Nanosphere-mediated delivery of vascular endothelial growth factor gene for therapeutic angiogenesis in mouse ischemic limbs
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)
- et al.
Pharmacological regulation of protein expression from adeno-associated viral vectors in the eye
Mol Ther
(2002) - et al.
Recent advances in liposome technologies and their applications for systemic gene delivery
Adv Drug Del Rev
(1998) - et al.
Gene therapy for tissue repair and regeneration
Adv Drug Del Rev
(1998) - et al.
A DNA controlled-release coating for gene transfer: transfection in skeletal and cardiac muscle
J Pharm Sci
(1998) - et al.
Gene transfection using biodegradable nanospheres: results in tissue culture and a rat osteotomy model
Colloids Surf B: Biointerfaces
(1999) - 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) - et al.
Biodegradable polyesters for controlled release of trypanocidal drugs: in vitro and in vivo studies
Biomaterials
(1998) The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices
Biomaterials
(2000)- et al.
Cellular interactions and degradation of aliphatic poly(ester amide)s derived from glycine and/or 4-amino butyric acid
Biomaterials
(2003) - 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)
Vascular endothelial growth factor gene delivery by magnetic DNA nanospheres ameliorates limb ischemia in rabbits
J Surg Res
Angiogenesis
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
Cited by (69)
ROS-responsive nanoparticle-mediated delivery of CYP2J2 gene for therapeutic angiogenesis in severe hindlimb ischemia
2022, Materials Today BioCitation Excerpt :All these results strongly suggest that 3S-PLGA-po-PEG is a promising delivery vector for gene therapy for lower limb ischemia with a certain degree of novelty. It is well known that efficient VEGF expression is essential for the application of therapeutic angiogenesis treatment in CLI [54]. VEGF is one of the target genes of HIF-1α, an inducible transcription regulator that plays an important role in intracellular oxygen homeostasis [57].
Gene-Loaded Nanoparticle-Coated Sutures Provide Effective Gene Delivery to Enhance Tendon Healing
2019, Molecular TherapyCitation Excerpt :PEI has been widely used for vectors with a wide range of molecular weights (MW), and its toxicity is mainly dependent on its MW. When PEI is used to modify nanoparticles, its cytotoxicity was significantly reduced.36 Our previous study also showed that PEI-modified nanoparticle/plasmid complexes have no significant cytotoxicity compared with PBS.16
Local pharmacological induction of angiogenesis: Drugs for cells and cells as drugs
2019, Advanced Drug Delivery ReviewsControllable preparation of SB-3CT loaded PLGA microcapsules for traumatic-brain-injury pharmaco-therapy
2018, Chemical Engineering JournalDegradable polymers
2017, Comprehensive Biomaterials II
- 1
These authors contributed equally to this work.