A Neuroprotective Sericin Hydrogel As an Effective Neuronal Cell

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A neuroprotective sericin hydrogel as an effective neuronal cell carrier for the repair of ischemic stroke Zheng Wang, Jian Wang, Jin Yang, Zhen Luo, Wen Yang, Hongjian Xie, Kai Huang, and Lin Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b06804 • Publication Date (Web): 19 Oct 2015 Downloaded from http://pubs.acs.org on October 26, 2015

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A neuroprotective sericin hydrogel as an effective neuronal cell carrier for the repair of ischemic stroke Zheng Wang,1,2,*,#, Jian Wang1,#, Yang Jin3,4,#, Zhen Luo1, Wen Yang1, Hongjian Xie1, Kai Huang5,*, Lin Wang1,4,6,* 1

Research Center for Tissue Engineering and Regenerative Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 430022. 2

Department of Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 430022. 3

Department of Respiration, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 430022. 4

Medical Research Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China 430022. 5

Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China 430022.

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Department of Clinical Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China 430022.

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, These authors contributed equally to this work.

*Correspondence to: Lin Wang, Phone: 86-27-85726612. E-mail: [email protected] Or to: Kai Huang, Phone: 86-27-85726612. E-mail: [email protected] Or to: Zheng Wang, Phone: 86-27-85726612. E-mail: [email protected]

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ABSTRACT Ischemic stroke causes extensive cellular loss that impairs brain functions, resulting in severe disabilities. No effective treatments are currently available for brain tissue regeneration. The need to develop effective therapeutic approaches for treating stroke is compelling. A tissue engineering approach employing a hydrogel carrying both cells and neurotropic cytokines to damaged regions is an encouraging alternative for neuronal repair. However, this approach is often challenged by low in vivo cell survival rate, and low encapsulation efficiency and loss of cytokines. To address these limitations, we propose to develop a biomaterial that can form a matrix capable of improving in vivo survival of transplanted cells and reducing in vivo loss of cytokines. Here, we report that using sericin, a natural protein from silk, we have fabricated a genipin-crosslinked sericin hydrogel (GSH) with porous structure and mild swelling ratio. The GSH supports the effective attachment and growth of neurons in vitro. Strikingly, our data reveal that sericin protein is intrinsically neurotropic and neuroprotective, promoting axon extension and branching as well as preventing primary neurons from hypoxia-induced cell death. Notably, these functions are inherited by the GSH’s degradation products, which might spare a need of incorporating costly cytokines. We further demonstrate that this neurotropic effect is dependent on the Lkb1-Nuak1 pathway, while the neuroprotective effect is realized through regulating the Bcl-2/Bax protein ratio. Importantly, when transplanted in vivo, the GSH gives high cell survival rate and allows the cells to continuously proliferate. Together, this work unmasks the neurotropic and neuroprotective functions for sericin and provides strong evidence justifying the GSH’s suitability as a potential neuronal cell delivery vehicle for ischemic stroke repair.

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KEYWORDS: Sericin, Hydrogel, Stroke, Neuronal cell carrier, Neuroprotection.

INTRODUCTION Stroke is the fifth leading cause of death worldwide.1 Ischemic stroke accounting for nearly 75-80% of stroke is caused by disruption in blood flow to brain,2 resulting in the deprivation of the energy of neuronal cells such as oxygen and glucose, thus leading to cell death and corresponding behavioral impairment. Despite its importance, effective ischemic stroke therapies are limited. The only available treatment approved by the Food and Drug Administration (FDA) is the intravenous administration of tissue-type plasminogen activator (tPA) for thrombolysis.3-4 However, its narrow therapeutic time window only allows selected patients to benefit from it. Other treatments are mainly supportive, such as supporting ventilation, controlling blood pressure, reducing brain edema and preventing infection.5-6 Currently, there are still no effective therapies that can reverse neural tissue damage once stroke has occurred.7-8 Tissue engineering is thought to be a promising alternative. Hydrogels incorporated with different neuroprotective factors and stem cells for neuronal tissue repair is a common approach for facilitating brain tissue regeneration.9 However, this strategy is often challenged by relatively low in vivo cell survival, low encapsulation efficiency and significant reduction of neurotropic factors’ activity.10 A possible way to overcome these drawbacks would be to identify a biomaterial that possesses intrinsic neurotropic function while suitable for fabricating a hydrogel improving in vivo cell survival. A number of synthetic polymers and natural materials have been explored for their

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utility in neuronal tissue repair, such as hyaluronan/methyl cellulose (HAMC) hydrogel,11 alginate,12 PLA/PLGA8, 13 and matrigel.14 However, these materials are not neurotropic and have their own limitations. The HAMC15 and alginate16 hydrogels are hardly adhesive to cells, while the degraded products of PLA/PLGA alter pH value of microenvironment in host tissues17. As basement membrane extracts from tumor cells, matrigel’s origin14 concerns its clinical applications. Thus, in addition to requiring a biomaterial with intrinsic neurotropic activity, it is highly desired that this biomaterial is naturally cell-adhesive and its degraded products are neuronal biocompatible without inducing adverse effects on neuronal regeneration. Silk produced by silkworms consists of two major protein components, fibroin and sericin. While fibroin has been extensively studied in the field of tissue engineering and regenerative medicine,18-21 sericin has just begun to be explored. An increasing number of studies are directed towards the applications of sericin in regenerative medicine, such as cartilage regeneration22 and wound dressing.23 Sericin consisting of 17-18 amino acids24-25 has a large number of polar side chains made of hydroxyl, carboxyl and amino groups. The degraded products of sericin contain abundant glycine and serine, which are the precursors for tryptophan ethanolamine and chlorophy II that play an important role in neurotransmission.26-27 Given sericin’s diverse biological activities, including anti-oxidation,28 anti-bacterium,29 anti-coagulation30-31 and promoting cell migration32 and differentiation,33-35 we hypothesize that sericin might possess neuron-favorable activity and its degradation products might be beneficial to neurons. We previously reported that sericin is naturally cell-adhesive and can be crosslinked by glutraldehyde to form an injectable hydrogel,36 which

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offers a basis for further testing whether a sericin hydrogel can serve as a vehicle delivering neurons for neuronal tissue repair. Utilizing genipin, a biocompatible crosslinker,37 we have generated a new crosslinked sericin hydrogel (GSH). The physical and mechanical properties of this GSH have been investigated. The biocompatibility of the GSH with primary cortical neurons has been assessed in vitro. By analyzing sericin’s interactions with primary neurons, we have uncovered sericin’s intrinsic neurotropic function and the underlying molecular mechanisms. Employing an in vitro model imitating stroke damage, we have revealed the neuroprotective effects of the sericin protein and the GSH’s degradation products. Further, the effectiveness of the GSH on delivering neuronal cells (SHSY-5Y) in vivo and improving cell survival has been assessed. This work provides strong evidence demonstrating the suitability and potentiality of this GSH with intrinsic neurotropic and neuroprotective functions as an in vivo effective cell delivery platform for brain tissue repair.

EXPERIMENTAL SECTION Preparation of the genipin-crosslinked sericin hydrogel (GSH) and the GSH degraded products. Sericin protein was isolated from a mutant silkworm strain, Bombyx mori, 140 Nd-s silk cocoons (Sericultural Research Institute, China Academy of Agricultural Sciences, Zhenjiang, Jiangsu, China) using the LiBr extraction method as described previously.36 To fabricate a GSH, 6 mL sericin solution (2%, w/v; distilled water) and 1 mL genipin solution (1%, w/v; distilled water) were mixed gently at room temperature for 30 minutes. The sericin-to-genipin ratio of 6:1 and the 30-minute reaction time for crosslinking were

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determined by the crosslinking and gelation analysis using a series of sericin-to-genipin ratios and different reaction time (Supplementary Experimental Section; Figure S1 and Table S1). Characterizations of morphology, secondary structures and mechanical properties. The surface of GSH scaffolds was examined using a scanning electron microscope (SEM) (JSM-5610LV, Japan) with the voltage of 25 kV. The fourier transform infrared (FTIR) spectra of sericin protein and the GSH were obtained by using an FTIR spectroscopy (Nexus, Thermal Nicolet, USA) with a ZnSe ATR cell for a spectral region of 4000-650 cm-1. The mechanical property of the GSH was recorded by a universal testing machine (Instron 5848 MicroTester, MA, USA) equipped with a 100 N load cell. The hydrogels were molded into a cylindrical structure (8 mm in height, 12 mm in diameter) before they were tested. Swelling behavior of the GSH. To calculate the formation swelling ratio, the newly-formed hydrogels were weighed (Wf) using an electronic balance (Ohous, NJ, USA). These hydrogels were then subject to oven dry. Their dry weight was recorded (Wf’). To calculate the equilibrium swelling ratio calculation, the newly-formed hydrogels were equilibrated in 50 mL PBS at 37°C for 24 hours. These well-swollen hydrogels were weighed (We) and then subject to oven dry to obtain their dry weight (We’). The swelling ratios of the hydrogels upon the formation (swellingf) and the equilibrium (swellinge) could thus be calculated using the following equations:38 ‫݈݈݃݊݅݁ݓݏ‬௙ (%) =

ௐ೑ ିௐ೑ᇲ ௐ೑ᇲ

‫݈݈݃݊݅݁ݓݏ‬௘ (%) =

ௐ೐ ିௐ೐ᇲ ௐ೐ᇲ

In vitro degradation of the GSH. The hydrogels were fabricated in cylindrical molds (10.2 mm in diameter and 5 mm in height) and then were immersed in PBS at 37°C. PBS was replaced daily. At given time points, the hydrogels were taken out, washed, lyophilized and

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re-weighed. The degradation was calculated as the difference between the initial and following dry mass at indicated times divided by the initial dry mass. To obtain the GSH degraded products, 6 mL sericin solution (2%, w/v) and 1 mL genipin solution (1%, w/v) were mixed in a 50 mL tube. After gelling for 30 minutes, 43 mL PBS was added into the tube. Then the tube was shaken at a speed of 200 rpm at 37°C. After 4 weeks, the solution was collected and filtered through a 0.22-um filter. The BCA assay described previously39 was performed to calculate the concentrate of original GSH degraded products. The original GSH degraded products were adjusted using culture medium to obtain desired concentrations. Primary cortical neurons and SHSY-5Y cell culture. Primary neurons were isolated from cerebral cortices of C57BL/6J mouse embryos (E16-18). Primary cortical neurons were cultured as described previously.40 Cells were washed with D-Hank’s solution and re-suspended in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone, UT, USA) containing 10% fetal bovine serum (FBS; Gibco, CA, USA). After cells were seeded at the density of 2,000 cells/mm2 on the plates coated with poly-L-lysine (Sigma, TX, USA) for 4-6 hours, the medium was replaced by neurobasal medium (Gibco, CA, USA) supplemented with 2% B27, 1% glutamine and 1% penicillin/streptomycin (Hyclone, UT, USA). Human neuronal cells (SHSY-5Y) (ATCC, CRL-2266) were cultured in high glucose DMEM containing 10% FBS or 2% FBS for long-term culture. All cells were maintained at 37°C in a humidified incubator with 5% CO2. Live & Dead staining and CCK-8 assay. The Live & Dead staining was performed according to the manufacture instructions (Biovision, CA, USA). Primary neurons were

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seeded onto a 96-well plate. 24 or 48 hours after indicated treatments, the cells were incubated in the staining solution at 37°C for 15 minutes, and then observed immediately using a fluorescence microscope (Olympus IX71, Japan). CCK-8 assay (Dojindo, Japan) was performed as previously described.41 Briefly, the cells growing in a 96-well plate or a 96-well plate with GSHs pre-formed at the bottom of each well were washed three times. 100 uL DMEM mixed with 10 uL CCK-8 solution was added into each well of the 96-well plate and incubated for 3 hours at 37°C in the incubator. The absorbance was measured at 450 nm with a microplate reader (TECAN, Männedorf, Switzerland). SHSY-5Y cell long-term growth on the GSH. SHSY-5Y cells were seeded at the density of 100 cells/mm2 onto 24- and 96-well plates with the GSHs pre-formed at the bottom of the wells and cultured for 20 days in DMEM with 2% FBS. The cells on the 24-well plates were imaged at different time points under phase contrast using a compound microscope (Olympus IX71, Japan). The cells growing in the 96-wells were subject to CCK-8 assay described above to obtain the growth curve. F-actin cytoskeleton staining. Primary neurons were seeded on a sterilized GSH in a glass 35-mm culture plate. After cultured for 48 hours, cells were fixed with 4% paraformaldehyde for 15 minutes. F-actin and cell nuclei were stained with rhodamine-phalloidin (YEASEN, Shanghai, China) and 4',6-diamidino-2-phenylindole (DAPI, Sigma, TX, USA), respectively. The stained cells were visualized with a confocal laser microscope (Nikon A1Si, Japan). Oxygen glucose deprivation (OGD) assay. The OGD model was used as previously described.42 Briefly, isolated primary mouse cortical neurons were cultured in a glucose-free bicarbonate buffer (120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 0.8 mM MgCl2, 25 mM

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NaHCO3) at 37°C for 1 hour in a humidified incubator filled with an anoxic gas mixture (5% CO2 and 95% N2). The neurons that were subject to the OGD treatment were incubated in the indicated solution for additional 24 hours. The cells were then harvested for the Live & Dead staining and CCK-8 assay described above. The lactate dehydrogenase (LDH) release was detected using a commercially available kit (Jiancheng, Nanjing, China) as previously described.40 Analysis of axon extension and branching of primary neurons. Primary neurons were isolated from fetal mouse brain and cultured as described above. After isolated primary neurons were plated at the density of 2,000 cells/mm2 onto a 24-well plate for 4-6 hours, the sericin solution (0.05 mg/ml) was added to the wells along with neurobasal medium described above. The neurons were cultured in the neurobasal medium containing sericin (0.05 mg/ml) for 4 days. The neurons in the 24-well plate were imaged at different time points. Axon lengths of individual neurons were measured using the software ImageJ and axon branches were manually counted. To inhibit Nuak1 activity, the inhibitor WZ4003 was added to the isolated primary neurons 4-6 hours after they were plated at the density of 2,000 cells/mm2 onto a 24-well plate. After 24-hour incubation with WZ4003, the neurobasal medium was replaced. Axon length and branching were assessed on Day 4. Real-time PCR and western blotting. mRNA was extracted from primary cortical neurons by Trizol (Invitrogen, CA, USA). RT-PCR was performed using the ThermoScript RT-PCR System (Invitrogen, CA, USA). Primers were designed using the software Primer Premier 5.0 (Table S2). Western blot analyses were performed as previously described.43 The protein concentrations of cell lysates were analyzed using the BCA protein assay reagent (Pierce,

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USA). Then samples were subjected to SDS-PAGE, transferred to nitrocellulose membrance (Amersham, Piscataway, NJ, USA). The membranes were blocked and incubated with the antibodies: anti-Bax (Proteintech, Wuhan, China), anti-Bcl-2, anti-β-actin (Santa Cruz Technology, CA, USA). Blots were analyzed using a Luminescent Image Analyzer (Fujifilm LAS-4000, Tokyo, Japan). Generation of SHSY-5Y cells stably expressing GFP. The GFP (green fluorescence protein) gene was cloned into pFB-Neo vector (Haoran, Shanghai, China) at the sites of SalI/XhoI. The plasmid pVPack-GP, pVPack-VSV-G and the modified vector pFB-Neo-GFP were co-transfected into 293T cells. The supernatants containing retrovirus were collected. SHSY-5Y cells were infected with the retrovirus for 48 hours. The monoclonal cells were selected and allowed to proliferate in the medium supplemented with 300 µg/mL G418 for 4 weeks. Analysis of cell survival in the GSH in vivo. GFP-expressing SHSY-5Y cells were seeded on the hydrogels at the density of 2,500 cells/mm2. After cultured for 24 hours, the hydrogels with SHSY-5Y cells were transplanted into the lower abdominal cavity of nude mice. At Day 1, 2, 3, 5 or 7, 10, the mice were sacrificed and the hydrogels were taken out for analysis. Each isolated hydrogel sample was cut into the halves, with one for imaging using a confocal laser-scanning microscope (Nikon A1Si, Japan) and the other being digested in 0.25% trypsin solution for cell collecting. The collected cell suspension was re-suspended with PBS and the total GFP-positive cells were counted. To further examine the viability of the GFP-positive cells, PI staining was performed. The cell samples were then immediately analyzed on an FACS Calibur flow cytometer (BD Canto II, NJ, USA).

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Statistics. Data were presented as mean ± standard deviation. Two-tailed student’s t-tests were performed for statistical comparisons. P

A Neuroprotective Sericin Hydrogel As an Effective Neuronal Cell

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