Therapeutic Gene Delivery Systems Research Articles

A mini-review of Nanocarriers in drug delivery systems

The application of nanotechnology indrug delivery systems (DDS) has been researched widely and seen an advancementover the past three decades. Since the 1970s, nanoparticles were primarilyutilised in vaccine deliveries and cancer chemotherapy. In more recent years,they have been found to hold promises for broader applications such as inproteins and therapeutic gene delivery systems. To date, there have been only ahandful of nanocarrier-loaded drugs commercialised into the pharmaceuticalmarket. More research is thus needed to facilitate a breakthrough of theseproducts into the current market. This mini-review mainly focuses on four typesof commonly utilised organic nanocarriers including micelles, compactpolymerics, solid-lipid nanoparticles and liposomal vesicles and discusses theprogress and some challenges associated with these nanoparticles (NP). 

Chitosan/hyaluronic acid/plasmid-DNA nanoparticles encoding interleukin-1 receptor antagonist attenuate inflammation in synoviocytes induced by interleukin-1 beta

Synovial inflammation mainly resulting from interleukin-1 beta (IL-1β) plays a crucial role in the early and late stage of osteoarthritis. Recent progress in therapeutic gene delivery systems has led to promising strategies for local sustained target gene expression. The aim of this study was to design a nanoparticle made of chitosan (CS)/hyaluronic acid (HA)/plasmid-DNA (pDNA) encoding IL-1 receptor antagonist gene (pIL-1Ra) and furtherly use it to transfect the primary synoviocytes, and then investigate whether CS/HA/pIL-1Ra nanoparticles could make the synoviocytes overexpress functional IL-1Ra to attenuate inflammation induced by IL-1β. In this study, CS was modified with HA to generate CS/HA nanoparticles and then combined with pIL-1Ra to form CS/HA/pIL-1Ra nanoparticles. The physicochemical characteristics results showed that CS/HA nanoparticles exhibited an appropriate particle size (144.9 ± 2.8 nm) and positive zeta potential ( + 28 mV). The gel retardation assay revealed that pDNA was effectively protected and released in a sustained manner more than 15 days. Cytotoxicity results showed that CS/HA/pIL-1Ra nanoparticles had a safe range (0-80 μg/ml) for the application to synoviocytes. RT-qPCR and western blot analysis demonstrated that CS/HA/pIL-1Ra nanoparticles were able to increase IL-1Ra expression in primary synoviocytes, and reduce the mRNA and protein levels of matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in IL-1β-induced synoviocytes. Our findings indicated that CS/HA/pIL-1Ra nanoparticles efficiently transfected synoviocytes and attenuated synovitis induced by IL-1β, which will provide a potential strategy for OA synovitis.

PELA microspheres with encapsulated arginine–chitosan/pBMP-2 nanoparticles induce pBMP-2 controlled-release, transfected osteoblastic progenitor cells, and promoted osteogenic differentiation

Repair of the bone injury remains a challenge in clinical practices. Recent progress in tissue engineering and therapeutic gene delivery systems have led to promising new strategies for successful acceleration of bone repair process. The aim of this study was to create a controlled-release system to slowly release the arginine–chitosan/plasmid DNA nanoparticles encoding BMP-2 gene (Arg-CS/pBMP-2 NPs), efficiently transfect osteoblastic progenitor cells, secrete functional BMP-2 protein, and promote osteogenic differentiation. In this study, chitosan was conjugated with arginine to generate arginine–chitosan polymer (Arg-CS) for gene delivery. Mix the Arg-CS with pBMP-2 to condense pBMP-2 into nano-sized particles. In vitro transfection assays demonstrated that the transfection efficiency of Arg-CS/pBMP-2 nanoparticles and the expression level of BMP-2 was obviously exceed control groups. Further, PELA microspheres as the controlled-release carrier for the nanoparticles were used to encapsulate Arg-CS/pBMP-2 NPs. We demonstrated that the Arg-CS/pBMP-2 NPs could slowly release from the PELA microspheres at least for 42 d. During the co-culture with the PELA microspheres, the content of BMP-2 protein secreted by MC3T3-E1 reached the peak at 7 d. After 21d, the secretion of BMP-2 protein still maintain a higher level. The alkaline phosphatase activity, alizarin red staining, and osteogenesis-related gene expression by real-time quantitative PCR analysis all showed the PELA microspheres entrapping with Arg-CS/pBMP-2 NPs can obviously induce the osteogenic differentiation. The results indicated that the Arg-CS is a suitable gene vector which can promote the gene transfection. And the novel PELA microspheres-nanoparticle controlled-release system has potential clinical application in the future after further research.

Porous chitosan scaffolds with embedded hyaluronic acid/chitosan/plasmid-DNA nanoparticles encoding TGF-β1 induce DNA controlled release, transfected chondrocytes, and promoted cell proliferation.

Cartilage defects resulting from traumatic injury or degenerative diseases have very limited spontaneous healing ability. Recent progress in tissue engineering and local therapeutic gene delivery systems has led to promising new strategies for successful regeneration of hyaline cartilage. In the present study, tissue engineering and local therapeutic gene delivery systems are combined with the design of a novel gene-activated matrix (GAM) embedded with hybrid hyaluronic acid(HA)/chitosan(CS)/plasmid-DNA nanoparticles encoding transforming growth factor (TGF)-β1. A chitosan scaffold functioned as the three-dimensional carrier for the nanoparticles. Results demonstrated that scaffold-entrapped plasmid DNA was released in a sustained and steady manner over 120 days, and was effectively protected in the HA/CS/pDNA nanoparticles. Culture results demonstrated that chondrocytes grown in the novel GAM were highly proliferative and capable of filling scaffold micropores with cells and extracellular matrix. Confocal laser scanning microscopy indicated that chondrocytes seeded in the GAM expressed exogenous transgenes labeled with green fluorescent protein. ELISA results demonstrated detectable TGF-β1 expression in the supernatant of GAM cultures, which peaked at the sixth day of culture and afterwards showed a moderate decline. Histological results and biochemical assays confirmed promotion of chondrocyte proliferation. Cell culture indicated no affects on phenotypic expression of ECM molecules, such as GAG. The results of this study indicate the suitability of this novel GAM for enhanced in vitro cartilage tissue engineering.

Enhanced gene expression promoted by the quantized folding of pDNA within polyplex micelles

Selective packaging of plasmid DNA (pDNA) into folded rod or collapsed sphere structures in polyplex micelles was demonstrated by modulating the PLys segment length of poly(ethylene glycol)- block- poly( l-lysine) (PEG-PLys) block catiomers used for micelle formation. The two basic packaging structures correlated well to the integrity of double-stranded DNA contained within the micelles. Rod structures formed by the quantized folding mechanism, which results in dissociation of double-stranded DNA only at each fold. Collapsed sphere structures formed by substantial random disruption of the double-stranded DNA structure. Analysis of gene expression in a cell-free transcription/translation system, cultured cells and also skeletal muscle of mice showed that micelles containing pDNA packaged by quantized folding exhibited higher gene expression than naked pDNA and micelles containing collapsed pDNA. These results indicate that controlled packaging of pDNA into an appropriate structure is critical for achieving effective gene expression. Improved gene transfection and expression resulting from the quantized folding of pDNA within polyplex micelles is promising for application in therapeutic gene delivery systems.

Lentivirus transduction of human osteoclast precursor cells and differentiation into functional osteoclasts

Gene transfer into stem cells has been an ongoing priority as a treatment for genetic disease and cancer for more than two decades. Methods described herein, form the basis for providing the cell source to determine if osteoclast precursor cells (OcP) can be used as therapeutic gene delivery systems in vivo. Osteoclasts and tumor associated macrophages or OcP, support survival, tumor progression and osteolysis in bone cancers. Two sources of precursor cells are compared: CD14+ cells, the standard OcP, found abundantly in peripheral blood and CD34+ cells, hematopoietic stem cells that are rare, but which can be expanded into OcP. Our findings characterize cell yield at each step of the transduction process and thus provide essential data for planning future in vivo experiments. In addition we demonstrate that essential functions of OcP are preserved following lentiviral transduction. Specifically, neither the transduction method nor the lentiviral transduction influence the OcP's ability to form osteoclasts, express the marker gene, EGFP, or resorb bone. Finally, we conclude that CD34+ cells yield significantly more transduced cells and form functionally superior osteoclasts in vitro. This study represents a step towards considering human gene therapy for bone cancer by demonstrating successful transduction of human OcP for use as cellular delivery vehicles to sites of bone cancer.

Identification of cell-type-specific promoters within the brain using lentiviral vectors

The development of cell-type-specific mini-promoters for genetic studies is complicated by a number of issues. Here, we describe a general method for the relatively rapid screening of specific promoter activity in cell culture, in acute brain slice preparations and in vivo. Specifically, we examine the activity of an approximately 3 kb promoter region from the neuroactive peptide cholecystokinin (CCK) compared to the commonly used cytomegalovirus promoter. We find a high degree of cell-type selectivity in vivo using lentiviral approaches in rats and traditional transgenic approaches in mice. Appropriate colocalization of Cre-recombinase and CCK gene expression is found within the hippocampus, when the CCK promoter is driving either the expression of Cre-recombinase or green fluorescent protein. We also demonstrate fluorescent identification of CCK-positive interneurons that allows for cell-type-specific electrophysiologic studies in rats and mice. In conclusion, these studies identify a functional mini-promoter for the CCK gene and outline a novel and sensitive general method to test activity of selective promoters in vitro and in vivo. This approach may allow for the more rapid identification of specific promoters for use with transgenic animals, in genetically modified viruses, and in the design of targeted, therapeutic gene-delivery systems.

Porous chitosan-gelatin scaffold containing plasmid DNA encoding transforming growth factor- β1 for chondrocytes proliferation

Cartilage defects as a result of disease or injury have a very limited ability to heal spontaneously. Recently, tissue engineering and local therapeutic gene delivery systems have been paid much attention in the cartilage natural healing process. Gene-activated matrix (GAM) blends these two strategies, serving as local bioreactor with therapeutic agents expression and also providing a structural template to fill the lesion defects for cell adhesion, proliferation and synthesis of extracellular matrix (ECM). In the current study, we used chitosan-gelatin complex as biomaterials to fabricate three-dimensional scaffolds and plasmid DNA were entrapped in the scaffolds encoding transforming growth factor- β1 (TGF- β1), which has been proposed as a promoter of cartilage regeneration for its effect on the synthesis of matrix molecules and cell proliferation. The plasmid DNA incorporated in the scaffolds showed a burst release in the first week and a sustained release for the other 2 weeks. The gene transfectd into chondrocytes expresses TGF- β1 protein stably in 3 weeks. The histological and immunohistochemical results confirmed that the primary chondrocytes cultured into the chitosan-gelatin scaffold maintained round and owned characters of high secretion of specific ECM. From this study, it can be concluded that this gene-activated chitosan-gelatins matrix has a potential in the application of cartilage defects regeneration.

Therapeutic potential of retroviral vectors

Therapeutic gene delivery systems based on disabled murine retroviruses are now in clinical trials for the treatment of disorders ranging from rare genetic diseases to cancer. Here I provide an overview of the retrovirus life cycle and recombinant retrovirus production, and discuss recent modifications in retroviral vector design that may translate into improvements in infection efficiency of target cell populations and maintenance of high-level gene expression in humans.

Molecular conjugates: a targeted gene delivery vector for molecular medicine.

The use of gene therapy to treat diseases of both genetic and infectious origins has increased rapidly over the past 5 years. Accordingly, the number of delivery systems, based on either recombinant viruses or nonviral vectors, has also increased. As a result most research in this field continues to be in the area of vector development. Crucial to this aspect is the development of efficient, multipurpose therapeutic gene delivery systems which should have, but not be limited to, the following characteristics: (a) the ability to target specific cells, (b) no limitation on the size or the type of nucleic acid that can be delivered, (c) no intact viral component and therefore safe for the recipient, (d) the ability to transduce a large number of cells regardless of the mitotic status; and (e) the potential to be completely synthetic. As a step toward the development of this type of delivery system, molecular conjugates have been created. Molecular conjugates are ligands to which a nucleic acid or DNA-binding agent has been attached for the specific targeting of nucleic acids (i.e., plasmid DNA) to cells. The resulting protein/DNA complex can consist of at least four components: (a) a ligand for cell-specific targeting and receptor-mediated endocytosis, (b) a nucleic acid or DNA-binding agent (than is chemically attached to the ligand) for the binding of the DNA by ionic or other nondamaging interactions, (c) a nucleic acid, i.e., a plasmid for gene expression, and (d) an endosomal lysis agent to enhance the re-