CN103987379B - Bone targeting liposome and preparation method thereof - Google Patents
Bone targeting liposome and preparation method thereof Download PDFInfo
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- CN103987379B CN103987379B CN201280029973.7A CN201280029973A CN103987379B CN 103987379 B CN103987379 B CN 103987379B CN 201280029973 A CN201280029973 A CN 201280029973A CN 103987379 B CN103987379 B CN 103987379B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0014—Skin, i.e. galenical aspects of topical compositions
- A61K9/0017—Non-human animal skin, e.g. pour-on, spot-on
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- 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
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- 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
Abstract
Bone targeting liposome, unilamellar vesicle is formed by phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate, the polyethyleneglycol lipid conjugate with active group, medicine is coated with, the Bone targeting functional component that the active group being connected with the polyethyleneglycol lipid conjugate by having active group outside it connects in the inner side of unilamellar vesicle.The invention also discloses the preparation method of Bone targeting liposome.
Description
Technical field
The present invention relates to medical art, particularly relate to a kind of Bone targeting liposome and preparation method thereof.
Background technology
Osseous tissue organizes hardness large owing to having, the physiological structure that poor permeability etc. are special, and general medicine is difficult to effectively arrive diseased region, makes the drug level of diseased region on the low side, can not reach good therapeutic effect.Improving the method for concentration by improving dosage, although the concentration of medicine at diseased region can be improved to a certain extent, also can increase the toxic and side effects of medicine to its hetero-organization.
3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one is a kind of flavone compound with the effect of phytoestrogen sample, is Herba Epimedii major secondary metabolite in vivo, is a kind of compound of effective promoting bone growing.Nearest in vitro study display, 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one can promote osteoblastic propagation and differentiation, strengthens calcified bone matrix function, can also suppress differentiation and the bone resorption function of osteoclast simultaneously, reduces the motility of osteoclast.In In vivo study, the osteoporotic mouse model utilizing removal ovary to induce, finds that 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one can mediate dependency and dependent/non-dependent estrogen receptor pathway, all has protective effect to the skeleton of mice and muscle.In addition, 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one can also reduce the sickness rate that family exempts from the osteonecrosis that steroid hormone in model brings out, and its mechanism inhibits the formation of Intravascular Thrombus and the deposition of extravascular lipid for 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one.
Although 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one has very strong biological activity in vivo, but vivo medicine-feeding is made to be faced with stern challenge due to the physical property of the slightly solubility of 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one.The pharmacokinetic being given the rabbit of 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one by oral and vein is found, strengthens oral 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one dosage 20,40and80mg/kg gradually, corresponding pharmacokinetic parameter C
maxand AUC
0-∞also corresponding increase, but the bioavailability in its body but obviously declines, and is respectively 17.29%, 13.80% and be respectively 10.70%.Vein gives 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one 20mg/kg, its AUC
0-∞for 2470ngh/mL, and oral 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one dosage reaches the AUC of 80mg/kg
0-∞be only 495.67ngh/mL, the AUC of intravenously administrable
0-∞larger than oral administration 4 times, result shows that 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one is oral and not easily absorbs, intravenously administrable may be alternative optimum way (LiuHP, MengFH, GuoJF, SiDY, ZhuXW, ZhaoYM (2010) .Pharmacokineticsoficaritininrats.ChinesePharmaceuticalJ ournal45 (7), 539-543.).
Liposome has good biocompatibility, is widely used as pharmaceutical carrier, the medicine of encapsulating can be made to have good stability, can improve the stability of medicine, and can reduce poisonous side effect of medicine to a certain extent.
But general Bone targeting liposome can not improve the drug level of bone surface in use.
Summary of the invention
Based on this, provide a kind of Bone targeting liposome that can improve the drug level of bone surface in use and preparation method thereof.
A kind of Bone targeting liposome, comprises medicine, phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate, the polyethyleneglycol lipid conjugate with active group and Bone targeting functional component;
Described phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate and have active group polyethyleneglycol lipid conjugate formed unilamellar vesicle, described medicine is covered by the inner side of described unilamellar vesicle, and described Bone targeting functional component is connected to outside described unilamellar vesicle and described Bone targeting functional component is connected with the described active group with the polyethyleneglycol lipid conjugate of active group;
Described phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate, the mol ratio of polyethyleneglycol lipid conjugate and Bone targeting functional component with active group are 70 ~ 95:5 ~ 20:0.5 ~ 5:1 ~ 10:1 ~ 8.
In one embodiment, described Bone targeting functional component is alendronate, Asp8 and (DSS)
6in at least one.
In one embodiment, described phosphatidylcholine is at least one in soybean lecithin, dimyristoyl phosphatidyl choline, DOPC and disaturated phosphatidyl choline.
In one embodiment, described polyethyleneglycol lipid conjugate is at least one in C12Ceremide-mPEG, C16Ceremide-mPEG, C20Ceremide-mPEG12 and DSPE-mPEG.
In one embodiment, the polyethyleneglycol lipid conjugate described in active group is DSPE-PEG-Mal.
In one embodiment, the diameter range of described Bone targeting liposome is for being less than 500nm.
In one embodiment, the diameter of described Bone targeting liposome is 50nm ~ 200nm.
In one embodiment, described phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate and there is the total mole number of polyethyleneglycol lipid conjugate of active group and the ratio of the molal quantity of described medicine is 1:5 ~ 1:25.
A preparation method for Bone targeting liposome, comprises the steps:
Prepared the unilamellar vesicle of medicine coated, described unilamellar vesicle is formed by phosphatidylcholine, cholesterol and polyethyleneglycol lipid conjugate; Wherein, the mol ratio of described phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate is 70 ~ 95:5 ~ 20:0.5 ~ 5;
Preparation has the micelle of the polyethyleneglycol lipid conjugate material of active group, and described micelle is mixed with Bone targeting functional component, described Bone targeting functional component is connected, the composite micelle obtained with the described active group with the polyethyleneglycol lipid conjugate of active group; Wherein, the mol ratio of the polyethyleneglycol lipid conjugate and Bone targeting functional component described in active group is 1 ~ 10:1 ~ 8;
The unilamellar vesicle of described coated medicine is mixed with described composite micelle, described composite micelle is embedded in described unilamellar vesicle, obtains described Bone targeting liposome; Wherein, described phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate, the mol ratio of polyethyleneglycol lipid conjugate and Bone targeting functional component with active group are 70 ~ 95:5 ~ 20:0.5 ~ 5:1 ~ 10:1 ~ 8.
A preparation method for Bone targeting liposome, comprises the steps:
Prepared the unilamellar vesicle of medicine coated, described unilamellar vesicle is formed by phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate and the polyethyleneglycol lipid conjugate with active group; Wherein, the mol ratio of described phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate and the polyethyleneglycol lipid conjugate with active group is 70 ~ 95:5 ~ 20:0.5 ~ 5:1 ~ 10;
The unilamellar vesicle of described coated medicine is mixed with Bone targeting functional component, described Bone targeting functional component is connected with the described active group with the polyethyleneglycol lipid conjugate of active group, obtains described Bone targeting liposome; Wherein, described phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate, the mol ratio of polyethyleneglycol lipid conjugate and Bone targeting functional component with active group are 70 ~ 95:5 ~ 20:0.5 ~ 5:1 ~ 10:1 ~ 8.
During this Bone targeting liposome transporting drugs, by the guide effect of Bone targeting function composition, drug targeting can be transported to bone surface, play the effect of the drug level improving bone surface.
Accompanying drawing explanation
Fig. 1 is the structural representation of Bone targeting liposome;
Fig. 2 is the overall shape appearance figure of Bone targeting liposome prepared by embodiment 1;
Fig. 3 is the scanning electron microscope comparison diagram of Bone targeting liposome prepared by embodiment 1;
Fig. 4 is the In-vitro release curves comparison diagram of Bone targeting liposome prepared by embodiment 1;
Fig. 5 is the fluorescence intensity figure that Bone targeting liposome prepared by embodiment 1 carries out organ after mouse tail vein injection.
Detailed description of the invention
Below by way of the drawings and specific embodiments, Bone targeting liposome and preparation method thereof is described further.
The Bone targeting liposome of an embodiment as shown in Figure 1, comprises medicine, phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate, the polyethyleneglycol lipid conjugate with active group and Bone targeting functional component.
Phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate and have active group polyethyleneglycol lipid conjugate formed unilamellar vesicle, medicine is covered by the inner side of unilamellar vesicle, Bone targeting functional component is connected to outside described unilamellar vesicle, and Bone targeting functional component is connected with the active group of the polyethyleneglycol lipid conjugate with active group.
Generally speaking, the diameter of this Bone targeting liposome should be less than 500nm.
In the present embodiment, the diameter of this Bone targeting liposome can be 50nm ~ 200nm, thus is conducive to this Bone targeting liposome from vascular leakage to osseous tissue.
Phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate, the mol ratio of polyethyleneglycol lipid conjugate and Bone targeting functional component with active group are 70 ~ 95:5 ~ 20:0.5 ~ 5:1 ~ 10:1 ~ 8.
During this Bone targeting liposome transporting drugs, by the guide effect of Bone targeting function composition, drug targeting can be transported to bone surface, play the effect of the drug level improving bone surface.
This Bone targeting liposome, when the transport agent as 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one, can extend the 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one holdup time in blood, and keeps with constant speed release 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one.By the guide effect of Bone targeting function composition, 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one targeting can be transported to bone surface, play the effect of the 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one concentration improving bone surface.
Phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate and the ratio with the total mole number of polyethyleneglycol lipid conjugate of active group and the molal quantity of medicine can be 1:5 ~ 1:25.
The unilamellar vesicle of this Bone targeting liposome not only can coated 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one, can also the bone target medicine of coated other types as required, thus plays and improve the function of medicine in bone surface concentration.
Bone targeting functional component can be alendronate, Asp8 (8 aspartic acid repetitive sequences) or (DSS)
6(repetitive sequences of 6 (aspartic acid-Ser-Sers)).
Phosphatidylcholine can be soybean lecithin, dimyristoyl phosphatidyl choline, DOPC or disaturated phosphatidyl choline.
Polyethyleneglycol lipid conjugate can be polyethyleneglycol modified ceramide, DSPE derivant, as: C12Ceremide-mPEG, C16Ceremide-mPEG, C20Ceremide-mPEG12 or DSPE-mPEG.
The polyethyleneglycol lipid conjugate with active group can for the DSPE derivant (DSPE-PEG-Mal) with maleic amide group.
In a preferred embodiment, also cryoprotective agent is added with in this Bone targeting liposome, so that extended refrigerated storage.The Bone targeting liposome that with the addition of cryoprotective agent only needs again to dissolve before practicality, just can form uniform particles, encapsulate good Bone targeting liposome solutions.
Cryoprotective agent can be sucrose, mannitol, glucose or trehalose.The ratio of phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate and the total mole number of polyethyleneglycol lipid conjugate and the molal quantity of cryoprotective agent with active group is 2:1 ~ 10:1.
In concrete use procedure, this Bone targeting liposome can adopt intradermal injection, the mode of subcutaneous injection or intramuscular injection is sent in body.
Above-mentioned Bone targeting liposome can be prepared by multiple method, common as film evaporation method, ultrasonic method, squeezing and pressing method, high-pressure homogenization and infusion process etc.
The preparation method of the above-mentioned Bone targeting liposome of one embodiment is provided, comprises the steps:
S110, the unilamellar vesicle having prepared medicine coated.
Unilamellar vesicle is formed by phosphatidylcholine, cholesterol and polyethyleneglycol lipid conjugate.Wherein, the mol ratio of phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate is 70 ~ 95:5 ~ 20:0.5 ~ 5.
Phosphatidylcholine can be soybean lecithin, dimyristoyl phosphatidyl choline, DOPC or disaturated phosphatidyl choline.
Polyethyleneglycol lipid conjugate can be C12Ceremide-mPEG, C16Ceremide-mPEG, C20Ceremide-mPEG12 or DSPE-mPEG.
The concrete preparation process of the unilamellar vesicle of coated medicine is as follows:
Medicine, phosphatidylcholine, cholesterol and polyethyleneglycol lipid conjugate being dissolved in volume ratio is, in the methanol of 1:1 and the mixed liquor of chloroform, utilize rotatory vacuum drying machine, make organic solvent evaporation, obtain lipid membrane.
After making the lipid membrane bone dry obtained, this lipid membrane is dipped in aquation in PBS solution, then water-bath is hatched, and obtains lipid suspension.
With nitrogen pressurization, with squeezer (Lipex
tMextruder) this lipid suspension is extruded, makes lipid suspension be the filter membrane of 200nm and 100nm by aperture successively, continuously extruded repeatedly after, the unilamellar vesicle having obtained medicine coated.By being the filter membrane of 200nm and 100nm by aperture successively by lipid suspension, the diameter of the unilamellar vesicle obtained is made to be 50nm ~ 200nm.
S120, preparation have the micelle of the polyethyleneglycol lipid conjugate material of active group, and micelle is mixed with Bone targeting functional component, Bone targeting functional component is connected, the composite micelle obtained with the active group of the polyethyleneglycol lipid conjugate with active group.
Wherein, the mol ratio of the polyethyleneglycol lipid conjugate and Bone targeting functional component with active group is 1 ~ 10:1 ~ 8.
The polyethyleneglycol lipid conjugate with active group can be DSPE-PEG-Mal.
Bone targeting functional component can be alendronate, Asp8 or (DSS)
6.
The concrete preparation process of composite micelle is as follows:
Be dissolved in chloroform by the polyethyleneglycol lipid conjugate with active group, volatilization, except desolventizing, obtains lipid film.
In the lipid film obtained, add aquation after PBS, form the micelle with the polyethyleneglycol lipid conjugate material of active group.
The PBS solution of Bone targeting functional component is joined in the micelle of the polyethyleneglycol lipid conjugate material with active group, after reaction, Bone targeting functional component is connected with the active group of the polyethyleneglycol lipid conjugate with active group, the composite micelle obtained.
Also comprise interpolation Cys in S120, neutralize the operation of unreacted active group.
S130, the unilamellar vesicle of coated medicine obtained by S110 mix with the composite micelle that S120 obtains, and make composite micelle be embedded in unilamellar vesicle, obtain Bone targeting liposome.
Wherein, phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate, the mol ratio of polyethyleneglycol lipid conjugate and Bone targeting functional component with active group are 70 ~ 95:5 ~ 20:0.5 ~ 5:1 ~ 10:1 ~ 8.
The unilamellar vesicle of the coated medicine obtained by S110 mixes with the composite micelle that S120 obtains, and water-bath is also hatched.Composite micelle is before mixing with unilamellar vesicle, and hydrophilic segment (PEG and target head) is aqueous phase outwards, and lipophilic portion (DSPE) is inside.After mixing with unilamellar vesicle, in the lipid end of composite micelle and unilamellar vesicle, phospholipid bilayer has similarity, so the DSPE partial insertion lipid bilayer of composite micelle, and hydrophilic PEG and target head part distribute to aqueous phase, are distributed in the surface of unilamellar vesicle.Finally make composite micelle be embedded in unilamellar vesicle, obtain Bone targeting liposome.
The preparation method of the above-mentioned Bone targeting liposome of another embodiment is provided, comprises the steps:
S210, the unilamellar vesicle having prepared medicine coated.
Unilamellar vesicle is formed by phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate and the polyethyleneglycol lipid conjugate with active group.Wherein, the mol ratio of phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate and the polyethyleneglycol lipid conjugate with active group is 70 ~ 95:5 ~ 20:0.5 ~ 5:1 ~ 10.
Phosphatidylcholine can be soybean lecithin, dimyristoyl phosphatidyl choline, DOPC or disaturated phosphatidyl choline.
Polyethyleneglycol lipid conjugate can be C12Ceremide-mPEG, C16Ceremide-mPEG, C20Ceremide-mPEG12 or DSPE-mPEG.
The polyethyleneglycol lipid conjugate with active group can be DSPE-PEG-Mal.
In S210, the concrete preparation process of the unilamellar vesicle of coated medicine is as follows:
Medicine, phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate and the polyethyleneglycol lipid conjugate with active group being dissolved in volume ratio is in the methanol of 1:1 and the mixed liquor of chloroform, utilize rotatory vacuum drying machine, make organic solvent evaporation, obtain lipid membrane.
After making the lipid membrane bone dry obtained, this lipid membrane is dipped in aquation in PBS solution, then water-bath is hatched, and obtains lipid suspension.
With nitrogen pressurization, with squeezer (Lipex
tMextruder) this lipid suspension is extruded, makes lipid suspension be the filter membrane of 200nm and 100nm by aperture successively, continuously extruded repeatedly after, the unilamellar vesicle having obtained medicine coated.By being the filter membrane of 200nm and 100nm by aperture successively by lipid suspension, the diameter of the unilamellar vesicle obtained is made to be 50nm ~ 200nm.
S220, the unilamellar vesicle of coated medicine to be mixed with Bone targeting functional component, Bone targeting functional component is connected with the active group of the polyethyleneglycol lipid conjugate with active group, obtains Bone targeting liposome.
Wherein, phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate, the mol ratio of polyethyleneglycol lipid conjugate and Bone targeting functional component with active group are 70 ~ 95:5 ~ 20:0.5 ~ 5:1 ~ 10:1 ~ 8.
Bone targeting functional component can be alendronate, Asp8 or (DSS)
6.
Also comprise after obtaining Bone targeting liposome in S220, by this Bone targeting liposome by agarose gel CL-4B column chromatography, remove the operation of the Bone targeting functional component be not connected with active group.
Be specific embodiment below.
Embodiment 1
With round-bottomed flask, methanol-nitrogen that 21umol 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one and 325umol lipid are dissolved in (being made up of according to mol ratio 85:11:4 SPC, cholesterol, C16Ceremide-mPEG200) 6ml is imitated in mixed liquor (1:1, V/V).Regulate temperature to be 55 DEG C, utilize rotatory vacuum drying machine, the organic solvent in round-bottomed flask is volatilized, obtains lipid membrane.Subsequently lipid membrane is placed in vacuum drying oven and spends the night, make it completely dry.Then drying completely lipid membrane be dipped in 10mlPBS (10mM, pH7.4), 55 DEG C of water-baths are hatched, and obtain liposome suspension.With nitrogen pressurization, by the liposome suspension squeezer (Lipex obtained
tMextruder, NorthernLipidsInc., Vancouver, BC, Canada) extrude, liposome suspension is made to be the polycarbonate leaching film (NucleporeTrack-EtchMembrane) of 200nm and 100nm successively by aperture, continuously extruded six times, the unilamellar vesicle having obtained 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one coated.
Join in the test tube of 1.5ml by 50ul relative to the chloroformic solution of the DSPE-PEG2000-Mal of TL 2mol%, volatilization removing organic solvent obtains lipid film.In the lipid film obtained, add aquation after PBS, form the micelle of DSPE-PEG2000-Mal material.According to DSPE-PEG2000-Mal and (DSS)
6mol ratio be 2:1, will containing (DSS) of sulfydryl
6pBS solution add the micelle of DSPE-PEG2000-Mal material, vibration 4h makes (DSS)
6react with DSPE-PEG2000-Mal and form DSPE-PEG2000-(DSS)
6composite micelle.Add Cys after reaction terminates, make final concentration be 1mM, in room temperature, hatch at least 10min, for neutralizing the group of non-conjugated.
By DSPE-PEG2000-(DSS)
6composite micelle mixes with the unilamellar vesicle of coated 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one, hatches 2h, make DSPE-PEG2000-(DSS) under 37 DEG C of water-baths
6embed in unilamellar vesicle with micelle form, obtain Bone targeting liposome.
The float of Bone targeting liposome mixed with the distilled water containing mannitol, the mol ratio of mannitol and lipid is 5, and dry 48h in freezer dryer obtains the Bone targeting liposome of lyophilizing.The Bone targeting liposome aquation before the use of lyophilizing, the sterilised membrane filter then with 0.22 μm is degerming.
The overall shape appearance figure of the Bone targeting liposome of embodiment 1 preparation as shown in Figure 2.Fig. 2 A is the overall shape appearance figure of the Bone targeting liposome before lyophilization, Fig. 2 B is the overall shape appearance figure of the long-term Bone targeting liposome placed after lyophilization, and Fig. 2 C is the overall shape appearance figure of the Bone targeting liposome obtained after long-term Bone targeting liposome rehydration of placing.
As can be seen from Figure 2B, the cryodesiccated Bone targeting liposome after long-term placement is pie.The Bone targeting liposome obtained after can finding out long-term Bone targeting liposome rehydration of placing by Fig. 2 A and Fig. 2 C compared with before lyophilization without significant change.
As seen from Figure 2, lyophilization can not affect the overall appearance of Bone targeting liposome.
Fig. 3 is the scanning electron microscope comparison diagram of Bone targeting liposome prepared by embodiment 1.Fig. 3 A is the scanning electron microscope (SEM) photograph of the Bone targeting liposome in Fig. 2 A, and Fig. 3 B is the scanning electron microscope (SEM) photograph of the Bone targeting liposome in Fig. 2 C.
Bone targeting liposome in Fig. 3 A is unilamellar vesicle shape, and the Bone targeting liposome in Fig. 3 B after aquation is also unilamellar vesicle shape.
Comparison diagram 3A and Fig. 3 B can find out, the structure of the Bone targeting liposome before and after lyophilization under Electronic Speculum is without significant change.
As seen from Figure 3, lyophilization can not affect the structure of Bone targeting liposome.
Release profiles is evaluated by the dialysis membrane method of fine setting, concrete operations are as follows: in vial, and the liposome (approximately containing 800ug 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one) 1mL embodiment 1 prepared is added in 50mL release medium (PBS (pH7.4) containing 10% ethanol).The release medium of 12 deciles is placed in (12,000 ~ 14,000 macromolecule sieve cuts) in the bag filter of anticipating respectively, is dipped in identical vial by bag filter respectively simultaneously.Vial is placed on constant-temperature table, vibrates at 37 DEG C with 100 ± 10rpm.Sample reclaimed in predetermined time interval (more than 72h), and the fresh medium of in addition same volume.By the concentration of Ultra Performance Liquid Chromatography systems axiol-ogy 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one, obtain the In-vitro release curves of 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one as shown in Figure 4.
As seen from Figure 4, Bone targeting liposome prepared by embodiment 1 can control the release of 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one, and its control time is up to 72h.Meanwhile, the release of 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one in liposome is not affected adding of Bone targeting molecule.
Embodiment 2
With round-bottomed flask, methanol-nitrogen that 21umol 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one and 360umol lipid (being made up of according to mol ratio 75:18:3:4 DOPC, Cholesterol, DSPE-mPEG2000 and DSPE-PEG2000-Mal) are dissolved in 6ml is imitated in mixed liquor (1:1, V/V).Regulate temperature to be 55 DEG C, utilize rotatory vacuum drying machine, the organic solvent in round-bottomed flask is volatilized, obtains lipid membrane.Subsequently lipid membrane is placed in vacuum drying oven inner drying to spend the night.Then drying completely lipid membrane be dipped in aquation in 10mlPBS (10mM, pH7.4), obtain liposome suspension.With nitrogen pressurization, by the liposome suspension Lipex obtained
tMextruder extrudes, and makes liposome suspension be the polycarbonate leaching film of 200nm and 100nm by aperture successively, continuously extruded six times, the unilamellar vesicle having obtained 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one coated.
Then about 7umol is had (DSS) of sulfydryl
6add in unilamellar vesicle, shaken at room temperature 4h, obtain the suspension of Bone targeting liposome.The suspension of the Bone targeting liposome obtained removes non-conjugated (DSS) by agarose gel CL-4B column chromatography
6and purification.
Embodiment 3
With round-bottomed flask, by 21umol 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one and 325umol lipid (by SPC, cholesterol, C16Ceremide ?mPEG200 form according to mol ratio 85:11:4) be dissolved in 6ml Jia Chun ?nitrogen imitate in mixed liquor (1:1, V/V).Regulate temperature to be 55 DEG C, utilize rotatory vacuum drying machine, the organic solvent in round-bottomed flask is volatilized, obtains lipid membrane.Subsequently lipid membrane is placed in vacuum drying oven and spends the night, make it completely dry.Then drying completely lipid membrane be dipped in 10mlPBS (10mM, pH7.4), 55 DEG C of water-baths are hatched, and obtain liposome suspension.With nitrogen pressurization, by the liposome suspension squeezer (Lipex obtained
tMextruder, NorthernLipidsInc., Vancouver, BC, Canada) extrude, make liposome suspension be the polycarbonate leaching film (NucleporeTrack ?EtchMembrane) of 100nm ~ 200nm by aperture, continuously extruded six times, obtain icaritin liposome.
By the Bone targeting liposome prepared by fluorescently-labeled icaritin liposome and embodiment 1 respectively in the female mice body that tail vein injection enters 86 moonrat ages (n=4, namely often organizes four mices), dosage is 1mg/kg.
After 4h and 24h, respectively by 4 sacrifice, core, liver, spleen, lung, kidney, the major organs such as bilateral femur.Utilization XenogenIVIS imaging system detects the fluorescence intensity in each organ, obtains Fig. 5.
In Fig. 5, blue expression fluorescence intensity is high, and red fluorescence intensity is low.Fluorescence intensity is high shows that this liposome is more in the distribution of this tissue, and vice versa.
As seen from Figure 5,4h after tail vein injection, relative to other organ of mice, the fluorescence signal in femur is the strongest.24h after tail vein injection, the femur place of the mice of the Bone targeting liposome injection still can prepared in embodiment 1 detects fluorescence signal, but the femur of the mice of being injected by the icaritin liposome of chip mark does not detect fluorescence signal.
It can be said that bright, Bone targeting liposome prepared by embodiment 1 is easier to targeting osseous tissue, extends the holdup time of 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one in bone simultaneously.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (7)
1. a Bone targeting liposome, is characterized in that, comprises medicine, phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate, the polyethyleneglycol lipid conjugate with active group and Bone targeting functional component;
Described phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate and have active group polyethyleneglycol lipid conjugate formed unilamellar vesicle, described medicine is covered by the inner side of described unilamellar vesicle, and described Bone targeting functional component is connected to outside described unilamellar vesicle and described Bone targeting functional component is connected with the described active group with the polyethyleneglycol lipid conjugate of active group;
Described phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate, the mol ratio of polyethyleneglycol lipid conjugate and Bone targeting functional component with active group are 70 ~ 95:5 ~ 20:0.5 ~ 5:1 ~ 10:1 ~ 8;
Described Bone targeting functional component is (DSS)
6;
Described medicine is 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one;
Described polyethyleneglycol lipid conjugate is at least one in C12Ceremide-mPEG, C16Ceremide-mPEG, C20Ceremide-mPEG12 and DSPE-mPEG;
The described polyethyleneglycol lipid conjugate with active group is DSPE-PEG-Mal.
2. Bone targeting liposome according to claim 1, is characterized in that, described phosphatidylcholine is at least one in soybean lecithin, dimyristoyl phosphatidyl choline, DOPC and disaturated phosphatidyl choline.
3. Bone targeting liposome according to claim 1, is characterized in that, the diameter range of described Bone targeting liposome is for being less than 500nm.
4. Bone targeting liposome according to claim 3, is characterized in that, the diameter of described Bone targeting liposome is 50nm ~ 200nm.
5. Bone targeting liposome according to claim 1, it is characterized in that, described phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate and there is the total mole number of polyethyleneglycol lipid conjugate of active group and the ratio of the molal quantity of described medicine is 1:5 ~ 1:25.
6. a preparation method for Bone targeting liposome, is characterized in that, comprises the steps:
Prepared the unilamellar vesicle of medicine coated, described unilamellar vesicle is formed by phosphatidylcholine, cholesterol and polyethyleneglycol lipid conjugate; Wherein, the mol ratio of described phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate is 70 ~ 95:5 ~ 20:0.5 ~ 5; Described medicine is 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one; Described polyethyleneglycol lipid conjugate is at least one in C12Ceremide-mPEG, C16Ceremide-mPEG, C20Ceremide-mPEG12 and DSPE-mPEG;
Preparation has the micelle of the polyethyleneglycol lipid conjugate material of active group, and described micelle is mixed with Bone targeting functional component, described Bone targeting functional component is connected, the composite micelle obtained with the described active group with the polyethyleneglycol lipid conjugate of active group; Wherein, described in there is active group the mol ratio of polyethyleneglycol lipid conjugate and Bone targeting functional component be 1 ~ 10:1 ~ 8, described Bone targeting functional component is (DSS)
6; The described polyethyleneglycol lipid conjugate with active group is DSPE-PEG-Mal;
The unilamellar vesicle of described coated medicine is mixed with described composite micelle, described composite micelle is embedded in described unilamellar vesicle, obtains described Bone targeting liposome; Wherein, described phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate, the mol ratio of polyethyleneglycol lipid conjugate and Bone targeting functional component with active group are 70 ~ 95:5 ~ 20:0.5 ~ 5:1 ~ 10:1 ~ 8.
7. a preparation method for Bone targeting liposome, is characterized in that, comprises the steps:
Prepared the unilamellar vesicle of medicine coated, described unilamellar vesicle is formed by phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate and the polyethyleneglycol lipid conjugate with active group; Wherein, the mol ratio of described phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate and the polyethyleneglycol lipid conjugate with active group is 70 ~ 95:5 ~ 20:0.5 ~ 5:1 ~ 10; Described medicine is 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one; Described polyethyleneglycol lipid conjugate is at least one in C12Ceremide-mPEG, C16Ceremide-mPEG, C20Ceremide-mPEG12 and DSPE-mPEG; The described polyethyleneglycol lipid conjugate with active group is DSPE-PEG-Mal;
The unilamellar vesicle of described coated medicine is mixed with Bone targeting functional component, described Bone targeting functional component is connected with the described active group with the polyethyleneglycol lipid conjugate of active group, obtains described Bone targeting liposome; Wherein, described phosphatidylcholine, cholesterol, polyethyleneglycol lipid conjugate, the mol ratio of polyethyleneglycol lipid conjugate and Bone targeting functional component with active group are 70 ~ 95:5 ~ 20:0.5 ~ 5:1 ~ 10:1 ~ 8, and described Bone targeting functional component is (DSS)
6.
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| KR101742862B1 (en) * | 2015-02-11 | 2017-06-15 | 인하대학교 산학협력단 | Liquid Filtration Structure Integrated with Artificial Membrane |
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| CN108236603B (en) * | 2016-12-23 | 2020-06-05 | 深圳先进技术研究院 | Pyrrole imidazole polyamide liposome and preparation method and application thereof |
| CN107028885B (en) * | 2017-03-23 | 2022-05-13 | 内蒙古自治区人民医院 | Bone targeting liposome for treating osteoporosis and preparation method thereof |
| CN106913525A (en) * | 2017-04-26 | 2017-07-04 | 温州医科大学附属口腔医院 | It is a kind of to carry the preparation method for promoting bone synthetic drug nano particle lipopolymer |
| CN107412164B (en) * | 2017-04-26 | 2020-10-20 | 温州医科大学附属口腔医院 | Preparation method of double-targeting drug-loaded nanoparticle lipid-polymer for osteoporosis |
| CN107007552A (en) * | 2017-04-26 | 2017-08-04 | 温州医科大学附属口腔医院 | A kind of preparation method for carrying anti-bone information medicament nano particle lipopolymer |
| CN114096238A (en) * | 2019-07-25 | 2022-02-25 | 北京盛诺基医药科技股份有限公司 | Nanometer micelle preparation of aclidinum and preparation method and application thereof |
| CN113967262B (en) * | 2021-10-21 | 2022-12-23 | 中国人民解放军空军军医大学 | Bone-level targeting-ultrasonic triggering type drug delivery system, preparation method and application |
| CN116396358B (en) * | 2023-06-08 | 2023-08-29 | 时夕(广州)生物科技有限公司 | Preparation and application of bone-targeted nano lipid particles |
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