Nano-composite for promoting thermalization of cold tumors as well as preparation method and application of nano-composite

Abstract

The invention relates to a nano-composite for promoting thermalization of cold tumors as well as a preparation method and application of the nano-composite. The nano-composite comprises capsaicin and graphene oxide. According to the invention, capsaicin is combined with graphene oxide, so that immunogenic cell death and active oxygen mediated pyroptosis can be promoted, and infiltration and activation of CD4 < + > and CD8 < + > T cells can be remarkably enhanced by the combined immunoreaction, so that potent anti-tumor immunoreaction is induced, and remarkable tumor inhibition is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor, and particularly relates to a nano-complex for promoting the "thermization" of "cold" tumors, a preparation method thereof, and an application thereof. Background Art

[0002] Immunotherapy has revolutionized cancer treatment by harnessing the immune system's ability to recognize and kill malignant cells. However, its clinical efficacy in breast cancer remains limited, especially in "cold" tumors with low immune cell infiltration and weak immunogenicity. To address this challenge, developing novel strategies to enhance cancer cell immunogenicity and trigger a pro-inflammatory tumor microenvironment has become a research hotspot. Although most forms of cell death, such as apoptosis, are difficult to initiate an immune response due to lack of immunogenicity, types such as immunogenic cell death (ICD), ferroptosis, partial necroptosis, and recently discovered pyroptosis have strong immunogenicity. ICD transmits "phagocytosis signals" to the immune system by exposing tumor antigens and releasing damage-associated molecular patterns (DAMPs). Pyroptosis, as a highly inflammatory form of cell death, is characterized by the cleavage of the GSDM protein family and the release of pro-inflammatory cytokines such as IL-1β, IL-18, and IL-6, which can significantly enhance the immune activation effect. Although pyroptosis shares commonalities with ICD in the release of DAMPs such as HMGB1 and ATP, its individual action is difficult to fully activate adaptive immunity due to limited antigen presentation ability.

[0003] Regarding the current treatment of immune cold tumors, drug treatment is indispensable. Natural compounds have the characteristics of low toxicity and wide sources, and are an important source of innovative drug lead compounds. Studies have found that natural compounds can kill tumor cells through various pathways to achieve the effect of treating cancer. Pyroptosis is a new type of cell death in recent years. The pyroptosis process activates Cleaved Caspase 3 (abbreviated as CC3). CC3 cleaves GSDME to produce a toxic N-terminus, which punches holes in the cell membrane, leading to cell swelling, blistering, and cell death, accompanied by the release of a large number of pro-inflammatory factors, thereby triggering a series of immune responses.

[0004] According to research, a variety of natural compounds have been found to be able to kill cancer cells by inducing pyroptosis, achieving the purpose of tumor treatment. Although many natural compounds have been reported to be able to induce pyroptosis to kill tumor cells, they have many deficiencies, such as weak efficacy, insufficient targeting, low bioavailability, or large side effects. Exploring and developing other natural compounds with strong targeting and high biocompatibility for tumor treatment is of positive significance. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a nanocomposite for promoting the "thermalization" of "cold" tumors, a preparation method and applications thereof. The present invention combines capsaicin with graphene oxide, which can promote immunogenic cell death and reactive oxygen species-mediated pyroptosis. This combined immune response significantly enhances CD 4+ and CD8 + T cell infiltration and activation, thereby inducing a potent anti-tumor immune response and achieving significant tumor suppression.

[0006] To achieve this purpose, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a nanocomposite for promoting the "thermalization" of "cold" tumors, and the nanocomposite comprises capsaicin and graphene oxide.

[0008] In the present invention, a nanocomposite capable of synchronously inducing pyroptosis and ICD has been developed. It is worth noting that the C═C-rich characteristics of GO and Cap (both containing benzyl structures) enable them to be assembled into a Cap / GO nanocomposite. This composite exhibits dual advantages in the classical breast cancer research model EMT-6 syngeneic model by synergistically inducing pyroptosis and ICD: directly killing tumor cells while enhancing anti-cancer immunity, providing a new idea for the development of long-term breast cancer treatment regimens.

[0009] Preferably, the connection mode between the capsaicin and the graphene oxide includes any one of π-π stacking interaction, van der Waals force or electrostatic interaction.

[0010] In the present invention, capsaicin (Cap, chemical name: (E)-N-(4-hydroxy-3-methoxybenzyl)-8-methylnon-6-enamide) is a natural compound that can induce reactive oxygen species (ROS)-mediated pyroptosis and bind to graphene oxide (GO) through π-π stacking interaction, thereby promoting immunogenic cell death (ICD) under near-infrared (NIR) irradiation. Intratumoral injection of this physically bound Cap / GO nanocomposite can simultaneously trigger pyroptosis and ICD, forming an immunologically active tumor microenvironment rich in IL-6 and IL-1β (released by pyroptosis) as well as CRT and HMGB1 (released by ICD). This combined immune response significantly enhances CD 4+ and CD 8+ T cell infiltration and activation, thereby inducing a potent anti-tumor immune response and achieving significant tumor suppression.

[0011] Preferably, the mass ratio of capsaicin to graphene oxide in the nanocomposite is 1:(0.5-5). The (0.5-5) can be, for example, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5, etc.

[0012] Preferably, the carbonyl content of the graphene oxide is 0.5%-50%, for example, it can be 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%, etc.

[0013] Preferably, the particle size of the graphene oxide is 500-1500 nm, for example, it can be 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm or 1500 nm, etc.

[0014] In a second aspect, the present invention provides a method for preparing the nano-complex for promoting the "thermalization" of "cold" tumors described in the first aspect, the preparation method includes mixing the graphene oxide solution and the capsaicin solution, and then centrifuging to obtain.

[0015] Preferably, the mass ratio of capsaicin to graphene oxide in the mixed solution is 1:(0.5-5). The (0.5-5), for example, can be 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5, etc.

[0016] During the mixing process, the stirring temperature is 20-30 °C and the time is 2-30 h. The 20-30 °C, for example, can be 20 °C, 22 °C, 24 °C, 26 °C, 28 °C or 30 °C, etc. The 2-30 h, for example, can be 2 h, 5 h, 10 h or 30 h, etc.

[0017] Preferably, the centrifugation speed is 12000-18000 g and the time is 10-20 min. The 12000-18000 g, for example, can be 12000 g, 13000 g, 14000 g, 15000 g, 16000 g, 17000 g or 18000 g, etc. The 10-20 min, for example, can be 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 20 min, etc.

[0018] In a third aspect, the present invention provides a pharmaceutical composition, the pharmaceutical composition includes the nano-complex for promoting the "thermalization" of "cold" tumors described in the first aspect and pharmaceutically acceptable excipients.

[0019] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of carriers, diluents, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, bacteriostatic agents or buffers.

[0020] Fourthly, the present invention provides an application of the nano - composite for promoting the "thermalization" of "cold" tumors described in the first aspect and / or the pharmaceutical composition described in the second aspect in the preparation of anti - tumor drugs, wherein the tumors include any one or at least two combinations of breast cancer, pancreatic cancer, prostate cancer, glioblastoma, gastric cancer, colorectal cancer, hepatocellular carcinoma or ovarian cancer.

[0021] Compared with the prior art, the present invention has at least the following beneficial effects:

[0022] The present invention combines capsaicin and graphene oxide to construct a nano - composite that can achieve the combination of immunogenic cell death and pyroptosis. It can not only directly eliminate tumor cells, but also convert "cold" tumors into "hot" tumors, thereby enhancing immune cell infiltration and significantly amplifying the anti - tumor immune response.

[0023] ICD enhances the recognition of cancer cells by the immune system by releasing DAMPs and tumor - specific antigens. At the same time, the pro - inflammatory factors released by pyroptosis can reshape the tumor immune microenvironment, recruit and activate immune cells to strengthen the anti - tumor immunity. This dual mechanism can effectively reverse the immunosuppressive microenvironment, while stimulating a strong local immune response and supporting systemic anti - tumor immunity. Description of the Drawings

[0024] Figure 1 It is a schematic diagram for the preparation of the nano - composite.

[0025] Figure 2 It is a graph of the cancer cell killing rate of different graphene oxides.

[0026] Figure 3 It is a graph of the relative fluorescence intensity of reactive oxygen species of different graphene oxides.

[0027] Figure 4 It is a structural diagram of graphene oxides with different sizes.

[0028] Figure 5 It is a graph of the characterization results of the nano - composite. Among them, Figure A is the surface potential result graph, and Figure B is the TLC detection result graph.

[0029] Figure 6 It is a schematic diagram of the anti - tumor effect of the Cap / GO nano - composite.

[0030] Figure 7 It is a Western blot result graph of the pyroptosis index.

[0031] Figure 8 It is a graph of the change in the expression level of the immunogenic cell death index.

[0032] Figure 9 It is a graph of the cell viability experiment results.

[0033] Figure 10 The figure shows the volume change results of small tumors in different treatment groups.

[0034] Figure 11 The figure shows the curve of the tumor volume of mice in different treatment groups changing with time.

[0035] Figure 12 The figure shows the statistical analysis of the tumor weights of mice in different treatment groups on the 14th day of the experiment. Detailed implementation manners

[0036] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and through specific implementation manners. However, the following examples are merely simple examples of the present invention and do not represent or limit the scope of the protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0037] Example 1

[0038] In this example, a nanocomposite is prepared

[0039] (1) Explore the preparation of nanocomposites with graphene oxide of different sizes and carbonyl contents

[0040] In order to explore that the lateral size and carbonyl (C=O) content of graphene oxide significantly affect its biological effects on cells, in this example, graphene oxides with carbonyl contents of ~5%, ~10% and ~15%, and particle sizes of ~500 nm, ~1000 nm and ~1500 nm are used as research objects respectively, and their potential as ICD inducers in cancer cells is screened and compared to prepare more effective nanocomposites. The specific parameters are shown in Table 1. The structural detection diagrams of particles with sizes of 500 nm, 1000 nm and 1500 nm are as Figure 1 shown.

[0041] Table 1

[0042]

[0043]

[0044] The results of cancer cell killing rate and relative fluorescence intensity of reactive oxygen species are as Figure 2 and Figure 3 shown. Considering that a larger lateral size can adsorb more Cap, and at the same time a higher C=O content can induce the generation of a large amount of ROS to kill cancer cells. Combining the results of cancer cell killing and the change amount of reactive oxygen species in the above table, we choose GO-3 to evaluate its binding ability with Cap.

[0045] Specifically, the above-screened graphene oxide (GO-3) was prepared into a graphene oxide solution with a concentration of 1 mg / mL, and mixed with 2.5 mL of Cap solution pre-dissolved in dimethyl sulfoxide (DMSO) and adjusted to 1 mg / mL. The mixture was stirred at 25 °C for 19 h to promote the physical adsorption between GO and Cap. Subsequently, the solution was centrifuged at 15,000 g for 15 min to separate the Cap / GO nanocomposite and the supernatant.

[0046] The structure of the Cap / GO nanocomposite prepared in the present invention is as Figure 4 shown. Due to the complementarity of the interaction mechanism between Cap and GO, the aromatic ring of Cap interacts with the π-electron system of GO through π-π stacking, and at the same time, van der Waals forces may also participate in the adsorption process. In addition, the negative charge conferred by the oxygen-containing groups on the GO surface can produce electrostatic interactions with the positively charged or polar regions in the Cap molecule, promoting the formation of the nanocomposite through the above binding methods.

[0047] The precipitate obtained after centrifugation was washed twice with deionized water to remove unbound capsaicin. The surface potential changes of GO and its Cap / GO composite were analyzed by dynamic light scattering (DLS). At the same time, supernatant samples were taken before (0 h) and after (19 h) mixing for thin-layer chromatography (TLC) analysis: using a developing agent system with ethyl acetate and n-hexane mixed in a ratio of 2:1, and the sample application amount was 2 μL to verify the adsorption efficiency of Cap on GO-3. The specific results are as Figure 5 shown. Through dynamic light scattering (DLS) and thin-layer chromatography (TLC), the successful construction of the physical Cap / GO composite was verified: the negative surface potential of GO decreased after incubation with Cap, and the Cap content in the supernatant of the nanocomposite continuously decreased during the 0-19 hour adsorption process. The measurement showed that the mass ratio of Cap to GO in the composite was approximately 1:2.

[0048] Example 2

[0049] In this example, immunoblotting experiments were performed for detecting pyroptosis

[0050] Capsaicin (Cap) in the Cap / GO nanocomposite prepared in the present invention is a natural compound that can induce reactive oxygen species (ROS)-mediated pyroptosis and bind to graphene oxide (GO) through π-π stacking, thereby promoting immunogenic cell death (ICD) under near-infrared (NIR) irradiation. As Figure 6 shown, intratumoral injection of this physically bound Cap / GO nanocomposite can simultaneously trigger pyroptosis and ICD, forming an immunologically active tumor microenvironment rich in GSDME-NT (a key driving protein for pyroptosis) and CRT and HMGB1 (released by ICD). This combined immune response significantly enhances CD4+ and CD 8+ The infiltration and activation of T cells, thereby inducing a potent anti-tumor immune response and achieving significant tumor suppression.

[0051] To explore the pyroptosis-promoting effect of the Cap / GO nanocomposite, in this example, breast cancer EMT6 cells were continuously incubated with 25 μg / mL of Cap monomer, GO monomer, or Cap / GO nanocomposite for 24 hours, and then the proteins in the cells were collected. Western blot (WB) was used to analyze the expression of GSDME-NT to evaluate the cell pyroptosis. The experimental results are as Figure 7 shown. The Cap / GO composite, which was used to simultaneously activate pyroptosis and ICD, significantly upregulated the expression of GSDME-NT in EMT6 cells, while GO combined with NIR (only inducing ICD) failed to produce this effect, and this upregulation was significantly stronger than that of Cap alone (only inducing pyroptosis), which further demonstrated the potential advantages of the nanocomposite.

[0052] Example 3

[0053] This example characterized the immunogenic cell death effect

[0054] To verify the effect of the Cap / GO nanocomposite in inducing ICD, in this example, an EMT6 cell-bearing mouse model was established on Balb / C mice, with 6 mice in each group. Intratumoral administration was carried out in groups: control group, Cap monomer, GO monomer, or Cap / GO nanocomposite group. Among them, 24 hours after administration in the GO and Cap / GO nanocomposite groups, an 808 nm infrared laser was used to irradiate the tumor site of the mice. The maximum power (about 5.8 W) was used to raise the irradiation temperature to 55 °C, and the power was adjusted to maintain the temperature and continuously irradiate for 5 minutes. Three days after administration, mouse serum and tumor tissues were obtained. The serum was used for ELISA to detect inflammatory factors, and the tumor tissues were fixed, embedded, sectioned, and immunohistochemically (IHC) stained to analyze key ICD markers (such as CRT membrane translocation and HMGB1 release). The semi-quantitative results of immunohistochemistry are as Figure 8 shown. By immunohistochemistry, the Cap / GO nanocomposite could significantly increase the expression levels of CRT and HMGB1, inducing ICD; while inducing pyroptosis alone failed to achieve this effect, and it had a stronger effect than only inducing ICD (GO monomer group).

[0055] Example 4

[0056] This example evaluated the anti-tumor cell effect

[0057] After evaluating pyroptosis by analyzing the expression of GSDME-NT through Western blot (WB) and simultaneously detecting the levels of the key immunogenic cell death (ICD) markers CRT and HMGB1 in EMT6 tumor-bearing mice by immunohistochemistry (IHC), this example further used the CCK-8 cell viability assay to quantify the killing effect of each treatment regimen on cancer cells. In the experiment, EMT6 breast cancer cells were seeded in 96-well plates and cultured until the logarithmic growth phase, and then the following treatment groups were given respectively: Cap / GO complex (25 μg / mL) (combining pyroptosis and ICD induction); Cap treatment group (only inducing pyroptosis); GO-3 + NIR treatment group (only inducing ICD); control group. After 24 hours of treatment, according to the instructions of the cell viability detection kit (CCK-8), the optical density (OD value) at 450 nm was measured, and the cell survival rate was calculated. The cell viability results, as Figure 9 shown, compared with using Cap or GO alone, the Cap / GO nanocomposite could significantly enhance the cancer cell killing effect, and the cell survival rate was significantly reduced, suggesting that the synergistic induction of pyroptosis and ICD has higher efficacy in anti-cancer treatment.

[0058] Example 5

[0059] In this example, an anti-tumor experiment of the nanocomposite was carried out

[0060] To further explore the synergistic therapeutic potential of the Cap / GO complex, this example evaluated the anti-tumor effect of the Cap / GO nanocomposite in an EMT6 tumor-bearing mouse model. After the tumor-bearing mice received intratumoral injection of the Cap / GO complex, NIR irradiation was carried out for 5 minutes to raise the local temperature to about 50 °C. The control groups included PBS, Cap alone, and GO alone treatment groups. During the 14-day treatment period, the changes in tumor size, volume, and weight of each group were monitored. The expression levels of ICD markers (CRT, HMGB1), immune cell infiltration (CD 4+ , CD 8+ ) and anti-tumor immune factor (IFN-γ) in tumor tissues were analyzed by IHC staining. The integrated optical density percentage of the IHC images was semi-quantitatively analyzed using ImageJ software to quantify the expression intensity of the above markers. The comparison of tumor volumes in the mouse breast cancer (EMT6) xenograft model under different treatment conditions, as Figure 10 shown, there were 6 mice in each of the control group (untreated), capsaicin (Cap) group, graphene oxide (GO) group, and Cap / GO nanocomposite group. The tumor volume of the Cap / GO nanocomposite group was significantly reduced (green tick mark), indicating that it has strong anti-tumor activity against the breast cancer model. The comparison of tumor volumes, as Figure 11As shown, the nano - composite group showed the most obvious tumor - suppressing effect. Compared with other groups, the tumor volume increased most slowly. On the 14th day of the experiment, the statistical analysis of the tumor weights of mice in each group was as Figure 12 shown. The tumor weight of the nano - composite group was significantly lower than that of the control group, the graphene oxide group, and the capsaicin group, indicating its potent anti - tumor effect in the breast cancer model.

[0061] In summary, by adsorbing and combining capsaicin and graphene oxide to construct the Cap / GO nano - composite, the present invention can promote immunogenic cell death and pyroptosis of tumors, showing dual advantages in the classical EMT - 6 syngeneic model of breast cancer research: directly killing tumor cells while enhancing anti - cancer immunity, providing a new idea for the development of long - acting breast cancer treatment regimens.

[0062] The applicant declares that the above - mentioned is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A nanocomposite for promoting the "thermalization" of "cold" tumors, characterized in that, The nano - composite includes capsaicin and graphene oxide.

2. The nano - composite for promoting the "thermalization" of "cold" tumors according to claim 1, wherein The connection mode between the capsaicin and the graphene oxide includes at least one of π - π stacking interaction, van der Waals force or electrostatic interaction.

3. The nano - composite for promoting the "thermalization" of "cold" tumors according to claim 1 or 2, characterized in that, The mass ratio of capsaicin to graphene oxide in the nano - composite is 1:(0.5 - 5).

4. The nano - composite for promoting the "thermalization" of "cold" tumors according to any one of claims 1 - 3, characterized in that, The carbonyl content of the graphene oxide is 0.5% - 50%; Preferably, the particle size of the graphene oxide is 500 - 1500 nm.

5. A method for preparing a nanocomposite for promoting the "thermalization" of "cold" tumors according to any one of claims 1-4, characterized in that, The preparation method includes mixing a graphene oxide solution and a capsaicin solution and obtaining the product after centrifugation.

6. The preparation method of the nanocomposite for promoting the "thermalization" of "cold" tumors according to claim 5, characterized in that, The mass ratio of capsaicin to graphene oxide in the mixed solution is 1:(0.5 - 5).

7. The preparation method of the nano - composite for promoting the "thermalization" of "cold" tumors according to claim 5 or 6, characterized in that, During the mixing process, the stirring temperature is 20 - 30 °C and the time is 2 - 30 h; Preferably, the centrifugation speed is 12000 - 18000 g and the time is 10 - 20 min.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes the nano - composite for promoting the "thermalization" of "cold" tumors described in any one of claims 1 - 4 and pharmaceutically acceptable excipients.

9. The pharmaceutical composition according to claim 8, wherein The pharmaceutically acceptable excipients include any one or a combination of at least two of carriers, diluents, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, bacteriostatic agents or buffers.

10. Use of the nanocomposite for promoting "thermalization" of "cold" tumors as described in any one of claims 1-4 and / or the pharmaceutical composition as described in claim 8 or 9 in the preparation of a tumor therapeutic drug, characterized in that, The tumors include any one or a combination of at least two of breast cancer, pancreatic cancer, prostate cancer, glioblastoma, gastric cancer, colorectal cancer, hepatocellular carcinoma or ovarian cancer.

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