CY-09-loaded albumin nanoparticles as well as preparation method and application thereof
By preparing the optimized albumin nanoparticle carrier, the problems of low bioavailability and liver targeting efficiency in the treatment of acute liver injury were solved, and the efficient liver targeted delivery and significant therapeutic effects of CY-09 were achieved.
Patent Information
- Application Number
- CN202510299390.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-13
- Publication Date
- 2025-06-06
AI Technical Summary
The prior art is difficult to effectively improve the bioavailability and liver targeting efficiency of CY-09, and the lack of reports on the use of albumin nanoparticles as CY-09 delivery system, limiting its application in the treatment of acute liver injury.
By preparing albumin nanoparticles with CY-09 as the active ingredient, using albumin and soybean oil as auxiliary materials, the mass ratio of CY-09 to albumin and soybean oil is optimized, the encapsulation rate and stability are improved, and the liver targeted delivery of CY-09 is achieved.
It significantly improved the bioavailability and liver targeting efficiency of CY-09, significantly inhibited the necrosis of liver tissue induced by LPS/D-GalN and increased ALT and AST activities, improved liver function, and had the prospect of promotion and application.
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Figure CN120093696A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of pharmaceutical preparations, and in particular relates to albumin nanoparticles loaded with CY-09, and a preparation method and application thereof. Background Art
[0002] Acute liver injury is one of the most common liver diseases in the world. Viral infection, specific drug toxicity, autoimmune hepatitis, and excessive drinking can all lead to acute liver injury. Due to its acute and destructive characteristics, as well as the lack of timely and effective liver support treatment, many cases of acute liver injury can rapidly develop into severe hepatitis or even life-threatening acute liver failure. However, the current treatment options for acute liver injury are limited, so there is an urgent need to develop new therapeutic drugs for acute liver injury.
[0003] In recent years, studies have found that in the occurrence of liver diseases including acute liver injury, the activation of inflammasomes, especially the activation of NLPR3 inflammasomes, plays an important role. Inhibition of NLRP3 inflammasome activation has become an important target for treatment (PMID: 34752711). CY-09 is an NLRP3 inhibitor. Studies have found that CY-09 can alleviate acute liver injury caused by thioacetamide treatment during severe hyperglycemia (PMID: 31625631). However, CY-09 has poor water solubility, low bioavailability, and cannot act specifically on the liver in vivo, which hinders its in vivo effect. In order to overcome these shortcomings, it is necessary to use an appropriate drug delivery system to improve the bioavailability of CY-09 and achieve liver targeting.
[0004] Albumin is a protein present in plasma with hydrophobic regions that can bind to poorly soluble compounds through hydrophobic interactions. The preparation of albumin nanoparticles is a feasible method to improve drug solubility and create effective carriers to regulate drug delivery. In addition, as a nanoparticle, most of the nanoparticles passively accumulate in the liver after systemic administration, which gives albumin nanoparticles an inherent advantage in targeting drugs to the liver for liver disease intervention. In addition, due to the biodegradability and biocompatibility of albumin and good safety, paclitaxel albumin nanoparticles have been approved by the FDA to enter the market, and a large number of drug-loaded albumin nanoparticles have been developed and entered preclinical studies and clinical trials. However, different drugs have different encapsulation rates and stability due to differences in hydrophobicity, molecular size and charge properties. If there is no optimized method for preparing albumin nanoparticles loaded with the drug to improve the encapsulation rate and stability for a specific drug, its subsequent clinical application will be greatly limited.
[0005] At present, there is no application of delivery system to improve the bioavailability and liver targeting efficiency of CY-09 and its therapeutic effect on acute liver injury, and there is no report on using albumin nanoparticles as a CY-09 delivery system to improve the therapeutic effect on acute liver injury. Summary of the invention
[0006] In order to solve the above problems, the present invention provides an albumin nanoparticle loaded with CY-09, which is an albumin nanoparticle prepared by taking CY-09 as an active ingredient and adding pharmaceutically acceptable excipients.
[0007] Furthermore, the auxiliary material consists of albumin and soybean oil.
[0008] Furthermore, the mass ratio of CY-09 to soybean oil is 1:1 to 10, preferably 1:2.
[0009] Furthermore, the mass ratio of CY-09 to albumin is 1:10 to 40, preferably 1:20.
[0010] Furthermore, the albumin is human serum albumin or bovine serum albumin.
[0011] The present invention also provides a method for preparing the above-mentioned medicine, which comprises the following steps:
[0012] 1) Dissolve CY-09 and soybean oil in an organic solvent according to the proportion;
[0013] 2) Weigh albumin in proportion and dissolve it in PBS, add it to the mixture obtained in step 1), sonicate, remove the organic solvent by rotary evaporation under reduced pressure, centrifuge, and filter with a 0.22 μm pore size filter membrane to obtain the product.
[0014] Furthermore, in step 1), the organic solvent is dichloromethane-ethyl acetate; the volume ratio of the dichloromethane-ethyl acetate is 0.5-2:1, preferably 1:1.
[0015] Furthermore, in step 2), the ultrasound is performed in an ice bath for 5 to 15 minutes, during which the ultrasound is stopped for 3 seconds every 3 seconds; the centrifugation speed is 18000×g, the time is 5 to 20 minutes, and the temperature is 2 to 8°C; the albumin is dissolved in PBS so that the albumin concentration is 5 to 20 mg / mL, preferably 10 mg / mL.
[0016] Finally, the present invention provides a use of the above-mentioned drug in preparing a drug for treating liver injury.
[0017] Furthermore, the drug is a drug that has a therapeutic effect on acute chemical liver injury.
[0018] Furthermore, the drug is a drug that has a therapeutic effect on acute liver injury caused by LPS / D-GalN.
[0019] The albumin nanoparticles loaded with CY-09 developed by the present invention through a specific auxiliary material composition have high encapsulation efficiency and good stability. They are applied to acute liver injury, enhance the targeting of the active ingredient CY-09 to the liver, and improve its bioavailability in vivo. Animal experiments show that compared with free CY-09, the albumin nanoparticles loaded with CY-09 of the present invention significantly inhibit LPS / D-GalN-induced liver tissue necrosis and increased ALT and AST activities, improve liver function, and have a prospect for promotion and application.
[0020] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.
[0021] The following is a further detailed description of the above contents of the present invention through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 Characterization of CY-09 loaded albumin nanoparticles (A: appearance; B: particle size distribution; C: zeta potential).
[0023] Figure 2 Stability analysis of CY-09 loaded albumin nanoparticles at 4°C (A: particle size; B: PDI; C: Zeta potential).
[0024] Figure 3 Biodistribution of DiD-loaded albumin nanoparticles in mice with acute liver injury induced by LPS / D-GalN. A) In vivo imaging of free DiD and DiD@ANPs groups; B) Distribution of free DiD and DiD@ANPs in major organs.
[0025] Figure 4 Effects of CY-09-loaded albumin nanoparticle intervention on mice with acute liver injury induced by LPS / D-GalN (A: liver macroscopically; B: H&E staining; C: quantification of necrotic areas in liver sections by H&E staining; D: ALT; E: AST). ****P<0.0001, ***P<0.001, **P<0.01. DETAILED DESCRIPTION
[0026] Example 1 Preparation of the drug of the present invention
[0027] 1) CY-09 and soybean oil were dissolved in dichloromethane-ethyl acetate (v / v=1:1) at a mass ratio of 1:2;
[0028] 2) HSA was weighed at a mass ratio of CY-09 to HSA of 1:20, dissolved in PBS (pH 7.4) to a final albumin concentration of 10 mg / mL, and then added to the mixture obtained in step 1), and ultrasonicated for 10 min (on: 3 s, off: 3 s) using a probe sonicator at 250 W under an ice bath, followed by removing the organic solvent by rotary evaporation under reduced pressure, followed by centrifugation for 10 min (18000×g, 4°C) to remove unencapsulated drugs, and then filtered through a 0.22 μm pore size filter membrane to obtain the product.
[0029] The beneficial effects of the present invention are described below through test examples.
[0030] Experimental Example 1 Preparation of CY-09-loaded albumin nanoparticles and their application in acute liver injury
[0031] 1. Methods
[0032] 1. Materials and Methods
[0033] 1.1 Materials
[0034] CY-09 was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (Shanghai, China), human serum albumin (HSA) was purchased from Sigma-Aldrich (USA), bovine serum albumin (BSA) and soybean oil were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China), and DiD was purchased from Suzhou Youyi Landi Biotechnology Co., Ltd. (Suzhou, China). All other chemical reagents used were of AR grade.
[0035] 1.2 Preparation of nanoparticles
[0036] CY-09 and soybean oil were dissolved in dichloromethane-ethyl acetate (v / v=1:1), and an albumin solution prepared in PBS (pH 7.4) was added. Ultrasonication was performed with a probe sonicator at 250W for 10 min (on: 3s, off: 3s) under an ice bath, and then the organic phase was removed by rotary evaporation under reduced pressure, followed by centrifugation for 10 minutes (18000×g, 4°C) to remove the unencapsulated drug, and then filtered with a 0.22μm pore size filter membrane. The obtained CY-09-loaded albumin nanoparticles (CY-09@ANPs) ( Figure 1 A) Store at 4°C.
[0037] The albumin nanoparticles loaded with fluorescent dye DiD (DiD@ANPs) required for the biodistribution experiment of nanoparticles were also prepared according to the above method, in which the fluorescent dye DiD was used instead of CY-09.
[0038] 1.3 Characterization of prepared nanoparticles
[0039] 1.3.1 Particle size, polydispersity index (PDI) and zeta potential
[0040] The sample was diluted 10 times with ultrapure water and placed in a cuvette. The particle size and PDI of the nanoparticles were detected using a nanoparticle size potentiometer (Zetasizer Pro, UK). The sample was then placed in a potential detection cuvette to measure the Zeta potential.
[0041] 1.3.2 Encapsulation efficiency and drug loading
[0042] 0.9 mL DMSO was added to 0.1 mL nanoparticles, ultrasonicated for 1 h, and then centrifuged at 4 ° C and 18000 × g for 10 min. The supernatant was aspirated and the mass of CY-09 loaded in the nanoparticles was obtained by measuring the absorbance at 390 nm (maximum absorption wavelength of CY-09) with a spectrophotometer. The encapsulation efficiency and drug loading were calculated using the following formula:
[0043] Encapsulation efficiency = loaded CY09 mass / added CY-09 mass × 100%
[0044] Drug loading = loaded CY09 mass / nanoparticle mass × 100%
[0045] 1.3.3 Stability
[0046] The method for evaluating the stability of nanoparticles stored at 4°C is as follows: samples are taken out at different time points, and the particle size, PDI and Zeta potential are detected according to the method described in 1.3.1 to monitor their changes.
[0047] 2. Animal experiments
[0048] Male C57BL / 6 mice aged 6–8 weeks were used for the experiments. All animal experiments were in accordance with the Guiding Principles for the Care and Use of Laboratory Animals of West China Hospital, Sichuan University. The experimental protocol of this study was evaluated and approved by the Ethics Committee of Sichuan University.
[0049] 2.1 Biodistribution of nanoparticles
[0050] Male C57BL / 6 mice aged 6-8 weeks were intraperitoneally injected with 10 μg / kg LPS and 500 mg / kg D-GalN to establish an acute liver injury model. DiD@ANPs and free DiD (dosage of 40 μg / kg DiD) were injected through the tail vein 0.5 h later. In vivo imaging (SI Imaging, US) was performed 4 h after modeling, and the main organs of the mice were collected for fluorescence analysis.
[0051] 2.2 Detection of the efficacy of nanoparticles on mice with acute liver injury induced by LPS / D-GalN
[0052] The animals were randomly divided into 5 groups, of which 4 groups were injected intraperitoneally with 10 μg / kg LPS and 500 mg / kg D-GalN to induce acute liver injury, and 0.5 h after induction, 1) PBS, 2) empty nanoparticles (ANPs), 3) 0.1 mg / kg CY-09, and 4) 0.1 mg / kg CY-09@ANPs were injected into the tail vein respectively; healthy mice without any treatment were set as the normal group. After 4 h of LPS / D-GalN induction, liver tissue and blood samples were collected for subsequent analysis.
[0053] 2.3 Histological analysis
[0054] Liver injury was analyzed by hematoxylin-eosin (H&E) staining. The tissue was placed in 4% paraformaldehyde, then dehydrated, paraffin-embedded, and sectioned. Thereafter, it was stained with H&E. It was then observed under an optical microscope (Nikon Eclipse 80i, Nikon, Japan).
[0055] 2.4 Serum ALT and AST determination
[0056] Liver function was assessed by measuring the levels of ALT and AST in serum. Serum was obtained from blood by centrifugation at 3500 rpm and 4°C for 10 minutes. Serum ALT and AST levels were determined according to the instructions of the kit ((Nanjing Jiancheng, China).
[0057] 2.5 Statistical analysis
[0058] Data are expressed as mean ± SD. Statistical analysis was performed using Prism 6 (GraphPad Software, USA). Differences between groups were analyzed by one-way analysis of variance (ANOVA) and t-test.
[0059] 2. Results
[0060] 1. Optimization of preparation conditions of albumin nanoparticles loaded with CY-09
[0061] Albumin nanoparticles are nanoparticles made of albumin as the main material, and may also include additives such as cross-linking agents, surfactants and stabilizers to improve the encapsulation efficiency and stability of the nanoparticles. Due to the different physical and chemical properties of the loaded active pharmaceutical ingredients, the selected additives are different.
[0062] Through a large number of preliminary screenings, it was finally determined that the combination of albumin and soybean oil as excipients can form CY-09 nanoparticles with high encapsulation efficiency and stable storage. Further screening of the soybean oil addition ratio found that the soybean oil addition ratio significantly affected the effect of albumin nanoparticles loading CY-09. As can be seen from Table 1, with the increase in the soybean oil addition ratio, the encapsulation rate increased. When the mass ratio of CY-09 to soybean oil was 1:2, the encapsulation rate was higher than 90%. With the increase in the amount of soybean oil added, the encapsulation rate did not increase significantly, but the drug loading decreased. We chose a mass ratio of CY-09 to soybean oil of 1:2 for subsequent experiments.
[0063] Table 1 Effect of soybean oil addition ratio on albumin nanoparticles loaded with CY-09
[0064]
[0065] The ratio of drug to albumin will also affect the encapsulation rate. As can be seen from Table 2, when the mass ratio of CY-09 to albumin is 1:10, the encapsulation rate is less than 60%, which may be due to insufficient albumin binding to the drug. When the amount of albumin added increases and the mass ratio of CY-09 to albumin is 1:20, the encapsulation rate can reach more than 90%. However, as the amount of albumin added continues to increase, when the mass ratio of CY-09 to albumin is 1:40, the drug loading decreases. Therefore, we chose a mass ratio of CY-09 to albumin of 1:20 for subsequent experiments.
[0066] Table 2 Effect of the ratio of drug to albumin on albumin nanoparticles loaded with CY-09
[0067]
[0068] The albumins used to prepare albumin nanoparticles generally include human serum albumin and bovine serum albumin. As can be seen from Table 3, there is no significant difference in the encapsulation efficiency and drug loading of the albumin nanoparticles loaded with CY-09 prepared by the two albumins.
[0069] Table 3 Effect of albumin type on albumin nanoparticles loaded with CY-09
[0070]
[0071] 2. Characterization of optimized CY-09 loaded albumin nanoparticles
[0072] like Figure 1 As shown in B, the particle size of the optimized CY-09-loaded albumin nanoparticles is 160.3±1.2nm, and the PDI is 0.1289±0.013, indicating that the particle size distribution is uniform. The Zeta potential is -18.54±0.32mV ( Figure 1C), indicating that the nanoparticles are not easy to aggregate due to electrostatic repulsion, and obtain better dispersion stability. The nanoparticles were stored at 4°C for 4 weeks, and the particle size, PDI and Zeta potential did not change significantly ( Figure 2 ).
[0073] 3. Biodistribution of albumin nanoparticles
[0074] The biodistribution of albumin nanoparticles loaded with DiD in animals was studied. Figure 3 As shown in A, compared with free DiD, DiD-loaded albumin nanoparticles showed higher fluorescence intensity in the upper abdomen of mice. Figure 3 As shown in B, the fluorescence accumulated in the liver of DiD-loaded albumin nanoparticles was significantly higher than that in other organs and stronger than that of the free DiD group, indicating that the albumin nanoparticles used in this study can promote the targeted delivery of the loaded drugs to the damaged liver.
[0075] 4. Therapeutic effect of CY-09-loaded albumin nanoparticles on LPS / D-GalN-induced mice
[0076] The LPS / D-GalN-induced acute liver injury mouse model was used to investigate the therapeutic effect of albumin nanoparticles loaded with CY-09. Figure 4 As shown in A, the liver tissue turned black after modeling, indicating severe liver damage. The gross condition of the liver was significantly improved in the group injected with albumin nanoparticles loaded with CY-09. We used hematoxylin and eosin (H&E) stained liver sections to assess liver damage. Figure 4 As shown in BC, LPS / D-GalN induced liver congestion, massive cell necrosis, and liver structural damage. The intervention of albumin nanoparticles loaded with CY-09 greatly reduced liver necrosis and improved liver structure, and the effect was significantly better than that of free CY-09. LPS / D-GalN-induced hepatocyte necrosis also released hepatocyte enzymes such as ALT and AST. Figure 4 As shown in Figures DE, compared with the normal group, the levels of ALT and AST in serum were significantly increased after LPS / D-GalN. The intervention of albumin nanoparticles loaded with CY-09 significantly inhibited the increase of ALT and AST activity induced by LPS / D-GalN, and the effect was stronger than that of free CY-09.
[0077] In summary, the albumin nanoparticles loaded with CY-09 developed by the present invention with albumin and soybean oil as excipients in a specific ratio have high encapsulation efficiency and good stability. When applied to acute liver injury, the targeting of the active ingredient CY-09 to the liver is enhanced, and its bioavailability in the body is improved, with significant therapeutic effect on acute liver injury.
Claims
1. A CY-09 loaded albumin nanoparticle, characterized in that: The albumin nanoparticles are prepared by taking CY-09 as the active ingredient and adding pharmaceutically acceptable excipients.
2. The drug according to claim 1, characterized in that: The auxiliary material consists of albumin and soybean oil.
3. The drug according to claim 1 or 2, characterized in that: The mass ratio of CY-09 to soybean oil is 1:1-10, preferably 1:2; the mass ratio of CY-09 to albumin is 1:10-40, preferably 1:20; and the albumin is human serum albumin or bovine serum albumin.
4. A method for preparing the drug according to any one of claims 1 to 3, characterized in that: It includes the following steps: 1) Dissolve CY-09 and soybean oil in an organic solvent according to the proportion; 2) Weigh albumin in proportion and dissolve it in PBS, add it to the mixture obtained in step 1), sonicate, remove the organic solvent by rotary evaporation under reduced pressure, centrifuge, and filter with a 0.22 μm pore size filter membrane to obtain the product.
5. The method according to claim 4, characterized in that: In step 1), the organic solvent is dichloromethane-ethyl acetate; the volume ratio of dichloromethane-ethyl acetate is 0.5-2:1, preferably 1:
1.
6. The method according to claim 4, characterized in that: Step 2) The ultrasound is carried out in an ice bath for 5 to 15 minutes, during which the ultrasound is stopped for 3 seconds every 3 seconds; the centrifugation speed is 18000×g, the time is 5 to 20 minutes, and the temperature is 2 to 8°C; the albumin is dissolved in PBS to make the albumin concentration 5 to 20 mg / mL, preferably 10 mg / mL.
7. Use of the drug according to any one of claims 1 to 3 in the preparation of a drug for treating liver injury.
8. The use according to claim 7, characterized in that: The drug is a drug having a therapeutic effect on acute chemical liver injury, preferably a drug having a therapeutic effect on acute liver injury caused by LPS / D-GalN.