Preparation and application of a gambogeylic acid preparation targeting malignant tumors

Abstract

This application relates to the preparation and application of a gambogeylic acid formulation targeting malignant tumors. It employs a three-layer structure: the core is a gambogeylic acid-black phosphorus quantum dot complex, possessing both chemotherapy and photothermal therapy functions; the middle layer consists of cryogenically frozen NK92 MI cell-derived nanovesicles, providing natural immune targeting and killing capabilities; the outer shell is a hyaluronic acid chelate, enabling active targeting and pH / photothermal responsive drug release. The core of its preparation method lies in using coaxial electrospray technology to form the three layers in one step, resulting in a highly efficient process and good formulation stability. This formulation can achieve synergistic photothermal-chemo-immunotherapy and simultaneously possesses near-infrared fluorescence and photoacoustic multimodal imaging capabilities, effectively solving the problems of poor targeting, significant toxic side effects, and lack of real-time monitoring associated with traditional chemotherapy. It demonstrates significant advantages and application potential in the precision treatment and diagnosis of solid tumors.

Description

Technical Field

[0001] This application belongs to the technical field of pharmaceutical preparations, specifically relating to the preparation and application of a gambogeylic acid preparation targeting malignant tumors. Background Technology

[0002] Malignant tumors are a major disease threatening human health. Traditional chemotherapy drugs suffer from poor targeting, significant toxic side effects, and easy development of drug resistance, limiting their clinical application. Gambogic acid (GA), a natural active ingredient extracted from plants of the Clusium family, possesses significant anti-tumor activity and can inhibit tumor growth through multiple mechanisms, including inducing apoptosis and inhibiting angiogenesis. However, GA suffers from poor water solubility, low bioavailability, poor in vivo stability, and certain toxicity to normal tissues, limiting its further clinical application.

[0003] In recent years, nanomedicine delivery systems (NDDS) have provided a new strategy for improving the targeting and efficacy of antitumor drugs. By constructing nanocarriers with specific structures, controlled drug release, prolonged circulation time, enhanced accumulation at tumor sites, and reduced damage to normal tissues can be achieved. Currently, various nanocarriers (such as liposomes, polymer nanoparticles, and quantum dots) have been used in the delivery research of gambogeylic acid, but problems such as low targeting efficiency, low drug loading rate, and lack of multimodal synergistic therapeutic functions still exist.

[0004] Furthermore, vesicles derived from immune cells (such as NK cell membrane vesicles) are widely used to construct biomimetic nanomedicine carriers due to their excellent biocompatibility and natural targeting properties. The synergistic strategy of combining photothermal therapy (PTT) with chemotherapy has also become a research hotspot for improving tumor treatment efficacy. However, how to efficiently integrate gambogeylic acid with immune cell membrane vesicles, photothermal materials (such as black phosphorus quantum dots), and targeting molecules (such as hyaluronic acid) to construct intelligent formulations with a multi-layered "core-middle-shell" structure remains a technological gap.

[0005] Therefore, developing a multifunctional gamboge acid preparation with high targeting efficiency, good stability, and the ability to achieve synergistic treatment integrating photothermal, chemical, and immunomodulatory therapies is of significant scientific research value and clinical application value. Summary of the Invention

[0006] This application provides a preparation and application of a gamboge acid preparation targeting malignant tumors. By constructing a "core-middle-shell" structure, a synergistic therapeutic core is constructed using black phosphorus quantum dots and gamboge acid, and dual targeting is achieved by using NK cell membrane vesicles and a hyaluronic acid shell. This invention provides a multifunctional integrated preparation that can simultaneously achieve photothermal-chemical-immunotherapy three-in-one multimodal imaging, solving the problems of insufficient targeting, low efficiency of single therapy, and lack of real-time imaging.

[0007] This application proposes a gamboge acid preparation targeting malignant tumors. The gamboge acid preparation is a preparation with a core-middle-shell structure, wherein: the core is a gamboge acid complex; the middle layer is a cryogenic NK92 MI cell-nanovesicle; and the shell is a hyaluronic acid chelate.

[0008] Preferably, the gamboge acid complex is a complex of gamboge acid surface modified with black phosphorus quantum dots.

[0009] Preferably, the mass ratio of gambogeylic acid to black phosphorus quantum dots is 9:1.

[0010] Preferably, the hyaluronic acid chelate is a complex of hyaluronic acid and a chelating agent.

[0011] Preferably, the preparation method employs coaxial electrospray technology to encapsulate the gambogeylic acid complex in the core, forming a formulation with a core-middle-shell structure, specifically including the following steps:

[0012] (1) Core preparation: A complex of gamboge acid surface modified with black phosphorus quantum dots was prepared to obtain gamboge acid complex. It should be noted that: under irradiation with 808nm near-infrared light, black phosphorus quantum dots rapidly convert light energy into heat energy, raising the local temperature to ≥45℃, causing tumor cells to thermally coagulate, denature proteins and rupture cell membranes. Gamboge acid can enhance cell membrane fluidity and endocytic activity through thermal effects, making it easier for gamboge acid to enter the cell. High temperature can also accelerate the binding of gamboge acid to intracellular targets, producing photothermal-chemical synergistic cell killing. The two work together to form a photothermal-chemical dual attack, achieving efficient cell apoptosis in a short time and reducing the risk of drug resistance to single drugs.

[0013] (2) Preparation of the middle layer: The above-mentioned gambogeylic acid complex and the frozen NK92 MI cell-nanovesicle complex were mixed at a mass ratio of 1:1 to form a middle layer complex. The NK92 MI cell receptor can recognize the stress ligands regulated on the surface of tumor cells, triggering the specific binding of the NK92 MI cell-nanovesicle complex to tumor cells. When the photothermal-chemical action causes tumor cell stress and surface antigen upregulation, the receptor binding of the NK92 MI cell-nanovesicle complex is tighter, further activating the cytotoxic function of NK cells. The bilayer structure of the vesicle membrane prolongs the drug circulation time and increases the in vivo half-life, achieving synergistic killing of the three-in-one photothermal-chemical-immune system, and significantly improving the overall anti-tumor efficiency.

[0014] (3) Shell preparation: A complex of hyaluronic acid and chelating agent is prepared as the shell material. Hyaluronic acid actively targets CD44 receptor, increases the hydrophilicity of particles in blood, and reduces non-specific adsorption. The metal chelate partially dissociates after photothermal heating in the acidic tumor microenvironment, actively targets and releases in a controlled manner to achieve precise delivery. It disintegrates under photothermal stimulation and releases the middle-core complex simultaneously to trigger controlled release so that the drug is activated only at the tumor site.

[0015] (4) The core of step 1), the middle layer of step 2), and the outer shell of step 3) are respectively used as three layers of fluid in a coaxial electro-spraying device. The temperature is 20±2°C, the relative humidity is 30%~40%, and coaxial electro-spraying is performed at voltage (10kV±1kV), flow rate (core 1.0mL / h, middle layer 2.0mL / h, outer shell 2.0mL / h) and collection (rotation speed 200-400rpm) to obtain a gamboge acid preparation with a core-middle-shell structure.

[0016] (5) The obtained preparation is dried, cross-linked and quality tested to obtain the final gamboge acid preparation.

[0017] Preferably, the preparation method of the gamboge acid surface-modified black phosphorus quantum dot complex is as follows:

[0018] (1) Synthesis of black phosphorus quantum dots: black phosphorus powder was dispersed in N-methylpyrrolidone by low-frequency ultrasonication (20kHz~50kHz) for 30min, and then centrifuged to remove large pieces to obtain black phosphorus quantum dots with a diameter of 2-5nm.

[0019] (2) Garcinia acid dissolution: Garcinia acid is dissolved in dimethyl sulfoxide at 10 mg / mL and kept at 4°C to prevent degradation;

[0020] (3) Surface modification: Black phosphorus quantum dots were slowly added dropwise to gamboge acid solution and magnetically stirred for 2 hours to form gamboge acid complex;

[0021] (4) Purification: Unbound gamboge acid was removed by dialysis (10 kDa) to obtain a homogeneous gamboge acid complex solution.

[0022] Preferably, the preparation method of the middle layer complex is as follows:

[0023] (1) Culture and cryopreservation of NK92-MI cells:

[0024] 1) NK92-MI cells were seeded in RPMI-1640 medium containing 10% fetal bovine serum and IL-2 and cultured at 37°C and 5% CO2 until the logarithmic growth phase. This cell line expressed NK cells and was able to recognize and kill a variety of malignant tumor cells.

[0025] 2) Wash the cells twice with PBS, collect the cell pellet and resuspend it in cryoprotectant (10% dimethyl sulfoxide + 90% cell culture medium), place it in a -80℃ freezer for 30 min to achieve rapid freezing and form partially ruptured cell membrane structures, thus obtaining cryopreserved NK92 MI cells;

[0026] (2) Cryogenic NK92 MI cells-nanovesicles:

[0027] 1) Quickly transfer the frozen NK92 MI cells to an ice bath at 4°C to thaw for 2 min, then add an equal volume of low osmotic pressure lysis buffer (10 mM HEPES, pH 7.4, 1 mM EDTA) and mix gently.

[0028] 2) Centrifuge at 1000 rpm / min for 5 min at 4℃ to remove cell nuclei and unruptured cell debris, and collect the supernatant;

[0029] 3) Centrifuge the supernatant at 35,000 rpm for 2 hours at 4°C. The precipitate is cell membrane fragments.

[0030] 4) Resuspend the cell membrane pellet in PBS (pH 7.4) and sonicate it three times for 10–15 seconds each time at 30W to allow the membrane to reorganize and form NK92 MI cell-derived nanovesicles.

[0031] 5) By extruding 10 times in succession: first pre-treat with a 200nm filter membrane, then extrude 10 times with a 100nm filter membrane to obtain uniform nanovesicles with a particle size distribution of 30-150nm.

[0032] 6) Dialyze at 4°C for 6–8 hours using a dialysis bag with a 10kDa cutoff molecular weight, changing the PBS 3–4 times. This will effectively remove uncoated proteins, salt ions, and residual lysis buffer, ultimately yielding a purified NK92 MI nanovesicle suspension.

[0033] (3) Assembly of the middle layer complex:

[0034] 1) Mix the NK92 MI nanovesicle suspension with the gambogeylic acid complex solution at a mass ratio of 1:1;

[0035] 2) Gentle sonication at 20W for 3 minutes under ice bath conditions to allow the gamboge acid complex to be uniformly embedded in the NK92 MI nanovesicle membrane, forming a mixed system of gamboge acid complex / NK92 MI nanovesicle suspension complex.

[0036] 3) Centrifuge the mixture at 15000 rpm, 30 min, and 4 °C to remove the unloaded gamboge acid complex, collect the precipitate and resuspend it with PBS to obtain the middle layer complex.

[0037] Preferably, the method for preparing the outer shell is as follows:

[0038] (1) Hyaluronic acid activation: Adjust the pH of the hyaluronic acid solution to 4.5-5.0, add NHS at 1.2 times the molar amount of hyaluronic acid COOH, stir at 4°C for 30 min, add EDC at 1.5 times the molar amount of hyaluronic acid COOH, stir at 4°C for 2 h, and adjust the pH to 7.2-7.6 to obtain the carboxyl-activated hyaluronic acid reaction solution;

[0039] (2) Zinc-rich metal ion chelation:

[0040] 1) Slowly add zinc nitrate or zinc chloride dropwise to PBS at pH 7.4-8.0, with a hyaluronic acid to zinc ion mass ratio of 1:0.2. Stir gently for 30 min to allow zinc ions to form coordinate bonds with activated carboxyl groups, thus obtaining hyaluronic acid chelates.

[0041] 2) Purification: Dialyze using a dialysis bag with an MWCO of 10 kDa, changing the water 3–4 times (4 hours each time) to remove unreacted EDC, NHS, zinc-rich metal ions and small molecule byproducts. After dialysis, freeze the solution at -80°C and freeze-dry it under vacuum to obtain hyaluronic acid chelate powder.

[0042] 3) Crosslinking: Dissolve hyaluronic acid chelate powder in PBS with pH 7.4-7.6, add EDC and NHS mixture, and gently stir at 4℃ for 30 min to obtain crosslinked hyaluronic acid-chelate. The mass ratio of EDC to NHS mixture is EDC:NHS=1:1.

[0043] Preferably, the formulation is used in the preparation of a photothermal-chemo-immunotherapy drug for solid tumors. After the tumor site is irradiated with near-infrared light (808nm), the formulation utilizes the photothermal effect generated by gambogeylic acid-black phosphorus quantum dots to achieve a local temperature increase, while releasing gambogeylic acid in the core to exert a chemical pharmacological effect. With the help of natural NK cell receptors provided by NK92 MI cell-derived nanovesicles in the middle layer, it achieves immune recognition and cytotoxic killing of tumor cells, thereby forming a photothermal-immunotherapy synergistic mode for the local eradication of solid tumors and the inhibition of residual micrometastases after surgery.

[0044] Preferably, the formulation also includes its application in the preparation of a multimodal imaging diagnostic reagent for solid tumors. This formulation is used for multimodal imaging diagnosis of tumors: near-infrared fluorescence imaging is achieved through the fluorescence properties (emission wavelength 900-1100nm) of black phosphorus quantum dots in the core; high-contrast photoacoustic signals are obtained under ultrasound / photoacoustic instruments by combining the acoustic properties of the outer shell hyaluronic acid chelate; and targeted enrichment of tumor sites is achieved by utilizing the specific receptors on the surface of the middle NK92 MI nanovesicles, which can be used for preoperative tumor localization, intraoperative real-time navigation, and postoperative efficacy evaluation.

[0045] The beneficial effects of the embodiments in this application are as follows:

[0046] (1) Multimodal synergistic therapy: Photothermal-chemical dual therapy is achieved through the photothermal effect of black phosphorus quantum dots and the chemical toxicity of gamboge; cryogenic NK92MI cell-derived nanovesicles provide natural NK cell receptors to achieve immune recognition and cytotoxic effects of tumor cells, forming a synergistic therapy mode of photothermal-chemical-immune integration.

[0047] (2) The hyaluronic acid shell actively binds to the CD44 receptor highly expressed on the surface of tumor cells, significantly increasing the enrichment of the preparation in tumor tissue; the zinc-rich metal ion chelating layer partially dissociates after the acidic tumor microenvironment and photothermal heating, achieving controlled and precise drug release and reducing exposure to normal tissues.

[0048] (3) The double membrane structure of nanovesicles prolongs the blood circulation half-life and slows down the rapid clearance of drugs. The three-layer structure of core-middle-shell constructed by coaxial electro-spraying avoids particle aggregation and drug leakage caused by multiple processing during the preparation process, ensuring the uniformity and stability of the formulation.

[0049] (4) Multimodal imaging diagnosis: Black phosphorus quantum dots have strong fluorescence in the near-infrared (900-1100nm) band and can be used for real-time near-infrared fluorescence imaging; Hyaluronic acid chelate layer generates significant acoustic signals under ultrasound / photoacoustic conditions, realizing ultrasound / photoacoustic dual-mode imaging; Targeted enrichment of tumor sites is further enhanced through specific receptors on the surface of NK92 MI nanovesicles, which helps with preoperative localization, intraoperative navigation and postoperative efficacy evaluation. Attached Figure Description

[0050] Figure 1 This is a schematic diagram of the release curve in Embodiment 4 of this application;

[0051] Figure 2 This is a schematic diagram showing the tumor inhibition rate of each group in Example 6 of this application. Detailed Implementation

[0052] The technical solutions of this application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0053] Example 1

[0054] Preparation of gambogeylic acid complex - kernel

[0055] (1) Material preparation: black phosphorus powder (Aladdin); gamboge acid (Aladdin, purity ≥97%); dimethyl sulfoxide, N-methylpyrrolidone (Sinopharm Group).

[0056] (2) Synthesis of black phosphorus quantum dots:

[0057] 1) Weigh 5 mg of black phosphorus powder and dissolve it in 10 mL of N-methylpyrrolidone. Disperse the powder by sonication at 40 kHz for 30 min to overcome the van der Waals forces between the black phosphorus layers. Then centrifuge at 10,000 rpm for 5 min to remove undispersed large pieces and collect the supernatant.

[0058] 2) The particle size was confirmed to be in the range of 2-5 nm by transmission electron microscopy.

[0059] (3) Garcinia galanga acid dissolution: Dissolve 10 mg of garcinia galanga acid in 1 mL of dimethyl sulfoxide to a concentration of 10 mg / mL. Store at 4-8℃ to prevent degradation and protect from light for later use.

[0060] (4) Surface modification: On a magnetic stirrer, the speed is set to 100–200 rpm. The above black phosphorus quantum dot solution is slowly added dropwise to the gamboge acid solution. The mass ratio of gamboge acid to black phosphorus quantum dots is strictly controlled at 9:1. Stirring is continued at room temperature for 2 hours so that gamboge acid can be stably modified on the surface of black phosphorus quantum dots through π-π stacking and hydrophobic interaction to form gamboge acid complex.

[0061] (5) Purification: The mixture was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed in deionized water at 4°C for 6 hours. The dialysate was replaced every 2 hours to completely remove unbound gambogey acid molecules. Finally, a homogeneous and stable aqueous solution of gambogey acid complex was obtained and stored at 4°C for later use.

[0062] (6) Characterization and Results

[0063] 1) UV-Vis absorption spectrum: The complex exhibits the characteristic absorption peak of gamboge acid at 380 nm and shows a broad absorption band of black phosphorus quantum dots in the range of 600-900 nm, confirming the successful formation of the complex;

[0064] 2) Photothermal performance test: Take 1 mL of gambogeylic acid complex solution and irradiate it with an 808 nm laser (power density: 1 W / cm²). Record the temperature change using an infrared thermal imager. The solution temperature rises from 25 °C to 48.2 °C within 5 min, indicating that the complex has excellent photothermal conversion efficiency and can meet the requirements of tumor photothermal therapy.

[0065] Example 2

[0066] Preparation of cryogenic NK92 MI cells-nanovesicles-middle layer preparation

[0067] (1) Experimental materials: NK92 MI cell line was obtained from Pronosei Biotechnology Co., Ltd.; culture medium: RPMI-1640 medium supplemented with 10% (v / v) fetal bovine serum (FBS) and 100 IU / mL human recombinant IL-2;

[0068] (2) Experimental methods:

[0069] 1) Culture and cryopreservation of NK92 MI cells:

[0070] a. Cell culture: NK92 MI cells were seeded in RPMI-1640 medium containing 10% fetal bovine serum and IL-2 and cultured at 37°C and 5% CO2 saturated humidity until the logarithmic growth phase.

[0071] b. Cell washing: Collect cells and wash twice with pre-cooled PBS buffer (pH 7.2-7.6);

[0072] c. Cryotherapy: The cell pellet was resuspended in pre-cooled cryoprotectant (10% DMSO + 90% cell culture medium) and placed directly in an ultra-low temperature freezer at -80℃ for 30 minutes to achieve rapid cryotherapy, forming partially ruptured cell membrane structures, and obtaining cryotherapy NK92 MI cells.

[0073] 2) Preparation of cryopreserved NK92 MI cells-nanovesicles: Thawing and lysis: The cryopreserved NK92 MI cells were rapidly transferred to an ice bath at 4°C to thaw, and then an equal volume of low osmotic pressure lysis buffer (10 mM HEPES, 1 mM EDTA, pH 7.4-7.6) was added and gently mixed to obtain NK92 MI cell lysis buffer.

[0074] (3) Removal of cell nuclei and unruptured cells: Centrifuge the lysate at 1000 rpm / min and 4℃ for 5 min, discard the precipitate, and collect the supernatant.

[0075] (4) Membrane fragment collection: The supernatant was subjected to ultracentrifugation at 35,000 rpm for 2 hours at 4°C. The supernatant was discarded, and the resulting precipitate was cell membrane fragment.

[0076] (5) Vesicle remodeling: Cell membrane fragments were resuspended in PBS (pH 7.4-7.6). The cells were sonicated three times at a frequency of 30-50 kHz using an ultrasonic cell disruptor to allow the membrane to remodel itself into NK92 MI nanovesicles.

[0077] (6) Vesicle homogenization: NK92 MI nanovesicles were obtained by continuous extrusion: first pre-treatment with a 200 nm filter membrane, and then extrusion with a 100 nm filter membrane 10 times to obtain uniform nanovesicles with a particle size distribution of 30–150 nm.

[0078] (7) Purification: The nanovesicle suspension was placed in a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed against PBS at 4°C for 6–8 h, with the solution changed 3–4 times to remove free protein and small molecule impurities. The purified NK92 MI nanovesicle suspension was finally obtained, and the protein concentration was determined by the BCA method.

[0079] (8) Assembly of the middle layer complex: The NK92 MI nanovesicle suspension and the gamboge acid complex solution were mixed at a mass ratio of 1:1. The mixture was then subjected to mild sonication (20 W power, 3 min time) under ice bath conditions to allow the gamboge acid complex to be uniformly embedded in the NK92 MI nanovesicle membrane, forming a gamboge acid complex / NK92 MI nanovesicle suspension complex mixed system. The mixed system was centrifuged at 15000 rpm, 30 min, and 4 °C to remove the unloaded gamboge acid complex. The precipitate was collected and resuspended in PBS to obtain the middle layer complex.

[0080] (9) Characterization and results: The particle size is 70-120nm and the distribution is uniform.

[0081] Example 3

[0082] Overall assembly and characterization of gambogeylic acid formulations (core-middle-shell)

[0083] (1) Experimental materials: Hyaluronic acid (molecular weight 800kDa, Aladdin); Zinc nitrate (Sinopharm Group); EDC (Sigma-Aldrich); Other materials are the same as in Examples 1 and 2;

[0084] (2) Experimental methods:

[0085] 1) Shell preparation:

[0086] a. Hyaluronic acid activation: Weigh 0.1g of hyaluronic acid and dissolve it in 10mL of deionized water. Adjust the pH to 4.5-5.0 with 0.1mol / L HCl. Add NHS (0.03g) at 1.2 times the molar amount of COOH in the hyaluronic acid solution and stir at 4℃ for 30min. Then add EDC (0.05g) at 1.5 times the molar amount of COOH and continue stirring at 4℃ for 2h. Finally, adjust the pH to 7.2-7.6 with 0.1mol / L NaOH to obtain the carboxyl-activated hyaluronic acid reaction solution.

[0087] b. Metal ion chelation: In PBS at pH 7.4-8.0, zinc nitrate solution was slowly added dropwise, and the mass ratio of hyaluronic acid to zinc ions was controlled at 1:0.2. The mixture was gently stirred for 40 min to allow the zinc ions to form stable coordinate bonds with the activated carboxyl groups, thus obtaining a crude solution of hyaluronic acid-zinc chelate.

[0088] c. Purification and Cross-linking: The crude solution was placed in a 10kDa dialysis bag and dialyzed against PBS at 4°C, with the water changed every 4 hours for a total of 4 times to remove unreacted reagents and small molecule impurities. After dialysis, the solution was transferred to a beaker, and a mixture of EDC and NHS (mass ratio 1:1, total mass of 5% of hyaluronic acid) was added. The mixture was gently stirred at 4°C for 40 minutes to enhance the cross-linking degree of the chelate. Finally, the solution was frozen at -80°C and freeze-dried under vacuum to obtain hyaluronic acid-zinc chelate powder, with a 40% increase in cross-linking degree.

[0089] d. Characterization: The hyaluronic acid-zinc chelate has a particle size of 50-70 nm, is spherical or slightly elliptical, has a smooth surface, a narrow particle size distribution, and shows a weak absorption peak in the 200-400 nm region under ultraviolet light.

[0090] 2) Formulation assembly: Using coaxial electrospray technology, the core of Example 1, the middle layer of Example 2, and the above-mentioned hyaluronic acid-zinc chelate (dissolved in PBS at pH 7.4-7.6) were used as three-layer fluids. Electrospraying was performed under the following conditions: voltage 10kV±1kV, core flow rate 1.0mL / h, middle layer flow rate 2.0mL / h, shell flow rate 2.0mL / h, temperature 20±2℃, relative humidity 30%-40%, and collection speed 300rpm. After drying, cross-linking, and quality inspection, the final gambogeylic acid formulation was obtained.

[0091] Example 4

[0092] In vitro drug release of gambogeylic acid preparations

[0093] (1) Experimental materials: Garcinia cambogia preparation prepared in Example 3, with 10 mg of cambogia cambogia powder accurately weighed; Release medium: Simulated normal body fluid: PBS buffer at pH 7.4 (containing 0.1% Tween 80 to increase the solubility of cambogia cambogia); Simulated tumor microenvironment: PBS buffer at pH 5.5 (containing 0.1% Tween 80).

[0094] (2) Experimental steps:

[0095] 1) System construction for release:

[0096] Take four equal portions of the formulation sample (each containing 2.5 mg of gambogeylic acid), and place them into four dialysis bags. Seal the bags with dialysis bag clips to ensure no leakage. Immerse each of the four dialysis bags into a conical flask containing 50 mL of release medium. Label the flasks as follows:

[0097] Group 1: pH 7.4 PBS, without laser irradiation;

[0098] Group 2: pH 7.4 PBS, with laser irradiation (once a day for 5 minutes each time);

[0099] Group 3: pH 5.5 PBS, without laser irradiation;

[0100] Group 4: pH 5.5 PBS, with laser irradiation (once a day for 5 minutes each time).

[0101] 2) Construction of the release system: Place all conical flasks in a constant temperature water bath shaker, set the temperature to 37±0.5℃, the shaker speed to 100rpm, and keep them away from light throughout the process (except for the laser irradiation period).

[0102] 3) Sample Collection and Detection: At 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h, and 48h of release, 2mL of the release solution was taken from each conical flask, and 2mL of fresh release medium was added simultaneously (to maintain a constant system volume). The concentration of gambogeylic acid in the release solution was determined by HPLC. The HPLC detection conditions were as follows: Column: C18 column (250mm×4.6mm, 5μm); Mobile phase: methanol-water (85:15, v / v); Flow rate: 1.0 mL / min; Detection wavelength: 380nm (characteristic absorption peak of gambogeylic acid); Column temperature: 30℃; Injection volume: 20μL.

[0103] 4) Data Calculation: Based on the concentration determined by HPLC, calculate the cumulative release of gambogeylic acid at each time point using the following formula: Cumulative Release Rate (%) = (V0 × C) n +ΣVᵢ×Cᵢ-1) / m×100% where V0 is the total volume of the release medium (50mL), C n Vᵢ represents the concentration at the nth sampling, Vᵢ represents the volume of each sampling (2 mL), Cᵢ-1 represents the concentration at the (i-1)th sampling, and m represents the total mass of gamboge acid in the preparation (2.5 mg).

[0104] (3) Experimental Results: The release results are shown in Table 1, and the release curves are as follows: Figure 1 As shown, pH sensitivity is the main driving factor of this system. Acidic conditions significantly enhance drug release. Photostimulation plays a synergistic amplification role in acidic environments, while having little effect on release in neutral environments. Dual stimulation (acidity + near-infrared light) can achieve efficient and controllable drug release in the tumor microenvironment, which is consistent with "precise release at the tumor site".

[0105] Table 1. Cumulative release rate over 48 hours

[0106] Group condition 48h cumulative release rate Results Explanation Group 1 pH 7.4, no laser 18.2% The formulation is stable in normal bodily fluids, with minimal drug leakage and reduced toxicity to normal tissues. Group 2 pH 7.4, with laser 25.6% The release was slightly higher than in group 1, but remained low, indicating that laser alone has a limited effect on neutral pH environments. Group 3 pH 5.5, no laser 52.3% An acidic environment promotes the dissociation of the hyaluronic acid shell, resulting in significantly higher drug release compared to neutral conditions. Group 4 pH 5.5, with laser 81.5% The synergistic effect of acid and laser significantly enhances release; a 20%-30% release peak occurs within 1 hour after laser irradiation, achieving precise release at the tumor site.

[0107] Comparative Example

[0108] Targeted hydrophobic antitumor drug nanoformulation

[0109] According to Embodiment 3 of Patent CN105412024B:

[0110] (1) Preparation of hyaluronic acid-albumin conjugate:

[0111] 1.4319 g of hyaluronic acid (molecular weight 5 kDa) and 3.1741 g of human serum albumin (molar amount 1 / 6 of hyaluronic acid) were dissolved in 18 mL of 0.01 M PBS. The pH of the solution was adjusted to 5.4. Then, 1.6470 g of EDCI and 3.3579 g of Sulfo-NHS were added to the above solution and reacted for 15 min. The pH of the solution was adjusted to 7.5, and the reaction was continued to be stirred at room temperature for 4 h. After the reaction was completed, the unbonded hyaluronic acid, unreacted EDCI, Sulfo-NHS and other byproducts were removed by dialyzing. The hyaluronic acid-albumin conjugate was obtained by freeze drying.

[0112] (2) Preparation of carrier solution:

[0113] 1.0594 g of human serum albumin and 0.4606 g of hyaluronic acid-albumin conjugate were dissolved in 150 mL of sterile water to obtain a carrier solution;

[0114] (3) Preparation of drug solution: Dissolve 0.08g of paclitaxel in about 4.3mL of a mixed solvent of chloroform and ethanol (9:1) to obtain the drug solution;

[0115] (4) Preparation of nano-formulations:

[0116] Under high-speed homogenization conditions, the drug solution prepared in step (3) above was added to the carrier solution prepared in step (2) to obtain the primary emulsion. Then, it was circulated 12 times under high pressure homogenization (pressure: 17000psi, material flow rate: 15L / h) to obtain the secondary emulsion. Then, the organic solvent was removed by rotary evaporation under reduced pressure at 15-25℃ and -0.1MPa. The solution was filtered through a 0.22μm filter, and the filtrate was freeze-dried to obtain the paclitaxel-targeted anti-tumor nano-formulation.

[0117] Example 5

[0118] Comparison of stability

[0119] (1) Comparison of reconstitution stability time:

[0120] Take 10.0 mg of each of the lyophilized formulations obtained in Examples 1-3 and the comparative examples, and reconstitute them with 4.0 mL of sterile filtered physiological saline. Observe the state of the suspension, the presence or absence of precipitation, and the time of precipitation at room temperature.

[0121] (2) Stability comparison: 50 mg of the lyophilized formulations obtained in Examples 1-3 and the comparative example were placed in a dialysis bag with a molecular weight cutoff of 5000 and placed in 500 mL of PBS buffer solution (0.01 M, containing 0.5 wt% 80 wt%) at pH 7.4 at 37 °C. A certain amount of PBS buffer solution was taken out periodically, filtered through a 0.22 μm filter membrane, and then determined by high performance liquid chromatography. The results are shown in Table 2.

[0122] Table 2. Reconstitution stability and 24h release rate of the formulation

[0123] sample 24h release rate / % Reconstitution stabilization time / h Example 1 1.71 ＞72 Example 2 1.32 ＞72 Example 3 1.15 ＞72 Comparative Example 2.3 ＞72

[0124] As shown in Table 2, less than 2% of the drug in the formulation of this application was released within 24 hours in PBS buffer solution, which is much lower than that in the comparative example. This indicates that the binding force between the drug and the carrier is stronger, which is more conducive to avoiding or reducing leakage of the drug from the carrier during circulation in vivo and before reaching the target site after administration. This can reduce drug toxicity, improve drug utilization, and facilitate a reduction in dosage. Based on the reconstitution dispersion stability time, the targeted formulation of this application exhibits good particle dispersibility and a weak tendency to aggregate. This result is consistent with the finding that the particle size did not significantly increase before and after reconstitution.

[0125] Example 6

[0126] Evaluation of tumor suppression effect

[0127] (1) Animal models: BALB / c nude mice with colorectal cancer subcutaneous model and breast cancer subcutaneous model were purchased from the National Laboratory Animal Center. The source and use of the animals were approved by the Animal Ethics Committee. The animals were raised and experimented in accordance with the "Regulations on the Management of Laboratory Animals". All animals were raised under SPF conditions, with a temperature of 22±2℃ and a relative humidity of 55±10%. The light and dark conditions were alternated for 12h / 12h. Sterile drinking water and standard feed were provided. The experiment was conducted after 1 week of adaptation.

[0128] (2) Experimental grouping: The two types of tumor model mice were randomly divided into 6 groups, with 10 mice in each group. The grouping and treatment schemes are shown in Table 3.

[0129] Table 3. Drug grouping and treatment methods

[0130] Grouping Handling method control group Serous saline was injected into the tail vein, without laser irradiation. Garcinia cambogia alone The patient was given a tail vein injection of a preparation containing gambogeylic acid (5 mg / kg), without laser irradiation. Example 1 Group Tail vein injection of the formulation of Example 1, without laser irradiation. Example 2 group The formulation of Example 2 was administered via tail vein injection without laser irradiation. Example 3 Group Example 3: Compound preparation (garcinia cambogia 5 mg / kg) administered via tail vein injection, without laser irradiation. Example 3 + Photothermal Group Example 3: The compound preparation (garcinia cambogia, dose 5 mg / kg) was injected via tail vein. 24 hours after injection, the tumor site was irradiated with an 808 nm near-infrared laser (power density 1 W / cm²) for 5 minutes, twice a week.

[0131] (3) Experimental protocol: For colorectal cancer and breast cancer, drug administration was initiated when the tumor volume reached 80-100 mm³, with a 3-day interval between each administration to ensure the drug's half-life and metabolic recovery in the body, avoiding cumulative toxicity. Five administrations were administered, and the tumor inhibition rate was calculated. ×100%, of which, : Average volume at the time of initial administration (80-100 mm³); : Average volume of each group on day 21; The average volume of each group in the control group on day 21; the weight of mice was weighed and recorded weekly, and biochemical tests were performed after the administration ended on day 22.

[0132] (4) Experimental results: SPSS 15.0 software was used for analysis, and the data were presented in the form of... ±s indicates the mean. Analysis of variance was used for comparisons of means among multiple groups, and the LSD-t test was used for pairwise comparisons. P < 0.05 indicated statistical significance, *: P < 0.05, **: P < 0.01; the tumor inhibition rate is as follows: Figure 2 As shown: Control group: 0%; Garcinia cambogia alone group: 36.9%; Example 1 group: 42.6%; Example 2 group: 46.9%; Example 3 group: 66.7%; Example 3 + photothermal group: 84.8%; Mouse weight changes are shown in Table 4: All groups showed weight changes <5%, and no significant toxicity was observed; Blood biochemistry & routine blood tests (day 22) are shown in Table 5: All indicators were within the normal reference range; Immunohistochemical results and HE staining are shown in Table 6: Ki-67 decreased, TUNEL increased, CD31 decreased, and necrotic area increased, indicating the dual effect of photothermal-chemotherapy: inhibiting proliferation, promoting apoptosis, and destroying blood vessels. Histological examination showed that the tumor center necrosis rate in the Example 3 + photothermal group was ≥50%, significantly higher than that in the single-drug group.

[0133] Table 4. Changes in mouse body weight

[0134] Days control group Garcinia cambogia alone Example 1 Group Example 2 group Example 3 Group Example 3 + Photothermal Group 0 0±0 0±0 0±0 0±0 0±0 0±0 7 +2.1±0.8 +1.8±0.7 +1.7±0.6 +1.6±0.6 +1.5±0.5 +1.3±0.5 14 +3.4±1.0 +2.9±0.9 +2.7±0.8 +2.5±0.8 +2.2±0.7 +1.8±0.6 21 +4.0±1.2 +3.2±1.0 +3.0±0.9 +2.8±0.9 +2.4±0.8 +2.0±0.7

[0135] Table 5 Blood Biochemistry & Complete Blood Count Tests

[0136] project control group Garcinia cambogia alone Example 3 + Photothermal Group ALT(U / L) 32±5 34±6 31±5 AST(U / L) 45±7 48±8 41±5 BUN (mmol / L) 5.8±0.4 5.9±0.5 5.4±0.3 CREA (µmol / L) 62±6 63±5 58±4 <![CDATA[WBC(×10 9 / L)]]> 6.2±0.8 6.0±0.7 5.6±0.5 RBC (×10¹² / L) 7.8±0.3 7.7±0.3 8.0±0.3

[0137] Table 6 Immunohistochemical results and HE staining results

[0138] mark control group Garcinia cambogia alone Example 3 Group Example 3 + Photothermal Group Ki-67 (Proliferation Index %) 68±4 45±5 28±3 12±2 TUNEL (apoptosis rate %) 6±1 14±2 24±3 38±4 CD31 (vascular density / field of view) 22±2 18±2 12±1 7±1 HE (necrotic area %) <5 15±3 35±4 55±5

[0139] In summary, through the synergistic effect of "photothermal-chemical-immune", a tumor inhibition effect far exceeding that of single therapy is achieved (tumor inhibition rate as high as 84.8%). It can effectively induce tumor cell apoptosis, inhibit proliferation and destroy blood vessels. The three-layer structure formed in one step using coaxial electro-spraying technology effectively avoids drug leakage and particle aggregation. The formulation has good uniformity and stability, long reconstitution stability time, and extended blood circulation time.

[0140] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application. All equivalent changes and improvements made within the scope of this application shall still fall within the patent coverage of this application.

Claims

1. A gambogeylic acid preparation targeting malignant tumors, characterized in that, The gambogeylic acid formulation is a formulation with a core-middle-shell structure, and the formulation is a multifunctional integrated formulation for photothermal-chemical-immunoassay three-modal imaging, wherein: The core is a gamboge acid complex; The middle layer is composed of cryogenic NK92 MI cells-nanovesicles; The outer shell is a hyaluronic acid chelate; The gamboge acid complex is a complex of gamboge acid surface modified with black phosphorus quantum dots; The hyaluronic acid chelate is a complex of hyaluronic acid and a chelating agent, wherein the chelating agent is zinc nitrate or zinc chloride; The middle layer is prepared by mixing the gamboge acid complex with frozen NK92 MI cells-nanoves at a mass ratio of 1:

1.

2. The gambogeylic acid preparation targeting malignant tumors as described in claim 1, characterized in that, The mass ratio of gambogeylic acid to black phosphorus quantum dots is 9:

1.

3. A method for preparing a gambogeylic acid preparation targeting malignant tumors, characterized in that, The preparation method employs coaxial electrospray technology to encapsulate the gamboge acid complex within a core, thereby creating a formulation with a core-middle-shell structure. The specific steps include: Core preparation: A complex of gambogey acid surface-modified with black phosphorus quantum dots was prepared to obtain the gambogey acid complex; Preparation of the middle layer: The above gambogeylic acid complex and the frozen NK92 MI cell-nanovesicle complex were mixed at a mass ratio of 1:1 to form the middle layer complex; Shell preparation: A complex of hyaluronic acid and a chelating agent was prepared as the shell material; The core of step 1), the middle layer of step 2), and the outer shell of step 3) are respectively used as three layers of fluid in a coaxial electro-spraying device, and coaxial electro-spraying is performed to obtain a gamboge acid preparation with a core-middle-outer shell structure. The obtained preparation was dried, cross-linked, and subjected to quality inspection to obtain the final gamboge acid preparation.

4. The method for preparing a gambogeylic acid preparation targeting malignant tumors as described in claim 3, characterized in that, The preparation method of the gamboge acid surface-modified black phosphorus quantum dot complex is as follows: Synthesis of black phosphorus quantum dots: Black phosphorus powder was dispersed in N-methylpyrrolidone by low-frequency ultrasonication for 30 min, and then centrifuged to remove large pieces to obtain black phosphorus quantum dots with a diameter of 2-5 nm. The low-frequency ultrasonication range was 30 kHz to 50 kHz. Garcinia galanga dissolution: Dissolve garcinia galanga in dimethyl sulfoxide to a concentration of 10 mg / mL and store at 4-8°C. Surface modification: The black phosphorus quantum dots were added dropwise to the gamboge acid solution, and stirred for 2 hours with a magnetic force of 100-200 rpm to form a gamboge acid complex; Purification: Unbound gambogeylic acid was removed by passing the solution through a 10 kDa dialysis bag to obtain a homogeneous gambogeylic acid complex solution.

5. The method for preparing a gambogeylic acid preparation targeting malignant tumors as described in claim 3, characterized in that, The preparation method of the middle layer complex is as follows: Culture and cryopreservation of NK92 MI cells: NK92 MI cells were seeded in RPMI-1640 medium containing 10% fetal bovine serum and IL-2 and cultured at 37°C and 5% CO2 until the logarithmic growth phase. The cells were washed twice with PBS, and the cell pellet was collected and resuspended in cryoprotectant. The cells were placed in a -80°C freezer for 30 min to achieve rapid cryopreservation and form partially ruptured cell membrane structures, thus obtaining cryopreserved NK92 MI cells. The pH of the PBS was 7.2-7.

6. Cryogenic NK92 MI cells-nanovesicles: The frozen NK92 MI cells were rapidly transferred to an ice bath at 4°C to thaw, and then an equal volume of low osmotic pressure lysis buffer was added and gently mixed to obtain NK92 MI cell lysis buffer. The low osmotic pressure lysis buffer was a 10 mM HEPES and 1 mM EDTA buffer with a pH of 7.4-7.

6. The NK92 MI cell lysis buffer was centrifuged at 1000 rpm / min for 5 min at 4℃ to remove cell nuclei and unruptured cell debris, and the supernatant was collected. The supernatant was subjected to ultracentrifugation at 35,000 rpm for 2 hours at 4°C, and the precipitate was collected as cell membrane fragments. The cell membrane fragments were resuspended in PBS at pH 7.4-7.6 and subjected to ultrasonic disruption three times to allow the membrane to reorganize into NK92MI nanovesicles. The ultrasonic frequency was 30kHz-50kHz. The NK92 MI nanovesicles were continuously extruded to obtain uniform nanovesicles with a particle size distribution of 30-150 nm. Free proteins and small molecule impurities were removed using a 10kDa dialysis bag, and finally purified NK92 MI nanovesicle suspension was obtained. Assembly of the middle layer complex: The NK92 MI nanovesicle suspension and the gambogeylic acid complex solution were mixed at a mass ratio of 1:

1. The gambogia complex was uniformly embedded in the NK92 MI nanovesicle membrane by gentle sonication at 20W for 3 minutes under ice bath conditions, forming a mixed system of gambogia complex / NK92 MI nanovesicle suspension complex. The mixture of the gamboge acid complex / NK92 MI nanovesicle suspension complex was centrifuged at 15000 rpm for 30 min at 4 °C to remove the unloaded gamboge acid complex. The precipitate was collected and resuspended in PBS to obtain the middle layer complex.

6. The method for preparing a gambogeylic acid preparation targeting malignant tumors as described in claim 3, characterized in that, The method for preparing the outer shell is as follows: Hyaluronic acid activation: Adjust the pH of the hyaluronic acid solution to 4.5-5.0, add NHS at 1.2 times the molar amount of hyaluronic acid COOH, stir at 4°C for 30 min, add EDC at 1.5 times the molar amount of hyaluronic acid COOH, stir at 4°C for 2 h, and adjust the pH to 7.2-7.6 to obtain the carboxyl-activated hyaluronic acid reaction solution; Zinc-rich metal ion chelation: Slowly add zinc nitrate or zinc chloride dropwise to PBS at pH 7.4-8.0, with a hyaluronic acid to zinc ion mass ratio of 1:0.2, and gently stir for 30 min to obtain hyaluronic acid chelate; Purification: Dialysis was performed using a dialysis bag with an MWCO of 10 kDa, with water changes 3–4 times, each time for 4 hours, to remove unreacted EDC, NHS, zinc-rich metal ions and small molecule byproducts. After dialysis, the solution was frozen at -80°C and freeze-dried under vacuum to obtain hyaluronic acid chelate powder. Crosslinking: The hyaluronic acid chelate powder was dissolved in PBS with a pH of 7.4-7.6, and a mixture of EDC and NHS was added. The mixture was gently stirred at 4°C for 30 min to obtain the crosslinked hyaluronic acid-chelate. The mass ratio of the components in the EDC and NHS mixture was EDC:NHS = 1:

1.

7. The gambogeylic acid formulation targeting malignant tumors as described in any one of claims 1-2, characterized in that, The formulation is used in the preparation of a photothermal-chemo-immunotherapy drug for solid tumors.

8. The gambogeylic acid formulation targeting malignant tumors as described in any one of claims 1-2, characterized in that, The formulation also includes its use in the preparation of multimodal imaging diagnostic reagents for solid tumors.

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