Deciphering the Role of Sulfonated Unit in Heparin ... - ACS Publications

Subscriber access provided by Columbia University Libraries

Article

Deciphering the Role of Sulfonated Unit in HeparinMimicking Polymer to Promote Neural Differentiation of ESCs Jiehua Lei, Yuqi Yuan, Zhonglin Lyu, Mengmeng Wang, Qi Liu, Hongwei Wang, Lin Yuan, and Hong Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b08034 • Publication Date (Web): 07 Aug 2017 Downloaded from http://pubs.acs.org on August 8, 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.

ACS Applied Materials & Interfaces 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 52

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 Applied Materials & Interfaces

Deciphering the Role of Sulfonated Unit in Heparin-Mimicking Polymer to Promote Neural Differentiation of ESCs Jiehua Lei, Yuqi Yuan, Zhonglin Lyu, Mengmeng Wang, Qi Liu, Hongwei Wang,* Lin Yuan,* and Hong Chen

State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, P. R. China.

KEYWORDS: Heparin-mimicking polymer, unit-recombination strategy, sulfonated unit, embryonic stem cells (ESCs), neural differentiation.

ABSTRACT. Glycosaminoglycans (GAGs), especially heparin and heparan sulfate (HS) hold great potential for inducing the neural differentiation of embryonic stem cells (ESCs) and have brought new hope for the treatment of neurological diseases. However, the disadvantages of natural heparin/HS, such as difficulty in isolating with a sufficient amount, highly heterogeneous in structure and the risk of immune responses, have limited their further therapeutic applications. Thus, there is a great demand for stable, controllable and well-defined synthetic alternatives of heparin/HS with more effective biological functions. In this study, based upon a previously 1

ACS Paragon Plus Environment


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 52

proposed unit-recombination strategy, several heparin-mimicking polymers were synthesized by integrating glucosamine-like 2-methacrylamido glucopyranose monomers (MAG) with three sulfonated units in different structural forms, and their effects on cell proliferation, the pluripotency and the differentiation of ESCs were carefully studied. The results showed that all the copolymers had good cytocompatibility and displayed much better bioactivity in promoting the neural differentiation of ESCs as compared to natural heparin; copolymers with different sulfonated units exhibited different levels of promoting ability; among them, copolymer with 3-sulfopropyl acrylate (SPA) as a sulfonated unit was the most potent in promoting the neural differentiation of ESCs; the promoting-effect is dependent on the molecular weight and concentration of P(MAG-co-SPA), with the highest levels occurring at the intermediate molecular weight and concentration. These results clearly demonstrated that the sulfonated unit in the copolymers played an important role in determining the promoting-effect on ESCs neural differentiation; SPA was identified as the most potent sulfonated unit for copolymer with the strongest promoting ability. The possible reason for sulfonated unit structure as a vital factor influencing the ability of the copolymers may be attributed to the difference in electrostatic and steric hindrance effect. The synthetic heparin-mimicking polymers obtained here can offer an effective alternative to heparin/HS and have great therapeutic potential for nervous system diseases.

1. INTRODUCTION

2


Page 3 of 52

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


Glycosaminoglycans (GAGs), as linear polyanionic heteropolysaccharides, are the major components of the extracellular matrix.1 GAGs are generally involved in the regulation of numerous essential cellular processes, of which the most prominent is they interact with some key proteins that are of physiological importance in many fundamental

cell-to-cell

communication,

cell

signaling

recognition

and

transduction.2-4 These interactions are often critical to the basic development processes of cell proliferation and cytodifferentiation.5,6 Recent findings have shown that specific GAGs could help regulate stem cell differentiation, especially the neural differentiation of embryonic stem cells (ESCs) into neural cells or neurons.3,7-9 The differentiation of neural cells from ESCs plays an important role in the cell therapy of nervous system diseases, such as Parkinson's disease, Alzheimer's disease, and spinal injuries.10-13 GAGs, including heparin / heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), and keratan sulfate (KS), are among the most complex polysaccharide. And the differences in the number of repeat units, charge distribution, sulfation, sugar conformation, and three-dimensional structure of GAGs endow them with rich structural diversity.14,15 The extraordinary structural diversity of GAGs is the basis for the wide range of their biological activities.16 A slight difference in the molecular structure of GAGs would have an impact on the mode and degree of their interaction with the key proteins in signaling pathways8,17 and therefore lead to different inducing effects on ESCs neural differentiation. For instance, compared with heparin and HS, the differentiation-promoting activities of CS and DS are relatively lower, mostly 3



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 52

caused by the absence of glucosamines and N-acetylglucosamines in their polysaccharide backbones;17 undersulfated GAGs or GAGs removal of sulfate groups significantly decreased or even lose their effect on neural differentiation.17,18 Of all the GAGs, heparin and HS are the focus of research and concern owing to the fact that they show a remarkable capacity to induce and promote the neural differentiation of ESCs

3,17-19

and have great potential for therapeutic application toward many

diseases that depend on replacement therapy and transplantation of neural cells. However, since the in vivo biosynthetic pathway of heparin (or HS) in the Golgi apparatus is very complicated, the structures of heparin (or HS), derived from different organisms, different tissues, or even the same cells at different growth stages are highly variable and different, which leads to the great instability in their biological activities.8 Moreover, as natural products from living organisms, heparin (or HS) is susceptible to desulfation and difficult to isolate in quantities sufficient for research, and may also bring the risk to introduce potentially hazardous pathogens and induce cellular immune responses. All these distinctive disadvantages have limited further applications of natural heparin (or HS) in inducing ESCs differentiation to neural cells. It is necessary to replace natural heparin (or HS) with fully defined and more effective synthetic alternatives, which can provide more selectively synthetic control of the structure and has great therapeutic potential for neural tissue replacement therapy and regenerative medicine in treating nervous system diseases. Efforts to obtain synthetic heparin (or HS) with high bioactivities have employed various methods and strategies. One of the commonly used approaches is to prepare 4


Page 5 of 52

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


synthetic heparin (or HS) directly by chemical or chemoenzymatic methods. Synthetic heparin (or HS) obtained by chemical synthesis has accurate and well-defined structure, however, the chemical method suffers from the problem of tedious process, a relative poor yield and the difficulty in synthesizing large molecular weight polysaccharides.16 In these respects, chemoenzymatic method is more efficient, but it needs to pay highly for lots of different enzymes and complex purification processes in aiming to get a highly heterogeneous structure of heparin (or HS).19,20 Considering the practical difficulties of chemical and chemoenzymatic synthesis methods, molecular simulation strategy to fabricate heparin-mimicking materials has been proposed.21-24 The bioactivity of heparin is determined by its specific molecular structure, which is a linear highly sulfated glycosaminoglycan with an alternating sequence of disaccharide units consisting of repeating 1→4 linked D-glucosamines (GlcN) and L-iduronic acid (IdoA) residues with varying degrees of sulfation.25,26 It is now known that specifically sulfated sequences along heparin (or HS) chains cluster in groups along the side-chain to act as the functional domains.26 A convenient way for molecular simulation is to introduce functional domains, sulfated groups, into natural non-GAG polysaccharides that possess sugar backbone structures similar to heparin, to mimic the structures and the biological effects of heparin. As a linear natural polysaccharide, chitosan is an excellent candidate for simulating and substituting for the saccharide chain in heparin. It has been reported that the sulfated chitosan, by introducing sulfate groups to specific sites in glucosamine residues of chitosan, acquires biological activities similar to heparin (or HS),27-29 and has been 5



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 52

widely used in many biomedical applications.27,30,31 Ding et al. reported that the regioselectively sulfated chitosan could show heparin mimicking ability and promote the differentiation of ESCs into neurons.32 Yet, this strategy is still limited by the impressive structural heterogeneity and the not-well-controlled degree of sulfation in natural non-GAG polysaccharides produced by organisms, which resulted in the instability of physiological activity. To overcome these limitations, a novel unit-recombination strategy was proposed to synthesize heparin-mimicking polymer by utilizing independent units containing similar functional groups to naturally produced heparin (or HS). This strategy was inspired by the crucial roles of the overall carbohydrate backbone and the sulfated chain on determine the bioactivity of heparin (or HS),17,18,26,33 and for the first time proposed

to

split

the

basic

sulfated

saccharide

building

blocks

of

heparin/HS-mimetics into two functional components: sulfonated unit (S unit) and glyco unit (G unit).34 The heparin-mimicking polymers can be obtained by copolymeration of 2-methacrylamido glucopyranose (MAG) as the G unit and sodium 4-styrenesulfonate (SS) as the S unit. The copolymer p(SS-co-MAG) exhibited both higher proliferation of mouse embryonic stem cells (mESCs) and higher promotion of neural differentiation in mESCs better than either pSS or the pMAG homopolymer. Although the aforementioned study clearly demonstrated that the synergistic effects between the sulfonated units and the sugar units in the copolymer contribute to the high biological activity of this copolymer, the influence of sulfonated unit structure on the neural-inducing activity had been less emphasized and not explored in depth. 6


Page 7 of 52

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


Increasing evidences suggest that GAGs regulate complex processes in the form of a ‘sulfation code’; interaction between heparin (or HS) and key proteins (e.g. growth factors) is driven through the sulfate groups along the heparin (or HS) chains.35 And as a consequence, the sulfonated unit in the heparin (or HS) analogues may play essential role in determining the synergistic effects on the neural differentiation in ESCs. However, the sulfonated unit in heparin-mimetics mentioned above is SS, the constitutional unit containing aromatic groups, among diverse sulfonated monomers, such as 3-sulfopropyl acrylate (SPA) and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), used widely in biomedical research.36-38 Are these sulfonated groups with distinct sulfonated structures suitable and effective to promote neural differentiation of ESCs? Which kind of sulfonated group is the most competent? What molecular weight and concentration of the copolymer is most effective for the best effect on neural differentiation in ESCs? To explore answers to these questions will certainly help to decipher the sulfonate-containing unit in the heparin (or HS) analogue and search for the competent sulfonated groups, which are critical for understanding the mechanisms and exploiting the full potential of heparin-mimicking polymers for ESC-based therapy. In the present paper, several sulfonated monomers, representative of different chemical structures and different steric hindrance effects, SS, SPA and AMPS, were used as the candidates for sulfonated unit (S unit). A series of heparin-mimicking polymers were prepared by recombination of nonsulfated MAG (G unit) with the three different sulfonated monomers, respectively. Their influences on cell 7



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 52

proliferation, and cell differentiation, especially neural differentiation were studied in detail as compared to natural heparin. It can be found that all the three copolymers exhibit good ability in facilitating mESCs differentiate into neural cells compared with heparin and the copolymer P(MAG-co-SPA) with SPA as the sulfonated unit show the greatest potential in neural-differentiation-promoting effect on ESCs. Furthermore, both the molecular weight-dependent and the concentrations-dependent characteristic of differentiation-promoting effects of P(MAG-co-SPA) were studied for better exploiting the full potential of the heparin-mimicking and the critical design variables for neural differentiation of ESCs-based therapies. Such synthetic heparin-mimicking polymers not only offer a cost-effective alternative to heparin, but also circumvent problems associated with batch-to-batch variations and other shortcomings observed in natural heparin. 2. MATERIALS AND METHODS Materials D-glucosamine hydrochloride and 2, 2-azobisisobutyronitrile (AIBN) were purchased

from

TCI.

N-methacryloyl

4-cyano-4-(phenylcarbonothioylthio)pentanoic 4-styrenesulfonate

(SS),

3-sulfopropyl

acids,

acrylate

chloride,

paraformaldehyde, potassium

salt

sodium

(SPA)

and

2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) were purchased from Sigma-Aldrich. Dulbecco's modified Eagle’s medium (DMEM) high glucose medium, Roswell Park Memorial Institute (RPMI-1640) medium, 0.25 % (1 ×)

8


Page 9 of 52

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


Trypsin and 10 × phosphate buffer solution (PBS) were purchased from Hyclone. Fetal bovine serum (FBS), nonessential amino acids (NEAA), GlutaMAX™ and 0.1 % TritonX-100 were purchased from Gibco. Leukemia inhibitory factor (LIF), PlasmocinTM prophlactic, bovine serum albumin (BSA) and β-mercaptoethanol were purchased from Merck Millipore, Invivogen, Solarbio and Beijing Dingguo Biotechnology Co. Ltd., respectively. N, N-dimethylformamide (DMF), methyl alcohol and heparin (MW≈ 3.5~8.0 kDa) were purchased from Shanghai Chemical Reagent Co.. Methyl alcohol and AIBN were purified by distillation and recrystallization before using. All aqueous solutions were prepared in 18.2 MΩ·cm purified water (deionized water, DIW) from a Milli-Q water purification system (Millipore, Bedford, MA, USA). Synthesis of MAG Monomer D-glucosamine hydrochloride (5.00 g, 2.32 × 10-2 mol) and potassium carbonate (3.20 g, 4.64 × 10-2 mol) were dissolved in 120 mL methyl alcohol at a 250 mL single neck flask, with magnetic stirring under -10 °C for half an hour, followed by the dropwise addition of 2.0 mL methacryloyl chloride (2.08 × 10-2 mol) with vigorous stirring under an ethanol/ice bath. Four hours later, filtrate was obtained with the suction filter and concentrated with vacuum distillation. The products were purified via column chromatography with dichloromethane/methanol (4:1) as the eluent. Synthesis of Copolymers

9



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 52

A series of copolymers were synthesized (see Table 1) by reversible addition-fragmentation chain transfer (RAFT) polymerization through adjusting the concentration and ratio of each monomer, chain transfer agent (CTA) and initiator (I). Table 1. Details in synthesizing copolymers by RAFT polymerization.

Monomer Copolymer

Ratio [MG]:[MS]

G unit

S unit

[M]:[CTA]:[I]*

P(MAG-co-SS)

MAG

SS

2:1

100:2:1

P(MAG-co-SPA)

MAG

SPA

1:1

100:2:1

P(MAG-co-AMPS)

MAG

AMPS

1:1

100:2:1

*CTA: 4-cyano-4-(phenylcarbonothioylthio) pentanoic acids; I: AIBN

Monomers, CTA and initiator were dissolved in 5 mL mixed solvent (DMF: DIW=1: 1, V/V). Which was bubbled nitrogen for 30 min to remove oxygen in the container before transferring into gloves box for further reaction at 70 °C for 24 h. Copolymers was obtained via lyophilization after dialysis. Characterization of Copolymers Fourier-transform infrared (FTIR) spectra were obtained with a Thermo Scientific Nicolet 6700 spectrometer. 1H nuclear magnetic resonance (1H-NMR) was measured with Agilent 300 MHz nuclear magnetic resonance spectroscopy. The molecular weight and polymer dispersity index (PDI) of polymers were measured with an

10


Page 11 of 52

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


Agilent PL Aquagel-OH column in Waters 1515 gel-permeation chromatography system. The zeta potential of polymer was measured by Malvin Potentiometer. Culture and Differentiation of mESCs Primary generation of mESCs was purchased from Shanghai Institutes for Biological Science, CAS. Cells were cultured in a T25 culture flask on the feeder layer at humidified atmosphere at 37 °C incubator (Eppendorf Galaxy 170 R) with 5 % (V/V) CO2. As previous reported,34 feeder layers are mitomycin C-inactivated mouse embryonic fibroblasts (mEFs), cultured in DMEM high glucose medium supplemented with 10% FBS, 1% penicillin/streptomycin, 5 µg/mL PlasmocinTM prophlactic, 2.0 mM GlutaMAX™, 0.1 mM NEAA, 0.1 mM β-mercaptoethanol and 1000 U/mL LIF. Medium was replaced every 2 days. mESCs with the number of 6 × 103 were seeded in each well at 96-well plate that was coated with gelatin in mESC medium overnight as mentioned above, then medium was replaced with fresh medium containing 10 µg/mL polymer and changed every other day. We used Cell Count Kit-8 (CCK-8) to test cell viability at day 1, 3 and 7, respectively. mESCs at the density of 2.6 × 103 cells/cm2 were seeded in gelatin pre-coated 6-well culture plate in mESC medium overnight. Finally, differentiation medium (DMEM high glucose medium containing 10 % FBS, 1 % penicillin/streptomycin, 5 µg/mL PlasmocinTM prophlactic, 2.0 mM GlutaMAX™, 0.1 mM NEAA and 0.1 mM β-mercaptoethanol) was added after cells rinsed with PBS. Differentiation medium was changed every second day and passage was carried out every four days. 11



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 52

Immunofluorescence Assay Cells were fixed with 4 % paraformaldehyde solution for 10 min after washed with PBS for 3 times and 0.1 % TritonX-100 solution was used to increase cell membrane permeability, after that cells were blocked with 3 % BSA (in PBS). Cells were incubated with 1% BSA (in PBS) containing anti-β3-tubulin antibody (Cell Signaling Technology) at 4 °C overnight. Then primary antibody was removed and residual was rinsed with PBS, secondary antibody, fluorescein isothiocyanate (FITC)-labeled goat anti-Rabbit IgG (Abcom), was diluted in 1 % BSA to treat cells for 1 h at room temperature (RT). After thorough rinsing, the nuclei of cells were stained with 4', 6-diamidino-2-phenylindole (DAPI) (Invitrogen) and observed with microscopy (Olympus IX71 fluorescence microscope). Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Real-time qPCR Total RNAs were extracted from cells using Total RNA Kit (Tiangen) following the manufacturer’s instruction. Before total RNAs extraction, cells cultured on the surface of cell culture plate were rinsed with PBS for 3 times. The extracted RNAs were reverse transcribed into cDNA using REVERTAID 1ST CDNA SYNTH KIT (Fermentas). Quantitative real-time PCR reactions were performed on a Step One Plus real-time PCR system and monitored using the Fast SYBR Green PCR Master Mix with a ROX reference dye (ABI). cDNA samples were analyzed for target genes (Oct-4, Foxa2, Sox17, Flk1, Brachyury, Nestin and β3-tubulin) and for the reference

12


Page 13 of 52

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


gene β-Actin. RT-PCR was performed at 95 °C for 20 s followed by 50 cycles of 30 s denaturation at 95 °C, 45 s annealing at 60 °C, and 45 s elongation at 72°C. The level of expression of each target gene was then calculated as 2-∆∆CT. All the samples were normalized to heparin-supplemented medium, which was set to 1. FGF Binding Assay mESCs at the density of 4 × 104 cells/cm2 were seeded on 24-well plate coated with gelatin. After overnight incubation, cells were washed three times with PBS and fixed in 4% paraformaldehyde (PFA) for 10 min at RT, then washed three times with PBS to remove residual PFA. For FGF binding, cells were blocked with 3% BSA for 1h at 4 °C and then incubated with FGF2 and copolymers for 2h at 4°C. After that, cells were washed with PBS to removed unbound FGF2 and copolymers. Then, immunofluorescence assay was used to detect FGF2 bound to FGFR on the cell surface with primary antibody rabbit anti-mouse FGF2 and second antibody Alexa fluor 555 goat anti-rabbit IgG (red). Cytotoxicity Evaluation L929 cells were seeded in 96-well plate (2 × 103 cells/well) for CCK-8 assay and cells were seeded as 5 × 103 cells/cm2 in 48-well plate for Live/Dead cell viability assay. Cells were cultured in 100 µL RPMI-1640 medium containing 10 % FBS, 1 % penicillin/streptomycin and 5 µg/mL PlasmocinTM prophlactic in the 37°C incubator with 5 % (V/V) CO2. Then polymer was added in culture medium with the concentration of 10 µg/mL 4 h later. The medium was changed every other day. The 13



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 52

cell viability was analyzed using Cell Counting kit-8 (CCK-8) at days 1, 3, and 5, respectively. For LIVE/DEAD viability/cytotoxicity assay, cells cultured for 7 days were first washed with PBS for three times and incubated with 100 µL mix solution containing 2 µM calcein-AM and 4 µM propidium iodide (PI) for 20 min at 37 °C. Then, cells were washed with PBS and fluorescence images were obtained by Olympus IX71 inverted fluorescence microscope. Statistical Analysis The data are expressed as the mean ± SD. One-way ANOVA test was used for statistical analysis with p less than 0.05 considered statistically significant. 3. RESULTS AND DISCUSSION Synthesis of Heparin-Mimicking Polymers with Different Sulfonated Units Monomers bearing saccharide groups (MAG), which has glucosamine-containing carbohydrate unit, were synthesized via nucleophilic substitution of methacryloyl chloride to glucosamine hydrochloride. The MAG monomer was then copolymerized with three structurally different sulfonated unit (S unit) monomers. The feed ratio of MAG monomer to sulfonated unit monomer used for the preparation of copolymers was 2:1 or 1:1 considering different reactivity of these monomers in the polymerization, which could promise their similar ratios of around 1:1 in the final products, according to our previous study.34 And the expected molecular weight of the produced copolymers is approximately 9 kDa, close to the molecular weights of commercial heparins (the best known hyper-sulfated form of HS). For the sake of 14


Page 15 of 52

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


simplicity,

the

obtained

copolymers

P(MAG-co-SS),

P(MAG-co-SPA)

and

P(MAG-co-APMS) are correspondingly named as pGS1, pGS2 and pGS3, respectively.

Their

structural

characteristics were

first

studied

using

the

Fourier-transform infrared (FT-IR) spectroscopy in the 4,000-500 cm-1 region. The characteristic peaks of the functional groups correspondingly present in glyco unit (MAG) and sulfonated unit are presented in Figure 1. The three copolymers obviously displayed one peak at around 3300 cm-1 attributes to the N–H/O–H bonds of MAG, and at least two absorption peaks at 1665 and 1530 cm-1, which can be assigned to the amide I band (mainly due to the C=O stretching vibration) and the amide II band (a combination of the N–H bending vibration and C–N stretching vibration ) of MAG, respectively.39 The absorption peaks around 1200 cm-1 and 1050 cm-1 in the three copolymers correspond to the SO stretches from S unit.40 The characteristic absorption peaks were 833 cm-1, 774 cm-1 and 674 cm-1 for SS, 1718 cm-1 for SPA, and 1380 cm-1 corresponds to the two methyl absorption peak signal on one carbon atom for AMPS. The FT-IR results clearly indicated the successful synthesis of all the copolymers that contain both the glyco unit (MAG) and sulfonated unit.

15



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 52

Figure 1. The infrared spectra of copolymers. PGS1, PGS2 and PGS3 represents respectively P(MAG-co-SS), P(MAG-co-SPA), and P(MAG-co-AMPS). The exact structure of each copolymer can be further proved by the 1H nuclear magnetic resonance (NMR) spectrum of the purified copolymers (Figure 2), from which the characteristic peaks of MAG and sulfonated unit are clearly visible. The practical ratios of the glyco units and sulfonated units in all the copolymers can be calculated as close to 1:1 according to 1H NMR data (see Table 2). Moreover, information about the number-average molecular weights (Mn) and polymer dispersity index (PDI) values of all copolymers were acquired by gel permeation chromatography (GPC) experiments as shown in Table 2. As shown, each copolymer has a molecular weight (Mn) of around 9 kDa and extremely narrow polydispersity (PDI) in the range of 1.22-1.23, suggesting good control in the RAFT polymerization. The detail results from FT-IR, 1H-NMR and GPC all demonstrated that the successful synthesis of heparin-mimicking polymers that have pre-designed structures.

16


Page 17 of 52

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


Figure 2. 1H NMR spectra of MAG (A), PGS1 (B), PGS2 (C), and PGS3 (D). Table 2.GPC results of synthetic copolymers by RAFT polymerization. Ratio of constitutional unit* Copolymer

Mn (×103 Da)

PDI G unit : S unit

PGS1

9.9

1.23

1.00:1.00

PGS2

9.2

1.22

1.00:1.11

PGS3

9.5

1.23

1.00:1.02

* The ratio of constitutional unit of each copolymer measured through integrals of their 1H-NMR spectra. Effect of Heparin-Mimicking Polymers with Different Sulfonated Units on the Proliferation of mESCs

17



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 52

ESCs can be expanded to large numbers infinitely as undifferentiated entities through a process of self-renewal, and have been used as a model system for studying early development and the generation of desired cells for regenerative medicine. The normal proliferation of ESCs is an important prerequisite for maintaining their potential to differentiate into various somatic cell types and regulating ESCs efficient differentiation. It is known that proper amount of heparin supplementation in medium can maintain the growth and proliferation of ESCs, 41,42 on account of the fact that heparin can bind and activate a large number of mitogenic factors that mediate proliferation of progenitor cells.43 As synthetic copolymers simulating the structure of natural heparin, do they have the similar impact on the proliferation of mESCs? The “YES” answer to this question was given by CCK-8 cell proliferation assay on the three heparin-mimicking polymers with different S unit. The results obtained at day 1, day 3 and day 7 showed that each synthetic copolymer displayed the ability to maintain the normal proliferation of mESCs similar to the effect of heparin (Figure 3), without obvious stimulative or restraining effect, indicating that all the synthetic copolymers displayed the basic functions of natural heparin in mESCs growth and made it possible for us to investigate their potential in inducing differentiation of stem cells into desired cell types.

18


Page 19 of 52

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


Figure 3. Influence of synthetic copolymers on the proliferation of mESCs. mESCs were treated with synthetic copolymers and heparin respectively for 1, 3 and 7 days. The copolymers studied in this experiment all have a controlled molecular weight of about 9 kDa. Data are presented as the mean ± SD (n = 3).

Effect of Heparin-Mimicking Polymers with Different Sulfonated Units on the Pluripotency of mESCs ESCs are undifferentiated cells that are capable of in vitro self-renewal and proliferation while maintaining their pluripotency with the potential to give rise to all lineages of the developing embryo. Once pluripotent ESCs initiate differentiation, they are considered to decrease and lose their pluripotency rapidly, prior to any evidence of morphological change. Previous studies have demonstrated that Oct-4, a transcription factor which plays an important role in maintaining the pluripotent state 19



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 52

of ESCs and may prevent expression of genes activated during differentiation, is one of the acknowledged specific markers for pluripotency in ESCs. The difference in the amount of Oct-4 transcriptional expression can reflect the change in stem cell pluripotency.44-46 The effect of heparin-mimicking polymers with different sulfonate units on the pluripotency of ESCs were evaluated by the transcriptional expression level of pluripotency marker Oct-4 determined by quantitative RT-PCR as reflected by their mRNA levels in the cells during differentiation. Since heparin is a factor that controls ESCs fate commitment and is required for ESCs to exit from self-renewal in vivo, 19,47 it was used as a positive control and all the samples were normalized to heparin-supplemented medium, which was set to 1, in order to assess the relative ability of the heparin-mimicking polymers in cell differentiation. As seen from Figure 4, after ten days of incubation, results showed the mRNA level for Oct-4 was significantly decreased in mESCs treated by all the heparin-mimicking polymers with different sulfonate units compared with cells treated by heparin. The results indicated that the transcription of gene Oct-4 is downregulated by heparin-mimicking polymers treatment and differentiation of copolymers-treated cells occurred more frequently than that of heparin-treated cells; the copolymers, which have both glyco unit and sulfonated unit in the structures, could stimulate stem cells exit from pluripotency, and have higher efficiency in inducing mESCs differentiation compared to heparin. Furthermore, from Figure 4 it could be observed that the mRNA level for Oct-4 in ESCs treated by PGS1, PGS2 and PGS3 compared with heparin was 0.23, 0.20 and

20


Page 21 of 52

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


0.41, respectively, indicating that copolymer with the SPA as sulfonated unit exhibited the strongest capability to induce the differentiation of mESC.

Figure 4. Influence of synthetic copolymers on the relative transcript level of pluripotency marker Oct-4 gene in mESCs. mESCs were treated with each synthetic copolymer and heparin respectively for 10 days. The copolymers studied in this experiment all have a controlled molecular weight of about 9 kDa. Data are presented as the mean ± SD (n = 3); *** p

Deciphering the Role of Sulfonated Unit in Heparin ... - ACS Publications

Recommend Documents