Controlled Gene Delivery Research Articles

422: Enhanced Efficacy of Radiation Sensitivity by Controlled Gene Delivery of PTEN Expression Vector Conjugated with Cationized Gelatin in Prostate Cancer Cells

You have accessJournal of UrologyDiscussed Poster, Sunday, May 22, 2005, 1:00 - 5:00 pm1 Apr 2005422: Enhanced Efficacy of Radiation Sensitivity by Controlled Gene Delivery of PTEN Expression Vector Conjugated with Cationized Gelatin in Prostate Cancer Cells Motoyoshi Tanaka, Satoshi Takada, Atsushi Tomioka, Yoshiaki Matsumura, Hirotsugu Uemura, and Yoshihiko Hirao Motoyoshi TanakaMotoyoshi Tanaka More articles by this author , Satoshi TakadaSatoshi Takada More articles by this author , Atsushi TomiokaAtsushi Tomioka More articles by this author , Yoshiaki MatsumuraYoshiaki Matsumura More articles by this author , Hirotsugu UemuraHirotsugu Uemura More articles by this author , and Yoshihiko HiraoYoshihiko Hirao More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(18)34675-5AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "422: Enhanced Efficacy of Radiation Sensitivity by Controlled Gene Delivery of PTEN Expression Vector Conjugated with Cationized Gelatin in Prostate Cancer Cells." The Journal of Urology, 173(4S), p. 115 © 2016 by American Urological AssociationFiguresReferencesRelatedDetails Volume 173Issue 4SApril 2005Page: 115 Advertisement Copyright & Permissions© 2016 by American Urological AssociationMetricsAuthor Information Motoyoshi Tanaka More articles by this author Satoshi Takada More articles by this author Atsushi Tomioka More articles by this author Yoshiaki Matsumura More articles by this author Hirotsugu Uemura More articles by this author Yoshihiko Hirao More articles by this author Expand All Advertisement PDF downloadLoading ...

Molecular Engineering of Silk-Elastinlike Polymers for Matrix-Mediated Gene Delivery: Biosynthesis and Characterization

The unique advantage of genetic engineering techniques for the design and development of polymers for controlled gene delivery lies in exquisite control over polymer structure. In this article we report the biosynthesis and characterization of a series of new silk-elastinlike protein polymers (SELPs), namely, SELP415K, with larger elastin blocks per monomer unit than SELP47K previously studied for matrix-mediated gene delivery. A new cloning strategy was used, where a block of eight elastin units (8E) was integrated into the existing DNA sequence of SELP47K monomer genes using appropriate restriction endonuclease recognition sites. Following random multimerization, multimer gene segments of desired size were selected, expressed, and purified on Ni-agarose columns. The molecular weight and sequence composition of the purified SELPs were determined by MALDI-TOF and amino acid analysis, respectively. The influence of structural changes on the rheological properties of the polymers was investigated. In addition, hydrogel disks were prepared from 47K and 415K-8mer polymer solutions, and the effects of cure time and environmental conditions on the hydrogel equilibrium swelling ratio as a function of polymer composition were studied. DNA sequencing and agarose gel electrophoresis confirmed the successful cloning of the monomer gene segment of SELP415K consisting of 312 bp. Random concatemerization of SELP415K monomer gene segments resulted in a library of SELP415K multimer sequences of 6, 8, and 10 repeats respectively, each yielding a polymer with exact molecular weight and sequence. Rheometric measurements showed that both complex shear modulus (G*) and gelation point were influenced by polymer composition. Equilibrium swelling studies on hydrogel disks prepared from 47K and 415K-8mer polymer solutions showed that changes in polymer composition resulted in different gelation patterns and increased sensitivity toward changes in temperature and ionic strength but not pH. Together these results demonstrate the potential of recombinant techniques in engineering polymers with defined structures which allows the study of the structural parameters affecting matrix-mediated delivery of genes and bioactive agents.

Novel cationic pentablock copolymers as non-viral vectors for gene therapy

New cationic pentablock copolymers of poly(diethylaminoethylmethacrylate) (PDEAEM), poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO)—PDEAEM-b-PEO-b-PPO-b-PEO-b-PDEAEM—synthesized in our laboratory were investigated for their potential as non-viral vectors for gene therapy. Agarose gel studies showed that the copolymers effectively condensed plasmid DNA to form polyplexes, and also protected plasmids against nuclease degradation. Light scattering and transmission electron microscopy were used to analyze the apparent size, molecular weight and morphology of these polyplexes. Lactate dehydrogenase assay was employed to find the cytotoxicity limits of the polymers and polyplexes on a human ovarian cancer cell line. The polymers showed much less cytotoxicity than commercially available ExGen 500 (linear polyethyleneimine). By changing the relative lengths of the blocks in the copolymers, it was found that the cytotoxicity of these copolymers could be tailored. The micellar structures of these copolymers in aqueous solutions and their pH-sensitive protonation were added advantages. In vitro transfection efficiencies of the polymers using green fluorescent protein (pEGFP-N1) and luciferase (pRL-CMV) reporter genes were found comparable to ExGen 500. Besides, aqueous solutions of these pentablock copolymers have been shown to exhibit thermodynamic phase transitions and thermoreversible gelation, a quality that could allow subcutaneous/intramuscular injections of these polymers for controlled gene delivery over time.

DNA delivery from hyaluronic acid-collagen hydrogels via a substrate-mediated approach

Efficient and controlled gene delivery from biodegradable materials can be employed to stimulate cellular processes that lead to tissue regeneration. In this report, a substrate-mediated approach was developed to deliver DNA from hyaluronic acid-collagen hydrogels. The hydrogels were formed by crosslinking HA with poly(ethylene glycol) diglycidyl ether. Poly(ethylene imine)(PEI)/DNA complexes were immobilized to the substrate using either biotin/neutravidin or non-specific adsorption. Complexes were formed in the presence or absence of salt to regulate complex size, and resulted in complexes with z-average diameters of 1221.7±152.3 and 139.4±1.3 nm, respectively. During 48-h incubation in PBS or hyaluronidase, DNA was released slowly from the hydrogel substrate (<30% of immobilized DNA), which was enhanced by incubation with conditioned media (≈50% of immobilized DNA). Transgene expression mediated by immobilized, large diameter complexes was 3 to 7-fold greater than for small diameter complexes. However, the percentage of cells expressing the transgene was greater for small diameter complexes (48.7%) than for large diameter complexes (22.3%). Spatially controlled gene transfer was achieved by topographically patterning the hydrogel to pattern cell adhesion. Biomaterial-based gene delivery can be applicable to numerous tissue engineering applications, or as a tool to examine tissue formation.

845. Enhanced Efficacy of Radiation Sensitivity by Controlled Gene Delivery of PTEN Expression Vector Conjugated with Cationized Gelatin in Prostate Cancer Cells

We have previously demonstrated the superiority of copolymer-protected gene vectors (COPROGs) over naked DNA and standard nonviral vectors in matrix-mediated gene delivery (Scherer et al. 2002, J. Gene Med. 4:634-643). Here we present a novel carrier formulation based on lyophilized fibrinogen-vector compositions. Upon rehydration and local mixing with thrombin, these preparations can be used as fibrin glues for the local in situ delivery of genes and nucleic acids, particularly of growth factor genes for tissue engineering. As fibrin is an excellent matrix for cell growth, the preparations can be formulated with cells to fill tissue defects such as cavities in cartilage or bone or to cover skin defects. Naked DNA is readily released from fibrin clots (70 % within 5 days) while COPROGs remain tightly immobilized in the fibrin matrix over extended periods of time with a linear release of 0.29 % per day. Naked DNA was essentially inactive in mediating gene delivery to primary cells including chondrocytes and keratinocytes incorporated in vector-loaded fibrin clots. COPROGs mediated efficient and sustained transfection resulting in transgene expression over at least three weeks. The transfection of the BMP-2 gene in chondrocytes enclosed in COPROG-loaded fibrin clots in vitro led to sustained BMP-2 expression and to the induction of alkaline phosphatase as well as to increased production of extracellular matrix. Sustained reporter gene expression was achieved in critical cartilage defects in rabbit knee joints filled with autologous chondrocyte-COPROG-fibrin glue composites. In conclusion, COPROG-loaded fibrinogen preparations used as gene-activated fibrin glue display a strong potential as injectable implants for tissue repair and also as growth factor producing matrices for tissue engineering.

Gene delivery to the mouse brain with adeno-associated virus.

The efficient transduction of postmitotic cells by adeno-associated virus (AAV) makes it an excellent vector to deliver marker, functional, or therapeutic genes to the mammalian brain. An attractive feature of AAV is that all the viral-coding sequences are removed when engineering the recombinant genome, thereby limiting the extent of cell toxicity and immune response that are often associated with viral gene transcription. Of the seven described AAV serotypes, AAV serotype-2 (AAV2) is the most studied gene-transfer vehicle for in the mammalian brain. A feature of AAV2 transduction in the brain is that the vector remains confined to the injection site and predominately infects neurons rather than glia (2-8). The limited diffusion of AAV2 vectors is beneficial for controlled gene delivery. For instance, targeting therapeutic genes only to brain structures showing pathology would eliminate complications associated with vector diffusion and subsequent expression in healthy structures, and is an important consideration when designing treatment strategies for localized neurodegenerative diseases. The same is true for other experimental paradigms, such as investigating the function of genes in specific brain structures or using marker genes in tract-tracing experiments. Although AAV2 vectors were shown to remain predominately at the injection site, one study demonstrated that the vector itself may undergo axonal transport in inter-regional systems (9).

Controlled gene delivery system based on thermosensitive biodegradable hydrogel.

Currently, most pDNA delivery systems based on synthetic polymers are either nonbiodegradable or not sensitive to the release environment. The primary objective of this study was to develop and evaluate an aqueous-based, thermosensitive, biodegradable and biocompatible triblock copolymer to control pDNA delivery in vitro and in vivo. The triblock copolymers, poly[ethylene glycol-b-(D, L-lactic acid-co-glycol acid)-b-ethylene glycol] (PEG-PLGA-PEG), were synthesized as previously described. The molecular weight and polydispersity of PEG-PLGA-PEG were monitored by gel permeation chromatography (GPC). The cytotoxicity of PEG-PLGA-PEG was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The release of 32P-labeled pDNA entrapped in aqueous dispersion of PEG-PLGA-PEG in 0.1 mol/L sodium phosphate buffer solution (pH 7.4) was studied at 37 degrees C under agitation. Gene transfection efficiency was evaluated in a skin wound model in CD-1 mice. The aqueous dispersion of PEG-PLGA-PEG flows freely at room temperature but form a gel at 37 degrees C body temperature. The in vitro degradation of PEG-PLGA-PEG lasted for more than 30 days. The cytotoxicity of PEG-PLGA-PEG evaluated in HEK 293 cells was significantly lower than that of poly-L-lysine hydrochloride. The release profile of supercoiled pDNA from the polymer followed the zero-order kinetics up to 12 days. Maximal gene expression of luciferase was at 24 h in the skin wound of CD-1 mice and by 72 h, the expression dropped by nearly 94%. These results suggest hydrogel formed by PEG-PLGA-PEG could be a promising platform for delivery of pDNA, which represents a novel strategy that may serve as a non-viral vector for gene therapy in wound healing.

Enhanced gene expression in mouse muscle by sustained release of plasmid DNA using PPE-EA as a carrier.

Delivery of plasmid DNA by nanoparticles improves the DNA bioavailability, for instance in intramuscular administration, by localizing the DNA in the muscle tissue. Extracellular sustained release of the DNA may lead to more prolonged transgene expression. The present study describes a novel controlled gene delivery system based on a water soluble and biodegradable polyphosphoester, poly(2-aminoethyl propylene phosphate) (PPE-EA). The polymer degraded in PBS at 37 degrees C through the cleavage of the backbone phosphate bonds, and it was synthesized with a relative high molecular weight to ensure a suitable hydrolytic stability as a gene carrier. The tissue response and cytotoxicity study demonstrated a better tissue compatibility of PPE-EA in mouse muscle compared with commonly used polyethylenimine and poly-L-lysine. PPE-EA condensed DNA efficiently and protected DNA from nuclease and serum degradation. Sustained release of plasmid was achieved from PPE-EA/DNA complexes as a result of PPE-EA degradation. The DNA release profiles appear to be predominantly controlled by carrier degradation and the release rate of plasmid could be adjusted by varying the charge ratio of PPE-EA to DNA. At an N/P (amino to phosphate groups) ratio of 1, a 46% burst was observed for the first day, followed by about 4% release per day (24 microg DNA/day/mg of complex) for 12 days. Higher charge ratios reduced both the DNA release rate and the burst effect. The released DNA retained its structural and functional integrity. Intramuscular injection of PPE-EA-p43-LacZ complexes at N/P ratios of 0.5 and 1 resulted in enhanced beta-galactosidase expression in anterior tibialis muscle in Balb/c mice, as compared with naked DNA injections. Similarly, PPE-EA/IFN(alpha)2b DNA complexes generated an increased systemic level of interferon-alpha2b in mouse serum following intramuscular injection, as compared with naked DNA injection.

Controlled release of plasmid DNA from a genetically engineered silk-elastinlike hydrogel.

The purpose of this study was to evaluate the potential of a genetically engineered silk-elastinlike polymer (SELP) as a matrix for the controlled release of plasmid DNA. The influences of SELP concentration, DNA concentration, SELP cure time, and buffer ionic strength on the release of DNA from SELP hydrogels were investigated. To calculate the average effective diffusivity of DNA within the hydrogels, the release data were fitted to a known equation. DNA was released from SELP hydrogels by an ion-exchange mechanism. Under the conditions studied, the release rate was influenced by buffer ionic strength, SELP concentration, and SELP cure time but not DNA concentration. The apparent diffusivity of pRL-CMV plasmid DNA in SELP hydrogels ranged from 3.78 +/- 0.37 x 10(-10) cm2/s (for hydrogels containing 12% w/w SELP and cured for 4 h) to 4.69 +/- 2.81 x 10(-9) cm2/s (for hydrogels containing 8% w/w SELP and cured for 1 h). The ability to precisely customize the structure and physicochemical properties of SELPs using recombinant techniques, coupled with their ability to form injectable, in situ hydrogel depots that release DNA, renders this class of polymers an interesting candidate for further evaluation in controlled gene delivery.

Controlled gene delivery by DNA-gelatin nanospheres.

A novel system for gene delivery, based on the use of DNA-gelatin nanoparticles (nanospheres) formed by salt-induced complex coacervation of gelatin and plasmid DNA, has been developed. These particles were spherical, with a size range of 200-700 nm, contained 25-30% (w/w) DNA, and were stabilized by cross-linking of gelatin. As a consequence of being controlled by the cross-linking density of the gelatin matrix, the average release rate of DNA from nanospheres synthesized under standard conditions was 2.2%/day in serum. Nanosphere DNA incubated in bovine serum was more resistant to nuclease digestion than was naked DNA. Various bioactive agents could be encapsulated in the nanospheres by ionic interaction with the matrix components, physical entrapment, or covalent conjugation. Transfection of cultured cells with a luciferase plasmid was enhanced by conjugating human transferrin onto the nanosphere and coencapsulating the endolysolytic agent chloroquine. Under our experimental conditions, gene expression in mice subsequent to intramuscular injection of nanospheres containing 1 microg of a beta-galactosidase plasmid was greater and more prolonged than was observed after injection of an equal amount of naked DNA or DNA complexed with Lipofectamine.

Polyvinyl derivatives as novel interactive polymers for controlled gene delivery to muscle.

DNA plasmids (pDNA) can be taken up by and expressed in striated muscle after direct intramuscular injection. We have developed interactive polymeric gene delivery systems that increase pDNA bioavailability to muscle cells by both protecting pDNA from nucleases and controlling the dispersion and retention of pDNA in muscle tissue. A DNA plasmid, containing a CMV promoter and a galactosidase reporter gene (CMV-beta-gal), was injected either in saline or formulated in polyvinyl pyrrolidone (PVP) and polyvinyl alcohol (PVA) solutions. Interactions between PVP and pDNA were assessed by dynamic dialysis, Isothermal Titration Calorimetry (ITC), and Fourier Transformed Infra Red (FT-IR) spectroscopy. Formulations (50 mu l) were injected into rat tibialis muscles after surgical exposure. Immunohistochemistry for beta-gal was used to visualize the sites of expression in muscle. Beta-gal expression using pDNA in saline reached a plateau while beta-gal expression using PVP formulations increased linearly in the dose range studied (12.5-150 mu g pDNA injected) and resulted in an increase in the number and distribution of cells expressing beta-gal. The interaction between PVP and pDNA was found to be an endothermic process governed largely by hydrogen-bonding and results in protection of pDNA from extracellular nucleases. Significant enhancement of gene expression using interactive polyvinyl-based delivery systems has been observed. The improved tissue dispersion and cellular uptake of pDNA using polyvinyl-based systems after direct injection into muscle is possibly due to osmotic effects.