99results about How to "High transfection rate" patented technology

The invention discloses a gold nanorods based drug carrier. The carrier is characterized in that: gold nanorods is wrapped with a polyanion film layer, a polycation film layer and an hTERT gene antisense oligonucleotide layer from inside to outside. The gold nanorods based drug carrier is prepared by the layer-by-layer self-assembly principle by utilizing the electrostatic force among radicals, and can be used for preparing inhibitors for malignant tumor cell telomerase. The invention causes hTERT gene antisense oligonucleotide to be loaded on the gold nanorods for the first time to solve the problem of low transfection rate occurred in naked antisense oligonucleotide. The prepared gold nanorods based drug carrier does not only has the advantages of high transfection rate, strong targeting property, obvious effect of inhibiting the activity of the malignant tumor cell telomerase, and the like, but also has the advantages of safety and innocuity, which opens up a new way for the application of the gold nanorods in the biomedicine field; in addition, the invention has the advantages of simple operation for the preparation technology and low cost, and is suitable for scale production.

A method of delivering an oligonucleotide or a plasmid expressing an oligonucleotide into a target cell comprises introducing an oligonucleotide into a donor cell, particularly a stem cell, and contacting the target cell with the donor cell under conditions permitting the donor cell to form a gap junction with the target cell, whereby the oligonucleotide or a product of the oligonucleotide is delivered into the target cell from the donor cell.

The invention relates to a novel cationic graft copolymer, and a preparation method and an application of a multiple composite non-viral gene vector. The novel cationic graft copolymer PCL-g-LPEI (polycaprolactone-g-linear polyethylenimine) is synthesized by ring-opening copolymerization and amidation reaction. The multiple composite non-viral gene vector comprises: (A) a plasmid DNA (deoxyribonucleic acid)-supported PCL-g-LPEI cationic compound serving as a kernel of a multiple gene compound; (B) a tumor-targeting ligand HA (hyaluronic acid) wrapping the cationic compound under the interaction of positive and negative charges to form a shell of the multiple gene compound. A multiple composite gene constructed by the non-viral gene vector has the advantages of low cytotoxicity, high blood compatibility, high transfection efficiency, tumor targeting effects and the like, and is expected to be applied to clinical gene therapy.

A method for using the ultrasonic microvesicle contrast medium as the carrying tool of gene to locate the gene and control to release it to target tissue for transfection. After the contrast medium reaches the target tissue under themonitor of audio-video image, the ultrasonic waves are used to destroy the microvesicle for releasing the gene and performing the gene transfection.

The invention relates to an engineering CD20 targeting NKT cell and a preparation method and application thereof. The cell is an NKT cell modified by chimeric antigen receptor CD20ScFv-CD8-CD137-CD3 sigma, an amino acid sequence of the chimeric antigen receptor is as shown by SEQID NO. 1 in a sequence list. The preparation method comprises the following steps: firstly establishing a chimeric antigen receptor pWPT-CD20ScFv-CD8-CD137-CD3 sigma, infecting the NKT cell, performing in-vitro induction and multiplication culture, and obtaining the engineering CD20 targeting NKT cell. When the engineering CD20 targeting NKT cell is used for treating a CD20 positive malignant tumor at a progressive stage, the survival time of the immune cell in the body of a patient can be obviously prolonged, the capacity of the immune cell for target identifying a tumor antigen is improved, and the killing activity for the tumor cell is improved.

The invention discloses a cavitation-bubble-mediated laser transfection method. According to the method, laser is used to irradiate nano particles with especially strong light absorptivity, the nano particles are combined with a specific cell through a specific ligand and a specific receptor, and cavitation bubbles are generated at the peripheries of the nano particles which are irradiated by the laser. The generation of the cavitation bubbles causes a cell membrane to be temporarily permeable, and thus the transfection of an allogenic material is achieved. The laser is contactless and free from pollution and cytotoxicity, so that the safe gene transfection method is realized; and moreover, as the cell membrane is caused to be permeable through generating the cavitation bubbles, the method is a physical method, can realize the transfection of any type of cells, can realize transfection in large area, and does not need special, expensive and precision instruments.

A method of delivering an oligonucleotide or a plasmid expressing an oligonucleotide into a target cell comprises introducing an oligonucleotide into a donor cell, particularly a stem cell, and contacting the target cell with the donor cell under conditions permitting the donor cell to form a gap junction with the target cell, whereby the oligonucleotide or a product of the oligonucleotide is delivered into the target cell from the donor cell.

This invention relates to an antisense oligonucleotide-carbon nanotubes medicine carrier which comprises carbon nanotube casing and antisense oligonucleotide. The preparation method comprises oxidizing multi-wall carbon nanotube with strong acid to carry carboxyl on the shell of pipe, cutting to 50-200nm with diameter of 10-50nm with ultrasound, adding poly-cationization bichloride to embellish on shell of carbon nanotube, and assembling antisense oligonucleotide to carbon nanotube. The preparation method is simple and rapid. The medicine carrier has high transfection efficiency, high target effect, and good effect in inhibiting tumor cell. Targeted bimolecular or medicine with carbon nanotube as carrier material aim at different biological system can be applied for treating tumor or genetic diseases.

The invention discloses a non-viral gene carrier of autofluorescence degradable poly-citrate and a preparation method thereof. Citric acid, 1,8-octylene glycol and polyethylene glycol are subjected to thermal polymerization to obtain a POCG prepolymer; then, the POCG and PEI (polyethyleneimine) are subjected to catalytic polymerization to obtain a POCG-PEI polymer; the POCG-PEI polymer and various genes (DNA / siRNA / miRNA) can form a stable nanocomposite in HEPES buffer liquid. The used thermal polymerization method has the advantages that the environment is protected; the operation is convenient; the raw material cost is low. The prepared POCG-PEI has good biocompatibility and high gene transfection efficiency; meanwhile, the POCG-PEI also shows certain fluorescence characteristics and optical stability, so that the polymer has good application prospects in the gene treatment process.

This invention provides a method for delivery of agents such as genes, plasmids, and other active DNA-related molecules useful for treatment peritoneal disease, including peritoneal fibrosis or postoperative adhesion specifically using an ultrasound-triggered disruption of inducible Smad7 gene-bearing microbubble system.

The invention discloses an amphipathic triblock copolymer containing a morpholine-2,5-diketone derivative copolymer as well as a preparation method and application thereof. The copolymer is formed by connecting methoxy polyethylene glycol, the morpholine-2,5-diketone derivative copolymer and polyethylene imine in sequence, wherein molecular weight of the amphipathic triblock copolymer is 2000-105000; the morpholine-2,5-diketone copolymer is a copolymer of a morpholine-2,5-diketone derivative monomer and one or two cyclic monomers. An experiment proves that the copolymer disclosed by the invention is low in cell toxicity and high in transfection efficiency. The preparation method disclosed by the invention is simple and pollution-free. The surface of a nano-particle prepared by the copolymer disclosed by the invention is linked with a plurality of low molecular weight PEI (Polyetherimide) and hydrophilic PEG (Polyethylene Glycol) chain segments, so that transfection efficiency of the nano-particle is improved while cell toxicity of the nano-particle is lowered. Besides, the amphipathic triblock copolymer has good biocompatibility and can effectively carry genes to enter cells.

Disclosed are compositions comprising carbohydrate-modified polymers, such as cyclodextrin-modified polyethyleneimines, for the delivery of drugs and other active agents, such as nucleic acids. Compositions containing carbohydrate-modified polymeric carriers that release the agent under controlled conditions are also disclosed. The present invention also discloses compositions comprising carbohydrate-modified polymeric carriers coupled to biorecognition molecules for targeted delivery of drugs to their sites of action.

The invention belongs to the field of tumor cell immunotherapy, and particularly relates to a recombinant CAR gene and carrier, CAR-T cell and application thereof. The recombinant CAR gene includes a nucleotide sequence coded the following parts of an antigen-binding part of a CD19 antibody, a transmembrane part and a CD28 endochylema functional domain, a 41BB endochylema functional domain and a CD3zeta endochylema functional domain connected in a random sequence. The efficiency of using the recombination CAR gene and the carrier containing the recombinant CAR gene to transfect T cell is high, and the positive T cell through transfection has good multiplication capacity. Besides, the recombination CAR gene is high in specificity to B cell tumor antigen, and prompts CAR-T to kill and wound B cell tumor specifically.

The invention belongs to the technical field of biological medicines and aims at improving the gene transfection efficiency by adopting nano-particles NoNPs released through cells. The nano-particles exist extensively in a biosystem and an extracellular circulatory system, such as bronchoalveolar lavage fluid, body fluid, blood, saliva, cow milk or urine. A non-virus gene transfection vector is doped with the nano-particles so that the DNA (Desoxvribose Nucleic Acid) carrying capacity of the gene transfection vector can be improved remarkably without increase of cytotoxicity. Experimental results indicate that the NONPs, which are extracted from the cow milk and added to the PEI gene transfection vector, are capable of remarkably improving the gene transfection efficiency under the circumstance of hardly increasing the cytotoxicity. The major use of the nano-particles is to serve as the gene transfection reagent which can be used for cell biology research, gene transfection preparation production, gene therapy and the like.

A viral transduction and / or electroporation device has s a membrane separating two chambers and two electroporation electrodes for the chambers. An electrical voltage source is used for establishing an electrical field across the membrane and between the two electrodes. In operation, fluid is flowed into the chambers including fluid containing electroporation cargo and viral transduction solution and an electrical field is established across the membrane and between the electrodes to electroporate cells pinned to the membrane and transfecting the cells.

The invention discloses a preparation method of a difunctional liver cancer targeted nanobubble carrying NET-1siRNA. The method comprises the following steps: (1) synthesizing 3 pairs of NET-1siRNA sequences, and biotinylating the sequences; (2) preparing a biotinylated nanobubble; (3) synthesizing biotinylated galactosylated polylysine; and (4) coupling the biotinylated NET-1siRNA, the biotinylated nanobubble and the biotinylated galactosylated polylysine through a biotin-avidin system to obtain the difunctional liver cancer targeted nanobubble carrying NET-1siRNA. The liver cancer targeted nanobubble can be used in clinical ultrasonic diagnosis as a targeted ultrasonic contrast agent; and more importantly, the NET-1siRNA carried by the liver cancer targeted nanobubble can inhibit tumor growth, so the liver cancer targeted nanobubble can be used in gene therapy of the liver cancer.

The invention belongs to the field of genetic engineering techniques disclosing the recombinant and application of intelligent adenovirus vector and khp53 anti-oncogene. The invention specifically involves recombinant viral vector of human tumor-suppressing gene, especially involves human p53 tumor-suppressing gene recombination intellectual adenovirus vector conducted by Kozak sequence, and also involves drug combinations with the gene recombinant vector. Through the combination utilization of elements enhancing eukaryotic gene expression such as MCMV promoter, intron and Kozak regular sequence etc, the problem of relative shortage expression of the p53 gene is solved; and through adopting AdMAX HiIQ recombinant adenovirus vector system, the problem of effective packaging exogenous gene high expressing the recombined adenovirus effectively, and finally,through site-specific homologous recombination in eukaryotic cells, 5-recombinated adenovirus rAdMH-khp53 with E1, E3 deleted is packaged out. Utilizing recombinant adenovirus of the invention, clinical-used gene drugs can be prepared for prevention and treatment of a variety of cancer.

The present invention relates to process of leading plasmid carrier containing gene therapy segment into cell with nanometer particle. The process includes the following steps: inoculating cell in good growth condition to cell culturing plate and culturing; centrifuging nanometer inorganic salt suspension and dissolving the precipitate in deionized water in 500 microliter; dissolving 2 microliter of plasmid in 98 microliter of deionized water; compounding 1 ml of suspension with solution of nanometer particle in 5-10 mg and solution of plasmid in 4-15 microliter through shaking for best combination between the nanometer particle and the plasmid; centrifuging the suspension to obtain the precipitate, adding deionized water, adding to the cell culturing plate, and adding fresh culture liquid to culture for the nanometer particle well combined to the plasmid to transfect the cell; and finally observing the transfection effect in a microscope.

The present invention, pertaining to the field of biomedicine and bioengineering, relates to the gene delivery system technology related to the gene therapy, specifically to a mediated gene conversion method. The invention combines the ultrasonic targeted damage microbubble technology and gland relevant viral vector technology at the appropriate time and mode to realize advantage complementary, so that the target gene can be more safely and efficiently entered in the histiocytes to realize the long-term stable expression of the target gene. The method described in this invention can be used in the vivo experimental investigation of AAV carrying of different target genes for gene therapy, specifically in the non-AAV direction cells or tissues; the invention can safely improve the AAV carrying gene transfection efficiency, speed up gene body expression, increase the targeting of the transfection, reduce the virus dosage and diminish the toxic side effects.

The invention discloses a turbot muscle cell line establishment method. According to the turbot muscle cell line establishment method, turbot muscular tissues are adopted as materials, and primary culture is started by a single cell method; the turbot muscular tissues are cultured in an L15 culture medium with fetal calf serum and fibroblast growth factors, and subculture is performed by a trypsin digestion method; a cryopreservation culture medium with DMSO (dimethylsulfoxide) is adopted for cryopreservation. The turbot muscle cell line establishment method has the advantages that an established turbot muscle cell line has passed by 35 generations and is quick and stable in growth and capable of providing a large quantity of cells for researches on nutrition, immunity, environmental toxicity, gene function analysis and the like.

The invention discloses a parental plasmid for obtaining minicircle DNA, and an application thereof. The parental plasmid is constructed by inserting an attR segment and an attL segment and a [Phi]C31 integrase gene expression cassette in a starting vector pEGFP-N1-attB, wherein the attR segment is inserted between cleavage sites of restriction enzymes SpeI and DraIII of the starting vector; the attL segment is inserted in the cleavage sites of restriction enzymes Af1 III and Ase I of the starting vector; and the [Phi]C31 integrase gene expression cassette is inserted in a cleavage site of the restriction enzyme Af1 III of the starting vector. The minicircle DNA for stable integration can be obtained rapidly, efficiently and economically by using the parental plasmid; subsequent transgenosis operational method is simple and practicable; and the parental plasmid has important biology significance and practical application values.

The invention discloses ultrasonic microbubbles for treating arthritis and a use thereof. The microbubbles contain gene plasmids capable of expressing tumor necrosis factor receptor (TNFR) protein, wherein the gene plasmids are adsorbed to the surface of the microbubbles and / or packaged in the microbubbles; the gene plasmids are expression plasmids; the gene plasmids of the tumor necrosis factor receptor protein contain extracellular domain gene of the tumor necrosis factor receptor (TNFR) or contain gene segment of the protein or peptide fragment, which can be combined with a tumor necrosis factor (TNF); and the gene plasmids express soluble protein or peptide fragment which can prevent the tumor necrosis factor from combining with the tumor necrosis factor receptor on a cell membrane after transfecting the body cells. The ultrasonic microbubbles are not only low in manufacturing cost for greatly relieving the economic burden of the patients and families, but also have breakthrough development in the field of treating anthritis, and can be extensively popularized and used in the field.

The invention relates to an implanted in vivo electrotransfection device, which is used for transferring a photosensitive target gene into a target cell in a transplantation part. The electrotransfection device comprises a central control system, an electrode system and an injection system, wherein the central control system is connected with the electrode system and the injection system for controlling the electrode system and the injection system; the injection system is used for injecting the target gene into the target cell; and the electrode system generates electric pulses to transfer the target gene into the target cell and acquires an electrophysiological signal of a transplantation part and feeds the electrophysiological signal of the transplantation part back to the central control system. The implanted in vivo electrotransfection device combines a micro-injection technology, electrotransfection technology and microelectrode measurement technology and can realize the transfection of a target neuron, optogenetic control and electrophysiological signal measurement. As a pure physical method, electrotransfection avoids threats from chemical and biological toxicity. The transfection can be widely used in cells and the transfection is high.

The invention discloses construction and preparation of double-target chimeric antigen receptor T (CAR-T) of FLT3-NKG2D. CAR of the invention belongs to double-target second-generation CAR, and has the functions of overcoming tumor immune escape and enhancing the specific recognition and killing of tumor cells. By combination of the double-target CAR-T and gilteritinib, the treatment dilemma of refractory acute myeloid leukemia of FLT3<mut+> can be effectively solved, and a new treatment strategy is provided for treatment of acute myeloid leukemia.

The invention discloses a preparation method and application of an aminated graphene quantum dot. The preparation method includes: blending a uniform dispersion liquid of prepared graphene, ammonia water and deionized water, then transferring the mixture into an autoclave for heating, filtering out insoluble fragments by a porous inorganic membrane, then conducting heating to remove redundant amine, and then using a centrifugal filtration device of molecular weight interception membrane for ultrafiltration of the supernatant, and subjecting the obtained yellow suspension liquid to further dialysis by a Spectra / Por CE dialysis tube membrane, thus obtaining aminated graphene quantum dot. The aminated graphene quantum dot prepared by the method provided by the invention can be applied to gene delivery vectors, has no inhibiting effect on RNAnase, has protective effect on to-be-delivered miRNA, and has good selectivity and universality.

The invention discloses a targeting triblock copolymer containing a morpholine-2,5-dione derivative copolymer, a preparation method and application. The targeting triblock copolymer containing the morpholine-2,5-dione derivative copolymer is a copolymer which is composed of the morpholine-2,5-dione derivative copolymer, polyethyleneimine and CREDVW and possesses the molecular weight of 3600-98000. Experiments prove that the triblock copolymer containing the morpholine-2,5-dione derivative copolymer is low in cytotoxicity and high in transfection rate and possesses endothelial-cell targeting function. The method is simple and free of pollution, and a nanoparticle prepared from the triblock copolymer containing the morpholine-2,5-dione derivative copolymer possesses good biocompatibility, is capable of effectively carrying a gene and entering a cell and posseses blood-vessel endothelial-cell targeting property.

The invention belongs to the field of gene treatment, provides a new gene therapy product, and specifically relates to a cationic liposome compound of cancer suppressor gene LKB1 eukaryotic expression plasmid as well as a preparation method and an anti-tumor effect thereof. The gene expression carrier contains genes encoding human LKB1 protein and can express the human LKB1 protein in eukaryotic cells. Based on experiments, the cationic liposome compound of cancer suppressor gene LKB1 eukaryotic expression plasmid, disclosed by the invention, has excellent functions of resisting tumor growth and invasion and metastasis so as to provide a new selection for the tumor treatment.

The invention discloses a copolymerization micelle drug-loading nanoparticle. The copolymerization micelle drug-loading nanoparticle is a block copolymer comprising a discharged hydrophilic polymer chain block, a discharged hydrophobic polymer chain block and a cationic polymer chain block. The discharged hydrophilic polymer chain block and the cationic polymer chain block are positioned at two ends of the discharged hydrophobic polymer chain block through chemical bonds. The cationic polymer chain block is grafted with a lipid ligand. The nano drug-loading particle as a triblock copolymer self-assembled micelle system is capable of realizing co-transmission of siRNA and a chemotherapy drug. A triblock copolymer is amphipathic, and can be self-assembled into the micelle nanoparticle. Preparation cost is low and preparation is convenient. A hydrophilic part is capable of reinforcing circulation time. The discharged hydrophobic polymer chain block is used for wrapping adriamycin so as toform a hydrophobic kernel. The cationic polymer chain block carries positive charge, so that the negative charge siRNA can be carried. Cholesterol is connected to the cationic polymer chain block, sothat efficiency of endocytosis is improved.

The invention relates to the technical field of molecular biology, in particular to a mir-124 and HER2-shRNA double-gene expression cassette virus vector, a construction method, a virus and application. The double-gene expression cassette virus vector is constructed by integratively connecting a mir-124 sequence and a HER2-shRNA sequence in a virus expression vector, wherein the mir-124 sequence is shown as SEQ ID NO.1; the HER2-shRNA sequence is shown as SEQ ID NO.2 and SEQ ID NO.3. The transfection rate of the mir-124 and HER2-shRNA double-gene expression cassette virus vector for breast cancer cells is as high as 80 to 90 percent. The mir-124 and HER2-shRNA double-gene expression cassette virus vector can express mir-124 and HER2-shRNA efficiently and stably in the breast cancer cells,plays a synergistic role, can remarkably inhibit the proliferation of the breast cancer cells and promote apoptosis,and can be applied to gene therapy of HER2-positive tumors such as breast cancer andovarian cancer.