Elsevier

Biomaterials

Volume 31, Issue 14, May 2010, Pages 4194-4203
Biomaterials

Polycation/DNA complexes coated with oligonucleotides for gene delivery

https://doi.org/10.1016/j.biomaterials.2010.01.116Get rights and content

Abstract

Ternary nanoparticles with negatively charged surface were prepared by coating single-stranded oligonucleotides (5′–C10A20–3′) on histidine-conjugated polyallylamine (PAA-HIS)/DNA complexes for gene delivery. Characterization of PAA-HIS/DNA/oligonucleotide complexes demonstrated that nanoparticles possessed the negative surface charge −27 mV and size of around 100 nm when the molar ratio of oligonucleotide/PAA-HIS exceeded 1.5. The negatively charged oligonucleotide-coated PAA-HIS/DNA complexes could be entirely internalized by the living HeLa cells to exhibit high gene expression with low cytotoxicity and the resistance against erythrocyte agglutination and serum inhibition. Since the gene expression of PAA-HIS/DNA complexes was significantly inhibited by coating other polyanions and oligonucleotides, the ternary PAA-HIS/DNA/deoxyadenosine-rich oligonucleotide complexes were uptaken by specific receptor-mediated process. Additionally, the deposition of a layer of oligonucleotides onto the binary PAA-HIS/DNA complexes could effectively transfect various types of cells including HEK-293, HepG2 and Hs68 cells, indicating the technology of coating specific oligonucleotides on PAA-HIS/DNA complexes or other cationic binary DNA complexes might facilitate the use of nanoparticles for safe and efficient gene delivery and eventual therapy.

Introduction

Gene therapy offers a potential method to cure inherited or acquired diseases by transferring exogenous nucleic acids into cells to alter protein expression profiles. Although recombinant viruses are currently used for this purpose because of their high transfection activity, the use for nonviral vectors such as cationic polymers is still desired from the viewpoint of safety. A variety of polyamines have been widely investigated in DNA delivery such as polyethylenimine (PEI), poly(L-lysine) (PLL), poly (2-dimethyl amino ethyl) methacrylate (PDMAEMA) and so on [1], [2]. Polyallylamine (PAA), also a synthetic cationic polymer, possesses high density of primary amino groups which are suitable for binding and packaging negatively charged DNA. However, the utility of PAA for gene delivery application is limited by its cytotoxicity of too strong polycationic character [3]. Different chemical modifications have been used to decrease cytotoxicity and enhance transfection efficiency of PAA. For example, Boussif et al. showed glycolated PAA not only decreased the cytotoxicity of PAA/DNA complex but also increased the transfection efficiency of PAA/DNA complex [4]. Pathak et al. demonstrated that PAA modified with imidazole and poly ethylene glycol (PEG)-bis (phosphate) could reduce the positive surface charge to decrease cytotoxicity and to achieve enhanced transfection efficiency [3]. In addition, Nimesh et al. showed PAA complexed with dextran and DNA could simultaneously improve upon transfection efficiency and cell viability [5].

On the other hand, to achieve desired therapeutic goals, efficient delivery of a therapeutic gene to target cells is an important issue. Cationic polymers can be readily modified with antibodies, aptamers, peptides, or small molecules, which induce endocytosis upon binding to their cognate cell surface receptors to enhance cell selectivity and delivery [6], [7], [8], [9]. To this end, the technique DNA hybridization was used to prepare nanoparticles containing tumor-targeting antibodies in our laboratory. At first, oligonucleotides were coated onto the cationic polyplexes led to the surface incorporation of negatively charged single-stranded DNA. The cationic polymers used were PAA and histidine-conjugated PAA (PAA-HIS). Subsequently, we employed complementary oligonucleotides to conjugate with specific antibodies, and assembled them with oligonucleotide-coated polyplexes into multilayered nanoparticles by nucleic acid–base-paired interaction, which allowed for the surface conjugation of biomolecules to mediate internalization in a cell-specific manner. Generally, the uptake of polycation/DNA complexes is mediated by electrostatic interactions of positively charged complexes with negatively charged cellular surface, so the anionic oligonucleotide-coated polyplexes should show minimal cellular transfection if specific targeting ligands were not exposed on the multilayered nanoparticle surfaces. Accidentally, we discovered that the negatively charged oligonucleotide-coated PAA-HIS/DNA complexes could enter cells to exhibit high gene expression without inducing cytotoxicity and agglutination of erythrocytes even they did not conjugate any specific targeting molecules. Therefore, the purpose of this work was to study whether the technology of coating oligonucleotides on PAA-HIS/DNA complexes could facilitate the use of nanoparticles for safe and efficient gene delivery.

Section snippets

Synthesis of PAA-HIS

All chemicals were reagent grade and used as received unless noted otherwise. PAA-HIS was synthesized according to Fig. 1(a). Briefly, 62.6 mg/mL N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC) (Aldrich, St. Louis, MO) and 24.3 mg/mL N-hydroxysuccinimide (NHS) (Fluka, St. Louis, MO) were added to 53.7 mg/mL N-Boc histidine (Fluka, St. Louis, MO) in dimethyl sulfoxide (DMSO) (Aldrich, St. Louis, MO). Reaction was carried out under stirring for 30 min at room temperature.

The physicochemical properties of oligonucleotide-coated PAA-HIS/DNA complexes

The buffering capacity of gene delivery system has been demonstrated to improve the transfection efficiency during endocytosis, so histidine was conjugated with PAA to improve the buffering capacity of the polycation in this study [3], [12]. The chemical structure of imidazole-containing PAA-HIS was characterized by ATR-FTIR and 1H-NMR (Fig. 2). The ATR–FITR spectra shows the carbonyl group (Cdouble bondO) from amide was at 1645 cm−1. The characteristic peaks of imidazole at 1000–1200 cm−1 show the

Discussion

Generally, naked DNA or oligonucleotides is hardly transferred into cells because of enzymatic degradation and electrostatic repulsion. Thus, cationic polymers, such as PEI, and PLL have been employed to condense and protect DNA to efficient gene delivery [1], [2]. However, high density amino groups of cationic molecules are well known for their cytotoxicity to limit the application [4]. Previous reports have described various techniques to decrease cytotoxicity and enhance transfection

Conclusion

Negatively charged oligonucleotide (5′–C10A20–3′) was firstly used to prepare a ternary anionic gene delivery system with low cytotoxicity, high transfection efficiency and the resistance against erythrocyte agglutination and serum inhibition. The technique of assembling oligonucleotides with binary polycation/DNA complexes developed in this study could be applied to other delivery system, such as drug delivery system. Furthermore, the ternary polyplexes can be easily modified with specific

Acknowledgements

The authors thank National Science Council of the Republic of China and Industrial Technology Research Institute for their financial support of this research. We are also grateful to the staffs of TC5 Bio-Image Tools, Technology Commons, College of Life Science, NTU for help with the confocal laser scanning microscopy (CLSM).

References (24)

  • T. Niidome et al.

    Gene therapy progress and prospects: nonviral vectors

    Gene Ther

    (2002)
  • A. Pathak et al.

    Engineered polyallylamine nanoparticles for efficient in vitro transfection

    Pharm Res

    (2007)
  • Cited by (44)

    • Effects of nanoparticle-mediated Co-delivery of bFGF and VEGFA genes to deep burn wounds: An in vivo study

      2022, Colloids and Surfaces B: Biointerfaces
      Citation Excerpt :

      In recent years, molecular regulation has been the focus of attention, and many studies have reported success experimentally [59,60]. For example, the application of nanoparticles as carriers in the treatment of various diseases has become a popular strategy, which has the advantages of biodegradability and controlled release [26–28]. The size of nanoparticles is less than 100 nm, and nanoparticles can load nucleic acids and penetrate the cell membrane easily [61,62].

    • Advanced polymers for nonviral gene delivery

      2019, Nucleic Acid Nanotheranostics: Biomedical Applications
    View all citing articles on Scopus
    View full text