Elsevier

Biomaterials

Volume 31, Issue 7, March 2010, Pages 1830-1838
Biomaterials

Low molecular weight polyethylenimine cross-linked by 2-hydroxypropyl-γ-cyclodextrin coupled to peptide targeting HER2 as a gene delivery vector

https://doi.org/10.1016/j.biomaterials.2009.11.012Get rights and content

Abstract

Gene delivery is one of the critical steps for gene therapy. Non-viral vectors have many advantages but suffered from low gene transfection efficiency. Here, in order to develop new polymeric gene vectors with low cytotoxicity and high gene transfection efficiency, we synthesized a cationic polymer composed of low molecular weight polyethylenimine (PEI) of molecular weight of 600 Da cross-linked by 2-hydroxypropyl-γ-cyclodextrin (HP γ-CD) and then coupled to MC-10 oligopeptide containing a sequence of Met-Ala-Arg-Ala-Lys-Glu. The oligopeptide can target to HER2, the human epidermal growth factor receptor 2, which is often over expressed in many breast and ovary cancers. The new gene vector was expected to be able to target delivery of genes to HER2 positive cancer cells for gene therapy. The new gene vector was composed of chemically bonded HP γ-CD, PEI (600 Da), and MC-10 peptide at a molar ratio of 1:3.3:1.2. The gene vector could condense plasmid DNA at an N/P ratio of 6 or above. The particle size of HP γ-CD-PEI-P/DNA complexes at N/P ratios 40 was around 170–200 nm, with zeta potential of about 20 mV. The gene vector showed very low cytotoxicity, strong targeting specificity to HER2 receptor, and high efficiency of delivering DNA to target cells in vitro and in vivo with the reporter genes. The delivery of therapeutic IFN-α gene mediated by the new gene vector and the therapeutic efficiency were also studied in mice animal model. The animal study results showed that the new gene vector HP γ-CD-PEI-P significantly enhanced the anti-tumor effect on tumor-bearing nude mice as compared to PEI (25 kDa), HP γ-CD-PEI, and other controls, indicating that this new polymeric gene vector is a potential candidate for cancer gene therapy.

Introduction

Gene delivery is one of the critical steps for gene therapy. Viral vectors have been proven to have high gene transfection efficiency. However, they have some disadvantages, such as immunogenicity, potential of insertional mutagenesis in the host genome [1], limited DNA delivery capacity and scale of production. Non-viral vectors are promising alternatives to viral vectors due to the advantages that they will not elicit immune responses or randomly integrate DNA into the host genome [2], [3]. Cationic polymers are the major type of the non-viral vectors investigated in the past decade. The most attractive feature of the polymeric vectors is that they can be easily tailored and synthesized to suit the special requirements encountered by gene delivery [4]. Polyethylenimine (PEI) is one of the best known and most widely studied cationic polymers as non-viral gene delivery vectors, [2], [5]. It has been documented that the transfection efficiency of PEI depends on its molecular weight. High molecular weight (HMW) PEI has high gene transfection efficiency but high cytotoxicity; Low molecular weight (LMW) PEI (below 2000 Da) shows low cytotoxicity but low gene transfection efficiency [6], [7], [8]. Cyclodextrins (CDs) are a series of natural cyclic oligosaccharides, which have low immunogenicity and toxicity in animals and humans [9], [10], and can enhance the absorption and resistance to nucleases through binding and interacting with oligonucleotides [11]. CDs have been incorporated into cationic polymers as new gene delivery vectors [9], [10]. Tang et al. employed β-CD to crosslink LMW PEI (MW 600 Da) to form a high MW cationic polymer, which showed lower cytotoxicity and the high transfection efficiency close to PEI 25 kDa in vitro and in vivo [12]. Li's group synthesized cationic star-shaped polymers by conjugating oligoethylenimine to an α-CD core [13] and cationic supramolecular structures by threading cationic α- or β-CD on polymer chain as new non-viral gene delivery vectors [14], [15], [16], [17]. The star-shaped polymers and the cationic supramolecules showed excellent gene transfection efficiency in HEK293 and Cos7 cells.

It is necessary to improve the specificity of polymeric vectors for gene uptake by tumor cells for efficient cancer gene therapy. The distinctive markers of tumor are candidate targets for cancer therapy. HER2, the human epidermal growth factor receptor 2, has been reported to be a good target [18], [19], which is over expressed in breast cancers and ovarian cancers. More importantly, HER2 expression is related to the malignancy and progress of tumor [19], [20]. Some studies selected Trastuzumab, a monoclonal antibody targeting HER2, and linked it with PEI or PEI-polyethylene glycol as gene delivery vector targeting HER2 positive cells in vitro [21], [22].

In this study, a polymer matrix was synthesized by cross-linking LMW PEI by 2-hydroxypropyl-γ-cyclodextrin (HP γ-CD-PEI). An oligopeptide targeting HER2 was conjugated to the polymer matrix to form a new polymeric gene vector (HP γ-CD-PEI-P). The sequence of the oligopeptide is Met-Ala-Arg-Ala-Lys-Glu, which has been identified by Houimel et al. [23]. We investigated the specificity and efficiency of this polymeric gene vector in HER2 positive human ovary cancer SKOV-3 cells in vitro and in vivo. To further evaluate the potential of the new vector in cancer gene therapy, we utilized the polymeric gene vector to delivery interferon-α (IFN-α) expression plasmid in vivo and tested its anti-tumor effect. The results showed that the therapeutic effect of the new gene vector on tumor-bearing nude mice was significantly enhanced as compared to the control groups.

Section snippets

Chemicals and oligopeptide

Polyethylenimine (PEI, MW 600 Da and 25 kDa), 2-hydroxypropyl-γ-cyclodextrin (HP γ-CD), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma–Aldrich (USA). 1,1-Carbonyldiimidazole (CDI) and N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (MW 312) were purchased from Pierce Corporation (Rockford, USA). The oligopeptide NH2-Met-Ala-Arg-Ala-Lys-Glu-Gly-Gly-Gly-Cys-COOH (MC-10, MW 979.1 Da) was synthesized by GL Biochem, Shanghai, China, and the amino

Synthesis and 1H NMR measurement of polymeric gene vector

We previously developed the non-viral polymeric gene vector HP γ-CD-PEI by cross-linking low molecular branch PEI (600 Da) with HP γ-CD, which has low cytotoxicity and reasonably high gene transfection efficiency [24]. To improve the targeting ability, MC-10 peptide targeting HER2 was chosen to be coupled to HP γ-CD-PEI, to give the final gene vector HP γ-CD-PEI-P (Scheme 1). The structures of HP γ-CD-PEI and HP γ-CD-PEI-P were confirmed by 1H NMR spectroscopy (Fig. 1). The ratio between

Conclusions

We developed a new polymeric gene vector, HP γ-CD-PEI-P, by cross-linking low molecular weight PEI with HP γ-CD and further coupling the peptide MC-10 to the polymer for targeting HER2 receptor, a receptor over expressed in many breast and ovary cancers. The new gene vector was characterized in terms of its chemical structure and composition, biophysical parameters, and in vitro and in vivo gene transfection efficiency. It was found that the new gene vector was composed of chemically bonded HP

Acknowledgments

This work was co-funded by the National High Technology Development Program of China (863 Program, No. 2007AA03Z355), the National Nature Science Foundation of China (No. 30571068, No. 30800227), the Science and Technology Department of Zhejiang Province (2009R10003), and the Singapore Ministry of Education Academic Research Fund Tier 2 Grant (R-397-000-031-112). Finally, the Foundation of National Excellent Doctoral Dissertation of China (FANEDD, 200364) is also acknowledged.

References (29)

  • Y.Y. Yang et al.

    Polymeric core–shell nanoparticles for therapeutics

    Clin Exp Pharmacol Physiol

    (2006)
  • O. Boussif et al.

    A versatile vector for gene and oligonucleotide transfer into cells in culture and in-vivo –s polyethylenimine

    Proc Natl Acad Sci U S A

    (1995)
  • W.T. Godbey et al.

    Size matters: molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle

    J Biomed Mater Res

    (1999)
  • M.E. Davis et al.

    Cyclodextrin-based pharmaceutics: past, present and future

    Nat Rev Drug Discov

    (2004)
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