WO2013044734A1

WO2013044734A1 - Bone targeted liposome and preparation method therefor

Info

Publication number: WO2013044734A1
Application number: PCT/CN2012/081355
Authority: WO
Inventors: 吴蘅; 秦岭; 杨智钧; 张戈; 王新峦
Original assignee: 中国科学院深圳先进技术研究院; 香港中文大学
Priority date: 2011-09-28
Filing date: 2012-09-13
Publication date: 2013-04-04
Also published as: CN103987379B; CN103987379A

Abstract

In a bone targeted liposome, a monolayer vesicle is formed by phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugates, and polyethylene glycol lipid conjugates having an active radical, a drug is wrapped inside the monolayer vesicle, and a bone targeted functional ingredient is connected outside the monolayer vesicle through the active radical of the polyethylene glycol lipid conjugates having an active radical. Also disclosed is a preparation method for a bone targeted liposome.

Description

Bone-targeted liposome and preparation method thereof

[Technical Field]

The invention relates to the technical field of medicine, in particular to a bone targeting liposome and a preparation method thereof.

【Background technique】

Because of the special physiological structure such as large tissue hardness and poor permeability, the bone tissue is difficult to reach the lesion site effectively, and the drug concentration in the lesion site is low, which can not achieve a good therapeutic effect. The method of increasing the concentration by increasing the dose of administration, while increasing the concentration of the drug at the lesion site to a certain extent, also increases the toxic side effects of the drug on other tissues.

Icariin is a flavonoid compound with phytoestrogen-like effects. It is the main secondary metabolite of Epimedium in the body and is an effective compound for promoting bone formation. Recent in vitro studies have shown that icariin can promote the proliferation and differentiation of osteoblasts, enhance the calcification of bone matrix, inhibit the differentiation and bone resorption of osteoclasts, and reduce the motility of osteoclasts. In vivo studies, using a mouse model of ovariectomized osteoporosis, it was found that icariin can mediate a dependent and independent estrogen receptor pathway and protect both bone and muscle of mice. In addition, icariin can reduce the incidence of steroid hormone-induced osteonecrosis in the model, which is the mechanism by which icariin inhibits the formation of intravascular thrombus and extravascular lipid deposition.

Although icariin has strong biological activity in the body, the in vivo administration of icariin poses a serious challenge due to the insoluble physical properties of icariin. The pharmacokinetic study of rabbits administered orally and intravenously with icariin found that the oral doses of icariin were gradually increased by 20, 40 and 80 mg/kg, and the corresponding pharmacokinetic parameters C _max and AUC. _{0- ∞} also increased accordingly, but the bioavailability in the body decreased significantly, 17.29%, 13.80% and 10.70% respectively. Intravenous administration of Icariin 20 mg/kg, AUC _0-∞ was 2470 ng•h/mL, and oral administration of etoposide at a dose of 80 mg/kg of AUC _{0-∞ was} only 495.67 ng•h/mL, intravenously The AUC _0-∞ is 4 times larger than oral administration, and the results indicate that icariin is not easily absorbed orally, and intravenous administration may be the best route to choose ( Liu HP, Meng FH, Guo JF, Si DY, Zhu XW) , Zhao YM (2010). Pharmacokinetics of icaritin in rats. Chinese Pharmaceutical Journal 45(7), 539-543. ).

The liposome has good biocompatibility and has been widely used as a drug carrier, which can make the encapsulated drug have better stability, can improve the stability of the drug, and can reduce the toxicity of the drug to a certain extent. .

However, general bone-targeting liposomes do not increase the drug concentration of the bone surface when in use.

[Summary of the Invention]

Based on this, there is provided a bone-targeting liposome capable of increasing the drug concentration of a bone surface at the time of use and a preparation method thereof.

A bone-targeting liposome comprising a drug, phosphatidylcholine, cholesterol, a polyethylene glycol lipid conjugate, a polyethylene glycol lipid conjugate having a reactive group, and a bone targeting functional component;

The phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate, and polyethylene glycol lipid conjugate having a reactive group form a monolayer vesicle, and the drug is coated on the monolayer Inside the vesicle, the bone targeting functional component is attached to the outside of the unilamellar vesicle and the bone targeting functional component and the reactive group of the reactive group-containing polyethylene glycol lipid conjugate connection;

The molar ratio of the phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate, polyethylene glycol lipid conjugate having a reactive group, and bone targeting functional component is 70 to 95: 5-20 :0.5~5:1~10:1~8.

In one embodiment, the bone targeting functional component is at least one of alendronate, Asp8, and (DSS) ₆ .

In one embodiment, the phosphatidylcholine is at least one of soy lecithin, dimyristoylphosphatidylcholine, dioleoyl lecithin, and disaturated lecithin.

In one embodiment, the polyethylene glycol lipid conjugate is C12 Ceremide-mPEG, C16 At least one of Ceremide-mPEG, C20 Ceremide-mPEG 12 and DSPE-mPEG.

In one embodiment, the polyethylene glycol lipid conjugate having a reactive group is DSPE-PEG-Mal.

In one embodiment, the bone targeting liposomes have a diameter in the range of less than 500 nm.

In one embodiment, the bone-targeting liposome has a diameter of from 50 nm to 200 nm.

In one embodiment, the total number of moles of the phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate, and polyethylene glycol lipid conjugate having a reactive group and the number of moles of the drug The ratio is 1:5~1:25.

A method for preparing a bone-targeted liposome, comprising the steps of:

Preparing a drug-coated monolayer vesicle formed of phosphatidylcholine, cholesterol, and a polyethylene glycol lipid conjugate; wherein the phosphatidylcholine, cholesterol, polyethylene The molar ratio of the alcohol lipid conjugate is 70~95:5~20:0.5~5;

Preparing a micelle of a polyethylene glycol lipid conjugate material having a reactive group, and mixing the micelle with a bone targeting functional component such that the bone targeting functional component and the active group are The reactive group of the polyethylene glycol lipid conjugate is linked to obtain a composite micelle; wherein the molar ratio of the polyethylene glycol lipid conjugate having a reactive group to the bone targeting functional component is 1 ~10:1~8;

Mixing the drug-coated monolayer vesicle with the composite micelle such that the composite micelle is embedded in the unilamellar vesicle to obtain the bone-targeting liposome; wherein The molar ratio of phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate, polyethylene glycol lipid conjugate having active group and bone targeting functional component is 70-95:5-20:0.5 ~5:1~10:1~8.

A method for preparing a bone-targeted liposome, comprising the steps of:

Preparing a drug-coated monolayer vesicle formed of phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate, and a polyethylene glycol lipid conjugate having a reactive group Wherein the molar ratio of the phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate and the polyethylene glycol lipid conjugate having a reactive group is 70 to 95: 5 to 20: 0.5 5:1~10;

Mixing the drug-coated monolayer vesicle with a bone targeting functional component such that the bone targeting functional component is linked to the reactive group of the reactive group-containing polyethylene glycol lipid conjugate Obtaining the bone-targeting liposome; wherein the phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate, polyethylene glycol lipid conjugate having a reactive group, and bone targeting The molar ratio of functional components is 70~95:5~20:0.5~5:1~10:1~8.

When the bone-targeting liposome transports the drug, the drug can be targeted to the bone surface by the guiding action of the bone targeting functional component, thereby increasing the drug concentration on the bone surface.

[Description of the Drawings]

Figure 1 is a schematic view showing the structure of a bone-targeting liposome;

2 is an overall topographical view of the bone-targeting liposome prepared in Example 1;

3 is a scanning electron microscope comparison diagram of bone-targeted liposomes prepared in Example 1;

4 is a comparison diagram of in vitro release profiles of bone-targeted liposomes prepared in Example 1;

Figure 5 is a graph showing the fluorescence intensity of the organ after the tail vein injection of the bone-targeted liposome prepared in Example 1.

【detailed description】

Bone-targeted liposomes and methods for their preparation are further illustrated by the accompanying figures and specific examples.

A bone-targeting liposome according to one embodiment as shown in Figure 1, comprising a drug, phosphatidylcholine, cholesterol, a polyethylene glycol lipid conjugate, a polyethylene glycol lipid conjugate having a reactive group And bone-targeting functional components.

Phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate, and polyethylene glycol lipid conjugate having a reactive group form a monolayer vesicle, and the drug is coated on the inner side of the monolayer vesicle, bone A targeting functional component is attached to the outside of the unilamellar vesicle, and the bone targeting functional component is linked to a reactive group of a polyethylene glycol lipid conjugate having a reactive group.

In general, such bone-targeted liposomes should have a diameter of less than 500 nm.

In this embodiment, the bone-targeting liposome may have a diameter of 50 nm to 200 nm, thereby facilitating leakage of the bone-targeted liposome from the blood vessel to the bone tissue.

The molar ratio of phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate, polyethylene glycol lipid conjugate having active group and bone targeting functional component is 70-95:5-20:0.5 ~5:1~10:1~8.

Such bone-targeted liposomes, when used as a transport vehicle for icariin, can prolong the residence time of icariin in the blood and maintain the release of icariin at a constant rate. Through the targeting of bone-targeting functional components, it is possible to target the transport of icariin to the bone surface and to increase the concentration of icariin on the bone surface.

The ratio of the total number of moles of phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate and polyethylene glycol lipid conjugate having a reactive group to the number of moles of the drug may be 1:5 to 1: 25.

The single-layered vesicles of the bone-targeting liposome can not only coat the icariin, but also coat other types of bone-targeting drugs as needed, thereby improving the concentration of the drug on the bone surface.

The bone targeting functional component can be alendronate, Asp8 (8 aspartate repeats) or (DSS) ₆ (6 (aspartate-serine-serine) repeats).

The phosphatidylcholine may be soy lecithin, dimyristoyl phosphatidylcholine, dioleoyl lecithin or di-saturated lecithin.

The polyethylene glycol lipid conjugate may be a polyethylene glycol modified ceramide, a distearoylphosphatidylethanolamine derivative such as: C12 Ceremide-mPEG, C16 Ceremide-mPEG, C20 Ceremide-mPEG 12 or DSPE-mPEG.

The polyethylene glycol lipid conjugate having a reactive group may be a distearoylphosphatidylethanolamine derivative (DSPE-PEG-Mal) bearing a maleimide group.

In a preferred embodiment, a cryoprotectant is added to the bone targeting liposomes for long term cryopreservation. The bone-targeted liposome to which the cryoprotectant is added only needs to be re-dissolved before application, and a bone-targeted liposome solution with uniform particles and good encapsulation can be formed.

The cryoprotectant can be sucrose, mannitol, glucose or trehalose. The ratio of the total number of moles of phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate and polyethylene glycol lipid conjugate having a reactive group to the number of moles of cryoprotectant is 2:1~10 :1.

Such bone-targeted liposomes can be delivered to the body by intradermal injection, subcutaneous injection or intramuscular injection during specific use.

The above-mentioned bone-targeting liposome can be prepared by various methods such as a thin film evaporation method, an ultrasonic method, an extrusion method, a high pressure homogenization method, and a dipping method.

A method for preparing the above bone-targeted liposome according to an embodiment comprises the following steps:

S110, preparing a monolayer vesicle coated with a drug.

Monolayer vesicles are formed from phosphatidylcholine, cholesterol, and polyethylene glycol lipid conjugates. The molar ratio of phosphatidylcholine, cholesterol, and polyethylene glycol lipid conjugate is 70-95:5-20:0.5-5.

The polyethylene glycol lipid conjugate can be C12 Ceremide-mPEG, C16 Ceremide-mPEG, C20 Ceremide-mPEG 12 or DSPE-mPEG.

The specific preparation process of the drug-coated monolayer vesicle is as follows:

The drug, phosphatidylcholine, cholesterol and polyethylene glycol lipid conjugate were dissolved in a mixture of methanol and chloroform in a volume ratio of 1:1, and the organic solvent was evaporated by a rotary vacuum dryer to obtain a lipid. film.

After the obtained lipid film was completely dried, the lipid film was immersed in a PBS solution to be hydrated, followed by incubation in a water bath to obtain a lipid suspension.

At nitrogen pressure, squeezing with an extruder (Lipex ^TM Extruder) the lipid suspension, liposome suspension was such that the pore size of 200nm and 100nm by the membrane after repeated continuous extrusion, to give coated A single layer of vesicles of the drug. The obtained monolayer vesicles have a diameter of 50 nm to 200 nm by sequentially passing the lipid suspension through a filter having a pore diameter of 200 nm and 100 nm.

S120, preparing a micelle of a polyethylene glycol lipid conjugate having a reactive group, and mixing the micelle with a bone targeting functional component, so that the bone targeting functional component and the polyethylene glycol having the active group The reactive groups of the lipid conjugate are linked to obtain a composite micelle.

The molar ratio of the polyethylene glycol lipid conjugate having a reactive group to the bone targeting functional component is from 1 to 10:1 to 8.

The polyethylene glycol lipid conjugate having a reactive group may be DSPE-PEG-Mal.

The bone targeting functional component can be alendronate, Asp8 or (DSS) ₆ .

The specific preparation process of the composite micelles is as follows:

The polyethylene glycol lipid conjugate having a reactive group was dissolved in chloroform, and the solvent was evaporated to obtain a lipid film.

After adding PBS to the obtained lipid film, it was hydrated to form a micelle of a polyethylene glycol lipid conjugate having a reactive group.

The bone-targeting functional component PBS solution is added to the micelle of the polyethylene glycol lipid conjugate material having the active group, and the bone-targeting functional component and the polyethylene glycol lipid having the active group are reacted after the reaction. The reactive groups of the conjugate are linked to obtain a composite micelle.

Also included in S120 is the addition of L-cysteine to neutralize unreacted reactive groups.

S130, mixing the drug-coated single-layer vesicle obtained by S110 with the composite micelle obtained by S120, so that the composite micelle is embedded in the single-layer vesicle to obtain a bone-targeting liposome.

Wherein, the molar ratio of phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate, polyethylene glycol lipid conjugate having active group and bone targeting functional component is 70-9:5-20 :0.5~5:1~10:1~8.

The drug-coated monolayer vesicle obtained in S110 was mixed with the composite micelle obtained in S120, and incubated in a water bath. Before the composite micelles are mixed with the monolayer vesicles, the hydrophilic portion (PEG and target) is out of the aqueous phase, and the lipophilic portion (DSPE) is inward. When mixed with a single layer of vesicles, the lipid ends of the composite micelles are similar to the phospholipid bilayers in the monolayer vesicles, so the DSPE portion of the composite micelles is inserted into the lipid bilayer, while the hydrophilic PEG and target The head portion is distributed to the aqueous phase and is distributed on the surface of the single layer vesicle. Eventually, the composite micelles are embedded in the monolayer vesicles to obtain bone-targeted liposomes.

A method for preparing the above bone-targeted liposome of another embodiment is provided, comprising the steps of:

S210, preparing a monolayer vesicle coated with a drug.

The unilamellar vesicles are formed from phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugates, and polyethylene glycol lipid conjugates having reactive groups. Wherein, the molar ratio of phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate and polyethylene glycol lipid conjugate having a reactive group is 70 to 95:5 to 20:0.5 to 5:1. ~10.

In S210, the specific preparation process of the drug-coated monolayer vesicle is as follows:

The drug, phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate, and polyethylene glycol lipid conjugate having a reactive group are dissolved in a mixture of methanol and chloroform in a volume ratio of 1:1. The organic solvent was evaporated using a rotary vacuum dryer to obtain a lipid film.

S220, mixing the drug-coated monolayer vesicle with a bone targeting functional component, so that the bone targeting functional component is linked to the active group of the polyethylene glycol lipid conjugate having a reactive group to obtain a bone target. To liposomes.

The bone targeting functional component can be alendronate, Asp8 or (DSS) ₆ .

S220 also includes obtaining bone-targeted liposomes, and then targeting the bone to liposomes through agarose gel CL-4B Column chromatography removes the operation of bone-targeting functional components that are not linked to reactive groups.

The following is a specific embodiment.

Example 1

In a round bottom flask, 21 um of icariin and 325 um of lipid (composed of SPC, cholesterol, C16 Ceremide-mPEG 200 in a molar ratio of 85:11:4) were dissolved in 6 ml of a methanol-nitrogen mixture (1:1). , V/V). The temperature was adjusted to 55 ° C, and the organic solvent in the round bottom flask was volatilized by a rotary vacuum dryer to obtain a lipid film. The lipid film was then placed in a vacuum oven overnight to allow it to dry completely. The dried lipid membrane was then immersed in 10 ml of PBS (10 mM, pH 7.4) and incubated in a 55 ° C water bath to obtain a liposome suspension. Pressurized with nitrogen, and the resulting liposome suspension was extruder ^{(Lipex TM Extruder, Northern Lipids Inc.} , Vancouver, BC, Canada) extrusion, the liposome suspension that passes through a pore size of 200nm and 100nm A polycarbonate filter (Nuclepore Track-Etch Membrane) was continuously extruded six times to obtain a single layer of vesicles coated with icariin.

50 ul of a chloroform solution of DSPE-PEG2000-Mal relative to 2 mol% of total lipid was added to a 1.5 ml test tube, and the organic solvent was evaporated to obtain a lipid film. After adding PBS to the obtained lipid film, it was hydrated to form a micelle of DSPE-PEG2000-Mal. According to the molar ratio of DSPE-PEG2000-Mal and (DSS) ₆ of 2:1, the thiol-containing (DSS) ₆ PBS solution was added to the micelle of DSPE-PEG2000-Mal and shaken for 4 h to make (DSS) ₆ and DSPE. - PEG2000-Mal reacts to form DSPE-PEG2000-(DSS) ₆ composite micelles. After the end of the reaction, L-cysteine was added to a final concentration of 1 mM and incubated at room temperature for at least 10 min for neutralization of unconjugated binding groups.

The DSPE-PEG2000-(DSS) ₆ composite micelles were mixed with the epithelial-coated single-layer vesicles and incubated for 2 h at 37 °C in a water bath to embed DSPE-PEG2000-(DSS) ₆ in a micelle. Within the vesicles, bone-targeted liposomes are obtained.

The suspension of bone-targeted liposomes was mixed with distilled water containing mannitol, and the molar ratio of mannitol to lipid was 5, and dried in a freeze dryer for 48 hours to obtain lyophilized bone-targeted liposomes. Lyophilized bone-targeted liposomes were hydrated prior to use and then sterilized using a 0.22 μm sterile filter.

An overall topographical view of the bone-targeted liposomes prepared in Example 1 as shown in Figure 2. 2A is an overall topographical view of bone-targeted liposomes before freeze-drying, FIG. 2B is a generalized topography of bone-targeted liposomes after long-term freeze-drying, and FIG. 2C is a long-term placed bone-targeting lipid. The overall morphology of the bone-targeted liposomes obtained after rehydration.

As can be seen from Figure 2B, the freeze-dried bone-targeted liposomes after long-term placement were in the form of a cake. It can be seen from Fig. 2A and Fig. 2C that the bone-targeted liposomes obtained after rehydration of the bone-targeted liposomes for a long period of time have no significant change compared with that before lyophilization.

As can be seen from Figure 2, freeze drying does not affect the overall appearance of the bone targeting liposomes.

3 is a scanning electron microscope comparison of bone-targeted liposomes prepared in Example 1. 3A is a scanning electron micrograph of the bone-targeting liposome of FIG. 2A, and FIG. 3B is a scanning electron micrograph of the bone-targeting liposome of FIG. 2C.

The bone-targeting liposomes in Figure 3A are in the form of a single layer of vesicles, and the hydrated bone-targeting liposomes in Figure 3B are also in the form of a single layer of vesicles.

Comparing Fig. 3A with Fig. 3B, it can be seen that the structure of the bone-targeted liposome before and after freeze-drying did not change significantly under electron microscope.

As can be seen from Figure 3, freeze drying does not affect the structure of the bone targeting liposomes.

The release profile was evaluated by a fine-tuned dialysis membrane method as follows: In a glass vial, 1 mL of the liposome prepared in Example 1 (about 800 ug of icariin) was dissolved in 50 mL of release medium (containing 10% ethanol). In PBS (pH 7.4)). The 12 aliquots of the release medium were placed in pre-treated dialysis bags (12,000 to 14,000 large molecular weight sieves), and the dialysis bags were separately immersed in the same glass bottles. The glass bottle was placed on a constant temperature shaker and shaken at 37 ± 10 rpm at 100 ± 10 rpm. Samples were recovered at predetermined time intervals (over 72 h) and the same volume of fresh medium was applied. The in vitro release profile of icariin was determined by ultra performance liquid chromatography system to determine the concentration of icariin.

As can be seen from Figure 4, the bone-targeted liposomes prepared in Example 1 were able to control the release of icariin with a control time of up to 72 h. At the same time, the addition of bone targeting molecules did not affect the release of icariin in liposomes.

Example 2

In a round bottom flask, 21 μmol of icariin and 360umol lipid (composed of DOPC, Cholesterol, DSPE-mPEG2000 and DSPE-PEG2000-Mal in a molar ratio of 75:18:3:4) were dissolved in 6 ml of methanol-nitrogen imitation. Mixture (1:1, V/V). The temperature was adjusted to 55 ° C, and the organic solvent in the round bottom flask was volatilized by a rotary vacuum dryer to obtain a lipid film. The lipid film was then dried in a vacuum oven overnight. The dried lipid film was then immersed in 10 ml of PBS (10 mM, pH 7.4) to obtain a liposome suspension. Pressurized with nitrogen, and the resulting liposome suspension was extruded by Lipex ^TM Extruder, liposome suspension was filtrated through a pore size of 200nm and 100nm polycarbonate filters of six continuous extrusion, to give a coated Monolayer vesicles of icariin.

Approximately 7 umol of sulfhydryl-containing (DSS) _{6 was} then added to the monolayer vesicles and shaken at room temperature for 4 h to obtain a suspension of bone-targeted liposomes. The resulting suspension of bone-targeted liposomes was purified by agarose gel CL-4B column chromatography to remove unconjugated binding (DSS) ₆ .

Example 3

In a round bottom flask, 21 um of icariin and 325 umol of lipid (composed of SPC, cholesterol, C16 Ceremide-mPEG 200 in a molar ratio of 85:11:4) were dissolved in 6 ml of a methanol-nitrogen mixture (1:1) , V/V). The temperature was adjusted to 55 ° C, and the organic solvent in the round bottom flask was volatilized by a rotary vacuum dryer to obtain a lipid film. The lipid film was then placed in a vacuum oven overnight to allow it to dry completely. The dried lipid membrane was then immersed in 10 ml of PBS (10 mM, pH 7.4) and incubated in a 55 ° C water bath to obtain a liposome suspension. Pressurized with nitrogen, and the resulting liposome suspension was extruder ^{(Lipex TM Extruder, Northern Lipids Inc.} , Vancouver, BC, Canada) extrusion, such liposome suspension through a pore size of 100nm ~ 200nm poly The carbonate filter (Nuclepore Track-Etch Membrane) was continuously extruded six times to obtain epimedium liposomes.

The fluorescently labeled icariin liposome and the bone-targeted liposome prepared in Example 1 were injected into the tail of 8 6-month-old female mice, respectively (n=4, ie, four groups per group). Only mice), the dose is 1 mg / kg.

After 4h and 24h, 4 mice were sacrificed, and the main organs such as heart, liver, spleen, lung, kidney and bilateral femur were taken. Using Xenogen The IVIS imaging system detects the fluorescence intensity in each organ and obtains Figure 5.

In Fig. 5, blue indicates high fluorescence intensity and low red fluorescence intensity. A high fluorescence intensity indicates that the liposome is more distributed in the tissue and vice versa.

As can be seen from Figure 5, the fluorescent signal in the femur was the strongest relative to other organs of the mouse 4 h after the tail vein injection. Fluorescence signals were still detectable at the femur of the bone-targeted liposome-injected mice prepared in Example 1 24 h after the tail vein injection, but the mice were injected with the chip-labeled icariin liposome. No fluorescent signal was detected at the femur.

It can be explained that the bone-targeted liposome prepared in Example 1 is easier to target bone tissue while prolonging the residence time of icariin in the bone.

The above-mentioned embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims

A bone-targeting liposome comprising a drug, phosphatidylcholine, cholesterol, a polyethylene glycol lipid conjugate, a polyethylene glycol lipid conjugate having a reactive group, and a bone target Functional ingredient

The phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate, and polyethylene glycol lipid conjugate having a reactive group form a monolayer vesicle, and the drug is coated on the monolayer Inside the vesicle, the bone targeting functional component is attached to the outside of the unilamellar vesicle and the bone targeting functional component and the reactive group of the reactive group-containing polyethylene glycol lipid conjugate connection;

The molar ratio of the phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate, polyethylene glycol lipid conjugate having a reactive group, and bone targeting functional component is 70 to 95: 5-20 :0.5~5:1~10:1~8.
The bone-targeting liposome according to claim 1, wherein the bone-targeting functional component is at least one of alendronate, Asp8, and (DSS) 6 .
The bone-targeted liposome according to claim 1, wherein the phosphatidylcholine is in soybean lecithin, dimyristoyl phosphatidylcholine, dioleoyl lecithin, and di-saturated lecithin. At least one.
The bone-targeted liposome according to claim 1, wherein the polyethylene glycol lipid conjugate is C12 Ceremide-mPEG, C16 At least one of Ceremide-mPEG, C20 Ceremide-mPEG 12 and DSPE-mPEG.
The bone-targeting liposome according to claim 1, wherein the polyethylene glycol lipid conjugate having a reactive group is DSPE-PEG-Mal.
The bone-targeting liposome of claim 1 wherein the bone-targeting liposome has a diameter in the range of less than 500 nm.
The bone-targeting liposome according to claim 6, wherein the bone-targeting liposome has a diameter of 50 nm to 200 nm.
The bone-targeting liposome according to claim 1, wherein the phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate, and polyethylene glycol lipid having a reactive group are conjugated The ratio of the total number of moles of the substance to the number of moles of the drug is from 1:5 to 1:25.
A method for preparing a bone-targeted liposome, comprising the steps of:

Preparing a drug-coated monolayer vesicle formed of phosphatidylcholine, cholesterol, and a polyethylene glycol lipid conjugate; wherein the phosphatidylcholine, cholesterol, polyethylene The molar ratio of the alcohol lipid conjugate is 70~95:5~20:0.5~5;

Preparing a micelle of a polyethylene glycol lipid conjugate material having a reactive group, and mixing the micelle with a bone targeting functional component such that the bone targeting functional component and the active group are The reactive group of the polyethylene glycol lipid conjugate is linked to obtain a composite micelle; wherein the molar ratio of the polyethylene glycol lipid conjugate having a reactive group to the bone targeting functional component is 1 ~10:1~8;

Mixing the drug-coated monolayer vesicle with the composite micelle such that the composite micelle is embedded in the unilamellar vesicle to obtain the bone-targeting liposome; wherein The molar ratio of phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate, polyethylene glycol lipid conjugate having active group and bone targeting functional component is 70-95:5-20:0.5 ~5:1~10:1~8.
A method for preparing a bone-targeted liposome, comprising the steps of:

Preparing a drug-coated monolayer vesicle formed of phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate, and a polyethylene glycol lipid conjugate having a reactive group Wherein the molar ratio of the phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate and the polyethylene glycol lipid conjugate having a reactive group is 70 to 95: 5 to 20: 0.5 5:1~10;

Mixing the drug-coated monolayer vesicle with a bone targeting functional component such that the bone targeting functional component is linked to the reactive group of the reactive group-containing polyethylene glycol lipid conjugate Obtaining the bone-targeting liposome; wherein the phosphatidylcholine, cholesterol, polyethylene glycol lipid conjugate, polyethylene glycol lipid conjugate having a reactive group, and bone targeting The molar ratio of functional components is 70~95:5~20:0.5~5:1~10:1~8.