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Article Cite This: ACS Omega 2019, 4, 4376−4383
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Selective Cell Adhesion on Peptide−Polymer Electrospun Fiber Mats Gagandeep Kaur,†,∥ Savita Kumari,§ Piyali Saha,‡ Rafat Ali,† Sandip Patil,§ Subramaniam Ganesh,‡ and Sandeep Verma*,† †
Department of Chemistry and ‡Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016, India § E-Spin Nanotech Pvt. Ltd., Indian Institute of Technology, Kanpur 208016, India
ACS Omega 2019.4:4376-4383. Downloaded from pubs.acs.org by 5.189.206.242 on 03/21/19. For personal use only.
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ABSTRACT: Electrospun polymer fibers are valuable for a number of applications ranging from catalysis to drug delivery. At times, lack of biocompatibility, biodegradability, and hydrophobicity presents hindrance in their use in biological applications. Aromatic amino acids are veritable precursors for biocompatible nanofibers, which could also be chemically modified with suitable addressable recognition tags to invoke specific binding events. This study presents an attractive strategy for constructing electrospun fibrous mats from dityrosine folic acid conjugate and polycaprolactone to afford a new biomaterial displaying excellent tensile properties, biocompatibility, and cell adhesion. We demonstrate that appropriate choice of peptide-to-polymer ratio gave mats with sufficient hydrophilic and better mechanical properties and allowed favorable interaction of folate receptor presenting cells with electrospun mats, while the ones lacking folate receptor did not exhibit binding. Such selectivity could be possibly invoked for separation and also for custom synthesis of nanomats for healthcare applications.
1. INTRODUCTION Solution-assisted electrospinning, a simple and versatile technology, can be used to produce two-dimensional as well as three-dimensional nanofibers of varied diameter and length depending upon experimental conditions.1 Electrospun fibers are highly useful scaffolds that offer added scope for fine-tuning physical properties such as surface area and mechanical strength and chemical functionalization to afford materials for cell culture, tissue engineering, and regenerative medicine.2−5 More specifically, such fibers have been used in filtration systems, protective clothing, medical prostheses, energy storage, space applications, nano-optoelectronics, wound healing, tissue engineering scaffolds, etc.6−15 Advantages of using polymer fibrous materials as tissue scaffolds are ascribed to their nontoxic nature and the possibility of creating structural features similar to extracellular matrices.16−19 Biological applications necessitate fibers that are biocompatible and present favorable interactions with cells, extracellular matrix, growth factors, and other biomolecules. Exploration in this domain includes modification of hydrophobic/hydrophilic properties, creation of a robust meshlike structure enabling optimal fluid sorption, and modification of transport and delivery properties supporting the overarching aim to achieve cell interaction and adhesion. It is desirable that biocompatible fibers also exhibit mechanical strength and present a large enough surface area to allow for high-volume cell attachment and favorable interactions.20,21 For example, poly(lactic-co© 2019 American Chemical Society
glycolic acid), a biodegradable polymer, has been extensively used in electrospinning to create scaffolds with appropriate physical and mechanical properties.22,23 Poly-ε-caprolactone (PCL) is a biocompatible synthetic polymer with potential uses in tissue engineering and regenerative therapies due to its affordability, biocompatibility, and mechanical strength.24−27 However, hydrophobicity and cell binding incapability of PCL demands attention before it could be developed as an advanced biomaterial. These issues were addressed by the incorporation of peptides and peptide amphiphiles along with PCL in the electrospinning process.28−30 Fibrous networks derived from peptide-modified PCL displayed improved cell adhesion and proliferation.31−34 Folic acid (FA) is a vitamin crucial for DNA synthesis, cell division and replication, and the formation of red and white blood cells. As a strategy to achieve cell interaction and uptake, folate-drug conjugates exploit binding of folic acid to its cognate folate receptor (FR) protein present on the cell surface of many human cancers, through endocytosis uptake mechanism.35−38 Recent applications in this direction include folic acid-conjugated poly(ε-caprolactone)-polypeptide copolymer vesicles as antibacterial agents,39 polyethylenimine-graf tpolycaprolactone-block-poly(ethylene glycol)-folate ternary Received: December 13, 2018 Accepted: February 14, 2019 Published: February 27, 2019 4376
DOI: 10.1021/acsomega.8b03494 ACS Omega 2019, 4, 4376−4383
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Scheme 1. Synthetic Scheme for 3: (i) 75% Trifluoroacetic Acid (TFA)−Dichloromethane (DCM), 4 h, N2 Atmosphere; (ii) N,N′-Dicyclohexylcarbodiimide, Pyridine, Dark
Figure 1. (A) UV−vis spectra of 2, FA, and 3 (10 μM); (B) FTIR spectra of 2, FA, and 3. Morphology of self-assembled structures of 3 (1 mM): (C, D) scanning electron microscopy (SEM) images.
copolymer for targeted siRNA delivery,40,41 and folatefunctionalized poly(ethylene glycol)-b-polycaprolactone (folate PEG-b-PCL) for intracellular and prolonged delivery to retinal pigment epithelium cells,42 to name a few. Our interest in peptide conjugates and functional peptide self-assembly concerns the creation of soft compartments for cellular delivery and amyloid modeling.43−48 We have recently described synthesis of a folic acid containing phenylalanine, which was further used to study self-assembly, gross morphology of ensuing supramolecular structures, and their cell delivery properties.49 It was determined that covalent conjugation of folic acid to the dipeptide afforded predictable assemblies and that both macropinocytosis-mediated pathway and clathrin-mediated endocytosis were possibly involved in the cellular uptake process. To expand the applications of folate-conjugated amino acids and peptides, we became interested in developing a new conjugate as an additive for electrospun PCL fiber mats to seek separation of cancer cells,
given the propensity of overexpression of folate receptors on certain cancer cell surfaces. This study is expected to serve as a step toward developing adaptive electrospun mats for detection and selective capture of FR-positive cancer cells.
2. RESULTS AND DISCUSSION 2.1. Solution-Phase Self-Assembly of Folate LTyrosyl-L-tyrosine Methyl Ester (3). Tyrosine is an important aromatic amino acid that serves as a versatile precursor for the biosynthesis of certain neurotransmitters and plays a key role in signal transduction through phosphorylation catalyzed by tyrosine kinases. We have recently demonstrated that Tyr can afford formation of well-ordered assemblies, which could be influenced by parameters such as concentration, duration of aggregation, pH, etc., forming morphologies ranging from nanoribbons to branched, fernlike structures.48 4377
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Figure 2. Morphology of Mat I fibers (1 mg/mL in 10% w/v PCL): (A, B) SEM images, (C) FM image, and (D) CLSM image. The green emission (λem = 488 nm) in (C) and (D) is ascribed to 3.
These supramolecular structures could find potential applications such as biosensors, which leads us to surmise their potential use as molecular scaffolds in regenerative medicine by chemical modification via Tyr cross-linking. With this as a backdrop, 3 was designed and synthesized by standard procedures and protocols using N,N′-dicyclohexylcarbodiimide (DCC) coupling method (Scheme 1). The UV−vis spectrum of 3 shows strong peaks at 220 and 282 nm, whereas the UV−vis spectrum of folic acid (FA) shows a peak at 286 nm (Figure 1A). This shift in peak from 288 to 282 nm is attributed to the formation of amide bond between −COOH group of FA and −NH2 group of L-tyrosyl-L-tyrosine methyl ester.50 However, a dominating contribution from aromatic stacking interactions must also be taken into account in conjugate 3. The Fourier transform infrared (FTIR) spectrum of 3 shows peaks at 1694 and 1603, which are characteristic bands for folic acid: the peak at 1694 corresponds to carbonyl (CO stretch) group of COOH, whereas the peak at 1603 is due to aromatic CC bending (Figure 1B). Furthermore, the FTIR spectrum of 3 shows increase in the absorbance and broadening of band at 1649 and appearance of a new band around 1514, arising from the amide bands. The increase and broadening of amide bands in 3 suggests the formation of amide bond by the conjugation of folic acid with L-tyrosyl-L-tyrosine methyl ester in 3.51 A stock solution of 3 (5 mg/mL dimethyl sulfoxide (DMSO)) was diluted with DCM to afford 1 mM final concentration, which was used to study the self-assembly behavior. The choice of solvent system for studying the selfassembly was taken by considering the solvent conditions used for peptide−polymer material for electrospinning. Compound 3 afforded fibrous networks as determined by scanning electron microscopy studies on silicon wafers (100) (Figure 1C,D). These structures were somewhat different from the ones obtained for pure Tyr, suggesting a role of folic acid in giving rise to aggregate structures. 2.2. Preparation and Characterization of Peptide− PCL-Based Mats. Three different concentrations of peptide− PCL solutions (Mat I = 1 mg/mL; Mat II = 2.5 mg/mL; and Mat III = 5 mg/mL, peptide in 10% w/v PCL solution in DCM) were used to make three different mats through the
electrospinning process. It was characterized through microscopy tools such as scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and fluorescence microscopy (FM) to understand gross morphology (Figure 2). Peptide−PCL mats (with all three peptide concentrations: Mat I, Mat II, and Mat III) showed notable change in fiber thickness (600 nm to 2.5 μm) compared to PCL control fibers. The peptide structures were uniformly distributed throughout the PCL fibers in the mats, which was confirmed by FM and CLSM (Figure 2C,D). Since the mechanical strength and hydrophilic nature with high surface area for high-volume cell attachment are desirable conditions for biocompatible fibrous mats, peptide−polymer mats were characterized for these attributes. Surface hydrophobicity of mats was found to decrease with the addition of 1 mg of peptide per mL of 10% w/v PCL solutions, compared to PCL control fibers (Figure 3A). Tyrosine aromatic ring offers hydrophobicity to fibrous mats, which could lead to high contact angle of Mat II and Mat III. However, a more precise explanation on the nonmonotonous behavior of hydrophobicity needs better physical understanding of the composite. Further, the mechanical properties were studied with PCL control mat along with Mat I, Mat II, and Mat III. The results obtained in Figure 3B showed that Mat I had better stress-tostrain limits and higher Young’s modulus compared to PCL control mat, Mat, Mat II, and Mat III (Figure 3B,C). The Young’s modulus of Mat I increased ∼5 times compared to that of the PCL control mat. It was hypothesized that dispersion and orientation of peptide fibers in polymer matrix affected the mechanical properties of PCL. Hydrogen-bonding, π−π stacking, and hydrophobic interactions could be the major reasons for increase in the mechanical strength of Mat I compared to PCL control mats. Further increase in the concentration of peptide in Mat II and Mat III results in decrease in the tensile properties of the respective mats. Similar trends have been reported in many literature studies.52 As Mat I showed the desirable characteristics such as higher mechanical strength and less hydrophobic nature with high surface area for cell 4378
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Low-cost, reliable, and early detection and diagnosis of cancer and other folate overexpressed infections would lead to increased chances of treatment and survival. Mat I, with high surface area, flexibility, and low-cost manufacturing had provided the solution for problems facing the methods of detection of cancer cells. To test such potential, Mat I was studied for its differential affinity toward cells that overexpress FR. PCL fibers are known to be biocompatible in nature.24−27 To test if Mat I can promote adhesion of cancerous cells, HeLa cells, a human cervical cancer line with high FR expression,56 were seeded on a glass surface coated with Mat I fibrous mat. The cells were seeded and grown on a glass surface coated with 0.2% gelatin (Figure 5A), and the PCL control mat (Figure 5B) served as the control. The seeded cells were incubated at 37 °C in regular culture medium for 24 h, washed with phosphate-buffered saline (PBS), and fixed, and the glass surfaces were analyzed using SEM and CLSM (Figure 5C−F). Mat I favored better attachment and growth of HeLa cells as the adhered cells seem to have aligned along the direction of the Mat I fibers (Figure 5C−F). It was noted that very few cells are attached to PCL control mat compared to Mat I. To check if the observed adhesive property of HeLa cells is dependent on FR, equal number of HeLa cells or HEK 293 cells, a noncancerous cell line derived from the human embryonic kidney with very low-level FR expression,57 were seeded onto Mat I (Figure 5H) and allowed to settle for 24 h at 37 °C, and the number of cells attached to the surface (0.5 mm2) were counted. While Mat I surface showed twofold increase in the number of HeLa cells (627 cells/0.5 mm2 area) compared to the gelatin-coated surface (304 cells/0.5 mm2 area), HEK 293 cells were hardly detected on Mat I surface (Figure 5H), thus establishing a selective affinity for Mat I surface to FR-positive cells. The number of cells in different conditions are summarized in the graph shown in Figure 5I. It was revealed that Mat I selectively bound to folate overexpressed cells.
Figure 3. (A) Contact angles of PCL control and peptide−PCL mats with different concentrations of peptide (Mat I, Mat II, and Mat III with concentrations 1, 2.5, and 5 mg peptide/mL of 10% w/v PCL solution in DCM, respectively). (B) Stress−strain curves for PCL control mat, Mat I, Mat II, and Mat III. (C) Young’s modulus of PCL control mat, Mat I, Mat II, and Mat III.
adhesion and diagnostics, further studies were carried out with Mat I only. 2.3. Cytotoxicity of 3. To test the biocompatibility of 3, HeLa cells were grown in a medium container containing five different concentrations of 3 (0.01, 0.02, 0.08, 0.12, and 0.16 mM) and its cytotoxicity is measured using the established 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Figure 4). There was no statistically significant
3. CONCLUSIONS We have designed and synthesized a covalently linked dityrosine−folic acid conjugate, followed by its use in creating electrospun mats with PCL. Hydrophilicity of these mats could be fine-tuned by choosing the correct ratio of peptide conjugate−PCL mixture, leading to the formation of homogeneous fibers with folate display. The applicability of these recognition element functionalized mat was confirmed by analyzing selective adhesion of HeLa cells (FR-positive cells) and the lack of adhesion of HEK 293 cells (FR-negative cells). These electrospun mats can be engineered further for selective detection and retention of specific cancerous cells and parasites, such as leishmania. Optimization of the aforementioned objectives is in progress and will be reported in future.
Figure 4. Bar diagram showing viability of HeLa cells cultured in various concentrations of 3, as measured by MTT assay. Each bar represents mean of three independent experiments. There was no significant difference in cell viability at the five different concentrations of 3.
difference between control and treated sets (p < 0.05). As shown in Figure 4, the peptide was nontoxic to HeLa cells up to 24 h incubation and, hence, biocompatible. 2.4. Cell Adhesion Properties of Mat I. Certain cancerous cells have higher levels of folate receptor (FR), the cell surface protein that binds to and promotes the cellular uptake of FA. Therefore, FR is often considered as a target for cancer diagnosis and therapeutics.53 For example, electrodes modified with folate-conjugated peptide have been previously studied to successfully detect HeLa cancerous cells with 250 cells per mL detection limit in very low-concentration samples,54 and L-tyrosine-based folic acid-decorated nanoparticles have been studied for their uptake in HeLa cells.55
4. EXPERIMENTAL SECTION 4.1. Materials. Poly-ε-caprolactone (PCL, average Mn = 80 000) used to synthesize electrospun mats was purchased from Sigma-Aldrich (Bengaluru, India). L-Tyrosine (Tyr), N,N′-dicyclohexylcarbodiimide (DCC), and trifluoroacetic acid (TFA) were purchased from Spectrochem Pvt. Ltd., Mumbai, India; N-tert-butoxycarbonyl (Boc anhydride), thionyl chloride, N-hydroxybenzotriazole (HOBt), and folic acid (FA) were purchased from S. D. Fine-Chem Ltd., 4379
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Figure 5. (A) SEM image showing HeLa cell adhesion on 0.2% gelatin-coated glass surface. (B) SEM image showing HeLa cell adhesion on PCL control mat. Images showing HeLa cell adhesion on Mat I: (C) SEM image, (D) false-colored SEM image, and (E, F) CLSM images. (G) CLSM image showing HEK 293 cell adhesion on glass surface coated with poly-L-lysine. (H) CLSM image showing HEK 293 cell adhesion on Mat I. The green emission (λem = 488 nm) in (E)−(G) is ascribed to 3; HeLa cells were stained with deep red plasma membrane dye, which exhibits red emission (λem = 633 nm) in (E) and (F); HEK 293 nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI), which exhibits blue emission (λem = 405 nm) in (G) and (H). (I) Bar diagram showing the average number of cells (HeLa or HEK 293) on the glass surface compared to Mat I and PCL control mat. Average number of cells were taken in 0.5 mm2 area each time.
× 30 mL), and brine (30 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude compound was purified on a silica gel column by using DCM and MeOH to get pure 1 (1.90 g, 64% yield), Rf [7% MeOH in DCM] 0.5, HRMS (M + Na)+ for C24H30N2O7: 481.1951 (calcd), 481.1959 (anal.), 1H NMR (500 MHz, CDCl3, TMS, δ ppm): 1.39 (s, 9H), 2.84− 3.19 (m, 4H), 3.63 (s, 3H) 4.28 (t, 1H), 4.71 (t, 1H), 5.26 (m, 2H), 6.51−7.34 (m, 8H); 13C NMR (125 MHz; CDCl3, δ ppm): 28.34, 37.05, 37.73, 52.57, 56.06, 60.59, 80.91, 115.76, 126.88, 130.38, 155.40, 155.84, 171.67, 171.79. 4.2.2. L-Tyrosyl-L-tyrosine Methyl Ester (2). Compound 1 (1 g) was dissolved in 75% TFA−DCM (10 mL) and stirred for 6 h under nitrogen atmosphere. After completion of the reaction, the solvent was evaporated in vacuo and the resulting solid was subsequently washed with diethyl ether (3 × 20 mL) to afford a white solid. The latter was dissolved in MeOH and passed through Dowex resin, followed by evaporation of fractions under reduced pressure to obtain pure 2 (0.822 g, 82.2% yield), which was used as such in the next step. 4.2.3. Folate L-Tyrosyl-L-tyrosine Methyl Ester (3). The folate dityrosine conjugate was synthesized by a modified method reported in the literature.58 Compound 2 (0.203 g, 0.556 mmol) was added to an FA (0.25 g, 0.556 mmol) and DCC (0.701 g, 3.39 mmol) mixture dissolved in DMF (5 mL), in the presence of dry pyridine (10 μL). The mixture was stirred overnight at room temperature under nitrogen atmosphere and dark conditions. The resulting mixture was diluted with deionized water (10 mL) and centrifuged at 1000 rpm for 30 min to separate insoluble DCU. The supernatant was collected and washed with diethyl ether to afford a yellow precipitate, which was filtered and dried. The yellow compound was further purified by dialysis using cellulose acetate dialysis membrane with MWCO 500 Da, which
Mumbai, India, and used without further purification. Dichloromethane (DCM), methanol (MeOH), N,N-dimethylformamide (DMF), methanol, trimethylamine, and pyridine were distilled according to standard procedures prior to use. Merck precoated TLC plates (TLC Silica gel 60 F254) were used for thin-layer chromatography (TLC), and compounds were visualized with UV light at 254 nm; 100−200 mesh silica gel (S. D. Fine-Chem Pvt. Ltd.) was used for chromatographic separation. Dulbecco’s modified Eagle’s medium (DMEM), 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypsin−ethylenediaminetetraacetic acid, penicillin− streptomycin, and gelatin (from coldwater fish skin) were purchased from Sigma-Aldrich (Bengaluru, India). Dimethyl sulfoxide (DMSO) was obtained from Merck (Bengaluru, India). Fetal bovine serum (FBS), 4,6-diamidino-2-phenylindole (DAPI), and CellMask deep red plasma membrane stains were purchased from Gibco Life Technologies. 4.2. Peptide Conjugate Synthesis. Dityrosine folic acid conjugate 3 was prepared by the standard solution-phase peptide synthesis methodology (Scheme 1). 4.2.1. Boc-L-Tyrosyl-L-tyrosine Methyl Ester (1). N-(Boc)-Ltyrosine (3 g, 10.7 mmol) and HOBt (1.44 g, 10.7 mmol) were dissolved in dry DMF (25 mL) under nitrogen atmosphere. The reaction mixture was cooled to 0 °C, and a solution of DCC (2.64 g, 12.7 mmol) dissolved in DCM was added in small portions. L-Tyrosine methyl ester hydrochloride (2.50 g, 12.8 mmol) was further added into the reaction mixture followed by dropwise addition of triethylamine (7.44 mL, 53.3 mmol) after 55 min, while stirring at 0 °C. The reaction mixture was further stirred for 24 h at room temperature. N,N′Dicyclohexylurea (DCU) was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate, and the organic layer was washed with 1 N HCl (2 × 30 mL), 10% Na2CO3 solution (2 4380
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separates the final compound (MW ∼ 781) from the unreacted starting materials such as folic acid (MW ∼ 441). The pure compound was dried under high vacuum (0.167 g, 65% yield). HRMS (M + H)+ for C38H39N9O10: 782.2898 (calcd), 782.2897 (anal.), 1H NMR (400 MHz, DMSO-d6, δ ppm): 1.79−2.49 (m, 4H), 2.50−2.90 (m, 4H), 3.54 (s, 3H), 4.34− 4.47 (m, 3H), 4.49 (s, 2H), 6.46−6.71 (m, 6H), 6.81−7.39 (m, 7H), 7.57−7.78 (m, 2H), 7.76−8.57 (m, 4H), 8.64 (s, 1H), 9.14 (s, 1H), 9.24 (s, 1H), 11.45 (s, 1H); 13C NMR (125 MHz; DMSO-d6, δ ppm): 25.84, 31.29, 34.65, 36.69, 42.57, 46.40, 52.26, 52.77, 55.00, 111.69, 115.32, 115.57, 121.73, 128.47, 129.53, 130.53, 149.10, 149.90, 151.29, 156.22, 156.53, 161.44, 162.87, 166.90, 172.06, 174.32, 174.45. 4.3. Field Emission Scanning Electron Microscopy (FE-SEM). Electrospun mats were deposited on a glass surface at 25 °C, and 10 μL aliquots of peptide samples (1 mM solution) were deposited on a silicon wafer (100) and allowed to dry at room temperature. The samples were dried in vacuo for 30 min prior to imaging. The samples were gold-coated, and SEM images were acquired on an FEI Quanta 200 microscope equipped with a tungsten filament gun, operating at a 4 mm WD and an operating voltage of 10 kV. 4.4. Electrospinning Method. Electrospun fibrous mats were synthesized using peptide−PCL polymer solution on a Super ES-2 model electrospinning unit (E-Spin Nanotech Pvt. Ltd., SIDBI Innovation and Incubation Centre, IIT Kanpur). High voltage (10−30 kV) was applied onto peptide−PCL, and its flow rate was controlled and maintained by a syringe pump. When the applied voltage exceeded the critical limit, the former stretches, leading to solvent evaporation and deposition of peptide−PCL in the form of nanofibers on a grounded collector. 4.5. Preparation of Peptide−Polymer Mats. A stock solution of 3 (50 mg/mL DMSO) was prepared and diluted with 10% w/v PCL solution in DCM to afford a final concentration of 1 mg/mL. This solution was used for the fiber mat containing a fiber network structure using an electrospinning machine. Three different concentrations of peptide− PCL solutions (Mat I = 1 mg/mL; Mat II = 2.5 mg/mL; and Mat III = 5 mg/mL, peptide in 10% w/v PCL solution in DCM) were used to make three different mats through the electrospinning process. A blend tip 20-G needle and a drum collector were used during the electrospinning process. The syringes were filled with peptide−PCL solution and connected to the silicon tube of a connector. The solution was released at 1 mL/h by a pump operating at a voltage of 14 kV. Fibrous mat was synthesized on a glass surface attached to aluminum foil wrapped around a drum collector (2000 rpm), with a diameter of 10 cm, at a distance of 14 cm from the needle, at room temperature (25 °C). It was washed with absolute ethanol for 20 min and vacuum-dried prior to use. 4.6. Contact Angle Measurements. The surface hydrophobicity was measured on Drop Shape Analyser DSA25 (Krüss advancing), equipped with a high-resolution USB 3.0 camera for the drop shape analysis, and used at 25 °C and 65% relative humidity. Peptide−PCL solutions were electrospun on a glass surface, and ultrapure water was applied on different areas of the mats. The experiments were performed in triplicate, and the average of the data was reported. 4.7. Mechanical Analysis. Mechanical studies were performed using samples (PCL control Mat, Mat I, Mat II and Mat III) with dimensions of 6 cm × 2 cm × 0.34 mm
using ZwickRoellzwickiLine materials testing machine Z5.0 TS. 4.8. Cell Lines, Culture Conditions, and Treatments. HeLa cells (cervical cancer cells) and HEK 293 cells (human embryonic kidney cells) were maintained at 37 °C, 5% CO2, and 95% relative humidity in DMEM and DMEM with 100 mM sodium pyruvate medium, respectively, supplemented with 10% heat-inactivated FBS, penicillin (100 U/mL), and streptomycin (100 U/mL). The cells were seeded for 24 h prior to treatment. All of the treatments were performed at 37 °C and at a cell density allowing exponential growth. 4.9. MTT Cell Viability Assay. In vitro biocompatibility studies of 3 peptide were carried out with HeLa cells by MTT assay.59 The cells were maintained with DMEM and supplemented with 10% fetal bovine serum in a humid incubator (37 °C and 5% CO2). The cells (104 cells/well) were plated onto 96-well glass-bottom culture plates. After 12 h, 3 was added to final concentrations of 0.01, 0.02, 0.08, 0.12, and 0.16 mM to the wells and incubated for ∼24 h. MTT in DMEM (0.5 mg/mL) was prepared and stored in the dark. After discarding the old media, 200 μL of freshly prepared MTT solution was added to each of the cellcontaining wells, followed by incubation for 4 h. After incubation, basal DMEM (having MTT) was removed and 200 μL of DMSO was added. Cell viability was determined by measuring their absorbance at 570 nm. The optical density of absorbance is directly proportional to the number of live cells. All in vitro cytotoxicity experiments were performed in triplicate, and the three consistent readings were used. 4.10. Cell Adhesion Study. The cells (104 cells/well) were seeded on a sterilized glass surface (13 × 13 mm2, 0.2% gelatin-coated) for 24 h. To study the cell adhesion on Mat I (1 mg/mL peptide in 10% w/v PCL) by confocal laser scanning microscopy (CLSM), HeLa cells were incubated on Mat I in the cell culture media for ∼24 h at 37 °C in a 5% CO2 humidified incubator. After incubation, the cells were washed thrice with phosphate-buffered saline (PBS) and fixed with 4% formaldehyde solution for 15 min. After washing, the cells were stained with deep red plasma membrane dye or DAPI and washed again with PBS. Coverslips were then mounted on the slides coated with buffered mounting medium to prevent fading and drying.
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S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b03494. FTIR spectra of 2, FA, and 3; SEM images of PCL nanofiber, Mat II, and Mat III; and HRMS image, 1H NMR spectrum, 13C NMR spectrum, and analytical high-performance liquid chromatography image of 3 (PDF)
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[email protected]. ORCID
Sandeep Verma: 0000-0002-2478-8109 4381
DOI: 10.1021/acsomega.8b03494 ACS Omega 2019, 4, 4376−4383
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Department of Pharmaceutical Sciences, College of Pharmacy, Texas A&M University, TAMU Mailstop 1114, College Station, Texas 77843, United States (G.K.). Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding
This work was supported by a DST Nano Mission project, Uchchatar Avishkar Yojana (MHRD, India), and J. C. Bose National Fellowship, DST, India (S.V.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge Center for Nanoscience, IIT Kanpur, for instrumentation. G.K. acknowledges IIT Kanpur and UGC, India, for financial support. P.S. acknowledges IIT Kanpur for postdoctoral fellowship. R.A. acknowledges SERB, India, for a National Postdoctoral Fellowship. E-Spin Pvt. Ltd. is acknowledged by S.K. and S.P. for financial support.
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ABBREVIATIONS PCL, polycaprolactone; FA, folic acid; DCC, dicyclohexylcarbodiimide; DCU, N,N′-dicyclohexylurea; TFA, trifluoroacetic acid; FR, folate receptor; TLC, thin-layer chromatography; HOBt, N-hydroxybenzotriazole; DCM, dichloromethane; MeOH, methanol; DMF, N,N-dimethylformamide; DMEM, Dulbecco’s modified Eagle’s medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; DAPI, 4,6diamidino-2-phenylindole; DNA, deoxyribonucleic acid; PEG, polyethylene glycol; FE-SEM, field emission scanning electron microscopy; PBS, phosphate-buffered saline; OM, optical microscopy; FM, fluorescence microscopy
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