Tailored Chemical Properties of 4-Arm Star Shaped Poly(d,l-lactide

Mar 27, 2017 - Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhavan, 2 Rafi Marg, New Delhi-110001, India. ⊥ Department of Chemi...
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Tailored Chemical Properties of 4 arm star shaped poly(D,LLactide) as Cell Adhesive Three-Dimensional Scaffolds Chelladurai Karthikeyan Balavigneswaran, Sanjeev Kumar Mahto, Subia Bano, Arumugam Prabhakar, Kheyanath Mitra, Vivek Rao, Munia Ganguli, Biswajit Ray, Pralay Maiti, and Nira Misra Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00071 • Publication Date (Web): 27 Mar 2017 Downloaded from http://pubs.acs.org on March 28, 2017

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Bioconjugate Chemistry

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Tailored Chemical Properties of 4 arm star

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shaped poly(D,L-Lactide) as Cell Adhesive

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Three-Dimensional Scaffolds

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Chelladurai Karthikeyan Balavigneswaran,† Sanjeev Kumar Mahto,‡ Subia Bano,§

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Arumugam Prabhakar,§,ǁ Kheyanath Mitra,ǂ Vivek Rao,§,ǁ Munia Ganguli,§,ǁ Biswajit Ray,ǂ

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Pralay Maiti,┴ and Nira Misra*†

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Polymer Engineering Laboratory, School of Biomedical Engineering, Indian Institute of

Technology (Banaras Hindu University), Varanasi-221005, India ‡

Tissue Engineering and Biomicrofluidics Laboratory, School of Biomedical Engineering,

Indian Institute of Technology (Banaras Hindu University), Varanasi-221005, India §

CSIR-Institute of Genomics and Integrative Biology, Mathura road, New Delhi - 110025,

India ǁ

Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhavan, 2 Rafi Marg,

New Delhi – 110001 ǂ

Department of Chemistry, Institute of Science, Banaras Hindu University (BHU), Varanasi-

221005, India ┴

School of Materials Science and Technology,, Indian Institute of Technology (BHU),

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Varanasi-221005, India

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E.mail : [email protected]

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Abstract

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Biodegradable poly(lactic acid) (PLA) is widely used to fabricate 3D scaffolds for tissue

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regeneration. However, PLA lacks cell adhering functional moieties, which limit its

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successful application in tissue engineering. Herein, we have tailored the cell adhesive

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properties of star shaped poly(D,L-Lactide) (ss-PDLLA) by grafting gelatin to their 4 arms.

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Grafting of gelatin on PDLLA backbone was confirmed by 1H NMR and FTIR. The

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synthesized star shaped poly(D,L-Lactide)-b-Gelatin (ss-pLG) exhibited enhanced wettability

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and protein adsorption. The modification also facilitated better cell adhesion and proliferation

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on their respective polymer coated 2D substrates, compared to their respective unmodified ss-

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PDLLA. Further, 3D scaffolds were fabricated from gelatin grafted and unmodified

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polymers. The fabricated scaffolds were shown to be cytocompatible to 3T3-L1 cells and

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heamocompatible to Red Blood Cells (RBCs). Cell proliferation was increased upto 2.5 folds

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in ss-pLG scaffolds compared to ss-PDLLA scaffolds. Furthermore, a significant increase in

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cell number reveals, high degree of infiltration of cells into the scaffolds, forming a viable

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and healthy 3D interconnected cell community. In addition to that, burst release of docetaxal

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(DTX) was observed from ss-pLG scaffolds. Hence, this new system of grafting polymers

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followed by fabricating 3D scaffolds could be utilized as a successful approach in variety of

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applications where cells containing depots are used.

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Key words

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Ring opening polymerization, Carbodimide Chemistry, Poly (D,L-Lactide), Gelatin, 3D

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scaffolds, Cell Depots

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INTRODUCTION

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The ultimate goal of an artificial 3D scaffold is to mimic the natural extracellular

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environment of cells for successful tissue regeneration. Over the past few years, researchers

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have been strongly focusing on studies related to cell-material interactions to understand the

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cellular functions and behaviour in response to the materials1. Such materials initially serve

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as a physical support for the cells to facilitate adhesion followed by manifestation of

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subsequent cellular functions, including proliferation, migration and differentiation

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Plethora of literatures is available on biodegradable polymers with an increasing research

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effort particularly in the last ten years. Yet there has been no corresponding rise in their final

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commercial end products useful for tissue regeneration4 indicating, cell-material interface

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science needs more attention towards developing such novel biomaterials in a better and

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efficient way. Therefore, cell − material interface science needs more attention towards

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developing novel biomaterials that meets the industrial demands. Therefore, designing a

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scaffold to control the cell-material interactions could be highly important for the successful

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development of 3D matrix for tissue engineering applications.

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Synthetic biodegradable polymer poly(lactic acid) (PLA) is widely used to fabricate 3D

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scaffolds for tissue regeneration due to their biodegradable nature, good mechanical

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properties, and importantly their FDA approval for its clinical use. 4 arm star shaped PLA

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representing one of the branched structures of PLA,4 exhibits various important properties of

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tissue engineering scaffolds such as chemical multifunctionality 5, low viscosity6 and higher

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degradation rate7, 8 compared to their linear counterpart. However, the hydrophobic nature of

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PLA surface does not promote cell adhesion thereby hampering its applications in tissue

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engineering9. Initial response of the cells to the scaffold is dependent on the surface

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properties since material’s surface is the first site of cellular contact. Thus, the ability of

2, 3

.

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supporting cell adhesion is the prerequisite for any material to serve as the tissue engineering

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scaffold. In a natural environment, cell-membrane interaction occurs through integrin binding

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sequences of ECM proteins, including RGD, YIGSR, REDV, etc10. Therefore, immobilizing

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adhesive ECM proteins such as fibronectin, laminin and their functional putative peptides

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(Arg-Gly-Asp (RGD) and Tyr-Ile-GlySer-Arg (YIGSR)) as well as other biocompatible

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molecules including gelatin and chitosan to the surface of materials is considered to be an

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innovative approach to control cell adhesion11, 12.

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Many surface-modification techniques have been reported to enhance the biocompatibility

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and cellular properties of PLA, that include polymerization grafting13, 14, ozone oxidization15,

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plasma modification16,

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aminolysis,13,

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modification onto PLA substrates has been reported by photografting, surface initiated Atom

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Transfer Radical Polymerization (ATRP) based chemical grafting and plasma treatment23-25.

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All such modifications are usually done after the fabrication of either films or scaffolds. To

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the best of our knowledge very few articles report the scaffold fabrication using copolymers

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of RGD / RGD mimicking peptides coupled biodegradable polymers such as PLA and PCLA

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(poly(3-caprolactone-co-lactide))26, 27. Here, we have assessed a hypothesis that 3D scaffolds

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fabricated from gelatin grafted 4 arm star shaped poly(D,L-Lactide) (ss-PDLLA) copolymer

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may support the cell growth and proliferation more appropriately, as explained in Figure 1.

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In particular, cells can migrate deep into the fabricated 3D scaffolds and form the 3D

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interconnected cell community; whereas such interconnected cell community is highly

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unlikely to be achieved through other surface modification techniques. As of now, no such

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study has been reported for controlling the cell adhesion by gelatin grafted on ss-PDLLA(

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star-shaped PDLLA-b-Gelatin (ss-pLG)).

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, physical entrapment of cell adhering molecules18, hydrolysis,19,

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, surface coating20 and layer-by-layer self-assembly21,

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. Notably, gelatin

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Figure 1 Schematic representation of cell proliferation within the surface treated scaffolds and the scaffolds fabricated from gelatin grafted polymers. In case of surface-treated 3D scaffolds, it is likely that distribution of modified entities will be largely non-uniform throughout the scaffold. In such scenarios, introduction of treating moieties is generally found hindered towards the core region of the scaffold. Whereas in case of 3D scaffolds fabricated from gelatin coupled polymers, gelatin is expected to be present in situ within the entire region of the scaffold uniformly since it is already grafted with the backbone of PDLLA. Thus, the fabricated scaffolds are thought to facilitate interconnected 3D cellular networks throughout the matrix.

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In the present study, tailoring the chemical properties of star shaped PDLLA (ss-PDLLA) by

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gelatin grafting and their ability of supporting cellular growth on the modified polymers is

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demonstrated. It is known that one can control the wettability of PLA by branching

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coupling it with some hydrophilic polymers such as poly(ethylene glycol)29, poly(N-

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vinylpyrrolidone)30, 31, gelatin12, 23, etc through chemical reactions. As a result, PLA can be

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tuned preferentially from super-hydrophobic to moderately hydrophilic. We used gelatin (a

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denatured protein of collagen that has the integrin binding receptor such as DGEA32), for

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modulating the wettability and cell adhesion behaviour of ss-PDLLA polymers. Therefore,

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and

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herein we report the synthesis and characterization of ss-PDLLA and ss-pLG, followed by

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fabricating them into 3D scaffolds by freeze drying technique. Considering the advantage of

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hydrophilicity after gelatin grafting to different arms of PDLLA, we propose that ss-pLG

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could be a versatile platform for the proliferation of cells in 3D environment with tuneable

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cellular behaviour and responses. In order to evaluate our hypothesis and to use our ss-pLG

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scaffolds for tissue engineering applications, we cultured 3T3-L1 cells on ss-pLG polymeric

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scaffold; unmodified ss-PDLLA scaffold was used for the comparative study. We observed

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that ss-pLG polymeric scaffolds prominently modulated the cell adhesion property.

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Therefore, such tuning of cellular compatibility properties of polymers synthesized by simple

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chemical reactions of gelatin grafting will certainly be useful for 3D culturing of cells,

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especially required in various tissue engineering applications.

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RESULTS AND DISCUSSION

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Synthesis and characterization of gelatin grafted copolymer (ss-PDLLA-b-Gelatin)

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Methods for the synthesis of ss-PDLLA followed by ss-pLG are shown in Scheme 1, where

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we used combined ring opening polymerization (ROP) followed by end functionalization of

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gelatin by carbodimide chemistry. Initially, 4 arm ss-PDLLA was synthesized via our earlier

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reported method,31 where ROP of D,L-Lactide was done in bulk at 150ᵒC using

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pentaerythritol as initiator and Sn(Oct)2 as catalyst with the molar feed ratio of D,L-Lactide

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to pentaerythritol at 693.3. The 1H NMR confirms its formation; Mn (NMR) and conversion

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(NMR) was found to be 1,86,300 g mol-1 and 97.2% respectively (Figure S1). Mn (GPC) and

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Mn/Mw of the synthesized polymer calibrated against PMMA standard was 3,79,000 g mol-1

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and 1.9 respectively (Table 1). To graft the gelatin into ss-PDLLA arm, carboxyl terminated

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PDLLA was first synthesized by ring opening of succinic anhydride using ss-PDLLA-OH.

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The appearance of new peaks at 3.6 (g+h) comes from the methylene protons of succinic 6

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anhydride and the disappearance of end terminal methine proton of ss-PDLLA at 4.36

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confirms successful ring opening reaction (Figure 2A & Figure S1). The end terminal –

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COOH group was activated by EDC.HCl and NHS followed by coupling with gelatin was

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done at unit molar feed ratio of 1 (PDLLA-COOH to Gelatin). The chemical coupling was

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confirmed by 1H NMR (Figure S1), where apart from the characteristic peaks of gelatin,

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characteristic peaks of ss-PDLLA at 5.1-5.2 and 1.46 ppm corresponding to the backbone

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methine and pendent methyl protons, respectively, are present (Figure 2A & Figure S1).

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Detailed description about synthesis and conversion is given in supporting information.

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Scheme 1. Synthesis of gelatin grafted star shaped PDLLA by ROP using pentaerythritol as initiator and carbodimide mediated end functionalization methods

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Gelatin grafting to ss-PDLLA backbone was further analysed by FTIR spectroscopic

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technique. Figure 2B shows the respective FTIR spectra of ss-PDLLA-COOH, gelatin and 7

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cm-1 which

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ss-pLG. ss-PDLLA showed its characteristic peaks at 2998, 2939 and 1765

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corresponds to asymmetric, symmetric stretching of CH2 and carbonyl stretching,

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respectively33. (Figure S2). When ss-PDLLA-OH opened succinic anhydride ring,

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characteristic shifting was found from 1765 to 1760 cm-1. Pure gelatin showed the

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characteristic peaks at 1537 (amide II), 1661(amide I) and 3462 cm-1 for its distinct C=O

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bond vibrations and coupling of bending of N–H bond and stretching of C–N bonds,

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respectively

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peaks shifted to lower frequencies at 3284 and 1627 cm-1. This observation suggests that –

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NH2 group of gelatin has successfully coupled with the -COOH group of ss-PDLLA. ss-pLG

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also showed the characteristic peaks of ss-PDLLA at 2916, 2851 and 1743 cm-1 which further

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supports that ss-PDLLA backbone is present in ss-pLG polymer, whereas the intensity of

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peak at 1743 cm-1 corresponding to the carbonyl stretch of ss-PDLLA backbone was weak. It

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might be due to the higher content of gelatin or wrapping of ss-PDLLA by gelatin in the ss-

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pLG

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XPS confirmed the presence of gelatin in the ss-pLG polymer as the large increase in

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intensity of nitrogen and the shift in the binding energy, whereas ss-PDLLA did not show any

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peak respective for the nitrogen in XPS spectrum (Data not shown) (Figure 2C).26 Wide XPS

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spectrum of ss-pLG scaffolds were presented in Figure S3.

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Table 1 GPC Characteristic data star shaped PDLLA

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. After coupling of gelatin into ss-PDLLA back bone, these amide stretching

Sample

Conversion (%)a

Mn(NMR)b

Mn(GPC)c

Mn/Mw

ss-PDLLA

97.2

1,86,300

379000

1.9

a

Conversion was determined by comparing the peak area of the residual poly(D,L-Lactide) ~ 5.1-5.2

and monomer D,L-Lactide ~ 5.3 ppm using 1H NMR

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b

Determined from 1H NMR by comparing the methylene proton of pentaerythritol and methine proton

of ss-PDLLA at ~ 4.2 and ~ 5.1-5.2 ppm for ss-PDLLA c

Determined by GPC (DMF, 0.5 mL/min, 40 ºC) calibrated against PMMA standards

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Figure 2. Characterization of polymers by 1H NMR, FTIR, XPS, TGA and DSC: (A) 1H NMR spectra of star shaped polymers namely ss-PDLLA-COOH, Gelatin, ss-pLG. (B) FTIR spectra of the respective series of star shaped polymers. (C) XPS spectrum of ss-pLG. (D) TGA and (E) DSC of series of star shaped polymers namely gelatin, ss-PDLLA and ss-pLG.

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The thermogravimetric analysis (TGA) of ss-PDLLA, Gelatin, ss-pLG are presented in

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Figure 2D. Thermal degradation profile of gelatin modified and unmodified polymers were

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quite different. It can be seen that unmodified polymers ss-PDLLA and gelatin showed Tonset

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at 241 and 285 ᵒC whereas Tmax of the polymers were found to be 300 and 457 ᵒC

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respectively. On the other hand, the modified copolymer ss-pLG showed improved thermal

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properties than their original unmodified polymers. Tmax of ss-pLG was found to be 455 ᵒC

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which almost equivalent to the gelatin (Figure 2D). This thermal behaviour may be attributed

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due to the strong coupling of gelatin onto the ss-PDLLA back bone. Moreover, ss-pLG show

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two different distinct degradation peaks owing to the PDLLA and gelatin block. From this

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observation, it is clear that the modified ss-pLG has better thermal stability than their

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unmodified ss-PDLLA.

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DSC thermograms of ss-PDLLA, gelatin and modified ss-pLG are shown in Figure 2E. In

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second heating run, Tgs of ss-PDLLA, gelatin, and ss-pLG were observed at 30.36, 142.9, and

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112.59 ᵒC, respectively. None of the star shaped evaluated polymers showed melting

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endotherms or crystallization exotherms which suggests that the ss-PDLLA or gelatin

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coupled ss-PDLLA (ss-pLG) are highly amorphous in nature. Moreover, because of the

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grafting of gelatin into PDLLA, clear shifting of Tg was observed from 30.36 to 112.59 ᵒC for

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ss-pLG. The observed higher shifting in ss-pLG clearly indicates the presence of higher

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amount of gelatin in the PDLLA arm (ss-pLG).

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Influence of gelatin grafting on protein adsorption and cell adhesion 11

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Gelatin is a natural protein that possesses inherent integrin binding sites and known for the

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cell adhesion properties. Therefore, we demonstrate this property of gelatin by chemically

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grafting it to star PDLLA (ss-PDLLA) backbone. Before cell culturing, effects of gelatin on

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different polymer arms were examined for the protein adsorption efficiency. 12 % polymer

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films were made from ss-PDLLA and ss-pLG polymers, and further evaluated for their

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protein adsorption property. Both the films were incubated in BSA after they were wet in

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PBS completely. ss-pLG film showed higher adsorption, which was 2.6 folds greater owing

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to its hydrophilicity compared to ss-PDLLA (Figure 3A). It is important to note that ss-

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PDLLA also exhibits noticeable adsorption of BSA, most likely due to its hydrophilic

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property compared to linear PDLLA (data not shown)

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indicate that gelatin modified PDLLA (ss-pLG) facilitate higher degree of protein adsorption

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which is a crucial parameter required during cell culturing process.

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In order to assess the effects of cell−material interactions as well as cell-cell interactions,

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3T3-L1 cells were cultured on three different substrates (gelatine, ss-PDLLA and ss-pLG) at

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low seeding densities. Typically, the focal adhesions contain transmembrane proteins i.e.

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integrin αvβ3 as well as plaque proteins (paxillin, vinculin etc.), which connect the cells to

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underlying substrates. Hence, determination of focal contacts can be used as an index of cell

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adhesion strength. Vinculin is a cytoskeletal protein associated with cell-cell and cell-matrix

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junctions. It is involved in the anchoring of filamentous actin (F-actin) to the membrane

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adhesion molecules. To evaluate the cell adhesions associated with normal physiological

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proteins, we chose vinculin as a model protein that was costained with actin filament and cell

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nuclei. . Post 24 h seeding, 3T3-L1 cells cultured on polymer coated cover glass were

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observed to be speroidal in shape and did not exhibit a proper shape of well-spread healthy

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cells. Also, actin staining revealed the intense, mesh-like red stains extended from the cell

28, 35

(Figure 3B). These results

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perimeter; indicating the compromised status of the cells (Figure 3C). The intensified

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vinculin expression at the edge of a cell indicated the presence of strong integrated adhesive

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sites in case of gelatin (control) and gelatin coupled PDLLA (ss-pLG) coated cover glass.

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Gelatin modified ss-pLG were found more effective than the unmodified polymer in terms of

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establishing focal contacts, since the vinculin expression was clearly seen in almost all the

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cells and also the cell shape were comparable with the gelatin coated cover glass. In contrast,

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very weak fluorescence was observed on unmodified ss-PDLLA (Figure 3C).

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Figure 3. Demonstration of cell adhesion behaviour on polymer coated 2D cover glass; (A) BSA protein adsorption on to the 2D polymer film after 4 h of incubation. Protein estimation was done by using micro BCA kit. Experiments were done in triplicate and results are expressed as mean ±SD (n=3). *p