Pharmaceutical Nanotechnology
Development of glycyrrhetinic acid-modified stealth cationic liposomes for gene delivery

https://doi.org/10.1016/j.ijpharm.2010.06.029Get rights and content

Abstract

The glycyrrhetinic acid-modified stealth cationic liposomes (GA–PEG–CLs) loaded with pDNA (GA–PEG–CLPs) were developed and found to transfect human hepatocellular carcinoma cell line HepG2 with high efficiency. GA–PEG–CLs were comprised of DOTAP, cholesterol (Chol) and glycyrrhetinic acid–polyethyleneglycol–cholesterol conjugate (GA–PEG–Chol). Agarose gel electrophoresis revealed that 5% GA–PEG–CLs constituted by DOTAP/Chol/GA–PEG–Chol at molar ratio of 50:45:5 could completely entrap pDNA at a lower liposomes/pDNA weight ratios of 4:1 (N/P ratio: 1.14). Compared to ordinary cationic liposomes (CLs), steric cationic liposomes (PEG–CLs) and 1% GA–PEG–CLs made from DOTAP/Chol/MPEG2000-Chol/GA–PEG–Chol at molar ratio of 50:45:4:1, 5% GA–PEG–CLs were found to possess the highest transfection efficiency as gene vectors in serum-free or serum-containing medium in PKCα over-expressed HepG2 cells but no significance difference in human embryonic kidney cell line HEK 293. Additionally, 5% GA–PEG–CLs have the lowest cytotoxicity on human normal hepatocyte cell line L02. The competitive inhibition experiments mediated by GA were carried out in HepG2 cells, which demonstrated that GA–PEG–CLs could deliver selectively pDNA to hepatoma cells by the targeting moiety GA. In conclusion, GA–PEG–CLs containing 5% GA–PEG–Chol might be one of the most potential gene vectors as hepatoma targeting therapy.

Introduction

Cationic liposomes, one of the promising and the most representative non-viral gene delivery vectors, have many advantages over viral ones such as less-immunogenicity, non-oncogenicity, large DNA capacity, non-restriction on the size of the DNA molecule, ability of evading random integration of vector DNA into host chromosome (avoiding any insertional mutagenesis) and large-scale production (Templeton, 2003, Lv et al., 2006, Karmali and Chaudhuri, 2007). Cationic liposomes could interact with negatively charged plasmids via electrostatic attractions at the physiological pH to form positively charged lipid–DNA complexes (lipoplexes) and get endocytosed by the negatively charged cell membrane (Karmali and Chaudhuri, 2007). Lipoplexes represent one kind of attractive alternative to viral vectors for cell transfection in vitro and in vivo but still suffer from relatively low efficiency (Duan et al., 2009) and certain toxicity. The non-specific electrostatic interactions between the lipoplexes and cells or tissues leaded to non-specific cell or tissue uptake, non-specific immune response (a toxic response), a shorter half-life in vivo (Lv et al., 2006, Karmali and Chaudhuri, 2007, Morille et al., 2008, Ditto et al., 2009). Therefore, many researchers focused on the development of novel, effective, long-circulating and targeted-delivery cationic liposomes.

According to literatures, cationic liposomes tagged with appropriate targeting ligands could function as an efficient targeted gene delivery system (Sudimack and Lee, 2000, Karmali and Chaudhuri, 2007, Morille et al., 2008, Yan and Qi, 2008, Pathak et al., 2009). The published ligands used to modify cationic liposomes included folate (Yan and Qi, 2008), galactosyl (Fumoto et al., 2003), integrins (Hood et al., 2002), sigma ligands (Mukherjee et al., 2005), estradiol (Reddy and Banerjee, 2005), transferrin (Penacho et al., 2008), TAT (MacKay et al., 2008), and so on. In this study, glycyrrhetinic acid (GA) was selected as the specific ligand of cationic liposomes. GA is one of the main compounds extracted from the root of Glycyrrhiza glabra L. (licorice) (Mao et al., 2007), which could inhibit liver carcinogenesis and cell proliferation of the human hepatocellular carcinoma (HCC) cell line HepG2 (Satomi et al., 2005). It has been proved that protein kinase C (PKC) α, the target binding sites of GA, expressed more highly in HCC cells than that in the adjacent non-tumor liver cells (O’Brian et al., 1990, Yang et al., 2003, Ying et al., 2008). Therefore, we hypothesized that GA-modified cationic liposomes could target selectively to HCC cells by the specific interaction of GA with PKCα over-expressed in HCC cells.

To increase the HCC cells-targeted efficiency in vivo probably by decreasing the clearance of reticuloendothelial system, poly(ethyleneglycol) (PEG) was selected as the linker between GA and cationic liposomes according to literatures (Green et al., 2007, Morille et al., 2009). PEG could dissimulate the positive charges by shielding cationic liposomes surface with hydrophilic chain, thereby weakening the toxic response caused by non-specific electrostatic interactions of lipoplexes with normal cells. Additionally, the introduction of PEG to targeted cationic liposomes would have many other advantages such as extending the half-life of lipoplexes by reducing opsonization and enhancing serum stabilization (Bombelli et al., 2007), avoiding the tendency of lipoplexes to form large aggregates (de Lima et al., 2001), improving their biocompatibility with biological fluids (Sudimack and Lee, 2000) and achieving the higher targeting efficiency (Green et al., 2007, Morille et al., 2009).

To our knowledge, no gene vectors modified with GA have been published so far. Thus, we aimed to develop GA-modified stealth cationic liposomes (GA–PEG–CLs) for gene delivery. In this paper, cholesterol (Chol) and PEG2000 were covalently linked to form PEG–Chol, and then GA was covalently conjugated to the distal end of PEG–Chol to produce GA–PEG–Chol. GA–PEG–CLs were made from GA–PEG–Chol, cholesterol and 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP). The toxicity of GA–PEG–CLs as gene vector was investigated. Additionally, in vitro cell transfection efficiency of gene loaded GA–PEG–CLs (GA–PEG–CLPs) was assessed on HCC HepG2 cell line and human embryonic kidney cell line HEK 293.

Section snippets

Materials

1,2-Dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Cholesterol (Chol) was obtained from Shanghai Bio Life Science & Technology Co. Ltd. (Shanghai, China). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and dimethylaminopyridine (DMAP) were purchased from Sigma. Green fluorescent protein plasmid DNA (pDNA) was kindly donated by Dr. Hong Xin Deng (Sichuan University). All reagents and solvents were

Synthesis of GA–PEG–Chol conjugates

Fig. 1 shows the 1H NMR spectra of GA–PEG–Chol, mGA-suc and PEG–Chol in CDCl3. The single peaks at δ 5.69 and 5.60 (a) were attributed to the protons of olefinic bond (–(Cdouble bondO)–CHdouble bondC–) in GA. The single peak at δ 5.30 (b) was attributed to the protons of olefinic bond (–CH2–CHdouble bondC–) in cholesterol. The peaks at δ 2.58–2.66 (c) came from the protons of succinate linkage (–(Cdouble bondO)–CH2–CH2–(Cdouble bondO)–). The peaks at δ 3.52–3.76 (d) were attributed to the protons from the glycol unit (–O-CH2–CH2–O–) in PEG chain.

Conclusion

As a novel hepatoma targeting gene vector, GA–PEG–CLs could entrap pDNA with high efficiency and enhance the uptake of pDNA in hepatocellular carcinoma cells and then achieve high transfection efficiency. The gene entrapment and transfection efficiencies of GA–PEG–CLs were dependent on the amount of GA ligand and had a positive relationship. 5% GA–PEG–CLs, comprising DOTAP/Chol/GA–PEG–Chol at molar ratio of 50:45:5, had higher gene transfection efficiency and lower cytotoxicity of normal

Acknowledgements

This research has received financial support from the National Natural Science Foundation of China (Nos. 30901868 and 30772668), National 863 Project (No. 2007AA021810) and the Doctoral Program of Higher Education of China (No. 20090181120114).

References (30)

  • C. Bombelli et al.

    PEGylated lipoplexes: preparation protocols affecting DNA condensation and cell transfection efficiency

    J. Med. Chem.

    (2007)
  • M.C.P. de Lima et al.

    Cationic lipid-DNA complexes in gene delivery: from biophysics to biological applications

    Adv. Drug Deliv. Rev.

    (2001)
  • A.J. Ditto et al.

    Non-viral gene delivery using nanoparticles

    Expert Opin. Drug Deliv.

    (2009)
  • Y. Duan et al.

    The biological routes of gene delivery mediated by lipid-based non-viral vectors

    Expert Opin. Drug Deliv.

    (2009)
  • S. Fumoto et al.

    Enhanced hepatocyte-selective in vivo gene expression by stabilized galactosylated liposome/plasmid DNA complex using sodium chloride for complex formation

    Mol. Ther.

    (2003)
  • Cited by (101)

    • Advanced drug delivery systems in hepatocellular carcinoma

      2021, Advanced Drug Delivery Systems in the Management of Cancer
    • Enhanced targeted delivery of adenine to hepatocellular carcinoma using glycyrrhetinic acid-functionalized nanoparticles in vivo and in vitro

      2020, Biomedicine and Pharmacotherapy
      Citation Excerpt :

      Such systems may prolong drug blood circulation by evading clearance by the reticulo-endothelial system and specifically deliver drugs to liver cells via receptor-mediated endocytosis-based ligand-receptor specific binding [18]. Glycyrrhetinic acid (GA) is a pentacyclic triterpenoid glycoside obtained from the roots of Glycyrrhiza glabra L [21], exhibits high levels of specific binding on rat and human hepatocyte membranes at protein kinase Cα, which is expressed more highly in HCC cells than in adjacent non-tumor liver cells [22,23]. In addition, abundant GA receptors have been identified on the cell membrane of hepatocytes, GA receptors also showed 1.5- to 5-fold higher expression in tumor tissue than the normal [24].

    View all citing articles on Scopus
    1

    These authors contributed equally.

    View full text