Polyester with Pendent Acetylcholine-Mimicking Functionalities

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A polyester with pendent acetylcholinemimicking functionalities promotes neurite growth Shaofei Wang, Eric Jeffries, Jin Gao, Lijie Sun, Zhengwei You, and Yadong Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b12379 • Publication Date (Web): 24 Mar 2016 Downloaded from http://pubs.acs.org on March 29, 2016

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ACS Applied Materials & Interfaces ,

A polyester with pendent acetylcholine-mimicking functionalities promotes neurite growth Shaofei Wanga, Eric Jeffriesb, Jin Gaob, Lijie Suna, Zhengwei Youa,*, Yadong Wangb,* a

State Key Laboratory for Modification of Chemical Fibers and Polymer

Materials, College of Materials Science and Engineering, Donghua University, 2999 North Renmin Road, Shanghai 201620, P. R. China b

Departments of Bioengineering, Chemical Engineering, Surgery, and the

McGowan Institute, University of Pittsburgh, 3700 O’Hara Street, Pittsburgh, PA 15261, USA. *

Corresponding authors. Tel: 86-21-67874253; fax: 86-21-67792855 (Z. You).

Tel.: 1-412-624-7196;fax: 1-412-383-8788 (Y. Wang). E-mail addresses: [email protected] (Z. You), [email protected] (Y. Wang).

1

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ABSTRACT Successful regeneration of nerves can benefit from biomaterials that provide a supportive biochemical and mechanical environment while also degrading with controlled inflammation and minimal scar formation. Herein, we report a neuroactive polymer functionalized by covalent attachment of the neurotransmitter acetylcholine (Ach). The polymer was readily synthesized in two steps from poly(sebacoyl diglyceride)

(PSeD),

which

previously

demonstrated

biocompatibility

and

biodegradation in vivo. Distinct from prior acetylcholine-biomimetic polymers, PSeD-Ach contains both quaternary ammonium and free acetyl moieties, closely resembling native acetylcholine structure. The polymer structure was confirmed via 1

H nuclear magnetic resonance and Fourier-transform infrared spectroscopy.

Hydrophilicity, charge, and thermal properties of PSeD-Ach were determined by tensiometer, zetasizer, differential scanning calorimetry and thermal gravimetric analysis respectively. PC12 cells exhibited the greatest proliferation and neurite outgrowth on PSeD-Ach and laminin substrates, with no significant difference between these groups. PSeD-Ach yielded much longer neurite outgrowth than the control polymer containing ammonium but no the acetyl group, confirming the importance of the entire acetylcholine-like moiety. Furthermore, PSeD-Ach supports adhesion of primary rat dorsal root ganglions and subsequent neurite sprouting and extension. The sprouting rate is comparable to the best conditions from previous report. Our findings are significant in that they were obtained with acetylcholine-like functionalities in 100% repeating units, a condition shown to yield significant toxicity in prior publications. Moreover, PSeD-Ach exhibited favorable mechanical and degradation properties for nerve tissue engineering application. Humidified 2


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PSeD-Ach had an elastic modulus of 76.9 kPa, close to native neural tissue, and could well recover from cyclic dynamic compression. PSeD-Ach showed a gradual in vitro degradation under physiologic conditions with a mass loss of 60% within 4 weeks. Overall, this simple and versatile synthesis provides a useful tool to produce biomaterials for creating the appropriate stimulatory environment for nerve regeneration. KEYWORDS: acetylcholine, neurotransmitter, poly(glycerol sebacate), neurite extension, nerve regeneration, biomimetic material, neuron, dorsal root ganglion

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1.

INTRODUCTION Neurological diseases affect every age group and all segments of society. In

the USA alone, an estimated 50 million people are struck by neurological disorders each year, leading to an annual economic cost of billions of dollars in medical expenses and lost productivity.1 Functional restoration of damaged nerves remains a significant challenge in medicine due to the limited mitotic capacity of adult neurons and the prohibitive biochemical environment following injury.2-3 Consequently, there is not yet an effective treatment for spinal cord injury. Biomaterials play a key role in regenerative medicine approaches, serving as scaffolds for tissue regrowth and vehicles for cell transplantation and drug delivery.4-5 Synthetic polymeric biomaterials have received significant attention due to their reproducible and controllable structures, wide range of tunable properties, easy processing, and low risks of immunogenicity and disease transmission.6

However,

most

existing

synthetic

polymers

including

polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), poly(hydroxy butyrate) (PHB), and poly(ethylene glycol) (PEG) are biologically inert, which significantly compromises their role in guiding the regenerative response. Therefore, bioactive polymers that can elicit specific neuron cell responses, facilitate neurite outgrowth, and promote nerve regeneration are needed for future development.5 A common approach to create neural biomaterials is the integration of proteins such as laminin7 and neurotrophic factors8 or their bioactive epitopes5. However, due to their complex structures and sensitivity to temperature and chemicals, it is relatively difficult to purify and process these biomolecules.9 Recently, the use of 4


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bioactive small functional moieties instead of complex biomacromolecules to control the bioactivity has been recognized as an efficient and economical alternative to develop bioactive materials.9-10 Neurotransmitters represent an important class of biomolecules in the nervous system. Neurotransmitters direct cell survival, proliferation, differentiation and are essential for neuronal outgrowth during embryonic and neonatal development and after injury.5 Accordingly, incorporating neurotransmitters is a promising strategy to impart corresponding bioactivity to resultant biomaterials for nerve regeneration.11-12 Acetylcholine (Ach) is an important neurotransmitter associated with signaling within the neuromuscular junction. The activation of acetylcholine receptors also induces neurite outgrowth,13 promotes the formation and strengthening of synapses.14 Furthermore, acetylcholine can induce the turning of nerve growth cone.15 Polymers with acetylcholine-like functionalities (ALFs) have shown the ability to modulate neuronal responses and promote the neurite growth as well.16-20 For example, biomimetic

polymers

dimethylaminoethyl

made

from

methacrylate

the

(bioactive

free

radical

ALF)

and

polymerization PEG

of

monomethyl

ether-glycidyl methacrylate (bioinert unit) modulate the growth of hippocampal neurons in an ALF concentration-dependent manner.16 Similarly, when the appropriate amount of ALFs (2-acetoxy-N,N,N-trimehtylethanaminium) are tethered into non-adhesive PEG-based hydrogels via radical co-polymerization, the resultant hydrogels enhance the adhesion and viability of hippocampal neurons and glial cells.17 However, these polymers are constructed with polymethacrylate backbone and PEG side chain, which are both non-degradable, posing challenges for use in vivo. 16-18

Recently, biodegradable poly(ester-carbonate)s with pendent acetylcholine 5



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analogs were shown to promote the cell viability and neurite outgrowth of PC12 cell.19 However, syntheses of these polymers are complex processes (more than 5 steps) and require toxic/hazardous reagents (e.g. stannous 2-ethyl hexanoate, trimethylamine). Furthermore, all aforementioned reports attach ALFs to the polymer chain via modified acetyl end, possibly altering the biological activity of the free acetyl group in acetylcholine. To this end, we previously reported a series of biodegradable poly(aminoglycerol ester)s with varying ALFs containing free acetyl ends.20 Dorsal root ganglia (DRG) cultured on polymers with 70% ALFs exhibited neurite sprouting and extension comparable to those grown on laminin. However, in these polymers, tertiary amine was used to mimic the quaternary ammonium of acetylcholine. At physiological condition (neutral pH), only a fraction of these tertiary amine groups will be protonated, while the quaternary ammonium of acetylcholine are permanently charged. We hypothesized that more closely mimicking native acetylcholine would further improve results in nerve regeneration applications. Thus, the objective of this work was to develop a facile synthesis of a biodegradable polymer with ALFs both quaternary ammonium and free acetyl groups. In

this

study,

we

designed,

synthesized,

and

characterized

a

new

acetylcholine-like biomimetic polymer PSeD-Ach (Figure 1). The backbone is a biodegradable polymer poly(sebacoyl diglyceride) (PSeD) that was developed in our group and exhibits excellent cell adhesion and proliferation compared to tissue culture polystyrene (TCPS) and minimal host response during subcutaneous implantation in rats.21-22 In addition, PSeD maintains many of the desirable properties of its predecessor, poly(glycerol sebacate) (PGS), which demonstrated favorable outcomes in both sciatic nerve and retinal regeneration applications.23-29 6


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We employed a simple two-step modification of the free hydroxyl groups in PSeD to readily attach ALFs and produce PSeD-Ach. Similar to our first generation of acetylcholine-biomimetic polymers, the free acetyl moiety is entirely retained in the ALFs in PSeD-Ach, which was distinct from previous reports.16-19 In contrast to our prior work, quaternary ammonium is tethered to a pendent side chain rather than a tertiary amine group integrated directly into the backbone.20 Thus, we anticipate that the PSeD-Ach will have greater bioactivity and increased accessibility to cells. After material characterization, we performed in vitro testing with PC12 cells and rat dorsal root ganglions (DRGs) to evaluate the biocompatibility and bioactivity of PSeD-Ach. Additionally, we contrasted our results with a similar control polymer PSeD-B, which lacked the acetyl group (Figure 1).

Figure 1. The structure of Ach-mimetic polymer PSeD-Ach and its resemblance to acetylcholine (Ac = Acetyl). To verify whether the acetyl functional groups had effect on the bioactivity, we design a control material (PSeD-B) with similar ammonium structure to PSeD-Ach, but without acetyl related moiety (2-acetoxy-ethyl groups), which is replaced with inert butyl groups. 2.

EXPERIMENTAL SECTION

2.1. Synthesis and characterization Chemical Reagents: The chemicals were purchased from Acros Organics unless otherwise specified. Sebacoyl chloride (TCI, 90%) and glycidol (96%) were distilled 7



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under reduced pressure. Triethylamine (Alfa Aesar, 99%) was dried by anhydrous NaOH and distilled. Sebacic acid (Alfa Aesar, 98%) was recrystallized three times from ethanol and dried under vacuum. Tetrabutylammonium hydroxide (Alfa Aesar, 55% aqueous solution), chloroacetyl chloride (ClCH2COCl, 98%), pyridine (99.5%), 2,2-dimethylaminoethyl acetate (>99%), N-butyldimethylamine, anhydrous sodium sulfate, and all solvents were used without further purification. Deuterated solvents for NMR analysis were purchased from Cambridge Isotope Laboratories, Inc. Characterization: The molecular weight was determined via gel permeation chromatography (GPC) on a Viscotek GPCmax VE2001 system equipped with a Viscotek 270 dual detector (differential refractive index and right angle light scattering). For PSeD and PSeD-Ach, the measurement was performed on a Viscotek organic I-MBMMW-3078 column using N,N-dimethyl formamide (DMF, HPLC) as the eluent at room temperature. For PSeD-B, the measurement was performed on a PSS GRAM 100 Å and 1000 Å two columns system using a dimethylacetamide (HPLC) solution of 3 g L-1 lithium bromide (Alfa Aesar, 99.9%) and 6 mL L− 1 acetic acid (HPLC) at 80 oC. Polystyrene (American polymer standards PS34K) was used for calibration. 1H nuclear magnetic resonance (NMR) spectra were recorded on a Varian Mercury-400 NMR (400-MHz) or a Bruker Avance 600 NMR (600-MHz). Fourier transformed infrared (FTIR) spectrum was recorded on a Thermo Nicolet IR-100 spectrometer via sample film coated on NaCl crystal window. Differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA) of the polymers were performed on Seiko DSC 220C and TG/DTA 320 instruments, respectively, at a heating rate of 10 °C/min under a nitrogen atmosphere. Static air-water contact angle of PSeD-Ach film coated on glass was recorded on AST PRODUCTS INC. 8


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VCA2000 instrument at room temperature. Five measurements were performed and averaged. Zeta potential of PSeD-Ach methanol solution was recorded on a Malvern Zetasizer Nano-ZS90 instrument. Four measurements were performed and averaged. Synthesis of PSeD: PSeD was synthesized as our previous report.30 Briefly, an equimolar amount of sebacic acid and diglycidyl sebacate (prepared by the esterification reaction between glycidyl and sebacoyl chloride), and 0.6 mol% bis(tetrabutylammonium) sebacate (TBAS, prepared by the neutralization reaction between sebacic acid and tetrabutylammonium hydroxide as we described previously31) in anhydrous DMF under a nitrogen atmosphere were stirred at 100 oC for 26 hours. The reaction mixture was purified via dilution in acetone, precipitation in ethyl ether, and vacuum-dried at ambient temperature to yield PSeD. 1H NMR (400 MHz, CDCl3) δ 5.26-3.58 (m, 5H), 3.34 (br s, 1H), 2.35 (t, J = 7.4 Hz, 4H), 1.62 (m, 4H), 1.30 (m, 8H). GPC: number-averaged molecular weight (Mn) = 75.2 kDa, polydispersity index (PDI) = 1.31. Synthesis of PSeD 2-chloroacetate: It was synthesized as our previous report.30 Briefly, PSeD (1 eq based on the theoretical amount of hydroxyl groups) and pyridine (3 eq) were dissolved in anhydrous dichloromethane and cooled to around -78 oC via a dry ice/isopropanol bath under a nitrogen atmosphere. A ClCH2COCl (3.2 eq) solution in anhydrous dichloromethane was added dropwise to the mixture. After addition, the cooling bath was changed to ice/isopropanol bath (-15 oC) and the reaction continued for another 3 h before quenched by methanol. Then the reaction mixture was diluted by ethyl acetate, washed by deionized water for three times, dried by anhydrous Na2SO4, filtered, concentrated under a vacuum to furnish PSeD

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2-chloroacetate. 1H NMR (600 MHz, CDCl3) δ 5.38-5.24 (m, 1H), 4.50-4.16 (m, 4H), 4.08 (s, 2H), 2.32 (t, J = 7.5 Hz, 4H), 1.61 (m, 4H), 1.30 (m, 8H). Synthesis of PSeD-Ach: PSeD 2-chloroacetate (1 eq based on the theoretical amount of chloromethyl groups) and CH3CO2CH2CH2N(CH3)2 (2 eq) were dissolved in acetone, stirred at room temperature for 17 h. The precipitate was redissolved in methanol and precipitated in acetone for 3 time, then vacuum dried at room temperature to provide PSeD-Ach. 1H NMR (400 MHz, DMSO-d6) δ 5.41-5.16 (m, 1H), 4.70 (m, 2H), 4.44-4.43 (m, 2H), 4.35-4.14 (m, 4H), 3.92 (m, 2H), 3.35 (s, 6H), 2.31 (t, J = 7.2 Hz, 4H), 1.50 (m, 4H), 1.23 (m, 8H). GPC: Mn = 117.5 kDa, PDI = 1.35. Synthesis of PSeD-B: It was synthesized as our previous report.30 Briefly, PSeD 2-chloroacetate (1 eq based on the theoretical amount of chloromethyl groups) and N-butyldimethylamine (2 eq) were dissolved in acetone, stirred at room temperature for 19 h. The precipitate was redissolved in methanol and precipitated in acetone for 3 time, then vacuum dried at room temperature to provide PSeD-B. 1H NMR (600 MHz, DMSO-d6) δ 5.40-5.17 (m, 1H), 4.58-3.97 (m, 6H), 3.57-3.48 (m, 2H), 3.35 (s, 6H), 2.31-2.27 (m, 4H), 1.69-1.64 (m, 2H), 1.50 (m, 4H),1.31-1.27 (m, 2H), 1.24 (m, 8H), 0.92 (t, J = 7.2 Hz, 3H). GPC: Mn = 111.4 kDa, PDI = 1.21. 2.2. Mechanical test of PSeD-Ach Disk-shaped PSeD-Ach samples (typical size was 5 mm in diameter and 1.34 mm in height) were casted in a Teflon mold after softening with mild heat. The compression test was conducted at room temperature on an MTS insight mechanical analyzer equipped with a 5 Newton load cell. Samples were compressed at a crosshead speed of 1 mm/min. For simple compression test, dry samples were 10


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compressed to 20% and the initial slopes of the strain-stress curves were recorded as the modulus. Five samples were tested and averaged. In cyclic mechanical test, the sample was preconditioned at 37 oC in a humidified incubator overnight, and compressed to 30% then allowed to recover to 10% before immediately compressed to 30% for 10 times. Elastic modulus was determined from the initial part of the stress-strain curve in the first cycle. 2.3. In vitro degradation of PSeD-Ach Disk-shaped (diameter = 5 mm, height = 2 mm) PSeD-Ach samples were evenly cut into 4 pieces of fan-shaped specimens. Every specimen was weighted and submerged in 1 ml Dulbecco’s phosphate buffered saline (DPBS) solution, and incubated at 37 oC. At the predetermined period of times, the samples were retrieved, washed with deionized water, and lyophilized. The degree of degradation was determined by dry-weight change. At each time point three specimens were used and averaged. 2.4. The interaction between PSeD-Ach and PC12 cells Preparation of culture substrates: Tissue culture treated polystyrene (TCPS) surfaces were coated with polymers as follows: A methanol solution of PSeD or PSeD-Ach or PSeD-B (1 g/l) was filtered through a sterilized 0.2 µm filter and added to 24-well (80 µl/well) or 96-well (13 µl/well) TCPS plates. After evaporation of the solvent, the plates were dried under vacuum overnight. The surface morphology of the polymer coatings were characterized by scanning electron microscopy (SEM; Hitachi S-3000N, 15 kV). Before cell culture, the polymer surfaces were sterilized by UV light for 30 minutes, washed with DPBS (three times) and culture medium [Dulbecco's

Modified

Eagle

Medium

(DMEM)

supplemented

with

10% 11



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heat-inactivated horse serum, and 5% fetal bovine serum (FBS)] once (1 ml/well). Laminin control (BD Biosciences, #354232) was coated on TCPS by gently shaking overnight [200 µl of laminin in molecular grade water (50 mg/l) per well]. To test the effect of soluble acetylcholine, a solution of acetylcholine chloride (MP Biomedicals, Solon, OH) was added to the culture media to maintain a final concentration of 1 mM. Cell culture: PC12 cells were cultured in 85% DMEM medium supplemented with 10% heat-inactivated horse serum and 5% FBS, and maintained in a humid 5% CO2 incubator at 37 °C. The cells were primed for 24 h in differentiation medium [DMEM, 1% heat-inactivated horse serum, 0.5% FBS, 50 ng/ml nerve growth factor (NGF)] before seeding at a density of 1 × 104 per cm-1. Cell viability: Primed PC12 cells were seeded on 96-well TCPS plates. The number

of

metabolically

active

cells

was

determined

with

a

[3-(4,5)-dimethylthiazol-2,5-diphenyltetrazolium bromide] (MTT) assay at day 0, 1, 3, and 5 as previous reported protocol.32 Absorbance at a wavelength of 570 nm was measured using a Synergy MX plate reader (Biotek, Winooski, VT). Cell morphology and neurite outgrowth: Primed PC12 cells seeded on 24-well TCPS plates. At day 1, 3, and 5, the phase contrast images were taken on an inverted microscope Eclipse Ti (Nikon, Melville, NY) with a RETIGA-SRV digital camera (QImaging, BC, Canada). The neurite lengths, defined as the distance from the tip of the neurite to the junction between the cell body and neurite base were analyzed by NIH ImageJ (version 1.42). The length of the longest branch was used for branched neurites. The longest 20 neurites from 10 images under each condition per day were used for statistical analysis. 2.5. Ex vivo biocompatibility: DRG culture 12


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The isolation and culture of DRG were performed as we previously described20. Briefly, DRGs from spinal levels L4-L6 of postnatal day-3 Sprague Dawley rats were dissected and collected in Hanks' balanced salt solution (Mediatech, Inc., Herndon, VA). The ganglia were washed with Hanks' balanced salt solution twice and seeded in neurobasal medium supplemented with B-27 (2%; Invitrogen), 2 mM L-glutamine, and 50 ng/ml NGF on a precoated 24-well TCPS plate (PSeD-Ach and laminin were coated as described in section 2.4) at one ganglion per well. Glial proliferation was inhibited with 5-fluorodeoxyuridine (7.5 µg/ml) and uridine (17.5 µg/ml) (MP Biomedicals, Solon, OH). Half of the medium was first changed at day 4 and once every 3 days afterwards. The culture was maintained in a humid 5% CO2 incubator at 37 °C. The neurite outgrowth was photographed using an inverted phase contrast microscope [Nikon TE-2000 U microscope (Melville, NY) equipped with a 4 MP Diagnostics Spot Flex digital camera (Sterling Heights, MI)]. Adobe Photoshop CS2 (San Josc. CA) was used to merge the photos and outline the neurites using the "find edge" function. DRG with neurite projections around the majority of the perimeter on day 4 were considered to have neurite sprouting. DRG were fixed and stained for neurofilament using rabbit anti-neurofilament M (145 kDa) polyclonal antibody (Millipore) and Alexa Fluor®594 fluorescently labeled goat anti-rabbit IgG antibody (Invitrogen). 2.6. Statistical analysis For PC12 assay, statistical analysis was performed using one-way ANOVA and a LSD Post Hoc multiple comparison with a minimum confidence level of p < 0.05 for statistical significance. All values are reported as the mean ± standard deviation. For DRG assay, error bars for sprouting percentages were calculated as the 95 % 13



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confidence interval for the proportion being estimated using the normal approximation to the binomial distribution. Chi-squared homogeneity testing was used to evaluate statistical significance of the difference in substrates’ ability to promote neurite sprouting. 3.

RESULTS

3.1. Synthesis and structural characterization The backbone PSeD with a number-averaged molecular weight (Mn) of 75.2 kDa and a polydispersity index (PDI) of 1.31 was prepared by acid-induced epoxide ring-opening polymerization between equimolar amounts of diglycidyl sebacate and sebacic acid in the presence of catalytic amount of TBAS according to our previous report30. PSeD-Ach was synthesized from PSeD in 2 steps (Figure 2). The esterification of the hydroxyl groups in PSeD was performed with excessive chloroacetyl chloride using pyridine as the base to absorb produced hydroxyl chloride. Subsequently, a Menshutkin reaction using 2-dimethylaminoethyl acetate as tertiary amine efficiently built the pendent ALFs at room temperature. The resultant PSeD-Ach had a Mn of 117.5 kDa and a PDI of 1.35. The control polymer PSeD-B (Mn = 111.4 kDa, PDI = 1.21) was synthesized via a similar way to PSeD-Ach as our previous report.30

Figure 2. Synthetic route of PSeD-Ach. 14


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The structure of PSeD-Ach was analyzed by 1H NMR and FTIR spectroscopies (Figure 3). 1H NMR spectrum of PSeD-Ach showed all protons ascribe to PSeD-Ach structure (Figure 3A). The three signals marked ‘H1, H2, H3’ at chemical shift δ 2.31, 1.50 and 1.23 ppm were assigned to the sebacoyl moiety in PSeD backbone. Signal 'H4' at δ 4.44 ppm corresponded to CH2 protons in the glyceryl moiety. The CH protons of the glyceryl moiety in PSeD usually appear at δ < 4.5 ppm.21 But the OH groups in PSeD-Ach were esterified, thus the corresponding chemical shift of CH protons shifted to downfield (5.22-5.35 ppm) because of the electron withdrawing effect of the carbonyl group. The signals at δ 4.19-4.35ppm marked ‘H6’ were attributed to the CH2 protons in the glycine moiety, the chemical shift of which was significantly higher than the ones in typical glycine moiety due to the electron withdrawing effect of adjacent ammonium structure. The sharp peak at δ 3.35 ppm marked ‘H7’ corresponded to the methyl groups in ammonium moiety. The peaks at δ 3.92 ppm and 4.70 ppm marked ‘H8’ and ‘H9’ were attributed to CH2 protons adjacent to ammonium and acetate moieties in acetylcholine moiety, respectively. The signals at δ 2.03 ppm marked ‘H10’ corresponded to methyl groups in acetyl moiety. The comparison of relative integrations of protons in backbone [protons of sebacoyl group near carbonyl (H1, 4H) at δ 2.31] and side ALFs [protons of acetyl group (H10, 3H) at δ 2.03] revealed one repeat unit essentially had one ALF. It demonstrated the efficiency of the synthetic method. FTIR spectrum of PSeD-Ach displayed the characteristic stretch vibration of C-O bond in acetate group at 1240 cm-1 (arrow), which further confirmed the presence of ALFs (Figure 3B).

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Figure 3. Spectroscopic characterization of PSeD-Ach (A) 1H NMR spectrum of PSeD-Ach (DMSO-d6). The interpretation of all the peaks in spectrum is shown in the molecule structure. The relative integration of the protons in acetyl group (red, 3H) at δ 2.03 and the protons in sebacoyl group near carbonyl (purple, 4H) at δ 2.31 is around 9:4 indicating all the pendent hydroxyl groups in PSeD has been converted into ALFs. (B) FTIR spectrum of PSeD-Ach. The characteristic absorption of C-O bond of acetate ester at 1240 cm-1 (arrow) further confirmed the presence of ALF. 3.2. Physical properties of PSeD-Ach Physical properties of PSeD-Ach including hydrophilicity, charge, thermal and mechanical properties were investigated. The TGA revealed that the decomposition temperature of PSeD-Ach was 148.2 oC. Thus, PSeD-Ach was stable at body temperature (37 oC) and suitable for in vivo biomedical application. The DSC analysis showed PSeD-Ach had a Tg of -8.5 oC (Figure 4A). No apparent melting peak was observed indicating it may be total amorphous at room and body temperatures. In contrast, the backbone PSeD was semi-crystallized with a melt point at around 30 o

C.30 The increased structural irregularity induced by pendent ALFs may inhibit the

crystallization of PSeD-Ach. Hydrophilicity of a material can significantly alter protein adsorption and influence cell adhesion, spreading, and cytoskeletal organization.33-34 The water 16


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contact angle of PSeD-Ach film, which was 28.3 ± 1.4o revealing it was a relatively hydrophilic material compared to PSeD (45.0 ± 1.1o) (Figure 4B).22 Zeta potential of PSeD-Ach in methanol was 30.1 ± 1.8 mV, further confirming the existence of quaternary ammonium in PSeD-Ach (Figure 4C). PSeD-Ach is a soft and flexible material. Compression test showed that dry PSeD-Ach had an elastic modulus of 571 ± 58.5 kPa and could be compressed without any apparent crack at least up to 20% strain (Figure 4D). Since the material will be in a hydrated state for implanted applications, we ran a cyclic compression test on humidified PSeD-Ach sample (Figure 4E). It exhibited a low modulus of 76.9 kPa. Furthermore, PSeD-Ach exhibited limited hysteresis for 10 cycles of a strain up to 30%. Its recovery from cyclic dynamic compression deformation is likely influenced by strong adhesion to the test head combined with its compliant nature.

Figure 4. (A) The DSC curve of PSeD-Ach showed a glass transition temperature of -8.5 oC, and no apparent melting point. (B) The water contact angle of PSeD-Ach was 28.3 ± 1.4o revealing its good hydrophilicity. (C) Zeta potential of PSeD-Ach in 17



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methanol was 30.1 ± 1.8 mV confirmed the existence of quaternary ammonium in the molecule. (D) Simple compression of dry PSeD-Ach. (E) Cyclic compression test at 30% strain for 10 cycles of humidified PSeD-Ach samples. 3.3. In vitro degradation of PSeD-Ach The main chain and side groups of PSeD-Ach are all assembled by ester bonds. Thus, we expected that PSeD-Ach could be degraded by hydrolysis and investigated its in vitro degradation in DPBS solution at 37 oC. Mass loss of PSeD-Ach appeared gradual and linear, losing approximately 15% original mass per week. (Figure 5).

Figure 5. PSeD-Ach degradation in DPBS solution at 37 oC. 3.4. The interaction between PSeD-Ach and PC12 cells The polymers including PSeD, PSeD-A, and PSeD-B were coated on TCPS surfaces for cell culture to evaluate their biological properties. The surface 18


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morphology of biomaterials could affect cell behaviors. Thus, we investigated the polymer coatings via SEM (Figure S1 in support information). The original stripes of TCPS surfaces (Figure S1A) disappearred after coating. All the coatings were effectively smooth except a few scattered small defects (Figure SB-D). Thus, the potential effects of surface morphologies of different coatings on cell behaviors were negligible. Hence, these coatings provided suitable substrates to study the interactions between different functionalities including acetylcholine-mimicking moieties of polymers and cells. Rat pheochromocytoma (PC12) cells have been widely used in studying the interaction between biomaterials and neurons.11, 35-36 Therefore, we used PC12 cells as an in vitro model to evaluate the cytocompatibility and bioactivity of PSeD-Ach. The PC12 cells were primed with nerve growth factor for 24 h before seeding. PSeD and PSeD-B were used as control polymers. Furthermore, appropriate free acetylcholine chloride (1 mM) was added to the culture medium on PSeD surface to evaluate the effect of immobilized ALFs in PSeD-Ach compared with solution-phase acetylcholine.20 For positive control, we used laminin, a standard substrate for in vitro culturing many types of neurons.11, 20 3.4.1.

Cell viability

NGF-primed PC12 cells were seeded on the coatings of PSeD-Ach and control materials (PSeD, PSeD-B, and laminin) on TCPS. The cell viability was monitored by the MTT assay at different culture times (Figure 6). The number of metabolically active cells at day 1 was similar to the one at day 0 under all conditions indicating that the PC12 cells attached well on all materials. The metabolic activity of cells was similar among all groups at day 1 and significantly increased from day 1 to day 3 19



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under all conditions. By day 5, the PC12 cells proliferated significantly on both PSeD-Ach and PSeD-B, comparable to the laminin controls. In contrast, the unmodified PSeD with or without free acetylcholine showed much lower metabolic activity.

Figure 6. MTT assay of PC12 cells on different materials. PSeD-Ach exhibited to be a good substrate for the growth of PC12 cells and have robust ability to promote the proliferation of PC12 cells comparable to laminin. Metabolically active cells on PSeD-Ach significantly increase within 5 day's culture, more than the ones on PSeD. Addition of free acetylcholine in culture medium on PSeD surface did not significantly promote the proliferation of PC12 cells. # (p < 0.05), ## (p < 0.01), and ### (p < 0.001): statistically significant difference compared to the previous time point under same conditions. * (p < 0.05), ** (p < 0.01), and *** (p < 0.001): 20


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statistically significant difference between different condition at the same time point. 3.4.2.

Cell morphology and neurite outgrowth

To further evaluate the interaction between PSeD-Ach and PC12 cells, we investigated the cell morphology and quantified the length of neurites.

The images

of PC12 cells under different culture condition at day 5 are shown in Figure 7A-E. There were relatively more cells on PSeD-Ach than PSeD, which was consistent with the results of MTT assay. PC12 cells on PSeD-Ach exhibited more long neurites than the ones on control polymers PSeD and PSeD-B without ALFs. Addition of free acetylcholine in the culture medium induced more long neurites of PC12 cells on PSeD surface. PC12 cells on both PSeD with free acetylcholine and PSeD-Ach showed relatively more straight neurites with the less branches than cells plated on laminin.

21



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Figure 7. (A-E) Phase contrast images of PC12 cells after 5 days' culture on (A) PSeD, (B) PSeD + free Ach (1 mM acetylcholine was added to the culture medium), (C) PSeD-Ach, (D) PSeD-B, and (E) laminin. (Magnification100 ×, scale bar = 100 µm.) (F) PSeD-Ach significantly promoted the neurite extension of PC12 cells. Twenty longest neurites from 10 images under each condition per day were measured. The neurite length of PC12 cells on PSeD-Ach significantly increased in another day within 5 days' culture comparable to laminin, and was higher than the one on PSeD 22


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without pendent ALFs and PSeD-B with similar quaternary ammonium structure while using bioinert butyl groups instead of 2-acetoxy-ethyl groups in ALFs. In addition, PSeD-Ach showed better ability to promote neurite extension than combination of PSeD with free acetylcholine in culture medium (1 mM) at short term (1 day) and similar effect at long term (3 and 5 days). The quantitative analysis of the neurite length is shown in Figure 7F. Results indicate that all materials permitted neurite extension from PC12 cells, though to varying degrees. The neurites on PSeD-Ach are longer than those on unmodified PSeD and the control polymer PSeD-B for every time point. The addition of solution-phase acetylcholine to PSeD surfaces seemed not to have immediate effect on the extension of neurites at short term (e.g. day 1) but produced results similar to PSeD-Ach at later time points. Laminin and PSeD-Ach yielded the longest neurites and showed no statistical difference between the two groups. 3.5. Ex vivo biocompatibility of PSeD-Ach with nervous tissue Culture of DRG explants is a well-established model to evaluate the ex vivo biocompatibility with both nerve and glial cells.11,

20, 37

Preliminary ex vivo

biocompatibility of PSeD-Ach with neural tissue was evaluated via explanting DRGs. Around half of DRGs (50 ± 30%) on PSeD-Ach adhered well and significantly sprouted neurites, which was comparable to laminin (60 ± 29%), and significantly more (p = 0.05) than the TCPS (10 ± 18%) (Figure 8A). PSeD-Ach demonstrated no adverse effects on DRGs in long-term culture (up to 29 days). Additionally, numerous neurites sprouted from the DRGs on PSeD-Ach and formed extensive networks around the bodies (Figure 8B). Furthermore, immunostaining of neurofilaments showed positive staining both in the cell bodies and the sprouting neurites, suggesting 23



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that DRG neurons cultured on PSeD-Ach retained their neuronal phenotype. This further demonstrated PSeD-Ach was a good substrate for neuron culture and neurite outgrowth.

Figure 8. PSeD-Ach exhibited good ex vivo biocompatibility when tested using rat DRG explants. (A) PSeD-Ach showed strong ability to promote neurite sprouting. The percentages of DRGs having sprouted neurites on PSeD-Ach (50 ± 30%) was higher (p = 0.05) than the one on TPCS (10 ± 18%). There was no statistical difference between laminin (60 ± 29%) and PSeD-Ach (p > 0.05). Error bars indicate 95% confidence intervals. Chi-squared homogeneity test is used for statistical analysis. Ten DRGs were explanted on each surface. (B) Image of DRG on PSeD-Ach at day 29 revealed that PSeD-Ach well supported the adhesion, neurite sprouting and extension of rat DRG explants for a long-term culture. The figure is merged from a series of 100 × phase contrast images of the body and sprouting neurites of DRG at day 29 (scale bar = 1 mm). (C) The fluorescent images of neurofilement staining of DRG revealed PSeD-Ach supported neuronal phenotypic expression (day 29, 100 ×, scale bar = 200 µm). 4.

DISCUSSION In this study, we designed a new neuroactive polymer with functional groups

closely mimicking acetylcholine. PSeD-Ach promotes the proliferation and neurite 24


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extension of PC12 cells better than unmodified PSeD and PSeD with free acetylcholine in culture medium. In fact, PSeD-Ach is comparable to laminin in promoting PC12 cell neurite extension and proliferation. However, unlike laminin which is water soluble and typically used as coatings, PSeD-Ach is a solid material and can be used to construct 3-dimension scaffolds. The elastic modulus (76.9 kPa) is close to that of spinal cord (89 kPa),38 which can be also beneficial on modulating neural cell behaviors and tissue regeneration.39-40 We aimed to address two challenges presented by earlier work: degradation and toxicity. As mentioned previously, some acetylcholine-biomimetic polymers were constructed with non-degradable units such as polymethacrylate and PEG.16-18 Others contain biodegradable units such as ester and carbonate bonds but their degradation profiles have not yet been investigated.19-20 For PSeD-Ach, we used ester bonds to construct the polymer’s backbone and side chain. PSeD-Ach demonstrated biodegradation under physiologic conditions with approximately 40% remaining at 4 weeks (Figure 5). No toxicity was observed for PSeD-Ach at any time point for either PC12 cells or DRG. This suggests that neither PSeD-Ach nor its degradation by-products are harmful to these cells. This was significant considering that beneficial effects of ALFs were known to be countered by toxic effects at high ALF substitutions ratios in previous studies16-17, 19 where ALFs were incorporated into the polymers via substituted acetyl moiety. However, in this study ALFs were attached into the polymers via one substitution of ammonium, indicating the importance of free acetyls on ALFs for biological function. In our first generation and other acetylcholine-biomimetic polymers,16-17, 19-20 it has been shown that ALFs modulate the neuronal responses in a concentration-dependent manner. In contrast, our studies 25



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performed with 100% hydroxyl conversion to ALFs, resulted in half of the DRG showing neurite sprouting on PSeD-Ach surface (Figure 8A). This is significantly greater than the (18±14)% of DRG on our corresponding first generation Ach-mimetic polymer (PSA100L0) and comparable to the best first generation polymer (PSA70L30).20 Since the prior work used tertiary amine instead of the quaternary ammonium in free acetylcholine, this supports our reasoning that quaternary ammonium is an important moiety in ALF presentation. Our future studies plan to explore the role of varying degrees of ALF substitution for PSeD-Ach. In vitro evaluation comparing our polymer to appropriate controls revealed interesting trends. Metabolic activity results (Figure 6) closely matched the cell attachment (Figure 7A-E) and showed best results with modified PSeD and laminin. Both PSeD-Ach and PSeD-B produced similar results, suggesting the immobilized ammonium groups may greater influence cell attachment than the presence of free bioactive molecules. This was confirmed by the poor results seen in the unmodified PSeD supplemented with free acetylcholine. In contrast, results of neurite extension appear more dependent on the presence of acetylcholine, a powerful neural signaling molecule. Samples containing both free Ach and pendent-linked ALFs demonstrate increased neurite extension. Additionally, PSeD-Ach showed a stronger ability to promote neurite extension of PC12 cells than PSeD-B, which was also positively charged. This suggests that ALF’s role stimulating neurons is not simply charge interactions. This had not yet been revealed in the previous reports on acetylcholine-biomimetic polymers.16-20 In addition, primary rat DRGs adhere and extend long neurite networks, and retain phenotypic protein expression on PSeD-Ach surfaces. Overall, presenting acetylcholine in a manner closer to its native form may 26


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yield greater neural cell attachment and sprouting and result in enhanced capacity for promoting regeneration. Yet, since acetylcholine naturally occurs as a synaptic signaling molecule, it is unclear from these studies if acetylcholine can act when bound or only once released from the polymer backbone. Thus, future studies will employ experiments to elucidate this mechanism. PSeD-Ach is a practical neuroactive material. Compared to other neuroactive materials derived from proteins and peptides such as laminin7, neurotrophic factors8, and IKVAV41, the synthesis of PSeD-Ach is simple and well-controlled. Thus, scale up of PSeD-Ach is more cost-effective. Compared to free acetycholine, PSeD-Ach shows clear advantages. Acetylcholine is a water soluble small molecule, making it hard to localize or provide mechanical support to the growth of neuron under physiological conditions. In contrast, PSeD-Ach can be made into a tissue engineering scaffold for nerve regeneration. PSeD-Ach may provide a reservoir for sustained release of acetycholine-like molecules. This is significant since the enzyme cholinesterase can catalyze degradation of 25,000 acetylcholine molecules per second.42 The synthetic strategy of PSeD-Ach is distinct from previous reports about polymers with ALFs.16-20 The backbone polyester with pendent hydroxyl groups is readily produced via our recently developed new polyesterification method: acid-induced epoxide ring-opening polymerization.30 This polymerization is applied to various monomers with carboxylic acid and epoxy groups and can offer polyesters with a wide range of physical, thermal, mechanical properties. The following esterification and Menshutkin reaction is a general and efficient method to attach designed ALFs to pendent hydroxyl groups. Accordingly, we can obtain a series of acetylcholine-biomimetic polymers with diverse structures and consequent properties. 27



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They can provide a polymer pool to develop bioactive materials with the superior properties for nerve regeneration. For biomedical application, biocompatibility is the first concern. Thus the minimal toxicity/harshness of reagents used for synthesis is critical for biomaterial preparation. In our last step of synthesis of PSeD-Ach, we designed chloroacetate as the substrate for Menshutkin reaction to react with tertiary amine to provide the quaternary ammonium (Figure 2). The electron-withdrawing effect of carbonyl group in chloroacetate greatly enhances the reactivity of adjacent carbon-chloro bonds. Thus, the reaction can be performed in a mild condition (room temperature) to avoid potential side reactions in absence of catalyst. In contrast, the synthesis of previously reported acetylcholine-biomimetic polymers usually involve toxic reagents such as ABIN, CuBr, trimethylamine, stannous 2-ethyl hexanoate, and Mg(ClO4),16-20 which raise safety concerns. In this study, we demonstrated a new design principle of acetylcholine-biomimetic neuroactive polymers and developing a practical synthetic method of the polymer. As mentioned previously, concentration of ALFs will be optimized in future work. Crosslinking of the remaining free hydroxyl groups may also be performed to produce tougher elastomers for applications that require tougher surgical handling.21 We will also perform additional in vitro and in vivo investigations. For instance, the significant role of glial cells in the neural regenerative process has recently been recognized, thus glial cell response on PSeD-Ach should also be explored. Finally, in vivo work using PSeD-Ach is necessary to demonstrate the benefits and begin translation into clinical uses.

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5.

CONCLUSION We designed a new neuroactive polymer PSeD-Ach with immobilized

acetylcholine-mimicking functional groups. PSeD-Ach promoted the proliferation and neurite extension of PC12 cells and DRGs at a similar capacity as laminin. We demonstrated that a complete acetylcholine-like moiety is important to modulate the neuronal response not just the positive charge from the ammonium group. PSeD-Ach exhibited good biodegradability and its mechanical properties was similar to that of nerve tissue. The latter is important for tissue regeneration, but has been largely overlooked in previously reported Ach-based neuroactive polymers. In addition, the versatile synthetic method could readily tailor both the backbone and the concentration of ALFs. This design could yield a new family of bioactive materials for nerve regeneration. ACKNOWLEDGMENTS This research is funded by National Natural Science Foundation of China (21574019), the State Key Laboratory for Modification of Chemical Fibers and Polymer Materials (Donghua University, LK1412), Natural Science Foundation of Shanghai (13ZR1401200), Fundamental Research Funds for the Central Universities (2232014A3-01) and DHU Distinguished Young Professor Program (B201303), and the NIH grant #7R21EB008565-03. We thank Dr. Christiane B. Gumera for her help on DRG culture. REFERENCES 1. Http://Www.Ninds.Nih.Gov/About_Ninds/Ninds_Overview.Htm. 2. Evans, G. R., Challenges to Nerve Regeneration. Semin. Surg. Oncol. 2000, 19 (3), 312-318. 3. Chalfoun, C. T.; Wirth, G. A.; Evans, G. R. D., Tissue Engineered Nerve Constructs: Where Do We Stand? J. Cell. Mol. Med. 2006, 10 (2), 309-317. 4. Tam, R. Y.; Fuehrmann, T.; Mitrousis, N.; Shoichet, M. S., Regenerative Therapies for Central Nervous System Diseases: A Biomaterials Approach. Neuropsychopharmacology 2014, 39 (1), 169-188. 29



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Polyester with Pendent Acetylcholine-Mimicking Functionalities

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