Functionalized Biopolymer Particles Enhance Performance of a Tissue

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Functionalized biopolymer particles enhance performance of a tissue-protective peptide under proteolytic and thermal stress Kevin Dooley, Julie Devalliere, Basak E. Uygun, and Martin L Yarmush Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00280 • Publication Date (Web): 24 May 2016 Downloaded from http://pubs.acs.org on May 31, 2016

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Functionalized biopolymer particles enhance performance of a tissue-protective peptide under proteolytic and thermal stress Kevin Dooley1, Julie Devalliere1, Basak E. Uygun1, & Martin L. Yarmush1,2* 1

Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals for Children, Boston, Massachusetts, USA 2

Department of Biomedical Engineering, Rutgers University, Piscataway, New Jersey, USA

*

Corresponding author

ACS Paragon Plus Environment

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ABSTRACT Cutaneous burns are often exacerbated by poor perfusion and subsequent necrosis of the microvasculature surrounding the primary injury. Preservation of these vessels can reduce necrotic tissue expansion and increase success rates of skin graft procedures. Recent work has identified a peptide derived from erythropoietin, ARA290, with the ability to mediate tissue protection in a variety of cell types. Here we demonstrate the advantages of fusing ARA290 to an elastin-like polypeptide (ELP) to salvage microvascular endothelial cells in harsh proteolytic conditions following thermal shock. These fusion proteins were expressed recombinantly in bacterial hosts and rapidly purified by inverse transition cycling. They were shown to spontaneously aggregate into particles at sub-physiological temperatures. The bi-functional sub-micron particles were resistant to digestion in enzymes upregulated after burn injury. Furthermore, the data strongly suggest these ARA290-functionalized particles were superior to treatment with the peptide alone in preventing microvascular cell death in these conditions. The results bring to light an efficient and cost-effective strategy for the delivery therapeutic peptides to proteolytically active wound sites.

Keywords: elastin-like polypeptide, ARA290, erythropoietin, sub-micron particle, burn injury, drug delivery


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INTRODUCTION Engineered biopolymer materials offer several advantages over traditional synthetic polymer systems. Precise genetic tuning on a codon-by-codon basis affords an extraordinary level of control over the physiochemical properties of the resultant biopolymer. Morphology, binding affinity, catalytic activity, and rheological properties can be adjusted using both rational design and combinatorial protein engineering approaches.1-5 Coupling these design principles with recombinant protein expression in microbial hosts allows for the cost-effective production of functional biopolymers. This class of materials has achieved a tremendous amount of success in tissue engineering, drug and cellular delivery, and regenerative medicine, both in the laboratory and in clinical settings.6-12 One of the most successfully engineered biopolymers is the repetitive elastin-like polypeptide (ELP). The prototypical primary sequence was originally derived from an amino acid motif found in the hydrophobic domain of tropoelastin. ELPs are artificial, oligomeric peptides typically composed of the sequence [VPGXG]n where X, the guest residue, can represent any of the common amino acids except proline and n signifies the number of repeating pentapeptides. These biopolymers undergo an entropically-driven inverse phase transition in aqueous solutions in response to an external stimulus (temperature, ionic strength, pH). Conformationally, the ELPs shift from an extended, hydrated state to an insoluble, β-spiral aggregate.13 The number of pentapeptide repeats, guest residue composition, and hydrophilic-to-hydrophobic block ratios have been identified as tunable design parameters to specifically tailor ELP properties for a particular application. Additionally, fusing biologically active proteins and peptides to ELPs on the genetic level has led to chimeric fusions which retain the function of each component. Researchers have been able to exploit the stimulus-responsive phase transition inherent to ELPs to engineer fusion proteins for non-chromatographic protein purification, targeted cancer therapeutics, and functionalized nanoparticle self-assembly.14-16 A number of bio-inspired materials, including elastin, have been investigated as potential therapeutic treatment options for chronic and acute wounds.17 Cutaneous wound management, and in particular acute


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burn care, represents an enormous burden on healthcare systems worldwide. Mean total healthcare costs have been estimated at nearly $90,000 per patient and the American Burn Association reported 30,000 acute hospitalizations related to burn injury in 2015 in the United States.18 Cutaneous burn injuries can be classified into three distinct zones characterized by the severity of tissue destruction and retention of perfusion: coagulation, stasis, and hyperemia.19,20 All tissue in the coagulation zone is irreversibly destroyed as a direct effect of burn injury. This is surrounded by a potentially salvageable zone of stasis, characterized by poor perfusion and excessive inflammation. Within 48 hours, tissue in the zone of stasis including the dermal microvasculature undergoes necrosis due to ischemia and/or hypoxia, which results in a larger, deeper injury typically requiring surgical intervention.21 Furthermore, insufficient dermal vascularization is one of the most common reasons for skin graft failure when treating burn injuries.22,23 Preservation of the microvascular network in the zone of stasis could dramatically improve skin graft success rates. Recently, Koria et al. used ELP nanoparticles conjugated with keratinocyte growth factor (KGF) to promote epithelial morphogenesis and granulation tissue formation in full-thickness excisional wounds in diabetic mice.24 The ELP component was thought to help shield KGF from the highly proteolytic wound microenvironment although this was not investigated. In this work, we demonstrate the ability of ELPs to deliver a cellular protective peptide, ARA290, to endothelial cells in a simulated burn wound environment. The ARA290 peptide was originally derived from a contiguous stretch of amino acids found on a helical subunit of erythropoietin (EPO), a regulatory cytokine for erythrocyte production (Figure 1a). EPO and its derivative peptides have been previously shown to protect cells both in vitro and in vivo from a variety of stresses including ischemic stroke, peripheral nerve trauma, and thermal injury.21,25 This tissue protective activity is conferred by interaction with a heteroreceptor composed of the EPO receptor (EPO-R) monomeric subunits and CD131, the β-common receptor, both of which are expressed in endothelial cells.26-28 Here we demonstrate the bi-functionality of ARA290-ELP sub-micron particles; resistance to enzymatic degradation and endothelial cell preservation following thermal shock. Both of these activities


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are crucial to the development of translational therapies for the treatment and management of cutaneous burn injuries. It is generally accepted that topical application of biologically active moieties, including growth factors, cytokines, and chemokines, can stimulate different aspects of the tissue repair process in skin defects.29,30 However, these externally supplied factors are susceptible to swift proteolytic degradation due to the excessive secretion of matrix metalloproteinases (MMP) by leukocytes at the wound site, which include several isoforms of collagenases and elastases.31 This, in turn, necessitates repeated application in order to achieve the desired therapeutic effects, thereby inflating the cost of treatment. Thus, strategies to deliver these protein cargoes in such a way that offer a more persistent release are desirable. We demonstrate coupling the tissue protectant activity of the ARA290 peptide with aggregating ELP sub-micron particles to develop a proteolytically resilient delivery vehicle for preserving dermal microvascular cells.

EXPERIMENTAL SECTION Materials All enzymes for DNA cloning as well as BL21(DE3) E. coli were purchased from New England Biolabs (Ipswitch, MA). Kanamycin sodium salt was purchased from Gold Biotechnology (St. Louis, MO). All materials for polyacrylamide gel electrophoresis and membrane dialysis as well as the PrestoBlue® cell viability reagent were purchased from Thermo Fisher Scientific (Waltham, MA). The pET24a(+) plasmid and porcine pancreas elastase were purchased from EMD Millipore (Billerica, MA). Oligonucleotides were synthesized by the DNA Core at the Center for Computational & Integrative Biology at the Massachusetts General Hospital (Boston, MA). The ARA290 peptide was synthesized by GenScript (Piscataway, NJ). Human dermal microvascular endothelial cells (HMVEC) were purchased from Lonza (Walkersville, MD) along with EGM™-2MV BulletKit endothelial cell growth medium. All other reagents, including collagenase from Clostridium histolyticum and lysozyme from chicken egg white, were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.


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Cloning Expression Cassettes An empty pET24a(+) plasmid was modified to incorporate BseRI and AcuI endonuclease restriction sites as well as short leader and trailer sequences, as previously reported by McDaniel et al.32 A short cassette encoding five pentapeptide ELP repeats, (VPGVG)5, was generated by annealing two overlapping oligonucleotides with appropriate overhangs for ligation into the modified pET24a(+) vector linearized with BseRI. Valine was chosen for the guest residue position to create hydrophobic biopolymers that undergo phase transition at lower temperatures. ELP cassettes were concatenated by recursive directional ligation by plasmid reconstruction (PRe-RDL) until the desired lengths, 40 and 120 pentapeptide repeats, were achieved (termed V40, V120). ARA290 (QEQLERALNSS) was generated by annealing two short, overlapping oligonucleotides with appropriate overhangs for ligation into the modified pET24a(+) vector linearized with BseRI. The peptide gene was appended to the N-terminus of the ELP cassettes using the same PRe-RDL technique (termed A-V40, A-V120). A short myc epitope (EQKLISEEDL) was similarly generated and appended to the C-terminus of all constructs for staining and labeling purposes. The resulting plasmids were transformed into BL21(DE3) E. coli for expression. All oligonucleotide sequences used for cloning experiments are provided in the Supporting Information.

Expression & Purification of ELP Constructs All ELP and ELP fusion proteins were expressed identically in terrific broth (TB) media supplemented with 100 µg/mL kanamycin. Ten milliliter E. coli cultures containing the appropriate vector were grown to saturation overnight and used to inoculate 1 L of sterilized media. Expression was carried out at 37 °C and 220 RPM and relied solely on leaky expression of the T7 promoter, as previously described.33 The cells were harvested by centrifugation at 3,000 g for 10 minutes and resuspended in 30 mL phosphate-buffered saline (PBS). The cells were lysed via microtip sonication for 12 minutes on ice in 1 minute intervals with 1 minute recovery times in between. Cell debris was separated from the soluble protein by centrifugation at 15,000 g for 30 minutes at 4 °C. Polyethyleneimine (0.7 % w/v) was used to precipitate the nucleic acid contaminants which were separated from the soluble protein fraction by


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centrifugation at 20,000 g for 15 minutes at 4 °C. The ELP and ELP fusion proteins were further purified by inverse transition cycling (ITC).15 NaCl was added to the samples to a final concentration of 1 M and were then heated to 45 °C to induce precipitation of the ELP. The precipitated proteins were separated from host cell contaminants by centrifugation at 10,000 g for 15 minutes in a centrifuge warmed to 40 °C. The soluble fraction was discarded and the precipitated pellet was resuspended in 5 mL of ice cold PBS. Resuspension was achieved by pipetting and agitation on a rotator at 4 °C. The samples were then centrifuged at 4 °C at 15,000 g for 15 minutes to remove any insoluble contamination, thus completing one round of ITC. Typically, two additional rounds were required to achieve an acceptable purity, which was verified by SDS-PAGE (Figure S1). Expected molecular weights were calculated using the ExPASy ProtParam tool. Concentrations were determined by absorbance at 280 nm using a NanoDrop ND-1000 spectrophotometer. A calculated extinction coefficient of 1,490 cm-1·M-1 was used for all constructs based on a common C-terminal tyrosine residue.

ELP Characterization ELP and ELP fusion transition temperatures (Tt) were calculated on a temperature controlled BioRad Benchmark Plus microplate spectrophotometer. Two hundred and fifty microliter solutions of varying ELP concentrations (1-100 µM) were prepared in PBS in a 96 well plate. The plates were warmed from 25 to 45 °C over 20 minutes and optical density readings were taken at 350 nm each minute. All readings were made in triplicate. Only small decreases in transmittance were observed for the shorter biopolymer constructs V40 and A-V40 at concentrations up to 1 mM using these conditions. The resulting transmittance profiles were plotted for each construct and are given in the Supporting Information. The data were fit a four parameter logistic (4PL) curve and the Tt was defined as the inflection point temperature at half the minimal transmittance. Tt was also evaluated by dynamic light scattering (DLS) on a temperature controlled Brookhaven Instruments Particle Size Analyzer at 25 µM. A detector at 90° was used to measure changes in intensity while the temperature was raised from 25 to 65 °C, with readings taken in 1 °C increments. Changes in effective particle diameter are given as a function


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of temperature. The shorter polymers did undergo a phase transition at these conditions and Tts were calculated using a similar method. Mean effective particle diameter was also determined by DLS for all constructs. Twenty-five micromolar samples were incubated at 60 °C for 10 minutes to induce particle formation prior to measurement. Differential particle size distributions are given along with the effective diameter and polydispersity index. The data represent the average of 10 individual 15-second readings.

FITC Labeling To better visualize the ELP and ELP fusion proteins, purified constructs were labeled with fluorescein isothiocyanate (FITC). FITC was chemically conjugated to the free amine groups located on the N-terminus and the lysine residue in the myc-tag on the V40 and V120 constructs. A 10 mg/mL solution of FITC was prepared in dimethylformamide. Approximately 100 nmol of purified protein was incubated with 500 nmol of FITC in 100 mM sodium carbonate buffer, pH 8.5 in a total volume of 1 mL. The conjugation was allowed to proceed for 3 hours at room temperature or overnight at 4 °C, protected from light. Most of the unbound FITC was removed by a single cycle of ITC. The labeled protein was resuspended in PBS and any remaining unbound FITC was removed by overnight dialysis against PBS in 7 kDa MWCO cassettes at 4 °C.

Quantitative Fluorescence SDS-PAGE For endpoint experiments, 5 µg of FITC labeled V40 or V120 was incubated with each enzyme in PBS at 37 °C for 3 hours in a total volume of 30 µL. The tubes were rapidly cooled to dissociate any aggregation and the entire sample was run on a 4 – 12 % Bis-Tris gel and imaged on a UVP transilluminator. Cleaved ELP chains ran out as smears on the gel below the primary band. The intact ELP remaining after 3 hours was quantified by band intensity using ImageJ software (Bethesda, MD) and compared to uncut ELP as well as a blank lane containing only enzyme to normalize background intensity (Figure S4). Time course measurements were performed identically with fractions pulled at various points and stored at -20 °C to inhibit any further cleavage until analysis by SDS-PAGE. Lysozyme, collagenase,


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and elastase concentrations used for these experiments were 400 U/mL, 3.3 U/mL, and 1 U/mL, respectively.

Cell Culture and Viability Assay HMVEC were serially passaged 2 – 6 times using the EGM™-2MV BulletKit. 24-well plates with confluent monolayers of endothelium were sealed with Parafilm and immersed for 20 minutes in a circulating water bath thermoregulated at 55 °C to induce thermal shock. Culture media was then replaced with fresh media supplemented with ARA290, V40, V120, A-V40, or A-V120. Where indicated, cells were treated with collagenase (3.33 U/mL) or elastase (1 mU/mL) in serum-deprived EGM™-2 media. A substantially lower concentration of elastase was required in these experiments to maintain cell adhesion to the tissue culture plate. Cell viability was assessed 24 hours post-treatment using PrestoBlue® cell viability reagent according to the manufacturer’s instructions. Cell morphology was assessed by phase contrast on a Zeiss Axiovert 200 M inverted microscope at 20X magnification.

Statistical Analysis Cell viability results are expressed as mean ± SEM for replicate experiments. Statistical significance was determined using an unpaired, two-tailed T-test with a 95% confidence interval. Statistical calculations were performed using GraphPad Prisim software (GraphPad Software, San Diego, CA).

RESULTS & DISCUSSION Characterization of ELPs and ARA290-ELP fusion proteins Several biophysical techniques were used to investigate the Tt, particle size, and polydispersity of the ELP biopolymers (V40, V210) as well as the chimeric fusions to ARA290 (A-V40, A-V120). The results of this characterization process are given in Figure 2 and Table 1. The entropically-driven phase transition in response to increasing temperature was evaluated by turbidimetry over a range of


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concentrations for all four constructs (Figure 2A, S2). Concentration-dependent phase separation was only observed for the longer biopolymers, V120 and A-V120, as the shorter constructs did not produce appreciable changes in transmittance within the temperature limit of the spectrophotometer, at concentrations up to 1 mM (Figure S2). Tt was found to decrease with increasing concentration for both V120 and A-V120 by turbidimetry (Figure 2B). The Tt for all constructs was also calculated by DLS (Table 1). 25 µM samples were incubated at 1° C increments until particle formation was observed, indicated by a sharp spike in the calculated diameter (Figure 2C). The longer, more hydrophobic V120 constructs aggregated at much lower temperatures (~34 °C) than the V40 constructs (~52 °C), as was expected. DLS was also used to calculate the mean effective particle diameter and polydispersity index of each construct. 25 µM samples were incubated at 60 °C for 10 minutes prior to analysis. The lognormal size distributions for V40 and V120 are given in Figure 2C along with a 90 nm particle control. Distributions for A-V40 and A-V120 are given in Figure S3 in the Supporting Information. The average particle diameters were fairly similar for all four biopolymers, ranging from approximately 520 to 560 nm. However, there was a considerable difference in the polydispersity index. The V120 particles assembled into more monodisperse populations whereas the V40 particles exhibited a more polydisperse distribution (Figure 2D, Table 1). Two different length ELP blocks composed of the sequence [VPGVG]n were built in order to control ELP microstructure at physiological temperature. We opted to achieve this control by adjusting the biopolymer length as opposed to the hydrophobicity of the guest residue to maintain consistent substrate specificity across all ELPs used in the study. The longer, more hydrophobic V120 ELP rapidly forms sub-micron structures at low micromolar concentrations, whereas the shorter V40 ELP requires much higher temperature and/or concentration to achieve phase separation. This relationship between ELP chain length, concentration, and thermal responsive behavior is well established.34 Despite the difference in Tt, both biopolymers formed sub-micron particles approximately 540 nm in diameter at 60 °C. The large particle size indicates the formation of higher order supramolecular aggregates as opposed


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to micellar structures. ELPs of similar length and composition have been previously shown to separate into sub-micron particles, driven thermodynamically by induced conformational changes in the ELP and subsequent dehydration.24,34,35 It is important to note that appending the short ARA290 peptide to the ELP blocks minimally affected the biophysical properties of the resultant aggregates in terms of Tt, diameter, and polydispersity. This is unsurprising due to the disparate difference in molecular weight between the peptide and the larger ELP block. Short peptide fusions have been previously shown to have no effect on ELP properties in other studies as well.36

Proteolytic stability of ELP biopolymers In order to model the stability of the ELP biopolymers in the harsh wound microenvironment, they were incubated in an array of enzymes including lysozyme, collagenase, and elastase, which are all upregulated following a cutaneous burn injury.37-39 Fluorescently labeled ELPs were pre-incubated at 37 °C to induce aggregation of V120; at these conditions, V40 should remain in its soluble, monomeric form. The fraction of intact ELP following enzymatic digestion was calculated using quantitative SDS-PAGE analysis in ImageJ (Figure S4). Samples containing enzyme were compared to ELP alone and the endpoint comparison is given in Figure 3A for V40 and V120. Lysozyme, which catalyzes the hydrolysis of 1,4-β linkages in peptidoglycans, showed no activity toward either ELP, which was expected.40 Interestingly, the ELP stability in collagenase was quite different depending on the ELP chain length. The shorter V40 construct was susceptible to collagenase degradation with less than 40 % of the original sample remaining after 3 hours. The longer biopolymer was shown to remain largely stable throughout the entire time period assayed. Both V40 and V120 were shown to be susceptible to elastase activity, seemingly regardless of phase separation, as evidenced by a marked drop in fluorescence after the 3-hour incubation (Figure 3A). A time course experiment was also performed using the same format (Figure 3B, C) where individual fractions were pulled at various times and the amount of intact ELP was quantified. Again, lysozyme showed no activity toward either V40 or V120. Collagenase was shown to cut the soluble V40


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polymer at a fairly constant rate during the incubation. V120, however, was resistant to collagenase activity. Elastase catalyzed the cleavage of both constructs in a kinetically similar fashion with nearly all of the ELP degraded after 3 hours. Tuning these proteins on the genetic level to produce ELPs with widely different Tts allowed us to specifically investigate the effects of phase separation on proteolytic digestion. Previous studies have investigated ELP hydrogel erosion rates and micelle-forming ELP biodegradation in a variety of enzymes including elastase and collagenase.41-43 Elastases are serine proteases secreted by neutrophils and macrophages in cutaneous wound sites that cleave peptide bonds on the carbonyl side of small, hydrophobic amino acids including glycine, valine, and alanine.44 The ELPs used in this study are primarily composed glycine (40 %) and valine (40 %), so it is not surprising that elastase has high activity toward these substrates; each VPGVG repeat contains 4 potential cleavage sites. Collagenases represent a broad class of proteases which target collagen-rich extracellular structures and have been shown to act on a variety of substrates, both naturally occurring and synthetic.45,46 MMP-2 and MMP-9 are both type IV collagenases upregulated in wound exudate and are responsible for breaking down damaged matrix proteins as well as modulating inflammatory and cellular signaling in the wound bed.39 Although the prototypical cleavage site for collagenases is at the peptide bond between X-G in the sequence -P-X-G-P-, which is not present in the ELP pentapeptide sequence, specificity toward ELPs has been previously reported.41,43 Non-specific cleavage may partly be due to impurities present in the commercially purchased enzyme, which contains 7 isoforms of collagenase.47 Regardless, phase separation clearly protects V120 from collagenase degradation as nearly all the ELP remained intact throughout the entire experiment. This can most likely be attributed to a decrease in protease accessibility to the ELP chains when aggregated. Additionally, the elastase (25 kDa) used in this study is considerably smaller in size when compared to the collagenases (70 – 130 kDa), which may allow for greater ease of access to the ELP, even after aggregation. Protection from collagenase activity via phase separation is consistent with claims by Shah et al. for similar ELP-based materials.43


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Fusions to ELP Enhance Performance of ARA290 under Proteolytic and Thermal Stress Human dermal microvascular endothelial cells (HMVEC) were used to assess the cellular protectant activity of the ARA290 peptide following thermal shock. Cells were seeded in 24-well tissue culture plates and allowed to proliferate until confluent. The plates were submerged for 20 minutes in a 55 °C water bath to induce a thermal shock. After which ARA290, ELP, and ARA290-ELP fusions were added to the wells. The cells were allowed to recover for 24 hours and the number of dead cells were quantified and compared to control groups. In media, both fusion proteins as well as the peptide alone protected HMVEC from apoptosis following heat shock with the A-V120 construct salvaging over 80 % of the cells at 2 µM compared to untreated cells (Figure 4A). This protective activity was confirmed by microscopic observation of HMVEC 6 hours after heat shock. HMVEC cells treated with V120 only exhibited an elongated morphology due to cell shrinkage and a refringent aspect as cells began to detach from the tissue culture plates, all indicative of cell death (Figure 5A). Those treated with ARA290 fusions maintained a viable, confluent monolayer (Figure 5B). A dose-dependent response was observed for all active constructs tested. In media supplemented with collagenase, ELP fusions to ARA290 retained a dose-dependent response with A-V120 achieving up to 70 % cell survival at 2 µM (Figure 4B). The peptide alone performed similarly to the unfused ELP control groups, indicating its instability in collagenase. In the presence of elastase, a marked drop in cell survival was observed across all groups tested (Figure 4C). In the absence of proteases, all three active groups offered some degree of anti-apoptotic activity, presumably mediated through the alternative EPO signaling pathway. At low doses (0.1 – 0.2 µM), the peptide alone was more effective than A-V40 or A-V120, which may speak to the somewhat decreased bioavailability of the peptide when fused to the ELP. This is most likely caused by potential blockage of the EPO/CD131 heteroreceptor by elongated ELP chains (A-V40) or ligand burial beneath the particle surface (A-V210). There was an interesting discrepancy observed between A-V40 and A-V120 cellular protectant effects in the presence of proteinase. Despite equal molar loading, A-V120 significantly outperforms A-V40 at 2 µM doses (71 % cell survival vs 42 % in collagenase, p < 0.005; 42 % cell


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survival vs 28 % in elastase, p < 0.05). This may be due in part to the multivalent presentation of the ARA290 peptide on the V120 aggregate, leading to increased avidity and potential interaction with multiple EPO-R/CD131 heteroreceptors simultaneously. Similar effects were observed for micelleforming ELPs functionalized with an RGD ligand on the particle corona for recognition of αvβ3 cell surface integrins; although in our study the ligand is not specifically directed to the particle surface.36 The longer ELP chains also simply provide more non-active substrate for the enzymes to digest, which may also contribute to the enhanced activity of A-V120. The ARA290 peptide alone appeared to be susceptible to biodegradation by both proteases as evidenced by a drop in cell survival from 77 % in media alone to 21 % in collagenase and 16 % in elastase at 2 µM doses. The primary sequence contains several potential cleavage sites for elastase activity which most likely accounts for the more pronounced effect on cell viability when compared to collagenase. Fusion to the V120 ELP significantly enhanced the cellular protectant activity of ARA290 in the presence of both collagenase and elastase, when compared to the peptide alone at 2 µM treatments (70 % cell survival vs 24% in collagenase, p < 0.005; 42% cell survival vs 16% in elastase, p < 0.005).

CONCLUSIONS In this work, we investigate the advantages of creating ARA290 fusions to ELP sub-micron particle carriers. Genetically functionalized ELP sub-micron particles are excellent candidates for drug delivery vehicles and have been used to deliver biologically active cargoes to a variety of tissues.24,35,48,49 High yield recombinant expression, rapid purification by ITC, biocompatibility, and precise control over the biopolymer properties set these materials apart from synthetic polymer systems. We have demonstrated the ability of ARA290-ELP fusions to salvage thermally shocked microvascular cells in the presence of enzymes upregulated following burn injury. These promising results offer a new strategy for reliable delivery of the ARA290 peptide to harsh wound microenvironments.

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Supporting Information Oligonucleotide sequences for all cloning experiments. Primary sequences for all protein constructs. SDSPAGE results for all protein constructs. ELP and ELP fusion characterization by turbidimetry and DLS. This material is available free of charge via the Internet at http://pubs.acs.org

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was funded by the Shriners Hospitals for Children. J.D. was supported by a Shriners Hospital for Children postdoctoral fellowship award.

REFERENCES (1)

Huber, M. C.; Schreiber, A.; Wild, W.; Benz, K.; Schiller, S. M. Biomaterials 2014, 35,

8767. (2) Hassouneh, W.; Fischer, K.; MacEwan, S. R.; Branscheid, R.; Fu, C. L.; Liu, R.; Schmidt, M.; Chilkoti, A. Biomacromolecules 2012, 13, 1598. (3) Sengupta, D.; Heilshorn, S. C. Tissue Eng., Part B 2010, 16, 285. (4) Dooley, K.; Bulutoglu, B.; Banta, S. Biomacromolecules 2014, 15, 3617. (5) Dooley, K.; Kim, Y. H.; Lu, H. D.; Tu, R.; Banta, S. Biomacromolecules 2012, 13, 1758. (6) Hunt, N. C.; Grover, L. M. Biotechnol. Lett. 2010, 32, 733. (7) MacEwan, S. R.; Chilkoti, A. Pept. Sci. 2010, 94, 60. (8) Kim, Y. H.; Campbell, E.; Yu, J.; Minteer, S. D.; Banta, S. Angew. Chem. Int. Ed. 2013, 52, 1437. (9) Langer, R.; Tirrell, D. A. Nature 2004, 428, 487. (10) Hu, X.; Cebe, P.; Weiss, A. S.; Omenetto, F.; Kaplan, D. L. Mater. Today 2012, 15, 208. (11) Guvendiren, M.; Lu, H. D.; Burdick, J. A. Soft Matter 2012, 8, 260. (12) Stoppel, W. L.; Ghezzi, C. E.; McNamara, S. L.; Black III, L. D.; Kaplan, D. L. Ann. Biomed. Eng. 2015, 43, 657. (13) Chilkoti, A.; Dreher, M. R.; Meyer, D. E.; Raucher, D. Adv. Drug Delivery Rev. 2002, 54, 613.


Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 22

(14) Bidwell, G. L.; Fokt, I.; Priebe, W.; Raucher, D. Biochem. Pharmacol. 2007, 73, 620. (15) Hassouneh, W.; Christensen, T.; Chilkoti, A. Curr. Protoc. Protein Sci. 2010, 6.11. 1. (16) McDaniel, J. R.; Callahan, D. J.; Chilkoti, A. Adv. Drug Delivery Rev. 2010, 62, 1456. (17) Almine, J. F.; Bax, D. V.; Mithieux, S. M.; Nivison-Smith, L.; Rnjak, J.; Waterhouse, A.; Wise, S. G.; Weiss, A. S. Chem. Soc. Rev. 2010, 39, 3371. (18) Hop, M. J.; Polinder, S.; Vlies, C. H.; Middelkoop, E.; Baar, M. E. Wound Repair Regen. 2014, 22, 436. (19) Jackson, D. M. Br. J. Surg. 1953, 40, 588. (20) Nisanci, M.; Eski, M.; Sahin, I.; Ilgan, S.; Isik, S. Burns 2010, 36, 397. (21) Bohr, S.; Patel, S. J.; Shen, K.; Vitalo, A. G.; Brines, M.; Cerami, A.; Berthiaume, F.; Yarmush, M. L. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 3513. (22) Gibot, L.; Galbraith, T.; Huot, J.; Auger, F. A. Tissue Eng., Part A 2010, 16, 3199. (23) MacNeil, S. Nature 2007, 445, 874. (24) Koria, P.; Yagi, H.; Kitagawa, Y.; Megeed, Z.; Nahmias, Y.; Sheridan, R.; Yarmush, M. L. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 1034. (25) Brines, M.; Patel, N. S.; Villa, P.; Brines, C.; Mennini, T.; De Paola, M.; Erbayraktar, Z.; Erbayraktar, S.; Sepodes, B.; Thiemermann, C. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 10925. (26) Hand, C. C.; Brines, M. J. Investig. Med. 2011, 59, 1073. (27) Bohr, S.; Patel, S. J.; Vasko, R.; Shen, K.; Iracheta-Vellve, A.; Lee, J.; Bale, S. S.; Chakraborty, N.; Brines, M.; Cerami, A. J. Mol. Med. 2015, 93, 199. (28) Bennis, Y.; Sarlon-Bartoli, G.; Guillet, B.; Hubert, L.; Pellegrini, L.; Velly, L.; Blot-Chabaud, M.; Dignat-George, F.; Sabatier, F.; Pisano, P. J. Thromb. Haemostasis 2012, 10, 1914. (29) van Royen, N.; Hoefer, I.; Buschmann, I.; Heil, M.; Kostin, S.; Deindl, E.; Vogel, S.; Korff, T.; Augustin, H.; Bode, C. FASEB J. 2002, 16, 432. (30) Robson, M. C. Wound Repair Regen. 1997, 5, 12. (31) Eming, S. A.; Krieg, T.; Davidson, J. M. J. Invest. Dermatol. 2007, 127, 514. (32) McDaniel, J. R.; MacKay, J. A.; Quiroz, F. G.; Chilkoti, A. Biomacromolecules 2010, 11, 944. (33) Guda, C.; Zhang, X.; McPherson, D.; Xu, J.; Cherry, J.; Urry, D.; Daniell, H. Biotechnol. Lett. 1995, 17, 745. (34) Meyer, D. E.; Chilkoti, A. Biomacromolecules 2004, 5, 846. (35) Shamji, M. F.; Chen, J.; Friedman, A. H.; Richardson, W. J.; Chilkoti, A.; Setton, L. A. J. Controlled Release 2008, 129, 179. (36) Simnick, A. J.; Valencia, C. A.; Liu, R.; Chilkoti, A. ACS Nano 2010, 4, 2217. (37) Cutting, K. F. Br. J. Community Nurs. 2003, 8, 4. (38) Krejner, A.; Grzela, T. Cent. Eur. J. Immunol. 2015, 40, 391. (39) Nessler, M. B.; Puchała, J.; Chrapusta, A.; Nessler, K.; Drukała, J. Med. Sci. Monit. 2013, 20, 91. (40) Smith, L.; Mohr, L.; Raftery, M. J. Am. Chem. Soc. 1973, 95, 7497. (41) Nettles, D. L.; Chilkoti, A.; Setton, L. A. Adv. Drug Delivery Rev. 2010, 62, 1479. (42) Ong, S. R.; Trabbic-Carlson, K. A.; Nettles, D. L.; Lim, D. W.; Chilkoti, A.; Setton, L. A. Biomaterials 2006, 27, 1930. (43) Shah, M.; Hsueh, P. Y.; Sun, G.; Chang, H. Y.; Janib, S. M.; MacKay, J. A. Protein Sci. 2012, 21, 743. (44) Herrick, S.; Ashcroft, G.; Ireland, G.; Horan, M.; McCollum, C.; Ferguson, M. Lab. Invest. 1997, 77, 281. (45) Ågren, M. S.; Taplin, C. J.; Woessner, J. F.; Eagistein, W. H.; Mertz, P. M. J. Invest. Dermatol. 1992, 99, 709.


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(46) Dive, V.; Yiotakis, A.; Nicolaou, A.; Toma, F. Eur. J. Biochem. 1990, 191, 685. (47) Angleton, E. L.; Van Wart, H. E. Biochemistry 1988, 27, 7406. (48) Shamji, M. F.; Betre, H.; Kraus, V. B.; Chen, J.; Chilkoti, A.; Pichika, R.; Masuda, K.; Setton, L. A. Arthritis Rheum. 2007, 56, 3650. (49) MacEwan, S. R.; Chilkoti, A. J. Controlled Release 2014, 190, 314.

TABLES

Effective Particle Dia. (nm)

Construct

MW (kDa)

Tt (°C)

Polydispersity Index

V40

18.0

51.7

535 ± 10

0.337

A-V40

19.3

53.6

522 ± 8

0.351

V120

50.8

33.9

543 ± 16

0.052

A-V120

52.1

34.0

562 ± 7

0.015

Table 1. ELP and ARA290-ELP fusion protein properties. The expected molecular weights were obtained from the ExPASy ProtParam tool. The transition temperatures and particle sizing data were calculated using dynamic light scattering measurements at 25 µM.


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FIGURES

Figure 1. ARA290 peptide derivation and ELP fusion protein design. (A) Erythropoietin structure (PDB 1BUY) with the surface exposed residues from Helix B used to create the linear ARA290 peptide, highlighted in blue. The primary sequence for ARA290 is given. (B) The ARA290 peptide was appended to the N-terminus of both V40 and V120 ELP blocks. (C) The resulting fusion proteins reversibly aggregate into sub-micron particles in response to increasing temperature.


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Figure 2. ELP characterization. (A) Percent transmittance measurements at 350 nm for 1-100 µM samples of A-V120 over a range of temperatures. (B) Calculated Tt by turbidimetry for V120 and A-V120 over a range of concentrations. All measurements were made in triplicate and the error bars represent standard deviations. (C) Tt measurements via DLS at 25 µM for all constructs over a range of temperatures. (D) Lognormal particle size distributions at 25 µM for V120, V40, and a 90 nm size standard at 60 °C. Characterization data for all constructs is tabulated in Table 1.


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Figure 3. Enzymatic degradation rates of ELPs. Fluorescently labeled V40 and V120 were incubated in lysozyme, collagenase, and elastase for 3 hours at 37 °C. Particle stability was assessed using quantitative SDS-PAGE. Endpoint data for both ELPs in each enzyme as well as a control with no enzyme are given in panel (A). Time course data for V40 and V120 in each enzyme are given in panels (B) and (C), respectively. All experiments were performed in triplicate and error bars represent standard deviations.

Figure 4. Cell survival following thermal shock. HMVEC cells were subjected to 55 °C thermal shock for 20 minutes. The ARA290 peptide, V40, V120 and ARA290-ELP fusions were added and the cell survival percentages were calculated after 24 hours in media (A), media supplemented with 3.3 U/mL collagenase (B), and 1.0 mU/mL elastase (C). All experiments were performed in triplicate and the error bars represent standard errors.


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Figure 5. HMVEC cell morphology 6 hours post heat shock. Cells treated with V120 (A) and A-V120 (B) were imaged at 6 hours after induced thermal shock at 55 °C. The cells treated with V120 alone began to detach from the tissue culture plate as indicated by the white outline around the cells. They also exhibited morphological abnormalities consistent with cell death. Cells treated with the ARA290 fusion maintained a confluent, viable monolayer at 6 hours. Scale bar = 20 µm.


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Table of Contents Graphic


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Functionalized Biopolymer Particles Enhance Performance of a Tissue

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