WO2017197726A1 - 一种骨靶向基因载体及其制备方法和应用 - Google Patents

一种骨靶向基因载体及其制备方法和应用 Download PDF

Info

Publication number
WO2017197726A1
WO2017197726A1 PCT/CN2016/087283 CN2016087283W WO2017197726A1 WO 2017197726 A1 WO2017197726 A1 WO 2017197726A1 CN 2016087283 W CN2016087283 W CN 2016087283W WO 2017197726 A1 WO2017197726 A1 WO 2017197726A1
Authority
WO
WIPO (PCT)
Prior art keywords
bone
phospholipid
alendronate
targeting gene
targeting
Prior art date
Application number
PCT/CN2016/087283
Other languages
English (en)
French (fr)
Inventor
赵晓丽
郑楚萍
Original Assignee
中国科学院深圳先进技术研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院深圳先进技术研究院 filed Critical 中国科学院深圳先进技术研究院
Publication of WO2017197726A1 publication Critical patent/WO2017197726A1/zh

Links

Images

Classifications

    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric

Definitions

  • the invention relates to the field of non-viral gene delivery systems, in particular to a bone targeting gene carrier and a preparation method and application thereof.
  • Gene therapy is a method of delivering a target gene into a target cell through a gene carrier, and treating the disease by adding, blocking, and correcting the gene.
  • Gene therapy offers a promising treatment for some major diseases.
  • the lack of efficient and targeted gene vectors restricts the wide clinical application of gene therapy. Undoubtedly the key to the success of gene therapy.
  • Non-viral vectors mainly include liposomes, polyethyleneimine (PEI), chitosan, etc., and these non-viral vectors are transfected by electrostatic interaction with DNA to form a gene delivery system.
  • liposome is the only nano drug-loading system approved by the FDA, which has good biocompatibility and degradability, and thus is widely used in non-viral transgenic vectors.
  • the transfection efficiency of liposome as a non-viral transgene vector is generally low and lacks targeting. Therefore, how to improve the targeting and transfection efficiency of liposome non-viral transgenic vectors has become the focus of research.
  • the present invention aims to provide a bone-targeting gene vector, a preparation method and application thereof.
  • the bone targeting gene carrier has a bisphosphonate group and has high targeting and transfection efficiency to bone tissue.
  • the vector has a good stabilizing effect on the genetic material, and the gene substance can be efficiently expressed in the vicinity of the bone tissue.
  • the gene vector is low in toxicity, safe and effective.
  • the present invention provides a bone-targeting gene vector, wherein the bone-targeting gene carrier is alendronate-modified liposome, and the alendronate-modified liposome comprises a cationic lipid, a neutral auxiliary lipid, a cholesterol, and an alendronate-modified phospholipid, the cationic lipid, a neutral auxiliary lipid, cholesterol constitute a phospholipid layer, and the alendronate-modified phospholipid Interspersed in the phospholipid layer and forming a vesicle structure with the phospholipid layer, the alendronate sodium being exposed outside of the phospholipid layer.
  • the bone-targeting gene carrier is alendronate-modified liposome
  • the alendronate-modified liposome comprises a cationic lipid, a neutral auxiliary lipid, a cholesterol, and an alendronate-modified phospholipid
  • the cationic lipid, a neutral auxiliary lipid, cholesterol constitute a phospholipid layer
  • the bone targeting gene carrier has a particle size of 40-200 nm.
  • the molar ratio of the alendronate-modified phospholipid to the cationic lipid, neutral auxiliary lipid, cholesterol is (0.01-0.07): (1-3): (0.5-1): (0.1-1).
  • Such a molar ratio can contribute to the formation of a bone-targeted gene carrier having a relatively regular morphology, good dispersibility, uniform particle size distribution, and stable structure between the components, which is not easily diluted and dissolved by the body fluid, and is disintegrated. It is advantageous to target to bone cells, and the use of the bone targeting gene carrier to encapsulate the genetic material has great advantages in biomedical applications.
  • the molar ratio of the alendronate-modified phospholipid to the cationic lipid, neutral auxiliary lipid, cholesterol is (0.01-0.07): (2-3): (0.5-1) :(0.5-1).
  • the alendronate sodium modified phospholipid comprises alendronate sodium and a phospholipid linked thereto by an amide bond.
  • the alendronate sodium modified phospholipid comprises a polyethylene glycol derivatized phospholipid and alendronate sodium linked to the polyethylene glycol derivatized phospholipid via an amide bond.
  • the cationic lipid comprises (2,3-dioleoyl-propyl)-trimethylammonium chloride (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP), (2,3-di N-[l-(2,3-dioleyloxy)propyl]-N,N,N-tri-methylammonium chloride,DOTMA) and dioctadecyldimethyl One or more of ammonium bromide (DODAB).
  • the cationic lipid enhances the positive charge of the entire liposome, plays a major role in the process of transporting the gene, and has the characteristics of good stability in vitro and biodegradability in vivo.
  • the hydrophobic tail chain of the cationic lipid affects the stability and fluidity of the formed liposome, while the charge characteristics of the hydrophilic cation head affect the surface characteristics of the formed liposome.
  • the cationic lipid is DOTAP.
  • the neutral auxiliary lipid comprises 1,2-dioleyl-sn-glycero-3-phosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (1,2-dioleoyl-sn) -glycero-3-phosphocholine, DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), bis(monoacylglycerol) phosphate (bis One or more of (monomyristoylglycero)phosphate, BMP) and phosphatidylglycerol (PG).
  • DOPE 1,2-dioleyl-sn-glycero-3-phosphatidylethanolamine
  • DOPC dioleoylphosphatidylcholine (1,2-dioleoyl-sn) -glycero-3-phosphocholine
  • DOPS 1,2-dioleoyl-sn-glycero-3-phospho-
  • the neutral auxiliary lipid is dioleoylphosphatidylethanolamine (DOPE).
  • DOPE has a strong cell membrane destabilization, and DOPE-rich cationic liposomes can assist DNA transfection and provide transfection efficiency.
  • DOPE can promote the formation of liposomes, especially under acidic conditions, to promote the transition of liposomes to the anti-hexagonal phase, which is conducive to fusion with cell membranes.
  • cholesterol can be embedded in a cationic lipid and a neutral helper lipid molecule to form a phospholipid layer, which can improve the gene carrier complex formed by coating the gene carrier with the gene. In vivo transfection activity of the substance.
  • the polyethylene glycol-derivatized phospholipid is obtained by linking polyethylene glycol to a phospholipid by a covalent bond, and the polyethylene glycol molecule has a molecular weight of 200 to 20,000 Daltons.
  • the phospholipids may be synthetic or naturally occurring phospholipids including, but not limited to, distearoylphosphatidylethanolamine (DSPE), distearoylphosphatidylglycerol (DSPG) or cholesterol.
  • DSPE distearoylphosphatidylethanolamine
  • DSPG distearoylphosphatidylglycerol
  • the molecular weight of the polyethylene glycol molecule may be 200, 500, 1000, 2000, 5000, 7000, 10000, 15000 or 20000.
  • Alendronate is a bisphosphonate compound containing a -NH 2 reactive functional group in its molecule. It is understood that the alendronate sodium modified phospholipid is amino and carboxylated by alendronate sodium. Polyethylene glycol-derivatized phospholipids are linked by an amide bond.
  • the carboxylated polyethylene glycol derivatized phospholipid is distearoylphosphatidylethanolamine-polyethylene glycol-carboxylic acid copolymer (DSPE-PEG-COOH).
  • the alendronate-modified phospholipid is alendronate sodium-polyethylene glycol-distearoylphosphatidylethanolamine (DSPE-PEG-Aln).
  • the molar ratio of DSPE-PEG-Aln to the DOTAP, DOPE, cholesterol is (0.01-0.07): (1-3): (0.5-1): (0.5-1).
  • the bone targeting gene carrier provided by the first aspect of the invention is modified with a main part of alendronate, and the gene carrier has good affinity for bone tissue, and the bisphosphonate and hydroxyphosphorus in the molecule
  • the high-efficiency combination of gray stone can deliver the target gene substance to the bone cells with high targeting, which is beneficial to the subsequent expression of the target gene.
  • the surface of the bone-targeting gene carrier is stably modified with alendronate-polyethylene glycol, which can effectively and stably stabilize the bone-targeting gene carrier, prolonging the time of circulation in the body, low cost and low Poisonous, safe and effective.
  • the present invention provides a method for preparing a bone-targeting gene vector, comprising the steps of:
  • the bone targeting gene carrier is alendronate sodium modified liposome comprising a cationic lipid, a neutral helper lipid, cholesterol, and a phosphonate-modified phospholipid, the cationic lipid, a neutral helper lipid, cholesterol constitutes a phospholipid layer, and the alendronate-modified phospholipid is interspersed in the phospholipid layer and is associated with the phospholipid layer A vesicle structure is formed, the alendronate sodium being exposed outside of the phospholipid layer.
  • the molar ratio of alendronate-modified phospholipid to said cationic lipid, neutral auxiliary lipid, cholesterol is (0.01-0.07): (1-3): (0.5-1): (0.1 -1).
  • the molar ratio of the alendronate-modified phospholipid to the cationic lipid, neutral auxiliary lipid, cholesterol is (0.01-0.07): (2-3): (0.5-1) :(0.5-1).
  • the bone targeting gene carrier has a particle size of 40-200 nm.
  • the bone-targeting gene carrier has a particle diameter of 50 to 150 nm.
  • the cationic lipid comprises one or more of DOTAP, DOTMA and DODAB.
  • the cationic lipid is DOTAP.
  • the neutral helper lipid comprises one or more of DOPE, DOPC, DOPS, BMP and PG.
  • the neutral auxiliary lipid is DOPE.
  • the alendronate sodium modified phospholipid comprises a polyethylene glycol derivatized phospholipid and alendronate sodium linked to the polyethylene glycol derivatized phospholipid via an amide bond.
  • the phospholipids include, but are not limited to, distearoylphosphatidylethanolamine (DSPE), distearoylphosphatidylglycerol (DSPG) or cholesterol.
  • the polyethylene glycol-derivatized phospholipid is obtained by linking polyethylene glycol to a phospholipid by a covalent bond, and the polyethylene glycol molecule has a molecular weight of 200 to 20,000 Daltons.
  • the alendronate sodium modified phospholipid is alendronate sodium-polyethylene glycol-distearoylphosphatidylethanolamine (DSPE-PEG-Aln).
  • the molar ratio of DSPE-PEG-Aln to the DOTAP, DOPE, cholesterol is (0.01-0.07): (1-3): (0.5-1): (0.5-1).
  • the first solvent comprises water, 2-(N-morpholine) ethanesulfonic acid buffer (referred to as "MES buffer solution”) having a pH of 5.5-6.0, and a pH value. a phosphate buffer solution of 7.0 to 7.4.
  • MES buffer solution 2-(N-morpholine) ethanesulfonic acid buffer
  • the separation and purification are performed by dialysis for 48-72 h using a dialysis bag having a molecular weight cut off of 500-1000 Da.
  • the method of the amidation reaction is well known to those skilled in the art.
  • the catalyst which may also be referred to as an activator, is often used in combination with a condensing agent for the amidation reaction.
  • the condensing agent comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (abbreviated as EDC).
  • the catalyst comprises N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide sodium salt (Sufo-NHS), 1-hydroxybenzotriazole (HOBT) One or more of them.
  • NHS N-hydroxysuccinimide
  • Sufo-NHS N-hydroxysulfosuccinimide sodium salt
  • HBT 1-hydroxybenzotriazole
  • the amidation reaction time is 8 h.
  • the activation time is 1-4 h. It can be 1h, 2h, 3h or 4h.
  • the organic solvent is chloroform or a mixed solution of chloroform and methanol.
  • the volume of the organic solvent is 1-3 mL.
  • the method further comprises: placing the flask in a vacuum drying oven for vacuuming for 4-8 h, and drying overnight.
  • the ultrasonic treatment is specifically:
  • the water bath was first ultrasonically irradiated for 30-60 min at a power of 50 W, and then ultrasonically applied for 4-8 min at a frequency of 20 kHz and a power of 750 W using a probe type ultrasonic system.
  • the microporous filtration membrane is a polycarbonate membrane having a pore diameter of 0.1 to 0.2 ⁇ m.
  • the second mixed solution is first extruded by a polycarbonate film having a pore size of 0.2 ⁇ m for 2 to 5 times, and then extruded through a polycarbonate film having a pore size of 0.1 ⁇ m. -5 times.
  • the preparation method of the bone-targeting gene carrier provided by the second invention of the present invention firstly forms an alendronate-modified phospholipid by an amide condensation reaction of a polyethylene glycol-derivatized phospholipid with alanine sodium, and then passes through a film.
  • Dispersion method Alendronate-modified phospholipids and cationic lipids, neutral helper lipids, and cholesterol are used to construct alendronate-modified liposomes, ie, bone-targeting gene carriers.
  • the gene vector obtained by the method has low cytotoxicity, good biocompatibility, high bone targeting efficiency, and remarkable transfection efficiency. Bone targeting gene vector
  • the preparation method is simple, the operation is convenient, the conditions are mild, and the application prospect is broad.
  • the present invention provides a gene delivery system, wherein the gene delivery system is a nanoparticle formed by electrostatic interaction of a genetic material and the bone targeting gene carrier described above, wherein the bone targeting gene carrier The ratio of nitrogen to phosphorus with the genetic material is (2-12):1.
  • the "nitrogen-phosphorus ratio” is the ratio of the number of moles of amino groups in the cationic lipid of the bone-targeting gene carrier to the number of moles of phosphate in the genetic material.
  • the ratio of nitrogen to phosphorus of the cationic lipid and the genetic material in the bone targeting gene carrier is (2-12):1, which can effectively improve the complex efficiency of the gene carrier and the genetic material, and the surface charge of the gene delivery system. , colloidal stability and particle size.
  • the gene delivery system has an average particle size of from 50 to 200 nm.
  • the genetic material includes, but is not limited to, one or more of deoxyribonucleic acid, plasmid DNA, microcircular DNA, and ribonucleic acid.
  • the genetic material comprises one or both of a pSDF-1 plasmid and a peSDF-1 plasmid.
  • the bone-targeting gene carrier is surface-modified with alendronate sodium, and the gene carrier has good affinity for bone tissue, and the bisphosphonate energy and hydroxyapatite in the molecule Efficiently combined, the target gene substance can be delivered to the bone cells with high targeting, which is beneficial to the subsequent expression of the target gene.
  • the present invention provides a method of preparing a gene delivery system comprising the following steps:
  • the concentration of the bone-targeting gene vector is from 0.7 to 2.1 mg/mL (concentration of the carrier itself, before mixing). It is measured by the mass content of cationic lipids (such as DOTAP) in the system. For example, it may be 0.7, 0.8, 1.0, 1.2, 1.5, 2.0, 2.1 mg/mL.
  • the volume ratio of the genetic material to the bone-targeting gene vector is 1:1.
  • the incubation time is between 10 and 50 min.
  • the incubation is carried out directly at room temperature without heating or cooling, and the incubation temperature is 20-37 °C.
  • the bone-targeting gene vector of the first aspect of the invention or the gene delivery system of the third aspect of the invention is for use in the preparation of a gene therapy drug, preferably a targeted delivery drug.
  • the bone-targeting gene vector provided by the present invention has a surface modified with alendronate sodium, and the bone-targeting gene carrier has good affinity and targeting to bone tissue, and the molecule thereof
  • the bisphosphonate group can be efficiently combined with hydroxyapatite
  • the bone-targeting gene carrier has good biocompatibility and bone targeting, can effectively combine with genetic substances to form a gene delivery system, and carries the genetic material into the bone cells, thereby promoting the genetic material in the bone tissue. Local high expression of targeted target substances, gene transfection efficiency is high, safe and effective;
  • the size and stability of the prepared bone-targeting gene vector can be regulated by adjusting the amount between the cationic lipid, the neutral auxiliary lipid, the cholesterol and the alendronate-modified phospholipid;
  • the preparation method of the bone targeting gene carrier is simple, convenient to operate, mild in condition, and has broad application prospects.
  • FIG. 1 is a schematic structural view of a bone-targeting gene vector prepared according to Example 1 of the present invention.
  • Example 2 is a characterization of a nuclear magnetic spectrum of alendronate-modified phospholipid prepared in Example 1 of the present invention
  • Example 3 is a transmission electron micrograph of a bone-targeting gene vector prepared in Example 1 of the present invention.
  • Example 4 is a diagram showing the targeting efficiency of a bone-targeting gene vector prepared in Example 2 of the present invention.
  • FIG. 5 is a diagram showing the effect of a bone-targeting gene vector prepared by the first embodiment of the present invention on a cell transfection effect of a GFP plasmid;
  • Figure 6 is a bar graph showing the transfection efficiency of the pGL-3 control plasmid prepared by the bone-targeting gene vector obtained in Example 1 of the present invention.
  • the plasmid DNA of pGL3-control contains the luciferase gene of the North American firefly tail, which can express luciferase in mammalian cells.
  • the generated luciferase as a biocatalyst can catalyze a certain type of chemical reaction, and bio-fluorescence is generated at the same time as a chemical reaction occurs. Therefore, the use of the plasmid DNA as a reporter gene can sufficiently ensure exclusion of experimental interference, and since the intensity of the emitted fluorescence can be detected by a fluorometer, the prepared cationic liposome can be quantitatively characterized by measuring the fluorescence intensity after transfection. Transfection efficiency.
  • a method for preparing a bone targeting gene vector comprising the steps of:
  • the obtained reaction solution was dialyzed in deionized water for 48 hours with a dialysis bag having a molecular weight cut off of 1000 Da to remove unreacted DSPE-PEG2000-COOH, sodium alenate, etc.; the dialysis product was freeze-dried to obtain a white solid, that is, Is DSPE-PEG2000-Aln; wherein, the molar ratio of the carboxyl group of DSPE-PEG2000-COOH to the amino group of alendronate sodium is 2.08:1;
  • the sonicated second mixed solution was placed at 4 ° C overnight, using an Avanti liposome extruder.
  • the particle size is controlled, and the second mixed solution is first extruded three times with a polycarbonate film having a pore size of 0.2 ⁇ m, and then extruded three times through a polycarbonate film having a pore size of 0.1 ⁇ m to obtain a bone-targeting gene.
  • Carrier
  • 1 is a schematic view showing the structure of a bone-targeting gene carrier prepared in Example 1.
  • 1 is alendronate-modified phospholipid
  • 2 is a phospholipid layer.
  • the bone targeting gene carrier is alendronate sodium modified liposome comprising a cationic lipid, a neutral auxiliary lipid, cholesterol, and alendronate.
  • the alendronate-modified phospholipid 1 comprises a polyethylene glycol-derivatized phospholipid (DSPE-PEG-COOH) and alenone linked to the polyethylene glycol-derivatized phospholipid via an amide bond a sodium phosphonate; the cationic lipid, a neutral helper lipid, cholesterol constitutes a phospholipid layer 2, and a phospholipid (ie, DSPE end) in the polyethylene glycol-derivatized phospholipid is interspersed in the phospholipid layer 2 The phospholipid layer 2 forms a vesicle structure, and the alendronate sodium is exposed outside the phospholipid layer 2.
  • DSPE-PEG-COOH polyethylene glycol-derivatized phospholipid
  • reaction formula of the step (1) is as follows (the structural formula of the product is shown in Fig. 2):
  • the DSPE-PEG2000-Aln prepared in the first step (1) of Example 1 was dissolved in deuterated chloroform, and the hydrogen spectrum was scanned on a 400 MHz Bruker ARX 400 nuclear magnetic resonance spectrometer for structural characterization. The results are shown in Fig. 2.
  • Fig. 2 As can be seen from Figure 2, compared with separate DSPE-PEG2000-COOH and NMR spectrum of alendronate, hydrogen spectrum [delta] 2.0 DSPE-PEG2000-Aln occurring (CONH-CHCH 2 CH 2 - ) wherein Peak, which indicates that alendronate has been modified to the backbone of DSPE-PEG2000-COOH.
  • FIG. 3 is a transmission electron micrograph (TEM) of a bone-targeting gene vector prepared in accordance with Example 1 of the present invention. As can be seen from Figure 3, the bone targeting gene vector has a particle size of 45 ⁇ 2 nm.
  • a method for preparing a bone-targeting gene carrier which differs from Example 1 in that a fluorescent label NBD-F (4-fluoro-7-nitro-2,1,3-benzoxazepine) is used.
  • NBD-F 4-fluoro-7-nitro-2,1,3-benzoxazepine
  • NBD-PC oxazolidine-labeled dioleoylphosphatidylcholine DOPC
  • the bone targeting gene carrier has a particle size of 50 nm.
  • alendronate-modified liposome (abbreviated as "no target gene carrier”) is also used as a control, that is, a phospholipid modified without alendronate is used.
  • a method for preparing a bone targeting gene vector comprising the steps of:
  • the chloroform in the first mixed solution was removed by rotary evaporation under vacuum at 40 ° C to form a uniform film on the inner wall of the flask, and then the flask was placed in a vacuum oven for 6 h, and dried under vacuum overnight. , obtaining a film material (thin layer of liposome);
  • the sonicated second mixed solution was allowed to stand at 4 ° C overnight to fully hydrate, and the liposome was sequentially passed through a polycarbonate membrane having a pore size of 0.2 ⁇ m and 0.1 ⁇ m for particle size control using an Avanti liposome extruder. , obtaining a bone targeting gene vector.
  • a method for preparing a bone targeting gene vector comprising the steps of:
  • the liquid gun accurately absorbs the components of each component, and is uniformly mixed into the flask to obtain a first mixed solution having a total volume of 1.5 mL.
  • the chloroform is removed by rotary evaporation under vacuum at 40 ° C to form a uniform layer on the inner wall of the flask. a film, and then placed the above flask in a vacuum drying oven to evacuate 6 h, and dried under vacuum overnight to obtain a film material (thin layer of liposome);
  • the sonicated second mixed solution was allowed to stand at 4 ° C overnight to fully hydrate, and the liposome was sequentially passed through a polycarbonate membrane having a pore size of 0.2 ⁇ m and 0.1 ⁇ m for particle size control using an Avanti liposome extruder.
  • a bone targeting gene vector is obtained.
  • a method for preparing a bone targeting gene vector comprising the steps of:
  • the sonicated second mixed solution was allowed to stand at 4 ° C overnight, and the particle size control was carried out using an Avanti liposome extruder, and the second mixed solution was first extruded 5 times with a polycarbonate film having a pore size of 0.2 ⁇ m. Thereafter, it was extruded five times through a polycarbonate membrane having a pore size of 0.1 ⁇ m to obtain a bone-targeting gene vector.
  • a method for preparing a bone targeting gene vector comprising the steps of:
  • the components were pipetted, added to the flask, and uniformly mixed to obtain a first mixed solution having a total volume of 3 mL.
  • the chloroform was removed by rotary evaporation under vacuum at 40 ° C to form a uniform film on the inner wall of the flask, and then the flask was placed. Placed in a vacuum drying oven for 5 h, and dried under vacuum overnight to obtain a film material (thin layer of liposome);
  • the sonicated second mixed solution was allowed to stand at 4 ° C overnight, and the particle size control was carried out using an Avanti liposome extruder, and the second mixed solution was first extruded 4 times with a polycarbonate film having a pore size of 0.2 ⁇ m. Thereafter, it was extruded four times through a polycarbonate membrane having a pore size of 0.1 ⁇ m to obtain a bone-targeting gene vector.
  • a method of preparing a gene delivery system comprising the steps of:
  • the bone-targeting gene vector (concentration: 0.7 mg/mL) prepared in Example 1 and plasmid DNA (specifically, pSDF-1 plasmid DNA) were separately dissolved in sterile pure water, and then sterilized by filtration through a 0.22 ⁇ m filter.
  • the bone targeting gene carrier solution and the plasmid DNA solution are mixed in an equal volume ratio to obtain a mixed solution, and the mixed solution is rapidly blown 30 times, and incubated at room temperature for 30 minutes to obtain a gene delivery system;
  • the final concentration of the bone-targeting gene vector in the mixture is 0.35 mg/mL, the ratio of nitrogen to phosphorus of the bone-targeting gene vector and the plasmid DNA is 4:1, and the gene delivery system is plasmid DNA and the above
  • the nano-spheres formed by the electrostatic interaction of the bone-targeting gene carrier are mixed in an equal volume ratio to obtain a mixed solution, and the mixed solution is rapidly blown 30 times, and incubated at room temperature for 30 minutes to obtain a gene delivery system;
  • the final concentration of the bone-targeting gene vector in the mixture is 0.35 mg/mL, the ratio of nitrogen to phosphorus of the bone-targeting gene vector and the plasmid DNA is 4:1, and the gene delivery system is plasmid DNA and the above
  • a method of preparing a gene delivery system comprising the steps of:
  • the bone-targeting gene vector prepared in the third embodiment (concentration: 2.0 mg/mL) and the plasmid DNA (specifically, peSDF-1 plasmid DNA) were separately dissolved in sterilized pure water, and then sterilized by filtration through a 0.22 ⁇ m filter.
  • the bone targeting gene carrier solution and the plasmid DNA solution are mixed in an equal volume ratio to obtain a mixed solution, and the mixed solution is rapidly blown 30 times, and incubated at room temperature for 30 minutes to obtain a gene delivery system;
  • the final concentration of the bone-targeting gene carrier in the mixture is 1.0 mg/mL, the ratio of nitrogen to phosphorus of the bone-targeting gene vector and the plasmid DNA is 12:1, and the gene delivery system is plasmid DNA and the above
  • HAP hydroxyapatite
  • A is the target efficiency map of fluorescent untargeted liposomes (ie, the fluorescence intensity of the supernatant before and after HAP binding)
  • B in Figure 4 is the target efficiency map of the fluorescent bone-targeted gene vector.
  • the bone-targeting gene carrier prepared in the examples is modified with alendronate, and the bisphosphonate in the alendronate molecule can efficiently bind to the hydroxyapatite, the bone targeting is further improved.
  • the affinity of the gene vector for HAP the above results indicate that the bone-targeting gene vector prepared by the invention has good targeting property, and further proves that the alendronate-modified phospholipid is successfully embedded in the liposome, and Exposed to the outside.
  • COS-1 cells were collected, divided into 96-well plates at a cell concentration of 1 ⁇ 10 4 /well, and the volume was 100 uL, and the marginal wells were filled with sterile PBS. The cells were cultured at 37 ° C in a 5% CO 2 incubator.
  • the bone-targeting gene vector prepared in Example 1 (initial concentration: 0.7 mg/mL) was diluted with DMEM basal medium to a final concentration of 2, 6, 10, 50 ⁇ g/mL and a volume of 100 ⁇ L.
  • the commercial liposome Lipofectamine 2000 was used as a positive control, and the normal cultured COS-1 cells were used as a blank control. And set up 3 duplicate wells containing only the culture solution to subtract background absorption.
  • OD absorbance of pores containing cell, cck-8 and bone-targeting gene vector (or Lipofectamine 2000);
  • OD background: absorbance of only the pores containing DMEM, cck-8;
  • OD (blank): The absorbance of a hole containing only cell and cck-8.
  • the experimental results showed that the bone-targeting gene vector prepared in Example 1 showed good cytocompatibility to COS-1 cells.
  • concentration of the carrier was as high as 50 ⁇ g/mL, the cell survival rate reached 91.45%, which was much higher than the cell survival rate of 4.43% under the same concentration of the positive control Lipofectamine 2000 liposome.
  • COS-1 cells (1 ⁇ 10 5 /well) were seeded in 24-well culture plates, and 0.5 mL of DMEM medium containing 10% fetal bovine serum was added, and cultured in a CO 2 incubator at 37 ° C overnight.
  • the bone-targeting gene vector (abbreviated as Aln-lipo) prepared in Example 1 was sterilized by filtration at 0.22 ⁇ m, diluted with sterile pure water, and mixed with GFP plasmid DNA solution (liposome-DNA mixed with nitrogen and phosphorus). The molar ratio was 4:1), and after 30 minutes of incubation, nanospheres, the gene delivery system, were obtained.
  • the commercial liposome Lipofectamine TM 2000 was used as a positive control, and the NP plasmid DNA was also complexed with a nitrogen to phosphorus molar ratio of 4:1, and plasmid DNA was simply added as a negative control.
  • the cells were gently washed with PBS, and 0.5 mL of serum-free and antibiotic-free DMEM medium was added to each well, and 50 ⁇ L of the above different treatment drugs (Aln-lipo composite DNA, Lipo2000 composite DNA, simple plasmid DNA), each well.
  • the DNA content was 1 ⁇ g.
  • the transfection solution was removed, and the transfection solution was replaced with DMEM medium containing 10% fetal bovine serum, and culture was continued for 42 hours. After 48 hours, the expression of GFP green fluorescent protein was observed by an inverted fluorescence microscope, and the results are shown in Fig. 5.
  • Results Figure 5 shows that the present invention is prepared nano bone targeting transgenic vector GFP transfection efficiency of plasmid DNA than Lipofectamine TM 2000.
  • COS-1 cells (1 ⁇ 10 5 /well) were seeded in 24-well culture plates and cultured overnight until the cell fusion degree was 70% to 80%, washed with PBS, and 450 ⁇ L of antibiotic-free medium was added.
  • a bone prepared in Example embodiments will be targeted nano commonly used gene transfer vector and liposome Lipofectamine TM 2000 commercially sterilized by filtration with 0.22 ⁇ m filter, and diluted with the sterile water, different ratios of nitrogen and phosphorus Mixed with the pGL-3 control plasmid DNA solution (wherein the nitrogen-to-phosphorus ratio of the bone-targeting gene vector and the pGL-3 control plasmid DNA prepared in Example 1 were 2:1, 4:1, 6:1, and 8: 1, 10:1, 12:1, Lipofectamine TM 2000 and pGL-3 control plasmid DNA have a nitrogen to phosphorus ratio of 2:1), and a complex is obtained after 30 minutes of incubation.
  • a solution containing 50 ⁇ L of the total volume of the different complexes was added to the COS-1 cells, and the DNA content per well was 1 ⁇ g.
  • Liposomal Lipofectamine 2000 was used as a positive control, and water and plasmid DNA (cDNA) were used as negative controls.
  • the transfection solution was aspirated, and the luciferase expressed by pGL3-control was measured after adding the intact medium for further 48 hours.
  • the aspirate medium was washed with PBS, 150 ⁇ L of the cell lysate was added and lysed for 30 minutes, and then transferred to a 1.5 mL centrifuge tube and centrifuged at 12,000 rpm for 5 minutes.
  • the present invention is prepared in the bone targeted gene carrier nanocomposite transfection efficiency pGL-3control plasmid DNA transfection efficiency is higher than when the nanocomposite Lipofectamine TM 2000 as a support, and preferably The ratio of nitrogen to phosphorus using the bone-targeting gene vector and the plasmid DNA was 4:1.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biochemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Dispersion Chemistry (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

本发明提供了一种骨靶向基因载体，所述骨靶向基因载体为阿仑膦酸钠改性的脂质体，所述阿仑膦酸钠改性的脂质体包括阳离子类脂、中性辅助类脂、胆固醇以及阿仑膦酸钠改性的磷脂，所述阳离子类脂、中性辅助类脂、胆固醇构成磷脂层，所述阿仑膦酸钠改性的磷脂穿插于所述磷脂层中并与所述磷脂层形成囊泡结构，所述阿仑膦酸钠暴露在所述磷脂层之外。该骨靶向基因载体对骨组织有较高的靶向性和转染效率，可以负载基因物质在骨组织附近高效表达。本发明还提供了该骨靶向基因载体的制备方法及应用。

Description

一种骨靶向基因载体及其制备方法和应用
本发明要求2016年05月20日向中国专利局递交的发明名称为“一种骨靶向基因载体及其制备方法和应用”、申请号为201610339273.8的在先申请的优先权，上述在先申请的内容以引入的方式并入本文本中。
技术领域
本发明涉及非病毒基因传递系统领域，具体涉及一种骨靶向基因载体及其制备方法和应用。
背景技术
基因治疗是一种通过基因载体将目标基因传递到靶细胞内，通过添加、阻断、纠正基因等方法实现治疗疾病的目的。基因治疗为一些重大疾病提供了一种很有发展前途的治疗方法。然而，高效、靶向性的基因载体的匮乏，制约着基因治疗在临床上的广泛应用。无疑成为基因治疗成功与否的关键所在。
目前，基因载体主要有病毒型载体和非病毒型载体。应用较多的是无免疫反应、造价低廉、可大量重复生产的非病毒型载体。非病毒载体主要包括脂质体、聚乙烯亚胺(PEI)、壳聚糖等，这些非病毒载体通过静电作用与DNA复合形成基因传输系统进行基因转染。其中，脂质体是唯一被FDA批准的纳米载药体系，具有良好的生物相容性、可降解性，因而广泛地应用于非病毒转基因载体。但脂质体作为非病毒转基因载体的转染效率一般较低、缺乏靶向性，因此，如何提高脂质体非病毒转基因载体的靶向性以及转染效率成为研究重点。
发明内容
为解决上述问题，本发明旨在提供一种骨靶向基因载体及其制备方法和应用。该骨靶向基因载体具有双磷酸基，对骨组织有较高的靶向性和转染效率。该载体对基因物质有较好的稳定作用，可使基因物质在骨组织附近高效表达。该基因载体的毒性低、安全有效。
第一方面，本发明提供了一种骨靶向基因载体，所述骨靶向基因载体为阿仑膦酸钠改性的脂质体，所述阿仑膦酸钠改性的脂质体包括阳离子类脂、中性辅助类脂、胆固醇以及阿仑膦酸钠改性的磷脂，所述阳离子类脂、中性辅助类脂、胆固醇构成磷脂层，所述阿仑膦酸钠改性的磷脂穿插于所述磷脂层中并与所述磷脂层形成囊泡结构，所述阿仑膦酸钠暴露在所述磷脂层之外。
优选地，所述骨靶向基因载体的粒径为40-200nm。
优选地，所述阿仑膦酸钠改性的磷脂与所述阳离子类脂、中性辅助类脂、胆固醇的摩尔比为(0.01-0.07)：(1-3)：(0.5-1)：(0.1-1)。这样的摩尔比，可有助于各个组分之间形成形貌较规则、分散性良好、粒径分布较均匀、结构稳定的骨靶向基因载体，不易被人体体液稀释、溶解而解体，有利于靶向到骨细胞，用该骨靶向基因载体包裹基因物质在生物医学应用有较大优势。
进一步优选地，所述阿仑膦酸钠改性的磷脂与所述阳离子类脂、中性辅助类脂、胆固醇的摩尔比为(0.01-0.07)：(2-3)：(0.5-1)：(0.5-1)。
本发明中，所述阿仑膦酸钠改性的磷脂包括阿仑膦酸钠以及通过酰胺键与其相连的磷脂。
优选地，所述阿仑膦酸钠改性的磷脂包括聚乙二醇衍生化磷脂和通过酰胺键与聚乙二醇衍生化磷脂连接的阿仑膦酸钠。
优选地，所述阳离子类脂包括(2,3-二油酰基-丙基)-三甲基氯化铵(1,2-dioleoyl-3-trimethylammonium-propane,DOTAP)、(2,3-二油氧基丙基)三甲基氯化铵(N-[l-(2,3-dioleyloxy)propyl]-N,N,N-tri-methylammonium chloride,DOTMA)和双十八烷基二甲基溴化胺(DODAB)中的一种或多种。
本发明中，所述阳离子类脂为整个脂质体提高正电荷，在转运基因的过程中起主要作用，且具有在体外稳定性好、在体内可生物降解的特点。阳离子类脂的疏水尾链会影响所形成脂质体的稳定性与流动性，而亲水的阳离子头部的电荷特性则会影响到所形成脂质体的表面特性。
更优选地，所述阳离子类脂为DOTAP。
优选地，所述中性辅助类脂包括二油酰磷脂酰乙醇胺(1,2-dioleyl-sn-glycero-3-phosphatidylethanolamine,DOPE)、二油酰磷脂酰胆碱(1,2-dioleoyl-sn-glycero-3-phosphocholine,DOPC)、二油酰基甘油基磷脂酰丝氨酸(1,2-dioleoyl-sn-glycero-3-phospho-L-serine,DOPS)、双(单酰基甘油)磷酸酯(bis(monomyristoylglycero)phosphate,BMP)和卵磷脂(phosphatidylglycerol,PG)中的一种或多种。
更优选地，所述中性辅助类脂为二油酰磷脂酰乙醇胺(DOPE)。DOPE具有很强的细胞膜去稳定化作用，富含DOPE的阳离子脂质体可辅助DNA转染，提供转染效率。DOPE能促进脂质体的形成，尤其是在酸性条件下促进脂质体向反六角形相的过渡，有利于与细胞膜进行融合。
本发明的所述述骨靶向基因载体中，胆固醇可镶嵌于阳离子类脂、中性辅助类脂分子之间，共同形成磷脂层，可以提高所述基因载体包覆基因后形成的基因载体复合物的体内转染活性。
优选地，所述聚乙二醇衍生化磷脂是由聚乙二醇通过共价键和磷脂类物质相连得到，所述聚乙二醇分子的分子量为200～20000道尔顿。所述磷脂类物质可以为人工合成的或自然界存在的磷脂，所述磷脂类物质包括但不限于二硬脂酰磷脂酰乙醇胺(DSPE)、二硬脂酰磷脂酰甘油(DSPG)或胆固醇。具体地，聚乙二醇分子的分子量可以为200、500、1000、2000、5000、7000、10000、15000或20000。
阿仑膦酸钠为双磷酸类化合物，其分子中含有-NH2活性官能团，可以理解的是，所述阿仑膦酸钠改性的磷脂是通过阿仑膦酸钠的氨基与羧基化的聚乙二醇衍生化磷脂通过酰胺键连接而成。
更优选地，所述羧基化的聚乙二醇衍生化磷脂为二硬脂酰磷脂酰乙醇胺-聚乙二醇-羧酸共聚(DSPE-PEG-COOH)。此时，所述阿仑膦酸钠改性的磷脂为阿仑膦酸钠-聚乙二醇-二硬脂酰磷脂酰乙醇胺(DSPE-PEG-Aln)。
进一步优选地，DSPE-PEG-Aln与所述DOTAP、DOPE、胆固醇的摩尔比为(0.01-0.07)：(1-3)：(0.5-1)：(0.5-1)。
本发明第一方面提供的所述骨靶向基因载体表面修饰有阿仑膦酸的主体部分，该基因载体对骨组织有较好的亲和性，其分子中的双磷酸基能与羟基磷灰石高效地结合，能够高靶向性地将目的基因物质递送到骨细胞内，有利于目的基因后续的表达。此外，所述骨靶向基因载体的表面稳定地修饰有阿伦膦酸-聚乙二醇，可以有效、长时间地稳定所述骨靶向基因载体，延长体内循环的时间，低成本，低毒，安全有效。
第二方面，本发明提供了一种骨靶向基因载体的制备方法，包括以下步骤：
(1)将聚乙二醇衍生化磷脂溶于第一溶剂，并加入催化剂、缩合剂活化， 再加入溶解在第一溶剂中的阿伦膦酸钠，在室温下进行酰胺化反应8-12h，得到反应液，将所述反应液经分离纯化后进行冷冻干燥，得到阿仑膦酸钠改性的磷脂，其中，所述聚乙二醇衍生化磷脂的羧基与阿仑膦酸钠的氨基的摩尔比为(1-10):1；
(2)取上述阿仑膦酸钠改性的磷脂，与阳离子类脂、中性辅助类脂、胆固醇加入到反应器中，加入有机溶剂，混合均匀后得到第一混合溶液，通过旋转蒸发法去除所述第一混合溶液中的溶剂，得到膜状材料；
(3)加入磷酸盐缓冲溶液溶解所述膜材料，并进行超声处理，得到第二混合溶液，将所述第二混合溶液采用微孔过滤膜来回挤压过滤多次，得到骨靶向基因载体，其中，所述骨靶向基因载体为阿仑膦酸钠改性的脂质体，所述阿仑膦酸钠改性的脂质体包括阳离子类脂、中性辅助类脂、胆固醇以及阿仑膦酸钠改性的磷脂，所述阳离子类脂、中性辅助类脂、胆固醇构成磷脂层，所述阿仑膦酸钠改性的磷脂穿插于所述磷脂层中并与所述磷脂层形成囊泡结构，所述阿仑膦酸钠暴露在所述磷脂层之外。
优选地，阿仑膦酸钠改性的磷脂与所述阳离子类脂、中性辅助类脂、胆固醇的摩尔比为(0.01-0.07)：(1-3)：(0.5-1)：(0.1-1)。
进一步优选地，所述阿仑膦酸钠改性的磷脂与所述阳离子类脂、中性辅助类脂、胆固醇的摩尔比为(0.01-0.07)：(2-3)：(0.5-1)：(0.5-1)。
优选地，所述骨靶向基因载体的粒径为40-200nm。
进一步优选地，所述骨靶向基因载体的粒径为50-150nm。
优选地，所述阳离子类脂包括DOTAP、DOTMA和DODAB的一种或多种。
更优选地，所述阳离子类脂为DOTAP。
优选地，所述中性辅助类脂包括DOPE、DOPC、DOPS、BMP和PG中的一种或多种。
更优选地，所述中性辅助类脂为DOPE。
优选地，所述阿仑膦酸钠改性的磷脂包括聚乙二醇衍生化磷脂和通过酰胺键与聚乙二醇衍生化磷脂连接的阿仑膦酸钠。所述磷脂类物质包括但不限于二硬脂酰磷脂酰乙醇胺(DSPE)、二硬脂酰磷脂酰甘油(DSPG)或胆固醇。
进一步优选地，所述聚乙二醇衍生化磷脂是由聚乙二醇通过共价键和磷脂类物质相连得到，所述聚乙二醇分子的分子量为200～20000道尔顿。
更优选地，所述阿仑膦酸钠改性的磷脂为阿仑膦酸钠-聚乙二醇-二硬脂酰磷脂酰乙醇胺(DSPE-PEG-Aln)。
进一步优选地，DSPE-PEG-Aln与所述DOTAP、DOPE、胆固醇的摩尔比为(0.01-0.07)：(1-3)：(0.5-1)：(0.5-1)。
优选地，步骤(1)中，所述第一溶剂包括水、pH值为5.5～6.0的2-(N-吗啡啉)乙磺酸缓冲液(简称为“MES缓冲溶液”)、pH值为7.0～7.4的磷酸盐缓冲液等。
优选地，步骤(1)中，所述分离纯化为采用截留分子量为500-1000Da的透析袋进行透析48-72h。
步骤(1)中，所述酰胺化反应的方法为本领域的技术人员所熟知。催化剂又可称为活化剂，常与缩合剂联用，用于酰胺化反应。
优选地，步骤(1)中，所述缩合剂包括1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(简称EDC)。
优选地，步骤(1)中，所述催化剂包括N-羟基琥珀酰亚胺(NHS)、N-羟基硫代琥珀酰亚胺钠盐(Sufo-NHS)、1-羟基苯并三唑(HOBT)中的一种或多种。
进一步优选地，所述酰胺化反应的时间为8h。
优选地，所述活化的时间为1-4h。可以为1h、2h、3h或4h。
优选地，步骤(2)中，所述有机溶剂为氯仿或者为氯仿和甲醇的混合溶液。
进一步优选地，所述有机溶剂的体积为1-3mL。
优选地，步骤(2)中，在所述去除第一混合溶液中的溶剂之后，还包括：将烧瓶置于真空干燥箱中进行抽真空4-8h，并干燥过夜。
优选地，步骤(3)中，所述超声处理具体为：
先在功率为50W下进行水浴超声30-60min，然后采用探针式超声仪在20kHz的频率和750W的功率下进行超声4-8min。
优选地，步骤(3)中，所述微孔过滤膜为孔径为0.1-0.2μm的聚碳酸酯膜。
进一步优选地，步骤(3)中，将所述第二混合溶液先采用孔径为0.2μm的聚碳酸酯膜挤压2～5次后，再通过孔径为0.1μm的聚碳酸酯膜挤压2-5次。
本发明第二发明提供的所述骨靶向基因载体的制备方法，先通过聚乙二醇衍生化磷脂与阿伦磷酸钠的酰胺缩合反应形成阿仑膦酸钠改性的磷脂，再通过薄膜分散法将阿仑膦酸钠改性的磷脂与阳离子类脂、中性辅助类脂、胆固醇构建阿伦磷酸修饰的脂质体，即骨靶向基因载体。此方法获得的基因载体的细胞毒性低、生物相容性好、骨靶向效率高、转染效率显著。所述骨靶向基因载体 的制备方法简单，操作方便，条件温和，具有广阔的应用前景。
第三方面，本发明提供了一种基因递送系统，所述基因递送系统为基因物质和上述所述的骨靶向基因载体通过静电作用形成的纳米微球，其中，所述骨靶向基因载体与所述基因物质的氮磷比为(2-12)：1。
如本发明所述的，所述“氮磷比”为骨靶向基因载体的阳离子类脂中氨基的摩尔数与所述基因物质中磷酸根的摩尔数之比。
本发明中，骨靶向基因载体中的阳离子类脂与基因物质的氮磷比为(2-12)：1，可以有效地提高基因载体与基因物质的复合效率，以及基因递送系统的表面电荷、胶体稳定性及粒径大小。
优选地，所述基因递送系统的平均粒径为50-200nm。
所述基因物质包括但不限于脱氧核糖核酸、质粒DNA、微环DNA、核糖核酸中的一种或多种。
更优选地，所述基因物质包括pSDF-1质粒和peSDF-1质粒中的一种或两种。
所述基因递送系统中，所述骨靶向基因载体表面修饰有阿仑膦酸钠，该基因载体对骨组织有较好的亲和性，其分子中的双磷酸基能与羟基磷灰石高效地结合，能够高靶向性地将目的基因物质递送到骨细胞内，有利于目的基因后续的表达。
第四方面，本发明提供了一种基因递送系统的制备方法，包括以下步骤：
将基因物质和上述所述的骨靶向基因载体溶解于灭菌纯水后，得到混合液，然后室温静置，所述混合液中的骨靶向基因载体和所述基因物质通过静电作用形成纳米微球，得到所述基因递送系统，其中，所述骨靶向基因载体与所 述基因物质的氮磷比为(2-12)：1。
优选地，所述骨靶向基因载体的浓度为0.7-2.1mg/mL(载体自身的浓度，未混合前)。以体系中阳离子类脂(如DOTAP)的质量含量进行计量。例如，可以是0.7、0.8、1.0、1.2、1.5、2.0、2.1mg/mL。
优选地，所述基因物质和所述骨靶向基因载体的体积比为1:1。
优选地，所述孵育的时间为10-50min。所述孵育直接在室温下进行反应，无须加热或降温，所述孵育温度为20-37℃。
第五方面，本发明第一方面所述的骨靶向基因载体或如本发明第三方面所述的基因递送系统在制备基因治疗药物(优选为靶向投递药物)中的应用。
本发明的有益效果在于：
(1)本发明提供的所述骨靶向基因载体，其表面修饰有阿仑膦酸钠，所述骨靶向基因载体对对骨组织有较好的亲和性、靶向性，其分子中的双磷酸基能与羟基磷灰石高效地结合；
(2)所述骨靶向基因载体具有良好的生物相容性和骨靶向性，能够有效与基因物质进行复合形成基因递送系统，将基因物质携带进入骨细胞，进而促进基因物质在骨组织局部高表达靶向目标物质，基因转染效率高，安全有效；
(3)可以通过调节阳离子类脂、中性辅助类脂、胆固醇与及阿仑膦酸钠改性的磷脂之间的用量来调控制备的骨靶向基因载体的大小、稳定性；
(4)所述骨靶向基因载体的制备方法简单，操作方便，条件温和，具有广阔的应用前景。
附图说明
图1为本发明实施例一制得的骨靶向基因载体的结构示意图；
图2为本发明实施例一中制得的阿伦膦酸钠改性的磷脂的核磁谱图表征；
图3为本发明实施例一制得的骨靶向基因载体的透射电镜图；
图4为本发明实施例二制得的骨靶向基因载体的靶向效率图；
图5为本发明实施例一制得的骨靶向基因载体对GFP质粒的细胞转染效果图；
图6为本发明实施例一制得的骨靶向基因载体对pGL-3 control质粒的转染效率柱状图。
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。
pGL3-control这种质粒DNA中包含有北美萤火虫尾部的荧光素酶基因，它可以在哺乳动物细胞中表达出荧光素酶。而生成的荧光素酶作为一种生物催化剂可以催化某类化学反应，在发生化学反应的同时会产生生物荧光。所以采用该质粒DNA作为报告基因可以充分保证排除实验干扰，并且由于所发出荧光的强度可以用荧光计检测，从而可以通过测量转染后的荧光强度来定量的表征所制备的阳离子脂质体的转染效率。
实施例一
一种骨靶向基因载体的制备方法，包括以下步骤：
(1)制备阿伦膦酸钠改性的磷脂DSPE-PEG2000-Aln：
以1-(3-二甲基氨基丙基)-3-乙基碳化二亚胺(EDC)和N-羟基琥珀酰亚胺 (NHS)作为活化剂，将0.24mmol的DSPE-PEG2000-COOH在室温下溶于MES缓冲溶液(0.05mol/L，pH＝5.5)中，向上述溶液中加入EDC和NHS在冰水浴条件下反应活化羧基2h，其中，DSPE-PEG2000-COOH:EDC:NHS的摩尔比为0.03：1.25：0.8；
将0.5mmol的阿伦磷酸钠预先溶于适量的MES缓冲溶液(0.05mol/L，pH＝5.5)中，再加入上述活化后的反应液中，在搅拌条件下进行酰胺化缩合反应8h，将得到的反应液用截留分子量为1000Da的透析袋在去离子水中透析48小时，以除去未反应的DSPE-PEG2000-COOH、阿伦磷酸钠等；将透析后的产品进行冷冻干燥得到白色固体，即为DSPE-PEG2000-Aln；其中，DSPE-PEG2000-COOH的羧基与阿仑膦酸钠的氨基的摩尔比为2.08：1；
(2)骨靶向基因载体的制备：
将DOTAP、DOPE、胆固醇Chol与上述制得的DSPE-PEG2000-Aln溶于氯仿，并加到烧瓶中，其中各物料的摩尔比为DSPE-PEG2000-Aln：DOTAP:DOPE:Chol:＝0.02:2:0.5:0.5；混合均匀后得到第一混合溶液，在40℃的真空条件下通过旋转蒸发法去除得到第一混合溶液中的氯仿，在烧瓶内壁形成一层均匀的薄膜，然后抽真空6h，并将上述烧瓶放置于真空干燥箱中真空干燥过夜，得到薄膜材料(脂质体薄层)；
(3)用pH为7.4的PBS缓冲溶液对上述干燥的薄膜材料进行水化，然后先在功率为50W下进行40℃的水浴超声60min，使烧瓶壁上的薄膜溶解于PBS溶液中；再采用探针式超声仪以20kHz的频率和750W的功率进行超声4min，超声间隔为2s，直至溶液呈现蓝色乳光状态，得到第二混合溶液；
将超声后的第二混合溶液于4℃下放置过夜，采用Avanti脂质体挤出器进 行粒径控制，将所述第二混合溶液先采用孔径为0.2μm的聚碳酸酯膜挤压3次后，再通过孔径为0.1μm的聚碳酸酯膜挤压3次，获得骨靶向基因载体。
图1为实施例一制备的骨靶向基因载体的结构示意图，图中1为阿仑膦酸钠改性的磷脂，2为磷脂层。所述骨靶向基因载体为阿仑膦酸钠改性的脂质体，所述阿仑膦酸钠改性的脂质体包括阳离子类脂、中性辅助类脂、胆固醇以及阿仑膦酸钠改性的磷脂1，所述阿仑膦酸钠改性的磷脂1包括聚乙二醇衍生化磷脂(DSPE-PEG-COOH)和通过酰胺键与聚乙二醇衍生化磷脂连接的阿仑膦酸钠；所述阳离子类脂、中性辅助类脂、胆固醇构成磷脂层2，所述聚乙二醇衍生化磷脂中的磷脂(即DSPE端)穿插于所述磷脂层中2并与所述磷脂层2形成囊泡结构，所述阿仑膦酸钠暴露在所述磷脂层2之外。
本实施例中，步骤(1)的反应式如下所示(产物的结构式示出在图2中)：
Figure PCTCN2016087283-appb-000001
对实施例一步骤(1)中制备的DSPE-PEG2000-Aln溶于氘代氯仿中，在400MHz Bruker ARX 400的核磁共振光谱仪上扫描氢谱以进行结构表征，结果见图2。从图2可以看出，与单独的DSPE-PEG2000-COOH和阿伦磷酸钠的核磁谱图相比，DSPE-PEG2000-Aln的氢谱出现的δ2.0(CONH–CHCH2CH2–)特征峰，这表明阿仑膦酸钠已经被修饰到DSPE-PEG2000-COOH的主链上。
实施例一的骨靶向基因载体的TEM表征：
用10μL的移液枪吸取1滴实施例一制备的骨靶向基因载体样品悬液，滴在含有碳膜的铜网上，用镊子夹着铜网，滴液后静置数分钟，然后用滤纸从铜 网边缘吸去多余的液体，滴上1％的磷钨酸负染色液，染色1～2分钟后用滤纸吸去负染色液，再用蒸馏水滴在铜网上洗1～2次，用滤纸吸去水，待干后用透射电镜观察。图3为本发明实施例一制得的骨靶向基因载体的透射电镜图(TEM)。从图3中可以看出，所述骨靶向基因载体的粒径为45±2nm。
实施例二
一种骨靶向基因载体的制备方法，与实施例1的不同之处在于，采用荧光标记物NBD-F(4-氟-7-硝基-2,1,3-苯并氧杂恶二唑)标记的二油酰磷脂酰胆碱DOPC(简写为NBD-PC)替换实施例1中的DOPE，其他条件同实施例一。
本发明实施例二所制得的骨靶向基因载体中，各物料的摩尔比为DSPE-PEG2000-Aln:DOTAP:NBD-PC:Chol＝0.02:2:0.5:0.5。所述骨靶向基因载体的粒径为50nm。
同时为了突出本发明的有益效果，还将未采用阿仑膦酸改性的脂质体(简称为“无靶向基因载体”)作为对照，即采用未经阿伦膦酸钠改性的磷脂DSPE-PEG2000替换实施例二的DSPE-PEG2000-Aln，其中，无靶向基因载体中各物料的摩尔比为DSPE-PEG2000:DOTAP:NBD-PC:Chol＝0.02:2:0.5:0.5。
实施例三
一种骨靶向基因载体的制备方法，包括以下步骤：
(1)制备阿伦膦酸钠改性的磷脂DSPE-PEG2000-Aln：同实施例1；
(2)骨靶向基因载体的制备：
将DOTAP、DOPE、胆固醇Chol与上述制得的DSPE-PEG2000-Aln溶于 氯仿，并加入烧瓶中，混合均匀后得到总体积为2mL的第一混合溶液，其中，各物料的摩尔比为DOTAP:DOPE:Chol:DSPE-PEG2000-Aln＝0.02:2:1:1；在40℃真空条件下通过旋转蒸发法去除所述第一混合溶液中的氯仿，在烧瓶内壁形成一层均匀的薄膜，然后将上述烧瓶放置于真空干燥箱中抽真空6h，并真空状态下干燥过夜，得到薄膜材料(脂质体薄层)；
(3)用pH为7.4的PBS缓冲溶液对上述干燥的薄膜材料进行水化，然后先在功率为50W下进行水浴超声30min，使烧瓶壁上的薄膜溶解于PBS溶液中；再采用探针式超声仪以20kHz的频率和750W的功率进行超声5min，超声间隔为2s，直至溶液呈现蓝色乳光状态，得到第二混合溶液；
将超声后的第二混合溶液于4℃下放置过夜以充分水化，采用Avanti脂质体挤出器，将脂质体依次通过孔径为0.2μm、0.1μm的聚碳酸酯膜进行粒径控制，获得骨靶向基因载体。
实施例四
一种骨靶向基因载体的制备方法，包括以下步骤：
(1)制备阿伦膦酸钠改性的磷脂DSPE-PEG7000-Aln：同实施例1；
(2)骨靶向基因载体的制备：
将DOTAP、DOPE、胆固醇Chol与上述制得的DSPE-PEG7000-Aln溶于氯仿，以各物料的摩尔比为DSPE-PEG7000-Aln：DOTAP:DOPE:Chol＝0.05：3:1:1；用移液枪准确吸取各方组分物质，加入烧瓶中混合均匀后，得到总体积为1.5mL的第一混合溶液，在40℃真空条件下通过旋转蒸发法去除氯仿，在烧瓶内壁形成一层均匀的薄膜，然后将上述烧瓶放置于真空干燥箱中抽真空6 h，并真空状态下干燥过夜，得到薄膜材料(脂质体薄层)；
(3)用pH为7.4的PBS缓冲溶液对上述干燥的薄膜材料进行水化，然后先在功率为50W下进行40℃的水浴超声45min，使烧瓶壁上的薄膜溶解于PBS溶液中；再采用探针式超声仪以20kHz的频率和750W的功率进行超声4min，超声间隔为2s，直至溶液呈现蓝色乳光状态，得到第二混合溶液；
将超声后的第二混合溶液于4℃下放置过夜充分水化，采用Avanti脂质体挤出器，将脂质体依次通过孔径为0.2μm、0.1μm的聚碳酸酯膜进行粒径控制，获得骨靶向基因载体。
实施例五
一种骨靶向基因载体的制备方法，包括以下步骤：
(1)制备阿伦膦酸钠改性的磷脂DSPE-PEG10000-Aln：同实施例1；
(2)骨靶向基因载体的制备：
将DOTMA、DOPS、胆固醇Chol与上述制得的DSPE-PEG10000-Aln溶于氯仿，加入烧瓶中，其中各物料的摩尔比为DSPE-PEG10000-Aln：DOTMA:DOPS:Chol＝0.01:1:0.6:0.1；混合均匀后得到总体积为1mL的第一混合溶液，在40℃的真空条件下通过旋转蒸发法去除得到第一混合溶液中的氯仿，在烧瓶内壁形成一层均匀的薄膜，然后将上述烧瓶放置于真空干燥箱中抽真空4h，并真空状态下干燥过夜，得到薄膜材料(脂质体薄层)；
(3)用pH为7.4的PBS缓冲溶液对上述干燥的薄膜材料进行水化，然后先在功率为50W下进行40℃的水浴超声40min，使烧瓶壁上的薄膜溶解于PBS溶液中；再采用探针式超声仪以20kHz的频率和750W的功率进行超声 5min，超声间隔为2s，直至溶液呈现蓝色乳光状态，得到第二混合溶液；
将超声后的第二混合溶液于4℃下放置过夜，采用Avanti脂质体挤出器进行粒径控制，将所述第二混合溶液先采用孔径为0.2μm的聚碳酸酯膜挤压5次后，再通过孔径为0.1μm的聚碳酸酯膜挤压5次，获得骨靶向基因载体。
实施例六
一种骨靶向基因载体的制备方法，包括以下步骤：
(1)制备阿伦膦酸钠改性的磷脂DSPE-PEG20000-Aln：同实施例1；
(2)骨靶向基因载体的制备：
将DODAB、BMP、胆固醇Chol与上述DSPE-PEG20000-Aln溶于氯仿，以各物料的摩尔比为DSPE-PEG20000-Aln：DODAB:BMP:Chol＝0.07:2:0.8:0.6；用移液枪准确吸取各组分，加入烧瓶中，混合均匀后得到总体积为3mL的第一混合溶液，在40℃真空条件下通过旋转蒸发法去除氯仿，在烧瓶内壁形成一层均匀的薄膜，然后将上述烧瓶放置于真空干燥箱中抽真空5h，并真空状态下干燥过夜，得到薄膜材料(脂质体薄层)；
(3)用pH为7.4的PBS缓冲溶液对上述干燥的薄膜材料进行水化，然后先在功率为50W下进行40℃的水浴超声50min，使烧瓶壁上的薄膜溶解于PBS溶液中；再采用探针式超声仪以20kHz的频率和750W的功率进行超声6min，超声间隔为2s，直至溶液呈现蓝色乳光状态，得到第二混合溶液；
将超声后的第二混合溶液于4℃下放置过夜，采用Avanti脂质体挤出器进行粒径控制，将所述第二混合溶液先采用孔径为0.2μm的聚碳酸酯膜挤压4次后，再通过孔径为0.1μm的聚碳酸酯膜挤压4次，获得骨靶向基因载体。
实施例七
一种基因递送系统的制备方法，包括以下步骤：
将实施例一制得的骨靶向基因载体(浓度为0.7mg/mL)与质粒DNA(具体为pSDF-1质粒DNA)分别溶解在灭菌纯水后，用0.22μm滤膜过滤除菌得到骨靶向基因载体溶液、质粒DNA溶液，将两种溶液以等体积比进行混合，得到混合溶液，快速吹打上述混合溶液30次，并在室温下共同孵育30min，获得基因递送系统；其中，所述骨靶向基因载体在混合液中的最终浓度为0.35mg/mL，所述骨靶向基因载体与所述质粒DNA的氮磷比为4：1，所述基因递送系统为质粒DNA和上述骨靶向基因载体通过静电作用形成的纳米微球。
实施例八
一种基因递送系统的制备方法，包括以下步骤：
将实施例三制得的骨靶向基因载体(浓度为2.0mg/mL)与质粒DNA(具体为peSDF-1质粒DNA)分别溶解在灭菌纯水后，用0.22μm滤膜过滤除菌得到骨靶向基因载体溶液、质粒DNA溶液，将两种溶液以等体积比进行混合，得到混合溶液，快速吹打上述混合溶液30次，并在室温下共同孵育30min，获得基因递送系统；其中，所述骨靶向基因载体在混合液中的最终浓度为1.0mg/mL，所述骨靶向基因载体与所述质粒DNA的氮磷比为12：1，所述基因递送系统为质粒DNA和上述骨靶向基因载体通过静电作用形成的纳米微球。
效果实施例：
1、骨靶向基因载体的靶向效率测定
将本发明实施例二制得的荧光骨靶向基因载体与荧光无靶向基因载体使用荧光分光光度计在激发波长＝533nm下，分别检测DOTAP终浓度相同(均为0.7mg/mL)的靶向脂质体、无靶向脂质体的荧光强度a1；
取20mg的羟基磷灰石(HAP)分散至2mL的PBS配成10mg/mL的分散液；将10μL上述荧光靶向脂质体、荧光无靶向脂质体分别与HAP在缓慢震荡下共培养5h，在5000rpm下高速离心后，分别检测其上清液的荧光强度a2，其中：HAP的结合率＝(a1-a2)/a1×100％。靶向效率的测试结果如图4所示。其中，图4中A为荧光无靶向脂质体的靶向效率图(即结合HAP前后，上清液的荧光强度变化)，图4中B为荧光骨靶向基因载体的靶向效率图。
从图4可以明显看出，在荧光骨靶向基因载体与HAP共培养之后，两者高效结合，在离心之后，荧光骨靶向基因载体与HAP的复合物基本位于下层沉淀中，使得上清液的荧光强度大大降低。由图4可以看出，连接有DSPE-PEG2000-Aln的脂质体与HAP的结合能力显著高于无靶向脂质体，经计算，其结合率从13.7％提高到61.4％。由于实施例制得的骨靶向基因载体的外面修饰有阿仑膦酸，而阿仑膦酸钠分子中的双磷酸基能与羟基磷灰石高效结合，也进一步提高了所述骨靶向基因载体对HAP的亲和性，以上结果表明本发明制得的骨靶向基因载体具有良好的靶向性，也进一步佐证了阿仑膦酸改性的磷脂成功嵌入到脂质体上，并暴露在外面。
2、骨靶向基因载体的细胞相容性研究：
收集COS-1细胞，以1×104/孔的细胞浓度分于96孔板，体积100uL，边缘孔用无菌PBS填充。置37℃、5％CO2温箱培养使细胞贴壁。
将实施例一制得的骨靶向基因载体(起始浓度为0.7mg/mL)用DMEM基础培养基进行稀释，终浓度分别为2,6,10,50μg/mL,体积为100μL。以商业脂质体Lipofectamine 2000为阳性对照，正常培养的COS-1细胞为空白对照。并设置3个仅含培养液的复孔以扣除背景吸收。
弃去原有培养液，分别加入含各浓度载体药物的培养液，做好标记，放入培养箱培养48h。然后每孔加入10μL的cck-8溶液，继续培养4h。终止培养，使用酶标仪在450nm处测量各孔的吸光值OD。
细胞活性(％)＝[OD(加药)-OD(背景)]/[OD(0加药)-OD(背景)]×100％；
OD(加药)：含cell、cck-8和骨靶向基因载体(或Lipofectamine 2000)的孔的吸光值；
OD(背景)：仅含DMEM、cck-8的孔的吸光值；
OD(空白)：仅含cell、cck-8的孔的吸光值。
实验结果显示，实施例一制得的骨靶向基因载体对COS-1细胞显示出良好的细胞相容性。当载体的作用浓度高达50μg/mL时，细胞存活率达到91.45％，远远高于阳性对照Lipofectamine 2000脂质体相同浓度作用下的细胞存活率4.43％。
3、骨靶向基因载体的细胞转染效率研究
(1)GFP质粒转染细胞：
将COS-1细胞(1×105/孔)接种在24孔培养板中，加入0.5mL含有10％胎牛血清的DMEM培养基，置于CO2孵箱中37℃下培养过夜。
将实施例一制得的骨靶向基因载体(简写为Aln-lipo)用0.22μm过滤除菌后，用灭菌纯水稀释后与GFP质粒DNA溶液混合(脂质体与DNA混合的氮磷摩尔比为4:1)，培育30分钟后得到纳米微球，即基因递送系统。同时以商业脂质体LipofectamineTM 2000作为阳性对照，也以氮磷摩尔比为4:1与GFP质粒DNA复合，并单纯加质粒DNA为阴性对照。
用PBS轻轻洗涤细胞，每孔中加入无血清无抗生素的DMEM培养基0.5mL，以及50μL的以上不同处理药物(Aln-lipo复合的DNA、Lipo2000复合的DNA、单纯的质粒DNA)，每孔的DNA含量为1μg。轻轻摇晃混匀，置于CO2孵箱中37℃孵育6h。移去转染液，用10％胎牛血清的DMEM培养基替换转染液，继续培养42h。48h后利用倒置荧光显微镜观察GFP绿色荧光蛋白的表达，结果见图5。
图5的结果表明，本发明制得的骨靶向纳米转基因载体对GFP质粒DNA的转染效率高于Lipofectamine TM 2000。
(2)pGL-3 control转染细胞：
将COS-1细胞(1×105/孔)接种于24孔培养板，过夜培养至细胞融合度为70％～80％时，用PBS洗涤后加入450μL不含抗生素的培养基。将实施例一制得的骨靶向纳米转基因载体和商业上常用的脂质体LipofectamineTM 2000用0.22μm滤膜进行过滤除菌后，并用灭菌纯水稀释后，按不同的氮磷比分别与pGL-3 control质粒DNA溶液混合(其中，实施例1制得的骨靶向基因载体和pGL-3 control质粒DNA的氮磷比分别为2:1、4:1、6:1、8:1、10:1、12:1， LipofectamineTM 2000与pGL-3 control质粒DNA的氮磷比为2：1)，培育30分钟后得到复合物。将含不同复合物总体积为50μL的溶液加入到COS-1细胞中，每孔的DNA含量为1μg。并以脂质体Lipofectamine 2000为阳性对照，水和单纯加质粒DNA(cDNA)为阴性对照。培养24h后，吸去转染液后，加入完整培养基继续培养48h后测量pGL3-control表达的荧光素酶。吸去培养基用PBS洗涤后，加入150μL的细胞裂解液裂解30分钟后，转移至1.5mL的离心管中，12000rpm高速离心5分钟。取50μL上清液与50μL荧光素酶检测试剂(Promega)反测定相对光单位。另取20μL上清液进行BCA蛋白含量测试。以每毫克蛋白的相对光度值(RLU/mg，荧光素酶的表达量)作为转染效率的评价指标，结果见图6。
从图6中可以看出，本发明制备的骨靶向基因载体的纳米复合物对pGL-3control质粒DNA的转染效率高于Lipofectamine TM 2000作为载体时的纳米复合物的转染效率，而且优选采用骨靶向基因载体与质粒DNA的氮磷比为4:1。

Claims (10)

  1. 一种骨靶向基因载体，其特征在于，所述骨靶向基因载体为阿仑膦酸钠改性的脂质体，所述阿仑膦酸钠改性的脂质体包括阳离子类脂、中性辅助类脂、胆固醇以及阿仑膦酸钠改性的磷脂，所述阳离子类脂、中性辅助类脂、胆固醇构成磷脂层，所述阿仑膦酸钠改性的磷脂穿插于所述磷脂层中并与所述磷脂层形成囊泡结构，所述阿仑膦酸钠暴露在所述磷脂层之外。
  2. 如权利要求1所述的骨靶向基因载体，其特征在于，所述阿仑膦酸钠改性的磷脂包括聚乙二醇衍生化磷脂和通过酰胺键与聚乙二醇衍生化磷脂连接的阿仑膦酸钠。
  3. 如权利要求1所述的骨靶向基因载体，其特征在于，所述骨靶向基因载体的粒径为40-200nm。
  4. 如权利要求1所述的骨靶向基因载体，其特征在于，所述阿仑膦酸钠改性的磷脂与所述阳离子类脂、中性辅助类脂、胆固醇的摩尔比为(0.01-0.07)：(1-3)：(0.5-1)：(0.1-1)。
  5. 如权利要求1所述的骨靶向基因载体，其特征在于，所述阳离子类脂包括(2,3-二油酰基-丙基)-三甲基氯化铵、(2,3-二油氧基丙基)三甲基氯化铵和双十八烷基二甲基溴化胺中的一种或多种；所述中性辅助类脂包括二油酰磷脂酰乙醇胺和二油酰磷脂酰胆碱中的一种或两种。
  6. 如权利要求2所述的骨靶向基因载体，其特征在于，所述聚乙二醇衍生化磷脂是由聚乙二醇通过共价键和磷脂类物质相连得到，所述聚乙二醇分子的分子量为200～20000道尔顿。
  7. 一种骨靶向基因载体的制备方法，其特征在于，包括以下步骤：
    (1)将聚乙二醇衍生化磷脂溶于第一溶剂，并加入催化剂、脱水剂活化，再加入溶解在第一溶剂中的阿伦膦酸钠，在室温下进行酰胺化反应8-12h，得到反应液，将所述反应液经分离纯化后进行冷冻干燥，得到阿仑膦酸钠改性的磷脂，其中，所述聚乙二醇衍生化磷脂的羧基与阿仑膦酸钠的氨基的摩尔比为(1-10):1；
    (2)取上述阿仑膦酸钠改性的磷脂，与阳离子类脂、中性辅助类脂、胆固醇加入到反应器中，加入有机溶剂，混合均匀后得到第一混合溶液，通过旋转蒸发法去除所述第一混合溶液中的溶剂，干燥得到膜状材料；
    (3)加入磷酸盐缓冲溶液溶解所述膜材料，并进行超声处理，得到第二混合溶液，将所述第二混合溶液采用微孔过滤膜来回挤压过滤多次，得到骨靶向基因载体，其中，所述骨靶向基因载体为阿仑膦酸钠改性的脂质体，所述阿仑膦酸钠改性的脂质体包括阳离子类脂、中性辅助类脂、胆固醇以及阿仑膦酸钠改性的磷脂，所述阳离子类脂、中性辅助类脂、胆固醇构成磷脂层，所述阿仑膦酸钠改性的磷脂穿插于所述磷脂层中并与所述磷脂层形成囊泡结构，所述阿仑膦酸钠暴露在所述磷脂层之外。
  8. 一种基因递送系统，其特征在于，所述基因递送系统为基因物质和权利要求1所述的骨靶向基因载体通过静电作用形成的纳米微球，其中，所述骨靶向基因载体与所述基因物质的氮磷摩尔比为(2-12)：1。
  9. 一种基因递送系统的制备方法，其特征在于，包括以下步骤：
    将基因物质和如权利要求1所述的骨靶向基因载体溶解于灭菌水后，得到混合液，然后室温静置，所述混合液中的骨靶向基因载体和所述基因物质通过静电作用形成纳米微球，得到所述基因递送系统，其中，所述骨靶向基因载体与所述基因物质的氮磷比为(2-12)：1。
  10. 如权利要求1-6任一项所述的骨靶向基因载体或如权利要求8所述的基因递送系统在制备基因治疗药物中的应用。
PCT/CN2016/087283 2016-05-20 2016-06-27 一种骨靶向基因载体及其制备方法和应用 WO2017197726A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610339273.8A CN106047935B (zh) 2016-05-20 2016-05-20 一种靶向基因载体及其制备方法和应用
CN201610339273.8 2016-05-20

Publications (1)

Publication Number Publication Date
WO2017197726A1 true WO2017197726A1 (zh) 2017-11-23

Family

ID=57177435

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/087283 WO2017197726A1 (zh) 2016-05-20 2016-06-27 一种骨靶向基因载体及其制备方法和应用

Country Status (2)

Country Link
CN (1) CN106047935B (zh)
WO (1) WO2017197726A1 (zh)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113599861A (zh) * 2021-08-13 2021-11-05 广东石油化工学院 一种利用修饰基底分离磷脂膜中胆固醇的方法
CN114699390A (zh) * 2022-04-15 2022-07-05 高州市人民医院 一种具备主动靶向性的纳米缓释递药系统及其制备方法
CN114712310A (zh) * 2022-01-18 2022-07-08 中国人民解放军空军军医大学 可高效入胞的智能骨靶向递送药物的制备方法及其应用
US11464792B2 (en) * 2020-01-28 2022-10-11 Applaud Medical, Inc. Phospholipid compounds and formulations
US11554094B2 (en) 2015-06-18 2023-01-17 California Institute Of Technology Synthesis and application of microbubble-forming compounds
US11642410B2 (en) 2011-08-24 2023-05-09 California Institute Of Technology Targeting microbubbles
JP2023525599A (ja) * 2020-01-28 2023-06-16 アプロード メディカル， インコーポレイテッド リン脂質化合物および製剤
CN117045811A (zh) * 2023-08-14 2023-11-14 深圳市儿童医院 一种骨靶向纳米载药体系及其制备方法和应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109771663B (zh) * 2017-11-10 2022-03-18 中国科学院宁波材料技术与工程研究所 一种酸响应性抗癌纳米药物的制备及应用
CN109010089B (zh) * 2018-07-13 2021-06-29 安徽医科大学 一种用于龋齿预防和浅龋治疗的纳米材料的制备方法
CN112704742A (zh) * 2019-10-24 2021-04-27 华东理工大学 一种用于恶性肿瘤治疗的包载质粒的阳离子脂质体复合物
CN111001011B (zh) * 2019-11-05 2021-07-20 中国药科大学 一种低分子量肝素修饰的骨靶向脂质体及其制备方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102824647A (zh) * 2011-06-13 2012-12-19 香港中文大学 基于小核酸药物成骨治疗的骨靶向递送系统及其制备方法
WO2013044734A1 (zh) * 2011-09-28 2013-04-04 中国科学院深圳先进技术研究院 骨靶向脂质体及其制备方法
CN104027811A (zh) * 2014-04-03 2014-09-10 西南交通大学 帕米膦酸二钠修饰Brij78为关键成分的钙亲和纳米粒及其制备方法
CN104174033A (zh) * 2014-08-14 2014-12-03 武汉大学 一种骨靶向rna干扰复合物及其合成方法
CN104274407A (zh) * 2013-07-08 2015-01-14 广东医学院 一种丹参素脂质体骨靶向药物制剂及其制备方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102824647A (zh) * 2011-06-13 2012-12-19 香港中文大学 基于小核酸药物成骨治疗的骨靶向递送系统及其制备方法
WO2013044734A1 (zh) * 2011-09-28 2013-04-04 中国科学院深圳先进技术研究院 骨靶向脂质体及其制备方法
CN104274407A (zh) * 2013-07-08 2015-01-14 广东医学院 一种丹参素脂质体骨靶向药物制剂及其制备方法
CN104027811A (zh) * 2014-04-03 2014-09-10 西南交通大学 帕米膦酸二钠修饰Brij78为关键成分的钙亲和纳米粒及其制备方法
CN104174033A (zh) * 2014-08-14 2014-12-03 武汉大学 一种骨靶向rna干扰复合物及其合成方法

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11642410B2 (en) 2011-08-24 2023-05-09 California Institute Of Technology Targeting microbubbles
US11554094B2 (en) 2015-06-18 2023-01-17 California Institute Of Technology Synthesis and application of microbubble-forming compounds
US11464792B2 (en) * 2020-01-28 2022-10-11 Applaud Medical, Inc. Phospholipid compounds and formulations
JP2023525599A (ja) * 2020-01-28 2023-06-16 アプロード メディカル， インコーポレイテッド リン脂質化合物および製剤
US11844807B2 (en) 2020-01-28 2023-12-19 Applaud Medical, Inc. Methods of manufacturing microspheres
US11903954B2 (en) 2020-01-28 2024-02-20 Applaud Medical, Inc. Method of treating urolithiasis
CN113599861A (zh) * 2021-08-13 2021-11-05 广东石油化工学院 一种利用修饰基底分离磷脂膜中胆固醇的方法
CN113599861B (zh) * 2021-08-13 2023-06-27 广东石油化工学院 一种利用修饰基底分离磷脂膜中胆固醇的方法
CN114712310A (zh) * 2022-01-18 2022-07-08 中国人民解放军空军军医大学 可高效入胞的智能骨靶向递送药物的制备方法及其应用
CN114699390A (zh) * 2022-04-15 2022-07-05 高州市人民医院 一种具备主动靶向性的纳米缓释递药系统及其制备方法
CN117045811A (zh) * 2023-08-14 2023-11-14 深圳市儿童医院 一种骨靶向纳米载药体系及其制备方法和应用

Also Published As

Publication number Publication date
CN106047935B (zh) 2019-08-23
CN106047935A (zh) 2016-10-26

Similar Documents

Publication Publication Date Title
WO2017197726A1 (zh) 一种骨靶向基因载体及其制备方法和应用
Noureddine et al. Engineering of monosized lipid-coated mesoporous silica nanoparticles for CRISPR delivery
JP6137894B2 (ja) リポソーム−エキソソームハイブリッドベシクル及びその調製法
CN105801668B (zh) 一种寡聚精氨酸修饰的磷脂与其组装的纳米粒及制备方法和应用
AU2003205049B2 (en) Efficient nucleic acid encapsulation into medium sized liposomes
Li et al. A novel cationic liposome formulation for efficient gene delivery via a pulmonary route
JP2003535832A (ja) ポリヌクレオチドおよび薬物の標的化リポソームへのカプセル化
CN109288794B (zh) 一种蜂毒素脂质体纳米制剂及其制备方法与应用
JPWO2006101201A1 (ja) 目的物質を効率的に核内に送達可能なリポソーム
Wang et al. Construction of a novel cationic polymeric liposomes formed from PEGylated octadecyl‐quaternized lysine modified chitosan/cholesterol for enhancing storage stability and cellular uptake efficiency
Zhu et al. Strategies for engineering exosomes and their applications in drug delivery
EP2200586A2 (en) Improved liposomes and uses thereof
CN107648617A (zh) 用于基因递送的壳聚糖纳米粒微泡复合物及其制备方法
US11560575B2 (en) High efficient delivery of plasmid DNA into human and vertebrate primary cells in vitro and in vivo by nanocomplexes
CN116763757A (zh) 一种包裹型复合物及其制剂的制备方法和应用
CN102031269A (zh) 含阳离子氨基酸多肽修饰的纳米磷酸钙基因载体的制备方法
CN102517332A (zh) Egf修饰的pamam自组装转基因组合物及其制备方法与应用
CN115252555B (zh) 一种膜融合性脂质体、制备方法及其在蛋白质递送中的应用
CN115645550A (zh) 一种脂质金磁纳米粒子及其制备方法和应用
CN112089849B (zh) 一种靶向脑胶质瘤的治疗基因递送载体和递送系统
CN116082179B (zh) 基于内源性二羧酸的可电离脂质及其制备方法与应用
KR101437885B1 (ko) Ｐｃｒ 기반의 유전자 전달체 및 그 제조방법
CN116869968B (zh) 一种靶向大脑和脑胶质瘤的纳米粒及其合成方法、应用
CN112717140B (zh) 一种含HP1γ的胍基化聚氨基胺类聚合物基因载体复合物的制备及其应用
KR100986604B1 (ko) 신규한 아미노지질을 함유하는 에스아이알엔에이 수송용 유전자 조성물 및 제조방법

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16902106

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 16902106

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 28/03/2019)

122 Ep: pct application non-entry in european phase

Ref document number: 16902106

Country of ref document: EP

Kind code of ref document: A1