CN114099533A - Nucleic acid drug delivery system, preparation method, pharmaceutical composition and application - Google Patents
Nucleic acid drug delivery system, preparation method, pharmaceutical composition and application Download PDFInfo
- Publication number
- CN114099533A CN114099533A CN202111413537.7A CN202111413537A CN114099533A CN 114099533 A CN114099533 A CN 114099533A CN 202111413537 A CN202111413537 A CN 202111413537A CN 114099533 A CN114099533 A CN 114099533A
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- delivery system
- drug delivery
- complex
- polyphenol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5115—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5123—Organic compounds, e.g. fats, sugars
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5192—Processes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Nanotechnology (AREA)
- Epidemiology (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Optics & Photonics (AREA)
- General Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Zoology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Wood Science & Technology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Inorganic Chemistry (AREA)
- Plant Pathology (AREA)
- Condensed Matter Physics & Semiconductors (AREA)
- Manufacturing & Machinery (AREA)
- Botany (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Medical Informatics (AREA)
Abstract
The invention discloses a nucleic acid drug delivery system, a preparation method, a pharmaceutical composition and application, wherein the drug delivery system is formed by forming a complex by a nucleic acid drug and polyphenol or polyphenol metal ion complex through non-covalent interaction, then coating lipid on the surface of the complex, forming a drug delivery system with high encapsulation efficiency, delivery efficiency, stability, safety and preparation simplicity, and reducing the functional requirements on lipid, transfection materials and the like. The drug delivery system can be prepared into a liquid preparation by mixing and dispersing technologies, and can be further added with a protective agent to prepare a freeze-dried preparation, so that the drug delivery system is suitable for drugs and diagnostic agents of various administration routes such as oral administration, inhalation, injection, ophthalmic administration, transdermal administration, mucous membrane and the like.
Description
Technical Field
The invention relates to a nucleic acid drug delivery system based on non-covalent action, a preparation method, a pharmaceutical composition and application, in particular to a nucleic acid drug delivery system with significantly improved drug encapsulation efficiency, delivery efficiency, stability and biological safety, a preparation method, a pharmaceutical composition and application.
Background
The problems of poor physicochemical stability and bioavailability of nucleic acid drugs bring difficulty to the research and development of traditional oral and injection administration dosage forms. In order to realize the high-efficiency delivery of nucleic acid drugs, nano drug delivery systems such as liposomes, polymeric micelles, dendrimers, magnetic nanoparticles and the like are widely researched to enhance the stability of nucleic acids in vivo and retain the activity of the nucleic acids to the maximum extent. However, only three varieties of nucleic acid drug complex preparations based on nano drug delivery systems have been marketed up to now (Onpattro, 2018; BNT162b2, 2020; mRNA1273, 2020). The reasons for this are that the target preparation has high requirements for the functional structure of the biological material, the delivery efficiency is limited, the preparation process is complex, and the process is difficult to amplify.
The successful marketing of Onpattetro is critically benefited by the use of the weakly acid ionizable lipid DLin-MC 3-DMA. DLin-MC3-DMA has pKa of 6.44, so that it may be positively charged by regulating pH value in the preparation process to raise the encapsulating rate of nucleic acid medicine, and the final preparation has no charge under neutral physiological pH condition, is stable in organism circulation, has no cationic toxicity and has proper transfection efficiency after entering cell. Based on the successful application of weak acid ionizable lipids, BNT162b2 and mRNA1273, which were marketed in 2020, were delivered using ionizable lipids Acuitas ALC-0315 and SM-102 as well. The existing research on nucleic acid drug delivery systems almost utilizes the adsorption and encapsulation effects of cationic materials or acid-ionizable materials. In addition, the cationic material has the problems of high cytotoxicity, easy removal by an immune system and the like, and has strong restriction requirements on the functional structure of the material, so that the practical application of the nucleic acid medicament is limited by a plurality of limitations.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the defects of the existing nucleic acid drug delivery system, the invention aims to provide a nucleic acid drug delivery system, a preparation method, a pharmaceutical composition and application, which can obviously improve the drug encapsulation efficiency, the delivery efficiency, the stability and the biological safety.
The technical scheme is as follows: as a first aspect of the present invention, in the nucleic acid drug delivery system of the present invention, the nucleic acid drug forms a complex with polyphenol or polyphenol metal ion complex by non-covalent interaction, and the surface of the complex is coated with one or more of lipid and biofilm.
The nucleic acid drug delivery system fully utilizes the structural characteristics and physical characteristics of polyphenol, and the delivery system sequentially comprises nucleic acid drug, polyphenol or polyphenol metal ion complex and stabilizer from inside to outside. The polyphenol compounds have gallic acid or catechol group, so that the polyphenol compounds have natural binding force with nucleic acid molecules (such as DNA, RNA and the like) and can form a complex through non-covalent interaction. Furthermore, the catechol group in the polyphenol can form a complex with metal ions through coordination bonds, and a metal organic framework is formed on the surface of the nucleic acid medicament. Furthermore, polyphenols can weaken the activity of nucleases in vivo by adsorption. More importantly, by utilizing the bioadhesion formed by the polyphenol based on hydrophobic effect, hydrogen bond effect and the like, various types of stabilizers such as lipid, biomembrane and the like can be coated on the surface of the compound formed by the nucleic acid medicament and the polyphenol through simple operation, and the preparation method can be suitable for different disease treatment requirements.
Preferably, the nucleic acid drug comprises one or more of DNA, antisense oligonucleotide, mRNA, small interfering RNA, microrna, short hairpin RNA, aptamer, CpG ODN, poly I: C, 2 '3' -cGAMP, 3 '3' -cGAMP, cAMP, cGMP, or CRISPR/Cas9, CRISPR/Cas12a, CRISPR/Cas13a, or CRISPR/dCas9 gene editing systems based CRISPR-Cas-sgRNA plasmid or Cas protein mRNA + sgRNA co-delivery system.
Preferably, the lipid includes soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, dipalmitoylphosphatidylserine, dioleoylphosphatidylserine, lysophosphatidylethanolamine, palmitoyl lysolecithin, myristoyl lysolecithin, stearoyl lysolecithin, dimyristoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine-polyethylene glycol 2000, dioleoylphosphatidylglycerol, egg yolk phosphatidylglycerol, 1-palmitoyl-2 oleoylphosphatidylglycerol, 1, 2-palmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dimyristoylphosphatidylglycerol, distearoylphosphatidic acid, dipalmitoylphosphatidic acid, dilauroyllecithin, hydrogenated soybean lecithin, dipalmitoylphosphatidylphosphatidylserine phosphatidylserine, dioleoylphosphatidylserine phosphatidylserine, dioleoylphosphatidylethanolamine, distearoylphosphatidylethanolamine-2000, dioleoylphosphatidylglycerol, stearoyl phosphatidylglycerol, dioleoylphosphatidylglycerol, distearoylphosphatidylglycerol, and a, One or more of erucylphosphatidylcholine, dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, cholesterol, or a cholesterol derivative.
Preferably, the biological membrane comprises one or more of a cell membrane, a platelet membrane, a bacterial outer membrane, a cell-derived vesicle, a fused cell membrane.
Preferably, the polyphenol comprises one or more of quercetin, kaempferol, myricetin, anthocyanin, luteolin, catechol, gallocatechol gallate, epigallocatechin gallate (EGCG), digallacyl glucose, trigallacyl glucose, tetragalloyl glucose, pentagalloyl glucose, gallic acid, digallic acid, Tannic Acid (TA), ellagitannin, ellagic acid, hydrolyzed tannins, polydopamine; the polyphenol metal ion complex comprises a complex formed by polyphenol and one or more metal ions of Mn (II), Fe (II), Co (II), Ni (II), Cu (II), Zn (II), Ca (II), Mg (II), Sr (II), Sn (II), Cd (II), Pb (II), Ba (II), Fe (III), Al (III), Co (III), Cr (III), Ce (III), Au (III), Tb (III), Eu (III), Pb (IV), Pt (IV), Ti (IV), Sn (IV), V (IV) and Cr (VI).
Preferably, the mass ratio of the nucleic acid medicament to the polyphenol or polyphenol metal ion complex in non-covalent binding is 10:1-1:10, and the molar ratio of the polyphenol to the metal ion in coordination binding is 1:1-1: 5.
Preferably, the particle size of the nucleic acid drug delivery system is 50-1000 nm.
As a second aspect to which the present invention relates, a method for producing a nucleic acid drug delivery system of the present invention comprises the steps of:
(1) preparation of the Complex
Dissolving nucleic acid medicine, polyphenol and metal ions respectively, and mixing the nucleic acid medicine solution with the polyphenol solution and the metal ion solution in sequence to prepare a compound solution;
or dissolving nucleic acid medicine and polyphenol separately, mixing the nucleic acid medicine solution and polyphenol solution to obtain compound solution;
(2) preparation of nucleic acid drug delivery systems
The method comprises the following steps: preparing a film from lipid or a biological membrane, redissolving or dispersing the film by using a compound solution to form a uniform system, and removing a solvent to prepare a nucleic acid drug delivery system;
the second method comprises the following steps: mixing the solvent dispersed lipid or biological membrane with the complex solution to form a uniform system, and removing the solvent to obtain a nucleic acid drug delivery system;
the third method comprises the following steps: dispersing part of lipid or biomembrane with solvent as oil phase, using the compound solution as internal water phase, mixing oil phase and internal water phase to obtain W/O type emulsion, dissolving or dispersing the rest of lipid or biomembrane in external water phase, mixing W/O type emulsion and external water phase to obtain W/O/W type multiple emulsion, and removing solvent to obtain nucleic acid drug delivery system.
The mixing process of the method step (1), the mixing process of the method step (2) and the mixing process of the method step three for preparing the W/O type emulsion and the W/O/W type multiple emulsion comprise one or more process methods of ultrasonic, homogenization, vortex, stirring and microfluidic technology so as to realize common or rapid mixing of insoluble and insoluble solutions. The micro-fluidic technology is characterized in that an oil phase and a water phase are respectively injected into different channels, the relative volume flow rate ratio of the oil phase and the water phase is controlled, and rapid mixing is realized.
Specifically, the mass ratio of the nucleic acid medicament to the polyphenol or polyphenol metal ion complex in the step (1) in the non-covalent binding is 10:1-1: 10; the molar ratio of the coordination combination of the polyphenol and the metal ions is 1:1-1: 5. The ultrasonic frequency is 300W-1000W, and the ultrasonic time is 1-10 min; homogenizing at 200-1000 Pa and 2-15 times of homogenizing cycle; the vortex or stirring treatment is realized by a vortex instrument and a stirrer, the vortex or stirring rotating speed is generally 1000-.
As a third aspect to which the present invention relates, the pharmaceutical composition of the present invention comprises the above-described nucleic acid drug delivery system and a pharmaceutically acceptable carrier.
The pharmaceutical composition is a liquid injection, an oral solution, an eye drop, an aerosol, a spray, a powder spray or a freeze-dried preparation composition. Wherein the freeze-drying protective agent can be added into the composition, and comprises one or more of glucose, lactose, sorbitol, xylitol, sucrose, trehalose, mannitol, inositol, lactobionic acid, arginine, aspartic acid and other amino acids, dextrin, dextran, soluble starch, albumin, gelatin, polyvidone, polyethylene glycol, sodium chloride, sodium glutamate, citrate, acetate and phosphate.
As a fourth aspect of the present invention, the nucleic acid drug delivery system or the pharmaceutical composition can be prepared into a disease preventive/therapeutic agent or a disease diagnostic agent administered orally, by inhalation, by injection, through eyes, transdermally or through mucous membrane, and is particularly suitable for the treatment of inflammatory infection, tumor, autoimmune disease, and other related diseases.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages:
(1) the encapsulation efficiency of the nucleic acid drug is remarkably improved to be more than 99 percent, the high stability and the safety of a drug delivery system enhance the delivery efficiency, the intracellular drug delivery can be realized safely and efficiently, and the nucleic acid drug has good application prospect in the treatment of related diseases such as inflammatory infection, tumor, autoimmune disease and the like;
(2) the functional requirements on lipid, transfection materials and the like are reduced, the selection range of the stabilizer is wide, and the biological safety is high;
(3) the preparation process has strong operability and repeatability and does not have harsh preparation conditions.
Drawings
FIG. 1 shows epigallocatechin gallate Fe3+Nucleic acid electrophoresis of siRNA inner core;
FIG. 2 shows tannin-Fe3+Nucleic acid electrophoresis of siRNA inner core;
FIG. 3 shows tannin-Fe3+Force of siRNA core;
FIG. 4 shows tannin-Fe3+Nucleic acid electrophoresis of mRNA inner core;
FIG. 5 shows tannin-Fe3+Nucleic acid electrophoresis of the/CRISPR/Cas 9 pDNA core;
FIG. 6 shows tannin-Fe3+siRNA liposome transmission electron microscopy image;
FIG. 7 is tannin-Fe3+siRNA liposome, siRNA/protamine/EGCG/hyaluronic acid complex, EGCG-Fe3+Stability of the culture medium of the/siRNA/polyethyleneimine complex and the cationic lipid DOTAP/siRNA complex;
FIG. 8 is tannin-Fe3+siRNA liposome, siRNA/protamine/EGCG/hyaluronic acid complex, EGCG-Fe3+Serum PBS stability of/siRNA/polyethyleneimine complex, cationic lipid DOTAP/siRNA complex;
FIG. 9 shows tannin-Fe3+siRNA liposome, siRNA/protaminewhite/EGCG/hyaluronic acid complex, EGCG-Fe3+The serum stability of the/siRNA/polyethyleneimine complex and the cationic lipid DOTAP/siRNA complex is 48 h;
FIG. 10 shows tannin-Fe3+siRNA liposome encapsulation efficiency;
FIG. 11 shows tannin-Fe3+The encapsulation efficiency of mRNA liposome;
FIG. 12 is tannin-Fe3+siRNA liposome, siRNA/protamine/EGCG/hyaluronic acid complex, EGCG-Fe3+Cytotoxicity of/siRNA/polyethyleneimine complex, cationic lipid DOTAP/siRNA complex in different cells;
FIG. 13 shows tannin-Fe3+siRNA liposome, siRNA/protamine/EGCG/hyaluronic acid complex, EGCG-Fe3+Transfection efficiencies of/siRNA/polyethyleneimine complex and cationic lipid DOTAP/siRNA complex in 4T1-luc cells;
FIG. 14 is tannin-Fe3+siRNA liposome, siRNA/protamine/EGCG/hyaluronic acid complex, EGCG-Fe3+The transfection efficiency of the/siRNA/polyethyleneimine complex and the cationic lipid DOTAP/siRNA complex in Panc02-luc cells.
Detailed Description
The technical scheme of the invention is further explained by combining the attached drawings.
Example 1
(1) Preparation of Epigallocatechin gallate-Fe3+Inner core of siRNA
Mixing epigallocatechin gallate (EGCG) and FeCl3·6H2O prepared with RNase Free water as a stock solution, and siRNA prepared with RNase Free water as a 20. mu.M solution. Taking siRNA and EGCG solution to mix for 2h by vortex, and then mixing simply under vortex condition or rapidly mixing Fe by micro-fluidic3+Solution of Fe3+The molar ratio to EGCG was 2: 1. Standing at room temperature and incubating for 30 min.
Specifically, the sample particle size and polydispersity index (PDI) were determined using a Zetasizer 3000HS instrument particle size analyzer (Malvern Instruments, Malvern, UK). Meanwhile, the loading efficiency of siRNA was examined by 1% agarose gel electrophoresis.
The specific results are as follows: the average particle diameter of the preparation can be controlled at 187 + -3.5 nm, and PDI is less than 0.3. The result of agarose gel electrophoresis is shown in FIG. 1, when Fe3+When the molar ratio of the siRNA to EGCG is 2:1, the siRNA has obvious blockage, and when Fe is used3+The complete entrapment of siRNA is basically realized when the molar ratio of the siRNA to EGCG is 3: 1.
(2) Preparation of tannin-Fe3+Inner core of siRNA
Mixing Tannic Acid (TA) and FeCl3·6H2Dissolving O in RNase Free water to prepare a mother solution, and preparing the siRNA into a 20 mu M solution by using the RNase Free water. Mixing siRNA solution with equal volume of TA diluted solution according to the mass ratio of 1:10, slightly swirling for 20s, and then simply mixing under the swirling condition or rapidly mixing Fe through microfluidics3+Solution of Fe3+The molar ratio to TA was 3: 1. Standing at room temperature for 30min to obtain the final product. Meanwhile, the sample particle size and polydispersity index (PDI) were measured using a Zetasizer 3000HS instrument particle size analyzer (Malvern Instruments, Malvern, UK). Meanwhile, the loading efficiency of siRNA was examined by 1% agarose gel electrophoresis. In addition, four chemical substances are selected to investigate the acting force, namely Tween 20, urea, EDTA and NaCl, and the influence of the chemical substances on the electrophoresis result of the target preparation under the condition of different concentrations of compounds is investigated.
The specific results are as follows: the average particle diameter of the preparation is 100 +/-2.8 nm, and the PDI is less than 0.3. The result of agarose gel electrophoresis is shown in FIG. 2, when Fe3+At a molar ratio of 1:3 to TA, the nucleic acid was almost completely entrapped, and Fe was further increased3+In the amount of (B), the nucleic acid bands are rather brightened, and TA and Fe are observed3+The optimum ratio of the molar ratio is 1: 3. The force results are shown in FIG. 3, and when EDTA is added, the electrophoresis results are obviously changed, which can indicate that Fe3+Complexing with TA through coordination bonds and entrapping genes; the fluorescence of the gene strip is also enhanced after the urea is added, and the hydrogen bond action between TA and siRNA is probably destroyed; the gene has no obvious leakage after NaCl is added, and the gene has slight leakage after Tween 20 is added, which probably influences the hydrophobic acting force between TA and siRNA. Thus, it can be stated that the main force for loading the gene in the target preparation is the bindingThe site bonds, hydrogen bonds, and then hydrophobic forces, are all non-covalent interactions.
(3) Preparation of tannin-Fe3+mRNA core
Mixing TA and FeCl3·6H2Dissolving O in RNase Free water to prepare a mother solution, and preparing mRNA into a 20 mu M solution by using the RNase Free water. Mixing mRNA solution with equal volume of TA diluted solution at a mass ratio of 1:10, gently vortexing for 20s, and then simply mixing under vortexing condition or rapidly mixing Fe by microfluidics3+Solution of Fe3+The molar ratio to TA was 3: 1. Standing at room temperature for 30min to obtain the final product. Meanwhile, the sample particle size and polydispersity index (PDI) were measured using a Zetasizer 3000HS instrument particle size analyzer (Malvern Instruments, Malvern, UK). Meanwhile, the entrapment efficiency of mRNA was examined by electrophoresis on 1% agarose gel.
The specific results are as follows: the average particle diameter of the preparation is 120 +/-2.0 nm, and the PDI is less than 0.3. The result of agarose gel electrophoresis is shown in FIG. 4, when Fe3+At a molar ratio of 1:5 to TA, the nucleic acid was almost completely entrapped, indicating TA and Fe3+The optimum ratio of the molar ratio is 1: 5.
(4) Preparation of tannin-Fe3+CRISPR-Cas9 pDNA inner core
Mixing TA and FeCl3·6H2Dissolving O in RNase Free water to prepare a mother solution, and preparing the CRISPR-Cas9 pDNA into a 20 mu M solution by using the RNase Free water. Mixing CRISPR-Cas9 pDNA solution with equal volume of TA dilution solution according to the mass ratio of 1:10, slightly vortexing for 20s, and then simply mixing under vortexing condition or rapidly mixing Fe through microfluidics3+Solution of Fe3+The molar ratio to TA was 3: 1. Standing at room temperature for 30min to obtain the final product. Meanwhile, the sample particle size and polydispersity index (PDI) were measured using a Zetasizer 3000HS instrument particle size analyzer (Malvern Instruments, Malvern, UK). And meanwhile, the entrapment efficiency of the CRISPR-Cas9 pDNA is examined through 1% agarose gel electrophoresis.
The specific results are as follows: the average particle diameter of the preparation is 125 +/-2.1 nm, and the PDI is less than 0.3. The result of agarose gel electrophoresis is shown in FIG. 5, when Fe3+At a 1:5 molar ratio to TA, there was significant retardation of pDNA, but further increase in Fe3+When measured inPrecipitate appeared, and TA and Fe were observed3+The optimum ratio of the molar ratio is 1: 5.
In summary, the molar ratio of the polyphenol to the metal ion in coordination can be 1:1 to 1: 5.
Example 2
(1) Preparation of EGCG-Fe3+siRNA/liposomes
Soybean lecithin, cholesterol or cholesterol derivatives were dissolved in chloroform in the prescribed amounts as the oil phase. EGCG-Fe3+The siRNA solution was used as internal aqueous phase W1. Adding the inner water phase into the oil phase, and carrying out ultrasonic treatment by a 300W probe under the ice bath condition to form W/O type emulsion. Dripping W/O type emulsion into external water phase containing prescription amount of yolk lecithin and DSPE-PEG2000, performing ultrasonic treatment in 1000W water bath for 5-10min to form multiple emulsion, and evaporating under reduced pressure to remove organic solvent to obtain EGCG-Fe3+siRNA/liposomes. Meanwhile, the sample particle size and polydispersity index (PDI) were measured using a Zetasizer 3000HS instrument particle size analyzer (Malvern Instruments, Malvern, UK).
The specific results are as follows: the average particle diameter of the preparation is 120 +/-2.8 nm, and the PDI is less than 0.3.
Or dissolving the prescribed amount of lipid in chloroform, evaporating under reduced pressure to obtain thin film, adding EGCG-Fe3+Dispersing film with siRNA solution, and performing water bath ultrasound for 5-10min to obtain EGCG-Fe3+siRNA/liposomes.
Or EGCG-Fe3+Injecting the siRNA solution, the lipid solution and the buffer solution into a microfluidic device at a relative volume flow rate of 1:1:2, and putting the newly prepared lipid nanoparticles into the buffer solution for dialysis to obtain the lipid nanoparticle.
(2) Preparation of TA-Fe3+siRNA/liposomes
Egg yolk lecithin, cholesterol or cholesterol derivatives are dissolved in chloroform in prescribed amounts as an oil phase. Mixing TA-Fe3+The siRNA solution was used as internal aqueous phase W1. Adding the inner water phase into the oil phase, and carrying out ultrasonic treatment by a 300W probe under the ice bath condition to form W/O type emulsion. Dripping W/O type emulsion into external water phase containing prescription amount of lecithin and DSPE-PEG2000, performing ultrasonic treatment in 1000W water bath for 5-10min to form multiple emulsion, and evaporating under reduced pressure to remove organic solvent to obtain TA-Fe3+A siRNA liposome. At the same time, useThe Zetasizer 3000HS Instrument particle size Analyzer (Malvern Instruments, Malvern, UK) measures the sample particle size and polydispersity index (PDI). In addition, the liposome solution prepared above is dropped on a copper net, dried and then characterized by a transmission electron microscope, observed and photographed.
The specific results are as follows: the average particle diameter of the preparation is 110 +/-2.5 nm, and the PDI is less than 0.3. As shown in FIG. 6, the size distribution of liposome particles was uniform, which was helpful for the uptake of siRNA-loaded liposomes by cells and the exertion of drug effect.
(3) Preparation of TA-Fe3+mRNA/liposomes
Dissolving DOPE, DSPE-PEG2000, cholesterol or cholesterol derivative in chloroform according to prescription amount to obtain oil phase, evaporating under reduced pressure to obtain film, adding TA-Fe3+Dispersing film with mRNA solution, and performing water bath ultrasound for 5-10min to obtain TA-Fe3+mRNA/liposomes.
The specific results are as follows: the average particle diameter of the preparation is 120 +/-2.3 nm, and the PDI is less than 0.3.
(4) Preparation of TA-Fe3+CRISPR-Cas9 pDNA/liposome
Dioleoyl lecithin, DSPE-PEG2000, cholesterol or cholesterol derivatives were dissolved in chloroform in prescribed amounts as a lipid solution, followed by TA-Fe3+Injecting the/CRISPR-Cas 9 pDNA solution, the lipid solution and the buffer solution into a microfluidic device at a relative volume flow rate of 1:1:2, and then putting the newly prepared lipid nanoparticles into the buffer solution for dialysis to obtain the nanoparticle.
The specific results are as follows: the average particle size of the preparation is 118 +/-2.1 nm, and the PDI is less than 0.3.
(5) Preparing liposome freeze-dried powder injection
And (3) adding 8% of sucrose into the liposome solution prepared in the steps (1), (2), (3) and (4), and freeze-drying to obtain the liposome powder injection. The liposome can be dissolved in 0.9% physiological saline or 5% glucose before administration, and the obtained liposome can be used for intravenous injection.
Example 3
(1) Preparation of TA-Fe3+siRNA/biofilm complexes
Will be derived from whole bloodSuspending the extracted erythrocyte in hypotonic buffer solution, treating with ultrasound for 5min, centrifuging at low temperature to collect cell membrane, resuspending the collected erythrocyte membrane at 2mg/ml in water, and mixing with TA-Fe3+Mixing the siRNA/solution in equal volume, and carrying out water bath ultrasound for 2 min. Then centrifuging at 4 deg.C and 10000g for 5min to collect nanoparticles. The average particle diameter of the preparation is 140 +/-2.5 nm, and the PDI is less than 0.3.
(2) Preparation of TA-Fe3+mRNA/biofilm complexes
Extracting platelet membrane from plasma by repeated freeze thawing, suspending in water at a concentration of 2mg/ml, and mixing the platelet membrane solution with TA-Fe3+The mRNA solution is mixed in equal volume, incubated for 30min and sequentially passed through a polycarbonate porous membrane of 1000nm, 400 nm and 200nm by using a micro-extruder. Centrifuging at 4 deg.C and 10000g for 5min, and collecting nanoparticles. The average particle diameter of the preparation is 135 + -1.2 nm, and PDI is less than 0.3.
(3) Preparation of TA-Fe3+CRISPR-Cas9 pDNA/biofilm complex
Separating bacterial outer membrane vesicle from Escherichia coli, and mixing with TA-Fe3+the/CRISPR-Cas 9 pDNA solution is mixed in equal volume, water bath ultrasound is carried out for 2min, centrifugation is carried out at 4 ℃ for 5min at 10000g, and the nano particles are collected. The average particle diameter of the preparation is 146 +/-2.4 nm, and the PDI is less than 0.3.
(4) Preparing cell membrane compound freeze-dried powder injection
And (3) adding 8% of sucrose into the liposome solution prepared in the steps (1), (2) and (3), and freeze-drying to obtain the liposome powder injection. The liposome can be used for intravenous injection by re-dissolving with 0.9% physiological saline or 5% glucose before use.
Example 4: TA-Fe3+siRNA/liposome, siRNA/protamine/EGCG/hyaluronic acid complex, EGCG-Fe3+Stability examination of/siRNA/polyethyleneimine Complex and cationic lipid DOTAP/siRNA Complex
siRNA/protamine/EGCG/hyaluronic acid complexes (Ding J, Liang T, Min Q, Jiang L, Zhu JJ. "Stealth and Fully-Laden" Drug Carriers: Self-isolated Nanogels Encapsulated with Epigallocatechin Gallate and siRNA for Drug-resist Cancer therapy. ACS apply Mather Interfaces.2018; 10(12): 9938-. Further mixing and stirring the hyaluronic acid solution and the siRNA/protamine/EGCG compound for 24 hours according to the siRNA mass ratio of 10:1 to obtain the iRNA/protamine/EGCG/hyaluronic acid compound.
Preparation of EGCG-Fe3+the/siRNA/polyethyleneimine complex (Shen W, Wang Q, Shen Y, et al, Green Tea healthcare proteins RNAi media by Low-Molecular-Weight polymers. ACS Cent Sci.2018; 4(10): 1326) 1333), 0.5 μ g siRNA and EGCG were mixed at room temperature for 20min, and PEI solution was added at a PEI and siRNA mass ratio (W/W) of 5:1 for further incubation for 30min to form TA-Fe3+a/siRNA/polyethyleneimine complex.
DOTAP cationic lipid/siRNA complexes (Buck J, Mueller D, Metal U, et al. improvement of DNA Vector Delivery of DOTAP Lipopexes by Short-Chain amino lipids. ACS omega.2020; 5(38):24724 and 24732) were prepared, cholesterol, DOTAP were dissolved in chloroform in a 1:1 ratio and dried overnight under a nitrogen stream. The mixture was then hydrated with a 5% solution of D (+) -glucose and stirred and vortexed. After the hydrate is subjected to circulating freeze thawing and high-pressure extrusion, the hydrate and siRNA are mixed according to the proportion of 8 mu M: 1 mu g of the mixture is incubated for 30 minutes to obtain the DOTAP cationic lipid/siRNA complex.
Investigating TA-Fe by using particle size variation as index3+siRNA/liposome, siRNA/protamine/EGCG/hyaluronic acid complex, EGCG-Fe3+The stability of the/siRNA/polyethyleneimine complex and the DOTAP cationic lipid/siRNA complex in phosphate buffer containing 10% serum and basal medium at 37 ℃ are shown in FIGS. 7 and 8, respectively. The results show that TA-Fe3+The particle size of the siRNA/liposome solution preparation is not obviously changed within 48 hours in two media, and the stability is good. The siRNA/protamine/EGCG/hyaluronic acid compound has loose and expanded structure and EGCG-Fe in the ionic environment3+siRNA/polyethyleneimine Complex, DOThe TAP cationic lipid/siRNA complex is aggregated in serum conditions due to protein adsorbed by positive electricity on the surface, so that the particle size is obviously increased.
Investigation of TA-Fe by agarose gel electrophoresis3+siRNA/liposome, siRNA/protamine/EGCG/hyaluronic acid complex, EGCG-Fe3+Serum stability of the/siRNA/polyethyleneimine complex, DOTAP cationic lipid/siRNA complex after 48h incubation in 50% serum, respectively. The results are shown in FIG. 9. The results show that TA-Fe3+The siRNA/liposome solution preparation has no obvious leakage of siRNA after 48 hours under serum condition and has good stability. siRNA/protamine/EGCG/hyaluronic acid complex, EGCG-Fe3+The siRNA/polyethyleneimine complex and the DOTAP cationic lipid/siRNA complex have obvious nucleic acid leakage after 48 hours in serum.
Example 5
(1)TA-Fe3+Perfect examination of siRNA/liposomes
TA-Fe prepared as in example 23+siRNA/liposomes, adding Triton and EDTA treatment sequentially, then with the loading buffer mixed, 1% agarose gel electrophoresis blocking experiment. Results encapsulation efficiency was quantitatively estimated using Image J.
As a result, as shown in fig. 10, the encapsulation efficiencies of the liposomes prepared using cholesterol or cholesterol derivatives were 78.7% and 84.71%, respectively.
(2)TA-Fe3+PermRNA/Liposome encapsulation efficiency Studies
TA-Fe prepared as in example 23+siRNA/liposomes, adding Triton and EDTA treatment sequentially, then with the loading buffer mixed, 1% agarose gel electrophoresis blocking experiment. Results encapsulation efficiency was quantitatively estimated using Image J.
As a result, as shown in FIG. 11, the encapsulation efficiencies of the liposomes prepared using cholesterol or cholesterol derivatives were 96.12% and 99.31%, respectively.
Example 6: TA-Fe3+siRNA/liposome, siRNA/protamine/EGCG/hyaluronic acid complex, EGCG-Fe3+Evaluation of cytotoxicity of siRNA/polyethyleneimine Complex and DOTAP cationic lipid/siRNA Complex
4T1-luc, Panc02-luc cells at 5X 103Cell density per well was seeded in 96-well plates, and the plates were placed at 37 ℃ in 5% CO2The culture box is used for culturing for 24 hours. After 24h, the culture medium was aspirated from the wells and the cells were washed 3 times with PBS. TA-Fe was added to the siRNA at a dose of 200nM3+siRNA/liposome, siRNA/protamine/EGCG/hyaluronic acid complex, EGCG-Fe3+the/siRNA/polyethyleneimine complex, the DOTAP cationic lipid/siRNA complex, and the incubation for 48 hours under the serum-free condition. After the incubation was completed, MTT was added and the mixture was incubated for 4 hours, washed off, and dissolved in DMSO to measure absorbance.
The specific results are shown in FIG. 12, TA-Fe3+The siRNA/liposome and siRNA/protamine/EGCG/hyaluronic acid compound do not show obvious cytotoxicity under the administration concentration in two cell lines, and the EGCG-Fe3+the/siRNA/polyethyleneimine complex and the DOTAP cationic lipid/siRNA complex show obvious cytotoxicity under the same concentration.
Example 7: simulating TA-Fe under in vivo conditions3+/siLuc/liposome, siLuc/protamine/EGCG/hyaluronic acid complex, EGCG-Fe3+Complex of/siLuc/polyethyleneimine, complex of DOTAP cation lipid/siLuc, TA-Fe3+Transfection efficiency of/siCon LNP in 4T1-luc, Panc02-luc model cells
4T1-luc, Panc02-luc cells at 5X 104Cell density per well was seeded in 24-well plates, and the plates were placed at 37 ℃ in 5% CO2The culture was carried out in the incubator for 24 hours. After 24h, the culture medium was aspirated from the wells and the cells were washed 3 times with PBS. Distributed addition of TA-Fe3+/siLuc/liposome, siLuc/protamine/EGCG/hyaluronic acid complex, EGCG-Fe3+Complex of/siLuc/polyethyleneimine, complex of DOTAP cation lipid/siLuc, TA-Fe3+the/siCon LNP was incubated in a medium containing 10% serum for 12h, and the Yangsheng preparation Lipofectamine3000/siLuc complex was incubated in a medium containing serum for 12 h. The incubation was then continued for 36h with complete medium change. After the incubation, the liquid in the wells was aspirated and luciferase activity was measured using a luciferase assay kit.
The specific results are shown in the figure13. 14, TA-Fe3+The silencing efficiency of the/siLuc/liposome is about 60% at the administration concentration of 200nM, while the siLuc/protamine/EGCG/hyaluronic acid complex, EGCG-Fe3+The transfection efficiency of the/siLuc/polyethyleneimine complex and the DOTAP cationic lipid/siLuc complex is obviously reduced because the complexes are unstable or aggregated.
Claims (10)
1. A nucleic acid drug delivery system, wherein the nucleic acid drug forms a complex with a polyphenol or polyphenol metal ion complex via non-covalent interactions, and the surface of the complex is coated with one or more of a lipid and a biological membrane.
2. The nucleic acid drug delivery system of claim 1, wherein the nucleic acid drug comprises one or more of DNA, antisense oligonucleotide, mRNA, small interfering RNA, microRNA, short hairpin RNA, aptamer, CpG ODN, poly I: C, 2 '3' -cGAMP, 3 '3' -cGAMP, cAMP, cGMP, or CRISPR-Cas-sgRNA plasmid or Cas protein mRNA + sgRNA co-delivery system based on CRISPR/Cas9, CRISPR/Cas12a, CRISPR/Cas13a, or CRISPR/dCas9 gene editing system.
3. The nucleic acid drug delivery system of claim 1, wherein the lipid comprises soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, dipalmitoylphosphatidylserine, dioleoylphosphatidylserine, lysophosphatidylethanolamine, palmitoyl lysolecithin, myristoyl lysolecithin, stearoyl lysolecithin, dimyristoyl phosphatidylethanolamine, distearoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, distearoylphosphatidylethanolamine-polyethylene glycol 2000, dioleoylphosphatidylglycerol, egg yolk phosphatidylglycerol, 1-palmitoyl-2 oleoylphosphatidylglycerol, 1, 2-palmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dimyristoylphosphatidylglycerol, hydrogenated soybean lecithin, dipalmitoylphosphatidylserine phosphatidylserine, dioleoylphosphatidylserine, lysophosphatidylserine, dioleoylphosphatidylglycerol, myristoylphosphatidylglycerol lysophosphatidylglycerol, stearoyl lysophosphatidylcholine, stearoylphosphatidylcholine 2000, stearoylphosphatidylglycerol, and phosphatidylglycerol One or more of distearoylphosphatidic acid, dipalmitoylphosphatidic acid, dilauroyl lecithin, erucyl phosphatidylcholine, dioleoyl lecithin, dimyristoyl lecithin, 1-palmitoyl-2-oleoyl lecithin, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, cholesterol, or cholesterol derivatives.
4. The nucleic acid drug delivery system of claim 1, wherein the biological membrane comprises one or more of a cell membrane, a platelet membrane, a bacterial outer membrane, a cell-derived vesicle, a fused cell membrane.
5. The nucleic acid drug delivery system of claim 1, wherein the polyphenol comprises one or more of quercetin, kaempferol, myricetin, anthocyanin, luteolin, catechol, gallocatechol gallate, epigallocatechin gallate, digallacyl glucose, trigallacyl glucose, tetragalloyl glucose, pentagalloyl glucose, gallic acid, digallic acid, tannic acid, ellagitannin, ellagic acid, hydrolyzed tannin, polydopamine; the polyphenol metal ion complex comprises a complex formed by polyphenol and one or more metal ions of Mn (II), Fe (II), Co (II), Ni (II), Cu (II), Zn (II), Ca (II), Mg (II), Sr (II), Sn (II), Cd (II), Pb (II), Ba (II), Fe (III), Al (III), Co (III), Cr (III), Ce (III), Au (III), Tb (III), Eu (III), Pb (IV), Pt (IV), Ti (IV), Sn (IV), V (IV) and Cr (VI).
6. The nucleic acid drug delivery system of claim 1, wherein the mass ratio of the non-covalent binding of the nucleic acid drug to the polyphenol or polyphenol metal ion complex is 10:1 to 1:10, and the molar ratio of the coordination binding of the polyphenol to the metal ion is 1:1 to 1: 5.
7. The nucleic acid drug delivery system of claim 1, wherein the particle size is 50-1000 nm.
8. A method of preparing a nucleic acid drug delivery system according to any one of claims 1 to 7, comprising the steps of:
(1) preparation of the Complex
Dissolving nucleic acid medicine, polyphenol and metal ions respectively, and mixing the nucleic acid medicine solution with the polyphenol solution and the metal ion solution in sequence to prepare a compound solution;
or dissolving nucleic acid medicine and polyphenol separately, mixing the nucleic acid medicine solution and polyphenol solution to obtain compound solution;
(2) preparation of nucleic acid drug delivery systems
The method comprises the following steps: preparing a film from lipid or a biological membrane, redissolving or dispersing the film by using a compound solution to form a uniform system, and removing a solvent to prepare a nucleic acid drug delivery system;
the second method comprises the following steps: mixing the solvent dispersed lipid or biological membrane with the complex solution to form a uniform system, and removing the solvent to obtain a nucleic acid drug delivery system;
the third method comprises the following steps: dispersing part of lipid or biomembrane with solvent as oil phase, using the compound solution as internal water phase, mixing oil phase and internal water phase to obtain W/O type emulsion, dissolving or dispersing the rest of lipid or biomembrane in external water phase, mixing W/O type emulsion and external water phase to obtain W/O/W type multiple emulsion, and removing solvent to obtain nucleic acid drug delivery system.
9. A pharmaceutical composition comprising the nucleic acid drug delivery system of any one of claims 1 to 7 and a pharmaceutically acceptable carrier.
10. Use of the nucleic acid drug delivery system of any one of claims 1 to 7 or the pharmaceutical composition of claim 9 for the preparation of a disease preventive/therapeutic agent or a disease diagnostic agent for oral, inhalation, injection, ophthalmic, transdermal or mucosal administration.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111413537.7A CN114099533A (en) | 2021-11-25 | 2021-11-25 | Nucleic acid drug delivery system, preparation method, pharmaceutical composition and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111413537.7A CN114099533A (en) | 2021-11-25 | 2021-11-25 | Nucleic acid drug delivery system, preparation method, pharmaceutical composition and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114099533A true CN114099533A (en) | 2022-03-01 |
Family
ID=80373082
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111413537.7A Pending CN114099533A (en) | 2021-11-25 | 2021-11-25 | Nucleic acid drug delivery system, preparation method, pharmaceutical composition and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114099533A (en) |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114404388A (en) * | 2022-03-31 | 2022-04-29 | 天九再生医学(天津)科技有限公司 | Method for loading mRNA in vitro by extracellular vesicles |
| CN114668742A (en) * | 2022-03-17 | 2022-06-28 | 中国科学技术大学 | Bacteria-like nano-drug delivery system and preparation method and application thereof |
| CN114712520A (en) * | 2022-04-15 | 2022-07-08 | 中国药科大学 | Nanocrystalline drug stabilizing system, preparation method, pharmaceutical composition and application |
| CN114870025A (en) * | 2022-04-28 | 2022-08-09 | 云南大学 | Response slow-release polydopamine-Zn-natural polyphenol coordination nano-medicament and preparation method thereof |
| CN115317461A (en) * | 2022-08-05 | 2022-11-11 | 常州大学 | Adriamycin delivery system Cu-GA-DOX NPs and preparation method thereof |
| CN115804761A (en) * | 2022-08-05 | 2023-03-17 | 暨南大学 | Bionic nano gene vector, preparation method and application |
| CN117467708A (en) * | 2023-10-17 | 2024-01-30 | 中山大学孙逸仙纪念医院 | Nucleic acid delivery complex and preparation method and application thereof |
| CN117582511A (en) * | 2022-08-09 | 2024-02-23 | 湖南健瑞医药科技有限公司 | Metal-phospholipid composite particles, and preparation method and application thereof |
| CN117582510A (en) * | 2022-08-09 | 2024-02-23 | 湖南健瑞医药科技有限公司 | Use of metal-polyphenol complexes in nucleic acid delivery systems |
| CN117582512A (en) * | 2022-08-09 | 2024-02-23 | 湖南健瑞医药科技有限公司 | Metal-polyphenol composite particles, and preparation method and application thereof |
| CN117582417A (en) * | 2022-08-09 | 2024-02-23 | 湖南健瑞医药科技有限公司 | Medicine-lipid particle and preparation method and application thereof |
| WO2024099251A1 (en) * | 2022-11-08 | 2024-05-16 | 山东省创启先进材料研究院 | Imaging agent containing myricetin nanomaterial, preparation method therefor, and use thereof |
| WO2024099250A1 (en) * | 2022-11-08 | 2024-05-16 | 山东省创启先进材料研究院 | Myricetin nanomaterial preparation, preparation method therefor, and use thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110237264A (en) * | 2019-01-31 | 2019-09-17 | 华东理工大学 | A kind of PLGA nano particle and preparation method thereof for TA-Fe (III) modification containing doxorubicin hydrochloride |
| CN111714643A (en) * | 2019-03-22 | 2020-09-29 | 约翰霍普金斯大学 | tannin/Fe3+Nanoparticle system, drug delivery method |
| CN113521298A (en) * | 2021-07-01 | 2021-10-22 | 东华大学 | Responsive dendrimer drug-loaded material coated by tannic acid/iron complex |
| CN113616812A (en) * | 2021-09-18 | 2021-11-09 | 康健仿生(清远)科技有限公司 | Polyphenol modified liposome nano system and preparation method and application thereof |
-
2021
- 2021-11-25 CN CN202111413537.7A patent/CN114099533A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110237264A (en) * | 2019-01-31 | 2019-09-17 | 华东理工大学 | A kind of PLGA nano particle and preparation method thereof for TA-Fe (III) modification containing doxorubicin hydrochloride |
| CN111714643A (en) * | 2019-03-22 | 2020-09-29 | 约翰霍普金斯大学 | tannin/Fe3+Nanoparticle system, drug delivery method |
| CN113521298A (en) * | 2021-07-01 | 2021-10-22 | 东华大学 | Responsive dendrimer drug-loaded material coated by tannic acid/iron complex |
| CN113616812A (en) * | 2021-09-18 | 2021-11-09 | 康健仿生(清远)科技有限公司 | Polyphenol modified liposome nano system and preparation method and application thereof |
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114668742A (en) * | 2022-03-17 | 2022-06-28 | 中国科学技术大学 | Bacteria-like nano-drug delivery system and preparation method and application thereof |
| CN114404388A (en) * | 2022-03-31 | 2022-04-29 | 天九再生医学(天津)科技有限公司 | Method for loading mRNA in vitro by extracellular vesicles |
| CN114712520A (en) * | 2022-04-15 | 2022-07-08 | 中国药科大学 | Nanocrystalline drug stabilizing system, preparation method, pharmaceutical composition and application |
| CN114712520B (en) * | 2022-04-15 | 2024-02-27 | 中国药科大学 | Nanocrystalline drug stabilization system, preparation method, pharmaceutical composition and application |
| CN114870025B (en) * | 2022-04-28 | 2023-06-30 | 云南大学 | Responsive slow-release polydopamine-Zn-natural polyphenol coordination nano-drug and preparation method thereof |
| CN114870025A (en) * | 2022-04-28 | 2022-08-09 | 云南大学 | Response slow-release polydopamine-Zn-natural polyphenol coordination nano-medicament and preparation method thereof |
| CN115317461A (en) * | 2022-08-05 | 2022-11-11 | 常州大学 | Adriamycin delivery system Cu-GA-DOX NPs and preparation method thereof |
| CN115804761A (en) * | 2022-08-05 | 2023-03-17 | 暨南大学 | Bionic nano gene vector, preparation method and application |
| CN117582511A (en) * | 2022-08-09 | 2024-02-23 | 湖南健瑞医药科技有限公司 | Metal-phospholipid composite particles, and preparation method and application thereof |
| CN117582510A (en) * | 2022-08-09 | 2024-02-23 | 湖南健瑞医药科技有限公司 | Use of metal-polyphenol complexes in nucleic acid delivery systems |
| CN117582512A (en) * | 2022-08-09 | 2024-02-23 | 湖南健瑞医药科技有限公司 | Metal-polyphenol composite particles, and preparation method and application thereof |
| CN117582417A (en) * | 2022-08-09 | 2024-02-23 | 湖南健瑞医药科技有限公司 | Medicine-lipid particle and preparation method and application thereof |
| WO2024099251A1 (en) * | 2022-11-08 | 2024-05-16 | 山东省创启先进材料研究院 | Imaging agent containing myricetin nanomaterial, preparation method therefor, and use thereof |
| WO2024099250A1 (en) * | 2022-11-08 | 2024-05-16 | 山东省创启先进材料研究院 | Myricetin nanomaterial preparation, preparation method therefor, and use thereof |
| CN117467708A (en) * | 2023-10-17 | 2024-01-30 | 中山大学孙逸仙纪念医院 | Nucleic acid delivery complex and preparation method and application thereof |
| CN117467708B (en) * | 2023-10-17 | 2024-06-14 | 中山大学孙逸仙纪念医院 | Nucleic acid delivery complex and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114099533A (en) | Nucleic acid drug delivery system, preparation method, pharmaceutical composition and application | |
| Xi et al. | Drug loading techniques for exosome-based drug delivery systems | |
| EP1017365B1 (en) | Liposome-entrapped polynucleotide composition and method | |
| JP2024023853A (en) | Use of exosomes for treatment of disease | |
| TWI618548B (en) | Method of producing lipid nanoparticles for drug delivery | |
| Algarni et al. | In vivo delivery of plasmid DNA by lipid nanoparticles: the influence of ionizable cationic lipids on organ-selective gene expression | |
| Koh et al. | Delivery of antisense oligodeoxyribonucleotide lipopolyplex nanoparticles assembled by microfluidic hydrodynamic focusing | |
| EP2892505B1 (en) | Lipid assemblies comprising anionic lysolipids and use thereof | |
| JP2008520600A (en) | Improvements in or relating to pharmaceutical compositions for topical administration | |
| CA2742831A1 (en) | Fusogenic properties of saposin c and related proteins and peptides for application to transmembrane drug delivery systems | |
| US20220347109A1 (en) | Exosome-Mediated Transfection for Delivery of Nucleic Acids | |
| CN113058042B (en) | Preparation method of lipid nanoparticle capable of being subjected to nasal spraying and used for stably delivering RNA molecules | |
| JP2021506795A (en) | Methods and Compositions for Treating Cancer Using Exosome-Related Gene Editing | |
| Zhu et al. | Strategies for engineering exosomes and their applications in drug delivery | |
| WO2018072646A1 (en) | Lipid nanoparticle of antisense oligonucleic acid for inhibiting bcl-2 and preparation method therefor | |
| WO2014030601A1 (en) | Method for producing novel nano-bubble poly-lipo-plex having anionic property | |
| CN110898231B (en) | Functionalized Lalotxel liposome and preparation method and application thereof | |
| CN106913880B (en) | RSPO 1-containing targeted drug delivery system and preparation and application thereof | |
| CN112263565B (en) | Sorafenib-gene co-loaded nano-drug for cancer treatment and preparation method and application thereof | |
| Jing-Jing et al. | Fabrication of a folic acid-modified arsenic trioxide prodrug liposome and assessment of its anti-hepatocellular carcinoma activity | |
| WO2015160271A1 (en) | Lipid composition used for construction of liposomal genetic drug carrier targeted with antibodies, and use thereof | |
| EP4342496A1 (en) | Lipid nanoparticles | |
| CN113577305B (en) | Liposome containing albumin and cationic amphiphilic compound | |
| CN111920960B (en) | Protoporphyrin liposome for chemotherapy drug cell nucleus delivery and preparation method and application thereof | |
| Soleimani et al. | CD73 Downregulation by EGFR-Targeted Liposomal CD73 siRNA Potentiates Antitumor Effect of Liposomal Doxorubicin (Doxil) in 4T1 Tumor-Bearing Mice |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |