Gene Delivery and Tissue Engineering of Polymer Vectors

Summary * Gene release and tissue engineering of polymer carriers Liu Wenguang, Zhang Xin, Sun Guangjie, and Yao Kangde proposed the core issue of their technology is the sustained release of genes using biodegradable macromolecules as carriers; Molecular gene-based gene release and its research progress in tissue engineering applications, and refers to the existing problems.

With the development of the disciplines of cell biology, molecular biology, clinical medicine, materials science and related physical chemistry, researchers have put forward the concept of organizational engineering on the basis of multidisciplinary research. The purpose of tissue engineering is to directly regenerate normal tissues from living cells to replace damaged tissues or organs. Typical methods used in typical tissue engineering include: (1) Transplanting in vitro cultured cells onto biodegradable macromolecules and cells growing on three-dimensional scaffolds of the matrix material; the disadvantage of this method is the need for in vitro separation and amplification. Cells, and many types of cells survive after transplantation; (2) release of tissue-inducing proteins such as bone morphogenetic protein (P-P) and platelet-derived growth factor (PDGF). The obvious disadvantage of this method is that the protein is prone to denaturation under the influence of humidity, temperature, chemical environment, or the degradation products of polymer matrix. W. Contrast to the shortcomings of the existing tissue engineering technology, Mooney research group in gene therapy and tissue engineering The concept is based on the concept of macromolecules as carriers, sustained-release coding for inducible tissue proteins (tissuei- 03-29; revision date : 2002-09-04 Funded Project: National Key Fund (No. G1999054305) and National Postdoctoral Fund Support ( No. 2(1) 2031165) To study biopolymer materials.

Ductive) plasmid DNA method to achieve tissue reconstruction. The core issue of this technology is the slow release and transfection of genes using biodegradable polymers as carriers. In order to increase the transfection rate of genes, researchers use bacteria as a carrier of DNA, but the infectivity, carcinogenicity, and immunogenicity of bacterial vectors have limited their use. From the perspective of the safety of living organisms, domestic and foreign researchers have developed a large number of non-viral vectors, such as cationic liposomes, polylysine, diethylaminodextran, polyethylenediamine, dendrimers, and shell polymers. Sugar and so on. This article will review the progress of gene release using macromolecules as a carrier and its application in tissue engineering.

1 Liposomes/DNA complexes Negatively charged DNAs can form complexes with positively charged liposomes through electrostatic interactions. Researchers conducted extensive and in-depth studies of their structure. Ridler et al. studied the structure of human phage DNA and lipid complexes by simultaneous irradiation small angle X-ray diffraction (SAXS). They proposed a multi-sandwich structure in which DNA was alternately embedded in lipid bilayers to form two-dimensional, smectic liquid crystals. A model diagram of the complex is shown. The round bar represents the DNA double helix. The head of the lipid is represented by black and white circles. The dmcm is a long multi-sandwich cycle and the dDNA is the distance between the DNA helixes in the same layer. In vitro experimental curves for cumulative release of plasmid DNA from PLG. In vitro experiments showed that DNA can maintain sustained release in all three PLGs. Electrophoresis experiments showed that the released DNA structure remained intact in the range of 1 to 28 days. In vitro experiments Plasmid DNA encoding nt 6-gal was transfected into 293T cells and a high percentage of cells could be transfected, indicating that the released DNA retained its function. The PLG encoding the nt 6-gal plasmid DNA was loaded into Lewis rats subcutaneously, and after 4 weeks a large number of cells around the implant were observed to be transfected. PLGs loaded with PDGF were implanted subcutaneously in rats. Within 2 weeks to 4 weeks, the increase in the number of blood vessels and the thickness of the granules was observed, suggesting the release of gene expression and the effect on tissue formation. The low-temperature, solvent-free gas foaming method pioneered by Mooney et al. to prepare porous three-dimensional carriers avoids the disadvantages of organic solvents and the inactivation and inflammatory reactions of bioactive molecules caused by high temperatures. By comparison, it was confirmed that direct injection of DNA into Kchaidson et al. studied the gene release characteristics of the low relative molecular weight in more detail. The experimental results showed that chitosan showed low toxicity to CCRF-CEM and L132 cells and no hemolysis in the selected relative molecular weight range. Chitosan and DNA all blocked the enzymolysis of the three groups. After intravenous injection, chitosan was quickly cleared in the blood. 125I labeling experiments showed that the intrahepatic accumulation of chitosan was molecular weight dependent. The intrahepatic accumulation of chitosan with a relative molecular weight of less than 5000 is very low, and the intrahepatic accumulation of chitosan with a relative molecular weight of more than 5,000 is higher. Therefore, when choosing a gene carrier, the factors that affect the relative molecular weight cannot be ignored.

According to the latest study, chitosan/DNA nanoparticles can cross cell membranes without ligand-receptor interactions. Chan et al. studied the interaction between chitosan and dipalmitoyl-glycero-3-phosphocholoroyl (DPPC) membrane bilayer and found that the intermolecular and intramolecular interaction forces of hydrophobic chains in DPPC are due to chitosan and membranes. The strong effect and significant reduction, chitosan also reduces the order of the two-dimensional accumulation of acyl chains, DPPC membrane perturbation increased, plus DPPC double fluidity. This experimental result reveals the mechanism of chitosan transmembrane.

3 Dendrimer/DNA composites Dendrimers are a class of hyperbranched molecules. Their highly branched structures make their physicochemical properties very different from those of linear molecules. Recently, cationic polyamino dendrimers have been tried as vectors for transgenes. Under physiological pH conditions, the amino-terminated dendrimer chain can be partially protonated so that it can form a complex with negatively charged DNA through electrostatic interaction. Choi et al. found that only the fourth-generation dendrimer copolymer forms spherical particles with DNA and protects the DNA from DNase attack. In vitro experiments indicate that dendrimers from the fifth to tenth generations can carry DNA into the cell membrane to obtain highly efficient gene expression and exhibit only low toxicity to a wide range of mammalian cell lines. The dendrimer regulates gene expression in vitro by transfecting antisense oligonucleotides or antisense expression plasmid DNA into cells. The dendrimers of the seventh to tenth generations have a diameter of 8 to 130 A) and are similar in size and shape to histones. Therefore, the high-order dendrimer-DNA complexes are more stable and more conducive to improvement. Gene transfection. Bielindca found that the dendrimers/DNA complexes remained active after drying, and that the complexes were bound to bioabrasive membranes for gene transfection of skin cells in vitro and in vivo. M. 4 Collagen/DNA Complex system 6] The technique of releasing genes using macromolecules as the carrier was first used to transfect K-2-80 cells in tissue tissues; while slow-released DNA from PLG can transfect 105-106 cells, with only a large amount of transfer. Stained cells produce enough gene-encoded proteins that eventually form observable physiological effects, such as the formation of blood vessels and granular tissue.

6 Conclusions On the basis of the existing concept of tissue engineering and gene release, the use of macromolecules as carriers to release genes encoding induced tissue proteins has shown potential applications in tissue repair and reconstruction. According to the US "Science" magazine, scientists have fixed the parathyroid hormone (PTH) gene plasmid DNA DNA fragment into a polymer matrix, and then transplanted in the canine leg defect, the plasmid DNA expression PTH up to 6 Weeks, the bones are completely repaired and are currently being applied for Phase I clinical trials. However, objectively speaking, this technical method still has many problems to be solved, such as the degradation rate of macromolecules, cell and genotoxicity, enzymatic hydrolysis of DNA, barriers across cell membranes, and dissociation of DNA from carriers. Release and translocation of DNA into target cell nuclei, etc. It is precisely for the problems that have been recognized and the strong interest in gene release and its application in tissue engineering. Researchers in various countries are making unremitting efforts to introduce gene therapy into tissue engineering and promote the clinical application of tissue engineering as soon as possible.