A multifunctional tumor-targeted cryotherapy nanoplatform and its applications

Abstract

This invention discloses a multifunctional tumor-targeted cryotherapy nanoplatform and its applications, belonging to the field of nanobiomedicine technology. The multifunctional tumor-targeted cryotherapy nanoplatform of this invention uses two-dimensional Ti3C2 nanomaterials as a carrier, loaded with indocyanine green as a photosensitizer, surface-modified with polydopamine to improve stability, and loaded with gambogeylic acid as an anti-tumor drug and a heat shock protein inhibitor. It is covalently coupled with nucleic acid aptamers that can recognize cell surface-specific proteins. The multifunctional tumor-targeted cryotherapy nanoplatform has small particle size, high dispersion, and good biocompatibility, and exhibits excellent photothermal conversion ability, photodynamic therapy effect, and chemotherapy effect in tumor-targeted therapy. It can effectively induce tumor cell apoptosis under relatively low temperature conditions, significantly reducing the damage of high temperature to surrounding healthy tissues. It also has the ability to specifically recognize tumor lesions, achieving high enrichment at the tumor site and enhancing the therapeutic effect.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomedicine technology, specifically relating to a multifunctional tumor-targeted cryotherapy nanoplatform and its applications. Background Technology

[0002] Two-dimensional Ti3C2 nanomaterials, as a high-performance photothermal agent (PTA), have shown great potential in tumor therapy. Their unique near-infrared (NIR) light absorption and efficient photothermal conversion properties enable Ti3C2 nanomaterials to generate high temperatures under NIR laser irradiation, thus achieving photothermal therapy (PTT) for tumors. However, significant drawbacks remain in the current application of Ti3C2 nanomaterials in single-treatment PTT for tumors. To achieve effective tumor ablation, the temperature of the tumor region typically needs to reach above 50°C, requiring the use of high-concentration PTA or high-power-density laser irradiation. However, the high temperatures generated during this process can not only damage surrounding healthy tissues but also trigger inflammatory responses in the body, leading to the release of intracellular biomolecules and increasing the risk of tumor metastasis.

[0003] To address the limitations of single-modality photodynamic therapy (PTT), researchers have begun exploring combination therapies, such as combining PTT with photodynamic therapy (PDT) and chemotherapy (CHT). For instance, by loading photosensitizers onto Ti3C2 nanomaterials, tumor PDT properties can be imparted to nanotherapeutic platforms, enabling combined PDT and PTT treatment of tumors. Furthermore, anti-tumor drugs can be loaded onto nanoplatforms to achieve combined PTT and CHT treatment. However, while these combination therapies have improved treatment outcomes to some extent, issues such as less than ideal therapeutic effects and low targeting precision remain. In particular, when PTT is used as the primary treatment method, the high temperatures it generates can still damage surrounding tissues, limiting its widespread clinical application.

[0004] In the prior art, patent CN111153405B discloses a method for preparing Ti3C2 MXene nanosheet materials, including the following steps: Step 1, adding titanium aluminum carbon powder, hydrofluoric acid solution, and hexadecyltrimethylammonium bromide to a corrosion-resistant container and stirring at 20-60℃ for 6-60 hours to obtain a CTAB-intercalated Ti3C2 mixture; Step 2, centrifuging the CTAB-intercalated Ti3C2 mixture, washing with water until the supernatant is neutral, filtering to collect the precipitate, and obtaining a CTAB-intercalated Ti3C2 multilayer material; Step 3, adding water to the CTAB-intercalated Ti3C2 multilayer material, sonicating for 30-120 minutes, and freeze-drying to obtain a CTAB-intercalated Ti3C2 nanosheet powder material. However, this method for preparing Ti3C2 nanosheet powder material requires sonication for 30-120 minutes after CTAB intercalation, making the preparation process cumbersome and consuming more energy.

[0005] In the prior art, patent CN118384272A discloses a targeted nanomedicine for combination therapy of breast cancer, its preparation method, and its application. This invention uses Fe-TCPP as a carrier and therapeutic agent, indocyanine green as a photosensitizer and photothermal agent loaded onto the Fe-TCPP surface, and nucleic acid aptamers as targeting molecules coupled to the Fe-TCPP surface. However, this invention lacks sufficient functional development of indocyanine green and nucleic acid aptamers, and does not deeply utilize the in vivo fluorescence imaging capability of indocyanine green, nor does it conduct in-depth research on the targeting effect and tissue distribution of nucleic acid aptamers in mice. Although existing technologies also use indocyanine green and nucleic acid aptamers, their research on targeting effects and optimal targeting time in mice is insufficient.

[0006] In existing technologies, the paper "Study on the Application of Garcinia Cambogia-Indocyanine Green Co-loaded Nanostructured Liposomes in Cervical Cancer Treatment Based on Pyroptosis Pathway" utilizes nanostructured liposomes to co-load the photosensitizer indocyanine green and the natural drug component garcinia Cambogia, and modifies its surface with the tumor-targeting peptide cTMTP1 for anti-tumor application. However, this method provides a targeted therapy strategy for cervical cancer, and it does not deeply utilize the in vivo fluorescence imaging capability of indocyanine green to study its targeting effect in mice. Furthermore, it does not deeply utilize garcinia Cambogia's ability to inhibit the expression of tumor cell heat shock proteins (HSPs) for tumor cryotherapy. Additionally, this study uses a simple garcinia Cambogia loading method and does not achieve the triple-response garcinia Cambogia drug release function based on the pH / GSH / NIR-I environment in the tumor microenvironment.

[0007] In existing technologies, the paper "A Highly Biocompatible Nanoparticle Therapy Platform Encapsulated with Polydopamine for Enhanced Photothermal Therapy of Cervical Cancer" utilizes polydopamine and monoamino polyethylene glycol to encapsulate and modify the photothermal material indocyanine green, forming nanoparticles. The therapeutic effects on cervical cancer cells and cervical cancer-bearing mice were evaluated using photothermal therapy. However, this method does not fully utilize the fact that indocyanine green can also perform photothermal therapy (PDT) on tumors under near-infrared 808nm laser irradiation.

[0008] Given the challenges of tumor treatment and a comprehensive evaluation of existing technologies, integrating multiple treatment modalities and relying on active targeting technologies to achieve precise tumor treatment under cryogenic conditions has become a current research hotspot. Cryotherapy not only avoids damage to normal tissues caused by high temperatures, improving treatment safety, but also reduces the heat resistance of tumor cells by inhibiting the expression of HSPs within the tumor, thereby improving the treatment efficiency of PTT (post-treatment telescope). Therefore, there is an urgent need to develop a superior multifunctional nanotherapy platform that integrates multiple treatment modalities such as PTT, PDT, and CHT into a single nanosystem and increases its targeted enrichment at the tumor site to achieve targeted cryogenic precision treatment of tumors. Summary of the Invention

[0009] To address the issues of excessively high temperatures during PTT treatment damaging surrounding healthy tissue; the limited therapeutic effect of a single PTT treatment modality; and the lack of specificity in active tumor targeting, this invention provides a multifunctional tumor-targeting cryotherapy nanoplatform and its applications.

[0010] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0011] A multifunctional tumor-targeted cryotherapy nanoplatform uses two-dimensional Ti3C2 nanomaterials as a carrier, and is subsequently modified with indocyanine green (ICG), polydopamine (PDA), and gamboge acid (GA) to form a surface, and is coupled with a nucleic acid aptamer (Apt-M) that can recognize cell surface-specific proteins.

[0012] Furthermore, the average size of the two-dimensional Ti3C2 nanomaterial is ~100nm, and the average thickness is 5.0nm.

[0013] Furthermore, the indocyanine green (ICG) is loaded onto two-dimensional Ti3C2 nanomaterials as a photosensitizer via electrostatic interaction.

[0014] Furthermore, the nucleic acid aptamer is covalently coupled to the surface of the two-dimensional Ti3C2 nanomaterial.

[0015] A method for preparing a multifunctional tumor-targeted cryotherapy nanoplatform includes the following steps:

[0016] Step 1, ICG loading: Ti3C2 nanosheets were added to an aqueous solution of indocyanine green (ICG), and the precipitate was collected by centrifugation to prepare Ti3C2 / ICG (TI) nanosheets;

[0017] Step 2, PDA surface modification: Ti3C2 / ICG (TI) nanosheets were dispersed in Tris-HCl buffer, then dopamine hydrochloride was added, and the Ti3C2 / ICG / PDA (TIP) nanosheets were collected by centrifugation;

[0018] Step 3, GA loading: First, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) are used to activate gamboge acid (GA). The activated gamboge acid (GA) is then reacted with mPEG-SS-NH2 to obtain PEG-SS-GA. PEG-SS-GA is then added to Ti3C2 / ICG / PDA (TIP) nanosheets to prepare Ti3C2 / ICG / PDA / GA (TIPG) nanosheets.

[0019] Step 4, Apt-M Coupling: Ti3C2 / ICG / PDA / GA (TIPG) nanosheets were dispersed in Tris-HCl buffer containing nucleic acid aptamer (Apt-M) and stirred to obtain Ti3C2 / ICG / PDA / GA / Apt-M (TIPGA) nanoplatform.

[0020] Furthermore, the preparation method of the two-dimensional Ti3C2 material is as follows: Ti3AlC2 powder is mixed with HF aqueous solution, stirred and reacted at room temperature, and the precipitate is washed with deionized water after centrifugation. The precipitate is then dispersed in tetrapropylammonium hydroxide (TPAOH) aqueous solution and stirred and reacted. After centrifugation, the precipitate is washed with deionized water to obtain two-dimensional Ti3C2 nanomaterials.

[0021] An application of a multifunctional tumor-targeted cryotherapy nanoplatform in tumor-targeted therapy. This multifunctional tumor-targeted cryotherapy nanoplatform can actively identify specific tumor cells, has the ability to specifically identify tumor lesions, can achieve high enrichment at the tumor site, and can perform photothermal / photodynamic / chemotherapy combination therapy at low temperature (≤45℃), effectively inhibiting tumor growth, thereby achieving precision medicine.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] (1) This invention ingeniously loads water-insoluble GA onto the TIPGA nanoplatform via covalent bonding. Leveraging the acidic pH and high glutathione (GSH) concentration in the tumor microenvironment, it achieves precise controlled release of the chemotherapeutic drug GA at the tumor site, enhancing the therapeutic effect. Furthermore, the precisely released GA at the tumor site reduces the expression of HSPs in tumor cells, allowing the TIPGA nanoplatform to effectively induce tumor cell apoptosis at lower temperatures (≤45℃), improving the efficiency of tumor treatment under low-temperature conditions, reducing damage to surrounding healthy tissues from high temperatures, and lowering the risk of tumor metastasis.

[0024] (2) The TIPGA nanoplatform prepared by the present invention has multimodal combined therapy characteristics. By integrating PTT, PDT and CHT, it not only enhances the therapeutic effect of deep tumors, but also overcomes the limitations of single photothermal therapy in completely eradicating tumors.

[0025] (3) The TIPGA nanoplatform prepared by this invention has high-efficiency targeting performance. Through Apt-M functionalization modification, the TIPGA nanoplatform has excellent active targeting performance at both the in vitro tumor cell level and the in vivo mouse tumor tissue level, thereby improving the specificity of treatment and drug utilization, reducing the impact on normal cells and tissues, and reducing side effects.

[0026] (4) Unlike the existing patent CN111153405B, the Ti3C2 nanosheets of this invention can be prepared without ultrasound after treatment with TPAOH aqueous solution, which is simple and avoids consuming more energy. Unlike the existing patent CN118384272A, although this invention also loads indocyanine green and nucleic acid aptamers, this invention utilizes the characteristic of indocyanine green to perform in vivo fluorescence imaging to conduct in-depth research on the targeting effect of nucleic acid aptamers in mice. Compared with the existing research paper "Study on the application of gambogeic acid-indocyanine green co-loaded nanostructured liposomes in the treatment of cervical cancer based on the pyroptosis pathway", this invention not only provides a treatment strategy for specific breast tumors, but also makes in-depth use of the fluorescence imaging characteristics of indocyanine green to study the targeting effect in mice, and innovatively designs the gambogeic acid loading method to achieve the triple-response drug release performance of gambogeic acid, thereby improving the treatment's specificity and effectiveness. Compared with the existing research paper "Highly Biocompatible Nanotherapy Platform Encapsulated indocyanine Green for Enhanced Photothermal Therapy of Cervical Cancer", this invention delves deeper into the PDT therapeutic potential of indocyanine green and combines it with PTT to enable indocyanine green to achieve a dual PTT / PDT treatment mode. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a schematic diagram illustrating the preparation and application of the multifunctional cryotherapy nanoplatform in this invention;

[0029] Figure 2 This is a TEM image of the Ti3C2 material in Example 1 of the present invention;

[0030] Figure 3 AFM image of Ti3C2 material in Example 1 of this invention;

[0031] Figure 4 The XRD analysis results are shown for the Ti3C2 material in Example 1 of this invention.

[0032] Figure 5 The results of Zata potential analysis for each nanomaterial in Example 1 of this invention;

[0033] Figure 6 The results of dynamic light scattering analysis of each nanomaterial in Example 1 of this invention;

[0034] Figure 7 The results show the temperature changes of the TIPGA material dispersion under different 808nm laser power densities in Example 2 of this invention.

[0035] Figure 8 The results of photodynamic performance analysis of TIPGA material in Example 3 of this invention;

[0036] Figure 9 The results of the GSH consumption capacity analysis of TIPGA material in Example 4 of this invention;

[0037] Figure 10 The results of the effects of pH, GSH and NIR-I laser irradiation on the release of GA from the TIPGA material in Example 5 of the present invention are shown.

[0038] Figure 11 This is a comparison chart of the uptake of MCF-7 cells by different nanomaterials after 4 hours of incubation in Example 6 of the present invention;

[0039] Figure 12 These are Western blotting images of HSP90 protein expression levels in MCF-7 cells under different treatments in Example 7 of this invention.

[0040] Figure 13The results show the survival rates of MCF-7 and HepG2 cells after treatment with various nanomaterials in Example 8 of this invention.

[0041] Figure 14 This is a graph showing the change in material enrichment at the tumor site of mice after tail vein injection of various nanomaterials in Example 9 of the present invention over time.

[0042] Figure 15 These are digital infrared thermographic images of mice during treatment with various nanomaterials in Example 10 of this invention.

[0043] Figure 16 This is a graph showing the changes in tumor volume in mice after treatment with various nanomaterials in Example 10 of the present invention;

[0044] Figure 17 These are images showing the anatomical results of mouse tumors treated with various nanomaterials in Example 10 of this invention.

[0045] Figure 18 These are digital photographs of mice treated with various nanomaterials in Example 10 of this invention within 15 days.

[0046] Figure 19 This is a graph showing the changes in mouse body weight during treatment with various nanomaterials in Example 10 of the present invention. Detailed Implementation

[0047] To gain a deeper understanding of this invention, we will provide a comprehensive and detailed description. However, this invention has various implementations and is not limited to the specific examples listed herein. These examples are presented to enhance a full understanding of the disclosure of this invention.

[0048] Example 1

[0049] Depend on Figure 1 As shown, a multifunctional tumor-targeting cryotherapy nanoplatform uses two-dimensional Ti3C2 nanomaterials as a carrier; ICG is loaded as a photosensitizer with an ICG loading of 38.94%; PDA is then modified on the surface to improve stability; GA is loaded as an anti-tumor drug and HSP inhibitor with a GA loading of 9.02%; finally, Apt-M, which can recognize cell surface-specific proteins, is covalently coupled to give it targeting ability.

[0050] The preparation method of this multifunctional nanoplatform includes the following steps:

[0051] Step 1, Preparation of two-dimensional Ti3C2 material: 1.0 g of Ti3AlC2 powder was slowly added to 20 mL of 40% HF aqueous solution (please take personal protective measures when using this hazardous reagent), and stirred at room temperature for 3 days. After centrifugation, the precipitate was washed with deionized water and dispersed in 20 mL of LTPAOH aqueous solution (25 wt.%), and stirred at room temperature for 3 days. The precipitate was then collected by centrifugation (3500 rpm, 10 min) and washed 6–8 times with deionized water to adjust the pH of the solution to be greater than 6.0. Finally, Ti3C2 nanosheets were prepared by centrifugation at 3500 rpm for 60 min.

[0052] Depend on Figure 2 The TEM image shown indicates that the particle size of the two-dimensional Ti3C2 material is 100 nm. Figure 3 The AFM image shown indicates that the average thickness of the two-dimensional Ti3C2 material is approximately 5.0 nm. Figure 4 The XRD analysis results show that the obtained two-dimensional Ti3C2 material has almost no characteristic peak at 2θ≈39.0° (104), and its characteristic peak at 2θ≈9.5° (002) is broadened and moves to a lower angle (6.0°), which proves the successful preparation of two-dimensional Ti3C2 nanomaterials.

[0053] Step 2, ICG loading: The prepared Ti3C2 nanosheets (5.0 mg) were slowly added to an ICG aqueous solution (5.0 mL, 0.4 mg / mL). -1 The mixture was stirred at room temperature for 4 hours. The precipitate was then collected by centrifugation (12000 rpm, 5 min) and washed three times with deionized water to remove residual ICG, thus preparing Ti3C2 / ICG (TI) nanosheets.

[0054] Depend on Figure 5 and Figure 6 As can be seen, due to the loading of ICG, the hydrated particle size of TI increased from 93.7 nm to 119.2 nm, and the Zeta potential decreased from -21.27 mV to -27.80 mV. These results indicate that ICG was successfully loaded into two-dimensional Ti3C2 nanomaterials.

[0055] Step 3, PDA surface modification: TI nanosheets (5.0 mg) were dispersed in Tris-HCl buffer (5.0 mL, 10 mM, pH 8.5), and then dopamine hydrochloride (25 μL, 100 mg / mL) was added. -1 The mixture was stirred at room temperature and in the dark for 4 hours. The Ti3C2 / ICG / PDA (TIP) nanosheets were then collected by centrifugation (12000 rpm, 5 min) and washed three times with deionized water.

[0056] Depend on Figure 5and Figure 6 As can be seen, due to the modification of the TI surface with PDA, the hydrated particle size of TIP increased to 139.9 nm, while the Zeta potential decreased to -29.17 mV. These results indicate that the TIP surface was successfully modified with PDA.

[0057] Step 4, GA loading: First, add 0.4 mg of GA to 200 μL of DMSO, and activate it for 30 min under light-protected conditions with EDC (900 μL, 200 mM) and NHS (900 μL, 50 mM). Then wash the activated GA three times with deionized water. Next, add the activated GA to 1.0 mL of mPEG-SS-NH2 (2.5 mg / mL). -1 The reaction was carried out under shaking conditions for 5 hours. The product was then encapsulated in a dialysis bag with a molecular weight cutoff of 3000 Daltons, and the bag was immersed in 1000 mL of deionized water. Dialysis was performed at room temperature for 24 hours to obtain PEG-SS-GA. Finally, PEG-SS-GA was slowly added to a TIP solution (5.0 mL, 1 mg / mL). -1 The reaction was carried out in the dark for 8 hours. Ti3C2 / ICG / PDA / GA (TIPG) nanosheets were collected by centrifugation (12000 rpm, 10 min), washed twice with deionized water, and stored at 4 °C for later use.

[0058] Depend on Figure 5 and Figure 6 As can be seen, due to the loading of GA on the TIP surface, the hydrated particle size of TIPG increased to 164.2 nm, while the Zeta potential decreased to -33.13 mV. These results indicate that GA was successfully loaded onto the TIPG surface.

[0059] Step 5, Apt-M Coupling: TIPG (2.0 mg) was dispersed in Tris-HCl buffer (2 mL, 10 ml, pH 8.5) containing Apt-M (10 OD) and stirred for 4 h at room temperature and in the dark. The product was then centrifuged and washed three times with deionized water. The obtained Ti3C2 / ICG / PDA / GA / Apt-M (TIPGA) was stored at 4°C for use. Furthermore, to verify the targeting performance of the nanotherapy platform, Ti3C2 / ICG / PDA / GA / Apt-C (TIPGC) was prepared under the same conditions as above, using Apt-C (without targeting ability) as a control sequence.

[0060] Depend on Figure 5 and Figure 6As can be seen, due to the coupling of Apt-M with targeting properties on the TIPG surface, the hydrated particle size of TIPGA increased to 177.8 nm, while the Zeta potential decreased to -38.27 mV. These results indicate that Apt-M was successfully modified and coupled to the TIPGA surface.

[0061] Example 2

[0062] To study the photothermal properties of nanomaterials, a TIPGA dispersion (30 μg mL) was prepared. -1 Irradiate the sample with an 808nm laser at different power densities for 10 minutes, while recording the temperature changes.

[0063] Depend on Figure 7 It is evident that under 808nm laser irradiation, the photothermal temperature rise of the TIPGA nanotherapy platform increases with increasing laser power density, exhibiting laser power density-dependent photothermal characteristics. Moreover, even at 0.8W / cm²... 2 After laser irradiation, the temperature of the TIPGA dispersion rapidly increased to about 45°C, indicating that TIPGA has good photothermal properties and can meet the needs of tumor photothermal therapy.

[0064] Example 3

[0065] To evaluate the photodynamic properties of TIPGA nanosheets, DPBF was used as a detection method. 1 O2 indicator. TIPGA and Ti3C2 / PDA / GA / Apt-M (TPGA) were added to DPBF (1.0 mL, 14 μg / mL) solution, respectively. Subsequently, an 808 nm laser (0.8 W cm⁻¹) was used in the laser irradiation group. -2 The mixture was irradiated and the UV-vis absorption spectrum of the mixture was recorded.

[0066] Depend on Figure 8 As can be seen, when TIPGA was exposed to 808nm laser irradiation for 10 minutes, the DPBF absorbance decreased sharply, indicating that the process can efficiently produce... 1 O2; moreover, under the same conditions, no decrease in DPBF absorbance was detected in TPGA, indicating that TIPGA loaded with ICG can perform photodynamic therapy on tumors under 808nm laser irradiation.

[0067] Example 4

[0068] To detect the ability of TIPGA to consume GSH, different concentrations of Ti3C2 / ICG / PDA / Apt-M (TIPA) or TIPGA nanomaterials and DTNB solution (5 μL, 100 mM) were added to GSH solutions of a certain concentration. After incubation for 10 min, the absorbance of the solution at 405 nm was measured to calculate the amount of GSH consumed.

[0069] Depend on Figure 9 It is evident that as the concentration of TIPGA increases, the consumption of GSH also increases; conversely, no significant GSH consumption was observed in TIPA, which does not contain disulfide bonds. These results indicate that the TIPGA nanotherapy platform can reduce intracellular GSH concentration, which may help improve the efficacy of PDT.

[0070] Example 5

[0071] To verify the effects of pH, GSH, and NIR-I on GA release, TIPGA (1.0 mL, 1.0 mg / mL) was prepared. -1 The GA was encapsulated in a dialysis bag with a molecular weight cutoff of 3000 Daltons. The dialysis bag was then immersed in 40 mL of PBS with different pH values ​​(pH 7.4 or pH 5.0) and dialyzed at room temperature in the dark. The absorbance of the dialysate was measured using UV-Vis spectroscopy, and the GA release rate was calculated. To investigate the effects of GSH and NIR-I on GA release, additional GSH (10 mM) was added to the PBS, and GA was released at 5 h and 8 h using an 808 nm laser (1.0 W cm⁻¹). -2 The dialysis bag immersed in PBS was irradiated for 10 min. The absorbance of the dialysate at 360 nm was measured by UV-vis at different time points.

[0072] Depend on Figure 10 It is evident that GA release from TIPGA is affected by pH, GSH, and NIR-I laser irradiation. This triple-response GA release mode of pH / GSH / NIR-I endows the TIPGA nanotherapy platform with great potential for specific GA controlled release in the tumor microenvironment (TME) and lays the foundation for active tumor-targeted PTT / PDT / CHT at low temperatures.

[0073] Example 6

[0074] MCF-7 cells were seeded in 6-well plates (2 × 10⁶ cells / well). 5 Cells were cultured in wells for 24 h. Then, they were treated with nanosheets containing Ti3C2 / ICG / PDA / GA / Apt-Mf (TIPGA-f) at 30 μg / mL. -1 ), TIPGA-f (30μgmL) -1)+2ODApt-M, TIPGA-f (30μg mL) -1 Fresh medium containing 2+ODApt-C was used instead of the original medium. Cells were analyzed by flow cytometry after 4 hours of culture.

[0075] Depend on Figure 11 It is evident that Apt-M targets the MUC1 protein on the surface of MCF-7 cells, thus increasing the uptake of TIPGA by MCF-7 cells. However, when MCF-7 cells were first co-incubated with Apt-M and then with TIPGA, the fluorescence signal significantly decreased, indicating that Apt-M can shield the MUC1 protein site on the surface of MCF-7 cells, leading to a sharp reduction in the amount of TIPGA entering the cells and preventing TIPGA from effectively exerting its targeting effect.

[0076] Example 7

[0077] To investigate the mechanism by which TIPGA reduces cell thermostability, Western blot was used to detect the expression of HSP90 protein in MCF-7 cells. MCF-7 cells were seeded in 6-well plates (5 × 10⁵ cells / well) and incubated overnight. Then, the cells were divided into six treatment groups: (1) Control group, (2) TIPGA (30 μg / mL) treatment. -1 (3) TIP+Laser group (TIP = 30 μg mL) -1 (4) TIPG+Laser group (TIPG = 30 μg mL) -1 (5) TIPGC+Laser group (TIPGC = 30 μg mL) -1 ), (6) TIPGA+Laser group (TIPGA=30μg mL -1 After incubating MCF-7 cells with different nanomaterials for 4 hours, each group of cells was slowly washed three times with PBS. Then, each laser irradiation group was treated with an 808nm laser (0.8W cm⁻¹). -2 Irradiation for 10 min. After 12 h, total cellular protein was separated by SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was pre-incubated with primary antibody for 12 h, followed by incubation with secondary antibody for 1 h. Finally, HSP90 protein was imaged.

[0078] Depend on Figure 12As can be seen, the expression level of HSP90 in MCF-7 cells of the TIPG+L and TIPGC+L groups was significantly reduced, indicating that GA can effectively downregulate cellular HSP90 expression. Since Apt-M modified TIPGA can actively target MCF-7 cells and increase their intracellular accumulation, the HSP90 expression level in the TIPGA+L group was significantly lower than that in other experimental groups. These results demonstrate that TIPGA can inhibit the heat resistance of cancer cells by suppressing HSP90 expression, thereby enabling the TIPGA nanotherapy platform to kill tumor cells at relatively low temperatures.

[0079] Example 8

[0080] MCF-7 cells were seeded in 96-well plates and cultured for 24 hours. The original culture medium was discarded, and fresh culture medium containing the respective drugs and nanomaterials was added, followed by another 4 hours of culture. Subsequently, the cells were slowly washed with PBS. All laser treatment groups used a power intensity of 0.8 W / cm². -2 Cells were irradiated with an 808nm laser for 10 minutes. After culturing for 12 hours, cell viability was assessed using the MTT assay.

[0081] Depend on Figure 13 It is evident that TIP can perform combined PTT and PDT treatment on tumor cells, thus it is effective in laser irradiation (0.8W cm⁻¹). -2 At 10 min, only 73.53% of MCF-7 cells survived in the TIP+L group, while over 96.86% of MCF-7 cells survived in the TIP group without laser irradiation. When TIPG was irradiated with an 808 nm laser, the survival rate of MCF-7 cells significantly decreased to 44.87%, indicating that the TIPG+L group had a good combined therapy effect of tumor hypothermia PTT / PDT / CHT. A similar combined therapy effect of tumor hypothermia PTT / PDT / CHT was also observed in HepG2 cells. With the help of Apt-M's excellent active targeting capability, the survival rate of MCF-7 cells after PTT / PDT / CHT in the TIPGA+L group was only 19.74%; while as a control, there was no significant difference in the survival rate of HepG2 cells after treatment in the TIPG+L group and the TIPGA+L group. The above results indicate that the TIPGA nanotherapy platform can induce downregulation of HSP90 expression and reduce the heat resistance of tumors, thereby enabling tumor cells to actively target PTT / PDT / CHT at low temperatures.

[0082] Example 9

[0083] To evaluate the accumulation efficiency of TIPGA in mouse tumor tissues, the biological distribution of TIPGA was analyzed based on in vivo fluorescence imaging. A unilateral tumor-bearing nude mouse model was established by subcutaneous injection of MCF-7 cells into the right groin of mice. The tumor volume was measured to be ~100 mm². 3 Subsequently, each mouse was intravenously injected with TIPGA (10 mg / kg). -1 ) or TIPGC (10mg kg) -1 In vivo fluorescence imaging was performed at fixed time points after injection.

[0084] Depend on Figure 14 As can be seen, compared with intravenous injection of TIPGC, the fluorescence intensity of the tumor in mice after intravenous injection of TIPGA gradually and continuously increased and reached a peak at 4h, indicating that TIPGA also has excellent targeting ability in mice.

[0085] Example 10

[0086] A nude mouse tumor model was established using MCF-7 cells. 100 μL of MCF-7 cells (5 × 10⁻⁶ cells) were suspended in PBS solution. 7 Cells / mL were injected subcutaneously into mice. When the tumor volume reached approximately 100 mm², the tumor was treated. 3 Mice were randomly divided into six groups (n=5 per group). Each group of mice was injected with the same dose of different nanomaterials (10 mg / kg) via the tail vein according to their body weight. -1 Four hours after injection, mice in the laser irradiation group were treated with 808nm (0.8W cm⁻¹) laser light. -2 The mice were irradiated with laser for 10 minutes, during which time the temperature changes and thermal images of the tumor sites were recorded using an infrared thermal imager.

[0087] Depend on Figure 15 Thermographic imaging of mouse tumors showed that the surface temperature of the tumors in the TIPGA+L group rapidly increased to 44.7℃ within 10 minutes, significantly higher than that in other laser treatment groups. This result further confirms that TIPGA can actively target MCF-7 cells, thereby increasing the accumulation of nanomaterials within the mouse tumor region.

[0088] Depend on Figure 16 and Figure 17As can be seen, both the TIP+L and TPG+L groups showed very limited tumor growth inhibition, indicating that neither the PTT / PDT nor the PTT / CHT combination therapy could effectively inhibit tumor growth in mice. However, both the TIPG+L and TIPGC+L groups showed significant tumor inhibition, indicating that the PTT / PDT / CHT combination therapy strategy could inhibit tumor growth in mice. Similar to the cell experiments, the tumor volume in the TIPGA+L group showed a stable trend with a slight decrease, indicating that its active targeting of PTT / PDT / CHT had the best inhibitory effect on tumor growth.

[0089] It is worth noting that, by Figure 18 It is evident that the mice in the TIPGA+L group did not show obvious burn marks on their skin surface, indicating the feasibility of the TIPGA+L group's low-temperature targeted PTT / PDT / CHT strategy in mice.

[0090] During tumor treatment in mice, the mice's body weight was recorded every two days. Figure 19 As can be seen, the weight of mice in each group increased steadily, and there was no significant difference in weight gain between the groups, which means that the TIPGA nanotherapy platform has good biosafety.

[0091] The sequences of Apt-M, Apt-C, Apt-Mf, and Apt-Cf in the above embodiments are as follows:

[0092] The Apt-M sequence is:

[0093] NH2-(CH2)6-GCAGTTGATCCTTTGGATACCCTGGGTTTTTTTTTT;

[0094] The Apt-C sequence is:

[0095] NH2-(CH2)6-ATTGCACTTACTATATTGCACTTACTATATTGCAC;

[0096] The Apt-Mf sequence is:

[0097] NH2-(CH2)6-GCAGTTGATCCTTTGGATACCCTGGGTTTTTTTTTT-FAM;

[0098] The Apt-Cf sequence is:

[0099] NH2-(CH2)6-ATTGCACTTACTATATTGCACTTACTATATTGCAC-FAM.

[0100] Contents not described in detail in this specification are prior art known to those skilled in the art. Although illustrative specific embodiments of the invention have been described above to facilitate understanding by those skilled in the art, it should be understood that the invention is not limited to the scope of the specific embodiments. Various modifications are readily apparent to those skilled in the art as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims, and all inventions utilizing the concept of this invention are protected.

Claims

1. A multifunctional tumor-targeting cryotherapy nanoparticle, characterized in that: Using two-dimensional Ti3C2 nanomaterials as a carrier, Ti3C2 / ICG / PDA / GA nanosheets were prepared by sequentially modifying the surface with indocyanine green, polydopamine, and gambogeylic acid. Then, nucleic acid aptamers that can recognize the cell surface specific protein MUC1 were coupled to obtain Ti3C2 / ICG / PDA / GA / Apt-M nanoparticles. The indocyanine green is loaded onto the two-dimensional Ti3C2 nanomaterial as a photosensitizer via electrostatic interaction; the nucleic acid aptamer is coupled to the surface of the two-dimensional Ti3C2 nanomaterial via covalent binding.

2. The multifunctional tumor-targeting cryotherapy nanoparticle according to claim 1, characterized in that: The two-dimensional Ti3C2 nanomaterial has an average size of 100 nm and an average thickness of 5.0 nm.

3. A method for preparing the multifunctional tumor-targeting cryotherapy nanoparticles according to claim 1, characterized in that, Includes the following steps: Step 1, ICG loading: Ti3C2 nanosheets were added to an aqueous solution of indocyanine green, and the precipitate was collected by centrifugation to prepare Ti3C2 / ICG nanosheets; Step 2, PDA surface modification: Ti3C2 / ICG nanosheets were dispersed in Tris-HCl buffer, then dopamine hydrochloride was added, and the Ti3C2 / ICG / PDA nanosheets were collected by centrifugation. Step 3, GA loading: First, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are used to activate gamboge acid. The activated gamboge acid is then reacted with mPEG-SS-NH2 to obtain PEG-SS-GA. PEG-SS-GA is then added to Ti3C2 / ICG / PDA nanosheets to prepare Ti3C2 / ICG / PDA / GA nanosheets. Step 4, Apt-M coupling: Ti3C2 / ICG / PDA / GA nanosheets were dispersed in Tris-HCl buffer containing nucleic acid aptamers and stirred to obtain Ti3C2 / ICG / PDA / GA / Apt-M nanoparticles.

4. The method for preparing multifunctional tumor-targeting cryotherapy nanoparticles according to claim 3, characterized in that: The preparation method of the two-dimensional Ti3C2 material is as follows: Ti3AlC2 powder is mixed with HF aqueous solution, stirred and reacted at room temperature, and the precipitate is washed with deionized water after centrifugation. The precipitate is then dispersed in tetrapropylammonium hydroxide aqueous solution and stirred and reacted. After centrifugation, the precipitate is washed with deionized water to obtain two-dimensional Ti3C2 nanomaterials.

5. The use of the multifunctional tumor-targeting cryotherapy nanoparticles of claim 1 in the preparation of tumor-targeting therapeutic drugs.

Images