WO2014005314A1 - 基于小核酸药物成骨治疗的骨靶向递送***及其制备方法 - Google Patents
基于小核酸药物成骨治疗的骨靶向递送***及其制备方法 Download PDFInfo
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- WO2014005314A1 WO2014005314A1 PCT/CN2012/078243 CN2012078243W WO2014005314A1 WO 2014005314 A1 WO2014005314 A1 WO 2014005314A1 CN 2012078243 W CN2012078243 W CN 2012078243W WO 2014005314 A1 WO2014005314 A1 WO 2014005314A1
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- GRVDJDISBSALJP-UHFFFAOYSA-N methyloxidanyl Chemical group [O]C GRVDJDISBSALJP-UHFFFAOYSA-N 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000009818 osteogenic differentiation Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
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- 229920000573 polyethylene Polymers 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 239000002342 ribonucleoside Substances 0.000 description 1
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- 238000012360 testing method Methods 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 229940040944 tetracyclines Drugs 0.000 description 1
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- 210000003462 vein Anatomy 0.000 description 1
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Classifications
-
- 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
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
- A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
-
- 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
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1277—Processes for preparing; Proliposomes
- A61K9/1278—Post-loading, e.g. by ion or pH gradient
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
Definitions
- Bone-targeted delivery system based on small nucleic acid drug osteogenic treatment and preparation method thereof
- the invention belongs to the field of molecular biology and relates to a bone targeting delivery system based on small nucleic acid drug osteogenic treatment and a preparation method thereof. Background technique
- the basic processes of bone tissue formation include bone tissue formation and bone tissue absorption. Osteoblasts and osteoclasts are responsible for bone formation and bone resorption, respectively, and the balance between the two ensures a constant bone mass in adult animals and humans.
- bone-targeted drug delivery systems are primarily used for the delivery of small molecule drugs, usually consisting of bone targeting molecules and small molecule drug/polymer compounds.
- Common bone-targeting molecules include tetracyclines, diterpenoids, and polypeptides containing aspartic acid repeats.
- These targeting molecules can be directly linked to small molecule drugs such as estradiol, ibuprofen, etc., or can be linked with polymer compounds such as polylactic acid-glycolic acid copolymer, polyethylene glycol, etc. to form a carrier system to carry drugs. .
- These bone targeting molecules mediate the distribution of bone tissue. It has been reported that bisphosphonates target both the bone forming surface and the bone resorption surface, while the aspartate repeats primarily target the bone resorption surface.
- drug delivery systems capable of specifically targeting bone formation surfaces have not been seen.
- osteoblast-like cells are mainly distributed on the surface of bone formation, and osteoblast-like cells are the main target for osteogenic treatment using small nucleic acid drugs, so specific targeting of bone-forming surfaces and osteoblast-like cells is particularly important.
- the present invention provides a bone-targeting molecule for bone-forming surfaces and osteoblast-like cells, a small nucleic acid drug that inhibits negative regulation of bone formation, and a liposome junction that facilitates transfection of small nucleic acid drugs.
- a bone-targeted delivery system based on small nucleic acid drug osteogenic treatment and a preparation method thereof.
- the invention has stronger specificity and specificity, and the small nucleic acid drug has high transfection efficiency and can achieve higher silencing efficiency.
- the present invention provides a bone-targeted delivery system based on small nucleic acid drug osteogenic treatment, comprising liposomes, a bone targeting molecule and a small nucleic acid drug, wherein the bone targeting molecule is selected from the group consisting of a sclerophyllin, SEQ ID NO: 1 (8 aspartic acid polypeptide repeats (Asp) 8 ), SEQ ID NO: 2 (6) One or more of the aspartate-serine-serine polypeptide repeats (Asp-Ser-Ser) 6 , ie (DSS) 6 ) and the aptamers screened for osteoblast-like cells, small nucleic acid drugs One or more selected from the group consisting of a small interfering ribonucleic acid (siRNA) that promotes bone formation function, a microRNA mimic of microRNA (miRNA), and a blocker of tiny ribonucleic acid (Antagomir).
- the liposome contains a non-cationic lipid or a mixture of a non-cationic lipid and a cationic lipid.
- the cationic lipid in the liposome accounts for 0% to 50% of the total lipid content, and the non-cationic lipid accounts for 50% to 100% of the total lipid content.
- Cationic lipids are a class of positively charged phospholipids, usually composed of one or several neutral lipids.
- the neutral lipid component used in the cationic liposome is similar to conventional liposomes, such as cholesterol (chol), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and the like.
- the cationic lipid is selected from the group consisting of 1,2-dioleyloxy-3-trimethylaminopropane (DOTAP), N-[l-(2,3-dioleyloxy)propyl]-N, N, N, - one or more of trimethylammonium chloride (DOTMA), and DC-cholesterol (DC-Chol).
- DOTAP 1,2-dioleyloxy-3-trimethylaminopropane
- DOTMA trimethylammonium chloride
- DC-Chol DC-cholesterol
- Non-cationic lipids include uncharged and negatively charged lipids.
- the non-cationic lipid is selected from the group consisting of dioleyldimethylammonium propane (DODAP), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), one or more of dioleoylphosphatidylcholine (DOPC) and DLin-KC2-DMA.
- DODAP dioleyldimethylammonium propane
- DOPE dioleoylphosphatidylethanolamine
- DSPC distearoylphosphatidylcholine
- DPPC dipalmitoylphosphatidylcholine
- DOPC dioleoylphosphatidylcholine
- DOPC dioleoylphosphatidylcholine
- DLin-KC2-DMA DLin-KC2-DMA
- the liposome may contain an auxiliary lipid such as neutral lipid cholesterol and/or a modifying agent such as polyethylene glycol (PEG).
- PEG-modified liposomes have good water solubility, low toxicity, and no immunogenicity. Since the PEG terminal can be modified with a reactive group such as a carboxylic acid, an amine or a maleimide group to facilitate further attachment to a bone targeting molecule, the PEG-modified lipid is suitable for use as a starter for the ligation reaction.
- the PEG-modified lipid carries a methoxy terminus, such as DSPE-mPEG.
- the bone targeting delivery system comprises distearyl betethanolamine-polyethylene glycol 2000-maleimide.
- the liposome PEG-modified lipid
- DSPE-PEG-MAL distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide
- the bone targeting molecule may be pre-diaryl acyl Phosphatidylethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG-MAL) is covalently linked and then inserted into the lipid bilayer in the form of micelles.
- the bone targeting molecule can be selected from one or more of a double-tallow acid salt, an aspartic acid polypeptide repeat sequence, or an aptamer selected for osteoblast-like cells.
- the aspartic acid polypeptide repeat may be selected from the group consisting of SEQ ID NO: 1 (8 aspartic acid polypeptide repeats (Asp) 8 ) and SEQ ID NO: 2 (6 aspartate-serine-serine polypeptide repeats) One or more of (DSS) 6 ).
- the bone targeting delivery system comprises distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide and The ends of the bone targeting molecules are subjected to thiol modification.
- the bone-targeting molecule is directly linked to the surface of the liposome: the base of the bone-targeting molecule may be combined with distearyl glycolamine-polyethylene glycol 2000-maleimide The maleimide group in the reaction is directly reacted.
- the bone targeting molecule is linked to other molecules and inserted into the liposome bilayer in the form of micelles: the sulfhydryl group at the end of the bone targeting molecule can be combined with distearyl alcohol glycol-polyethylene Alcohol 2000-maleimide stump reacted with maleimide to form a thioether bond, a bone targeting molecule and distearoyl alcohol glycolamine-polyethylene glycol 2000-maleimide
- the covalently linked compound is inserted into the liposome bilayer (the distearyl glycolamine moiety is inserted into the lipid bilayer;) in the form of micelles, thereby allowing the liposome to have bone targeting properties.
- the bone targeting molecule is SEQ ID NO: 2 (6 aspartate-serine-serine polypeptide repeats (DSS) 6 ).
- the bone targeting molecule comprises from 2 to 10 mol% of the liposome concentration.
- An aptamer is a single-stranded DNA or RNA that is specifically selected for osteoblast-like cells to specifically bind thereto.
- the aptamer having specific binding to osteoblast-like cells is selected from the group consisting of: SEQ ID NO: 4 (5, -GTACTTCCGGCGGGTTCTATGGGCCCTGTCTCCCTTCCC
- the small nucleic acid drug is selected from a small interfering ribonucleic acid (siRNA), a micro 'j, a ribonucleic acid (miRNA) mimic or a microribonucleic acid blocker (Antagomir) that promotes bone formation.
- small nucleic acid drugs are encapsulated in the delivery system and act on targeted targets to inhibit gene expression that negatively regulates bone formation.
- the lipid mass ratio of the small nucleic acid drug to the liposome is 2% to 20%.
- the small interfering ribonucleic acid is a small interfering ribonucleic acid directed against a negative regulatory gene of osteogenesis. More preferably, the small interfering ribonucleic acid is a small interfering ribonucleic acid directed against the casein kinase interacting protein K CKIP-1), WIF-1 or Hoxc8. And preferably, the small interfering ribonucleic acid is a small interfering ribonucleotide directed against casein kinase interacting protein 1 (CKIP-1). Still preferably, the small interfering ribonucleotide for CKIP-1 comprises SEQ ID NO: 3 (5-CCUGAGUGACUAUGAGAAG-3).
- the bone targeting vector carrying the small interfering ribonucleic acid The same applies to mimic microRNAs and microRNA cleavage agents.
- the present invention provides a method for preparing a bone-targeted delivery system based on small nucleic acid drug osteogenic treatment, comprising the following steps:
- the molecule is selected from the group consisting of a double-telephonic acid salt, SEQ ID NO: 1, SEQ ID NO: 2, and one or more of the aptamers selected for osteoblast-like cells;
- step (1) is to prepare a liposome containing a small nucleic acid drug.
- the lipid is dissolved in ethanol in a certain proportion, and slowly added to the citrate buffer solution (10-50 mM, pH 3.5-5.5) containing the small nucleic acid drug under rapid stirring; stirring After 10 ⁇ 30 minutes, the liposome is extruded by an extruder, and dialysis is carried out for 2 to 4 hours in a phosphate buffer solution having a pH of 6.8 to 7.5 at room temperature to remove the ethanol, thereby obtaining a small nucleic acid drug.
- the proportion of ethanol in the total volume is 30% to 40% in the preparation of liposomes containing small nucleic acid drugs. The high content of ethanol makes the liposome deformable and reduces the interfacial tension of the liposome, thereby increasing its fluidity.
- the liposome comprises a non-cationic lipid or a mixture of a non-cationic lipid and a cationic lipid. More preferably, the cationic lipid in the liposome accounts for 0% to 50% of the total lipid content, and the non-cationic lipid accounts for 50 to 100% of the total lipid content.
- Cationic lipids are a class of positively charged phospholipids, usually composed of one or several neutral lipids.
- the neutral phospholipids used in the cationic liposome are similar to conventional liposomes, such as cholesterol (chol), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and the like.
- the cationic lipid is selected from the group consisting of 1,2-dioleyloxy-3-trimethylaminopropane (DOTAP), N-[l-(2,3-dioleyloxy)propyl]-N, N, N, - one or more of trimethylammonium chloride (DOTMA) and DC-cholesterol (DC-Chol).
- DOTAP 1,2-dioleyloxy-3-trimethylaminopropane
- DOTMA trimethylammonium chloride
- DC-Chol DC-cholesterol
- Non-cationic lipids include uncharged and negatively charged lipids.
- the non-cationic lipid is selected from the group consisting of dioleyldimethylammonium propane (DODAP), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), one or more of dioleoylphosphatidylcholine (DOPC) and DLin-KC2-DMA.
- DODAP dioleyldimethylammonium propane
- DOPE dioleoylphosphatidylethanolamine
- DSPC distearoylphosphatidylcholine
- DPPC dipalmitoylphosphatidylcholine
- DOPC dioleoylphosphatidylcholine
- DOPC dioleoylphosphatidylcholine
- DLin-KC2-DMA DLin-KC2-DMA
- the liposome may contain an auxiliary lipid such as neutral lipid cholesterol and/or a modifying agent such as polyethylene glycol (PEG).
- PEG polyethylene glycol
- the PEG-modified liposome comprises distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG-MAL), and the terminal maleimide group facilitates liposome Surface modification.
- the mass ratio of the small nucleic acid drug to the lipid in the liposome containing the small nucleic acid drug is 2% to 20%.
- the small nucleic acid drug is selected from one or more of a small interfering ribonucleic acid having a function of promoting bone formation, a mimetic of a small ribonucleic acid, and a blocking agent for microribonucleic acid.
- the small interfering ribonucleic acid is a small interfering ribonucleic acid directed against a negative regulatory gene of osteogenesis. More preferably, the small interfering ribonucleic acid is a small interfering ribonucleic acid directed against the casein kinase interacting protein K CKIP-1), WIF-1 or Hoxc8. And preferably, the small interfering ribonucleic acid is a small interfering ribonucleic acid directed against casein kinase interacting protein 1 (CKIP-1). Still preferably, the small interfering ribonucleic acid for CKIP-1 comprises SEQ ID NO: 3 (5-CCUGAGUGACUAUGAGAAG-3).
- the bone targeting molecule is selected from the group consisting of a double-tallow acid salt, an aspartic acid polypeptide repeat sequence, and one or more of the aptamers selected for osteoblast-like cells.
- the aspartic acid polypeptide repeat sequence may be selected from the group consisting of SEQ ID NO: 1 (8 aspartic acid polypeptide repeats (Asp) 8 ) or SEQ ID NO: 2 (6 aspartate-serine-serine polypeptide repeats) (DSS) 6 ).
- the end of the bone targeting molecule is thiol modified.
- the liposome containing the small nucleic acid drug contains polyethylene glycol (PEG) modified liposome-distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG- At MAL), the bone targeting molecule is directly attached to the surface of the liposome.
- PEG polyethylene glycol
- DSPE-PEG- At MAL polyethylene glycol modified liposome-distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide
- the mixing of the step (2) further comprises adding distearoylethanolamine-polyethylene glycol 2000-maleimide, and chemically reacting the bone targeting molecule with DSPE-PEG-MAL to form a bone target.
- PEG polyethylene glycol
- DSPE-PEG-MAL modified liposome-distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide
- a compound is covalently bonded to a molecule and DSPE-PEG-MAL, which forms a micelle in an aqueous solution, and is incubated with a liposome containing a small nucleic acid drug for 4 to 12 hours, and then inserted into a lipid bilayer to make a bone target.
- a molecule is attached to the surface of the liposome.
- the bone targeting molecule is SEQ ID NO: 2 (6 aspartate-serine-serine polypeptide repeats (DSS) 6 ).
- the bone targeting molecule comprises from 2 to 10 mol% of the liposome concentration.
- An aptamer is a single-stranded DNA or RNA that specifically binds to osteoblast-like cells.
- the aptamer having specific binding to osteoblast-like cells is selected from the group consisting of: SEQ ID NO: 4 (5, -GTACTTCCGGCGGGTTCTATGGGCCCTGTCTCCCTTCCC
- Step (3) is to remove unreacted bone targeting molecules and micelle molecules that are not inserted into the liposomes.
- the purification method may be molecular size exclusion chromatography by a Sepharose CL-4B column.
- the purified bone-targeting liposome in step (3) can be further lyophilized to form a lyophilized powder.
- a lyoprotectant is added during lyophilization, one or more selected from the group consisting of sucrose, mannitol, lactose, trehalose, albumin, glucose, and dextran.
- the present invention provides a preparation method of another bone-targeted delivery system based on small nucleic acid drug osteogenic treatment.
- the method differs from the second aspect of the present invention in that a blank liposome is prepared in step (1), the bone-targeting molecule is ligated, lyophilized, and a small nucleic acid drug phosphate buffer solution is added for hydration. Specifically, the following steps are included:
- the bone-targeting molecule is mixed with the blank liposome to form a bone-targeting blank liposome, and the bone-targeting molecule is selected from the group consisting of double-stone salt, SEQ ID NO: 1 (8 aspartic acid polypeptide repeats (Asp) 8 ), SEQ ID NO: 2 (6 aspartate-serine-serine polypeptide repeats (DSS) 6 ) and screening for osteoblast-like cells One or several of the aptamers;
- Encapsulating small nucleic acid drugs Dissolving small nucleic acid drugs in DEPC-treated distilled water, adding freeze-dried bone-targeting blank liposomes for hydration, and preparing a bone-targeting small nucleic acid drug liposome delivery system,
- the small nucleic acid drug is selected from one or more of a small interfering ribonucleic acid having a function of promoting bone formation, a mimetic of a small ribonucleic acid, and a blocker of microribonucleic acid.
- the end of the bone-targeting molecule is subjected to a radical modification.
- the blank liposomes comprise distearoylethanolamine-polyethylene glycol 2000-maleimide, and the bone targeting molecule is directly incubated with the blank liposome to attach to the surface of the blank liposome.
- step (2) further comprises adding two hard Fatty acid glycolylethanolamine-polyethylene glycol 2000-maleimide, chemically reacting a bone targeting molecule with distearyl betethanolamine-polyethylene glycol 2000-maleimide in advance, A covalently bonded compound of a bone targeting molecule with disteoyl succinylethanolamine-polyethylene glycol 2000-maleimide is formed which forms micelles in aqueous solution and is incubated with blank liposomes 4 The lipid bilayer was inserted after ⁇ 12 hours.
- the step (2) bone targeting molecule is SEQ ID NO: 2.
- the step (2) bone targeting molecule comprises 2 to 10 mol% of the lipid concentration in the blank liposome.
- a small nucleic acid drug buffer salt solution is prepared, the lyophilized powder is hydrated, and after 20 to 40 minutes of incubation, a bone-targeting small nucleic acid drug liposome system is obtained.
- the small nucleic acid drug buffer salt solution is a 10 to 50 mM phosphate buffer having a pH of 6.8 to 7.5.
- the mass ratio of the small nucleic acid drug to the lipid in the blank lipid is 2% to 20%.
- the small nucleic acid drug is selected from one or more of a small interfering ribonucleic acid having a function of promoting bone formation, a mimetic of a microribonucleic acid, and a blocker of a microribonucleic acid.
- the small interfering ribonucleic acid is a small interfering ribonucleic acid directed against a negative regulatory gene of osteogenesis. More preferably, the small interfering ribonucleic acid is a small interfering ribonucleic acid directed against the casein kinase interacting protein K CKIP-1), WIF-1 or Hoxc8. And preferably, the small interfering ribonucleic acid is a small interfering ribonucleoside directed against casein kinase interacting protein 1 (CKIP-1) Acid. Still preferably, the small interfering ribonucleic acid for CKIP-1 comprises SEQ ID NO: 3 (5-CCUGAGUGACUAUGAGAAG-3).
- the present invention provides a small based on the use of bone targeting molecules for bone formation surfaces and osteoblast-like cells, small nucleic acid drugs that inhibit negative regulation of bone formation, and liposome linkages that facilitate transfection of small nucleic acid drugs.
- the bone-targeted delivery system for nucleic acid drug osteogenic treatment and the preparation method thereof have the following beneficial effects: mainly targeting the bone forming surface and the osteoblast-like cells, having strong specificity; preventing small nucleic acid drugs from being in vivo Degradation, it can be specifically transmitted to target cells, and the transfection efficiency is high, which is beneficial to the high silencing efficiency of small nucleic acid drugs.
- Figure 1 is a graph showing the fluorescence distribution of FITC-labeled (DSS) 6 and Asp 8 and a control group on the surface of bone formation;
- Figure 2 is a graph showing the fluorescence intensity of each organ in the bone targeting verification test of the bone-targeting small nucleic acid drug delivery system of the present invention carrying FAM fluorescent label;
- Figure 3 to Figure 7 are the results of immunohistochemical analysis of various cells labeled with FAM fluorescently labeled siRNA and corresponding antibodies (anti-RUNX2, C0L1A1, anti-ALP, anti-osteocalcin and OSCAR). detailed description
- Example 1 (DSS) 6 is capable of selectively targeting bone forming surfaces
- FITC, Asp 8 -FITC and (DSS) 6 -FITC were administered by tail vein injection, respectively, 27 ⁇ /13 ⁇ 4/ 0.2 ⁇ 1 physiological saline.
- the rats were sacrificed 24 hours later to observe the uncalcified tissue fraction.
- the femur and tibia were dehydrated with a gradient of ethanol and embedded with modified methyl methacrylate (no decalcification).
- the femur was sliced to a thickness of 15 ⁇ using a Leica SM2500E mocrotome (Leica Microsystems).
- the liposome was extruded by an extruder and passed through a double-layer polycarbonate membrane having a pore size of 0.08 ⁇ m to control the particle diameter at 50 to 100 nm.
- the liposome was dialyzed against a buffered saline solution of pH 7.4 for 3 hours at room temperature to remove ethanol and unencapsulated small interfering ribonucleic acid.
- the lipid concentration of the liposome was measured by phosphate analysis, and then 2 mol% (relative total lipid concentration) of DSPE-PEG2000-(Asp) 8 was inserted into the surface of the liposome in the form of micelles, and incubated at 37 ° C in a water bath. After 6 hours, a bone targeting small nucleic acid drug liposome delivery system was prepared.
- Molecular size exclusion chromatography was performed on a Sepharose CL-4B column, and elution was carried out with phosphate buffer to remove micelles that were not inserted into the liposome to unligated bone targeting molecules.
- the liposome was extruded by an extruder and passed through a double-layer polycarbonate membrane having a pore size of 0.08 ⁇ to control the particle diameter of 50 to 100 nm.
- the liposome was dialyzed against a buffered saline solution of pH 7.4 for 3 hours at room temperature to remove ethanol and unencapsulated small interfering ribonucleic acid.
- the lipid concentration of the liposome was detected by the acid salt analysis, and then 2 mol% (relative total lipid concentration) of DSPE-PEG2000-aptamer was inserted into the surface of the liposome in the form of micelles, and incubated at 37 ° C in a water bath. After 6 hours, a bone targeting small nucleic acid drug liposome delivery system was prepared.
- Molecular size exclusion chromatography was performed on a Sepharose CL-4B column, and elution was carried out with phosphate buffer to remove micelles that were not inserted into the liposome to unligated bone targeting molecules.
- the bone was targeted to a small nucleic acid drug liposome delivery system for in vivo bone targeting validation.
- the vector carrying the FAM fluorescently labeled SEQ ID NO: 3 was injected intravenously into the rat, respectively, with siRNA carrying no vector, a commercially available vector.
- the system (in vivo jetPEI) and the homemade carrier system liposomes that do not contain bone targeting molecules are compared.
- Two hours after the intravenous injection the rats were sacrificed, and the main organs (heart, liver, spleen, lung, kidney and femur) were collected, and the fluorescence intensity in each organ was observed with a fluorescence imaging system (Xenogen Imaging Technologies, Alameda, CA).
- the bone-targeting small nucleic acid drug liposome delivery system of the present invention can significantly increase the accumulation of small nucleic acid drugs in bone tissues while reducing exposure to liver organs and reducing side effects.
- the (DSS) 6 -liposome and the liposome carrying the FAM fluorescently labeled SEQ ID NO: 3 and the FAM fluorescently labeled SEQ ID NO: 3 without the vector are injected intravenously into the rat, 4 After the hour, the distal femur and the proximal tibia were removed for cryosection and immunohistochemical analysis.
- anti-RUNX2 and anti-type I collagen al (COL1A1) antibodies to label immature and mature bone precursor cells in the early stages of osteogenic differentiation
- anti-ALP and anti-osteocalcin antibodies to pre-differentiated pre-osteogenesis Cells and osteoblasts were labeled
- OSCAR antibodies labeled osteoclast-like cells (pre-osteoclasts and mature osteoclasts).
- FAM fluorescently labeled siRNA and corresponding antibody labeled various cells was observed to demonstrate the cellular selectivity of the bone targeting vector.
- the green fluorescence of (DSS) 6 -liposome-loaded siRNA has a good coincidence with the red fluorescence of osteoblast-like cells, and the coincidence probability is greater than that of liposome-carrying siRNA, but with osteoclast
- the red fluorescence of the sample cells did not coincide, and the green fluorescence of the siRNA carried by the liposome coincided with the red fluorescence of the osteoblast-like cells and osteoclast-like cells, indicating that the (DSS) 6 -liposome has cell selectivity. Conducive to small nucleic acid targeting to osteoblast-like cells.
- the present invention mainly targets bone formation surfaces and osteoblast-like cells, and has strong specificity; it can prevent small nucleic acid drugs from being degraded by in vivo substances, and can specifically transmit them to target cells.
- the high dyeing efficiency is beneficial to the high silencing efficiency of small nucleic acid drugs.
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Abstract
一种基于小核酸药物成骨治疗的骨靶向递送***及其制备方法。该骨靶向递送***包括脂质体、骨靶向分子和小核酸药物,所述骨靶向分子选自双磷酸盐、8个天门冬氨酸多肽重复序列、6个天门冬氨酸-丝氨酸-丝氨酸多肽重复序列和针对成骨样细胞筛选出的适配子中的一种或几种,所述小核酸药物选自具有促进骨形成功能的小干扰核糖核酸、微小核糖核酸的模拟物和微小核糖核酸的阻断剂中的一种或几种。
Description
基于小核酸药物成骨治疗的骨靶向递送***及其制备方法 技术领域
本发明属于分子生物学领域, 涉及一种基于小核酸药物成骨治疗的骨靶向递送***及其 制备方法。 背景技术
骨组织发生的基本过程包括骨组织形成和骨组织吸收两个方面。 成骨细胞和破骨细胞分 别负责骨形成和骨吸收, 两者之间的平衡保证了成年动物及人类骨量的恒定。
目前, 骨靶向药物递送***主要运用于小分子药物的递送, 通常由骨靶向分子和小分子 药物 /高分子化合物组成。 常见的骨靶向分子包括四环素类, 双碑酸盐类, 以及含有天门冬氨 酸重复序列的多肽。 这些靶向分子可直接与小分子药物如***, 布洛芬等共价连接, 也可 以与高分子化合物如聚乳酸 -羟基乙酸共聚物, 聚乙二醇等连接形成载体***携载药物。 这些 骨靶向分子介导的骨组织分布情况有所不同。 据报道, 双磷酸盐类既靶向骨形成表面又靶向 骨吸收表面, 而天门冬氨酸重复序列主要靶向骨吸收表面。 然而, 能够特异性靶向骨形成表 面的药物递送***还未见艮道。
过去的几年里, 骨生物学得到了快速的发展, 一系列负调控骨形成且不激活骨吸收的基 因被鉴定出来, 这包括酪蛋白激酶相互作用蛋白 1 ( CKIP-1 )、 WIF-1和 Hoxc8等。 这使得通 过核糖核酸干扰(RNAi )等技术手段利用小核酸药物沉默上述负调控骨形成的基因, 从而进 行骨治疗成为可能。 然而, 成骨样细胞主要分布在骨形成表面, 而且在使用小核酸药物进行 成骨治疗时, 成骨样细胞是主要的作用靶点, 所以特异性靶向骨形成表面以及成骨样细胞尤 为重要。
另外, 由于小核酸药物需要在细胞浆里释放并与 mRNA作用, 普通的高分子材料很难确 保有较高的转染效率, 所以递送小分子药物的骨靶向递送***并不适用于小核酸药物。 发明内容
为解决上述问题, 本发明通过釆用针对骨形成表面以及成骨样细胞的骨靶向分子、 抑制 负调控骨形成的小核酸药物和有助于小核酸药物转染的脂质体连接, 提供了一种基于小核酸 药物成骨治疗的骨靶向递送***及其制备方法。 本发明相比传统的骨靶向递送***具有更强 的专属性和特异性, 小核酸药物转染效率高, 能够达到较高的沉默效率。
一方面, 本发明提供了一种基于小核酸药物成骨治疗的骨靶向递送***, 包括脂质体、
骨靶向分子和小核酸药物, 其中, 骨靶向分子选自双碑酸盐、 SEQ ID NO: 1 ( 8个天门冬氨 酸多肽重复序列(Asp)8 )、 SEQ ID NO: 2 ( 6 个天门冬氨酸-丝氨酸-丝氨酸多肽重复序列 (Asp-Ser-Ser)6, 即 (DSS)6 )和针对成骨样细胞筛选出的适配子中的一种或几种, 小核酸药物 选自具有促进骨形成功能的小干扰核糖核酸(siRNA )、 微小核糖核酸(miRNA ) 的模拟物 ( microRNA mimic )和微小核糖核酸的阻断剂 (Antagomir ) 中的一种或几种。
其中, 脂质体含有非阳离子脂质, 或者含有非阳离子脂质和阳离子脂质的混合物。 优选 地, 所述脂质体中阳离子脂质占总脂质含量的 0%~50% , 非阳离子脂质占总脂质含量的 50%~100%。
阳离子脂质为一类带正电荷的磷脂, 通常与一种或几种中性脂质组成阳离子脂质体。 阳 离子脂质体中使用的中性脂质成分上与常规脂质体相似,如胆固醇( chol )、磷脂酰胆碱( PC )、 磷脂酰乙醇胺(PE )等。优选地, 阳离子脂质选自 1,2-二油烯氧基 -3-三甲氨基丙烷(DOTAP )、 N-[l- ( 2, 3-二油基氧)丙基] -N, N, N, -氯化三甲铵(DOTMA )、 和 DC-胆固醇( DC-Chol ) 的一种或几种。
非阳离子脂质包括不带电荷和带负电荷的脂质。 优选地, 非阳离子脂质选自二油酰二甲 基铵丙烷(DODAP )、 二油酰磷脂酰乙醇胺(DOPE )、 二硬脂酰磷脂酰胆碱( DSPC )、 二棕 榈酰磷脂酰胆碱( DPPC ), 二油酰磷脂酰胆碱 (DOPC)和 DLin-KC2-DMA中的一种或几种。
脂质体中可含有中性脂质胆固醇等辅助脂质和 /或聚乙二醇(PEG )等修饰剂。 PEG修饰 脂质体具有良好的水溶性、 毒性低、 无免疫原性等优势。 因为 PEG末端可以进行羧酸、 胺或 马来酰亚胺基团等活性基团修饰, 便于进一步与骨靶向分子连接, 所以 PEG修饰脂质适合用 作于连接反应的起始剂。 优选地, PEG修饰脂质带有甲氧基末端, 例如 DSPE-mPEG。
优选地, 骨靶向递送***含有二硬脂酰碑脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺。 优选 地, 脂质体 (PEG 修饰脂质) 含有二硬脂酰磷脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺 ( DSPE-PEG-MAL ), 末端的活性马来酰亚胺基团有利于在脂质体表面进行修饰。 也优选地, 当脂质体中不含有二硬脂酰磷脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺 ( DSPE-PEG-MAL )时, 骨靶向分子可预先与二硬脂酰磷脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺 ( DSPE-PEG-MAL ) 进行共价键连接, 然后以胶束的形式***脂质双分子层中。
骨靶向分子可以选自双碑酸盐、 天门冬氨酸多肽重复序列或针对成骨样细胞筛选出的适 配子中的一种或几种。 天门冬氨酸多肽重复序列可以选自 SEQ ID NO: 1 ( 8个天门冬氨酸多 肽重复序列 (Asp)8 )和 SEQ ID NO: 2 ( 6个天门冬氨酸-丝氨酸-丝氨酸多肽重复序列(DSS)6 ) 中的一种或几种。
优选地, 骨靶向递送***含有二硬脂酰磷脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺以及所
述骨靶向分子末端进行疏基修饰。 此时, 优选地, 骨靶向分子直接连接于所述脂质体表面: 骨靶向分子末端的疏基可以与二硬脂酰碑脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺中的马来酰 亚胺基团直接反应。 以及优选地, 骨靶向分子与其它分子连接后以胶束的形式***所述脂质 体双分子层: 骨靶向分子末端的疏基可以与二硬脂酰碑脂酰乙醇胺-聚乙二醇 2000-马来酰亚 胺残端的顺丁烯二酞亚胺反应, 形成硫醚键, 骨靶向分子与二硬脂酰碑脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺的共价键连接化合物以胶束的形式***脂质体双分子层(二硬脂酰碑脂酰乙 醇胺部分***脂质双分子层中;), 从而使脂质体具有骨靶向性。
优选地, 骨靶向分子为 SEQ ID NO: 2 ( 6 个天门冬氨酸-丝氨酸-丝氨酸多肽重复序列 (DSS)6 )。
优选地, 骨靶向分子占脂质体浓度的 2~10mol%。
适配子 (aptamer)是针对成骨样细胞进行 选获得的与其具有特异性结合的单链 DNA或 者 RNA。 优选地, 和成骨样细胞具有特异性结合地适配子选自: SEQ ID NO : 4 ( 5, -GTACTTCCGGCGGGTTCTATGGGCCCTGTCTCCCTTCCC
AAAAGTGCACGCTAC-3' ) , 以及 SEQ ID NO: 5 ( 5'-GTACTTCCGACTTGGGGC
GGGTTTGTTGCTGGCTGTCGGC AAAAGTGCA-3' )。
小核酸药物选自具有促进骨形成功能的小干扰核糖核酸 ( siRNA )、 微 ' j、核糖核酸 ( miRNA ) 的模拟物 ( microRNA mimic )或微小核糖核酸的阻断剂 (Antagomir )。 小核酸药 物被包封在递送***中, 可作用于靶向目标, 抑制负调控骨形成的基因表达。 优选地, 小核 酸药物与脂质体的脂质质量比为 2%~20%。
优选地, 小干扰核糖核酸为针对成骨的负调控基因的小干扰核糖核酸。 更优选地, 小干 扰核糖核酸为针对酪蛋白激酶相互作用蛋白 K CKIP-1 )、 WIF-1或 Hoxc8的小干扰核糖核酸。 以及优选地, 小干扰核糖核酸为针对酪蛋白激酶相互作用蛋白 1 ( CKIP-1 ) 的小干扰核糖核 酸。 再优选地 , 针对 CKIP-1 的 小 干扰核糖核 酸 包含 SEQ ID NO: 3 ( 5-CCUGAGUGACUAUGAGAAG-3 )。
由于微小核糖核酸的模拟物和微小核糖核酸的阻断剂是较短的碱基序列, 与小干扰核糖 核酸在化学性质上具有较高的相似性, 所以携带小干扰核糖核酸的骨靶向载体同样适用于微 小核糖核酸的模拟物和微小核糖核酸的阻断剂。
第二方面, 本发明提供了一种基于小核酸药物成骨治疗的骨靶向递送***的制备方法, 包括以下步骤:
( 1 )制备含有小核酸药物的脂质体: 取脂质溶解在乙醇中, 在搅拌状态下緩慢加至含有 小核酸药物的緩冲液溶液中, 釆用挤出仪对脂质体进行挤出, 透析去除乙醇, 小核酸药物选
自具有促进骨形成功能的小干扰核糖核酸、 微小核糖核酸的模拟物和微小核糖核酸的阻断剂 中的一种或几种;
( 2 )连接骨靶向分子和含有小核酸药物的脂质体: 将骨靶向分子与含有小核酸药物的脂 质体混合连接, 制得骨靶向小核酸药物脂质体, 骨靶向分子选自双碑酸盐、 SEQ ID NO: 1、 SEQ ID NO: 2和针对成骨样细胞筛选出的适配子中的一种或几种;
( 3 ) 纯化骨靶向小核酸药物脂质体。
其中, 步骤(1 )为制备含有小核酸药物的脂质体。 优选地, 将脂质按一定比例溶解在乙 醇中, 緩慢加入快速搅拌状态下的含有小核酸药物的枸橼酸盐緩冲液溶液( 10~50 mM, pH 值为 3.5~5.5 )中;搅拌 10~30分钟后,釆用挤出仪对脂质体进行挤出,室温下在 pH值为 6.8~7.5 的磷酸盐緩冲溶液中透析 2~4小时去除乙醇, 即得含有小核酸药物的脂质体。 优选地, 在制 备含有小核酸药物的脂质体时乙醇在总体积中所占比例为 30%~40%。高含量的乙醇使得脂质 体具有变形能力, 降低脂质体的界面张力, 从而使其流动性增加。
优选地, 所述脂质体含有非阳离子脂质, 或者含有非阳离子脂质和阳离子脂质的混合物。 更优选地, 所述脂质体中阳离子脂质占总脂质含量的 0%~50% , 非阳离子脂质占总脂质含量 的 50 ~100 „
阳离子脂质为一类带正电荷的磷脂, 通常与一种或几种中性脂质组成阳离子脂质体。 阳 离子脂质体中使用的中性磷脂成分上与常规脂质体相似,如胆固醇( chol )、磷脂酰胆碱( PC )、 磷脂酰乙醇胺(PE )等。优选地, 阳离子脂质选自 1,2-二油烯氧基 -3-三甲氨基丙烷(DOTAP )、 N-[l- ( 2, 3-二油基氧) 丙基] -N, N, N, -氯化三甲铵 ( DOTMA )和 DC-胆固醇(DC-Chol ) 的一种或几种。
非阳离子脂质包括不带电荷和带负电荷的脂质。 优选地, 非阳离子脂质选自二油酰二甲 基铵丙烷(DODAP )、 二油酰磷脂酰乙醇胺(DOPE )、 二硬脂酰磷脂酰胆碱( DSPC )、 二棕 榈酰磷脂酰胆碱( DPPC ), 二油酰磷脂酰胆碱 (DOPC)和 DLin-KC2-DMA中的一种或几种。
脂质体中可含有中性脂质胆固醇等辅助脂质和 /或聚乙二醇(PEG )等修饰剂。 优选地, PEG修饰脂质体含有二硬脂酰磷脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺 ( DSPE-PEG-MAL ), 末端的马来酰亚胺基团有利于脂质体的表面修饰。
优选地, 小核酸药物与含有小核酸药物的脂质体中脂质的质量比为 2%~20%。 小核酸药 物选自具有促进骨形成功能的小干扰核糖核酸、 微小核糖核酸的模拟物和微小核糖核酸的阻 断剂中的一种或几种。
优选地, 小干扰核糖核酸为针对成骨的负调控基因的小干扰核糖核酸。 更优选地, 小干 扰核糖核酸为针对酪蛋白激酶相互作用蛋白 K CKIP-1 )、 WIF-1或 Hoxc8的小干扰核糖核酸。
以及优选地, 小干扰核糖核酸为针对酪蛋白激酶相互作用蛋白 1 ( CKIP-1 ) 的小干扰核糖核 酸。 再优选地 , 针对 CKIP-1 的 小 干扰核糖核 酸 包含 SEQ ID NO: 3 ( 5-CCUGAGUGACUAUGAGAAG-3 )。
将含有小核酸药物的脂质体通过双层聚碳酸酯膜挤出, 从而控制粒径在 50~150nm。 步骤( 2 ) 中, 骨靶向分子选自双碑酸盐、 天门冬氨酸多肽重复序列和针对成骨样细胞筛 选出的适配子中的一种或几种。 天门冬氨酸多肽重复序列可以选自 SEQ ID NO: 1 ( 8个天门 冬氨酸多肽重复序列 (Asp)8 )或 SEQ ID NO: 2 ( 6个天门冬氨酸 -丝氨酸-丝氨酸多肽重复序列 (DSS)6 )。
优选地, 骨靶向分子末端进行巯基修饰。
优选地, 当含有小核酸药物的脂质体中含有聚乙二醇(PEG )修饰脂质体-二硬脂酰磷脂 酰乙醇胺-聚乙二醇 2000-马来酰亚胺 ( DSPE-PEG-MAL ) 时, 骨靶向分子直接连接于所述脂 质体表面。
优选地, 当脂质体中不含有聚乙二醇(PEG )修饰脂质体 -二硬脂酰磷脂酰乙醇胺-聚乙二 醇 2000-马来酰亚胺(DSPE-PEG-MAL ) 时, 步骤(2 )所述混合进一步包括加入二硬脂酰碑 脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺, 预先将骨靶向分子与 DSPE-PEG-MAL进行化学反 应, 生成骨靶向分子与 DSPE-PEG-MAL 的共价键连接化合物, 该化合物可在水溶液中形成 胶束, 与含有小核酸药物的脂质体孵育 4~12小时后***脂质双分子层,使得骨靶向分子连接 于所述脂质体表面。
优选地, 骨靶向分子为 SEQ ID NO: 2 ( 6 个天门冬氨酸-丝氨酸-丝氨酸多肽重复序列 (DSS)6 )。
优选地, 骨靶向分子占脂质体浓度的 2~10mol%。
适配子 (aptamer)是针对成骨样细胞进行筛选获得的与其具有特异性结合的单链 DNA或 者 RNA。 优选地, 和成骨样细胞具有特异性结合地适配子选自: SEQ ID NO : 4 ( 5, -GTACTTCCGGCGGGTTCTATGGGCCCTGTCTCCCTTCCC
AAAAGTGCACGCTAC-3' ) , 以及 SEQ ID NO: 5 ( 5'-GTACTTCCGACTTGGGGC
GGGTTTGTTGCTGGCTGTCGGC AAAAGTGCA-3' )。
步骤(3 )为除去未反应的骨靶向分子以及未***脂质体的胶束分子。 纯化方法可以为通 过 Sepharose CL-4B柱进行分子大小排除层析。 优选地, 步骤( 3 ) 中的纯化的骨靶向脂质体 可进一步进行冻干, 制成冻干粉末保存。 更优选地, 冻干时加入冻干保护剂, 选自蔗糖, 甘 露醇, 乳糖, 海藻糖, 白蛋白, 葡糖糖, 右旋糖酐中的一种或几种。
第三方面, 本发明提供了另外一种基于小核酸药物成骨治疗的骨靶向递送***的制备方
法, 与本发明第二方面的区别在于, 在步骤(1 ) 中先制备空白脂质体, 连接骨靶向分子后冻 干, 加入小核酸药物磷酸盐緩冲溶液进行水化。 具体包括以下步骤:
( 1 )制备空白脂质体: 将脂质溶解在氯仿中, 旋转蒸发除去有机溶剂后, 加緩冲液进行 水化, 釆用挤出仪对多层脂质体进行挤出, 制得空白脂质体;
( 2 )连接骨靶向分子和空白脂质体: 取骨靶向分子与空白脂质体混合连接, 反应生成骨 靶向空白脂质体, 骨靶向分子选自双碑酸盐、 SEQ ID NO: 1 ( 8个天门冬氨酸多肽重复序列 (Asp)8 )、 SEQ ID NO: 2 ( 6个天门冬氨酸-丝氨酸-丝氨酸多肽重复序列(DSS)6 )和针对成骨 样细胞筛选出的适配子中的一种或几种;
( 3 ) 纯化骨靶向空白脂质体以及冻干;
( 4 ) 包封小核酸药物: 将小核酸药物溶于经 DEPC处理的蒸馏水中, 加入冻干骨靶向空 白脂质体进行水化, 制得骨靶向小核酸药物脂质体递送***, 小核酸药物选自具有促进骨形 成功能的小干扰核糖核酸、微小核糖核酸的模拟物和微小核糖核酸的阻断剂中的一种或几种。
步骤(2 ) 中, 优选地, 骨靶向分子末端进行疏基修饰。 优选地, 空白脂质体含有二硬脂 酰碑脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺, 骨靶向分子直接与空白脂质体孵化连接于所述 空白脂质体表面。
也优选地, 当步骤(2 )所述空白脂质体不含有二硬脂酰磷脂酰乙醇胺-聚乙二醇 2000-马 来酰亚胺时, 步骤(2 )所述混合进一步包括加入二硬脂酰碑脂酰乙醇胺-聚乙二醇 2000-马来 酰亚胺, 预先将骨靶向分子与二硬脂酰碑脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺进行化学反 应, 生成骨靶向分子与二硬脂酰碑脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺的共价键连接化合 物, 所述化合物在水溶液中形成胶束, 与空白脂质体孵育 4~12小时后***脂质双分子层。
优选地, 步骤(2 )骨靶向分子为 SEQ ID NO: 2。
优选地, 步骤(2 )骨靶向分子占所述空白脂质体中脂质浓度的 2~10mol%。
步骤(3 ) 中, 先制备小核酸药物緩冲盐溶液, 水化所述冻干粉末, 孵育 20~40分钟后, 制得骨靶向小核酸药物脂质体***。 优选地, 小核酸药物緩冲盐溶液为 10~50 mM磷酸盐緩 冲液, pH值为 6.8~7.5。
优选地, 小核酸药物与所述空白脂质中脂质质量比为 2%~20%。 小核酸药物选自具有促 进骨形成功能的小干扰核糖核酸、 微小核糖核酸的模拟物和微小核糖核酸的阻断剂中的一种 或几种。
优选地, 小干扰核糖核酸为针对成骨的负调控基因的小干扰核糖核酸。 更优选地, 小干 扰核糖核酸为针对酪蛋白激酶相互作用蛋白 K CKIP-1 )、 WIF-1或 Hoxc8的小干扰核糖核酸。 以及优选地, 小干扰核糖核酸为针对酪蛋白激酶相互作用蛋白 1 ( CKIP-1 ) 的小干扰核糖核
酸。 再优选地 , 针对 CKIP-1 的 小 干扰核糖核 酸 包含 SEQ ID NO: 3 ( 5-CCUGAGUGACUAUGAGAAG-3 )。
其它物质组分、 含量和连接过程均同前文所述。
本发明通过釆用针对骨形成表面以及成骨样细胞的骨靶向分子、 抑制负调控骨形成的小 核酸药物和有助于小核酸药物转染的脂质体连接, 提供了一种基于小核酸药物成骨治疗的骨 靶向递送***及其制备方法, 具有以下有益效果: 主要针对骨形成表面以及成骨样细胞进行 靶向, 具有较强的专属性; 可防止小核酸药物被体内物质降解, 可将其特异性传递到靶细胞 中, 转染效率高, 有利于小核酸药物达到较高的沉默效率。 附图说明
图 1为标记 FITC的 (DSS)6和 Asp8以及对照组在骨形成表面的荧光分布图;
图 2为携带 FAM荧光标记的本发明骨靶向小核酸药物脂质体递送***以及对照组在体内 骨靶向性验证实验中各器官的荧光强度图;
图 3〜图 7为 FAM荧光标记的 siRNA和相应抗体(anti-RUNX2、 C0L1A1、 anti-ALP、 anti-osteocalcin和 OSCAR )标记各种细胞的免疫组化分析结果图。 具体实施方式
以下所述是本发明的优选实施方式, 应当指出, 对于本技术领域的普通技术人员来说, 在不脱离本发明原理的前提下, 还可以做出若干改进和润饰, 这些改进和润饰也视为本发明 的保护范围。 下列具体实施方式中如果未注明具体条件的实验方法, 通常按照本领域技术内 的分子生物学的常规方法和条件, 这种技术和条件在文献中有完整解释。 参见例如 Sambrook 等人, 分子克隆: 实验手册中所述的技术和条件, 或按照制造厂商所建议的条件。
实施例一 (DSS)6能够选择性靶向骨形成表面
选用 18只 6月龄雌性大鼠( Sprague-dawley ), 分成 FITC组(异硫氰酸荧光素, n=6 )、 Asp8组(n=6 )和 (DSS)6组( n=6 )。 Asp8和 (DSS)6均分别标记上 FITC。 提前 3天对大鼠进行皮 下注射, 注射 Xylenol Orange ( XO, 30mg/kg )标记骨形成表面。 使用*** ( 75mg/kg )和 二甲苯胺瘗 ( 10mg/kg )麻醉大鼠后, 将 FITC、 Asp8-FITC和 (DSS)6-FITC 分别通过尾部静 脉注射给药, 27 μ Μ/1¾/0.2ηι1生理盐水。 24小时后杀死大鼠观察未钙化组织部分。 用梯度浓 度的乙醇将股骨和胫骨脱水, 用改性甲基丙烯酸甲酯包埋(不脱钙)。 使用 Leica SM2500E mocrotome ( Leica Microsystems ) 将股骨切片成 15 μ ηι的厚度。 然后, 通过共聚焦显 ^:镜 ( LSM510, Carl Zeiss, Oberkocken, Germany )观察 FITC的荧光分布。
结果如图 1 所示, FITC自身在切片中没有显示出绿色荧光, 表明没有骨靶向性, 然而 (DSS)6-FITC的绿色荧光带与标记骨形成表面的红色荧光带有很好的重合, 表明 (DSS)6主要到 达骨形成表面。 Asp8-FITC的绿色荧光带没有与红色荧光带重合, 主要分布在表面呈蚕食状和 坑状凹陷的骨吸收表面。
实施例二 制备骨靶向小核酸药物脂质体递送***
( 1 )制备含有小核酸药物的脂质体: 取二油酰二甲基铵丙烷(DODAP ), 二油酰碑脂酰 乙醇胺(DOPE )、 胆固醇(Chol )、 DSPE-mPEG2000 (甲氧基为末端的 PEG )按 40:10:45:5 的摩尔比溶解在乙醇中, 緩慢加入快速搅拌中的 lmg/mL 的小干扰核糖核酸随机序列的橼酸 盐緩冲液(pH值为 4 ), 乙醇占总体积的 35% , siRNA与脂质的质量比为 10%。 在室温下搅 拌 20分钟后釆用挤出仪对脂质体进行挤出,通过孔径为 0.08 μ m的双层聚碳酸酯膜,控制粒 径在 50~100nm。在室温下将该脂质体在 pH7.4的緩冲盐溶液中透析 3小时去除乙醇和未包封 的小干扰核糖核酸。
( 2 )连接骨靶向分子和含有小核酸药物的脂质体: 将二硬脂酰磷脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺(DSPE-PEG-MAL )溶于氯仿中, 在氮气环境中蒸发除去有机溶剂后, 取末 端带有疏基基团 (-SH ) 的骨靶向分子 8个天门冬氨酸多肽重复序列 (Asp)8, 按摩尔比为 4:1 与 DSPE-PEG-MAL混合, 在 10 mM磷酸盐緩冲液中 4°C孵育过夜。 釆用高效液相色谱法和 Ellman试剂监测反应的进行。 通过磷酸盐分析检测脂质体的脂质浓度, 然后取 2mol% (相对 总脂质浓度)的 DSPE-PEG2000-(Asp)8以胶束的形式***到脂质体表面, 37°C水浴孵育 6小时 后, 制得骨靶向小核酸药物脂质体递送***。
( 3 ) 纯化骨靶向小核酸药物脂质体递送***
通过 Sepharose CL-4B柱进行分子大小排除层析, 釆用磷酸盐緩冲液进行洗脱, 除去未插 入脂质体的胶束以未连接的骨靶向分子。
( 4 )冷冻干燥: 取骨靶向小核酸药物脂质体 0.5ml, 用 0.5ml含有乳糖的蒸馏水稀释(乳 糖: 脂质体摩尔比为 10 )后, 使用冷冻冻干机( Labconco, Freezezone 6, USA )冻干 48小时, 制得骨靶向小核酸药物脂质体冻干粉。
实施例三 制备骨靶向小核酸药物脂质体递送***
( 1 )制备空白脂质体:将 N-( 1-( 2,3-二油酰氧基 )丙基)-N,N,N-三甲基氯化铵( DOTAP )、 二油酰碑脂酰乙醇胺 (DOPE )、 胆固醇 (Chol )、 二硬脂酰碑脂酰乙醇胺-聚乙二醇 2000 ( DSPE-mPEG2000 )和二硬脂酰磷脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺( DSPE-PEG-MAL ) 按摩尔比 42:15:38:3:2溶解于氯仿中, 釆用薄膜分散法, 旋转蒸发除去氯仿后, 加入 10 mM 磷酸盐緩冲液进行水化形成多层脂质体, 釆用挤出仪对多层脂质体进行挤出, 制得大单室空
白脂质体;
( 2 )连接骨靶向分子和空白脂质体: 取末端带有疏基的骨靶向分子 (DSS)6-SH (相对于空 白脂质体中 DSPE-PEG-MAL摩尔比为 3:1)与空白脂质体混合, 在室温条件下孵化 3小时, 形 成骨靶向空白脂质体。
( 3 )纯化骨靶向空白脂质体以及冻干: 釆用 Sepharose CL-4B柱进行分子大小排除层析或 者在磷酸盐緩冲液(pH 7.4 )透析除去游离的 (DSS)6。 取经纯化后的骨靶向空白脂质体 0.5ml, 脂质浓度约 20umol/ml, 釆用 0.5ml含有蔗糖的蒸馏水稀释(蔗糖: 脂质体摩尔比为 8 )后, 使用冷冻冻干机 ( Labconco, Freezezone 6, USA ) 冻干 48小时, 制得骨靶向空白脂质体冻干 粉。
( 4 ) 包封小核酸药物:
加入 0.75 mg/ml 的 SEQ ID NO: 3 DEPC水溶液 0.5 ml水化冻干脂质体, 室温下孵育 20 分钟, 制得骨靶向小核酸药物脂质体递送***。
实施例四 制备骨靶向小核酸药物脂质体递送***
( 1 )制备含有小核酸药物的脂质体: 取 DLin-KC2-DMA,二棕榈酰磷脂酰胆碱 ( DPPC )、 胆固醇(Chol )、 DSPE-mPEG2000 (甲氧基为末端的 PEG )按 42:10:44:4的摩尔比溶解在乙 醇中, 緩慢加入快速搅拌中的 0.5 mg/mL SEQ ID NO: 3的橼酸盐緩冲液( pH值为 4 ), 乙醇 占总体积的 35% , siRNA与脂质的质量比为 8%。 在室温下搅拌 20分钟后釆用挤出仪对脂质 体进行挤出, 通过孔径为 0.08 μ ηι的双层聚碳酸酯膜, 控制粒径在 50~100nm。 在室温下将该 脂质体在 pH7.4的緩冲盐溶液中透析 3小时去除乙醇和未包封的小干扰核糖核酸。
( 2 )连接骨靶向分子和含有小核酸药物的脂质体: 将二硬脂酰磷脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺(DSPE-PEG-MAL )溶于氯仿中, 在氮气环境中蒸发除去有机溶剂后, 取末 端带有疏基基团( -SH )的骨靶向分子适配子序列 SEQ ID 4,按摩尔比为 2:1与 DSPE-PEG-MAL 混合, 在 10 mM磷酸盐緩冲液中 4°C孵育过夜。 通过碑酸盐分析检测脂质体的脂质浓度, 然 后取 2mol% (相对总脂质浓度) 的 DSPE-PEG2000-适配子以胶束的形式***到脂质体表面, 37 °C水浴孵育 6小时后, 制得骨靶向小核酸药物脂质体递送***。
( 3 ) 纯化骨靶向小核酸药物脂质体递送***
通过 Sepharose CL-4B柱进行分子大小排除层析, 釆用磷酸盐緩冲液进行洗脱, 除去未插 入脂质体的胶束以未连接的骨靶向分子。
实施例五 对骨靶向小核酸药物脂质体递送***的体内险证
将该骨靶向小核酸药物脂质体递送***进行体内骨靶向性的验证。将该载体携带 FAM荧 光标记的 SEQ ID NO: 3尾静脉注射于大鼠体内, 分别与没有载体携带的 siRNA, 市售载体
*** (in vivo jetPEI) 以及自制的不含有骨靶向分子的载体***脂质体(liposome )进行比较。 静脉注射 2小时后, 处死大鼠, 收集主要器官(心, 肝, 脾, 肺, 肾和股骨) , 釆用荧光成 像*** (Xenogen Imaging Technologies, Alameda, CA)观察每个器官中的荧光强度。
由图 2可见, 该发明中的骨靶向小核酸药物脂质体递送***能显著提高小核酸药物在骨 组织中的累积, 同时降低在肝脏器官中的暴露, 减少副作用。
实施例六 评价骨靶向载体***的在体细胞选择性
将 (DSS)6-脂质体(liposome ) 以及脂质体携载 FAM荧光标记的 SEQ ID NO: 3 和没有载 体携带的 FAM荧光标记的 SEQ ID NO: 3尾静脉注射于大鼠体内, 4小时后取出远端股骨和近 端胫骨,进行水冻切片和免疫组化分析。釆用 anti-RUNX2和 anti-type I collagen al (COL1A1) 抗体对骨源细胞分化早期阶段的未成熟和成熟骨前体细胞进行标记, anti- ALP和 anti-osteocalcin抗体对分化后期的前成骨细胞和成骨细胞进行标记, 以及 OSCAR抗体对破骨 样细胞(前破骨细胞和成熟的破骨细胞)进行标记。 观察 FAM荧光标记的 siRNA和相应抗体 标记各种细胞的重合情况以说明骨靶向载体的细胞选择性。
由图 3〜图 7可见, (DSS)6-脂质体携带的 siRNA的绿色荧光与成骨样细胞的红色荧光有很 好的重合, 重合机率大于脂质体携带的 siRNA, 但与破骨样细胞的红色荧光没有重合, 而脂 质体携带的 siRNA绿色荧光与成骨样细胞和破骨样细胞的红色荧光都有一定的重合, 表明 (DSS)6-脂质体具有细胞选择性, 有利于小核酸靶向对成骨样细胞。 DAPI 荧光和 H&E染色指 示被成功转染的成骨样细胞主要分布于骨形成表面及其周边骨髓区域, 进一步证实 (DSS)6- 脂质体可以特异靶向骨形成表面, 并具有一定的细胞选择性。 综上所述, 本发明主要针对骨 形成表面以及成骨样细胞进行靶向, 具有较强的专属性; 可防止小核酸药物被体内物质降解, 可将其特异性传递到靶细胞中, 转染效率高, 有利于小核酸药物达到较高的沉默效率。
Claims
1、 一种基于小核酸药物成骨治疗的骨靶向递送***, 包括脂质体、 骨靶向分子和小核酸 药物, 其特征在于, 骨靶向分子选自双碑酸盐、 SEQ ID NO: 1、 SEQ ID NO: 2和针对成骨 样细胞筛选出的适配子中的一种或几种, 小核酸药物选自具有促进骨形成功能的小干扰核糖 核酸、 微小核糖核酸的模拟物和微小核糖核酸的阻断剂中的一种或几种。
2、 如权利要求 1所述的骨靶向递送***, 其特征在于, 所述脂质体中阳离子脂质占总脂 质含量的 0%~50% , 非阳离子脂质占总脂质含量的 50%~100%。
3、 如权利要求 2所述的骨靶向递送***, 其特征在于, 所述阳离子脂质选自 1,2-二油烯 氧基 -3-三甲氨基丙烷、 N-[l- ( 2, 3-二油基氧) 丙基] -N, N, N, -氯化三甲铵和 DC-胆固醇 中的一种或几种。
4、 如权利要求 2所述的骨靶向递送***, 其特征在于, 所述非阳离子脂质选自二油酰二 甲基铵丙烷、 二油酰磷脂酰乙醇胺、 二硬脂酰磷脂酰胆碱、 二棕榈酰磷脂酰胆碱、 二油酰磷 脂酰胆碱和 DLin-KC2-DMA中的一种或几种。
5、 如权利要求 1所述的骨靶向递送***, 其特征在于, 所述骨靶向递送***含有二硬脂 酰碑脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺以及所述骨靶向分子末端进行疏基修饰。
6、 如权利要求 1所述的骨靶向递送***, 其特征在于, 所述骨靶向分子以胶束的形式插 入所述脂质体双分子层。
7、 如权利要求 1所述的骨靶向递送***, 其特征在于, 所述骨靶向分子占所述脂质体脂 质浓度的 2~10mol%。
8、 如权利要求 1所述的骨靶向递送***, 其特征在于, 所述小核酸药物与所述脂质体脂 质质量比为 2%~20%。
9、 如权利要求 1所述的骨靶向递送***, 其特征在于, 所述小干扰核糖核酸为针对成骨
的负调控基因的小干扰核糖核酸。
10、 如权利要求 9所述的骨靶向递送***, 其特征在于, 所述针对成骨的负调控基因的 小干扰核糖核酸为针对 CKIP-1、 WIF-1或 Hoxc8的小干扰核糖核酸。
11、 如权利要求 10所述的骨靶向递送***, 其特征在于, 所述针对 CKIP-1的小干扰核 糖核酸包含 SEQ ID NO: 3。
12、 一种基于小核酸药物成骨治疗的骨靶向递送***的制备方法, 其特征在于, 包括以 下步骤:
( 1 )制备含有小核酸药物的脂质体: 取脂质溶解在乙醇中, 在搅拌状态下緩慢加至含有 小核酸药物的緩冲液溶液中, 釆用挤出仪对脂质体进行挤出, 透析去除乙醇, 小核酸药物选 自具有促进骨形成功能的小干扰核糖核酸、 微小核糖核酸的模拟物和微小核糖核酸的阻断剂 中的一种或几种;
( 2 )连接骨靶向分子和含有小核酸药物的脂质体: 将骨靶向分子与含有小核酸药物的脂 质体混合连接, 制得骨靶向小核酸药物脂质体, 骨靶向分子选自双碑酸盐、 SEQ ID NO: 1、 SEQ ID NO: 2和针对成骨样细胞筛选出的适配子中的一种或几种;
( 3 ) 纯化骨靶向小核酸药物脂质体。
13、 如权利要求 12所述的骨靶向递送***的制备方法, 其特征在于, 步骤(1 ) 中所述 脂质体中阳离子脂质占总脂质含量的 0%~50%, 非阳离子脂质占总脂质含量的 50%~100%。
14、 如权利要求 12所述的骨靶向递送***的制备方法, 其特征在于, 步骤(1 ) 中所述 乙醇在总体积中所占比例为 30%~40%。
15、 如权利要求 12所述的骨靶向递送***的制备方法, 其特征在于, 步骤(1 ) 中所述 緩冲液溶液为枸橼酸盐緩冲液溶液, 10~50 mM, pH值为 3.5~5.5。
16、 如权利要求 12所述的骨靶向递送***的制备方法, 其特征在于, 步骤(1 ) 中所述 小核酸药物与所述脂质体脂质质量比为 2%~20%。
17、 如权利要求 12所述的骨靶向递送***的制备方法, 其特征在于, 步骤(1 ) 中所述
小干扰核糖核酸为针对成骨的负调控基因的小干扰核糖核酸。
18、 如权利要求 12所述的骨靶向递送***的制备方法, 其特征在于, 步骤(2 ) 中所述 骨靶向分子末端进行巯基修饰。
19、 如权利要求 12所述的骨靶向递送***的制备方法, 其特征在于, 步骤(2 ) 中所述 含有小核酸药物的脂质体含有二硬脂酰磷脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺, 骨靶向分 子直接连接于所述脂质体表面。
20、 如权利要求 12所述的骨靶向递送***的制备方法, 其特征在于, 步骤(2 )所述含 有小核酸药物的脂质体不含有二硬脂酰磷脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺, 步骤(2 ) 所述混合进一步包括加入二硬脂酰碑脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺, 预先将骨靶向 分子与二硬脂酰碑脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺进行化学反应, 生成骨靶向分子与 二硬脂酰磷脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺的共价键连接化合物, 所述化合物在水溶 液中形成胶束, 与含有小核酸药物的脂质体孵育 4~12小时后***脂质双分子层。
21、 如权利要求 12所述的骨靶向递送***的制备方法, 其特征在于, 步骤(2 )所述骨 靶向分子占所述含有小核酸药物的脂质体脂质浓度的 2~10mol%。
22、 如权利要求 12所述的骨靶向递送***的制备方法, 其特征在于, 步骤(3 )进一步 包括冻干。
23、 一种基于小核酸药物成骨治疗的骨靶向递送***的制备方法, 其特征在于, 包括以 下步骤:
( 1 )制备空白脂质体: 将脂质溶解在氯仿中, 旋转蒸发除去有机溶剂后, 加緩冲液进行 水化, 釆用挤出仪对多层脂质体进行挤出, 制得空白脂质体;
( 2 )连接骨靶向分子和空白脂质体: 取骨靶向分子与空白脂质体混合连接, 反应生成骨 靶向空白脂质体, 骨靶向分子选自双碑酸盐、 SEQ ID NO: 1、 SEQ ID NO: 2和针对成骨样 细胞筛选出的适配子中的一种或几种;
( 3 ) 纯化骨靶向空白脂质体以及冻干;
( 4 ) 包封小核酸药物: 将小核酸药物溶于 DEPC处理过的蒸馏水中, 加入冻干骨靶向空 白脂质体进行水化, 制得骨靶向小核酸药物脂质体递送***, 小核酸药物选自具有促进骨形
成功能的小干扰核糖核酸、微小核糖核酸的模拟物或微小核糖核酸的阻断剂中的一种或几种。
24、 如权利要求 23所述的骨靶向递送***的制备方法, 其特征在于, 步骤(1 ) 中所述 空白脂质体中阳离子脂质占总脂质含量的 0%~50% , 非阳离子脂质占总脂质含量的 50%~100%。
25、 如权利要求 23所述的骨靶向递送***的制备方法, 其特征在于, 步骤(2 ) 中所述 骨靶向分子末端进行巯基修饰。
26、 如权利要求 23所述的骨靶向递送***的制备方法, 其特征在于, 步骤(2 ) 中所述 空白脂质体含有二硬脂酰磷脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺, 骨靶向分子直接连接于 所述空白脂质体表面。
27、 如权利要求 23所述的骨靶向递送***的制备方法, 其特征在于, 步骤(2 )所述空 白脂质体不含有二硬脂酰磷脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺, 步骤(2 )所述混合进一 步包括加入二硬脂酰碑脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺, 预先将骨靶向分子与二硬脂 酰碑脂酰乙醇胺-聚乙二醇 2000-马来酰亚胺进行化学反应, 生成骨靶向分子与二硬脂酰碑脂 酰乙醇胺-聚乙二醇 2000-马来酰亚胺的共价键连接化合物,所述化合物在水溶液中形成胶束, 与空白脂质体孵育 4~12小时后***脂质双分子层。
28、 如权利要求 23所述的骨靶向递送***的制备方法, 其特征在于, 步骤(2 )所述骨 靶向分子占所述空白脂质体脂质浓度的 2~10mol%。
29、 如权利要求 23所述的骨靶向递送***的制备方法, 其特征在于, 步骤(4 ) 中所述 小核酸药物与所述空白脂质体脂质质量比为 2%~20%。
30、 如权利要求 23所述的骨靶向递送***的制备方法, 其特征在于, 步骤(4 ) 中所述 小干扰核糖核酸为针对成骨的负调控基因的小干扰核糖核酸。
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CN107998406A (zh) * | 2017-11-30 | 2018-05-08 | 中国科学院苏州纳米技术与纳米仿生研究所 | 一种级联靶向药物递送***及其制备方法与应用 |
CN107998406B (zh) * | 2017-11-30 | 2020-09-01 | 中国科学院苏州纳米技术与纳米仿生研究所 | 一种级联靶向药物递送***及其制备方法与应用 |
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CN110548007A (zh) * | 2018-05-31 | 2019-12-10 | 北京泰德制药股份有限公司 | 阳离子脂质体、基于其的核酸类药物递送***及其制备方法与用途 |
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