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Enzymatic Formation of an Injectable Hydrogel from a Glycopeptide as a Biomimetic Scaffold for Vascularization Jingjing Qi, Yufei Yan, Baochang Cheng, Lianfu Deng, Zeng-Wu Shao, Ziling Sun, and Xinming Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18535 • Publication Date (Web): 30 Jan 2018 Downloaded from http://pubs.acs.org on February 1, 2018
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Enzymatic Formation of an Injectable Hydrogel from a Glycopeptide as a Biomimetic Scaffold for Vascularization ⊥
Jingjing Qi,†,¶ Yufei Yan,‡,¶ Baochang Cheng,† Lianfu Deng,‡ Zengwu Shao,§ Ziling Sun,*, and Xinming Li*,† †
College of Chemistry, Chemical Engineering and Materials Science, Soochow University,
Suzhou, 215123, China. E-mail:
[email protected]. ‡
Shanghai Key Laboratory for Bone and Joint Diseases, Shanghai Institute of Orthopaedics and
Traumatology, Shanghai Ruijin Hospital, Shanghai Jiaotong University, School of Medicine, Shanghai 200025, China. §
Department of Orthopedic, Union Hospital, Tongji Medical School, Huazhong University of
Science and Technology, Wuhan, 430022, China. ⊥
School of Biology and Basic Medical Science, Soochow University, Suzhou, 215123, China.
E-mail:
[email protected]. KEYWORDS: glycopeptide, hydrogel, self-assembly, DFO, tissue engineering.
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ABSTRACT: The construction of functional vascular networks in regenerative tissues is a crucial technology in tissue engineering to ensure the sufficient supply of nutrients. Although natural hydrogels are highly prevalent in fabricating 3D scaffolds to induce neovascular growth, their widespread applicability was limited by the potential risk of immunogenicy or pathogen transmission. Therefore, developing hydrogels with good biocompatibility and cell affinity is highly desirable for fabricating alternative matrices for tissue regeneration applications. Herein, we report the generation of a new kind of hydrogel from supramolecular assembling of a synthetic glycopeptide to mimic the glycosylated microenvironment of ECM. With the presence of a tyrosine phosphate group, this molecule can undergo supramolecualr self-assembling and gelation triggered by alkaline phosphatase under physiological conditions. Following supramolecular self-assembling, the glycopeptide gelator tended to form nanofiment structures displaying a high density of glucose moieties on their surface for endothelial cell adhesion and proliferation. Further incorporation with deferoxamine (DFO), the self-assembled hydrogel can serve as a reservoir for sustainably releasing DFO and inducing endothelial cell capillary morphogenesis in vitro. After subcutaneous injection in mice, the glycopeptide hydrogel encapsulating DFO can work as an effective matrix to trigger the generation of new blood capillaries in vivo.
1. INTRODUCTION Tissue engineering provides a promising approach to replace or regenerate diseased or damaged tissues.1 The long-term survival of engineered tissues relies on the formation of sufficient microvascular network for nutrient exchange and oxygen delivery.2-3 In vivo, vascularization involves a cascade of events regulated by complex signals from both extracellular matrix (ECM) and growth factors, and the spatial and temporal arrangement of these signals in ECM
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orchestrated the in-situ assembly of endothelial cells into capillary-like structures.4-5 Therefore, designing a scaffold which can mimic the structures and biological functions of native ECM for regulating the spatial arrangement of pro-angiogenic signals and supporting cell morphogenesis remains a major challenge in tissue engineering. Over the last decade, hydrogels from different origins (e.g., natural and synthetic polymers) have received a considerable interest as desirable candidates for fabricating biomaterials to support cell proliferation and growth, owing to their high water content and structural similarity to the natural ECM.6-7 Although hydrogels derived from natural ECM components (e.g., collagen, laminin, fibronectin and glycoaminoglycans) exhibited good capabilities to adhere endothelial cells and support their transformation to microvessels in vitro,8 their widespread applicability of was bothered with the difficulty of purification and the risk of immunogenicy or pathogen transmission. Therefore, development of a hydrogel to mimic the structural morphologies or chemical compositions of ECM is desirable for fabricating alternative matrices for tissue regeneration applications. Peptide-based hydrogels from supramolecular self-assembling have emerged as an important class of biomimetic materials for biomedical applications in recent years, because of their inherent biocompatibility, biodegradability and morphological similarity to fibrous proteins in ECM.9-15 Moreover, further decoration with certain bioactive cues (such as RGD, YIGSR, IKVAV, REDV and PHSRN) derived from natural ECM, can greatly improve the biological performance of peptide-based hydrogels in inducing cell adhesion, migration, and differentiation.16-20 On the other hand, besides the presence of fibrous proteins to work as structural supports for cells and tissues, the microenvironment of ECM also contains varied glycidic molecules, such as glycosaminoglycans and proteoglycans, which play a significant role
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in modulating cell adhesion, migration and proliferation or controlling cellular behaviours via interactions between sugars and cell surface receptors.21-22 Therefore, in order to generate a hydrogel for mimicking the glycosylated microenvironment of ECM, we designed and synthesized a new kind of glycopeptide molecule which consisted of a naphthyl group, a tetra-peptide segment (Phe-Phe-Asp-Tyr(H2PO3)) and a sugar moiety (Dglucosamine) on the side chain of Asp. Then we used this molecule as building-block to prepare a supramolecular hydrogel with glucose decoration on its surface (Scheme 1). The naphthyl moiety and Phe-Phe dipeptide segment acted as a rigid scaffold to drive supramolecular selfassembling of our designed glycopeptide gelator via extended aromatic−aromatic interactions; the Asp group was exploited for site-specific glucose modifications; and the tyrosine-phosphate (e.g., Tyr(H2PO3)) worked as an excellent substrate of alkaline phosphatase for catalytic supramolecular gelation. Because of its good sensitivity towards alkaline phosphatase for dephosphorylation reaction, this glycopeptide molecule can undergo quick supramolecular selfassembly in water at physiological conditions. Following supramolecular self-assembling, the glucose moiety on the surface of hydrogel can work as biomolecular cues for promoting cell attachment and growth. Further incorporation with DFO, an organic drug with potentials to provoke vascularization, the self-assembled hydrogel can work as an effective matrix to support rapid formation of blood capillaries both in vitro and in vivo. 2. RESULTS AND DISCUSSION Scheme 2 shows the synthetic route and molecular structure of gelator precursor 1, which was prepared by following solid phase synthesis protocols with the application of Fmoc-Tyr(PO3)OH, Fmoc-Asp(Glc)-OH, Fmoc-Phe-OH and 2-(Naphthalen-6-yl) acetic acid. Fmoc-Asp(Glc)-
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OH was obtained through the coupling reaction between Fmoc-Asp-OtBu and D-glucosamine (Figure S1).23-24 After HPLC purification, we got gelator precursor 1 in yield around 63%. NMR and MS analysis confirmed the accuracy of the molecular structures of both Fmoc-Asp(Glc)-OH and gelator precursor 1 (Figure S2-S5). After obtaining gelator precursor 1, we tested its solubility in water and its sensitivity towards alkaline phosphatase for supramolecular gelation. Typically, we dissolved 1.2 mg of 1 in water, adjusted the pH of this solution to 7.4 and obtained a clear solution with a final concentration at 0.6 wt% (Figure 1A). Followed by the addition of alkaline phosphatase, the solution of 1 transformed to a transparent hydrogel of glycopeptide (namely, Gp gel) in 20 min at room temperature (Figure 1B), which is due to the enzymatic dephosphoryaltion on 1 and the generation of gelator 2. The afforded gelator 2 then underwent supramolecular self-assembling for the formation of Gp gel, relying on extended aromaticaromatic and hydrogen bonding interactions between gelator 2. The transmission electron microscopy (TEM) image shown in Figure 1C revealed the formation of one-dimensional nanofibrous structures from gelator 2 during the process of gelation, with several micrometers in length and 5 nm in width. These high-aspect-ratio nanofibers physically cross-linked with each other to form homogeneous networks as a gel matrix. Scanning electron micrograph (SEM) images showed the presence of three-dimensional porous structures self-assembled from gelator 2 at microscales (Figure 1D). Dynamic rheological studies revealed that Gp gel self-assembled from 2 exhibited a dominantly elastic property of hydrogels (Figure 2A and Figure S6), as exemplified by its much higher storage modulus (G') than its loss modulus (G") within the investigated oscillating frequency limits (0.1-20 Hz). These results indicated that gelator precursor 1 containing a glucose moiety can work as a good building block for the preparation of a stable supramolecular hydrogel of a
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glycopeptide via an enzymatic dephosphorylation reaction. Circular dichroism (CD) was used to examine the molecular conformations of gelator 2 during its supramolecular self-assembling. As shown in Figure 2B, the solution of gelator precursor 1 displayed a positive peak at 201 nm and a negative peak at 232 nm, indicating the absence of regular secondary structures of gelator precursor 1 in a solution without the presence of alkaline phosphatase.25 However, after adding alkaline phosphatase to the solution of 1 for supramolecular gelation, we observed the generation of a negative peak near 217 nm and a positive peak around 192 nm in the CD spectrum of Gp gel, which was consistent with the characteristic of a β-sheet conformation.26 Besides, the additional negative peak at 204 nm, and bisignated Cotton effects around 279 and 297 nm could be ascribed to the highly ordered arrangements of carbonyl groups and phenyl residues within its selfassembled structures.27 FTIR analysis provided further information about the secondary structures of gelator 2 during the process of supramolecular assembling. For example, its FTIR spectrum showed a major amide I peak at 1629 cm-1 and a shoulder peak around 1665 cm-1, indicating the formation of a typical β-sheet structure, together with a small amount of a α-helixlike conformation (Figure 2C).28-29 The absorption at 1591 cm-1 was ascribed to the vibration of aromatic rings. The broad peak near 1458 cm-1 was assigned to the bending and stretching absorptions of N-H and C-N groups on gelator 2.30 One of important features of peptide glycosylation in nature is to increase the biostability of peptide residues by resisting proteolytic digestion.31-32 According to biostability tests shown in Figure 2D, we found that gelator precursor 1 showed a well resistance to proteinase K digestion, as more than 61 % of 1 remained intact after 24 h of incubation with proteinase K. In contrast, the peptide moiety without glucose modification (e.g., NapFFDYp) was hydrolysed completely in 8 h by proteinase K, indicating
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the important role of a glucose moiety on gelator precursor 1 for protecting its peptide backbone from proteinase digestion. The ECM microenvironment is composed of a variety of glycidic molecules ranging from glycosaminoglycans to proteoglycans, which play a significant role in modulating cell adhesion, migration, proliferation, and other cellular processes via the interactions between carbohydrate epitopes and cell surface receptors.33-34 Because of the high densities of glucose moieties on the scaffold of Gp gel, we decided to examine the effects of glucose decoration on cell adhesion and proliferation when using Gp gel as a cell culturing matrix. Cell viability assays confirmed the good biocompatibility of 1 towards human umbilical vein endothelial cells (HUVEC) (Figure S7). In vitro gel stability tests indicated that Gp gel can maintain good stability in cell culture media for more than 7 days (Figure S8). After planting HUVECs on the surface of Gp gel, we examined cell attachment and proliferation behaviors of HUVECs over the course of 5-day culture. Based on live-dead assays shown in Figure 3A, HUVECs were able to adhere and proliferate well on the surface of Gp gel, as exemplified by the polyhedral and spindle-like morphologies of cells after one day of incubation, and the steady increase of cell densities over the course of 5-day culture. Cytoskeletal F-actin staining assays revealed the formation of welldefined and elongated stress actin filaments inside HUVECs (Figure 3B and 3C), due to the establishment of glucose-mediated interactions between HUVECs and the gel matrix. Quantitative analysis confirmed the gradual increase of cell numbers of HUVECs from day-1 (1.53×104/cm2) to day-5 (4.57×104/cm2) when they were cultured on the surface of Gp gel (Figure 3D). Therefore, the supramolecular hydrogel from gelator precursor 1 can work as an effective scaffold to promote endothelial cell adhesion and proliferation via glucose-mediated cell interactions.
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Establishing vascular systems in tissue constructs is a key challenge in tissue engineering for the survival and function of regenerative tissues.2 The angiogenic process in nature is achieved by complex interactions between endothelial cells, ECM and growth factors.4-5 Particularly, physical interactions between endothelial cells and ECM play a critical role in cell adhesion, proliferation, differentiation and vascular formations.35-36 Although hydrogels derived from different naturally occurring polymers are highly prevalent in fabricating 3D scaffolds for neovascular growth, their applicability was limited by the risk of potential immunogenicy or pathogen transmission. Because of high affinity of Gp gel to HUVECs for cell adhesion and proliferation, we further examined the potentials of Gp gel to support microvasculature formations from HUVECs with the presence of a pro-angiogenic agent. Deferoxamine (DFO) is a clinical drug originally used for treating diseases of excess iron, but recent studies revealed that DFO can also work as a pro-angiogenic agent to promote the formation of new blood vessels by up-regulating the expression of HIF-1α and vascular endothelial growth factor.37-38 In comparison with naturally occurring or recombinant growth factors, DFO exhibited numerous advantages in inducing angiogenesis, such as quickly angiogenic process, long-term stability and efficiency.39-40 In addition, encapsulation of DFO in hydrogels can regulate the spatial and temporal delivery of DFO through sustained release.41 The pro-angiogenic activity of DFO released from Gp gel was checked by an in vitro tube formation assay. After HUVECs were seeded on the surface of Gp gel encapsulating DFO (1 mg/mL), the morphogenetic changes of cells were examined over time. As shown in Figure 4A, HUVECs attached well on the surface of Gp gel and tended to form cell clusters and capillary sprouts in 1 day. With the extension of incubation time to 2 and 3 days, most of cells underwent microvascular morphogenesis and formed capillary tube networks. Quantitative analysis revealed the gradual increase of closed
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meshes, junctions and total tubule length with time (Figure 4B-4D). For example, the average numbers of meshes, junctions and tubule length were about 6, 59 and 4922 µm at day-1, and gradually increased to 41, 142 and 8394 µm at day-3. In comparison, no obvious capillary tubes or short cord structures were observed in Gp gel without encapsulating DFO over the course 3day culture, indicating the bioactivity of DFO for inducing capillary morphogenesis of endothelial cells. During this process, Gp gel can not only provide a suitable growth support for effective cell attachment and colonization, but also served as a reservoir for sustained DFO delivery to induce the formation of capillary tubes (Figure S11). Injectable hydrogels from different origins (e.g., natural and synthetic polymers) have received increasing interests in recent years for drug delivery, cell encapsulation and wound dressings, because of numerous advantages such as in-situ formability, simple incorporation of drugs/cells, and minimally invasive delivery.42-43 The in-situ gelation property of gelator precursor 1 under the catalysis of alkaline phosphatase encouraged us to explore the potentials of Gp gel for injectable applications in inducing microvascularization in vivo. First, we prepared a Gp gel (0.6 wt%), and loaded it in a 1 mL syringe. After extrusion through a needle with diameter in 0.4 mm, the sample was collected in a vial and it can transform into a stable gel properly within 15 min (Figure 5A). Its sheer-recovery property was further tested by a dynamic shear-shining and recovery experiment. As shown in Figure 5B, when Gp gel (0.6 wt%) was subject to a dynamic oscillatory deformation at a low strain amplitude (1.0%), its storage modulus (G’) was much higher than its loss modulus (G''), indicating the dominantly elastic property of Gp gel at a low oscillatory strain. After the shear amplitude was increased to 100%, both G' and G" decreased significantly to 27.6 and 12.6 Pa, due to the disruption of physically cross-linked networks of Gp gel. However, when the amplitude of oscillatory strain was changed back to 1%, both G' and G''
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values recovered to 85% of their original values, suggesting the sheer-recovery property of GP gel. In addition, Gp gel loaded with DFO (1 mg/mL) also exhibited similar sheer-recovery properties to Gp gel, but its G’ value was 10 times higher than that of Gp gel without DFO (Figure 5C and Figure S12), implying that the incorporation of DFO could enhance the mechanical strength of Gp gel via intermolecular interactions. These results indicated that Gp gel possessed tunable shear-shining and recovery properties (Figure S13) which could be exploited for injectable applications. After DFO-loading Gp gel was implanted into the dorsal side of mice through subcutaneous injection, its biodegradability, biocompatibility and capabilities to induce microvascularization in vivo were evaluated at day-3, day-7, and day-10. The DFO-loaded hydrogel remained localized in the implantation site and was easily identifiable through 10 days, but its volume or size decreased gradually with time (Figure S14). H&E staining confirmed that the areas of implanted Gp gel were decreased gradually with time due to slow bioresorption and degradation of Gp gel and cell invasion and tissue growth (Fig. S15). In addition, within the course of implantation, the implanted Gp gel did not induce significant inflammatory responses to the surrounding tissue, such as the absence of red or swollen features on mice, suggesting a negative inflammatory response. More importantly, after implantation of DFO-loading Gp gel, we observed an obviously increased numbers of blood vessels in connective tissues surrounding DFO-loading Gp gel at day-3, compared with the group injected with blank Gp gel (Figure 6). In addition, the densities of blood capillaries in connective tissues were further increased within the course of 10day implantation, which could be ascribed to the long bioavailability of DFO sustainably released from Gp gel, and its good biostability in vivo. Meanwhile, we also identified the presence of red blood cells within these blood vessels, indicating the biofunctionality of these
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newly generated blood vessels. Immunohistochemical staining with CD31 antibodies indicated that the blood vessels identified in the periphery of the implanted gel constructs originated from endothelial cells (Figure 7). Quantitative analyses confirmed the steady increase of blood vessels over the course of implantation of DFO-loading Gp gel. For example, the average areas of blood vessels for DFO-loading group were around 1.98 % per observed field at day-3, and increased to 3.3 % at day-10 (Figure S16). In comparison, the groups implanted with the blank Gp gel showed a much lower vessel densities (e.g., 0.50 % at day-3 and 1.06 % at day-10) over the course of 10-day implantation. These results suggested Gp gel can work as an effective matrix for supporting microvasculature formations in vivo through efficient encapsulation and sustained deliveries of DFO. 3. CONCLUSIONS In summary, we reported the generation of a new self-assembled hydrogel from a glycopeptide for mimicking the highly glycosylated microenvironment of ECM. With the presence of tyrosine phosphate group, this molecule can undergo in-situ supramolecualr self-assembly and gelation triggered by alkaline phosphatase under physiological condition. Following supramolecular selfassembling, the glucose moieties on the surface of hydrogel worked as biomolecular cues for improving cell attachment and growth of HUVECs via sugar-receptor interactions. Further incorporation with a pro-angiogenic factor (DFO), the self-assembled Gp gel severed as a reservoir for sustainably releasing DFO and inducing endothelial cell capillary morphogenesis in vitro. After subcutaneously injection in mice, the self-assembled Gp gel encapsulating DFO can trigger the generation of new blood capillaries in vivo. The results of this study suggested that glucose decoration of a peptide gelator proved to be an effective means for the generation of a
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novel supramolecular hydrogelator and hydrogel with improved cell adhesion properties for regenerative medicine applications. 4. EXPERIMENTAL SECTION Synthesis of gelator precursor 1. Gelator precursor 1 was synthesized by following standard solid-phase peptide synthesis protocols with the application of Fmoc-Tyr(H2PO3)-OH, FmocAsp(Glc)-OH, Fmoc-Phe-OH and 2-(naphthalen-6-yl) acetic acid. Fmoc-Asp(Glc)-OH was prepared according to a previously reported method. Firstly, 2-chlorotritylchloride resin (1.0 g) was suspended in dry dichloromethane (DCM) with N2 bubbling for 30 min, and then was washed by dry dimethylformamide (DMF) three times. Afterward, the solution containing FmocTyr(H2PO3)-OH and N,N-diisopropylethylamine (DIEA) in DMF was added to a reactor. After reaction for 1 h, the resin was washed by dry DMF three times and quenched by a blocking solution (80:15:5 of DCM/methanol/DIEA) for 10 min. Then, the resin was treated with 20% piperidine in DMF for 0.5 h, to remove Fmoc-protecting groups on amino acids. The designed peptide chain was elongated step-by-step by using HBTU as a coupling reagent and following standard Fmoc SPPS protocols. Finally, the synthetic glycopeptide was removed from the resin and purified with HPLC by using H2O−CH3CN as eluents (from 80:20 to 0:100). The final yield of gelator precursor 1 is around 63%. 1H NMR (400 MHz, DMSO-d6): 8.30−8.22 (s, 4H), 8.14−8.09 (d, 1H), 7.83−7.79 (d, 1H), 7.76−7.72 (d, 1H), 7.72−7.65 (d, 1H), 7.58−7.52 (s, 1H), 7.47−7.39 (m, 3H), 7.24−7.02 (m, 14H),7.02−6.96 (m, 1H), 4.58−4.38 (m, 4H), 3.58−3.15 (m, 14H), 3.04-2.84 (m, 4H), 2.80−2.57 (m, 4H). MS: calcd M+=999.3, obsd (M+H)+=1000.3. TEM and SEM characterizations. 10 µL of Gp gel (0.6 wt%) was loaded on a carbon-coated Cu grid, followed by phosphotungstic acid (1.0%) staining. Then TEM images of the
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nanostructures within Gp gel were recorded on a transmission electron microscope (Tecnai G220). The sample for SEM characterization was prepared by loading 5 µL of Gp gel (0.6 wt%) on a single-crystal silica plate and dried under vacuum, followed by coating with a thin layer of gold before SEM measurement. SEM images of the microstructures within Gp gel were obtained from a scanning electron microscope (Hitachi S-4800). Rheological tests. 200 µL of Gp gel (0.6 wt%) was loaded on a 20 mm parallel plate and subjected to rheological experiments on a Thermo Scientific HAAKE ReoStress 6000 rheometer. Dynamic strain sweep experiments were carried out from 0.1 to 10% strain with a fixed frequency at 6.282 rad/s at 25 °C. Dynamic frequency sweep tests were conducted from 20 to 0.1 rad/s with a fixed strain at 0.5% to ensure dynamic viscoelastic linearity. Shear-shining and recovery tests were firstly tested at low oscillatory strain amplitude (1.0%) with a fixed frequency at 6.28 rad/s, followed by the application of a high oscillatory strain at 100% for 30 s. And then, the amplitude of oscillatory strain was changed back to 1% for gel recovery. Circular dichroism and FTIR characterizations. 20 µL of Gp gel (0.6 wt%) was placed evenly on a 1 mm quartz cuvette and analysed on a Jasco J-810 spectrometer from 185 to 400 nm. The FTIR spectrum of Gp gel (0.6 wt%) was collected on a Perkin-Elmer spectrophotometer by loading 20 µL of the hydrogel into a KBr cuvette. The hydrogel for FTIR characterization was prepared by using deuterium oxide, deuterium chloride and deuterium hydroxide as reagents. In vitro biostability assays. 1.0 mg of gelator precursor 1 was dissolved in 5 mL of a HEPES buffer solution (pH=7.4), followed by treatment with proteinase K (3.2 units/L). Then the mixed solution was incubated at 37 °C for 24 h. At each specific time, 100 µL of solution was taken out from the sample and analysed by HPLC.
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Cytocompatibility tests in vitro. Cytocompatibility assays were carried out by following CCK8 protocols. Human umbilical vein endothelial cells (HUVEC) were seeded in a 96-well microplate at a density of 4×104 cells/mL and incubated in a 5% CO2 humidified incubator at 37 °C. After 24 h, the medium was replenished with complete media supplementation with varied concentrations of gelator precursor 1 (10.0, 20.0, 50.0, 100.0 and 200.0 µM) and incubated for 24 and 72 h. Then, the fresh medium containing 10% CCK-8 solution was added to each well and incubated for 2 h. The optical absorbance at 450 nm was read using a Thermo Scientific Varioskan Flash spectral scanning multimode reader. Cell viabilities were expressed as a percentage of the control (untreated) cells and viabilities in the control group were designated as 100%. All experiments were performed in triplicate. Cell viability in vitro. The viability of HUVEC planted on Gp gel was examined by live-dead assays according to manufacturer’s procedures. Approximately, 100 µL of a sterilized solution containing both gelator precursor 1 (0.6 wt%) and alkaline phosphatase (5 unit/mL) was added in a 48-well plate. After gelation, the hydrogel sample was subjected to buffer exchange with 200 µL of Dulbecco’s Modified Eagle’s Medium (DMEM). After incubation for 24 h, the medium was replaced with fresh medium. Then 200 µL of cell suspension (8×104 cells/mL) was transferred into each well and placed in an incubator at 37 °C under 5% CO2 atmosphere. The medium was changed every other day. After incubation for 1, 3 and 5 days, the cells were stained with calcein-AM/propidium Iodide (PI) and imaged by an Olympus IX71 fluorescence microscope. Microfilament Staining. Morphologies and cytoskeleton structures of HUVEC cultured on surface of Gp gel (0.6 wt%) were examined with FITC-phalloidin staining according to manufacturer’s instruction. After 1-day culture, the cells on the surface of Gp gel were washed
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by PBS buffer (pH=7.4) and fixed by 4% paraformaldehyde. After incubation at room temperature for 10 minutes, the fixed cells were washed three times with PBS buffer containing 0.1% triton X-100 (5 minutes each), and then stained with a mixed solution containing FITCphalloidin (40 µg/mL) and Hoechst (20 µM) for 60 minutes. The stained cells were imaged by a confocal laser scanning microscopy with excitation filters at 488 nm (green, FITC) and 346 nm (blue, Hoechst). In vitro angiogenesis tests. 100 µL of Gp gel (0.6 wt%) containing DFO (1 mg/mL) was coated on a 48-well plate, then 200 µL of cell suspension (8.0×103 cells) was seeded on the surface of Gp gel (0.6 wt%) in each well and incubated at 37 °C under 5% CO2 atmosphere. Cell status and morphologies were recorded by an OLYMPUS IX71 fluorescence microscope every 12 hours. The numbers of meshes, junctions and length of each tubule formed by HUVECs were quantified by using Image J software in 5 random fields. Evaluation of DFO release behaviours in vitro. Three groups of Gp gel (500 µL, 0.6 wt%) encapsulating DFO (0.5 mg) were immersed in 2 mL of PBS buffer (pH=7.4), and incubated at 37 °C for DFO release. At each specific time (1, 2, 4, 8, 12, 24, 48, 72 and 96 h), 200 µL of PBS buffer was taken and treated with a certain amount of FeCl3. The amount of DFO released was determined by UV absorbance at 485 nm and a standard DFO calibration curve. In vivo angiogenesis tests. Mice were purchased from laboratory animal centre of Soochow University and in vivo tests were performed in full compliance with the Animal Care and Use regulations of Soochow University. Each experimental group consisted of 9 female BALB/c mice (8-week old). Mice were anesthetized with 4% chloral hydrate before in vivo tests. Then, 100 µL of Gp gel (0.6 wt%) encapsulating 100 µg of DFO was subcutaneously injected into the
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dorsal side of mice with a 1 mL syringe. After 3, 7 and 10 days, the mice were sacrificed and subcutaneous tissues containing implanted gels were excised, fixed in formaldehyde solution (4%), processed to paraffin blocks, and sectioned and stained with hematoxylin and eosin (H&E) or anti-CD31 antibody. Blood vessel areas in surrounding tissues were determined by using Image Pro Plus software from three different fields. Statistical analysis. All the data were averaged from the measurements in more than three groups, and analysed by using GraphPad Prism version (6.00) software. Values were displayed as mean±standard deviation of the mean. The statistical significance of the difference was measured by using a Student’s t-test and one-way ANOVA (*P＜0.05, **P＜0.01, and ***P＜ 0.001). P＜0.05 was considered significant.
ASSOCIATED CONTENT Supporting Information. Materials and methods, synthesis and characterizations of gelator precursor 1, sustained release behavior of DFO from Gp gel. AUTHOR INFORMATION Corresponding Author * E-mail:
[email protected];
[email protected]. Author Contributions ¶
These two authors contribute equally.
Notes The authors declare no competing financial interests.
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ACKNOWLEDGMENT This work was supported financially by the National Key R&D Program of China (Ministry of Science and Technology of China, 2016YFC1100100); the National Natural Science Foundation of China (51673142) to X. Li, the Natural Science Foundation of Jiangsu Province (BK20151218) to X. Li; the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). REFERENCES 1. Vacanti, J. P.; Langer, R., Tissue Engineering: the Design and Fabrication of Living Replacement Devices for Surgical Reconstruction and Transplantation. Lancet 1999, 354, SI32SI34. 2. Jain, R. K.; Au, P.; Tam, J.; Duda, D. G.; Fukumura, D., Engineering Vascularized Tissue. Nat. Biotechnol. 2005, 23, 821-823. 3. Levenberg, S.; Rouwkema, J.; Macdonald, M.; Garfein, E. S.; Kohane, D. S.; Darland, D. C.; Marini, R.; van Blitterswijk, C. A.; Mulligan, R. C.; D'Amore, P. A.; Langer, R., Engineering Vascularized Skeletal Muscle Tissue. Nat. Biotechnol. 2005, 23, 879-884. 4. Phelps, E. A.; Garcia, A. J., Engineering More Than a Cell: Vascularization Strategies in Tissue Engineering. Curr. Opin. Biotechnol. 2010, 21, 704-709. 5. Rouwkema, J.; Khademhosseini, A., Vascularization and Angiogenesis in Tissue Engineering: Beyond Creating Static Networks. Trends Biotechnol. 2016, 34, 733-745. 6. Peppas, N. A.; Hilt, J. Z.; Khademhosseini, A.; Langer, R., Hydrogels in Biology and Medicine: From Molecular Principles to Bionanotechnology. Adv. Mater. 2006, 18, 1345-1360.
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29. Xu, X.-D.; Liang, L.; Cheng, H.; Wang, X.-H.; Jiang, F.-G.; Zhuo, R.-X.; Zhang, X.-Z., Construction of Therapeutic Glycopeptide Hydrogel as a New Substitute for Antiproliferative Drugs to Inhibit Postoperative Scarring Formation. J. Mater. Chem. 2012, 22, 18164-18171. 30. Hamley, I. W.; Brown, G. D.; Castelletto, V.; Cheng, G.; Venanzi, M.; Caruso, M.; Placidi, E.; Aleman, C.; Revilla-Lopez, G.; Zanuy, D., Self-Assembly of a Designed Amyloid Peptide Containing the Functional Thienylalanine Unit. J. Phys. Chem. B 2010, 114, 10674-10683. 31. Sola, R. J.; Griebenow, K., Effects of Glycosylation on the Stability of Protein Pharmaceuticals. J. Pharm. Sci. 2009, 98, 1223-1245. 32. Culyba, E. K.; Price, J. L.; Hanson, S. R.; Dhar, A.; Wong, C.-H.; Gruebele, M.; Powers, E. T.; Kelly, J. W., Protein Native-State Stabilization by Placing Aromatic Side Chains in NGlycosylated Reverse Turns. Science 2011, 331, 571-575. 33. Taylor, M. E.; Drickarner, K., Paradigms for Glycan-Binding Receptors in Cell Adhesion. Curr. Opin. Cell Biol. 2007, 19, 572-577. 34. Hughes, R. C., Complex Carbohydrates of Mammalian-Cell Surfaces and Their Biological Roles. Essays in Biochemistry 1975, 11, 1-36. 35. Stromblad, S.; Cheresh, D. A., Integrins, Angiogenesis and Vascular Cell Survival. Chemistry & Biology 1996, 3, 881-885. 36. Stromblad, S.; Cheresh, D. A., Cell Adhesion and Angiogenesis. Trends Cell Biol. 1996, 6, 462-468. 37. Jiang, X.; Malkovskiy, A. V.; Tian, W.; Sung, Y. K.; Sun, W.; Hsu, J. L.; Manickam, S.; Wagh, D.; Joubert, L.-M.; Semenza, G. L.; Rajadas, J.; Nicolls, M. R., Promotion of Airway
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Scheme 1. Illustration of the molecular structure of a glycopeptide molecule and its selfassembling process for the generation of a supramolecular hydrogel with glucose decoration which could be exploited for inducing angiogenesis in vivo.
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Scheme 2. Synthetic routes for the preparation of gelator precursor 1 and its correspondingly enzymatic transition to gelator 2 by alkaline phosphatase.
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Figure 1. Optical images of (A) the solution of gelaor precursor 1 (0.6 wt %, pH=7.4) and (B) the resulting Gp gel triggered by alkaline phosphatase (10 units/mL); (C) TEM and (D) SEM of the self-assembled structures from gelator 2 within Gp gel shown in Figure 1B.
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Figure 2. (A) Frequency dependence of the dynamic storage moduli (G’) and the loss moduli (G’’) of Gp gel shown in Figure 1B. (B) CD and (C) FTIR spectra of the solution of gelator precursor 1 (0.6 wt %, pH=7.4) and the corresponding Gp gel shown in Figure 1B. (D) timedependent course of the digestion of gelator precursor 1 and a peptide derivative without glucose modification by proteinase K.
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Figure 3. (A) Fluorescence images of the live/dead assays of HUVECs cultured on the surface of Gp gel over the course of 5 days. (B) Cytoskeletal F-actin staining of HUVECs cultured on the surface of Gp gel by fluorescein-phalloidin. (C) A high magnification image of the boxed area shown in Figure 3C. (D) Cell densities of HUVECs determined by cell counting with a hemocytometer over the course of 5-day culture on Gp gel.
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Figure 4. (A) Light microscopy images of the morphologies of HUVECs cultured on the surface of blank Gp gel and Gp gel loaded with DFO over the course 72 h culture. Scale bar=50 µm. Quantitative analysis of the numbers of (B) meshes, (C) the numbers of junctions and (D) total length of vessels formed on the surface of Gp gel and Gp gel loaded with DFO over the course 72 h culture. The error bars indicates the SD.
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Figure 5. (A) Optical images of (A) Gp gel (0.6 wt%), (B) the process of extruding Gp gel through a needle with a diameter of 0.4 mm, (C) the state of sample collected from the process shown in Figure 5B, (D) Gp gel recovered from the sample shown in Fig. 3C. Time-dependent of repetitive cycles of the step-strain analysis of (E) blank Gp gel and (F) Gp gel encapsulating DFO (1 mg/mL).
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Figure 6. (A, (B) and (C) H&E stained sections of the implanted Gp gel without loading DFO at day-3, day-7 and day-10, respectively; (D), (E) and (F) high magnification images of the boxed areas in Figure 6A, 6B and 6C, respectively. (G), (H) and (I) H&E stained sections of Gp gel loaded with DFO at day-3, day-7 and day-10, respectively; (J), (K) and (L) high magnification images of the boxed areas in Figure 6G, 6H and 6I, individually. Scale bars=100 µm. Arrows indicate blood vessels.
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Figure 7. (A) and (B) Representative images of the sections of Gp gel without loading DFO at day-3 and day-10, which were stained with CD31 antibodies (red). Nuclei were stained with DAPI (blue); (C) and (D) high magnification images of the boxed areas in Figure 7A and 7B, respectively. (E) and (F) Representative images of the sections of implanted Gp gel loaded with DFO at day-3 and day-10, which were stained with CD31 antibodies (red); (G) and (H) high magnification images of the boxed areas in Figure 7E and 7F, individually. Scale bar=50 µm.
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TOC Graphic
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