A biodegradable triblock copolymer poly(ethylene glycol)-b-poly(l-lactide)-b-poly(l-lysine): Synthesis, self-assembly, and RGD peptide modification

Abstract

A novel biodegradable triblock copolymer poly(ethylene glycol)-b-poly(l-lactide)-b-poly(l-lysine) (PEG–PLA–PLL) was synthesized by acidolysis of poly(ethylene glycol)-b-poly(l-lactide)-b-poly(ɛ-benzyloxycarbonyl-l-lysine) (PEG–PLA–PZLL) obtained by the ring-opening polymerization (ROP) of ɛ-benzyloxycarbonyl-l-lysine N-carboxyanhydride (ZLys NCA) with amino-terminated PEG–PLA–NH₂ as a macroinitiator, and the pendant amino groups of the lysine residues were modified with a peptide known to modulate cellular functions, Gly-Arg-Gly-Asp-Ser-Tyr (GRGDSY, abbreviated as RGD) in the presence of 1,1′-carbonyldiimidazole (CDI). The structures of PEG–PLA–PLL/RGD and its precursors were confirmed by ¹H NMR, FT-IR, amino acid analysis and XPS analysis. The cell adhesion and cell spread on the PEG–PLA–PLL/RGD film were enhanced compared to those on pure PLA film. Therefore, the novel RGD-grafted triblock copolymer is promising for cell or tissue engineering applications. Both copolymers PEG–PLA–PZLL and PEG–PLA–PLL showed an amphiphilic nature and could self-assemble into micelles of homogeneous spherical morphology. The micelles were determined by fluorescence technique, dynamic light scattering (DLS), and field emission scanning electron microscopy (ESEM) and could be expected to find application in drug and gene delivery systems.

Introduction

In the past three decades biodegradable polymers have become more and more important for pharmaceutical and biomedical fields [1], [2]. Among them, polylactide (PLA), a very important synthetic biodegradable material, has been widely used in surgical repair, carriers in drug delivery, and temporary matrixes or scaffolds in tissue engineering [3], [4], [5] due to its biodegradability, biocompatibility, high mechanical properties, and excellent shaping and molding properties. However, an important problem is inadequate interaction between the polymers and cells, leading to in vivo foreign body reactions, such as inflammation, infections, local tissue necrosis, and implant encapsulation as well as thrombosis [6], [7], [8]. Moreover, due to lack of functional groups, they cannot be modified easily with biologically active moieties.

Recently, many investigations have attempted to improve the hydrophilicity of polyester and provide functional groups for polyesters. Poly(ethylene glycol) is often introduced for its hydrophilicity, nontoxcity, biocompatibility and nonimmunogenicity. Polypeptides are very important biological macromolecules and suitable for biomedical applications such as sutures, artificial tissues, implants and drug delivery [9], [10], [11], [12] because of their excellent physical properties, biocompatibility and biodegradability. Among all polypeptides, poly(l-lysine) (PLL) is unique due to its hydrosolubility and functional side NH₂ groups which can help to improve its affinity to proteins and cells, or to covalently or ionically combine with drugs, antibodies or DNAs, and thus may lead to breakthrough in the fields of targeting drug delivery and gene delivery [13], [14]. Therefore, chemical modifications of aliphatic polyesters are realized by preparing hyperbranched polyesters [15], [16], [17], star block copolymer [18], and preparing co-polyesters [19], such as PEG–polyester, polyester–poly(amino acid) [20], [21], [22].

Advances in tissue engineering require biofunctional scaffolds that can provide not only physical support for cells, but also chemical and biological cues needed in forming functional tissues. Since Pierschbacher and Ruoslahti found that the tripeptide arginine-glycine-aspatic acid (Arg-Gly-Asp or RGD) was the minimal cell-recognizable sequence in many extracellular matrix protein and blood protein [23], RGD-containing peptides have been incorporated into synthetic nondegradable polymers [24], [25], [26], [27], [28], [29], [30], [31]. However, degradability may be very important so that implanted cells can eventually obtain a completely natural environment, thereby eliminating the possibility of long-term detrimental tissue responses [32]. Barrera et al. have synthesized biodegradable copolymer poly(l-lactic acid-co-l-lysine) and attached the peptide sequence GRGDY to the lysine residue in the copolymer [32]. But the yield of 3-(ɛ-benzyloxycarbonyl-l-lysine)–6-l-methyl-2,5-morpholinedione used to synthesize poly(l-lactic acid-co-l-lysine) is low and only 2% lysine can be incorporated into the copolymer [33]. Yamaoka et al. have prepared biodegradable malic acid-containing functional polymers poly(l-lactic acid-co-malic acid-co-glycolic acid) [34], however, the situation of low yield of monomer and low content of malic acid still exists. Recently, we have obtained a new biodegradable triblock copolymer, poly(ethylene glycol)-b-poly(l-lactide)-b-poly(l-glutamic acid), containing side carboxyl groups and modified it with RGD peptide [35], [36].

In this paper, a novel structure of poly(ethylene glycol)-b-poly(l-lactide)-b-poly(l-lysine) (PEG-b-PLA-b-PLL) triblock copolymer containing side amino groups was synthesized with PEG-b-PLA-NH₂ diblock copolymer as a macroinitiator for the ring-opening polymerization (ROP) of NCA by a convenient way. The pendent amino groups of the lysine residues were modified with an RGD peptide in the presence of 1,1′-carbonyldiimidazole (CDI). The triblock copolymer combined the characters of polyester, PEG and poly(amino acid). Number of the grafted RGD peptide could be adjusted either by changing the length of PLL block or by changing the grafting rate of RGD. Therefore, this polymer is expected to have enhanced cell adhesion and can serve as biodegradable scaffold for cell and tissue engineering. Recently, much interest has been concentrated on the self-assembly behavior of the block copolymers containing polypeptide and hydrophilic block [37], [38], [39], [40], [41], [42], [43]. Here, the micelles behavior of triblock copolymer PEG–PLA–PZLL with PEG as hydrophilic block and PLA and PZLL as hydrophobic blocks, and PEG–PLA–PLL with PEG and PLL as hydrophilic blocks and PLA as hydrophobic block was investigated by fluorescence technique, DLS, and ESEM. Such micelles are potential biomedical materials and can be applied in carrier systems in drug and gene delivery.