Kukoamine A liver targeting delivery system based on ZIF-8 nano-carrier as well as preparation method and application of Kukoamine A liver targeting delivery system

Abstract

The invention relates to a Kukoamine A liver targeting delivery system based on a ZIF-8 nano-carrier as well as a preparation method and application of the Kukoamine A liver targeting delivery system. The system comprises a ZIF-8 nano carrier and a Kukoamine A, wherein the Kukoamine A is packaged in the ZIF-8 nano carrier; the surface of the ZIF-8 nano-carrier is modified with a tumor cell membrane and / or folic acid. According to the system, ZIF-8 is used as a carrier, and tumor cell membranes and folic acid modification are combined, so that the liver targeting property and the treatment effect of Kukoamine A are remarkably improved. The nano-carrier system not only improves the drug delivery efficiency of Kukoamine A, but also shows a remarkable anti-tumor effect in vivo, and can effectively inhibit tumor growth and prolong the lifetime of mice. After the system is combined with anti-PD-1 immunotherapy, the treatment effect is further enhanced, which indicates that the system can promote the sensitivity of tumors to immunotherapy. The invention provides a novel liver targeting delivery platform, and has a potential clinical application prospect, especially in the combined application of tumor therapy and immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of life science research, specifically to a liver-targeted delivery system for Kukoamine A based on ZIF-8 nanocarriers, its preparation method, and its application. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, and its treatment methods mainly include surgical resection, local ablation, chemotherapy, radiotherapy, and targeted therapy. However, despite some progress in existing treatment methods, the efficacy of HCC treatment remains limited due to the complexity of liver tumors, low drug targeting, drug side effects, and the emergence of drug resistance. Therefore, developing new, highly effective, targeted treatments with reduced side effects has become a pressing challenge in the field of HCC treatment.

[0003] Kukoamine A, a natural plant compound, has been shown to possess multiple biological activities, including antitumor, anti-inflammatory, and immunomodulatory effects. However, the application of kukoamine A still faces many challenges, mainly including its low water solubility (<0.1 mg / mL), poor oral absorption, metabolic instability, and poor targeting. These factors limit the clinical application and efficacy of kukoamine A as a drug. Therefore, improving the drug delivery efficiency of kukoamine A, enhancing its targeting, and reducing side effects have become current research hotspots.

[0004] Advances in nanotechnology have offered new possibilities for solving these problems. Nanocarrier systems can effectively encapsulate drugs within nanoparticles and enhance drug targeting through modification, thereby increasing drug concentration at specific sites and reducing side effects in normal tissues. Zeolite imidazole framework-8 (ZIF-8), as a novel metal-organic framework (MOF) material, has shown great potential in drug delivery due to its high drug loading capacity, good biocompatibility, stable structure, and responsiveness to pH changes. ZIF-8 can release drugs in acidic tumor microenvironments and is therefore widely used in targeted drug delivery systems for cancer treatment.

[0005] Although existing research has shown the potential of ZIF-8 vectors in drug delivery, further improving their targeting of the liver and tumors, and combining them with immunotherapy to enhance anti-tumor effects, remains a challenge for current research. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a liver-targeted delivery system for Kukoamine A based on a ZIF-8 nanocarrier, its preparation method, and its applications. This system, by employing ZIF-8 as a carrier and combining it with surface modifications to tumor cell membranes and folic acid, significantly enhances the targeted delivery efficiency of Kukoamine A, improves its drug utilization, and effectively reduces drug side effects. The innovation of this invention lies in enhancing its targeting ability in the liver and tumor microenvironment through modification of the ZIF-8 nanocarrier, and further improving its anti-tumor effect when used in combination with immunotherapy.

[0007] The technical solution adopted in this invention is as follows:

[0008] A liver-targeted delivery system for Kukoamine A based on a ZIF-8 nanocarrier includes: a ZIF-8 nanocarrier; Kukoamine A encapsulated in the ZIF-8 nanocarrier; and the surface of the ZIF-8 nanocarrier is modified with tumor cell membranes and / or folic acid.

[0009] Selection of ZIF-8 nanocarriers:

[0010] ZIF-8, as a metal-organic framework (MOF) material, possesses excellent drug loading capacity, good biocompatibility and stability, and can achieve controlled drug release in acidic environments. By loading Kukoamine A into ZIF-8 nanocarriers, this invention can effectively improve the solubility and stability of Kukoamine A while retaining its antitumor activity.

[0011] Packaging and Loading of Kukoamine A:

[0012] Kukoamine A is loaded onto ZIF-8 nanocarriers via electrostatic adsorption, coordination, or hydrogen bonding, achieving an encapsulation efficiency of 75.95%. This loading method not only protects the efficacy of Kukoamine A but also increases its concentration in tumor tissues, thereby enhancing the therapeutic effect of the drug.

[0013] ZIF-8 Surface Finishing:

[0014] To enhance the system's targeting specificity, this invention modifies the surface of the ZIF-8 nanocarrier by incorporating tumor cell membranes and folic acid molecules. Tumor cell membranes can improve drug targeting to tumors through their specific receptor recognition mechanism, while folic acid further enhances targeting to the liver by binding to folic acid receptors on the surface of liver cells. This modification allows the ZIF-8 nanocarrier to more effectively target the liver and tumor tissues while reducing impact on normal tissues.

[0015] pH-responsive drug release:

[0016] The ZIF-8 nanocarrier of this invention exhibits excellent pH responsiveness, accelerating drug release in acidic tumor microenvironments while maintaining a slower release rate under neutral or physiological pH conditions. This characteristic allows for concentrated drug release within the tumor region, thereby improving drug targeting and therapeutic efficacy.

[0017] Combined use with immunotherapy:

[0018] This invention also evaluated the combined effect of Kukoamine A and anti-PD-1 immunotherapy. Studies showed that Kukoamine A can enhance the sensitivity of tumors to anti-PD-1 therapy, thereby significantly inhibiting tumor growth and prolonging the survival of mice. Therefore, the system provided by this invention not only has independent efficacy in tumor treatment but also synergistically enhances therapeutic effects when combined with immunotherapy.

[0019] The Kukoamine A liver-targeted delivery system based on ZIF-8 nanocarriers provided by this invention can effectively increase the concentration of the drug in target organs (such as the liver and tumor tissue), reduce the side effects of the drug in healthy tissues, and significantly enhance the therapeutic effect on tumors. Furthermore, the controlled release mechanism of the drug and its combined application with immunotherapy give this invention broad potential for clinical application.

[0020] The advantages of this invention lie in its simple and effective preparation method, high drug loading capacity, good biocompatibility and targeting, and its ability to significantly enhance the clinical application value of Kukoamine A in tumor treatment, providing a novel solution for the treatment of hepatocellular carcinoma and other tumors. Attached Figure Description

[0021] Figure 1 The standard curve of kukoamine A was established by high performance liquid chromatography.

[0022] Figure 2 Transmission electron microscopy images of ZIF-8@kukoamine A (ZIF-8@KukA) nanoparticles with different surface modifications.

[0023] Figure 3 X-ray diffraction patterns of ZIF-8@KukA nanoparticles under different conditions.

[0024] Figure 4 Particle size distribution of ZIF-8@KukA, ZIF-8@KukA-EPC and ZIF-8@KukA-MEM-FA nanoparticles.

[0025] Figure 5Zeta potential measurement of ZIF-8@KukA, ZIF-8@KukA-EPC and ZIF-8@KukA-MEM-FA nanoparticles.

[0026] Figure 6 Cumulative drug release curves of kukoamine A from nanoparticles under different pH conditions.

[0027] Figure 7 A schematic diagram of the experimental design for animal therapy research.

[0028] Figure 8 Bioluminescence images of in situ liver tumors in each treatment group on days 7, 14, and 21 post-transplantation.

[0029] Quantitative analysis of bioluminescence intensity.

[0030] Figure 9 Macroscopic morphological images of in situ liver tumors harvested from treated mice and statistical data on tumor size in each treatment group.

[0031] Figure 10 Kaplan-Meier survival analysis of orthotopic tumor mice established from hepatocellular carcinoma cell lines.

[0032] Figure 11 A schematic diagram of the experimental design for an animal study on the enhancement of anti-PD-1 treatment effects by liver-targeted ZIF-8-encapsulated kukoamine A.

[0033] Figure 12 Quantitative analysis of bioluminescence images and bioluminescence intensity of in situ liver tumors in each treatment group on days 7 and 21 post-transplantation.

[0034] Figure 13 Macroscopic morphological images of in situ liver tumors harvested from treated mice and statistical data on tumor size in each treatment group.

[0035] Figure 14 Kaplan-Meier survival analysis of orthotopic tumor mice established from hepatocellular carcinoma cell lines.

[0036] Figure 15 Serum biochemistry showed no significant differences between the kukoamine A group and the DMSO control group in creatinine (CRE), urea, creatine kinase (CK), CK-MB, AST, ALT, albumin (ALB), and total bilirubin (TBIL).

[0037] Figure 16 Representative H&E staining images of major organs (heart, liver, lungs, kidneys, spleen, and intestines) did not show significant histological abnormalities.

[0038] Figure 17 Representative tumor images of Hepa1-6 cells after treatment with DMSO and kukoamine A.

[0039] Figure 18 Quantitative analysis of tumor volume.

[0040] In all statistical charts, data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significant difference. Detailed Implementation

[0041] Example 1: ZIF-8 nanocarrier enhances liver-targeted delivery of Kukoamine A and inhibits tumor progression

[0042] ZIF-8 nanocarrier enhances liver-targeted delivery of Kukoamine A and inhibits tumor progression. Zeolite imidazole framework-8 (ZIF-8) is an effective drug delivery carrier due to its high drug loading capacity, excellent pH response, and mild synthesis conditions. It can achieve controlled release of drugs in acidic tumor environments while maintaining drug activity, making it very suitable for delivering small molecule drugs and nucleic acids. Therefore, this invention studies the encapsulation and delivery of Kukoamine A using ZIF-8 carrier with additional surface modification. The preparation of ZIF-8 carrier encapsulating Kukoamine A and the surface modification are as follows: (1) Cell membrane extraction of Huh7 cells (approximately 5 × 10⁻⁶ cells). 8 Cells were first digested, centrifuged, and then washed once with PBS buffer. The cell pellet was resuspended in an extraction solution containing 225 mM mannitol, 0.5 mM EDTA, 30 mM Tris-HCl, and a 1% (w / v) mixture of protease inhibitors. The cell suspension was placed on ice and intermittently sonicated at 90 W for 10 minutes using a probe sonicator. The lysis buffer was then centrifuged at 800 × g for 10 minutes at 4°C. The resulting supernatant was then centrifuged at 10,000 × g for 30 minutes at 4°C to precipitate cell membrane fragments, which were collected and stored for subsequent use.

[0043] (2) Drug loading procedure: A stock solution of 2.5 mg / mL Kukoamine A was prepared using anhydrous ethanol. For drug loading, 2 mL of the drug solution was mixed with 50 mg of nano-sized ZIF-8 particles and stirred continuously overnight to ensure complete adsorption of the drug into the mesoporous framework, yielding drug-loaded KukA@ZIF-8 particles. Subsequently, an appropriate amount of lecithin was added to the suspension to seal the surface pores. The particles were collected by centrifugation at 5,000 × g for 10 minutes to remove unencapsulated drug molecules, and then washed three times with pure water to obtain lecithin-coated KukA-EPC@ZIF-8 particles. The final product was lyophilized and stored at 4°C for later use.

[0044] (3) Membrane Coating and Folic Acid Modification: Huh7 cell membrane fragment suspension was mixed with KukA-EPC@ZIF-8 solution at a mass ratio of 2:1. The mixture was then extruded sequentially through 400 nm and 200 nm polycarbonate membranes using an Avanti mini extruder, 10 times for each pore size. Uncoated cell membrane fragments were removed by centrifugation at 5,000 × g, and the resulting membrane-coated drug-loaded particles (KukA@ZIF-8@MEM) were resuspended in deionized water. For folic acid modification, DSPE-PEG2000-FA was added to the particle suspension at a mass ratio of 1:80 and incubated overnight at 45°C. Unbound DSPE-PEG2000-FA was then removed by centrifugation, finally yielding folic acid-mediated membrane-coated Kukoamine A ZIF-8 particles (KukA@ZIF-8@MEM-FA). Based on the standard curve generated in the range of 0–30 μg / mL (R² = 0.9985), the encapsulation efficiency was calculated to be 75.95%, confirming the suitability of ZIF-8 for Kukoamine A delivery. Figure 1 The targeting of tumors and liver can be enhanced by using tumor cell membranes and folic acid through coating and modified carriers. Therefore, a ZIF-8-based carrier was designed and modified with tumor cell membranes and folic acid. Transmission electron microscopy (TEM) confirmed that Kukoamine A was successfully encapsulated within the ZIF-8 framework. Compared with uncoated ZIF-8, ZIF-8@KukA-EPC (egg yolk lecithin) and ZIF-8@KukA-MEM (cell membrane)-FA (folic acid) showed a significant "thickened thin layer" structure on the particle surface, indicating an effective coating of lipids or cell membranes. Figure 2X-ray diffraction analysis further confirmed that the drug loading and surface coating did not disrupt the crystal structure of ZIF-8. Characteristic diffraction peaks corresponding to the 011, 002, 112, 022, 013, and 222 planes were retained in the drug-loaded particles (2θ = 7.2°, 10.3°, 12.7°, 14.6°, 16.2°, and 18.8°, respectively). Notably, ZIF-8@KukA retained the characteristic diffraction peaks of Kukoamine A, indicating that some drug is locally present on the surface. The peak intensity in the ZIF-8@KukA-EPC formulation further decreased, indicating that the successful EPC coating effectively masked the characteristic signals of the drug crystals. Figure 3 Dynamic light scattering measurements showed a slight increase in particle size after loading Kukoamine A, with further particle enlargement following EPC or MEM-FA coating, confirming the success of membrane encapsulation. Figure 4 Supplemental potential measurements further validated the surface modification, demonstrating that it improved colloidal stability and dispersibility in biological media. Figure 5 High-performance liquid chromatography (HPLC) revealed significant differences in drug release under different pH conditions. Particularly under acidic conditions (pH 5.0), simulating the tumor microenvironment, drug release was faster and more complete; in contrast, release was slower under water-soluble conditions. Nonlinear regression fitting confirmed the controlled-release behavior of this nanocarrier system. Figure 6 Treatment workflow as follows: Figure 7 As shown. An orthotopic HCC model was established in C57BL / 6 mice. One week after tumor implantation, animals were randomly divided into two groups, receiving either DMSO or ZIF-8@KukA-MEM-FA treatment every 3 days. Tumor progression was monitored weekly using small animal visible light imaging. Imaging results showed that ZIF-8@KukA-MEM-FA significantly inhibited tumor growth compared to the DMSO control group. Figure 8 At the end of the experiment, the final tumor volume measurement further validated these imaging observations. Figure 9 Parallel survival analyses showed that ZIF-8@KukA-MEM-FA treatment prolonged survival, superior to the vector control group (…). Figure 10 ).

[0045] Example 2: Liver-targeted delivery of Kukoamine A enhances the efficacy of anti-PD-1 immunotherapy

[0046] Combined with reference Figure 11-14 Further evaluation of trials of Kukoamine A in combination with anti-PD-1 therapy ( Figure 11 Imaging results showed that, compared with anti-PD-1 therapy alone, combination therapy significantly inhibited tumor growth. Figure 12At the end of the experiment, the final tumor volume measurement further validated these imaging observations. Figure 13 Parallel survival analyses showed that combination therapy prolonged survival, superior to the group receiving anti-PD-1 therapy alone. Figure 14 The results indicate that Kukoamine A can enhance the sensitivity of tumors to anti-PD-1 therapy. Overall, these findings demonstrate that the ZIF-8@KukA-MEM-FA nanocarrier can enhance liver targeting, inhibit tumor growth, and improve the efficacy of anti-PD-1 immunotherapy, highlighting its potential translational prospects in clinical applications.

[0047] Example 3: Kukoamine A had no significant effect on the health and organ function of mice and significantly inhibited tumor growth.

[0048] In the experimental procedure, healthy mice received daily intraperitoneal injections of Kukoamine A for 21 days. Subsequently, the heart, liver, lungs, kidneys, spleen, and intestines of the mice were stained with Hematoxylin and Escherichia coli (H&E), and their serum was subjected to biochemical analysis to assess liver, kidney, and heart function. Serum biochemical analysis showed no significant differences between the Kukoamine A group and the DMSO control group in creatinine (CRE), urea, creatine kinase (CK), creatine kinase isoenzyme (CK-MB), aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (ALB), and total bilirubin (TBIL) (Figure 15). Observation of H&E-stained tissue sections of major organs showed no significant pathological changes in the tissue structure of the heart, liver, lungs, kidneys, spleen, and intestines in the Kukoamine A-treated group compared to the DMSO control group, demonstrating good tissue compatibility (Figure 16). Further tumor experiments showed that the Hepa1-6 cell tumors in the Kukoamine A treatment group were significantly smaller (Figure 17), and quantitative analysis showed that Kukoamine A significantly inhibited tumor growth (p < 0.01) (Figure 18), indicating that it has potential anti-tumor effects.

Claims

1. A Kukoamine A liver-targeted delivery system based on ZIF-8 nanocarriers, characterized by , the system comprises: ZIF-8 nanocarriers; Kukoamine A encapsulated in the ZIF-8 nanocarriers; The surface of the ZIF-8 nanocarriers is modified with tumor cell membranes and / or folic acid.

2. The system of claim 1, wherein , wherein the ZIF-8 nanocarriers can control the release of drugs in an acidic tumor microenvironment.

3. The system of claim 1, wherein, The drug encapsulation efficiency of the ZIF-8 nanocarriers is 75.95%.

4. The system of claim 1, wherein, The drug loading of the Kukoamine A does not affect the crystal structure of ZIF-8, and the characteristic diffraction peaks of ZIF-8 are retained.

5. The system of claim 1, wherein, The size of the ZIF-8 nanocarriers is in the range of 200-300 nm.

6. The system of claim 1, wherein, The release of the Kukoamine A is regulated by pH value, and the release speed is faster and more complete in an acidic environment at pH 5.

0.

7. Use of the system of claim 1 in the preparation of an anti-PD-1 immunotherapy drug.

8. A method for preparing the Kukoamine A liver-targeted delivery system according to claim 1, characterized in that, The method comprises the following steps: Providing ZIF-8 nanocarriers; Loading Kukoamine A into the ZIF-8 nanocarriers; Modifying the surface of the ZIF-8 nanocarriers by adding tumor cell membranes and / or folic acid.

9. The production method according to claim 8, characterized by, The modification step is achieved by co-precipitation or self-assembly.

10. The preparation method according to claim 8, characterized in that, The combination of Kukoamine A and ZIF-8 is achieved by electrostatic adsorption, coordination or hydrogen bonding.

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