WO2022227927A1 - Matériau polymère dégradable et nanocomposite auto-assemblé et son application - Google Patents

Matériau polymère dégradable et nanocomposite auto-assemblé et son application Download PDF

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WO2022227927A1
WO2022227927A1 PCT/CN2022/081850 CN2022081850W WO2022227927A1 WO 2022227927 A1 WO2022227927 A1 WO 2022227927A1 CN 2022081850 W CN2022081850 W CN 2022081850W WO 2022227927 A1 WO2022227927 A1 WO 2022227927A1
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cpd
det
cells
polydisulfide
monomer
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平渊
郭家晶
万涛
辛虎虎
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浙江大学
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G75/00—Macromolecular compounds obtained by reactions forming a linkage containing sulfur with or without nitrogen, oxygen, or carbon in the main chain of the macromolecule
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

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  • the invention belongs to the fields of organic chemistry, polymer chemistry, molecular biology, cell biology, biotechnology and the like, and relates to a degradable polymer material and a self-assembled nanocomposite and its application.
  • nucleic acid refers to the transfer of exogenous genetic material into cells, so as to realize the regulation of cell biological functions and the treatment of diseases.
  • nucleic acid includes various types of nucleic acid polymers such as, but not limited to, plasmid DNA (pDNA), messenger RNA (mRNA), small interfering RNA (siRNA) or antisense oligonucleotides (ASO).
  • pDNA plasmid DNA
  • mRNA messenger RNA
  • siRNA small interfering RNA
  • ASO antisense oligonucleotides
  • Nucleic acid delivery systems can be divided into three distinct categories: (i) physical methods, (ii) viral delivery systems, and (iii) non-viral delivery systems.
  • Non-viral DNA delivery systems include lipid or liposome materials, inorganic materials and organic polymer materials, which have obvious advantages over physical methods and viral delivery systems: 1. With hydrophilic and hydrophobic ends, it is easy to form small nanovesicles Or micelles; 2. Degradable, low toxicity; 3. No immunogenicity; 4. The surface can be targeted modified to improve the delivery efficiency of drugs; Design; 6. Stable in nature and easy to prepare on a large scale; 7. It can form stable nanocomplexes with biological macromolecules such as DNA, and is easy to deliver.
  • Gene editing or genome engineering is a technique for the targeted insertion, replacement or deletion of specific DNA sequences in the genome of a host cell.
  • nucleases There are four different nucleases available for this strategy so far: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system.
  • the DNA vector can encode both Cas9 protein and target sequence-specific guide RNA (gRNA). After transcribing the gRNA and translating Cas9 mRNA, Cas9 will combine with the gRNA to form a ribonucleoprotein (RNP) complex.
  • gRNA target sequence-specific guide RNA
  • the formed ribonucleoprotein complex is guided by the nuclear localization sequence (NLS), which can bring the RNP to the nucleus, while the gRNA can pair with a specific gene sequence in the nuclear genome and guide the Cas9 protein to sequence specificity. Cut, create site-specific double-strand breaks and insert the donor gene through homology-directed repair (HDR) mechanisms, or through non-homologous end joining (NHEJ), which can silence, delete, or repair the gene of interest.
  • NLS nuclear localization sequence
  • HDR homology-directed repair
  • NHEJ non-homologous end joining
  • CRISPR/Cas9 technology is a very promising method for the treatment of various genetic diseases, especially single-gene genetic diseases.
  • the Cas9 protein is relatively large (170kDa)
  • the plasmid required to encode its protein is relatively large (about 10.6kb)
  • the virus can only carry a 4.7kb plasmid.
  • the virus has the danger of inserting DNA into the host cell gene.
  • problems such as high immunogenicity and difficulty in large-scale preparation, so the development of organic non-viral gene editing delivery systems has become very critical.
  • the purpose of the present invention is to provide a degradable macromolecular material, which is a polydisulfide cation material, specifically a guanidine group-containing material.
  • a cationic polymer material with a disulfide backbone is provided.
  • A S or Se
  • n 1 and n 2 are integers between 0 and 20;
  • R 1 is a small molecule containing a thiol group that can be used as an initiator or a macromolecule of a thiol polyethylene glycol, for example:
  • PEGSH mercapto polyethylene glycol, MW (relative molecular mass): 200, 400, 600, 800, 1000, 2000, 5000, 10000
  • 4-arm-PEGSH (4-arm-mercapto polyethylene glycol) or 8-arm-PEGSH (8-arm-mercaptopolyethylene glycol) (MW: 2000, 5000, 10000).
  • R 4 is: H, COOH,
  • R3 is: or
  • Another object of the present invention is to provide a guanidine-containing disulfide backbone polymer composite, which is a nanoparticle formed by self-assembly of polydisulfide cation materials and biological macromolecules such as nucleotides or proteins, wherein Nucleotide chains include but are not limited to plasmids, mRNAs, and proteins include but are not limited to bovine serum albumin (BSA), ⁇ -galactosidase ( ⁇ -gal), purpurin (R-Pe), ribonuclease A ( RNase A), Cas9 protein, etc.
  • BSA bovine serum albumin
  • ⁇ -gal ⁇ -galactosidase
  • R-Pe purpurin
  • RNase A ribonuclease A
  • Cas9 protein etc.
  • the dosage of the nucleotide and the polydisulfide cation material is: every 400ng of nucleotides and 2 ⁇ l (1mg/ml) of polydisulfide cation The materials were mixed thoroughly and the nanocomplexes were formed after 30 min of incubation.
  • the dosage of the protein and the polydisulfide cation material is as follows: every 500 ng of the protein is fully mixed with 2 ⁇ l of the polydisulfide cation material, and the polymer complex can be formed after incubation for 30 min.
  • Another object of the present invention is to provide the application of the guanidine group-containing disulfide backbone polymer complex as a carrier for intracellular delivery of plasmids, mRNAs, and proteins.
  • the polydisulfide cation material of the present invention acts as a carrier to form nanoparticles through self-assembly with biological macromolecules such as nucleotides and proteins, which can effectively deliver these biological macromolecules to the cytoplasm of cells without any Make any chemical modifications so as not to alter its structure nor affect its biological activity.
  • the present invention is used for intracellular CRISPR/Cas9 gene delivery by synthesizing a cationic macromolecular carrier with a guanidine group-bearing disulfide as the main chain.
  • the positively charged guanidine functional group carried on the carrier can interact with negatively charged biological macromolecules to form stable nanoparticles and at the same time help the nanoparticles to pass through the barrier of the cell membrane and enter the cell.
  • the disulfide-containing backbone helps nanoparticles through thiosulfhydryl exchange reactions with thiol groups carried by membrane proteins on the cell membrane surface.
  • the nanoparticles can chemically react with the disulfide backbone under the action of GSH in the cytoplasm to degrade the biomacromolecule to achieve traceless release of biological macromolecules and reduce the toxicity of the carrier, thus solving the problem of macromolecular cellularity.
  • the present invention obtains this type of cationic macromolecule through design, and further obtains an intracellular delivery carrier of biological macromolecules with high efficiency and low toxicity.
  • guanidine-containing cationic carriers are currently used for intracellular delivery such as cell-penetrating peptides (arginine-rich cell-penetrating peptides, CPPs), a major drawback is that they cannot be degraded intracellularly and have greater toxicity.
  • these penetrating peptides must be covalently linked with the delivered functional biomacromolecules, which will affect their function. Due to the existence of these defects, the research and application of cell penetrating peptides are limited.
  • the designed and synthesized carrier of the present invention has a brand-new chemical structure, belongs to a new delivery carrier and has a new application.
  • the cationic material has the following characteristics: the biomacromolecule intracellular delivery vector proposed in the present invention has high intracellular delivery efficiency and expression efficiency, and for the intracellular delivery of plasmids, the efficiency is significantly better than the commercial transfection agent PEI 25K (gold standard) and Lipofectamine 2000; for the intracellular delivery of mRNA, the efficiency was significantly better than the commercial transfection agent PEI 25K and Lipofectamine 3000.
  • the efficiency is basically on par with the commercial delivery vehicle Lipofectamine CRISPR MAX (CMAX).
  • CMAX Lipofectamine CRISPR MAX
  • the delivery efficiency of nanoparticles was characterized by the expression of a reporter gene labeled on the nucleotide chain or a Cas9-fused fluorescent protein, and quantitatively compared by flow cytometry.
  • the cytotoxicity test (MTT) shows that the cationic disulfide polymer provided by the present invention has low toxicity. Under the delivery conditions of biological macromolecules, the survival rate of the experimental cells is higher than 90%, and it has a good biological phase. Capacitance.
  • the cationic disulfide material provided by the invention has high efficiency in the intracellular delivery process, low production cost, low material toxicity, and can effectively deliver various biological macromolecules into cells without chemical modification, and does not require chemical modification. affect its biological activity.
  • Fig. 1 is the quantitative result of EGFP expression of CMV-Cas9-GFP-luciferase plasmid containing part of disulfide cationic polymer material delivered to 293T cells in Example 2, and compared with the commercial formula PEI 25K, lipo2000.
  • Fig. 2 is the result of quantitative expression of green fluorescent protein of CMV-Cas9-GFP-luciferase plasmid delivered to 293T cells containing part of disulfide cationic polymer material in Example 2, and compared with commercial formula PEI 25K, lipo2000 .
  • Example 3 shows the size and surface potential of the complex formed by the polycationic material DET-CPD-12 and CMV-Cas9-GFP-luciferase in Example 3.
  • Figure 4 shows the intracellular delivery efficiency of different N/P complexes formed by DET-CPD-12 and CMV-Cas9-GFP-luciferase plasmids in 293T cell line in Example 4.
  • Figure 5 shows the intracellular delivery efficiency of the complex formed by the cationic polymeric material DET-CPD-12 and CMV-Cas9-GFP-luciferase plasmid in four mammalian cell lines of 293T, Hela, HepG2 and A549 in Example 5.
  • Example 6 is the evaluation of the biodegradability of the cationic polymer material DET-CPD-12 in Example 6.
  • Figure 7 shows the complexes formed by the cationic polymer material DET-CPD-12 and the CMV-Cas9-GFP-luciferase plasmid in Example 7 in four types: 293T (A), Hela (B), HepG2 (C), and A549 (D). Mammalian cell toxicity assessment.
  • FIG. 8 shows the delivery efficiency of the nanocomplex formed by DET-CPD-12 and EGFP-plasmid (4.3kb) in 293T cells in Example 8.
  • Figure 9 shows the efficiency of the complex formed by DET-CPD-12 and CRISPR-Ca9 plasmid in Example 9 for CCNE1 gene knockout in 293T cells.
  • Figure 10 shows the knockout of EGFP gene in 293T-EGFP cells by DET-CPD-12 delivering CRISPR-Ca9 plasmid in Example 10.
  • Example 11 shows the delivery efficiency of the complex formed by DET-CPD-12 and EGFP-mRNA in Example 11 in 293T cells.
  • Example 12 shows the delivery efficiency of the complex formed by DET-CPD-12 and Cas9-GFP-mRNA in Example 12 in 293T cells.
  • FIG. 13 is the size and surface potential of the complex formed by DET-CPD-12 and Cas9-GFP-mRNA in Example 13.
  • Figure 14 shows that DET-CPD-12 delivers Cas9-mRNA in 293T cells for knockout of CCNE-1 gene locus in Example 14.
  • Figure 15 shows that DET-CPD-12 delivers Cas9-mRNA in 293T-EGFP cells for knockout of the EGFP gene locus in Example 15.
  • FIG. 16 shows the intracellular delivery efficiency of the complex formed by DET-CPD-12 and rhodamine-labeled Cas9 protein in 293T cells in Example 16.
  • Example 17 is the size and surface potential of the complex formed by DET-CPD-12 and the gene editing ribonucleic acid complex (CRISPR-Cas9) in Example 17.
  • Figure 18 shows that the complex formed by DET-CPD-12 and gene editing ribonucleoprotein complex (CRISPR-Cas9) in Example 18 is used for CCNE1 gene knockout in 293T cells.
  • CRISPR-Cas9 gene editing ribonucleoprotein complex
  • Figure 19 shows that the complex formed by DET-CPD-12 and gene editing ribonucleoprotein complex (CRISPR-Cas9) in Example 19 is used for EGFP gene knockout in 293T-EGFP cells.
  • CRISPR-Cas9 gene editing ribonucleoprotein complex
  • Figure 20 shows DET-CPD-12, DET-CPD-13, DET-CPD-14, DET-CPD-15, DET-CPD-16, DET-CPD-17, DET-CPD-18, DET in Example 20 -CPD-19, DET-CPD-20 complexes formed by nine materials and phycoerythrin (R-PE) for intracellular delivery and efficiency evaluation in HeLa cells.
  • R-PE phycoerythrin
  • Figure 21 shows DET-CPD-12, DET-CPD-13, DET-CPD-14, DET-CPD-15, DET-CPD-16, DET-CPD-17, DET-CPD-18, DET in Example 21 -Complexes of nine materials CPD-19, DET-CPD-20 and BSA-FITC were used for intracellular delivery and efficiency evaluation in HeLa cells.
  • Figure 22 shows the complexes formed by five materials DET-CPD-12, DET-CPD-13, DET-CPD-14, DET-CPD-15, DET-CPD-16 and RNase-A in Example 22 for MDA - Evaluation of intracellular delivery and apoptosis in MB-231 cells.
  • Figure 23 shows that the complexes formed by DET-CPD-12, DET-CPD-13, DET-CPD-14 and ⁇ -Gal protein in Example 23 were used for intracellular delivery and activity evaluation in HeLa cells.
  • Figure 24 shows that the complexes formed by the three materials DET-CPD-12, DET-CPD-13 and DET-CPD-14 and HRP protein in Example 24 were used for intracellular delivery in HeLa cells.
  • Figure 25 shows the complexes formed by DET-CPD-12, DET-CPD-13, DET-CPD-14 and GFP protein in Example 25 for intracellular delivery and efficiency evaluation in HeLa cells.
  • Figure 26 shows that the complexes formed by the three materials DET-CPD-12, DET-CPD-13, DET-CPD-14 and Cyt C protein in Example 26 were used for intracellular delivery in HeLa cells.
  • Figure 27 shows the complexes formed by DET-CPD-12, DET-CPD-13, DET-CPD-14 and OVA protein in Example 27 for intracellular delivery and efficiency evaluation in HeLa cells.
  • Figure 28 shows the complexes formed by DET-CPD-12, DET-CPD-13, DET-CPD-14 and LGG protein in Example 28 for intracellular delivery and efficiency evaluation in HeLa cells.
  • Figure 29 shows the complexes formed by DET-CPD-12, DET-CPD-13, DET-CPD-14 and Lysozyme in Example 29 for intracellular delivery and efficiency evaluation in HeLa cells.
  • Example 1 Specific synthesis method of monomer and polydisulfide cationic polymer material CPD (Cell-penetrating poly(disulfide)s).
  • lipoic acid 1 (2.06g, 10mmol) was dissolved in 40ml of anhydrous dichloromethane (DCM), carbonyldiimidazole (CDI, 2.43g, 15mmol) was added, stirred at room temperature, triethylenediamine 2 (8.24g, 80mmol) was dissolved in 10ml of anhydrous dichloromethane, stirred in an ice bath for 0.5h, and then the dichloromethane solution dissolved with lipoic acid and CDI was added dropwise.
  • DCM anhydrous dichloromethane
  • CDI carbonyldiimidazole
  • lipoic acid 1 (2.06 g, 10 mmol) was dissolved in 20 ml of anhydrous DMF, carbonyldiimidazole (CDI, 2.43 g, 15 mmol) was added, and the mixture was stirred at room temperature for 1 h.
  • CDI carbonyldiimidazole
  • Arginine methyl ester hydrochloride 3 (1.05g, 5mmol) and DIEA (0.645g, 5mmol) were dissolved in 10ml of anhydrous DMF, and then the arginine hydrochloride solution was added dropwise to the lipoic acid solution and stirred at room temperature 4h, after the reaction is completed, the solvent is removed to obtain the crude product, which is separated and purified by a silica gel column, and the mobile phase is a methanol and dichloromethane system to obtain the monomer M2.
  • compound 5 refers to the synthesis method of monomer M1
  • compound 5 (1.68g, 5mmol) and 1H-pyrazole-1-carboxamidine hydrochloride (0.56g, 5mmol) are dissolved in anhydrous dichloromethane
  • the mixture was stirred at room temperature for 4 h, the solvent was removed, and the silica gel column was separated and purified.
  • the mobile phase was a methanol and dichloromethane system to obtain monomer M4.
  • 4-guanidinobenzoic acid hydrochloride 6 (1.08g, 5mmol) and triethylamine (0.5g, 5mmol) were added to 20ml of dichloromethane and stirred at room temperature for 1h, then (1.24g, 5mmol) of M3, EDCI (0.96g, 5mmol) and DMAP (0.122g, 1mmol) were reacted at room temperature overnight, the solvent was removed after the reaction was completed, the silica gel column was separated and purified, and the mobile phase was methanol and dichloromethane system to obtain monomer M5 .
  • lipoic acid 1 (2.06g, 10mmol) was dissolved in 20ml DCM, followed by adding EDCI (2.3g, 12mmol), DMAP (0.244g, 2mmol) and 4-aminophenylboronic acid (1.6g, 12 mmol), stirred at room temperature overnight, stopped the reaction, extracted three times with saturated brine, dried over anhydrous sodium sulfate, removed the solvent, separated and purified on a silica gel column, and the mobile phase was a methanol and dichloromethane system to obtain monomer M6.
  • Monomer M7 is referenced to the synthesis of M6 and M4.
  • TEOA triethanolamine
  • the molar ratio of monomer I and monomer II is 1:2, and the initiator cysteine methyl ester is dissolved in TEOA buffer (the sum of the molar amount of monomer I and monomer II is 20 times that of the initiator, and added to In the reaction solution with monomers dissolved, react at room temperature for 1.5h, take out the reaction solution and add dropwise to deionized water dissolved in iodoacetamide (equivalent to 5 times the total monomer amount) to terminate, and terminate after half an hour , and fully dialyzed to obtain the material DET-CPD-9.
  • the molar ratio of monomer I and monomer II is 1:2, and the initiator cysteine methyl ester is dissolved in TEOA buffer (the sum of the molar amount of monomer I and monomer II is 40 times that of the initiator, and added to In the reaction solution with monomers dissolved, react at room temperature for 1.5h, take out the reaction solution and add dropwise to deionized water dissolved in iodoacetamide (equivalent to 5 times the total monomer amount) to terminate, and terminate after half an hour , and fully dialyzed to obtain the material DET-CPD-10.
  • the molar ratio of monomer I and monomer II is 1:2, and the initiator cysteine methyl ester is dissolved in TEOA buffer (the sum of the molar amounts of monomer I and monomer II is 160 times that of the initiator, and added to In the reaction solution with monomers dissolved, react at room temperature for 1.5h, take out the reaction solution and add dropwise to deionized water dissolved in iodoacetamide (equivalent to 5 times the total monomer amount) to terminate, and terminate after half an hour , and fully dialyzed to obtain the material DET-CPD-11.
  • the molar ratio of monomer I and monomer II is 1:2, and the initiator PEGSH (MW: 2000) is dissolved in TEOA buffer (the sum of the molar amounts of monomer I and monomer II is the initiator.
  • Add 80 times of the amount to the reaction solution dissolved in the monomer react at room temperature for 1.5h, take out the reaction solution and add it dropwise to deionized water dissolved in iodoacetamide (equivalent to 5 times the total monomer amount) to terminate, After half an hour, it was terminated and fully dialyzed to obtain the material DET-CPD-12.
  • the molar ratio of monomer M3 and monomer M2 is 1:2, and the initiator PEGSH (MW: 2000) is dissolved in TEOA buffer (the sum of the molar amounts of monomer M2 and monomer M3 is the initiator.
  • Add 80 times of the amount to the reaction solution dissolved in the monomer react at room temperature for 1.5h, take out the reaction solution and add it dropwise to deionized water dissolved in iodoacetamide (equivalent to 5 times the total monomer amount) to terminate, After half an hour, it was terminated and fully dialyzed to obtain the material DET-CPD-13.
  • the molar ratio of monomer M1 and monomer M5 is 1:1, and the initiator PEGSH (MW: 1000) is dissolved in TEOA buffer (the sum of the molar amounts of monomer M1 and monomer M5 is the initiator.
  • Add 80 times of the amount to the reaction solution dissolved in the monomer react at room temperature for 1.5h, take out the reaction solution and add it dropwise to deionized water dissolved in iodoacetamide (equivalent to 5 times the total monomer amount) to terminate, After half an hour, it was terminated and fully dialyzed to obtain the material DET-CPD-14.
  • the molar ratio of monomer M4 and monomer M7 is 1:1, and the initiator PEGSH (MW: 5000) is dissolved in TEOA buffer (the sum of the molar amounts of monomer M4 and monomer M7 is the initiator.
  • Add 80 times of the amount to the reaction solution dissolved in the monomer react at room temperature for 1.5h, take out the reaction solution and add it dropwise to deionized water dissolved in iodoacetamide (equivalent to 5 times the total monomer amount) to terminate, After half an hour, it was terminated and fully dialyzed to obtain the material DET-CPD-15.
  • the molar ratio of monomer M5 and monomer M6 is 1:1, and the initiator PEGSH (MW: 2000) is dissolved in TEOA buffer (the sum of the molar amounts of monomer M5 and monomer M6 is the initiator.
  • Add 80 times of the amount to the reaction solution dissolved in the monomer react at room temperature for 1.5h, take out the reaction solution and add it dropwise to deionized water dissolved in iodoacetamide (equivalent to 5 times the total monomer amount) to terminate, After half an hour, it was terminated and fully dialyzed to obtain the material DET-CPD-16.
  • the molar ratio of monomer M1 and monomer M2 is 1:2, and the initiator I5 is dissolved in TEOA buffer solution (the sum of the molar amounts of monomer M1 and monomer M2 is 80 times that of the initiator, adding In the reaction solution with monomers dissolved, react at room temperature for 1.5h, take out the reaction solution and add dropwise to deionized water dissolved in iodoacetamide (equivalent to 5 times the total monomer amount) to terminate, stop after half an hour Completed, fully dialyzed to obtain material DET-CPD-17.
  • the molar ratio of the monomer M1 and the monomer M2 is 1:2, and the initiator I4 is dissolved in the TEOA buffer (the sum of the molar amounts of the monomer M1 and the monomer M2 is 80 times that of the initiator, adding In the reaction solution with monomers dissolved, react at room temperature for 1.5 h, take out the reaction solution and add dropwise to deionized water dissolved in iodoacetamide (equivalent to 5 times the total monomer amount) to terminate, and terminate after half an hour After completion, full dialysis, the material DET-CPD-18 is obtained.
  • the molar ratio of monomer M4 and monomer M7 is 1:1, and the initiator I3 is dissolved in the TEOA buffer (the sum of the molar amounts of monomer M4 and monomer M7 is 80 times that of the initiator, adding In the reaction solution with monomers dissolved, react at room temperature for 1.5h, take out the reaction solution and add dropwise to deionized water dissolved in iodoacetamide (equivalent to 5 times the total monomer amount) to terminate, stop after half an hour After completion, full dialysis, the material DET-CPD-19 is obtained.
  • the molar ratio of monomer M1 and monomer M5 is 1:1, and the initiator I2 is dissolved in the TEOA buffer solution (the sum of the molar amounts of monomer M1 and monomer M5 is 80 times that of the initiator, adding In the reaction solution with monomers dissolved, react at room temperature for 1.5h, take out the reaction solution and add dropwise to deionized water dissolved in iodoacetamide (equivalent to 5 times the total monomer amount) to terminate, stop after half an hour After completion, full dialysis, the material DET-CPD-20 is obtained.
  • Example 2 Comparison of plasmid intracellular delivery efficiency of cationic polymer materials DETs, CPDs, and DET-CPDs series vectors
  • the efficiency of intracellular delivery of the cationic polymer material was evaluated on 293T cells by photographing the brightness of intracellularly expressed green fluorescent protein and detecting the intracellular fluorescence. Screening best material.
  • the specific method is as follows: inoculate 293 T cells into a 48-well plate overnight, and start the cell transfection experiment when the cell density in the well reaches about 60%-80% on the second day. Dissolve 400ng CMV-Cas9-GFP-luciferase plasmid in 50ul ddH 2 O, then add DETs, CPDs, DET-CPDs series cationic materials prepared in Example 1 of the invention, and the amount added is respectively 1ul, 2ul, 4ul, 8ul , 16ul, incubate for 30min, add an appropriate amount of serum-free medium to make the total volume 250uL, replace the medium in the original well, after incubating in the cell incubator for 6h, replace it with 250uL of medium containing 10% FBS, after 48h, pass Fluorescence microscopy was used to observe protein expression or directly collect cells for protein expression identification. PEI and Lipofectamine 2000 were used as positive controls.
  • Fig. 1 is the flow quantification result of the expression of green fluorescent protein in the cells of which the material obtained in Example 1 of the present invention was delivered with CMV-Cas9-GFP-luciferase plasmid on 293T cells.
  • Fig. 2 is the quantitative result of luciferin expression by the material obtained in Example 1 of the present invention in 293T cells that deliver the CMV-Cas9-GFP-luciferase plasmid. It shows that the cationic polymer material can obtain higher delivery efficiency only when a certain reaction time and a certain proportion of monomers are used.
  • DET-CPD-12 has the highest plasmid delivery efficiency, and is significantly better than the commercially available positive transfection reagents PEI25K and Lipofectamine 2000, indicating that this DET-CPD-12 material is a good intracellular delivery plasmid. vector.
  • Example 3 The cationic polymer material DET-CPD-12 was complexed with the CMV-Cas9-GFP-luciferase plasmid, and the size and surface potential of the complex were measured using dynamic light scattering (DLS).
  • DLS dynamic light scattering
  • the specific method is as follows: Dissolve 2ug CMV-Cas9-GFP-luciferase plasmid in 200ul ddH 2 O, then add 10ul of cationic material, incubate for 30min, dilute the solution to 1ml, and use a laser nanoparticle analyzer to detect the size of nanoparticles in the solution The distribution and surface potential, and the size of the nanoparticles were measured using a transmission electron microscope.
  • Figure 3 shows the size distribution and surface potential of the complex formed by the DET-CPD-12 and CMV-Cas9-GFP-luciferase plasmid prepared by the present invention, characterized by DLS.
  • the results show that the polycationic material DET-CPD-12 can form nanoparticles with a size of about 80 nm with the Cas9 plasmid, and the surface potential of the particles is positive.
  • Example 4 Screening the intracellular delivery efficiency of DET-CPD-12 and CMV-Cas9-GFP-luciferase plasmids different N/P-forming complexes in 293T cell line.
  • the N/P of the material DET-CPD-12 and the CMV-Cas9-GFP-luciferase plasmid were 3, 5, 7, 9, and 11, respectively, and PEI 25K and Lipofectamine 2000 were used as positive controls.
  • Figure 4 shows that when the N/P of the material and the plasmid is 5, the fluorescent expression of the cells is photographed by a fluorescence microscope, and the positive rate of the fluorescent protein expression of the cells is quantitatively counted by flow cytometry. The transfection efficiency is the highest, and the obvious Better than PEI 25K and Lipofectamine 2000.
  • Example 5 Intracellular delivery efficiency of complexes between DET-CPD-12 and CMV-Cas9-GFP-luciferase plasmids in various mammalian cell lines, including 293T, Hela, HepG2, and A549 cell lines.
  • the specific method is as follows: (Take Hela cells as an example) cell culture (take a 48-well plate as an example, other culture dishes can refer to 48-well plates), one day (18-24 hours) before transfection, cells (the specific number of cells should be (depending on the type, size and growth rate of cells) were seeded into the wells and cultured so that the cell density on the second day could reach about 60%-80%.
  • Figure 5 shows that the complex formed by the DET-CPD-12 cationic material prepared by the present invention and the CMV-Cas9-GFP-luciferase plasmid was delivered to a variety of mammalian cell lines, and the cytofluorescent protein was quantitatively counted by flow cytometry positive rate of expression.
  • the material efficiency of DET-CPD-12 cation was significantly higher.
  • Example 6 Evaluation of the biodegradability of the cationic polymer material DET-CPD-12.
  • the specific method is as follows: add 10mM GSH (glutathione) to the DET-CPD-12 material, stir overnight at room temperature, measure the molecular weight of the material by GPC, and compare the GPC results of the material without GSH.
  • GSH glutthione
  • Example 7 Cytotoxicity evaluation of the complex formed by cationic polymeric material DET-CPD-12 and CMV-Cas9-GFP-luciferase plasmid
  • the specific operation is as follows: Take 293T as an example, inoculate an appropriate amount of 293T cells into a 96-well plate and culture overnight. Remove the medium, add 100ul DET-CPD-12/plasmid complex serum-free medium with different concentrations respectively, after incubation for 4 hours, remove the medium, add an equal amount of 10% serum-containing medium, and continue to culture for 20 hours . The cell viability was then determined according to the standard procedure of the MTT method.
  • Example 8 DET-CPD-12 cationic polymer complexed with EGFP-plasmid (4.3 kb) for intracellular delivery.
  • the specific method is as follows: inoculate the cells (take 293T cells as an example) into a 48-well plate overnight (take a 48-well plate as an example, other culture dishes can refer to the 48-well plate), so that the cell density in the wells on the second day reaches about 60 % ⁇ 80%, start the cell transfection experiment, dissolve 400ng of plasmid in 50ul ddH2O, then add 2ul of DET-CPD-12 cationic material, incubate for 30min, add 200ul of serum-free medium, the total volume is 250ul, replace the medium in the original well, after 6h incubation in the cell incubator, replace it with 250ul of medium containing 10% FBS, after 48h, observe the protein expression by fluorescence microscope or directly collect the cells for protein expression identification.
  • Figure 8 shows that the complex formed by the DET-CPD-12 cationic material prepared by the present invention and the EGFP-plasmid (4.3kb) was delivered to 293T cells, and the fluorescence expression of the cells was photographed by a fluorescence microscope. The positive rate of cytofluorescent protein expression was quantitatively calculated. Comparable to the commercially available positive controls PEI 25K and Lipofectamine 2000.
  • Example 9 DET-CPD-12 intracellular delivery of CRISPR-Ca9 plasmid for knockout of CCNE-1 locus.
  • the specific operation method is as follows: inoculate 293T cells into a 24-well plate overnight, and start the cell transfection experiment when the cell density in the well reaches about 60%-80% on the second day. Dissolve 800ng of the CRISPR-Cas9 plasmid knocking out the CCNE-1 gene in 100ul ddH 2 O (sgCCNE1:TTTCAGTCCGCTCCAGAAAA), then add 4ul of the DET-CPD-12 cationic material prepared in Example 1 of the invention, incubate for 30min, add an appropriate amount of The total volume of serum medium was 500 ul, which replaced the medium in the original well.
  • Figure 9 shows that the material DET-CPD-12 prepared by the present invention is used to detect the mutation rate of mutants by T7E1 endonuclease method after delivering CRISPR-Cas9 plasmid on 293T cells to edit the CCNE1 gene locus, using commercial PEI25K Compared with Lipofectamine 2000 as a positive control, it can be seen that the efficiency of DET-CPD-12 is significantly better than that of PEI 25K and Lipofectamine 2000.
  • Example 10 The cationic polymeric material DET-CPD-12 delivered the CRISPR-Cas9 plasmid into 293T-EGFP cells for knockout of the EGFP locus.
  • the specific operation method is as follows: Refer to Example 9 for the implementation method, inoculate 293T cells into a 24-well plate overnight, and start the cell transfection experiment when the cell density in the well reaches about 60%-80% the next day. 800ng of EGFP gene knockout CRISPR-Cas9 plasmid was dissolved in 100ul ddH2O (sgEGFP:GTGAACCGCATCGAGCTGAA, EGFP-FP:ATGGTGAGCAAGGGCGAG, EGFP-FP:TTACTTGTACAGCTCGTCCATGC).
  • Example 1 of the invention add 4 ul of the DET-CPD-12 cationic material prepared in Example 1 of the invention, incubate for 30 min, add an appropriate amount of serum-free medium to make the total volume 500 ul, replace the medium in the original well, and incubate in the cell incubator for 6 hours, use The medium containing 10% FBS was replaced by 500ul, and the culture was continued for 48h.
  • Figure 10 shows that the material DET-CPD-12 prepared in the present invention delivers the CRISPR-Cas9 plasmid on 293T-EGFP cells to knock out the EGFP gene locus, and the intracellular GFP expression was observed by a fluorescence microscope. GFP-expressing cells were quantified. Using commercial PEI 25K and Lipofectamine 2000 as positive controls, it can be seen that the knockout effect of DET-CPD-12 delivery CRISPR-Cas9 plasmid is significantly better than that of PEI 25K and Lipofectamine 2000.
  • Example 11 DET-CPD-12 cationic polymer complexed with EGFP-mRNA for intracellular delivery of 293T cell line.
  • Figure 11 shows that the complex formed by the DET-CPD-12 cationic material prepared by the present invention and EGFP-mRNA was delivered to 293T cells, the fluorescence expression of the cells was photographed by a fluorescence microscope, and the cells were quantitatively counted by flow cytometry. Positive rate of fluorescent protein expression. Compared with the positive controls PEI 25K and Lipofectamine 3000, the DET-CPD-12 cationic material was significantly more efficient.
  • Example 12 DET-CPD-12 cationic polymer complexed with Cas9-GFP-mRNA for intracellular delivery of 293T cell line.
  • Figure 12 shows that the complex formed by the DET-CPD-12 cationic material prepared by the present invention and Cas9-GFP-mRNA was delivered to 293T cells, and the fluorescence expression of the cells was photographed by a fluorescence microscope and quantified by flow cytometry The positive rate of cytofluorescent protein expression was counted. Compared with the positive controls PEI 25K and Lipofectamine 3000, the DET-CPD-12 cationic material was significantly more efficient.
  • Example 13 The cationic polymer material DET-CPD-12 formed a complex with Cas9-GFP-mRNA, and the size and surface potential of the complex were measured by DLS, and the size of nanoparticles was measured by TEM.
  • the specific method is as follows: Dissolve 2ug Cas9-GFP-mRNA in 200ul ddH 2 O, then add 10ul cationic material, incubate for 30min, dilute the solution to 1ml, use a laser nanoparticle analyzer to detect the size distribution and surface of nanoparticles in the solution electric potential.
  • Figure 13 shows the size distribution and surface potential of the complex formed by DET-CPD-12 and Cas9-GFP-mRNA prepared by the present invention, characterized by DLS.
  • the results show that the polycationic material DET-CPD-12 can form nanoparticles with a size of about 120 nm with Cas9-mRNA, and the surface potential of the particles is positive.
  • Example 14 DET-CPD-12 intracellular delivery of Cas9-mRNA for knockout of the CCNE-1 locus.
  • Figure 14 shows that the material DET-CPD-12 prepared by the present invention delivers Ca9-mRNA on 293T cells to edit the CCNE1 gene locus using the T7E1 endonuclease method to detect the mutation rate of the mutants, using commercial PEI 25K Compared with Lipofectamine 3000 as a positive control, it can be seen that the knockout efficiency of DET-CPD-12 is significantly better than that of PEI25K and Lipofectamine 3000.
  • Example 15 DET-CPD-12 cationic polymer delivers Cas9-mRNA into 293T-EGFP cells for knockout of the EGFP locus.
  • Figure 15 shows that the material DET-CPD-12 prepared by the present invention delivers Cas9-mRNA on 293T-EGFP cells to knock out the EGFP gene locus, using a fluorescence microscope to observe the intracellular GFP expression, and use a flow cytometer to measure the expression. GFP cells were quantified.
  • Example 16 DET-CPD-12 delivers rhodamine-tagged Cas9 protein intracellularly.
  • the specific method is as follows: inoculate 293 T cells into a 48-well plate overnight, and start the cell transfection experiment when the cell density in the well reaches about 60%-80% on the second day. Dissolve 0.5ug of Rhodamine-labeled Cas9 in 50ul ddH 2 O, then add 4ul of DET-CPD-12 prepared in Example 1 of the invention, incubate for 30min, add an appropriate amount of serum-free medium to make the total volume 250ul, Instead of the medium in the original well, after 6 hours of incubation in the cell incubator, the protein uptake by cells was observed by fluorescence microscopy or the cells were directly collected and the fluorescence intensity in the cells was detected by flow cytometry.
  • the commercial formulations Lipofectamine CRISPRMAX (CMAX) and PEI 25K were used as positive controls.
  • FIG. 16 is the fluorescence photo and flow cytometry results of DET-CPD-12 prepared in the present invention delivering Cas9 protein. The results showed that DET-CPD-12 had better protein delivery effect.
  • Example 17 The complex of DET-CPD-12 and gene editing ribonucleic acid complex (CRISPR-Cas9) was prepared, and the size and surface potential of the complex were characterized by DLS, and the size of nanoparticles was characterized by TEM.
  • CRISPR-Cas9 gene editing ribonucleic acid complex
  • the specific operation method is as follows: mix an appropriate amount of CRISPR-Cas9 protein and sgRNA in an appropriate amount of PBS solution, incubate at 37°C for 10 minutes to form RNP, then add an appropriate amount of DET-CPD-12 solution and mix evenly to form nanoparticles, incubate at room temperature After 30 minutes, 1 mL of deionized water was added for dilution, and the size distribution and surface potential of the nanoparticles in the solution were detected using a laser nanoparticle analyzer.
  • Figure 17 shows the size distribution and surface potential of the complex formed by DET-CPD-12 and CRISPR-Cas9 protein prepared by the present invention, characterized by DLS.
  • the results show that the polycationic material DET-CPD-12 can form nanoparticles with a size of about 150 nm with the CRISPR-Cas9 protein, and the surface potential of the particles is positive.
  • Example 18 Intracellular delivery of DET-CPD-12 gene editing ribonucleoprotein complex (CRISPR-Cas9) to edit the CCNE1 gene.
  • CRISPR-Cas9 ribonucleoprotein complex
  • the specific operation method is as follows: inoculate 293 T cells into a 24-well plate overnight, and start the cell transfection experiment when the cell density in the well reaches about 60% to 80% on the second day. Dissolve 1 ⁇ g of Cas9 protein in 100ul of PBS, add the synthesized gRNA (sgCCNE1:TTTCAGTCCGCTCCAGAAAA), incubate at 300ng at 37°C for 10min to form RNP, then add 4ul of DET-CPD-12 cationic material prepared in Example 1 of the invention, and incubate 30min, add an appropriate amount of serum-free medium to make the total volume to 500ul, replace the medium in the original well, after incubating in the cell incubator for 4h, replace it with 500ul of medium containing 10% FBS, and continue to culture for 48h.
  • CCNE1-FP TCCAAGCCCAAGTCCTGAGCCA
  • CCNE1-RP TGGCCTGCAGCTCTGTTTTGGG
  • T7E1 endonuclease after 30 minutes of reaction at 37°C, run 2% agarose gel electrophoresis to detect and analyze the results of enzyme cleavage.
  • Figure 18 shows that the material DET-CPD-12 prepared by the present invention delivers the gene editing ribonucleoprotein complex (CRISPR-Cas9) on 293T cells to edit the CCNE1 gene locus using the T7E1 endonuclease method to detect the mutants. Mutation rate, using commercial PEI 25K and CMAX as positive controls, the results showed that DET-CPD-12 has a better CCNE1 knockout effect.
  • CRISPR-Cas9 gene editing ribonucleoprotein complex
  • Example 19 DET-CPD-12 delivers gene editing ribonucleoprotein complex (CRISPR-Cas9) into 293T-EGFP cells to knock out the EGFP gene.
  • CRISPR-Cas9 gene editing ribonucleoprotein complex
  • Figure 19 shows that the material DET-CPD-12 prepared by the present invention delivers the gene editing ribonucleoprotein complex (CRISPR-Cas9) on 293T-EGFP cells to knock out the EGFP gene locus using a fluorescence microscope to observe intracellular GFP expression Quantification of GFP-expressing cells was performed using flow cytometry. Using commercialized PEI 25K and CMAX as positive controls, the results showed that DET-CPD-12 had a good EGFP knockout effect.
  • CRISPR-Cas9 gene editing ribonucleoprotein complex
  • Example 20 DET-CPD-12, DET-CPD-13, DET-CPD-14, DET-CPD-15, DET-CPD-16, DET-CPD-17, DET-CPD-18, DET-CPD- 19.
  • DET-CPD-20 delivers phycoerythrin (R-PE) into HeLa cells.
  • the specific operations are as follows: inoculate HeLa cells into a 24-well plate overnight, and when the cell density in the well reaches about 60% to 80% on the second day, start the protein transfection experiment, now remove the medium in the well, and use PBS The cells were washed three times, then 1 ⁇ g of phycoerythrin was dissolved in 100 ul dd H 2 O, and 4 ul of DET-CPD-12, DET-CPD-13, DET-CPD-14, DET-CPD-15 were added to each.
  • Figure 20 shows the nanocomposite formed by DET-CPD-12, DET-CPD-13, DET-CPD-14, DET-CPD-15, DET-CPD-16 cationic materials and phycoerythrin prepared by the present invention
  • the uptake of the protein in HeLa cells was measured, the protein uptake of the cells was photographed by a fluorescence microscope, and the mean fluorescence intensity of the cells after the uptake of the protein was quantitatively calculated by flow cytometry.
  • the results show that DET-CPD-12 has a good ability to deliver phycoerythrin to HeLa cells.
  • Example 21 DET-CPD-12, DET-CPD-13, DET-CPD-14, DET-CPD-15, DET-CPD-16, DET-CPD-17, DET-CPD-18, DET-CPD- 19.
  • DET-CPD-20 delivers bovine serum albumin (BSA-FITC) into HeLa cells.
  • the specific operations are as follows: inoculate HeLa cells into a 24-well plate overnight, and when the cell density in the well reaches about 60% to 80% on the second day, start the protein transfection experiment, now remove the medium in the well, and use PBS The cells were washed twice, then 1 ⁇ g of BSA-FITC was dissolved in 100ul dd H 2 O, and 4ul of DET-CPD-12, DET-CPD-13, DET-CPD-14, DET-CPD-15 were added to each.
  • Figure 21 shows the nanocomposite formed by the DET-CPD-12, DET-CPD-13, DET-CPD-14, DET-CPD-15, DET-CPD-16 cationic materials prepared by the present invention and BSA-FITC
  • the uptake of the protein in HeLa cells was measured, the protein uptake of the cells was photographed by a fluorescence microscope, and the mean fluorescence intensity of the cells after the uptake of the protein was quantitatively calculated by flow cytometry.
  • the results show that DET-CPD-12 has a good ability to deliver bovine serum albumin to HeLa cells
  • Example 22 Intracellular delivery of ribonuclease A (RNase A) by DET-CPD-12, DET-CPD-13, DET-CPD-14, DET-CPD-15, DET-CPD-16 into MDA-MB-231 cells .
  • RNase A ribonuclease A
  • the specific operations are as follows: Inoculate MDA-MB-231 cells into a 48-well plate overnight, and when the cell density in the well reaches about 60% to 80% on the second day, start the protein transfection experiment. Remove, wash the cells three times with PBS, then dissolve 500ng of RNase A in 50ul dd H2O , add 2ul of DET-CPD-12, DET-CPD-13, DET-CPD-14, DET- CPD-15, DET-CPD-16, incubate for 30min after mixing, then add an appropriate amount of serum-free medium to make the total volume 250ul, instead of the PBS solution in the original well, after incubating in the cell incubator for 6h, use 250ul containing 10 The medium in each well was replaced by fresh DMEM with % fetal bovine serum, and the incubation was continued for 42 h. The toxicity of MDA-MB-231 cells was evaluated with CCK-8 formula kit.
  • Figure 22 shows the nanocomplexes formed by DET-CPD-12, DET-CPD-13, DET-CPD-14, DET-CPD-15, DET-CPD-16 cationic materials and RNase A prepared by the present invention
  • DET-CPD-12/RNase A nanocomplex was taken up by MDA-MB-231 cells, the cell activity was lower than 20%, and it had obvious cytotoxicity, indicating that DET-CPD-12 has a good ability to convert RNA.
  • Example 23 DET-CPD-12, DET-CPD-13, DET-CPD-14 deliver ⁇ -galactosidase ( ⁇ -Gal) into HeLa cells.
  • the specific operations are as follows: inoculate HeLa cells into a 24-well plate overnight, and when the cell density in the well reaches about 60% to 80% on the second day, start the protein transfection experiment, now remove the medium in the well, and use PBS Wash the cells twice, then dissolve 1 ⁇ g of ⁇ -galactosidase in 100 ul dd H 2 O, add 4 ul of DET-CPD-12, DET-CPD-13, DET-CPD-14 to each, and mix well.
  • Figure 23 shows the uptake of the nanocomplexes formed by the DET-CPD-12, DET-CPD-13, DET-CPD-14 cationic materials and ⁇ -galactosidase prepared by the present invention in HeLa cells, This indicated that DET-CPD-12 could deliver ⁇ -galactosidase into HeLa cells with high efficiency.
  • Example 24 DET-CPD-12, DET-CPD-13, DET-CPD-14 deliver horseradish peroxidase (HRP) into HeLa cells.
  • HRP horseradish peroxidase
  • the specific operations are as follows: inoculate HeLa cells into a 24-well plate overnight, and when the cell density in the well reaches about 60% to 80% on the second day, start the protein transfection experiment, now remove the medium in the well, and use PBS Wash the cells twice, then dissolve 1 ⁇ g of HRP in 100ul dd H2O, add 4ul of DET-CPD-12, DET-CPD-13, DET-CPD-14 to each, mix well and incubate for 30min, then add an appropriate amount The total volume of serum-free medium was 500 ⁇ l, and the PBS solution in the original well was replaced.
  • Figure 24 shows the nanocomplexes formed by the DET-CPD-12, DET-CPD-13, DET-CPD-14 cationic materials prepared by the present invention and horseradish peroxidase (HRP) in HeLa cells.
  • HRP horseradish peroxidase
  • Example 25 DET-CPD-12, DET-CPD-13, DET-CPD-14 deliver green fluorescent protein (GFP) into HeLa cells.
  • GFP green fluorescent protein
  • Figure 25 shows the uptake of the nanocomplexes formed by the DET-CPD-12, DET-CPD-13, DET-CPD-14 cationic materials and GFP prepared by the present invention in HeLa cells, and the cells were photographed by a fluorescence microscope The protein uptake was quantified by flow cytometry, and the mean fluorescence intensity after cell uptake of the protein was calculated. The results showed that DET-CPD-12 had a good ability to deliver green fluorescent protein to HeLa cells.
  • Example 26 DET-CPD-12, DET-CPD-13, DET-CPD-14 deliver cytochrome C protein (Cyt C-FITC) into HeLa cells.
  • Cyt C-FITC cytochrome C protein
  • Figure 26 shows the uptake of the nanocomplexes formed by DET-CPD-12, DET-CPD-13, DET-CPD-14 cationic materials and cytochrome C protein prepared by the present invention in HeLa cells. Microscopic images of protein uptake by cells. The results show that DET-CPD-12 has a good ability to deliver Cyt C-FITC to HeLa cells.
  • Example 27 DET-CPD-12, DET-CPD-13, DET-CPD-14 deliver chicken ovalbumin (OVA-FITC) into HeLa cells.
  • Figure 27 shows the uptake of the nanocomplexes formed by DET-CPD-12, DET-CPD-13, DET-CPD-14 cationic materials and chicken ovalbumin prepared by the present invention in HeLa cells.
  • the protein uptake of cells was photographed by microscope, and the mean fluorescence intensity after cell uptake of protein was quantified by flow cytometry.
  • Example 28 DET-CPD-12, DET-CPD-13, DET-CPD-14 deliver murine immunoglobulin (IgG-FITC) into HeLa cells.
  • IgG-FITC murine immunoglobulin
  • Figure 28 shows the uptake of the nanocomplexes formed by the DET-CPD-12, DET-CPD-13, DET-CPD-14 cationic materials and IgG-FITC prepared by the present invention in HeLa cells, by fluorescence microscope The protein uptake of the cells was photographed, and the mean fluorescence intensity after the cell uptake of the protein was quantified by flow cytometry.
  • DET-CPD-12 has a good ability to deliver murine-derived immunoglobulins to HeLa cells.
  • Example 29 DET-CPD-12, DET-CPD-13, DET-CPD-14 delivery of lysozyme to HeLa cells (Lysozyme-RBITC)
  • Figure 29 shows the uptake of the nanocomplexes formed by the DET-CPD-12, DET-CPD-13, DET-CPD-14 cationic materials and lysozyme prepared by the present invention in HeLa cells, photographed by a fluorescence microscope
  • the protein uptake of cells was quantified by flow cytometry to calculate the mean fluorescence intensity of cells after protein uptake.
  • the results showed that DET-CPD-12 had a good ability to deliver lysozyme to HeLa cells.

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Abstract

La présente invention concerne un matériau polymère dégradable et un nanocomposite auto-assemblé et une application de celui-ci. Le matériau polymère est un matériau cationique polydisulfure et un composite de polymère avec un disulfure contenant un groupe guanidine en tant que chaîne principale est formé par auto-assemblage du matériau cationique polydisulfure et de macromolécules biologiques de nucléotides et protéines. Après que le composite de polymère selon la présente invention a pénétré dans des cellules, sous l'action du GSH dans le cytoplasme, la chaîne principale d'un support peut être rapidement dégradée pour libérer dans le cytoplasme des macromolécules biologiques encapsulées. Par conséquent, le matériau selon la présente invention est moins toxique pour les cellules et a une bonne cytocompatibilité. Le composite de polymère selon l'invention a une efficacité élevée dans un processus d'administration intracellulaire, un faible coût de production et une faible toxicité, il permet d'administrer efficacement diverses macromolécules biologiques dans des cellules sans modification chimique et sans incidence sur son activité biologique, et il peut être appliqué en tant que support dans l'administration intracellulaire de plasmides, d'ARNm et de protéines. La structure du matériau cationique polydisulfure est représentée dans la formule (1).
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