WO2012139469A1 - Delivery system and preparation of low-interference rna drug - Google Patents

Delivery system and preparation of low-interference rna drug Download PDF

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WO2012139469A1
WO2012139469A1 PCT/CN2012/073366 CN2012073366W WO2012139469A1 WO 2012139469 A1 WO2012139469 A1 WO 2012139469A1 CN 2012073366 W CN2012073366 W CN 2012073366W WO 2012139469 A1 WO2012139469 A1 WO 2012139469A1
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sirna
pla
peg
nanoparticles
composition according
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PCT/CN2012/073366
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French (fr)
Chinese (zh)
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王均
杨显珠
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苏州瑞博生物技术有限公司
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    • 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
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • 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
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to a polyethylene glycol-polylactic acid or a polyethylene glycol-poly(lactic-glycolic acid) two-block/triblock copolymer (amphiphilic polymer) and a cationic lipid material Small interfering RNA (siRNA) delivery systems and formulations.
  • siRNA Small interfering RNA
  • RA Small interfering RA has the ability to specifically inhibit the expression of disease-causing genes as well as high-efficiency and diverse characteristics. In recent years, it has shown good application prospects in the treatment of a large number of diseases such as hepatitis, AIDS, age-related macular degeneration, avian influenza and cancer. .
  • the RA-interfering CALAA-01 formulation developed by Calendo Pharmaceuticals shows the effect of treating cancer in a phase I clinical trial after systemic administration. There is reason to believe that siRNA-based RNA interference therapy may be used for clinical treatment in the near future. However, there are significant challenges in the delivery of siRNA in vivo for the purpose of treating human diseases.
  • siRNA molecule itself has a very poor ability to penetrate the cell membrane, has no targeting function, and is extremely unstable in a physiological environment, the current bottleneck in the development of siRNA drugs lies in the delivery system and technology of siRNA. How to enhance the stability of siRNA in vivo and the ability to penetrate cell membranes, as well as enhance the cell and tissue targeting of disease treatment, are urgent problems to be solved by siRNA delivery systems. Therefore, the in vivo drug delivery system of siRNA has also become a key to the development and implementation of R A interference therapy.
  • siRNA delivery systems Materials currently used in the construction of siRNA delivery systems include polyethyleneimine (PEI), degraded collagen with terminal peptides, some cationic liposomes, and various cationic polymers synthesized chemically. These carrier molecules are mixed with the siRNA solution to form a complex of the two by charge interaction, thereby achieving the purpose of delivering siRNA.
  • PKI polyethyleneimine
  • Such drug delivery systems face problems that are difficult to scale up and are less reproducible.
  • some studies have reported the in vivo administration of siRNA by encapsulating siRNA in a polymer to prepare a nanoscale formulation. Such nanoparticles are generally prepared by using polylactic acid, polyglycolic acid or a copolymer of the two.
  • the disadvantage is that the encapsulation efficiency of siRNA is very low (less than 30%), and the drug loading is very low, which is difficult to meet clinical application. Claim. Summary of the invention
  • the object of the present invention is to provide a nucleic acid drug delivery carrier, in particular to a drug delivery vehicle containing an amphiphilic polymer and a cationic lipid, and a use thereof, a preparation method and a drug combination thereof Things.
  • This drug delivery vehicle composition has an extremely high siRNA encapsulation efficiency and a large drug loading.
  • siRNA is added to a solution of a degradable amphiphilic polymer and a cationic lipid, and the siRNA is embedded in the nanoparticle formed by the amphiphilic polymer and the cationic lipid by a double emulsion method. .
  • the added cationic lipid increased the encapsulation efficiency of siRNA to over 90%.
  • This method can not only prepare nanoparticles with high efficiency of encapsulating siRNA, but also the prepared nanoparticles can effectively enter cells and escape from endosomes, thereby effectively silencing the expression of pathogenic target genes and inhibiting the growth of breast cancer in vivo.
  • the carrier composition i.e., the nucleic acid drug-administered carrier
  • the carrier composition contains an amphiphilic polymer (component A) and a cationic lipid (component B).
  • the amphiphilic polymer refers to an amphiphilic block copolymer containing both hydrophilic and hydrophobic segments on one macromolecular chain.
  • the hydrophilic segment in the amphiphilic block copolymer may be polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylamide, polyamidomethacrylate, polyacrylic acid and polymethyl. Any homopolymer or copolymer formed from one or two or more of acrylic acid.
  • the hydrophobic segment in the amphiphilic block copolymer may be any homopolymer formed of one or more of polylactic acid, polycaprolactone, polyglycolide, polyamino acid and polyphosphazene. Or a copolymer.
  • the amphiphilic polymer is polyethylene glycol (PEG)-polylactic acid (PLA) or polyethylene glycol-poly(lactic-glycolic acid) (PLGA).
  • the diblock or triblock copolymer may, for example, be PEG-PLA, PEG-PLGA, PLA-PEG-PLA, PLGA-PEG-PLGA.
  • the block copolymer can self-assemble into micelles or nanoparticles in an aqueous medium, and the relatively hydrophobic PLA or PLGA aggregates into a hydrophobic core, and the PEG block assembles into a hydrophilic shell, which has stable micelles and effectively evades.
  • the primary role of cationic lipids in this delivery system is to increase the drug loading and encapsulation efficiency of the nucleic acid drug in the carrier by electrostatic interaction.
  • the cationic lipid is preferably an amphiphilic lipid material of the ammonium salt type, and the present invention is not particularly limited to the ammonium salt type amphiphilic lipid material as long as it is pharmacologically acceptable.
  • DDAB dimethyl dioctadecyl ammonium bromide
  • DOTAP 1,2-dimyristoyl-3-trimethylammonium propane
  • DOTAP 1,2-dioleyl-3-trimethyl Radium-based propane
  • 1,2-dipalmitoyl-3-trimethylammonium propane 1,2-distearyl Acyl-3-trimethylammonium propane, N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), dimyristoyl Oxypropyl dimethyl hydroxyethyl ammonium bromide (DMRIE), dioleoyloxypropyl dimethyl hydroxyethyl ammonium bromide (DORIE), dimethyl di(dodecyl) bromination Ammonium, N-(a-trimethylammonioacetyl)-di(do
  • the cationic lipid is preferably N,N-dihydroxyethylmethyl-N-2-(cholesteryloxycarbonylamino)ethylammonium bromide (BHEM-Chol), (2,3-dimethoxypropyl)trimethylammonium chloride (DOTAP) and N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethyl At least one of the ammonium chlorides.
  • BHEM-Chol cholesteryloxycarbonylamino)ethylammonium bromide
  • DOTAP (2,3-dimethoxypropyl)trimethylammonium chloride
  • N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethyl At least one of the ammonium chlorides.
  • the compounding ratio of the component A and the component B in the carrier composition is not particularly limited, and the amphiphilic polymer and the cationic lipid may be in a range of from the viewpoint of efficiently transporting the nucleic acid into the cell.
  • the amphiphilic high molecular polymer is 100 parts by weight, and the cationic lipid is 0.001 to 20 parts by weight, preferably 0.01 to 10 parts by weight; preferably, the amphiphilic property is high based on the total weight of the carrier composition.
  • the molecular polymer content is
  • the cationic lipid content is 0.001 to 16.7% by weight, further preferably, the amphiphilic high molecular polymer is contained in an amount of 90.9 to 99.999% by weight, and the cationic lipid is contained in an amount of 0.001 to 9.1% by weight. %.
  • the number average molecular weight of the polyethylene glycol monoblock is preferably 550 to 10000 g/mol, and the number average molecular weight of the polylactic acid or poly(lactic acid-glycolic acid) block is preferably 4800 to 51000 g/mol, wherein The number average molecular weight of the poly(lactic acid-glycolic acid) block is more preferably from 10,000 to 50,000 g/mol.
  • the degree of polymerization of the lactide/glycolide monomer may be from 75/25 to 50/50.
  • the molecular weight of the cationic lipid BHEM-Chol is 655.5 g/mol
  • the molecular weight of the cationic lipid DOTAP is 698.5 g/mol
  • the molecular weight of the cationic lipid DOTMA is 670.6 g/mol.
  • the above block copolymer may be PEG 55 Q-PLA 286 QQ, PEG 2 QQQ-PLA 125 QQ, PEG 5 QQQ-PLA 5000 , PEG5000-PLA25000, PEG5000-PL A51000, PEG10000-PLA15000, PEGioooo-PLGAioooo (5o/ 50), PEGioooo-PLGA5oooo (75/25), PLA 6 3oo-PEGi2oo-PLA 6 3oo, PLA4800-PEG5000-PLA4800 and PLA4 8 oo-PEG 10 ooo-PLA4 8 oo, wherein the subscript indicates the number average molecular weight,
  • the ratio in the lower brackets is the ratio of the degree of polymerization of the lactide monomer/glycolide monomer in the poly(lactic-glycolic acid) block (hereinafter the same as this), and the present invention is not limited to the above-described composition. Segment copolymer.
  • Polyethylene glycol-polylactic acid, polyethylene glycol-poly(lactic-glycolic acid), polyethylene glycol-polylactic acid triblock copolymer or polyethylene glycol-poly(lactic-glycolic acid) triblock copolymer The amphiphilic substance, these block copolymers and cationic lipids can form nano-scale particles by a double-emulsification method, such nanoparticles have a hydrophilic polyethylene glycol outer shell, and the size of the particles and the composition of the copolymer Related, can be regulated.
  • the administration carrier containing the above amphiphilic high molecular polymer and cationic lipid can form nanoparticles, and the nanoparticles may have a diameter of 50 to 250 nm.
  • the surface of the nanoparticles of the present invention may have chemical modifications, antibody modifications or ligand modifications.
  • the method of modification can be a variety of conventional methods for modifying the surface of nanoparticles.
  • cationic lipids significantly increased the encapsulation efficiency and drug loading of siR A.
  • the use of such nanoparticle as a carrier enables endocytosis of the encapsulated siRNA nanoparticles and achieves gene silencing.
  • the carrier composition of the present invention may further contain an oily base (hereinafter referred to as a component C) in addition to the components A and B described above.
  • a component C an oily base
  • the nucleic acid introduction efficiency of the carrier composition for nucleic acid transport can be controlled by utilizing its characteristics. For example, by adjusting the specific gravity of the carrier composition for nucleic acid transport by blending an oily base, cells and carriers can be controlled.
  • the contact properties of the body composition improve the efficiency of introduction in vitro.
  • the core of the nucleic acid carrier can be broken under a predetermined temperature condition, the fluctuation of the cell surface can be induced, and the introduction efficiency of the nucleic acid can be improved.
  • the core of the nucleic acid carrier composition can be broken by external stimulation, the fluctuation of the cell surface can be induced, and the introduction efficiency of the nucleic acid can be improved.
  • the oily base material to be used in the carrier composition of the present invention may, for example, be a perfluorocarbon, perfluoropentane, brominated perfluorooctane, perfluorohexane, perfluorotributylamine or soybean oil.
  • soybean oil refined soybean oil, hydrogenated soybean oil, soybean oil unsaponifiable, squalene, castor oil, clove oil, sorbitan trioleate, turpentine, safflower oil, safflower oil fatty acid, oleic acid, palm oil , rapeseed oil, fusel oil, olive oil, linseed oil, sesame oil, chlorophyll oil, croton oil, bergamot oil, cedar oil, orange oil, fennel oil, eucalyptus oil, corn oil, lavender oil, marjoram oil, lemon Oil, cottonseed oil, coconut oil, egg butter, rose oil, pine oil, almond oil, peanut oil, camellia oil, white eucalyptus oil, chamomile oil, cinnamon oil, peppermint oil, esterified corn oil, ginger oil, Roman spring Chamomile oil, snake oil, spearmint oil, sunflower oil, cocoa butter, wheat germ oil, zinc oxide oil, hydrogenated oil, hydrogenated vegetable oil, light
  • perfluoropentane has temperature sensitivity and has a property of being vaporized by boiling at 29.5 °C.
  • perfluorohexane, brominated perfluorooctane, and perfluorotributylamine have the following characteristics, that is, external stimuli-breaking property, and the carrier is stimulated by external stimuli such as stimulation by ultrasonic irradiation.
  • the core of the composition creates a cavity that breaks it.
  • the ratio of the oily base is not particularly limited as long as it does not impair the effects of the present invention, and may be, for example, a ratio of the above-mentioned A component and B component to 100 parts by weight.
  • the total amount of the oily substrate is from 0.1 to 50 parts by weight, preferably from 1 to 30 parts by weight, more preferably from 5 to 20 parts by weight.
  • the carrier composition of the present invention may contain a membrane-fused lipid (auxiliary lipid) as needed.
  • a membrane-fused lipid auxiliary lipid
  • the efficiency of transport of nucleic acid into cells can be further improved.
  • the membrane-fused lipid include dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine, transphosphatidylethanolamine, and 1,2-bis(10,12-docosanedioyl).
  • the ratio of the membrane-fused lipid is not particularly limited as long as the effect of the present invention is not impaired, and the following ratio is used, that is, the above-mentioned ratio with respect to 100 parts by weight.
  • the film-fused lipid is added in an amount of 1 to 500 parts by weight, preferably 10 to 250 parts by weight, more preferably 25 to 100 parts by weight, based on the total amount of the component and the component B.
  • the carrier composition of the present invention may contain various additives such as an isotonic agent, an excipient, a diluent, a thickener, a stabilizer, a buffer, and a preservative depending on the form of use.
  • the compounding amount of the above additives can be appropriately set depending on the form of use of the nucleic acid transport carrier.
  • the carrier composition for nucleic acid delivery of the present invention is prepared by mixing the above-mentioned A component, B component, and, if necessary, mixing other components.
  • the vector composition of the present invention can be used for the transport of exogenous nucleic acids, wherein the present invention does not specifically limit the kind or structure of the exogenous nucleic acid.
  • the nucleic acid may be siR A, mRNA, tRNA, rRNA, cDNA, miR A (microRA), ribozyme, antisense oligonucleotide, plasmid DNA, peptide nucleic acid, and triple-stranded oligonucleoside Acid (Triplex Forming Oligonucleotide, TFO), genes, etc.
  • the vector composition of the present invention is particularly suitable for transporting siRNA into cells.
  • the nucleic acid to which the vector composition of the present invention is applied may be a nucleic acid derived from a human, an animal, a plant, a bacterium, a virus or the like, or may be a nucleic acid prepared by chemical synthesis. Further, the nucleic acid may be any of a single chain, a double chain, and a triple chain, and the molecular weight thereof is not particularly limited. Further, in the present invention, the nucleic acid may be a nucleic acid modified by a chemical, an enzyme or a peptide. In the present invention, one type of nucleic acid may be used alone or two or more types may be used in combination as appropriate. In a preferred embodiment, the vector composition of the invention preferably transports small interfering nucleic acids (siRNA) or analogs thereof.
  • siRNA small interfering nucleic acids
  • the present invention also provides a pharmaceutical composition comprising the above carrier composition and nucleic acid.
  • the present invention is not particularly limited to the kind or structure of the nucleic acid to be used in the pharmaceutical composition.
  • Specific examples of the nucleic acid may be siRNA, mR A, tRNA, rRNA, cDNA, miRNA (microRA), ribozyme, antisense oligonucleotide, plasmid DNA, peptide nucleic acid, and triple-stranded oligonucleotide. (Triplex Forming Oligonucleotide, TFO), genes, etc.
  • the nucleic acid is preferably a small interfering nucleic acid (siR A).
  • the mass ratio of the nucleic acid, the cationic lipid to the amphiphilic high molecular polymer may be 0.04: 0.0001: 100.0-7.2: 20.0: 100.0, preferably 0.2/1.0/25.0-1.8/1.0/ 25.0, most preferably 0.2/1.0/25.0.
  • other drugs having synergistic therapeutic effects or reducing toxic side effects may be simultaneously contained.
  • the present invention also provides a method for preparing the above pharmaceutical composition, the method comprising the steps of: dissolving the amphiphilic high molecular polymer and the cationic lipid in an oil phase (such as chloroform, etc.), and adding the aqueous nucleic acid solution to perform the first step.
  • an oil phase such as chloroform, etc.
  • Secondary ultrasound An initial emulsion is formed, the initial emulsion is added to the aqueous phase and a second ultrasonication is performed to form the emulsion, the emulsion is added to the aqueous phase, the organic solvent is removed under reduced pressure (e.g., 1000 Pa), and the nanoparticles are collected by centrifugation.
  • the aqueous phase in the present invention includes, but is not limited to, an aqueous PVA solution.
  • the condition of the first ultrasound preferably comprises: a power of 80 watts and a time of 30 seconds; and the condition of the second ultrasound preferably comprises: a power of 80 watts and a time of 2 minutes.
  • the conditions of the centrifugation preferably include: a temperature of 4 ° C, a speed of 3000 g, and a time of 1 h.
  • the specific conditions for the preparation of the above pharmaceutical composition of the present invention are not limited to the above conditions.
  • the present invention also provides a nucleic acid introduction method for introducing a nucleic acid in a pharmaceutical composition into a cell by bringing the above pharmaceutical composition into contact with a cell.
  • the cell is preferably a mammalian cell, more preferably a mammalian cell in a pathological state or an abnormal physiological state, and the nucleic acid is preferably a small interfering nucleic acid (siR A).
  • the invention also provides the use of the carrier composition for the preparation of an anti-tumor drug, preferably a breast tumor or a liver tumor.
  • the block copolymer used in the present invention has good biocompatibility and degradability, and its physical and chemical properties can be adjusted by adjusting the composition of the polymer. For example, when increasing the proportion of PLA in a polymer, the ability of the nanoparticles to transport siRNA into the cell increases.
  • the present invention provides a delivery system and preparation for encapsulating siRNA by a double emulsion method using an amphiphilic polymer.
  • the prepared nanoparticles have good stability, simple preparation method, high siRNA encapsulation efficiency and drug loading amount, can protect siRNA from degradation, and can efficiently transport nucleic acid drugs into cells.
  • the drug delivery carrier of the nucleic acid drug provided by the present invention has high biocompatibility and degradability, it is less toxic to the organism and has high biosafety.
  • the drug delivery system and preparation are mainly applicable to the fields of small interference R A and administration preparations similar to small nucleic acid drugs.
  • the present invention utilizes the above-described administration systems and preparations to deliver specific siRNA, and has demonstrated its efficacy in silencing target gene expression at both the cellular and animal levels, as well as having the effect of silencing the oncogene Plkl expression and inhibiting the growth of breast cancer.
  • Fig. 1 is a biocompatibility test of a drug delivery system prepared by a double emulsion method for a polymer and a cationic lipid.
  • Fig. 2 is a laser distribution of a cell containing FAM-siRNA-loaded nanoparticles and HepG2 cells after 2 hours of culture. Focus on the microscope photo. Among them, the intracellular red fluorescence was derived from the Alexa 568-phalloidin-labeled cell skeleton; the green fluorescence was derived from FAM-siRNA; the blue fluorescence was derived from the DAPI-labeled nuclei.
  • Figure 3 is a graph showing the effect of down-regulating the mR A level of Plkl after the inclusion of siP/W nanoparticles into HepG2 cells.
  • Figure 4 is a graph showing the effect of the biological effect evaluation of the drug delivery system at the animal level.
  • 4A is a diagram showing the effect of the tail vein injection-loaded siLuci administration system on inhibiting the expression of luciferase in a mouse model of liver cancer implantation;
  • FIG. 4B is a tail vein injection of a siP d-administered system to inhibit the in situ implantation of mice. The effect map of breast cancer growth.
  • Figure 5 is a diagram showing the construction of a galactose-modified targeted drug delivery system and the effect of biological effect evaluation.
  • Figure 5A 1H NMR analysis of HOOC-PEG 5 QQQ-PLA 21 Q3Q and Gal-PEG 5 QQQ-PLA 21 Q 3 Q
  • Figure 5B is a flow cytometric assay for targeted drug delivery system in Hepa 1-6 cells
  • Figure 5C is a graph showing the effect of down-regulating the mRNA level of apoB after entering the siapoB-targeted drug delivery system into Hepa 1-6 cells
  • Figure 5D is a targeted delivery system containing siapoB-loaded siRNA silencing mouse hepatocyte apoB Effect map of protein expression.
  • FIG. 6 is a diagram showing the construction of a single-stranded fragment antibody-modified targeted drug delivery system and the effect of biological effect evaluation.
  • 6A is a 1H NMR analysis map of Mal-PEG 5 (xxrPLA 22()7() ;
  • FIG. 6B is a flow cytometric assay targeted drug delivery system for endocytosis of BT474 cells;
  • FIG. 6C is an entrapped siPl! The effect of d-targeted drug delivery system on the down-regulation of Plkl mRNA levels after entry into BT474 cells;
  • Figure 6D is a graph showing the effect of tail-injection of siPlkl-targeted drug delivery system to inhibit the growth of mice implanted in situ in breast cancer.
  • Racemic lactide (d, l-LA), purity ⁇ 99%, purified by sublimation under reduced pressure before use.
  • PEG550-PLA28600 PEG2000-PLA12500 PEGioooo-PLAi5ooo PLA63oo-PEGi2oo-PLA 6 3oo PLA 4 8oo-PEG 5 ooo-PLA 4 8oo ⁇ PLA 4800 -PEG 10000 -PLA 4800 was purchased from Polymer Source.
  • PEGKKKXTPLGAKKKKXSO/SQ) and PEG 1Q ( KKrPLGA 5( KKK) (75/25) were purchased from Jinan Co., Ltd.
  • a heterofunctional PEG with one end group being a carboxyl group and a hydroxyl group at another end (Mn of 5000 g/) Mol, HOOC-PEG 50 oo-OH)
  • hetero-functional PEG with end group of maleimide and hydroxyl group (Mn is 5000 g/mol, Mal-PEG 5 ooo-OH) purchased from Creative PEGWorks .
  • DCC ⁇ , ⁇ '-Dicyclohexylcarbodiimide
  • NHS ⁇ -hydroxysuccinimide
  • DMAP 4-dimethylaminopyridine
  • 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide ( ⁇ ), galactose (Gal-NH 2 ) and 4',6-didecyl- 2-Phenylhydrazine (DAPI) was purchased from Aldrich.
  • DOTAP and DOTMA were purchased from Avanti Polar Lipids.
  • BHEM-Chol is synthesized in the present invention, and the specific synthetic steps are as follows: 2-bromoethylamine hydrobromide (17.4 g, 85.0 mmol) and cholesteryl chloroformate (34.7 g, 77.3 mmol) were added to a 500 ml single-mouth bottle, dissolved. Triethylamine (24 mL, 172 mmol) was added dropwise to the above solution at -30 ° C in chloroform. After reacting at room temperature overnight, it was washed three times with a saturated sodium chloride solution (150 mL) containing 1 M hydrochloric acid and once with a saturated sodium chloride solution (150 mL).
  • the organic phase was dried over anhydrous magnesium sulfate and the organic solvent was evaporated under reduced pressure.
  • the crude product was recrystallized from ethanol and acetone to give the product N-(2-bromoethyl)carbamic acid cholesteryl ester in a yield of 73%.
  • the obtained N-(2-bromoethyl)carbamic acid cholesteryl ester (4.8 g, 7.8 mmol) and N-methyldiethanolamine (1.2 g, 9.7 mmol) was added to 50 mL of dry toluene and refluxed overnight.
  • the reaction solution was added to a large amount of diethyl ether to precipitate, and after filtration, the precipitate was collected and dried in vacuo.
  • the obtained crude product was recrystallized twice from ethanol to give a white solid (yield: 62%).
  • Pretreatment of stannous isooctanoate (National Pharmaceutical Group Chemical Reagent Co., Ltd.): Azeotrope twice with p-xylene and then distilled under reduced pressure to collect a fraction of 152 ° C (20-40 Pa) for polymerization.
  • Alexa 568-phalloidin Lipofectamine 2000 is hooked to Invitrogen. R easy mini-kits is hooked to Qiagen. RimeScriptTM 1st Strand cDNA Synthesis Kit, SYBR® Premix Ex Taq was purchased from Takara. The Lipoprotein B (apoB) Quantitative Assay Kit was purchased from R&D Systems. D-luciferin was purchased from Xenogen.
  • HepG2 cells, MDA-MB-435s cells, and BT474 cells (HER2 receptor high expressing cell line) stably expressing luciferase were purchased from the cell bank of the Culture Collection Committee of the Chinese Academy of Sciences.
  • the corresponding antisense strand sequence is: 5'-UAAGGAGGGUGAUCUUCUUCAdTdT-3 ' ( SEQ ID No :
  • siLucil the corresponding antisense strand sequence is: 5 '-CUUACGCUGAGUACUUCGAdTdT-3 ' ( SEQ ID No:
  • siapoB corresponding to the antisense strand sequence: 5 ' - AUUGGUAUUC AGUGUGAUGAC ACdTdT-3 ' ( SEQ ID No: 3
  • sequence corresponding to the antisense strand of siN.C is: 5 '-AACCACUCAACUUUUUCCCAAdTdT-3, (SEQ ID No: 4), as a negative control siRNA.
  • FAM-siR A is a fluorescent dye FAM-labeled siN.C.
  • siRNAs were synthesized by Suzhou Ruibo Biotechnology Co., Ltd.
  • the siRNA-encapsulated nanoparticles were prepared by a double emulsion method using an amphiphilic block polymer and a cationic lipid.
  • the amphiphilic block copolymer used is polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic-glycolic acid) diblock/triblock copolymer, specifically PEG 55 Q-PLA 286 QQ, PEG 2 QQQ-PLA 125 QQ, PEG 5 QQQ-PLA 5000 , PEG5000-PLA25000, PEG5000-PL A51000, PEG10000-PLA15000, PEGioooo-PLGAioooo (5o/50),
  • PEGl0000-PLGA50000 75/25
  • PLA6300-PEGl200-PLA 6 300 PLA48OO-PEG50OO-PLA48OO
  • PLA 4800 - PEG 10000 -PLA 4800 The cationic lipids used were BHEM-Chol, DOTAP and DOTMA, respectively.
  • An siRNA delivery system having different properties is prepared by changing the quality of the added cationic lipid, the quality of the siRNA, the kind of the polymer, and the type of the cationic lipid. 1. Effect of the quality of cationic lipid BHEM-Chol on siRNA nanoparticles
  • the siRNA-loaded nanoparticles were prepared by a double emulsion method using different masses of cationic lipid BHEM-Chol.
  • the polymer PEG 5 QQQ-PLA 25 QQ was used.
  • FAM-siR A (0.2 mg) are given as examples.
  • the mass of the cationic lipid added was 0.0 mg 0.1 mg 0.5 mg 1.0 mg, 2.5 mg and 5.0 mg, respectively, to investigate the effect of adding different quality cationic lipids on the properties of the prepared encapsulated siRNA nanoparticles.
  • the siRNA-loaded nanoparticles were prepared by double emulsification by: dissolving the polymer PEG 5 ooo-PLA 250 oo ( 25 mg ) and different masses of BHEM-Chol in 0.5 mL chloroform, and adding FAM-siRNA ( After the solution was 0.025 mL, 0.2 mg), the initial emulsion was formed under ultrasound (80 watts, 30 seconds), then the initial emulsion was added to 1.5 mL of 1% PVA aqueous solution and emulsified again (80 watts, 2 minutes) to obtain an emulsion.
  • the HPLC consisted of a Waters 1525 bi-directional pump, a Waters 2475 fluorescence detector, a 1500 column oven and a Symmetry® C18 separation column.
  • the mobile phase was acetonitrile/triethylamine acetate buffer (0.1 M pH 7.4) and the flow ratio was 28:72.
  • Drug loading (100%) (M , S siRNA -C XV /M xl 00%
  • M S siRNA indicates the mass of the total siRNA added in the preparation
  • M iS e ⁇ indicates the total mass of the nanoparticles. It can be seen from Table 1 that the encapsulation efficiency of siRNA was only 26.2% when no cationic lipid was added, and the encapsulation efficiency of siRNA was significantly increased after the addition of cationic lipid. For example, when 10 mg of BHEM-Chol was added, the encapsulation efficiency of siRNA increased to 95.7%. When the cationic lipid is continuously increased, the encapsulation efficiency of the siRNA is maintained at 95% or more.
  • the particle size and particle size distribution of the nanoparticles carrying the siRNA were detected by a dynamic light scattering instrument Model Malvern Zetasizer Nanao ZS90, wherein the concentration of the nanoparticles was 0.1 mg/mL.
  • the drug delivery system of the present invention is capable of optimizing a suitable system for siRNA administration.
  • the siRNA when the cationic lipid was added in an amount of 1.0 mg, the siRNA was substantially contained in the nanoparticles. However, when the amount of cationic lipid is increased, the drug loading is decreased, so the weight ratio of siRNA/BHEM-Chol/PEGsfxxrPLA fKK) is 0.2/1.0/25.0 when BHEM-Chol and PEG 5000 -PLA 25000 are fixed at 1.0 mg and 25.0 mg.
  • the ideal siRNA drug delivery system when the cationic lipid was added in an amount of 1.0 mg, the siRNA was substantially contained in the nanoparticles. However, when the amount of cationic lipid is increased, the drug loading is decreased, so the weight ratio of siRNA/BHEM-Chol/PEGsfxxrPLA fKK) is 0.2/1.0/25.0 when BHEM-Chol and PEG 5000 -PLA 25000 are fixed at 1.0 mg and 25.0 mg.
  • the ideal siRNA drug delivery system when the amount of cationic lipid
  • the siRNA-loaded nanoparticles were prepared by a double emulsification method using different kinds of cationic lipids. Among them, the use of polymers (25 mg) and FAM-siRNA (0.2 mg) were used as examples. At the time of preparation, the mass of the cationic lipid added was 1.0 mg, and the cationic lipids used were BHEM-Chol, DOTAP and DOTMAo, respectively.
  • the specific method is as follows: The polymer PEGSOOO-PLA, (25 mg) and cationic lipid (10 mg) are dissolved in 0.5 mL of chloroform, and after adding siRNA (0.025 mL, 0.2 mg) solution, under ultrasound (80 watts, 30 Second) The initial emulsion is formed. Then, the initial emulsification is added to 1.5 mL of 1% PVA aqueous solution and emulsified again (80 watts, 2 minutes) to obtain an emulsion.
  • the emulsion is added to 25 mL of 0.3% PVA aqueous solution, minus The organic solvent was depressed (1000 white), and the nanoparticles were collected by centrifugation (4 ° C, 30000 g, lh); and resuspended twice with water and collected by centrifugation to remove PVA, and the sample was lyophilized and used.
  • the siRNA encapsulation efficiency is about 95%.
  • the prepared nanoparticles have a substantially constant particle size between 160 nm and 170 nm, and the potential of the nanoparticles entrained with siRNA is close.
  • the type of cationic lipid has little effect on the properties of the nanoparticle-encapsulated siRNA.
  • the main function of the cationic lipid in the drug delivery system is to increase the drug loading amount and encapsulation efficiency of the nucleic acid drug in the carrier by electrostatic interaction, and therefore, cations having the above properties can be used in the present invention.
  • the molecular weights of the three types of cationic lipids used are close to the charge, so the resulting siRNA-encapsulated nanoparticles are similar in nature. Also, this also indicates that the cationic lipid which can be used in the administration system of the present invention is not limited to the ones used herein, and other cationic lipids can be also applied to this system.
  • Table 2 Specific composition and properties of individual components of encapsulated siRNA nanoparticles prepared by different cationic lipids
  • the siRNA-loaded nanoparticles were prepared by a double emulsion method using different quality siRNAs.
  • the polymer and cationic lipid used were PEG 5 Q (KrPLA 25 QQQ (25 mg) and BHEM-Chol (1.0 mg), respectively.
  • the quality of FAM-siRNA added was 0.0 mg, 0.2 mg, respectively. 0.6 mg and 1.8 mg were studied to investigate the effect of the addition of different masses of siRNA on the performance of the prepared encapsulated siRNA nanoparticles.
  • the specific method is as follows: Dissolve the polymer PEGsooo-PLA ⁇ oo (25 mg) and BHEM-Chol (1.0 mg) in 0.5 mL chloroform, add FAM-siRNA (0.025 mL) solution, and under ultrasound (80 watts, 30 Second) The initial emulsion was formed, then the initial emulsion was added to 1.5 mL of 1% PVA aqueous solution and emulsified again (80 watts, 2 minutes) to obtain an emulsion. The emulsion was added to 25 mL of 0.3% PVA aqueous solution, and decompressed.
  • the organic solvent was evaporated (1000 Pa), and the nanoparticles were collected by centrifugation (4 ° C, 30000 g, lh); and resuspended twice with water and collected by centrifugation to remove PVA, and the sample was lyophilized and used.
  • Nanoparticles entrapped in siRNA were prepared using different polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic-glycolic acid) diblock/triblock copolymers.
  • the siRNA-coated nanoparticles were prepared by double emulsification by: dissolving the polymer (25 mg) and BHEM-Chol (1.0 mg) in 0.5 mL of chloroform and adding siRNA (0.025 mL, 0.2 mg) solution. Thereafter, an initial emulsion was formed under ultrasound (80 watts, 30 seconds), and then the initial emulsification was added to 1.5 mL of a 1% PVA aqueous solution and emulsified again (80 watts, 2 minutes) to obtain an emulsion, and the emulsion was added thereto.
  • the polymer used is PEG 55 Q-PLA 286 QQ, PEG 2 QQQ-PLA 125 QQ, PEG 5 QQQ-PLA 5000 ,
  • PEG5000-PLA25000 PEG5000-PL A51000, PEG10000-PLA15000, PEGioooo-PLGAioooo (5o/50), PEGi0000"PLGA50000 (75/25), PLA6300"PEGi200-PLA6300, PLA4800-PEG5000-PLA4800 and PLA4800-PEG10000-PLA4800.
  • the siRNA when the amount of BHEM-Chol and the polymer added was fixed, the siRNA was substantially contained in the nanoparticles, and the encapsulation efficiency was substantially above 90%. However, when the PEG ratio is high, the encapsulation efficiency of siRNA will decrease to some extent. For example, when the polymer used is PEG ⁇ KKXTPLGAKKKK ⁇ /SO) or PLA 4800-PEG10000-PLA4800, the encapsulation efficiency of siRNA is reduced to about 85%.
  • the zeta potential of the obtained siRNA-loaded nanoparticles varies depending on the polymer. It can be seen from the above embodiments that the potential of the nanoparticles affects the circulation of the nanoparticles in the body, and the potential of the nanoparticles is more neutral to avoid being removed by the immune system in the body, thereby being better enriched to the tumor site. Therefore, from the results of Table 1, Table 3 and Table 4, it is known that the drug delivery system can optimize a suitable system for siRNA administration by adjusting the quality of the added cationic lipid and siRNA and the polymer used.
  • the prepared nanoparticles have a large particle size variation between 82 nm and 227 nm.
  • the hydrophobic segment of the polymer used in the preparation has a great influence on the particle size of the prepared encapsulated siRNA nanoparticles.
  • a polymer PEG 5 grown using a hydrophobic segment is used. . . -PLA 5 . . . , PEG 5 . . . -PLA 25 . .
  • the particle size of the obtained siRNA nanoparticles was 82, 167 and 225 nm, respectively.
  • Example 1 when the mass ratio of siRNA/cationic lipid/polymer was 0.2/1.0/25.0, the obtained siRNA delivery system had a high siRNA encapsulation efficiency and drug loading amount. This example is used to illustrate the biological effects of this drug delivery system.
  • the polymers used were PEG 550 -PLA 286 oo , PEG 2 ooo-PLA 1250 o , PEG 5 ooo-PLA 50 oo , PEG5000-PLA25000, PEG5000-PL A51000, PEG10000-PLA15000, PEGioooo-PLGAioooo (5o/50),
  • the cationic lipid used in PLA 4 8oo-PEGioooo-PLA48oo' is BHEM-Chol. 1. Biocompatibility evaluation of nanoparticles prepared by different polymers
  • Nanoparticles not containing siRNA were prepared by the method described in Example 1. The toxicity of nanoparticles on human hepatoma cell line HepG2 was detected by MTT (3-(4, 5-dimethylthiazole-2)-2, 5-diphenyltetrazolium bromide) colorimetric assay.
  • the specific method for preparing the non-encapsulated siRNA nanoparticles is as follows: The polymer (25 mg) and BHEM-Chol (1.0 mg) are dissolved in 0.5 mL of chloroform, and after adding distilled water (0.025 mL) solution, under ultrasound (80 watts, 30 seconds) The initial emulsion was formed. Then, the initial emulsion was added to 1.5 mL of 1% PVA aqueous solution and emulsified again (80 watts, 2 minutes) to obtain an emulsion, and the emulsion was added to 25 mL of 0.3% PVA aqueous solution.
  • the organic solvent was evaporated under reduced pressure (1000 Pa), and the nanoparticles were collected by centrifugation (4 ° C, 30000 g, lh); and resuspended twice with water and collected by centrifugation to remove PVA, and the sample was lyophilized and used.
  • Polymer used was PEG 550 -PLA 28600, PEG 2 ooo -PLAi 250 o, PEG 5000 -PLA 5000, PEG5000-PLA25000, PEG5000-PL A51000, PEG10000-PLA15000, PEGioooo-PLGAioooo (5o / 5o), PEGi0000 "PLGA50000 (75/25), PLA6300"PEGi200-PLA6300, PLA4800-PEG5000-PLA4800 and PLA4800-PEG10000-PLA4800.
  • the obtained siRNA-free nanoparticles were NP (PEG 55Q -PLA 286(X) ), NP (PEG 2 ooo-PLAi 250 o), NP (PEG 5000 -PLA 5000 ), NP (PEG 5000 -PLA 25000). ), NP (PEG 50 oo-PLA 5 iooo), NP (PEG 10 . . .
  • NP PEGioooo-PLGAioooo ( 5o/50)
  • NP PEGioooo-PLGA 50 ooo (75/) 25
  • NP PEGi 6300 - PEG 1200 - PLA 6300
  • NP PEG 4800 - PEG ⁇ - PLA shed
  • NP PEG ⁇ 0 - PLA 4800
  • the toxicity of the above prepared nanoparticles to human liver cancer cell HepG2 was examined by MTT colorimetry.
  • the specific method is as follows: First, inoculate 10000 cells/ ⁇ DMEM medium (10% newborn bovine serum) per well in a 96-well plate, and culture in a C0 2 incubator (37 ° C, C0 2 concentration of 5%). After the hour, after removing the medium, add the above Nanoparticles of ⁇ fresh DMEM medium, wherein the concentration of the above nanoparticles in the medium is
  • Abs is the absorbance of the solution at 570 nm
  • Abs(sample) is the absorbance of the sample well
  • Nanoparticles coated with FAM-siR A were prepared by the method described in Example 1 to study the endocytosis behavior of this drug delivery system. Among them, the mass ratio of siRNA/cationic lipid/polymer was 0.2/1.0/25.0.
  • FAM-labeled siRNA FAM-labeled siRNA
  • cationic lipid was ⁇ -Chol
  • the polymers used were PEG550-PLA28600, PEG2000-PLA12500, PEG5000-PLA5000, PEG5000-PLA25000, PEG5ooo-PLA5iooo PEGioooo-PLAisooo PEGioooo-PLGAioooo ( 5o/so) PEGioooo-PLGA5oooo(75/25)
  • PLA63oo-PEGi2oo-PLA 6 3oo PLA4800-PEG5000-PLA4800 and PLA tsoo—PEGioooo—PLA tsoo c
  • the obtained encapsulated FAM-siRNA nanoparticles are separated into NP (PEG 550 -PLA 28600 ),
  • NP PEG 2 ooo-PLAi 250 o
  • NP PEG 5000 -PLA 5000
  • NP PEG 5000 -PLA 25000
  • NP PEG 50 oo-PLA 5 iooo
  • NP PEG 10 ⁇ -PLA 15 . . .
  • NP PEGioooo-PLGAioooo ( 5o/50)
  • NP PEGioooo-PLGA 50 ooo (75/25)
  • NP PEG 6300 -PEG 1200 -PLA 6300
  • NP PEG 4800 -PEG ⁇ ) -PLA shed
  • NP PAG shed - PEG ⁇ 0 - PLA 4800 ).
  • the FAM-siRNA-loaded nanoparticles (final concentration of FAM-siRNA was 200 nM) and human hepatoma HepG2 cells (24-well plates, 5 ⁇ 10 4 cells/well) were co-cultured at 37 ° C for 2 hours, using 4% more The cells were fixed in polyoxymethylene for 15 min. After washing three times with PBS, the cells were permeabilized with acetone (-20 ° C, 5 min); finally, the cytoskeleton was stained with Alexa 568-phalloidin, using 4',6-diamidino-2-phenylindole The nucleus was counterstained by DAPI and mounted after laser confocal microscopy with the model Zeiss LSM 710. The results are shown in Figure 2.
  • intracellular red fluorescence is derived from the cytoskeleton of Alexa 568-phalloidin labeling; green fluorescence is derived from FAM-siRNA; blue fluorescence is derived from DAPI-labeled nuclei.
  • NPiPEGsQQQ-PLAsQQQ NPiPEGKKKxrPLGAKKKxxso/so)) and NP LA ⁇ QQ-PEGnKxxrPLA ⁇ Q had the lowest fluorescence intensity.
  • the above three nanoparticles used the same amount of siRNA and cationic lipid, except that the polymer used was PEGsfxxrPLAscxK ⁇ PEG 10000 - PLGA 10000 (50/50 ) and PLA 4800 - PEG 10000 - PLA 4800 , the PEG was higher (50%) in all three polymers.
  • PEG may have the ability to reduce the endocytosis of nanoparticles.
  • Nanoparticles coated with siRNA were prepared by the method described in Example 1 to investigate the effect of silencing target gene expression in this drug delivery system. Among them, the mass ratio of siRNA/cationic lipid/polymer was 0.2/1.0/25.0.
  • the siRNA used was siPlkl and s ⁇ N.C., the cationic lipid was ⁇ -Chol, and the polymer was
  • PEGioooo-PLAi5ooo PEGioooo-PLGAioooo (5o/so)
  • PEGioooo-PLGA5oooo 75/25
  • the obtained siRNA-loaded nanoparticles were NP (PEG 55 Q-PLA 286 QQ), NP (PEG 2 QQQ-PLA 12500 ), NP (PEG 5 ooo-PLA 50 oo), NP (PEG 5000 -PLA 25000 ).
  • NP PEG 5000 -PLA 51000
  • NP PEGioooo-PLAi 5 ooo
  • NP PEG 10000 -PLGA 10000 (50/50)
  • NP PEG 10000 -PLGA 50000 (75/25)
  • NP PEG 6 3oo-PEGi 2 oo-PLA 6 3oo
  • NP PEG 48 ..- PEG 5 ...- PLA 48 ..
  • NP PA 4 8oo-PEGioooo- PLA 4 8oo) °
  • Plkl helps promote and accelerate mitosis in mammalian cells and is highly expressed in a variety of tumor cells. By silencing its expression, tumor growth can be inhibited.
  • HepG2 cells were seeded in a 6-well plate at a density of 1 ⁇ 10 5 cells/well, and cultured at 37° C. for 24 hours, the medium was removed, and the following treatments were carried out, respectively, and in each group, the medium was replaced with the following groups.
  • set one duplicate hole per treatment group :
  • Treatment 1 100 ⁇ l of PBS solution was added to 1.9 mL of fresh medium.
  • Treatment 2 Dilute 5 Lipofectamine 2000 to 50 ⁇ with sterile water, add to 50 ⁇ L of an aqueous solution containing 100 pmol of siPlkl, and after combining for 15 minutes at room temperature, add 100 ⁇ L of the solution to 1.9 mL. In fresh medium. Among them, the final concentration of siP/ is 50 nM.
  • Treatment 3 100 ⁇ L of siPlkJ solution was added to 1.9 mL of fresh medium with a final concentration of si n/W of 200 nM.
  • Treatment 4 (NP (PEG 55 Q-PLA 286 QQ) group: Add 100 ⁇ L of siPlkl-coated nanoparticle NP (PEG 55Q -PLA 286(K) ) solution to 1.9 mL of fresh medium, siPlkl The final concentration is 200 nM o
  • the NP (PEG 55Q -PLA 286(K) ) solution was added to 1.9 mL of fresh medium with a final concentration of siN.C. of 200 nM.
  • Treatment 6 (NP(PEG-PLA 12500 ) group): Add 100 ⁇ of siPlkl-loaded nanoparticle NP (PEG 2 -PLA 125 ) solution to 1.9 mL of fresh medium, where siP/W is final Concentration is 200 nM o
  • Treatment 7 (NP (PEG 2 QQQ-PLA 125 QQ) group: 100 ⁇ of nanoparticles loaded with siN. C.
  • the NP (PEG 2 QQQ-PLA 125 QQ) solution was added to 1.9 mL of fresh medium with a final concentration of siN.C. of 200 nM.
  • Treatment 8 NP (PEG 5 QQQ-PLA 5 QQQ) group: 100 ⁇ L of siPlld-loaded nanoparticles NPCPEG 5 ( KKrPLA 5 ( KK solution was added to 1.9 mL of fresh medium, where the final concentration of siPlkl was 200
  • treatment 9 NP (PEG 5 QQQ-PLA 5 QQQ) group: Add 100 ⁇ L of the siN.
  • C. nanoparticle NP (PEG 5 QQQ-PLA 5 QQQ) solution to 1.9 mL of fresh medium.
  • the final concentration of siN.C. was 200 nM.
  • Treatment 10 100 ⁇ l of SiPlkl-loaded nanoparticles NPCPEG 5 was loaded. . . -PLA 25 . . . The solution was added to 1.9 mL of fresh medium, where the final concentration of siP/W was 200 for treatment 1 1 (NP (PEG 5 QQQ-PLA 25 QQQ) group): 100 ⁇ L of nano-coated SiN. C. The granule NP (PEG 5 QQQ-PLA 25 QQQ) solution was added to 1.9 mL of fresh medium with a final concentration of siN.C. of 200 nM.
  • Treatment 12 (NP(PEG ⁇ -PLA ⁇ ) group): Add 100 ⁇ L of the siPlkl-loaded nanoparticle NP (PEG 5 . . . -PLA 51 ) solution to 1.9 mL of fresh medium, The final concentration of siP/W was 200 treatment 13 (NP (PEG 5 QQQ-PLA 51 QQQ) group): 100 of the packaged siN.
  • C nanoparticle NP (PEG 5 QQQ-PLA 51 QQQ) solution was added to 1.9 In a mL fresh medium, the final concentration of siN.C. is 200 nM.
  • Treatment 14 (NP(PEGioooo-PLAi 5 ooo) ia ) : Add 100 ⁇ g of siPlW nanoparticle NPCPEGKKKXTPLAC) solution to 1.9 mL of fresh medium, where the final concentration of siPl!d is 200 treatment 15 (NP ( PEGioooo-PLA 150 oo) ia ) : 100 ⁇ of nano-packaged siN.C.
  • NPCPEG 1 KKKrPLA 15 ( KK solution added to 1.9 mL of fresh medium, where the final concentration of siN.C.
  • Treatment 16 (NP EGKKKKrPLGAKKKx ⁇ Q/sQ)): A 100 ⁇ solution of the siPlkl-coated nanoparticle NPCPEGKKKxrPLGAKKKKxsQ/sQ)) was added to 1.9 mL of fresh medium with a final concentration of si n n n n 200 nM.
  • Treatment 18 (NP EGKKKxrPLGAsQQQQ ⁇ / ⁇ ): Add 100 ⁇ L of the siPlkl-coated nanoparticle NPCPEGKKKxrPLGAsfKKKXTO ⁇ )) solution to 1.9 mL of fresh medium with a final concentration of 200 nM for siPlkl.
  • Treatment 19 (NP EGKKKxrPLGAsQQQQ ⁇ / ⁇ ) group: 100 ⁇ l of the solution containing the siN. C. nanoparticle NPCPEGKKKxrPLGAsfKKK ⁇ )) was added to 1.9 mL of fresh medium, wherein the final concentration of siN.C. was 200 nM.
  • NP PEG 63 QQ- PEG 12 QQ-PLA 63 QQ
  • a 100 ⁇ entrapped siPlW nanoparticles NP PEG 12 ..- PLA 63 ..
  • the final concentration of siP/W was 200 nM.
  • Treatment 21 NP (PLA 63 QQ-PEG 12 QQ-PLA 63 QQ) group: 100 ⁇ L of the coated siN.C. nanoparticle NPCPLA 63 (xr PEG 12 ( KrPLA 63 ( K solution added to 1.9 mL fresh) In the medium, wherein the final concentration of siN.C. was 200 nM.
  • NP PEG 48 QQ- PEG 5 QQQ-PLA 48 QQ
  • a 100 ⁇ entrapped siPlW nanoparticles NP PEG 48 ..- PEG 5 ...- PLA 48 ..
  • the final concentration of siP/W was 200 nM.
  • NP PEG 48 QQ- PEG 5 QQQ-PLA 48 QQ
  • Processing 23 The 100 ⁇ L of nanoparticles entrapped siN.C. NP (PLA 48 ..- PEG 5 ...- PLA 48 .. The solution was added to 1.9 mL of fresh medium with a final concentration of siN.C. of 200 nM.
  • Treatment 24 (NP LA ⁇ QQ-PEGK!QQQ-PLA QQ) group: 100 ⁇ L of nanoparticles loaded with siPlkl
  • the NPCPLA ⁇ oo-PEGKKKxrPLA ⁇ oo) solution was added to 1.9 mL of fresh medium, where the final concentration of siPl!d was 200 nM.
  • Treatment 25 (NP(PLA 4 8oo-PEGioooo-PLA 4 8oo) ia): Add 100 ⁇ of the siN.C. nanoparticle NPCPLA ⁇ oo-PEGKKKxrPLA ⁇ oo) solution to 1.9 mL of fresh medium, The final concentration of siN.C. was 200 nM.
  • RA concentration ( ⁇ ⁇ / ⁇ ) 0.04xOD 26Q x dilution factor, calculate the concentration of RA samples, then synthesize cDNA using PrimeScriptTM 1st Strand cDNA Synthesis Kit (Takara, Dalian, China), using 2 g total mR per sample A. After cDNA synthesis, real-time PCR was performed according to the SYBR® Premix Ex TaqTM (Takara) kit. Among them, PCR primers for Plkl and glycerol triphosphate dehydrogenase GAPDH genes are as follows:
  • Plkl-upstream primer 5'-AGCCTGAGGCCCGATACTACCTAC-3' (SEQ ID No: 5); Plkl-downstream primer: 5'-ATTAGGAGTCCCACACAGGGTCTTC-3' (SEQ ID No: 6); GAPDH-upstream primer: 5'-TTCACCACCATGGAGAAGGC-3' (SEQ IDNo) : 7) ;
  • GAPDH-downstream primer 5'-GGCATGGACTGTGGTCATGA-3' (SEQ ID No: 8).
  • the PCR reaction conditions are as follows:
  • NP PEG 5 QQQ-PLA 5 QQQ
  • NPiPEGKKKxrPLGAKKKxxsQ/sQ NPiPLA ⁇ QQ-PEGKKxxrPLA ⁇ Q
  • the experimental group was caused by poor endocytosis.
  • the NP EGscxxrPLA ⁇ oo) experimental group had the best silencing effect and was able to down-regulate the expression of Plk1 gene to 45% of the PBS group.
  • Example 3 Evaluation of biological effects of the drug delivery system at the animal level
  • the polymer used as the lipid for BHEM-ChoK is PEGsooo-PLA ⁇ oo
  • the nanoparticles coated with siRNA can be efficiently inserted into the cell, and the expression of the target gene can be significantly silenced, and the nanometer containing the siRNA can be prepared by using the formula.
  • Granule The biological effects of this drug delivery system at the animal level were investigated as an example.
  • Luciferase is a firefly luciferase that catalyzes the emission of visible light through a substrate injected intraperitoneally.
  • the expression of the Luciferase gene can be qualitatively determined based on the uniformity of the enzyme reaction.
  • HepG2-luciferase cells (density 2.0xl 7 /mL, 25 ⁇ ) suspension and 25 Matrigel
  • the tumor can be formed after about 10 days of feeding.
  • the drug is administered according to the following treatment method.
  • the tail vein is injected once a day for 2 times:
  • Treatment 1 PBS control group: 25 ( ⁇ L of PBS was administered as a control.
  • Treatment 2 (naked siL d group): 250 ⁇ l of naked siL d was administered, wherein the dose of siL d was 20 ⁇ .
  • Treatment 3 (NP(PEG 5 ooo-PLA 250 oo)/siN. C. group): Dosing nanoparticles containing siN. C.
  • NP PEG 5 Q (KrPLA 25 QQQ), wherein the dose of siN. C. is 20 ⁇ ⁇ .
  • Treatment 4 (NP (PEG 5 QQQ-PLA 25 QQQ) / siL d group): The nanoparticle NP (PEG 5 QQQ-PLA 25 QQQ) containing siLuci was administered, wherein the dose of siLwd was 2 ( ⁇ g.
  • mice were intraperitoneally injected with 200 ⁇ firefly luciferase substrate D-luciferin (15 mg/mL, Xenogen), and the liver site luciferase was detected by Xenogen IVIS® Lumina system (Caliper Life Sciences, Hopkinton, MA) small animal in vivo imager. Expression before and after administration, the results are shown in Figure 4A.
  • Fig. 4A from the pre-dose (day 0) image, the expression of firefly luciferase in the liver of mice was significant, and the expression level was basically equivalent. Two days after dosing (2 days), the expression of firefly luciferase was increased in the negative control group (PBS, naked siLuci, NP (PEG 5 ooo-PLA 250 oo)/siN. C. control group), derived from HepG2 -Proliferation of luciferase in the liver.
  • PBS negative control group
  • NP PEG 5 ooo-PLA 250 oo
  • C. control group derived from HepG2 -Proliferation of luciferase in the liver.
  • NPCPEG 5Q ( xrPLA 25()()() ysiL d administration group of nude mice liver
  • the expression of firefly luciferase was significantly decreased, indicating that NPiPEGscxxrPLA ⁇ ooysiL d silenced the expression of firefly luciferase in liver cancer cells.
  • MDA-MB-435S cells (0.5 ⁇ 10 7 ) were inoculated in situ under the fat pad of the second mammary gland of nude mice.
  • the tumor was formed in about 14 days.
  • the tumor volume was about 50 mm 3 .
  • the nude mice were randomly divided into five groups.
  • the tail vein injection treatment (treatment 1-treatment 5) was administered once every two days.
  • Treatment 1 PBS control group: Each nude mouse was injected with 250 ⁇ l of PBS.
  • Treatment 2 naked siPlkl group: Each nude mouse was injected with 250 ⁇ of bare siPlkl, and the dose of siPlkl was 2 ( ⁇ g.
  • Treatment 3 NP (PEG 5 ooo-PLA 250 oo) group: Each nude mouse was injected with blank nanometer Particle NP (PEG 5 ooo-PLA 250 oo), the mass of the nanoparticles was 2.70 mg.
  • Treatment 4 (NP(PEG 5 ooo-PLA 250 oo)/siN.C. M ): Each nude mouse was injected with nanoparticle NP (PEG 5 ooo-PLA 250 oo) encapsulating siN. C., siN.C. The dose is 20 g and the mass of the nanoparticles is 2.70 mg.
  • NP PEG 5 ooo-PLA 250 oo
  • siP/L The dose of NP (PEG 5 ooo-PLA 250 oo), siP/L? was 20 g, and the mass of the nanoparticles was 2.70 mg.
  • Fig. 4B Tumor volume was measured every other day after the start of treatment. As a result, as shown in Fig. 4B, the tumor growth rate was faster in all the negative control groups, and in the treatment group using the siPlkl nanoparticles (NPiPEGsooo-PLA ⁇ oo), the tumor growth rate was significantly inhibited compared with the negative control group. . It is indicated that the siRNA-encapsulated nanoparticles provided by the present invention can effectively silence oncogenes in vivo, thereby inhibiting tumor growth.
  • the siRNA delivery system is capable of effectively silencing the expression of a target gene at a tumor site at an animal level, and the down-regulation of expression of the target gene is caused by sequence-specific gene silencing.
  • Example 4 Targeted modification of this drug delivery system and evaluation of biological effects
  • a galactose group is a targeting ligand that specifically recognizes the asialoglycoprotein receptor (ASGP-R, a glycoprotein overexpressed on the surface of mammalian hepatocytes), so the present invention passes this
  • ASGP-R asialoglycoprotein receptor
  • the galactose-modified, siRNA-encapsulated nanoparticles were prepared by encapsulating siRNA by a double emulsion method using galactose-modified PEG-PLA ( Gal-PEG-PLA) and cationic lipids.
  • Gal-PEG-PLA is a hetero-functional polyethylene glycol with terminal groups of carboxyl and hydroxyl groups as initiators, and the monomer polymerization of lactide monomer is initiated by using polyethylene glycol terminal hydroxyl groups under bulk conditions.
  • HOOC-PEG-PLA which is obtained by bonding an aminogalactose to a PEG segment.
  • the aminogalactose is further bonded to the HOOC-PEG-PLA obtained by the synthesis to obtain an aminogalactose-modified polyethylene glycol-polylactic acid (Gal-PEG-PLA).
  • the specific test is as follows: lg HOOC-PEG 5 QQQ-PLA 21 Q 3 Q is dissolved in 5 mL of dichloromethane, and N,N-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinyl group is added.
  • Figure 5A shows the synthesized HOOC-PEG 5 QQQ-PLA 21 Q 3 .
  • the results of nuclear magnetic resonance spectroscopy of Gal-PEG 5 QQQ-PLA 2K) 3 Q are analyzed as follows: The letters a to d mark the proton signal belonging to PEG-PLA.
  • the molecular weight of polylactic acid passes through a multiple peak of 5.17 ppm (-C(0)OC- assigned to polylactic acid, H in italics, the following analogy) and a single peak of 3.63 ppm (integral area attributed to polyethylene glycol) Compared with the calculation. Compare Figure 5A
  • siRNA-loaded nanoparticles were prepared by the method of Example 1 to study the biological effects of this drug delivery system.
  • the mass ratio of siRNA/cationic lipid/polymer was 0.2/1.0/25.0
  • the siRNA used was siPlkl and siN.C
  • the cationic lipid was BHEM-Chol.
  • the polymer used was HOOC-PEG 5 ( KKrPLA 21Q3() and
  • NP HOOC-PEG 5 . . . -PLA 21 . 3
  • NP Gal-PEG 5000-PLA2103o
  • FAM-siRNA-encapsulated NP (HOOC-PEG 5 QQQ-PLA 21 Q 3 Q) and NP (Gal-PEG 5 . . . -PLA 21 . 3 ) were prepared in the above system to study the targeted drug delivery system. Endocytosis of cells. Hepa 1-6 cells were seeded in a 24-well plate at a density of 5 ⁇ 10 4 cells/well, and cultured at 37° C. for 24 hours, and then the medium was removed, and the following treatments were carried out, respectively. In each treatment, the medium was changed to the following Solution in each group of treatments:
  • PBS Treatment 1
  • FAM-siRNA Treatment 2
  • Treatment 3 50 ⁇ of the loaded FAM-siRNA-coated nanoparticle NP (HOOC-PEG 5 QQQ-PLA 21 Q 3 Q) solution was added to 450 ⁇ fresh In DMEM medium, the final concentration of FAM-siRNA was 200 nM.
  • Treatment 4 NP(Gal-PEG 5 QQQ-PLA 21 Q 3 Q) group: 50 ⁇ of the loaded FAM-siRNA-loaded nanoparticle NP (Gal-PEG 5 . . . -PLA 21 . 3 ) solution was added to 450 ⁇ In a fresh DMEM medium, the final concentration of FAM-siRNA was 200 nM.
  • each group of cells was digested and analyzed, and the cells were subjected to phagocytosis of the nanoparticles by flow cytometry (FACS, BD Bioscience, Bedford, MA), and the results are shown in Fig. 5B.
  • FAM-siRNA is basically unable to enter cells, targeting drug delivery systems.
  • NP Gal-PEG 50 oo-PLA 2 io3o
  • non-targeted drug delivery system NP HOOC-PEG 5 QQQ-PLA 2 K 3 Q
  • the experimental group of the targeted drug delivery system detected that the FAM-siRNA fluorescence signal in the cells was significantly stronger than the non-targeted drug delivery system, indicating that the galactose-modified nanoparticles could better transport the siRNA to the liver cancer cells.
  • the nanoparticles cannot mediate the endocytosis of the nanoparticles through the specific binding of ASGP-R, so the transported FAM-siRNA is reduced. Causes a decrease in the fluorescent signal.
  • the encapsulated siRNA nanoparticles prepared by the ligand-modified polymer have the targeting ability.
  • a ligand modification may be a galactose modification, and is not limited thereto, such as folic acid, a short peptide containing arginine-glycine-aspartic acid (RGD), and the like.
  • folic acid a galactose modification
  • RGD arginine-glycine-aspartic acid
  • apoB is a cholesterol transport-related protein in hepatocytes and has been widely used as a model target gene for liver gene silencing; the present invention can be better evaluated by silencing Hepa 1 -6 cell apoB gene expression to evaluate the galactose-modified targeting nanoparticle.
  • the transport siRNA enters the liver cancer cells and successfully releases the siRNA to silence the expression of the target gene.
  • the NP HOOC-PEG 5 QQQ-PLA 21 Q 3 Q
  • NP Gal-PEG 5 QQQ-PLA 21030
  • siapoB and siN.C. were prepared by the above system to study the targeted drug delivery system at the cellular level. Silencing the effect of the target gene.
  • Hepa 1 -6 cells were seeded in a 6-well plate at a density of lxl O 5 cells/well, cultured at 37 ° C for 24 hours, and then the medium was removed, and the following treatments were carried out, respectively. In each treatment, the medium was changed to the following For each group of treated solutions, set one duplicate hole per treatment group:
  • Treatment 1 100 PBS solution was added to 1.9 mL of fresh DMEM medium.
  • Treatment 2 Free skywfi group: Add 100 skywfi solution, where the final concentration of skywfi is 200 nM.
  • Treatment 3 (Lipofectamine group): After diluting 5 Lipofectamine 2000 to 50 with sterile water, adding it to 50 L of an aqueous solution containing lOOpmolskywfi, and after combining for 15 minutes at room temperature, the 100 solution was added to 1.9 mL of fresh medium. . Among them, the final concentration of siapoB was 50 nM.
  • Treatment 4 (NP(HOOC-PEG 5 QQQ-PLA 21 Q 3 Q)/siN.C. group): 100-packed siN.C.-targeted nanoparticle NP (HOOC-PEG 5 . . . -PLA 21 3. ) Add to 1.9 mL of fresh DMEM medium, where the final concentration of siN.C. is 200 nM.
  • Treatment 5 100 ⁇ of siapoB-loaded non-targeted nanoparticle NP (HOOC-PEG 5 . . . -PLA 21 . 3 ) was added to In 1.9 mL of fresh DMEM medium, the final concentration of siapoB was 200 nM.
  • Treatment 6 (NP(Gal-PEG 5 QQQ-PLA 21 Q 3 Q)/siN.C. group): 100-packaged siN.C.-targeted nanoparticle NP (Gal-PEG 5 . . . -PLA 21 3 )) Add to 1.9 mL of fresh DMEM medium, where the final concentration of siN.C. is 200 nM.
  • Treatment 7 100-packaged sia/wfi-targeted nanoparticle NP (Gal-PEG 5 . . . -PLA 21 . 3 ) was added to 1.9 mL of fresh DMEM culture In the base, wherein the final concentration of siapoB is 200 nM.
  • RA concentration ( ⁇ ⁇ / ⁇ ) 0.04xOD 26Q x dilution factor, calculate the concentration of RA sample, then synthesize cDNA using PrimeScriptTM 1st Strand cDNA Synthesis Kit (Takara, Dalian, China), using 2 ⁇ g total mRNA per sample .
  • real-time PCR was performed according to the SYBR® Premix Ex TaqTM (Takara) kit.
  • the PCR primer of the apoB gene is as follows, and the PCR primer of the glycerol triphosphate dehydrogenase GAPDH gene is as shown in SEQ ID No: 7 and B SEQ ID No: 8:
  • apoB-upstream primer 5'-TTCCAGCCATGGGCAACTTTACCT-3' SEQ ID No: 9
  • apoB-downstream primer 5'-TACTGCAGGGCGTCAGTGACAAAT-3' SEQ ID No: 10.
  • the PCR reaction conditions are as follows:
  • the difference in apoB gene expression between different experimental groups was analyzed by 2- AACT.
  • GAPDH was used as an internal reference to analyze the expression level of apoB gene in different experimental groups.
  • the expression in the PBS experimental group was 100%, and the expressions of other experimental groups were expressed as relative to the PBS group.
  • the experimental results are shown in Fig. 5C.
  • NP HOOC-PEG 50 oo-PLA 2 io3o
  • ⁇ (0- ⁇ 0 5 . . . - ⁇ 21 .3 ) / 8 ⁇ experimental group
  • NP HOOC-PEG 50 oo-PLA 2 io3o
  • ⁇ (0- ⁇ 0 5 . . . - ⁇ 21 .3 ) / 8 ⁇ experimental group
  • the Gal-NP/siapofi group ⁇ (0&1- ⁇ 0 5 . ⁇ - ⁇ 21 . 3 )/8 ⁇
  • apoB gene was as high as 80%, which was significantly better than that of NP/skywB experimental group ( ⁇ ( ⁇ [5 ⁇ ( 5 ⁇ )- ⁇ 2 ⁇ )3() )/8 ⁇ , 39% silencing efficiency).
  • the experimental control group containing siN.C. had no significant effect on the expression of apoB mRNA in the cells. This indicates that downregulation of apoB expression is due to sequence-specific gene silencing.
  • the effectiveness and targeting of the vector as a liver-targeted siRNA vector was observed by in vivo experiments by injecting a targeted delivery system of siapoB and a non-targeted drug delivery system into the mouse via the tail vein.
  • the targeted drug delivery system and the non-targeted drug delivery system as described above were injected into C57BL/6 mice via the tail vein at a dose of 4 mg/kg. After 48 hours, the mice were sacrificed by eyeballs and collected after coagulation. Serum, serum apoB protein levels were measured using a mouse apolipoprotein B (apoB) quantitative assay kit (ELISA; R&D systems, Minneapolis, MN, USA). For each group of 6 mice, the experimental group was set as follows:
  • Treatment 1 PBS control group: Each nude mouse was injected with 400 L of PBS.
  • Treatment 2 (Free siapoB group): Each nude mouse was injected with 400 ⁇ L of naked siapoB, and the dose of ⁇ was 80 ⁇ treatment 3 (NP(HOOC-PEG 5 QQQ-PLA 21 Q 3 Q)/siN. C. group): Each nude mouse was injected with nanoparticle NP (HOOC-PEG 5 QQQ-PLA 21 Q 3 Q) encapsulating siN.C., wherein the dose of siN.C. was 80 g.
  • Treatment 4 NP HOOC-PEGsQQQ-PLA Q ⁇ ysifl/wB group: Each nude mouse was injected with nanoparticle NP (HOOC-PEG 5 QQQ-PLA 21 Q 3 Q) containing skywfi, wherein the dose of skywfi was 80 g.
  • NP Gal-PEG 5 (xxrPLA 21 Q 3 QysiN.C. group): Each nude mouse was injected with nanoparticles containing siN.C.
  • NP (Gal-PEG 50 oo-PLA 2 io3o), wherein the dose of siN.C. is 80 g.
  • the present invention further utilizes single-stranded fragment antibody modification to develop another targeted siRNA delivery system.
  • Single-stranded fragment antibody Single-stranded fragment antibody
  • Anti-Her2 scFv-cys is capable of specifically recognizing the HER2 receptor on the surface of breast cancer cells, and thus the present invention prepares a targeted drug delivery system by modifying the single-stranded fragment antibody on the surface of the nanoparticles.
  • Single-stranded fragment antibody-modified, siRNA-loaded nanoparticles are PEG-PLA modified with maleimide groups
  • Mal-PEG-PLA cationic lipids entrap siRNA nanoparticles by double emulsion method, and then single chain The fragment antibody binds to the maleimide group on the surface of the nanoparticle to give a targeted delivery system.
  • Mal-PEG-PLA is a hetero-functional polyethylene glycol with terminal groups of maleimide group and hydroxyl group as initiator, and the lactide is initiated by using polyethylene glycol terminal hydroxyl group under bulk conditions.
  • the bulk monomer is polymerized to obtain Mal-PEG-PLA.
  • Gal-PEG-PLA By adjusting the ratio of polyethylene glycol to lactide, it is theoretically possible to obtain Gal-PEG-PLA of different molecular weight.
  • stannous isooctylate is the most widely used catalyst for ring-opening polymerization of lactone and lactide ring-shaped monomers, and has been approved by the US FDA as a food additive.
  • Figure 6A shows the results of NMR spectroscopy of the synthesized Mal-PEGscxxrPLA ⁇ oTM (subscript molecular weight).
  • the analysis is as follows: The letters a to d mark all proton signals belonging to PEG-PLA, and the molecular weight of polylactic acid passes 5.17 ppm.
  • the multiple peaks (-C(0)OC - attributed to polylactic acid) were calculated from the integrated area ratio of a single peak of 3.63 ppm (-CH2CH2- assigned to polyethylene glycol).
  • the prepared siRNA delivery system can be very Goodly transport siRNA into tissue cells and play a role. Therefore, the present invention also uses this formulation to study the biological effects of this targeted drug delivery system.
  • the mass ratio of siRNA/cationic lipid/polymer was 0.2/1.0/25.0
  • the siRNA used was siPlkl and siN.C.
  • the cationic lipid was BHEM-Chol.
  • the polymer used was Mal-PEG 5 . . . -PLA 22 . 7 .
  • the prepared siRNA-loaded nanoparticles are NP (Mal-PEG 5000"PLA2207o).
  • the siRNA-coated nanoparticles are obtained as NP (Mal-PEG 5 ( KKrPLA 22Q7Q ) and further single-stranded fragment antibody is bonded to the nanoparticles.
  • maleimide groups on the surface of the targeted delivery system to give siRNA NP-specific experimental steps (scFv-PEG 5 ...- PLA 22 7..):.
  • FAM-siRNA-encapsulated NP (Mal-PEG 5000 - PLA 22070 ) and NPCscFv-PEG 5 were prepared in the above system. . . -PLA 22 . 7 .
  • BT474 cells HER2 receptor high expression cell line purchased from the Chinese Academy of Sciences' Type Culture Collection Cell Bank
  • the density was inoculated into a 24-well plate, and after culturing at 37 ° C for 2 hours, the medium was removed, and the following treatments were carried out, respectively. In each group of treatments, the medium was replaced with the solution in each of the following groups.
  • Treatment 1 PBS: 50 L of PBS solution was added to 450 ⁇ l of fresh DMEM medium.
  • Treatment 2 (FAM-siR A): An equal volume of FAM-siR A solution was added, wherein the final concentration of FAM-siR A was 200 nM.
  • Nanoparticle NP (Mal-PEG 5 QQQ-PLA 22 Q 7 Q) containing FAM-siRNA was added to 450 ⁇ l of fresh DMEM medium. Among them, the final concentration of FAM-siRNA was 200 nM.
  • Nanoparticle NP (SCFV-PEG 5 QQQ-PLA 22 Q7Q) containing FAM-siRNA was added to 450 ⁇ l of fresh DMEM medium, The final concentration of FAM-siRNA was 200 nM.
  • Treatment 5 NP(scFv-PEG 50 oo-PL A 2 207o) + Anti-Her2 scFv-cys group
  • Nanoparticle NP-encapsulated FAM-siRNA scFv-PEG 5 . . . -PLA 22 . 7 .
  • each group of cells was digested and collected by flow cytometry (FACS, BD).
  • FAM-siRNA is substantially incapable of entering cells, targeting the drug delivery system NP (scFv-PEG 5 ooo-PLA 220 7o) and the non-targeted drug delivery system NP (Mal-PEG 5() ( xrPLA 22 ( 7() ) can successfully transport FAM-siRNA into cells.
  • the experimental group of targeted drug delivery system detected that the intracellular FAM-siRNA fluorescence signal was significantly stronger than the non-targeted drug delivery system, indicating that the single The chain fragment antibody Anti-H e r2 scFv-cys modified nanoparticles can better transport the siR A to the HER2 receptor-expressing breast cancer cell line BT474.
  • the targeting inhibitor Anti-Her2 scFv-cys is added. (final concentration of 7 ⁇ ) in the experimental group (NP (scF V -PEG 5 QQQ-PLA 22 Q 7 Q) + Anti-Her2 scFv-cys group), low fluorescence signal, and non-targeted experimental group NP (Mal -PEG 5 QQQ-PLA 22 Q 7 Q) is equivalent.
  • NP scF V -PEG 5 QQQ-PLA 22 Q 7 Q
  • NP anti-Her2 scFv-cys modified nanoparticles are specific to the HER2 receptor on the surface of cancer cells by fragment antibody on the surface of the nanoparticles.
  • the nanoparticles after binding mediated endocytosis of the nanoparticles added targeted inhibitors Anti-H e r2 scFv-cys , the nanoparticles can not by antibody to the receptor Laid Binding mediated endocytosis of the nanoparticles, is reduced transport FAM-siRNA, resulting in decreased fluorescence signal.
  • the encapsulated siRNA nanoparticle prepared by the polymer modified by the single-stranded fragment antibody has a targeting ability.
  • a ligand modification may be a single-chain fragment antibody modification without being limited thereto, such as transferrin or the like.
  • Plkl helps promote and accelerate mitosis in mammalian cells and is highly expressed in a variety of tumor cells. By silencing its expression, tumor growth can be inhibited; the present invention evaluates the single-stranded fragment antibody Anti-Her2 scFv-cys modified targeting nanoparticle by silencing the expression of Plk1 gene of BT474 cells to better transport siRNA into milk Adenocarcinoma cells, and successfully release siRNA, silence the expression of target genes.
  • the targeted system was prepared by preparing NP (Mal-PEG 5QQ ( rPLA 22Q7() ) and NP (scF V -PEG 5Q (X rPLA 22()7() )) containing siPlkl and siN.C. in the above system. The system silences the effect of the target gene at the cellular level.
  • BT474 cells were seeded at a density of 1 ⁇ 10 5 cells/well in a 6-well plate, cultured at 37° C. for 24 hours, and then the medium was removed, and the following treatments were carried out, respectively, and the medium was replaced with the following groups in each treatment. In the solution, one duplicate hole is set for each treatment group.
  • Treatment 1 PBS: 100 ⁇ l of PBS solution was added to 1.9 mL of fresh DMEM medium.
  • Treatment 2 Free siPlkl group: Add 100 ⁇ L of a separate siPlld solution, wherein the final concentration of siPlld is 200 nM.
  • Treatment 3 Dilute 5 Lipofectamine 2000 to 50 with sterile water, add to 50 ⁇ L of an aqueous solution containing 100 pmol of siPlkl, and after combining for 15 minutes at room temperature, add 100 ⁇ L of the solution to 1.9 mL of fresh In the medium. Among them, the final concentration of siPlkJ was 50 nM.
  • Treatment 4 (NP(Mal-P EG 5 QQQ-PLA 22 Q 7 Q)/siN.C. group): 100-packed siN.C.-targeted nanoparticle NP (Mal-PEG 5 . . . - PLA 22. 7 )) Add to 1.9 mL of fresh DMEM medium with a final concentration of siN.C. of 200 nM.
  • Treatment 5 100 ⁇ of non-targeted nanoparticle NP (Mal-PEG 5 . . . -PLA 22. 7 ) containing siPlkl. Add to 1.9 mL of fresh DMEM medium with a final concentration of si n npl of 200 nM.
  • NP scFv-PEG 5000 -PLA 22070
  • SiN.C Group. 100 package carrier siN.C. targeted nanoparticle NP (. ScFv-PEG 5 ...- PLA 22 7.)
  • Treatment 7 100 ⁇ of the coated nanoparticle NP (scFv-PEG 5 . . . -PLA 22 . 7 ) To 1.9 mL of fresh DMEM medium, the final concentration of siPlkl was 200 nM.
  • the PCR reaction conditions are as follows:
  • the effectiveness and targeting of the vector as a targeted siRNA delivery system was observed by in vivo experiments by injecting the siP/W targeted drug delivery system and the non-targeted drug delivery system into the mouse via the tail vein.
  • BT474 cells (0.5 ⁇ 10 7 ) were inoculated in situ under the fat pad of the second mammary gland of nude mice.
  • the tumor was formed in about 10 days.
  • the tumor volume was about 50 mm 3 .
  • the nude mice were randomly divided into six groups of 8 rats each.
  • the tail vein injection is administered once every three days.
  • the administration method is as follows:
  • Treatment 1 PBS control group: Each nude mouse was injected with 250 L of PBS.
  • Treatment 2 (naked siPlkl group): Each nude mouse was injected with 250 ⁇ l of bare siPlkl, wherein the siPU dose was 20 g.
  • NP Mal-PEG 5 (KKrPLA 22Q7Q ysiN C. Group): each nude mouse injected entrapped siN C. nanoparticles NP (Mal-PEG 5 ...- PLA 22 7),... Among them, the dose of siN. C. was 20 g.
  • Treatment 5 NP(scFv-PEG 5( xKrPLA 22 Q 7 Q)/siN. C. group: Each nude mouse was injected with nanoparticles containing siN. C. (NP (scFv-PEG 5 ( KKrPLA 22 Q 7) Q), wherein the dose of siN. C. is 20 g.
  • Treatment 6 NP ⁇ cFv-PEGsooQ-PLA ⁇ TOysiP/L? group: Each nude mouse was injected with a nanoparticle NP (scFv-PEG 5 . . . -PLA 22 . 7 ) containing siP/L? The siP/W dose is 20 g.
  • Tumor volume was measured every other day after the start of treatment.
  • Fig. 6D tumor growth rates were faster in all PBS groups and in all negative control groups, while using siPlkl nanoparticles NP (Mal-PEG 5 QQQ-PLA 22 Q 7 Q) and NP (SCFV-) were used.
  • NP Mal-PEG 5 QQQ-PLA 22 Q 7 Q
  • SCFV- NP
  • the tumor growth rate was significantly inhibited compared with the negative control group, but the NP ( SC F V -PEG 5 . . . -PLA 22Q7Q ) treatment group was able to inhibit more significantly.
  • the drug delivery system can utilize a targeted modified block polymer and a cationic lipid to prepare a targeted siR A delivery system by double emulsification, or can utilize block polymers and cationic lipids.
  • the double emulsification method first prepares siRNA nanoparticles, and then performs targeted modification to prepare a targeted siRNA delivery system.
  • the targeting group may be a targeting group of a small molecule such as galactose, folic acid, a short peptide containing arginine-glycine-aspartate (RGD), or a fragment antibody, a transferrin antibody, or the like.
  • a targeting group of a small molecule such as galactose, folic acid, a short peptide containing arginine-glycine-aspartate (RGD), or a fragment antibody, a transferrin antibody, or the like.

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Abstract

A delivery system and preparation of siRNA, with nanoparticles enclosing siRNA as an active ingredient, wherein the nano-particles are formed by a polymer and a cationic lipid, the polymer is a di-block or tri-block copolymer of polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic acid-glycolic acid), the cationic lipid is N,N-dihydroxyethyl-N-methyl-N-2-(cholesteroloxy carbonylamino) ethylammonium bromide or (2,3-dioleyloxy propyl) trimethylammonium chloride. The nanoparticle and preparation can desirably deliver siRNA to a cell and effectively silence expression of a target gene. Moreover, after being modified by a ligand or antibody, the siRNA delivery system can desirably silence the target gene at both a cellular level and an animal level.

Description

一种小干扰 RNA药物的给药系统和制剂 技术领域  Drug delivery system and preparation for small interfering RNA drugs
本发明涉及一种聚乙二醇 -聚乳酸或者聚乙二醇-聚（乳酸-乙醇酸）两嵌段 /三嵌段共 聚物（两亲性高分子聚合物）和阳离子脂质材料共同制备的小干扰 RNA (siRNA) 的给 药系统和制剂。 背景技术  The invention relates to a polyethylene glycol-polylactic acid or a polyethylene glycol-poly(lactic-glycolic acid) two-block/triblock copolymer (amphiphilic polymer) and a cationic lipid material Small interfering RNA (siRNA) delivery systems and formulations. Background technique
小干扰 R A具有特异性抑制致病基因表达的能力以及高效、多样化的特征， 近年来 已经在肝炎、 艾滋病、 老年性黄斑病变、 禽流感以及癌症等大量疾病的治疗中显示出良 好的应用前景。例如 Calendo制药公司研发的基于 R A干扰的 CALAA-01制剂经系统给药 后在一期临床试验中显示出治疗癌症的效果。 有理由相信， 基于 siRNA的 RNA干扰疗法 在不久的将来就可能用于临床治疗。 然而， 在以治疗人类疾病为目的的体内 siRNA给药 方面面临着巨大挑战。由于 siRNA分子本身穿透细胞膜的能力极差，也不具备靶向功能， 而且在生理环境中极不稳定， 因此目前 siRNA药物研发的瓶颈在于 siRNA的给药系统和 技术。 如何增强 siRNA在体内的稳定性和穿透细胞膜的能力， 以及增强疾病治疗的细胞 和组织靶向性等都是 siRNA给药系统急切需要解决的问题。 因此 siRNA的体内给药系统 也已成为发展和实施 R A干扰疗法的一个关键所在。  Small interfering RA has the ability to specifically inhibit the expression of disease-causing genes as well as high-efficiency and diverse characteristics. In recent years, it has shown good application prospects in the treatment of a large number of diseases such as hepatitis, AIDS, age-related macular degeneration, avian influenza and cancer. . For example, the RA-interfering CALAA-01 formulation developed by Calendo Pharmaceuticals shows the effect of treating cancer in a phase I clinical trial after systemic administration. There is reason to believe that siRNA-based RNA interference therapy may be used for clinical treatment in the near future. However, there are significant challenges in the delivery of siRNA in vivo for the purpose of treating human diseases. Since the siRNA molecule itself has a very poor ability to penetrate the cell membrane, has no targeting function, and is extremely unstable in a physiological environment, the current bottleneck in the development of siRNA drugs lies in the delivery system and technology of siRNA. How to enhance the stability of siRNA in vivo and the ability to penetrate cell membranes, as well as enhance the cell and tissue targeting of disease treatment, are urgent problems to be solved by siRNA delivery systems. Therefore, the in vivo drug delivery system of siRNA has also become a key to the development and implementation of R A interference therapy.
目前用于 siRNA给药系统构建的材料包括聚乙烯亚胺（PEI)、去了端肽的精制胶原、 一些阳离子脂质体， 和化学合成的各种阳离子高分子等。 这些载体分子与 siRNA溶液互 混后通过电荷相互作用得到二者的复合物， 从而实现传递 siRNA的目的。 这类给药系统 面临难以放大规模的问题， 重复性较差。 除了通过电荷相互作用与 siRNA形成复合物从 而实现 siRNA给药的系统和技术外， 有部分研究报道了将 siRNA包载在高分子中制备纳 米尺度制剂的方法， 来实现 siRNA的体内给药。 这类纳米颗粒一般利用聚乳酸、 聚乙醇 酸或者二者的共聚物来制备， 不足之处在于 siRNA的包封率非常低（低于 30%)， 载药量 很低， 难以满足临床应用的要求。 发明内容  Materials currently used in the construction of siRNA delivery systems include polyethyleneimine (PEI), degraded collagen with terminal peptides, some cationic liposomes, and various cationic polymers synthesized chemically. These carrier molecules are mixed with the siRNA solution to form a complex of the two by charge interaction, thereby achieving the purpose of delivering siRNA. Such drug delivery systems face problems that are difficult to scale up and are less reproducible. In addition to systems and techniques for siRNA administration by complex interaction with siRNA by charge interaction, some studies have reported the in vivo administration of siRNA by encapsulating siRNA in a polymer to prepare a nanoscale formulation. Such nanoparticles are generally prepared by using polylactic acid, polyglycolic acid or a copolymer of the two. The disadvantage is that the encapsulation efficiency of siRNA is very low (less than 30%), and the drug loading is very low, which is difficult to meet clinical application. Claim. Summary of the invention
本发明的目的是提供一种核酸药物给药载体，具体涉及一种含有两亲性高分子聚合 物和阳离子脂质的核酸药物的给药载体及其应用、 该给药载体制备方法和药物组合物。 这一给药载体组合物具有极高的 siRNA包封率， 载药量也大为提高。  The object of the present invention is to provide a nucleic acid drug delivery carrier, in particular to a drug delivery vehicle containing an amphiphilic polymer and a cationic lipid, and a use thereof, a preparation method and a drug combination thereof Things. This drug delivery vehicle composition has an extremely high siRNA encapsulation efficiency and a large drug loading.
本发明将 siRNA加入到可降解的两亲性高分子聚合物和阳离子脂质的溶液中，通过 双乳化的方法将 siRNA包埋在两亲性高分子聚合物和阳离子脂质形成的纳米颗粒中。加 入的阳离子脂质将 siRNA的包封率提高到 90%以上。 这种方法不但可以制备高效包载 siRNA的纳米颗粒， 而且制得的纳米颗粒能有效进入细胞， 并从内涵体逃逸， 从而有效 沉默致病靶基因的表达， 并在体内抑制乳腺癌的生长。 本发明所述的载体组合物 （即核酸药物给药载体）含有两亲性高分子聚合物 （A成 分） 和阳离子脂质 （B 成分）。 所述两亲性高分子聚合物是指在一个大分子链上同时含 有亲水性和疏水性链段的两亲性嵌段共聚物。所述两亲性嵌段共聚物中的亲水性链段可 以是聚乙二醇、 聚乙烯醇、 聚乙烯吡咯烷酮、 聚丙烯酰胺类、 聚甲基丙烯酸氨基酯类、 聚丙烯酸和聚甲基丙烯酸中的一种或两种以上形成的任意均聚物或共聚物。所述两亲性 嵌段共聚物中的疏水性链段可以是聚乳酸、 聚己内酯、 聚乙交酯、 聚氨基酸和聚磷腈中 的一种或两种以上形成的任意均聚物或共聚物。 In the present invention, siRNA is added to a solution of a degradable amphiphilic polymer and a cationic lipid, and the siRNA is embedded in the nanoparticle formed by the amphiphilic polymer and the cationic lipid by a double emulsion method. . The added cationic lipid increased the encapsulation efficiency of siRNA to over 90%. This method can not only prepare nanoparticles with high efficiency of encapsulating siRNA, but also the prepared nanoparticles can effectively enter cells and escape from endosomes, thereby effectively silencing the expression of pathogenic target genes and inhibiting the growth of breast cancer in vivo. The carrier composition (i.e., the nucleic acid drug-administered carrier) of the present invention contains an amphiphilic polymer (component A) and a cationic lipid (component B). The amphiphilic polymer refers to an amphiphilic block copolymer containing both hydrophilic and hydrophobic segments on one macromolecular chain. The hydrophilic segment in the amphiphilic block copolymer may be polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylamide, polyamidomethacrylate, polyacrylic acid and polymethyl. Any homopolymer or copolymer formed from one or two or more of acrylic acid. The hydrophobic segment in the amphiphilic block copolymer may be any homopolymer formed of one or more of polylactic acid, polycaprolactone, polyglycolide, polyamino acid and polyphosphazene. Or a copolymer.
在一种较佳的实施方式中， 所述两亲性高分子聚合物为聚乙二醇 （PEG) -聚乳酸 (PLA) 或聚乙二醇-聚 （乳酸-乙醇酸） （PLGA) 的两嵌段或三嵌段共聚物， 例如可以 为 PEG-PLA、 PEG-PLGA、 PLA-PEG-PLA、 PLGA-PEG-PLGA。 这种嵌段共聚物能够 在水介质中自组装成胶束或纳米颗粒，相对疏水性的 PLA或 PLGA聚集成疏水性的核， PEG嵌段组装成亲水性的壳， 具有稳定胶束、有效躲避生物体内质网系统的捕捉和蛋白 质吸附的作用。阳离子脂质在此给药系统中的主要作用是通过静电相互作用提高核酸药 物在载体中的载药量和包封率。  In a preferred embodiment, the amphiphilic polymer is polyethylene glycol (PEG)-polylactic acid (PLA) or polyethylene glycol-poly(lactic-glycolic acid) (PLGA). The diblock or triblock copolymer may, for example, be PEG-PLA, PEG-PLGA, PLA-PEG-PLA, PLGA-PEG-PLGA. The block copolymer can self-assemble into micelles or nanoparticles in an aqueous medium, and the relatively hydrophobic PLA or PLGA aggregates into a hydrophobic core, and the PEG block assembles into a hydrophilic shell, which has stable micelles and effectively evades. The capture and protein adsorption of the endoplasmic reticulum system in the organism. The primary role of cationic lipids in this delivery system is to increase the drug loading and encapsulation efficiency of the nucleic acid drug in the carrier by electrostatic interaction.
在一较佳的实施方案中， 阳离子脂质优选为铵盐型的两亲性脂质材料， 只要在药理 学上允许，本发明对铵盐型的两亲性脂质材料没有特别的限制。例如可以为二甲基二 (十 八烷基)溴化铵盐（DDAB)、 1，2-二肉豆蔻酰基 -3-三甲基铵丙烷、 1，2-二油酰基 -3-三甲基 铵丙烷（DOTAP)、 1，2-二油酰基 -3-三甲基铵丙烷甲基硫酸盐、 1，2-二棕榈酰基 -3-三甲基 铵丙烷、 1，2-二硬脂酰基 -3-三甲基铵丙烷、 N-(l-(2，3-二油酰氧基)丙基) -N，N，N-三甲基氯 化铵 （DOTMA)、 二肉豆蔻酰基氧基丙基二甲基羟基乙基溴化铵盐 （DMRIE)、 二油酰 氧基丙基二甲基羟基乙基溴化铵 （DORIE)、 二甲基二 (十二烷基)溴化铵、 N-(a-三甲基 铵基乙酰基) -二（十二烷基) -D-谷氨酰胺盐酸盐、 N-(a-三甲基铵基乙酰基) -0，0'-双 -(1H，1H，2H，2H-全氟癸烷基) -L-谷氨酰胺盐酸盐、 0，0'-二 (十二烷酰基) -N-(a-三甲基铵基 乙酰基)二乙醇胺盐酸盐、 甲基烯丙基二 (十二烷基)溴化铵、 N- {p-(w-三甲基铵基丁基氧 基) -苯甲酰基 } -二 (十二烷基) -L-谷氨酰胺盐酸盐、 9-(w-三甲基铵基丁基) -3,6-双 (十二烷酰 基)咔唑溴化物、二甲基二 (十八烷基)铵盐酸盐、 N-w-三甲基铵基癸酰基-二 (十六烷基) -D- 谷氨酰胺溴化物、 N- {p-(w-三甲基铵基己基氧基)-苯甲酰基} -二 (十四烷基) -L-谷氨酰胺 溴化物、 p-(w-三甲基铵基癸基氧基) -ρ'-辛氧基偶氮苯溴化物盐 （MC-1-0810)、 p-{w-(b- 羟基乙基)二甲基-铵基-癸基氧基 }-ρ'-辛氧基偶氮苯溴化物盐 （MC-3-0810)、 Ο,Ο',Ο"- 三 (十二烷酰基) -N-(w-三甲基-铵基癸酰基) -三 (羟基甲基)氨基甲烷澳化物盐 （TC-1-12)、 1，2-二月桂基 -甘油 -3-乙基磷酸胆碱、 1，2-二肉豆蔻酰基 -甘油 -3-乙基磷酸胆碱、 1，2-二棕 榈酰基 -甘油 -3-乙基磷酸胆碱、 1，2-二硬脂酰基 -甘油 -3-乙基磷酸胆碱、 1，2-二油酰基-甘 油 -3-乙基磷酸胆碱、 1-棕榈酰基 -2-油酰基 -甘油 -3-乙基磷酸胆碱、 N，N-二羟乙基 甲基 -N-2- (胆固醇氧羰基氨基） 乙基溴化铵 （BHEM-Chol)、 （2,3-二油氧基丙基）三甲基氯 化铵 （DOTAP) 和 N-(l-(2，3-二油酰氧基)丙基) -N，N，N-三甲基氯化铵等。 上述铵盐型的 阳离子脂质可以单独使用 1种， 也可以任意组合 2种以上使用。 在本发明的更佳的实施方案中， 所述阳离子脂质优选为 N，N-二羟乙基 甲基 -N-2- (胆固醇氧羰基氨基)乙基溴化铵 （BHEM-Chol)、 （2,3-二油氧基丙基)三甲基氯化铵 (DOTAP) 和 N-(l-(2，3-二油酰氧基)丙基) -N，N，N-三甲基氯化铵中的至少一种。 In a preferred embodiment, the cationic lipid is preferably an amphiphilic lipid material of the ammonium salt type, and the present invention is not particularly limited to the ammonium salt type amphiphilic lipid material as long as it is pharmacologically acceptable. For example, it may be dimethyl dioctadecyl ammonium bromide (DDAB), 1,2-dimyristoyl-3-trimethylammonium propane, 1,2-dioleyl-3-trimethyl Radium-based propane (DOTAP), 1,2-dioleyl-3-trimethylammonium propane methyl sulfate, 1,2-dipalmitoyl-3-trimethylammonium propane, 1,2-distearyl Acyl-3-trimethylammonium propane, N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), dimyristoyl Oxypropyl dimethyl hydroxyethyl ammonium bromide (DMRIE), dioleoyloxypropyl dimethyl hydroxyethyl ammonium bromide (DORIE), dimethyl di(dodecyl) bromination Ammonium, N-(a-trimethylammonioacetyl)-di(dodecyl)-D-glutamine hydrochloride, N-(a-trimethylammonioacetyl)-0,0 '-Bis-(1H,1H,2H,2H-perfluorodecyl)-L-glutamine hydrochloride, 0,0'-bis(dodecanoyl)-N-(a-trimethyl Ammonylacetyl)diethanolamine hydrochloride, methallyl bis(dodecyl)ammonium bromide, N-{p-(w-trimethylammonium butyloxy)-benzoyl} - bis(dodecyl)-L-glutamine hydrochloride, 9-(w-tripa Alkyl ammonium butyl) -3,6-bis(dodecanoyl)carbazole bromide, dimethyldi(octadecyl)ammonium hydrochloride, Nw-trimethylammonium decanoyl-di Cetyl)-D-glutamine bromide, N-{p-(w-trimethylammonium hexyloxy)-benzoyl}-bis(tetradecyl)-L-glutamine Bromide, p-(w-trimethylammonium decyloxy)-ρ'-octyloxyazobenzene bromide salt (MC-1-0810), p-{w-(b-hydroxyethyl Dimethyl-ammonium-fluorenyloxy}-ρ'-octyloxyazobenzene bromide salt (MC-3-0810), hydrazine, Ο', Ο"-tris(dodecanoyl)- N-(w-trimethyl-ammoniodecanoyl)-tris(hydroxymethyl)aminomethane alkoxide (TC-1-12), 1,2-dilauryl-glycerol-3-ethylphosphonate Base, 1,2-dimyristoyl-glycerol-3-ethylphosphocholine, 1,2-dipalmitoyl-glycero-3-ethylphosphocholine, 1,2-distearoyl-glycerol- 3-ethylphosphocholine, 1,2-dioleoyl-glycerol-3-ethylphosphocholine, 1-palmitoyl-2-oleoyl-glycero-3-ethylphosphocholine, N,N- Dihydroxyethylmethyl-N-2-(cholesteryloxycarbonylamino)ethylammonium bromide (BHEM-Chol), (2,3-dimethoxypropyl)trimethylammonium chloride (DOTAP) and N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethyl The above-mentioned ammonium salt type cationic lipid may be used singly or in combination of two or more kinds. In a further preferred embodiment of the invention, the cationic lipid is preferably N,N-dihydroxyethylmethyl-N-2-(cholesteryloxycarbonylamino)ethylammonium bromide (BHEM-Chol), (2,3-dimethoxypropyl)trimethylammonium chloride (DOTAP) and N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethyl At least one of the ammonium chlorides.
本发明对载体组合物中的 A成分和 B成分的配合比例没有特殊的限制， 从将核酸 有效地转运至细胞内的观点考虑， 两亲性高分子聚合物和阳离子脂质的含量范围可以 为： 两亲性高分子聚合物为 100重量份， 阳离子脂质为 0.001-20重量份，优选为 0.01-10 重量份； 优选地， 以载体组合物的总重量为基准， 所述两亲性高分子聚合物的含量为 The compounding ratio of the component A and the component B in the carrier composition is not particularly limited, and the amphiphilic polymer and the cationic lipid may be in a range of from the viewpoint of efficiently transporting the nucleic acid into the cell. The amphiphilic high molecular polymer is 100 parts by weight, and the cationic lipid is 0.001 to 20 parts by weight, preferably 0.01 to 10 parts by weight; preferably, the amphiphilic property is high based on the total weight of the carrier composition. The molecular polymer content is
83.3-99.999重量％， 阳离子脂质的含量为 0.001-16.7重量％， 进一步优选地， 所述两亲 性高分子聚合物的含量为 90.9-99.999重量％， 阳离子脂质的含量为 0.001-9.1重量％。 83.3-99.999% by weight, the cationic lipid content is 0.001 to 16.7% by weight, further preferably, the amphiphilic high molecular polymer is contained in an amount of 90.9 to 99.999% by weight, and the cationic lipid is contained in an amount of 0.001 to 9.1% by weight. %.
根据本发明，聚乙二醇单嵌段的数均分子量优选为 550-10000 g/mol,聚乳酸或聚（乳 酸-乙醇酸） 嵌段的数均分子量优选为 4800-51000 g/mol, 其中， 聚 （乳酸-乙醇酸） 嵌 段的数均分子量进一步优选为 10000-50000 g/mol。  According to the present invention, the number average molecular weight of the polyethylene glycol monoblock is preferably 550 to 10000 g/mol, and the number average molecular weight of the polylactic acid or poly(lactic acid-glycolic acid) block is preferably 4800 to 51000 g/mol, wherein The number average molecular weight of the poly(lactic acid-glycolic acid) block is more preferably from 10,000 to 50,000 g/mol.
所述聚（乳酸-乙醇酸）嵌段中， 丙交酯 /乙交酯单体的聚合度比例可为 75/25-50/50。 所述阳离子脂质 BHEM-Chol的分子量为 655.5 g/mol, 所述阳离子脂质 DOTAP的 分子量为 698.5 g/mol, 所述阳离子脂质 DOTMA的分子量为 670.6 g/mol 本发明的实 施方式基于这三种阳离子脂质， 但本发明并不限于这三种脂质， 类似阳离子脂质均可用 于制备本发明所述的核酸药物给药载体， 实现本发明的目的。  In the poly(lactic acid-glycolic acid) block, the degree of polymerization of the lactide/glycolide monomer may be from 75/25 to 50/50. The molecular weight of the cationic lipid BHEM-Chol is 655.5 g/mol, the molecular weight of the cationic lipid DOTAP is 698.5 g/mol, and the molecular weight of the cationic lipid DOTMA is 670.6 g/mol. Three kinds of cationic lipids, but the present invention is not limited to these three kinds of lipids, and similar cationic lipids can be used for preparing the nucleic acid drug-administering carrier of the present invention, achieving the object of the present invention.
上述嵌段共聚物， 可以为 PEG55Q-PLA286QQ、 PEG2QQQ-PLA125QQ、 PEG5QQQ-PLA5000、 PEG5000-PLA25000、 PEG5000-PL A51000、 PEG10000-PLA15000、 PEGioooo-PLGAioooo(5o/50)、 PEGioooo-PLGA5oooo(75/25) 、 PLA63oo-PEGi2oo-PLA63oo 、 PLA4800-PEG5000-PLA4800 禾口 PLA48oo-PEG10ooo-PLA48oo, 其中， 下标表示数均分子量， 下标括号中的比例为聚 （乳酸- 乙醇酸） 嵌段中的丙交酯单体 /乙交酯单体的聚合度比例 （以下与此类同）， 本发明并不 限于上述组成的嵌段共聚物。 The above block copolymer may be PEG 55 Q-PLA 286 QQ, PEG 2 QQQ-PLA 125 QQ, PEG 5 QQQ-PLA 5000 , PEG5000-PLA25000, PEG5000-PL A51000, PEG10000-PLA15000, PEGioooo-PLGAioooo (5o/ 50), PEGioooo-PLGA5oooo (75/25), PLA 6 3oo-PEGi2oo-PLA 6 3oo, PLA4800-PEG5000-PLA4800 and PLA4 8 oo-PEG 10 ooo-PLA4 8 oo, wherein the subscript indicates the number average molecular weight, The ratio in the lower brackets is the ratio of the degree of polymerization of the lactide monomer/glycolide monomer in the poly(lactic-glycolic acid) block (hereinafter the same as this), and the present invention is not limited to the above-described composition. Segment copolymer.
聚乙二醇-聚乳酸、 聚乙二醇-聚 （乳酸 -乙醇酸）、 聚乙二醇-聚乳酸三嵌段共聚物或 聚乙二醇-聚 （乳酸-乙醇酸） 三嵌段共聚物具有两亲性， 这些嵌段共聚物和阳离子脂质 通过双乳化方法可以形成纳米尺度的颗粒， 这类纳米颗粒具有亲水性的聚乙二醇外壳， 而且颗粒的尺寸与共聚物的组成相关， 可以调控。  Polyethylene glycol-polylactic acid, polyethylene glycol-poly(lactic-glycolic acid), polyethylene glycol-polylactic acid triblock copolymer or polyethylene glycol-poly(lactic-glycolic acid) triblock copolymer The amphiphilic substance, these block copolymers and cationic lipids can form nano-scale particles by a double-emulsification method, such nanoparticles have a hydrophilic polyethylene glycol outer shell, and the size of the particles and the composition of the copolymer Related, can be regulated.
具体地， 含有上述两亲性高分子聚合物和阳离子脂质的给药载体能够形成纳米颗 粒， 所述纳米颗粒的直径可以为 50-250 nm。 在较佳的实施方式中， 本发明的纳米颗粒 表面可具有化学修饰、抗体修饰或者配体修饰。 所述修饰的方法可为本领域各种常规的 对纳米颗粒的表面进行修饰的方法。  Specifically, the administration carrier containing the above amphiphilic high molecular polymer and cationic lipid can form nanoparticles, and the nanoparticles may have a diameter of 50 to 250 nm. In a preferred embodiment, the surface of the nanoparticles of the present invention may have chemical modifications, antibody modifications or ligand modifications. The method of modification can be a variety of conventional methods for modifying the surface of nanoparticles.
有意义的是，加入阳离子脂质能够显著提高 siR A的包封率和载药量。采用这种纳 米颗粒作为载体实现了包载 siRNA纳米颗粒的内吞， 并达到基因沉默的效果。  Significantly, the addition of cationic lipids significantly increased the encapsulation efficiency and drug loading of siR A. The use of such nanoparticle as a carrier enables endocytosis of the encapsulated siRNA nanoparticles and achieves gene silencing.
本发明的载体组合物， 除上述 A成分及 B成分之外， 还可以含有油性基剂 （以下 称为 C成分）。 配合油性基剂， 利用其特性， 能够控制核酸转运用载体组合物的核酸导 入效率。例如通过配合油性基剂调整核酸转运用载体组合物的比重， 可以控制细胞和载 体组合物的接触性， 改善体外的导入效率。 另外， 例如通过配合具有温度感受性功能的 油剂作为油性基剂， 能够在规定的温度条件下使核酸载体的芯破碎， 诱发细胞表面的波 动， 提高核酸的导入效率。 进而， 例如通过配合具有外部刺激破碎性的油剂作为油性基 剂， 可以通过外部刺激使核酸载体组合物的芯破碎， 诱发细胞表面的波动， 提高核酸的 导入效率。 The carrier composition of the present invention may further contain an oily base (hereinafter referred to as a component C) in addition to the components A and B described above. When the oily base is blended, the nucleic acid introduction efficiency of the carrier composition for nucleic acid transport can be controlled by utilizing its characteristics. For example, by adjusting the specific gravity of the carrier composition for nucleic acid transport by blending an oily base, cells and carriers can be controlled. The contact properties of the body composition improve the efficiency of introduction in vitro. Further, for example, by blending an oil agent having a temperature-sensing function as an oily base, the core of the nucleic acid carrier can be broken under a predetermined temperature condition, the fluctuation of the cell surface can be induced, and the introduction efficiency of the nucleic acid can be improved. Further, for example, by blending an oil agent having external stimuli disruptability as an oily base, the core of the nucleic acid carrier composition can be broken by external stimulation, the fluctuation of the cell surface can be induced, and the introduction efficiency of the nucleic acid can be improved.
本发明的载体组合物中配合使用的油性基材，例如可以举出，全氟化碳、全氟戊烷、 溴代全氟辛烷、 全氟己烷、 全氟三丁基胺、 大豆油、 精制大豆油、 氢化大豆油、 大豆油 非皂化物、 角鲨烯、 蓖麻油、 丁香油、 三油酸脱水山梨糖醇酯、 松节油、 红花油、 红花 油脂肪酸、 油酸、 棕榈油、 菜子油、 杂醇油、 橄榄油、 亚麻仁油、 芝麻油、 叶绿素油、 巴豆油、 佛手油、 雪松油、 橙油、 茴香油、 桉树油、 玉米油、 熏衣草油、 马郁兰油、 柠 檬油、 棉籽油、 椰子油、 蛋黄油、 玫瑰花油、 松油、 杏仁油、 花生油、 山茶油、 白樟油、 春黄菊油、 桂皮油、 薄荷油、 酯化玉米油、 生姜油、 罗马春黄菊油、 蛇油、 留兰香油、 向日葵油、 可可脂、 小麦胚芽油、 氧化锌油、 氢化油、 氢化植物油、 轻质液体石蜡、 液 体石蜡、 中链脂肪酸三甘油酯、貂油、苦橙油、聚氧乙烯蓖麻油、聚氧乙烯氢化蓖麻油、 聚氧乙烯氢化蓖麻油 10、聚氧乙烯氢化蓖麻油 100、聚氧乙烯氢化蓖麻油 20、聚氧乙烯 氢化蓖麻油 40、聚氧乙烯氢化蓖麻油 5、聚氧乙烯氢化蓖麻油 50、聚氧乙烯氢化蓖麻油 60、 聚氧基 35蓖麻油、 操作油等。 上述油性基剂中， 全氟戊烷具有温度感受性， 具有 在 29.5°C下通过沸腾气化的特性。 另外， 全氟己烷、 溴代全氟辛烷、 及全氟三丁基胺具 有下述特性， 即具有外部刺激破碎性， 在由超声波照射产生的刺激等来自外部的刺激作 用下， 使载体组合物的芯产生空腔， 使其破碎。  The oily base material to be used in the carrier composition of the present invention may, for example, be a perfluorocarbon, perfluoropentane, brominated perfluorooctane, perfluorohexane, perfluorotributylamine or soybean oil. , refined soybean oil, hydrogenated soybean oil, soybean oil unsaponifiable, squalene, castor oil, clove oil, sorbitan trioleate, turpentine, safflower oil, safflower oil fatty acid, oleic acid, palm oil , rapeseed oil, fusel oil, olive oil, linseed oil, sesame oil, chlorophyll oil, croton oil, bergamot oil, cedar oil, orange oil, fennel oil, eucalyptus oil, corn oil, lavender oil, marjoram oil, lemon Oil, cottonseed oil, coconut oil, egg butter, rose oil, pine oil, almond oil, peanut oil, camellia oil, white eucalyptus oil, chamomile oil, cinnamon oil, peppermint oil, esterified corn oil, ginger oil, Roman spring Chamomile oil, snake oil, spearmint oil, sunflower oil, cocoa butter, wheat germ oil, zinc oxide oil, hydrogenated oil, hydrogenated vegetable oil, light liquid paraffin, liquid paraffin, medium chain fat Triglyceride, eucalyptus oil, bitter orange oil, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, polyoxyethylene hydrogenated castor oil 10, polyoxyethylene hydrogenated castor oil 100, polyoxyethylene hydrogenated castor oil 20, polyoxygen Ethylene hydrogenated castor oil 40, polyoxyethylene hydrogenated castor oil 5, polyoxyethylene hydrogenated castor oil 50, polyoxyethylene hydrogenated castor oil 60, polyoxyl 35 castor oil, process oil, and the like. Among the above oily bases, perfluoropentane has temperature sensitivity and has a property of being vaporized by boiling at 29.5 °C. Further, perfluorohexane, brominated perfluorooctane, and perfluorotributylamine have the following characteristics, that is, external stimuli-breaking property, and the carrier is stimulated by external stimuli such as stimulation by ultrasonic irradiation. The core of the composition creates a cavity that breaks it.
含有该油性基剂时， 作为该油性基剂的比例， 只要不妨碍本发明的效果即可， 没有 特殊的限制， 例如可以为下述比例， 即相对于 100重量份的上述 A成分及 B成分的总 量， 该油性基材为 0.1-50重量份， 优选为 1-30重量份， 更优选为 5-20重量份。  When the oily base is contained, the ratio of the oily base is not particularly limited as long as it does not impair the effects of the present invention, and may be, for example, a ratio of the above-mentioned A component and B component to 100 parts by weight. The total amount of the oily substrate is from 0.1 to 50 parts by weight, preferably from 1 to 30 parts by weight, more preferably from 5 to 20 parts by weight.
进而， 根据需要， 本发明的载体组合物中也可以含有膜融合性脂质 （辅助脂质）。 通过含有上述膜融合性脂质， 能够进一步提高核酸向细胞内的转运效率。 作为上述膜融 合性脂质， 例如可以举出， 二油酰基磷脂酰乙醇胺、 二油酰基磷脂酰胆碱、 反式磷脂酰 基乙醇胺、 1，2-双 (10， 12-二十三烷二酰基)-磷酸乙醇胺、 1，2-二反油酰基磷酸乙醇胺、 1，2-二 (十六烷基)磷酸乙醇胺、 1，2-二己酰基磷酸乙醇胺、 1，2-二月桂酰基磷酸乙醇胺、 1，2-二亚油酰基磷酸乙醇胺、 1，2-二肉豆蔻酰基磷酸乙醇胺、 1，2-二油酰基磷酸乙醇胺、 1，2-二棕榈油酰基磷酸乙醇胺、 1，2-二棕榈酰基磷酸乙醇胺、 1，2-二植烷酰磷酸乙醇胺、 1，2-二硬脂酰基磷酸乙醇胺、 1-棕榈酰基 -2-油酰基磷酸乙醇胺、 1-棕榈酰基 -2-(10，12-二 十三烷二酰基)磷酸乙醇胺、 1，2-二油酰基磷酸乙醇胺 -N-己酰胺、 1，2-二棕榈酰基磷酸乙 醇胺 -N-己酰胺、 N，N-二甲基 -1,2-二油酰基磷酸乙醇胺、 N，N-二甲基 -1,2-二棕榈酰基磷酸 乙醇胺、 N-十二烷酰基 -1,2-二棕榈酰基磷酸乙醇胺、 N-十二烷酰基 -1,2-二油酰基磷酸乙 醇胺、 1，2-二油酰基磷酸乙醇胺 -N-十二烷基胺、 1，2-二棕榈酰基磷酸乙醇胺 -N-十二烷基 胺、 1，2-二油酰基磷酸乙醇胺 -N-戊二酰、 1，2-二棕榈酰基磷酸乙醇胺 -N-戊二酰、 1，2-二 油酰基磷酸乙醇胺 -N-乳糖、 1，2-二油酰基磷酸乙醇胺 -N-[4(p-马来酰亚胺甲基)环己烷- 羧酸盐]、 二棕榈酰磷酸乙醇胺 -N-[4-(p-马来酸亚胺甲基)环己烷 -羧酸盐]、 1，2-二棕榈酰 基磷酸乙醇胺 -N-[4-(p-马来酰亚胺苯基)丁酰胺]、 1，2-二油酰基磷酸乙醇胺 -N-[4-(p-马来 酰亚胺苯基)丁酸盐]、 N-甲基 -1,2-二油酰基磷酸乙醇胺、 N-甲基 -二棕榈酰基磷酸乙醇胺、 1，2-二油酰基磷酸乙醇胺 -N-[3-(2-吡啶二硫)丙酸盐、 1，2-二棕榈酰基磷酸乙醇胺 -N-[3-(2- 吡啶二硫)丙酸盐]、 N- (琥珀酰) -1,2-二油酰基磷酸乙醇胺、 N- (琥珀酰) -1,2-二棕榈酰基磷 酸乙醇胺等。 其中， 在本发明的载体组合物中优选使用二油酰基磷脂酰乙醇胺。 Further, the carrier composition of the present invention may contain a membrane-fused lipid (auxiliary lipid) as needed. By containing the above membrane-fused lipid, the efficiency of transport of nucleic acid into cells can be further improved. Examples of the membrane-fused lipid include dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine, transphosphatidylethanolamine, and 1,2-bis(10,12-docosanedioyl). )-phosphoethanolamine, 1,2-di-oleoylphosphoethanolamine, 1,2-di(hexadecyl)phosphoethanolamine, 1,2-dihexanoylphosphoethanolamine, 1,2-dilauroylphosphoethanolamine, 1,2-dilinoleylphosphoethanolamine, 1,2-dimyristoylphosphoethanolamine, 1,2-dioleoylphosphoethanolamine, 1,2-dipalmitoylphosphoethanolamine, 1,2-dipalmitoyl Phosphoethanolamine, 1,2-diphytanoylphosphoethanolamine, 1,2-distearoylphosphoethanolamine, 1-palmitoyl-2-oleoylphosphoethanolamine, 1-palmitoyl-2-(10,12-di Tridecanedioylphosphoethanolamine, 1,2-dioleylphosphoethanolamine-N-caproamide, 1,2-dipalmitoylphosphoethanolamine-N-hexanamide, N,N-dimethyl-1,2 -Dioleylphosphoethanolamine, N,N-dimethyl-1,2-dipalmitoyl phosphate ethanolamine, N-dodecanoyl-1,2-dibrown Acylphosphoethanolamine, N-dodecanoyl-1,2-dioleoylphosphoethanolamine, 1,2-dioleoylphosphoethanolamine-N-dodecylamine, 1,2-dipalmitoyl phosphate ethanolamine-N -dodecylamine, 1,2-dioleoylphosphoethanolamine-N-glutaryl, 1,2-dipalmitoyl phosphate ethanolamine-N-glutaryl, 1,2-di Oleoylphosphoethanolamine-N-lactose, 1,2-dioleoylphosphoethanolamine-N-[4(p-maleimidomethyl)cyclohexane-carboxylate], dipalmitoyl phosphate ethanolamine-N -[4-(p-maleimidomethyl)cyclohexane-carboxylate], 1,2-dipalmitoyl phosphate ethanolamine-N-[4-(p-maleimidophenyl) Butyramide], 1,2-dioleylphosphoethanolamine-N-[4-(p-maleimidophenyl)butyrate], N-methyl-1,2-dioleylphosphoethanolamine, N-methyl-dipalmitoyl phosphate ethanolamine, 1,2-dioleoyl phosphate ethanolamine-N-[3-(2-pyridinedithio)propionate, 1,2-dipalmitoyl phosphate ethanolamine-N-[ 3-(2-pyridyldithio)propionate], N-(succinyl)-1,2-dioleylphosphoethanolamine, N-(succinyl)-1,2-dipalmitoylphosphoethanolamine, and the like. Among them, dioleoylphosphatidylethanolamine is preferably used in the carrier composition of the present invention.
含有该膜融合性脂质时， 作为该膜融合性脂质的比例， 只要不妨碍本发明的效果即 可， 没有特殊的限制， 可以举出下述比例， 即相对于 100重量份的上述 A成分及 B成 分的总量， 该膜融合性脂质的加入量为 1-500重量份， 优选为 10-250重量份， 更优选为 25-100重量份。  When the membrane-fused lipid is contained, the ratio of the membrane-fused lipid is not particularly limited as long as the effect of the present invention is not impaired, and the following ratio is used, that is, the above-mentioned ratio with respect to 100 parts by weight. The film-fused lipid is added in an amount of 1 to 500 parts by weight, preferably 10 to 250 parts by weight, more preferably 25 to 100 parts by weight, based on the total amount of the component and the component B.
根据使用形态， 本发明的载体组合物可以含有等渗剂、 赋形剂、 稀释剂、 增稠剂、 稳定剂、 缓冲剂、 保存剂等各种添加剂。 上述添加剂的配合量， 可以根据核酸转运用载 体的使用形态适当地设定。  The carrier composition of the present invention may contain various additives such as an isotonic agent, an excipient, a diluent, a thickener, a stabilizer, a buffer, and a preservative depending on the form of use. The compounding amount of the above additives can be appropriately set depending on the form of use of the nucleic acid transport carrier.
本发明的核酸转运用载体组合物通过混合上述 A成分、 B成分以及根据需要混合其 他成分而制成。  The carrier composition for nucleic acid delivery of the present invention is prepared by mixing the above-mentioned A component, B component, and, if necessary, mixing other components.
本发明的载体组合物可以用于转运外源核酸， 其中， 本发明对所述外源核酸的种类 或结构没有特殊的限定。 作为该核酸的具体例， 可以为 siR A、 mRNA、 tRNA、 rRNA、 cDNA、 miR A (微 R A)、 核酶、 反义寡核苷酸、 质粒 DNA、 肽核酸、 三链形成型寡 核苷酸 （Triplex Forming Oligonucleotide, TFO)、 基因等。 其中， 本发明的载体组合物 特别适用于将 siRNA转运至细胞内。 本发明的载体组合物适用的核酸， 可以是来自人、 动物、 植物、 细菌、 病毒等的核酸， 另外， 也可以是通过化学合成制备的核酸。 进而， 上述核酸可以是单链、 双链、 三链中的任一种， 并且对其分子量也没有特殊的限定。 另 夕卜， 本发明中， 核酸可以为被化学、 酶或肽修饰的核酸。 本发明中， 核酸可以单独使用 1种， 也可以 2种以上适当地组合使用。 在一较佳的实施方式中， 本发明的载体组合物 优选转运小干扰核酸 （siRNA) 或其类似物。  The vector composition of the present invention can be used for the transport of exogenous nucleic acids, wherein the present invention does not specifically limit the kind or structure of the exogenous nucleic acid. Specific examples of the nucleic acid may be siR A, mRNA, tRNA, rRNA, cDNA, miR A (microRA), ribozyme, antisense oligonucleotide, plasmid DNA, peptide nucleic acid, and triple-stranded oligonucleoside Acid (Triplex Forming Oligonucleotide, TFO), genes, etc. Among them, the vector composition of the present invention is particularly suitable for transporting siRNA into cells. The nucleic acid to which the vector composition of the present invention is applied may be a nucleic acid derived from a human, an animal, a plant, a bacterium, a virus or the like, or may be a nucleic acid prepared by chemical synthesis. Further, the nucleic acid may be any of a single chain, a double chain, and a triple chain, and the molecular weight thereof is not particularly limited. Further, in the present invention, the nucleic acid may be a nucleic acid modified by a chemical, an enzyme or a peptide. In the present invention, one type of nucleic acid may be used alone or two or more types may be used in combination as appropriate. In a preferred embodiment, the vector composition of the invention preferably transports small interfering nucleic acids (siRNA) or analogs thereof.
本发明还提供了一种药物组合物， 该组合物含有上述的载体组合物及核酸。 本发明 对药物组合物中适用的核酸的种类或结构没有特殊的限定。 作为该核酸的具体例， 可以 为 siRNA、 mR A、 tRNA、 rRNA、 cDNA、 miRNA (微 R A)、 核酶、 反义寡核苷酸、 质粒 DNA、 肽核酸、 三链形成型寡核苷酸（Triplex Forming Oligonucleotide, TFO)、 基 因等。在一较佳的实施方式中，所述核酸优选为小干扰核酸（siR A)。在药物组合物中， 所述核酸、 阳离子脂质与两亲性高分子聚合物的质量比可以为 0.04: 0.0001： 100.0-7.2： 20.0： 100.0，优选为 0.2/1.0/25.0-1.8/1.0/25.0，最优选为 0.2/1.0/25.0。上述药物组合物中， 除核酸类药物外， 还可同时包载其它具有协同治疗作用或者降低毒副作用的药物。  The present invention also provides a pharmaceutical composition comprising the above carrier composition and nucleic acid. The present invention is not particularly limited to the kind or structure of the nucleic acid to be used in the pharmaceutical composition. Specific examples of the nucleic acid may be siRNA, mR A, tRNA, rRNA, cDNA, miRNA (microRA), ribozyme, antisense oligonucleotide, plasmid DNA, peptide nucleic acid, and triple-stranded oligonucleotide. (Triplex Forming Oligonucleotide, TFO), genes, etc. In a preferred embodiment, the nucleic acid is preferably a small interfering nucleic acid (siR A). In the pharmaceutical composition, the mass ratio of the nucleic acid, the cationic lipid to the amphiphilic high molecular polymer may be 0.04: 0.0001: 100.0-7.2: 20.0: 100.0, preferably 0.2/1.0/25.0-1.8/1.0/ 25.0, most preferably 0.2/1.0/25.0. In the above pharmaceutical composition, in addition to the nucleic acid drug, other drugs having synergistic therapeutic effects or reducing toxic side effects may be simultaneously contained.
本发明还提供了上述药物组合物的制备方法， 所述方法包括如下步骤： 将两亲性高 分子聚合物和阳离子脂质溶于油相 （如氯仿等） 中， 加入核酸水溶液后进行第一次超声 形成初始乳液， 将初始乳液加入到水相中并进行第二次超声形成乳液， 将乳液加入到水 相中， 减压 （如 1000帕） 下除去有机溶剂， 离心收集纳米颗粒。 本发明中所述水相包 括但不限于 PVA水溶液。 所述第一次超声的条件优选包括： 功率为 80瓦， 时间为 30 秒； 所述第二次超声的条件优选包括： 功率为 80瓦， 时间为 2分钟。 所述离心的条件 优选包括： 温度为 4°C， 速度为 3000g， 时间为 lh。 本发明制备上述药物组合物的具体 条件不限于上述条件。 The present invention also provides a method for preparing the above pharmaceutical composition, the method comprising the steps of: dissolving the amphiphilic high molecular polymer and the cationic lipid in an oil phase (such as chloroform, etc.), and adding the aqueous nucleic acid solution to perform the first step. Secondary ultrasound An initial emulsion is formed, the initial emulsion is added to the aqueous phase and a second ultrasonication is performed to form the emulsion, the emulsion is added to the aqueous phase, the organic solvent is removed under reduced pressure (e.g., 1000 Pa), and the nanoparticles are collected by centrifugation. The aqueous phase in the present invention includes, but is not limited to, an aqueous PVA solution. The condition of the first ultrasound preferably comprises: a power of 80 watts and a time of 30 seconds; and the condition of the second ultrasound preferably comprises: a power of 80 watts and a time of 2 minutes. The conditions of the centrifugation preferably include: a temperature of 4 ° C, a speed of 3000 g, and a time of 1 h. The specific conditions for the preparation of the above pharmaceutical composition of the present invention are not limited to the above conditions.
本发明还提供了一种核酸导入方法， 通过使上述药物组合物与细胞接触， 将药物组 合物中的核酸导入到细胞内。所述细胞优选为哺乳动物细胞， 更优选为病理状态下或非 正常生理状态下的哺乳动物细胞， 所述核酸优选为小干扰核酸 （siR A)。  The present invention also provides a nucleic acid introduction method for introducing a nucleic acid in a pharmaceutical composition into a cell by bringing the above pharmaceutical composition into contact with a cell. The cell is preferably a mammalian cell, more preferably a mammalian cell in a pathological state or an abnormal physiological state, and the nucleic acid is preferably a small interfering nucleic acid (siR A).
本发明还提供了所述载体组合物在制备抗肿瘤药物中的应用，所述肿瘤优选为乳腺 肿瘤或者肝脏肿瘤。  The invention also provides the use of the carrier composition for the preparation of an anti-tumor drug, preferably a breast tumor or a liver tumor.
本发明使用的嵌段共聚物具有良好的生物相容性和可降解性， 其物理、 化学性能可 通过调节聚合物的组成而调节。例如，增加聚合物中 PLA比例时， 纳米颗粒输运 siRNA 进入细胞的能力增加。  The block copolymer used in the present invention has good biocompatibility and degradability, and its physical and chemical properties can be adjusted by adjusting the composition of the polymer. For example, when increasing the proportion of PLA in a polymer, the ability of the nanoparticles to transport siRNA into the cell increases.
有益效果  Beneficial effect
本发明提供了一种利用两亲性高分子聚合物通过双乳化的方法制备包载 siRNA 的 给药系统和制剂。 制备得到的纳米颗粒具有良好的稳定性， 制备方法简单， siRNA包封 率和载药量高，能保护 siRNA免于降解，并能将核酸药物高效率地转运至细胞内。另外， 由于本发明提供的核酸药物的给药载体具有较高的生物相容性和可降解性， 因此对生物 体的潜在毒性较低， 具有高的生物安全性。 该给药系统和制剂主要适用于小干扰 R A 和类似小核酸药物的给药制剂等领域。  The present invention provides a delivery system and preparation for encapsulating siRNA by a double emulsion method using an amphiphilic polymer. The prepared nanoparticles have good stability, simple preparation method, high siRNA encapsulation efficiency and drug loading amount, can protect siRNA from degradation, and can efficiently transport nucleic acid drugs into cells. In addition, since the drug delivery carrier of the nucleic acid drug provided by the present invention has high biocompatibility and degradability, it is less toxic to the organism and has high biosafety. The drug delivery system and preparation are mainly applicable to the fields of small interference R A and administration preparations similar to small nucleic acid drugs.
本发明利用上述给药系统和制剂， 输送特异性 siRNA, 在细胞和动物水平都证明了 其具有沉默靶基因表达的功效， 以及具有沉默癌基因 Plkl 表达并抑制乳腺癌生长的效 果。 附图说明  The present invention utilizes the above-described administration systems and preparations to deliver specific siRNA, and has demonstrated its efficacy in silencing target gene expression at both the cellular and animal levels, as well as having the effect of silencing the oncogene Plkl expression and inhibiting the growth of breast cancer. DRAWINGS
图 1 为聚合物和阳离子脂质通过双乳化方法制备得到的给药系统的生物相容性检 图 2为包载 FAM-siRNA的纳米颗粒与 HepG2细胞培养 2小时后在细胞内分布的激 光共聚焦显微镜照片。其中， 细胞内红色荧光来源于 Alexa 568-phalloidin标记的细胞骨 架； 绿色荧光来源于 FAM-siRNA; 蓝色荧光来源于 DAPI标记的细胞核。  Fig. 1 is a biocompatibility test of a drug delivery system prepared by a double emulsion method for a polymer and a cationic lipid. Fig. 2 is a laser distribution of a cell containing FAM-siRNA-loaded nanoparticles and HepG2 cells after 2 hours of culture. Focus on the microscope photo. Among them, the intracellular red fluorescence was derived from the Alexa 568-phalloidin-labeled cell skeleton; the green fluorescence was derived from FAM-siRNA; the blue fluorescence was derived from the DAPI-labeled nuclei.
图 3为包载 siP/W的纳米颗粒进入 HepG2细胞后下调 Plkl的 mR A水平的效果图。 图 4为给药系统在动物水平的生物效应评价效果图。 其中， 图 4A为尾静脉注射包 载 siLuci的给药系统抑制肝癌原位种植模型小鼠表达 luciferase的效果图； 图 4B为尾静 脉注射包载 siP d的给药系统抑制小鼠原位植入乳腺癌生长的效果图。  Figure 3 is a graph showing the effect of down-regulating the mR A level of Plkl after the inclusion of siP/W nanoparticles into HepG2 cells. Figure 4 is a graph showing the effect of the biological effect evaluation of the drug delivery system at the animal level. 4A is a diagram showing the effect of the tail vein injection-loaded siLuci administration system on inhibiting the expression of luciferase in a mouse model of liver cancer implantation; FIG. 4B is a tail vein injection of a siP d-administered system to inhibit the in situ implantation of mice. The effect map of breast cancer growth.
图 5为半乳糖修饰的靶向给药系统的构建以及生物效应评价的效果图。其中，图 5A 为 HOOC-PEG5QQQ-PLA21Q3Q和 Gal-PEG5QQQ-PLA21Q3Q的 1H NMR分析图谱； 图 5B为流式 细胞计数检验靶向给药系统在 Hepa 1-6细胞的内吞；图 5C为包载 siapoB的靶向给药系 统进入 Hepa 1-6细胞后下调 apoB的 mRNA水平的效果图；图 5D为包载 siapoB的靶向 给药系统输运 siRNA沉默小鼠肝细胞 apoB蛋白表达的效果图。 Figure 5 is a diagram showing the construction of a galactose-modified targeted drug delivery system and the effect of biological effect evaluation. Among them, Figure 5A 1H NMR analysis of HOOC-PEG 5 QQQ-PLA 21 Q3Q and Gal-PEG 5 QQQ-PLA 21 Q 3 Q; Figure 5B is a flow cytometric assay for targeted drug delivery system in Hepa 1-6 cells Figure 5C is a graph showing the effect of down-regulating the mRNA level of apoB after entering the siapoB-targeted drug delivery system into Hepa 1-6 cells; Figure 5D is a targeted delivery system containing siapoB-loaded siRNA silencing mouse hepatocyte apoB Effect map of protein expression.
图 6 为单链片段抗体修饰的靶向给药系统的构建以及生物效应评价的效果图。 其 中， 图 6A为 Mal-PEG5(xxrPLA22()7()的 1H NMR分析图谱； 图 6B为流式细胞计数检验靶 向给药系统在 BT474 细胞的内吞； 图 6C为包载 siPl!d的靶向给药系统进入 BT474细 胞后下调 Plkl的 mRNA水平的效果图； 图 6D为尾静脉注射包载 siPlkl的的靶向给药 系统抑制小鼠原位植入乳腺癌生长的效果图。 具体实施方式 Figure 6 is a diagram showing the construction of a single-stranded fragment antibody-modified targeted drug delivery system and the effect of biological effect evaluation. 6A is a 1H NMR analysis map of Mal-PEG 5 (xxrPLA 22()7() ; FIG. 6B is a flow cytometric assay targeted drug delivery system for endocytosis of BT474 cells; FIG. 6C is an entrapped siPl! The effect of d-targeted drug delivery system on the down-regulation of Plkl mRNA levels after entry into BT474 cells; Figure 6D is a graph showing the effect of tail-injection of siPlkl-targeted drug delivery system to inhibit the growth of mice implanted in situ in breast cancer. Detailed ways
下面将结合实施例进一步详细描述本发明。 应当理解， 列举这些实施例只是为了起 说明作用， 并不是用来限制本发明的范围。 除非特别说明， 本发明所用到的试剂、 培养 基等均为市售商品。  The invention will be described in further detail below with reference to examples. It is to be understood that the examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the reagents, the medium, and the like used in the present invention are all commercially available.
实施例中所用原料来源及处理方法：  Sources and treatment methods used in the examples:
外消旋丙交脂 （d，l-LA) ， 纯度≥99 %， 使用前减压下升华纯化。  Racemic lactide (d, l-LA), purity ≥ 99%, purified by sublimation under reduced pressure before use.
PEG550-PLA28600 PEG2000-PLA12500 PEGioooo-PLAi5ooo PLA63oo-PEGi2oo-PLA63oo PLA48oo-PEG5ooo-PLA48oo 禾卩 PLA4800-PEG10000-PLA4800 购于 Polymer Source 公司。 PEG5。。。-PLA25。。。和 PEG5。。。-PLA51。。。购于 Alkermes公司； PEG5。。。-PLA5。。。购于 Aldrich 公司； PEGKKKXTPLGAKKKKXSO/SQ)和 PEG1Q(KKrPLGA5(KKK)(75/25)购于济南岱罡公司。 一端基为 羧基另一端基为羟基的异官能团的 PEG ( Mn为 5000 g/mol， HOOC-PEG50oo-OH) ， 一 端基为马来酰亚胺另一端基为羟基的异官能团的 PEG ( Mn 为 5000 g/mol， Mal-PEG5ooo-OH) 购于 Creative PEGWorks公司。 PEG550-PLA28600 PEG2000-PLA12500 PEGioooo-PLAi5ooo PLA63oo-PEGi2oo-PLA 6 3oo PLA 4 8oo-PEG 5 ooo-PLA 4 8oo 禾卩PLA 4800 -PEG 10000 -PLA 4800 was purchased from Polymer Source. PEG 5 . . . -PLA 25 . . . And PEG 5 . . . -PLA 51 . . . Purchased from Alkermes; PEG 5 . . . -PLA 5 . . . Purchased from Aldrich; PEGKKKXTPLGAKKKKXSO/SQ) and PEG 1Q ( KKrPLGA 5( KKK) (75/25) were purchased from Jinan Co., Ltd. A heterofunctional PEG with one end group being a carboxyl group and a hydroxyl group at another end (Mn of 5000 g/) Mol, HOOC-PEG 50 oo-OH) , hetero-functional PEG with end group of maleimide and hydroxyl group (Mn is 5000 g/mol, Mal-PEG 5 ooo-OH) purchased from Creative PEGWorks .
Ν,Ν'-二环己基碳二亚胺（DCC )、 Ν-羟基琥珀酰亚胺（NHS )和 4-二甲氨基吡啶（DMAP ) 购于国药集团化学试剂有限公司。  Ν,Ν'-Dicyclohexylcarbodiimide (DCC), Ν-hydroxysuccinimide (NHS) and 4-dimethylaminopyridine (DMAP) were purchased from Sinopharm Chemical Reagent Co., Ltd.
3-(4，5-二甲基噻唑 -2)-2，5-二苯基四氮唑溴盐 （ΜΤΤ ) 、 氨基半乳糖 （Gal-NH2 ) 和 4'，6-二脒基 -2-苯基吲哚 （DAPI ) 购于 Aldrich公司。 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (ΜΤΤ), galactose (Gal-NH 2 ) and 4',6-didecyl- 2-Phenylhydrazine (DAPI) was purchased from Aldrich.
聚乙烯醇 （PVA， 88%水解， Mw = 22000 ) 购于 Acros Organics公司。  Polyvinyl alcohol (PVA, 88% hydrolyzed, Mw = 22000) was purchased from Acros Organics.
DOTAP和 DOTMA购于 Avanti Polar Lipids公司。 DOTAP and DOTMA were purchased from Avanti Polar Lipids.
BHEM-Chol为本发明合成， 具体合成步骤如下： 在 500毫升单口瓶中加入 2-溴乙 胺氢溴酸盐（17.4 g， 85.0 mmol)和氯甲酸胆固醇酯（34.7 g， 77.3 mmol) , 溶解于 -30°C 的氯仿溶液中， 然后将三乙胺 （24 mL， 172 mmol) 滴加到上述溶液中。 在室温下反应 过夜后， 用含有 1 M盐酸的饱和氯化钠溶液（150 mL)洗涤三次并用饱和氯化钠溶液洗 涤一次 （150 mL)。 有机相用无水硫酸镁干燥并在减压下除去有机溶剂。 粗产物用乙醇 和丙酮各重结晶一次后得到产物 N-(2-溴乙基） 氨基甲酸胆固醇酯， 产率为 73%。 将得 到的 N-(2-溴乙基） 氨基甲酸胆固醇酯 （4.8 g， 7.8 mmol) 和 N-甲基二乙醇胺 （1.2 g， 9.7 mmol) 加入到 50毫升干燥的甲苯中， 回流过夜。 将反应溶液加入到大量的乙醚中 沉淀，过滤后收集沉淀并真空干燥。得到的粗产物在乙醇中重结晶两次后得到白色固体， 产率为 62%。 BHEM-Chol is synthesized in the present invention, and the specific synthetic steps are as follows: 2-bromoethylamine hydrobromide (17.4 g, 85.0 mmol) and cholesteryl chloroformate (34.7 g, 77.3 mmol) were added to a 500 ml single-mouth bottle, dissolved. Triethylamine (24 mL, 172 mmol) was added dropwise to the above solution at -30 ° C in chloroform. After reacting at room temperature overnight, it was washed three times with a saturated sodium chloride solution (150 mL) containing 1 M hydrochloric acid and once with a saturated sodium chloride solution (150 mL). The organic phase was dried over anhydrous magnesium sulfate and the organic solvent was evaporated under reduced pressure. The crude product was recrystallized from ethanol and acetone to give the product N-(2-bromoethyl)carbamic acid cholesteryl ester in a yield of 73%. The obtained N-(2-bromoethyl)carbamic acid cholesteryl ester (4.8 g, 7.8 mmol) and N-methyldiethanolamine (1.2 g, 9.7 mmol) was added to 50 mL of dry toluene and refluxed overnight. The reaction solution was added to a large amount of diethyl ether to precipitate, and after filtration, the precipitate was collected and dried in vacuo. The obtained crude product was recrystallized twice from ethanol to give a white solid (yield: 62%).
异辛酸亚锡 （国药集团化学试剂有限公司）预处理： 先用对二甲苯共沸两次， 然后 再减压蒸馏， 收集 152°C ( 20-40 Pa) 的馏分用于聚合反应。  Pretreatment of stannous isooctanoate (National Pharmaceutical Group Chemical Reagent Co., Ltd.): Azeotrope twice with p-xylene and then distilled under reduced pressure to collect a fraction of 152 ° C (20-40 Pa) for polymerization.
Alexa 568-phalloidin Lipofectamine 2000贝勾于 Invitrogen公司。 R easy mini-kits贝勾 于 Qiagen公司。 RimeScript™ 1st Strand cDNA Synthesis Kit、 SYBR® Premix Ex Taq购 于 Takara公司。 脂蛋白 B ( apoB ) 定量检测试剂盒购于 R&D systems公司。 D-luciferin 购于 Xenogen公司。  Alexa 568-phalloidin Lipofectamine 2000 is hooked to Invitrogen. R easy mini-kits is hooked to Qiagen. RimeScriptTM 1st Strand cDNA Synthesis Kit, SYBR® Premix Ex Taq was purchased from Takara. The Lipoprotein B (apoB) Quantitative Assay Kit was purchased from R&D Systems. D-luciferin was purchased from Xenogen.
稳定表达 luciferase的 HepG2细胞、 MDA-MB-435s细胞、 BT474细胞（HER2受体高 表达细胞系） 购于中国科学院典型培养物保藏委员会细胞库。  HepG2 cells, MDA-MB-435s cells, and BT474 cells (HER2 receptor high expressing cell line) stably expressing luciferase were purchased from the cell bank of the Culture Collection Committee of the Chinese Academy of Sciences.
以下实验中：  In the following experiment:
ήΡΜ , 对应反义链序列为： 5'-UAAGGAGGGUGAUCUUCUUCAdTdT-3 ' ( SEQ ID No :  ήΡΜ , the corresponding antisense strand sequence is: 5'-UAAGGAGGGUGAUCUUCUUCAdTdT-3 ' ( SEQ ID No :
siLucil，对应反义链序列为： 5 '-CUUACGCUGAGUACUUCGAdTdT-3 ' ( SEQ ID No: siLucil, the corresponding antisense strand sequence is: 5 '-CUUACGCUGAGUACUUCGAdTdT-3 ' ( SEQ ID No:
2)。 2).
siapoB,对应反义链序列为： 5 ' - AUUGGUAUUC AGUGUGAUGAC ACdTdT-3 ' ( SEQ ID No: 3  siapoB, corresponding to the antisense strand sequence: 5 ' - AUUGGUAUUC AGUGUGAUGAC ACdTdT-3 ' ( SEQ ID No: 3
siN. C, 对应反义链序列为： 5 '-AACCACUCAACUUUUUCCCAAdTdT-3， ( SEQ ID No : 4)， 作为阴性对照 siRNA。  The sequence corresponding to the antisense strand of siN.C is: 5 '-AACCACUCAACUUUUUCCCAAdTdT-3, (SEQ ID No: 4), as a negative control siRNA.
FAM-siR A为荧光染料 FAM标记的 siN.C.。  FAM-siR A is a fluorescent dye FAM-labeled siN.C.
以上 siRNA均由苏州瑞博生物技术有限公司合成。  The above siRNAs were synthesized by Suzhou Ruibo Biotechnology Co., Ltd.
未经说明的其它试剂均可直接使用。  Other reagents not described may be used directly.
下述实施例中的实验方法， 如无特殊说明， 均为常规方法。 商品化试剂按照试剂附 带的说明书进行。 实施例 1、 siRNA给药系统和制剂的制备  The experimental methods in the following examples are conventional methods unless otherwise specified. The commercial reagents are carried out in accordance with the instructions attached to the reagents. Example 1. Preparation of siRNA delivery system and preparation
利用两亲性嵌段聚合物和阳离子脂质通过双乳化方法制备包载 siRNA的纳米颗粒。 使用的两亲性嵌段共聚物为聚乙二醇 -聚乳酸或者聚乙二醇-聚 （乳酸-乙醇酸） 两嵌段 / 三嵌段共聚物， 具体为 PEG55Q-PLA286QQ、 PEG2QQQ-PLA125QQ、 PEG5QQQ-PLA5000、 PEG5000-PLA25000、 PEG5000-PL A51000、 PEG10000-PLA15000、 PEGioooo-PLGAioooo(5o/50)、The siRNA-encapsulated nanoparticles were prepared by a double emulsion method using an amphiphilic block polymer and a cationic lipid. The amphiphilic block copolymer used is polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic-glycolic acid) diblock/triblock copolymer, specifically PEG 55 Q-PLA 286 QQ, PEG 2 QQQ-PLA 125 QQ, PEG 5 QQQ-PLA 5000 , PEG5000-PLA25000, PEG5000-PL A51000, PEG10000-PLA15000, PEGioooo-PLGAioooo (5o/50),
PEGl0000-PLGA50000(75/25) 、 PLA6300-PEGl200-PLA6300 、 PLA48OO-PEG50OO-PLA48OO 禾口PEGl0000-PLGA50000 (75/25), PLA6300-PEGl200-PLA 6 300, PLA48OO-PEG50OO-PLA48OO
PLA4800-PEG10000-PLA4800。使用的阳离子脂质分别为 BHEM-Chol、 DOTAP禾卩 DOTMA。 通过改变加入的阳离子脂质的质量、 siRNA的质量、聚合物的种类和阳离子脂质的种类 来制备得到具有不同性质的 siRNA给药系统。 1、 阳离子脂质 BHEM-Chol的质量对包载 siRNA纳米颗粒的影响 PLA 4800 - PEG 10000 -PLA 4800 . The cationic lipids used were BHEM-Chol, DOTAP and DOTMA, respectively. An siRNA delivery system having different properties is prepared by changing the quality of the added cationic lipid, the quality of the siRNA, the kind of the polymer, and the type of the cationic lipid. 1. Effect of the quality of cationic lipid BHEM-Chol on siRNA nanoparticles
通过双乳化的方法使用不同质量的阳离子脂质 BHEM-Chol制备包载 siRNA的纳米 颗粒。其中， 使用聚合物 PEG5QQQ-PLA25QQ。 (25 mg)和 FAM-siR A (0.2 mg)作为实例。 制备时，加入的阳离子脂质的质量分别为 0.0mg 0.1mg 0.5mg l.Omg, 2.5mg和 5.0mg 以研究加入不同质量的阳离子脂质对制备得到的包载 siRNA纳米颗粒的性能的影响。 The siRNA-loaded nanoparticles were prepared by a double emulsion method using different masses of cationic lipid BHEM-Chol. Among them, the polymer PEG 5 QQQ-PLA 25 QQ was used. (25 mg) and FAM-siR A (0.2 mg) are given as examples. At the time of preparation, the mass of the cationic lipid added was 0.0 mg 0.1 mg 0.5 mg 1.0 mg, 2.5 mg and 5.0 mg, respectively, to investigate the effect of adding different quality cationic lipids on the properties of the prepared encapsulated siRNA nanoparticles.
通过双乳化的方法制备包载 siRNA 的纳米颗粒， 具体方法为： 将聚合物 PEG5ooo-PLA250oo ( 25 mg ) 和不同质量的 BHEM-Chol 溶于 0.5 mL 氯仿中， 加入 FAM-siRNA (0.025 mL, 0.2 mg) 溶液后， 在超声下 （80瓦， 30秒） 形成初始乳液， 然后将初始乳液加入到 1.5 mL的 1% PVA水溶液中并再次超声乳化 （80瓦， 2分钟） 得到乳液， 将乳液加入到 25 mL的 0.3% PVA水溶液中， 减压下 （1000 帕） 挥发有机 溶剂， 离心 （4 V， 30000 g 1 h) 收集纳米颗粒； 并用水重悬 2次并离心收集以除去 PVA, 样品冻干。 The siRNA-loaded nanoparticles were prepared by double emulsification by: dissolving the polymer PEG 5 ooo-PLA 250 oo ( 25 mg ) and different masses of BHEM-Chol in 0.5 mL chloroform, and adding FAM-siRNA ( After the solution was 0.025 mL, 0.2 mg), the initial emulsion was formed under ultrasound (80 watts, 30 seconds), then the initial emulsion was added to 1.5 mL of 1% PVA aqueous solution and emulsified again (80 watts, 2 minutes) to obtain an emulsion. Add the emulsion to 25 mL of 0.3% PVA aqueous solution, volatilize the organic solvent under reduced pressure (1000 Pa), centrifuge (4 V, 30000 g 1 h) to collect the nanoparticles; resuspend twice with water and collect by centrifugation to remove PVA, samples were lyophilized.
a) siRNA包封率的测定  a) Determination of siRNA encapsulation efficiency
将制备的包载 FAM-siRNA的纳米颗粒的离心后， 收集并精确定量上清液体积， 记 为 V 通过高效液相色谱（HPLC)来检测上清液中 FAM-siRNA的浓度， 记为 C HPLC由 Waters 1525双向泵、 Waters 2475荧光检测器、 1500柱温箱及 Symmetry® C18 分离柱组成，流动相为乙腈 /三乙胺乙酸缓冲液 (0.1 M pH 7.4)，流动相比例为 28:72(V/v)， 流速为 0.5 mL min- 检测器温度为 30°C， 荧光检测器激发波长为 485 nm， 发射波长为 535 nm。通过 Breeze软件对结果进行分析，各组成纳米颗粒的载药率与载药效率见表 1 包封率 （100%) = ( 1-C XV /M slRNA) l 00% After centrifugation of the prepared FAM-siRNA-loaded nanoparticles, the supernatant volume was collected and accurately quantified, and V was measured by high performance liquid chromatography (HPLC) to determine the concentration of FAM-siRNA in the supernatant, which was recorded as C. The HPLC consisted of a Waters 1525 bi-directional pump, a Waters 2475 fluorescence detector, a 1500 column oven and a Symmetry® C18 separation column. The mobile phase was acetonitrile/triethylamine acetate buffer (0.1 M pH 7.4) and the flow ratio was 28:72. ( V / v), flow rate 0.5 mL min - detector temperature is 30 ° C, fluorescence detector excitation wavelength is 485 nm, emission wavelength is 535 nm. The results were analyzed by Breeze software. The drug loading rate and drug loading efficiency of each component nanoparticle are shown in Table 1. Encapsulation efficiency (100%) = ( 1-C XV /M slRNA ) l 00%
载药量 （100%) = (M ,S siRNA-C XV /M xl 00% Drug loading (100%) = (M , S siRNA -C XV /M xl 00%
M ,S siRNA表示制备中加入的总的 siRNA的质量， M iSe ^表示纳米颗粒的总质量。 从表 1可见， 不加入阳离子脂质时， siRNA的包封率仅为 26.2%， 而加入阳离子脂 质后， siRNA的包封率明显增加。 譬如， 加入 l.O mg的 BHEM-Chol时， siRNA的包封 率增加到 95.7%。 继续增加阳离子脂质时， siRNA的包封率保持在 95%以上。 M, S siRNA indicates the mass of the total siRNA added in the preparation, and M iS e ^ indicates the total mass of the nanoparticles. It can be seen from Table 1 that the encapsulation efficiency of siRNA was only 26.2% when no cationic lipid was added, and the encapsulation efficiency of siRNA was significantly increased after the addition of cationic lipid. For example, when 10 mg of BHEM-Chol was added, the encapsulation efficiency of siRNA increased to 95.7%. When the cationic lipid is continuously increased, the encapsulation efficiency of the siRNA is maintained at 95% or more.
b)纳米颗粒的粒径和 zeta电势  b) particle size and zeta potential of the nanoparticles
利用型号为 Malvern Zetasizer Nanao ZS90的动态光散射仪检测包载 siRNA的纳米 颗粒的粒径与粒径分布， 其中， 所述纳米颗粒的浓度为 0.1 mg/mL  The particle size and particle size distribution of the nanoparticles carrying the siRNA were detected by a dynamic light scattering instrument Model Malvern Zetasizer Nanao ZS90, wherein the concentration of the nanoparticles was 0.1 mg/mL.
从表 1可见，改变加入的 siRNA和阳离子脂质的量时，制备得到的纳米颗粒的粒径 基本不变， 在 160 nm-180 nm之间。 表 1.不同 BHEM-Chol制备的包载 siRNA纳米颗粒各个组份具体组成及性质  It can be seen from Table 1 that when the amount of siRNA and cationic lipid added is changed, the particle size of the prepared nanoparticles is substantially constant, ranging from 160 nm to 180 nm. Table 1. Specific composition and properties of various components of siRNA nanoparticles prepared by BHEM-Chol
Figure imgf000010_0001
0.20 1.0 25.0 95.7 0.736 167 13.8
Figure imgf000010_0001
0.20 1.0 25.0 95.7 0.736 167 13.8
0.20 2.5 25.0 96.7 0.698 175 21.80.20 2.5 25.0 96.7 0.698 175 21.8
0.20 5.0 25.0 96.9 0.642 168 35.6 从表 1可见， 加入的 siRNA和聚合物的量固定时， zeta电势随着加入的阳离子脂质 的量增加。 纳米颗粒的电势会影响纳米颗粒在体内的循环， 纳米颗粒的电势接近中性能 够更有效的避免其被体内的免疫系统清除， 从而能够更好的富集到肿瘤部位。 因此， 从 表 1可知， 通过调节加入的阳离子脂质的质量， 本发明的给药系统能够优化出合适的体 系用于 siRNA给药。 0.20 5.0 25.0 96.9 0.642 168 35.6 It can be seen from Table 1 that when the amount of siRNA and polymer added is fixed, the zeta potential increases with the amount of cationic lipid added. The potential of the nanoparticles affects the circulation of the nanoparticles in the body. The potential of the nanoparticles is close to the medium performance. It is more effective to avoid being removed by the immune system in the body, so that it can be better enriched in the tumor site. Thus, it can be seen from Table 1 that by adjusting the quality of the added cationic lipid, the drug delivery system of the present invention is capable of optimizing a suitable system for siRNA administration.
从表 1可知， 当阳离子脂质的加入量为 1.0 mg时， siRNA基本被包载在纳米颗粒 中。 但是增加阳离子脂质的量， 载药量降低， 因此 siRNA/BHEM-Chol/PEGsfxxrPLA fKK) 的重量比为 0.2/1.0/25.0是 BHEM-Chol和 PEG5000-PLA25000固定为 1.0 mg和 25.0 mg时 比较理想的 siRNA载药体系。 As can be seen from Table 1, when the cationic lipid was added in an amount of 1.0 mg, the siRNA was substantially contained in the nanoparticles. However, when the amount of cationic lipid is increased, the drug loading is decreased, so the weight ratio of siRNA/BHEM-Chol/PEGsfxxrPLA fKK) is 0.2/1.0/25.0 when BHEM-Chol and PEG 5000 -PLA 25000 are fixed at 1.0 mg and 25.0 mg. The ideal siRNA drug delivery system.
2、 不同的阳离子脂质制备包载 siRNA的纳米颗粒 2. Preparation of siRNA-loaded nanoparticles by different cationic lipids
通过双乳化的方法使用不同种类的阳离子脂质制备包载 siRNA的纳米颗粒。 其中， 使用聚合物
Figure imgf000011_0001
(25 mg)和 FAM-siRNA (0.2 mg)作为实例。 制备时， 加 入的阳离子脂质的质量为 1.0 mg, 使用的阳离子脂质分别为 BHEM-Chol、 DOTAP和 DOTMAo The siRNA-loaded nanoparticles were prepared by a double emulsification method using different kinds of cationic lipids. Among them, the use of polymers
Figure imgf000011_0001
(25 mg) and FAM-siRNA (0.2 mg) were used as examples. At the time of preparation, the mass of the cationic lipid added was 1.0 mg, and the cationic lipids used were BHEM-Chol, DOTAP and DOTMAo, respectively.
具体方法为： 将聚合物 PEGSOOO-PLA , (25 mg)和阳离子脂质（l.O mg)溶于 0.5 mL氯仿中， 加入 siRNA (0.025 mL, 0.2 mg) 溶液后， 在超声下 （80瓦， 30秒） 形成 初始乳液， 然后， 将初始乳化加入到 1.5 mL的 1% PVA水溶液中并再次超声乳化 （80 瓦， 2分钟），得到乳液，将乳液加入到 25 mL的 0.3% PVA水溶液中，减压下（ 1000 巾白） 挥发有机溶剂， 离心（4 °C， 30000 g, l h)收集纳米颗粒； 并用水重悬 2次并离心收集 以除去 PVA， 样品冻干后备用。  The specific method is as follows: The polymer PEGSOOO-PLA, (25 mg) and cationic lipid (10 mg) are dissolved in 0.5 mL of chloroform, and after adding siRNA (0.025 mL, 0.2 mg) solution, under ultrasound (80 watts, 30 Second) The initial emulsion is formed. Then, the initial emulsification is added to 1.5 mL of 1% PVA aqueous solution and emulsified again (80 watts, 2 minutes) to obtain an emulsion. The emulsion is added to 25 mL of 0.3% PVA aqueous solution, minus The organic solvent was depressed (1000 white), and the nanoparticles were collected by centrifugation (4 ° C, 30000 g, lh); and resuspended twice with water and collected by centrifugation to remove PVA, and the sample was lyophilized and used.
siRNA包封率的测定、纳米颗粒的粒径和 zeta电势的测定方法如上所述， 结果列入 表 2中。  The measurement of the siRNA encapsulation ratio, the particle size of the nanoparticles, and the zeta potential were measured as described above, and the results are shown in Table 2.
从表 2可见， 加入不同质量的阳离子脂质时， siRNA包封率基本在 95%左右。 而且 制备得到的纳米颗粒的粒径基本不变，在 160 nm-170 nm之间， 并且包载 siRNA的纳米 颗粒的电势接近。  It can be seen from Table 2 that when different concentrations of cationic lipids are added, the siRNA encapsulation efficiency is about 95%. Moreover, the prepared nanoparticles have a substantially constant particle size between 160 nm and 170 nm, and the potential of the nanoparticles entrained with siRNA is close.
从表 2可知， 阳离子脂质的种类对包载 siRNA的纳米颗粒性质的影响不大。阳离子 脂质在给药系统中的主要作用是通过静电相互作用提高核酸药物在载体中的载药量和 包封效率， 因此， 具有上述性质的阳离子均可用于本发明。 使用的这三类阳离子脂质的 分子量接近和电荷一致，所以得到的包载 siRNA的纳米颗粒性质类似。同样，这也说明， 可以用于本发明的给药系统的阳离子脂质并不限于此处所用的这几种，其他阳离子脂质 同样可以应用于此体系。 表 2.不同的阳离子脂质制备的包载 siRNA纳米颗粒各个组份具体组成及性质
Figure imgf000012_0001
As can be seen from Table 2, the type of cationic lipid has little effect on the properties of the nanoparticle-encapsulated siRNA. The main function of the cationic lipid in the drug delivery system is to increase the drug loading amount and encapsulation efficiency of the nucleic acid drug in the carrier by electrostatic interaction, and therefore, cations having the above properties can be used in the present invention. The molecular weights of the three types of cationic lipids used are close to the charge, so the resulting siRNA-encapsulated nanoparticles are similar in nature. Also, this also indicates that the cationic lipid which can be used in the administration system of the present invention is not limited to the ones used herein, and other cationic lipids can be also applied to this system. Table 2. Specific composition and properties of individual components of encapsulated siRNA nanoparticles prepared by different cationic lipids
Figure imgf000012_0001
3、 siRNA的质量对包载 siRNA的纳米颗粒的影响 3. Effect of siRNA quality on nanoparticles loaded with siRNA
通过双乳化的方法使用不同质量的 siRNA制备包载 siRNA的纳米颗粒。 其中， 使 用的聚合物和阳离子脂质分别以 PEG5Q(KrPLA25QQQ (25 mg)和 BHEM-Chol ( 1.0 mg)作 为实例。 制备时， 加入的 FAM-siRNA质量分别为 0.0 mg、 0.2 mg、 0.6 mg禾 B 1.8 mg， 以研究加入不同质量的 siRNA对制备得到的包载 siRNA纳米颗粒的性能影响。 The siRNA-loaded nanoparticles were prepared by a double emulsion method using different quality siRNAs. Among them, the polymer and cationic lipid used were PEG 5 Q (KrPLA 25 QQQ (25 mg) and BHEM-Chol (1.0 mg), respectively. The quality of FAM-siRNA added was 0.0 mg, 0.2 mg, respectively. 0.6 mg and 1.8 mg were studied to investigate the effect of the addition of different masses of siRNA on the performance of the prepared encapsulated siRNA nanoparticles.
具体方法为： 将聚合物 PEGsooo-PLA^oo (25 mg) 和 BHEM-Chol ( 1.0 mg) 溶于 0.5 mL氯仿中， 加入 FAM-siRNA (0.025 mL) 溶液后， 在超声下 （80瓦， 30秒） 形成 初始乳液， 然后将初始乳液加入到 1.5 mL的 1% PVA水溶液中并再次超声乳化（80瓦， 2分钟）， 得到乳液， 将乳液加入到 25 mL的 0.3% PVA水溶液中， 减压下 （1000 帕） 挥发有机溶剂， 离心（4 °C， 30000 g, l h)收集纳米颗粒； 并用水重悬 2次并离心收集 以除去 PVA， 样品冻干后备用。  The specific method is as follows: Dissolve the polymer PEGsooo-PLA^oo (25 mg) and BHEM-Chol (1.0 mg) in 0.5 mL chloroform, add FAM-siRNA (0.025 mL) solution, and under ultrasound (80 watts, 30 Second) The initial emulsion was formed, then the initial emulsion was added to 1.5 mL of 1% PVA aqueous solution and emulsified again (80 watts, 2 minutes) to obtain an emulsion. The emulsion was added to 25 mL of 0.3% PVA aqueous solution, and decompressed. The organic solvent was evaporated (1000 Pa), and the nanoparticles were collected by centrifugation (4 ° C, 30000 g, lh); and resuspended twice with water and collected by centrifugation to remove PVA, and the sample was lyophilized and used.
siRNA包封率的测定、纳米颗粒的粒径和 zeta电势的测定方法如上所述， 结果列入 表 3中。  The measurement of the siRNA encapsulation ratio, the particle size of the nanoparticles, and the zeta potential were measured as described above, and the results are shown in Table 3.
从表 3可见， 当固定 BHEM-Chol和聚合物 PEG5。。。-PLA25。。Q的加入量时，包封率随 着加入的 siRNA的质量增加而略有降低。当加入的 siRNA的质量低于 0.2 mg时， siRNA 包载效率高于 95%。 而继续增加 siRNA质量， siRNA包封率降低， 但是载药量增加。 As seen from Table 3, when BHEM-Chol and polymer PEG 5 were immobilized. . . -PLA 25 . . When the amount of Q added, the encapsulation efficiency slightly decreased as the mass of the added siRNA increased. When the quality of the added siRNA was less than 0.2 mg, the siRNA entrapment efficiency was higher than 95%. While continuing to increase siRNA quality, the siRNA encapsulation efficiency decreased, but the drug loading increased.
从表 3可见， 改变加入的 siRNA量时， 制备得到的纳米颗粒的粒径基本不变。 从表 3可见， 当固定 BHEM-Chol和聚合物 PEG5。。。-PLA25。。Q的加入量时，得到的包 载 siRNA纳米颗粒的 zeta电势随着加入的 siRNA的质量增加而降低。 纳米颗粒的电势 会影响纳米颗粒在体内的循环，纳米颗粒的电势接近中性能够更有效的避免其被体内的 免疫系统清除， 从而能够更好的富集到肿瘤部位。 因此， 由表 1和表 3的结果可知， 通 过调节加入的阳离子脂质和 siRNA 的质量， 此给药系统能够优化出合适的体系用于 siRNA给药。 表 3.包载不同质量的 siRNA纳米颗粒各个组份具体组成及性质 As can be seen from Table 3, when the amount of siRNA added was changed, the particle diameter of the prepared nanoparticles was substantially unchanged. As seen from Table 3, when BHEM-Chol and polymer PEG 5 were immobilized. . . -PLA 25 . . When the amount of Q is added, the zeta potential of the obtained encapsulated siRNA nanoparticles decreases as the mass of the added siRNA increases. The potential of the nanoparticles affects the circulation of the nanoparticles in the body. The potential of the nanoparticles is close to neutral and can be more effectively prevented from being cleared by the immune system in the body, so that it can be better enriched in the tumor site. Therefore, from the results of Tables 1 and 3, it is known that by adjusting the quality of the added cationic lipid and siRNA, the drug delivery system can optimize a suitable system for siRNA administration. Table 3. Specific composition and properties of each component of siRNA nanoparticles loaded with different masses
Figure imgf000012_0002
4、 使用不同分子量和组成的聚合物制备包载 siRNA的纳米颗粒
Figure imgf000012_0002
4. Preparation of siRNA-loaded nanoparticles using polymers of different molecular weights and compositions
使用不同的聚乙二醇-聚乳酸或聚乙二醇-聚（乳酸-乙醇酸）两嵌段 /三嵌段共聚物制 备包载 siRNA的纳米颗粒。  Nanoparticles entrapped in siRNA were prepared using different polyethylene glycol-polylactic acid or polyethylene glycol-poly(lactic-glycolic acid) diblock/triblock copolymers.
通过双乳化的方法制备包载 siRNA的纳米颗粒， 具体方法为： 将聚合物 （25 mg) 禾卩 BHEM-Chol ( 1.0 mg)溶于 0.5 mL氯仿中， 加入 siRNA ( 0.025 mL, 0.2 mg)溶液后， 在超声下 （80瓦， 30秒） 形成初始乳液， 然后， 将初始乳化加入到 1.5 mL的 1% PVA 水溶液中并再次超声乳化（80瓦， 2分钟），得到乳液，将乳液加入到 25 mL的 0.3% PVA 水溶液中， 减压下 （1000 帕） 挥发有机溶剂， 离心 （4 °C， 30000 g, l h) 收集纳米颗 粒； 并用水重悬 2次并离心收集以除去 PVA， 样品冻干后备用。  The siRNA-coated nanoparticles were prepared by double emulsification by: dissolving the polymer (25 mg) and BHEM-Chol (1.0 mg) in 0.5 mL of chloroform and adding siRNA (0.025 mL, 0.2 mg) solution. Thereafter, an initial emulsion was formed under ultrasound (80 watts, 30 seconds), and then the initial emulsification was added to 1.5 mL of a 1% PVA aqueous solution and emulsified again (80 watts, 2 minutes) to obtain an emulsion, and the emulsion was added thereto. In 25 mL of 0.3% PVA aqueous solution, the organic solvent was evaporated under reduced pressure (1000 Pa), and the nanoparticles were collected by centrifugation (4 °C, 30000 g, lh); resuspended twice with water and collected by centrifugation to remove PVA, and the sample was frozen. After the dry, spare.
其中，制备时，使用的聚合物为 PEG55Q-PLA286QQ、 PEG2QQQ-PLA125QQ、 PEG5QQQ-PLA5000、Among them, when preparing, the polymer used is PEG 55 Q-PLA 286 QQ, PEG 2 QQQ-PLA 125 QQ, PEG 5 QQQ-PLA 5000 ,
PEG5000-PLA25000、 PEG5000-PL A51000、 PEG10000-PLA15000、 PEGioooo-PLGAioooo(5o/50)、 PEGi0000"PLGA50000(75/25) 、 PLA6300"PEGi200-PLA6300 、 PLA4800-PEG5000-PLA4800 禾口 PLA4800-PEG10000-PLA4800。 PEG5000-PLA25000, PEG5000-PL A51000, PEG10000-PLA15000, PEGioooo-PLGAioooo (5o/50), PEGi0000"PLGA50000 (75/25), PLA6300"PEGi200-PLA6300, PLA4800-PEG5000-PLA4800 and PLA4800-PEG10000-PLA4800.
siRNA包封率的测定、纳米颗粒的粒径和 zeta电势的测定方法如上所述， 结果列入 表 4中。  The measurement of the siRNA encapsulation ratio, the particle size of the nanoparticles, and the zeta potential were measured as described above, and the results are shown in Table 4.
从表 4可见， 当固定 BHEM-Chol和聚合物的加入量时， siRNA基本包载在纳米颗 粒中， 包封率基本在 90%以上。 但是， 当 PEG比例较高时， siRNA的包封率会有一定 程度下降。 例如当使用的聚合物为 PEG^KKXTPLGAKKKK^/SO)或 PLA 4800-PEG10000-PLA4800 时， siRNA的包封率下降到 85%左右。  As can be seen from Table 4, when the amount of BHEM-Chol and the polymer added was fixed, the siRNA was substantially contained in the nanoparticles, and the encapsulation efficiency was substantially above 90%. However, when the PEG ratio is high, the encapsulation efficiency of siRNA will decrease to some extent. For example, when the polymer used is PEG^KKXTPLGAKKKK^/SO) or PLA 4800-PEG10000-PLA4800, the encapsulation efficiency of siRNA is reduced to about 85%.
从表 4可见， 得到的包载 siRNA的纳米颗粒的 zeta电势随聚合物不同而有所变化。 从上述实施例可知， 纳米颗粒的电势会影响纳米颗粒在体内的循环， 纳米颗粒的电势接 近中性能够更有效的避免其被体内的免疫系统清除， 从而能够更好的富集到肿瘤部位。 因此， 从表 1、 表 3以及表 4的结果可知， 通过调节加入的阳离子脂质和 siRNA的质量 以及使用的聚合物， 此给药系统能够优化出合适的体系用于 siRNA给药。  As can be seen from Table 4, the zeta potential of the obtained siRNA-loaded nanoparticles varies depending on the polymer. It can be seen from the above embodiments that the potential of the nanoparticles affects the circulation of the nanoparticles in the body, and the potential of the nanoparticles is more neutral to avoid being removed by the immune system in the body, thereby being better enriched to the tumor site. Therefore, from the results of Table 1, Table 3 and Table 4, it is known that the drug delivery system can optimize a suitable system for siRNA administration by adjusting the quality of the added cationic lipid and siRNA and the polymer used.
从表 4可见， 改变加入的聚合物时， 制备得到的纳米颗粒的粒径变化较大， 在 82 nm-227 nm之间。制备时使用的聚合物疏水链段对制备得到的包载 siRNA纳米颗粒的粒 径影响较大。 例如使用疏水链段增长的聚合物 PEG5。。。-PLA5。。。、 PEG5。。。-PLA25。。(）禾口 PEGsooo-PLAsiooo时， 得到的包载 siRNA纳米颗粒的粒径分别为 82、 167和 225 nm。 It can be seen from Table 4 that when the added polymer is changed, the prepared nanoparticles have a large particle size variation between 82 nm and 227 nm. The hydrophobic segment of the polymer used in the preparation has a great influence on the particle size of the prepared encapsulated siRNA nanoparticles. For example, a polymer PEG 5 grown using a hydrophobic segment is used. . . -PLA 5 . . . , PEG 5 . . . -PLA 25 . . When () and PEGsooo-PLAsiooo, the particle size of the obtained siRNA nanoparticles was 82, 167 and 225 nm, respectively.
表 4.使用不同嵌段聚合物制备的包载 siRNA纳米颗粒的性质  Table 4. Properties of encapsulated siRNA nanoparticles prepared using different block polymers
Figure imgf000013_0001
PLA4800-PEG5000-PLA4800 91.8 0.687 137 11.4
Figure imgf000013_0001
PLA4800-PEG5000-PLA4800 91.8 0.687 137 11.4
PLA4800-PEG10000-PLA4800 85.1 0.637 84 19.3 从上述实施例可知，包载 siR A的纳米颗粒的电势可以通过加入的阳离子脂质的量 或者加入的 siRNA量来调节； 包载 siRNA的纳米颗粒的粒径可以通过使用的聚合物来 调控。 因此， 可以根据疾病的不同， 来调节不同因素来制备理想的给药系统。 实施例 2、 此给药系统在细胞水平的效应评价 PLA4800-PEG10000-PLA4800 85.1 0.637 84 19.3 It can be seen from the above examples that the potential of the siR A-loaded nanoparticles can be adjusted by the amount of cationic lipid added or the amount of siRNA added; the particle size of the nanoparticles carrying the siRNA It can be regulated by the polymer used. Therefore, different factors can be adjusted to prepare an ideal drug delivery system depending on the disease. Example 2. Evaluation of the effect of this drug delivery system at the cellular level
根据实施例 1可知， siRNA/阳离子脂质 /聚合物的质量比为 0.2/1.0/25.0时， 得到的 siRNA给药系统具有较高的 siRNA包封率和载药量。 故以此实例来说明此给药系统的 生物学效应。  According to Example 1, when the mass ratio of siRNA/cationic lipid/polymer was 0.2/1.0/25.0, the obtained siRNA delivery system had a high siRNA encapsulation efficiency and drug loading amount. This example is used to illustrate the biological effects of this drug delivery system.
使用 的聚合物为 PEG550-PLA286oo 、 PEG2ooo-PLA1250o 、 PEG5ooo-PLA50oo 、 PEG5000-PLA25000、 PEG5000-PL A51000、 PEG10000-PLA15000、 PEGioooo-PLGAioooo(5o/50)、The polymers used were PEG 550 -PLA 286 oo , PEG 2 ooo-PLA 1250 o , PEG 5 ooo-PLA 50 oo , PEG5000-PLA25000, PEG5000-PL A51000, PEG10000-PLA15000, PEGioooo-PLGAioooo (5o/50),
PEGi0000-PLGA50000(75/25) 、 PLA63oo-PEGi2oo-PLA63oo 、 PLA48OO-PEG500O-PLA48OO 禾口PEGi0000-PLGA50000(75/25), PLA 6 3oo-PEGi2oo-PLA 6 3oo, PLA48OO-PEG500O-PLA48OO
PLA48oo-PEGioooo-PLA48oo' 使用的阳离子脂质为 BHEM-Chol。 1、 不同聚合物制备的纳米颗粒生物相容性评价 The cationic lipid used in PLA 4 8oo-PEGioooo-PLA48oo' is BHEM-Chol. 1. Biocompatibility evaluation of nanoparticles prepared by different polymers
用实施例 1中所述方法制备未包载 siRNA的纳米颗粒。通过 MTT (3-(4， 5-二甲基 噻唑 -2)-2， 5-二苯基四氮唑溴盐） 比色法检测纳米颗粒对人肝癌细胞 HepG2的毒性。  Nanoparticles not containing siRNA were prepared by the method described in Example 1. The toxicity of nanoparticles on human hepatoma cell line HepG2 was detected by MTT (3-(4, 5-dimethylthiazole-2)-2, 5-diphenyltetrazolium bromide) colorimetric assay.
制备未包载 siRNA的纳米颗粒具体方法为： 将聚合物（25 mg)和 BHEM-Chol ( 1.0 mg) 溶于 0.5 mL氯仿中， 加入蒸馏水 （0.025 mL) 溶液后， 在超声下 （80瓦， 30秒） 形成初始乳液， 然后， 将初始乳液加入到 1.5 mL的 1% PVA水溶液中并再次超声乳化 ( 80瓦， 2分钟），得到乳液，将乳液加入到 25 mL的 0.3% PVA水溶液中，减压下（ 1000 帕）挥发有机溶剂， 离心 （4 °C， 30000 g, l h) 收集纳米颗粒； 并用水重悬 2次并离心 收集以除去 PVA， 样品冻干后备用。  The specific method for preparing the non-encapsulated siRNA nanoparticles is as follows: The polymer (25 mg) and BHEM-Chol (1.0 mg) are dissolved in 0.5 mL of chloroform, and after adding distilled water (0.025 mL) solution, under ultrasound (80 watts, 30 seconds) The initial emulsion was formed. Then, the initial emulsion was added to 1.5 mL of 1% PVA aqueous solution and emulsified again (80 watts, 2 minutes) to obtain an emulsion, and the emulsion was added to 25 mL of 0.3% PVA aqueous solution. The organic solvent was evaporated under reduced pressure (1000 Pa), and the nanoparticles were collected by centrifugation (4 ° C, 30000 g, lh); and resuspended twice with water and collected by centrifugation to remove PVA, and the sample was lyophilized and used.
使用 的聚合物为 PEG550-PLA28600 、 PEG2ooo-PLAi250o 、 PEG5000-PLA5000 、 PEG5000-PLA25000、 PEG5000-PL A51000、 PEG10000-PLA15000、 PEGioooo-PLGAioooo(5o/5o ) 、 PEGi0000"PLGA50000(75/25) 、 PLA6300"PEGi200-PLA6300 、 PLA4800-PEG5000-PLA4800 禾口 PLA4800-PEG10000-PLA4800。 Polymer used was PEG 550 -PLA 28600, PEG 2 ooo -PLAi 250 o, PEG 5000 -PLA 5000, PEG5000-PLA25000, PEG5000-PL A51000, PEG10000-PLA15000, PEGioooo-PLGAioooo (5o / 5o), PEGi0000 "PLGA50000 (75/25), PLA6300"PEGi200-PLA6300, PLA4800-PEG5000-PLA4800 and PLA4800-PEG10000-PLA4800.
得 到 的 不 包载 siRNA 的 纳 米 颗粒 分 别 为 NP(PEG55Q-PLA286(X)) 、 NP(PEG2ooo-PLAi250o) 、 NP(PEG5000-PLA5000) 、 NP(PEG5000-PLA25000) 、 NP(PEG50oo-PLA5iooo) 、 NP(PEG10。。。-PLA15。。。） 、 NP(PEGioooo-PLGAioooo(5o/50)) 、 NP(PEGioooo-PLGA50ooo(75/25))、 NP(PLA6300-PEG1200-PLA6300)、 NP(PLA4800-PEG誦 -PLA棚） 和 NP(PLA棚 -PEG誦 0-PLA4800)。 The obtained siRNA-free nanoparticles were NP (PEG 55Q -PLA 286(X) ), NP (PEG 2 ooo-PLAi 250 o), NP (PEG 5000 -PLA 5000 ), NP (PEG 5000 -PLA 25000). ), NP (PEG 50 oo-PLA 5 iooo), NP (PEG 10 . . . -PLA 15 ), NP (PEGioooo-PLGAioooo ( 5o/50)), NP (PEGioooo-PLGA 50 ooo (75/) 25)), NP (PLA 6300 - PEG 1200 - PLA 6300 ), NP (PLA 4800 - PEG 诵 - PLA shed) and NP (PLA shed - PEG 诵0 - PLA 4800 ).
MTT比色法检测上述制备的纳米颗粒对人肝癌细胞 HepG2的毒性。具体方法如下： 首先在 96孔板上每孔接种 10000个细胞 /ΙΟΟμΙ^ DMEM培养基 （ 10%初生牛血清）， 在 C02培养箱中 （37°C， C02浓度为 5%) 培养 24小时后， 除去培养基后， 加入包含上述 纳米颗粒的 ΙΟΟμΙ^新鲜 DMEM培养基， 其中上述纳米颗粒在培养基中的浓度分别为The toxicity of the above prepared nanoparticles to human liver cancer cell HepG2 was examined by MTT colorimetry. The specific method is as follows: First, inoculate 10000 cells/ΙΟΟμΙ^ DMEM medium (10% newborn bovine serum) per well in a 96-well plate, and culture in a C0 2 incubator (37 ° C, C0 2 concentration of 5%). After the hour, after removing the medium, add the above Nanoparticles of ΙΟΟμΙ^ fresh DMEM medium, wherein the concentration of the above nanoparticles in the medium is
0.25、 0.5、 1禾卩 2 g L- 禾 B HepG2细胞共培养。 24小时后， 于每孔中加入 25 MTT 储液 （5 mg mL- 1于 PBS溶液中） ， 并在空白孔 （blank) 中加入 25 PBS缓冲液， 继 续培养 2 h; 在每孔中加入 100 L萃取缓冲液（20% SDS， 溶解在 50% N，N-二甲基甲酰 胺中， pH 4.7， 37 °C ) ， 然后 37°C培养过夜； 用 Bio-Rad酶标仪在 570 nm下测定孔中 溶液的吸光值。 用以下公式计算细胞活力： 0.25, 0.5, 1 and 2 g L-W HepG2 cells were co-cultured. After 24 hours, add 25 MTT stock solution (5 mg mL- 1 in PBS) to each well, and add 25 PBS buffer to the blank for 2 h; add 100 to each well. L extraction buffer (20% SDS, dissolved in 50% N, N-dimethylformamide, pH 4.7, 37 °C), then cultured overnight at 37 °C; using Bio-Rad microplate reader at 570 nm The absorbance of the solution in the well was measured. Calculate cell viability using the following formula:
Abs(sample) - Abs(blank) ,  Abs(sample) - Abs(blank) ,
X 100%  X 100%
Abs{control) - Abs{blank)  Abs{control) - Abs{blank)
其中 Abs为溶液在 570 nm的吸光值， Abs(sample)为样品孔的吸光度值， Abs(control)  Abs is the absorbance of the solution at 570 nm, Abs(sample) is the absorbance of the sample well, Abs(control)
为对照孔的吸光度值。  The absorbance value of the control well.
结果见图 1。 由图 1可见， 当纳米颗粒浓度为 2 g L-1时， 90%以上的细胞仍存活， 证明这种脂质聚合物共同制备的纳米颗粒具有良好的生物相容性。 The results are shown in Figure 1. It can be seen from Fig. 1 that when the concentration of the nanoparticles is 2 g L- 1 , more than 90% of the cells still survive, which proves that the nanoparticles prepared by the lipid polymer have good biocompatibility.
2、 不同聚合物制备的包载 siRNA纳米颗粒进入细胞的能力 2. The ability of the encapsulated siRNA nanoparticles prepared by different polymers to enter the cell
用实施例 1中所述方法制备包载 FAM-siR A的纳米颗粒来研究此给药系统的细胞 内吞行为。 其中， siRNA/阳离子脂质 /聚合物的质量比为 0.2/1.0/25.0。  Nanoparticles coated with FAM-siR A were prepared by the method described in Example 1 to study the endocytosis behavior of this drug delivery system. Among them, the mass ratio of siRNA/cationic lipid/polymer was 0.2/1.0/25.0.
使用 FAM标记的 siRNA ( FAM-siRNA) , 阳离子脂质为 ΒΗΕΜ-Chol, 使用的聚合 物为 PEG550-PLA28600 、 PEG2000-PLA12500 、 PEG5000-PLA5000 、 PEG5000-PLA25000 、 PEG5ooo-PLA5iooo PEGioooo-PLAisooo PEGioooo-PLGAioooo(5o/so) PEGioooo-PLGA5oooo(75/25) PLA63oo-PEGi2oo-PLA63oo PLA4800-PEG5000-PLA4800禾卩 PLA tsoo—PEGioooo—PLA tsoo c FAM-labeled siRNA (FAM-siRNA), cationic lipid was ΒΗΕΜ-Chol, and the polymers used were PEG550-PLA28600, PEG2000-PLA12500, PEG5000-PLA5000, PEG5000-PLA25000, PEG5ooo-PLA5iooo PEGioooo-PLAisooo PEGioooo-PLGAioooo ( 5o/so) PEGioooo-PLGA5oooo(75/25) PLA63oo-PEGi2oo-PLA 6 3oo PLA4800-PEG5000-PLA4800 and PLA tsoo—PEGioooo—PLA tsoo c
得 到 的 包 载 FAM-siRNA 纳 米 颗 粒 分 另 lj 为 NP(PEG550-PLA28600) 、The obtained encapsulated FAM-siRNA nanoparticles are separated into NP (PEG 550 -PLA 28600 ),
NP(PEG2ooo-PLAi250o) 、 NP(PEG5000-PLA5000) 、 NP(PEG5000-PLA25000) 、 NP(PEG50oo-PLA5iooo) 、 NP(PEG10。。。-PLA15。。。） 、 NP(PEGioooo-PLGAioooo(5o/50)) 、 NP(PEGioooo-PLGA50ooo(75/25))、 NP(PLA6300-PEG1200-PLA6300)、 NP(PLA4800-PEG誦 -PLA棚) 和 NP(PLA棚 -PEG誦 0-PLA4800)。 NP (PEG 2 ooo-PLAi 250 o), NP (PEG 5000 -PLA 5000 ), NP (PEG 5000 -PLA 25000 ), NP (PEG 50 oo-PLA 5 iooo), NP (PEG 10 。 -PLA 15 . . . , NP (PEGioooo-PLGAioooo ( 5o/50)), NP (PEGioooo-PLGA 50 ooo (75/25)), NP (PLA 6300 -PEG 1200 -PLA 6300 ), NP (PLA 4800 -PEG诵) -PLA shed) and NP (PLA shed - PEG 诵0 - PLA 4800 ).
将包载 FAM-siRNA的纳米颗粒 （FAM-siRNA终浓度为 200 nM) 与人肝癌 HepG2 细胞 （24孔板， 5x l04细胞 /孔）在 37°C共同培养 2小时后， 使用 4%多聚甲醛固定细胞 15 min。 以 PBS 洗涤三遍后， 用丙酮对细胞穿膜 （-20°C， 5 min ) ； 最后用 Alexa 568-phalloidin对细胞骨架进行染色， 用 4'，6-二脒基 -2-苯基吲哚 （DAPI ) 对细胞核进行 复染， 封片后使用型号为 Zeiss LSM 710的激光共聚焦显微镜进行观察。 结果见图 2。 The FAM-siRNA-loaded nanoparticles (final concentration of FAM-siRNA was 200 nM) and human hepatoma HepG2 cells (24-well plates, 5×10 4 cells/well) were co-cultured at 37 ° C for 2 hours, using 4% more The cells were fixed in polyoxymethylene for 15 min. After washing three times with PBS, the cells were permeabilized with acetone (-20 ° C, 5 min); finally, the cytoskeleton was stained with Alexa 568-phalloidin, using 4',6-diamidino-2-phenylindole The nucleus was counterstained by DAPI and mounted after laser confocal microscopy with the model Zeiss LSM 710. The results are shown in Figure 2.
图 2中， 细胞内红色荧光来源于 Alexa 568-phalloidin标记的细胞骨架； 绿色荧光来 源于 FAM-siRNA; 蓝色荧光来源于 DAPI标记的细胞核。 通过三种颜色叠加的结果可 知， 2小时培养后， 纳米颗粒均能有效进入细胞胞浆中， 呈现出颗粒状分布。  In Figure 2, intracellular red fluorescence is derived from the cytoskeleton of Alexa 568-phalloidin labeling; green fluorescence is derived from FAM-siRNA; blue fluorescence is derived from DAPI-labeled nuclei. By superimposing the results of the three colors, it is known that after 2 hours of culture, the nanoparticles can effectively enter the cytoplasm of the cells and exhibit a granular distribution.
从图 2中绿色荧光强度对比可知， NPiPEGsQQQ-PLAsQQQ NPiPEGKKKxrPLGAKKKxxso/so)) 和 NP LA^QQ-PEGnKxxrPLA^Q)三组的荧光强度最低。 而上述三个纳米颗粒使用的 siRNA 量和阳离子脂质量均一样， 不同的是使用的聚合物分别为 PEGsfxxrPLAscxK^ PEG10000-PLGA10000(50/50)禾卩 PLA4800-PEG10000-PLA4800， 这三个聚合物中 PEG 均较高 ( 50%) 。 由此可见， PEG可能具有降低纳米颗粒被内吞的能力。 From the comparison of the green fluorescence intensity in Fig. 2, NPiPEGsQQQ-PLAsQQQ NPiPEGKKKxrPLGAKKKxxso/so)) and NP LA^QQ-PEGnKxxrPLA^Q) had the lowest fluorescence intensity. The above three nanoparticles used the same amount of siRNA and cationic lipid, except that the polymer used was PEGsfxxrPLAscxK^ PEG 10000 - PLGA 10000 (50/50 ) and PLA 4800 - PEG 10000 - PLA 4800 , the PEG was higher (50%) in all three polymers. Thus, PEG may have the ability to reduce the endocytosis of nanoparticles.
3、 包载 siRNA的纳米颗粒沉默靶基因的表达 3. Expression of silencing target genes in nanoparticle-encapsulated siRNA
用实施例 1中所述方法制备包载 siRNA的纳米颗粒来研究此给药系统的沉默靶基因 表达的效果。 其中， siRNA/阳离子脂质 /聚合物的质量比为 0.2/1.0/25.0。  Nanoparticles coated with siRNA were prepared by the method described in Example 1 to investigate the effect of silencing target gene expression in this drug delivery system. Among them, the mass ratio of siRNA/cationic lipid/polymer was 0.2/1.0/25.0.
使用的 siRNA 分别为 siPlkl 和 s\N.C.， 阳离子脂质为 ΒΗΕΜ-Chol , 聚合物为 The siRNA used was siPlkl and s\N.C., the cationic lipid was ΒΗΕΜ-Chol, and the polymer was
PEG55O-PLA286OO PEG20OO-PLA125OO PEG50OO-PLA50OO PEG50OO-PLA250OO PEG50OO-PL A51000PEG55O-PLA286OO PEG20OO-PLA125OO PEG50OO-PLA50OO PEG50OO-PLA250OO PEG50OO-PL A51000
PEGioooo-PLAi5ooo 、 PEGioooo-PLGAioooo(5o/so) 、 PEGioooo-PLGA5oooo(75/25) 、 PLA63oo-PEGi2oo-PLA63oo PLA4800-PEG5000-PLA4800和 PLA4800-PEG10000-PLA4800。 PEGioooo-PLAi5ooo, PEGioooo-PLGAioooo (5o/so), PEGioooo-PLGA5oooo (75/25), PLA 6 3oo-PEGi2oo-PLA 63 oo PLA 4800 -PEG 5000 -PLA 4800 and PLA 4800 -PEG 10000 -PLA 4800 .
得到的包载 siRNA的纳米颗粒分别为 NP(PEG55Q-PLA286QQ)、 NP(PEG2QQQ-PLA12500)、 NP(PEG5ooo-PLA50oo) 、 NP(PEG5000-PLA25000) 、 NP(PEG5000-PLA51000) 、 NP(PEGioooo-PLAi5ooo)、 NP(PEG10000-PLGA10000(50/50))、 NP(PEG10000-PLGA50000(75/25))、 NP(PLA63oo-PEGi2oo-PLA63oo) 、 NP(PLA48。。-PEG5。。。-PLA48。。） 和 NP(PLA48oo-PEGioooo-PLA48oo) ° The obtained siRNA-loaded nanoparticles were NP (PEG 55 Q-PLA 286 QQ), NP (PEG 2 QQQ-PLA 12500 ), NP (PEG 5 ooo-PLA 50 oo), NP (PEG 5000 -PLA 25000 ). , NP (PEG 5000 -PLA 51000 ), NP (PEGioooo-PLAi 5 ooo), NP (PEG 10000 -PLGA 10000 (50/50) ), NP (PEG 10000 -PLGA 50000 (75/25) ), NP (PLA 6 3oo-PEGi 2 oo-PLA 6 3oo), NP (PLA 48 ..- PEG 5 ...- PLA 48 ..) and NP (PLA 4 8oo-PEGioooo- PLA 4 8oo) °
Plkl有助于促进和加速哺乳动物细胞的有丝分裂， 并在多种肿瘤细胞中高表达。通 过沉默其表达， 可以抑制肿瘤生长。  Plkl helps promote and accelerate mitosis in mammalian cells and is highly expressed in a variety of tumor cells. By silencing its expression, tumor growth can be inhibited.
将 HepG2细胞以 l x lO5细胞 /孔的密度接种于 6孔板， 37°C培养 24小时后， 除去培 养基， 分别进行如下处理， 在各组处理中， 将培养基更换成如下各组处理中的溶液， 每 个处理组设置 1个复孔： HepG2 cells were seeded in a 6-well plate at a density of 1×10 5 cells/well, and cultured at 37° C. for 24 hours, the medium was removed, and the following treatments were carried out, respectively, and in each group, the medium was replaced with the following groups. In the solution, set one duplicate hole per treatment group:
处理 1 (对照组） ： 100 μ 的 PBS溶液加入到 1.9 mL新鲜培养基中。  Treatment 1 (control): 100 μl of PBS solution was added to 1.9 mL of fresh medium.
处理 2 ( Lipofectamine组） ： 将 5 的 Lipofectamine 2000用无菌水稀释到 50 μΐ 后， 加入到 50 μL含 100 pmol siPlkl的水溶液中， 室温下结合 15分钟后， 将此 100 μL 溶液加入到 1.9 mL新鲜培养基中。 其中， siP/ 的终浓度为 50 nM。  Treatment 2 (Lipofectamine group): Dilute 5 Lipofectamine 2000 to 50 μΐ with sterile water, add to 50 μL of an aqueous solution containing 100 pmol of siPlkl, and after combining for 15 minutes at room temperature, add 100 μL of the solution to 1.9 mL. In fresh medium. Among them, the final concentration of siP/ is 50 nM.
处理 3 (裸 siPlkJ组） ： 将 100 μL的 siPlkJ溶液加入到 1.9 mL新鲜培养基中， 其 中， siP/W的终浓度为 200 nM。  Treatment 3 (naked siPlkJ group): 100 μL of siPlkJ solution was added to 1.9 mL of fresh medium with a final concentration of si n/W of 200 nM.
处理 4 ( NP(PEG55Q-PLA286QQ)组） ： 将 100 μL 的包载 siPlkl 的纳米颗粒 NP(PEG55Q-PLA286(K))溶液加入到 1.9 mL新鲜培养基中， 其中， siPlkl 的终浓度为 200 nM o Treatment 4 (NP (PEG 55 Q-PLA 286 QQ) group): Add 100 μL of siPlkl-coated nanoparticle NP (PEG 55Q -PLA 286(K) ) solution to 1.9 mL of fresh medium, siPlkl The final concentration is 200 nM o
处理 5 NP(PEG55Q-PLA286QQ)组） ： 将 100 μ 的包载 siN. C.的纳米颗粒Treatment of 5 NP (PEG 55 Q-PLA 286 QQ) group): 100 μ of nanoparticles loaded with siN. C.
NP(PEG55Q-PLA286(K))溶液加入到 1.9 mL新鲜培养基中，其中， siN.C.的终浓度为 200 nM。 The NP (PEG 55Q -PLA 286(K) ) solution was added to 1.9 mL of fresh medium with a final concentration of siN.C. of 200 nM.
处理 6 ( NP(PEG -PLA12500)组） ： 将 100 μ 的包载 siPlkl 的纳米颗粒 NP(PEG2 -PLA125。。)溶液加入到 1.9 mL新鲜培养基中， 其中， siP/W的终浓度为 200 nM o Treatment 6 (NP(PEG-PLA 12500 ) group): Add 100 μ of siPlkl-loaded nanoparticle NP (PEG 2 -PLA 125 ) solution to 1.9 mL of fresh medium, where siP/W is final Concentration is 200 nM o
处理 7 ( NP(PEG2QQQ-PLA125QQ)组） ： 将 100 μ 的包载 siN. C.的纳米颗粒Treatment 7 (NP (PEG 2 QQQ-PLA 125 QQ) group): 100 μ of nanoparticles loaded with siN. C.
NP(PEG2QQQ-PLA125QQ)溶液加入到 1.9 mL新鲜培养基中，其中， siN.C.的终浓度为 200 nM。 处理 8 ( NP(PEG5QQQ-PLA5QQQ)组） ： 将 100 μL 的包载 siPlld 的纳米颗粒 NPCPEG5(KKrPLA5(KK溶液加入到 1.9 mL新鲜培养基中， 其中， siPlkl 的终浓度为 200 处理 9 ( NP(PEG5QQQ-PLA5QQQ)组） ： 将 100 μL 的包载 siN. C.的纳米颗粒 NP(PEG5QQQ-PLA5QQQ)溶液加入到 1.9 mL新鲜培养基中，其中， siN.C.的终浓度为 200 nM。 The NP (PEG 2 QQQ-PLA 125 QQ) solution was added to 1.9 mL of fresh medium with a final concentration of siN.C. of 200 nM. Treatment 8 (NP (PEG 5 QQQ-PLA 5 QQQ) group): 100 μL of siPlld-loaded nanoparticles NPCPEG 5 ( KKrPLA 5 ( KK solution was added to 1.9 mL of fresh medium, where the final concentration of siPlkl was 200 treatment 9 (NP (PEG 5 QQQ-PLA 5 QQQ) group): Add 100 μL of the siN. C. nanoparticle NP (PEG 5 QQQ-PLA 5 QQQ) solution to 1.9 mL of fresh medium. Among them, the final concentration of siN.C. was 200 nM.
处理 10 ( NP(PEG5QQQ-PLA25QQQ)组） ： 将 100 μ 的包载 siPlkl 的纳米颗粒 NPCPEG5。。。-PLA25。。。)溶液加入到 1.9 mL新鲜培养基中， 其中， siP/W的终浓度为 200 处理 1 1 ( NP(PEG5QQQ-PLA25QQQ)组） ： 将 100 μL 的包载 siN. C.的纳米颗粒 NP(PEG5QQQ-PLA25QQQ)溶液加入到 1.9 mL新鲜培养基中，其中， siN.C.的终浓度为 200 nM。 Treatment 10 (NP (PEG 5 QQQ-PLA 25 QQQ) group): 100 μl of SiPlkl-loaded nanoparticles NPCPEG 5 was loaded. . . -PLA 25 . . . The solution was added to 1.9 mL of fresh medium, where the final concentration of siP/W was 200 for treatment 1 1 (NP (PEG 5 QQQ-PLA 25 QQQ) group): 100 μL of nano-coated SiN. C. The granule NP (PEG 5 QQQ-PLA 25 QQQ) solution was added to 1.9 mL of fresh medium with a final concentration of siN.C. of 200 nM.
处理 12 ( NP(PEG誦 -PLA誦)组） ： 将 100 μL 的包载 siPlkl 的纳米颗粒 NP(PEG5。。。-PLA51。。。)溶液加入到 1.9 mL新鲜培养基中， 其中， siP/W的终浓度为 200 处理 13 ( NP(PEG5QQQ-PLA51QQQ)组） ： 将 100 的包载 siN. C.的纳米颗粒 NP(PEG5QQQ-PLA51QQQ)溶液加入到 1.9 mL新鲜培养基中，其中， siN. C.的终浓度为 200 nM。 Treatment 12 (NP(PEG诵-PLA诵) group): Add 100 μL of the siPlkl-loaded nanoparticle NP (PEG 5 . . . -PLA 51 ) solution to 1.9 mL of fresh medium, The final concentration of siP/W was 200 treatment 13 (NP (PEG 5 QQQ-PLA 51 QQQ) group): 100 of the packaged siN. C. nanoparticle NP (PEG 5 QQQ-PLA 51 QQQ) solution was added to 1.9 In a mL fresh medium, the final concentration of siN.C. is 200 nM.
处理 14 ( NP(PEGioooo-PLAi5ooo) ia ) ： 将 100 的包载 siPlW 的纳米颗粒 NPCPEGKKKXTPLAC)溶液加入到 1.9 mL新鲜培养基中， 其中， siPl!d的终浓度为 200 处理 15 ( NP(PEGioooo-PLA150oo) ia ) ： 将 100 μ 的包载 siN.C.的纳米颗粒 NPCPEG1(KKKrPLA15(KK溶液加入到 1.9 mL新鲜培养基中， 其中， siN.C.的终浓度为 200 处理 16 (NP EGKKKKrPLGAKKKx^Q/sQ))组） ： 将 100 μ 的包载 siPlkl 的纳米颗粒 NPCPEGKKKxrPLGAKKKKxsQ/sQ))溶液加入到 1.9 mL新鲜培养基中， 其中， siPlld的终浓度 为 200 nM。 Treatment 14 (NP(PEGioooo-PLAi 5 ooo) ia ) : Add 100 μg of siPlW nanoparticle NPCPEGKKKXTPLAC) solution to 1.9 mL of fresh medium, where the final concentration of siPl!d is 200 treatment 15 (NP ( PEGioooo-PLA 150 oo) ia ) : 100 μ of nano-packaged siN.C. NPCPEG 1 ( KKKrPLA 15 ( KK solution added to 1.9 mL of fresh medium, where the final concentration of siN.C. is 200) Treatment 16 (NP EGKKKKrPLGAKKKx^Q/sQ))): A 100 μ solution of the siPlkl-coated nanoparticle NPCPEGKKKxrPLGAKKKKxsQ/sQ)) was added to 1.9 mL of fresh medium with a final concentration of si n n n n 200 nM.
处理 17 (NPiPEGKKxxrPLGAKKKK^Q/sQ))组） ： 将 100 μL 的包载 siN. C.的纳米颗粒 Treatment 17 (NPiPEGKKxxrPLGAKKKK^Q/sQ))): 100 μL of nanoparticles coated with siN. C.
NPiPEGKKKxrPLGAKKKxxsQ/sQ))溶液加入到 1.9 mL新鲜培养基中， 其中， siN.C.的终浓度为 200 nM。 NPiPEGKKKxrPLGAKKKxxsQ/sQ)) The solution was added to 1.9 mL of fresh medium with a final concentration of siN.C. of 200 nM.
处理 18 (NP EGKKKxrPLGAsQQQQ^/^)组） ： 将 100 μL 的包载 siPlkl 的纳米颗粒 NPCPEGKKKxrPLGAsfKKKXTO^))溶液加入到 1.9 mL新鲜培养基中， 其中， siPlkl的终浓度 为 200 nM。  Treatment 18 (NP EGKKKxrPLGAsQQQQ^/^)): Add 100 μL of the siPlkl-coated nanoparticle NPCPEGKKKxrPLGAsfKKKXTO^)) solution to 1.9 mL of fresh medium with a final concentration of 200 nM for siPlkl.
处理 19 (NP EGKKKxrPLGAsQQQQ^/^)组） ： 将 100 μ 的包载 siN. C.的纳米颗粒 NPCPEGKKKxrPLGAsfKKK^^))溶液加入到 1.9 mL新鲜培养基中， 其中， siN.C.的终浓度为 200 nM。  Treatment 19 (NP EGKKKxrPLGAsQQQQ^/^) group: 100 μl of the solution containing the siN. C. nanoparticle NPCPEGKKKxrPLGAsfKKK^^)) was added to 1.9 mL of fresh medium, wherein the final concentration of siN.C. was 200 nM.
处理 20 (NP(PLA63QQ-PEG12QQ-PLA63QQ)组） ： 将 100 μ 的包载 siPlW 的纳米颗粒 NP(PLA63。。-PEG12。。-PLA63。。)溶液加入到 1.9 mL新鲜培养基中， 其中， siP/W的终浓度 为 200 nM。 处理 21 (NP(PLA63QQ-PEG12QQ-PLA63QQ)组） ： 将 100 μL 的包载 siN.C.的纳米颗粒 NPCPLA63(xrPEG12(KrPLA63(K溶液加入到 1.9 mL新鲜培养基中，其中， siN.C.的终浓度为 200 nM。 Processing 20 (NP (PLA 63 QQ- PEG 12 QQ-PLA 63 QQ) group): A 100 μ entrapped siPlW nanoparticles NP (PLA 63 ..- PEG 12 ..- PLA 63 ..) was added to In a 1.9 mL fresh medium, the final concentration of siP/W was 200 nM. Treatment 21 (NP (PLA 63 QQ-PEG 12 QQ-PLA 63 QQ) group): 100 μL of the coated siN.C. nanoparticle NPCPLA 63 (xr PEG 12 ( KrPLA 63 ( K solution added to 1.9 mL fresh) In the medium, wherein the final concentration of siN.C. was 200 nM.
处理 22 (NP(PLA48QQ-PEG5QQQ-PLA48QQ)组） ： 将 100 μ 的包载 siPlW 的纳米颗粒 NP(PLA48。。-PEG5。。。-PLA48。。)溶液加入到 1.9 mL新鲜培养基中， 其中， siP/W的终浓度 为 200 nM。 Processing 22 (NP (PLA 48 QQ- PEG 5 QQQ-PLA 48 QQ) group): A 100 μ entrapped siPlW nanoparticles NP (PLA 48 ..- PEG 5 ...- PLA 48 ..) was added In a 1.9 mL fresh medium, the final concentration of siP/W was 200 nM.
处理 23 (NP(PLA48QQ-PEG5QQQ-PLA48QQ)组） ： 将 100 μL 的包载 siN.C.的纳米颗粒 NP(PLA48。。-PEG5。。。-PLA48。。)溶液加入到 1.9 mL新鲜培养基中，其中， siN.C.的终浓度为 200 nM。 Processing 23 (NP (PLA 48 QQ- PEG 5 QQQ-PLA 48 QQ) group): The 100 μL of nanoparticles entrapped siN.C. NP (PLA 48 ..- PEG 5 ...- PLA 48 .. The solution was added to 1.9 mL of fresh medium with a final concentration of siN.C. of 200 nM.
处理 24 (NP LA^QQ-PEGK!QQQ-PLA QQ)组） ： 将 100 μL的包载 siPlkl的纳米颗粒 Treatment 24 (NP LA^QQ-PEGK!QQQ-PLA QQ) group: 100 μL of nanoparticles loaded with siPlkl
NPCPLA^oo-PEGKKKxrPLA^oo)溶液加入到 1.9 mL新鲜培养基中， 其中， siPl!d的终浓 度为 200 nM。 The NPCPLA^oo-PEGKKKxrPLA^oo) solution was added to 1.9 mL of fresh medium, where the final concentration of siPl!d was 200 nM.
处理 25 (NP(PLA48oo-PEGioooo-PLA48oo)ia) ： 将 100 μ 的包载 siN.C.的纳米颗粒 NPCPLA^oo-PEGKKKxrPLA^oo)溶液加入到 1.9 mL新鲜培养基中， 其中， siN.C.的终浓度 为 200nM。 Treatment 25 (NP(PLA 4 8oo-PEGioooo-PLA 4 8oo) ia): Add 100 μ of the siN.C. nanoparticle NPCPLA^oo-PEGKKKxrPLA^oo) solution to 1.9 mL of fresh medium, The final concentration of siN.C. was 200 nM.
在转染培养 24 h后， 用 R easy mini-kits (Qiagen, Valencia, CA) 提取细胞中的总 R A, 用紫外分光光度计测定提取的 R A样品的 OD28Q和 OD26Q的吸光度， 并利用公 式： R A 浓度 （μ§/μί) = 0.04xOD26Qx稀释倍数， 计算 R A 样品的浓度， 然后用 PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara, Dalian, China)合成 cDNA, 每个样 品使用 2 g总 mR A。 在合成 cDNA后， 按照 SYBR® Premix Ex Taq™ (Takara)试剂盒 进行实时荧光定量 PCR反应。 其中， Plkl和甘油三磷酸脱氢酶 GAPDH基因的 PCR引 物如下： After 24 h of transfection, the total RA in the cells was extracted with R easy mini-kits (Qiagen, Valencia, CA), and the absorbance of OD 28Q and OD 26Q of the extracted RA samples was determined by UV spectrophotometer, and the formula was used. : RA concentration (μ § /μί) = 0.04xOD 26Q x dilution factor, calculate the concentration of RA samples, then synthesize cDNA using PrimeScriptTM 1st Strand cDNA Synthesis Kit (Takara, Dalian, China), using 2 g total mR per sample A. After cDNA synthesis, real-time PCR was performed according to the SYBR® Premix Ex TaqTM (Takara) kit. Among them, PCR primers for Plkl and glycerol triphosphate dehydrogenase GAPDH genes are as follows:
Plkl-上游引物： 5'-AGCCTGAGGCCCGATACTACCTAC-3' (SEQIDNo: 5) ； Plkl-下游引物： 5'-ATTAGGAGTCCCACACAGGGTCTTC-3' (SEQIDNo: 6) ； GAPDH-上游引物： 5'-TTCACCACCATGGAGAAGGC-3' (SEQIDNo: 7) ； Plkl-upstream primer: 5'-AGCCTGAGGCCCGATACTACCTAC-3' (SEQ ID No: 5); Plkl-downstream primer: 5'-ATTAGGAGTCCCACACAGGGTCTTC-3' (SEQ ID No: 6); GAPDH-upstream primer: 5'-TTCACCACCATGGAGAAGGC-3' (SEQ IDNo) : 7) ;
GAPDH-下游引物： 5'-GGCATGGACTGTGGTCATGA-3' (SEQIDNo: 8) 。 GAPDH-downstream primer: 5'-GGCATGGACTGTGGTCATGA-3' (SEQ ID No: 8).
PCR反应条件如下：  The PCR reaction conditions are as follows:
1) 95 °C加热预变性 30秒。  1) Pre-denaturation at 95 °C for 30 seconds.
2) 95°C加热变性 5秒。  2) Heat denaturation at 95 ° C for 5 seconds.
3) 60°C加热延伸 30秒。 40个循环  3) Heat at 60 ° C for 30 seconds. 40 cycles
4) 95°C加热变性 15秒。  4) Heat denaturation at 95 ° C for 15 seconds.
5) 60°C加热延伸 60秒。  5) Heat at 60 ° C for 60 seconds.
6) 95°C加热变性 15秒。  6) Heat denaturation at 95 ° C for 15 seconds.
利用 2- MCT对不同实验组中 Plkl基因表达差异进行了分析，其中以 GAPDH为内参， 分析不同实验组中 Plkl基因的表达水平。 以 PBS实验组为 100%，其他实验组的表达表 示为相对于 PBS组的相对表达量， 实验结果见图 3。 图 3中， 不经过处理的 PBS组细胞内的 Plkl表达量高， 包载 siPlld的实验组均可 以有效抑制 PM基因的表达， 而所有包载 siN. C.的实验对照组对细胞中 Plkl的基因表 达没有明显影响。 说明 Plkl表达下调是由于序列特异性的基因沉默造成的。 2- MCT Plkl using differences in gene expression in the different experimental groups were analyzed, which GAPDH as internal control, expression level of the different experimental groups Plkl gene. The expression was expressed as 100% in the PBS experimental group, and the expression in the other experimental groups was expressed as relative expression with respect to the PBS group. The experimental results are shown in Fig. 3. In Fig. 3, the expression level of Plk1 in the untreated PBS group was high, and the experimental group containing siPlld could effectively inhibit the expression of PM gene, and all the experimental groups containing siN.C. were in the cell Plkl. Gene expression had no significant effect. This indicates that down-regulation of Plkl expression is due to sequence-specific gene silencing.
同时， 从实验结果可知， NP(PEG5QQQ-PLA5QQQ)、 NPiPEGKKKxrPLGAKKKxxsQ/sQ))和 NPiPLA^QQ-PEGKKxxrPLA^Q)三组沉默效果较差， 结合上述实施例推测， 这是由于这三 个实验组内吞效果较差造成的。 而 NP EGscxxrPLA^oo)实验组的沉默效果最佳， 能够 将 Plkl基因的表达下调到 PBS组的 45%。 实施例 3、 此给药系统在动物水平的生物效应评价 At the same time, it can be seen from the experimental results that NP (PEG 5 QQQ-PLA 5 QQQ), NPiPEGKKKxrPLGAKKKxxsQ/sQ)) and NPiPLA^QQ-PEGKKxxrPLA^Q) have poor silencing effects, which is inferred from the above examples. The experimental group was caused by poor endocytosis. The NP EGscxxrPLA^oo) experimental group had the best silencing effect and was able to down-regulate the expression of Plk1 gene to 45% of the PBS group. Example 3: Evaluation of biological effects of the drug delivery system at the animal level
从细胞水平实验可知，用实施例 1中所述的方法制备包载 siRNA的纳米颗粒时， 当 各组分的质量比例为 siRNA/阳离子脂质 /聚合物 =0.2/1.0/25.0、 使用的阳离子脂质为 BHEM-ChoK 使用的聚合物为 PEGsooo-PLA^oo时， 制备得到包载 siRNA的纳米颗粒能 够有效进入细胞，并且能够显著沉默靶基因的表达， 以此配方来制备包载 siRNA的纳米 颗粒
Figure imgf000019_0001
并作为实例来研究此给药系统在动物水平的生物效应。 From the cell level experiments, it was found that when the siRNA-loaded nanoparticles were prepared by the method described in Example 1, when the mass ratio of each component was siRNA/cationic lipid/polymer=0.2/1.0/25.0, the used cation When the polymer used as the lipid for BHEM-ChoK is PEGsooo-PLA^oo, the nanoparticles coated with siRNA can be efficiently inserted into the cell, and the expression of the target gene can be significantly silenced, and the nanometer containing the siRNA can be prepared by using the formula. Granule
Figure imgf000019_0001
The biological effects of this drug delivery system at the animal level were investigated as an example.
1、 包载 siLuci的纳米颗粒对肝原位植入肿瘤细胞中萤火虫荧光素酶表达的抑 制 1. Inhibition of firefly luciferase expression in liver cells implanted in situ by siLuci-coated nanoparticles
Luciferase 是萤火虫荧光素酶， 能够催化通过腹腔注射的底物发出可见光， 根据酶 的反应均一性， 可定性测定 Luciferase基因的表达。  Luciferase is a firefly luciferase that catalyzes the emission of visible light through a substrate injected intraperitoneally. The expression of the Luciferase gene can be qualitatively determined based on the uniformity of the enzyme reaction.
将 HepG2-luciferase细胞 （密度为 2.0x l 07个 /mL， 25 μ∑) 悬液和 25 的 MatrigelHepG2-luciferase cells (density 2.0xl 7 /mL, 25 μ∑) suspension and 25 Matrigel
( BD Biosciences ) 混合均匀后， 注射至裸鼠肝叶， 伺养 10天左右可形成肿瘤， 按照下 面的处理方法进行给药， 尾静脉注射， 每天一次， 共注射 2次： (BD Biosciences) After mixing evenly, it is injected into the liver lobe of nude mice. The tumor can be formed after about 10 days of feeding. The drug is administered according to the following treatment method. The tail vein is injected once a day for 2 times:
处理 1 ( PBS对照组） ： 给药 25(^L的 PBS , 作为对照。  Treatment 1 (PBS control group): 25 (^L of PBS was administered as a control.
处理 2 (裸 siL d组） ： 给药 250μ 的裸 siL d, 其中， siL d的剂量为 20μ§。 处理 3 ( NP(PEG5ooo-PLA250oo)/siN. C.组） ： 给药包载 siN. C.的纳米颗粒Treatment 2 (naked siL d group): 250 μl of naked siL d was administered, wherein the dose of siL d was 20 μ§. Treatment 3 (NP(PEG 5 ooo-PLA 250 oo)/siN. C. group): Dosing nanoparticles containing siN. C.
NP(PEG5Q(KrPLA25QQQ)， 其中， siN. C.的剂量为 20μ§。 NP (PEG 5 Q (KrPLA 25 QQQ), wherein the dose of siN. C. is 20 μ § .
处理 4 ( NP(PEG5QQQ-PLA25QQQ)/siL d 组） ： 给药包载 siLuci 的纳米颗粒 NP(PEG5QQQ-PLA25QQQ)， 其中， siLwd的剂量为 2(^g。 Treatment 4 (NP (PEG 5 QQQ-PLA 25 QQQ) / siL d group): The nanoparticle NP (PEG 5 QQQ-PLA 25 QQQ) containing siLuci was administered, wherein the dose of siLwd was 2 (^g.
在小鼠腹腔注射 200 μ∑萤火虫荧光素酶底物 D-luciferin ( 15 mg/mL , Xenogen) ， 通过 Xenogen IVIS® Lumina system (Caliper Life Sciences, Hopkinton, MA)小动物活体成 像仪检测肝部位 luciferase在给药前后的表达， 结果如图 4A所示。  The mice were intraperitoneally injected with 200 μ∑ firefly luciferase substrate D-luciferin (15 mg/mL, Xenogen), and the liver site luciferase was detected by Xenogen IVIS® Lumina system (Caliper Life Sciences, Hopkinton, MA) small animal in vivo imager. Expression before and after administration, the results are shown in Figure 4A.
如图 4A所示， 从给药前 （0天） 图像可知， 小鼠肝部萤火虫荧光素酶表达明显， 表达水平基本相当。 给药两天后 （2 天） ， 阴性对照组 （PBS、 裸 siLuci、 NP(PEG5ooo-PLA250oo)/siN. C.对照组） 中萤火虫荧光素酶的表达均有增加， 源于 HepG2-luciferase在肝部位的增殖。 使用 NPCPEG5Q(xrPLA25()()()ysiL d给药组的裸鼠肝部 位的萤火虫荧光素酶表达明显降低，说明 NPiPEGscxxrPLA^ooysiL d沉默肝癌细胞中的 萤火虫荧光素酶的表达。 As shown in Fig. 4A, from the pre-dose (day 0) image, the expression of firefly luciferase in the liver of mice was significant, and the expression level was basically equivalent. Two days after dosing (2 days), the expression of firefly luciferase was increased in the negative control group (PBS, naked siLuci, NP (PEG 5 ooo-PLA 250 oo)/siN. C. control group), derived from HepG2 -Proliferation of luciferase in the liver. NPCPEG 5Q ( xrPLA 25()()() ysiL d administration group of nude mice liver The expression of firefly luciferase was significantly decreased, indicating that NPiPEGscxxrPLA^ooysiL d silenced the expression of firefly luciferase in liver cancer cells.
2、 包载 siPlkl纳米颗粒对乳腺癌的生长抑制 2. Encapsulation of growth inhibition of breast cancer by siPlkl nanoparticles
在裸鼠第二个乳腺的脂肪垫下原位接种 MDA-MB-435S细胞 （0.5x l07) ， 14天左 右形成可见肿瘤， 肿瘤体积约 50 mm3 , 将裸鼠随机分成五组， 分别进行尾静脉注射治 疗 （处理 1-处理 5 ) ， 每两天注射一次。 肿瘤的体积按公式计算： V=0.5xaxb2计算， 其 中 a指肿瘤长径， b指肿瘤短径。 MDA-MB-435S cells (0.5×10 7 ) were inoculated in situ under the fat pad of the second mammary gland of nude mice. The tumor was formed in about 14 days. The tumor volume was about 50 mm 3 . The nude mice were randomly divided into five groups. The tail vein injection treatment (treatment 1-treatment 5) was administered once every two days. The volume of the tumor is calculated according to the formula: V = 0.5 x axb 2 , where a refers to the long diameter of the tumor and b refers to the short diameter of the tumor.
处理 1 ( PBS对照组） ： 每只裸鼠注射 250μ 的 PBS。  Treatment 1 (PBS control group): Each nude mouse was injected with 250 μl of PBS.
处理 2 (裸 siPlkl组） ： 每只裸鼠注射 250μ 的裸 siPlkl， siPlkl的剂量为 2(^g。 处理 3 ( NP(PEG5ooo-PLA250oo)组 ） ： 每只裸 鼠注射空 白 纳米颗粒 NP(PEG5ooo-PLA250oo), 纳米颗粒的质量为 2.70mg。 Treatment 2 (naked siPlkl group): Each nude mouse was injected with 250 μ of bare siPlkl, and the dose of siPlkl was 2 (^g. Treatment 3 (NP (PEG 5 ooo-PLA 250 oo) group): Each nude mouse was injected with blank nanometer Particle NP (PEG 5 ooo-PLA 250 oo), the mass of the nanoparticles was 2.70 mg.
处理 4 ( NP(PEG5ooo-PLA250oo)/siN.C. M ) ： 每只裸鼠注射包载 siN. C.的纳米颗粒 NP(PEG5ooo-PLA250oo), siN.C.的剂量为 20 g， 纳米颗粒的质量为 2.70 mg。 Treatment 4 (NP(PEG 5 ooo-PLA 250 oo)/siN.C. M ): Each nude mouse was injected with nanoparticle NP (PEG 5 ooo-PLA 250 oo) encapsulating siN. C., siN.C. The dose is 20 g and the mass of the nanoparticles is 2.70 mg.
处理 5 (NP(PEG5ooo-PLA250oo)/siP/W 组） ： 每只裸鼠注射包载 siPlkl 的纳米颗粒Treatment 5 (NP(PEG 5 ooo-PLA 250 oo)/siP/W group): Each nude mouse was injected with nanoparticles containing siPlkl
NP(PEG5ooo-PLA250oo), siP/L?的剂量为 20 g， 纳米颗粒的质量为 2.70 mg。 The dose of NP (PEG 5 ooo-PLA 250 oo), siP/L? was 20 g, and the mass of the nanoparticles was 2.70 mg.
在治疗开始后， 每隔一天对肿瘤体积进行测量。 结果如图 4B所示， 所有阴性对照 组中肿瘤生长速度均较快， 而在使用包载 siPlkl 纳米颗粒 NPiPEGsooo-PLA^oo)的治疗 组中， 肿瘤生长速度与阴性对照组相比受到明显抑制。 说明本发明提供的包载 siRNA 的纳米颗粒在体内能够有效沉默致癌基因， 从而抑制肿瘤生长。  Tumor volume was measured every other day after the start of treatment. As a result, as shown in Fig. 4B, the tumor growth rate was faster in all the negative control groups, and in the treatment group using the siPlkl nanoparticles (NPiPEGsooo-PLA^oo), the tumor growth rate was significantly inhibited compared with the negative control group. . It is indicated that the siRNA-encapsulated nanoparticles provided by the present invention can effectively silence oncogenes in vivo, thereby inhibiting tumor growth.
从实施例 3可知，此 siRNA给药系统在动物水平能够有效沉默肿瘤部位靶基因的表 达， 而且这种靶基因表达的下调是由于序列特异性的基因沉默造成的。 实施例 4、 此给药系统的靶向修饰以及生物效应的评价  As is apparent from Example 3, the siRNA delivery system is capable of effectively silencing the expression of a target gene at a tumor site at an animal level, and the down-regulation of expression of the target gene is caused by sequence-specific gene silencing. Example 4. Targeted modification of this drug delivery system and evaluation of biological effects
1、 半乳糖修饰、 包载 siRNA的纳米颗粒构建肝靶向的 siRNA给药系统 为了提高该 siRNA给药系统在疾病治疗应用中的潜力，本发明在这种纳米颗粒表面 修饰了靶向基团， 将此给 siRNA药系统发展成为一种具有靶向性的 siRNA给药系统。 半乳糖基团是一种靶向性配体， 其能够特异性的识别去唾液酸糖蛋白受体 （ASGP-R, 一种哺乳动物肝细胞表面过量表达的糖蛋白）， 因此本发明通过这一实例说明在纳米颗 粒表面修饰半乳糖基团， 可制备靶向给药系统。  1. Galactose-Modified, siRNA-Coated Nanoparticles Constructing a Liver-Targeted siRNA Delivery System To enhance the potential of the siRNA delivery system in disease therapeutic applications, the present invention modifies a targeting group on the surface of such nanoparticles. This has led to the development of a siRNA drug system into a targeted siRNA delivery system. A galactose group is a targeting ligand that specifically recognizes the asialoglycoprotein receptor (ASGP-R, a glycoprotein overexpressed on the surface of mammalian hepatocytes), so the present invention passes this An example illustrates the modification of a galactose group on the surface of a nanoparticle to prepare a targeted drug delivery system.
半乳糖修饰、 包载 siRNA 的纳米颗粒是利用氨基半乳糖修饰的 PEG-PLA ( Gal-PEG-PLA)和阳离子脂质通过双乳化方法包载 siRNA而制备。其中， Gal-PEG-PLA 是以端基分别为羧基和羟基的异官能团的聚乙二醇为引发剂，在本体条件下利用聚乙二 醇端羟基引发丙交酯单体的单体聚合得到 HOOC-PEG-PLA, 再将氨基半乳糖键合到 PEG链段上从而得到。通过调节聚乙二醇与丙交酯的投料比， 理论上可以得到不同分子 量的 Gal-PEG-PLA。异辛酸亚锡因其高催化效率及无毒性， 是被最广泛应用的催化内酯 及交酯环状单体开环聚合反应的催化剂， 已被美国 FDA批准作为食品添加剂。 The galactose-modified, siRNA-encapsulated nanoparticles were prepared by encapsulating siRNA by a double emulsion method using galactose-modified PEG-PLA ( Gal-PEG-PLA) and cationic lipids. Among them, Gal-PEG-PLA is a hetero-functional polyethylene glycol with terminal groups of carboxyl and hydroxyl groups as initiators, and the monomer polymerization of lactide monomer is initiated by using polyethylene glycol terminal hydroxyl groups under bulk conditions. HOOC-PEG-PLA, which is obtained by bonding an aminogalactose to a PEG segment. By adjusting the feed ratio of polyethylene glycol to lactide, it is theoretically possible to obtain Gal-PEG-PLA of different molecular weight. Stannous isooctylate is the most widely used catalytic lactone due to its high catalytic efficiency and non-toxicity. And a catalyst for ring-opening polymerization of a lactide cyclic monomer, which has been approved by the US FDA as a food additive.
具体合成步骤如下： 首先， 将进行反应的圆底烧瓶经多次的抽真空干燥和充氮气处 理后， 放入手套箱。 然后， 以 HOOC-PEGSQQQ-OH/丙交酯 /Sn(Oct)2=l/320/0.09的摩尔配 比进行投料。 向烧瓶中加入 HOOC-PEG5(KKrOH、 单体和异辛酸亚锡， 在 130°C搅拌下反 应。 反应 24小时后， 将产物移出手套箱， 用少量的 CH2C12溶解， 将溶液沉淀到冷的乙 醚中， 重复两次， 收集沉淀物， 用油泵抽干至恒重为止， 即得产物（HOOC-PEG-PLA) 。 The specific synthesis steps are as follows: First, the round bottom flask subjected to the reaction is subjected to a plurality of vacuum drying and nitrogen treatment, and then placed in a glove box. Then, the charge was carried out at a molar ratio of HOOC-PEGSQQQ-OH/lactide/Sn(Oct) 2 = l/320/0.09. To the flask was added HOOC-PEG 5 ( KKrOH, monomer and stannous isooctanoate, and reacted at 130 ° C with stirring. After 24 hours of reaction, the product was removed from the glove box, dissolved with a small amount of CH 2 C 1 2 , and the solution was precipitated. In a cold diethyl ether, the mixture was repeated twice, and the precipitate was collected and dried with an oil pump until constant weight to obtain a product (HOOC-PEG-PLA).
以合成得到的 HOOC-PEG-PLA进一步键合氨基半乳糖得到氨基半乳糖修饰的聚乙 二醇-聚乳酸（Gal-PEG-PLA) 。 具体试验如下： 将 lg的 HOOC-PEG5QQQ-PLA21Q3Q溶解在 5 mL的二氯甲烷中， 并加入 N，N-二环己基碳二亚胺（DCC)、 N-羟基琥珀酰亚胺（NHS ) 和氨基半乳糖 （Gal-NH2) ， 按 HOOC-PEG5QQQ-OH/DCC/NHS/Gal-NH2=l/2/2/2的摩尔配 比进行投料。 室温下反应 12小时后， 将溶液过滤， 然后收集得到的滤液并加入到冷的 乙醚中进行沉淀， 收集沉淀物， 用油泵抽干至恒重为止， 即得产物 （Gal-PEG-PLA) 。 The aminogalactose is further bonded to the HOOC-PEG-PLA obtained by the synthesis to obtain an aminogalactose-modified polyethylene glycol-polylactic acid (Gal-PEG-PLA). The specific test is as follows: lg HOOC-PEG 5 QQQ-PLA 21 Q 3 Q is dissolved in 5 mL of dichloromethane, and N,N-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinyl group is added. The imine (NHS) and the aminogalactose (Gal-NH 2 ) were charged at a molar ratio of HOOC-PEG 5 QQQ-OH/DCC/NHS/Gal-NH 2 = l/2/2/2. After reacting for 12 hours at room temperature, the solution was filtered, and the obtained filtrate was collected and added to cold diethyl ether to precipitate, and the precipitate was collected and dried with an oil pump until constant weight to obtain a product (Gal-PEG-PLA).
图 5A为合成得到的 HOOC-PEG5QQQ-PLA21Q3。和 Gal-PEG5QQQ-PLA2K)3Q的核磁共振氢谱 结果， 分析如下： 字母 a到 d标记了归属 PEG-PLA的质子信号。 聚乳酸的分子量通过 5.17 ppm的多重峰 （归属于聚乳酸的 -C(0)OC -， 斜体标识的 H， 以下类同） 与 3.63 ppm的 单峰 （归属于聚乙二醇的 的积分面积比计算得到。 对比图 5A中 Figure 5A shows the synthesized HOOC-PEG 5 QQQ-PLA 21 Q 3 . The results of nuclear magnetic resonance spectroscopy of Gal-PEG 5 QQQ-PLA 2K) 3 Q are analyzed as follows: The letters a to d mark the proton signal belonging to PEG-PLA. The molecular weight of polylactic acid passes through a multiple peak of 5.17 ppm (-C(0)OC- assigned to polylactic acid, H in italics, the following analogy) and a single peak of 3.63 ppm (integral area attributed to polyethylene glycol) Compared with the calculation. Compare Figure 5A
HOOC-PEG50oo-PLA2io3o和 Gal-PEG5QQQ-PLA2K)3Q的核磁共振氢谱可知，键合后 d信号峰消 失， 因为键合后化学环境变化导致其信号峰和 3.63 ppm的单峰 （归属于聚乙二醇的 -CH2CH2- ) 重合。 从核磁谱图可以推断成功合成得到 Gal-PEG5(KKrPLA21Q3Q聚合物。 HOOC-PEG 50 oo-PLA 2 io3o and Gal-PEG 5 QQQ-PLA 2K) 3 The nuclear magnetic resonance spectrum of Q shows that the d signal peak disappears after bonding, because the chemical environment changes after bonding and its signal peak and 3.63 ppm The single peak (-CH2CH2- assigned to polyethylene glycol) coincides. From the nuclear magnetic resonance spectrum, it can be inferred that Gal-PEG 5 ( KKrPLA 21Q3Q polymer) was successfully synthesized.
用实施例 1中的方法制备包载 siRNA的纳米颗粒来研究此给药系统的生物效应。其 中， siRNA/阳离子脂质 /聚合物的质量比为 0.2/1.0/25.0， 使用的 siRNA分别为 siPlkl和 siN.C , 阳离子脂质为 BHEM-Chol。 使用的聚合物为 HOOC-PEG5(KKrPLA21Q3()和 The siRNA-loaded nanoparticles were prepared by the method of Example 1 to study the biological effects of this drug delivery system. The mass ratio of siRNA/cationic lipid/polymer was 0.2/1.0/25.0, the siRNA used was siPlkl and siN.C, and the cationic lipid was BHEM-Chol. The polymer used was HOOC-PEG 5 ( KKrPLA 21Q3() and
Gal-PEG5ooo-PLA2103o> 制备得到的包载 siRNA的纳米颗粒分别为 Gal-PEG 5 ooo-PLA 210 3o> The prepared siRNA-loaded nanoparticles are respectively
NP(HOOC-PEG5。。。-PLA21。3。)和 NP(Gal-PEG 5000-PLA2103o) ° a) 流式细胞检测包载 FAM-siRNA靶向纳米颗粒的细胞内吞 NP (HOOC-PEG 5 . . . -PLA 21 . 3 ) and NP (Gal-PEG 5000-PLA2103o) ° a) Flow cytometry for intracellular endocytosis of FAM-siRNA-targeted nanoparticles
以 上 述 体 系 制 备 包载 FAM-siRNA 的 NP(HOOC-PEG5QQQ-PLA21Q3Q)和 NP(Gal-PEG5。。。-PLA21。3。)来研究此靶向给药体系的细胞内吞。 将 Hepa 1-6细胞以 5x l04 细胞 /孔的密度接种于 24孔板， 37°C培养 24小时后， 除去培养基， 分别进行如下处理， 在各组处理中， 将培养基更换成如下各组处理中的溶液： FAM-siRNA-encapsulated NP (HOOC-PEG 5 QQQ-PLA 21 Q 3 Q) and NP (Gal-PEG 5 . . . -PLA 21 . 3 ) were prepared in the above system to study the targeted drug delivery system. Endocytosis of cells. Hepa 1-6 cells were seeded in a 24-well plate at a density of 5 ×10 4 cells/well, and cultured at 37° C. for 24 hours, and then the medium was removed, and the following treatments were carried out, respectively. In each treatment, the medium was changed to the following Solution in each group of treatments:
处理 1 (PBS ) ： 50μ 的 PBS溶液加入到 450μ 的新鲜 DMEM培养基中。  Treatment 1 (PBS): 50 μl of PBS solution was added to 450 μl of fresh DMEM medium.
处理 2 (FAM-siRNA) ： 50μL的 FAM-siRNA溶液加入到 450μΙ^的新鲜 DMEM培 养基， 其中， FAM-siRNA的终浓度为 200 ηΜ。  Treatment 2 (FAM-siRNA): 50 μL of the FAM-siRNA solution was added to 450 μM of fresh DMEM medium, wherein the final concentration of FAM-siRNA was 200 ηΜ.
处理 3 (NP(HOOC-PEG5QQQ-PLA21Q3Q)组） ： 50μ 的包载 FAM-siRNA的纳米颗粒 NP(HOOC-PEG5QQQ-PLA21Q3Q)溶液加入到 450μ 的新鲜 DMEM 培养基中， 其中， FAM-siRNA的终浓度为 200 nM。 处理 4 ( NP(Gal-PEG5QQQ-PLA21Q3Q)组） ： 50μ 的包载 FAM-siRNA 的纳米颗粒 NP(Gal-PEG5。。。-PLA21。3。)溶液加入到 450μ 的新鲜 DMEM培养基中，其中， FAM-siRNA 的终浓度为 200 nM。 Treatment 3 (NP(HOOC-PEG 5 QQQ-PLA 21 Q 3 Q) group): 50 μ of the loaded FAM-siRNA-coated nanoparticle NP (HOOC-PEG 5 QQQ-PLA 21 Q 3 Q) solution was added to 450 μ fresh In DMEM medium, the final concentration of FAM-siRNA was 200 nM. Treatment 4 (NP(Gal-PEG 5 QQQ-PLA 21 Q 3 Q) group): 50 μ of the loaded FAM-siRNA-loaded nanoparticle NP (Gal-PEG 5 . . . -PLA 21 . 3 ) solution was added to 450 μ In a fresh DMEM medium, the final concentration of FAM-siRNA was 200 nM.
处理 5 (NP(Gal-PEG5QQQ-PLA21Q3Q)+Gal-NH2组） ： 50μ 的包载 FAM-siRNA的纳米 颗粒 NP(Gal-PEG5。。。-PLA21。3。)溶液加入到 450μ 的含靶向抑制剂 Gal-NH2 的新鲜 DMEM培养基中， 其中， 靶向抑制剂 Gal-NH2的终浓度 60 mM， FAM-siRNA的终浓度 为 200 nM。 Process 5 (NP (Gal-PEG 5 QQQ-PLA 21 Q 3 Q) + Gal-NH 2 group): entrapped FAM-siRNA nanoparticle NP 50μ in (Gal-PEG 5 ...- PLA 21 3. ) was added to a solution of the targeted inhibitors 450μ fresh DMEM medium Gal-NH 2, wherein Gal-NH targeted inhibitors to a final concentration of 60 mM 2, FAM-siRNA final concentration of 200 nM.
分别经过以上几种条件处理 2小时后，消化收集各组细胞，用流式细胞仪 ( FACS， BD Bioscience, Bedford, MA) 分析细胞吞噬纳米颗粒， 结果见图 5B。  After 2 hours of treatment under the above conditions, each group of cells was digested and analyzed, and the cells were subjected to phagocytosis of the nanoparticles by flow cytometry (FACS, BD Bioscience, Bedford, MA), and the results are shown in Fig. 5B.
从图 5B 可知， FAM-siRNA 基本不能够进入细胞， 靶向给药体系 As can be seen from Figure 5B, FAM-siRNA is basically unable to enter cells, targeting drug delivery systems.
NP(Gal-PEG50oo-PLA2io3o)和非靶向给药体系 NP(HOOC-PEG5QQQ-PLA2 K)3Q)均可以将 FAM-siRNA 成功输运到细胞中。 其中， 靶向给药体系实验组检测到细胞内的 FAM-siRNA荧光信号明显强于非靶向给药体系， 说明该半乳糖修饰的纳米颗粒能够更 好的将 siRNA输运到肝癌细胞。 同时， 加入靶向抑制剂 Gal-NH2 (终浓度 60 mM )的实 验组 （ NP(Gal-PEG5Q(xrPLA2K)3())+Gal-NH2组） ， 荧光信号较低， 和非靶向实验组 NP(HOOC-PEG5(xKrPLA21()3())相当。这一现象说明这种半乳糖修饰的纳米颗粒是通过纳米 颗粒表面的半乳糖配体与肝细胞表面 ASGP-R特异性结合介导纳米颗粒的内吞，加入靶 向抑制剂 Gal-NH2后， 纳米颗粒不能够通过 ASGP-R特异性结合介导纳米颗粒的内吞， 所以输运的 FAM-siRNA降低， 导致荧光信号降低。 Both NP (Gal-PEG 50 oo-PLA 2 io3o) and non-targeted drug delivery system NP (HOOC-PEG 5 QQQ-PLA 2 K) 3 Q) can successfully transport FAM-siRNA into cells. Among them, the experimental group of the targeted drug delivery system detected that the FAM-siRNA fluorescence signal in the cells was significantly stronger than the non-targeted drug delivery system, indicating that the galactose-modified nanoparticles could better transport the siRNA to the liver cancer cells. At the same time, the experimental group (NP(Gal-PEG 5Q( xrPLA 2K)3() )+Gal-NH 2 group) with the targeted inhibitor Gal-NH 2 (final concentration 60 mM) was added, and the fluorescence signal was low, and The targeted experimental group NP (HOOC-PEG 5( xKrPLA 21()3() ) is equivalent. This phenomenon indicates that this galactose-modified nanoparticle is a galactose ligand on the surface of the nanoparticle and a hepatocyte surface ASGP-R The specific binding mediates the endocytosis of the nanoparticles. After the addition of the targeted inhibitor Gal-NH 2 , the nanoparticles cannot mediate the endocytosis of the nanoparticles through the specific binding of ASGP-R, so the transported FAM-siRNA is reduced. Causes a decrease in the fluorescent signal.
从细胞内吞结果可知，这种经过配体修饰后聚合物制备的包载 siRNA纳米颗粒具有 靶向能力。 同样， 这种配体修饰可以是半乳糖修饰而不限于此， 例如叶酸、 含精氨酸- 甘氨酸-天冬氨酸 （RGD ) 的短肽等。 b) 靶向纳米颗粒输运 siRNA在细胞水平沉默靶基因表达  From the results of endocytosis, the encapsulated siRNA nanoparticles prepared by the ligand-modified polymer have the targeting ability. Also, such a ligand modification may be a galactose modification, and is not limited thereto, such as folic acid, a short peptide containing arginine-glycine-aspartic acid (RGD), and the like. b) targeting nanoparticle transport siRNA silencing target gene expression at the cellular level
apoB是肝细胞中胆固醇转运相关蛋白， 已广泛被用作肝脏基因沉默的模式靶基因； 本发明通过沉默 Hepa 1 -6细胞 apoB基因表达来评价该半乳糖修饰的靶向纳米颗粒可以 更好的输运 siRNA进入肝癌细胞， 并成功释放 siRNA, 沉默靶基因的表达。 以上述体 系制备包载 siapoB和 siN. C.的 NP(HOOC-PEG5QQQ-PLA21Q3Q)和 NP(Gal-PEG5QQQ-PLA21030) 来研究此靶向给药体系在细胞水平沉默靶基因的效果。 apoB is a cholesterol transport-related protein in hepatocytes and has been widely used as a model target gene for liver gene silencing; the present invention can be better evaluated by silencing Hepa 1 -6 cell apoB gene expression to evaluate the galactose-modified targeting nanoparticle. The transport siRNA enters the liver cancer cells and successfully releases the siRNA to silence the expression of the target gene. The NP (HOOC-PEG 5 QQQ-PLA 21 Q 3 Q) and NP (Gal-PEG 5 QQQ-PLA 21030 ) containing siapoB and siN.C. were prepared by the above system to study the targeted drug delivery system at the cellular level. Silencing the effect of the target gene.
将 Hepa 1 -6细胞以 l x l O5细胞 /孔的密度接种于 6孔板， 37 °C培养 24小时后， 除去 培养基， 分别进行如下处理， 在各组处理中， 将培养基更换成如下各组处理中的溶液， 每个处理组设置 1个复孔： Hepa 1 -6 cells were seeded in a 6-well plate at a density of lxl O 5 cells/well, cultured at 37 ° C for 24 hours, and then the medium was removed, and the following treatments were carried out, respectively. In each treatment, the medium was changed to the following For each group of treated solutions, set one duplicate hole per treatment group:
处理 1 ( PBS ) ： 100 的的 PBS溶液加入到 1.9 mL的新鲜 DMEM培养基中。 处理 2 ( Free skywfi组） ： 加入 100 的 skywfi溶液， 其中， skywfi的终浓度为 200 nM。 处理 3 (Lipofectamine组） ： 将 5 的 Lipofectamine 2000用无菌水稀释到 50 后， 加入到 50 L含 lOOpmolskywfi的水溶液中， 室温下结合 15分钟后， 将此 100 溶液加入到 1.9 mL新鲜培养基中。 其中， siapoB终浓度为 50 nM。 Treatment 1 (PBS): 100 PBS solution was added to 1.9 mL of fresh DMEM medium. Treatment 2 (Free skywfi group): Add 100 skywfi solution, where the final concentration of skywfi is 200 nM. Treatment 3 (Lipofectamine group): After diluting 5 Lipofectamine 2000 to 50 with sterile water, adding it to 50 L of an aqueous solution containing lOOpmolskywfi, and after combining for 15 minutes at room temperature, the 100 solution was added to 1.9 mL of fresh medium. . Among them, the final concentration of siapoB was 50 nM.
处理 4 (NP(HOOC-PEG5QQQ-PLA21Q3Q)/siN.C.组） ： 100 的包载 siN.C.的靶向纳米 颗粒 NP(HOOC-PEG5。。。-PLA21。3。)加入到 1.9 mL的新鲜 DMEM培养基中， 其中， siN.C. 的终浓度为 200 nM。 Treatment 4 (NP(HOOC-PEG 5 QQQ-PLA 21 Q 3 Q)/siN.C. group): 100-packed siN.C.-targeted nanoparticle NP (HOOC-PEG 5 . . . -PLA 21 3. ) Add to 1.9 mL of fresh DMEM medium, where the final concentration of siN.C. is 200 nM.
处理 5 (NP(HOOC-PEG50oo-PLA2io3o)/siflpofi组） ： 100 μ 的包载 siapoB的非靶向 纳米颗粒 NP(HOOC-PEG5。。。-PLA21。3。)加入到 1.9 mL的新鲜 DMEM培养基中， 其中， siapoB的终浓度为 200 nM。 Treatment 5 (NP(HOOC-PEG 50 oo-PLA 2 io3o)/siflpofi group): 100 μ of siapoB-loaded non-targeted nanoparticle NP (HOOC-PEG 5 . . . -PLA 21 . 3 ) was added to In 1.9 mL of fresh DMEM medium, the final concentration of siapoB was 200 nM.
处理 6 (NP(Gal-PEG5QQQ-PLA21Q3Q)/siN.C.组） ： 100 的包载 siN.C.的靶向纳米颗 粒 NP(Gal-PEG5。。。-PLA21。3。)加入到 1.9 mL的新鲜 DMEM培养基中， 其中， siN.C.的终 浓度为 200 nM。 Treatment 6 (NP(Gal-PEG 5 QQQ-PLA 21 Q 3 Q)/siN.C. group): 100-packaged siN.C.-targeted nanoparticle NP (Gal-PEG 5 . . . -PLA 21 3 )) Add to 1.9 mL of fresh DMEM medium, where the final concentration of siN.C. is 200 nM.
处理 7 (NPiGal-PEGsQQQ-PLA Q^yskywB组） ： 100 的包载 sia/wfi的靶向纳米 颗粒 NP(Gal-PEG5。。。-PLA21。3。)加入到 1.9 mL的新鲜 DMEM培养基中， 其中， siapoB的 终浓度为 200nM。 Treatment 7 (NPiGal-PEGsQQQ-PLA Q^yskywB group): 100-packaged sia/wfi-targeted nanoparticle NP (Gal-PEG 5 . . . -PLA 21 . 3 ) was added to 1.9 mL of fresh DMEM culture In the base, wherein the final concentration of siapoB is 200 nM.
在转染培养 24 h后， 用 R easy mini-kits (Qiagen, Valencia, CA) 提取细胞中的总 R A, 用紫外分光光度计测定提取的 R A样品的 OD28Q和 OD26Q的吸光度， 并利用公 式： R A 浓度 （μ§/μί) = 0.04xOD26Qx稀释倍数， 计算 R A 样品的浓度， 然后用 PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara, Dalian, China)合成 cDNA, 每个样 品使用 2 μg总 mRNA。 在合成 cDNA后， 按照 SYBR® Premix Ex Taq™ (Takara)试剂盒 进行实时荧光定量 PCR反应。 其中， apoB基因的 PCR引物如下， 甘油三磷酸脱氢酶 GAPDH基因的 PCR引物如 SEQIDNo: 7禾 B SEQIDNo: 8所示： After 24 h of transfection, the total RA in the cells was extracted with R easy mini-kits (Qiagen, Valencia, CA), and the absorbance of OD 28Q and OD 26Q of the extracted RA samples was determined by UV spectrophotometer, and the formula was used. : RA concentration (μ § /μί) = 0.04xOD 26Q x dilution factor, calculate the concentration of RA sample, then synthesize cDNA using PrimeScriptTM 1st Strand cDNA Synthesis Kit (Takara, Dalian, China), using 2 μg total mRNA per sample . After cDNA synthesis, real-time PCR was performed according to the SYBR® Premix Ex TaqTM (Takara) kit. Wherein, the PCR primer of the apoB gene is as follows, and the PCR primer of the glycerol triphosphate dehydrogenase GAPDH gene is as shown in SEQ ID No: 7 and B SEQ ID No: 8:
apoB-上游引物 5'-TTCCAGCCATGGGCAACTTTACCT-3' (SEQIDNo: 9) apoB-下游引物 5'-TACTGCAGGGCGTCAGTGACAAAT-3' (SEQIDNo: 10)  apoB-upstream primer 5'-TTCCAGCCATGGGCAACTTTACCT-3' (SEQ ID No: 9) apoB-downstream primer 5'-TACTGCAGGGCGTCAGTGACAAAT-3' (SEQ ID No: 10)
PCR反应条件如下：  The PCR reaction conditions are as follows:
1) 95 °C加热预变性 30秒。  1) Pre-denaturation at 95 °C for 30 seconds.
2) 95°C加热变性 5秒。  2) Heat denaturation at 95 ° C for 5 seconds.
3) 60°C加热延伸 30秒。 40个循环  3) Heat at 60 ° C for 30 seconds. 40 cycles
4) 95°C加热变性 15秒。  4) Heat denaturation at 95 ° C for 15 seconds.
5) 60°C加热延伸 60秒。  5) Heat at 60 ° C for 60 seconds.
6) 95°C加热变性 15秒。  6) Heat denaturation at 95 ° C for 15 seconds.
利用 2- AACT对不同实验组中 apoB基因表达差异进行了分析， 其中以 GAPDH为内 参， 分析不同实验组中 apoB基因的表达水平。 以 PBS实验组为 100%， 其他实验组表 达表示为相对于 PBS组表达量， 实验结果见图 5C。 The difference in apoB gene expression between different experimental groups was analyzed by 2- AACT. GAPDH was used as an internal reference to analyze the expression level of apoB gene in different experimental groups. The expression in the PBS experimental group was 100%, and the expressions of other experimental groups were expressed as relative to the PBS group. The experimental results are shown in Fig. 5C.
图 5C 中， 以不经过处理的 PBS 组细胞内 apoB m NA 表达量作为 100%， In Figure 5C, the amount of apoB m NA expressed in the untreated PBS group was 100%.
NP(HOOC-PEG50oo-PLA2io3o)/siflpoJS实验组和 ΝΡ(0&1-ΡΕ05。。。-ΡΙ 21。3。)/8ΰφοβ实验组均 可以有效抑制 apoB mR A 的表达， 但在相同剂量的 siapoB ( 200 nM ) 情况下， Gal-NP/siapofi组 （ΝΡ(0&1-ΡΕ05。οο-ΡΙ 21。3。)/8ΰφοβ ) 可以成功沉默 apoB基因表达高达 80%,明显优于 NP/skywB实验组（ΝΡ(ΗΟΟ〔-ΡΕ05(Κχ)-ΡΙ 2Κ)3())/8ΰφοβ， 39%沉默效率）。 而所有包载 siN.C.的实验对照组对细胞中 apoB mRNA的表达没有明显影响。 说明 apoB 表达下调是由于序列特异性的基因沉默造成的。 c) 靶向纳米颗粒输运 siRNA沉默小鼠肝细胞靶基因的表达 NP (HOOC-PEG 50 oo-PLA 2 io3o)/siflpo J S experimental group and ΝΡ (0-ΡΕ0 5 . . . -ΡΙ 21 .3 ) / 8 ΰφοβ experimental group It can effectively inhibit the expression of apoB mR A, but in the same dose of siapoB (200 nM ), the Gal-NP/siapofi group (ΝΡ(0&1-ΡΕ0 5 .οο-ΡΙ 21 . 3 )/8ΰφοβ ) can be successfully silenced. The expression of apoB gene was as high as 80%, which was significantly better than that of NP/skywB experimental group (ΝΡ(ΡΕ[5ΡΕ ( 5 χ)-ΡΙ 2Κ)3() )/8ΰφοβ, 39% silencing efficiency). The experimental control group containing siN.C. had no significant effect on the expression of apoB mRNA in the cells. This indicates that downregulation of apoB expression is due to sequence-specific gene silencing. c) targeting nanoparticle transport siRNA to silence mouse hepatocyte target gene expression
通过将 siapoB的靶向给药系统和非靶向给药系统经尾静脉注入小鼠体内，通过体内 实验观察该载体作为肝靶向 siRNA载体的有效性和靶向性。  The effectiveness and targeting of the vector as a liver-targeted siRNA vector was observed by in vivo experiments by injecting a targeted delivery system of siapoB and a non-targeted drug delivery system into the mouse via the tail vein.
将如上所述的靶向给药系统和非靶向给药系统经尾静脉注入 C57BL/6 小鼠体内， siRNA剂量为 4 mg/kg， 48小时后眼球取血法处死小鼠， 凝血后收集血清， 使用小鼠载 脂蛋白 B(apoB)定量检测试剂盒（ELISA; R&D systems, Minneapolis, MN， USA)检测血 清中 apoB蛋白水平。 每组 6只小鼠， 实验组设置如下：  The targeted drug delivery system and the non-targeted drug delivery system as described above were injected into C57BL/6 mice via the tail vein at a dose of 4 mg/kg. After 48 hours, the mice were sacrificed by eyeballs and collected after coagulation. Serum, serum apoB protein levels were measured using a mouse apolipoprotein B (apoB) quantitative assay kit (ELISA; R&D systems, Minneapolis, MN, USA). For each group of 6 mice, the experimental group was set as follows:
处理 1 ( PBS对照组） ： 每只裸鼠注射 400 L的 PBS。  Treatment 1 (PBS control group): Each nude mouse was injected with 400 L of PBS.
处理 2 ( Free siapoB组）：每只裸鼠注射 400 μL的裸 siapoB, ήαροΒ的剂量为 80 μ 处理 3 (NP(HOOC-PEG5QQQ-PLA21Q3Q)/siN. C.组） ： 每只裸鼠注射包载 siN.C.的纳米 颗粒 NP(HOOC-PEG5QQQ-PLA21Q3Q)， 其中， siN. C.的剂量为 80 g。 Treatment 2 (Free siapoB group): Each nude mouse was injected with 400 μL of naked siapoB, and the dose of ήαροΒ was 80 μ treatment 3 (NP(HOOC-PEG 5 QQQ-PLA 21 Q 3 Q)/siN. C. group): Each nude mouse was injected with nanoparticle NP (HOOC-PEG 5 QQQ-PLA 21 Q 3 Q) encapsulating siN.C., wherein the dose of siN.C. was 80 g.
处理 4 (NP HOOC-PEGsQQQ-PLA Q^ysifl/wB组） ： 每只裸鼠注射包载 skywfi的纳 米颗粒 NP(HOOC-PEG5QQQ-PLA21Q3Q)， 其中， skywfi的剂量为 80 g。 Treatment 4 (NP HOOC-PEGsQQQ-PLA Q^ysifl/wB group): Each nude mouse was injected with nanoparticle NP (HOOC-PEG 5 QQQ-PLA 21 Q 3 Q) containing skywfi, wherein the dose of skywfi was 80 g.
处理 5 (NP(Gal-PEG5(xxrPLA21Q3QysiN.C.组） ： 每只裸鼠注射包载 siN.C.的纳米颗粒Treatment 5 (NP (Gal-PEG 5 (xxrPLA 21 Q 3 QysiN.C. group): Each nude mouse was injected with nanoparticles containing siN.C.
NP(Gal-PEG50oo-PLA2io3o), 其中， siN.C.的剂量为 80 g。 NP (Gal-PEG 50 oo-PLA 2 io3o), wherein the dose of siN.C. is 80 g.
处理 6 (NPiGal-PEGsQQQ-PLA Q^yskywB组）： 每只裸鼠注射包载 skywfi的纳米颗 粒 NP(Gal-PEG5QQQ-PLA21Q3Q)， 其中， skywfi的剂量为 80 g。 Treatment 6 (NPiGal-PEGsQQQ-PLA Q^yskywB group): Each nude mouse was injected with skywfi-loaded nanoparticle NP (Gal-PEG 5 QQQ-PLA 21 Q 3 Q), wherein the dose of skywfi was 80 g.
血清中 apoB蛋白水平的检测结果如图 5D， 所有包载 siN.C.的实验对照组对细胞中 apoB mRNA的表达没有明显影响， NP(Gal-PEG50oo-PLA2103o)/siflpofi可以高效下调 apoB 蛋白表达量的 70%， 而 NPCHOOC-PEGsfxxrPLA ^yskywB实验组靶基因表达只能下调 35% , 说明 apoB 表达下调是由于序列特异性的基因沉默造成的。 说明本发明提供的 siRNA靶向纳米颗粒在体内能够更有效地沉默肝细胞基因的表达， 具有应用潜力。 2、 抗体修饰、 包载 siRNA的纳米颗粒构建靶向的 siRNA给药系统 The results of serum apoB protein levels are shown in Figure 5D. All experimental groups containing siN.C. had no significant effect on the expression of apoB mRNA in cells. NP (Gal-PEG 50 oo-PLA 210 3o)/siflpofi can be highly effective. The expression of apoB protein was down-regulated by 70%, while the target gene expression of NPCHOOC-PEGsfxxrPLA^yskywB experimental group was only down-regulated by 35%, indicating that down-regulation of apoB expression was caused by sequence-specific gene silencing. The siRNA targeting nanoparticle provided by the invention can more effectively silence the expression of hepatocyte genes in vivo, and has potential application. 2. Antibody-modified, siRNA-loaded nanoparticles to construct a targeted siRNA delivery system
如上所述，为了提高该 siRNA给药系统在疾病治疗应用中的潜力，本发明进一步利 用单链片段抗体修饰来发展另一种可以靶向的 siRNA给药系统。 单链片段抗体  As described above, in order to increase the potential of the siRNA delivery system in disease therapeutic applications, the present invention further utilizes single-stranded fragment antibody modification to develop another targeted siRNA delivery system. Single-stranded fragment antibody
(Anti-Her2 scFv-cys )能够特异性的识别乳腺癌细胞表面的 HER2受体， 因此本发明通 过在纳米颗粒表面修饰这种单链片段抗体， 从而制备一种靶向给药系统。  (Anti-Her2 scFv-cys) is capable of specifically recognizing the HER2 receptor on the surface of breast cancer cells, and thus the present invention prepares a targeted drug delivery system by modifying the single-stranded fragment antibody on the surface of the nanoparticles.
单链片段抗体修饰、包载 siRNA的纳米颗粒是利用马来酰亚胺基团修饰的 PEG-PLA Single-stranded fragment antibody-modified, siRNA-loaded nanoparticles are PEG-PLA modified with maleimide groups
( Mal-PEG-PLA) 和阳离子脂质通过双乳化方法包载 siRNA纳米颗粒、 然后再将单链 片段抗体键合到纳米颗粒表面的马来酰亚胺基团而得到靶向的给药系统。 (Mal-PEG-PLA) and cationic lipids entrap siRNA nanoparticles by double emulsion method, and then single chain The fragment antibody binds to the maleimide group on the surface of the nanoparticle to give a targeted delivery system.
其中， Mal-PEG-PLA 是以端基分别为马来酰亚胺基团和羟基的异官能团的聚乙二 醇为引发剂， 在本体条件下利用聚乙二醇端羟基引发丙交酯单体单体聚合得到 Mal-PEG-PLA。 通过调节聚乙二醇与丙交酯投料比， 理论上可以得到不同分子量的 Gal-PEG-PLA。 异辛酸亚锡因其高催化效率及无毒性， 是被最广泛应用的内酯及交酯环 状单体开环聚合反应的催化剂， 已被美国 FDA批准作为食品添加剂。  Among them, Mal-PEG-PLA is a hetero-functional polyethylene glycol with terminal groups of maleimide group and hydroxyl group as initiator, and the lactide is initiated by using polyethylene glycol terminal hydroxyl group under bulk conditions. The bulk monomer is polymerized to obtain Mal-PEG-PLA. By adjusting the ratio of polyethylene glycol to lactide, it is theoretically possible to obtain Gal-PEG-PLA of different molecular weight. Because of its high catalytic efficiency and non-toxicity, stannous isooctylate is the most widely used catalyst for ring-opening polymerization of lactone and lactide ring-shaped monomers, and has been approved by the US FDA as a food additive.
具体合成步骤如下： 首先， 将进行反应的圆底烧瓶经多次的抽真空干燥和充氮气处 理后， 放入手套箱。 然后， 按 Mal-PEG5。。。-OH/丙交酯 /Sn(Oct)2=l/360/0.1的摩尔配比进 行投料。 向烧瓶中加入 HOOC-PEG5(KKrOH、 单体和异辛酸亚锡， 在 130°C搅拌下反应。 反应 24小时后，将产物移出手套箱，用少量的 CH2C12溶解，将溶液沉淀到冷的乙醚中， 重复两次， 收集沉淀物， 用油泵抽干至恒重为止， 即得产物 （Mal-PEG-PLA) 。 The specific synthesis steps are as follows: First, the round bottom flask subjected to the reaction is subjected to a plurality of vacuum drying and nitrogen treatment, and then placed in a glove box. Then, press Mal-PEG 5 . . . The molar ratio of -OH/lactide/Sn(Oct) 2 = l/360/0.1 was charged. To the flask was added HOOC-PEG 5 ( KKrOH, monomer and stannous isooctanoate, and reacted under stirring at 130 ° C. After 24 hours of reaction, the product was removed from the glove box and dissolved with a small amount of CH 2 C 1 2 to precipitate the solution. In a cold diethyl ether, the mixture was repeated twice, and the precipitate was collected and dried with an oil pump until constant weight to obtain a product (Mal-PEG-PLA).
图 6A为合成得到的 Mal-PEGscxxrPLA^o™ (下标为分子量） 核磁共振氢谱结果， 分 析如下： 字母 a到 d标记了归属 PEG-PLA的所有质子信号， 聚乳酸的分子量通过 5.17 ppm 的多重峰 （归属于聚乳酸的 -C(0)OC - ) 与 3.63 ppm的单峰 （归属于聚乙二醇的 -CH2CH2- ) 的积分面积比计算得到。  Figure 6A shows the results of NMR spectroscopy of the synthesized Mal-PEGscxxrPLA^oTM (subscript molecular weight). The analysis is as follows: The letters a to d mark all proton signals belonging to PEG-PLA, and the molecular weight of polylactic acid passes 5.17 ppm. The multiple peaks (-C(0)OC - attributed to polylactic acid) were calculated from the integrated area ratio of a single peak of 3.63 ppm (-CH2CH2- assigned to polyethylene glycol).
如实施例 3和 4可知，当各组分质量比例为 siRNA/阳离子脂质 /聚合物 =0.2/1.0/25.0、 使用的阳离子脂质为 BHEM-Chol时， 制备得到的 siRNA给药系统能够很好地将 siRNA 输运到组织细胞中， 并发挥作用。 因此， 本发明同样以此配方来来研究此靶向给药系统 的生物效应。 其中， siRNA/阳离子脂质 /聚合物的质量比为 0.2/1.0/25.0， 使用的 siRNA 分别为 siPlkl 和 siN.C.， 阳离子脂质为 BHEM-Chol。 使用的聚合物为 Mal-PEG5。。。-PLA22。7。，制备得到的包载 siRNA的纳米颗粒为 NP(Mal-PEG 5000"PLA2207o)° 得到包载 siRNA的纳米颗粒为 NP(Mal-PEG5(KKrPLA22Q7Q)再进一步单链片段抗体键 合到纳米颗粒表面的马来酰亚胺基团而得到靶向的 siRNA 给药系统 NP(scFv-PEG5。。。-PLA22。7。)。 具体实验步骤如下： 向 1.5 mL 离心管中加入 6.25 μ§ 的 Anti-Her2 scFv-cys禾 B 1.0 mg包载 siRNA纳米颗粒 NP(Mal-PEG5000-PLA22070)， 其中， Anti-Her2 scFv-cys中的巯基基团和纳米颗粒表面的马来酰亚胺基团投料摩尔比为 1/10， 反应体系为 pH=6.8的 PB缓冲液， 终体积为 1.0 mL。 室温下以 40 rpm的转速在旋转式 混匀仪上反应。反应 4小时后， 5000 rpm离心 20 min。收集上清， 并使用 1.0 mL pH=6.8 的 PB(O.OIM) 缓 冲 液 重 悬 沉 淀 ， 得 到 的 包 载 siRNA 的 纳 米 颗 粒 NP(scFv-PEG5000-PLA a) 流式细胞检测包载 FAM-siRNA靶向纳米颗粒的细胞内吞 As can be seen from Examples 3 and 4, when the mass ratio of each component is siRNA/cationic lipid/polymer=0.2/1.0/25.0 and the cationic lipid used is BHEM-Chol, the prepared siRNA delivery system can be very Goodly transport siRNA into tissue cells and play a role. Therefore, the present invention also uses this formulation to study the biological effects of this targeted drug delivery system. The mass ratio of siRNA/cationic lipid/polymer was 0.2/1.0/25.0, the siRNA used was siPlkl and siN.C., and the cationic lipid was BHEM-Chol. The polymer used was Mal-PEG 5 . . . -PLA 22 . 7 . The prepared siRNA-loaded nanoparticles are NP (Mal-PEG 5000"PLA2207o). The siRNA-coated nanoparticles are obtained as NP (Mal-PEG 5 ( KKrPLA 22Q7Q ) and further single-stranded fragment antibody is bonded to the nanoparticles. maleimide groups on the surface of the targeted delivery system to give siRNA NP-specific experimental steps (scFv-PEG 5 ...- PLA 22 7..):. 6.25 μ § was added to 1.5 mL centrifuge tube Anti-Her2 scFv-cys Wo B 1.0 mg coated siRNA nanoparticle NP (Mal-PEG 5000 -PLA 22070 ), wherein the thiol group in Anti-Her2 scFv-cys and the maleimide on the surface of the nanoparticles The molar ratio of the group was 1/10, the reaction system was PB buffer with pH=6.8, and the final volume was 1.0 mL. The reaction was carried out at 40 rpm on a rotary mixer at room temperature. After 4 hours, 5000 rpm. Centrifuge for 20 min. Collect the supernatant and resuspend the pellet using 1.0 mL of PB(O.OIM) buffer pH=6.8 to obtain the siRNA-coated nanoparticle NP (scFv-PEG 5000 -PLA a) flow cytometry. Endocytosis of FAM-siRNA-targeted nanoparticles
以 上 述 体 系 制 备 包 载 FAM-siRNA 的 NP(Mal-PEG5000-PLA22070) 和 NPCscFv-PEG5。。。-PLA22。7。)来研究此靶向给药体系的细胞内吞。 将 BT474 细胞 （HER2 受体高表达细胞系， 购于中国科学院典型培养物保藏委员会细胞库） 以 5x l04细胞 /孔的 密度接种于 24孔板， 37°C培养 2小时后， 除去培养基， 分别进行如下处理， 在各组处 理中， 将培养基更换成如下各组处理中的溶液。 FAM-siRNA-encapsulated NP (Mal-PEG 5000 - PLA 22070 ) and NPCscFv-PEG 5 were prepared in the above system. . . -PLA 22 . 7 . To study the endocytosis of this targeted drug delivery system. BT474 cells (HER2 receptor high expression cell line purchased from the Chinese Academy of Sciences' Type Culture Collection Cell Bank) at 5x10 4 cells/well The density was inoculated into a 24-well plate, and after culturing at 37 ° C for 2 hours, the medium was removed, and the following treatments were carried out, respectively. In each group of treatments, the medium was replaced with the solution in each of the following groups.
处理 1 (PBS ) ： 将 50 L的 PBS溶液加入到 450μ 的新鲜 DMEM培养基中。 处理 2 (FAM-siR A) ： 加入等体积的 FAM-siR A溶液， 其中， FAM-siR A的 终浓度为 200 nM。  Treatment 1 (PBS): 50 L of PBS solution was added to 450 μl of fresh DMEM medium. Treatment 2 (FAM-siR A): An equal volume of FAM-siR A solution was added, wherein the final concentration of FAM-siR A was 200 nM.
处理 3 ( NP(Mal-PEG5ooo-PLA2207o)组） ： 包载 FAM-siRNA 的纳米颗粒 NP(Mal-PEG5QQQ-PLA22Q7Q)加入到 450μ 的新鲜 DMEM培养基中， 其中， FAM-siRNA 终浓度为 200 nM。 Treatment 3 (NP (Mal-PEG 5 ooo-PLA 220 7o) group): Nanoparticle NP (Mal-PEG 5 QQQ-PLA 22 Q 7 Q) containing FAM-siRNA was added to 450 μl of fresh DMEM medium. Among them, the final concentration of FAM-siRNA was 200 nM.
处理 4 ( NP(scFv-PEG5ooo-PLA2207o)组） ： 包载 FAM-siRNA 的纳米颗粒 NP(SCFV-PEG5QQQ-PLA22Q7Q)加入到 450μ 的新鲜 DMEM培养基中， 其中， FAM-siRNA 终浓度为 200 nM。 Treatment 4 (NP(scFv-PEG 5 ooo-PLA 220 7o) group): Nanoparticle NP (SCFV-PEG 5 QQQ-PLA 22 Q7Q) containing FAM-siRNA was added to 450 μl of fresh DMEM medium, The final concentration of FAM-siRNA was 200 nM.
处理 5 ( NP(scFv-PEG50oo-PL A2207o)+ Anti-Her2 scFv-cys组） ： 包载 FAM-siRNA 的纳米颗粒 NP(scFv-PEG5。。。-PLA22。7。)加入到 450μ 的新鲜 DMEM培养基中， 并加入 靶向抑制剂 Anti-Her2 scFv-cys (终浓度 7 μΜ)，其中， FAM-siRNA终浓度为 200 nM。 Treatment 5 (NP(scFv-PEG 50 oo-PL A 2 207o) + Anti-Her2 scFv-cys group): Nanoparticle NP-encapsulated FAM-siRNA (scFv-PEG 5 . . . -PLA 22 . 7 .) Add to 450 μl of fresh DMEM medium and add the targeting inhibitor Anti-Her2 scFv-cys (final concentration 7 μΜ) with a final concentration of FAM-siRNA of 200 nM.
分别经过以上几种条件处理 1小时后，消化收集各组细胞，用流式细胞仪 (FACS，BD After 1 hour of treatment under the above conditions, each group of cells was digested and collected by flow cytometry (FACS, BD).
Bioscience, Bedford, MA) 方法分析细胞吞噬纳米颗粒， 结果见图 6B。 Bioscience, Bedford, MA) Methods Cellular phagocytosis of nanoparticles, the results are shown in Figure 6B.
从图 6B 可知， FAM-siRNA 基本不能够进入细胞， 靶向给药体系 NP(scFv-PEG5ooo-PLA2207o)和非靶向给药体系 NP(Mal-PEG5()(xrPLA22()7())均可以将 FAM-siRNA 成功输运到细胞中。 其中， 靶向给药体系实验组检测到细胞内的 FAM-siRNA 荧光信号明显强于非靶向给药体系， 说明该单链片段抗体 Anti-Her2 scFv-cys修饰的纳米颗粒能够更好的将 siR A输运到 HER2受体高表达的乳腺癌细胞系 BT474。 同时， 加入靶向抑制剂 Anti-Her2 scFv-cys (终浓度为 7 μΜ ) 的实验组 (NP(scFV-PEG5QQQ-PLA22Q7Q)+ Anti-Her2 scFv-cys组）， 荧光信号较低， 和非靶向实验组 NP(Mal-PEG5QQQ-PLA22Q7Q)相当。 这一现象说明这种单链片段抗体 Anti-Her2 scFv-cys修 饰的纳米颗粒是通过纳米颗粒表面的片段抗体与癌细胞表面 HER2受体特异性结合介导 纳米颗粒的内吞， 加入靶向抑制剂 Anti-Her2 scFv-cys后， 纳米颗粒不能够通过抗体与 受体特异性结合介导纳米颗粒的内吞， 所以输运的 FAM-siRNA降低， 导致荧光信号降 低。 As can be seen from Fig. 6B, FAM-siRNA is substantially incapable of entering cells, targeting the drug delivery system NP (scFv-PEG 5 ooo-PLA 220 7o) and the non-targeted drug delivery system NP (Mal-PEG 5() ( xrPLA 22 ( 7() ) can successfully transport FAM-siRNA into cells. Among them, the experimental group of targeted drug delivery system detected that the intracellular FAM-siRNA fluorescence signal was significantly stronger than the non-targeted drug delivery system, indicating that the single The chain fragment antibody Anti-H e r2 scFv-cys modified nanoparticles can better transport the siR A to the HER2 receptor-expressing breast cancer cell line BT474. At the same time, the targeting inhibitor Anti-Her2 scFv-cys is added. (final concentration of 7 μΜ) in the experimental group (NP (scF V -PEG 5 QQQ-PLA 22 Q 7 Q) + Anti-Her2 scFv-cys group), low fluorescence signal, and non-targeted experimental group NP (Mal -PEG 5 QQQ-PLA 22 Q 7 Q) is equivalent. This phenomenon indicates that the single-stranded fragment antibody Anti-Her2 scFv-cys modified nanoparticles are specific to the HER2 receptor on the surface of cancer cells by fragment antibody on the surface of the nanoparticles. after binding mediated endocytosis of the nanoparticles added targeted inhibitors Anti-H e r2 scFv-cys , the nanoparticles can not by antibody to the receptor Laid Binding mediated endocytosis of the nanoparticles, is reduced transport FAM-siRNA, resulting in decreased fluorescence signal.
从细胞内吞结果可知，这种经过单链片段抗体修饰后聚合物制备的包载 siRNA纳米 颗粒具有靶向能力。 同样， 这种配体修饰可以是单链片段抗体修饰而不限于此， 例如转 铁蛋白等。 b) 靶向纳米颗粒输运 siRNA在细胞水平沉默靶基因表达  From the results of endocytosis, the encapsulated siRNA nanoparticle prepared by the polymer modified by the single-stranded fragment antibody has a targeting ability. Also, such a ligand modification may be a single-chain fragment antibody modification without being limited thereto, such as transferrin or the like. b) targeting nanoparticle transport siRNA silencing target gene expression at the cellular level
Plkl有助于促进和加速哺乳动物细胞有丝分裂， 并在多种肿瘤细胞中高表达。通过 沉默其表达， 可以抑制肿瘤生长； 本发明通过沉默 BT474细胞的 Plkl基因表达来评价 该单链片段抗体 Anti-Her2 scFv-cys修饰的靶向纳米颗粒可以更好的输运 siRNA进入乳 腺癌细胞，并成功释放 siRNA,沉默靶基因的表达。以上述体系制备包载 siPlkl和 siN.C. 的 NP(Mal-PEG5QQ(rPLA22Q7())和 NP(scFV-PEG5Q(XrPLA22()7())来研究此靶向给药体系在细胞 水平沉默靶基因的效果。 Plkl helps promote and accelerate mitosis in mammalian cells and is highly expressed in a variety of tumor cells. By silencing its expression, tumor growth can be inhibited; the present invention evaluates the single-stranded fragment antibody Anti-Her2 scFv-cys modified targeting nanoparticle by silencing the expression of Plk1 gene of BT474 cells to better transport siRNA into milk Adenocarcinoma cells, and successfully release siRNA, silence the expression of target genes. The targeted system was prepared by preparing NP (Mal-PEG 5QQ ( rPLA 22Q7() ) and NP (scF V -PEG 5Q (X rPLA 22()7() )) containing siPlkl and siN.C. in the above system. The system silences the effect of the target gene at the cellular level.
将 BT474细胞以 l x lO5细胞 /孔的密度接种于 6孔板， 37°C培养 24小时后， 除去培 养基， 分别进行如下处理， 在各组处理中， 将培养基更换成以下各组处理中的溶液， 每 个处理组设置 1个复孔。 BT474 cells were seeded at a density of 1×10 5 cells/well in a 6-well plate, cultured at 37° C. for 24 hours, and then the medium was removed, and the following treatments were carried out, respectively, and the medium was replaced with the following groups in each treatment. In the solution, one duplicate hole is set for each treatment group.
处理 1 (PBS ) ： 100 μ 的 PBS溶液加入到 1.9 mL的新鲜 DMEM培养基中。 。 处理 2 (Free siPlkl组） ： 加入单独的 siPlld溶液 100 μL, 其中， siPlld的终浓度 为 200 nM。  Treatment 1 (PBS): 100 μl of PBS solution was added to 1.9 mL of fresh DMEM medium. . Treatment 2 (Free siPlkl group): Add 100 μL of a separate siPlld solution, wherein the final concentration of siPlld is 200 nM.
处理 3 (Lipofectamine组） ： 将 5 的 Lipofectamine 2000用无菌水稀释到 50 后， 加入到 50 μL含 100 pmol siPlkl的水溶液中， 室温下结合 15分钟后， 将此 100 μL 溶液加入到 1.9 mL新鲜培养基中。 其中， siPlkJ终浓度为 50 nM。  Treatment 3 (Lipofectamine group): Dilute 5 Lipofectamine 2000 to 50 with sterile water, add to 50 μL of an aqueous solution containing 100 pmol of siPlkl, and after combining for 15 minutes at room temperature, add 100 μL of the solution to 1.9 mL of fresh In the medium. Among them, the final concentration of siPlkJ was 50 nM.
处理 4 (NP(Mal-P EG5QQQ-PLA22Q7Q)/siN.C.组） ： 100 的包载 siN.C.的靶向纳米颗 粒 NP(Mal-PEG5。。。-PLA22。7。)加入到 1.9 mL的新鲜 DMEM培养基中， 其中， siN.C.的终 浓度为 200 nM。 Treatment 4 (NP(Mal-P EG 5 QQQ-PLA 22 Q 7 Q)/siN.C. group): 100-packed siN.C.-targeted nanoparticle NP (Mal-PEG 5 . . . - PLA 22. 7 )) Add to 1.9 mL of fresh DMEM medium with a final concentration of siN.C. of 200 nM.
处理 5 (NP(Mal-PEG5ooo-PLA2207o)/siP/W组） ： 100 μ 的包载 siPlkl的非靶向纳米 颗粒 NP(Mal-PEG5。。。-PLA22。7。)加入到 1.9 mL的新鲜 DMEM培养基中， 其中， siPlld的 终浓度为 200 nM。 Treatment 5 (NP(Mal-PEG 5 ooo-PLA 220 7o)/siP/W group): 100 μ of non-targeted nanoparticle NP (Mal-PEG 5 . . . -PLA 22. 7 ) containing siPlkl. Add to 1.9 mL of fresh DMEM medium with a final concentration of si n npl of 200 nM.
处理 6 (NP(scFv-PEG5000-PLA22070)/siN.C.组） ： 100 的包载 siN.C.的靶向纳米颗 粒 NP(scFv-PEG5。。。-PLA22。7。)加入到 1.9 mL的新鲜 DMEM培养基中， 其中， siN.C.的终 浓度为 200 nM。 Processing 6 (NP (scFv-PEG 5000 -PLA 22070) /siN.C Group.): 100 package carrier siN.C. targeted nanoparticle NP (. ScFv-PEG 5 ...- PLA 22 7.) Add to 1.9 mL of fresh DMEM medium with a final concentration of siN.C. of 200 nM.
处理 7 (NP(scFv-PEG5ooo-PLA2207o)/siP/W组）： 100 μ 的包载 siPlkl的靶向纳米颗 粒 NP(scFv-PEG5。。。-PLA22。7。)加入到 1.9 mL的新鲜 DMEM培养基中，其中， siPlkl的终 浓度为 200 nM。 Treatment 7 (NP(scFv-PEG 5 ooo-PLA 220 7o)/siP/W group): 100 μ of the coated nanoparticle NP (scFv-PEG 5 . . . -PLA 22 . 7 ) To 1.9 mL of fresh DMEM medium, the final concentration of siPlkl was 200 nM.
在转染培养 24 h后， 用 R easy mini-kits ( Qiagen, Valencia, CA) 提取细胞中的总 After 24 h of transfection, extract total cells from the cells using R easy mini-kits (Qiagen, Valencia, CA)
R A, 用紫外分光光度计测定提取的 R A样品的 OD28Q和 OD26Q的吸光度， 并利用公 式： R A 浓度 （μ§/μί ) = 0.04xOD26Qx稀释倍数， 计算 R A 样品的浓度， 然后用 PrimeScript™ 1st Strand cDNA Synthesis Kit ( Takara, Dalian, China)合成 cDNA, 每个样 品使用 2 μg总 mRNA。 在合成 cDNA后， 按照 SYBR® Premix Ex Taq™ (Takara)试剂 盒进行实时荧光定量 PCR反应。 其中， Plkl和甘油三磷酸脱氢酶 GAPDH基因的 PCR 引物如 SEQ ID No: 5至 SEQ ID No: 8所示。 RA, the absorbance of OD 28Q and OD 26Q of the extracted RA sample was measured by an ultraviolet spectrophotometer, and the concentration of the RA sample was calculated using the formula: RA concentration (μ § /μί ) = 0.04xOD 26Q x dilution factor, and then using PrimeScript cDNA was synthesized by TM 1st Strand cDNA Synthesis Kit ( Takara, Dalian, China), and 2 μg of total mRNA was used per sample. After cDNA synthesis, real-time PCR was performed according to the SYBR® Premix Ex TaqTM (Takara) kit. Among them, PCR primers for the Plk1 and glycerol triphosphate dehydrogenase GAPDH genes are shown in SEQ ID No: 5 to SEQ ID No: 8.
PCR反应条件如下：  The PCR reaction conditions are as follows:
1 ) 95 °C加热变性 30秒。  1) Heat denaturation at 95 °C for 30 seconds.
2) 95 °C加热变性 5秒。  2) Heat denaturation at 95 °C for 5 seconds.
3 ) 60°C加热退火 30秒。 30个循环  3) Heat annealing at 60 ° C for 30 seconds. 30 cycles
4) 95 °C加热变性 15秒。 5 ) 95 °C加热变性 60秒。 4) Heat denaturation at 95 °C for 15 seconds. 5) Heat denaturation at 95 °C for 60 seconds.
6 ) 60 °C加热退火 15秒。  6) Heat annealing at 60 °C for 15 seconds.
利用 2- MCT对不同实验组中 Plkl基因表达差异进行了分析，其中以 GAPDH为内参， 分析不同实验组中 Plkl基因的表达水平。 以 PBS实验组为 100%，其他实验组表达表示 为相对于 PBS组表达量， 实验结果见图 6C。 2- MCT Plkl using differences in gene expression in the different experimental groups were analyzed, which GAPDH as internal control, expression level of the different experimental groups Plkl gene. The expression in the PBS experimental group was 100%, and the expression in the other experimental groups was expressed relative to the PBS group. The experimental results are shown in Fig. 6C.
图 6C中，不经过处理的 PBS组细胞内 Plkl的表达量高，包载 siPlkJ的实验组均可 以有效抑制 Plkl 基因的表达。 但是， 在相同剂量的 siPlkl ( 200 nM ) 情况下， NP(scFv-PEG5ooo-PLA2207o)/siP^ 组可以成功沉默 PM 基因表达高达 70%， 明显优于 NP(Mal-PEG5ooo-PLA2207o)/siP^ 实验组 （40%沉默效率） 。 而所有包载 siN. C.的实验对 照组对细胞中 Plkl mR A表达没有明显影响。 说明 Plkl表达下调是由于序列特异性的 基因沉默造成的。 c) 靶向纳米颗粒输运 siRNA对乳腺癌生长抑制 In Fig. 6C, the expression level of Plk1 in the untreated PBS group was high, and the experimental group containing siPlkJ could effectively inhibit the expression of Plk1 gene. However, in the same dose of siPlkl (200 nM), the NP (scFv-PEG 5 ooo-PLA 220 7o)/siP^ group can successfully silence PM gene expression up to 70%, which is significantly better than NP (Mal-PEG 5 ooo). -PLA 220 7o)/siP^ experimental group (40% silencing efficiency). All experimental controls containing siN.C. had no significant effect on Plkl mR A expression in cells. This indicates that down-regulation of Plkl expression is due to sequence-specific gene silencing. c) targeting nanoparticle transport siRNA for breast cancer growth inhibition
通过将 siP/W的靶向给药系统和非靶向给药系统经尾静脉注入小鼠体内，通过体内 实验观察该载体作为靶向 siRNA给药系统的有效性和靶向性。  The effectiveness and targeting of the vector as a targeted siRNA delivery system was observed by in vivo experiments by injecting the siP/W targeted drug delivery system and the non-targeted drug delivery system into the mouse via the tail vein.
在裸鼠第二个乳腺的脂肪垫下原位接种 BT474细胞 （0.5X 107) ， 10天左右形成可 见肿瘤， 肿瘤体积约 50 mm3， 将裸鼠随机分成六组， 每组 8只， 进行尾静脉注射治疗， 每三天给药一次， 给药方法如下： BT474 cells (0.5×10 7 ) were inoculated in situ under the fat pad of the second mammary gland of nude mice. The tumor was formed in about 10 days. The tumor volume was about 50 mm 3 . The nude mice were randomly divided into six groups of 8 rats each. The tail vein injection is administered once every three days. The administration method is as follows:
处理 1 ( PBS对照组） ： 每只裸鼠注射 250 L的 PBS。  Treatment 1 (PBS control group): Each nude mouse was injected with 250 L of PBS.
处理 2 (裸 siPlkl组） ： 每只裸鼠注射 250 μ 的裸 siPlkl， 其中， siPU 剂量为 20 g。  Treatment 2 (naked siPlkl group): Each nude mouse was injected with 250 μl of bare siPlkl, wherein the siPU dose was 20 g.
处理 3 (NP(Mal-PEG5(KKrPLA22Q7QysiN. C.组）： 每只裸鼠注射包载 siN. C.的纳米颗粒 NP(Mal-PEG5。。。-PLA22。7。)， 其中， siN. C.剂量为 20 g。 Processing 3 (NP (Mal-PEG 5 (KKrPLA 22Q7Q ysiN C. Group): each nude mouse injected entrapped siN C. nanoparticles NP (Mal-PEG 5 ...- PLA 22 7),... Among them, the dose of siN. C. was 20 g.
处理 4
Figure imgf000028_0001
siP/L?组） ： 每只裸鼠注射包载 siP/W的纳米颗 粒 NP(Mal-PEG5QQQ-PLA22Q7Q)， 其中， siP/W剂量为 20 g。 Processing 4
Figure imgf000028_0001
siP/L? group): Each nude mouse was injected with siP/W-loaded nanoparticle NP (Mal-PEG 5 QQQ-PLA 22 Q 7 Q), wherein the siP/W dose was 20 g.
处理 5 (NP(scFv-PEG5(xKrPLA22Q7Q)/siN. C.组） ： 每只裸鼠注射包载 siN. C.的纳米颗 粒 （NP(scFv-PEG5(KKrPLA22Q7Q)， 其中， siN. C.剂量为 20 g。 Treatment 5 (NP(scFv-PEG 5( xKrPLA 22 Q 7 Q)/siN. C. group): Each nude mouse was injected with nanoparticles containing siN. C. (NP (scFv-PEG 5 ( KKrPLA 22 Q 7) Q), wherein the dose of siN. C. is 20 g.
处理 6 (NP^cFv-PEGsooQ-PLA^TOysiP/L?组） ： 每只裸鼠注射包载 siP/L?的纳米颗 粒 NP(scFv-PEG5。。。-PLA22。7。)， 其中， siP/W剂量为 20 g。 Treatment 6 (NP^cFv-PEGsooQ-PLA^TOysiP/L? group): Each nude mouse was injected with a nanoparticle NP (scFv-PEG 5 . . . -PLA 22 . 7 ) containing siP/L? The siP/W dose is 20 g.
在治疗开始后， 每隔一天对肿瘤体积进行测量。 结果如图 6D所示， 所有 PBS组以 及所有阴性对照组中肿瘤生长速度均较快， 而在使用包载 siPlkl 纳米颗粒 NP(Mal-PEG5QQQ-PLA22Q7Q)和 NP(SCFV-PEG5QQQ-PLA22Q7Q)治疗组中， 肿瘤生长速度与阴性 对照组相比受到明显抑制， 但是 NP(SCFV-PEG5。。。-PLA22Q7Q)治疗组能够更显著的抑制肿 瘤的生长，说明单链片段抗体修饰的 siRNA靶向给药系统能够更有效的治疗 HER2阳性 的乳腺癌。 所有包载 siN. C.的实验对照组肿瘤的生长速度并未受到影响。 对肿瘤生长的 抑制是由于序列特异性的基因沉默造成的。 从实施例 4可知，此给药系统可以利用靶向修饰嵌段聚合物和阳离子脂质通过双乳 化的方法制备靶向的 siR A给药系统，也可以利用嵌段聚合物和阳离子脂质通过双乳化 的方法先制备出 siRNA纳米颗粒后， 再进行靶向修饰而制备靶向的 siRNA给药系统。 并且靶向基团可以是小分子的靶向基团如半乳糖、 叶酸、 含精氨酸 -甘氨酸-天冬氨酸 (RGD) 的短肽等， 也可以是片段抗体、 转铁蛋白抗体等。 Tumor volume was measured every other day after the start of treatment. As a result, as shown in Fig. 6D, tumor growth rates were faster in all PBS groups and in all negative control groups, while using siPlkl nanoparticles NP (Mal-PEG 5 QQQ-PLA 22 Q 7 Q) and NP (SCFV-) were used. PEG 5 QQQ-PLA 22 Q 7 Q) In the treatment group, the tumor growth rate was significantly inhibited compared with the negative control group, but the NP ( SC F V -PEG 5 . . . -PLA 22Q7Q ) treatment group was able to inhibit more significantly. Tumor growth indicates that single-stranded fragment antibody-modified siRNA-targeted drug delivery systems are more effective in treating HER2-positive breast cancer. The growth rate of all tumors in the experimental control group containing siN.C. was not affected. Inhibition of tumor growth is due to sequence-specific gene silencing. As can be seen from Example 4, the drug delivery system can utilize a targeted modified block polymer and a cationic lipid to prepare a targeted siR A delivery system by double emulsification, or can utilize block polymers and cationic lipids. The double emulsification method first prepares siRNA nanoparticles, and then performs targeted modification to prepare a targeted siRNA delivery system. And the targeting group may be a targeting group of a small molecule such as galactose, folic acid, a short peptide containing arginine-glycine-aspartate (RGD), or a fragment antibody, a transferrin antibody, or the like. .

Claims

权利要求 Rights request
1、 一种载体组合物， 其特征在于， 该载体组合物含有两亲性高分子聚合物和阳离 子脂质。 A carrier composition characterized in that the carrier composition contains an amphiphilic polymer and a cationic lipid.
2、 根据权利要求 1所述的载体组合物， 其中， 以载体组合物的总重量为基准， 所 述两亲性高分子聚合物的含量为 83.3-99.999重量％， 阳离子脂质的含量为 0.001 -16.7重 量0 /0。 The carrier composition according to claim 1, wherein the amphiphilic high molecular polymer is contained in an amount of 83.3 to 99.999% by weight and the cationic lipid content is 0.001 based on the total weight of the carrier composition. -16.7 weight 0 / 0 .
3、 根据权利要求 1或 2所述的载体组合物， 其中， 所述阳离子脂质为铵盐型的阳 离子脂质。 The carrier composition according to claim 1 or 2, wherein the cationic lipid is an ammonium salt type cation lipid.
4、 根据权利要求 3所述的载体组合物， 其中， 所述阳离子脂质选自 N，N-二羟乙基 甲基 -N-2- (胆固醇氧羰基氨基）乙基溴化铵、 （2,3-二油氧基丙基)三甲基氯化铵和4. The carrier composition according to claim 3, wherein the cationic lipid is selected from the group consisting of N,N-dihydroxyethylmethyl-N-2-(cholesteryloxycarbonylamino)ethylammonium bromide, ( 2,3-dioleyloxypropyl)trimethylammonium chloride and
N-(l-(2，3-二油酰氧基)丙基) -N，N，N-三甲基氯化铵中的至少一种。 At least one of N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride.
5、 根据权利要求 1 -4中任意一项所述的载体组合物， 其中， 所述两亲性高分子聚 合物为含有亲水性嵌段和疏水性嵌段的嵌段共聚物； 所述亲水性嵌段选自聚乙二醇嵌 段、聚乙烯醇嵌段、聚乙烯吡咯烷酮嵌段、聚丙烯酰胺嵌段、聚甲基丙烯酸氨基酯嵌段、 聚丙烯酸嵌段和聚甲基丙烯酸嵌段中的至少一种； 所述疏水性嵌段选自聚乳酸嵌段、 聚 己内酯嵌段、 聚乙交酯嵌段、 聚氨基酸嵌段和聚磷腈嵌段中的至少一种。 The carrier composition according to any one of claims 1 to 4, wherein the amphiphilic high molecular polymer is a block copolymer containing a hydrophilic block and a hydrophobic block; The hydrophilic block is selected from the group consisting of polyethylene glycol blocks, polyvinyl alcohol blocks, polyvinylpyrrolidone blocks, polyacrylamide blocks, polyalkyl methacrylate blocks, polyacrylic acid blocks, and polymethacrylic acid. At least one of the blocks; the hydrophobic block is selected from at least one of a polylactic acid block, a polycaprolactone block, a polyglycolide block, a polyamino acid block, and a polyphosphazene block. .
6、 根据权利要求 5所述的载体组合物， 其中， 所述两亲性高分子聚合物选自聚乙 二醇-聚乳酸两嵌段共聚物、 聚乙二醇-聚乳酸三嵌段共聚物、 聚乙二醇-聚 （乳酸 -乙醇 酸） 两嵌段共聚物和聚乙二醇-聚 （乳酸-乙醇酸） 三嵌段共聚物中的至少一种。 The carrier composition according to claim 5, wherein the amphiphilic polymer is selected from the group consisting of polyethylene glycol-polylactic acid diblock copolymer and polyethylene glycol-polylactic acid triblock copolymer. At least one of a polyethylene glycol-poly(lactic-glycolic acid) diblock copolymer and a polyethylene glycol-poly(lactic-glycolic acid) triblock copolymer.
7、 根据权利要求 6 所述的载体组合物， 其中， 聚乙二醇嵌段的数均分子量为 550-10000 g/mol o 7. The carrier composition according to claim 6, wherein the polyethylene glycol block has a number average molecular weight of 550 to 10000 g/mol.
8、 根据权利要求 6 所述的载体组合物， 其中， 聚乳酸嵌段的数均分子量为 4800-51000 g/mol o The carrier composition according to claim 6, wherein the polylactic acid block has a number average molecular weight of 4,800 to 5,1000 g/mol.
9、 根据权利要求 6所述的载体组合物， 其中， 聚（乳酸-乙醇酸）嵌段的数均分子 量为 10000-50000 g/mol oThe carrier composition according to claim 6, wherein the poly(lactic acid-glycolic acid) block has a number average molecular weight of 10,000 to 50,000 g/mol.
10、根据权利要求 1 -9中任意一项所述的载体组合物， 其中， 所述载体组合物在水 溶液中形成直径为 50-250纳米的纳米颗粒。 The carrier composition according to any one of claims 1 to 9, wherein the carrier composition is in water Nanoparticles having a diameter of 50 to 250 nm are formed in the solution.
11、 根据权利要求 10所述的载体组合物， 其中， 所述纳米颗粒的表面具有化学修 饰、 抗体修饰和配体修饰中的至少一种。 The carrier composition according to claim 10, wherein the surface of the nanoparticles has at least one of a chemical modification, an antibody modification, and a ligand modification.
12、 权利要求 1-11中任意一项所述的载体组合物在转运外源核酸中的应用。 12. Use of the vector composition of any of claims 1-11 for the transport of exogenous nucleic acids.
13、 根据权利要求 12所述的应用， 其中， 所述外源核酸为小干扰核酸 （siR A)。 13. The use according to claim 12, wherein the exogenous nucleic acid is a small interfering nucleic acid (siR A).
14、 一种药物组合物， 其特征在于， 该药物组合物含有核酸及权利要求 1-11中任 意一项所述的载体组合物。 A pharmaceutical composition comprising a nucleic acid and the carrier composition according to any one of claims 1-11.
15、 根据权利要求 14所述的药物组合物， 其中， 该药物组合物中， 核酸、 阳离子 脂质与两亲性高分子聚合物的质量比为 0.04: 0.0001： 100.0-7.2： 20.0： 100.0。 The pharmaceutical composition according to claim 14, wherein the pharmaceutical composition has a mass ratio of the nucleic acid, the cationic lipid to the amphiphilic high molecular polymer of 0.04: 0.0001: 100.0-7.2: 20.0: 100.0.
16、根据权利要求 14或 15所述的药物组合物， 所述核酸为小干扰核酸（siR A)。 The pharmaceutical composition according to claim 14 or 15, wherein the nucleic acid is a small interfering nucleic acid (siR A).
17、权利要求 14-16中任意一项所述的药物组合物的制备方法，将两亲性高分子聚 合物和阳离子脂质溶于油相中， 加入核酸水溶液后进行第一次超声形成初始乳液， 将初 始乳液加入到水相中并进行第二次超声形成乳液， 将乳液加入到水相中， 减压下除去有 机溶剂， 离心收集纳米颗粒。 The method for preparing a pharmaceutical composition according to any one of claims 14 to 16, wherein the amphiphilic high molecular polymer and the cationic lipid are dissolved in the oil phase, and the initial ultrasonic formation is performed after the aqueous nucleic acid solution is added. The emulsion, the initial emulsion is added to the aqueous phase and subjected to a second ultrasonication to form an emulsion, the emulsion is added to the aqueous phase, the organic solvent is removed under reduced pressure, and the nanoparticles are collected by centrifugation.
18、一种核酸导入方法， 其特征在于， 通过使权利要求 14-16中任意一项所述的药 物组合物与细胞接触， 使该核酸药物组合物中的核酸导入到细胞内。 A method of introducing a nucleic acid, which comprises introducing a nucleic acid in the nucleic acid pharmaceutical composition into a cell by contacting the drug composition according to any one of claims 14-16 with a cell.
19、 权利要求 1-11中任意一项所述的载体组合物在制备抗肿瘤药物中的应用。 Use of the carrier composition according to any one of claims 1 to 11 for the preparation of an antitumor drug.
20、 根据权利要求 19所述的应用， 其中， 所述肿瘤为肝脏肿瘤或乳腺肿瘤。 20. The use according to claim 19, wherein the tumor is a liver tumor or a breast tumor.
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