Array-Based High-Throughput Analysis of Silk-Elastinlike Protein

Apr 25, 2016 - Departments of Phamaceutics and Pharmaceutical Chemistry, and Bioengineering, Center for Nanomedicine, Nano Institute of Utah, Universi...
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Array-Based High-Throughput Analysis of Silk-Elastinlike Protein Polymer Degradation and C‑Peptide Release by Proteases Hye-Yoon Jeon,†,¶ Se-Hui Jung,†,¶ Young Mee Jung,‡ Young-Myeong Kim,† Hamidreza Ghandehari,*,§,∥ and Kwon-Soo Ha*,† †

Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, Korea ‡ Department of Chemistry, Kangwon National University, Chuncheon, Kangwon-Do 200-701, Korea § Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, Korea ∥ Departments of Phamaceutics and Pharmaceutical Chemistry, and Bioengineering, Center for Nanomedicine, Nano Institute of Utah, University of Utah, Salt Lake City, Utah 84112, United States S Supporting Information *

ABSTRACT: The objective of this study was to utilize an onchip degradation assay to evaluate polymer depots and the predicted drug release from the depots. We conjugated four silkelastinlike protein (SELP) polymers including SELP-815K, SELP-815K-RS1, SELP-815K-RS2, and SELP-815K-RS5 with a Cy5-NHS ester and fabricated SELP arrays by immobilizing the conjugated polymers onto well-type amine arrays. SELP polymer degradation rates were investigated by calculating the halfmaximal effective concentration (EC50). Eight cleavage enzymes were applied, all of which exhibited distinctive EC50 values for SELP-815K and its three analogues. We successfully utilized this assay to study the in vitro release of the Cy5-conjugated Cpeptide from SELP-815K hydrogel arrays. Additionally, cumulative C-peptide release from the SELP-815K depots was also demonstrated using repetitive elastase treatments. Therefore, this array-based on-chip degradation assay could potentially be used for evaluating depot degradation and controlled drug release from polymer depots at the molecular level.

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Drug release from hydrogel matrices is triggered by swelling, erosion, and/or enzymatic degradation of polymer strands.6,7 Thus, hydrogel degradation and drug release are dependent on environmental conditions, including pH, ionic composition, and/or enzyme concentration near the implantation site.3 Drug release has been widely investigated by in vitro assays using hydrogel discs;8−10 however, this approach is not suitable for high-throughput analyses. In vivo drug release has also been studied using animal models implanted with hydrogel containing drugs,11,12 but in vivo studies require animal use and are expensive, labor-intensive, and time-consuming.13,14 Thus, an in vitro assay suitable for analyzing drug release and depot degradation in a high-throughput manner is necessary. To this end, we selected silk-elastinlike protein polymers (SELPs) composed of amino acid motifs in the form of silkworm silk fibroin (GAGAGS) and mammalian elastin (GVGVP) (Figure 1A).8 The physicochemical properties of

ocal delivery systems have significant clinical utility due to their controlled release of drugs for a longer period of time, which enables reduced administration frequency.1,2 A number of polymer-based matrices have been developed over the past few decades to improve the efficacy and duration of drug delivery and to minimize associated side effects.3 These delivery systems are often implanted at the target site in a variety of shapes, including film, gel, wafer, rod, and particle.1 Most depots are designed to provide spatiotemporally controlled delivery and to be clearly eliminated from the body after drug delivery.4 The polymers used for localized delivery can be broadly divided into synthetic and natural materials.5 Synthetic polymers are suitable for the design of customized depots according to their application by fine chemical tuning, but accumulation of acidic degradation products can cause immune response near the implantation site.1 Conversely, natural polymers can form hydrogels via cross-linking or self-assembly and can thus be used in vivo as biocompatible and biodegradable matrices.1,5 © 2016 American Chemical Society

Received: February 24, 2016 Accepted: April 24, 2016 Published: April 25, 2016 5398

DOI: 10.1021/acs.analchem.6b00739 Anal. Chem. 2016, 88, 5398−5405

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Analytical Chemistry

Figure 1. Schematic diagram of amino acid sequences of SELP-815K and its three analogues (A) and the array-based enzymatic degradation profiling (B).

drug release from SELP arrays as a function of enzyme concentration. This array-based on-chip degradation assay provides for simple and rapid characterization of polymeric systems and for simulation of drug release.

SELPs, including thermosensitive sol−gel transition and slow strand degradation, enable their utilization as injectable bioactive agent-loaded delivery systems and ensures the spatiotemporally controlled release of bioactive agents.15 These physicochemical properties are dictated by the ratio and sequence of silk to elastin components, which can be tightly controlled by using recombinant DNA technology.8 Hydrogels made from the SELP analogue SELP-815K showed capability for sustained delivery of adenoviruses to solid tumors up to 12 weeks.15,16 To increase the degradation rate of SELPs and to enhance drug delivery, three types of SELP-815K analogues were synthesized via the insertion of amino acid sequences known to be cleaved specifically by matrix metalloproteinases (MMPs).8,9,17 Further, the three SELP-815K analogues containing an MMP-responsive sequence were characterized by investigating the effect of the inset location on physicochemical properties.8,9,17 However, the rates of polymer degradation and drug release at the molecular level remain unclear. In this study, we used an array-based high-throughput assay system to determine depot degradation and drug release. We fabricated SELP arrays using Cy5-conjugated SELP-815K and its three analogues in a well-type amine array and determined SELP analogue degradation rates using eight cleavage enzymes and a fluorescence scanner. Further, we determined C-peptide

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EXPERIMENTAL SECTION

Chemical Reagents. Recombinant SELP-815K, SELP815K-RS1, SELP-815K-RS2, and SELP-815K-RS5 were synthesized and purified as previously described.8,9 The structures and sequences of these copolymers are shown in Figure 1A. (3-Aminopropyl)trimethoxysilane, pepsin, thrombin, and trypsin were purchased from Sigma-Aldrich (St. Louis, MO). Elastase-2 was purchased from Elastin Products Company (Owensville, MO). Collagenase-2 was obtained from Worthington Biochemical Corporation (Lakewood, NJ). Calpain-1 and caspase-9 were obtained from Calbiochem (Darmstadt, Germany). The catalytic domain (Phe100-Pro273) of human matrix metalloproteinase-3 (cMMP-3) was kindly provided by Professor Seung-Taek Lee (Department of Biochemistry, Yonsei University, South Korea). Poly(dimethylsiloxane) (PDMS) solution was acquired from Sewang Hitech (Gimpo, Korea). Cy3 mono N-hydroxysuccinimide (NHS)-ester and Biospin columns were purchased from GE Healthcare (Little Chalfont, UK) and Bio-Rad (Hercules, CA), respectively. 5399

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Analytical Chemistry Fabrication of Well-Type Amine Arrays Using PDMS Gaskets. Well-type amine arrays were fabricated as previously reported.18 Briefly, glass slides (75 × 25 mm) were cleaned with an H2O2/NH4OH/H2O solution (1:1:5, v/v), immersed in 1.5% (3-aminopropyl)trimethoxysilane (v/v) for 2 h and baked at 110 °C. PDMS prepolymer solution was prepared by mixing 5 g of PDMS base and 0.5 g of curing agent until it became cloudy with bubbles. Degassing was performed for 30 min, and the mixture was poured into a chrome-coated copper mold with arrayed poles (1.5 mm diameter and 0.3 mm height; Amogreen Tech, Kimpo, Korea). The mold was incubated at 84 °C for 90 min, and PDMS gaskets containing 3 mm-diameter arrayed holes were detached. Well-type amine arrays were then fabricated by mounting the PDMS gaskets onto the aminemodified glass slides. Conjugation of SELP Analogues with Cy5-NHS Ester. SELP analogues, including SELP-815K, SELP-815K-RS1, SELP-815K-RS2, and SELP-815K-RS5 were labeled with Cy5 NHS-ester as previously reported.19 Briefly, 1 mL of each 100 μg/mL SELP solution in 100 mM sodium bicarbonate buffer (pH 8.3) was mixed with 10 μL of 1.2 mg/mL Cy5 mono NHS-ester in 10% dimethyl sulfoxide and incubated for 2 h on ice. To quench the reaction, 50 μL of 1 M Tris-HCl (pH 8.0) was added to the reaction solution. Reaction mixtures were loaded onto 1.5 mL Sephadex G-25 columns, and Cy5conjugated SELPs were eluted by centrifugation for 3 min at 1050g. To calculate molar concentrations of Cy5 and SELP in the Cy5-conjugated SELPs, reactions containing designated concentrations of pure SELP and BCA solution were conducted for 30 min at 37 °C (Pierce, Rockford, IL). The absorbances of SELP and Cy5 at 562 and 633 nm, respectively, were then obtained using the Molecular Devices VersaMax Tunable Microplate reader (Sunnyvale, CA). SELP concentrations in the conjugates were directly determined by measurement of absorption at 562 nm using eq 1: y = [(A1 − A 2 )/(1 + (x /x0) p )] + A 2

equipped with a liquid nitrogen-cooled MCT detector. The thin film of SELP strands was formed by applying 40 μL of 50 μg/mL SELP-815K solution to platinum-coated silicon wafer and incubating for 3 h at 37 °C in a humidity chamber. Chilled 100 μg/mL elastase-2 solution (40 μL) was applied to the SELP-coated silicon wafer and incubated for 2 h. The resulting wafers were set on the Bruker A513 reflection attachment with an incidence angle at 80°, which includes a heating block attachment. A total of 1024 scans were coadded for each IR spectral measurement. Degradation Profiling of SELPs Using Eight Cleavage Enzymes. For enzymatic degradation profiling of SELP polymers, we used eight cleavage enzymes including seven enzymes in circulating blood (elastase-2, collagenase-2, trypsin, calpain-1, caspase-9, cMMP-3, and thrombin) and pepsin, which is secreted into the stomach. A 10 μg/mL aliquot of each of the Cy5-conjugated SELPs was applied to the well-type amine arrays for 3 h at 37 °C to allow network formation. The arrays were incubated for 15 min at 37 °C with various concentrations of elastase-2 in buffer A (100 mM Tris-HCl and 100 mM NaCl, pH 7.5), collagenase-2 in buffer B (50 mM TrisHCl, 150 mM NaCl, 5 mM CaCl2, 0.2 mM NaN3, and 0.002% Brij-35, pH 7.6), trypsin in buffer C (50 mM Tris-HCl, 20 mM CaCl2, and 0.02% Tween-20, pH 8.1), calpain-1 in buffer D (50 mM Tris-HCl, 5 mM CaCl2, 0.5 mM ZnCl2, 5 mM βmercaptoethanol, and 0.002% Brij-35, pH 7.4), caspase-9 in buffer E (50 mM HEPES, 1 mM EDTA, 100 mM NaCl, 10 mM DTT, and 0.002% Brij-35, pH 7.2), cMMP-3 in buffer F (20 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, 0.5 mM ZnCl2, and 0.002% Brij-35, pH 7.4), thrombin in buffer G (20 mM Tris-HCl, 100 mM NaCl, 0.3 mM CaCl2, 1 mM DTT, and 0.1% Tween-20, pH 8.5), and pepsin in buffer H (10 mM HCl, pH 2.0). The arrays were washed with phosphate-buffered saline (PBS) containing 0.1% Tween-20 for 10 min and then with Milli-Q water for 5 min, dried under air, and analyzed using a fluorescence scanner equipped with a 633 nm laser (ScanArray Express GX, PerkinElmer, Waltham, MA). The amounts of SELP degradation by cleavage enzymes were calculated by subtracting the fluorescence intensities of spots incubated with the enzymes from the fluorescence intensities following incubation with the buffers and were expressed as relative fluorescence intensity (RFI). Determination of Half-Maximal Effective Concentrations for Cleavage Enzymes. We calculated the half-maximal effective concentrations (EC50) for cleavage enzymes by the following modified Langmuir isotherm using GraphPad PRISM (GraphPad Software; San Diego, CA):

(1)

The molar extinction coefficients of Cy5 at 562 and 633 nm were then measured, and the Cy5 concentrations in the conjugates were determined using eq 2, which was corrected for the absorbance of SELP reacted with the BCA solution at 633 nm: [Cy5] = (A 633 − (a × [SELP])/b)

(2)

where a is the molar extinction coefficient of SELP reacted with BCA solution at 633 nm, [SELP] is the concentration of SELP in the conjugates, and b is the molar extinction coefficient of Cy5 at 633 nm. Atomic Force Microscopy Imaging. SELP networks were formed by applying 40 μL of 50 μg/mL SELP-815K solution to freshly cleaved mica and incubating for 3 h at 37 °C in a humidity chamber. Chilled 100 μg/mL elastase-2 solution (40 μL) was applied to the SELP-coated mica and incubated for 2 h. The atomic force microscopy (AFM) imaging was then performed under air using a multimode microscope with a Nanoscope V controller installed with an E-type scanner (15 × 15 μm) (Bruker Nano) in tapping mode at a scanning rate of 0.1−1 Hz as described previously.4 Silicon cantilevers with a spring constant of 42 N/m were used for imaging. Characterization of SELP Degradation by Fourier Transform Infrared (FT-IR) Spectroscopy. The infraredreflection absorbance (IRRAS) spectra were collected at a 4 cm−1 resolution with a Bruker Vertex 80v FT-IR spectrometer

Fobs = (Fmax × [enzyme]/EC50 + [enxyme]) + background (3)

where Fobs is the fluorescence intensity of triplicate spots, Fmax is the maximum fluorescence at saturation, [enzyme] is the enzyme concentration, and EC50 is the apparent half-maximal effective concentration. C-Peptide Release from SELP-815K Hydrogel. Cy5conjugated C-peptide (100 μg/mL) was mixed (1:1, v/v) with 100 μg/mL SELP-815K and applied to the well-type amine arrays for 3 h. Reaction mixtures containing various concentrations of the cleavage enzymes elastase-2, collagenase-2, and trypsin were then applied to the SELP arrays for 2 h at 37 °C, and the arrays were analyzed using a fluorescence scanner. The amount of C-peptide released from the hydrogels was calculated by subtracting the fluorescence intensities of 5400

DOI: 10.1021/acs.analchem.6b00739 Anal. Chem. 2016, 88, 5398−5405

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Figure 2. Characterization of the on-chip degradation assay. (A, B) On-chip degradation of SELP-815K by elastase. Reaction mixtures containing the indicated concentrations of elastase-2 were applied to the SELP arrays and incubated at 37 °C for 15 min. The resulting arrays were analyzed using a fluorescence scanner. (A) A representative image of SELP degradation by elastase. (B) SELP degradation by elastase was determined by analyzing fluorescence intensities of the array spots. The results are expressed as the mean ± SD of three independent experiments. (C) Morphological analysis of SELP-815K degradation by elastase using AFM. The SELP depots were incubated in the absence or presence of 100 μg/mL elastase and subjected to the AFM imaging tapping mode. Scale bar, 300 nm. (D) FT-IR spectra of SELP-815K in the absence or presence of elastase-2.

spots treated with an enzyme from the fluorescence intensities of spots treated with reaction buffer only. Data Analyses. ScanArray Express software (PerkinElmer) was used for quantifying fluorescence intensities of array spots and for data extraction.

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evaluation of drug release at the molecular level is limited and is also expensive, labor-intensive, and time-consuming.13,14 Arrays are a promising technology for the high-throughput analysis of enzyme activity and molecular interactions, because they require very small sample volumes and have reduced reaction times and sensitivity is often increased due to shorter diffusion distances.20 Accordingly, array technology has been used to determine the activities of various cleavage enzymes including caspase-3, caspase-9, calpain-1, and cMMP-3.21−23 SELPs are recombinant polymers with precise control over molecular weight and sequence. Inclusion of MMP responsive sequences in the polymers renders them degradable under conditions where MMP is overexpressed. To investigate SELP

RESULTS AND DISCUSSION

Characterization of Cy5-Conjugated SELP-815K. Local depots have emerged as a significant clinical solution for efficient drug delivery due to their sustained drug release for a long period of time with only a single implantation.1 Polymer depots are predominantly evaluated by measuring drug levels or therapeutic effects of the drugs in animal models, but in vivo 5401

DOI: 10.1021/acs.analchem.6b00739 Anal. Chem. 2016, 88, 5398−5405

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Analytical Chemistry

Figure 3. Quantitative analysis of the enzymatic degradation of SELP-815K by seven cleavage enzymes. SELP-815K arrays were fabricated by immobilizing 10 μg/mL Cy5-conjugated SELP-815K onto well-type amine arrays. Reaction mixtures containing the indicated concentrations of seven representative cleavage enzymes (collagenase-2, trypsin, calpain-1, caspase-9, cMMP-3, thrombin, and pepsin) were applied to the arrays and incubated at 37 °C for 15 min. The resulting arrays were analyzed using a fluorescence scanner and the amount of SELP polymers cleaved by each of the seven cleavage enzymes was expressed as the RFI. Data are expressed as mean ± SD from three independent experiments.

degradation and drug release from the SELP hydrogels in a high-throughput manner, we developed an array-based on-chip degradation assay and used four SELP polymers and eight cleavage enzymes to characterize the assay. SELP-815K and its three analogues SELP-815K-RS1, SELP-815K-RS2, and SELP815K-RS5 containing MMP-responsive degradable sequences strategically located in the polymer backbone8,9,17 were conjugated with a Cy5 NHS-ester to visualize their degradation by cleavage enzymes as illustrated in Figure 1. To calculate molar concentrations of Cy5 and SELP-815K in Cy5-conjugated SELP polymers, we obtained a standard curve by measuring the absorbance at 562 nm as a function of pure SELP concentration after reacting with a BCA solution. We then measured the molar extinction coefficients of SELP-815K at 633 nm and those of Cy5 at 562 and 633 nm and determined that the molar extinction coefficients of SELP and of Cy5 at 633 nm were 219 500 and 109 600 M−1cm−1, respectively. However, the molar extinction coefficient of Cy5 at 562 nm was very low (3600 M−1cm−1, approximately 3% of the Cy5 extinction coefficient at 633 nm). We then reacted 1.27 pmole of SELP-815K with 2-, 5-, 10-, 20-, and 40-fold molar excesses of the Cy5 NHS-ester and determined the concentrations of SELP-815K and Cy5 in Cy5-conjugated SELP-815K. The concentration of Cy5 in the Cy5-conjugates increased in proportion to the molar excess of the Cy5 NHS ester, and the molar ratio of Cy5 to SELP-815K increased from 1.8 to 4.7.

The SELP-815K concentration in the Cy5 conjugates increased slightly according to the molar ratio of Cy5 to SELP-815K. Covalent conjugation of SELP polymers with a fluorophore might affect the three-dimensional structure and degradability of hydrogels because the lysine residues used for the conjugation are important for interaction of SELP strands;24 thus, we determined the optimal molar ratio of Cy5 NHS-ester to SELP-815K for conjugation by using the arrays to measure the degradation rate of the conjugates by elastase-2. The degradation of SELP conjugates by elastase-2 was determined by calculating the EC50 based on the amounts of Cy5conjugated SELP-815K that detached from the arrays. The EC50 value was the lowest (0.004 μg/mL) at the 10:1 ratio (Cy5 NHS-ester: SELP-815K), which suggested a minimal conjugation effect on enzymatic cleavage sites. Thus, the molar ratio of 10:1 was considered optimal for SELP-815K conjugation with the Cy5 NHS-ester. We then analyzed the purity of the four SELP conjugates (815K, 815K-RS1, 815KRS2, and 815K-RS5) using SDS-PAGE and observed single clear bands located slightly above those of the pure analogues (data not shown), which suggested that the SELP conjugates were pure and that Cy5 conjugation of the SELP analogues had no effect on their structure. Characterization of the Array-Based on-Chip Degradation Assay. To characterize the array-based on-chip degradation assay, SELP arrays were fabricated by applying Cy5-conjugated SELP-815K to the amine-modified arrays. 5402

DOI: 10.1021/acs.analchem.6b00739 Anal. Chem. 2016, 88, 5398−5405

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Analytical Chemistry Reaction mixtures containing various concentrations of elastase-2, ranging from 1 to 500 μg/mL, were applied to the SELP arrays, and the amounts of SELP-815K degraded by elastase-2 were determined by analyzing the RFIs of the array spots. The RFI increased in an elastase-2 concentrationdependent manner, with saturation at 50 μg/mL (Figure 2A,B). We confirmed the elastase-2-induced degradation of the SELP strands using AFM and scanning electron microscopy. To structurally characterize the SELP fibers and their degradation by elastase-2 (formation and degradation of SELP fibers), SELP-815K was loaded onto freshly cleaved mica and the resulting mica was incubated with elastase-2. The formation of cross-linked, high-density SELP networks by the hydrogen bonding of silk units was observed, and the crosslinked SELP networks were cleaved by elastase-2 (Figures 2C and S1), which was consistent with the results obtained from the on-chip degradation analysis (Figure 2A,B). Further, the observed formation of SELP networks by the cross-linking of each SELP strand and their resulting morphology was very similar to that of other SELP analogues, such as SELP-47K and SELP-415 K.4 Interestingly, a number of aggregates were observed after the enzymatic reaction, which might have occurred by the binding of the degradation fragments to the networks. Additionally, we analyzed changes in the functional groups of SELP-815K by elastase-2 using FT-IR spectroscopy. The most prominent changes concern components at 1674 and 1541 cm−1 assigned to amide I and II, respectively, of which absorbances were decreased by elastase-2 (Figure 2D), indicating conformational changes in SELP networks by enzymatic degradation. These results demonstrate that changes in the array spot fluorescence intensities were induced by the enzymatic degradation of the SELP-815K networks and indicated that this on-chip degradation assay could be applied to the enzymatic profiling of SELP polymers. Quantitative Analysis of the Degradation of Four SELP Polymers by Cleavage Enzymes. To quantify the degradation of SELP-815K by cleavage enzymes, we incubated the Cy5-conjugated SELP-815K arrays with various concentrations of eight cleavage enzymes including elastase-2 and plotted the resulting fluorescence intensities against the cleavage enzyme concentrations (Figure 3). Four enzymes, including elastase-2, collagenase-2, trypsin, and pepsin, increased SELP-815K degradation in an enzyme concentration-dependent manner; however, calpain-1, caspase-9, cMMP3, and thrombin had no effect on the degradation of SELP815K (Figure 3). The resulting EC50 values of the four SELP815K cleavage enzymes were 21.05, 2.99, 1.80, and 30.54 μg/ mL for elastase-2, collagenase-2, trypsin, and pepsin, respectively (Table 1), which was consistent with previous reports describing the preferential cleavage sites of each enzyme.25−32 Elastase-2, collagenase-2, trypsin, and pepsin can recognize and cleave repetitive silk and elastin units of SELPs; thus, the fragmentation of SELP strands into very small molecules may be helpful for clearing a SELP depot from the body after drug delivery. Interestingly, SELP-815K was not cleaved by calpain-1 despite the fact that it contains a calpain-1 cleavage site. One possible explanation is the steric hindrance introduced during network formation by the location of only one cleavable site prior to repetitive units. On the contrary, the three SELP-815K analogues containing an MMP-responsive sequence were cleaved by calpain-1, because calpain-1 can also cleave the MMP-responsive sequence. These results suggested that the

Table 1. Degradation Profiles (EC50 Values) of Eight Cleavage Enzymes against SELP-815K and Its Three Analoguesa EC50 (μg/mL)

a

enzymes

815K

815K-RS1

815K-RS2

815K-RS5

elastase-2 collagenase-2 trypsin calpain-1 caspase-9 cMMP-3 thrombin pepsin

21.05 2.99 1.80 N.D. N.D. N.D. N.D. 30.54

16.28 0.02 9.97 40.55 N.D. 0.83 N.D. 39.34

15.61 0.01 16.11 102.9 0.18 0.05 N.D. 36.93

18.46 0.04 9.02 223.3 0.27 0.09 0.28 58.28

N.D., nondetectable.

MMP-responsive sequence might be helpful for more rapid drug release from hydrogels in areas with increased MMP or calpain-1 expression levels. Further, four SELP analogues were cleaved by pepsin, indicating that SELP analogues can potentially be used for sustained drug delivery by oral administration. We also measured the EC50 values of the eight cleavage enzymes for the three SELP-815K analogues (Table 1). The EC50 values of elastase-2 for the three SELP analogues ranged from 15.61 to 18.46 μg/mL, which indicated that the elastase-2 degradation rates of the four SELP-815K polymers were similar. The pepsin EC50 values were also similar for SELP-815K and its three analogues. However, the EC50 value of trypsin against SELP-815K was 5- to 10-times lower than corresponding values of its three analogues, whereas the EC50 value of collagenase-2 against SELP-815K was 75- to 300-fold higher than values observed in the analogues, indicating that the insertion of the MMP-responsive sequence might have affected the threedimensional structure of the SELP strands adjacent to the trypsin and collagenase-2 cleavage sites. As expected, SELP815K was not degraded by cMMP-3; however, three SELP analogues containing the MMP-responsive sequence were degraded. In particular, cMMP-3 exhibited the lowest EC50 value against SELP-815K-RS2. Thus, the cleavage enzymes displayed distinctive degradation rates for SELP-815K and its analogues. On-Chip C-Peptide Release from SELP-815K Hydrogel Arrays. C-peptide, which is known as a potential therapeutic molecule for diabetic complications such as retinopathy, neuropathy, and cardiovascular disease,33−35 was used as the model drug for the on-chip study of drug release from SELP hydrogels. The SELP arrays used to study on-chip C-peptide release were fabricated by applying the mixture of Cy5conjugated C-peptide and SELP-815K to well-type amine arrays. Reaction mixtures containing cleavage enzymes were then applied to the SELP-815K arrays, and the amounts of Cpeptide released from the SELP hydrogel arrays were determined by the analysis of the RFIs of array spots (Figure 4). We analyzed time-course release of C-peptide by elastase-2 from the SELP-815K hydrogel arrays. C-peptide was released from the hydrogel in an incubation time-dependent manner, with saturation at 2 h (Figure S2). We then applied the reaction mixtures containing various concentrations of elastase-2, collagenase-2, and trypsin, the cleavage enzymes that yielded higher degradation rates (Table 1), to the SELP-815K arrays and noted that the amount of Cy5-conjugated C-peptide 5403

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Figure 5. Cumulative C-peptide release from the SELP-815K arrays by repetitive elastase treatment. Arrays for the in vitro C-peptide release assay were fabricated by applying a mixture containing 50 μg/mL SELP-815K and 50 μg/mL Cy5-conjugated C-peptide to well-type amine arrays. The reaction mixture containing 100 μg/mL elastase was applied five times to the arrays for 30 min for each application. Cpeptide release (%) was calculated as described. Data are expressed as mean ± SD from three independent experiments.

further supported by the conventional in vitro release assay using cylindrical SELP-815K hydrogel discs (data not shown). In the absence of elastase, the amount of C-peptide released increased according to the number of buffer treatments from 5.4% (after the first treatment) to 22.2% (after the fifth treatment), indicating that repetitive washing with a buffer containing detergent slightly affected the C-peptide release from the SELP arrays. Thus, C-peptide was caged in the SELP815K hydrogels and released by the blood circulating cleavage enzyme elastase-2. These results suggest that, in clinical application, continuous enzymatic degradation of SELPs located near the implantation sites can increase drug release from the hydrogels. Thus, our array-based on-chip degradation assay was useful for evaluating polymer degradation and drug release from SELPs and for simulating drug release depending on the implantation sites. Such array-based systems can save materials and time for elucidation of network properties and the drug release profile.

Figure 4. In vitro release of Cy5-conjugated C-peptide from the SELP815K arrays by cleavage enzymes. Arrays for the in vitro release assay were fabricated by applying a mixture containing 50 μg/mL SELP815K and 50 μg/mL Cy5-conjugated C-peptide to well-type amine arrays. Reaction mixtures containing the indicated concentrations of the three cleavage enzymes including elastase-2 (A), collagenase-2 (B), and trypsin (C) were applied to the arrays. C-peptide release (RFI) and EC50 values were calculated as described. Data are expressed as mean ± SD from three independent experiments.

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CONCLUSION In this study, we presented a high-throughput array-based assay system for evaluating depot degradation and drug release from polymer depots using Cy5-conjugated SELP-815K and its three analogues. We successfully applied this assay to the study of the in vitro release of Cy5-conjugated C-peptide from the SELP815K hydrogel arrays. Eight cleavage enzymes displayed distinctive EC50 values for the SELPs, and cumulative Cpeptide release from the SELP-815K gels was demonstrated by repetitive elastase treatments. Consequently, this array-based on-chip degradation assay has a potential for evaluating in vitro polymer depot degradation and drug release at the molecular level and for simulating drug release in in vivo environments.

released increased in an enzyme concentration-dependent manner (Figure 4). For the quantitative analysis of Cy5conjugated C-peptide release, RFIs were plotted against the cleavage enzyme concentrations and the EC50 values were calculated. The EC50 values of elastase-2, collagenase-2, and trypsin for SELP-815K were 9.85, 0.30, and 5.22 μg/mL, respectively. To simulate drug release from the SELP hydrogels in the presence of enzymes, we measured cumulative release of Cy5conjugated C-peptide from the SELP-815K hydrogel arrays by repetitive elastase-2 treatments (Figure 5). We found that 45.0% of the C-peptide was released from the arrays after the first treatment and that saturation at 86.0% occurred after the fourth treatment, which indicated that the amount of C-peptide released increased with the number of elastase treatments. The C-peptide release by elastase-2 from the hydrogel arrays was

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b00739. Morphological analysis of SELP-815K degradation by elastase using scanning electron microscopy; time-course 5404

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release of C-peptide from the SELP-815K arrays by elastase-2 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel.: 801 587-1566. Fax: 801 581-6321. E-mail: hamid. [email protected]. *Tel.: +82-33-250-8833. Fax: +82-33-250-8807. E-mail: ksha@ kangwon.ac.kr. Author Contributions ¶

H.-Y.J. and S.-H.J. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Research Foundation of Korea (2013-008193 and 2015R1A4A1038666), Global Innovative Research Center program of the National Research Foundation of Korea (2012K1A1A2A01055811), and the Intramural Research Program (Global RNAi Carrier Initiative) of Korean Institute of Science and Technology, as well as a grant from the US National Institutes of Health (R01CA107621, H.G.).

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DOI: 10.1021/acs.analchem.6b00739 Anal. Chem. 2016, 88, 5398−5405