Tunable, injectable hydrogels based on peptide ... - ACS Publications

§Center for Neuroregeneration and Department of Neurosurgery, Houston Methodist Research. Institute, Houston, TX 77030. ┴Molecular Engineering and ...
0 downloads 0 Views 2MB Size
Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES

Article

Tunable, injectable hydrogels based on peptide-crosslinked, cyclized polymer nanoparticles for neural progenitor cell delivery Tianyu Zhao, Drew L. Sellers, Yilong Cheng, Philip J. Horner, and Suzie H. Pun Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00510 • Publication Date (Web): 16 Aug 2017 Downloaded from http://pubs.acs.org on August 18, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Tunable, injectable hydrogels based on peptidecrosslinked, cyclized polymer nanoparticles for neural progenitor cell delivery Tianyu Zhao†, Drew L. Sellers†,‡, Yilong Cheng†, Philip J. Horner†,‡,§*, Suzie H. Pun†,┴* †Department of Bioengineering, University of Washington, Seattle, WA 98195. ‡Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98195. §Center for Neuroregeneration and Department of Neurosurgery, Houston Methodist Research Institute, Houston, TX 77030. ┴Molecular Engineering and Sciences Institute, University of Washington, Seattle, WA 98195.

ABSTRACT: A PEG-based cyclized vinyl polymer was synthesized via one-step RAFT polymerization and used as a precursor of injectable hydrogels. Dithiol linkers including laminin-derived peptides containing IKVAV and YIGSR sequences and DTT were used for gelation. Fast and adjustable gelation rate was achieved through nucleophile-initiated thiolMichael reaction under physiological conditions. Low swelling ratio and moderate degradation rate of the formed hydrogels were observed. 3D encapsulation of neural progenitor cells in the synthetic hydrogel showed good cell viability over 8 days. The long-term cell survival and

ACS Paragon Plus Environment

1

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 29

proliferation were promoted by the introduction of laminin-derived peptides. This hydrogel platform based on peptide-crosslinked, cyclized vinyl polymers can be used as a universal hydrogel template for 3D cell encapsulation.

INTRODUCTION The limited regeneration capacity of the damaged central nervous system has necessitated innovative strategies such as cell therapies for tissue and functional repair. Cell-based therapies aim to replace damaged cells or alter the local environment to be conducive for regeneration.1 Adult neural progenitor cells (aNPCs) are self-renewing, multipotent progenitors in the central nervous system (CNS) capable of differentiation into neurons, oligodendrocytes and astrocytes and are therefore key cells of interest in CNS cell therapy applications for regeneration.2 However, the effectiveness of direct cell transplantation into the injured spinal cord has been in part limited by low cell survival, strong gliogenic signaling of the injured nervous system that restricts cell fate, and ineffective integration into the host tissue.3-6 Hydrogels, which have tunable porosity, permeability and mechanical properties, are being extensively studied as a support material in NPCs delivery. Extraordinary works have been reported using natural materials, such as collagen,7 laminin,8 fibrin,9, 10 chitosan,11, 12 alginate,13, 14

hyaluronic acid,15-18 to encapsulate NPCs for improved cell survival, host-tissue integration

and functional repair. Compared to natural molecules, synthetic polymers can be used to form hydrogels with controlled composition, architecture, mechanical properties, and bioactive functionalities.19 Poly(ethylene glycol) (PEG) is often employed as a component in synthetic hydrogels due to its hydrophilic, protein-resistant property.20 Furthermore, chemically crosslinked PEG-based hydrogels can be fabricated from different precursors, including PEG diacrylate (PEGDA) monomers,21 multi-arm star-PEGs,22 branched PEG-based polymers23 and

ACS Paragon Plus Environment

2

Page 3 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

even microspheres.24 Crosslinking of PEGDA monomer usually occurs by light, thermal or oxidation induced free radical polymerization; however, the high concentration of reactive vinyl groups in the monomer solution is not ideal for cell encapsulation. Multi-arm star-PEGs have recently become popular precursors to fabricate hydrogels due to their precise structure and multi-functionality.22, 25, 26 However, large-scale production of multi-arm star-PEGs is limited by their tailored synthesis and thus restricts their industrial translation. Cyclized vinyl polymers, developed recently by Wenxin Wang’s group, are single polymer chains linked repeatedly and wrapped around themselves in an interlaced pattern.27 Cyclized vinyl polymers are synthesized by controlled/living polymerization of divinyl monomers resulting in intramolecular reaction between the propagating free radicals and the pendent vinyl groups on the same polymer chain, forming nanoparticles (NPs) as visualized by atomic-force microscopy (AFM).28 Importantly, most of the pendent vinyl groups on the polymer chains are consumed during the polymerization, leaving only a low content of vinyl groups that can be utilized for crosslinking or further modification with bioactive molecules. We hypothesized that cyclized vinyl polymers can be used as versatile hydrogel building blocks to crosslink with bioactive peptides. Potential advantages using cyclized polymer NPs compared to commercially available PEG-based vinyl functional monomers (e.g. PEGDA) or polymers (e.g. 4-arm-PEG-Acr), include tunable vinyl content, rapid gelation capability, better stability due to low swelling ratio and sufficient degradation time to support the host tissue ingrowth. In this study, hydrogels with flexible gelation and mechanical properties were developed by crosslinking a PEG-based cyclized vinyl polymer via nucleophile-initiated thiol-Michael reaction using dicysteine peptide and dithiothreitol (DTT) crosslinkers catalyzed by lysine29 (Scheme 1).

ACS Paragon Plus Environment

3

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 29

We show that rapid chemical gelation can be achieved within ten minutes. Given the importance of laminin in the neural extracellular matrix (ECM), laminin-derived peptides (YIGSR and IKVAV) were applied to promote neural cell adhesion.30,31 Murine neural progenitor cells embedded into the 3D hydrogel environment show good cell viability and increased cell metabolic activity, supporting the potential of these materials for stem and progenitor cell delivery for CNS regeneration. MATERIALS AND METHODS Materials and reagents. Oligo(ethylene glycol) diacrylate (Average Mn=700, OEGDA700), 4-Cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic

acid

(CDSP,

≥97%),

2,2’-

Azobis(2-methylpropionitrile) (AIBN, ≥98%), DL-dithiothreitol (DTT, ≥99%), DL-Lysine (≥95%) were purchased from Sigma Aldrich. Butanone (ACS reagent grade, Fisher), diethyl ether (ACS reagent grade, Fisher), d-Chloroform (99.8%, Aldrich), dichloromethane (ACS reagent grade, Fisher), dimethylflormamide (DMF, HPLC grade, Fisher) were used as received. Cyclized vinyl polymer synthesis and characterization. The polymer was synthesized by RAFT homopolymerization of oligo(ethylene glycol) diacrylate (OEGDA, Mn = 700 g/mol) at low monomer concentration ([M] = 0.1M). OEGDA700 monomer (7 g, 100 equiv), CDSP (40.4 mg, 1 equiv), butanone (93.5 ml) and AIBN (8.2 mg, 0.5 equiv) were added into a 250 ml twonecked round bottom flask. Oxygen was removed by bubbling argon through the solutions for 20 mins before the flask was sealed and immersed in a preheated oil bath at 60 °C. The solution was stirred at 800 rpm and the polymerization was conducted at 60 °C for the desired reaction time. The experiment was stopped by opening the flask and exposing the solution to air. About half of the butanone was evaporated and the rest of the solution was precipitated into a large excess of cold diethyl ether to remove the monomers. The precipitated mixture was dried under vacuum.

ACS Paragon Plus Environment

4

Page 5 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Molecular weights and molecular weight distributions (Mw/Mn) of the polymer samples were determined using GPC using a Tosoh SEC TSK-GEL α-3000 and α-4000 columns connected in series to an Agilent 1200 Series Liquid Chromatography System, along with a Wyatt Technology miniDAWN TREOS, 3 angle MALS light scattering instrument and Optilab T-rEX, refractive index detector. A mobile phase of dimethylformamide containing 0.1% w/v LiBr was used with a flowrate of 1mL/min at 60 °C. 30 kDa polystyrene standards were used for GPC calibration. 1H NMR spectra were recorded on a 500 MHz Bruker instrument using CDCl3 as the solvent and processed with MestReNova software Peptide synthesis. Biotinylated peptides were synthesized by standard FMOC solid phase peptide synthesis, and NHS-biotin (Pierce Chemicals) was conjugated to the amine terminus in DMF before trifluoroacetic acid deprotection and cleavage from the resin. Peptides were purified by HPLC to >90% purity. HPLC purification was performed using a Phenomenex Fusion-RP C18 semi-preparative column (Torrance, CA) at the flow rate of 5 mL/min with H2O (0.1% TFA) and ACN (0.1% TFA) as a mobile phase. Yields for all peptides are about 10-13 mg per 0.1 mmol scale synthesis. Desired peptides were confirmed by MALDI mass spectrometry analysis. Two peptides were synthesized: NH2-CGIKVAVEGC-CONH2 (IKVAV) and NH2CGEYIGSRGC-CONH2 (YIGSR). Injectable hydrogel fabrication Three dithiol linkers including DTT, NH2-CGIKVAVEGCCONH2 (IKVAV) and NH2-CGEYIGSRGC-CONH2 (YIGSR) were used in the present study and four combinations including DTT, DTT/IKVAV (85/15), DTT/YIGSR (85/15), DTT/IKVAV/YIGSR (85/7.5/7.5) were used for gelation. The polymer was dissolved in HBSS (Sigma) and mixed with dithiol linker H2O solution to reach final polymer concentrations of 7.5 % (w/v). The concentration of dithiol linker solution was fixed at an equivalent moles of thiol

ACS Paragon Plus Environment

5

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 29

groups to acrylate in the polymer (acrylate concentration was calculated from 1H NMR) and 10 mol% of lysine to thiol groups were pre-mixed with dithiol linkers. Laminin was physically mixed in the DTT crosslinked hydrogel at a final concentration of 77µg/ml and used as a control. Crosslinking efficiency test. 100µl hydrogel precursor solution was pipetted onto a teflon surface and settled for 20mins to allow complete gelation. The hydrogel was then transferred into a 15ml tube followed by adding 200µl distilled water into the tube. The hydrogel was then totally broken by votexing and the solution was settled overnight. The supernatant was then analyzed by Ellman assay to detect thiol concentration. Different concentration of DTT solution was made to plot the standard curve. Rheological assessments. An AR-G2 rheometer (TA Instruments, New Castle, DE) with steel parallel-plate geometry (8 mm diameter) was used for rheological characterization of all the hydrogel samples. For a real-time crosslinking rheological study, an oscillatory time sweep was performed at 20 °C and 37 °C respectively with different samples. The gelling solutions were prepared as described above and mixed quickly on the sample plate to start the testing directly. Morphological characterization. Scanning electron microscopy (SEM) was used to determine the morphology of freeze dried hydrogels. The hydrogels (7.5 wt%) were prepared as mentioned earlier and the samples were freeze-dried overnight. The freeze-dried samples were mounted on an aluminium stub using an adhesive carbon tab and sputter coated with gold before images were obtained using a SEM--FEI-Sirion-XL30 instrument. Swelling behavior. A volume of 0.5 mL of PBS 7.4 was added into a glass vial, with 40 µl hydrogel on the bottom prepared as mentioned earlier. The samples were incubated at 37 °C and constant agitation at 150 rpm. The weight of the hydrogel plus the vial was recorded, after careful removing of the surface water, at predetermined time interval. The weight of the original

ACS Paragon Plus Environment

6

Page 7 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

hydrogel (W0) and swollen hydrogel (Wt) was determined by substracting the vial weight from the total weight of the vial plus hydrogels. The swelling ratio of the hydrogel was defined as Wt/W0. Neural progenitor cell encapsulation. Neural progenitors isolated from the forebrain of 8 week old mice were encapsulated in 60µl hydrogel by mixing the cell suspension with gelling solution at a concentration of 1.25*106 cells/mL, pipetting onto a teflon surface, and leaving for 10 min to complete the formation of a hydrogel tablet at room temperature. With a diameter of 5±0.2mm and a height of 1.8±0.2mm, the hydrogel was then transferred into 24-well plate, rinsed with HBSS and immersed in 500µl of growth media (DMEM/F12 media with N2 supplement at 1X Heparin at 5µg, and EGF/FGF-2 at 20 ng/ml).32 The 24-well plate was then cultured at 37°C and 5% CO2 for predetermined time. Cell metabolic activity assay. AlamarBlue® assay was performed to evaluate the effect of polymer on cell metabolic activity. After a certain time of culture, the hydrogels were washed with HBSS, then 500 µl of alamarBlue® HBSS solution (10% v/v) was added to each well and cultured for another 24 hours. Then, the solution was used to assess cell metabolic activity. The absorbance at the lower wavelength filter (550 nm) was measured followed by the higher wavelength filter (595 nm) via a Varioskan Flash Plate Reader. To compensate the reducing effect of residue DTT in the gel, gels crosslinked by DTT without cells were used as a negative control. The percentage of alamarBlue reduced by the residue DTT as in the negative control was subtracted from all the testing groups. LIVE/DEAD® viability assay. The LIVE/DEAD® assay (Molecular Probes) was utilized to visualize the distribution of living and dead cells in the hydrogel. Fluorescence images were taken using an Nikon inverted microscope, while fluoresce green upon the reaction of

ACS Paragon Plus Environment

7

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 29

intracellular esterase and calcein-AM in living cells, and a dye of fluoresce red (ethidium homodimer-1) bound to the DNA of dead membrane compromised cells. RESULTES AND DISCUSSION Homopolymerization of divinyl monomer (OEGDA700) was controlled by RAFT (reversible addition fragmentation chain transfer) polymerization to synthesize the cyclized polymers. Compared to other controlled radical polymerization methods, RAFT polymerization uniquely offers competition between chain transfer and chain coupling when a propagating chain is in close proximity to another chain, thus reducing the chance of chain coupling and providing more opportunity for intramolecular reaction. To further promote cyclization versus chain coupling, a low monomer concentration ([M]0 = 0.1 M) and low ratio of chain transfer agent (CTA) to monomer ([CTA]0 : [M]0 = 1 : 100) was adopted. The kinetics of vinyl group conversion was monitored by NMR analysis, revealing a relatively constant reaction rate (Figure 1a). The evolution of molecular weight (MW) and associated polydispersity index (PDI) of cyclized vinyl polymers is shown in Figure 1b and recorded in Supporting information Table 1. Mw increased linearly with the vinyl conversion at the early reaction stage and PDI remained below 1.5 at 60% vinyl conversion with a unimodal molecular distribution. At later stages, a rapid and exponential increase in Mw occurred, which was accompanied by an increase of PDI, indicative of chain coupling. During the linear chain propagation period, the observed Mw was below the theoretical Mw, which can be attributed to the consumption of polymer vinyl groups by intramolecular cyclization that does not contribute to MW increase. The polymerization was stopped after 10 hours and the final product after purification possessed a Mw of 179 kDa with a PDI of 2.75 and an Rg of 34±1.2 nm. The multimodal GPC

ACS Paragon Plus Environment

8

Page 9 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

traces (Figure 1c) indicate the polymer product contained both single cyclized polymer chains and higher MW products resulting from chain coupling. The 1H NMR spectrum for the product demonstrates the existence of a low amount of vinyl groups at characteristic peaks between 6.5 ppm and 5.7 ppm (Figure 1d) while only 9.6% of the OEGDA units exist as pendent acrylate groups with the rest consumed intramolecularly during polymerization. The vinyl content value is calculated to be 0.14 mmol•g-1, which is lower than the previously reported branched polymer23 (0.4 mmol•g-1) or the 4-arm star-PEG-Acr24 (0.2 or 0.4 mmol•g-1) precursors used in cell encapsulating hydrogels. The low vinyl content is believed to be beneficial for cell survival by minimizing interactions between vinyl groups and amine or thiols on cell surface. Dithiol linkers, including cysteine-flanked, laminin-derived peptides33 (NH2-CGIKVAVEGCCONH2 and NH2-CGEYIGSRGC-CONH2) and DTT were used to crosslink the cyclized vinyl polymer for hydrogel fabrication. The peptide crosslinkers were synthesized by solid phase peptide synthesis, purified by HPLC and characterized by mass spectroscopy (Supporting Information Figure 1). Hydrogels were formed by nucleophile-initiated thiol-Michael reaction between the acrylate on the cyclized vinyl polymer and the thiol groups on the dithiol linkers (Scheme 1). L-lysine was selected as the primary amine catalyst, due to its low toxicity and absorbability. Crosslinking efficiency was determined by quantifying the amount of unreacted DTT and laminin peptides through measurement of free thiols by Ellman assay. In a typical gelation condition (polymer solution 7.5% w/v, dithiol crosslinkers with equivalent molar ratio of thiol groups to acrylate, and 10 mol% of L-lysine to thiol groups), crosslinking efficiency for DTT, DTT/YIGSR (85/15 mol/mol), DTT/IKVAV (85/15 mol/mol) are 94.5%, 96.6% and 96.3%, respectively, estimated by testing unreacted free dithiol linkers after crosslinking through Ellman assay.

ACS Paragon Plus Environment

9

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 29

The gelation behaviors of the resulting materials were determined by oscillation time sweep rheology analysis (Figure 2). Loss modulus (G’’) remained at a low level and the storage modulus (G’) increased significantly, demonstrating gelation occurred. Higher temperature and higher polymer concentration increases gelation rate (Figure 2a and 2b). Figure 2b also shows that higher polymer concentration significantly increases the storage modulus of the formed hydrogel. No gelation was observed for the 5 wt% polymer solution within 20 min. The gelation rate was also dependent on the catalyst concentration (Figure 2c). The gelling points for L-lysine concentration of 5 mol%, 10 mol% and 15 mol% are at 60s, 100s and 240s, respectively. The data reveal that gelation time can be flexibly tuned to accommodate the application by simply changing the catalyst concentration. The mixture of DTT/peptide linkers exhibited a lower gelation rate than the pure DTT linker (Figure 2d), possibly due to a lower reactivity of peptide than DTT. Nonetheless, gelation occurred within 10 minutes, which is a suitable time frame for local injection applications. Besides, the gelation on moist environments, such as pork chops, was also conducted (Supporting Information Figure 2) and showed similar gelation rate as on the hydrophobic surface. To address the concerning on the DTT cytotoxicity, viability of NPCs exposed at different DTT concentration over 24hrs were tested by live/dead assay (Supporting Information Figure 3). It shows that the NPCs viability is not significantly influenced until the DTT concentration reaches 7.5 mM, whereas in our typical crosslinking system, the DTT concentration at the starting point was calculate to be 3.6 mM. It would be expected that DTT should be quickly consumed by the crosslinking reaction and from the crosslinking efficiency test, only around 5% of DTT will be left, which is in a relatively safe concentration.

ACS Paragon Plus Environment

10

Page 11 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

The cyclized polymer-based hydrogel was imaged by scanning electron microscopy (SEM) using a cross-section of freeze-dried gel (Figure 3a), revealing a porous structure with average pore diameters of 10~20 µm. It is worth mentioning that the freeze-dried gel for SEM characterization shrinks significantly in size, thus the diameter of the real pore size could be much higher than the size characterized by SEM. The porous structure is desirable for cell therapy applications because it allows for tissue growth and facile diffusion of nutrients and waste products. The hydrogel swelling profile is shown in Figure 3b. The hydrogels that formed from the cyclized vinyl polymer maintained swelling ratios below 1.3, which could be attributed to the interlaced connection within the polymer structure. The hydrogel crosslinked by DTT alone showed the lowest swelling ratio. Inclusion of peptide linkers slightly increases the swelling ratio, possibly due to the longer and flexible linkages in the hydrogel. The degradation profile of the hydrogels was estimated by weight loss as a function of time (Figure 3c). Over 50% weight loss was observed after over 8 weeks, demonstrating a rapid degradation rate, comparable to PLGA materials that have been used as neural progenitor cell scaffolds.34 The DTT-crosslinked hydrogel showed a lower degradation rate compared to the hydrogel crosslinked by DTT/peptide. The supernatant containing degradation products was collected after a month and analyzed by mass spectrometry (Supporting Information Figure 4). The main products were detected as PEG chains and DTT derivatives from hydrolysis of hydrogels. Those degradation products showed very limited NPCs cytotoxicity at the tested concentration. To determine the biocompatibility of hydrogels, HeLa cells were first cultured in the hydrogel and cell viability analyzed at various time points by the Live/Dead viability assay (Supporting Information Figure 5). HeLa cells were viable for 2 weeks in all hydrogel samples with and without laminin peptide linkers. By counting from LIVE/DEAD staining micrographs, the

ACS Paragon Plus Environment

11

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 29

percentage of live cells was maintained above 80% and MTS assay showed the cell metabolic activity increased over time (Supporting Information Figure 6). The cell proliferation was also confirmed by the LIVE/DEAD image in Supporting Information Figure 5, showing the formation of large cell clusters at day 7 and 14. NPCs were then encapsulated in the hydrogels containing DTT alone, DTT with individual peptides (IKVAV or YIGSR) or DTT with both peptides at equimolar ratios. The viability of NPCs within the hydrogels was observed after 2, 5 and 8 days with the LIVE/DEAD viability assay (Figure 4). In the sample containing no laminin peptide linkers, survival of the encapsulated NPCs was poor at 5 and 8 days, with 44% and 35% live cells, respectively, in the hydrogel (Figure 5a). With laminin protein physically mixed into the hydrogel, the cell survival rate increased at all observed time points. However, a significant drop in survival rate at day 5 and 8 was also observed, possibly due to the diffusion of laminin from the hydrogel. The hydrogels containing IKVAV sequence showed slightly better survival rate than the hydrogel crosslinked by DTT alone but significant cell death were observed on day 8, possibly due to the lack of cell adhesion in the hydrogel. NPCs survival is maintained in hydrogels containing the cell adhesive YIGSR sequence. This indicated that the cell attachment to the scaffold played an important role in the cell survival. It was interesting to note that the hydrogels containing both YIGSR and IKVAV sequences showed the best survival after 8 days. This synergistic effect has been previously reported for survival of MIN6 β-cells.35 Cell clusters were also observed from the LIVE/DEAD images, indicating a certain level of cell proliferation.36 However, the cell metabolic activity did not significantly increase for NPCs as observed for the Hela cells (Figure 5b). These results emphasize the importance of immobilized laminin peptide for the long-term survival of the NPCs. The crosslinking reaction of acrylate-thiol addition did not induce

ACS Paragon Plus Environment

12

Page 13 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

additional cytotoxicity to the embedded NPCs, as no significant cell death was observed. There may be concerns that free laminin peptide crosslinkers in the hydrogels will have any antiadhesion effects on the cells. However, the high crosslinking efficiency (above 96%) could indicate cells would be exposed to low levels of free laminin peptide in the hydrogel. In addition, it would be expected that the free peptide would undergo some clearance due to cerebrospinal fluid circulation. The peptide sequences, which were immobilized at both termini in the hydrogel matrix as they are displayed in laminin, showed a strong effect on the NPCs survival. Immunohistochemical staining (Supporting Information Figure 8) shows that cells encapsulated in the hydrogel basically remain neural progenitor (SOX2 stain as progenitor marker) and no neuronal differentiation (TuJ1 stain as neuronal marker) was observed. Potential directions with the material to promote differentiation could include introducing longer laminin motifs, e.g. CDPGYIGSR and CQAASIKVAV, which have been shown to increase neurite outgrowth, myelination, astrogliosis inhibition compared to shorter motifs.37,38 Integrin-binding peptide RGD could also be introduced to promote cell adhesion and spreading, which are almost essential for cell differentiation. Alternatively, thiolated ECM components, such as thiolated hyaluronic acid or gelatin could be used to crosslink the acrylate-containing polymer in order to create a more recognized environment by the cells and thus accommodate better cell differentiation. CONCLUSIONS A PEG-based, intramolecularly cyclized vinyl polymer was synthesized by RAFT polymerization and used as a precursor to fabricate hydrogels with dithiol linkers through nucleophile-initiated thiol Michael addition under physiological conditions with a fast and adjustable gelation rate. From a practical standpoint, nucleophile-initiated thiol-Michael

ACS Paragon Plus Environment

13

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 29

reactions possess the same general features as the typical radical-mediated thiol-ene click reaction, facilitating the rapid, modular, orthogonal addition of thiols to electron-deficient vinyl groups in a quantitative manner, under nondemanding conditions (no heat or light needed), which is suitable for injectable delivery. 3D encapsulation of NPCs in the synthetic hydrogel showed good cell viability, promoted by the introduction of laminin-derived peptides. This hydrogel from cyclized vinyl polymer and dithiol linker system provides a blank palette on which thiol functional biomolecules and their combination can be painted for the study of 3D cell-biomaterial interactions and drug release.

ACS Paragon Plus Environment

14

Page 15 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

FIGURES

Figure 1 Kinetic study for the synthesis of cyclized vinyl polymer and characterization of the final polymer product: (a) Kinetic plots of ln(V0/V) versus time, showing a constant reaction rate, where V0 stand for the starting vinyl concentration and V stand for the real time vinyl concentration; (b) Kinetic plots of Mw versus vinyl concentration, showing a linear growth at the early stage of reaction and chain combination at the later stage; (c) GPC trace for the final polymer product at 10 hours after purification, detected by RI, UV and MALLS detectors; (d) 1H NMR spectra of the final polymer product at 10 hours after purification.

ACS Paragon Plus Environment

15

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 29

Figure 2: Time-sweep oscillatory rheology measuring the storage modulus (G’) and loss modulus (G’’) as a function of time after crosslinking the cyclized vinyl polymer with dithiol linkers: (a) Effect of crosslinking temperature, showing faster crosslinking at higher temperature; (b) The concentration of polymer solution (wt%) significantly affects the gelation rate and the gel stiffness; (c) The concentration of catalyst (L-lysine) affects the gelation rate but leads to similar gel modulus, showing that the gelation rate can be flexibly tuned; (d) Effect of dithiol linkers (DTT or DTT/peptide combination), showing DTT/peptide combinations lead to lower gelation rate than the pure DTT linker. Insertions are plots with logarithmic coordinate Y axis.

ACS Paragon Plus Environment

16

Page 17 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 3: Characterization of gel morphology, swelling ratio and degradation: (a) SEM image of freeze-dried gels samples prepared from 7.5 wt% cyclized vinyl polymer crosslinked by DTT at 37°C; (b) Swelling ratio of hydrogels from 7.5 wt% polymer by soaking in phosphate buffered saline (PBS, pH 7.4) at 37°C and measuring the change in weight of hydrated hydrogel at different time intervals; (c) Degradation of hydrogels from 7.5 wt% polymer by soaking in phosphate buffered saline (PBS, pH 7.4) at 37°C and measuring the weight of reserved freezedried hydrogels at different time intervals.

ACS Paragon Plus Environment

17

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 29

Figure 4. LIVE/DEAD viability assay for NPCs encapsulated in various hydrogels crosslinked by DTT or DTT/peptide linkers. Calcein AM (green) stain for live cells and ethidium homodimer-1 (red) for dead cells (scale bars in all cases represent 100 µm). The image was taken within a 1mm*1mm*0.2mm cubic space (as shown in Supporting Information Figure 7) and stacked in Z axis by ImageJ software.

ACS Paragon Plus Environment

18

Page 19 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 5. Cell viability and cell metabolic activity analysis of the NPCs encapsulated in various hydrogels crosslinked by DTT or DTT/peptide linkers: (a) Cell viability evaluated by percentage of live cells to total cell number calculated from LIVE/DEAD staining micrographs (mean ± SD, n = 3); (b) cell metabolic activity evaluated by percentage of reduced alamarBlue® absorbance at 550 and 595 nm (mean ± SD, n = 3, P < 0.05).

ACS Paragon Plus Environment

19

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 29

SCHEMES

Scheme 1: Cyclized vinyl polymer was synthesized through RAFT polymerization at low monomer concentration, where intramolecular cyclization is facilitated by the slow chain propagation and low chain concentration,27 resulting in highly intramolecularly crosslinked cyclized polymer. The vinyl containing polymer nanoparticles can be further crosslinked by dithiol linkers to form macroscopical gels for cell encapsulation.

ACS Paragon Plus Environment

20

Page 21 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: . Supporting Table 1: Polymerization conditions and molecular weight characteristics of cyclized vinyl polymers at different time point. Supporting Figure 1 Mass spectra of the synthesized peptide sequences. Supporting Figure 2: Gelation test on a pork chop, verifying the capability of gelation on a moist environment. Supporting Figure 3: Viability of NPCs exposed at different DTT concentration over 24hrs by live/dead assay. Supporting Figure 4: MALDI-TOF mass spectrometry of the degradation product of the hydrogel (left) and viability of NPCs exposed at different concentration of degradation products over 24hrs by live/dead assay (right). Supporting Figure 5: Live/dead staining of HeLa cells encapsulated in various hydrogels. Supporting Figure 6: HeLa cells viability by counting and cell proliferation by MTS assay. Supporting Figure 7: The 1mm*1mm*0.2mm cubic space within hydrogels for live/dead imaging. Supporting Figure 8: Immunohistochemical staining of NPCs encapsulated in the hydrogel crosslinked by DTT/IKVAV/YIGSR linkers at day 8. AUTHOR INFORMATION Corresponding Author * Suzie H. Pun ([email protected]) *Philip J. Horner ([email protected]) Notes

ACS Paragon Plus Environment

21

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 29

The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by a DOD SCIRP Investigator Initiated Award (SC130249). Part of this work was conducted at the Molecular Analysis Facility, a National Nanotechnology Coordinated Infrastructure site at the University of Washington which is supported in part by the National Science Foundation (grant ECC-1542101), the University of Washington, the Molecular Engineering & Sciences Institute, the Clean Energy Institute, and the National Institutes of Health. The authors also thank Prof. Patrick S. Stayton and Dr. Rick Edmark for assistance and use of equipment of rheology measurements. REFERENCES 1.

Watt, F. M.; Driskell, R. R., The therapeutic potential of stem cells. Philos. Trans. R. Soc.

Lond. B Biol. Sci. 2010, 365, (1537), 155-163. 2.

Gage, F. H., Mammalian neural stem cells. Science 2000, 287, (5457), 1433-1438.

3.

Horky, L. L.; Galimi, F.; Gage, F. H.; Horner, P. J., Fate of endogenous stem/progenitor

cells following spinal cord injury. J. Comp. Neurol. 2006, 498, (4), 525-538. 4.

Robel, S.; Berninger, B.; Gotz, M., The stem cell potential of glia: lessons from reactive

gliosis. Nat. Rev. Neurosci. 2011, 12, (2), 88-104. 5.

Rola, R.; Mizumatsu, S.; Otsuka, S.; Morhardt, D. R.; Noble-Haeusslein, L. J.; Fishman,

K.; Potts, M. B.; Fike, J. R., Alterations in hippocampal neurogenesis following traumatic brain injury in mice. Exp. Neurol. 2006, 202, (1), 189-199.

ACS Paragon Plus Environment

22

Page 23 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

6.

Sellers, D. L.; Maris, D. O.; Horner, P. J., Postinjury niches induce temporal shifts in

progenitor fates to direct lesion repair after spinal cord injury. J. Neurosci. 2009, 29, (20), 67226733. 7.

Cholas, R. H.; Hsu, H. P.; Spector, M., The reparative response to cross-linked collagen-

based scaffolds in a rat spinal cord gap model. Biomaterials 2012, 33, (7), 2050-2059. 8.

Tate, C. C.; Shear, D. A.; Tate, M. C.; Archer, D. R.; Stein, D. G.; LaPlaca, M. C.,

Laminin and fibronectin scaffolds enhance neural stem cell transplantation into the injured brain. J. Tissue. Eng. Regen. Med. 2009, 3, (3), 208-217. 9.

Johnson, P. J.; Tatara, A.; Shiu, A.; Sakiyama-Elbert, S. E., Controlled release of

neurotrophin-3 and platelet-derived growth factor from fibrin scaffolds containing neural progenitor cells enhances survival and differentiation into neurons in a subacute model of SCI. Cell Transplant. 2010, 19, (1), 89-101. 10. Asmani, M. N.; Ai, J.; Amoabediny, G.; Noroozi, A.; Azami, M.; Ebrahimi-Barough, S.; Navaei-Nigjeh, M.; Ai, A.; Jafarabadi, M., Three-dimensional culture of differentiated endometrial stromal cells to oligodendrocyte progenitor cells (OPCs) in fibrin hydrogel. Cell Biol. Int. 2013, 37, (12), 1340-1349. 11. Bozkurt, G.; Mothe, A. J.; Zahir, T.; Kim, H.; Shoichet, M. S.; Tator, C. H., Chitosan channels containing spinal cord-derived stem/progenitor cells for repair of subacute spinal cord injury in the rat. Neurosurgery 2010, 67, (6), 1733-1744. 12. Nomura, H.; Zahir, T.; Kim, H.; Katayama, Y.; Kulbatski, I.; Morshead, C. M.; Shoichet, M. S.; Tator, C. H., Extramedullary chitosan channels promote survival of transplanted neural

ACS Paragon Plus Environment

23

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 29

stem and progenitor cells and create a tissue bridge after complete spinal cord transection. Tissue Eng Part A. 2008, 14, (5), 649-665. 13. Banerjee, A.; Arha, M.; Choudhary, S.; Ashton, R. S.; Bhatia, S. R.; Schaffer, D. V.; Kane, R. S., The influence of hydrogel modulus on the proliferation and differentiation of encapsulated neural stem cells. Biomaterials 2009, 30, (27), 4695-4699. 14. Ashton, R. S.; Banerjee, A.; Punyani, S.; Schaffer, D. V.; Kane, R. S., Scaffolds based on degradable alginate hydrogels and poly(lactide-co-glycolide) microspheres for stem cell culture. Biomaterials 2007, 28, (36), 5518-5525. 15. Mothe, A. J.; Tam, R. Y.; Zahir, T.; Tator, C. H.; Shoichet, M. S., Repair of the injured spinal cord by transplantation of neural stem cells in a hyaluronan-based hydrogel. Biomaterials 2013, 34, (15), 3775-3783. 16. Ballios, B. G.; Cooke, M. J.; Donaldson, L.; Coles, B. L. K.; Morshead, C. M.; van der Kooy, D.; Shoichet, M. S., A hyaluronan-based injectable hydrogel improves the survival and integration of stem cell progeny following transplantation. Stem Cell Reports 2015, 4, (6), 10311045. 17. Seidlits, S. K.; Khaing, Z. Z.; Petersen, R. R.; Nickels, J. D.; Vanscoy, J. E.; Shear, J. B.; Schmidt, C. E., The effects of hyaluronic acid hydrogels with tunable mechanical properties on neural progenitor cell differentiation. Biomaterials 2010, 31, (14), 3930-3940. 18. Khaing, Z. Z.; Milman, B. D.; Vanscoy, J. E.; Seidlits, S. K.; Grill, R. J.; Schmidt, C. E., High molecular weight hyaluronic acid limits astrocyte activation and scar formation after spinal cord injury. J. Neural. Eng. 2011, 8, (4), Article Number: 046033.

ACS Paragon Plus Environment

24

Page 25 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

19. Battig, M. R.; Huang, Y.; Chen, N.; Wang Y.; Aptamer-functionalized superporous hydrogels for sequestration and release of growth factors regulated via molecular recognition. Biomaterials 2014, 35, (27), 8040-8048.20. Alcantar, N. A.; Aydil, E. S.; Israelachvili, J. N., Polyethylene glycol-coated biocompatible surfaces. J. Biomed. Mater. Res. 2000, 51, (3), 343351. 21. Lee, S.; Tong, X. M.; Yang, F., The effects of varying poly(ethylene glycol) hydrogel crosslinking density and the crosslinking mechanism on protein accumulation in threedimensional hydrogels. Acta Biomater. 2014, 10, (10), 4167-4174. 22. Missirlis, D.; Spatz, J. P., Combined effects of PEG hydrogel elasticity and cell-adhesive coating on fibroblast adhesion and persistent migration. Biomacromolecules 2014, 15, (1), 195205. 23. Dong, Y. X.; Saeed, A. O.; Hassan, W.; Keigher, C.; Zheng, Y.; Tai, H. Y.; Pandit, A.; Wang, W. X., "One-step" preparation of thiol-ene clickable PEG-based thermoresponsive hyperbranched copolymer for in situ crosslinking hybrid hydrogel. Macromol. Rapid Commun. 2012, 33, (2), 120-126. 24. Griffin, D. R.; Weaver, W. M.; Scumpia, P. O.; Di Carlo, D.; Segura, T., Accelerated wound healing by injectable microporous gel scaffolds assembled from annealed building blocks. Nat. Mater. 2015, 14, (7), 737-744. 25. Fairbanks, B. D.; Schwartz, M. P.; Halevi, A. E.; Nuttelman, C. R.; Bowman, C. N.; Anseth, K. S., A versatile synthetic extracellular matrix mimic via thiol-norbornene photopolymerization. Adv. Mater. 2009, 21, (48), 5005-5010.

ACS Paragon Plus Environment

25

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 29

26. Azagarsamy, M. A.; Marozas, I. A.; Spans, S.; Anseth, K. S., Photoregulated hydrazonebased hydrogel formation for biochemically patterning 3D cellular microenvironments. ACS Macro Lett. 2016, 5, (1), 24-28. 27. Zhao, T. Y.; Zheng, Y.; Poly, J.; Wang, W. X., Controlled multi-vinyl monomer homopolymerization through vinyl oligomer combination as a universal approach to hyperbranched architectures. Nat. Commun. 2013, 4, Article Number: 1873. 28. Gao, Y. S.; Zhou, D. Z.; Zhao, T. Y.; Wei, X.; McMahon, S.; Ahern, J. O.; Wang, W.; Greiser, U.; Rodriguez, B. J.; Wang, W. X., Intramolecular cyclization dominating homopolymerization of multivinyl monomers toward single-chain cyclized/knotted polymeric nanoparticles. Macromolecules 2015, 48, (19), 6882-6889. 29. Chan, J. W.; Hoyle, C. E.; Lowe, A. B.; Bowman, M., Nucleophile-intiated thiol-Michael reactions: Effect of organocatalyst, thiol, and ene. Macromolecules 2010, 43, (15), 6381-6388. 30. Graf, J.; Ogle, R. C.; Robey, F. A.; Sasaki, M.; Martin, G. R.; Yamada, Y.; Kleinman, H. K., A pentapeptide from the laminin-B1 chain mediates cell-adhesion and binds the 67000laminin peceptor. Biochemistry 1987, 26, (22), 6896-6900. 31. Tashiro, K.; Sephel, G. C.; Weeks, B.; Sasaki, M.; Martin, G. R.; Kleinman, H. K.; Yamada, Y., A synthetic peptide containing the IKVAV sequence from the a-chain of laminin mediates cell attachment, migration, and neurite outgrowth. J. Biol. Chem. 1989, 264, (27), 16174-16182.

ACS Paragon Plus Environment

26

Page 27 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

32. Petit, A.; Sellers, D. L.; Liebl, D. J.; Tessier-Lavigne, M.; Kennedy, T. E.; Horner, P. J., Adult spinal cord progenitor cells are repelled by netrin-1 in the embryonic and injured adult spinal cord. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, (45), 17837-17842. 33. Li, X. W.; Liu, X. Y.; Josey, B.; Chou, C. J.; Tan, Y.; Zhang, N.; Wen, X. J., Short laminin peptide for improved neural stem cell growth. Stem Cells Transl. Med. 2014, 3, (5), 662670. 34. Pan, Z.; Ding, J. D., Poly(lactide-co-glycolide) porous scaffolds for tissue engineering and regenerative medicine. Interface Focus 2012, 2, (3), 366-377. 35. Weber, L. M.; Hayda, K. N.; Haskins, K.; Anseth, K. S., The effects of cell-matrix interactions on encapsulated beta-cell function within hydrogels functionalized with matrixderived adhesive peptides. Biomaterials 2007, 28, (19), 3004-3011. 36. Shih, H.; Lin, C. C., Photoclick hydrogels prepared from functionalized cyclodextrin and poly(ethylene glycol) for drug delivery and in situ cell encapsulation. Biomacromolecules 2015, 16, (7), 1915-1923. 37. Tavakol S.; Saber R.; Hoveizi E.; Tavakol B.; Aligholi H.; Ai J.; Rezayat S.M., SelfAssembling Peptide Nanofiber Containing Long Motif of Laminin Induces Neural Differentiation, Tubulin Polymerization, and Neurogenesis: In Vitro, Ex Vivo, and In Vivo Studies. Mol. Neurobiol. 2016, 53, (8), 5288-5299. 38. Shaw D.; Shoichet M.S., Toward spinal cord injury repair strategies: peptide surface modification of expanded poly(tetrafluoroethylene) fibers for guided neurite outgrowth in vitro. J Craniofac. Surg. 2003, 14, (3), 308-316

ACS Paragon Plus Environment

27

Biomacromolecules

Page 28 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

28

Page 29 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

89x23mm (150 x 150 DPI)

ACS Paragon Plus Environment