Cholesterol rapamycin liposome as well as preparation and application thereof

Abstract

The invention belongs to the technical field of medicines, and relates to a cholesterolized rapamycin liposome as well as preparation and application thereof. The invention relates to a cholesterylated rapamycin liposome, which is prepared from cholesterylated rapamycin, phospholipid and a phospholipid PEG (Polyethylene Glycol) derivative. The liposome is mainly composed of a rapamycin cholesterol conjugate, the coupling effect of cholesterol and rapamycin indirectly enables a drug and phospholipid to be tightly combined, compared with free rapamycin, the entrapment efficiency is greatly improved, the leakage rate is reduced (the entrapment efficiency is improved by 8.05%, and the leakage rate is reduced by 100%), the entrapment efficiency of the preparation is as high as 95% or above, and the preparation has good application prospects. The particle size is about 120nm, the PDI is less than 0.2, the drug can be stably delivered in vivo, the circulation time of the drug in vivo is prolonged, the toxic and side effects are reduced, the safety is improved, the stability is good, and the storage, transportation and use are facilitated.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and relates to a cholesterolated rapamycin liposome and its preparation and use in treating or preventing transplant rejection, autoimmune diseases, food allergies and immune responses to therapeutic agents, and also in anti-tumor, anti-aging, treatment of heart failure, obesity, kidney disease, anti-angiogenesis and anti-fibrosis, and also in drugs for treating neurological diseases such as Alzheimer's disease, cerebral ischemia-reperfusion injury, etc. Background Art

[0002] Rapamycin (RAPA), also known as sirolimus, is a new type of macrolide immunosuppressant in the form of white solid crystals and a lipophilic compound. Rapamycin was developed in the 1970s and isolated from an island in the South Pacific. Rapamycin is produced by microorganisms of the genus Streptomyces and has antifungal activity. It can effectively inhibit the infection of a variety of pathogens, including Gram-positive bacteria, Gram-negative bacteria, mycobacteria, etc. It is often used to treat kidney transplantation, especially to slow down the immune rejection reaction after organ transplantation surgery, and can also be used to prevent and treat food allergies. Rapamycin blocks signal transduction through different cytokine receptors, blocks the process of T lymphocytes and other cells from G1 phase to S phase, and thus exerts an immunosuppressive effect. From the current clinical application, RAPA has a good anti-rejection effect and has a good synergistic effect with immunosuppressants such as cyclosporine A (CsA) and FK506. In addition, rapamycin has a potential therapeutic effect on anti-tumor by inhibiting the mTOR pathway, participating in cell growth, division and survival. Studies in recent years have shown that rapamycin can extend the lifespan of fruit flies and may be beneficial in delaying the progression of a variety of long-term diseases, although its use as an anti-aging drug has not yet been approved. In animal experiments, rapamycin has shown the potential to reverse or delay the progression of diseases such as heart failure, obesity, and kidney disease. In addition, scientists have recently discovered another use for it: it can be used to treat Alzheimer's disease (senile dementia). Many researchers have studied rapamycin from the perspective of neurological diseases and found that rapamycin also has a certain reversal effect on cerebral ischemia-reperfusion injury, and has a certain protective effect on neuronal cells at appropriate doses. mTOR plays a core role in cell death after cerebral ischemia. The mTORC1 inhibitor rapamycin can pharmacologically inhibit mTORC1 and is considered a potential treatment for stroke. Rapamycin can also prevent vascular stenosis and has a good effect on vascular stenosis caused by vascular intimal hyperplasia after stent placement. In addition, rapamycin can inhibit endothelial cell migration and new blood vessel formation, affect the proliferation and migration of smooth muscle cells, and the survival of endothelial cells, so it is used to control abnormal blood vessel growth in diabetic retinopathy and has anti-fibrotic effects. It can be seen that rapamycin has a wide range of therapeutic applications and is a drug with great application value.

[0003] Although rapamycin can treat a wide range of diseases, it also has significant toxic side effects and is generally referred to as a "double-edged sword." For example, as described in the article by Oak Z.Chi et al., rapamycin can show good brain cell protection at microdoses, but at medium and high doses, rapamycin will increase the infarct area of ​​cerebral ischemia. In addition, rapamycin can also cause significant hepatotoxicity, gastrointestinal tract, acute kidney injury and other side effects, which shows that rapamycin is highly toxic. And as a hydrophobic drug, rapamycin has extremely poor water solubility, which affects its absorption in the gastrointestinal tract, resulting in low bioavailability and poor efficacy. Therefore, how to reduce the toxicity of rapamycin and increase its solubility is an important problem that we need to solve.

[0004] Liposome (LIP) is an ultra-micro hollow spherical carrier preparation with one or more concentric lipid bilayer structures, and is the earliest first-generation drug carrier. Liposomes are mainly composed of phospholipids with a hydrophilic head and two hydrophobic carbon-terminal tails, and are amphiphilic. Its lipid bilayer is similar to the cell structure of organisms. By utilizing the special structure of liposomes, hydrophilic drugs can be encapsulated in the hollow hydrophilic region and hydrophobic drugs can be embedded in the lipid bilayer to improve the solubility of the drug. The preparation of general liposomes requires the addition of phospholipids, cholesterol and PEG.

[0005] Phospholipids are the most important membrane material in the preparation of liposomes. They are an important component of biological membranes. Their inherent hydrophilic and lipophilic properties allow phospholipids to spontaneously form a closed bilayer in an aqueous medium and become the skeleton of biological membranes. The addition of cholesterol will affect the rigidity of liposomes. The appropriate amount of cholesterol will improve the stability of liposomes, but for passive drug delivery, excessive cholesterol will cause cholesterol to occupy the site where drugs bind to phospholipids, thereby reducing the encapsulation rate. Therefore, a balance needs to be found in the addition of cholesterol.

[0006] After entering the body, liposomes are mainly phagocytosed by the reticuloendothelial system and concentrated in organs with rich reticuloendothelial cells such as the liver, spleen and bone marrow. The accumulation of drugs in the heart and kidneys is significantly lower than that of free drugs, which can reduce the toxicity of drugs to organs such as the heart and kidneys. Compared with other nanosystems, the advantage of using liposomes is that they are considered safe for clinical use, and more than 10 liposome drugs have been approved by the US Food and Drug Administration (FDA). Compared with other nanomaterials (such as polymer-based nanocarriers), liposomes control the colloidal stability of therapeutic drugs in the complex biological environment of diseased tissues through specific soft interactions. In terms of translational research, liposomes have shown excellent results, with almost no systemic toxicity and low immunogenicity. Therefore, using liposomes to deliver rapamycin to treat diseases may solve the problem of both improving its solubility, efficiently delivering drugs and reducing toxicity.

[0007] During the experiment, it was found that rapamycin is a low-solubility, lipophilic drug. When rapamycin is prepared into liposomes, it is very easy to precipitate or drug leakage occurs, and the liposomes are in a very unstable state. After research, O. Eloy, Josimar and others also pointed out in the article that the low encapsulation rate of rapamycin can be attributed to the instability of liposomes, which are retained on the extrusion membrane. The reason for this instability may be that phospholipids and rapamycin cannot be firmly combined. It is necessary to further solve the problem that phospholipids and rapamycin cannot be firmly combined. So far, no patents related to cholesterol rapamycin liposomes have been reported. Summary of the invention

[0008] The present invention aims to provide a cholesterol-containing rapamycin liposome and a preparation method and application thereof.

[0009] To achieve the above purpose, the present invention adopts the following technical solution:

[0010] A cholesterol-containing rapamycin liposome, wherein the liposome comprises cholesterol-containing rapamycin, phospholipids, and phospholipid PEG derivatives;

[0011] Among them, cholesterol-containing rapamycin is

[0012]

[0013] In the formula: R is cholesterol and its derivatives, L is a connecting arm, wherein L is -C(O)-, -C(O)CH2-, -C(O)CH2CH2-, -C(O)CH2CH2CH2- or -C(O)CH2CH2CH2-.

[0014] The cholesterol and its derivatives in R are C 28 H 45 ClO2, C 30 H51 NO2, C 31 H 52 O2、C 31 H 53 O2、C 34 H 59 NO2, the corresponding structural formula is as follows:

[0015]

[0016] C 34 H 59 NO2 structure C 28 H 45 ClO2 structure

[0017] The phospholipids in the liposomes are one or more of phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, phosphatidic acid, cardiolipin, acylcholine, and phosphatidylinositol; wherein the mass ratio of cholesterol-containing rapamycin to phospholipids is 1:30 to 8:30.

[0018] Preparation method of cholesterol rapamycin:

[0019] Rapamycin is reacted with dihydrofuran-2,5-dione at a certain temperature and time, a catalyst is added to the reaction, and after the reaction is complete, the product 1 is obtained by suction filtration. In addition, the cholesterol derivative and anhydrous ethylenediamine are dissolved in dichloromethane respectively, and the cholesterol derivative is dropped into anhydrous ethylenediamine under ice bath conditions, stirred for a certain time to room temperature, and the reaction solution is washed with Nacl several times, and the lower organic layer is collected, dried with anhydrous sodium sulfate, and the solvent is filtered to obtain the reaction product 2. After the product 1 is activated under ice bath conditions, the product 2 and the catalyst are added to react for a certain time. After the reaction is completed, the organic solvent is removed, and the residue is purified to obtain cholesterol rapamycin; wherein the reaction time of rapamycin and dihydrofuran-2,5-dione is 4 to 72 hours. The activation time of product 1 is 0.5 to 2 hours, and the reaction time of product 2 and the catalyst is 12 to 48 hours.

[0020] The reaction ratio of rapamycin to lipid is 1:40 to 10:1 (molar ratio), the reaction ratio of the reaction product to the catalyst is 1:40 to 10:1 (molar ratio), and the catalyst is generally triglycerol hydrolase, carboxylesterase, thioesterase, phosphomonoesterase, phosphodiesterase, etc. Preferably, triglycerol hydrolase is used.

[0021] A method for preparing liposomes:

[0022] (a) dissolving cholesterol-modified rapamycin and other lipids in anhydrous ethanol as an organic phase;

[0023] (b) slowly and uniformly dropping the organic phase into the buffer solution (aqueous phase) under stirring at 25 to 37° C., wherein the volume ratio of the organic phase to the buffer solution is 1:1 to 1:30;

[0024] (c) removing the organic solvent from the liposome suspension obtained after the above mixing to obtain the final cholesterol-containing rapamycin liposome.

[0025] The stirring time in the step (b) is 20 to 240 minutes.

[0026] The liposome preparation method may also include a thin film dispersion method, a reverse evaporation method, an ether injection method, an ultrasonic dispersion method, etc. Usually, the organic phase in which the membrane material and the drug are dissolved is slowly and uniformly dripped into the aqueous phase, because its longer side chains self-assemble into preliminary liposome nanoparticles through intermolecular forces under rapid stirring, and then the organic solvent is removed to obtain the final liposome. The method of removing the organic solvent includes but is not limited to rotary evaporation, reduced pressure evaporation, dialysis, ultrafiltration, solvent replacement, etc.

[0027] During the preparation process, rapamycin and the prepared cholesterol rapamycin are almost insoluble in water, and the drugs in the aqueous phase can be directly filtered. The liposome suspension is passed through a 0.22 micron microporous filter membrane to retain the drugs in the aqueous phase that are not encapsulated in the liposomes on the membrane, and a suspension of only liposomes is obtained, which is used to determine the encapsulation rate. About 1 ml of the liposome suspension is passed through a 0.22 micron microporous filter membrane, 75 μl is drawn from the filtrate and added to 1425 μl of anhydrous ethanol (20 times demulsification), the 20-fold diluted liposomes are vortexed for 5 to 10 minutes, and then centrifuged (speed 12000 rpm) for 40 minutes to completely demulsify, and 10 μl is taken into the high performance liquid phase to measure its peak area, which is the peak area after passing through the membrane C. Take 75 μl of the liposome suspension that has not passed through the microporous filter membrane and add it to 1425 μl of anhydrous ethanol (20 times demulsification). The peak area is measured by high performance liquid chromatography in the same way, which is the peak area of ​​C before passing through the membrane. The formula for calculating the liposome encapsulation efficiency of the present invention is EE (%) = peak area of ​​C after passing through the membrane / peak area of ​​C before passing through the membrane

[0028] An application of the liposome is the application of the liposome in the preparation of immunosuppressive and anti-tumor drugs.

[0029] The drug may be a liposomal formulation.

[0030] A cholesteryl rapamycin liposome preparation, comprising cholesteryl rapamycin, phospholipid, phospholipid PEG derivative, membrane stabilizer and buffer in the liposome;

[0031] The mass ratio of cholesterol-modified rapamycin to phospholipid is 1:30-8:30, the mass ratio of phospholipid PEG derivative to phospholipid is 1:5-1:150, and the mass ratio of membrane stabilizer to phospholipid is 0:30-15:30.

[0032] Wherein, the phospholipid is selected from one or more of phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, phosphatidic acid, cardiolipin, acylcholine, and phosphatidylinositol. The phospholipid PEG derivative is selected from one or more of polyethylene glycol lipid derivatives obtained by connecting polyethylene glycol with phospholipids or cholesterol. The membrane stabilizer is selected from one or more of cholesterol, ginsenoside, stigmasterol, β-sitosterol, squalene, phosphatidylinositol, phosphatidylethanolamine, sitosterol or glycolipids. The buffer solution is selected from one or more of phosphate buffer solution, acetate buffer solution, phthalate buffer solution, citric acid buffer solution, sodium carbonate buffer solution, glycine buffer solution, borax-sodium hydroxide buffer solution, ammonium chloride buffer solution, citrate buffer solution, and barbital buffer solution.

[0033] A lyoprotectant is added to the preparation, and the concentration of the lyoprotectant is 1 to 10% w / v, preferably 5 to 10% w / v; the lyoprotectant is selected from one or more of carbohydrate protectants, alcohol protectants, protein protectants, polymer protectants, amino acid protectants, salt protectants, antioxidants (ascorbic acid) and other small molecule protectants.

[0034] The carbohydrate protective agent includes one or more of sucrose, trehalose, lactose, maltopolysaccharide, fructan, raffinose, etc., the alcohol protective agent includes one or more of mannitol, sorbitol, propylene glycol, glycerol, the protein protective agent includes one or more of bovine serum albumin, skim milk, whey protein, gelatin, collagen, ovalbumin, etc., and other small molecule protective agents include one or more of betaine and L-tyrosine, etc.

[0035] The pre-freezing time is 2 to 24 hours, preferably 6 to 12 hours. The pre-freezing temperature is -20 to 80° C., preferably -40 to 60° C. The freeze-drying time is 8 to 48 hours, preferably 12 to 24 hours.

[0036] In the preparation, phospholipids are an important component, and their content has an important influence on the entire lipid system. The content of phospholipids in the entire lipid is 55.5-88.2% w / w, preferably 75.7-83.3% w / w. The phospholipids used in the present invention include but are not limited to phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin and other natural or synthetic phospholipids or their derivatives such as DSPC, DSPG, DSPE, DPPC, DPPG, DPPE, DOPC, DOPC, DOPE, HSPC, SPC, EPC. Preferably SPC, EPC, DOPC.

[0037] Furthermore, the addition of an appropriate amount of membrane stabilizer can improve the encapsulation efficiency and stability of the liposome. The content of the membrane stabilizer is 0-29.4% w / w, preferably 0-14.23% w / w. The membrane stabilizer includes but is not limited to cholesterol, ginsenoside, stigmasterol, β-sitosterol, squalene, phosphatidylinositol, phosphatidylethanolamine, sitosterol or glycolipids, etc. Cholesterol is preferred.

[0038] The content of the cholesteryl rapamycin is 2.70-18.10% w / w, preferably 2.70-10.0% w / w.

[0039] Further, polyethylene glycol modified liposomes can achieve long circulation of liposomes in vivo, and the content of phospholipid PEG derivatives is 0.5-15% w / w, preferably 2.0-10.5% w / w. The phospholipid PEG derivatives used in the present invention include but are not limited to polyethylene glycol modified distearoylphosphatidylethanolamine (PEG-DSPE) (PEG molecular weight 200-20000), polyethylene glycol modified distearoylphosphatidylglycerol (PEG-DSPG), polyethylene glycol modified cholesterol (PEG-CHOL), polyvinylpyrrolidone modified distearoylphosphatidylethanolamine (PVP-DSPE), polyvinylpyrrolidone modified distearoylphosphatidylglycerol (PVP-DSPG) or a combination thereof, more preferably polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) derivatives.

[0040] The volume of the buffer solution (aqueous phase) is 50.0-96.7% v / v, preferably 66.6-95.23% v / v. The buffer solution used in the present invention includes, but is not limited to, one or more of phosphate buffer solution, acetate buffer solution, phthalic acid buffer solution, citric acid buffer solution, sodium carbonate buffer solution, glycine buffer solution, borax-sodium hydroxide buffer solution, ammonium chloride buffer solution, citrate buffer solution, and barbital buffer solution.

[0041] The advantages of the present invention are:

[0042] The liposome of the present invention is mainly composed of rapamycin-cholesterol conjugate, and the coupling effect of cholesterol and rapamycin indirectly makes the drug and phospholipid tightly combined, and compared with free rapamycin, the encapsulation rate is greatly improved and the leakage rate is reduced (the encapsulation rate is improved by 8.05%, and the leakage rate is reduced by 100%), so that the encapsulation rate of the preparation is as high as more than 95%, the particle size is about 120nm, and the PDI is less than 0.2, so that the drug can be stably delivered in the body, the circulation time of the drug in the body is prolonged, the toxic side effects are reduced, the safety is improved, and the stability is good, which is conducive to storage, transportation and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the mass spectrum of the rapamycin prodrug provided in Example 1 of the present invention.

[0044] Figure 2 This is the appearance diagram of the cholesterol-containing rapamycin liposomes provided in Example 7 of the present invention.

[0045] Figure 3 This is a particle size diagram of the cholesterol-containing rapamycin liposomes provided in Example 7 of the present invention.

[0046] Figure 4 This is the pharmacodynamic diagram of anti-uricase antibody IgG for 42 days in cholesterol-containing rapamycin liposomes provided in Example 8 of the present invention.

[0047] Figure 5 This is a comparative diagram of the efficacy of free rapamycin and cholesterol-containing rapamycin liposomes for 42 days in the form of anti-uricase antibody IgG provided in Example 9 of the present invention.

[0048] Figure 6 The efficacy diagram of anti-AAV antibody IgG in cholesteryl rapamycin liposomes for 42 days provided in Example 10 of the present invention

[0049] Figure 7 This is a graph of mouse serum SEAP levels on day 42 provided in Example 10 of the present invention.

[0050] Figure 8 This is the freeze-dried appearance diagram of cholesterol-containing rapamycin liposomes provided in Example 13 of the present invention.

[0051] Fig. 9 This is the appearance diagram of the lyophilized and reconstituted cholesterol-containing rapamycin liposomes provided in Example 13 of the present invention. DETAILED DESCRIPTION

[0052] The present invention is described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0053] Embodiment 1:

[0054] 1) 4-(((1R,2R,4S)-4-((2R)-2-((3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29- Synthesis of pentaoxo-1,4,5,6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-tetradecahydro-3H-23,27-epoxypyridinyl[2,1-c][1]oxan-3-yl)propyl)-2-methoxycyclohexyl)oxy)-4-oxybutanoic acid.

[0055] Rapamycin (250 mg, 0.273 mmol), dihydrofuran-2,5-dione (125 mg, 1.24 mmol), lipase (molecular weight 35 KD, 1 mg) were added to the reaction bottle, and toluene (15 mL) was added. The reaction solution was heated to 45 ° C and reacted overnight. After the reaction was complete, it was filtered and the filter cake was washed with toluene three times (20 mL * 3). The filtrate was concentrated under reduced pressure and purified to obtain (Compound 1) High resolution mass spectrum: C 65 H 104 N2O 15 Na for[M+Na]+,calculated,1175.7334,found 1175.7360.

[0056] 2)(1R,2R,4S)-4-((R)-2-((3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,27-dihydroxy-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo-1,4,5,6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a- Synthesis of tetrahydro-3H-23,27-epoxypyrido[2,1-c][1]oxa[4]azepan-3-yl)propyl)-2-methoxycyclohexyl 4-(hexadecylamino)-4-(2-(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxycarbonyl)amino)ethyl)amino)-4-oxobutanoate.

[0057] Weigh cholesteryl chloroformate (0.449 g, 1 mmol) and dissolve it in dichloromethane (5 mL). Anhydrous ethylenediamine (0.93 mL, 14 mmol) is dissolved in dichloromethane (5 mL), added dropwise to the cholesteryl chloroformate solution at 0-5 ° C, and stirred overnight. The reaction solution is washed three times with a saturated NaCl solution and once with pure water. The system is layered, the lower layer is dried over anhydrous sodium sulfate, filtered, and the solvent is removed on a rotary evaporator to obtain (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylheptane-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta[a]phenanthrene-3-yl(2-aminoethyl)carbamate (Compound 2).

[0058] Compound 1 and p-dimethylaminopyridine (7.2 mg, 0.3 mmol) were activated in an ice bath for 15 min, then carbodiimide hydrochloride (57 mg, 1.5 mmol) and 1-hydroxybenzotriazole (40 mg, 1.5 mmol) were added and activated in an ice bath for 1 h, then compound 2 was added and reacted for 24 h. The final product was obtained after purification with a yield of (45.5%). High resolution mass spectrum:: C 85. H 133 N3O 17 Nafor[M+Na] + ,calculated,1490.952721,found 1490.952291.

[0059] Example 2

[0060] The above preparation obtains cholesterol rapamycin matching suitable phospholipid

[0061] prescription:

[0062]

[0063] The phospholipids of choice are DOPC, DMPC, DPPC, HSPC, SPC or EPC.

[0064] Preparation method: Dissolve the prescribed amount of membrane material and drug in 1 ml of anhydrous ethanol by ultrasonication as the organic phase, put 5 ml of PBS in a glass bottle as the aqueous phase, slowly and evenly drip the organic phase into the aqueous phase at room temperature (under stirring), and continue stirring for 30 minutes. Use a rotary evaporator at 37 rpm to remove the ethanol to obtain cholesterol-containing rapamycin liposomes.

[0065] The liposomes were diluted 20 times with PBS and the particle size of the liposomes was measured using a Malvern particle size detector. Table 1 shows the particle size, PDI and encapsulation efficiency of cholesterol-containing rapamycin liposomes prepared with different phospholipids:

[0066] Table 1 Investigation of cholesterol-containing rapamycin phospholipids

[0067]

[0068] It can be seen from Table 1 that when the phospholipids are DPPC and HSPC, the liposome particle sizes are 1302nm and 1947nm respectively, which are relatively large, even reaching the micron level, and the PDI is also relatively large. It can be seen that only by matching the appropriate auxiliary agents can the final effect be achieved.

[0069] In summary, when the phospholipids are natural phospholipids (soybean lecithin and egg yolk lecithin), the particle size and encapsulation efficiency of the prepared liposomes are acceptable.

[0070] Embodiment 3:

[0071] The matching formula of cholesterol and phospholipid in the cholesterol-containing rapamycin prepared above is as follows:

[0072]

[0073] The cholesterol to phospholipid mass ratios are 0:30, 1:30, 1:15, 1:7.5, 1:5, and the corresponding cholesterol masses are 0, 1, 2, 4, and 6 mg, respectively.

[0074] Preparation method: Dissolve the prescribed amount of membrane material and drug in 1 ml of anhydrous ethanol by ultrasonication as the organic phase, put 5 ml of PBS in a glass bottle as the aqueous phase, slowly and evenly drip the organic phase into the aqueous phase at room temperature (under stirring), and continue stirring for 30 minutes. Use a rotary evaporator at 37 rpm to remove the ethanol to obtain cholesterol-containing rapamycin liposomes.

[0075] The liposomes were diluted 20 times with PBS and the particle size of the liposomes was measured using a Malvern particle size detector. Table 2 shows the particle size, PDI and encapsulation efficiency of cholesterol-tagged rapamycin liposomes prepared with different cholesterol to phospholipid ratios.

[0076] Table 2 Study on the ratio of cholesterol to phospholipids in cholesteryl rapamycin

[0077]

[0078] It can be seen from Example 3 that the particle size of cholesterol-modified rapamycin liposomes increases with the increase of cholesterol content, and the encapsulation efficiency first increases and then decreases with the increase of cholesterol content. When the ratio of cholesterol to phospholipid is 1:15, the encapsulation efficiency of cholesterol-modified liposomes is higher than that of the same group, which is 94.40%, the particle size is 119.3nm, and the PDI is 0.168.

[0079] Embodiment 4:

[0080] Cholesterolized rapamycin liposome drug-lipid ratio screening and matching

[0081] prescription:

[0082]

[0083] The drug-lipid ratios used were 1:30, 1:15, 1:10, 1:7.5, and 1:5, and the corresponding rapamycin masses were 1, 2, 3, 4, and 6 mg, respectively.

[0084] Preparation method: Dissolve the prescribed amount of membrane material and drug in 1 ml of anhydrous ethanol by ultrasonication as the organic phase, put 5 ml of PBS in a glass bottle as the aqueous phase, slowly and evenly drip the organic phase into the aqueous phase at room temperature (under stirring), and continue stirring for 30 minutes. Use a rotary evaporator at 37 rpm to remove the ethanol to obtain cholesterol-containing rapamycin liposomes.

[0085] The liposomes were diluted 20 times with PBS and the particle size of the liposomes was measured using a Malvern particle size detector. Table 3 shows the particle size, PDI and encapsulation efficiency of cholesterol-containing rapamycin liposomes with different drug-lipid ratios:

[0086] Table 3 Study on the drug-lipid ratio of cholesterol-containing rapamycin

[0087]

[0088] It can be seen from Example 4 that with the increase of the amount of cholesteryl rapamycin, the particle size of cholesteryl rapamycin liposomes gradually increases and the encapsulation rate gradually decreases. When the drug-lipid ratio is 1:15, the encapsulation rate of cholesteryl rapamycin liposomes is higher than that of the same group, which is 95.10%, the particle size is 118.6nm, and the PDI is 0.122.

[0089] Embodiment 5:

[0090] Matching of dosage of cholesteryl rapamycin liposome DSPE-mPEG2000

[0091] prescription:

[0092]

[0093] The ratio of DSPE-mPEG2000 to lipid used was 2%, 5%, 8%, and 10%, and the corresponding DSPE-mPEG2000 masses were 0.7, 1.78, 2.95, and 4 mg, respectively.

[0094] Preparation method: Dissolve the prescribed amount of membrane material and drug in 1 ml of anhydrous ethanol by ultrasonication as the organic phase, put 5 ml of PBS in a glass bottle as the aqueous phase, slowly and evenly drip the organic phase into the aqueous phase at room temperature (under stirring), and continue stirring for 30 minutes. Use a rotary evaporator at 37 rpm to remove the ethanol to obtain cholesterol-containing rapamycin liposomes.

[0095] The liposomes were diluted 20 times with PBS and the particle size of the liposomes was measured using a Malvern particle size detector. Table 4 shows the particle size, PDI and encapsulation efficiency of cholesterol-modified rapamycin liposomes prepared with different concentrations of DSPE-mPEG2000:

[0096] Table 4 Dosage of cholesterol-containing rapamycin liposome DSPE-mPEG2000

[0097]

[0098] Study on the plasma stability of cholesterol-modified rapamycin liposomes at different concentrations of DSPE-mPEG2000:

[0099] Experimental method: add 50 μl liposomes to 500 μl 50% plasma, shake at 37 °C for 4 h, then dilute the liposomes 20 times to measure the particle diameter. Table 5 shows the particle size of DSPE-mPEG2000 free rapamycin liposomes at different concentrations in plasma at 0 h and 4 h, PDI:

[0100] Table 5 Plasma stability of cholesterol-modified rapamycin liposomes after adding DSPE-mPEG2000

[0101]

[0102] Through Example 5, it can be seen that with the addition of DSPE, the particle size of the cholesterol rapamycin liposomes decreases and the encapsulation rate increases. When DSPE is 10%, the encapsulation rate of the cholesterol rapamycin liposomes is high, which is 96.90%, and the particle size is 120nm and PDI is 0.163 respectively; when DSPE is 10%, the particle size of the cholesterol rapamycin liposomes in 37 alcohol plasma at 4h is about 3nm smaller than that at 0h, and there is basically no difference. According to the comprehensive evaluation of encapsulation rate, particle size, plasma stability and other factors of Example 5, the cholesterol rapamycin liposomes have a high encapsulation rate and good stability.

[0103] Embodiment 6:

[0104] Temperature matching for preparation of cholesteryl rapamycin liposomes

[0105] prescription:

[0106]

[0107] Preparation method: Dissolve the prescribed amount of membrane material and drug in 1 ml of anhydrous ethanol by ultrasonication as the organic phase, put 5 ml of PBS in a glass bottle as the aqueous phase, slowly and evenly drip the organic phase into the aqueous phase at 25% and 37% water respectively (under stirring), and continue stirring for 30 minutes. Use a rotary evaporator at 37% to remove the ethanol and obtain cholesterol-containing rapamycin liposomes.

[0108] The liposomes were diluted 20 times with PBS and the particle size of the liposomes was measured using a Malvern particle size detector. Table 6 shows the particle size, PDI and encapsulation efficiency of the cholesterol-containing rapamycin liposomes prepared at different temperatures:

[0109] Table 6 Study on the preparation temperature of cholesterol-containing rapamycin liposomes

[0110]

[0111] It can be seen from Example 6 that when the preparation temperature is 25°C, the cholesterol rapamycin liposome encapsulation efficiency is higher than that of the same group, which is 95.70%, the particle size is 117.1 nm, and the PDI is 0.152.

[0112] Embodiment 7:

[0113] Matching of volume ratio between organic phase and aqueous phase in cholesterol-modified rapamycin liposomes

[0114] prescription:

[0115]

[0116] The volume ratios of organic phase to aqueous phase used were 1:2, 1:5, 1:10, and 1:20, respectively, corresponding to 2, 5, 10, and 20 ml of PBS and 1 ml of anhydrous ethanol for fixation.

[0117] Preparation method: Dissolve the prescribed amount of membrane material and drug in 1 ml of anhydrous ethanol by ultrasonication as the organic phase, put different amounts of PBS in a glass bottle as the aqueous phase, slowly and evenly drip the organic phase into the aqueous phase at room temperature (under stirring), and continue stirring for 30 minutes. Remove the ethanol with a rotary evaporator at 37°C to obtain cholesterol-containing rapamycin liposomes.

[0118] The liposomes were diluted 20 times with PBS and the particle size of the liposomes was measured using a Malvern particle size detector. Table 7 shows the particle size, PDI and encapsulation efficiency of cholesterol-containing rapamycin liposomes with different organic phase and aqueous phase volumes:

[0119] Table 7 Cholesterol Rapamycin Organic Phase Compared with Water

[0120]

[0121] It can be seen from Example 7 that when the ratio of organic phase to water is 1:5, the encapsulation efficiency of cholesterol-containing liposomes is higher than that of the same group, which is 96.82%, the particle size is 125.9 nm, and the PDI is 0.136.

[0122] In summary, the optimal preparation of cholesterol-treated rapamycin liposomes is as follows: 30 mg egg yolk lecithin, 2 mg cholesterol, 4 mg DSPE-mPEG2000, and 2 mg cholesterol-treated rapamycin are completely dissolved in anhydrous ethanol as the organic phase, PBS is used as the aqueous phase, the organic phase is slowly injected into the aqueous phase, stirred at 25°C for 30 minutes, and after removing the ethanol, cholesterol-treated rapamycin liposomes are obtained, with a particle size of 125.9 nm, a PDI of 0.136, and an encapsulation efficiency of 96.82%. Figure 2 This is the appearance diagram of the prepared cholesterol-containing rapamycin liposomes. Figure 3 Figure 2 is a particle size diagram of the prepared cholesterol-containing rapamycin.

[0123] Comparative Example 1

[0124] The optimal formulation for free rapamycin (i.e., rapamycin added as the original drug) liposomes is:

[0125]

[0126] The volume ratio of the organic phase to the aqueous phase was 1:2.

[0127] Preparation method: Dissolve the prescribed amount of membrane material and drug in 1 ml of anhydrous ethanol by ultrasonication as the organic phase, put 2 ml of PBS in a glass bottle as the aqueous phase, slowly and evenly drip the organic phase into the aqueous phase at room temperature (under stirring), and continue stirring for 30 minutes. Remove the ethanol with a rotary evaporator at 37°C to obtain free rapamycin liposomes.

[0128] The liposomes were diluted 20 times with PBS and the particle size of the liposomes was measured by Malvern particle size detector. The particle size of the optimal formulation of free rapamycin liposomes was 109.5nm, the PDI was 0.068, and the encapsulation efficiency was 89.02%. The encapsulation efficiency was 8.05% lower than that of cholesterol rapamycin liposomes.

[0129] The liposomes prepared in the above examples were measured

[0130] Comparison of the 8-day leakage rate of free rapamycin and the cholesterol-modified rapamycin liposomes obtained in Example 7. The prepared liposomes were stored at 4°C and their encapsulation efficiency was measured after 8 days. The corresponding data are shown in Table 8.

[0131] Table 8 Leakage rate of free rapamycin liposomes and cholesterol rapamycin liposomes at 8 days

[0132]

[0133] It can be seen from the above that the stability of cholesterol-treated rapamycin liposomes is much higher than that of free rapamycin liposomes.

[0134] In summary, it can be seen that the encapsulation rate of cholesterol-treated rapamycin liposomes is much higher than that of free rapamycin liposomes, and the stability is also much better than that of free rapamycin liposomes. This is because the coupling of cholesterol and rapamycin indirectly makes the drug firmly bound to phospholipids, thereby improving the encapsulation rate and reducing the leakage rate; it can be seen that free rapamycin is not firmly bound to phospholipids, and the prepared liposomes leak seriously, which is not conducive to the preparation of preparations using it.

[0135] Example 8

[0136] In vivo pharmacodynamic evaluation of cholesterol-containing rapamycin (reducing anti-uricase antibody response)

[0137] The present invention investigates the inhibitory effect of cholesterol-containing rapamycin on anti-uricase antibodies. The experiment was divided into three groups, i cholesterol-containing rapamycin liposome group (RA-C@lipo), ii uricase group (Uricase), iii normal saline group. The experimental period was 6 weeks. In the first three weeks, group i injected RA-C@Lipo and uricase into C57 / BL6 male mice through the tail vein to induce immune tolerance (rapamycin dose of 50μg / mouse; uricase dose of 12.5μg / mouse), group ii was only injected with uricase (12.5μg / mouse), and group iii was only injected with normal saline. In the last three weeks, each group was only injected with uricase (25μg / mouse). During the six-week experiment, mouse serum was collected every week, and the level of anti-uricase antibody LgG in each group was examined using the Elisa detection method ( Figure 4 ). According to the experimental results, RA-C@Lipo weakened the anti-uricase antibody response by inducing immune tolerance and maintained the effectiveness and persistence of uricase.

[0138] Example 9

[0139] Comparison of the in vivo efficacy of free rapamycin and cholesterol-containing rapamycin liposomes (attenuation of anti-uricase antibody response)

[0140] The present invention investigates the comparison of the inhibitory effects of free rapamycin and cholesterol-containing rapamycin liposomes on anti-uricase antibodies. The experiment was divided into two groups, i cholesterol-containing rapamycin liposome group (RA-C@lipo), ii rapamycin group (RAPA). The experimental period was 6 weeks. In the first three weeks, group i injected RA-C@Lipo and uricase into C57 / BL6 male mice through the tail vein to induce immune tolerance (rapamycin dose of 50μg / mouse; uricase dose of 12.5μg / mouse), and group ii injected RAPA and uricase into C57 / BL6 male mice through the tail vein to induce immune tolerance (rapamycin dose of 50μg / mouse; uricase dose of 12.5μg / mouse). In the last three weeks, each group was only injected with uricase (25μg / mouse). During the six-week experiment, mouse serum was collected every week, and the level of anti-uricase antibody IgG in each group was examined using the Elisa detection method ( Figure 5 ). According to the experimental results, free rapamycin could not maintain the inhibitory effect on high-dose uricase injection in the last three weeks; on the contrary, RA-C@lipo could also reduce the level of anti-uricase antibodies under the same high-dose uricase injection, indicating that RA-C@lipo induced immune tolerance, and the effect of anti-drug antibodies was significantly better than free rapamycin. The effectiveness and sustainability of uricase were maintained.

[0141] Example 10

[0142] In vivo pharmacodynamic evaluation of cholesteryl rapamycin (reducing anti-AAV antibody response)

[0143] The present invention investigates the inhibitory effect of cholesterol-modified rapamycin on the capsid-specific antibody of adeno-associated virus (AAV) vector. The experiment was divided into three groups: i cholesterol-modified rapamycin liposome group (RA-C@lipo), ii AAV group, and iii blank group. The experimental period was 42 days. On the first day, group i injected RA-C@Lipo and AAV-luc into C57 / BL6 male mice through the tail vein to induce immune tolerance (rapamycin dose of 50 μg / mouse; AAV dose of 4x10 12 vg / kg, and group ii was injected with AAV-luc (4x10 12 vg / kg), and group iii was not treated. On day 21, group i injected RA-C@Lipo and AAV-SEAP into C57 / BL6 male mice through the tail vein (rapamycin dose was 50 μg / mouse; AAV-SEAP dose was 4x10 12 vg / kg, and group ii was injected with AAV-SEAP (4x10 12vg / kg), and group iii was not treated. During the 42-day experiment, mouse serum was collected weekly, and the level of anti-AAV antibody LgG in each group was examined using the Elisa method. Figure 6 The data results of this experiment were as follows. The serum collected at the last time was tested by Elisa method to investigate the SEAP level ( Figure 7 ). The experimental results demonstrated that RA-C@Lipo significantly reduced the level of anti-AAV antibody LgG and significantly increased the level of SEAP, indicating that RA-C@Lipo can allow AAV vector re-administration and establish long-term transgene expression.

[0144] Embodiment 11:

[0145] Preparation of liposomes by freeze drying

[0146] prescription:

[0147] Egg yolk lecithin 30mg

[0148]

[0149] Preparation method:

[0150] First, 500 mg of lyophilized protective agent was dissolved in 5 ml of HEPES. The prescribed amount of membrane material was ultrasonically dissolved in 1 ml of anhydrous ethanol as the organic phase. 5 ml of HEPES dissolved with lyophilized protective agent was placed in a glass bottle as the aqueous phase. The organic phase was slowly and evenly dripped into the aqueous phase at room temperature (under stirring), and then continued to stir for 30 minutes. The ethanol was removed by rotary evaporator at 37°C to obtain rapa-chol liposomes.

[0151] The prepared liposomes were pre-frozen at -60 for 12 hours, and then freeze-dried for 24 hours to obtain freeze-dried rapa-chol liposomes.

[0152] The particle size of the liposome before freeze-drying was 130.6 nm, and the particle size after freeze-drying and re-dissolving was 142.2 nm; the encapsulation efficiency before freeze-drying was 96.07%, and the encapsulation efficiency after re-dissolving was 94.07%.

[0153] Embodiment 12:

[0154] Preparation of liposomes by freeze drying

[0155] prescription:

[0156]

[0157] Preparation method:

[0158] First, 500 mg of lyophilized protective agent was dissolved in 5 ml of tris. The prescribed amount of membrane material was ultrasonically dissolved in 1 ml of anhydrous ethanol as the organic phase. 5 ml of tris dissolved with lyophilized protective agent was placed in a glass bottle as the aqueous phase. The organic phase was slowly and evenly dripped into the aqueous phase at room temperature (under stirring), and then continued to stir for 30 minutes. The ethanol was removed by rotary evaporator at 37°C to obtain rapa-chol liposomes.

[0159] The prepared liposomes were pre-frozen at -60 for 12 hours, and then freeze-dried for 24 hours to obtain freeze-dried rapa-chol liposomes.

[0160] The particle size of the liposome before freeze-drying was 117.5 nm, and the particle size after freeze-drying and reconstitution was 154.3 nm; the encapsulation efficiency before freeze-drying was 96.85%, and the encapsulation efficiency after reconstitution was 91.70%.

[0161] Embodiment 13:

[0162] prescription:

[0163]

[0164] Preparation method:

[0165] First, dissolve 250 mg sucrose and 250 mg trehalose in 5 ml HEPES. Use 1 ml of anhydrous ethanol to ultrasonically dissolve the prescribed amount of membrane material as the organic phase. Put 5 ml HEPES dissolved with lyophilized protective agent into a glass bottle as the aqueous phase. Slowly and evenly drip the organic phase into the aqueous phase at room temperature (under stirring), and continue stirring for 30 minutes. Use a rotary evaporator at 37°C to remove the ethanol to obtain rapa-chol liposomes.

[0166] The prepared liposomes were pre-frozen at -60 for 12 hours, and then freeze-dried for 24 hours to obtain freeze-dried rapa-chol liposomes.

[0167] The particle size of the liposome before lyophilization was 169.6 nm, and the particle size after reconstitution after lyophilization was 157.0 nm; the encapsulation efficiency was 93.21% before lyophilization, and 95.20% after reconstitution. The cholesterol-containing rapamycin liposome freeze-dried preparation prepared by the method was placed at 4°C for 2 months, and its particle size was 152.8 nm after reconstitution, and the encapsulation efficiency was 91.92%, which was basically unchanged. Figure 8 , Fig. 9 The following are the appearance pictures of cholesterol-treated rapamycin liposomes after freeze-drying and reconstitution. It can be seen that cholesterol-treated rapamycin liposomes can be stored for a long time.

Claims

1. A cholesterol-containing rapamycin liposome, characterized in that: The liposomes are cholesterol-containing rapamycin, phospholipids, and phospholipid PEG derivatives; Among them, cholesterol-containing rapamycin is In the formula: R is cholesterol and its derivatives, L is a connecting arm, wherein L is -C(O)-, -C(O)CH2-, -C(O)CH2CH2-, -C(O)CH2CH2CH2- or -C(O)CH2CH2CH2-.

2. The cholesterol-containing rapamycin liposome according to claim 1, characterized in that: The cholesterol and its derivatives in R are C 28 H 45 ClO2, C 30 H 51 NO2, C 31 H 52 O2、C 31 H 53 O2 or C 34 H 59 NO2.

3. The cholesterol-containing rapamycin liposome according to claim 1, characterized in that: The phospholipids in the liposomes are one or more of phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, phosphatidic acid, cardiolipin, acylcholine, and phosphatidylinositol; wherein the mass ratio of cholesterol-containing rapamycin to phospholipids is 1:30 to 8:

30.

4. A method for preparing liposomes according to claim 1, characterized in that: (a) dissolving cholesterol-modified rapamycin and other lipids in anhydrous ethanol as an organic phase; (b) slowly and uniformly dripping the organic phase into the buffer solution (aqueous phase) under stirring at 25 to 37° C., wherein the volume ratio of the organic phase to the buffer solution is 1:1 to 1:30; (c) removing the organic solvent from the liposome suspension obtained after the above mixing to obtain the final cholesterol-containing rapamycin liposome.

5. The method for preparing liposomes according to claim 4, wherein: The stirring time in the step (b) is 20 to 240 minutes.

6. A use of the liposome according to claim 1, characterized in that: The liposome is used in preparing immunosuppressive and anti-tumor drugs.

7. The use according to claim 6, characterized in that: The drug may be a liposomal formulation.

8. A cholesterol-containing rapamycin liposome preparation, characterized in that: The preparation is cholesterol-containing rapamycin, phospholipids, phospholipid PEG derivatives, membrane stabilizers and buffer in the liposomes of claim 1; The mass ratio of cholesterol-modified rapamycin to phospholipid is 1:30-8:30, the mass ratio of phospholipid PEG derivative to phospholipid is 1:5-1:150, and the mass ratio of membrane stabilizer to phospholipid is 0:30-15:

30.

9. The cholesterol-containing rapamycin liposome preparation according to claim 8, characterized in that: A freeze-dried protective agent is added to the preparation, and the concentration of the freeze-dried protective agent is 1 to 10% w / v; the freeze-dried protective agent is selected from one or more of carbohydrate protective agents, alcohol protective agents, protein protective agents, polymer protective agents, amino acid protective agents, salt protective agents, antioxidants (ascorbic acid) and other small molecule protective agents.

10. The cholesterol-containing rapamycin liposome preparation according to claim 9, characterized in that: The pre-freezing time is 2 to 24 hours, the pre-freezing temperature is -20 to 80° C., and the freeze-drying time is 8 to 48 hours.

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