Liposome-encapsulated human serum albumin drug-loaded nanoparticles and their preparation method and uses

Abstract

The present invention belongs to the technical field of pharmaceutical preparations, and particularly relates to liposome-encapsulated human serum albumin drug-loaded nanoparticles and a preparation method and use thereof. The liposome-encapsulated human serum albumin drug-loaded nanoparticles are obtained by encapsulating small-sized methotrexate-albumin complexes (MTX-HSA) in acid-sensitive liposomes. The liposome-encapsulated human serum albumin drug-loaded nanoparticles provided by the present invention can significantly increase the accumulation and retention time of drugs in the inflammatory microenvironment, and have higher therapeutic effects and smaller side effects compared with free drugs. In addition, the liposome-encapsulated human serum albumin drug-loaded nanoparticles can utilize the ELVIS effect of the inflammatory microenvironment, achieve passive targeting after intravenous injection, release smaller human serum albumin drug-loaded complexes at the inflammatory site, enhance the accumulation at the inflammatory site, and improve the therapeutic effect.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and in particular relates to liposome-encapsulated human serum albumin drug-loaded nanoparticles and a preparation method and application thereof. Background Art

[0002] Inflammation is a basic pathological process that occurs when living tissues with vascular systems are stimulated by various damaging factors and is dominated by defensive reactions. Inflammation is a dynamic process of damage, anti-damage and repair, which includes the following steps: ① Various damaging factors cause damage to the body's tissues and cells; ② Sentinel cells (such as macrophages) in the tissues surrounding the injury recognize the damaging factors and tissue necrosis and produce inflammatory mediators; ③ Inflammatory mediators activate the host's vascular response and leukocyte response, causing leukocytes and plasma proteins in the blood circulation of the injured area to infiltrate the site of the damaging factors, dilute, neutralize, kill and remove harmful substances; ④ The inflammatory reaction subsides and terminates; ⑤ Parenchymal cells and interstitial cells proliferate and repair damaged tissues. The persistent presence of inflammatory factors and damaged tissues are the root causes of chronic inflammation. The inflammatory focus is mainly infiltrated by macrophages, lymphocytes and plasma cells. Activated macrophages secrete a variety of biologically active products and are important mediators of tissue destruction and fibrosis in chronic inflammation. Therefore, promoting the apoptosis of inflammatory macrophages is a promising strategy for treating chronic inflammation.

[0003] At present, the drugs used clinically to treat inflammation include anti-inflammatory drugs and antibiotics. Methotrexate (MTX) has a chemical structure similar to folic acid, and has a competitive inhibitory effect on dihydrofolate reductase (DHFR), which can lead to the obstruction of tetrahydrofolate production. In addition, MTX can also inhibit thymic nucleotide synthetase, and at the same time inhibit the production of proinflammatory cytokines such as TNF-α, delaying the development of inflammation. However, MTX is not targeted in the body. If patients take it for a long time, it may cause more serious side effects, including gastrointestinal distress, bone marrow suppression, loss of appetite and hepatotoxicity. At the same time, when administered in vivo, the drug preparation will also be distributed throughout the body, resulting in impaired therapeutic effects in target tissues and increased risk of side effects. Most importantly, even if the drug preparation reaches the inflammatory microenvironment, it will still be quickly cleared from the site of inflammation.

[0004] According to literature reports, enhanced permeability and retention of EPR effects also exist in inflamed tissues, which is called extravasation through leaky vessels and subsequent inflammatory cell-mediated sequestration (ELVIS) effect. This is because as inflammation develops, vascular leakage occurs and the gap between endothelial cells in the inflammatory site is as high as 600nm. When combined with the characteristic pathophysiological characteristics of the microenvironment of the diseased site, NMs can also achieve active targeting strategies.

[0005] Compared with exogenous nano-drug delivery systems, natural biomimetic nano-drug delivery systems have advantages such as better biocompatibility, low cytotoxicity, and non-immunogenicity. Human serum albumin (HSA) is the main component of serum proteins, with natural biocompatibility, degradability, easy production, and low cost. It is a multifunctional therapeutic and diagnostic drug carrier with the ability to target inflammation. However, due to fibroplasia in inflamed tissues, dense fibroblasts prevent the penetration of large-sized carriers, reducing the therapeutic effect. Recently, a small-sized albumin-paclitaxel nanocomplex (10 nm) has been used to effectively penetrate tumor fibroblasts, which is consistent with the characteristics of fibrous cells in inflammation. Liposomes, as one of the most widely used nanoparticles, have characteristics such as high biocompatibility, easy availability, and surface modification, and have been widely used in clinical practice. In addition, due to the rapid proliferation of inflammatory cells leading to increased anaerobic glycolysis and lactate levels, an acidic environment is a typical feature of inflamed synovial tissue. Therefore, the specific pathological environment provides potential targets for designing on-demand drug delivery systems for the treatment of chronic inflammation.

[0006] In summary, how to solve the retention of small-sized drug-loaded nanoparticles in the blood and effectively accumulate the nanoparticles in inflamed tissues is the technical problem to be solved by the present invention. Summary of the Invention

[0007] In view of the above technical problems, the present invention provides liposome-encapsulated human serum albumin drug-loaded nanoparticles and their preparation methods and uses.

[0008] In the first aspect, the present invention provides a preparation method of liposome-encapsulated human serum albumin drug-loaded nanoparticles, including the following steps:

[0009] Dissolve methotrexate (MTX) in methanol to form test solution I with a concentration of 1 - 5 mg / mL, dissolve human serum albumin (HSA) in PBS buffer to form test solution II with a concentration of 3 - 6 mg / mL, mix test solution I and test solution II at a molar ratio of HSA:MTX of 1:1 - 6, react for 2 - 4 h, then remove free MTX and methanol, and obtain the MTX-HSA complex by freeze-drying.

[0010] 2) Dissolve egg yolk lecithin and cholesterol succinate monoesters in absolute ethanol to form an oil phase, and dissolve the MTX-HSA complex in PBS solution as the water phase; inject the oil phase into the water phase and react at 38 - 42 °C for 2 - 3 h, and filter the reaction solution to obtain the liposome-encapsulated human serum albumin drug-loaded nanoparticles.

[0011] Further, in step 1), free MTX and methanol are removed by ultrafiltration centrifugation, with parameters of 5000 - 7000 r / min and 20 - 40 min.

[0012] Further, in step 2), the mass ratio of egg yolk lecithin to cholesterol succinate in the oil phase is 3 - 5:1.

[0013] Further, in step 2), the injection rate of the oil phase into the aqueous phase is 0.5 - 2 mL / min.

[0014] Further, in step 2), the mass ratio of the MTX - HSA complex to cholesterol succinate is 1 - 2:1.

[0015] In a second aspect, the present invention provides liposome - encapsulated human serum albumin drug - loaded nanoparticles prepared by the described method.

[0016] In a third aspect, the present invention provides the use of the liposome - encapsulated human serum albumin drug - loaded nanoparticles in the preparation of a drug for treating inflammatory diseases.

[0017] Further, the inflammatory diseases include rheumatoid arthritis, osteoarthritis, atherosclerosis, myocardial infarction, sepsis, chronic nephritis, and pancreatitis.

[0018] In a fourth aspect, the present invention provides a drug for treating inflammatory diseases, which comprises the liposome - encapsulated human serum albumin drug - loaded nanoparticles and a pharmaceutically acceptable excipient or carrier.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The present invention obtains a human serum albumin MTX complex with a particle size of about 10 nm and a drug - loading amount of 2.4%.

[0021] 2. The present invention uses liposomes to encapsulate the human serum albumin MTX complex (Lipo / MTX - HSA), with an encapsulation efficiency of 85.1% and a drug - loading amount of 8.2%. In addition, the nanoparticles have a round and regular morphology, uniform size, a particle size of basically about 100 nm, and a Zeta potential of - 14.43 ± 0.39 mV. Lipo / MTX - HSA can be used as an excellent drug - delivery nanocarrier.

[0022] 3. The present invention prepares liposome - encapsulated human serum albumin - loaded MTX nanoparticles. By utilizing the leakage effect of the vasculature with increased permeability at the inflammatory site and the subsequent retention effect mediated by inflammatory cells (ELVIS), passive targeting can be achieved after intravenous injection, and small - particle - size MTX - HSA can be released at the inflammatory site, enhancing the accumulation at the inflammatory site and improving the therapeutic effect.

[0023] 4. The present invention selects human serum albumin loaded with MTX and liposome encapsulation as carrier materials to prepare a nano-drug targeting the inflammatory microenvironment with high drug loading, high biocompatibility, low toxicity and low immunogenicity. The results of in vitro experiments show that the nano-drug can target the inflammatory site, be targeted and taken up by activated macrophages, exhibit the effects of reducing pro-inflammatory factors TNF-α and IL-1β, increasing the anti-inflammatory factor IL-10, have the ability to convert the macrophage phenotype from M1 type to M2 type, regulate the inflammatory microenvironment, further inhibit the development of inflammation, and it can be found through JC-1 that the MTX-related preparation can lead to a decrease in mitochondrial membrane potential and induce mitochondrial dysfunction in macrophages; in a rat model of rheumatoid arthritis, in vivo imaging studies show that: the nano-drug can prolong the circulation time and exhibit good targeting to inflamed joints; in vivo experiments show that: the nano-drug shows excellent efficacy against diseased rats, the co-loaded drug nanoparticles can effectively improve the joint inflammatory microenvironment, regulate the secretion of inflammatory factors, and at the same time significantly improve the pathological conditions of diseased tissues; in vivo safety evaluation shows that: the nano-drug does not show any hemolysis and has good safety. Description of the Drawings

[0024] Figure 1 : The ultraviolet-visible light spectrum and fluorescence resonance energy transfer of the nanoparticles provided in the embodiments of the present invention. A. Ultraviolet-visible light spectrum diagrams of MTX, HSA, MTX-HSA, and Lipo / MTX-HSA; B. Fluorescence resonance energy transfer diagrams of DIO-Lipo / DIL-HSA at 1:1 / 2:1.

[0025] Figure 2 : Malvern particle size diagrams of the particles provided in the embodiments of the present invention. A. MTX-HSA; B. Lipo / MTX-HSA.

[0026] Figure 3 : Transmission electron microscope diagrams of the particles provided in the embodiments of the present invention. A. MTX-HSA; B. Lipo / MTX-HSA.

[0027] Figure 4 : Evaluation of the drug release ability. Release efficiency of Lipo / MTX-HSA under different pH conditions.

[0028] Figure 5 : Stability evaluation. A. Particle size change of Lipo / MTX-HSA within one week; B. PDI change of Lipo / MTX-HSA within one week.

[0029] Figure 6: In vitro biocompatibility evaluation provided by the embodiments of the present invention. Biocompatibility of MTX, MTX-HSA, and Lipo / MTX-HSA in RAW264.7 cells at concentrations of 0.5-30 μg / mL.

[0030] Figure 7 : Macrophage cell uptake evaluation provided by the embodiments of the present invention. A. Replace MTX with Cy5 and conduct macrophage uptake research using a confocal microscope; B. Conduct research on the drug endocytosis mechanism using a confocal microscope; C. Flow cytometry to detect the uptake of fluorescent agents by RAW264.7 before and after activation; D. Flow cytometry to detect the uptake mode of fluorescent agents by different endocytosis inhibitors.

[0031] Figure 8 : Synovial fibroblast uptake evaluation provided by the embodiments of the present invention. A. Replace MTX with Cy5 and conduct FLS cell uptake research using a confocal microscope; B. Magnification diagram of the uptake experiment in Figure A; C. Use JC-1 to study the mitochondrial damage of MTX-related preparations to FLS cells; D. Quantitative analysis of AM / PI calcein staining of FLS; E. Fluorescence microscope images of FLS cells treated with different AM / PI calcein staining.

[0032] Figure 9 : In vitro cell anti-inflammatory evaluation provided by the embodiments of the present invention. A-C. Use RT-qPCR to detect the expression of pro-inflammatory factors TNF-α, IL-1β, and anti-inflammatory factor IL-10.

[0033] Figure 10 : Mitochondrial damage evaluation provided by the embodiments of the present invention. Use JC-1 to study the mitochondrial damage of MTX-related preparations to RAW264.7 cells.

[0034] Figure 11 : In vivo tissue distribution evaluation provided by the embodiments of the present invention. After CIA rats are given DID fluorescent agents, in vivo imaging is performed. A. Fluorescent photos of the tail vein injection of Cy5-HSA and Lipo / Cy5-HSA are taken at 1, 6, 12, and 24 h respectively; B. After 24 h of giving the fluorescent agent, the rats are sacrificed, and the heart, liver, spleen, lungs, kidneys, joints, and blood are photographed for fluorescence; C. Fluorescent quantitative analysis of A.

[0035] Figure 12 : In vivo pharmacodynamic evaluation provided by the embodiments of the present invention. After treating CIA rats in the normal group, control group, MTX, MTX-HSA, and Lipo / MTX-HSA groups, A. Changes in joint swelling degree; B. Changes in foot volume; C. Joint score; D. Ankle joint diameter / foot sole thickness; E. Changes in rat body weight.

[0036] Figure 13 : In vivo anti-inflammatory evaluation provided by the embodiments of the present invention. A-C: Serum was taken from CIA rats before and after treatment with the normal group, control group, MTX, MTX-HSA, and Lipo / MTX-HSA groups, and ELISA was used to detect pro-inflammatory factors TNF-α and IL-1β; and anti-inflammatory factor IL-10.

[0037] Figure 14 : Histopathological evaluation of joint tissues provided by the embodiments of the present invention. Pathological studies were carried out on joint tissues, A-B, H&E staining; C-D, SO staining; E-F, toluidine blue staining.

[0038] Figure 15 : In vivo safety evaluation provided by the embodiments of the present invention. Hemolysis experiments of the MTX, MTX-HSA, and Lipo / MTX-HSA groups.

[0039] Figure 16 : In vivo distribution study of atherosclerotic mouse models provided by the embodiments of the present invention.

[0040] Figure 17 : Oil red O staining of atherosclerotic mouse blood vessels provided by the embodiments of the present invention.

[0041] Figure 18 : HE staining of atherosclerotic mouse blood vessels provided by the embodiments of the present invention. Detailed implementation manners

[0042] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0043] Example 1: Preparation and characterization of liposome-encapsulated human serum albumin drug-loaded (Lipo / MTX-HSA) nanoparticles

[0044] The preparation method of Lipo / MTX-HSA nanoparticles in this example is as follows:

[0045] Step 1: Synthesize the MTX-HSA complex. Specifically, ①Precisely weigh the prescribed amount of MTX and dissolve it in methanol / absolute ethanol / water to prepare a test solution with a concentration of 2 mg / mL; ②Precisely weigh the prescribed amount of HSA and dissolve it in PBS buffer to prepare a test solution with a concentration of 4 mg / mL; ③Measure the two test solutions according to the molar ratio of HSA:MTX = 1:1 / 1:2 / 1:4 / 1:6, mix and stir for 3 h, and then remove free MTX and methanol by ultrafiltration centrifugation (6000 r / min, 30 min); ④Then obtain the MTX-HSA complex by freeze-drying (MTX-HSA is dissolved in PBS solution before use).

[0046] Step 2: Synthesize Lipo / MTX-HSA nanoparticles. Specifically, ①Precisely weigh the egg yolk lecithin E80 and cholesterol succinate monoesters (CHS) described in the prescription, and use anhydrous ethanol as a solvent to vortex and dissolve to prepare 3 mL of an oil phase (egg yolk lecithin:CHS = 3:1 / 4:1 / 5:1, w / w). Dissolve the MTX-HSA complex described in the prescription in 5 mL of PBS solution with pH 7.4 as the aqueous phase; ②Set the injection speed to 0.5 / 1 / 2 mL / min, and slowly inject the prepared oil phase into the aqueous phase using a syringe with a needle (inject while stirring, MTX-HSA:CHS = 1:1 / 1.5:1 / 2:1, w / w), and slowly stir and react at 40 °C in a water bath at a rotation speed of 200 / 300 / 400 / 500 r / min for 2 h; ③Filter the reaction solution stirred for 2 h successively through 0.45 μm and 0.22 μm microporous membranes, and then the required Lipo / MTX-HSA solution can be obtained.

[0047] Characterize the MTX-HSA complex and Lipo / MTX-HSA nanoparticles by particle size (Size), Zeta potential, and polydispersity index (PDI).

[0048] Table 1 shows that for the MTX-HSA complex prepared in Step 1, the preferred organic solvent for preparation is methanol, and the molar ratio of HSA:MTX is 1:4.

[0049] Tables 2, 3, 4, and 5 show that for the Lipo / MTX-HSA nanoparticles prepared in Step 2, the preferred mass ratio of MTX-HSA:CHS for preparation is 1.5:1, the mass ratio of egg yolk lecithin E80:CHS is 4:1, the selected magnetic stirring speed is 300 r / min, and the selected injection speed of the syringe with a needle is 0.5 mL / min.

[0050] Table 6 is the final experimental prescription.

[0051] Table 1 Univariate study on the dosage ratio of MTX and HSA

[0052]

[0053] Table 2 Univariate study on the ratio of MTX-HSA to CHS (w / w)

[0054]

[0055]

[0056] Table 3 Univariate study on the dosage ratio of egg yolk lecithin E80 to CHS (w / w)

[0057]

[0058] Table 4 Univariate study on magnetic stirring speed

[0059]

[0060] Table 5 Univariate study on the injection speed of a syringe with a needle hole

[0061]

[0062] Table 6 Final experimental prescription

[0063]

[0064] Figure 1 A By full-wavelength scanning of Lipo / MTX-HSA with ultraviolet-visible light spectroscopy, an obvious absorption peak of MTX was observed at 302 nm, confirming the successful encapsulation of MTX in the liposome. Moreover, the specific absorption peaks of proteins of HSA and Lipo / MTX-HSA also existed, indicating that the protein structure was not affected by drug loading. Figure 1 B It can be confirmed that HSA was loaded on the liposome by fluorescence resonance energy transfer (FRET). DIO was used as the donor and bound to the liposome, while DIL was used as the acceptor and bound to HSA. As the concentration of DIL-HSA increased, the fluorescence intensity of the acceptor (DIL, 569 nm) increased, while the fluorescence intensity of the donor (DIO) at 501 nm decreased, indicating the existence of the FERT effect.

[0065] Figure 2 A showed that the particle size of MTX-HSA was about 10 nm, Figure 2 B showed that the particle size of Lipo / MTX-HSA was about 100 nm. In addition, the PDI of Lipo / MTX-HSA was 0.171 ± 0.013, and the Zeta potential was -14.43 ± 0.39 mV. The drug loading of the MTX-HSA complex was calculated to be about 2.4% based on the concentration of MTX, and the encapsulation efficiency of the prepared Lipo / MTX-HSA was about 85.1%, and the drug loading was about 8.2%.

[0066] Figure 3 Panel A shows that MTX-HSA was observed by transmission electron microscopy to be 20-nm spherical nanoparticles. Figure 3 Panel B shows that Lipo / MTX-HSA had a regular spherical morphology with uniform size, and the particle size was basically around 100 nm.

[0067] Figure 4 The release of Lipo / MTX-HSA under different pH conditions was shown. Compared with the pH = 7.4 group, the release rate of Lipo / MTX-HSA was faster at pH = 5.5. The release rate was faster in the pH = 5.5 group within 12 h, and 70% and 57% of MTX were released at pH = 5.5 and pH = 7.4, respectively. This indicates that Lipo / MTX-HSA can respond to the mildly acidic environment of inflammation and accelerate the release of MTX.

[0068] Figure 5 Panels A and B show that the particle size and PDI of Lipo / MTX-HSA hardly changed over 7 days, indicating good stability in vitro.

[0069] Example 2: In vitro cytological study of Lipo / MTX-HSA nanoparticles

[0070] (1) The cytotoxicity of different formulations was evaluated by the MTT method. Figure 6 It can be seen that after incubation with RAW264.7 cells for 24 h, MTX and MTX-HSA showed concentration-dependent inhibitory effects (0.5 - 30 μg / mL). At a concentration of 30 μg / mL, the survival rates of MTX and MTX-HSA were lower than 50% and 60%, respectively. However, after treatment with Lipo / MTX-HSA, we can see that the cell viability increased significantly, reaching more than 70% at a high concentration (30 μg / mL) (*P < 0.05). This confirmed that liposome coating improved the biocompatibility and selectivity of the nanoparticles for macrophages.

[0071] (2) The intracellular distribution of the fluorescent agent was studied. Figure 7 Panel A shows that the red fluorescence intensity in LPS-activated macrophages was significantly higher than that in non-activated macrophages. In RAW264.7 without LPS stimulation, the Cy5 fluorescence intensity in the Lipo / Cy5-HSA group was similar to that in the Cy5-HSA group and stronger than that in the free Cy5 group. Figure 7 Panel B shows that there was a difference in the fluorescence intensity between the Lipo / Cy5-HSA group and the Cy5-HSA group in LPS-stimulated RAW264.7 cells, consistent with flow cytometry ( Figure 7C) The detected fluorescence intensity data is consistent. This may be because smaller Cy5-HSA nanoparticles (7 nm) are more conducive to exocytosis than larger Lipo / Cy5-HSA (80 nm). Compared with Cy5-HSA, Lipo / Cy5-HSA reduces the extracellular secretion of Cy5-HSA to a certain extent and prolongs the residence time of Cy5-HSA in LPS-activated RAW264.7 cells.

[0072] Figure 8 A shows that in FLS cells, the Cy5 fluorescence intensity in the Lipo / Cy5-HSA group is similar to that in the Cy5-HSA group and stronger than that in the free Cy5 group. As Figure 8 Shown in B, the green fluorescence intensity of JC-1 monomers in the MTX formulation group is significantly higher than that in the control group, confirming that MTX-related formulations can induce mitochondrial dysfunction in macrophages and FLSs. Figure 8 C and D show that the cell viability after MTX-HSA treatment is significantly lower than that of MTX or Lipo / MTX-H SA at the same concentration (**P < 0.01). Figure 8 As shown in E, the red fluorescence of CIA-FLS treated with MTX-HSA and Lipo / MTX-HSA is the strongest. In contrast, CIA-FLS treated with MTX emits a large amount of green fluorescence, indicating a lower level of cell death. These data and results indicate that MTX-HSA can reduce inflammation by inhibiting the viability of FLSs.

[0073] (3) Explore the intracellular endocytosis pathway of the formulation. Methyl-β-CD (a small pore-mediated endocytosis inhibitor), amiloride hydrochloride (a macropinocytosis-mediated endocytosis inhibitor), and chlorpromazine (a clathrin-mediated endocytosis inhibitor) were used to study the cell entry pathway of the nanomaterials. As Figure 7 Shown in D, amiloride hydrochloride significantly reduces the uptake efficiency of Lipo / Cy5-HSA by activated macrophages. The results show that most of the Lipo / Cy5-HSA enters the cells through macropinocytosis, and this uptake method helps to bypass lysosomes.

[0074] Example 3: In vitro cell pharmacodynamics study of Lipo / MTX-HSA

[0075] (1) Study the cell anti-inflammatory pharmacodynamics of the formulation. By studying the effect of Lipo / MTX-HSA on the secretion of inflammatory factors, it was found that Lipo / MTX-HSA can inhibit the secretion of these cytokines in activated macrophages, which is comparable to the secretion of normal macrophages ( Figure 9 A - B). It is worth noting that Lipo / MTX-HSA increases the expression of the anti-inflammatory factor IL-10 ( Figure 9C). These data indicate that Lipo / MTX-HSA has a strong ability to inhibit inflammatory factors and increase the expression of anti-inflammatory factors, which is crucial for alleviating the inflammation of RA.

[0076] (2) Study the mitochondrial function of the cells in the preparation. To study whether MTX-related nano-preparations would damage the mitochondria of macrophages, JC-1 was used as a probe to detect the changes in mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 aggregates into polymers in the mitochondrial matrix, producing red fluorescence. On the contrary, the monomers formed by JC-1 emit green fluorescence when the membrane potential is low. The decrease in membrane potential was detected by the switch from red fluorescence to green fluorescence, which is considered a sign of mitochondrial damage. As Figure 10 shown, the green fluorescence intensity of JC-1 monomers in the MTX group was significantly higher than that in the control group, confirming that MTX-related preparations can induce mitochondrial dysfunction in macrophages.

[0077] Example 4: In vivo distribution study of Lipo / MTX-HSA

[0078] (1) Establishment of the CIA rat model method. After one week of adaptive feeding, on the 0th day, an emulsion obtained by fully emulsifying an equal volume of bovine type II collagen and complete Freund's adjuvant (CFA) was subcutaneously injected at the root of the rat tail, with 100 μL injected into each rat. On the 7th day after the primary immunization, each rat was injected with 100 μL of an emulsion obtained by fully emulsifying an equal volume of bovine type II collagen and incomplete Freund's adjuvant (IFA) for the second time to establish a collagen-induced arthritis (CIA) model in SD rats. The clinical symptom scores of arthritis in CIA rats are shown in Table 7.

[0079] Table 7 Clinical symptom scoring system for arthritis in CIA rats

[0080]

[0081] (2) Fluorescence in vivo imaging study. Six CIA rats were randomly divided into two groups, Cy5-HSA and Lipo / Cy5-HSA (n = 3 in each group), and a Cy5 dose of 5 μg was intravenously injected to observe the biodistribution of NPs in the inflamed joints. The rats were anesthetized at 1, 6, 12, and 24 hours after injection, and their feet were imaged using Carestream MI. The rats were sacrificed 24 hours after fluorescence imaging for in vitro tissue distribution analysis. The fluorescence of the collected organs and plasma was also imaged.

[0082] The results are as Figure 11 shown. The fluorescence intensity in the Lipo / Cy5-HAS group was significantly higher than that in the Cy5-HAS group 6 hours after injection.

[0083] Example 5: In vivo Pharmacodynamics Study of Lipo / MTX-HSA

[0084] (1) On the 16th, 19th, 22nd, 25th, and 28th days after the induction of arthritis and primary immunization, normal saline, MTX, MTX-HSA, or Lipo / MTX-HSA was intravenously injected into each group, and three normal rats were used as the control group. The dosing dose was 0.8 mg / kg for MTX, and the drug was administered once every three days for a total of five times. Before each administration, the joints ( Figure 12 A), foot volume ( Figure 12 B), joint score ( Figure 12 C), ankle joint diameter / foot sole thickness ( Figure 12 D), and rat body weight ( Figure 12 E) were photographed and recorded.

[0085] (2) After the administration was completed, serum was taken to complete the detection of inflammatory factors ( Figure 13 ), and histopathological studies were performed, and H&E staining ( Figure 14 A-B), SO staining ( Figure 14 C-D), and toluidine blue staining ( Figure 14 E-F)

[0086] Figures 12 - 15 The results proved that Lipo / MTX-HSA could effectively inhibit joint swelling, improve the joint inflammatory microenvironment, regulate the secretion of inflammatory factors, and at the same time significantly improve the pathological conditions of joint tissues.

[0087] Example 6: In vivo Safety Evaluation of Lipo / MTX-HSA

[0088] Figure 15 The results of the hemolysis experiment showed that even at such a high concentration as 500 μg / mL, no hemolysis occurred in Lipo / MTX-HSA, proving that Lipo / MTX-HSA is a safe and effective nanoparticle drug.

[0089] Example 7: In vivo Distribution and Pharmacodynamics Evaluation in an Atherosclerosis Disease Model

[0090] (1) The in vivo vascular distribution experiment in the atherosclerosis model showed that the distribution in the Lipo / MTX-HSA group was significantly higher than that in the free drug group ( Figure 16 ).

[0091] (2) The results of oil red staining showed that the plaques in the Lipo / MTX-HSA group were significantly lower than those in other groups ( Figure 17 ).

[0092] (3) The results of HE staining showed that the plaques in the Lipo / MTX-HSA group were significantly lower than those in other groupsFigure 18 )。

[0093] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0094] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. Use of a liposome-encapsulated human serum albumin drug-loaded nanoparticle with high biocompatibility in the preparation of a drug for treating atherosclerosis, characterized in that, The preparation method of the liposome-encapsulated human serum albumin drug-loaded nanoparticles comprises the following steps: 1) Dissolve methotrexate (MTX) in methanol to form a test solution I with a concentration of 1 - 5 mg / mL, and dissolve human serum albumin (HSA) in PBS buffer to form a test solution II with a concentration of 3 - 6 mg / mL. Mix test solution I and test solution II in a molar ratio of HSA:MTX of 1:4, react for 2 - 4 h, then remove free MTX and methanol, and obtain the MTX-HSA complex by freeze-drying; 2) Dissolve egg yolk lecithin E80 and cholesterol succinate in absolute ethanol to form an oil phase, and dissolve the MTX-HSA complex in PBS solution as the water phase; Inject the oil phase into the water phase at a rate of 0.5 mL / min, and react at 38 - 42°C and a rotation speed of 300 r / min for 2 - 3 h. Filter the reaction solution to obtain the liposome-encapsulated human serum albumin drug-loaded nanoparticles; The mass ratio of the MTX-HSA complex to cholesterol succinate is 1.5:1, and the mass ratio of egg yolk lecithin E80 to the MTX-HSA complex is 4:

1.

2. The use according to claim 1, characterized in that, The drug further comprises a pharmaceutically acceptable excipient or carrier.

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