Pharmaceutical composition as well as preparation method and application thereof

Abstract

The invention relates to a CD44 receptor and scavenger receptor-A (SR-A) dual-targeting pharmaceutical composition as well as a preparation method and application thereof. According to the invention, a certain proportion of carrier material with CD44 receptor targeting capability is adsorbed on the fatty acid albumin nanoparticles, and the albumin nanoparticles with CD44 receptor and SR-A dual-targeting capability are successfully prepared. The albumin nanoparticles can simultaneously deliver drugs to activated macrophages with high SR-A specificity expression and fibroblasts or tumor cells with high CD44 receptor specificity expression, provide new treatment schemes and ideas for various diseases including tissue fibrosis and tumors, and have great application value in the field of biological medicine.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials and pharmaceuticals, and more specifically, relates to a CD44 receptor and SR-A dual-targeted albumin nanoparticle, and a preparation method and application thereof. Background Art

[0002] The CD44 receptor is a complex transmembrane glycoprotein that is widely distributed in the body and participates in a variety of physiological processes in the body. Its abnormal expression and dysregulation are closely related to the occurrence and development of diseases. For example, the CD44 receptor is significantly upregulated on a variety of tumor cells and plays an important role in the development of tumors. At present, there are a large number of literature reports on the active delivery of anticancer drugs to tumor sites for tumor treatment using the CD44 receptor as a target; for example, the CD44 receptor is significantly upregulated on activated hepatic stellate cells (HSCs). HSCs are a type of non-parenchymal cell that is exclusive to the liver. They are in a quiescent state in normal liver tissue. When the liver is damaged and activated, they can secrete fibrogenic factors, promote the production of collagen, and lead to fibrosis. They play a key role in the initiation, progression, and regression of liver fibrosis. Therefore, the CD44 receptor can be used as an effective target for the treatment of tumors and tissue fibrosis.

[0003] SR-A is a type II transmembrane glycoprotein that is highly expressed on the surface of activated macrophages. Activated macrophages play an important role in the progression of many diseases. For example, tumor-associated macrophages (TAMs), as the most abundant immune cells in the tumor microenvironment (TME), play an important role in promoting the occurrence, development and metastasis of malignant tumors; for example, in the process of tissue fibrosis, activated macrophages promote fibrosis by secreting growth factors such as transforming growth factor-β (TGF-β), and secrete a variety of chemokines to recruit more inflammatory cells to the pathological site, aggravating tissue damage and fibrosis. Therefore, SR-A can be used as another effective target for the treatment of tumors and tissue fibrosis.

[0004] At present, most patents involving the treatment and research of chronic liver diseases by actively targeting cell-specific receptors only focus on the targeting of one type of cell. For example, the patent with publication number CN1743009A provides a dual-targeted anti-liver fibrosis drug carrier pseudoglycoprotein nanoparticle and its preparation method, which passively targets the nanoparticles loaded with anti-liver fibrosis drugs to the liver through the phagocytosis of macrophages of the reticuloendothelial system in the liver, spleen, etc.; through specific ligands and mannose-6-phosphate / insulin-like growth factor (M6P / IGFII) receptors in hepatic stellate cells, they enter the cells under the action of cell phagocytosis; for another example, the patent with publication number CN107854431A provides a HA nano-micelle targeted to hepatic stellate cells and its preparation method and application, which prepares nano-micelles based on HA, targets HSCs through CD44 receptors, and treats liver diseases caused by HSCs lesions. However, there is a close interaction between hepatic stellate cells and hepatic macrophages. For example, the chemokine CCL-5 produced by macrophages can induce the activation of HSCs through the NF-κB / ERK signal transduction pathway; and activated HSCs can in turn regulate the differentiation and function of macrophages. Therefore, the therapeutic effect of targeting HSCs or activated macrophages alone is relatively limited.

[0005] Currently, there are no reports on simultaneously targeting CD44 receptor and SR-A for disease treatment. Summary of the invention

[0006] The inventor team unexpectedly discovered in the study that the fatty acid albumin was used to encapsulate the drug to form nanoparticles, which had a strong negative charge on the surface, but still had some positive domains, and could relatively firmly adsorb negatively charged acidic mucopolysaccharides. Based on the above findings, the present invention creatively modified the targeting material acidic mucopolysaccharides on fatty acid albumin nanoparticles, providing a CD44 receptor and SR-A dual-targeted albumin nanoparticle to achieve targeted treatment of inflammation, fibrosis or tumors.

[0007] One of the purposes of the present invention is to provide a CD44 receptor and SR-A dual-targeting pharmaceutical composition.

[0008] One of the purposes of the present invention is to provide a carrier material modified with CD44 receptor targeting ability and fatty acid-modified albumin nanoparticles.

[0009] One of the purposes of the present invention is to provide albumin nanoparticles with dual cell targeting of CD44 receptor-specific high-expressing cells and SR-A-specific high-expressing cells; the CD44 receptor-specific high-expressing cells are various tumor cells and fibroblasts; preferably, the tumor cells are selected from melanoma cells B16F10, breast cancer cells 4T1, pancreatic cancer cells pan02, lung cancer cells LLC Lewis, liver cancer cells HepG2, and glioma cells C6; the fibroblasts are selected from activated hepatic stellate cells, synovial fibroblasts, and tumor fibroblasts; and the SR-A-specific high-expressing cells are activated macrophages.

[0010] One of the purposes of the present invention is to provide a method for preparing the above-mentioned nanoparticles.

[0011] One of the purposes of the present invention is to confirm the in vivo and in vitro targeting and therapeutic effects of the above-mentioned nanoparticles.

[0012] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0013] The present invention relates to CD44 receptor and SR-A dual-targeted albumin nanoparticles, characterized in that the albumin nanoparticles are prepared by adsorbing a targeting material on the surface of fatty acid-treated albumin nanoparticles, and contain the targeting material and the fatty acid-treated albumin; the fatty acid-treated albumin is prepared by connecting fatty acid and albumin through an acylation reaction.

[0014] The targeting material has CD44 receptor targeting capability, and is preferably one or more of chondroitin sulfate (CS), hyaluronic acid (HA), and low molecular weight heparin (LMWH).

[0015] The fatty acid is a saturated or unsaturated fatty acid having 6 to 24 carbon atoms, preferably one or more of myristic acid, palmitic acid, stearic acid, arachidic acid, oleic acid, linolenic acid, linoleic acid, and arachidonic acid.

[0016] The albumin is selected from one or more of bovine serum albumin, human serum albumin, ovalbumin, and recombinant human albumin.

[0017] Preferably, the targeting material contains 10 to 400 parts and the fatty acid-containing albumin contains 10 to 200 parts by weight.

[0018] The dual-targeted albumin nanoparticles further comprise a drug loaded on the nanoparticles, wherein the drug is an anti-inflammatory drug, an anti-fibrotic drug or an anti-cancer drug, preferably one or more of triptolide, triptolide, silybin, curcumin, glycyrrhetinic acid, paclitaxel, entecavir, dexamethasone, doxorubicin, pirarubicin, daunorubicin, indomethacin, cabazitaxel, oxaliplatin, teniposide, tanshinone IIA, hydroxycamptothecin, mitoxantrone, gemcitabine, trans-retinoic acid, schisandrin B, sorafenib, lenvatinib, regorafenib, cabozantinib, gemcitabine, cisplatin, 5-fluorouracil, and celecoxib; more preferably, one or more of sorafenib, lenvatinib, triptolide, paclitaxel, and pirarubicin, comprising 1 to 20 parts of the drug by weight.

[0019] The dual-targeted albumin nanoparticles further contain pharmaceutically acceptable excipients, which are selected from one or more of injection oil, polyethylene glycol derivatives, polyoxyethylene-polyoxypropylene block copolymers, injection phospholipids, and injection additives. The injection additives are selected from one or more of mannitol, lactose, glucose, maltose, trehalose, sucrose, sodium chloride, disodium hydrogen phosphate, and sodium dihydrogen phosphate. The injection oil is selected from one or more of soybean oil, medium-chain fatty acid glycerides, tea oil, and sesame oil. The amount of the injection oil is 0 to 100 parts by weight, preferably 10 to 80 parts, and the amount of the injectable oil is 0 to 50% (wt%) of the amount of the fatty acid-modified albumin, preferably 20 to 40% (wt%).

[0020] The pharmaceutically acceptable excipient is selected from polyethylene glycol derivatives, polyoxyethylene polyoxypropylene block copolymers, and preferably one or more of DSPE-PEG2000, HS15 or polysorbate 80. Preferably, the amount thereof is 0-25% (wt%) of the amount of the drug-loaded nanoparticles.

[0021] Preferably, the nanoparticles contain 20 to 200 parts of targeting materials, 20 to 200 parts of fatty acid-modified albumin, 1 to 40 parts of injection oil and other pharmaceutically acceptable excipients, and 1 to 20 parts of drugs loaded on the nanoparticles, in parts by weight; the drug loading of the nanoparticles is 0.1 to 40%.

[0022] In the present invention, the method for preparing the nanoparticles comprises the following steps:

[0023] (1) dissolving albumin in an alkaline solution and dissolving fatty acid-modified succinimide ester in an organic solvent, wherein the molar ratio of fatty acid succinimide ester to albumin is 10 to 100:1, reacting at 20 to 37° C. for 12 to 24 hours, dialyzing, centrifuging and freeze-drying to obtain fatty acid-modified albumin; (2) dissolving the fatty acid-modified albumin drug carrier in deionized water or in deionized water containing other excipients except the above-mentioned phospholipids for injection, adding an organic phase, high pressure homogenization or probe ultrasonic emulsification, and rotary evaporation to remove the organic solvent to prepare a fatty acid-modified albumin nanoparticle solution; (3) dissolving a targeting material in an aqueous solution, dripping the obtained solution into the fatty acid-modified albumin nanoparticle solution under uniform stirring, and continuing to stir uniformly for 3 to 60 minutes to obtain albumin nanoparticles.

[0024] The fatty acid-containing succinimide ester can be purchased from a commercial product or prepared by the following steps: dissolving a fatty acid in an organic solvent, adding dicyclohexylcarbodiimide and N-hydroxysuccinimide, reacting at 0°C to 37°C for 2 to 24 hours, and separating and purifying to obtain a fatty acid succinimide ester; the organic solvent is selected from N,N-dimethylformamide and dimethyl sulfoxide; the reaction temperature is preferably 20 to 37°C; and the reaction time is preferably 12 to 24 hours.

[0025] Furthermore, in the above-mentioned method for preparing nanoparticles, the alkaline solution in step (1) is preferably a carbonate buffer solution or a phosphate buffer solution with a pH of 8.0 to 10.0;

[0026] Furthermore, the organic solvent in step (1) is preferably N,N-dimethylformamide or dimethyl sulfoxide;

[0027] Furthermore, the organic phase in step (2) is preferably an organic phase in which a drug is dissolved, an organic phase in which injection oil and a drug are dissolved, or an organic phase in which a phospholipid complex of a drug is dissolved;

[0028] Furthermore, the organic solvent in step (2) is preferably dichloromethane or ethyl acetate;

[0029] Furthermore, in step (3), the mass ratio of the targeting material to the fatty acid-modified albumin is 5:1 to 1:5;

[0030] Furthermore, the uniform stirring speed in step (3) is 10 to 2000 rpm;

[0031] Furthermore, the uniform stirring time in step (3) is preferably 10 to 30 minutes;

[0032] Furthermore, the aqueous solution in step (3) can be deionized water, PBS buffer, physiological saline, 5% glucose solution, but is not limited to the above solutions.

[0033] The prepared albumin nanoparticles have a particle size of 50-500nm, preferably 50-200nm.

[0034] Preferably, the present invention provides a CD44 receptor and SR-A dual-targeted drug-loaded albumin nanoparticle, wherein the drug-loaded albumin nanoparticle comprises a step of adsorbing chondroitin sulfate (CS) onto the surface of palmitated human serum albumin nanoparticles.

[0035] The drug-loaded nanoparticles contain, by weight, 20 to 200 parts of palmitated human serum albumin, 20 to 200 parts of CS, 1 to 40 parts of soybean oil, and 1 to 20 parts of the drug loaded on the nanoparticles. Preferably, they contain 20 to 100 parts of palmitated human serum albumin, 20 to 100 parts of chondroitin sulfate, 5 to 20 parts of soybean oil, and 1 to 20 parts of the drug.

[0036] Preferably, the albumin nanoparticles are prepared by adsorbing CS on the surface of palmitated human serum albumin nanoparticles, and contain, by weight, 50 to 100 parts of palmitated human serum albumin, 50 to 100 parts of chondroitin sulfate, 10 to 40 parts of soybean oil, and 1 to 20 parts of the drug sorafenib or lenvatinib loaded on the nanoparticles.

[0037] The preparation method of the drug-loaded nanoparticles comprises dissolving the palmitate albumin in water or water containing the above-mentioned pharmaceutical excipients, adding an organic solvent dissolved with soybean oil and drugs, high-pressure homogenization or probe ultrasonic emulsification, and rotary evaporation to remove the organic solvent to obtain palmitate albumin nanoparticles, and dripping an aqueous solution dissolved with CS into the palmitate albumin nanoparticle solution under uniform stirring, and continuing to stir at a uniform speed for 10 to 30 minutes to obtain the drug-loaded nanoparticles.

[0038] The organic solvent is selected from dichloromethane, ethyl acetate and chloroform, preferably dichloromethane or ethyl acetate.

[0039] The use of CD44 receptor and SR-A dual-targeted albumin nanoparticles in disease treatment provided by the present invention is characterized in that the diseases include but are not limited to tissue fibrosis and tumors.

[0040] Furthermore, the tissue fibrosis includes, but is not limited to, liver fibrosis, pancreatic fibrosis, pulmonary fibrosis, cardiovascular system fibrosis, kidney fibrosis, spleen fibrosis, retinal fibrosis, and bone marrow fibrosis;

[0041] Furthermore, the tumor includes but is not limited to melanoma, breast cancer, pancreatic cancer, liver cancer, brain tumor, lung cancer, uterine cancer, prostate cancer, gastric cancer, and bladder cancer.

[0042] Beneficial Effects

[0043] (1) The CD44 receptor and SR-A dual-targeted albumin nanoparticles of the present invention target cells that specifically overexpress CD44 receptor and / or SR-A, have strong specificity, and good targeting effect.

[0044] (2) The CD44 receptor and SR-A dual-targeted albumin nanoparticles of the present invention can specifically deliver drugs to the endoplasmic reticulum and / or Golgi apparatus because some carrier materials with CD44 receptor targeting ability, such as HA and CS, have cell Golgi apparatus targeting, while fatty acids have cell endoplasmic reticulum targeting.

[0045] (3) The albumin nanoparticles with dual targeting of CD44 receptor and SR-A are prepared by first preparing fatty acid-modified albumin nanoparticles, and then the carrier material with CD44 receptor targeting ability can be adsorbed and modified on the nanoparticles by simple stirring. The preparation process is simple, with fewer steps and good repeatability. The nanoparticles have good safety and stability, and provide new treatment plans and ideas for various diseases including tissue fibrosis and tumors. They have good application prospects and broad development space in the medical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 .Fluorescence emission spectra of FITC-CS / Cy3-PSANPs, FITC-CS / PSANPs, and CS / Cy3-PSANPs.

[0047] Figure 2 .TEM characterization of nanoparticles.

[0048] Figure 3 .The experimental results of nanoparticle uptake on HSC-T6.

[0049] Figure 4 . Nanoparticle uptake results in Raw264.7 and activated macrophages.

[0050] Figure 5 .The results of the cytotoxicity experiment of nanoparticles on HSC-T6, Raw264.7 and activated macrophages.

[0051] Figure 6 .FRET experimental results at the cellular level.

[0052] Figure 7 .Colocalization of nanoparticles with the endoplasmic reticulum of cells.

[0053] Figure 8 .In vivo distribution of nanoparticles in a mouse model of ectopic hepatocellular carcinoma.

[0054] Fig. 9 .In vivo distribution of nanoparticles in a mouse model of liver fibrosis.

[0055] Fig.10 .Fluorescence images of complete targeting of the nanoformulation to the tumor and liver.

[0056] Fig.11 .Results of drug efficacy study on ectopic liver cancer.

[0057] Fig.12 .Results of efficacy study in cirrhotic mice.

[0058] Fig.13 .Dual targeting effect of nanoparticles on CD44 receptor and SR-A. DETAILED DESCRIPTION

[0059] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0060] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0061] Example 1

[0062] Preparation of palmitic acid (PA) and CS modified albumin nanoparticles (CS-PSANPs). Weigh 3.3 g of human serum albumin (HSA) and add 10 mL of sodium bicarbonate buffer (pH 8.4) to completely dissolve it. At the same time, weigh 350 mg of palmitic acid N-hydroxysuccinimide ester (PA-NHSE) and dissolve it in 1 mL of DMF. Under 37°C oil bath conditions, slowly add the DMF solution of PA-NHSE to the sodium bicarbonate buffer solution of HSA and stir the reaction for 24 hours. After the reaction is completed, the reaction solution is placed in a dialysis bag, dialyzed with deionized water at room temperature for two days, centrifuged at 10,000 rpm for 10 minutes, and the supernatant is collected. After centrifugation 4 to 5 times, the supernatant is freeze-dried to obtain the final product, palmitic acid albumin (PSA). Weigh 30 mg of medium-chain oil and dissolve it in 800 μL of a mixed solvent of dichloromethane and ethyl acetate as the oil phase; weigh 120 mg of the synthesized PSA and prepare a 3% PSA solution as the water phase. Mix the water phase and the oil phase, and ultrasonicate at a power of 250 W for 5 minutes (working for 3 seconds and resting for 7 seconds) to obtain a uniform colostrum solution, and then remove the organic solvent by rotary evaporation to obtain PSA nanoparticles (PSANPs). Then accurately weigh 120 mg of CS, prepare a CS solution with ultrapure water, add the CS solution to the PSANPs under rapid stirring, and stir for 20 minutes to obtain CS-PSANPs with a mass ratio of PSA to CS of 1:1.

[0063] Example 2

[0064] Stearic acid (SA) and CS modified albumin nanoparticles (CS 0.5 -SANPs). Weigh 0.005mM BSA, add 2mL sodium bicarbonate buffer (pH 8.49) to completely dissolve it. At the same time, weigh 10eq of stearic acid succinimidyl ester (SA-NHSE), add 200μLDMF to completely dissolve it. Under 28℃ water bath conditions, slowly add the DMF solution of SA-NHSE to the sodium bicarbonate buffer solution of BSA, and stir the reaction for 24 hours. After the reaction, the reaction solution was placed in a dialysis bag, dialyzed with deionized water at room temperature for two days, centrifuged at 10000rpm for 10min, collected the supernatant, centrifuged three times, and freeze-dried the supernatant to obtain the final product SAB. Weigh 15mg soybean oil and dissolve it in 800μL of a mixed solvent of dichloromethane and ethyl acetate as the oil phase; weigh 120mg of the synthesized SA and prepare a 3% SA solution as the aqueous phase. The water phase and the oil phase were mixed, and ultrasonicated at a power of 250W for 5 minutes (working for 3 seconds and resting for 7 seconds) to obtain a uniform colostrum solution, and then the organic solvent was removed by rotary evaporation to obtain SA nanoparticles (SANPs). Then 60 mg of CS was accurately weighed and prepared into a CS solution with ultrapure water. The CS solution was added to the PSANPs under rapid stirring and stirred for 20 minutes to obtain a CS with a mass ratio of PSA to CS of 1:0.5. 0.5 -SANPs.

[0065] Example 3

[0066] Preparation of albumin nanoparticles (HA3-OcAH NPs) modified with octanoic acid and HA. The synthesis method of octanoic acid-modified human serum albumin (OcAH) is the same as that in Example 1, with the only difference being that PA-NHSE in Example 1 is replaced with octanoic acid succinimidyl ester (OcA-NHSE). Weigh 30 mg of medium-chain oil and dissolve it in 800 μL of a mixed solvent of dichloromethane and ethyl acetate as the oil phase; weigh 120 mg of the synthesized OcAH and prepare a 3% OcAH solution as the aqueous phase. Mix the aqueous phase and the oil phase, and ultrasonicate at a power of 250 W for 5 min (working for 3 s and resting for 7 s) to obtain a uniform colostrum solution, and then remove the organic solvent by rotary evaporation to obtain OcAH nanoparticles (OcAH NPs). Then, 360 mg of HA was accurately weighed and prepared into HA solution with ultrapure water. The HA solution was added to OcAHNPs under rapid stirring and stirred for 20 min to obtain HA with a mass ratio of OcAH to HA of 1:3. 3- OcAH NPs.

[0067] Example 4

[0068] Albumin nanoparticles modified with arachidic acid (AA) and LMWH (LMWH 3-Preparation of AANPs). Weigh 0.63mM arachidic acid (AA), add DMF, and after complete dissolution, add 2eq dicyclohexylcarbodiimide (DCC) and 2eq N-hydroxysuccinimide (NHS), stir at room temperature for 12 hours, and recrystallize to obtain arachidic acid succinimide ester (AA-NHSE). Weigh 0.005mMBSA, add 2mL sodium bicarbonate buffer (pH 8.47) to completely dissolve it. At the same time, weigh 20eq of AA-NHSE and add 200μLDMF to completely dissolve it. Under 35℃ water bath conditions, the DMF solution of AA-NHSE is slowly added dropwise to the sodium bicarbonate buffer solution of HSA, and the reaction is stirred for 20 hours. After the reaction is completed, the reaction solution is placed in a dialysis bag, dialyzed with deionized water at room temperature for two days, centrifuged at 10000rpm for 10min, and the supernatant is collected. After centrifugation three times, the supernatant is freeze-dried to obtain the final product AAH. Weigh 22.5 mg of medium-chain oil and dissolve it in a mixed solvent of 1.2 mL of dichloromethane and ethyl acetate as the oil phase; weigh 80 mg of the synthesized AAH and prepare a 2% AAH solution as the water phase. Mix the water phase and the oil phase, ultrasonicate at a power of 250 W for 5 minutes (working for 3 seconds and resting for 7 seconds) to obtain a uniform colostrum solution, and then remove the organic solvent by rotary evaporation to obtain AA nanoparticles (AANPs). Then accurately weigh 240 mg of LMWH, prepare a LMWH solution with ultrapure water, add the HA solution to the AANPs under rapid stirring, and stir for 20 minutes to obtain LMWH3-AANPs with a mass ratio of OcAH to HA of 1:3.

[0069] Example 5

[0070] Preparation of PA and CS-modified albumin nanoparticles (SRF-CS-PSANPs) loaded with sorafenib (SRF). The synthesis method of PSA is the same as that in Example 1. Weigh 7.5 mg SRF and 30 mg soybean oil, dissolve in 1.2 mL of ethyl acetate as the oil phase; weigh 80 mg of the synthesized PSA and prepare a 2% PSA solution as the aqueous phase. Mix the aqueous phase and the oil phase, ultrasonicate at a power of 250 W for 5 min (working for 3 s, resting for 7 s) to obtain a uniform colostrum solution, and then remove the organic solvent by rotary evaporation to obtain PSA nanoparticles (SRF-PSANPs) loaded with SRF. Then accurately weigh 80 mg CS, prepare a CS solution with ultrapure water, add the CS solution to the SRF-PSANPs under rapid stirring, and stir for 20 min to obtain SRF-CS-PSANPs with a mass ratio of PSA to CS of 1:1.

[0071] Example 6

[0072] Preparation of PA loaded with fluorescent dye DiD and CS modified albumin nanoparticles (DiD-CS-PSANPs). The synthesis method of PSA is the same as that in Example 1. Take 37.5μg DiD and 16mg soybean oil, dissolve in 600μL of a mixed solvent of dichloromethane and ethyl acetate (volume ratio 1:1) to form an organic phase, weigh 80mg of the synthesized PSA, and prepare a 2% PSA solution as the aqueous phase. Mix the organic phase and the aqueous phase, ultrasonicate the probe in an ice bath for 10min (power 200W, working 4s, intermittent 6s), and remove the organic solvent by rotary evaporation to successfully prepare DiD-PSANPs. Then accurately weigh a certain amount of CS, configure it into a CS solution with ultrapure water, add 80mg of CS solution to DiD-PSANPs under rapid stirring, and stir for 20min to obtain DiD-CS-PSANPs with a mass ratio of PSA to CS of 1:1.

[0073] Example 7

[0074] Preparation of PA and CS-modified long-circulating albumin nanoparticles (SRF-PSA-PEG-CS NPs) loaded with sorafenib (SRF). The synthesis method of PSA is the same as that in Example 1. Weigh 7.5 mg SRF and 30 mg soybean oil, dissolve in 1.2 mL of ethyl acetate as the oil phase; weigh 80 mg of synthesized PSA and 10 mg DSPE-PEG2000, and prepare an aqueous solution containing 2% PSA and 0.25% DSPE-PEG2000 as the water phase. Mix the water phase and the oil phase, sonicate at a power of 250 W for 5 min (working for 3 s, resting for 7 s) to obtain a uniform colostrum solution, and then remove the organic solvent by rotary evaporation to obtain PSA nanoparticles (SRF-PSANPs) loaded with SRF. Then, 20 mg of CS was precisely weighed and prepared into a CS solution with ultrapure water. The CS solution was added to the SRF-PSANPs under rapid stirring and stirred for 20 min to obtain SRF-PSA-PEG-CSNPs with a mass ratio of PSA to CS of 0.25:1.

[0075] Example 8

[0076] Preparation of PA and CS-modified albumin nanoparticles (LEN-CS2-PSANPs) loaded with lenvatinib (LEN). The synthesis method of PSA is the same as that of Example 1. Weigh 15 mg of LEN and 20 mg of soybean oil, add 1.5 mL of ethyl acetate to dissolve, as the oil phase; weigh 80 mg of the synthesized PSA, and prepare a 3% PSA solution as the water phase. Mix 4 mL of the water phase and the oil phase, and ultrasonicate at a power of 200 W for 10 min (working 3 s, resting 7 s) to obtain a uniform colostrum solution, and then remove the organic solvent by rotary evaporation to obtain PSA nanoparticles (LEN-PSANPs) loaded with lenvatinib. Then accurately weigh 10 mg of CS, prepare a CS solution with deionized water, add the CS solution to the LEN-PSANPs under rapid stirring, and stir for 20 min to obtain CLT-CS2-PSANPs with a mass ratio of PSA to CS of 0.125:1.

[0077] Example 9

[0078] PA and CS-modified albumin nanoparticles loaded with tripterygium wilfordii (CLT) 0.5 -PSANPs). The synthesis method of PSA is the same as that in Example 1. Weigh 2.8 mg CLT and 19.6 mg soybean oil, add 400 μL ethyl acetate and 400 μL dichloromethane to dissolve as the oil phase; weigh 80 mg of the synthesized PSA and prepare a 2% PSA solution as the water phase. Mix 4 mL of the water phase and the oil phase, and ultrasonicate at a power of 250 W for 10 min (working 3 s, resting 7 s) to obtain a uniform colostrum solution, and then remove the organic solvent by rotary evaporation to obtain PSA nanoparticles (CLT-PSANPs) encapsulating tripterygium wilfordii. Then accurately weigh 40 mg CS, prepare a CS solution with deionized water, add the CS solution to the CLT-PSANPs under rapid stirring, and stir for 20 min to obtain CLT-CS-PSA with a mass ratio of PSA to CS of 1:0.5. 0.5 NPs.

[0079] Example 10

[0080] Preparation of PA and CS-modified albumin nanoparticles (PTX-CS-PSANPs) loaded with paclitaxel (PTX). The synthesis method of PSA is the same as that of Example 1. Weigh 3 mg of PTX and add it to 500 μL of chloroform to dissolve it as the oil phase; dissolve 45 mg of PSA in 3 mL of ultrapure water to obtain a PSA solution; add the prepared PSA solution to the organic solvent dissolved with PTX, perform probe ultrasound (200 W, 3 s working, 7 s intermittent) for 10 min under ice-water bath conditions, and remove the organic solvent by rotary evaporation to obtain PTX-PSANPs. Then accurately weigh 45 mg of CS, prepare a CS solution with ultrapure water, drip the CS solution into the PTX-PSANPs under rapid stirring conditions, and continue stirring for 20 min to obtain PTX-CS-PSANPs with a mass ratio of PSA to CS of 1:1.

[0081] Embodiment 11

[0082] Preparation of PA and CS-modified albumin nanoparticles (THP-HA-PAB NPs) loaded with pirarubicin (THP). The synthesis method of palmitic acid-modified bovine serum albumin (PAB) is the same as that in Example 1, except that HSA in Example 1 is replaced with bovine serum albumin (BSA). Weigh 10 mg of THP and 30 mg of soybean oil, dissolve in 1 mL of chloroform as the oil phase; weigh 80 mg of PAB, dissolve in 5 mL of ultrapure water as the water phase; then add the oil phase dropwise to the water phase under stirring, and continue stirring for 5 min; then emulsify by probe ultrasound at 200 W power for 10 min (working 5 s, resting 5 s); finally, completely remove the organic solvent by rotary evaporation in a water bath at 37 ° C to obtain THP-PSANPs. Then, 80 mg of HA was accurately weighed and prepared into HA solution with ultrapure water. The HA solution was dropped into THP-PSANPs under rapid stirring and continued to stir for 15 minutes to obtain THP-HA-PSANPs with a mass ratio of PSA to HA of 1:1.

[0083] Example 12

[0084] Preparation of paclitaxel (PTX)-loaded OcAH and HA-modified albumin nanoparticles (PTX-HA-OcAHNPs). The synthesis method of octanoic acid-modified human serum albumin (OcAH) is the same as that in Example 3, except that PA-NHSE in Example 1 is replaced with octanoic acid succinimidyl ester (OcA-NHSE). Weigh 3 mg of PTX and 9 mg of soybean oil, add them to 500 uL of chloroform to dissolve as the oil phase; dissolve 45 mg of OcAH in 3 mL of ultrapure water to obtain an OcAH solution; add the prepared OcAH solution to an organic solvent dissolved with soybean oil, perform probe ultrasound (200 W, 3 s working, 7 s intermittent) for 10 min under ice-water bath conditions, and remove the organic solvent by rotary evaporation to obtain PTX-OcAHNPs. Then, 45 mg of HA was precisely added to ultrapure water to prepare HA solution, and the HA solution was dropped into PTX-PSANPs under rapid stirring conditions. Stirring was continued for 15 min to obtain PTX-HA-OcAHNPs with a mass ratio of PSA to HA of 1:1.

[0085] Example 13

[0086] Preparation of AAH and CS-modified albumin nanoparticles (CUR-CS-AAH NPs) loaded with curcumin (CUR). The synthesis method of AAH is the same as that of Example 4. Take 80 mg AAH, dissolve it in 4 mL deionized water to form an aqueous phase, take 18 mg CUR and 28 mg soybean oil, dissolve it in 400 μL ethyl acetate, and then add 400 μL dichloromethane to form an organic phase. Mix the organic phase and the aqueous phase, perform probe ultrasound (200 W, 3 s working, 7 s intermittent) for 10 min under ice-water bath conditions, and remove the organic solvent by rotary evaporation to successfully prepare CUR-AAH NPs. Then accurately weigh 80 mg CS, prepare a CS solution with ultrapure water, drip the HA solution into the CUR-AAH NPs solution under rapid stirring conditions, and continue stirring for 30 min to obtain CUR-CS-AAH NPs with a mass ratio of PSA to CS of 1:1.

[0087] Embodiment 14

[0088] Preparation of AAH and LMWH-modified albumin nanoparticles (ETV-LMWH4-AAH NPs) loaded with entecavir (ETV). The synthesis method of AAH is the same as that of Example 4. Take 10 mg of ETV and 50 mg of lecithin E80 and dissolve them in 5 mL of a mixed solvent of isopropanol and ethanol with a volume ratio of 1:1, stir for 1 h in a water bath at 55°C, and remove the organic solvent by rotary evaporation under reduced pressure to obtain a phospholipid complex of entecavir. The obtained phospholipid complex and 30 mg of soybean oil are dissolved in 1 mL of dichloromethane to form an organic phase; weigh 120 mg of the synthesized AAH and prepare a 4% PSA solution as the aqueous phase. Mix 4 mL of the aqueous phase and the oil phase, and ultrasonicate at a power of 250 W for 10 min (working 3 s, resting 7 s) to obtain a uniform colostrum solution, and then rotary evaporate to remove the organic solvent to obtain PSA nanoparticles (ETV-AAH NPs) loaded with entecavir. Then, 480 mg of LMWH was accurately weighed and prepared into a LMWH solution with deionized water. The LMWH solution was added to the ETV-AAH NPs under rapid stirring and stirred for 20 min to obtain ETV-LMWH4-AAH NPs with a mass ratio of PSA to CS of 1:4.

[0089] Embodiment 15

[0090] Preparation of nanoparticles of fluorescent labeling carrier materials (FITC-CS / Cy3-PSANPs). (1) Synthesis of FITC-CS. Dissolve 0.1mmol of CS in 20mL of formamide solution, and 1.6mmol of EDC and NHS as catalysts in 5mL of DMF. Slowly drip into the CS solution under vigorous stirring conditions, activate at room temperature for 15min, slowly drip the solution after the reaction into 8mL of ethylenediamine, stir at room temperature overnight, dialyze for 24h, and freeze-dry to obtain amino-substituted CS; take the above-synthesized CS-NH2 and dissolve it in pH 9.8 carbonate buffer, add a certain amount of 1mg / mL FITC aqueous solution, stir at room temperature overnight, dialyze for 24h to remove unreacted FITC, and freeze-dry to obtain brown-yellow FITC-CS. (2) Synthesis of Cy3-PSA. The synthesis method of PSA is the same as that in Example 1. Take 100mg of the synthesized PSA and dissolve it in 5mL of ultrapure water. Add 1 mg / mL Cy3-NHS aqueous solution dropwise, react overnight, dialyze with pure water for 2 days to remove unreacted Cy3-NHS, and freeze-dry to obtain Cy3-PSA. (3) Weigh 30 mg soybean oil and dissolve it in 1.2 mL ethyl acetate as the oil phase; weigh 8 mg of synthesized Cy3-PSA and prepare a 2% Cy3-PSA solution as the water phase. Mix the water phase and the oil phase, and sonicate at a power of 250 W for 5 min (working 3 s, resting 7 s) to obtain a uniform colostrum solution, and then remove the organic solvent by rotary evaporation to obtain Cy3-labeled PSA nanoparticles (Cy3-PSANPs) without drug loading. Then, 80 mg of FITC-CS was accurately weighed and prepared into FITC-CS solution with ultrapure water. The FITC-CS solution was added to Cy3-PSANPs under rapid stirring and stirred for 20 min to obtain FITC-CS / Cy3-PSANPs (so that the mass ratio of CS to PSA was 1:1).

[0091] Example 16

[0092] Preparation of PA and CS modified albumin nanoparticles loaded with tripterygium wilfordii (CLT) (CLT-CS-PSANPs) The other conditions were the same as those in Example 9, except that 40 mg CS in Example 9 was replaced by 80 mg CS.

[0093] Embodiment 17

[0094] PA and CS-modified albumin nanoparticles loaded with fluorescent dye DiD (DiD-CS 0.5 The other conditions were the same as those for preparing DiD-CS-PSANPs in Example 6, except that 80 mg CS in Example 6 was replaced by 40 mg CS.

[0095] Embodiment 18

[0096] Preparation of PA and CS-modified albumin nanoparticles (SLB-CS-PSANPs) loaded with silybin (SLB). The synthesis method of PSA is the same as that of Example 1. Weigh 20 mg of SLB and add it to 500 μL of chloroform to dissolve it as the oil phase; dissolve 20 mg of PSA in 3 mL of ultrapure water to obtain a PSA solution; add the prepared PSA solution to the organic solvent in which SLB is dissolved, perform probe ultrasound (200 W, 3 s working, 7 s intermittent) for 10 min under ice-water bath conditions, and remove the organic solvent by rotary evaporation to obtain SLB-PSANPs. Then accurately weigh 20 mg of CS, prepare a CS solution with ultrapure water, drip the CS solution into the SLB-PSANPs under rapid stirring conditions, and continue stirring for 20 min to obtain SLB-CS-PSANPs with a mass ratio of PSA to CS of 1:1.

[0097] Comparative Example 1

[0098] Preparation of Sorafenib (SRF)-loaded Human Serum Albumin Nanoparticles (SRF-HSANPs) The other conditions were the same as those in the method for preparing SRF-PSANPs in Example 5, except that PSA was not required to be prepared and PSA was replaced with an equal mass of HSA.

[0099] Comparative Example 2

[0100] Preparation of human serum albumin nanoparticles loaded with tripterygium wilfordii (CLT) (CLT-HSANPs) The other conditions were the same as those in the method for preparing CLT-PSANPs in Example 9, except that PSA was not required to be prepared and was replaced with an equal mass of HSA.

[0101] Comparative Example 3

[0102] Preparation of human serum albumin nanoparticles loaded with fluorescent dye DiD (DiD-HSANPs). Other conditions were the same as those for preparing DiD-PSANPs in Example 6, except that PSA was not required to be prepared and PSA was replaced with an equal mass of HSA.

[0103] Experimental Example 1 Fluorescence Resonance Energy Transfer (FRET) Experiment

[0104] The inventors demonstrated that CS was successfully adsorbed on the surface of SANPs through fluorescence resonance energy transfer experiments.

[0105] FITC-CS / Cy3-PSANPs were prepared according to the method of Example 15 (so that the mass ratio of CS to PSA was 1:1); FITC-CS / PSANPs labeled with only FITC and CS / Cy3-PSANPs labeled with only Cy3 were prepared in the same way as a control group. The excitation and emission laser slit widths were set to 10 nm, and each preparation was excited at the maximum excitation wavelength Ex=485 nm of FITC at room temperature, and the emission spectrum of each preparation in the range of 497-650 nm was recorded.

[0106] The results are as follows Figure 1 As shown in the figure, after excitation, FITC-CS / PSANPs has an emission peak at 530nm, CS / Cy3-PSANPs has an emission peak at 565nm (located at the maximum emission wavelengths of FITC and Cy3, respectively), and the emission spectrum of FITC-CS / Cy3-PSANPs has two emission peaks at 530nm and 565nm. Compared with the emission peaks of FITC-CS / PSANPs and CS / Cy3-PSANPs of the same concentration, the fluorescence intensity at 530nm is reduced, and the fluorescence intensity at 565nm is increased, indicating that part of the fluorescence energy of FITC is transferred to Cy3, and an obvious fluorescence resonance energy transfer (FRET) phenomenon occurs between the two, which proves that CS is successfully adsorbed to the surface of PSANPs.

[0107] Experimental Example 2 Physicochemical Properties of Nanoparticles

[0108] The SRF-CS-PSANPs of Example 5 and the CLT-CS 0.5 -PSANPs and CLT-CS-PSANPs of Example 16 were diluted and the particle size, PDI and Zeta potential were measured by a laser particle size analyzer. As shown in Table 1, the results show that the above method can successfully prepare nanoparticles with a particle size of about 100 nm, uniform distribution and negative charge.

[0109] Table 1. Physicochemical properties of nanoformulations

[0110]

[0111] Experimental Example 3 TEM Characterization of Nanoformulations

[0112] The SRF-CS-PSANPs prepared in Example 5 were negatively stained with 1% phosphotungstic acid and dropped onto a dedicated copper grid. After natural drying, the morphology of the nanoparticles was observed using a transmission electron microscope (TEM) and photographs were taken.

[0113] Figure 2This is the TEM characterization result. It can be observed that the nanoparticles are spherical in shape, with a particle size of about 100nm and uniform distribution, which is consistent with the results measured by the laser particle size analyzer.

[0114] Experimental Example 4: Uptake experiment of nanoformulation on HSC-T6

[0115] The inventors determined the uptake of nanoparticles on HSC-T6 and preliminarily determined the effect of adsorbed CS on the uptake of cells expressing CD44 receptors.

[0116] DiD-PSANPs and DiD-CS-PSANPs were prepared according to the method in Example 6; DiD-CS 0.5 -PSANPs; DiD-HSANPs were prepared according to the method in Comparative Example 3.

[0117] HSC-T6 cells in the logarithmic growth phase were digested with 0.25% trypsin containing 0.02% EDTA, and resuspended in complete medium to obtain a cell suspension. 5 The cells were seeded at a density of 10 cells / mL on a 12-well plate. After incubation for 24 hours, the culture medium was replaced with DiD-HSANPs, DiD-PSANPs, DiD-CS containing DiD at a concentration of 0.4 μg / mL diluted in incomplete culture medium. 0.5 -PSANPs, DiD-CS-PSANPs, and incubate for 2 h, and the uptake of different preparations by HSC-T6 was analyzed by flow cytometry.

[0118] Figure 3 The results showed that the uptake of PSANPs was lower than that of HSANPs, and the uptake of HSC-T6 cells was increased after modification of CS.

[0119] Experimental Example 5: Uptake experiment of nanoparticles on macrophages and M1 macrophages stimulated by lipopolysaccharide

[0120] The inventors preliminarily determined the effect of PSA on the uptake capacity of cells expressing SR-A by measuring the uptake of nanoparticles in macrophages and M1 macrophages stimulated by lipopolysaccharide.

[0121] DiD-PSANPs and DiD-CS-PSANPs were prepared according to the method in Example 6; DiD-CS 0.5 -PSANPs; DiD-HSANPs were prepared according to the method in Comparative Example 3.

[0122] Take Raw264.7 cells in the logarithmic growth phase, resuspend them in complete medium to obtain a cell suspension, and add 4 μg / mL LPS to the medium to polarize Raw264.7 into M1 macrophages. 5 The cells were seeded at a density of 10 cells / mL on a 12-well plate. After incubation for 24 hours, the culture medium was replaced with DiD-HSANPs, DiD-PSANPs, DiD-CS containing DiD at a concentration of 0.4 μg / mL diluted in incomplete culture medium. 0.5 -PSANPs and DiD-CS-PSANPs were incubated for 2 h, and the uptake of different preparations by Raw264.7 and M1 macrophages was analyzed by flow cytometry.

[0123] Figure 4 (A) and Figure 4 (B) shows that compared with HSANPs, the uptake of PSANPs in Raw264.7, especially polarized M1 macrophages, was significantly increased. After adsorption of CS, there was almost no effect on the uptake of nanoparticles in macrophages, indicating that macrophages can hardly take up nanoparticles through the CD44 receptor.

[0124] Experimental Example 6 Cytotoxicity Experiment

[0125] CLT-PSANPs and CLT-CS-PSANPs were prepared according to the method in Example 9; CLT-HSANPs were prepared according to the method in Comparative Example 2.

[0126] HSC-T6 cells, Raw264.7 cells, and M1 macrophages in the logarithmic growth phase were resuspended in complete medium to obtain a cell suspension. 4 The cells were inoculated in a 96-well plate at a density of 100 cells / mL. When the cells covered 80% of the wells, the culture medium was replaced with CLT-HSANPs, CLT-PSANPs, and CLT-CS-PSANPs diluted with incomplete culture medium at different concentrations. The culture was continued for 24 h, and the cell survival rate was detected by the CCK-8 method.

[0127] Figure 5 It shows that for HSC-T6, CLT-CS-PSANPs is the most toxic, while for Raw264.7, especially M1 macrophages, PSA modification can enhance the cytotoxicity of nanoparticles.

[0128] Experimental Example 7 FRET experiment at the cell level

[0129] The inventors demonstrated through FRET experiments at the cellular level that CS-PSANPs can be completely engulfed by cells.

[0130] FITC-CS and Cy3-PSA were synthesized according to the method of Example 15, and FITC-CS / Cy3-PSANPs were prepared, wherein the mass ratios of FITC to Cy3 were 2:1, 1:1, and 1:2, respectively.

[0131] Raw264.7 in the logarithmic growth phase was taken, and the cell suspension was resuspended in complete culture medium, and inoculated in a confocal dish at an appropriate density. After incubation for 24 hours, the culture medium was replaced with FITC-CS / Cy3-PSANPs (2:1), FITC-CS / Cy3-PSANPs (1:1), and FITC-CS / Cy3-PSANPs (1:2) diluted with incomplete culture medium. After incubation at 37°C for 1 hour, the cells were fixed with 4% paraformaldehyde, the cell nucleus was labeled with the nuclear stain DAPI, and the fluorescence intensity of the preparation was observed using a laser confocal microscope.

[0132] Figure 6 It shows that with the increase of the proportion of Cy3 in the preparation, the fluorescence intensity of Cy3 as an energy acceptor gradually increases, while the fluorescence intensity of FITC as an energy donor gradually decreases, showing a fluorescence resonance energy transfer phenomenon, indicating that CS-PSANPs can be completely phagocytosed by cells.

[0133] Experimental Example 8 Endoplasmic reticulum targeting experiment

[0134] DiD-PSANPs and DiD-CS-PSANPs were prepared according to the method in Example 6; DiD-HSANPs were prepared according to the method in Comparative Example 3.

[0135] Raw and HepG2 cells were cultured at 1×10 4 The cells were inoculated at a density of 1000 / well in a confocal dish and cultured in an incubator for 24 h. The culture medium was discarded, and then 1 mL of incomplete culture medium containing DiD-HSANPs, DiD-PSANPs and DiD-CS-PSANPs was added to the dish respectively. After incubation for 1 h, the endoplasmic reticulum dye ER-Tracker Green working solution was added for light-proof staining, and then the cell nucleus stain DAPI was added for staining. The distribution of fluorescence in each group of cells was observed using a laser confocal microscope.

[0136] Figure 7 It shows that DiD-PSA NPs and DiD-CS-PSA NPs overlap significantly with the green fluorescence representing the endoplasmic reticulum, while DiD-HSANPs are dispersed in the cytoplasm and have no obvious overlap with the endoplasmic reticulum. This shows that compared with HSANPs, PSANPs can achieve endoplasmic reticulum targeting in Raw and HepG2 cells due to the endoplasmic reticulum targeting effect of PA.

[0137] Experimental Example 9 In vivo distribution experiment of ectopic liver cancer model

[0138] A mouse ectopic liver cancer model was constructed to determine whether CS-PSA nanoparticles can achieve higher accumulation in the tumor site.

[0139] 4-week-old male Kunming mice were inoculated with 0.1 mL of H22 cell suspension in their armpits. After 2 to 3 days, obvious protrusions could be felt in the armpits of the mice, indicating that the model was successfully constructed. DiD-PSANPs and DiD-CS-PSANPs were prepared according to the method in Example 6; DiD-CS 0.5 -PSANPs; DiD-HSANPs were prepared according to the method in Comparative Example 3.

[0140] When the tumor grows to about 300 mm 3 At the same time, 9 tumor-bearing Kunming mice were randomly divided into 3 groups, 3 mice in each group. DiD-HSANPs, DiD-PSANPs and DiD-CS-PSANPs were injected into the tail vein, and the dosage was 0.2 mg / kg calculated according to the amount of DiD. After 4 hours of administration, the mice were killed, the organs were dissected out, and the distribution of the preparations was observed with a live imaging device.

[0141] Figure 8 This indicates that compared with HSANPs, nanoparticles can achieve higher accumulation in tumor sites after modification with PSA and CS.

[0142] Experimental Example 10 In vivo distribution experiment of liver fibrosis model

[0143] A mouse liver fibrosis model was constructed to determine whether PSA-NPs could achieve higher accumulation in the liver.

[0144] Six-week-old male C57 mice were intraperitoneally injected with an olive oil solution containing 20% ​​CCl4 twice a week. The mice were killed 4 weeks after the injection, and the liver was removed. Masson staining and picrosirius red staining showed that the liver fibrosis model was successfully established. DiD-PSANPs were prepared according to the method in Example 6; DiD-CS-PSANPs were prepared according to the method in Example 17; and DiD-HSANPs were prepared according to the method in Comparative Example 3.

[0145] Nine C57 mice with successful modeling were randomly divided into three groups, with three mice in each group. Free DiD, DiD-HSANPs, DiD-PSANPs and DiD-CS-PSANPs were injected into the tail vein, and the dosage was 0.1 mg / kg calculated according to the amount of DiD. Fluorescence images at each time point were observed with a live imaging device at 2h, 4h, 8h, 12h and 24h after administration, and the mice were killed at 24h, and the main organs were dissected to observe the three-dimensional distribution images.

[0146] Fig. 9 It showed that at each time point, DiD-CS-PSANPs had a stronger accumulation in the liver than other groups, indicating that CS-PSANPs can improve the drug's targeting ability to the liver.

[0147] Experimental Example 11 Verification of the Complete Targeting of the Tumor and Liver by the Preparation

[0148] FITC-CS and Cy3-PSA were synthesized according to the method of Example 15, and FITC-CS / Cy3-PSANPs (the mass ratio of CS to PSA was 1:1) were prepared. The ectopic liver cancer mouse model was constructed according to the method of Experimental Example 9. Mice bearing ectopic tumors were injected with 0.2 mL of the above-mentioned nanoformulation through the tail vein. After 4 hours, the mice were killed and the tumor masses and liver tissues were removed. Frozen sections were prepared and observed under a laser confocal microscope.

[0149] Fig.10 This indicates that the red fluorescence of Cy3 and the green fluorescence of FITC almost completely overlap, that is, FITC-CS / Cy3-PSANPs can be completely targeted to the liver and tumors.

[0150] Experimental Example 12: Study on the efficacy of drugs for ectopic liver cancer

[0151] The treatment effects of SRF solution, SRF-HSANPs, SRF-PSANPs and SRF-CS-PSANPs on ectopic liver cancer in mice were compared. The preliminary indicators of the study were the growth of the tumor during the treatment process and the survival period of the mice.

[0152] An ectopic liver cancer mouse model was constructed according to the method of Experimental Example 9, SRF-PSANPs and SRF-CS-PSANPs were prepared according to the method of Example 5; SRF-HSANPs were prepared according to the method of Comparative Example 1, and SRF solution was prepared.

[0153] When the tumor grows to about 50 mm 3 At 14:00, 85 tumor-bearing mice were randomly divided into 5 groups, with 17 mice in each group. On the 10th, 13th, 16th, and 19th days after inoculation, each group of mice was injected with SRF solution, SRF-HSANPs, SRF-PSANPs, SRF-CS-PSANPs, and saline solution through the tail vein, and the dosage of each administration was 5 mg / kg calculated according to SRF. Eight mice were taken from each group, and the tumor volume of the mice was measured every two days during the experiment. After 25 days, the mice were killed and the tumor tissue was dissected. The remaining 9 mice in each group were raised normally after the administration, and the survival period was recorded.

[0154] Fig.11 (A), 11(B), and 11(C) show that SRF-CS-PSANPs has the best tumor inhibition effect. Fig.11(D) SRF-CS-PSANPs can maximally prolong the survival of mice.

[0155] Experimental Example 13: Drug efficacy study in cirrhotic mice

[0156] The treatment effects of SRF solution, SRF-HSANPs, SRF-PSANPs and SRF-CS-PSANPs on ectopic liver cancer mice were compared. The preliminary indicators of the study were the blood biochemical indicators in the mouse serum and the results of tissue pathology sections.

[0157] Six-week-old Kunming mice were given DEN aqueous solution as drinking water for 12 consecutive weeks. The mice were killed and their livers were dissected out. H&E staining showed that the cirrhosis model was successfully constructed.

[0158] SRF-PSANPs and SRF-CS-PSANPs were prepared according to the method in Example 5; SRF-HSANPs were prepared according to the method in Comparative Example 1, and an SRF solution was prepared.

[0159] Thirty-five cirrhotic mice were randomly divided into five groups, each with seven mice. Each group was injected with SRF solution, SRF-HSANPs, SRF-PSANPs, SRF-CS-PSANPs and saline via the tail vein. The dose of each administration was 5 mg / kg calculated based on SRF. The administration was carried out every two days after the first administration, for a total of four times. The blood biochemical indexes in the serum of each group of mice were measured and histopathological studies were performed.

[0160] Fig.12 (A) SRF-CS-PSANPs has the best effect in improving mouse fibrosis. Fig.12 (B) shows that after SRF-CS-PSANPs treatment, the blood biochemical indexes of mice were closest to the normal group compared with other groups, and the effect of improving liver function was the best.

[0161] Experimental Example 14: Verification of dual targeting of CD44 receptor and SR-A

[0162] The preparation method of DiD-CS-PSANPs was the same as that of Experimental Example 6. A mouse liver fibrosis model was constructed according to the method of Experimental Example 10, and free DiD and DiD-CS-PSANPs were injected into the tail vein, respectively. The mice were killed 4 hours later and the livers were dissected. The liver paraffin sections were prepared and the CD44 receptor and SR-A were stained, respectively. The distribution of fluorescence was observed using a laser confocal microscope.

[0163] Fig.13The results showed that in the DiD-CS-PSANPs group, DiD had a strong co-localization with both the CD44 receptor and SR-A, that is, CS-PSANPs had a dual targeting effect on the CD44 receptor and SR-A. This further demonstrated that CS-PSANPs could dually target the hepatic stellate cells and activated macrophages in the mouse liver fibrosis model through the CD44 receptor and SR-A, thereby achieving a more ideal therapeutic effect.

Claims

1. A dual-targeted albumin nanoparticle, characterized in that: The dual-targeted albumin nanoparticles comprise a targeting material and fatty acid-modified albumin, wherein the targeting material is an acidic mucopolysaccharide, and the fatty acid is a saturated or unsaturated fatty acid having 6 to 24 carbon atoms. The nanoparticles contain 10 to 400 parts of the targeting material and 10 to 200 parts of the fatty acid-modified albumin in parts by weight.

2. The dual-targeted albumin nanoparticles according to claim 1, characterized in that: The fatty acid-modified albumin is prepared by connecting fatty acid and albumin through an acylation reaction. The albumin is selected from one or more of bovine serum albumin, human serum albumin, ovalbumin, and recombinant human albumin. The fatty acid is selected from one or more of myristic acid, palmitic acid, stearic acid, arachidic acid, oleic acid, linolenic acid, linoleic acid, and arachidonic acid. The acidic mucopolysaccharide is preferably one or more of chondroitin sulfate (CS), hyaluronic acid (HA), and low molecular weight heparin (LMWH).

3. The dual-targeted albumin nanoparticles according to claim 1, characterized in that: The invention also comprises a drug, wherein the drug is selected from one or more of anti-inflammatory drugs, anti-fibrosis drugs or anti-cancer drugs, and is preferably one or more of triptolide, triptolide, silybin, curcumin, glycyrrhetinic acid, paclitaxel, entecavir, dexamethasone, dexamethasone palmitate, doxorubicin, pirarubicin, daunorubicin, indomethacin, cabazitaxel, oxaliplatin, teniposide, tanshinone IIA, hydroxycamptothecin, mitoxantrone, gemcitabine, trans-retinoic acid, schisandrin B, sorafenib, lenvatinib, regorafenib, cabozantinib, gemcitabine, cisplatin, 5-fluorouracil, and celecoxib; more preferably, one or more of sorafenib, lenvatinib, paclitaxel, triptolide, and pirarubicin, and comprises 1 to 20 parts of the drug by weight.

4. The dual-targeted albumin nanoparticle according to any one of claims 1 to 3, characterized in that: The dual-targeted albumin nanoparticles further contain pharmaceutically acceptable excipients, which are selected from one or more of injection oil, polyethylene glycol derivatives, polyoxyethylene-polyoxypropylene block copolymers, injection phospholipids, and injection additives, and the injection additives are selected from one or more of mannitol, lactose, glucose, maltose, trehalose, sucrose, sodium chloride, disodium hydrogen phosphate, and sodium dihydrogen phosphate, wherein the injection oil is selected from one or more of soybean oil, medium-chain fatty acid glycerides, tea oil, and sesame oil; in parts by weight, the amount of the injection oil is 0 to 100 parts, preferably 10 to 80 parts, and the amount of the injection oil is 0 to 50% (wt%) of the amount of the fatty acid-modified albumin, preferably 10 to 40% (wt%).

5. The dual-targeted albumin nanoparticles according to claim 4, characterized in that: The invention comprises, by weight, 20 to 200 parts of targeting materials, 20 to 200 parts of fatty acid-modified albumin, 1 to 40 parts of injection oil and other pharmaceutically acceptable excipients, and 1 to 20 parts of the drug loaded by the nanoparticles; the drug loading of the nanoparticles is 0.1 to 40%.

6. The dual-targeted albumin nanoparticles according to claim 5, characterized in that: The albumin nanoparticles are prepared by adsorbing CS on the surface of palmitated human serum albumin nanoparticles for modification, and contain, by weight, 20 to 200 parts of palmitated human serum albumin, 20 to 200 parts of chondroitin sulfate, 1 to 40 parts of soybean oil, and 1 to 20 parts of drugs carried by the nanoparticles. Preferably, the nanoparticles contain 20 to 100 parts of palmitated human serum albumin, 20 to 100 parts of chondroitin sulfate, 5 to 20 parts of soybean oil, and 1 to 20 parts of drugs.

7. The dual-targeted albumin nanoparticles according to claim 6, characterized in that: The albumin nanoparticles are prepared by adsorbing CS on the surface of palmitated human serum albumin nanoparticles, and contain 50-100 parts of palmitated human serum albumin, 50-100 parts of chondroitin sulfate, 10-40 parts of soybean oil, and 1-20 parts of sorafenib or lenvatinib loaded by the nanoparticles, in parts by weight.

8. The method for preparing dual-targeted albumin nanoparticles according to any one of claims 1 to 7, characterized in that: The preparation steps are as follows: (1) dissolving albumin in an alkaline solution and dissolving fatty acid succinimide ester in an organic solvent, reacting the two at a molar ratio of fatty acid succinimide ester to albumin of 10 to 100:1 at 20 to 37° C. for 12 to 24 hours, dialyzing, centrifuging and freeze-drying to obtain a fatty acid succinimide albumin drug carrier; the alkaline solution is preferably a carbonate buffer solution or a phosphate buffer solution at pH 8.0 to 10; the organic solvent is preferably N,N-dimethylformamide or dimethyl sulfoxide; (2) dissolving the fatty acid-modified albumin drug carrier in water or in water containing a pharmaceutically acceptable excipient, adding an organic phase, high pressure homogenization or ultrasonic emulsification by probe, and removing the organic solvent by rotary evaporation to prepare a fatty acid-modified albumin nanoparticle solution; the organic phase is preferably an organic phase containing a drug, an organic phase containing injection oil and a drug, or an organic phase containing a phospholipid complex of a drug; the organic solvent is preferably dichloromethane or ethyl acetate; (3) dissolving the targeting material in an aqueous solution to obtain a solution, and dripping the solution into the fatty acid-modified albumin nanoparticle solution under stirring, and continuing to stir for 3 to 60 minutes to obtain the dual-targeted albumin nanoparticles; the mass ratio of the targeting material to the fatty acid-modified albumin drug carrier is 5:1 to 1:5; the stirring speed is 10 to 2000 rpm; the uniform stirring time is preferably 10 to 30 minutes; the aqueous solution can be one or more of deionized water, PBS buffer, physiological saline, and 5% glucose solution.

9. Use of the dual-targeted albumin nanoparticles according to any one of claims 1 to 7 or the dual-targeted albumin nanoparticles prepared by the preparation method according to claim 8 in preparing a targeted preparation, characterized in that: The dual-targeted albumin nanoparticles target activated macrophages with high specific expression of SR-A and fibroblasts or tumor cells with high specific expression of CD44 receptors through the dual targeting effect of SR-A and CD44 receptors; preferably, the tumor cells are selected from melanoma cells B16F10, breast cancer cells 4T1, pancreatic cancer cells pan02, lung cancer cells LLC Lewis, liver cancer cells HepG2, and glioma cells C6; the fibroblasts are selected from activated hepatic stellate cells, synovial fibroblasts, and tumor fibroblasts; preferably, the dual-targeted albumin nanoparticles are used for the treatment of tissue fibrosis and tumors; the tissue fibrosis includes but is not limited to liver fibrosis, pancreatic fibrosis, pulmonary fibrosis, cardiovascular system fibrosis, renal fibrosis, spleen fibrosis, retinal fibrosis, and bone marrow fibrosis; the tumors include but are not limited to melanoma, breast cancer, pancreatic cancer, liver cancer, brain tumors, lung cancer, uterine cancer, prostate cancer, gastric cancer, and bladder cancer.

10. Use of the dual-targeted albumin nanoparticles according to any one of claims 1 to 7 or the dual-targeted albumin nanoparticles prepared by the preparation method according to claim 8 in preparing drugs for treating tumors and tissue fibrosis.

Images