Tailoring the supramolecular structure of amphiphilic glycopolypeptide analogue toward liver targeted drug delivery systems

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Abstract

Amphiphilic glycopolypeptide analogues have harboured great importance in the development of targeted drug delivery systems. In this study, lactosylated pullulan-graft-arginine dendrons (LP-g-G3P) was synthesized using Huisgen azide-alkyne 1,3-dipolar cycloaddition between lactosylated pullulan and generation 3 arginine dendrons bearing Pbf and Boc groups on the periphery. Hydrophilic lactosylated pullulan was selected for amphiphilic modification, aiming at specific lectin recognition. Macromolecular structure of LP-g-G3P combined alkyl, aromatic, and peptide dendritic hydrophobic moieties and was able to self-assemble spontaneously into core-shell nanoarchitectures with small particle sizes and low polydispersity in the aqueous media, which was confirmed by CAC, DLS and TEM. Furthermore, the polyaromatic anticancer drug (doxorubicin, DOX) was selectively encapsulated in the hydrophobic core through multiple interactions with the dendrons, including π-π interactions, hydrogen bonding and hydrophobic interactions. Such multiple interactions had the merits of enhanced drug loading capacity (16.89 ± 2.41%), good stability against dilution, and excellent sustained release property. The cell viability assay presented that LP-g-G3P nanoparticles had an excellent biocompatibility both in the normal and tumor cells. Moreover, LP-g-G3P/DOX nanoparticles could be effectively internalized into the hepatoma carcinoma cells and dramatically inhibited cell proliferation. Thus, this approach paves the way to develop amphiphilic and biofunctional glycopolypeptide-based drug delivery systems.

Introduction

Biological organisms, such as cells, were generated from precise self-assembly of natural carbohydrates, nucleic acids, and amino acids (Carlini et al., 2016). The self-assembly of amphiphilic molecules in a selective solvent also gives a versatile way to produce highly organized, well-defined micellar or vesicular nanostructures (Börner, 2009, Qiu et al., 2015, Wang et al., 2012). Numerous approaches have been developed for the self-assembly of natural or synthetic amphiphiles into soft core-shell nanoobjects which are of great interest in the field of drug delivery systems, wherein the hydrophobic nature of the micellar core can provide a safe harbor for hydrophobic drugs and the hydrophilic shell protects the core from an aqueous environment (Rösler et al., 2012). Among these amphiphilic molecules, amphiphiles containing natural blocks, such as polysaccharides or polypeptides, have attracted the most attention because they are non-toxic, non-immunogenic, biocompatible, biodegradable, and renewable (Akhlaghi et al., 2013, Hassani et al., 2012). Nanostructures obtained from the self-assembly of natural blocks are expected to have great potential in the development of biomedical applications, in particular, as nanocarriers for the delivery of drugs, proteins and genes. For example, polysaccharides-based amphiphilic polymers are the subjects of many studies (Alvarez-Lorenzo et al., 2013, Hassani et al., 2012, Saravanakumar et al., 2012) with the purpose of delivering hydrophobic anticancer drugs.

Polysaccharides, such as chitosan, dextran, pullulan, hyaluronic acid, heparin, and alginic acid, have a wide range of molecular weight and a large number of reactive groups (–COOH, –NH2, –OH, etc.). Therefore, polysaccharides can be easily chemically modified to be various amphiphilic derivatives (Akhlaghi et al., 2013, Hassani et al., 2012, Liu et al., 2011). Current hydrophobic moieties involved in creating amphiphilic polysaccharides derivatives that undergo self-assembly in aqueous solutions include linear molecules (long-chain fatty acids (Nichifor et al., 2014, Pramod et al., 2012) and poly(ε-caprolactone) (Chandel et al., 2016, Gu et al., 2014)), cyclic molecules (cholesterol (Kawasaki et al., 2016, Takahashi et al., 2011), bile acids (Li et al., 2012), and doxorubicin (Cao et al., 2010, Xu et al., 2015)), polyacrylate family (Poly(methyl methacrylate) (Dupayage et al., 2011, Dupayage et al., 2008) and poly(isobutyl cyanoacrylate) (Bravo-Osuna et al., 2007, Wang et al., 2011)), etc. The stability and polarity of amphiphilic polysaccharides based nanostructures are closely related to structural characteristics of amphiphiles, such as hydrophobicity and intra-/intermolecular interactions (Aschenbrenner et al., 2013). A promising alternative strategy for the generation of amphiphilic derivatives is to study the use of dendritic molecules (Hassan et al., 2004, Percec et al., 2004), such as peptide dendritic molecules (Li et al., 2013), mainly due to the unique radiate dendritic structures, low polydispersity and the amplification effect, which can be harnessed for providing large void space within the hydrophobic inner core. Generally, dendrons involving well-defined molecular structures allow multivalency in a tunable way upon selecting the peripheral chemical groups and the generation properly (Majoinen et al., 2014, Roglin et al., 2011, Rosen et al., 2009). However, the number of hydrophobically modified polysaccharides based systems that have been thoroughly investigated is limited and among existing studies only a few contain information about combination use of different types of hydrophobic molecules. Therefore, it provides us ample opportunities to modify polysaccharides hydrophobically with fine-tuned properties for drug delivery.

Herein, we present a straightforward and versatile synthesis of a glycopolypeptide analogue, amphiphilic polysaccharide-based graft copolymer, in which hydrophobic polypeptide blocks are covalently attached to a hydrophilic polysaccharide block. Macromolecular structures of this type combine alkyl, aromatic, and peptide dendritic hydrophobic moieties and are able to self-assemble spontaneously into core-shell nanocarriers in water by the fine-tuning of intra- and intermolecular interactions. As a proof of concept, we illustrate the synthesis of lactosylated pullulan-graft-arginine dendrons (LP-g-G3P) based on the conjugation of lactosylated pullulan with generation 3 arginine dendrons bearing Pbf and Boc groups on the periphery through Huisgen azide-alkyne 1,3-dipolar cycloaddition or “click” reaction. In this study, a hydrophilic polysaccharide derivative, lactosylated pullulan, was selected for hydrophobic modification. Pullulan is a neutral linear polysaccharide composed of α-(1–6)-linked maltotriosyl repeating units. The attractive properties of both lactose and pullulan not only lie in their water-solubility, biocompatibility, and non-immunogenicity, but also the higher affinity toward liver due to the presence of lectin like receptors on liver cells (hepatocytes and Kupffer cells), which possess biological affinity for sugar residues (Kawasaki et al., 2016, Singh et al., 2015). Harnessing these merits of pullulan and lactose in constructing amphiphilic blocks could be of great benefit for the development of liver targeted drug delivery systems.

AcronymsAlkyne-G1(K)PAlkyne-generation 1 (G1) lysine dendron bearing protecting groupsAlkyne-G2(K)PAlkyne-generation 2 (G2) lysine dendron bearing protecting groupsAlkyne-G3(R)PAlkyne-generation 3 (G3) arginine dendron bearing protecting groupsAzido-PAzidated pullulanAzido-LPAzido-lactosylated pullulanLP-g-G3PLactosylated pullulan graft G3(R)P

Section snippets

Materials

2-Propynylamine was purchased from J&K Company. Boc-l-Arg(Pbf)-OH, Boc-l-Lys(Boc)-OH, N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole hydrate (HOBt) and O-Benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate (HBTU) were commercially available from GL Biochem Ltd (Shanghai, China), where Pbf denotes 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, Boc denotes tertiary-butyloxycarbonyl. Sodium azide (NaN3), triphenylphosphine (PPh3),

Synthesis and characterisation of LP-g-G3P

An amphiphilic glycopolypeptide analogue, lactosylated pullulan-graft-arginine dendrons (LP-g-G3P), was prepared using azide-alkyne click chemistry between hydrophilic lactosylated pullulan and hydrophobic generation 3 arginine dendrons bearing Pbf and Boc groups on the periphery as shown in Fig. 1. Click chemistry has been proven as a highly efficient and convenient approach to obtain amphiphilic polysaccharides. The synthesis of alkynated peptide dendrons included the conjugation of two

Conclusions

In summary, we have developed an original amphiphilic glycopolypeptide analogue, lactosylated pullulan-graft-arginine dendrons (LP-g-G3P), using azide-alkyne click chemistry between hydrophilic lactosylated pullulan and hydrophobic generation 3 arginine dendrons bearing Pbf and Boc groups on the periphery. The resulting amphiphile LP-g-G3P not only owned the hydrophobic blocks consisting of alkyl, aromatic and peptide dendritic moieties, but also possessed the hydrophilic lactosylated pullulan

Acknowledgements

This work was financially supported by National Natural Science Foundation of China (NSFC, 81361140343, 81621003 and 31500810), International Science and Technology Cooperation Program of China (2015DFE52780), and the European Commission Research and Innovation (PIRSES-GA-2011-295218). The authors are grateful to the Young Scholar Program of Sichuan University (2015SCU11038) and the Foundation for Talent Introduction from Sichuan University (YJ201464).

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