Surface grafted glycopolymer brushes to enhance selective adhesion of HepG2 cells

https://doi.org/10.1016/j.jcis.2013.04.025Get rights and content

Highlights

  • Thickness of the initiator layer affects the thickness of poly(LAMA) brush.

  • Increase in polymer thickness achieved via second initiation of hydroxyl groups.

  • HepG2 cells on poly(LAMA) brush adapts extended structures.

  • High protein content along the cell periphery of HepG2 on poly(LAMA).

Abstract

This work demonstrates the application of carbohydrate based methacrylate polymer brush, poly(2-lactobionamidoethyl methacrylate), for the purpose of cell adhesion studies. The first part of the work illustrates the effects of the structure of the aminosilane based ATRP initiator layer on the polymerization kinetics of 2-lactobionamidoethyl methacrylate) (LAMA) monomer on thermally oxidized silicon wafer. Both monolayer and multilayered aminosilane precursor layers have been prepared followed by reaction with 2-bromoisobutyrylbromide to form the ATRP initiator layer. It is inferred from the kinetic studies that the rate of termination is low on a multilayered initiator layer compared to a disordered monolayer structure. However both initiator types results in similar graft densities. Furthermore, it is shown that thick comb-like poly(LAMA) brushes can be constructed by initiating a second ATRP process on a previously formed poly(LAMA) brushes. The morphology of human hepatocellular carcinoma cancer cells (HepG2) on the comb-like poly(LAMA) brush layer has been studied. The fluorescent images of the HepG2 cells on the glycopolymer brush surface display distinct protrusions that extend outside of the cell periphery. On the other hand the cells on bare glass substrate display spheroid morphology. Further analysis using ToF-SIMS imaging shows that the HepG2 cells on glycopolymer surfaces is enriched with protein fragment along the cell periphery which is absent in the case of cells on bare glass substrate. It is suggested that the interaction of the galactose units of the polymer brush with the asialoglycoprotein receptor (ASGPR) of HepG2 cells has resulted in the protein enrichment along the cell periphery.

Introduction

Methacrylate polymers containing carbohydrate pendant group represents an important class of macromolecules owing to their numerous biomedical applications such as polymeric drugs [1], [2], targeted drug delivery [3], [4], development of the membranes for specific protein binding [5], cell recognition [6], [7]. Often, the synthesis of surface grafted glycopolymers involves atom transfer radical polymerization (ATRP) of methacrylate derivative of protected carbohydrate moiety – the hydroxyl groups of the carbohydrate unit are protected as an isopropylidene derivative – on a suitable initiator layer followed by deprotection in acidic solution to convert back to free hydroxyl groups [8], [9], [10]. Both linear and hyperbranched surface-grafted glycopolymers [9] have been synthesized using this two-step procedure. In a recent work, Narain et al. has demonstrated that the glycomonomers such as 2-gluconamidoethyl methacrylate (GAMA) [11] and 2-lactobionamidoethyl methacrylate (LAMA) [12] can be polymerized without the necessity to protect the carbohydrate moieties. Due to the hydrophilic nature of these monomers, the polymerization has to be conducted in polar media such as water or its mixtures with methanol and N-methyl-2-pyrrolidone. The surface-initiated ATRP (SI-ATRP) of LAMA monomer on self-assembled monolayers (SAM) of thiol and disulfide based ATRP initiators on gold surfaces resulted in the thickness range of 12–40 nm [13], [14]. Such glycosurfaces were hydrophilic having contact angle values between 25° and 35°.

Adhesion of eukaryotic cells to various biomaterial interfaces is a ubiquitous phenomenon which is both a sought after and a deterred process. For instance, in the case of medical implants it is necessary to prevent protein adsorption and cell adhesion in order to avoid surface-induced thrombosis [15]. On the other hand, facilitation of cell adhesion to scaffolds [16] or to conjugated drug molecule is vital in tissue engineering and drug delivery systems [17] respectively. Thus, tailoring the interfacial properties to enhance or prevent cell adhesion is an important realm within biomaterial design and it is in this context that the surface grafted polymer brush layers are shown to be effective in modulating the cell adhesive behavior [18]. A plethora of approaches for tailoring of surface properties exist in which the cell–substrate interaction can be optimized. The physical characteristics of the material can be altered such that the cell adhesion is either increased or deprived. Such characteristics can involve the wettability [19] or the topographical design [20] of the surface. The magnitude of cells attaching can partially be controlled by the above mentioned physical properties of a substrate, however, the specific cell binding and recognition is more effectively controlled by the chemical characteristics by mimicking the extracellular matrix (ECM) and is achieved through ligand immobilization. Several approaches have been extensively investigated such as polymer–peptide [21] and polymer–carbohydrate conjugates [22] also referred to as glycopolymers. The hepatic asialoglycoprotein receptor (ASGPR) located on the cell surface is a lectin recognizing carbohydrate moieties and is responsible for uptake through the endocytotic pathway. Poly(2-lactobionamidoethyl methacrylate) poly(LAMA) having pendant galactose residues is a glycopolymer which has been widely used for recognition and adhesion of primary hepatocytes [23].

In this work we have studied the effect of initiator layer structure on the formation and structure of poly(LAMA) brushes on thermally oxidized silicon wafers. It is shown that the thickness of the glycopolymer brush layer can be significantly increased through post modification of the initial poly(LAMA) layer. Furthermore, we investigate the adhesive interaction of human hepatocellular carcinoma cancer cells (HepG2) on the synthesized glycopolymer brush surfaces.

Section snippets

Materials and methods

All chemicals were purchased from Sigma–Aldrich unless otherwise stated. Dichloromethane (DCM, ⩾99.8%), acetonitrile (MeCN), methanol (99.9%), ethanol (99.9%), pyridine (⩾99%), toluene (99.9%), water (Milli-Q, 18 MΩ cm) were used as solvents. 2-Aminoethyl methacrylate hydrochloride (90%), lactobionic acid (97%), (3-aminopropyl)trimethoxysilane (1, 97%), CuBr (99.999%), CuBr2 (99.999%), 2,2′-bipyridyl ⩾99%) were used as received. 2-Bromo-2-methylpropanoyl bromide (BMPB, 98%) was distilled under

Monolayer of aminosilane

The structure of the aminosilane precursor layer (Si-1) plays a crucial role in determining the structure of the final polymer brush layer. The thickness of the chemisorbed aminosilane layer, prepared in toluene, is found to be dependent on the adsorption time for a fixed concentration of 1 (=1.2 mM). Near monolayer thickness (1.2 nm) was achieved by fixing the adsorption time to 3 h in 1.2 mM solution of 1. This time was chosen based on the adsorption kinetics of 1 on thermally oxidized silicon

Conclusions

The thickness of poly(2-lactobionamidoethyl methacrylate) polymer brush obtained by surface initiated atom transfer radical polymerization method is significantly affected by the structure of the aminopropyltrimethoxy silane initiator layer. The polymer brush layer obtained on a disordered multi-layered initiator layer exhibits high thickness compared to the polymer brush initiated on a near monolayer thickness. On further modification of the pendant hydroxyl groups of the initial poly(LAMA)

Acknowledgments

J.I., K.D. and S.C. acknowledge Nordic Innovation Centre and project TOPNANO program for financial support.

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