GSH microspheres bound with GST–melittin and exposedto thrombin (1.8 μM) for 2 h. The data is presented as the absorbance at 600 nm fol-lowing 10 h of growth divided by the absorbance at 600 nm following 1 h of growth.Each bar represents the mean plus or minus the standard error of themean of three tofour technical replicates.

There are several possible reasons for the multilayer binding tothe PEGDA–GSH microspheres. Pendant GSH moieties would be ata distance from the visible surface of the microsphere dependingon the length of the growing acrylate backbone before GSH incorpo-ration. Pendant chains extending beyond the surface would greatlyincrease the amount of protein bound, but not change the apparentsize of the particles. GST–GFP could also partially enter the micro-sphere polymer mesh where the mesh is above the average value.The measured mesh size is based upon the bulk properties and thesurface of the microspheres, particularly when loops, entangledloops, and dangling chains are present. These small changes wouldbe expected to greatly increase the effective surface for binding.Based upon our results, it is clear that multilayers of GST fusion pro-teins are specifically binding.

To confirm the specificity, a 10 mM GSH solution added toPEGDA–GSH bound GST–GFP resulted in 50% protein elution from themicrospheres in 48 h and complete elution in 6 days (Fig. 4). LowGSH concentrations (below 0.1 μM) in buffered normal saline did notrelease the protein from the microspheres, further suggesting that theinteraction is specific. This is significant since plasma levels are signifi-cantly lower at 1.5 μg/mL, or 0.005 mM [43]. Intracellular glutathioneconcentrations have been measured to be between 1 mM and 10 mMin healthy cells [43], and can be further increased in disease [60,61]. Inthe future, we seek to also exploit this characteristic for intracellular re-lease of proteins. In the extracellular environment, the GST–GSH inter-action would be expected to be stable for periods of time at lowglutathione levels allowing for release specifically in the presence of pa-thology associated proteases, including thrombin.

Thrombin mediated release of GST fusion proteins is significantlyfaster than elution with GSH. The velocity of release of GFP wasachieved with thrombin concentrations similar to that necessaryfor clot formation (100 nM, [62]). Association with PEGDA micro-spheres slightly but significantly decreased the rate of thrombincleavage of GST–GFP (Fig. 5). This decrease in the enzymatic reactionwould be expected to be due to a reduced thrombin affinity for thesubstrate [63,64]. In this case, the reduced affinity may be due tounfavorable, rigid or steric limitations of the GST–GFP substrate onthe surface of the PEGDA microsphere. Though not further exploredin this manuscript, these phenomena may be another modifiable pa-rameter that can allow for more refined control of protein releasefrom immobilized GSH containing hydrogels.

Finally, we showed that GST–melittin could be loaded to thePEGDA–GSH microspheres as efficiently as GST–GFP, and thatS. pyogenes growth was inhibited by melittin cleaved from PEGDA–GSH microspheres by thrombin. Thrombin is upregulated duringbacterial infection, and was, therefore, deemed an appropriate enzymeto be used for activation of anti-bacterial coatings [22]. This would beparticularly true for blood-contactingmaterials. Under these conditions,themelittin released frommicrospheres was estimated to achieve 1 μMand achieved similar growth inhibition of S. pyogenes cells as 1 μM syn-thetic melittin (Fig. 6). Based upon the growth inhibition, melittin canbe loaded predictably on hydrogel microparticles and activated bythrombin. The loaded melittin inhibited growth of high-density cul-tures of pathogenic S. pyogenes cells. These densities of S. pyogenescells would be encountered during active infection, and suggest thatthe delivery potential of GST–melittin from surface coated materialmight find further utility as a treatment for active infection.

Bacterial infection coincides with a decrease in pH, and it hasbeen reported that pH values below 6 have been measured in effu-sions and abscesses caused by bacterial infection [65]. The behaviorof an anchor system in these environments would need consider-ation. The GST–GSH interaction has been shown to be favorable inpH values as low as 5 [66]. In addition, GST and GST–melittin areboth stable to denaturation at low pH [23]. It is, therefore, suggestedthat the GST anchor will maintain anti-bacterial protein immobilizedto a surface in low pH environments. We plan to verify the predicted

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response to decreased pH in the future to fully understand the possi-bly mechanisms of non-specific release from the microspheres.

Further, even if melittin is not released from the microspheres orif GST–melittin is released there may be non-specific toxicity. Duringthe development of a method to purify proteins from the insolubleprotein fraction, we noted that melittin fused to GST had significant-ly reduced toxicity [23]. Since this is true, there is already evidencethat toxicity would be minimized for the fusion protein. There issome activity, but not statistically different from the control, whenmicrospheres are incubated in the presence of bacteria. Two possibleexplanations can account for this biological activity: (1) the surfacebound melittin is active or (2) melittin is being released from theparticles based upon a bacterial mechanism. Unfortunately, our re-sults do not completely eliminate either option. Our observations(unpublished data) suggest that GST–GFP is not released from mi-crospheres in the presence of bacteria by proteolysis in the portionof the molecules that are equivalent or GST–GSH interaction disrup-tion. But, there may be proteolytic activity in the melittin specificportion of the protein.

As a proof of concept for the design of GST–GSH hydrogel proteindelivery systems, the fact that activation is necessary for full melittinactivity clearly confirms the system functions as designed. In addi-tion, utilization of the GST–GSH interaction as an anchor need notbe limited to peptides that show reduced toxicity when they arefused to GST. Proteins or peptides that show activity as fusion pro-teins may be anchored via a GST–GSH anchor within the hydrogelmesh. Hydrogel mesh size is an easily modifiable parameter [30]. Adelivery system that protects protein cargo within a hydrogel meshwould require control of mesh size large enough so that a suitable re-leasing enzyme, such as thrombin, could enter the hydrogel, cleavethe active protein from fused GST, and allow the cleaved product todiffuse out. On the other hand, the mesh size must be small enoughto keep therapeutic protein cargo protected from acting before it isrelease. Future developments of the delivery system will utilizeboth surface and interior loading of therapeutic proteins that maybe designed to allow or minimize activity while loaded.

In conclusion, we have shown that GSH immobilized on the sur-face of PEGDA microspheres can specifically and predictably interactwith GST fused GFP and melittin. The interaction is stable under ex-tracellular salt and GSH conditions. Further, thrombin can releaseproteins fused to the GST and allow them to exert biologic activity.In our case, melittin was released from the surface and was as func-tional as melittin at a similar concentration in inhibiting the growthof pathogenic S. pyogenes. This proof of concept study established thepossibility of using the GST–GSH interaction to load therapeutic pro-teins into a hydrogel-microsphere-based drug delivery system.

Acknowledgments

This investigation was conducted in a facility constructed withsupport from Research Facilities Improvement Program grant(RR15482) from the National Centre for Research Resources of theNational Institutes of Health (NIH). This research has been funded,in part, by the University of Illinois at Chicago Center for Clinicaland Translational Science (CCTS) award supported by the NCRR(UL1 TR000050, RAG), and the National Institute for Neurologic Dis-orders and Stroke (NS055095, RAG) and the National Institute of Al-lergy and Infectious Diseases (AI091779, MJF). The authors alsothank Dr. Debra A. Tonetti for use of instruments.

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