Article pubs.acs.org/journal/abseba
Synthetic Short Peptides for Rapid Fabrication of Monolayer Cell Sheets Yinan Ma,†,‡ Zhibo Li,*,†,§ and Keiji Numata*,‡ †
Laboratory of Polymer Physics and Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China Enzyme Research Team, Biomass Engineering Program Cooperation Division, Center for Sustainable Resource Science, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan § School of Polymer Science and Engineering, Qingdao University of Science and Technology, Qingdao 266042, China ‡
S Supporting Information *
ABSTRACT: Cell sheets are useful materials in regenerative medicine; however, the cell sheet fabrication processes developed to date are associated with several crucial challenges. The aim of this study was to develop a new and simple method for the rapid and efficient fabrication of transferable monolayer cell sheets. Chemoenzymatic synthesis mediated by proteinase K was used to synthesize short cooligopeptides for cell sheet fabrication, which showed high yield, well-defined structures, and a controllable composition. These co-oligopeptides predominantly adopted a random coil conformation in buffer. Histidine/cysteine co-oligopeptides with a disordered secondary structure displayed cysteine content-dependent esterase activity and cysteine content-independent protease activity. Taking advantage of this enzymatic activity, confluent cell monolayers were detached by simply adding the cooligopeptides solution to the culture media, and then an intact monolayer cell sheet was prepared with high cell viability and reattachment ability. The method proposed herein for preparing monolayer cell sheets represents a novel concept by using oligopeptides with enzymatic activity that show applied potential in cell sheet technology for tissue engineering and regenerative medicine. KEYWORDS: oligopeptides, chemoenzymatic synthesis, enzymatic activity, monolayer cell sheet
■
INTRODUCTION For cell-based regeneration of damaged organs and tissues, a cell sheet can be applied directly to the injury without implantation of any scaffolds, and the consequent preservation of the extracellular matrix (ECM) promotes cell survival and prevents instant diffusion.1 Since 1993 the first demonstration of the detachment of cell monolayers using temperatureresponsive surface polymer coatings,2 various cell sheetengineering approaches have been developed. Okano and colleagues used a thermoresponsive cell culture surface grafted with poly(N-isopropylacrylamide) to harvest an intact cell sheet by simply lowing the temperature,3−5 and successfully constructed multilayer cell sheets to mimic three-dimensional (3D) tissues.6−8 However, several of the fabrication processes commonly applied for cell sheet detachment and reattachment can potentially affect cellular growth and proliferation, which have thus far limited their utilization. A thermoresponsive hydrogel with high biocompatibility can also be used to prepare a transferable cell sheet, although modification of the hydrogel surface is required to improve cell attachment and nutrient supply.9,10 Polyelectrolyte surfaces formed by layer-by-layer deposition and controlled erosion of the polyelectrolyte layers can also be used to prepare a cell sheet, but the components required for effective cell attachment are quite limited.11 Alternatively, cell sheets can be harvested by impregnating © XXXX American Chemical Society
magnetic particles into the cells, which are then detached via the magnetic force; however, the fate of the internalized magnetic particles is not clear.12 Thus, development of a simple method for the rapid and efficient fabrication of transferable cell sheets is still required. In routine cell culture, the cells are usually harvested by using proteases, especially trypsin, and/or enzyme-free dissociating reagents composed of metal ion chelators, resulting in a singlecell suspension with damaged cell−cell junction proteins and receptor proteins on the cell membrane.13 In addition to trypsin, dispase has also been used for harvesting cell sheets in the clinical treatment of burn wounds.14 Dispase disrupted Ecadherin and laminin in ECM to detach cells, whereas residual dispase after the treatments was found to be harmful to the attachment efficiency and proliferation of cell sheet and transplantation of wound site.15 The remaining high enzymatic activity limited the application of dispase on cell sheet preparation, and several alternatives were achieved including thermal responsive culture surface.16 Protease catalyzes the hydrolytic cleavage of the peptide bonds of proteins for cell adhesion, while chelators bind with divalent cations (calcium, Received: February 26, 2016 Accepted: March 7, 2016
A
DOI: 10.1021/acsbiomaterials.6b00113 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
untreated TCPS surfaces with no residual components that would subsequently affect cell proliferation.
magnesium, etc.) to disrupt the stable structure of cell adhesion molecules, including integrin and cadherin. According to these mechanisms, a combination of protease activity and a metal chelator can be used for effective cell sheet preparation as long as these two functions can be appropriately controlled and balanced. To achieve this combination, synthetic peptides designed with protease catalytic triads and cation-binding amino acids are promising candidates. Because their structure and composition are identical to those of natural proteins, many efforts have been made to design and synthesize short peptides as artificial enzymes exhibiting catalytic activity. Histidine, cysteine/serine, and aspartic acid/glutamic acid have been shown to be the most promising amino acids exhibiting activity in a protease catalytic triad.17 The imidazole group of histidine serves as an important nucleophile in the catalytic triad by removing the proton for activation, as a proton shuttle by transferring a proton from a basic nitrogen atom to an acidic nitrogen atom, and as a coordinating ligand in metalloproteins by binding with metal ions.18,19 Moreover, the thiol group of cysteine acts as an oxyanion hole in the catalytic triad, binds with metal ions to form covalent bonds, and responds to redox stimuli by converting to disulfide, sulfoxide, or sulfone.20 The carboxyl group of glutamic acid or aspartic acid facilitates the proton-withdrawing effect of histidine, and displays good metal-binding properties together with cysteine residues as a mimic of phytochelatin.21,22 However, the synthesis of these copolypeptides using conventional methods is difficult because of the reactive sidechain groups of thiol, imidazole, and carboxyl. Solid-phase peptide synthesis is costly and achieves a relatively low yield,23 whereas recombinant DNA methods are associated with problems related to endotoxin as well as the requirement of purification steps.24 The ring-open polymerization of amino acid N-carboxyanhydrides shows productivity up to the kilogram scale and ability for side-chain functionalization, but also requires an extremely pure monomer, harsh reaction conditions, and side-chain groups protection.25 We recently showed the potential advantages of the chemoenzymatic synthesis of polypeptides catalyzed by proteases based on its atom economy, simplicity, relatively high yield and purity, and lack of requirement for protecting the side-chain active groups.26,27 In our previous work, the chemoenzymatic synthesis of linear homo-oligopeptides,28,29 co-oligopeptides,30−33 and star-shaped hybrid oligopeptides32 was successfully performed using proteases such as papain and proteinase K. For oligocysteine synthesis, oxidation of the thiol groups could be prevented with a high free thiol content without requiring any protection in aqueous solutions.34 In the present study, co-oligopeptides of histidine/cysteine and glutamic acid/cysteine with well-defined secondary structures and amino acid composition were chemoenzymatically synthesized by proteinase K. Monomers of cysteine, histidine, and glutamic acid were chosen for this study because of their proteolytic activity and ability to form complexes with metal cations, which suggested that their synthetic copolypeptides could be used as dissociating reagents in cell detachment and subsequent cell sheet fabrication.35 The esterase and protease activities of the synthesized oligopeptides were evaluated using small molecules and proteins as substrates. These co-oligopeptides were added to monolayer cells on tissue culture polystyrene plates (TCPS) for controlled cell detachment and preparation of monolayer cell sheets. This method allows for the facile and rapid removal of cell sheets from
■
EXPERIMENTAL SECTION
Materials. L-Cysteine ethyl ester hydrochloride (Cys-OEt·HCl) and proteinase K were purchased from Wako Pure Chemical Industries (Osaka, Japan). L-Histidine ethyl ester dihydrochloride (His-OEt·2HCl) was purchased from Watanabe Chemical Industries (Hiroshima, Japan). L-Glutamate diethyl ester hydrochloride (γEt-GluOEt·HCl) and deuterated trifluoroacetic acid (TFAd) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pyrogen-free deionized water (18.2 MΩ·cm) was obtained from a Millipore Milli-Q Biocel A10 purification system. All other chemicals were purchased from commercial suppliers and used without further purification unless otherwise noted. Proteinase K-Catalyzed Chemoenzymatic Synthesis of Oligo(His-co-Cys) (H/C) and Oligo(Glu-co-Cys) (E/C). The proteinase K-catalyzed co-oligomerization was carried out according to methods reported previously.32,33 In brief, Cys-OEt·HCl (464.18 mg, 2.5 mmol) and His-OEt·2HCl (640.35 mg, 2.5 mmol) or γEt-GluOEt·HCl (599.25 mg, 2.5 mmol) were dissolved in 1.0 M potassium phosphate buffer (pH 8) to a final volume of 5 mL, and potassium hydroxide was added to adjust the initial pH to 8.0. The solutions were then transferred to a 25 mL glass reaction tube and prewarmed at 40 °C before adding proteinase K (23 units/mL). Reactions were performed with gentle stirring in EYELA ChemiStation (Tokyo, Japan) at 40 °C for 3 h; white precipitates appeared for EγEt/C during the reaction, whereas transparent solutions turned from colorless to pale yellow for H/C. One milliliter of a 6 M HCl solution was added to inactivate the enzyme and the reaction mixture was cooled to room temperature. For E/C reactions, the precipitates were collected by centrifugation (10,000 × g, 25 °C for 10 min), and washed once with 0.1 M HCl and twice with Milli-Q water. Saponification of the precipitated oligo(γEt-Glu-co-Cys) (EγEt/C) to prepare fully watersoluble E/C was performed by reacting with 0.2 M NaOH at 60 °C for 2 h under constant nitrogen bubbling. The clear solution containing E/C was neutralized to pH 7 by adding a diluted HCl solution. For all H/C and E/C reaction mixtures, the salts and unreacted monomers in reaction mixture were removed by dialysis against 200 mM KCl and Milli-Q water using Spectra/Por Biotech CE tubing dialysis membrane MWCO 100−500 D (Spectrum Laboratories, Inc., Rancho Dominguez, CA, USA). Further purification was applied to remove any remaining proteinase K by Amicon Ultra-15 3k centrifugal filter devices, and the filtrates were collected and lyophilized to yield H/C and E/C as white powders. All the peptides were kept at −20 °C before use. Structural Characterization of Co-oligopeptides. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Varian NMR System 500 (500 MHz) spectrometer (Varian Medical Systems, Palo Alto, CA) at 25 °C, controlled with VnmrJ software. Oligopeptides (10 mg/mL) were dissolved in a 1:3 (v/v) mixture of TFA-d/TFA and 128 scans were collected. Tetramethylsilane (TMS) was used as an internal reference at 0.00 ppm. Data were processed and analyzed with MNova NMR software (Mestrelab Research SL, Santiago de Compostela, Spain). Matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectroscopy spectra were acquired on an ultrafleXtreme MALDI-TOF spectrophotometer (Bruker, Billerica, MA, USA). The instrument was operated in negative ion reflection mode with an accelerating voltage of +15 kV. Spectra were obtained by averaging at least 1000 laser shots. The spectrometer was calibrated using Peptide Calibration Standard (Bruker Daltonics GmbH, Leipzig, Germany) as external standards. Saturated α-cyano hydroxycinnamic acid prepared in a water:acetone mixture (2:1, v/v) with 0.1% TFA (TA solvent) was used as a matrix. Oligopeptides were dissolved in TA solvent (10 mg/mL) and mixed with the matrix at a 1:1 ratio. One microliter of prepared sample was spotted on a target plate and then allowed to air-dry at room temperature. The acquired data were analyzed with FLEX analysis software (Bruker Daltonics). B
DOI: 10.1021/acsbiomaterials.6b00113 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
uptake and fluorescence of calcein AM in response to intracellular esterase activity, whereas dead cells fluoresce red as a result of the entry of ethidium homodimer-1 through damaged cell membranes and its subsequent binding to nucleic acids. Time-course observations began with the addition of oligopeptide solution in DMEM without serum (pH 7.4) to a final concentration of 1 mg/mL in the presence of fluorescent dyes, whereas trypsin control cells were washed with DPBS (−) twice and then trypsin-EDTA was added. Fluorescent microscopy at different magnifications was performed with an Axio zoom V16 macroscope (Carl Zeiss, Oberkochen, Germany) composed of a motorized stereozoom microscope and a fluorescence light (HPX 200 C) coupled to a Zeiss MRc CCD camera. Images were also obtained with a confocal laser-scanning microscope (CLSM, Leica Microsystems, Wetzlar, Germany) with all stations controlled by ZEN 2011 software. Statistical Analysis. Experiments were performed in triplicate in at least three independent experiments. Data are reported as mean ± SD, and statistical comparisons using GraphPad Prism (GraphPad Software, San Diego, CA) were based on analysis of variance and Tukey’s test for pairwise comparisons, with a p-value
