WO2022122054A1 - 提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂及制备方法 - Google Patents

提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂及制备方法 Download PDF

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WO2022122054A1
WO2022122054A1 PCT/CN2022/074452 CN2022074452W WO2022122054A1 WO 2022122054 A1 WO2022122054 A1 WO 2022122054A1 CN 2022074452 W CN2022074452 W CN 2022074452W WO 2022122054 A1 WO2022122054 A1 WO 2022122054A1
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solution
preparation
tumor
nlc
organic solvent
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French (fr)
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刘志东
祁东利
张兵
李楠
郭盼
皮佳鑫
邓秀平
李佳玮
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天津中医药大学
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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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • A61K47/51—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 non-active ingredient being a modifying agent
    • A61K47/54—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 non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • 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
    • A61K47/51—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 non-active ingredient being a modifying agent
    • A61K47/62—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 non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the technical field of pharmacy, in particular to a nanostructured lipid preparation and a preparation method for improving the active targeting of doxorubicin tumor and kidney protection.
  • the purpose of the present invention is to overcome the shortcomings of poor tumor targeting and high nephrotoxicity in the prior art, and to provide a nanostructured lipid preparation that improves the active targeting of doxorubicin tumors and renal protection.
  • the second object of the present invention is to provide a preparation method of a nanostructured lipid preparation that improves the active tumor targeting and renal protection of adriamycin.
  • a preparation method of a nanostructured lipid preparation for improving active tumor targeting and renal protection of adriamycin comprising the following steps:
  • the first solution and the second solution are combined and stirred to make an oil phase;
  • the mass ratio of salvianolic acid A, doxorubicin hydrochloride, surfactant and water is (1-10):(1-5):(100-500):5000; preferably (2-5):(3 -4):(300-400):5000;
  • the surfactant is preferably at least one of poloxamer, polyoxyethylene fatty acid alcohol ether, polyoxyethylene stearate, sodium cholate, sodium deoxycholate, Mizre 52, Tween 80 and Span 80 .
  • the mass ratio of emulsifier, distearoyl phosphatidylethanolamine-polyethylene glycol-E-[c(RGDfK) 2 ] and the first organic solvent is (20-100):(1-5):1000; preferably The ground is (50-80):(2-3):1000;
  • the emulsifier is soybean lecithin, dipalmitoyl phosphatidyl serine, dioleoyl phosphatidyl serine, dimyristoyl phosphatidyl ethanolamine, distearoyl phosphatidyl ethanolamine, dipalmitoyl phosphatidyl ethanolamine, dioleoyl phosphatidyl ethanolamine , distearoyl phosphatidyl glycerol, dimyristoyl phosphatidyl glycerol, distearoyl phosphatidic acid, dipalmitoyl phosphatidic acid, dilauroyl lecithin, dimerucyl phosphatidyl choline, dioleoyl lecithin, At least one of dimyristoyl lecithin, distearoyl phosphatidyl choline and dipalmitoyl lecithin.
  • the first organic solvent is at least one of absolute ethanol, dimethyl sulfoxide and chloroform.
  • the lipids are stearic acid, glyceryl behenate, caprylic triglyceride, glyceryl monostearate, isopropyl palmitate, glyceryl palmitostearate, glyceryl myristate, isomyristate At least one of propyl ester, glyceryl monopalmitate, glyceryl laurate, caprylic acid capric acid macrogol glyceride, cholesterol, soybean oil and oleic acid; the second organic solvent is absolute ethanol, dimethyl at least one of sulfoxide and chloroform.
  • the volume ratio of the first solution to the second solution is (5-15):1; preferably: (8-10):1.
  • the volume ratio of the oil phase to the water phase is (1-4):5, preferably: (2-3):5.
  • the nanostructured lipid preparation prepared by the above method improves the active targeting of doxorubicin tumor and kidney protection.
  • the nanostructured lipid preparation prepared by the invention for improving the active targeting of doxorubicin tumor and the protection of kidneys has smaller and uniform particle size, strong tumor targeting, enhanced anti-tumor effect and reduced renal toxicity.
  • Fig. 1 is the particle size distribution diagram of E-[c(RGDfK) 2 ]/FA-Sal A/DOX-NLC prepared in Example 1;
  • Figure 3(a) is the biodistribution of E-[c(RGDfK) 2 ]/FA-Cou-6-NLC in tumor-bearing mice;
  • A physiological saline
  • B DiR solution
  • C DiR-NLC
  • D PEG-DiR-NLC
  • E FA-DiR-NLC
  • F E-[c(RGDfK) 2 ]-DiR-NLC
  • G E-[c(RGDfK) 2 ]/FA-DiR-NLC
  • Figure 3(b) is the IVIS images of organs and tumors in different preparation groups
  • A physiological saline
  • B DiR solution
  • C DiR-NLC
  • D PEG-DiR-NLC
  • E FA-DiR-NLC
  • F E-[c(RGDfK) 2 ]-DiR-NLC
  • G E-[c(RGDfK) 2 ]/FA-DiR-NLC
  • Figure 3(c) shows the fluorescence intensity in tumors and organs at 24h
  • Figure 4(b) is a picture of the isolated tumor of each group of mice after 12 days;
  • A physiological saline
  • B Sal A solution
  • C Sal A-NLC
  • D DOX solution
  • E DOX injection
  • F Sal A/DOX solution
  • G DOX-NLC
  • H Sal A/DOX- NLC
  • I E-[c(RGDfK) 2 ]/FA-Sal A/DOX-NLC.
  • Figure 5 shows the pathological results of isolated mouse kidneys after 12 days ( ⁇ 200);
  • A physiological saline
  • B DOX injection
  • C Sal A solution
  • D Sal A-NLC
  • E DOX solution
  • F Sal A/DOX solution
  • G DOX-NLC
  • H Sal A/DOX- NLC
  • I E-[c(RGDfK) 2 ]/FA-Sal A/DOX-NLC.
  • FIG. 6 shows the serum creatinine content of mice in each group after 12 days
  • Distearoylphosphatidylethanolamine-polyethylene glycol-E-[c(RGDfK) 2 ] was synthesized by Qiangyao Biotechnology Co., Ltd.
  • a preparation method of a nanostructured lipid preparation for improving active tumor targeting and renal protection of adriamycin comprising the following steps:
  • the first solution and the second solution are combined and stirred evenly to prepare an oil phase
  • the average molecular weight of polyethylene glycol was 2000.
  • the nanostructured lipid preparation for improving the active targeting of doxorubicin tumor and kidney protection of the present invention is small and uniform in particle size.
  • a preparation method of a nanostructured lipid preparation for improving active tumor targeting and renal protection of adriamycin comprising the following steps:
  • Dioleoylphosphatidylethanolamine and distearoylphosphatidylethanolamine-polyethylene glycol-E-[c(RGDfK) 2 ] were taken in a mass ratio of 20:1:1000 and dissolved in absolute ethanol to prepare into the first solution;
  • the first solution and the second solution are combined and stirred evenly to make an oil phase
  • the particle size of the nanostructured lipid preparation for improving the active tumor targeting and renal protection of adriamycin of the present invention is similar to that of Example 1.
  • the average molecular weight of the polyethylene glycol is 1000.
  • a preparation method of a nanostructured lipid preparation for improving active tumor targeting and renal protection of adriamycin comprising the following steps:
  • Dimyristoyl phosphatidyl glycerol and distearoyl phosphatidyl ethanolamine-polyethylene glycol-E-[c(RGDfK) 2 ] were taken in a mass ratio of 100:5:1000, dissolved in anhydrous ethanol-di In methyl sulfoxide (volume ratio 1:1), the first solution is made;
  • the first solution and the second solution are combined and stirred evenly to make an oil phase
  • the particle size of the nanostructured lipid preparation for improving the active tumor targeting and renal protection of adriamycin of the present invention is similar to that of Example 1.
  • the average molecular weight of the polyethylene glycol is 8000.
  • a preparation method of a nanostructured lipid preparation for improving active tumor targeting and renal protection of adriamycin comprising the following steps:
  • the particle size of the nanostructured lipid preparation for improving the active tumor targeting and renal protection of adriamycin of the present invention is similar to that of Example 1.
  • the average molecular weight of the polyethylene glycol is 5000.
  • surfactant of this embodiment is replaced by polyoxyethylene fatty acid alcohol ether, polyoxyethylene stearate or sodium deoxycholate (surfactant is composed of Tween 80 and Span 80 in a mass ratio of 1:1).
  • surfactant is composed of Tween 80 and Span 80 in a mass ratio of 1:1.
  • a nanostructured lipid preparation with properties and effects similar to this example is prepared to improve the active targeting of doxorubicin tumor and kidney protection.
  • mice breast cancer cells 4T1 cells purchased from Shanghai Cell Bank, Chinese Academy of Sciences
  • coumarin-6 Cou-6
  • the intracellular fluorescence intensity was used to investigate the cellular targeted uptake of the preparation.
  • the preparation methods of each group of medicines are as follows:
  • To form the second solution combine the first solution and the second solution in a ratio of 10:1 by volume to form an oil phase; drop the oil phase into the water phase, remove the organic solvent by heating, and cool it , to obtain a coumarin-6-encapsulated nanostructured lipid preparation (FA-Cou-6-NLC) targeted for modification of folic acid; the volume ratio of oil phase and water phase is 2:5.
  • F-Cou-6-NLC coumarin-6-encapsulated nanostructured lipid preparation
  • Figure 2 and Table 1 show that at 4h and 8h, FA-Cou-6-NLC, E-[c(RGDfK) 2 ]-Cou-6-NLC, E-[c(RGDfK) 2 ]/FA-Cou-6 -
  • the uptake of targeted modified nanoparticles in 4T1 cells in the three NLC groups was greater than that in the unmodified nanoparticle group Cou-6-NLC and Cou-6 solution groups, that is, the uptake of mouse breast cancer cells 4T1 cells to each group Enter size relationship: E-[c(RGDfK) 2 ]/FA-Cou-6-NLC>FA-Cou-6-NLC ⁇ E-[c(RGDfK) 2 ]-Cou-6-NLC>Cou-6- NLC>Cou-6 solution.
  • the nanostructured lipid preparation for improving the active targeting of doxorubicin tumor and renal protection of the present application has a very good ability of tumor cell targeting and uptake.
  • DiR(1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide, 1,1-octadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide, Iodide (DiR), purchased from AAT Bioquest Company) is a fluorescent molecular probe, and the probe is loaded into the nanostructured lipid preparation (prepared in Example 1) that improves the active targeting of doxorubicin tumor and kidney protection (prepared in Example 1), and the Tumor 4T1 cells were injected into the tail vein of nude mice, and the IVIS small animal in vivo imaging system was used to investigate the nanostructured lipid preparations that improve the active tumor targeting and renal protection of doxorubicin in BALB/c nude mice bearing tumor 4T1 cells.
  • the tumor targeting and organ distribution were determined.
  • One nude mouse in each group was sacrificed 24 hours after administration by tail vein injection.
  • the fluorescence intensity in the tumor is used to study the distribution of the preparation in the internal organs.
  • the preparation methods of each group of medicines are as follows:
  • Normal saline purchased from Shijiazhuang No. 4 Pharmaceutical Factory.
  • DiR and Maize 52 were taken in a mass ratio of 8:300:5000 and dissolved in deionized water to prepare a DiR solution.
  • the volume ratio of oil phase and water phase was 2:5.
  • the tumor-bearing nude mice were randomly divided into 7 groups, with 6 mice in each group, corresponding to the saline group, the DiR solution group, the DiR-encapsulated nanostructured lipid preparation (DiR-NLC), and the PEG-modified DiR-encapsulated nanostructures, respectively.
  • Lipid formulation PEG-DiR-NLC
  • E-[c(RGDfK) 2 ]-modified DiR-encapsulated nanostructured lipid formulation E-[c(RGDfK) 2 ]-DiR-NLC
  • folic acid-modified DiR-encapsulated nanostructured lipid formulations F-DiR-NLC
  • E-[c(RGDfK) 2 ] and folic acid-modified DiR-encapsulated nanostructured lipid formulations E-[c(RGDfK) 2 ]/FA -DiR-NLC, administered by tail vein injection at a dose of 200 ⁇ L/mouse.
  • Nude mice were anesthetized with isoflurane at five time points, 1h, 4h, 8h, 24h, and 36h after administration, respectively, and then the tumor-bearing nude mice were placed in the in vivo imaging system for observation, and pictures were collected. 24 hours after drug injection, the tumor-bearing nude mice were sacrificed, dissected, and the heart, liver, spleen, lung, kidney, tumor and other tissues were taken separately, and placed in a small animal in vivo imaging system for imaging to determine the active target of doxorubicin in tumors. Distribution of tropic and renoprotective nanostructured lipid formulations in various tissues, organs and tumors.
  • the PEG-DiR-NLC group showed stronger fluorescence at tumor sites than the DiR solution group over time, indicating that the drug could be continuously delivered to the tumor site by the PEG nanostructured lipid preparation through long-term circulation.
  • E-[c(RGDfK) 2 ]/FA-DiR-NLC group had stronger tumor sites than FA-DiR-NLC group and E-[c(RGDfK) 2 ]-DiR-NLC group over time Fluorescence, indicating that the drug can be continuously delivered to the tumor site through active targeting of folic acid, E-[c(RGDfK) 2 ] dual ligand, and has stronger tumor targeting in vivo than single tumor targeting modification.
  • the anti-in vivo anti-tumor effect of the nanostructured lipid preparation that improves the active tumor targeting and renal protection of doxorubicin was investigated.
  • the anti-tumor effect of the drug was verified by examining the cell proliferation and apoptosis in the tumor site.
  • the tumor-bearing mice were randomly divided into 9 groups.
  • the specific groupings are as follows:
  • Salvianolic acid A solution group (Sal A solution);
  • Nanostructured lipid formulation group (Sal A-NLC) encapsulated with salvianolic acid A;
  • Doxorubicin hydrochloride solution group (DOX solution);
  • Salvianolic acid A and doxorubicin hydrochloride solution group (Sal A/DOX solution);
  • Nanostructured lipid formulation group (DOX-NLC) encapsulated with doxorubicin hydrochloride;
  • Nanostructured lipid formulation group encapsulated with doxorubicin hydrochloride and salvianolic acid A (Sal A/DOX-NLC);
  • the preparation method of each group is as follows:
  • Normal saline group purchased from Shijiazhuang No. 4 Pharmaceutical Factory.
  • doxorubicin hydrochloride injection purchased from CSPC Group Ouyi Pharmaceutical Co., Ltd.
  • salvianolic acid A solution (Sal A solution): take salvianolic acid A and Meize 52 in a mass ratio of 5:300:5000, dissolve in deionized water, and prepare a salvianolic acid A solution.
  • Preparation of the nanostructured lipid preparation (Sal A-NLC) encapsulated with salvianolic acid A take salvianolic acid A and Maize 52 in a mass ratio of 5:300:5000, dissolve in deionized water, and prepare Aqueous phase; take soybean lecithin in a ratio of 50:1000 by mass, dissolve in absolute ethanol, and make a first solution; take glyceryl behenate in a ratio of 30:500 by mass, dissolve in chloroform , make the second solution: according to the volume ratio of 10:1, the first solution and the second solution are combined and stirred to make an oil phase; the oil phase is dropped into the water phase, and then the organic solvent is removed by heating , cooled to obtain a nanostructured lipid preparation (Sal A-NLC) encapsulated with salvianolic acid A; the volume ratio of oil phase and water phase is 2:5.
  • DOX solution doxorubicin hydrochloride solution
  • nanostructured lipid preparation (DOX-NLC) encapsulated with doxorubicin hydrochloride: take doxorubicin hydrochloride and Maize 52 in a ratio of 3:300:5000 by mass, dissolve in deionized water, and prepare water Phase; take soybean lecithin in a ratio of 50:3:1000 by mass, dissolve in absolute ethanol, and make a first solution; take glyceryl behenate in a ratio of 30:500 by mass, dissolve in chloroform , make the second solution: according to the volume ratio of 10:1, combine the first solution and the second solution and stir evenly to make an oil phase; drop the oil phase into the water phase, then heat to remove the organic Solvent, cooling, to obtain a nanostructured lipid preparation (DOX-NLC) loaded with doxorubicin hydrochloride; the volume ratio of oil phase and water phase is 2:5.
  • the administration dose was calculated according to body weight. About 200 ⁇ L of the solution or preparation was injected into the tail vein of the mice in each group, and the same volume of normal saline was injected in the control group. 1 injection every 2 days for a total of 6 doses.
  • the tumor-bearing mice were sacrificed the next day after the sixth administration, and the subcutaneously transplanted tumor and the cell membrane were completely removed from the tumor tissue as shown in Figure 4(b).
  • the tumor tissue volume was accurately weighed, and the relative tumor volume was calculated by the ratio of the experimental group tumor volume to the average tumor volume of the control group ( Figure 4(c)).
  • the tumor tissue weight was precisely weighed, and the average tumor weight of the experimental group and the control group was calculated The ratio of values was used to calculate relative tumor mass (Fig. 4(d)).
  • One-way analysis of variance was performed on the data using SPSS 22 software.
  • Tumor inhibitory potential was determined by measuring changes in relative tumor volume and relative tumor mass after administration.
  • the relative tumor volume and relative tumor mass of the DOX-NLC group were significantly lower than those of the DOX solution group (p ⁇ 0.01), indicating that the nanostructured lipid preparation can improve the effective accumulation of the drug at the tumor site and improve the bioavailability of the drug , to improve the anti-tumor effect of the drug in vivo.
  • the relative tumor volume and relative tumor mass of the Sal A/DOX-NLC group were decreased compared with the DOX-NLC group, and the relative tumor volume and relative tumor mass of the Sal A/DOX solution group were decreased compared with the DOX solution group.
  • the tumor volume inhibition rate and tumor weight inhibition rate also increased, indicating that Sal A can improve the anti-tumor effect of DOX.
  • the relative tumor volume and relative tumor mass of the E-[c(RGDfK) 2 ]/FA-Sal A/DOX-NLC group were significantly different from those of the positive drug group (DOX injection group) and DOX solution group (p ⁇ 0.01), and compared with the Sal A/DOX-NLC group, both the relative tumor volume and the relative tumor mass decreased, indicating that the E-[c(RGDfK) 2 ]/FA targeting modified nanostructured lipid preparations had a significant effect. The best anti-tumor effect.
  • the DOX injection group, the DOX solution group, and the DOX-NLC group exhibited renal lesions.
  • the renal tubular structure is disordered and swollen; glomerulosclerosis appears pyknosis, and the basement membrane thickens; inflammatory cell infiltration occurs, and the damage is more serious.
  • E-[c(RGDfK) 2 ]/FA-Sal A/DOX-NLC group (Example 1) had no obvious renal lesions, and the glomeruli and tubules were normal in shape. No obvious inflammation, lesion area was found, E-[c(RGDfK) 2 ]/FA-Sal A/DOX-NLC (Example 1) had no nephrotoxicity, indicating that E-[c(RGDfK) 2 ]/FA dual ligand The modified E-[c(RGDfK) 2 ]/FA-Sal A/DOX-NLC could significantly reduce the renal toxicity of DOX.
  • the circulating amount of free creatinine in serum is completely dependent on its excretion rate, so the amount of creatinine in serum can be measured, which can be used for the examination of renal function.
  • Increased creatinine levels are seen in: decreased excretion in chronic renal failure and in acromegaly.
  • Creatinine levels can be used to assess glomerular filtration rate to determine renal functional status. Studies have shown that doxorubicin can lead to the occurrence of nephrotic syndrome, resulting in elevated serum creatinine levels.

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Abstract

一种提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂及制备方法，制备方法为：取丹酚酸A、盐酸阿霉素和表面活性剂，溶于去离子水中，制成水相；取乳化剂和二硬脂酰基磷脂酰乙醇胺-聚乙二醇-E-[c(RGDfK)2]，溶于第一种有机溶剂中，制成第一溶液；取脂质和二硬脂酰基磷脂酰乙醇胺-聚乙二醇-叶酸，溶于第二种有机溶剂中，制成第二溶液；将第一溶液和第二溶液合并搅拌均匀，制成油相；将油相滴入到水相中，然后，加热去掉有机溶剂，冷却，即得。纳米结构脂质制剂粒径较小且均一，肿瘤靶向性强，增强抗肿瘤效果，肾毒性降低。

Description

提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂及制备方法 技术领域
本发明涉及制药技术领域，尤其涉及一种提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂及制备方法。
背景技术
恶性肿瘤已经成为当今世界威胁人类健康和导致人类死亡最严重的疾病之一，预测未来的十年中肿瘤患者总数仍会持续增长。盐酸阿霉素是现阶段临床医疗中广泛使用的抗肿瘤药物，具有高效广谱抗癌作用，作用机制是通过插入DNA碱基之间形成不规则键、抑制DNA合成关键酶(拓扑异构酶I和II)的活性及产生的自由基破坏DNA的形成有关，对于各个生长周期的肿瘤细胞均具有杀伤作用，广泛应用于多种恶性肿瘤。但是，越来越多的临床研究表明，盐酸阿霉素用药存在靶向性差、毒副作用大等问题，对心脏、肝脏和肾脏的正常细胞和组织有严重损伤，为肿瘤治疗增加了难度。因此，如何显著提高盐酸阿霉素的肿瘤靶向性和肿瘤抑制率，同时降低其毒副作用，成为肿瘤患者化疗疗效及预后的关键。
发明内容
本发明目的在于克服现有技术存在的肿瘤靶向性差和肾毒性大的不足，提供一种提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂。
本发明的第二个目的是提供一种提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂的制备方法。
本发明的技术方案概述如下：
提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂的制备方法，包括如下步骤：
(1)取丹酚酸A、盐酸阿霉素和表面活性剂，溶于去离子水中，制成水相；
取乳化剂和二硬脂酰基磷脂酰乙醇胺-聚乙二醇-E-[c(RGDfK) 2]，溶于第一种有机溶剂中，制成第一溶液；
取脂质和二硬脂酰基磷脂酰乙醇胺-聚乙二醇-叶酸，溶于第二种有机溶剂中，制成第二溶液：
将第一溶液和第二溶液合并搅拌均匀，制成油相；
(2)将油相滴入到水相中，然后，加热去掉有机溶剂，冷却，得到提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂；所述聚乙二醇的平均分子量为1000-8000，优选地选2000-5000。
丹酚酸A、盐酸阿霉素、表面活性剂和水的质量比为(1-10):(1-5):(100-500):5000； 优选地是(2-5):(3-4):(300-400):5000；
表面活性剂优选为泊洛沙姆、聚氧乙烯脂肪酸醇醚、聚氧乙烯硬脂酸酯、胆酸钠、脱氧胆酸钠、卖泽52、吐温80和司盘80中的至少一种。
乳化剂、二硬脂酰基磷脂酰乙醇胺-聚乙二醇-E-[c(RGDfK) 2]和第一种有机溶剂的质量比为(20-100):(1-5):1000；优选地是(50-80):(2-3):1000；
乳化剂为大豆卵磷脂、二棕榈酰磷脂酰丝氨酸、二油酰基磷脂酰丝氨酸、二肉豆蔻酰磷脂酰乙醇胺、二硬脂酰基磷脂酰乙醇胺、二棕榈酰基磷脂酰乙醇胺、二油酰磷脂酰乙醇胺、二硬脂酰磷脂酰甘油、二肉豆蔻酰磷脂酰甘油、二硬脂酰磷脂酸、二棕榈酰磷脂酸、二月桂酰基卵磷脂、二芥酰磷脂酰胆碱、二油酰基卵磷脂、二肉豆蔻酰基卵磷脂、二硬脂酰基磷脂酰胆碱和二棕榈酰基卵磷脂中至少一种。
所述第一种有机溶剂为无水乙醇、二甲基亚砜和氯仿中至少一种。
脂质、二硬脂酰基磷脂酰乙醇胺-聚乙二醇1000-8000-叶酸和第二种有机溶剂的质量比例为(10-50):(1-5):500，优选地是：(20-30):(2-3):500。
脂质为硬脂酸、山嵛酸甘油酯、辛酸癸酸甘油三酯、单硬脂酸甘油酯、棕榈酸异丙酯、甘油棕榈酸硬脂酸酯、豆蔻酸甘油酯、肉豆蔻酸异丙酯、单棕榈酸甘油酯、月桂酸甘油酯、辛酸癸酸聚乙二醇甘油酯、胆固醇、大豆油和油酸中至少一种；所述第二种有机溶剂为无水乙醇、二甲基亚砜和氯仿中至少一种。
第一溶液和第二溶液的体积比为(5-15):1；优选的是：(8-10):1。
所述油相与水相的体积比为(1-4):5，优选的是：(2-3):5。
上述方法制备的提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂。
本发明的优点：
本发明制备的提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂粒径较小且均一，肿瘤靶向性强，增强抗肿瘤效果，肾毒性降低。
附图说明
图1为实施例1制备的E-[c(RGDfK) 2]/FA-Sal A/DOX-NLC的粒径分布图；
图2为各组香豆素-6制剂在4T1细胞内的摄入荧光定量柱状图(mean±SD，n＝6)；
注：*0.01<p<0.05；**0.001<p<0.01；***p<0.001。
图3(a)为E-[c(RGDfK) 2]/FA-Cou-6-NLC在荷瘤小鼠中的生物分布；
(A：生理盐水；B：DiR溶液；C：DiR-NLC；D：PEG-DiR-NLC；E：FA-DiR-NLC；F：E-[c(RGDfK) 2]-DiR-NLC；G：E-[c(RGDfK) 2]/FA-DiR-NLC)
图3(b)为不同制剂组的器官和肿瘤的IVIS图像；
(A：生理盐水；B：DiR溶液；C：DiR-NLC；D：PEG-DiR-NLC；E：FA-DiR-NLC；F：E-[c(RGDfK) 2]-DiR-NLC；G：E-[c(RGDfK) 2]/FA-DiR-NLC)
图3(c)为24h时肿瘤和脏器中的荧光强度；
注：**0.001﹤p﹤0.01vs DiR-NLC；***p﹤0.001vs DiR-NLC； △p﹤0.05vs PEG-DiR-NLC； ##0.001﹤p﹤0.01vs FA-DiR-NLC； ○p﹤0.05vs E-[c(RGDfK) 2]-DiR-NLC。
图4(a)为荷瘤小鼠模型在治疗过程(第2-12天)的相对肿瘤体积变化(mean±SD，n＝6)；
图4(b)为12天后各组小鼠离体肿瘤图片；
A：生理盐水；B：Sal A溶液；C：Sal A-NLC；D：DOX溶液；E：DOX注射液；F：Sal A/DOX溶液；G：DOX-NLC；H：Sal A/DOX-NLC；I：E-[c(RGDfK) 2]/FA-Sal A/DOX-NLC。
图4(c)为12天后各组小鼠离体肿瘤相对肿瘤体积(mean±SD，n＝6)；
注：**0.001﹤p﹤0.01vs DOX注射液；***p﹤0.001vs DOX注射液； △△0.001﹤p﹤0.01vs DOX溶液； △△△p﹤0.001vs DOX溶液。
图4(d)为12天后各组小鼠离体肿瘤相对肿瘤质量(mean±SD，n＝6)
注：***p﹤0.001vs DOX注射液； △△△p﹤0.001vs DOX溶液； ☆☆0.001﹤p﹤0.01vs DOX-NLC。
图5为12天后小鼠离体肾脏病理学结果(×200)；
A：生理盐水；B：DOX注射液；C：Sal A溶液；D：Sal A-NLC；E：DOX溶液；F：Sal A/DOX溶液；G：DOX-NLC；H：Sal A/DOX-NLC；I：E-[c(RGDfK) 2]/FA-Sal A/DOX-NLC。
图6为12天后各组小鼠血清中肌酐的含量
注： △△△p﹤0.001vs DOX溶液； ☆☆☆p﹤0.001vs DOX-NLC； ###p﹤0.001vs Sal A/DOX-NLC。
具体实施方式
下面结合实施例，对本发明进行进一步的详细说明，但并不意于限制本发明的保护范围。
二硬脂酰基磷脂酰乙醇胺-聚乙二醇-E-[c(RGDfK) 2]委托强耀生物科技有限公司合成，其中的E-[c(RGDfK) 2]的氨基酸序列：H-Glu[cyclo(Arg-Gly-Asp-d-Phe-Lys)] 2。
实施例1
提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂的制备方法，包括如下步骤：
(1)按质量比为5:3:300:5000的比例取丹酚酸A、盐酸阿霉素和卖泽52，溶于去离子水中，制成水相；
按质量比为50:3:1000的比例取大豆卵磷脂和二硬脂酰基磷脂酰乙醇胺-聚乙二醇-E-[c(RGDfK) 2]，溶于无水乙醇中，制成第一溶液；
按质量比为30:3:500的比例取山嵛酸甘油酯和二硬脂酰基磷脂酰乙醇胺-聚乙二醇-叶酸，溶于氯仿中，制成第二溶液：
按体积比为10:1的比例，将第一溶液和第二溶液合并搅拌均匀，制成油相；
(2)将油相滴入到水相中，然后，加热去掉有机溶剂，冷却，得到提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂(E-[c(RGDfK) 2]/FA-Sal A/DOX-NLC)；油相和水相的体积比为2:5。
聚乙二醇的平均分子量为2000。
由图1可以看出，本发明的提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂粒径较小且均一。
实施例2
提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂的制备方法，包括如下步骤：
(1)按质量比为1:1:100:5000的比例取丹酚酸A、盐酸阿霉素和泊洛沙姆，溶于去离子水中，制成水相；
按质量比为20:1:1000的比例取二油酰磷脂酰乙醇胺和二硬脂酰基磷脂酰乙醇胺-聚乙二醇-E-[c(RGDfK) 2]，溶于无水乙醇中，制成第一溶液；
按质量比为10:1:500的比例取棕榈酸异丙酯和二硬脂酰基磷脂酰乙醇胺-聚乙二醇-叶酸，溶于氯仿-二甲基亚砜(体积比1:1)中，制成第二溶液：
按体积比为5:1的比例，将第一溶液和第二溶液合并搅拌均匀，制成油相；
(2)将油相滴入到水相中，然后，加热去掉有机溶剂，冷却，得到提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂；油相和水相的体积比为1:5。
经检测，本发明的提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂粒径与实施例1相似。
所述聚乙二醇的平均分子量为1000。
实施例3
提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂的制备方法，包括如下步骤：
(1)按质量比为10:5:500:5000的比例取丹酚酸A、盐酸阿霉素和胆酸钠，溶于去离子水中，制成水相；
按质量比为100:5:1000的比例取二肉豆蔻酰磷脂酰甘油和二硬脂酰基磷脂酰乙醇胺-聚乙二醇-E-[c(RGDfK) 2]，溶于无水乙醇-二甲基亚砜(体积比1:1)中，制成第一溶液；
按质量比为50:5:500的比例取月桂酸甘油酯和二硬脂酰基磷脂酰乙醇胺-聚乙二醇-叶酸， 溶于二甲基亚砜中，制成第二溶液：
按体积比为15:1的比例，将第一溶液和第二溶液合并搅拌均匀，制成油相；
(2)将油相滴入到水相中，然后，加热去掉有机溶剂，冷却，得到提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂；油相和水相的体积比为4:5。
经检测，本发明的提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂粒径与实施例1相似。
所述聚乙二醇的平均分子量为8000。
实施例4
提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂的制备方法，包括如下步骤：
(1)按质量比为2:4:400:5000的比例取丹酚酸A、盐酸阿霉素和表面活性剂(表面活性剂由质量比为1：1吐温80和司盘80组成)，溶于去离子水中，制成水相；
按质量比为80:2:1000的比例取二肉豆蔻酰基卵磷脂和二硬脂酰基磷脂酰乙醇胺-聚乙二醇-E-[c(RGDfK) 2]，溶于无水乙醇-氯仿(体积比1:1)中，制成第一溶液；
按质量比为20:2:500的比例取油酸和二硬脂酰基磷脂酰乙醇胺-聚乙二醇-叶酸，溶于无水乙醇中，制成第二溶液：
按体积比为8:1的比例，将第一溶液和第二溶液合并搅拌均匀，制成油相；
(2)将油相滴入到水相中，然后，加热去掉有机溶剂，冷却，得到提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂；油相和水相的体积比为3:5。
经检测，本发明的提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂粒径与实施例1相似。
所述聚乙二醇的平均分子量为5000。
实验证明，用聚氧乙烯脂肪酸醇醚、聚氧乙烯硬脂酸酯或脱氧胆酸钠替代本实施例的表面活性剂(表面活性剂由质量比为1：1吐温80和司盘80组成)，其它同本实施例，制备出性质、作用与本实施例相似的提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂。
实验证明，用二棕榈酰磷脂酰丝氨酸、二油酰基磷脂酰丝氨酸、二肉豆蔻酰磷脂酰乙醇胺、二硬脂酰基磷脂酰乙醇胺、二棕榈酰基磷脂酰乙醇胺、二硬脂酰磷脂酰甘油、二硬脂酰磷脂酸、二棕榈酰磷脂酸、二月桂酰基卵磷脂、二芥酰磷脂酰胆碱、二油酰基卵磷脂、二硬脂酰基磷脂酰胆碱和按质量比1：1的二棕榈酰基卵磷脂和二油酰基卵磷脂组成的混合物替代本实施例中的二肉豆蔻酰基卵磷脂，其它同本实施例，制备出性质、作用与本实施例相似的提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂。
实验证明，用硬脂酸、辛酸癸酸甘油三酯、单硬脂酸甘油酯、甘油棕榈酸硬脂酸酯、豆蔻酸甘油酯、肉豆蔻酸异丙酯、单棕榈酸甘油酯、辛酸癸酸聚乙二醇甘油酯、胆固醇、大豆油和质量比为1：1的大豆油与油酸组成的混合物替代本实施例中的油酸，其它同本实施例，制备出性质、作用与本实施例相似的提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂。
为了进一步了解药物，发明人还对本发明的提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂(实施例1制备的样品)进行了细胞摄入试验、肿瘤靶向性研究、抗肿瘤活性研究和肾脏保护作用研究，方法和结果如下：
1.体外细胞靶向摄入试验
本试验用小鼠乳腺癌细胞4T1细胞(购买自中国科学院上海细胞库)为试验对象，以香豆素-6(Cou-6)为荧光探针，测定包载香豆素-6的不同制剂在细胞内的荧光强度，考察制剂的细胞靶向摄入情况。各组药物制备方法如下：
香豆素-6溶液(Cou-6溶液)的制备：
按质量比为8:300:5000的比例取香豆素-6和卖泽52，溶于去离子水中，制成香豆素-6溶液(Cou-6溶液)。
包载香豆素-6的纳米结构脂质制剂(Cou-6-NLC)的制备：
按质量比为8:300:5000的比例取香豆素-6和卖泽52，溶于去离子水中，制成水相；按质量比为50:1000的比例取大豆卵磷脂，溶于无水乙醇中，制成第一溶液；按质量比为30:500的比例取山嵛酸甘油酯，溶于氯仿中，制成第二溶液：按体积比为10:1的比例，将第一溶液和第二溶液合并搅拌均匀，制成油相；将油相滴入到水相中，然后，加热去掉有机溶剂，冷却，得到包载香豆素-6的纳米结构脂质制剂(Cou-6-NLC)；油相和水相的体积比为2:5。
叶酸靶向修饰的包载香豆素-6的纳米结构脂质制剂(FA-Cou-6-NLC)的制备
按质量比为8:300:5000的比例取香豆素-6和卖泽52，溶于去离子水中，制成水相；按质量比为50:1000的比例取大豆卵磷脂，溶于无水乙醇中，制成第一溶液；按质量比为30:3:500的比例取山嵛酸甘油酯和二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000-叶酸，溶于氯仿中，制成第二溶液：按体积比为10:1的比例，将第一溶液和第二溶液合并搅拌均匀，制成油相；将油相滴入到水相中，然后，加热去掉有机溶剂，冷却，得到叶酸靶向修饰的包载香豆素-6的纳米结构脂质制剂(FA-Cou-6-NLC)；油相和水相的体积比为2:5。
E-[c(RGDfK) 2]靶向修饰的包载香豆素-6的纳米结构脂质制剂(E-[c(RGDfK) 2]-Cou-6-NLC)的制备：
按质量比为8:300:5000的比例取香豆素-6和卖泽52，溶于去离子水中，制成水相；按质量比为50:3:1000的比例取大豆卵磷脂和二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000-E-[c(RGDfK) 2]，溶于无水乙醇中，制成第一溶液；按质量比为30:500的比例取山嵛酸甘油酯，溶于氯仿中，制成第二溶液：按体积比为10:1的比例，将第一溶液和第二溶液合并搅拌均匀，制成油相；将油相滴入到水相中，然后，加热去掉有机溶剂，冷却，得到E-[c(RGDfK) 2]靶向修饰的包载香豆素-6的纳米结构脂质制剂(E-[c(RGDfK) 2]-Cou-6-NLC)；油相和水相的体积比为2:5。
E-[c(RGDfK) 2和叶酸靶向修饰的包载香豆素-6的纳米结构脂质制剂(E-[c(RGDfK) 2]/FA-Cou-6-NLC)的制备：
按质量比为8:300:5000的比例取香豆素-6和卖泽52，溶于去离子水中，制成水相；按质量比为50:3:1000的比例取大豆卵磷脂和二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000-E-[c(RGDfK) 2]，溶于无水乙醇中，制成第一溶液；按质量比为30:3:500的比例取山嵛酸甘油酯和二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000-叶酸，溶于氯仿中，制成第二溶液：按体积比为10:1的比例，将第一溶液和第二溶液合并搅拌均匀，制成油相；将油相滴入到水相中，然后，加热去掉有机溶剂，冷却，得到(E-[c(RGDfK) 2]/FA-Cou-6-NLC)；油相和水相的体积比为2:5。
将Cou-6溶液、Cou-6-NLC、FA-Cou-6-NLC、E-[c(RGDfK) 2]-Cou-6-NLC、E-[c(RGDfK) 2]/FA-Cou-6-NLC，用DMEM基础培养基(gibico公司)稀释1000倍，按照每孔100μL加入到4T1细胞中(n＝3)，5％CO 2、37℃孵育4h和8h。将孔中溶液吸出，用常温的PBS缓冲液(pH＝7.4)清洗细胞2遍，随后每孔加入100μL对应的DMEM基础培养基(gibico公司)，放入高内涵细胞成像分析系统(Operetta TM，Perkin Elmer公司，美国)中对细胞摄取进行定性定量分析。
图2和表1可知在4h和8h，FA-Cou-6-NLC、E-[c(RGDfK) 2]-Cou-6-NLC、E-[c(RGDfK) 2]/FA-Cou-6-NLC三组靶向修饰的纳米粒在4T1细胞的摄入量均大于未修饰的纳米粒组Cou-6-NLC和Cou-6溶液组，即小鼠乳腺癌细胞4T1细胞对各组的摄入大小关系：E-[c(RGDfK) 2]/FA-Cou-6-NLC>FA-Cou-6-NLC≈E-[c(RGDfK) 2]-Cou-6-NLC>Cou-6-NLC>Cou-6溶液。由此可见，本申请的提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂，具有非常好的肿瘤细胞靶向摄入能力。
表1小鼠乳腺癌细胞4T1细胞对不同制剂的细胞摄入的荧光强度(mean±SD，n＝6)
Figure PCTCN2022074452-appb-000001
2.体内肿瘤靶向性研究
本研究以DiR(1,1‘-dioctadecyl-3,3,3’,3‘-tetramethylindotricarbocyanine iodide，1,1-十八烷基-3,3,3,3-四甲基吲哚三碳菁碘化物(DiR)，购买自AAT Bioquest公司)为荧光分子探针，将探针载入提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂(实施例1制备)中，荷瘤4T1细胞裸鼠尾静脉注射给药，采用IVIS小动物活体成像系统，考察提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂在荷瘤4T1细胞的BALB/c裸鼠体内的肿瘤靶向与脏器分布情况，于尾静脉注射给药24h后每组处死1只裸鼠，将心、肝、脾、肺、肾以及肿瘤取出，在活体成像仪内观察各脏器和肿瘤中的荧光强度，研究制剂在体内脏器的分布情况。以评价提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂在体内的肿瘤靶向性以及在其他器官的分布。各组药物制备方法如下：
生理盐水：购买自石家庄第四制药厂。
DiR溶液的制备：
按质量比为8:300:5000的比例取DiR和卖泽52，溶于去离子水中，制成DiR溶液。
包载DiR的纳米结构脂质制剂(DiR-NLC)的制备：
按质量比为8:300:5000的比例取DiR和卖泽52，溶于去离子水中，制成水相；按质量比为50:1000的比例取大豆卵磷脂，溶于无水乙醇中，制成第一溶液；按质量比为30:500的比例取山嵛酸甘油酯，溶于氯仿中，制成第二溶液：按体积比为10:1的比例，将第一溶液和第二溶液合并搅拌均匀，制成油相；将油相滴入到水相中，然后，加热去掉有机溶剂，冷却，得到包载DiR的纳米结构脂质制剂(DiR-NLC)；油相和水相的体积比为2:5。
PEG修饰的包载DiR的纳米结构脂质制剂(PEG-DiR-NLC)的制备：
按质量比为8:300:5000的比例取DiR和卖泽52，溶于去离子水中，制成水相；按质量比为50:3:1000的比例取大豆卵磷脂和二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000，溶于无水乙醇中，制成第一溶液；按质量比为30:500的比例取山嵛酸甘油酯，溶于氯仿中，制成第二溶液：按体积比为10:1的比例，将第一溶液和第二溶液合并搅拌均匀，制成油相；将油相滴入 到水相中，然后，加热去掉有机溶剂，冷却，得到PEG修饰的包载DiR的纳米结构脂质制剂(PEG-DiR-NLC)；油相和水相的体积比为2:5。
E-[c(RGDfK) 2]修饰的包载DiR的纳米结构脂质制剂(E-[c(RGDfK) 2]-DiR-NLC)的制备：
按质量比为8:300:5000的比例取DiR和卖泽52，溶于去离子水中，制成水相；按质量比为50:3:1000的比例取大豆卵磷脂和二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000-E-[c(RGDfK) 2]，溶于无水乙醇中，制成第一溶液；按质量比为30:500的比例取山嵛酸甘油酯，溶于氯仿中，制成第二溶液：按体积比为10:1的比例，将第一溶液和第二溶液合并搅拌均匀，制成油相；将油相滴入到水相中，然后，加热去掉有机溶剂，冷却，得到E-[c(RGDfK) 2]修饰的包载DiR的纳米结构脂质制剂(E-[c(RGDfK) 2]-DiR-NLC)；油相和水相的体积比为2:5。
叶酸修饰的包载DiR的纳米结构脂质制剂(FA-DiR-NLC)的制备：
按质量比为8:300:5000的比例取DiR和卖泽52，溶于去离子水中，制成水相；按质量比为50:1000的比例取大豆卵磷脂，溶于无水乙醇中，制成第一溶液；按质量比为30:3:500的比例取山嵛酸甘油酯和二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000-叶酸，溶于氯仿中，制成第二溶液：按体积比为10:1的比例，将第一溶液和第二溶液合并搅拌均匀，制成油相；将油相滴入到水相中，然后，加热去掉有机溶剂，冷却，得到叶酸修饰的包载DiR的纳米结构脂质制剂(FA-DiR-NLC)；油相和水相的体积比为2:5。
E-[c(RGDfK) 2]和叶酸修饰的包载DiR的纳米结构脂质制剂E-[c(RGDfK) 2]/FA-DiR-NLC的制备：
按质量比为8:300:5000的比例取DiR和卖泽52，溶于去离子水中，制成水相；按质量比为50:3:1000的比例取大豆卵磷脂和二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000-E-[c(RGDfK) 2]，溶于无水乙醇中，制成第一溶液；按质量比为30:3:500的比例取山嵛酸甘油酯和二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000-叶酸，溶于氯仿中，制成第二溶液：按体积比为10:1的比例，将第一溶液和第二溶液合并搅拌均匀，制成油相；将油相滴入到水相中，然后，加热去掉有机溶剂，冷却，得到E-[c(RGDfK) 2]和叶酸修饰的包载DiR的纳米结构脂质制剂E-[c(RGDfK) 2]/FA-DiR-NLC；
油相和水相的体积比为2:5。
将荷瘤裸鼠随机分为7组，每组6只，分别对应生理盐水组、DiR溶液组、包载DiR的纳米结构脂质制剂(DiR-NLC)、PEG修饰的包载DiR的纳米结构脂质制剂(PEG-DiR-NLC)、E-[c(RGDfK) 2]修饰的包载DiR的纳米结构脂质制剂(E-[c(RGDfK) 2]-DiR-NLC)、叶酸修饰 的包载DiR的纳米结构脂质制剂(FA-DiR-NLC)、E-[c(RGDfK) 2]和叶酸修饰的包载DiR的纳米结构脂质制剂E-[c(RGDfK) 2]/FA-DiR-NLC，以200μL/只的剂量尾静脉注射给药。分别于给药后1h、4h、8h、24h和36h五个时间点以异氟烷麻醉裸鼠后，将荷瘤裸鼠置于活体成像系统内观察，采集图片。注射药物24h后，将荷瘤裸鼠处死，进行解剖，分别取心、肝、脾、肺、肾、肿瘤等组织，置于小动物活体成像系统中进行成像，确定提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂在各组织器官以及肿瘤的分布情况。
从图3(a)、图3(b)和图3(c)中可以看到，DiR溶液组肿瘤部位无荧光，说明药物溶液没有肿瘤靶向性。DiR-NLC组随着时间的延长，有肿瘤部位有很少荧光，说明药物可以由纳米结构脂质制剂少量递送至肿瘤部位。
PEG-DiR-NLC组随着时间的延长，有肿瘤部位有比DiR溶液组更强的荧光，说明药物可以由PEG纳米结构脂质制剂通过长循环持续递送至肿瘤部位。
FA-DiR-NLC组和E-[c(RGDfK) 2]-DiR-NLC组随着时间的延长，有肿瘤部位有比PEG-DiR-NLC组更强的荧光，说明药物可以由叶酸修饰的纳米结构脂质制剂、E-[c(RGDfK) 2]修饰的纳米结构脂质制剂通过长循环和主动靶向持续递送至肿瘤部位。
E-[c(RGDfK) 2]/FA-DiR-NLC组随着时间的延长，在肿瘤部位有比FA-DiR-NLC组和E-[c(RGDfK) 2]-DiR-NLC组更强荧光，说明药物可以通过叶酸、E-[c(RGDfK) 2]双配体主动靶向持续递送至肿瘤部位，而且在体内比单一的肿瘤靶向修饰具有更强的肿瘤靶向性。
3.抗肿瘤活性研究
通过构建荷瘤4T1细胞的BALB/c小鼠乳腺癌模型并给予不同药物，记录肿瘤体积与重量等方式，考察提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂在体内抗肿瘤的作用，并通过考察肿瘤部位的细胞增殖与凋亡情况对药物抗肿瘤效果加以验证。
将4T1细胞悬液以生理盐水稀释至1×10 7个/mL。用酒精棉擦拭雌性BALB/c小鼠腋下，将100μL细胞悬液用1mL注射器接种于小鼠右前肢腋下皮下，在接种时要注意细胞悬液的混匀。每天观察肿瘤生长情况，观察时注意脱毛，且用游标卡尺测量肿瘤大小，待肿瘤体积至30mm 3左右时(a：肿瘤宽度，b：肿瘤长度，肿瘤体积V＝ab 2/2)进行随机分组。
将荷瘤小鼠随机分成9组。具体分组情况如下：
生理盐水组；
阳性药物盐酸阿霉素注射液组(DOX注射液)；
丹酚酸A溶液组(Sal A溶液)；
包载丹酚酸A的纳米结构脂质制剂组(Sal A-NLC)；
盐酸阿霉素溶液组(DOX溶液)；
丹酚酸A和盐酸阿霉素溶液组(Sal A/DOX溶液)；
包载盐酸阿霉素的纳米结构脂质制剂组(DOX-NLC)；
包载盐酸阿霉素和丹酚酸A的纳米结构脂质制剂组(Sal A/DOX-NLC)；
E-[c(RGDfK) 2]和叶酸双配体修饰的包载丹酚酸A和盐酸阿霉素的纳米结构脂质制剂组(E-[c(RGDfK) 2]/FA-Sal A/DOX-NLC)。各组制备方法如下：
生理盐水组：购买自石家庄第四制药厂。
阳性药物盐酸阿霉素注射液(DOX注射液)：购买自石药集团欧意药业有限公司。
丹酚酸A溶液(Sal A溶液)的制备：按质量比为5:300:5000的比例取丹酚酸A和卖泽52，溶于去离子水中，制成丹酚酸A溶液。
包载丹酚酸A的纳米结构脂质制剂(Sal A-NLC)的制备：按质量比为5:300:5000的比例取丹酚酸A和卖泽52，溶于去离子水中，制成水相；按质量比为50:1000的比例取大豆卵磷脂，溶于无水乙醇中，制成第一溶液；按质量比为30:500的比例取山嵛酸甘油酯，溶于氯仿中，制成第二溶液：按体积比为10:1的比例，将第一溶液和第二溶液合并搅拌均匀，制成油相；将油相滴入到水相中，然后，加热去掉有机溶剂，冷却，得到包载丹酚酸A的纳米结构脂质制剂(Sal A-NLC)；油相和水相的体积比为2:5。
盐酸阿霉素溶液(DOX溶液)的制备：按质量比为3:300:5000的比例取盐酸阿霉素和卖泽52，溶于去离子水中，制成盐酸阿霉素溶液(DOX溶液)。
丹酚酸A和盐酸阿霉素溶液(Sal A/DOX溶液)的制备：按质量比为5:3:300:5000的比例取丹酚酸A、盐酸阿霉素和卖泽52，溶于去离子水中，制成水相；
包载盐酸阿霉素的纳米结构脂质制剂(DOX-NLC)的制备：按质量比为3:300:5000的比例取盐酸阿霉素和卖泽52，溶于去离子水中，制成水相；按质量比为50:3:1000的比例取大豆卵磷脂，溶于无水乙醇中，制成第一溶液；按质量比为30:500的比例取山嵛酸甘油酯，溶于氯仿中，制成第二溶液：按体积比为10:1的比例，将第一溶液和第二溶液合并搅拌均匀，制成油相；将油相滴入到水相中，然后，加热去掉有机溶剂，冷却，得到包载盐酸阿霉素的纳米结构脂质制剂(DOX-NLC)；油相和水相的体积比为2:5。
包载盐酸阿霉素和丹酚酸A的纳米结构脂质制剂(Sal A/DOX-NLC)的制备：按质量比为5:3:300:5000的比例取丹酚酸A、盐酸阿霉素和卖泽52，溶于去离子水中，制成水相；按质量比为50:1000的比例取大豆卵磷脂，溶于无水乙醇中，制成第一溶液；按质量比为30:500的比例取山嵛酸甘油酯，溶于氯仿中，制成第二溶液：按体积比为10:1的比例，将第一溶液 和第二溶液合并搅拌均匀，制成油相；将油相滴入到水相中，然后，加热去掉有机溶剂，冷却，得到包载盐酸阿霉素和丹酚酸A的纳米结构脂质制剂(Sal A/DOX-NLC)；油相和水相的体积比为2:5。
E-[c(RGDfK) 2]和叶酸双配体修饰的包载丹酚酸A和盐酸阿霉素的纳米结构脂质制剂(E-[c(RGDfK) 2]/FA-Sal A/DOX-NLC)：实施例1制备的样品)
每组分别标号后按体重计算给药剂量，给药各组小鼠尾静脉注射约200μL溶液或者制剂，对照组注射相同体积的生理盐水。每2天注射1次，共给药6次。
如图4(a)，用药后(第2、4、6、8、10、12天)用游标卡尺测量肿瘤长径(a)和短径(b)，肿瘤体积V＝ab 2/2。以给药时间为横坐标，以各组不同时间肿瘤体积与对照组给药12天后的肿瘤体积平均值之比为纵坐标(相对肿瘤体积)，绘制相对肿瘤体积-时间变化曲线。
第六次给药后次日处死荷瘤小鼠，把皮下移植瘤连同胞膜完整剥离出肿瘤组织如图4(b)。精密称定肿瘤组织体积，以实验组瘤体积与对照组肿瘤体积平均值之比计算相对肿瘤体积(图4(c))精密称定肿瘤组织重量，以实验组瘤质量与对照组肿瘤重量平均值之比计算相对肿瘤质量(图4(d))。采用SPSS 22软件对数据进行单因素方差分析。
Figure PCTCN2022074452-appb-000002
Figure PCTCN2022074452-appb-000003
通过测量给药后相对肿瘤体积和相对肿瘤质量的变化来确定肿瘤抑制潜力。
DOX-NLC组的相对肿瘤体积与相对肿瘤质量与DOX溶液组比均具有极显著下降(p<0.01)，说明纳米结构脂质制剂可以提高药物在肿瘤部位有效积累，提高了药物的生物利用度，提高药物的体内抗肿瘤作用。
Sal A/DOX-NLC组的相对肿瘤体积与相对肿瘤质量与DOX-NLC组比均下降，Sal A/DOX溶液组的相对肿瘤体积与相对肿瘤质量与DOX溶液组比均下降，肿瘤体积抑制率和肿瘤重量抑制率也有增高，说明在Sal A可以提高DOX的抗肿瘤作用。
E-[c(RGDfK) 2]/FA-Sal A/DOX-NLC组的相对肿瘤体积与相对肿瘤质量与阳性药物组(DOX注射液组)和DOX溶液组比，均具有极显著差异(p<0.01)，且与Sal A/DOX-NLC组相比，相对肿瘤体积与相对肿瘤质量均有下降趋势，说明E-[c(RGDfK) 2]/FA靶向修饰的纳米结构脂质制剂的抗肿瘤效果最好。
4.Sal A对DOX造成的肾脏损伤的保护作用研究
肾脏病理切片
由图5可知，DOX注射液组、DOX溶液组、DOX-NLC组表现出肾脏病变。表现为肾小管结构紊乱肿胀明显；肾小球硬化出现固缩，基底膜增厚；出现炎症细胞浸润，损伤较为严重。
通过对比DOX溶液组和DOX-NLC组，发现E-[c(RGDfK) 2]/FA-Sal A/DOX-NLC组(实施例1)肾脏无明显病变，肾小球，肾小管形态正常，未发现明显炎症、病灶区域，E-[c(RGDfK) 2]/FA-Sal A/DOX-NLC(实施例1)没有肾毒性，说明E-[c(RGDfK) 2]/FA双配体修饰而得的E-[c(RGDfK) 2]/FA-Sal A/DOX-NLC可以显著降低DOX对肾脏的毒性。
血清肌酐含量
血清中游离肌酐的循环量完全依赖于它的排泄速度，从而测定血清中的肌酐量，可用于肾功能的检查。肌酐含量的增高见于：慢性肾衰竭时排泄量的减少及肢端肥大症。肌酐含量可用于评价肾小球滤过率，以确定肾功能状态。研究表明，阿霉素可以导致肾病综合征的发生，导致血清肌酐的含量升高。通过测定给药12天后小鼠血清中肌酐的含量，评价各组药物对肾脏的损伤影响。由图6可知，与DOX溶液组相比，E-[c(RGDfK) 2]/FA-Sal A/DOX-NLC组(实施例1)血清中的肌酐含量显著降低；与DOX-NLC、Sal A/DOX-NLC组相比，E-[c(RGDfK) 2]/FA-Sal A/DOX-NLC组(实施例1)血清中的肌酐含量显著降低，说明本申请的经过E-[c(RGDfK) 2]/FA双配体靶向修饰而得的E-[c(RGDfK) 2]/FA-Sal A/DOX-NLC可以显著降低DOX和含DOX的普通非靶向修饰制剂对肾脏的毒性。
实验证明，实施例2、3、4制备的E-[c(RGDfK) 2]/FA-Sal A/DOX-NLC的体内肿瘤靶向性、抗肿瘤活性及对肾脏的保护与实施例1相似。

Claims (10)

  1. 提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂的制备方法，其特征是包括如下步骤：
    (1)取丹酚酸A、盐酸阿霉素和表面活性剂，溶于去离子水中，制成水相；
    取乳化剂和二硬脂酰基磷脂酰乙醇胺-聚乙二醇-E-[c(RGDfK) 2]，溶于第一种有机溶剂中，制成第一溶液；
    取脂质和二硬脂酰基磷脂酰乙醇胺-聚乙二醇-叶酸，溶于第二种有机溶剂中，制成第二溶液：
    将第一溶液和第二溶液合并搅拌均匀，制成油相；
    (2)将油相滴入到水相中，然后，加热去掉有机溶剂，冷却，得到提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂；所述聚乙二醇的平均分子量为1000-8000。
  2. 根据权利要求1所述的方法，其特征是所述丹酚酸A、盐酸阿霉素、表面活性剂和水的质量比为(1-10):(1-5):(100-500):5000。
  3. 根据权利要求1或2所述的方法，其特征是所述表面活性剂为泊洛沙姆、聚氧乙烯脂肪酸醇醚、聚氧乙烯硬脂酸酯、胆酸钠、脱氧胆酸钠、卖泽52、吐温80和司盘80中的至少一种。
  4. 根据权利要求1所述的方法，其特征是所述乳化剂、二硬脂酰基磷脂酰乙醇胺-聚乙二醇-E-[c(RGDfK) 2]和第一种有机溶剂的质量比为(20-100):(1-5):1000。
  5. 根据权利要求1或4所述的方法，其特征是所述乳化剂为大豆卵磷脂、二棕榈酰磷脂酰丝氨酸、二油酰基磷脂酰丝氨酸、二肉豆蔻酰磷脂酰乙醇胺、二硬脂酰基磷脂酰乙醇胺、二棕榈酰基磷脂酰乙醇胺、二油酰磷脂酰乙醇胺、二硬脂酰磷脂酰甘油、二肉豆蔻酰磷脂酰甘油、二硬脂酰磷脂酸、二棕榈酰磷脂酸、二月桂酰基卵磷脂、二芥酰磷脂酰胆碱、二油酰基卵磷脂、二肉豆蔻酰基卵磷脂、二硬脂酰基磷脂酰胆碱和二棕榈酰基卵磷脂中至少一种。
  6. 根据权利要求1或4所述的方法，其特征是所述第一种有机溶剂为无水乙醇、二甲基亚砜和氯仿中至少一种。
  7. 根据权利要求1所述的方法，其特征是所述脂质、二硬脂酰基磷脂酰乙醇胺-聚乙二醇1000-8000-叶酸和第二种有机溶剂的质量比例为(10-50):(1-5):500。
  8. 根据权利要求1或7所述的方法，其特征是所述脂质为硬脂酸、山嵛酸甘油酯、 辛酸癸酸甘油三酯、单硬脂酸甘油酯、棕榈酸异丙酯、甘油棕榈酸硬脂酸酯、豆蔻酸甘油酯、肉豆蔻酸异丙酯、单棕榈酸甘油酯、月桂酸甘油酯、辛酸癸酸聚乙二醇甘油酯、胆固醇、大豆油和油酸中至少一种；所述第二种有机溶剂为无水乙醇、二甲基亚砜和氯仿中至少一种。
  9. 根据权利要求1所述的方法，其特征是所述第一溶液和第二溶液的体积比为(5-15):1；所述油相与水相的体积比为(1-4):5。
  10. 权利要求1-9之一的方法制备的提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂。
PCT/CN2022/074452 2020-12-07 2022-01-28 提高阿霉素肿瘤主动靶向性和肾脏保护的纳米结构脂质制剂及制备方法 WO2022122054A1 (zh)

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