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Gene therapy is the introduction of an exogenous normal gene or a therapeutic gene into a target tissue or target cell by a vector or other means, and is appropriately expressed to treat a disease. The key to gene therapy is to obtain efficient and safe gene delivery vectors. Vectors for delivering genes are generally classified into viral vectors and non-viral vectors. Viral vectors are the most widely used gene delivery vectors, including retroviruses, adenoviruses, adeno-associated viruses, and lentiviruses. The biggest advantage of viral vector is the high transfection rate, but there are also many shortcomings, such as difficulty in preparation of virus, limitation of the size of foreign DNA loaded, cytotoxicity, immunogenicity, and carcinogenicity. The originality and tumorigenicity have not yet been completely solved, and these disadvantages have greatly limited the clinical application of the viral-type carrier.
At present, non-viral vectors do not reach the extremely high transfection efficiency of viral vectors, but non-viral vectors make up for the defects of viral vectors, with advantages like high safety, low immunogenicity, simple preparation, tight binding to DNA, no limitation of DNA fragment size, and the ability to deliver plasmid DNA to target cells by targeted modification, which makes gene therapy possible in clinical applications. Nano-gene delivery technology uses nanoparticles as vectors, and encapsulates DNA, RNA, etc. in nanoparticles or adsorbs on their surfaces, and couples specific targeting molecules (monoclonal antibodies, etc.) on the surface of the particles. It utilizes the interaction between the target molecules on the nanoparticles and the cell surface-specific receptor to target aimed cells. Under the action of lysosome, the nanoparticle is degraded to release the therapeutic gene, and the targeted gene therapy is realized. It can protect nucleic acid from enzymatic degradation, increase the amount of nucleic acid into the cell, enhance its stability in the cell, prolong the sustained expression time of the gene, help to improve the efficiency of cell transfection, and possibly achieve localization targeting delivery.
Inorganic Nanomaterial
1. Hydroxyapatite nanoparticles
Hydroxyapatite (HA) is one of the components of human bones and teeth and has good biology activity, chemical stability, and biocompatibility, which is a common biological material for clinical bone defect repair. The particle size of HA nanoparticles ranges from 1 to 100 nm, and has the common characteristics of general nanomaterials such as macroscopic quantum effect, small size effect, specific surface effect and interface effect. HA is a highly efficient adsorbent material widely used for the separation and purification of proteins and nucleic acids. The preparation of HA nanoparticles is mainly liquid phase synthesis, including hydrothermal reaction, precipitation, sol-gel and microemulsion methods. Compared with silica nanoparticles, HA has better biofilm compatibility, strong nucleic acid adsorption capacity, and can be designed according to needs. Further discussion of its transduction mechanism, toxicology and preparation process, and improvement of its transduction rate are the key to promoting the application of HA nanoparticles in gene therapy.
2. Gold nanoparticles
Gold nanoparticles (AuNPs) are usually 10-20 nm in diameter and are generally used in the form of gold sols, also known as colloidal gold. It is a new material with many biological functions, good biocompatibility and no obvious toxicity to cells. It has a variety of synthetic methods for preparing gold nanoparticles with particle sizes ranging from a few nanometers to hundreds of nanometers. AuNP has a strong adsorption effect on many biomacromolecules and does not denature biological macromolecules. It has been reported that AuNP transports DNA to the nucleus eight times more efficiently than PEI and has been successfully used in cancer therapy. AuNP crosses the cell membrane either by endocytosis or by direct infiltration into target cells, which may have cytotoxic effects during this process.
Therefore, in the design and preparation of nanogold, the balance between achieving an effective control of the amount of nanoparticles passing through the cell membrane and its potential toxicity is the key to the study. Au55 clusters are effective in interacting with DNA. This effect is related to the particle size, and the cluster of gold particles with small particle size can be inserted. The surface of the gold can be covalently linked to the thiol, so the DNA can be immobilized on the AuNP after thiolation. An AuNP can bind up to several hundred DNA molecules. Despite the broad application prospects of AuNP vectors, the cytotoxicity of AuNP still limits its biological applications.
3. Carbon nanotubes
Carbon nanotubes (CNTs) have typical layered hollow structure features, and their body part is composed of six sides. The carbon ring microstructure unit has a polygonal structure composed of a pentagon-shaped carbon ring. CNTs can be classified into single-walled carbon nanotubes (SWNTs) and multiwalled carbon nanotubes (MWNTs) according to their structure and dimension. The strong acid-treated MWNT was modified with PEI to react acetic anhydride and succinic anhydride with the amino group of PEI on the surface of MWNT to obtain MWNT with uncharged and negatively charged surface, respectively. It was introduced into FRO cells (thyroid cancer cell line) and KB cells (epidermal cancer cell line) in vitro, indicating that its biocompatibility is related to its surface charge. The CNTs used as gene carriers need to be functionalized and modified, mainly because unmodified CNTs are insoluble in body fluids and are toxic.
4. Quantum dots
Quantum dots (QDs) are particles with a particle size of a few nanometers and consist of II~IV or I~V semiconductors, such as CdSe, ZnSe, GaAs, InAs, and the like. QD is inherently toxic due to the presence of surface cations (such as Cd2+) and the formation of photoradicals. Surface modification can reduce QD toxicity. Combining amphiphilic polymers with QD produces a novel gene delivery system that is superior to liposomes in serum-free and complete cell culture media and is less toxic than PEI in gene silencing experiments. Due to the intrinsic emission of fluorescence and excellent optical properties, QD is widely used for siRNA delivery and in vitro and in vivo imaging. L-arginine-modified functionalized CdSe/ZnSe QD is an effective confrontation against nucleic acid degradation, which successfully delivers siRNA, and simultaneously acts as a fluorescent probe for real-time in vivo image tracking. At present, the research on QD shows that the transfection rate is low and has certain toxicity, but its excellent optical properties of intrinsic emission fluorescence make QD as a gene carrier with certain development prospects.
5. Silica nanoparticles
The synthesis of SiO2 is simple and the controllability is strong, and its outstanding advantages are that the surface modification range is wide, and the wide variety of silane reagents provides good conditions for the functionalization of SiO2. Taking advantage of the phenomenon that DNA is specifically adsorbed on the surface of SiO2 under high salt conditions, SiO2 nanoparticles without any functionalization can be used as an effective DNA extraction material. The positively modified SiO2 particles (aminosilane, divalent cations, etc.) on the surface can effectively protect genetic material from enzymatic degradation, and have a good gene transfection effect in vivo and in vitro. Antisense oligonucleotide-containing aminated SiO2 nanoparticles, with an average particle size of 25 nm, can protect antisense oligonucleotides from being decomposed by nucleases, improve transfection rate, prevent cancer cell differentiation and proliferation, and have better biocompatibility.
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