CN117084999A - Application of cationic lipid/polymer composite nano-particles in preparation of liver-targeted nucleic acid drug delivery carrier - Google Patents
Application of cationic lipid/polymer composite nano-particles in preparation of liver-targeted nucleic acid drug delivery carrier Download PDFInfo
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Classifications
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- 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
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
-
- 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
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Diabetes (AREA)
- Optics & Photonics (AREA)
- Biomedical Technology (AREA)
- Nanotechnology (AREA)
- Physics & Mathematics (AREA)
- Obesity (AREA)
- Molecular Biology (AREA)
- Emergency Medicine (AREA)
- Endocrinology (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to the technical field of targeted drug carriers. The invention provides a cationic lipid/polymer composite nanoparticle, a preparation method and application thereof in preparing a liver-targeted nucleic acid drug delivery carrier, wherein raw material components comprise a cationic polymer, DLin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol, dimyristoyl glycerol-polyethylene glycol, distearoyl phosphatidylethanolamine-polyethylene glycol-liver-targeted polypeptide, a 4, 8-disubstituted benzobisthiadiazole derivative and siRNA for specifically inhibiting target gene expression. The nano particles have the advantages of cationic lipid and cationic polymer, namely the advantages of both the cationic lipid and the cationic polymer, so that the prepared nucleic acid drug delivery carrier has the characteristics of high load and low toxicity.
Description
Technical Field
The invention relates to the technical field of targeted drug carriers, in particular to application of cationic lipid/polymer composite nano particles in preparation of a liver-targeted nucleic acid drug delivery carrier.
Background
The small interfering RNA (Small interfering RNA, siRNA) is double-stranded small molecule nucleic acid, plays an RNA interference Role (RNAi) after transcription, and the siRNA medicine is used as a hot spot for research and development of the small nucleic acid medicine, so that the siRNA has wide application by virtue of the advantages of high gene silencing efficiency, controllable adverse reaction, convenience in synthesis and the like, and the siRNA can specifically inhibit the expression of RNA of a disease-related target gene so as to play a role in treatment, so that the RNA interference can play the same role as a targeting medicine. With the elucidation of biological mechanisms of siRNA and the rapid development of synthetic methods of siRNA, most of genes can be silenced by an siRNA technology, while siRNA mainly acts on RNA, and can specifically inhibit the expression of target genes, the acting site is specific, and normal gene expression is not normally influenced, so that the siRNA has better selectivity and specificity when being applied as a targeted drug. At present, several siRNA drugs are approved internationally by FDA for marketing. The major challenges facing current in vivo applications of siRNA are: the naked siRNA sequence is unstable, has a short half-life, and is difficult to deliver in vivo. In recent years, the development of stability modification and efficient delivery systems of siRNA has greatly accelerated development of siRNA drugs, but the problem of in vivo targeted delivery has yet to be solved and overcome.
Lipid Nanoparticles (LNPs) are lipid vesicles with a homogeneous lipid core, have high biocompatibility and biodegradability, and thus are used to deliver a variety of active ingredients. Many non-viral delivery systems now also use cationic lipid-based or cationic polymer-based nanoparticles that are better biocompatible and less cytotoxic; nanoparticles based on cationic polymers can adjust the polarity, degradability, and molecular weight of the polymer to alter the efficiency of the polymer to deliver RNA into cells. Although cationic liposome nanoparticles have good biocompatibility and relatively low toxicity in vivo and in vitro transfection, the nanoparticles have limited siRNA quantity because of limited positive charges; while cationic polymer nanoparticles are strong in plasticity, the efficiency of delivering siRNA into cells can be changed by adjusting the polarity, degradability and molecular weight of the polymer, and more siRNA can be loaded than cationic liposome nanoparticles, but the cytotoxicity is large, so that the amount of transfection agent needs to be controlled in a lower range. Therefore, developing a delivery system having both cationic lipid and cationic polymer functions is important to improve siRNA loading rate and to improve siRNA delivery efficiency.
Type 2 diabetes (T2D) is a complex polygenic disease, insulin resistance and impaired insulin secretion being key factors in the pathophysiology of T2D. Indian and chinese populations are more susceptible to liver or skeletal muscle insulin resistance than others, resulting in a specific form of insulin deficiency. The incidence of T2D in the current Chinese population is still in an ascending trend, but the control rate and the standard rate are still optimistic. T2D is used as a non-infectious disease, in the course of natural disease, the functions of islet beta cells gradually decrease along with the extension of the course of disease, and the secretion amount of insulin is gradually reduced, so that the demand and the dependence degree of hypoglycemic drugs are gradually increased, the body is used for producing drug resistance for a long time, the drug dosage is increased, and simultaneously, the toxic and side effects of the drugs are increased, so that the development of a targeting delivery carrier capable of improving the drug effect, small in toxic and side effects and high in drug delivery efficiency is urgently needed.
Disclosure of Invention
The invention aims to provide cationic lipid/polymer composite nano particles and application thereof in preparing liver-targeted nucleic acid drug delivery carriers, and solves the problems that cationic lipid has low siRNA adsorption rate, and cationic polymer has high cytotoxicity and lacks a carrier for efficiently delivering diabetes drugs in the prior art.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a cationic lipid/polymer composite nanoparticle, which comprises the following components: cationic polymers, DLin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol, dimyristoyl glycerol-polyethylene glycol, distearoyl phosphatidylethanolamine-polyethylene glycol-liver targeting polypeptides, 4, 8-disubstituted benzobisthiadiazole derivatives, and sirnas that specifically inhibit target gene expression.
Preferably, the cationic polymer is 2-5 parts by weight, DLin-MC3-DMA is 30-35 parts by weight, distearoyl phosphatidylcholine is 5-9 parts by weight, cholesterol is 10-16 parts by weight, dimyristoyl glycerol-polyethylene glycol is 2-5 parts by weight, distearoyl phosphatidylethanolamine-polyethylene glycol-liver targeting polypeptide is 0.2-1 part by weight, and 4, 8-disubstituted benzobisthiadiazole derivative is 0.5-1.2 parts by weight.
Preferably, the ratio of the amount of the substance of P element in the siRNA specifically inhibiting the target gene expression to the amount of the substance of total N element in the cationic polymer is (2-10): 1.
Preferably, the cationic polymer has a structural formula shown in formula (I):
preferably, the cationic polymer is obtained by reacting polysuccinimide (pSI), oleylamine (OA), histamine dihydrochloride, 4-dimethylaminopyridine and N, N-Dimethylformamide (DMF), wherein the mass-to-volume ratio of polysuccinimide, oleylamine, histamine dihydrochloride, 4-dimethylaminopyridine and N, N-dimethylformamide is (1-3) g to (2.5-2.5) g to 0.1g to (20-40) mL.
The invention also provides a preparation method of the cationic lipid/polymer composite nanoparticle, which comprises the following steps:
(1) Dissolving a cationic polymer, DLin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol, dimyristoyl glycerol-polyethylene glycol, distearoyl phosphatidylethanolamine-polyethylene glycol-liver targeting polypeptide and a 4, 8-disubstituted benzobisthiadiazole derivative in an organic solvent to obtain a substance 1;
(2) Dissolving siRNA in Tris-HCLbuffer to obtain a substance 2;
(3) And mixing the substance 1 with the substance 2, and dialyzing to obtain the cationic lipid/polymer composite nano particles.
Preferably, the organic solvent in the step (1) is ethanol, and the volume ratio of the total volume of the cationic polymer, DLin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol, dimyristoyl glycerol-polyethylene glycol, distearoyl phosphatidylethanolamine-polyethylene glycol-liver targeting polypeptide and 4, 8-disubstituted benzobisthiadiazole derivative to the organic solvent is 1 (5-10).
Preferably, the addition volume of the Tris-HCLbuffer in the step (2) is 3-8 times of the volume of the organic solvent.
The invention also provides application of the cationic lipid/polymer composite nanoparticle in preparing a liver-targeted nucleic acid drug delivery carrier.
Preferably, the liver-targeted nucleic acid drug comprises a diabetes drug.
By adopting the technical scheme, the invention has the following beneficial effects:
according to the technical scheme, the cationic lipid/polymer composite nanoparticle has the characteristics of high load and low toxicity, and the prepared nucleic acid drug delivery carrier can efficiently and targetdly deliver diabetes drugs by scientifically matching and using cationic polymers, DLin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol, dimyristoyl glycerol-polyethylene glycol, distearoyl phosphatidylethanolamine-polyethylene glycol-liver targeting polypeptide, 4, 8-disubstituted benzobishiadiazole derivatives and siRNA with specificity for inhibiting target gene expression.
Drawings
FIG. 1 is a schematic illustration of cationic lipid/polymer complex nanoparticles;
FIG. 2 shows the particle size distribution and zeta potential of example 2 (A in FIG. 2 is the particle size distribution, and B in FIG. 2 is the zeta potential);
FIG. 3 shows the particle size distribution and zeta potential of example 3 (A in FIG. 3 is the particle size distribution, and B in FIG. 3 is the zeta potential);
FIG. 4 shows the particle size distribution and zeta potential of example 4 (A in FIG. 4 is the particle size distribution and B in FIG. 4 is the zeta potential);
FIG. 5 shows the binding of the cationic lipid of example 2 and the cationic lipid/polymer of example 4 to siRNA, respectively (A in FIG. 5 shows the binding of the cationic lipid/polymer of example 4 to siRNA, and B in FIG. 5 shows the binding of the cationic lipid of example 2 to siRNA);
FIG. 6 is a fluorescence detection of uptake of cationic liposome nanoparticles in cells in vitro;
FIG. 7 shows the result of transfection of cationic lipid/polymer complex nanoparticles (A in FIG. 7 shows the result of detection at 24h of incubation, and B in FIG. 7 shows the result of detection at 48h of incubation);
FIG. 8 shows the GFP expression levels in HepG2 and HUV cells (A in FIG. 8 shows the result of detection at 24 hours of culture, and B in FIG. 8 shows the result of detection at 48 hours of culture);
FIG. 9 shows the amount of GFP mRNA expressed by fluorescent quantitative PCR (A in FIG. 9 shows the result of detection at 24h of incubation, and B in FIG. 9 shows the result of detection at 48h of incubation);
FIG. 10 is a near infrared two-zone in vivo imaging of mice injected with the cationic lipid/polymer nanoparticles of example 4.
Detailed Description
The invention provides a cationic lipid/polymer composite nanoparticle, which comprises the following components: cationic polymers, DLin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol, dimyristoyl glycerol-polyethylene glycol, distearoyl phosphatidylethanolamine-polyethylene glycol-liver targeting polypeptides, 4, 8-disubstituted benzobisthiadiazole derivatives, and sirnas that specifically inhibit target gene expression.
In the present invention, the cationic polymer is preferably 2 to 5 parts by weight, more preferably 3 to 4 parts by weight, still more preferably 3.2 parts by weight; the weight part of DLin-MC3-DMA is preferably 30-35 parts, more preferably 31-33 parts, still more preferably 32 parts; the weight part of distearoyl phosphatidylcholine (DSPC) is preferably 5-9 parts, more preferably 6-8 parts, still more preferably 7.4 parts; the Cholesterol (CHOL) is preferably 10 to 16 parts by weight, more preferably 12 to 14 parts by weight, still more preferably 13 parts by weight; the weight part of the dimyristoyl glycerol-polyethylene glycol (DMG-PEG 2000) is preferably 2-5 parts, more preferably 3-4 parts, still more preferably 3.17 parts; the distearoyl phosphatidylethanolamine-polyethylene glycol-liver targeting polypeptide (DSPE-PEG-WSW or WSWGPYSC) is preferably 0.2-1 part, more preferably 0.5-0.7 part, still more preferably 0.64 part; the weight part of the 4, 8-disubstituted benzobisthiadiazole derivative is preferably 0.5 to 1.2 parts, more preferably 0.7 to 1 part, still more preferably 0.9 part.
In the present invention, the ratio of the amount of the substance of P element in the siRNA specifically inhibiting the target gene expression to the amount of the substance of total N element in the cationic polymer is preferably (2-10): 1, more preferably (6-8): 1.
In the invention, the structural formula of the cationic polymer is shown as a formula (I). The cationic polymer is prepared by reacting polysuccinimide, oleylamine, histamine dihydrochloride, 4-dimethylaminopyridine and N, N-dimethylformamide, and the chemical reaction formula is shown as a formula (II). The mass volume ratio of the polysuccinimide, the oleylamine, the histamine dihydrochloride, the 4-dimethylaminopyridine and the N, N-dimethylformamide is preferably (1-3) g, 0.5-2.5 g, 0.1g, 20-40 mL, more preferably (1.5-2.5) g, 2g, 1-2 g, 0.1g, 25-35 mL, still more preferably 2g, 1.5g, 0.1g and 30mL. The structural formula of the cationic polymer is shown as a formula (I).
In the present invention, the preparation method of the cationic polymer comprises the following steps: (a) Mixing polysuccinimide, oleylamine, histamine dihydrochloride and 4-dimethylaminopyridine, and adding N, N-dimethylformamide for ultrasonic dissolution; (b) After partial dissolution, adding magnetic beads, heating and stirring on a magnetic stirring oil bath pan for dissolution; (c) And (3) dialyzing, centrifuging and freeze-drying the dissolved solution to obtain the product.
In the present invention, the power of the ultrasound in the step (a) is preferably 50 to 1000W, more preferably 200 to 800W, still more preferably 500W. The heating temperature in step (b) in the present invention is preferably 50 to 100 ℃, more preferably 60 to 90 ℃, still more preferably 80 ℃. In the present invention, the dialysis in step (c) is performed by using 3500D dialysis bag, and the dialysis time is preferably 12-24 hours, more preferably 15-22 hours, and still more preferably 19 hours. The purpose of the dialysis according to the invention is to remove 4-dimethylaminopyridine, excess histamine dihydrochloride and solvent N, N dimethylformamide. The speed of centrifugation in the present invention is preferably 3500 to 4500rpm, more preferably 3700 to 4200rpm, still more preferably 4000rpm; the time of the centrifugation is preferably 20 to 40s, more preferably 25 to 35s, still more preferably 30s. The temperature of the freeze-drying is preferably-10 ℃ to-80 ℃, more preferably-20 ℃ to-70 ℃, and still more preferably-50 ℃.
In the invention, the structural formula of DLin-MC3-DMA (4- (N, N-dimethylamino) methyl butyrate) is shown as a formula (III).
In the invention, the structural formula of distearoyl phosphatidylcholine is shown as a formula (IV).
In the invention, the structural formula of the cholesterol is shown as a formula (V).
In the present invention, the distearoyl phosphatidylethanolamine-polyethylene glycol-liver targeting polypeptide (DSPE-PEG-WSW) is commercially available, and the DSPE-PEG-WSW is commercially available from the sierraxi biotechnology limited company.
In the invention, the structural formula of the dimyristoylglycerol-polyethylene glycol is shown as a formula (VI).
The invention also provides a preparation method of the cationic lipid/polymer composite nanoparticle, which comprises the following steps:
(1) Dissolving a cationic polymer, DLin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol, dimyristoyl glycerol-polyethylene glycol distearoyl phosphatidylethanolamine-polyethylene glycol-liver targeting polypeptide and a 4, 8-disubstituted benzobisthiadiazole derivative in an organic solvent to obtain a substance 1;
(2) Dissolving siRNA in Tris-HCLbuffer to obtain a substance 2;
(3) And mixing the substance 1 with the substance 2, and dialyzing to obtain the cationic lipid/polymer composite nano particles.
In the invention, each component of the cationic polymer, DLin-MC3-DMA, DSPC, chol, DMG-PEG2000, DSPE-PEG-WSW and 4, 8-disubstituted benzobisthiadiazole derivative is dissolved in organic solvent ethanol to obtain a substance 1, wherein the volume fraction of the ethanol is preferably 20% -99%, more preferably 45% -98%, even more preferably 92%. The volume ratio of the total volume of the components to the organic solvent in the present invention is preferably 1 (5-10), more preferably 1 (7-9), still more preferably 1:8. In the invention, the siRNA is dissolved in Tris-HCLbuffer to obtain a substance 2, wherein the Tris-HCLbuffer is acidic, and the addition volume of the Tris-HCLbuffer is preferably 3-8 times, more preferably 4-7 times, still more preferably 5 times of the volume of the organic solvent. In the invention, the substance 1 and the substance 2 are mixed and dialyzed, the substance 1 and the substance 2 are rapidly mixed in Tris-HCLbuffer by adopting an ethanol injection precipitation method, and then the mixture is dialyzed by ultrapure water to remove ethanol, so that the cationic lipid/polymer composite nano particles are obtained.
The invention also provides application of the cationic lipid/polymer composite nanoparticle in preparing a liver-targeted nucleic acid drug delivery carrier.
In the present invention, the liver-targeted nucleic acid drug includes a diabetes drug.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
The siRNA is a sequence of targeting GFP, and the sequence of 5'-3' is as follows: GCACCAUCUUCUCUCAAGGAdTdT.
Example 1
The preparation method of the cationic polymer comprises the following steps:
taking 2g of polysuccinimide pSI powder, OAm g of oleylamine, 1.5g of histamine dihydrochloride and 0.1g of 4-dimethylaminopyridine, placing the powder into a round-bottomed flask, adding 30mL of N, N-Dimethylformamide (DMF) into the flask, and performing ultrasonic dissolution by using an ultrasonic breaker; after part of the polymer is dissolved in N, N dimethylformamide, adding magnetic beads into a round-bottom flask, placing the round-bottom flask into a magnetic stirring oil bath, connecting a condensing tube at the upper end, and heating and stirring at 50-100 ℃ overnight until the polymer is completely dissolved; dialyzing the dissolved solution in 3500D dialysis bag for 12-24h, removing 4-dimethylaminopyridine, excessive histamine dihydrochloride and solvent N, N dimethylformamide; after dialysis, the liquid in the dialysis bag was transferred to a centrifuge tube at 4000rpm for 30s; and freeze-drying the residual liquid by using a freeze dryer to obtain the product cationic polymer.
Example 2
The cationic liposome nanoparticle comprises the following components: 32mg DLin-MC3-DMA, 7.4mg DSPC, 13mg Chol, 3.17mg DMG-PEG2000, 0.64mg DSPE-PEG-WSW, and siRNA specifically inhibiting target gene expression having a ratio of the amount of the substance of the P element in the composition to the amount of the substance of the total N element in the cationic polymer of 8:1.
The preparation of the cationic liposome nanoparticle comprises the following steps: the weighed 32mg DLin-MC3-DMA, 7.4mg DSPC, 13mg Chol, 3.17mg DMG-PEG2000 and 0.64mg DSPE-PEG-WSW are dissolved in 8mL ethanol with 92% volume concentration, the siRNA is dissolved in acid Tris-HCLbuffer with five times volume of an organic solvent, and the cationic liposome composite nano particles loaded with the siRNA are assembled by adopting an ethanol injection precipitation method.
Example 3
Cationic lipid/polymer composite nanoparticles, comprising the following components: 3.2mg of cationic polymer, 32mg of DLin-MC3-DMA, 7.4mg of DSPC, 13mg of Chol, 3.17mg of DMG-PEG2000, 0.64mg of DSPE-PEG-WSW, and siRNA specifically inhibiting the expression of a target gene in a ratio of the amount of the substance of the P element in the composition to the amount of the substance of the total N element in the cationic polymer of 6:1.
The preparation method of the cationic lipid/polymer composite nano-particles comprises the following steps: 3.2mg of the above-weighed cationic polymer, 32mg of DLin-MC3-DMA, 7.4mg of DSPC, 13mg of Chol, 3.17mg of DMG-PEG2000, 0.64mg of DSPE-PEG-WSW were dissolved in 8mL of 92% ethanol, the siRNA was dissolved in 5-fold volume of acidic Tris-HCLbuffer, and siRNA-loaded cationic lipid/polymer composite nanoparticles were assembled by ethanol injection precipitation, which rapidly mixed the lipid component dissolved in ethanol solution with siRNA in Tris-HCl buffer, and then the mixture was dialyzed with ultra-pure water to remove ethanol, thereby obtaining final cationic lipid/polymer composite nanoparticles (see FIG. 1).
Example 4
Cationic lipid/polymer composite nanoparticles, comprising the following components: 3.2mg of cationic polymer, 32mg of DLin-MC3-DMA, 7.4mg of DSPC, 13mg of Chol, 3.17mg of DMG-PEG2000, 0.64mg of DSPE-PEG-WSW and 0.9mg of 4, 8-disubstituted benzobisthiadiazole derivative, and siRNA specifically inhibiting the expression of a target gene, wherein the ratio of the amount of the substance of the P element to the amount of the substance of the total N element in the cationic polymer is 6:1.
The preparation method of the cationic lipid/polymer composite nano-particles comprises the following steps: the above weighed components of 6mg cationic polymer, 3.2mg cationic polymer, 32mg DLin-MC3-DMA, 7.4mg DSPC, 13mg Chol, 3.17mg DMG-PEG2000, 0.64mg DSPE-PEG-WSW and 0.9mg 4, 8-disubstituted benzobisthiadiazole derivative were dissolved in 8mL ethanol with 92% concentration by volume, the siRNA was dissolved in 5-fold volume of acidic Tris-HCLbuffer, and the cationic lipid/polymer nanoparticle loaded with siRNA was assembled by ethanol injection precipitation, by rapidly mixing the lipid component dissolved in ethanol solution with siRNA in Tris-HCl buffer, and then dialyzing the mixture with ultra-pure water to remove ethanol, thereby obtaining the final cationic lipid/polymer nanoparticle.
Comparative example 1
Cationic lipid/polymer composite nanoparticles, comprising the following components: 3.2mg of cationic polymer, 32mg of DLin-MC3-DMA, 7.4mg of DSPC, 12.6mg of Chol, 3.98mg of DMG-PEG2000, and siRNA specifically inhibiting the expression of a target gene in a ratio of 6:1 to the amount of the substance of the P element in the composition to the amount of the substance of the total N element in the cationic polymer.
The preparation method of the cationic lipid/polymer composite nano-particles comprises the following steps: 3.2mg of the weighed cationic polymer, 32mg of DLin-MC3-DMA, 7.4mg of DSPC, 12.6mg of Chol and 3.98mg of DMG-PEG2000 are dissolved in 8mL of ethanol with 92% volume concentration, the siRNA is dissolved in 5 times of the volume of acidic Tris-HCLbuffer, and the cationic lipid/polymer composite nano particles loaded with the siRNA are assembled by adopting an ethanol injection precipitation method.
Test example 1 determination of particle size and surface potential (Zeta potential) of cationic lipid/polymer composite nanoparticles
The particle sizes and charged sites of the cationic liposome nanoparticles, cationic lipid/polymer complex nanoparticles prepared in examples 2 to 4 were determined and analyzed using a Malvern ZetasizerNano ZS nm particle size potentiometric analyzer.
The method comprises the following specific steps:
(1) Three sets of samples of the prepared cationic liposome nanoparticles, cationic lipid/polymer complex nanoparticles described in examples 2-4 were prepared, respectively, wherein the mixing ratio of N/P in examples 2 and 4 was calculated as N/p=1:8, and the mixing ratio of N/P in example 3 was calculated as N/p=1:6 (N represents the amount of total nitrogen element-containing substance in the cationic polymer, and P represents the amount of total phosphorus element-containing substance in the disordered siRNA);
(2) Respectively taking 15 mu L of the three groups of samples, dripping the three groups of samples into a Markov micro sample pool, putting the samples into an instrument, measuring the particle size, and taking an average value for five times in each sample measurement;
(3) And respectively taking 50 mu L of the three groups of samples, fixing the volume to 1mL, dripping into a Markov potential sample pool, placing into an instrument, measuring the Zeta potential, and taking an average value three times for each sample measurement.
Analysis: the mean particle size and distribution of the cationic lipid/polymer complex nanoparticles was determined by DLS at 25 ℃. Each sample was tested in triplicate and averaged. The average particle size of the cationic lipid/polymer composite nanoparticles prepared from the three groups of samples was between 50nm and 200nm, and the potential was between 20mV and 40 mV. In general, LNPs are optimally between 20 and 200nm in size, considering that they need to be strong enough to withstand fluid flow (e.g., blood and lymph) while still allowing LNPs to pass through the matrix; the Zeta potential of the sample, which determines whether the particles in the liquid are stably present or tend to flocculate, is typically greater than-30 mV or greater than 30mV, and is generally sufficient to maintain interparticle repulsion and stable suspension of the particles. The nanoparticles prepared in examples 2-4 contain cations, and after the nanoparticles are compounded with siRNA with negative charges through the action of charge attraction, the nanoparticles are more stable and have more concentrated particle size distribution, so that the application of the cationic lipid/polymer composite nanoparticles in drug delivery is facilitated (the results are shown in figures 2-4).
Experimental example 2 adsorption experiment detection of siRNA
The method comprises the following specific steps:
(1) The cationic lipid of example 2 and the cationic lipid/polymer of example 4 were combined with a fragment of GFP-targeting siRNA,5'-3' with the following sequences: GCACCAUCUUCUCAAGGAdTdT is mixed according to a certain N/P mol ratio, and N/P is 1: 2. 1: 4. 1: 6. 1:8. 1:10, n represents the amount of the substance containing the total nitrogen element in the cationic polymer, and P represents the amount of the substance containing the total phosphorus element in the siRNA;
(2) Weighing 1g of agarose, adding 5mL of 10 Xelectrophoresis buffer, adding 45mL of distilled water, heating for dissolution, preparing into 2% agarose gel, cooling slightly, adding a chromogenic fluorescent nucleic acid stain GelStain, placing the agarose gel in an electrophoresis tank, and adding the newly prepared electrophoresis buffer into the electrophoresis tank;
(3) Samples of DNAmarker and mixed loading buffer were added dropwise to agarose gel lanes, run for 40 minutes at 220V,100A, and developed for detection by a gel developer.
As can be seen from fig. 5, the ratio N/P is 1: about 8, the binding rate of cationic lipid/polymer to siRNA is highest; at N/P1: 6 shows bands, meaning that now free siRNA migrates in the gel to the bands with electrostatic action; and at N/P1: 8, no band, means that all siRNA is adsorbed to the cationic lipid/polymer at this time, so N/P is considered to be 1: at 8, the binding rate of the cationic lipid/polymer and the siRNA is optimal; similarly, at N/P1: 6, the binding rate of cationic lipid to siRNA is optimal.
Test example 3 uptake assay of cationic liposome nanoparticles in cells in vitro
Selecting a dye cy3 modified GFP-targeting siRNA, and the sequence of 5'-3' is as follows: GCACCAUCUUCUCAAGGAdTdT, the cationic lipid nanoparticle prepared in the embodiment 2 of the invention is used for packaging siRNA and transfecting into a human hepatoma cell HepG2 (purchased from Shanghai enzyme research Biotechnology Co., ltd.); the human liver cancer cell line HepG2 with good growth state is digested by trypsin to 1X 10 4 cells/well density was inoculated in 8-well plates, DMEM medium containing 10% fetal bovine serum and 1% diabody (penicillin and streptomycin) was added at 37 ℃,5% co 2 Overnight culture, after 24h cell adherence, one group is blank control group, the blank control group is used for replacing DEME culture medium, the other group is experimental group, the experimental group is used for replacing DMEM culture medium containing prepared cationic liposome nano particles containing siRNA, and after incubation for 5h, the DMEM culture medium containing 10% fetal bovine serum is replaced; after 24h of siRNA transfection of HepG2 cells, observations were made using a fluorescent inverted microscope.
As can be seen from fluorescent chart 6 of the inverted microscope, the cationic liposome nanoparticle prepared by the invention is transfected into the human hepatoma cell HepG2, and after siRNA is transfected into the HepG2 cell for 24 hours, the bright field, blue light excitation and green light excitation of the fluorescent inverted microscope are respectively adopted to observe the blank control group and the experimental group respectively: the blank control group does not observe obvious red fluorescence under green light excitation, and the experimental group observes cy3 red fluorescence under green light excitation to be distributed in cells, which indicates that after the nanoparticles of the invention are loaded with siRNA, the siRNA is successfully delivered to target cells and enters cell membranes, and the delivery is completed.
Test example 4 cationic lipid/Polymer Complex nanoparticle transfection Effect detection
GFP-labeled human liver cancer cell line HepG2
mRNA of green jellyfish fluorescent protein (GFP) on the West coast of Victoria in the United states is selected as a target gene, a slow virus particle is utilized to enable a human liver cancer cell line HepG2 to stably express GFP, then the cationic lipid/polymer composite nano particles prepared in the embodiment 3 of the invention are utilized to transfect siRNA targeting GFP into the HepG2, and the sequence of 5'-3' is as follows: GCACCAUCUUCUCUCAAGGAdTdT.
(II) multifunctional enzyme-labeled instrument for detecting GFP expression level in HepG2 cells
The HepG2 cells were according to 1X 10 4 cell/well density was seeded in 96-well plates, 100. Mu.L of DMEM medium containing 10% fetal bovine serum and 1% diabody (penicillin and streptomycin) was added to each well, 37℃and 5% CO 2 After 24h incubation in a conditioning incubator, the medium was discarded, PBS was washed twice, serum-free DMEM was added, one group was the experimental group (Inp), 20. Mu.L of the cationic lipid/polymer complex nanoparticle of example 3 prepared by the present invention (siRNA (sequence 5 '-3': GCACCAUCUUCUUCAAGGAdTdT) was added to each well, the other group was the control group (naked siRNA), transfection was performed under otherwise identical conditions with naked siRNA without liposome carrier, the other group was the blank control group, i.e., transfection was performed under otherwise identical conditions without addition of liposome carrier and siRNA, incubation in the incubator was continued for 5h, the culture broth was aspirated, PBS was used for washing twice, and pre-warmed DMEM medium containing 10% fetal bovine serum and 1% diabody was added at 37℃and 5% CO 2 After the conditions are respectively cultured for 24 hours and 48 hours in a condition incubator, the incubator is placed in a multifunctional enzyme-labeled instrument BioTek, and after the incubator shakes for 10 seconds, excitation 488nm is set, and fluorescence intensity is detected by emitting 510 nm.
As can be seen from the results of FIG. 7, after 24 hours of incubation, the experimental group (Inp) had the weakest fluorescence intensity, the control group (naked siRNA) had the strongest fluorescence intensity, and the blank control group; after 48h of culture, the fluorescent intensity of the experimental group (Inp) is the weakest, the fluorescent intensity of the blank control group is the strongest, and the fluorescent intensity of the control group (naked siRNA) is slightly lower than that of the blank control group; the fluorescent intensities of the experimental groups (Inp) measured after 24h and 48h culture are the weakest, namely, the following is shown: the bare siRNA can not successfully cross cell membranes to enter cells to play the role of RNA interference, and the nano particles prepared by the embodiment 3 of the invention can wrap the siRNA in the cells and deliver the siRNA to human liver cancer cells, so that the siRNA successfully enters the cells, endosome escape is realized, and good delivery and release effects are realized.
Test example 5 multifunctional enzyme-labeled instrument verifies targeting effect of WSW modification
GFP-labeled human liver cancer cell line HepG2 and human umbilical vein endothelial cell HUV
Selecting mRNA of green jellyfish fluorescent protein (GFP) on the West coast of Victoria in the U.S. as a target gene, using slow virus particles to enable a human liver cancer cell line HepG2 and human umbilical vein endothelial cells HUV to stably express GFP, and then using the cationic lipid/polymer composite nano particles prepared in the embodiment 3 of the invention to transfer siRNA targeting GFP to the HepG2 and the HUV, wherein the sequence of 5'-3' is as follows: GCACCAUCUUCUCUCAAGGAdTdT.
(II) multifunctional enzyme-labeled instrument for detecting GFP expression level in HepG2 and HUV cells
HepG2 human liver cancer cell and HUV human umbilical vein endothelial cell were respectively prepared according to 1×10 4 cell/well density was seeded in 96-well plates with 100. Mu.L of DMEM medium containing 10% fetal bovine serum and 1% diabody at 37℃and 5% CO per well 2 Culturing in an incubator for 24 hours under the condition; taking the HepG2 and the HUV, discarding the culture medium, washing twice with PBS, adding serum-free DMEM, and taking the HepG2 (HepG 2-blank) and the HUV (HUV-blank) without adding the cationic lipid/polymer composite nano particles as blank control groups; GFP-siRNA-loaded cationic lipid/polymer complex nanoparticles (HepG2+LNP) provided in example 3 were added to HepG2 cells, and GFP-siRNA-loaded cationic lipid/polymer complex nanoparticles (HUV+LNP) provided in example 3 were added to HUV cells as experimental groups. The different treatments are carried outAfter 5h of further culture, the culture solution was aspirated, washed twice with PBS, and warmed-up DMEM medium containing 10% fetal bovine serum and 1% diabody was added at 37℃with 5% CO 2 Culturing in incubator for 24 hr and 48 hr; the sample to be tested is placed in a BioTek of an enzyme-labeled instrument, excitation 488nm is arranged after the plate is vibrated for 10 seconds, and fluorescence intensity is detected by emitting 510 nm.
As can be seen from the test results in FIG. 8, at the time points of 24 hours and 48 hours, the fluorescence intensity of the experiment group HepG2+LNP is the weakest, the fluorescence intensity of the blank group HepG2-blank and the HUV-blank is stronger, but the difference of the fluorescence intensities of the two blank control groups is not obvious; namely, compared with HUV human umbilical vein endothelial cells, the cationic lipid/polymer composite nano-particles prepared by the invention have better transfection effect on a human liver cancer cell line HepG2, which indicates that the cationic lipid/polymer composite nano-particles have better liver targeting.
Test example 6 fluorescent quantitative PCR analysis to verify the targeting effect of WSW modification
GFP-labeled human liver cancer cell line HepG2 and human umbilical vein endothelial cell HUV
mRNA of green jellyfish fluorescent protein (GFP) on the West coast of Victoria in the United states is selected as a target gene, a slow virus particle is utilized to enable a human liver cancer cell line HepG2 and a human umbilical vein endothelial cell HUV (purchased from Shanghai England Biotech Co., ltd.) to stably express GFP, and then siRNA targeting GFP is transfected into the HepG2 and the HUV by using the cationic lipid/polymer composite nano particles prepared in the embodiment 4 and the comparative example 1, wherein the sequence of 5'-3' is as follows: GCACCAUCUUCUCUCAAGGAdTdT.
(II) fluorescent quantitative PCR detection of GFP mRNA expression levels in HepG2 and HUV cells
The HepG2 and HUV cells were isolated according to 5X 10 5 cell/well density was seeded in 6-well plates, each well was supplemented with 2ml of DMEM medium containing 10% fetal calf serum and 1% diantigen (penicillin and streptomycin), 37℃and 5% CO 2 After culturing in a conditioned incubator for 24 hours, the medium was discarded, PBS was washed twice, serum-free DMEM was added, and HepG2 (HepG 2-blank) and HUV (HUV-blank) without cationic lipid/polymer complex nanoparticles were added as a blank pairA group is irradiated; adding GFP-siRNA-loaded cationic lipid/polymer complex nanoparticles (HepG2+LNP) provided in comparative example 1 to a group of HepG2 cells, and adding GFP-siRNA-loaded cationic lipid/polymer complex nanoparticles (HUV+LNP) provided in comparative example 1 to a group of HUV cells, as a negative control group; the cationic lipid/polymer complex nanoparticle (hepg2+lnp) provided in example 4 loaded with GFP-siRNA was added to another set of HepG2 cells, and the cationic lipid/polymer complex nanoparticle (huv+lnp) provided in example 4 loaded with GFP-siRNA was added to another set of HUV cells as an experimental group.
Culturing the above experimental samples for 5 hr, sucking off culture solution, washing with PBS twice, adding preheated DMEM medium containing 10% foetal calf serum and 1% double antibody, and adding 5% CO at 37deg.C 2 Culturing in incubator for 24 hr and 48 hr, and extracting total RNA from the sample. The specific operation of extracting the total RNA of the sample to be tested comprises the following steps: the culture medium in the 6-well plate is sucked and removed by a pipette, the cells are washed once by a 1xPBS buffer solution, PBS is sucked and removed, 1ml of TRIzol cell lysate is added to each well, the cells are blown and removed by the pipette, the lysate is sucked into a 1.5ml EP tube, the cells are kept standing and removed at room temperature for 5min,12000rpm,4 ℃ and centrifuged for 10min, the upper water phase is carefully sucked into a new 1.5ml EP tube, equal volume of isopropanol is added into the EP tube, the mixture is blown and removed by the pipette, after standing for 5min at room temperature, 12000rpm,4 ℃ and centrifuged for 15min, the supernatant is carefully sucked and removed by the pipette, 1ml of 75% ethanol diluted by DEPC water is added, the sediment is completely dissolved in ethanol by the pipetting, 12000rpm,4 ℃ and a proper amount of DEPC water is added for dissolving RNA sediment.
The concentration of the total RNA extracted above was measured by Nanodrop, and the total RNA was reverse transcribed into single-stranded cDNA using a reverse transcription kit (available from Shanghai Biotech Co., ltd.). When preparing the reverse transcription system, the reagent in the reverse transcription kit is added firstly, then 1.0 mug of RNA is added, and finally DEPC water is used for fixing the volume to 20 mug. After the system is configured, vortex mixing is carried out, the mixture is placed in a PCR instrument, the reaction time is 15min at 37 ℃, and then the reaction time is 5s at 85 ℃. And taking out the cDNA product after the reaction is finished, and placing the cDNA product at the temperature of-20 ℃ for standby.
Fluorescent quantitative PCR was then performed. When the fluorescent quantitative reaction system is prepared, 10. Mu.L of SYBR Green, 0.8. Mu.L of upstream primer, 0.8. Mu.L of downstream primer and 0.1. Mu.g of cDNA template are added, and DEPC water is finally added to fix the volume to 20. Mu.L. After the system is configured in the eight connecting pipes, the eight connecting pipes are centrifuged to ensure that no small bubbles exist in each pipe, otherwise, the system has a certain influence on fluorescence quantification, and the system is placed in a fluorescence quantitative PCR instrument, and the reaction procedure is as follows: stage one: reacting at 95 ℃ for 30s; stage two (cycle 40): 95 ℃ for 5s,60 ℃ for 30s,95 ℃ for 15s; stage three: the reaction was carried out at 60℃for 1min and at 95℃for 15s. After the reaction is finished, the original CT value is calculated into the expression quantity by a 2-delta Ct method, and the calculated result is processed by a normalization method and is drawn: the expression levels of the HepG2-blank and the HUV-blank are normalized, the knocking-down condition of the LNP-siRNA in the HepG2 cell can be seen from the expression level difference between the HepG2-blank and the HepG2+LNP group, and the knocking-down condition of the LNP-siRNA in the HUV cell can be seen from the expression level difference between the HUV-blank and the HUV+LNP group.
As can be seen from the results of fig. 9, the effect of 24h RNA interference was just shown, but it can be seen that the GFP mRNA expression level of the experimental group hepg2+lnp was lower than that of the negative control group and lower than that of the blank group, the GFP mRNA expression level of the experimental group huv+lnp was not significantly different from that of the negative control group, and the GFP mRNA expression level of both was not greatly different from that of the blank group; the RNA interference effect is obvious in 48 hours, and the GFP mRNA expression level of the HepG2+LNP of the experimental group is obviously lower than that of the HepG2-blank of the blank group, and the GFP mRNA expression level of the HepG2+LNP of the negative control group is lower than that of the blank group but higher than that of the experimental group; the GFP mRNA expression level of the HUV+LNP in the experimental group is not obviously different from that in the negative control group, and the GFP mRNA expression level of the HUV+LNP in the experimental group is slightly lower than that in the blank group. Namely, the cationic lipid/polymer composite nano-particles prepared by the method have better transfection effect on the HepG2 between the human hepatoma cell HepG2 and the human umbilical vein endothelial cell HUV under the same culture condition, and have better targeting on the liver after the cationic lipid/polymer composite nano-particles are subjected to targeted modification by using DSPE-PEG-WSW.
Experimental example 7 liver targeting effect verification of cationic lipid/polymer complex nanoparticle
To verify the liver targeting effect of the cationic liposome nanoparticle of the present invention, 6 week old female Balb/c mice (purchased from beijing villous laboratory animal technologies limited) were selected for in vivo imaging, and specific experimental procedures were as follows:
(1) All hairs except the head of the Balb/c mouse are shaved, anesthetic chloral hydrate is prepared in advance, the mouse is generally injected in an intraperitoneal mode according to 0.6mL/100g, and the mouse tail tip is pinched by forceps during anesthesia, so that the anesthetic state of the mouse is judged;
(2) After the mice were anesthetized, tail intravenous injection was performed using an insulin needle to aspirate the cationic lipid/polymer complex nanoparticles provided in example 4, with a dose of 100 μl per mouse;
(3) The mice were placed in a near infrared two-zone living animal imager 24 hours after injection, and photographed using a 808nm laser.
Analysis of experimental results: to verify the liver targeting effect of the cationic lipid/polymer nanoparticles of the present invention, 6 week old female Balb/c mice were selected for in vivo imaging. Before tail vein injection and 24 hours after injection, the mice are anesthetized and then are placed into a near infrared two-zone living animal imager for photographing, and the results show that (as shown in fig. 10) obvious fluorescence is visible at the liver part of the mice, and no fluorescence is observed at other parts of the mice, namely, the cationic lipid/polymer composite nano particles are mainly accumulated in the liver after 24 hours of tail vein injection; the targeting effect of the cationic lipid/polymer composite nanoparticle prepared in this example on liver after tail vein injection in vivo is illustrated.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. A cationic lipid/polymer composite nanoparticle comprising the following components: cationic polymers, DLin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol, dimyristoyl glycerol-polyethylene glycol, distearoyl phosphatidylethanolamine-polyethylene glycol-liver targeting polypeptides, 4, 8-disubstituted benzobisthiadiazole derivatives, and sirnas that specifically inhibit target gene expression;
2-5 parts of cationic polymer, 30-35 parts of DLin-MC3-DMA, 5-9 parts of distearoyl phosphatidylcholine, 10-16 parts of cholesterol, 2-5 parts of dimyristoyl glycerol-polyethylene glycol, 0.2-1 part of distearoyl phosphatidylethanolamine-polyethylene glycol-liver targeting polypeptide and 0.5-1.2 parts of 4, 8-disubstituted benzodithiadiazole derivative;
the ratio of the amount of the substance of the P element in the siRNA specifically inhibiting the target gene expression to the amount of the substance of the total N element in the cationic polymer is (2-10): 1.
2. The cationic lipid/polymer composite nanoparticle of claim 1, wherein the cationic polymer has a structural formula as shown in formula (i):
3. the cationic lipid/polymer composite nanoparticle according to claim 2, wherein the cationic polymer is obtained by reacting polysuccinimide, oleylamine, histamine dihydrochloride, 4-dimethylaminopyridine and N, N-dimethylformamide, and the mass-to-volume ratio of polysuccinimide, oleylamine, histamine dihydrochloride, 4-dimethylaminopyridine and N, N-dimethylformamide is (1-3) g:2g (0.5-2.5) g:0.1g (20-40) mL.
4. A method for preparing cationic lipid/polymer composite nanoparticles according to any one of claims 1 to 3, comprising the steps of:
(1) Dissolving a cationic polymer, DLin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol, dimyristoyl glycerol-polyethylene glycol, distearoyl phosphatidylethanolamine-polyethylene glycol-liver targeting polypeptide and a 4, 8-disubstituted benzobisthiadiazole derivative in an organic solvent to obtain a substance 1;
(2) Dissolving siRNA in Tris-HCLbuffer to obtain a substance 2;
(3) And mixing the substance 1 with the substance 2, and dialyzing to obtain the cationic lipid/polymer composite nano particles.
5. The method according to claim 4, wherein the organic solvent in the step (1) is ethanol, and the volume ratio of the total volume of the cationic polymer, DLin-MC3-DMA, distearoyl phosphatidylcholine, cholesterol, dimyristoyl glycerol-polyethylene glycol, distearoyl phosphatidylethanolamine-polyethylene glycol-liver targeting polypeptide, and 4, 8-disubstituted benzobisthiadiazole derivative to the organic solvent is 1 (5-10).
6. The method according to claim 4, wherein the Tris-HCl buffer in step (2) is added in an amount of 3 to 8 times the volume of the organic solvent.
7. Use of the cationic lipid/polymer complex nanoparticle of any one of claims 1-3 or the cationic lipid/polymer complex nanoparticle prepared by the preparation method of any one of claims 4-6 for the preparation of a liver-targeted nucleic acid drug delivery vehicle.
8. The use of claim 7, wherein the liver-targeted nucleic acid drug comprises a diabetes drug.
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| CN116425973A (en) * | 2023-04-03 | 2023-07-14 | 深圳市华元生物技术股份有限公司 | PH-responsive liver-targeted drug delivery carrier and preparation method and application thereof |
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| CN116425973A (en) * | 2023-04-03 | 2023-07-14 | 深圳市华元生物技术股份有限公司 | PH-responsive liver-targeted drug delivery carrier and preparation method and application thereof |
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