Nanosphere-mediated delivery of vascular endothelial growth factor gene for therapeutic angiogenesis in mouse ischemic limbs

doi:10.1016/j.biomaterials.2007.11.004

Biomaterials

Volume 29, Issue 8, March 2008, Pages 1109-1117

https://doi.org/10.1016/j.biomaterials.2007.11.004 Get rights and content

Abstract

Polymeric nanosphere-mediated gene delivery may sustain the duration of plasmid DNA (pDNA) administration. In this study, poly(lactic-co-glycolic acid) (PLGA) nanospheres were evaluated as a gene carrier. The pDNA-loaded PLGA nanospheres were formulated with high encapsulation efficiency (87%). The nanospheres sustained release of pDNA for 11 days. The released pDNA maintained its structural and functional integrity. Furthermore, the PLGA nanospheres showed lower cytotoxicity than polyethylenimine (PEI) in vitro and in vivo. The nanospheres with vascular endothelial growth factor (VEGF) gene were injected into skeletal muscle of ischemic limb model, and gene expression mediated by the PLGA nanospheres with VEGF gene was compared to that of PEI/pDNA or naked pDNA in vivo. PLGA nanosphere/pDNA had significantly higher VEGF expression levels in comparison to PEI/pDNA and naked pDNA at 12 days after administration. In addition, gene therapy using PLGA nanospheres resulted in more extensive neovascularization at ischemic sites than both naked pDNA and PEI/pDNA. These results indicated that PLGA nanosphere might be useful as a potential carrier for skeletal muscle gene delivery applications.

Introduction

Gene therapy has been developed as a potential cure for the treatment of genetic disorders and chronic diseases. In non-viral gene therapy, plasmid DNA (pDNA) has potential not only as a therapeutic agent but also as a new vaccination approach. The use of pDNA as drugs and vaccines will largely depend on a delivery method. Viral vectors are currently the most efficient gene delivery methods, taking advantage of the natural ability of viruses to penetrate into host cells and to transfer their genetic materials to the nucleus [1], [2], [3], [4]. However, viral vectors such as a retrovirus and adenovirus have serious safety concerns such as potential oncogenicity, toxicity and immunogenicity [5]. Non-viral strategies have been developed as an alternative for gene delivery.

Various types of non-viral vectors have been developed and evaluated for gene delivery [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. Polyethylenimine (PEI), a polycationic polymer, has been applied to gene delivery in a variety of cells in vitro and in vivo, since it has high transfection efficiency due to proton-sponge effect over a broad pH range [16], [17], [18]. However, high cytotoxicity of PEI has limited its application to clinical settings [18]. Therefore, development of non-toxic polymeric vectors with high transfection efficiency has been one of the goals in the non-viral gene carrier research.

Polymeric nanospheres formulated from biodegradable poly(lactic-co-glycolic acid) (PLGA) copolymer have been extensively investigated in drug and gene delivery [19], [20], [21]. PLGA nanospheres have advantages such as a high stability, easy uptake into the cells by endocytosis, and the targeting ability to specific tissue or organs by adsorption or coating with ligand materials at the surface of spheres. In addition, PLGA has good biocompatibility and has been approved by Food and Drug Administration for certain human clinical uses such as bioresorbable surgical sutures, surgical screws, plates and rods [22], [23]. Biodegradable PLGA nanospheres with entrapped pDNA have shown the potential for achieving sustained gene expression [24], [25]. This system has the advantage of pDNA protection along with the sustained release [24], [25]. In this study, we evaluated the possibility and efficiency of using PLGA nanospheres as a new vector for angiogenic gene therapy with vascular endothelial growth factor (VEGF) and investigated the efficacy of direct gene transfer of PLGA nanospheres into ischemic limbs of mice.

Section snippets

Preparation of PLGA nanospheres encapsulating VEGF gene

PLGA nanospheres encapsulating pDNA were prepared using a double emulsion–solvent evaporation method, as described previously [26]. Briefly, 1 ml of pDNA (pSV40–VEGF, 1 mg/ml) in Tris–EDTA buffer was emulsified in 30 ml of PLGA solution (5% w/v in methylene chloride) using homogenizer (T-18 basic, IKA, Tokyo, Japan) at 25,000 rpm for 1 min. The water-in-oil emulsion was further emulsified in 50 ml of a 2% (w/v) aqueous solution of polyvinyl alcohol (PVA, Mw 30,000–70,000, Sigma) using homogenizer for

Results

PLGA copolymer was synthesized by ring-opening polymerization of lactide and glycolide as reported previously [21]. A scanning electron microphotograph of fabricated PLGA nanospheres containing VEGF gene showed that the nanospheres were spherical and discrete particles without aggregation, and that they were smooth in surface morphology (Fig. 1A). The average diameter of the nanospheres was 201.9±36.3 nm (Fig. 1B). The theoretical loading amount was 65.5 μg pDNA/mg PLGA nanospheres. The loading

Discussion

A growing number of studies have demonstrated that non-viral vectors have therapeutic efficacy in gene delivery in vitro and in vivo [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. High molecular weight PEI (PEI25,000) has been an attractive carrier for its highly effective gene delivery, due to a proton-buffering effect [16], [17]. However, PEI has a serious problem for its application to human gene therapy such as high cytotoxicity, and non-degradability. Therefore, development of

Conclusion

PLGA nanospheres sustained release of pDNA with its structural and functional integrity for 11 days. PLGA nanospheres showed lower cytotoxicity than PEI in vitro and in vivo. VEGF gene delivery to mouse ischemic limbs with PLGA nanospheres resulted in significantly higher VEGF expression at 12 days and more extensive neovascularization at 4 weeks than VEGF gene delivery with either PEI or no carrier. With biodegradability, low toxicity and high transfection efficiency, PLGA nanosphere may be a

Acknowledgment

This study was supported by a Grant (A050082) from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea.

References (31)

A. Auricchio et al.
Pharmacological regulation of protein expression from adeno-associated viral vectors in the eye
Mol Ther
(2002)
A. Chonn et al.
Recent advances in liposome technologies and their applications for systemic gene delivery
Adv Drug Del Rev
(1998)
J. Bonadio et al.
Gene therapy for tissue repair and regeneration
Adv Drug Del Rev
(1998)
V. Labhasetwar et al.
A DNA controlled-release coating for gene transfer: transfection in skeletal and cardiac muscle
J Pharm Sci
(1998)
V. Labhasetwar et al.
Gene transfection using biodegradable nanospheres: results in tissue culture and a rat osteotomy model
Colloids Surf B: Biointerfaces
(1999)
O. Jeon et al.
Long-term and zero-order release of basic fibroblast growth factor from heparin-conjugated poly(l-lactide-co-glycolide) nanospheres and fibrin gel
Biomaterials
(2006)
Y. Lemmouchi et al.
Biodegradable polyesters for controlled release of trypanocidal drugs: in vitro and in vivo studies
Biomaterials
(1998)
R.A. Jain
The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices
Biomaterials
(2000)
S.I. Han et al.
Cellular interactions and degradation of aliphatic poly(ester amide)s derived from glycine and/or 4-amino butyric acid
Biomaterials
(2003)
O. Jeon et al.
Synergistic effect of sustained delivery of basic fibroblast growth factor and bone marrow mononuclear cell transplantation on angiogenesis in mouse ischemic limbs
Biomaterials
(2006)

H. Jiang et al.

Vascular endothelial growth factor gene delivery by magnetic DNA nanospheres ameliorates limb ischemia in rabbits

J Surg Res

(2005)

J. Folkman et al.

Angiogenesis

J Biol Chem

(1992)

M. Cavazzana-Calvo et al.

Gene therapy of human severe combined immune deficiency (SCID)-X1 disease

Science

(2000)

B.L. Davidson et al.

Recombinant adeno-associated virus type 2, 4, and 5 vectors: transduction of variant cell types and regions in the mammalian central nervous system

Proc Natl Acad Sci USA

(2000)

D.M. Krisky et al.

Deletion of multiple immediate-early genes from herpes simplex virus reduces cytotoxicity and permits long-term gene expression in neurons

Gene Ther

(1998)

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¹: These authors contributed equally to this work.

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Nanosphere-mediated delivery of vascular endothelial growth factor gene for therapeutic angiogenesis in mouse ischemic limbs

Abstract

Introduction

Section snippets

Preparation of PLGA nanospheres encapsulating VEGF gene

Results

Discussion

Conclusion

Acknowledgment

Mol Ther

Adv Drug Del Rev

Adv Drug Del Rev

J Pharm Sci

Colloids Surf B: Biointerfaces

Biomaterials

Biomaterials

Biomaterials

Biomaterials

Biomaterials

J Surg Res

J Biol Chem

Gene therapy of human severe combined immune deficiency (SCID)-X1 disease

Science

Recombinant adeno-associated virus type 2, 4, and 5 vectors: transduction of variant cell types and regions in the mammalian central nervous system

Proc Natl Acad Sci USA

Deletion of multiple immediate-early genes from herpes simplex virus reduces cytotoxicity and permits long-term gene expression in neurons

Gene Ther