Nanometer composition based on sequential induction and degradation and application thereof

Abstract

The invention discloses a nano composition based on sequential induction and degradation and application thereof, and belongs to the technical field of biological medicine. The nano composition based on sequential induction and degradation provided by the invention comprises first nano particles and second nano particles, the first nanoparticles comprise a first carrier and an STING signal pathway agonist loaded on the first carrier; the second nanoparticle comprises a second carrier, and an E3 ligase ligand and a PD-L1 protein ligand which are loaded on the second carrier; the first nanoparticle and the second nanoparticle are configured for sequential application, and the application time of the first nanoparticle is prior to the application time of the second nanoparticle. By constructing the two functional nanoparticles which are sequentially applied, immunosuppression feedback caused by treatment can be actively intercepted and eliminated, so that tumor drug resistance is effectively reversed, and the anti-tumor immunotherapy effect is remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a nanocomposite based on sequential induction and degradation and its applications. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Interferon gene-stimulating factor (STING) signaling pathway agonists, as an emerging strategy in tumor immunotherapy, can effectively activate the innate immune system, induce the production of type I interferon and various inflammatory factors, and reshape the tumor immune microenvironment. However, their clinical response rate as monotherapy is generally poor. Studies have shown that this is mainly due to a dynamic adaptive drug resistance mechanism in tumor cells: while activation of the STING pathway initiates a beneficial immune response, it also drives downstream signaling networks, leading to compensatory upregulation of key immunosuppressive molecules, including programmed death-ligand-1 (PD-L1). This process forms a "immune activation-feedback inhibition" cycle, where newly generated immunosuppressive signals offset the initial therapeutic effect, thus severely limiting the ultimate efficacy of STING signaling pathway agonists.

[0004] For this type of feedback inhibition triggered by treatment itself, existing intervention strategies mostly focus on using monoclonal antibodies and other agents to block the function of upregulated immune checkpoint molecules. However, these strategies have limitations in dealing with dynamic and continuous feedback upregulation. Their mechanisms of action are mostly reversible binding and inhibition, and their efficacy may not be durable; for example, antibody binding to the target can easily trigger endocytosis and recycling, resulting in the inability to completely remove inhibitory molecules from the surface of tumor cells, thus weakening the therapeutic effect. This makes it difficult for existing strategies to match the timeliness and depth of the continuously evolving negative feedback process. Therefore, how to design a treatment strategy that can systematically identify and break this negative feedback loop, especially to achieve efficient and synergistic intervention on newly upregulated immunosuppressive molecules, has become a key technical problem in improving the efficacy of STING pathway-targeted therapies. Summary of the Invention

[0005] In view of this, the present invention provides a nanocomposition based on sequential induction and degradation and its application. By constructing two functional nanoparticles for sequential application, the present invention can actively intercept and eliminate the immunosuppressive feedback caused by the treatment itself, thereby effectively reversing tumor drug resistance and significantly improving the effect of anti-tumor immunotherapy.

[0006] In a first aspect, the present invention provides a nanocomposition based on sequential induction and degradation, comprising: First nanoparticles and second nanoparticles; The first nanoparticle comprises a first carrier and a STING signaling pathway agonist loaded on the first carrier; the STING signaling pathway agonist is 2,5-hexanone theobromine (DMXAA). The second nanoparticle comprises a second carrier and an E3 ligase ligand and a PD-L1 protein ligand loaded on the second carrier; the E3 ligase ligand is thalidomide-NH-CH2-COOH; and the PD-L1 protein ligand is BMS-1166. The first nanoparticle and the second nanoparticle are configured to be applied sequentially, with the first nanoparticle being applied before the second nanoparticle.

[0007] Preferably, both the first and second carriers are core-shell structured nanoparticles formed by coating silicon dioxide (SiO2) with polyamide-amine dendritic polymer (PAMAM).

[0008] Preferably, the polyamide-amine dendritic polymer is covalently bonded to silicon dioxide.

[0009] Preferably, the first carrier and the STING signaling pathway agonist are connected by covalent bonds.

[0010] Preferably, the E3 ligase ligand and the PD-L1 protein ligand are covalently linked to the second vector.

[0011] Preferably, the time interval for the sequential application is 12 to 20 hours.

[0012] Secondly, the present invention provides a method for preparing the above-mentioned nanocomposition based on sequential induction and degradation, comprising the following steps: The STING signaling pathway agonist, the first activator, and the first carrier were mixed and reacted in the first solvent, and the first nanoparticles were obtained by separation and purification. The PD-L1 protein ligand, E3 ligase ligand, second activator, and second carrier were mixed and reacted in a second solvent, and the second nanoparticles were obtained after separation and purification.

[0013] Preferably, both the first activator and the second activator are combinations of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide.

[0014] Preferably, both the first carrier and the second carrier are core-shell structured nanoparticles formed by coating silica with polyamide-amine dendritic polymer.

[0015] Thirdly, the present invention provides the application of the above-mentioned nanocomposition based on sequential induction and degradation or the nanocomposition based on sequential induction and degradation prepared by the above-mentioned preparation method in the preparation of antitumor drugs.

[0016] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) In the nanocomposite based on sequential induction and degradation provided by the present invention, the first nanoparticle is responsible for activating immunity and inducing PD-L1 feedback upregulation; the second nanoparticle utilizes the loaded E3 ligase ligand and PD-L1 protein ligand to synergistically perform the protein degradation targeting chimera (PROTAC) function at the nanoscale to achieve targeted degradation of the upregulated PD-L1 protein. This sequential approach of "induction first, degradation later" can accurately match the dynamic process of PD-L1 expression, thereby blocking the drug resistance cycle of "immune activation-checkpoint feedback" at the source and synergistically enhancing the strength and persistence of the anti-tumor immune response.

[0017] (2) The polyamide-amine dendritic polymer (PAMAM) core-shell nanoparticles coated with silica are selected as a universal carrier in this invention. The abundant active groups on their surface enable stable and high-load chemical bonding between the STING signaling pathway agonist and PROTAC. This carrier design not only ensures the independence of the two functional components before delivery and avoids mutual interference, but also provides a reliable basis for efficient drug delivery and controlled release due to its good biocompatibility and modifiability.

[0018] (3) The preparation method provided by this invention can efficiently graft STING signaling pathway agonists, PD-L1 protein ligands, and E3 ligase ligands onto the surface of a carrier under mild conditions. This method has a clear process and good reproducibility, providing a practical and feasible technical path for the large-scale and standardized preparation of the sequential nanocomposite. Attached Figure Description

[0019] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0020] Figure 1 These are transmission electron microscope (TEM) images of monodisperse silica nanoparticles of Example 1, SPD nanoparticles and SPTAC nanoparticles of Example 2 of the present invention; wherein, A is a TEM image of monodisperse silica nanoparticles of Example 1, B is a TEM image of SPD nanoparticles of Example 2, and C is a TEM image of SPTAC nanoparticles of Example 2. Figure 2 These are dynamic light scattering (DLS) images of monodisperse silica nanoparticles of Example 1, SPD nanoparticles and SPTAC nanoparticles of Example 2 of the present invention; wherein, A is a DLS image of monodisperse silica nanoparticles of Example 1, B is a DLS image of SPD nanoparticles of Example 2, and C is a DLS image of SPTAC nanoparticles of Example 2. Figure 3 This is a Zeta potential diagram of SiO2, carboxyl-modified SiO2 (SiO2-COOH), PAMAM and SP nanoparticles in Example 1 of the present invention. Figure 4 Fourier transform infrared spectra of SiO2, carboxyl-modified SiO2 (SiO2-COOH), PAMAM and SP nanoparticles in Example 1 of this invention. Figure 5 This is the Fourier transform infrared spectrum of the SPD nanoparticles and DMXAA in Example 2 of the present invention; Figure 6 These are Fourier transform infrared (FTIR) spectra of the SPTAC nanoparticles of Example 2, the SP-T nanoparticles of Comparative Example 1, the SP-B nanoparticles of Comparative Example 2, the E3 ligase ligand (Thalidomide-NH-CH2-COOH), and the PD-L1 protein ligand (BMS-1166) of the present invention; wherein, A is the FTIR spectrum of the SPTAC nanoparticles of Example 2, the SP-T nanoparticles of Comparative Example 1, and the SP-B nanoparticles of Comparative Example 2; B is the FTIR spectrum of Thalidomide-NH-CH2-COOH; and C is the FTIR spectrum of BMS-1166. Figure 7 This is the result of Western blot analysis of proteins in the relevant pathways after CT26 cells were treated with phosphate-buffered saline (PBS), SP nanoparticles and SPD nanoparticles in the experimental examples of this invention; the relevant pathway proteins include phosphorylated TBK1 (p-TBK1) and phosphorylated IRF3 (p-IRF3), with GAPDH as the internal control protein. Figure 8 These are the results of Western blot analysis of CT26 cells treated with SPD nanoparticles at 0h, 24h and 48h in the experimental examples of this invention, with GAPDH as the internal reference protein. Figure 9 This is the Western blot result of PD-L1 protein after 12 hours of treatment of CT26 cells with PBS buffer, SP nanoparticles and SPTAC nanoparticles in the experimental example of this invention, with GAPDH as the internal reference protein. Figure 10This is the Western blot result of PD-L1 protein after CT26 cells were treated with SPTAC nanoparticles for 0-12 hours in the experimental example of this invention, with GAPDH as the internal reference protein. Figure 11 This is the Western blot result of PD-L1 protein after 12 hours of treatment of CT26 cells with PBS buffer, SP nanoparticles, commercial PD-L1 inhibitor (BMS-1166) and SPTAC nanoparticles in the experimental example of this invention, with GAPDH as the internal control protein. Figure 12 The results of Western blot analysis of PD-L1 protein obtained by pretreating CT26 cells with MG132 or MLN4924 and then treating them with SPTAC nanoparticles in the experimental examples of this invention are shown. No treatment is used as a control and GAPDH is used as an internal reference protein. Figure 13 The results of Western blot analysis of PD-L1 protein in CT26 cells treated with PBS, SPTAC nanoparticles, SP-T (Comparative Example 1), SP-B (Comparative Example 2), and a mixture of SP-T and SP-B (SP-T+SP-B) in the experimental examples of this invention are shown, with GAPDH as the internal reference protein. Figure 14 The images show immunofluorescence analysis of the PBS control group, SP group alone, SPD group alone, SPTAC group alone, SPD and SPTAC treatment group (SPD&SPTAC) and the sequential administration group (SPD+SPTAC) in the experimental examples of this invention; the scale bar is 20 μm. Figure 15 The cell viability of SP, SPD, SPTAC nanoparticles and the sequential combination of "SPD + SPTAC" at different concentrations in the experimental examples of this invention are shown in the figure. A represents SP nanoparticles, B represents SPD nanoparticles, C represents SPTAC nanoparticles, and D represents SPD + SPTAC. Figure 16 These are live and dead cell fluorescence images of the PBS control group, SP group alone, SPD group alone, SPTAC group alone, and the sequential drug administration group (SPD+SPTAC) in the experimental examples of this invention; the scale bar is 100 μm. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0022] This invention provides a nanocomposition based on sequential induction and degradation, comprising physically independent first and second nanoparticles. This design ensures the stability of the two functional components during storage and initial delivery, avoiding potential mutual interference or inactivation between the active ingredients, and provides the material basis for achieving controlled sequential drug delivery.

[0023] In this invention, the first nanoparticle includes a first carrier and a STING signaling pathway agonist loaded on the first carrier; its function is to effectively activate the interferon gene stimulating factor (STING) signaling pathway in tumor and immune cells in the initial stage of treatment, thereby inducing the production of a large number of cytokines such as type I interferon and initiating a strong innate anti-tumor immune response.

[0024] In an optional embodiment of the present invention, the STING signaling pathway agonist is 2,5-hexanone theobromine (DMXAA). DMXAA is a highly efficient and specific agonist of mouse STING protein and has been widely used in preclinical studies to mimic the activation of the human STING pathway, characterized by a clear mechanism of action and high activation efficiency. In other optional embodiments, the STING signaling pathway agonist may also be selected from known STING signaling pathway agonists such as cGAMP, ADU-S100, and diABZI.

[0025] In this invention, the second nanoparticle comprises a second carrier and an E3 ligase ligand and a PD-L1 protein ligand loaded on the second carrier. This design enables the second nanoparticle to function as an integrated nanoprotein degradation-targeting chimera (PROTAC): the PD-L1 protein ligand is responsible for recognizing and binding to the target protein PD-L1, and the E3 ligase ligand is responsible for recruiting specific E3 ubiquitin ligases within the cell. The two work closely together spatially through the second carrier, thereby achieving specific and irreversible degradation of the upregulated PD-L1 protein within the cell, effectively relieving immunosuppression.

[0026] In an optional embodiment of the present invention, the E3 ligase ligand is thalidomide-NH-CH2-COOH; this structure is based on a thalidomide derivative, and the terminal carboxyl group (-COOH) is the key functional group for chemical coupling with the second carrier. The PD-L1 protein ligand is BMS-1166. BMS-1166 is a known small molecule inhibitor of PD-L1, exhibiting high affinity and specificity for it. The preferred mass ratio of the E3 ligase ligand to the PD-L1 protein ligand is 1:(0.8~1.2).

[0027] In an optional embodiment of the present invention, both the first and second carriers are core-shell structured nanoparticles formed by coating silica (SiO2) with polyamide-amine dendritic polymer (PAMAM). The silica framework inside the carrier provides high specific surface area and rigid support, while the external PAMAM dendritic macromolecules provide a large number of terminal amino groups (-NH2), which can efficiently load drug molecules through chemical bonds. The active groups on the surface of PAMAM are easily chemically modified to achieve directional and stable coupling with STING signaling pathway agonists (DMXAA) or PD-L1 protein ligands and E3 ligase ligands. The positive charge of PAMAM facilitates the interaction between the nanoparticles and the negatively charged cell membrane, promoting endocytosis. The size of the nanoparticles can be controlled in the range of 100~400 nm, preferably 200~300 nm. The number of branched growth layers of PAMAM can be selected as needed, preferably 2~4 generations, more preferably 3 generations (i.e., G3), which has good biocompatibility. In this invention, the polyamide-amine dendritic polymer is covalently connected to silicon dioxide, for example, through covalent bonds formed by a silane coupling agent, which ensures the stability of the core-shell structure.

[0028] In the first nanoparticle of this invention, the first carrier and the STING signaling pathway agonist are covalently linked. In the second nanoparticle, the E3 ligase ligand and the PD-L1 protein ligand are covalently linked to the second carrier. The covalent bond can be an amide bond, etc. Compared with physical encapsulation, this covalent connection method can effectively prevent premature leakage of the drug during delivery, ensure that the drug remains stable before reaching the target site, and may enable release triggered by specific intracellular environments (such as enzyme digestion, pH changes).

[0029] In this invention, the first nanoparticle and the second nanoparticle are configured for sequential administration, with the first nanoparticle being administered before the second nanoparticle. Preferably, the time interval between the sequential administrations is 12-20 hours. Research in this invention shows that PD-L1 expression peaks approximately 18-24 hours after the action of a STING signaling pathway agonist. Therefore, setting the administration time of the second nanoparticle within the 12-20 hour range before the peak of PD-L1 expression ensures that the peak activity period of the second nanoparticle covers the peak expression period of PD-L1, thereby maximizing the clearance efficiency through "immediate expression and immediate degradation." A more preferred time interval is 16-20 hours.

[0030] The present invention also provides a method for preparing the above-mentioned nanocomposition based on sequential induction and degradation, comprising the following steps: The STING signaling pathway agonist, a first activator, and a first carrier are mixed and reacted in a first solvent, followed by separation and purification to obtain the first nanoparticles. The reaction is carried out at room temperature or under mild heating conditions. The reaction time can be 12 to 72 hours, for example 12, 24, 48, or 72 hours, preferably 24 to 48 hours to ensure sufficient reaction. The mass ratio of the STING signaling pathway agonist to the first carrier can be optimized in the range of 1:(1 to 5), and is further preferably 1:(1.5 to 2.5). The first solvent can be dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), or other polar aprotic solvents that can well dissolve the reactants. After the reaction, solid-liquid separation is performed by centrifugation, filtration, or ultrafiltration. The obtained solid precipitate needs to be repeatedly washed with a suitable washing solvent (e.g., water, ethanol, or a mixture thereof) to thoroughly remove unreacted raw materials, activator byproducts, etc., and finally freeze-dried or vacuum-dried to obtain the final product.

[0031] The PD-L1 protein ligand, E3 ligase ligand, second activator, and second carrier are mixed and reacted in a second solvent, and then purified to obtain the second nanoparticles. The specific preparation process of the second nanoparticles is similar to that of the first nanoparticles. The mass ratio of E3 ligase ligand, PD-L1 protein ligand, and second carrier can be optimized in the range of (0.8~1.2):(0.8~1.2):(0.8~1.2), and is further preferably (1.0~1.2):(1.0~1.2):(0.8~1.0).

[0032] In an optional embodiment of the present invention, the first activator and the second activator are both combinations of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS); the first carrier and the second carrier are both core-shell structured nanoparticles formed by coating silica with polyamide-amine dendritic polymer.

[0033] This invention does not impose any special restrictions on the specific preparation method of the core-shell structured nanoparticles formed by coating silica with the above-mentioned polyamide-amine dendritic polymer (PAMAM). Its preparation usually involves two key steps: the synthesis of the silica core and the modification of the PAMAM shell.

[0034] The silica core can be synthesized using various methods known in the art, such as the sol-gel method, template method, or microemulsion method. In one or more embodiments of the present invention, the silica core is prepared using the Stöber method in the sol-gel process to produce silica nanoparticles with uniform particle size and high specific surface area. The particle size is preferably controlled between 100 and 300 nm, for example, but not limited to 100, 120, 180, 200, 250, or 300 nm, or any value between the two, and more preferably 200 to 300 nm.

[0035] The preferred modification of the PAMAM shell includes the following steps: amylating the silica core with an amino-containing silane coupling agent, reacting it with an acid anhydride to obtain a carboxyl-modified silica core, and finally reacting it with PAMAM under the action of activators (EDC·HCl and NHS) to form a PAMAM shell, ultimately obtaining PAMAM-coated silica core-shell structured nanoparticles.

[0036] The present invention also provides the application of the above-mentioned nanocomposition based on sequential induction and degradation or the nanocomposition based on sequential induction and degradation prepared by the above-mentioned preparation method in the preparation of antitumor drugs.

[0037] Based on its "sequential induction and degradation" mechanism of action, this nanocomposition is particularly suitable for treating malignant tumors that are insensitive to single immune checkpoint inhibitors or STING signaling pathway agonists and are prone to adaptive resistance. This includes, but is not limited to, melanoma, non-small cell lung cancer, triple-negative breast cancer, colorectal cancer, and other cancers that have been shown to exhibit STING pathway activity and immune checkpoint feedback.

[0038] In this invention, the first and second nanoparticles can be prepared into two independent dosage form units, such as lyophilized powder for injection, and supplemented with a medication guide explaining their sequential use order and recommended time intervals, together constituting a therapeutic kit. The drug can be administered via intratumoral injection, intravenous injection, etc., wherein intratumoral injection can maximize the local drug concentration in the tumor and systematically stimulate anti-tumor immunity.

[0039] When the nanocomposition is applied, its beneficial effects are as follows: by first activating the STING pathway and then precisely degrading the PD-L1 induced by it, this strategy breaks the vicious cycle of "immune activation-feedback inhibition" from a temporal and climatic perspective; the use of PROTAC technology to achieve irreversible clearance of PD-L1 overcomes the defects of antibody therapy, which is reversible and easily endocytosed; the use of the same nanocarrier for separate delivery ensures the consistency and controllability of delivery efficiency, and ultimately synergistically enhances the depth and persistence of the anti-tumor immune response.

[0040] The technical solution of the present invention will be further described below with reference to specific embodiments. The present invention does not have any special restrictions on the source of reagents used in the following embodiments; commercially available products well known to those skilled in the art can be used. In the following embodiments, E3 ligase ligand (Thalidomide-NH-CH2-COOH), BMS-1166, and DMXAA were all purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0041] Example 1 This embodiment provides a method for preparing core-shell structured nanoparticles (SP nanoparticles) of silica coated with polyamide-amine dendritic polymer (PAMAM).

[0042] (1) Preparation of monodisperse silica nanoparticles Monodisperse silica nanoparticles were synthesized using a modified Stöber method. The specific steps are as follows: 1.5 mL of 25 wt% ammonia solution was mixed with 15 mL of anhydrous ethanol and stirred thoroughly in a constant temperature water bath at 45 ± 1 °C to form solution B. 1.5 mL of tetraethyl orthosilicate (TEOS) was dissolved in 15 mL of anhydrous ethanol to form solution A. Under vigorous stirring, solution A was pumped into the reactor containing solution B at a rate of 5.0 mL / min, and the reaction was continued at 45 °C for 6 hours. After the reaction, the resulting milky white suspension was centrifuged at 8000 rpm for 1 hour, the precipitate was collected, and washed three times repeatedly with anhydrous ethanol. Finally, it was dried in a 70 °C oven for 12 hours to obtain white powdered silica nanoparticles. Transmission electron microscopy (TEM) showed (see...). Figure 1 (A) The prepared silica nanoparticles exhibited uniform morphology and good dispersion. Dynamic light scattering (DLS) analysis revealed that the hydrated particle size of the silica nanoparticles was approximately 230 nm (see section A). Figure 2 The A in the figure is similar to the particle size measured by TEM.

[0043] (2) Surface amino functionalization of silica nanoparticles 0.4 mL of 3-aminopropyltriethoxysilane (APTES) was dissolved in 20 mL of anhydrous toluene, and then 0.2 g of the silica nanoparticles obtained in step (1) were added. The mixture was refluxed and stirred at 120 °C for 24 hours. After the reaction was completed, the product was separated by centrifugation and washed three times with ultrapure water and methanol, respectively. The product was then dried under vacuum at 50 °C to obtain aminated silica nanoparticles (denoted as SiO2-NH2).

[0044] (3) Grafting modification of PAMAM dendrimers The SiO2-NH2 obtained in step (2) was dispersed in 20 mL of acetone, and 3 g of succinic anhydride was added. The mixture was reacted at room temperature for 24 hours to modify the surface amino groups with carboxylation, resulting in carboxyl-modified SiO2 (denoted as SiO2-COOH). SiO2-COOH was dispersed in 10 mL of anhydrous dimethyl sulfoxide (DMSO), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 46 mg) and N-hydroxysuccinimide (NHS, 27.6 mg) were added for activation for 30 minutes. Subsequently, a DMSO solution of third-generation polyamide-amine dendritic macromolecules (G3 PAMAM, 41.6 mg) was added, and the mixture was stirred at room temperature for 24 hours. After the reaction was completed, the solid was collected by centrifugation at 12000 rpm for 10 minutes and washed three times with 50% ethanol solution. Finally, the solid was freeze-dried to obtain PAMAM-coated silica core-shell nanoparticles, i.e., SP nanoparticles.

[0045] Figure 3 Zeta potential analysis showed that the surface potential of PAMAM was positive and that of SiO2 was negative. After SiO2 was modified with carboxyl groups (SiO2-COOH), the surface potential remained negative and the absolute value increased. After further modification with PAMAM, the surface potential of the particles changed from negative to positive, confirming that the amino-rich PAMAM layer was successfully coated.

[0046] Fourier transform infrared (FT-IR) spectra of PAMAM, SiO2, SiO2-COOH, and SP nanoparticles, such as Figure 4 As shown, SiO2-COOH at 960 cm -1 1020~1110 cm -1 1640 cm -1 A distinct absorption peak appears at these three locations. These three absorption peaks are caused by the asymmetric bending and stretching vibrations of Si-OH, the asymmetric stretching vibrations of Si-O-Si, and the vibrations of -COOH, respectively. PAMAM absorption peaks are observed in the range of 1560–1640 cm⁻¹. -1 An absorption peak appears at 1560–1640 cm⁻¹, which is caused by the bending vibration of NH₄⁺. SP nanoparticles show an absorption peak at 1560–1640 cm⁻¹. -1 The presence of a characteristic absorption peak further confirms the successful grafting of PAMAM.

[0047] Example 2 This embodiment provides the preparation of a first nanoparticle (SPD nanoparticle) loaded with a STING signaling pathway agonist and a second nanoparticle (SPTAC nanoparticle) serving as a PD-L1 protein degradation targeting chimera.

[0048] (1) Preparation of SPD nanoparticles: At 25°C, NHS (45.0 mg), EDC·HCl (74.0 mg), and the STING signaling pathway agonist DMXAA (28.3 mg) were dissolved together in 5 mL of anhydrous DMSO, mixed, and incubated for 2 hours to activate the carboxyl groups on the DMXAA molecules, resulting in an activated mixture. Subsequently, SP nanoparticles (50.0 mg, dispersed in 5 mL DMSO) prepared in Example 1 were slowly added dropwise to the above activated mixture. The reaction was continuously stirred at 25°C for 2 days to ensure sufficient coupling between the activated DMXAA and the amino groups on the surface of the SP nanoparticles. After the reaction was completed, the mixture was centrifuged at 12000 rpm for 10 minutes. The resulting precipitate was washed three times with a 50% (v / v) ethanol solution to thoroughly remove unreacted raw materials. Finally, it was freeze-dried to obtain SPD nanoparticles.

[0049] Figure 1 Image B in the image is a TEM image of the SPD nanoparticles. It can be seen that their surface is rougher than that of SiO2 particles, which is attributed to the PAMAM layer covering the surface, reducing its surface smoothness. The particle size of the SPD nanoparticles is approximately 250 nm, a value that is... Figure 2 The DLS data in B matches. Figure 5 FT-IR characterization showed that SPD nanoparticles at 694 cm⁻¹ -1 and 1711cm -1 The presence of a characteristic absorption peak for DMXAA at this location proves that DMXAA was successfully loaded onto SP nanoparticles.

[0050] (2) Preparation of SPTAC nanoparticles: At 25°C, NHS (28.8 mg), EDC·HCl (47.9 mg), E3 ligase ligand (Thalidomide-NH-CH2-COOH, 56.3 mg), and PD-L1 protein ligand (BMS-1166, 51.3 mg) were dissolved together in 5 mL of anhydrous DMSO, mixed, and incubated for 2 hours to activate the carboxyl groups in the E3 ligase ligand and PD-L1 protein ligand, obtaining an activated mixture. Subsequently, a dispersion of SP nanoparticles (50.0 mg SP nanoparticles dispersed in 5 mL DMSO) was slowly added dropwise, and the reaction was stirred at 25°C for 2 days. After the reaction was completed, the mixture was centrifuged at 12000 rpm for 10 minutes, and the resulting precipitate was washed three times with 50% ethanol solution by centrifugation to completely remove unreacted raw materials. Finally, the precipitate was freeze-dried to obtain SPTAC nanoparticles.

[0051] Figure 1C in the image represents a TEM image of SPTAC nanoparticles. It can be seen that their surface is rougher than that of SiO2 particles, which is attributed to the PAMAM layer covering the surface, reducing its surface smoothness. The particle size of SPTAC nanoparticles is approximately 250 nm, a value that is... Figure 2 The DLS data for C are consistent. FT-IR characterization shows (see...) Figure 6 The infrared spectra of SPTAC (A) both showed characteristic peaks (1500~1600 cm⁻¹). -1 See Figure 6 The characteristic peaks (2210~2260 cm⁻¹) in B) and BMS-1166 -1 See Figure 6 The results (C) confirm that both ligands were successfully loaded onto the same SP nanoparticle.

[0052] Comparative Example 1 The difference between this comparative example and the SPTAC nanoparticles in Example 2 is that this comparative example does not include PD-L1 protein ligands. The preparation method of this comparative example is as follows: At 25°C, NHS (28.8 mg), EDC·HCl (47.9 mg), and E3 ligase ligand (Thalidomide-NH-CH2-COOH, 56.3 mg) were dissolved together in 5 mL of anhydrous DMSO, mixed, and incubated for 2 hours. Then, a dispersion of SP nanoparticles (50.0 mg SP nanoparticles dispersed in 5 mL DMSO) was slowly added dropwise, and the mixture was stirred at 25°C for 2 days. After the reaction was complete, the mixture was centrifuged at 12000 rpm for 10 minutes. The resulting precipitate was washed three times with 50% ethanol solution by centrifugation to completely remove unreacted raw materials. Finally, the precipitate was freeze-dried to obtain SP-T nanoparticles.

[0053] The infrared spectrum of SP-T nanoparticles is as follows: Figure 6 As shown in Figure A, it can be seen that it is between 1500 and 1600 cm. -1 The characteristic peak of the benzene ring appears at [location], and [is related to / ... Figure 6 The corresponding B in the text.

[0054] Comparative Example 2 The difference between this comparative example and the SPTAC nanoparticles in Example 2 is that this comparative example does not include E3 ligase ligands. The preparation method of this comparative example is as follows: At 25°C, NHS (28.8 mg), EDC·HCl (47.9 mg), and PD-L1 protein ligand (BMS-1166, 51.3 mg) were dissolved together in 5 mL of anhydrous DMSO and incubated for 2 hours. Then, a dispersion of SP nanoparticles (50.0 mg SP nanoparticles dispersed in 5 mL DMSO) was slowly added dropwise, and the mixture was stirred at 25°C for 2 days. After the reaction was complete, the mixture was centrifuged at 12000 rpm for 10 minutes. The resulting precipitate was washed three times with 50% ethanol solution by centrifugation to completely remove unreacted raw materials. Finally, the precipitate was freeze-dried to obtain SP-B nanoparticles.

[0055] The infrared spectrum of SP-B nanoparticles is as follows: Figure 6 As shown in Figure A, it can be seen that its range is 2210~2260 cm. -1 The characteristic peak of the C≡N triple bond appears at this location, and... Figure 6 The infrared spectrum of BMS-1166 in C corresponds to that in the image.

[0056] Test case 1. SPD nanoparticles activate the STING signaling pathway and induce PD-L1 protein upregulation. Mouse colon cancer cells (CT26 cells) were cultured at 2 × 10⁻⁶ cells per well. 6 Cells were cultured at a density of 100 μg / mL in 6-well cell culture plates for 24 h. Cells were treated with SPD nanoparticles at a concentration of 40 μg / mL for different time periods (0 h, 24 h, 48 h), with phosphate-buffered saline (PBS) and SP nanoparticles from Example 1 serving as controls. The expression levels of key proteins in the STING signaling pathway (phosphorylated TBK1, phosphorylated IRF3) and PD-L1 protein in cells were detected using Western blotting.

[0057] like Figure 7 As shown, compared with the control groups (PBS and SP), SPD nanoparticle treatment significantly increased the phosphorylation levels of TBK1 and IRF3, confirming its successful activation of the STING-TBK1-IRF3 signaling axis. Meanwhile, from... Figure 8 As can be seen, the expression level of PD-L1 protein increased with prolonged treatment time, reaching a peak after 24 hours of treatment. This result verifies that SPD nanoparticles have a dual function of "inducing" immune activation and feedback upregulating PD-L1.

[0058] 2. The effect, specificity and mechanism of SPTAC nanoparticles in degrading PD-L1 protein To evaluate the degradation effect, CT26 cells were treated with SPTAC nanoparticles (40 μg / mL) for different time periods (0–12 h). PD-L1 protein levels were detected by Western blotting, with PBS buffer and SP nanoparticles used as controls. Figure 9 As shown, SPTAC nanoparticles significantly reduced PD-L1 protein levels in CT26 cells, with the effect being pronounced within 4–6 hours. There was almost no difference in basal PD-L1 protein expression levels between the PBS control group and the SP group, confirming that the SP nanoparticles themselves had no significant effect on PD-L1 protein expression. This comparison clearly demonstrates that the observed PD-L1 protein degradation effect is entirely attributable to the protein degradation module successfully integrated into the SPTAC nanoparticles, rather than to non-specific effects from the material itself.

[0059] To assess the effect of exposure time on PD-L1 protein expression, CT26 cells were exposed to SPTAC nanoparticles for different durations. Results are as follows: Figure 10 As shown, the PD-L1 protein band gradually faded after 4 h, and the change in PD-L1 protein was not obvious after 6 h, demonstrating the strong degradation ability of SPTAC nanoparticles.

[0060] CT26 cells were treated with PBS, SP nanoparticles, a commercially available PD-L1 inhibitor (BMS-1166, 20 μg / mL), and SPTAC nanoparticles for 12 h. The Western blot results for PD-L1 protein are as follows: Figure 11 As shown in the figure, SPTAC nanoparticles exhibit the best degradation ability.

[0061] To investigate the degradation pathway, cells were pretreated with the proteasome inhibitor MG132 or the NEDD8 activator inhibitor MLN4924 before the addition of SPTAC nanoparticles. Figure 12 As shown, compared to direct exposure of CT26 cells to SPTAC nanoparticles, CT26 cells pretreated with MG132 and then exposed to SPTAC nanoparticles significantly inhibited PD-L1 protein degradation, revealing that SPTAC nanoparticle degradation of PD-L1 protein is related to the proteasome pathway. Similarly, CT26 cells pretreated with MLN4924 and then exposed to SPTAC nanoparticles also showed significantly inhibited PD-L1 protein degradation, indicating that SPTAC nanoparticle degradation of PD-L1 protein is also related to the ubiquitination pathway.

[0062] To clarify the specificity of SPTAC nanoparticles in inducing PD-L1 protein degradation, the effects of SP-T (without PD-L1 protein ligand), Comparative Example 1, SP-B (without E3 ligase ligand), and a mixture of both (SP-T+SP-B) on PD-L1 protein levels in CT26 cells were compared and investigated. The results are as follows: Figure 13 As shown, compared with the SPTAC nanoparticle treatment group, SP-T, SP-B, and their mixtures failed to induce significant degradation of PD-L1 protein. This result further confirms that SPTAC nanoparticles have significant specificity in inducing PD-L1 protein degradation, and their effect depends on the simultaneous presence of E3 ubiquitinase ligand and PD-L1 ligand on the same central material. This phenomenon also supports the following mechanism inference: in CT26 cells, SPTAC nanoparticles may guide PD-L1 protein into the ubiquitin-proteasome degradation pathway by simultaneously binding E3 ubiquitinase and PD-L1 protein to form a ternary complex.

[0063] 3. Implementation of sequential dosing strategies and evaluation of synergistic effects (1) The effect of sequential drug administration on the clearance of PD-L1 protein Based on the above results, a sequential dosing regimen was designed: CT26 cells were first treated with SPD nanoparticles (40 μg / mL) for 18 hours, followed by treatment with SPTAC nanoparticles (40 μg / mL) for another 6 hours; this group was designated as "SPD+SPTAC". Simultaneously, a PBS control group, a SP-only group, a SPD-only group, a SPTAC-only group, and a group treated with both SPD and SPTAC for 24 hours (SPD&SPTAC) were set up as controls. Immunofluorescence staining was used to visually observe and compare the fluorescence intensity of PD-L1 protein in each group of cells.

[0064] like Figure 14As shown, the expression of PD-L1 protein was evaluated using immunofluorescence imaging. In the figure, green fluorescence represents the PD-L1 signal, and blue fluorescence represents the signal of the nuclear dye DAPI (4',6-diamidinyl-2-phenylindole). The combined image shows the superposition of both. The results showed that in the sequential drug administration group (SPD+SPTAC), the green fluorescence signal representing PD-L1 was the weakest, significantly weaker than in the SPD&SPTAC treatment group. This result validates the effectiveness of the timing design of this invention: by setting the addition time of SPTAC nanoparticles (18 hours after SPD nanoparticle treatment) before the peak expression of PD-L1 (approximately 24 hours), and ensuring that the subsequent incubation process (6 hours) covers and spans this peak period, the maximum degradation activity window of SPTAC nanoparticles precisely coincides with the peak expression period of PD-L1. This design achieves "immediate expression and degradation" of the newly synthesized protein, thus most thoroughly clearing PD-L1 and breaking the immunosuppressive feedback.

[0065] (2) Synergistic antitumor cell activity of sequential administration The effect of different treatments on CT26 cell viability was evaluated using the MTT assay. CT26 cells were treated with different concentrations (0–40 μg / mL) of SP, SPD, SPTAC nanoparticles, and a sequential combination of SPD followed by SPTAC (SPD+SPTAC), and cell viability was detected. The sequential combination treatment method was the same as that in the SPD+SPTAC group in “(1) Sequential drug administration effect on PD-L1 protein clearance”, i.e., CT26 cells were first treated with SPD nanoparticles for 18 hours, and then treated with an equal concentration of SPTAC nanoparticles for another 6 hours. At the same time, the results were visually verified by a live-dead cell double staining fluorescence experiment. Calcein-AM was used to stain live cells (showing green fluorescence), and propidium iodide (PI) was used to stain dead cells (showing red fluorescence). The combined image was a superposition of the two to show the overall cell viability.

[0066] The results showed that SP nanoparticles had no significant effect on CT26 cell viability within the concentration range of 0-40 μg / mL. Figure 15 A in the text). SPD nanoparticles ( Figure 15 B), SPTAC nanoparticles ( Figure 15 As the concentration of C in the formula increased (0~40 μg / mL), the viability of CT26 cells gradually decreased. Among these, SPD+SPTAC showed the most significant inhibitory effect on CT26 cells. Figure 15 The presence of D in the figure indicates that it has a stronger activity in damaging tumor cells. Further experiments using CT26 cell live-death assays revealed that, as... Figure 16As shown, compared to the PBS group, the number of live cells (showing green fluorescence) and the number of dead cells (showing red fluorescence) were significantly reduced in the SPD+SPTAC-treated group, while the number of dead cells (showing red fluorescence) was significantly increased. Furthermore, the proportion of dead cells in the SPD+SPTAC-treated group was significantly higher than that in the SPD nanoparticle group and the SPTAC nanoparticle group. This result is consistent with the conclusions of the MTT assay, jointly confirming the excellent cytotoxic effect of SPD+SPTAC.

[0067] The above series of experimental examples confirm that, in this invention, the SPD component can effectively activate the immune system and induce PD-L1 feedback upregulation, while the SPTAC component can specifically degrade PD-L1 via the ubiquitin-proteasome pathway. Most importantly, the sequential dosing strategy of "administering SPD first, then SPTAC" achieves spatiotemporal synergy, maximizing the clearance of immunosuppressive molecules, thereby producing a significantly superior synergistic antitumor effect compared to a single component or a simple combination of both.

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A sequential induction and degradation-based nanocomposition, characterized in that, Comprise: a first nanoparticle and a second nanoparticle; the first nanoparticle comprises a first carrier and a STING signaling pathway agonist loaded on the first carrier; the STING signaling pathway agonist is 2,5-hexanone theobromine; the second nanoparticle comprises a second carrier and an E3 ligase ligand and a PD-L1 protein ligand loaded on the second carrier; the E3 ligase ligand is thalidomide-NH-CH2-COOH; the PD-L1 protein ligand is BMS-1166; the first nanoparticle and the second nanoparticle are configured to be sequentially administered, and the administration time of the first nanoparticle precedes that of the second nanoparticle.

2. The nanocomposition of claim 1, wherein, The first carrier and the second carrier are both core-shell structure nanoparticles formed by polyamidoamine dendrimer polymer coated silica.

3. The nanocomposition of claim 2, wherein, The polyamidoamine dendrimer polymer and the silica are connected by a covalent bond.

4. The nanocomposition of claim 1, wherein, The first carrier and the STING signaling pathway agonist are connected by a covalent bond.

5. The nanocomposition of claim 1, wherein, The E3 ligase ligand and the PD-L1 protein ligand are respectively connected to the second carrier by a covalent bond.

6. The nanocomposition of claim 1, wherein, The time interval of the sequential administration is 12-20h.

7. The method for preparing the sequential induction and degradation-based nanocomposition according to any one of claims 1 to 6, wherein, Comprise the following steps: mixing and reacting the STING signaling pathway agonist, the first activator and the first carrier in a first solvent, and obtaining the first nanoparticle by separation and purification; mixing and reacting the PD-L1 protein ligand, the E3 ligase ligand, the second activator and the second carrier in a second solvent, and obtaining the second nanoparticle by separation and purification.

8. The production method according to claim 7, wherein The first activator and the second activator are both a combination of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide.

9. The production method according to claim 7, wherein The first carrier and the second carrier are both core-shell structure nanoparticles formed by polyamidoamine dendrimer polymer coated silica.

10. The sequential induction and degradation-based nanoparticle composition according to any one of claims 1-6 or prepared by the preparation method according to any one of claims 7-9 for use in the preparation of an antitumor drug.

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