Biologically Triggered Delivery of EGF from ... - ACS Publications

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Biologically Triggered Delivery of EGF from Polymer Fiber Patches Si-Eun Kim,† Parker W. Lee, and Jonathan K. Pokorski* Department of Macromolecular Science and Engineering, Case Western Reserve University, Case School of Engineering, Cleveland, Ohio 44106, United States S Supporting Information *

ABSTRACT: Wound healing is modulated by complex spatial and temporal regulation of growth factors within a wound site. Regenerative medicine seeks to generate materials that can mimic this environment for the healing of chronic or traumatic wounds. Herein, we report a programmed release of epidermal growth factor (EGF) from coextruded polymer fiber patches, which is triggered by the natural biological cascade of wound healing. Genetically engineered EGF containing a matrix metalloproteinase (MMP) cleavage site was covalently conjugated to a nonwoven poly(ε-caprolactone) (PCL) fiber mat fabricated by multilayered melt coextrusion. The genetically modified EGF showed rapid release in the presence of a biological trigger, MMP-9, while a control protein showed negligible release. The biologically responsive fiber mat dramatically enhanced proliferation and migration of human keratinocytes in the presence of MMP-9. This study describes the release of a critical wound-healing growth factor as triggered by the biology inherent in the healing process.

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materials seek to control the delivery of therapeutic proteins based on the biological environment. Paramount among these materials are responsive hydrogels designed to specifically degrade in the presence of matrix metalloproteinases (MMPs),17,18 proteases secreted by the cellular components during the wound healing cascade.19,20 As the material degrades, the therapeutic proteins contained within the gel release at an accelerated rate to the environment. While these systems have seen great success, proteins embedded within the gels have an inherent chemical potential driving release. For growth factors like EGF, where tight regulation is needed, it is important to program a specific release mechanism to control the amount delivered. Genetic engineering of recombinant proteins has been an innovative approach to optimize therapeutic protein delivery and stability.21 These recombinant proteins have been used to supplement natural proteins that are deficient or absent in disease states, or to upregulate existing metabolic pathways to cure wounds and diseases.22,23 Thus, we sought to develop a wound healing patch where a genetically engineered EGF was covalently immobilized onto a polymeric fiber mat, thus eliminating the chemical driving force for release to the environment. A MMP-cleavage site was introduced between the active protein and the covalent attachment point on the mat, thus EGF would be liberated by MMP-9, a commonly upregulated MMP during the early wound healing stages (Scheme 1). The protein-conjugated fiber mat is envisioned as a topical depot of EGF, which can be tuned to provide the

ne of the most prevalent healthcare issues is severe and chronic wounds, including surgical incisions, trauma, burns, and diabetic ulcers.1 Wound healing is a complex process where wound healing cells migrate into the damaged site, vascularize, and regenerate the damaged tissue.2 The process is regulated by locally produced proteins that influence cells through autocrine and paracrine mechanisms.2,3 Such proteins are widely used as biopharmaceuticals due to their high biological activity, specificity, and therapeutic efficacy.4−6 Epidermal growth factor (EGF), a naturally occurring protein, is essential for wound healing and tissue repair, attracting inflammatory cells into the wound, stimulating cellular proliferation, enhancing neovascularization, and epithelial migration.7−9 EGF has been used successfully in the initial stages of wound healing from diabetic ulcers, burns, and skin trauma to accelerate wound closure.1,10,11 A recent study demonstrated that EGF contained in a polymer fiber patch greatly improved the wound healing response compared to existing wound dressing films.12 Both fiber mat porosity (increasing gas permeation) and EGF delivery contributed to rapid wound healing. However, uncontrolled delivery of EGF can result in malignancies via the overexpression of transforming growth factor alpha (TGF-α) and overstimulation of EGF receptors. Overstimulation of EGF receptors in many carcinomas correlates with the development of cancer metastasis, resistance to chemotherapy, and poor prognosis.13,14 Thus, it is critical that EGF be delivered with tight control over concentration and appropriate timing to correspond with wound healing biology. At a basic level, suitable biomaterials for wound healing should allow gaseous exchange, provide a barrier to microorganisms, and remove excess exudates.15,16 More complex © XXXX American Chemical Society

Received: March 19, 2017 Accepted: May 15, 2017

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DOI: 10.1021/acsmacrolett.7b00212 ACS Macro Lett. 2017, 6, 593−597

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ACS Macro Letters Scheme 1. Bioconjugation of EGF to an Aminooxy Decorated PCL Fiber Mat and MMP-Mediated Release

1C). MALDI-TOF mass spectrometry confirmed the molecular weight of the recombinant proteins, which are consistent with the theoretical molecular weights of control EGF and EGFMMP, 7344 and 8162 kDa (expected molecular weight 7344 and 8162 kDa), respectively (Figure 1D). The in vitro biological activity of the purified EGF-MMP protein was confirmed using both human keratinocytes (HaCaT) and epidermoid carcinoma (A431) cell lines. EGF is a potent mitogen in HaCaT cells, even at low concentrations, and improves further at high concentrations. EGF has the opposite biological effect on A431 cells, promoting apoptosis at high concentrations.28 Relative viability was evaluated utilizing the MTT proliferation assay on the two cell lines, when compared versus no addition of EGF. HaCaT cells demonstrated a ∼6-fold increase in cell viability from 100 to 587.7% at the highest concentration of EGF-MMP (Figure 2A). In contrast, the cell viability of A431 cells decreased from 100 to 38.2% as the concentration of EGF-MMP increased. These results were consistent with literature values and demonstrated that the purified recombinant EGF-MMP has the expected biological activity and the mutations introduced at the Nterminus do not affect the activity. In silico modeling predicted that the MMP reactive octapeptide would be cleaved by MMP-9 orders of magnitude faster than other amino acid sequences in EGF-MMP. Cleavage of EGF-MMP was performed via incubation with MMP-9 for 2 and 4 h (Figure 2B). The molecular weight of the protein was determined by MALDI-TOF following cleavage. After 2 h, the molecular weight decreased from 8.16 kDa to 6.64 kDa, in precise agreement with the theoretical molecular weight of cleaved EGF-MMP (6.64 kDa). After 4 h, the mass spectrum remained as a single peak at 6.64 kDa, indicating no proteolytic activity at other sites on the protein. This result indicated over 99% conversion to the cleaved product by integration of MALDI-TOF data, with no side products. The PLP-mediated transamination reaction was employed to introduce a ketone moiety at the N-terminus of EGF for conjugation to the fiber mat and conversion was verified using MALDI-TOF. Two significant peaks were found (Figure S1), one corresponding to the transamination product and another that correlates to an expected PLP adduct. The undesired product still contained a ketone group and was able to undergo oxime formation. Bioorthogonal ligation has been widely explored in chemical biology for the reaction of ketones with hydrazides and alkoxyamines to form hydrazones and oximes at acidic pH.29 Oxime chemistry prefers slightly acidic conditions, however proteins may precipitate under these conditions due to the relatively higher isoelectric points of proteins (pIEGF‑MMP =

optimal amounts of EGF for treatment of acute and chronic wounds. An enzymatically responsive recombinant EGF was synthesized with several features included at the N-terminus of the protein (EGF-MMP, Figure 1A). The N-terminus was modified

Figure 1. (A) EGF-MMP, (B) EGF control protein, (C) SDS-PAGE gel of purified control EGF (7.34 kDa) and EGF-MMP (8.16 kDa), and (D) MALDI-TOF MS analysis of control EGF protein and EGFMMP.

since the C-terminal sequence is responsible for receptor binding. The recombinant protein contained a N-terminal sequence (AKT) that is highly reactive for bioconjugation using pyridoxal 5′-phosphate (PLP) mediated bioconjugation.24 This method is a site-selective modification that utilizes N-terminal transamination of proteins to introduce a ketone or aldehyde, which could further undergo oxime ligation with a polymer substrate (see below). Next a polyhistidine tag was included for affinity purification. Finally, a MMP cleavage site for triggered release of the protein from the scaffold was included downstream of the remaining N-terminal extension. The chosen octapeptide sequence (VPLSLYSG) is cleavable by MMPs, including MMP-9 (kcat/KM = 49000 ± 3000),25,26 and is selectively cleaved by MMPs that are upregulated in early stages of the wound healing cascade.27 Only a 4-residue extension remains at the N-terminus following release, based on this domain orientation. The gene was synthesized and cloned into the pET28a(+) expression vector (pET28EGF-MMP), followed by standard IPTG-induced protein expression and purified via affinity chromatography. An EGF mutant without the MMP-cleavage site was synthesized in the same manner as a nonresponsive control (Figure 1B). The two proteins were analyzed via SDSPAGE to determine purity, resulting in a single band corresponding to a molecular weight of ∼10 kDa (Figure 594

DOI: 10.1021/acsmacrolett.7b00212 ACS Macro Lett. 2017, 6, 593−597

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Figure 3. (A) SEM micrograph, fiber thickness = 2.6 ± 1.5 μm, scale bar = 10 μm. (B) Pore size distribution of the fiber mat as measured via porimeter, mean pore diameter = 25.6 μm. (C) ATR-FTIR spectrum of EGF-MMP conjugated to fiber mats: black = unirradiated, red = irradiated. Arrows indicate (1) amide I and (2) amide II. (D) Kinetic study of released EGF and EGF-MMP from fiber mats after MMP-9 cleavage, *p < 0.05 and all samples had p ≤ 0.065, as determined by a two-tailed t test.

distribution (25.6 μm, Figure 3B). This pore size is in the range of commercially available wound healing patches.33 In this work, PCL was chosen due to its processing flexibility and biocompatibility, specifically as a wound dressing.34,35 Aminooxy benzophenone was synthesized as a linker for oxime chemistry, and deposited onto the PCL fiber mat through UV-initiated photochemistry. Ketone-modified EGFMMP was then covalently conjugated onto the aminooxy fiber mat via oxime ligation. Since protein adsorption without covalent attachment can provide false readings, nonspecific protein absorption onto the hydrophobic fiber mat was performed on unmodified mats. The protein modified PCL fiber mat was scanned using ATR-FTIR to confirm the presence of EGF-MMP (Figures 3C and S3). The unirradiated fiber mat showed no amide signal from the protein, while the UV irradiated side clearly showed EGF-MMP, as indicated by CO stretching vibrations at 1652 cm−1 from amide I, N−H bending vibration/C−N stretching vibration at 1558 cm−1 from amide II, and N−H stretching vibration near 3300 cm−1 from amide A. This result clearly indicated that protein conjugation only proceeds onto the aminooxy modified PCL fiber mat. We next explored whether incubation with MMP-9 releases EGF conjugated to the fiber mat. For comparison, a conjugated noncleavable control EGF was also studied. The release kinetics of EGF-MMP and control EGF from the fiber mats were determined by incubation with activated MMP-9 and quantification via ELISA. The concentration of released protein was normalized by the weight of each fiber mat. The ELISA results indicated that EGF-MMP was rapidly released, resulting in ∼11 ng EGF/mg PCL released over the first 10 min after introduction of the enzyme (Figure 3D). However, the control EGF conjugated fiber mat showed no significant protein release after introduction of the MMP enzyme. After 4 h of incubation, the control EGF protein showed a negligible amount of released protein while the EGF-MMP fiber mat released ∼20 ng of EGF per milligram of PCL fiber mat. Incubation was carried out for 24 h with MMP-9, with no further increase in released protein. Thus, the conjugation scheme yielded 20 ng

Figure 2. (A) MTT cell viability assay of HaCaT (human keratinocytes) and A431 (epidermoid carcinoma) cells in the presence of EGF-MMP. Values are displayed as a ratio versus no introduction of EGF. (B) MALDI-TOF MS indicating the molecular weight of EGFMMP protein before and after MMP-9 cleavage.

6.2) than those that are preferable for oxime ligation. Aniline catalysts effectively accelerate oxime formation by forming an in situ highly reactive protonated aniline Schiff base at neutral pH.30,31 Therefore, aniline was utilized as a nucleophilic catalyst for the bioconjugation reaction. EGF-MMP was modified with the ketone moiety at the N-terminus in the presence of PLP, as previously described. Excess PLP was removed via centrifugal spin filtration and the ketone functionalized protein was conjugated to aminooxy functionalized TAMRA dye with a minimal amount of aniline catalyst (Figure S2A). After removal of free dye and catalyst via centrifugal spin filtration, fast protein liquid chromatography (FPLC) was performed to monitor the elution volume of the PLP reacted protein and dye labeled EGF-MMP, both the modified and unmodified proteins showed identical elution volumes (Figure S2B). Furthermore, after oxime ligation a peak at 555 nm coeluted with the protein peaks, demonstrating successful bioconjugation. The polymeric material chosen was a microfibrous coextruded poly(ε-caprolactone) (PCL) nonwoven mat. Multilayered coextrusion of nano- and microfibers is a new manufacturing technique that is high throughput, allows for control over fiber dimensions and porosity, and is amenable to site-specific chemical modification strategies.32 Nonwoven poly(ε-caprolactone) (PCL) fiber mats were fabricated (Figure 3A) with a high porosity and near homogeneous pore size 595

DOI: 10.1021/acsmacrolett.7b00212 ACS Macro Lett. 2017, 6, 593−597

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Figure 4. (A) Optical micrographs of gap distance between HaCaT cells at 0 and 24 h after scratch test (scale bar = 200 μm). (B) Relative scratch closure distance after 24 h. Error bars are expressed as standard deviations, *p ≤ 0.03, as determined by a two-tailed t test.

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EGF/mg PCL that was accessible. When normalized per surface area, based on Brunner−Emmett−Teller (BET) surface measurement (43.2 cm2/mg of PCL fiber mat), ∼0.46 ng EGF/ cm2 of PCL fiber mat was available for release. The results indicated that EGF-MMP release was specifically triggered by MMP-9, while the control EGF remained conjugated to the polymer scaffold. Wound healing was simulated using a monolayer scratch test with HaCaT cells, a common model for migration and proliferation in the wound healing process (Figure 4). A control sample with no fiber mat and three different fiber mats were evaluated; unmodified PCL, control EGF-modified PCL, and EGF-MMP-modified PCL. Cells were grown to confluency and a scratch was introduced using a micropipette tip. The cells were then incubated with a fiber mat and MMP-9. After 24 h of incubation, the scratches were measured by optical microscopy. No scratch closure was observed for controls with neat PCL or no fiber mat (Figure 4). We observed a decreased gap for cells incubated with control EGF fiber mats. The improvement is likely due to a small amount of EGF released, either through nonspecific adsorption to the surface or low-level cleavage of unintended sites. Previous studies have shown EGF concentrations as low as 1 ng/mL can have a pronounced effect on gap closure.36 In contrast, the EGF-MMP mat demonstrated complete gap closure and significantly enhanced proliferation. Thus, the EGF-MMP fiber mat responded to MMP-9 to stimulate complete wound closure in the simulated healing. This study describes a new paradigm in triggered release of growth factors based on genetic engineering, where therapeutic protein release is triggered by the biology of wound healing. Recombinant EGF was engineered to release only in the presence of wound healing cues. The results presented here clearly indicate that EGF could be engineered for selective release by MMP-9. Moreover, released EGF from the EGFMMP conjugated fiber promoted proliferation and migration of cells. The amount of EGF delivered in response to MMP-9 can be tuned in the future via the amount of aminooxy groups coupled onto the fiber mat and release kinetics can be controlled by changing the MMP responsive sequence.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Parker W. Lee: 0000-0002-0491-3742 Jonathan K. Pokorski: 0000-0001-5869-6942 Present Address †

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, United States. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was funded through grants from the NIH (R00EB011530) and the NSF (DMR 0423914). REFERENCES

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00212. MALDI-TOF and ATR-FTIR spectra, FPLC chromatograms, extrusion and synthetic protocols (PDF). 596

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DOI: 10.1021/acsmacrolett.7b00212 ACS Macro Lett. 2017, 6, 593−597