Dual-functionalized graphene oxide for enhanced siRNA delivery to breast cancer cells

doi:10.1016/j.colsurfb.2016.08.015

Colloids and Surfaces B: Biointerfaces

Volume 147, 1 November 2016, Pages 315-325

https://doi.org/10.1016/j.colsurfb.2016.08.015 Get rights and content

Highlights

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Novel nano-carrier formulation based on reduced graphene oxide (rGO) is synthesized.
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Endosomal escape of nano-carrier is modified by R8 incorporation to deliver siRNA.
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The colloidal stability of rGO nanocarrier is significantly improved using PL-PEG.
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A superior internalization efficiency is observed compared to commercial agents.

Abstract

The aim of this study is to improve hydrocolloid stability and siRNA transfection ability of a reduced graphene oxide (rGO) based nano-carrier using a phospholipid-based amphiphilic polymer (PL-PEG) and cell penetrating peptide (CPPs). The dual functionalized nano-carrier is comprehensively characterized for its chemical structure, size, surface charge and morphology as well as thermal stability. The nano-carrier cytocompatibility, siRNA condensation ability both in the presence and absence of enzyme, endosomal buffering capacity, cellular uptake and intracellular localization are also assessed. The siRNA loaded nano-carrier is used for internalization to MCF-7 cells and its gene silencing ability is compared with AllStars Hs Cell Death siRNA as a model gene. The nano-carrier remains stable in biological solution, exhibits excellent cytocompatibility, retards the siRNA migration and protects it against enzyme degradation. The buffering capacity analysis shows that incorporation of the peptide in nano-carrier structure would increase the resistance to endo/lysosomal like acidic condition (pH 6–4) The functionalized nano-carrier which is loaded with siRNA in an optimal N:P ratio presents superior internalization efficiency (82 ± 5.1% compared to HiPerFect^®), endosomal escape quality and capable of inducing cell death in MCF-7 cancer cells (51 ± 3.1% compared to non-treated cells). The success of siRNA-based therapy is largely dependent on the safe and efficient delivery system, therefore; the dual functionalized rGO introduced here could have a great potential to be used as a carrier for siRNA delivery with relevancy in therapeutics and clinical applications.

Graphical abstract

Introduction

Gene therapy is a powerful and novel strategy that has a great potential to cure genetic based diseases particularly cancer [1]. Recently, ribonucleic acid interference (RNAi) has been attracted many attention as a promising candidate for the treatment of a variety of diseases that suffer from expression of undesired genes [2]. Small interfering RNA (siRNA) can suppress the expression of target pathogenic genes using degradation of the target messenger RNA (mRNA). However, duo to the limited delivery of siRNA through cell membrane, various nano-delivery systems have been developed to facilitate siRNA access to the cytoplasm of targeted cells [3], [4], [5]. A nano-carrier system is also essential to provide in vitro and in vivo biostability and to ease cellular uptake [6].

Graphene and its derivatives e.g. graphene oxide (GO) and reduced graphene oxide (rGO) have been considered as a biocompatible and conjugable nano-carriers for delivery of targeting compounds and biomolecules [7]. Lately, GO/rGO has been utilized for siRNA delivery and showed a great success in gene knockdown. Tripathi et al. covalently grafted polyethylenimine (PEI) to GO and evaluated delivery of GFP specific siRNA with 70% suppression of the target gene expression [8]. In another study, Cheng et al. have shown that a polyethylene glycol (PEG)-PEI-grafted graphene/Au composites could efficiently load anti-apoptosis Bcl-2 siRNA and successfully down-regulated the Bcl-2 expression in HL-60 cells [9]. Zhang et al. have used PEI for covalent functionalization of GO which was followed by loading of hTERT siRNA and targeting HeLa cells [10]. Feng et al. have reported synthesis of ultra-small size dual-polymer-functionalized GO (GO-PEG-PEI) and effective delivery of Polo-like kinase 1 (Plk1) siRNA using laser irradiation [11]. It is also demonstrated that GO-PEG-PEI is an excellent nano-carrier for delivery of Stat3 siRNA which could lead to significant regression in tumor growth [12]. Finally, Zhang et al. were investigated the sequential delivery of siRNA and anticancer drugs via PEI-functionalized GO [13].

Non-covalent functionalization of graphene with hydrophobic interactions or electrostatic is the most effective and non-destructive method as enables the modification of graphene without alteration of its chemical structure [14]. While much research have been devoted to covalent functionalization of graphene sheets using PEI or PEG for siRNA delivery, there has been little investigation of non-covalent functionalization of graphene as a siRNA nano-carrier using amphiphilic polymers.

We have previously synthesized GO, optimized the concentration of carboxylic acid on its surface [15], and successfully used it for plasmid delivery [16]. Here we introduced a novel reduced graphene oxide (rGO) based nano-carrier which was double functionalized by a phospholipid-based amphiphilic polymer and cell penetrating peptide (CPPs) for enhanced hydrocolloid stability and high siRNA transfection ability. After a comprehensive characterization, the positively charged nano-carrier was evaluated for delivery of siRNA into the MCF-7 breast cancer cells and the knockdown of the cell survival related genes.

Section snippets

Materials

All the reagents used for synthesis and functionalization of rGO nano-sheets were purchase from Sigma Aldrich, Canada except otherwise indicated specifically. The MCF-7 breast cancer cell line was purchased from American Type Cell Culture (ATCC, Manassas, USA). Fetal bovine serum (FBS) and Dulbecco’s modified eagle’s medium (DMEM) were purchased from Invitrogen (Montreal, CA). The MTS viability assay kit was obtained from Promega (Madison, USA). The FITC-labeled scrambled siRNA and AllStars Hs

FTIR spectroscopy

The chemical characteristic of as-prepared GON and rGON was confirmed by FT-IR spectroscopy (Fig. 1a). The base-treated GON showed multiple peaks around 1060, 1250, 1365, and 1720 cm⁻¹which are assigned to vibrational modes of C single bond O, COC, COH and C double bond O in carboxylic acid and carbonyl moieties [19]. After the reduction reaction, the peaks of the oxygenated groups e.g. carboxyl in rGON were disappeared and the intensity of peaks between 900 and 1500 cm⁻¹ decreased significantly [20]. In addition, the

Summary and conclusions

Gene therapy is based on the introduction of nucleic acids typically either siRNA or pDNA into targeted cells through gene knockdown and expression. However nucleic acids are highly unstable and have little cell penetration ability. In present study, an efficient gene nano-carrier is introduced based on reduced graphene oxide that co-functionalized with an amphiphilic polymer (PL-PEG) and R8 cell penetrating peptide. The rGON-PLPEG-R8 remained stable in biological solution and exhibited

Conflict of interest

There is no conflict of interest to declare.

Acknowledgments

The authors wish to acknowledge scientific help and guidance of Prof. Hojatollah Vali, McGill University, Montreal, Canada. SF also would like to thank partial financial support of this work by National Institute of Genetic Engineering and Biotechnology (940801-I-536).

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Dual-functionalized graphene oxide for enhanced siRNA delivery to breast cancer cells

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Materials

FTIR spectroscopy

Summary and conclusions

Conflict of interest

Acknowledgments

Curr. Opin. Biotechnol.

Carbon

Acta Biomater.

Carbon

Trends Biotechnol.

J. Controlled Release

J. Pharm. Sci.

Gene therapy clinical trials worldwide to 2012-an update

J. Gene Med.

Current prospects for RNA interference-based therapies

Nat. Rev. Genet.

Interfering with disease: a progress report on siRNA-based therapeutics

Nat. Rev. Drug Discov.

Delivery of siRNA therapeutics: barriers and carriers

AAPS J.

Polyethylenimine and chitosan carriers for the delivery of RNA interference effectors

Expert Opin. Drug Deliv.

Graphene: promises, facts, opportunities, and challenges in nanomedicine

Chem. Rev.

Highly dispersible PEGylated graphene/Au composites as gene delivery vector and potential cancer therapeutic agent

J. Mater. Chem. B