Modeling Glyco-Collagen Conjugates Using a Host–Guest Strategy To

Publication Date (Web): October 27, 2017 ... Herein, we present a simple and reliable host–guest approach to design a high-throughput glyco-collagen...
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Modeling Glyco-Collagen Conjugates Using a Host−Guest Strategy To Alter Phenotypic Cell Migration and in Vivo Wound Healing

Sivakoti Sangabathuni,# Raghavendra Vasudeva Murthy,# Madhuri Gade, Harikrishna Bavireddi, Suraj Toraskar, Mahesh V. Sonar, Krishna N. Ganesh,* and Raghavendra Kikkeri* Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pune 411008, India S Supporting Information *

ABSTRACT: The constructs and study of combinatorial libraries of structurally defined homologous extracellular matrix (ECM) glycopeptides can significantly accelerate the identification of cell surface markers involved in a variety of physiological and pathological processes. Herein, we present a simple and reliable host−guest approach to design a highthroughput glyco-collagen library to modulate the primary and secondary cell line migration process. 4-Amidoadamantyl-substituted collagen peptides and β-cyclodextrin appended with mono- or disaccharides were used to construct self-assembled glycocollagen conjugates (GCCs), which were found to be thermally stable, with triple-helix structures and nanoneedles-like morphologies that altered cell migration processes. We also investigated the glycopeptide’s mechanisms of action, which included interactions with integrins and cell signaling kinases. Finally, we report murine wound models to demonstrate the real-time application of GCCs. As a result of our observations, we claim that the host−guest model of ECM glycopeptides offers an effective tool to expedite identification of specific glycopeptides to manipulate cell morphogenesis, cell differentiation metastatic processes, and their biomedical applications. KEYWORDS: collagen, cyclodextrin, carbohydrates, self-assembly, wound healing, mouse model

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the host−guest strategy dictates high-throughput processes that yield dynamic combinatorial libraries in a short span. On the basis of such advantages, we used 4-adamantyl-capped peptides and sugar-appended β-cyclodextrin and created a versatile combinatorial library of glycopeptides. We have combined solid phase peptide synthesis, a high-throughput phenotypic cell migration assay, molecular biology, and a murine wound healing model to alter specific glycopeptide-mediated cell migration. Briefly, we designed a library of glyco-collagen conjugates (GCCs), one of the most abundant fibrous structural proteins in the extracellular matrix, which is extensively involved in the cell migration process.20−24 We modified the collagen peptide (CP) with 4-adamantyl derivatives to induce host−guest interactions with β-cyclodextrin derivatives, in a way that the triple helicity of the collagen remained intact. We have used these molecules for performing high-throughput cell migration assays using primary and secondary cell lines. Moreover, we have conducted mechanistic studies to identify specific combinations that can evidently be involved in murine wound healing processes. Our

rotein glycosylation is a post-translational process responsible for more than 50% of protein modifications in nature. Attaching glycans to proteins results in a dramatic increase in the bioavailability, stability, and solubility of proteins.1−5 The major roadblock in applying glycopeptides or glycoproteins to delineate the biology of disease pathways is the limited availability of affordable and accessible synthetic platforms useful for deciphering the biochemical basis of glycoprotein interactions. Recently, signature-based profiling of glycopeptides has provided a powerful strategy for mimicking the protein structure and understanding the functions of posttranslational modifications.6−10 Similar synthetic approaches were recently used to prepare tumor-associated MUC-1 conjugates, collagen,11 coiled coils, and antifreezing glycopeptides for studying structure−function relationships of native proteins.12−14 Synthesis of glycopeptides is usually carried out by incorporating glycosylated amino acid residues in the peptide skeleton following a stepwise process15 or via convergent synthesis, in which functionalization is performed in the final step.14,16,17 Host−guest interaction between sugar-functionalized β-cyclodextrin (β-CD) and adamantyl scaffolds has recently been used for the convergent assembly of multivalent glycodendrimers.18,19 In comparison to the stepwise process, © 2017 American Chemical Society

Received: March 15, 2017 Accepted: October 27, 2017 Published: October 27, 2017 11969

DOI: 10.1021/acsnano.7b01789 ACS Nano 2017, 11, 11969−11977

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Figure 1. Molecular structure of collagen peptides (C-1 to C-3) and sugar-capped β-cyclodextrin derivatives (G-1 to G-7).

combinatorial library of GCCs (Figure S5a). ESI-MS (soft ionization) data for C-3/G-1 reveal an m/z of 862 [M + 2K + 2H]+/4 (Figure S5b), corresponding to host−guest complexes of GCC. The peaks at m/z 768 and 1135, corresponding to individual C-3 and G-1, respectively, stem from partial dissociation of the C-3/G-1 complex due to collision of the analyte ions with neutral gas molecules during the ionization process. Similarly, mass spectral data of C-3/G-4 (m/z 1242; [M + K + 3H]+/4), C-3/G-5 (m/z 1213; [M + K + 3H]+/4), C3/G-10 (m/z 1525; [M + K + 3H]+/4), and C-3/G-6 (m/z 1289; [M + K]+/4) confirmed the existence of supramolecular assemblies of respective GCCs and β-CD derivatives (Figure S5c−i). 1H-NOESY NMR also supported the anchoring of βCD on the adamantyl moiety and formation of a supramolecular assembly. Overlapping of Pro-Hyp of C-3 and β-CD peaks in the range 4.0 to 3.2 ppm prevented the assignment of β-CD peaks required for identifying changes due to C-3/G-1 complex formation. However, the adamantyl Ha−Hc peaks of C-3 revealed strong NOE interactions with the protons in the β-CD region (Figure S6), confirming the formation of a supramolecular complex between adamantane and β-CD, not with the phenyl group. The morphology of the complexes was confirmed by SEM studies (Figure S8ii,iii and Table S5). GCC C-3/G-1 formed bifringent needle-like morphologies, with slightly different topologies of the host−guest complexes as compared to their native form. A similar morphology was observed for the C-3/G-2 complexes. To address the carbohydrate−protein interaction of GCCs, their binding affinity to mannose-specific ConA and galactose-specific PNA was examined. Inhibition of binding studies of these lectins with mannose-BSA and galactose-BSA in the presence of GCCs and the IC50 values were evaluated. As expected, C-3/G-4 exhibited strong inhibition as compared to other GCC combinations, while PNA lectin showed a strong binding preference to C-3/ G-3 glycopeptide. The results clearly indicated that glycopep-

convergent method of screening cell migration is highly scalable and allows identifying different targets. It may represent a platform that can be used in drug and inhibitor discovery.

RESULTS AND DISCUSSION Building the Glyco-collagen Library. Previous studies on functionalization of collagen have revealed that the middle site (Y-position) of the proline-hydroxyproline-glycine triad is ideal for bioconjugations without hampering the triple helix structure of collagen.14 In order to monitor the biological responses mediated by glyco-collagens, we synthesized two control CPs (C-1 and C-2) and adamantyl-CP (C-3) using a solid phase peptide synthesizer following standard protocols (Figures 1, S1, and S2). The CPs were purified by reverse phase semipreparative HPLC (Figure S4 and Table S1) and characterized by MALDI-TOF data. The binding events between specific glycans and their complementary receptors occur in a multivalent and cooperative manner.25,26 Hence, β-CD conjugates of common mono/ disaccharide derivatives, found in mammalian cell surfaces, were synthesized (G-2 to G-7) to mimic the collagen sugar microenvironment (Figure 1).27−29 Circular dichroism (CD) spectra and the corresponding thermal unfolding curves of peptides C-1 to C-3 and their β-CD complexes were used to verify the triple-helix formation of the CPs (Figure S7 and Tables S2 and S3). The morphological structures of peptides C-1, C-2, and C-3 were confirmed using scanning electron microscopy (SEM). As seen from SEM images, peptides C-1 and C-2 self-assembled into nanoneedle structures, whereas C3 displayed flat nanoneedles with an average size of 800 nm × 120 nm (Figure S8ii). Collagen peptide C-3 spontaneously formed a noncovalently conjugated complex by mixing it with stoichiometric amounts (1:1) of β-CD derivatives (G-1 to G-7) and formed a 11970

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Figure 2. (i) Schematic representation of the synthesis of a library of glyco-collagen peptides using host−guest methods and their effect on cell migration. (ii) Hierarchical clustering analysis (HCA) of cell migration assay with different GCC combinations after 6 h (HeLa, MDA-MB231), 10 h for (BJ, hUVEC, NIH-3T3), and 10 h for skin and ear cells extracted from a mouse; concentration of GCC (200 μM), concentration of C-1 to C-3 (200 μM), and concentration of G-1 to G-7 (200 μM). The origin of HCA of a specific cell line is associated with the interaction between specific glyco-collagen peptides and their receptors on cell surfaces. (iii) Bright field images of progressing wound healing of skin cell lines after 10 h: (a) C-3; (b) C-3/G-1; (c) C-3/G-2; (d) C-3/G-3; (e) C-3/G-4; (f) C-3/G-5; (g) C-3/G-6; (h) C-3/G-7.

were also used in our cell migration studies. The choice of the specific cell lines was based on the fact that fibroblasts and endothelial cells are directly involved in collagen-mediated wound-healing processes31 and cells isolated from primary murine tissues often mimic in vivo responses. Confirming the specific glyco-collagen molecule’s responsibility for cancer cell motility has implications in basic and translational research for targeting invasion and metastatic processes.32

tides display strong preferences toward specific lectins (Figure S9 and Table S6). Cell Migration Assays Using a Library of Combinatorial GCCs. We have carried out cell migration assays using synthetic GCCs. Primary fibroblast cells, isolated from murine tissues (ear and skin),30 and secondary fibroblast cell lines, such as NIH-3T3 and BJ cells, were used in our studies. A variety of human cancer cells, including cervical epithelial cancer (HeLa), breast cancer (MDA-MB-231), and endothelial cells (hUVEC), 11971

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Figure 3. (i) Fluorescence images of BJ, HeLa, and skin cells incubated for 6 h with (a) C-3-F, (b) C-3-F/G-1, (c) C-3-F/G-2, (d) C-3-F/G-5, and (e) C-3-F/G-7, respectively (green fluorescence corresponds to C-3-F, blue fluorescence corresponds to Hoechst33342). (ii) Flow cytometry of integrin (α1β1) expression in the BJ cell line after (a) no treatment and treatment with (b) C-3/G-2, (c) C-3/G-5, and (d) C-3/ G-7; integrin expression in the HeLa cell line after (e) no treatment and treatment with (f) C-3/G-2, (g) C-3/G-5, and (h) C-3/G-7; integrin 11972

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expression in the skin cell line after (i) no treatment and treatment with (j) C-3/G-2, (k) C-3/G-5, and (l) C-3/G-7. (iii) Expressions of FAK, pFAK, ERK, pERK, and α-tublin in (a) BJ cell lines and (b) skin cell lines (35 μg of whole-cell lysate of proteins) with (1) control, (2) C-3/G2, and (3) C-3/G-7 analyzed by Western blotting. Protein loading was assessed by probing the blots with anti-α-tublin.

(G-2) exhibited 90−100% wound healing in both primary cell lines (Figures 2ii,iii and S10vi,vii), while glucose (G-3) and lactose (G-7) displayed variable cell migration. Endothelial hUVEC cells displayed 90−100% wound-healing effects with complexes of galactose (C-3/G-2), and complexes with fucose (C-3/G-5) and lactose (C-3/G-7) exhibited 70−80% wound healing (Figure S10i).38 All these values were found to be superior to control collagen-II, suggesting that specific sugar− peptide combinations fine-tune the microenvironment essential for cell migration as compared to the full-length collagen peptide. In order to elucidate the mechanism of wound healing, we performed fluorescent imaging experiments with 5-carboxyfluorescein-conjugated C-3 peptide (C-3-F) with four distinct combinations of sugars (G-1, G-2, G-5, G-7) (Figures S2 and S8(i)). The data from confocal images with two secondary cell lines (HeLa and BJ) and a primary cell line isolated from skin, expressing different rates of migration with C-3-F GCCs, were generated (Figure 3i). After incubation for 6 h of C-3-F and different combinations of GCC, fluorescence was observed on the cell surfaces and inside the cells. Among them, C-3-F and C-3-F/G-1 did not bind to all three cell lines, while BJ cells express C-3-F/G-2 and C-3-F/G-7 glycopeptides on the cell surfaces in a more pronounced manner as compared to other glycopeptides. These findings suggest that C-3-F GCC derivatives bind to the cell adhesive molecules through specific sugar−peptide combinations. Thus, the sugar and collagen sequence of glycopeptides seems to be a crucial factor in cell uptake and specific interactions. We hypothesize that the glycopeptides bind to cell surface receptors, such as integrins, to regulate cell migration processes, while weak fluorescence intensity indicates less impact of GCCs on cell migration. A similar trend was also observed in the skin primary cell line and HeLa cells. To support this hypothesis, the level of integrins on the cell surfaces was examined in the presence of specific GCC combinations. Integrins are a major class of cell adhesion molecules, mediating cell−cell and cell−extracellular matrix interactions.39 X-ray crystal structure analysis has shown that the triple-helical structure of collagen and the presence of 4-hydroxyproline are essential for the binding of integrins.40−42 Integrins (α1β1) are collagen receptors involved in cell migration and metastatic processes found in fibroblast, endothelial, and epithelial cells.43 All collagens are expected to bind specific integrin receptors and up-regulate the expression levels, causing the production of matrix metalloproteinases (MMPs). Using multiple antibodies for fluorescence-activated cell sorting (FACS) analysis, we distinguished the level of integrin expression in the presence of specific GCCs in primary (skin) and secondary fibroblast cells (BJ cells) (Figure 3ii and Table S8). In the case of the BJ cells, the C-3/G-5 complex was found to have a small effect on integrin expression (31.7%), whereas C-3/G-2 expressed high levels (65.8%) of integrin compared to the C-3/G-7 combination (41.3%). In contrast, C-3/G-2 expressed 7.4% integrins in HeLa and 60.4% in primary skin fibroblast cells, demonstrating that the cell surface binding of GCCs results in

Cell migration assays were carried out using standard literature protocols.33 Phenotypic cells were seeded in 24-well plates and allowed to form a monolayer by maintaining at 37 °C in a CO2 incubator (Figure 2i), and each monolayer was scratched with a 1000 μL sterile tip, for generating wounds. Seven different combinations of GCCs (200 μM) were added, and the wells were assayed. Commercially available collagen-II (200 μM), carbohydrates (G-1 to G-7), and bare CPs (C-1 to C-3) were used as controls. The bright field images were recorded every single hour until all specific combinations of GCCs resulted in 100% wound healing as compared to untreated cells. We hypothesized that the disparity in cell surface sugar receptors on different cell types influences the rate of the wound-healing process. The hierarchical clustering results were used to trace particular combinations of GCCs responsible for specific cell type wound healing and to elucidate the mechanism(s) of these processes (Figure 2ii). For consistency, results were collected in duplicates and performed in triplicates. The hierarchical clustering analysis (HCA) responses of HeLa cell migration indicated the presence of several distinct clusters. Although many combinations of the sugar−peptide complexes revealed increased cell migration during the woundhealing process as compared to the individual components, particularly C-3 GCC addition induced stronger cell migration responses as compared to that of the C-1 and C-2 GCCs. Among the sugars, lactose conjugated to peptide (C-3/G-7) showed 90−100% wound healing after 6 h, maltose (C-3/G-6), 80−90%, and fucose (C-3/G-5), 70−80%. HeLa cells were shown to express several types of sugar receptors; among them lactose receptors are considered as “Trojan horses” (Figures 2ii and S10iv).34 Comparative studies of wound-healing behavior of two fibroblast cell lines from different origins (human and mouse), i.e., NIH-3T3 and BJ, were carried out (Figures 2ii and S10ii,iii). Both cell lines exhibited different sugar-based hierarchical clusters, indicating differences in cell surface sugar receptors. It has been shown that fibroblasts in general express growth factors, which selectively bind to heparin and glucosamine sugar moieties,35 while NIH-3T3 cells do not contain galactose-specific asialoglycoprotein receptors.36 Thus, even though both cells are of the same phenotype, they may display disparity in cell migration in the presence of glycopeptide conjugations. With regard to the NIH-3T3 cell line, the combination of C-3/G-4 exhibited 90−100% cell migration in comparison to other GCCs. In contrast C-3/G-3 and C-3/G-2 were effective in wound healing of BJ cells after 10 h (Figure S10ii). The breast cancer cell line MDA-MB-231 exhibited superior wound healing after 6 h when galactose (C3/G-2) and glucose (C-3/G-3) GCCs were administered (Figure S10v).37 On the basis of these results, we hypothesize that the origin of HCA of a specific cell line is associated with the interaction between specific glyco-collagen peptides and their receptors on the cell surfaces. The experiments with the primary cell lines isolated from different organs of mice (ear and skin) also demonstrated that the C-3 complex with β-cyclodextrins modified with galactose 11973

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Figure 4. (i) Change in total wound sizes with respect to time from day 0 to day 14 in mice. (ii) Respective pictures of wounds taken after treatment with collagen II (Col-II) and C-3/G-7 at different days. (iii) Histological analysis of wounds after treatment with hematoxylin and eosin stain in the presence of (a) normal (without wound); (b) control; (c) collagen II; and (d) C-3/G-7 after 8 days (arrow indicates the wound-healing process, scale bar = 200 μm, n = 6, data shown as mean ± SD, two-way ANOVA post-test for significance *p < 0.05, **p < 0.01, ***p < 0.01).

mice at postwounding days 3, 5, and 8, respectively. Complete wound closure was observed with C-3/G-7 on day ∼11, whereas natural collagen and the control showed similar wound healing on days ∼12 and ∼14, respectively (Figures 4i and S11). To further confirm the progress of the wound healing, histomorphometric analysis was performed on the wound biopsies on day 8 of postwounding. Histology images of the mouse showed substantial growth of the epidermis layer in the C-3/G-7-treated mouse as compared to the control and natural-collagen-treated mice, suggesting a faster wound-healing rate with C-3/G-7 peptide (Figure 4iii). In summary, our study shows that the use of the supramolecular strategy represents a valid step in identifying specific biomarkers for physiological and pathological processes.

up-regulation of integrins, which may influence MMP production and promote the wound-healing process. To examine the mechanism by which integrin induces cell migrations, the ERK/FAK pathways were investigated with BJ and skin fibroblast cells.44 Elevated expression levels of these proteins are considered as a general marker of cell migration. To test if the GCCs inducing integrin elevate the ERK and FAK, protein extracts of GCC (C-3/G-2 and C-3/G-7)-treated cells at 6 h were analyzed for the expression level of ERK and FAK. Western blot analysis of FAK and ERK showed that BJ cells treated with C-3/G-2 expressed a high level of ERK and FAK compared to C-3/G-7. In contrast, skin fibroblast cells express strong ERK and FAK in the presence of both C-3/G-2 and C-3/G-7 conjugates (Figure 3iii). Overall, these results suggest that the cell adhesion molecules (integrins) and sugar receptors synergistically work together to influence cell migration. On the basis of the in vitro screening experiments, we explored the wound-healing effect of C-3/G-7 in a wild-type mice model.45 Determination of wound-healing rate and histology of the wound is a useful method for studying the real-time application of these GCCs.46 Previously, hydrogelencapsulated fibroblast, collagen, and growth factor molecules showed wound-healing properties in a mice model.47−49 Hence, it is expected that self-assembled GCCs could also be a promising platform for in vivo wound healing. In order to study this phenomenon, a 5 mm excisional wound was created on the dorsal surface of the mice, and C-3/G-7 and collagen-II were applied on top of the wound; the decrease in the wound area was monitored for 14 days (Figure 4ii). As shown in Figure 4, the wound area of C-3/G-7-treated mice was significantly reduced as compared to that of collagen-II-treated and control

CONCLUSION A host−guest strategy was used to prepare glyco-peptides and utilized for studying high-throughput responses of cell migration in different cell lines. Validated by a series of biophysical and imaging techniques, it was shown that the cell adhesion molecules and carbohydrate receptors synergistically influence cell migration. The simplicity and effectiveness of the system underscore its potential in accelerating glycopeptide research. Expanding the scope of this platform and the synthesis of libraries are envisioned to be used for research toward understanding of the role of sugar−peptide combinations in various physiological and pathological processes for which no data are available. EXPERIMENTAL METHODS Host−Guest Complexes of Modified β-Cyclodextrins and the Peptides. The host−guest complexes were prepared by mixing 11974

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Western Blot Analysis. BJ and skin cells were grown in 100 mm Petri dishes and treated with C-3/G-2 and C-3/G-7 (200 μM) for 6 h. The cells were pelleted, washed with PBS buffer, and treated with protease inhibitors before treating with lysis buffer containing 150 mM NaCl, 1% NP-40, 0.25% sodium dodecyl sulfate (SDS), 1 mM ethylenediamine tetraacetic acid (EDTA), and 1 mM phenylmethane sulfonyl fluoride (PMSF) in 50 mM Tris-Cl (pH 7.4). After 1 h, the supernatant was collected by centrifugation (14 000 rpm) for 15 min and stored in aliquots. The protein content was quantified using the Bradford method. The protein (35 μg) was loaded on SDSpolyacrylamide gel electrophoresis (10%) and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated for 2 h with specific antibodies corresponding to FAK, phospho FAK (Tyr397), ERK1/2, and phospho ERK (Thr202/ Tyr204). Thereafter, the membranes were incubated with horse radish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature, and visualization was done using an Immobilon Western Chemiluminescent HRP substrate kit (Millipore Corporation, MA, USA) with α-tubulin as internal standard. BioRad’s Protein Ladder (Thermo, EU) was used to determine the molecular weights of the protein bands. Wound-Healing Analysis in Mice. Male C57BL mice (7−8 weeks old, from Reliance Biotech, Bombay) were used. Prior to experiments the mice were maintained in an animal house for 48 h in a 12 h/12 h light/dark cycle, with the proper amount of food and water. All experiments were performed in accordance with the relevant guidelines and regulations of the Institutional Animal Ethical Committee, set up by CPCSEA, Govt. of India. The mice (n = 6) were anesthetized by ketamine, and the dorsal flank was shaved and sterilized with 70% ethanol. An excisional wound was generated using 5 mm biopsy punch excision (surface area 28.27 mm2) and treated with native collagen II and with C-3/G-7 (500 μM). The progress of wound healing was followed by imaging and histology experiments over 3, 5, 8, 11, 12, and 14 days. Histology images were taken by cutting the tissue section of the wound and fixing in Bouin’s fixative followed by dehydration with ethanol and xylene. The tissues were embeded in Paraflast, and 10 μM sections were cut using a microtome. The tissue sections were fixed on a poly-L-lysine-coated glass plates followed by treatment with hematoxylin and eosin stain. The morphology of the section was observed with an EVOS microscope.

equimolar amounts of adamantyl-modified peptides (C-1 to C-3) and β-cyclodextrin or β-cyclodextrin substituted with sugar molecules (G-1 to G-7, Figures 1 and S3) followed by vortexing for 2 min (Figure S5a). Corroboration of the Formation of Inclusion Complexes by NMR. 1 H NMR NOESY spectra were recorded, and the intermolecular interactions of adamantyl protons with those of the β-cyclodextrin skeleton were probed by tracing the cross-peaks corresponding to the protons that are in close proximity to the protons of the β-cyclodextrin, indicating the incorporation of the adamantyl residue in the cavity. Peptide C-3 as guest and G-1 as host were used to study host−guest complex formation. The complex C-3/ G-1 was prepared using the protocol mentioned above, and the NOESY spectrum of the compound was recorded, in addition to the spectrum of the peptide C-3. The focus was put on the peaks of the adamantyl residues, labeled a, b, and c in Figure S6, and the crosspeaks, corresponding to adamantyl peaks in the C-3/G-1 complex, which are absent in C-3. The NMR data confirmed the formation of a host−guest inclusion complex between C-3 and G-1. Wound-Healing Analysis. Procedure for Isolation of Fibroblast Cells from Mouse Skin and Ear. Isolation of skin fibroblast cells from mice was done by following literature procedures.30 In more detail, after cleaning the mouse skin with 70% ethanol, the area was shaved and an incision cut was made by a sterilized scalpel blade. The tissues were washed with 70% ethanol and PBS for 3−4 min and transferred immediately into Dulbecco’s modified Eagle’s medium (DMEM). The tissue sections were further cut into tiny pieces and digested for 90 min, and the cells were collected by centrifugation and were cultured in DMEM medium containing 10% fetal bovine serum and 0.1% streptomycin. A similar protocol was followed for ear fibroblast cells from mice. The cells were seeded into 24-well tissue culture plates and allowed to form a monolayer. Details about the cell types, medium, and all other conditions are summarized in Table S7. The monolayers were gently scratched across the center of the well using 1000 μL pipet tips, and the wells were gently washed with phosphate-buffered saline (PBS) followed by medium, for removal of detached cells. Different combinations of glyco-collagen conjugates (200 μM) were added to the wells, and after incubation of 1 h, bright field microscopic images were recorded. The gap distance and area were quantitatively evaluated using ImageJ software. All measurements were done in triplicates. The results have shown that each cell line has a different growth rate and different wound-healing processes. For HeLa and MDA-MB-231 cells, we have observed complete wound healing after 6−7 h, whereas with BJ, hUVEC, and NIH-3T3 wound healing was observed after 9−10 h for skin and after 10−11 h for ear cells. Percentage of wound healing =

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b01789. Information about used materials and synthesis, as well as detailed analytical data of the compounds and data from the biophysical, in vitro, and in vivo assays (PDF)

(d0 − d1)× 100 d0

where “d1” corresponds to the average distance traveled by cells after adding GCC or sugars and “d0” represents the average distance traveled by cells in the absence of wound-healing compounds. Confocal Laser Scanning Microscope Images. HeLa/skin/BJ cells were cultured over an eight-well chambered cover glass (Sigma Aldrich) and were fed with fluorescein-conjugated C-3-F (200 μM) for 6 h, washed twice with PBS, and added DMEM). Further the cells were treated with Hoechst 33342 (10 μL of 2 μg/mL solution) for 30 min to stain nuclei and washed three times with PBS buffer. The fluorescence of Hoechst 33342 and fluorescein was excited with an argon laser at 405 and 450 nm, respectively, and the emission was collected through 403−452 nm and 500−530 nm filters, respectively. FACS Assay. HeLa/skin/BJ cells were cultured as described above and treated with C-3 and the complexes G-2, G-5, and G-7 (200 μM) for 6 h at 37 °C. The cells were washed with PBS, detached, and transferred to FACS tubes. Integrin α1β1 mouse antibody (Santa Cruz Biotechnology) was added and incubated for 1 h followed by a secondary antibody. The fluorescent channel FL-2 was used to detect integrin levels. All data were analyzed with the FlowJo software.

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Fax: +91-20-25899790. Tel: +91-20-25908207. ORCID

Raghavendra Kikkeri: 0000-0002-4451-6338 Author Contributions

All authors have given approval to the final version of the manuscript. Author Contributions #

S. Sangabathuni and R. V. Murthy contributed equally.

Notes

The authors declare no competing financial interest. 11975

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ACKNOWLEDGMENTS Financial support from the IISER, Pune, Max-Planck partner group, and DST (Grant No. SB/S1/C-46/2014) is gratefully acknowledged. S.S., H.B., and S.T. thank the CSIR-SRF and M.G. thanks UGC-SRF for supporting their fellowships. Special thanks to Prof. N. K. Subhedar for helping to set up mouse wound healing and histology experiments. We also thank Dr. Rina Arad Yellin for critical evaluation of the manuscript. REFERENCES (1) Dwek, R. A. Glycobiology: Toward Understanding the Function of Sugars. Chem. Rev. 1996, 96, 683−720. (2) Buskas, T.; Ingale, S.; Boons, G. J. Glycopeptides as Versatile Tools for Glycobiology. Glycobiology 2006, 16, 113R−136R. (3) Roth, J. Protein N-Glycosylation along the Secretory Pathway: Relationship to Organelle Topography and Function, Protein Quality Control, and Cell Interactions. Chem. Rev. 2002, 102, 285−303. (4) Wolfert, M. A.; Boons, G. J. Adaptive Immune Activation: Glycosylation Does Matter. Nat. Chem. Biol. 2013, 9, 776−784. (5) Harrison, K.; Hallett, J.; Burcham, T. S.; Feeney, R. E.; Kerr, W. L.; Yeh, Y. Ice Growth in Supercooled Solutions of Antifreeze Glycoprotein. Nature 1987, 328, 241−243. (6) Kotch, F. W.; Guzei, I. A.; Raines, R. T. Stabilization of the Collagen Triple Helix by O-Methylation of Hydroxyproline Residues. J. Am. Chem. Soc. 2008, 130, 2952−2953. (7) Yamazaki, C. M.; Nakase, I.; Endo, H.; Kishimoto, S.; Mashiyama, Y.; Masuda, R.; Futaki, S.; Koide, T. Collagen-Like Cell-Penetrating Peptides. Angew. Chem., Int. Ed. 2013, 52, 5497−5500. (8) Kim, W.; McMillan, R. A.; Snyder, J. P.; Conticello, V. P. A Stereoelectronic Effect on Turn Formation Due to Proline Substitution in Elastin-Mimetic Polypeptides. J. Am. Chem. Soc. 2005, 127, 18121−18132. (9) Wang, P.; Zhu, J.; Yuan, Y.; Danishefsky, S. J. Total Synthesis of the 2,6-Sialylated Immunoglobulin G Glycopeptide Fragment in Homogeneous Form. J. Am. Chem. Soc. 2009, 131, 16669−16671. (10) Andersson, I. E.; Andersson, C. D.; Batsalova, T.; Dzhambazov, B.; Holmdahl, R.; Kihlberg, J.; Linusson, A. Design of Glycopeptides Used to Investigate Class II MHC Binding and T-cell Responses Associated with Autoimmune Arthritis. PLoS One 2011, 6, e17881. (11) Gaidzik, N.; Westerlind, U.; Kunz, H. The Development of Synthetic Antitumour Vaccines from Mucin Glycopeptide Antigens. Chem. Soc. Rev. 2013, 42, 4421−4442. (12) Falenski, J. A.; Gerling, U. I.; Koksch, B. Multiple Glycosylation of de novo Designed Alpha-Helical Coiled Coil Peptides. Bioorg. Med. Chem. 2010, 18, 3703−3706. (13) Nagel, L.; Budke, C.; Dreyer, A.; Koop, T.; Sewald, N. Antifreeze Glycopeptide Diastereomers. Beilstein J. Org. Chem. 2012, 8, 1657−1667. (14) Erdmann, R. S.; Wennemers, H. Functionalizable Collagen Model Peptides. J. Am. Chem. Soc. 2010, 132, 13957−13959. (15) Westerlind, U. Synthetic Glycopeptides and Glycoproteins with Applications in Biological Research. Beilstein J. Org. Chem. 2012, 8, 804−818. (16) Russo, L.; Sgambato, A.; Lecchi, M.; Pastori, V.; Raspanti, M.; Natalello, A.; Doglia, S. M.; Nicotra, F.; Cipolla, L. Neoglucosylated Collagen Matrices Drive Neuronal Cells to Differentiate. ACS Chem. Neurosci. 2014, 5, 261−265. (17) Bini, D.; Russo, L.; Battocchio, C.; Natalello, A.; Polzonetti, G.; Doglia, S. M.; Nicotra, F.; Cipolla, L. Dendron Synthesis and Carbohydrate Immobilization on a Biomaterial Surface by a DoubleClick Reaction. Org. Lett. 2014, 16, 1298−1301. (18) Grunstein, D.; Maglinao, M.; Kikkeri, R.; Collot, M.; Barylyuk, K.; Lepenies, B.; Kamena, F.; Zenobi, R.; Seeberger, P. H. Hexameric Supramolecular Scaffold Orients Carbohydrates to Sense Bacteria. J. Am. Chem. Soc. 2011, 133, 13957−13966. (19) Bavireddi, H.; Vasudeva Murthy, R.; Gade, M.; Sangabathuni, S.; Chaudhary, P. M.; Alex, C.; Lepenies, B.; Kikkeri, R. Understanding 11976

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DOI: 10.1021/acsnano.7b01789 ACS Nano 2017, 11, 11969−11977