A cisplatin-kaempferol coordination type nano-drug and a preparation method and application thereof

Abstract

The application discloses a cisplatin-kaempferol coordination type nano drug and a preparation method and application thereof, and the method comprises the following steps: mixing cisplatin and a first part of kaempferol in a solvent to form a cisplatin-kaempferol coordination complex; then adding a second part of kaempferol and PEG two amphiphilic materials, so that the second part of kaempferol is self-assembled into a nano carrier under the action of the PEG two amphiphilic materials, and the cisplatin-kaempferol coordination complex is simultaneously loaded into the nano carrier in the self-assembly process to obtain the cisplatin-kaempferol coordination type nano drug; firstly, the cisplatin and a small amount of kaempferol are combined to form a stable coordination complex through a strong coordination bond, then the remaining kaempferol and the PEG two amphiphilic materials are added, the kaempferol is triggered to self-assemble into a nano carrier, and the coordination complex is physically embedded into the nano carrier; the nano drug with uniform particle size and excellent colloidal stability can be obtained, and flexible regulation of the total molar ratio of cisplatin and kaempferol is realized.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, specifically to a cisplatin-kaempferol coordination nanomedicine, its preparation method, and its application. Background Technology

[0002] Cisplatin is a widely used first-line platinum-based chemotherapy drug in clinical practice, demonstrating good therapeutic effects against various solid tumors such as lung cancer, breast cancer, and ovarian cancer. However, the clinical application of cisplatin is limited by its severe toxic side effects, including nephrotoxicity, neurotoxicity, and myelosuppression. Furthermore, the antitumor immune response induced by cisplatin-only chemotherapy is often insufficient to eliminate tumors, and the immunosuppressive mechanisms present in the tumor microenvironment weaken its immune-activating effect, thus limiting therapeutic efficacy.

[0003] Kaempferol is a natural flavonoid polyphenol compound with various biological activities, including anti-inflammatory, antioxidant, and immunomodulatory effects. Studies have shown that kaempferol can improve the tumor immunosuppressive microenvironment by regulating the expression of specific immune-related proteins and can exert a synergistic anti-tumor effect with cisplatin. However, free kaempferol has poor water solubility, a short half-life in vivo, and low tumor targeting, making it difficult to achieve simultaneous delivery with cisplatin in vivo.

[0004] Metal-polyphenol coordination self-assembly technology offers a novel approach for constructing carrier-free nanomedicines. For example, existing technology (CN121971486A) discloses a nanosystem based on metal-polyphenol coordination self-assembly, which forms a nanomedicine delivery system by self-assembling cerium ions with kaempferol in a PEG-8000 solution system. This technology utilizes the coordination interaction between polyvalent metal ions and polyphenol ligands to drive self-assembly, forming structurally stable nanoparticles.

[0005] However, when attempting to apply such metal-polyphenol coordination self-assembly schemes to the co-delivery nanosystem of cisplatin and kaempferol, significant difficulties arise: the products of direct self-assembly of cisplatin and kaempferol suffer from low encapsulation efficiency and poor colloidal stability, making it difficult to achieve effective loading and stable delivery of high doses of kaempferol, which severely restricts the application potential of this system.

[0006] Therefore, developing a nanomedicine capable of efficiently co-loading and stably delivering cisplatin and high-dose kaempferol is of significant clinical value and scientific importance for maximizing their synergistic anti-tumor effects and improving the efficacy of chemotherapy combined with immunotherapy. Summary of the Invention

[0007] The main objective of this invention is to provide a cisplatin-kaempferol coordination nanomedicine, its preparation method, and its application, in order to solve the technical problem that cisplatin and high-dose kaempferol are difficult to load simultaneously and efficiently.

[0008] To achieve the above objectives, this invention provides a method for preparing cisplatin-kaempferol coordination nanomedicines, comprising the following steps: Cisplatin and the first portion of kaempferol were mixed and reacted in a solvent to form a cisplatin-kaempferol coordination complex through coordination. Then, the second portion of kaempferol and a PEGylated amphiphilic material were added, allowing the second portion of kaempferol to self-assemble into a nanocarrier under the action of the PEGylated amphiphilic material. Simultaneously, the cisplatin-kaempferol coordination complex was loaded into the nanocarrier during the self-assembly of the second portion of kaempferol, resulting in the cisplatin-kaempferol coordination nanodrug KP@Ka NPs.

[0009] In the above technical solution, by first forming a stable coordination bond between the hydroxyl group in the first part of the kaempferol molecule and the Pt center of cisplatin, a kaempferol-cisplatin coordination complex is formed. Under the hydrophobic and hydrophilic modification of the PEGylated amphiphilic material, the second part of the kaempferol is triggered to self-assemble into a nanocarrier with uniform particle size, and the kaempferol-cisplatin coordination complex is embedded in it.

[0010] Compared with products obtained by traditional one-pot self-assembly, the nanomedicine KP@Ka NPs prepared by the method of this invention exhibits significantly improved encapsulation efficiency, uniform particle size, and excellent colloidal stability. Furthermore, this nanomedicine can induce immunogenic cell death in tumor cells, upregulate CX43 protein expression to promote ATP efflux, and inhibit the expression of CD39 and CD73, blocking the conversion of ATP to ADO, thereby enhancing the anti-tumor immune response.

[0011] Furthermore, the molar ratio of cisplatin to kaempferol is in the range of 1:3 to 1:14. Cisplatin has a limited Pt center coordination number, typically only able to coordinate meaningfully with the phenolic hydroxyl groups of 2 to 4 kaempferol molecules. If the traditional one-pot method is used for direct self-assembly, the excess kaempferol in the system cannot be stably integrated due to the lack of effective assembly driving force, resulting in the actual content of kaempferol in the product being strictly locked at a low proportion. This invention employs a stepwise cascade self-assembly strategy of first coordinating and then embedding, using kaempferol in two parts: the first part binds to cisplatin through coordination bonds to form a core complex, and the second part self-assembles through non-coordination to form a carrier and physically embed the core complex. Since the amount of carrier added is not limited by the coordination number of cisplatin, only the amount of kaempferol added in the second part needs to be adjusted to achieve flexible control of the total molar ratio of cisplatin to kaempferol in the final product, providing technical feasibility for screening the optimal synergistic therapeutic ratio. In this invention, the molar ratio of cisplatin to kaempferol in the nanomedicine can be flexibly controlled within a wide range of 1:3 to 1:14, breaking through the natural limitation of polyphenol content imposed by the fixed coordination number of cisplatin.

[0012] Furthermore, the PEGylated amphiphilic material is at least one of PEG-DSPE, PEG-lipid, and PEG-amphiphilic block copolymer. This PEGylated amphiphilic material possesses both hydrophobic segments and hydrophilic PEG chains, effectively promoting the self-assembly of kaempferol and stabilizing the nanoparticle structure. Kaempferol molecules have a multi-benzene ring hydrophobic framework and tend to spontaneously aggregate in aqueous solution through hydrophobic interactions and π-π stacking. The hydrophobic segments of the PEGylated amphiphilic material can embed into the hydrophobic core of the kaempferol aggregate, while its hydrophilic PEG chains extend into the aqueous phase, forming a hydration layer on the nanoparticle surface. This hydration layer prevents aggregation and precipitation between nanoparticles through steric hindrance, ensuring colloidal stability.

[0013] Furthermore, it includes the following steps: Cisplatin was dissolved in a solvent, silver nitrate solution was added dropwise and stirred in the dark, then centrifuged and the supernatant was removed to obtain a dechlorinated cisplatin solution; An ethanol solution containing the first portion of kaempferol was added to the dechlorinated cisplatin solution, and the reaction was carried out for 8-24 hours. The solution is prepared by adding 10-160 μL of an ethanol solution containing the second part of kaempferol and PEGylated amphiphilic material under ice bath conditions, followed by sonication, rotary evaporation under reduced pressure, and finally dialysis in water to remove free drug.

[0014] This preparation method employs a stepwise cascaded self-assembly strategy. First, a stable cisplatin-kaempferol coordination complex is formed through silver nitrate dechlorination activation and coordination reaction. In this step, the amount of kaempferol used is only sufficient to meet the coordination requirements, and the formed coordination complex serves as the core for subsequent embedding. Then, under ice bath conditions, the second part of kaempferol is triggered to self-assemble into a carrier and undergoes simultaneous physical embedding. The ice bath condition helps control the self-assembly rate. The newly added second part of kaempferol, unable to find any vacant cisplatin coordination sites, can only aggregate into a nanocarrier through non-coordination interactions such as hydrophobic interactions and π-π stacking, with the assistance of PEGylated amphiphilic materials. During this process, the coordination complex formed in the second step is simultaneously physically embedded into the carrier. Ultrasound provides energy to ensure uniform dispersion of the nanoparticles; rotary evaporation under reduced pressure removes organic solvents; and dialysis removes free, unencapsulated drugs and impurities.

[0015] This invention also provides a cisplatin-kaempferol coordination nanomedicine, prepared by the above-described method. The nanomedicine prepared by this method has a core-shell structure with the coordination complex as the core and a self-assembled carrier shell. When the nanomedicine is taken up by tumor cells, under the acidic intracellular environment and the action of enzymes, the shell gradually dissociates and releases kaempferol, simultaneously exposing the core coordination complex. The core complex further releases active cisplatin under acidic conditions, thereby achieving synchronous delivery of cisplatin and kaempferol, ensuring the synchronous release of the two drugs within the tumor cells and their synergistic effect.

[0016] Furthermore, the cisplatin-kaempferol coordination nanomedicine has a particle size of 50 nm to 250 nm and a zeta potential of -15 to -35 mV. The nanomedicine prepared by the method of this invention has a particle size that can be controlled within the range of 50 nm to 250 nm, and a zeta potential that can be controlled within the range of -15 mV to -35 mV. Under preferred conditions, the obtained nanomedicine particle size is concentrated in the range of 100 to 200 nm, and the zeta potential is concentrated in the range of -20 to -30 mV. This particle size and potential characteristic is beneficial for achieving efficient passive targeted enrichment by utilizing the EPR effect of tumor tissue, while ensuring good colloidal stability.

[0017] The present invention also provides the application of cisplatin-kaempferol coordination nanomedicine in the preparation of drugs for treating tumors.

[0018] Furthermore, the tumor includes at least one of triple-negative breast cancer, lung cancer, liver cancer, ovarian cancer, and pancreatic cancer.

[0019] Beneficial effects: This invention first involves forming a stable coordination complex between cisplatin and a small amount of kaempferol through strong coordination bonds. Then, the remaining kaempferol and a PEGylated amphiphilic material are added, triggering the self-assembly of kaempferol into a nanocarrier. Simultaneously, the coordination complex is physically embedded within the nanocarrier. Compared to products obtained through direct one-pot self-assembly, the nanomedicines (KP@Ka NPs) prepared by this method exhibit significantly improved encapsulation efficiency, uniform particle size, and excellent colloidal stability, yielding nanomedicines with a cisplatin to kaempferol molar ratio of 1:3 to 1:14.

[0020] This invention's nanomedicine achieves a synergistic effect of chemotherapy and immunomodulation by efficiently co-delivering cisplatin and high-dose kaempferol to the tumor site. Cisplatin efficiently induces immunogenic cell death in tumor cells, releasing immunostimulatory factors such as ATP; kaempferol promotes ATP efflux by upregulating CX43 channel protein, while inhibiting CD39 / CD73 protein expression to block the production of the immunosuppressive product adenosine. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the reaction mechanism of the cisplatin-kaempferol coordination nanomedicine (KP@Ka NPs) of the present invention.

[0022] Figure 2 The images shown are transmission electron microscope (TEM) images of KP@Ka NPs, Ka NPs, and KP NPs of the present invention.

[0023] Figure 3 The particle size and zeta potential distribution of KP@Ka NPs provided in Examples 1-3 of the present invention are shown.

[0024] Figure 4 The infrared spectra of KP@Ka NPs, Ka NPs and Cisplatin of the present invention are shown.

[0025] Figure 5 This is a comparison chart of the cisplatin encapsulation efficiency of KP@Ka NPs and KP NPs of the present invention.

[0026] Figure 6 The cumulative cisplatin release rate of KP@Ka NPs and KP NPs of the present invention at different pH values.

[0027] Figure 7 This is a flow cytometry result of KP@Ka NPs and Ka NPs inducing tumor cell apoptosis in vitro, according to the present invention.

[0028] Figure 8 This is a diagram showing the expression results of CD39 protein in tumor cells under the action of KP@Ka NPs and Ka NPs according to the present invention.

[0029] Figure 9 This is a diagram showing the expression results of CD73 protein in tumor cells under the action of KP@Ka NPs of the present invention.

[0030] Figure 10 This is a diagram showing the expression results of CX43 protein in tumor cells under the action of KP@Ka NPs of the present invention.

[0031] Figure 11 This is a graph showing the extracellular ATP levels in tumor cells under the action of KP@Ka NPs of the present invention.

[0032] Figure 12 This is a graph showing the extracellular ADO level in tumor cells under the action of KP@Ka NPs of the present invention.

[0033] Figure 13 This is a flow cytometry analysis result of CRT expression on the surface of tumor cells under the action of KP@Ka NPs of the present invention.

[0034] Figure 14 This is a graph showing the results of DC cell maturation analysis after the supernatant of tumor cells treated with KP@Ka NPs of the present invention.

[0035] The realization of the objective, functional characteristics and advantages of the present invention will be further explained with reference to the accompanying drawings and embodiments. Detailed Implementation

[0036] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials and commonly used chemical reagents used in the embodiments are commercially available products.

[0037] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention.

[0038] The following embodiments further describe the present invention, but these embodiments are not intended to limit the scope of protection of the present invention.

[0039] (I) Preparation of Nanomedicines Reference Figure 1 This invention provides three methods for preparing nanoparticles: the cisplatin-kaempferol coordination nanomedicine of this invention (KP@Ka NPs), the direct self-assembled nanoparticle comparative example (KP NPs), and the kaempferol nanoparticle comparative example (KaNPs).

[0040] 1. Activation by cisplatin dechlorination Weigh 3 mg of cisplatin and dissolve it in 9 mL of Tris solution (1 mM). Then, add 1 mL of Tris solution (1 mM) containing 1.5 mg / mL silver nitrate (cisplatin to silver nitrate molar ratio 1:2) dropwise to the solution and stir in the dark for 12 h. After stirring, centrifuge at 5000 rpm for 10 min, collect the supernatant, and discard the white silver chloride precipitate. Repeat the above steps twice to finally obtain the dechlorinated cisplatin solution (1 mM), i.e., the hydrated cisplatin solution.

[0041] 2. Preparation of cisplatin-kaempferol coordination nanomedicines (KP@Ka NPs) Example 1: (1) Transfer 2 mL of dechlorinated cisplatin solution (1 mM) into a reaction flask, and add 30 μL of anhydrous ethanol solution containing 150 mM kaempferol dropwise under stirring at room temperature. After the addition is complete, react for 24 h to allow the Pt center of cisplatin to combine with the hydroxyl group of kaempferol through a coordinate bond to form a cisplatin-kaempferol coordination complex.

[0042] (2) After the reaction was completed, under ice bath conditions, 10 μL of 150 mM kaempferol ethanol solution and 10 μL of 50 mg / mL DSPE-mPEG2000 chloroform solution were added dropwise to the reaction system, and the mixture was sonicated for 15 min at a power of 200 W. During this process, the newly added kaempferol self-assembled into a nanocarrier through non-coordination under the action of DSPE-mPEG2000, and simultaneously physically embedded the cisplatin-kaempferol coordination complex formed in step (1) inside the nanocarrier.

[0043] (3) After sonication, place the reaction flask on a rotary evaporator and evaporate under reduced pressure at 30°C for 10 min to remove the organic solvent.

[0044] (4) The reaction solution was transferred to a dialysis bag (MWCO 3500) and dialyzed in deionized water for 24 h to remove free drug. The water was changed every 6 h to obtain KP@Ka NPs, which were stored at 4℃ in the dark for later use. By adjusting the amount of kaempferol ethanol solution added, nanomedicines with a cisplatin to kaempferol molar ratio of 1:3 were obtained.

[0045] Example 2: (1) Transfer 2 mL of dechlorinated cisplatin solution (1 mM) into a reaction flask, and add 30 μL of anhydrous ethanol solution containing 150 mM kaempferol dropwise under stirring at room temperature. After the addition is complete, react for 24 h to allow the Pt center of cisplatin to combine with the hydroxyl group of kaempferol through a coordinate bond to form a cisplatin-kaempferol coordination complex.

[0046] (2) After the reaction was completed, under ice bath conditions, 157 μL of 150 mM kaempferol ethanol solution and 30 μL of 50 mg / mL DSPE-mPEG2000 chloroform solution were added dropwise to the reaction system, and the mixture was sonicated for 15 min at a power of 200 W. During this process, the newly added kaempferol self-assembled into a nanocarrier through non-coordination under the action of DSPE-mPEG2000, and simultaneously physically embedded the cisplatin-kaempferol coordination complex formed in step (1) inside the nanocarrier.

[0047] (3) After sonication, place the reaction flask on a rotary evaporator and evaporate under reduced pressure at 30°C for 10 min to remove the organic solvent.

[0048] (4) The reaction solution was transferred to a dialysis bag (MWCO 3500) and dialyzed in deionized water for 24 h to remove free drug. The water was changed every 6 h to obtain KP@Ka NPs, which were stored at 4℃ in the dark for later use. By adjusting the amount of kaempferol ethanol solution added, nanomedicine with a molar ratio of cisplatin to kaempferol of 1:14 was obtained.

[0049] Example 3: (1) Transfer 2 mL of dechlorinated cisplatin solution (1 mM) into a reaction flask, and add 30 μL of anhydrous ethanol solution containing 150 mM kaempferol dropwise under stirring at room temperature. After the addition is complete, react for 24 h to allow the Pt center of cisplatin to combine with the hydroxyl group of kaempferol through a coordinate bond to form a cisplatin-kaempferol coordination complex.

[0050] (2) After the reaction was completed, 77 μL of 150 mM kaempferol ethanol solution and 15 μL of 50 mg / mL DSPE-mPEG2000 chloroform solution were added dropwise to the reaction system under ice bath conditions. The mixture was sonicated for 15 min at a power of 200 W. During this process, the newly added kaempferol self-assembled into a nanocarrier through non-coordination under the action of DSPE-mPEG2000, and simultaneously physically embedded the cisplatin-kaempferol coordination complex formed in step (1) inside the nanocarrier.

[0051] (3) After sonication, place the reaction flask on a rotary evaporator and evaporate under reduced pressure at 30°C for 10 min to remove the organic solvent.

[0052] (4) The reaction solution was transferred to a dialysis bag (MWCO 3500) and dialyzed in deionized water for 24 h to remove free drug. The water was changed every 6 h to obtain KP@Ka NPs, which were stored at 4℃ in the dark for later use. By adjusting the amount of kaempferol ethanol solution added, nanomedicines with a molar ratio of cisplatin to kaempferol of 1:8 were obtained.

[0053] 3. Preparation of directly self-assembled nanoparticles (KP NPs) Comparative Example 1: (1) Transfer 2 mL of dechlorinated cisplatin solution (1 mM) into a reaction flask.

[0054] (2) Under ice bath conditions, add 40 μL of anhydrous ethanol solution containing 150 mM kaempferol and 10 μL of chloroform solution containing 50 mg / mL DSPE-mPEG2000 dropwise to the reaction flask, and sonicate for 15 min at a power of 200 W.

[0055] (3) After sonication, place the reaction flask on a rotary evaporator and evaporate under reduced pressure at 30°C for 10 min to remove the organic solvent.

[0056] (4) Transfer the reaction solution to a dialysis bag (MWCO 3500) and dialyze it in deionized water for 24 h to remove free drugs. Change the water every 6 h to obtain KP NPs. Store at 4℃ in the dark for later use.

[0057] 4. Preparation of Kaempferol Nanoparticles (Ka NPs) Comparative Example 2: (1) Transfer 2 mL of Tris solution (1 mM) into a reaction flask.

[0058] (2) Under ice bath conditions, add 107 μL of anhydrous ethanol solution containing 150 mM kaempferol and 15 μL of chloroform solution containing 50 mg / mL DSPE-mPEG2000 dropwise to the reaction flask, and sonicate for 15 min at a power of 200 W.

[0059] (3) After sonication, place the reaction flask on a rotary evaporator and evaporate under reduced pressure at 30°C for 10 min to remove the organic solvent.

[0060] (4) Transfer the reaction solution to a dialysis bag (MWCO 3500) and dialyze it in deionized water for 24 h to remove free kaempferol. Change the water every 6 h to obtain Ka NPs. Store at 4℃ in the dark for later use.

[0061] (II) Physicochemical characterization of nanomedicines 2.1 Transmission electron microscopy morphology characterization Appropriate amounts of KP@Ka NPs solution, KP NPs solution, and Ka NPs solution were taken respectively, diluted to suitable concentrations, and dropped onto a copper grid carrying a film. After static adsorption, the nanoparticles were allowed to air dry naturally, and the microstructure of each group of nanoparticles was observed using a transmission electron microscope. The results are as follows: Figure 2 As shown, the core-shell composite nanoparticles KP@Ka NPs with KP as the core and Ka as the shell were successfully prepared. The results showed no free small particles and that the Ka monomers were almost completely coated on the KP surface. This indicates that the core-shell structure effectively improves the stability of the nanomedicine, provides good coating, and prevents serious aggregation between particles, thus maintaining good colloidal stability.

[0062] 2.2 Particle size distribution and zeta potential detection Appropriate amounts of nanoparticle drug suspensions from Examples 1-3 were taken, and the particle size distribution, polydispersity index (PDI), and surface zeta potential of each group of nanoparticles were detected using a dynamic light scattering instrument. The detection temperature was controlled at 25°C, and each group of samples was detected in triplicate, with the average value taken. The results are as follows: Figure 3 As shown, KP@Ka NPs have a uniform particle size distribution, ranging from 50 to 250 nm, with a PDI < 0.3 and a Zeta potential of -15 to -35 mV, which meets the targeting requirements of EPR effect in tumor tissue.

[0063] 2.3 Infrared Spectroscopic Characterization Ka NPs, Cisplatin, and KP@Ka NPs were prepared as test samples using the KBr pellet method. The infrared spectra of each sample were detected using a Fourier transform infrared spectroscopy (FTIR) instrument to analyze functional group changes and verify coordination interactions. Results are as follows: Figure 4 As shown, in KP@Ka NPs at 1656 cm -1 and 1569 cm -1 The appearance of a new absorption peak at 831 cm⁻¹ confirms the successful coordination of platinum. Furthermore, a new absorption peak at 831 cm⁻¹ is observed in KP@Ka NPs. -1The characteristic peak at the point can be attributed to the strong stretching vibration band of Pt-O. The above results confirm that the hydroxyl group in the kaempferol molecule successfully forms a coordination bond with the Pt center of cisplatin.

[0064] 2.4 Encapsulation efficiency determination and comparison The KP@Ka NPs and KP NPs nanoparticles prepared by the methods of Example 1 and Comparative Example 1 were digested with nitric acid, and the Pt content loaded in the nanoparticles was detected by ICP-MS. The cisplatin encapsulation efficiency was calculated according to the following formula: Cisplatin encapsulation rate (%) = Mass of cisplatin loaded / Mass of cisplatin added × 100%.

[0065] Results, such as encapsulation rate comparison Figure 5 As shown, the cisplatin encapsulation efficiency of KP@Ka NPs was 86.6 ± 4.0%, significantly higher than that of directly self-assembled KP NPs (35.5 ± 0.8%). This demonstrates that the step-by-step assembly process of the present invention can effectively improve the encapsulation efficiency of cisplatin and solve the technical problem of low encapsulation efficiency in the direct self-assembly method.

[0066] (III) In vitro release experiment of nanomedicine Add the prepared KP@Ka NPs solution to 1.5 mL EP tubes (100 μL per tube), centrifuge at 12000 rpm, and discard the supernatant. Add 2 mL of 1 mM Tris solution (pH 7.4 and pH 5.4), respectively, and place in a 37℃ constant temperature water bath with shaking at 80 rpm. Take samples at 0, 1, 2, 4, 8, 12, 24, and 48 h, centrifuge at 12000 rpm, and discard the supernatant. Digest each tube with 1 mL of concentrated nitric acid, then dilute with 4 mL of 2% nitric acid solution. Detect the unreleased platinum content in the nanoparticles by ICP-MS. The cumulative cisplatin release rate at different time points is calculated using the following formula: Cumulative platinum release rate (%) = (Inherent platinum content in nanoparticles - Unreleased platinum content in nanoparticles) / Inherent platinum content in nanoparticles × 100%.

[0067] The cumulative release rate of cisplatin at different pH values ​​is as follows: Figure 6 As shown in the curve, under the simulated physiological environment of pH 7.4, the cumulative release rate of cisplatin by KP@Ka NPs within 48 h was extremely low, indicating that the Ka shell greatly enhances the structural stability of the nanoparticles in a neutral environment. Under the simulated tumor microenvironment of pH 5.4, the release rate of cisplatin was significantly accelerated, with a cumulative release rate of up to 78% within 48 h, which was significantly higher than that under pH 7.4 conditions. This is beneficial for the specific enrichment and release of nanomedicines at the tumor site and reduces toxic side effects on normal tissues.

[0068] (iv) Study on the in vitro antitumor cell efficacy of nanomedicines Log-phase triple-negative breast cancer 4T1 cells from mice were collected and processed at 2.5 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 μL in 6-well plates. After standard culture and adhesion, PBS, Ka NPs, Cisplatin, and KP@Ka NPs were added to the plates, respectively, for intervention culture at a cisplatin equivalent concentration of 3 μM. After 24 h of intervention, cells from each group were collected, stained with Annexin V-FITC / PI double staining, and apoptosis was detected by flow cytometry.

[0069] The results are as follows Figure 7 As shown, compared with the PBS control group, the Ka NPs group, and the Cisplatin group, the KP@Ka NPs group exhibited a significantly higher tumor cell apoptosis rate, demonstrating superior tumor cell killing effect. The apoptosis rate in the Ka NPs group was not significantly different from that in the PBS control group, indicating that the blank carrier itself had no significant cytotoxicity. These results demonstrate that the nanomedicine of this invention, through the synergistic effect of cisplatin and kaempferol, can effectively enhance the tumor cell killing ability.

[0070] (v) Detection of expression of immunomodulatory proteins related to nanomedicines Log-phase triple-negative breast cancer 4T1 cells from mice were collected and processed at a concentration of 6 × 10⁶ cells per dish. 5 Cells were seeded at a density of 1000 μm in 60 mm diameter cell culture dishes. After standard culture and adhesion, four intervention reagents were added: PBS, KaNPs, Cisplatin, and KP@KaNPs, respectively, with a cisplatin equivalent concentration of 3 μM for 16 h. Cells were collected, total cellular protein was extracted, and the expression levels of CD39, CD73, and CX43 proteins in each group of cells were detected by Western blotting (WB). The experiment was repeated three times.

[0071] CD39 protein expression results are as follows: Figure 8 As shown, compared to the PBS control group, the expression level of CD39 protein in tumor cells of the KP@Ka NPs group was significantly downregulated.

[0072] CD73 protein expression results are as follows: Figure 9 As shown, KP@Ka NPs nanomedicines can effectively inhibit the expression of CD73 protein in tumor cells.

[0073] CX43 protein expression results are as follows Figure 10 As shown, after intervention with KP@Ka NPs nanomedicine, the expression level of CX43 protein in tumor cells was significantly increased.

[0074] The above results confirm that the KP@Ka NPs nanomedicine of the present invention can simultaneously upregulate the expression of CX43 channel protein that promotes ATP efflux and inhibit the expression of CD39 and CD73 enzymes that mediate ATP hydrolysis, thereby performing bidirectional immune regulation from two directions: enhancing immune activation signals and blocking immunosuppressive metabolism.

[0075] (vi) Detection of extracellular ATP and ADO levels Log-phase triple-negative breast cancer 4T1 cells from mice were collected and processed at 2.5 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 mg / L in 6-well plates. After standard culture and adhesion, the cells were cultured for 18 h with a cisplatin equivalent concentration of 3 μM. Cell supernatants were collected from each group, and the ATP and ADO levels in the supernatants were measured using ATP and ADO assay kits, respectively, following the instructions of the kits. Multiple replicates were set up for each group.

[0076] The results are as follows Figure 11 , Figure 12 As shown, the extracellular ATP level in the KP@Ka NPs group was significantly higher than that in the PBS control group, the Ka NPs group, and the Cisplatin group, indicating that the nanomedicine can effectively promote the release of the immunostimulatory factor ATP. Simultaneously, the extracellular ADO content in the KP@Ka NPs group was significantly lower than that in the other groups, indicating that the nanomedicine effectively blocked the conversion of ATP into the immunosuppressive product ADO. These results demonstrate that the nanomedicine of this invention effectively increases the ATP / ADO ratio in the tumor microenvironment through a dual mechanism of upregulating CX43 to promote ATP efflux and inhibiting CD39 / CD73 to block ADO production, thereby transforming the immunosuppressive microenvironment into an immune-activating microenvironment.

[0077] (vii) Detection of tumor cell eversion using CRT Log-phase triple-negative breast cancer 4T1 cells from mice were collected and processed at 2.5 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 μL in 6-well plates. After standard culture and adhesion, cells were treated with cisplatin at an equivalent concentration of 3 μM for 12 h. Cells from each group were collected and stained with calreticulin (CRT) fluorescein-labeled antibody. The extravasation of CRTs on the cell surface was detected by flow cytometry to assess the immunogenic cell death (ICD) induction effect.

[0078] The results are as follows Figure 13 As shown, the CRT extravasation level on the surface of tumor cells in the KP@Ka NPs group was significantly increased, significantly higher than that in the free cisplatin group and the other control groups. CRT is a key biomarker of ICD, and the results confirm that the nanomedicine of this invention can efficiently induce immunogenic cell death in tumor cells and initiate the body's anti-tumor immune response.

[0079] (viii) Dendritic cell maturity detection Log-phase triple-negative breast cancer 4T1 cells from mice were collected and processed at 2.5 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 mcg / well in 6-well plates. After standard culture and adhesion, the cells were treated with cisplatin at an equivalent concentration of 3 μM for 24 h. The supernatant from each group after drug treatment was collected and diluted with 5 × 10⁶ cells / well. 5 Immature dendritic cells (DCs) were seeded at different densities and co-cultured for 24 h. After culture, DCs were collected, stained with fluorescein-labeled antibodies, and the expression levels of DC maturation markers (CD80, CD86) were detected by flow cytometry to analyze the DC maturation status.

[0080] The results are as follows Figure 14 As shown: Compared with the PBS control group, Ka NPs group, and Cisplatin group, the supernatant of the KP@Ka NPs nanomedicine group significantly promoted DC cell maturation and CD80. + CD86 + The proportion of double-positive cells was significantly increased. This result indicates that, compared to the control group, the supernatant of the nanomedicine of this invention can significantly promote the maturation of dendritic cells (DCs), induce tumor cells to release immune stimulation signals, effectively activate antigen-presenting cells, and initiate an adaptive anti-tumor immune response.

[0081] In summary, this invention successfully synthesized cisplatin-kaempferol coordination nanomedicines KP@Ka NPs through a stepwise cascade self-assembly strategy of first coordinating and then embedding. This nanomedicine effectively solves the technical challenges of low encapsulation efficiency and poor stability in direct self-assembly products of cisplatin and kaempferol, and exhibits acid-responsive release properties. In in vitro experiments, KP@Ka NPs efficiently induced tumor cell apoptosis and immunogenic cell death, upregulated CX43 protein expression to promote ATP efflux, and simultaneously inhibited CD39 / CD73 protein expression to block ADO generation, synergistically enhancing the anti-tumor immune response. This invention provides a novel nanomedicine solution for tumor immunotherapy that is structurally simple, controllable in preparation, and highly efficient.

[0082] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing a cisplatin-kaempferol coordination nanomedicine, characterized in that, Includes the following steps: Cisplatin and the first portion of kaempferol were mixed and reacted in a solvent to form a cisplatin-kaempferol coordination complex through coordination. Then, the second portion of kaempferol and a PEGylated amphiphilic material were added, allowing the second portion of kaempferol to self-assemble into a nanocarrier under the action of the PEGylated amphiphilic material. Simultaneously, the cisplatin-kaempferol coordination complex was loaded into the nanocarrier during the self-assembly of the second portion of kaempferol, resulting in the cisplatin-kaempferol coordination nanodrug KP@Ka NPs.

2. The method for preparing cisplatin-kaempferol coordination nanomedicine according to claim 1, characterized in that, The molar ratio of the total amount of cisplatin to kaempferol is in the range of 1:3 to 1:

14.

3. The method for preparing cisplatin-kaempferol coordination nanomedicine according to claim 1, characterized in that, The PEGylated amphiphilic material is at least one of PEG-DSPE, PEG-lipid, and PEG-amphiphilic block copolymer.

4. The method for preparing cisplatin-kaempferol coordination nanomedicine according to claim 1, characterized in that, Includes the following steps: Cisplatin was dissolved in a solvent, silver nitrate solution was added dropwise and stirred in the dark, then centrifuged and the supernatant was removed to obtain a dechlorinated cisplatin solution; An ethanol solution containing the first portion of kaempferol was added to the dechlorinated cisplatin solution, and the reaction was carried out for 8-24 hours. The solution is prepared by adding 10-160 μL of an ethanol solution containing the second part of kaempferol and PEGylated amphiphilic material under ice bath conditions, followed by sonication, rotary evaporation under reduced pressure, and finally dialysis in water to remove free drug.

5. A cisplatin-kaempferol coordination nanomedicine, characterized in that, It is prepared by any one of claims 1 to 4.

6. The cisplatin-kaempferol coordination nanomedicine according to claim 5, characterized in that, The cisplatin-kaempferol coordination nanomedicine has a particle size of 50 nm to 250 nm and a zeta potential of -15 to -35 mV.

7. The use of the cisplatin-kaempferol coordination nanomedicine according to claim 5 or 6 in the preparation of drugs for treating tumors.

8. The application according to claim 7, characterized in that, The tumors include at least one of triple-negative breast cancer, lung cancer, liver cancer, ovarian cancer, and pancreatic cancer.

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