Multifunctional tumor targeted low-temperature treatment nano platform and application thereof

Abstract

The invention discloses a multifunctional tumor targeted low-temperature treatment nano platform and application thereof, and belongs to the technical field of nano biological medicines. According to the multifunctional tumor targeted low-temperature treatment nano platform, the two-dimensional Ti3C2 nano material serves as a carrier, indocyanine green is loaded to serve as a photosensitizer, polydopamine is modified on the surface to improve the stability, and gambogic acid is loaded to serve as an anti-tumor drug and a heat shock protein inhibitor; and a nucleic acid aptamer capable of recognizing specific protein on the cell surface is coupled in a covalent binding manner. The multifunctional tumor targeted low-temperature treatment nano platform is small in particle size, high in dispersion degree and good in biocompatibility, and shows excellent photo-thermal conversion capacity, photodynamic treatment effect and chemotherapy effect in tumor targeted treatment. The apoptosis of tumor cells can be effectively induced under a relatively low temperature condition, and the damage of high temperature to surrounding healthy tissues is remarkably reduced. The tumor focus specific recognition capability is also realized, the high enrichment at the tumor part can be realized, and the treatment effect is enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano-biomedicine, and in particular relates to a multifunctional tumor-targeted cryotherapy nano-platform and an application thereof. Background Art

[0002] 2D Ti 3 C 2 Nanomaterials, as a photothermal agent (PTA) with excellent performance, have shown great application potential in tumor treatment. Their unique near-infrared (NIR) light absorption ability and efficient photothermal conversion performance make Ti 3 C 2 Nanomaterials can generate high temperatures under NIR laser irradiation, thereby achieving photothermal therapy (PTT) of tumors. 3 C 2 Nanomaterials still have significant disadvantages in the single treatment of tumor PTT. In order to achieve effective tumor ablation, the temperature of the tumor area usually needs to reach a high temperature of more than 50°C, which requires the use of high-concentration PTA or high-power density laser irradiation. However, the high temperature generated in this process may not only damage the surrounding healthy tissues, but also trigger the body's inflammatory response, leading to the release of intracellular biomolecules, thereby increasing the risk of tumor metastasis.

[0003] In order to solve the shortcomings of single PTT treatment, researchers began to explore combined treatment methods, such as combining PTT with photodynamic therapy (PDT), chemotherapy (CHT), etc. For example, by loading photosensitizers onto Ti 3 C 2 Nanomaterials can be used to give nanotherapeutic platforms tumor PDT properties, thereby achieving combined PDT and PTT treatment of tumors. In addition, anti-tumor drugs can be loaded onto nanoplatforms to achieve combined PTT and CHT treatment. However, although these combined treatment strategies have improved the treatment effect to a certain extent, there are still problems such as unsatisfactory treatment effect and low targeting accuracy. In particular, when PTT is used as the main treatment method, the high temperature it produces may still cause damage to surrounding tissues, limiting its wide clinical application.

[0004] In the prior art, patent CN111153405B discloses a Ti 3 C 2 The preparation method of MXene nanosheet material comprises the following steps: Step 1, adding titanium aluminum carbon powder, hydrofluoric acid solution and hexadecyl trimethyl ammonium bromide into a corrosion-resistant container, stirring at 20 to 60° C. for 6 to 60 hours, and obtaining CTAB intercalated Ti 3 C 2 Mixed solution; Step 2: intercalate CTAB into Ti 3 C2 The mixed solution was centrifuged, washed with water until the supernatant was neutral, and the precipitate was filtered to obtain CTAB intercalated Ti 3 C 2 Multilayer materials; Step 3: Insert Ti into CTAB 3 C 2 Water was added to the multilayer material for 30 to 120 minutes of ultrasound and freeze-dried to obtain CTAB intercalated Ti 3 C 2 Nanosheet powder material. However, the Ti 3 C 2 The preparation method of nanosheet powder material requires ultrasound for 30 to 120 minutes after CTAB intercalation. The preparation process is cumbersome and consumes more energy.

[0005] In the prior art, patent CN118384272A discloses a targeted nano drug for combined treatment of breast cancer and its preparation method and application. The invention uses Fe-TCPP as a carrier and therapeutic agent, indocyanine green as a photosensitizer and photothermal agent loaded on the Fe-TCPP surface, and nucleic acid aptamers as targeting molecules coupled to the Fe-TCPP surface. However, the invention is insufficient in the development of the functions of indocyanine green and nucleic acid aptamers, and does not make in-depth use of the characteristics of indocyanine green that can perform in vivo fluorescence imaging, and nucleic acid aptamers in mice. The targeting effect and tissue distribution of nucleic acid aptamers are studied. Although the prior art also uses indocyanine green and nucleic acid aptamers, it is insufficient to prove the targeting effect and optimal targeting time in mice.

[0006] In the prior art, the paper "Study on the application of garcinia acid-indocyanine green co-loaded nanostructured liposomes in the treatment of cervical cancer based on the cell pyroptosis pathway" uses nanostructured liposomes to co-load the photosensitizer indocyanine green and the natural drug ingredient garcinia acid, and modifies the tumor targeting peptide cTMTP1 on its surface for anti-tumor application. However, this method provides a targeted treatment strategy for cervical cancer, and the method does not make in-depth use of the characteristics of indocyanine green that can perform in vivo fluorescence imaging to study the targeting effect in mice. In addition, the method does not make in-depth use of garcinia acid to inhibit the expression of tumor cell heat shock proteins (HSPs) to study tumor hypothermia treatment. In addition, the design of the loading method of garcinia acid in this study is simple, and the triple response garcinia acid drug release function based on pH / GSH / NIR-I in the tumor microenvironment is not realized.

[0007] In the prior art, the paper "Polydopamine-encapsulated indocyanine green high biocompatibility nanotherapy platform for enhanced photothermal therapy of cervical cancer" uses polydopamine and monoamino polyethylene glycol to encapsulate and modify the photothermal material indocyanine green to form nanoparticles, and evaluates the therapeutic effect on cervical cancer cells and cervical cancer-bearing mice through photothermal therapy. However, this method does not make in-depth use of the fact that indocyanine green can also perform PDT treatment on tumors under near-infrared 808nm laser irradiation.

[0008] In view of the challenges faced by the above-mentioned tumor treatment and the comprehensive evaluation of existing technical solutions, integrating multiple treatment methods and relying on active targeting and other technologies to achieve precise tumor treatment under low temperature treatment conditions has become a hot topic in current research. Low temperature treatment can not only avoid the damage of high temperature to normal tissues and improve the safety of treatment, but also reduce the heat resistance of tumor cells by inhibiting the expression of HSPs in tumors, thereby improving the treatment efficiency of PTT. Therefore, it is urgent to develop a more excellent multifunctional nanotherapy platform that integrates multiple treatment methods such as PTT, PDT, and CHT into a single nanosystem, and increase its targeted enrichment at the tumor site to achieve targeted low temperature precision treatment of tumors. Summary of the invention

[0009] In order to solve the problems that excessively high temperature during PTT treatment damages surrounding healthy tissues; a single PTT treatment mode has limited therapeutic effect; and there is a lack of active tumor targeting specificity, the present invention provides a multifunctional tumor-targeted cryotherapy nanoplatform and its application.

[0010] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0011] A multifunctional tumor-targeted cryotherapy nanoplatform based on two-dimensional Ti 3 C 2 Nanomaterials were used as carriers, and indocyanine green (ICG), polydopamine (PDA), and gambogic acid (GA) were used for surface modification in sequence, and aptamers (Apt-M) that could recognize specific proteins on the cell surface were coupled;

[0012] Furthermore, the two-dimensional Ti 3 C 2 The average size of the nanomaterials is ~100 nm and the average thickness is 5.0 nm.

[0013] Furthermore, the indocyanine green (ICG) is loaded onto the two-dimensional Ti by electrostatic interaction as a photosensitizer. 3 C 2 On nanomaterials.

[0014] Furthermore, the nucleic acid aptamer is coupled to the two-dimensional Ti by covalent bonding. 3C 2 Nanomaterial surfaces.

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

[0016] Step 1, ICG loading: Ti 3 C 2 The nanosheets were added to an aqueous solution of indocyanine green (ICG), and the precipitate was collected by centrifugation to prepare Ti 3 C 2 / ICG(TI) nanosheets;

[0017] Step 2, PDA surface modification: Ti 3 C 2 / ICG(TI) nanosheets were dispersed in Tris-HCl buffer, and then dopamine hydrochloride was added, and Ti was collected by centrifugation. 3 C 2 / ICG / PDA(TIP) nanosheets;

[0018] Step 3, loading GA: 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were used to activate GA, and the activated GA was reacted with mPEG-SS-NH 2 The PEG-SS-GA was obtained by reaction, and then the PEG-SS-GA was added to Ti 3 C 2 / ICG / PDA(TIP) nanosheets to prepare Ti 3 C 2 / ICG / PDA / GA(TIPG) nanosheets;

[0019] Step 4, coupling of Apt-M: Ti 3 C 2 / ICG / PDA / GA (TIPG) nanosheets were dispersed in Tris-HCl buffer containing nucleic acid aptamer (Apt-M) and stirred to obtain Ti 3 C 2 / ICG / PDA / GA / Apt-M (TIPGA) nanoplatform.

[0020] Furthermore, the two-dimensional Ti 3 C 2 The material is prepared by: Ti 3 AlC 2The powder was mixed with HF aqueous solution, stirred and reacted at room temperature, centrifuged and washed with deionized water, and dispersed in tetrapropylammonium hydroxide (TPAOH) aqueous solution, stirred and reacted, centrifuged and washed with deionized water to obtain a two-dimensional Ti 3 C 2 Nanomaterials.

[0021] A multifunctional tumor-targeted cryogenic therapeutic nanoplatform is used in tumor targeted therapy. The multifunctional tumor-targeted cryogenic therapeutic nanoplatform can actively identify specific tumor cells, has tumor lesion-specific recognition capability, can achieve high enrichment at the tumor site, and can perform photothermal / photodynamic / chemotherapy combined treatment at low temperature (≤45°C), effectively inhibiting tumor growth, thereby achieving precision medicine.

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

[0023] (1) The present invention cleverly loads water-insoluble GA onto the TIPGA nano-platform through a covalent binding method, and relies on the acidic pH and high glutathione (GSH) concentration in the tumor microenvironment to achieve precise controlled release of the chemotherapy drug GA at the tumor site, thereby enhancing the tumor treatment effect. Moreover, GA precisely released at the tumor site can reduce the expression of HSPs in tumor cells, so that the TIPGA nano-platform can effectively induce tumor cell apoptosis at a relatively low temperature (≤45°C), improve the efficiency of tumor treatment under low temperature conditions, reduce the damage of high temperature to surrounding healthy tissues, and reduce the risk of tumor metastasis.

[0024] (2) The TIPGA nanoplatform prepared in the present invention has multimodal combined therapeutic properties. 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 the present invention has efficient targeting performance. Through Apt-M functional modification, the TIPGA nanoplatform has excellent active targeting performance at the tumor cell level in vitro and the mouse tumor tissue level in vivo, thereby improving the specificity of treatment and drug utilization, reducing the impact on normal cells and tissues, and reducing side effects.

[0026] (4) Different from the existing patent CN111153405B technology, the Ti 3 C 2Nanosheets can be prepared without ultrasound after being treated with TPAOH aqueous solution, and the process is simple and avoids consuming more energy; Different from the existing patent CN118384272A technology, although the present invention also loads indocyanine green and nucleic acid aptamers, the present invention uses the characteristics of indocyanine green that can 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 co-loaded nanostructured liposomes of garcinia acid-indocyanine green in the treatment of cervical cancer based on cell pyroptosis pathway", the present invention not only provides a treatment strategy for specific breast tumors, but also makes more in-depth use of the fluorescence imaging characteristics of indocyanine green to study the targeting effect in mice, and innovatively designs the loading method of garcinia acid to achieve the triple response drug release performance of garcinia acid, thereby improving the targetedness and effectiveness of treatment. Compared with the existing research paper "Study on the enhanced photothermal therapy of cervical cancer with a highly biocompatible nanotherapeutic platform of polydopamine-encapsulated indocyanine green", the present invention deeply explores the PDT therapeutic potential of indocyanine green, and combines PTT to enable indocyanine green to achieve a dual treatment mode of PTT / PDT. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 Schematic diagram of the preparation and application of the multifunctional cryotherapeutic nanoplatform of the present invention;

[0029] Figure 2 is Ti in Example 1 of the present invention 3 C 2 TEM images of materials;

[0030] Figure 3 is Ti in Example 1 of the present invention 3 C 2 AFM images of materials;

[0031] Figure 4 is Ti in Example 1 of the present invention 3 C 2 XRD analysis results of materials;

[0032] Figure 5 The Zata potential analysis results of the nanomaterials in Example 1 of the present invention;

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

[0034] Figure 7 The temperature variation results of the dispersion of the TIPGA material in Example 2 of the present invention under irradiation with different 808nm laser power densities;

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

[0036] Fig. 9 The results of the analysis of the GSH consumption capacity of the TIPGA material in Example 4 of the present invention;

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

[0038] Fig.11 This is a comparison diagram of the uptake of MCF-7 cells after incubation with different nanomaterials for 4 hours in Example 6 of the present invention;

[0039] Fig.12 The Western Blot images of the HSP90 protein expression levels of MCF-7 cells under different treatments in Example 7 of the present invention;

[0040] Fig.13 The survival rate results of MCF-7 cells and HepG2 cells after being treated with various nanomaterials in Example 8 of the present invention;

[0041] Fig.14 This is a graph showing the change of material enrichment in the tumor site of mice over time after tail vein injection of each nanomaterial in Example 9 of the present invention;

[0042] Fig.15 The infrared thermal imaging digital photos of mice during the treatment with each nanomaterial in Example 10 of the present invention;

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

[0044] Fig.17 This is a diagram showing the dissection results of mouse tumors after treatment with various nanomaterials in Example 10 of the present invention;

[0045] Fig.18 These are digital photos of mice treated with various nanomaterials in Example 10 of the present invention within 15 days;

[0046] Fig.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 DESCRIPTION

[0047] In order to gain a deeper understanding of the present invention, we will provide a comprehensive and detailed description of the present invention. However, the present invention has multiple implementations and is not limited to the specific examples listed herein. The presentation of these examples is intended to deepen the comprehensive understanding of the disclosure of the present invention.

[0048] Example 1

[0049] Depend on Figure 1 A multifunctional tumor-targeted cryotherapy nanoplatform based on two-dimensional Ti 3 C 2 Nanomaterials were used as carriers; ICG was loaded as a photosensitizer with an ICG loading of 38.94%; PDA was then surface modified to improve stability; GA was loaded as an anti-tumor drug and HSPs inhibitor with a GA loading of 9.02%; finally, Apt-M, which can recognize specific cell surface proteins, was coupled by covalent binding to give it targeting ability.

[0050] The preparation method of the multifunctional nano-platform comprises the following steps:

[0051] Step 1, 2D Ti 3 C 2 Material preparation: Ti 3 AlC 2 The powder reagent (1.0 g) was slowly added to 20 mL of 40% HF aqueous solution (please pay attention to personal protection 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 TPAOH 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 to 8 times with deionized water to make the pH value of the solution greater than 6.0. Finally, Ti was prepared after centrifugation at 3500 rpm for 60 min. 3 C 2 Nanosheets.

[0052] Depend on Figure 2 The TEM images shown show that the two-dimensional Ti 3 C 2 The particle size of the material is 100nm. Figure 3 The AFM images shown show that the two-dimensional Ti 3 C 2 The average thickness of the material is about 5.0nm. Figure 4 The XRD analysis results shown in the figure show that the obtained two-dimensional Ti 3 C 2 The material has almost no characteristic peak at 2θ≈39.0°(104), and its characteristic peak at 2θ≈9.5°(002) becomes broader and moves to a lower angle (6.0°), proving that the two-dimensional Ti 3 C2 Successful preparation of nanomaterials.

[0053] Step 2, ICG loading: 3 C 2 Nanosheets (5.0 mg) were slowly added to an ICG aqueous solution (5.0 mL, 0.4 mg mL -1 ) and stirred at room temperature for 4 h. The precipitate was then collected by centrifugation (12000 rpm, 5 min) and washed 3 times with deionized water to remove residual ICG. Finally, Ti 3 C 2 / ICG(TI) nanosheets.

[0054] Depend on Figure 5 and Figure 6 It can be seen that due to the loading of ICG, the hydrated particle size of TI increased from 93.7nm to 119.2nm, and the Zeta potential decreased from -21.27mV to -27.80mV. The above results show that ICG was successfully loaded into the two-dimensional Ti 3 C 2 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 -1 ), stirred at room temperature and protected from light for 4 h. Ti was then collected by centrifugation (12000 rpm, 5 min). 3 C 2 / ICG / PDA(TIP) nanosheets and washed three times with deionized water.

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

[0057] Step 4, loading GA: First, 0.4 mg of GA was added to 200 μL DMSO, and activated with EDC (900 μL, 200 mM) and NHS (900 μL, 50 mM) under light-proof conditions for 30 min. The activated GA was then washed three times with deionized water. Next, the activated GA was mixed with 1.0 mL of mPEG-SS-NH 2 (2.5mg mL -1) was reacted under oscillation for 5 h. The product was then encapsulated in a dialysis bag with a molecular weight cutoff of 3000 Daltons, and the dialysis bag was immersed in 1000 mL of deionized water and dialyzed at room temperature for 24 h to obtain PEG-SS-GA. Finally, PEG-SS-GA was slowly added to the TIP solution (5.0 mL, 1 mg mL -1 ) and reacted for 8 h in a dark environment. Ti was collected by centrifugation (12000 rpm, 10 min). 3 C 2 / ICG / PDA / GA(TIPG) nanosheets were washed twice with deionized water and stored at 4°C for future use.

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

[0059] Step 5, coupling of Apt-M: TIPG (2.0 mg) was dispersed in Tris-HCl buffer (2 mL, 10 mm, pH 8.5) containing Apt-M (10 OD) and stirred for 4 h at room temperature in the dark. The product was then centrifuged and washed three times with deionized water. 3 C 2 / ICG / PDA / GA / Apt-M (TIPGA) was stored at 4°C until use. In addition, in order to verify the targeting performance of the nanotherapeutic platform, Ti was prepared using Apt-C without targeting ability as a control sequence under the same conditions as above. 3 C 2 / ICG / PDA / GA / Apt-C(TIPGC).

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

[0061] Example 2

[0062] In order to study the photothermal properties of nanomaterials, TIPGA dispersion (30 μg mL -1 ) were irradiated with 808 nm laser at different power densities for 10 min and the temperature changes were recorded simultaneously.

[0063] Depend on Figure 7It can be seen that under 808nm laser irradiation, the photothermal temperature rise of the TIPGA nanotherapy platform increases with the increase of laser power density, showing the photothermal characteristics of laser power density dependence. 2 After laser irradiation, the temperature of the TIPGA dispersion rapidly increased by about 45°C, indicating that TIPGA has good photothermal properties and can meet the needs of tumor photothermal therapy.

[0064] Example 3

[0065] In order to evaluate the photodynamic performance of TIPGA nanosheets, DPBF was used as a detector. 1 O 2 TIPGA and Ti were added to the DPBF (1.0 mL, 14 μg / mL) solution. 3 C 2 / PDA / GA / Apt-M(TPGA). Subsequently, in the laser irradiation group, 808 nm laser (0.8 W cm -2 ) was irradiated and the UV-vis absorption spectrum of the mixture was recorded.

[0066] Depend on Figure 8 It can be seen that when TIPGA was exposed to 808 nm laser irradiation for 10 min, the absorbance of DPBF decreased sharply, indicating that the process can efficiently produce 1 O 2 ; Moreover, TPGA basically did not detect a decrease in DPBF absorbance under the same conditions. The results showed that TIPGA loaded with ICG can perform tumor photodynamic therapy under 808nm laser irradiation.

[0067] Example 4

[0068] In order to detect the ability of TIPGA to consume GSH, different concentrations of Ti 3 C 2 / ICG / PDA / Apt-M (TIPA) or TIPGA nanomaterials and DTNB solution (5 μL, 100 mM) were added to a certain concentration of GSH solution. After incubation for 10 min, the absorbance of the solution at 405 nm was measured to calculate the consumption of GSH.

[0069] Depend on Fig. 9 It can be seen that with the increase of TIPGA concentration, the consumption of GSH also increased; on the contrary, no obvious consumption of GSH was observed in TIPA without disulfide bonds. The results show that the TIPGA nanotherapy platform can reduce the intracellular GSH concentration and will 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 -1 ) 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 in a dark environment at room temperature. The absorbance of the dialysate was measured by UV-vis spectroscopy, and the release rate of GA was calculated. In order to study the effects of GSH and NIR-I on GA release, additional GSH (10 mM) was added to PBS, and 808 nm laser (1.0 W cm -2 The dialysis bag immersed in PBS was irradiated with UV-vis at different time points to measure the absorbance of the dialysate at 360 nm.

[0072] Depend on Fig.10 It can be seen that the release of GA from TIPGA is affected by pH, GSH and NIR-I laser irradiation. This pH / GSH / NIR-I triple-responsive GA release pattern gives the TIPGA nanotherapeutic platform 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 (2×10 5 cells / well) for 24 h. 3 C 2 / ICG / PDA / GA / Apt-Mf(TIPGA-f) nanosheets (30 μg mL -1 )、TIPGA-f(30μgmL -1 )+2ODApt-M, TIPGA-f (30 μg mL -1 The original culture medium was replaced with fresh culture medium containing 20 DAPt-C. After culturing for 4 h, the cells were analyzed by flow cytometry.

[0075] Depend on Fig.11 It can be seen that the MCF-7 cells increased their intake of TIPGA due to the targeting effect of Apt-M on the MUC1 protein on the surface of MCF-7 cells. When MCF-7 cells were first co-incubated with Apt-M and then with TIPGA, the fluorescence signal was significantly reduced, indicating that Apt-M can shield the MUC1 protein site on the surface of MCF-7 cells, resulting in a sharp decrease in the amount of TIPGA entering MCF-7 cells, making it impossible for TIPGA to effectively play its targeting role.

[0076] Example 7

[0077] In order to explore the mechanism by which TIPGA reduces cell thermotolerance, Western blot was used to detect the expression of HSP90 protein in MCF-7 cells. MCF-7 cells were seeded into 6-well plates (5×105 cells / well) and incubated overnight. Then, the cells were divided into six treatment groups: (1) Control group, (2) TIP (30 μg mL -1 ) group, (3) TIP+Laser group (TIP=30μg 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 h, the cells in each group were slowly washed with PBS for 3 times, and then each laser irradiation group was irradiated with 808 nm laser (0.8 W cm -2 ) for 10 min. After 12 h, total cell proteins were separated by SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was pre-incubated with primary antibody for 12 h and then incubated with secondary antibody for another 1 h. Finally, HSP90 protein was imaged.

[0078] Depend on Fig.12 It can be seen that the expression level of HSP90 in MCF-7 cells in the TIPG+L group and the TIPGC+L group was significantly reduced, indicating that GA can effectively downregulate the expression of cellular HSP90. Since TIPGA can actively target MCF-7 cells and increase their intracellular aggregation after Apt-M modification, the expression level of HSP90 in the TIPGA+L group is significantly lower than that in other experimental groups. The results of the study prove that TIPGA can inhibit the heat resistance of cancer cells by inhibiting the expression of HSP90, thereby enabling the TIPGA nanotherapy platform to kill tumor cells at relatively low temperatures.

[0079] Example 8

[0080] MCF-7 cells were inoculated in 96-well plates and cultured for 24 h. The original culture medium was discarded, and fresh culture medium containing each group of drugs and nanomaterials was added and cultured for another 4 h. Subsequently, the cells were slowly washed with PBS. All laser treatment groups used a power intensity of 0.8 W cm -2 The cells were irradiated with 808 nm laser for 10 min. After culturing for another 12 h, the cell viability was detected by MTT method.

[0081] Depend on Fig.13It can be seen that since TIP can perform PTT and PDT combined treatment on tumor cells, -2 , 10min), only 73.53% of MCF-7 cells in the TIP+L group survived, while more than 96.86% of MCF-7 cells in the TIP group survived without laser irradiation. When TIPG was irradiated with 808nm laser, the survival rate of MCF-7 cells was significantly reduced to 44.87%. The results showed that the TIPG+L group had a good tumor low-temperature PTT / PDT / CHT combined treatment effect. At the same time, similar tumor low-temperature PTT / PDT / CHT combined treatment effects can also be observed on HepG2 cells. With the help of Apt-M's excellent active targeting ability, the survival rate of the TIPGA+L group after targeted PTT / PDT / CHT of MCF-7 cells was only 19.74%; as a control, there was no significant difference in the survival rate of HepG2 cells treated with TIPG+L and TIPGA+L groups. These results indicate that the TIPGA nanotherapeutic platform can induce downregulation of HSP90 expression and reduce the thermotolerance of tumors, thereby enabling active targeted PTT / PDT / CHT of tumor cells at low temperatures.

[0082] Example 9

[0083] In order to evaluate the accumulation efficiency of TIPGA in mouse tumor tissue, the biological distribution of TIPGA was analyzed based on in vivo fluorescence imaging. MCF-7 cells were injected subcutaneously in the right groin of mice to establish a unilateral tumor-bearing nude mouse model. When the tumor volume reached ∼100 mm 3 Afterwards, each mouse was intravenously injected with TIPGA (10 mg kg -1 ) or TIPGC (10 mg kg -1 ). In vivo fluorescence imaging was performed at fixed time points after injection.

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

[0085] Example 10

[0086] MCF-7 cells were used to establish a nude mouse tumor model. 100 μL of MCF-7 cells (5×10 7 cells / ml) were injected subcutaneously into mice. When the tumor volume reached about 100 mm 3 The mice were randomly divided into six groups (5 mice in each group). Each group of mice was injected with the same dose of different nanomaterials (10 mg kg-1 ). 4 h after injection, mice in the laser irradiation group were exposed to 808 nm (0.8 W cm -2 ) laser irradiation for 10 min, during which time an infrared thermal imager was used to record the temperature changes and thermal images of the mouse tumor site.

[0087] Depend on Fig.15 The thermal images of mouse tumors showed that the surface temperature of the tumors in the TIPGA+L group rose rapidly to 44.7°C within 10 minutes, significantly higher than that in other laser treatment groups. This result further confirmed that TIPGA can actively target MCF-7 cells, thereby increasing the accumulation of nanomaterials in the mouse tumor area.

[0088] Depend on Fig.16 and Fig.17 It can be seen that both the TIP+L group and the TPG+L group showed very limited tumor growth inhibition, indicating that neither the PTT / PDT combination therapy nor the PTT / CHT combination therapy could effectively inhibit mouse tumor growth. However, significant mouse tumor inhibition effects were observed in both the TIPG+L group and the TIPGC+L group, indicating that the PTT / PDT / CHT combination therapy strategy can inhibit mouse tumor growth. Similar to the results of the cell experiment, the tumor volume of mice in the TIPGA+L group showed a stable change trend and slightly decreased, indicating that its active targeted PTT / PDT / CHT strategy had the best inhibitory effect on tumor growth.

[0089] It is worth noting that Fig.18 It can be seen that there were no obvious burn marks on the skin surface of mice in the TIPGA+L group, indicating the feasibility of the TIPGA+L group's low-temperature targeted PTT / PDT / CHT strategy for mice.

[0090] During the tumor treatment period, the body weight of mice was recorded every two days. Fig.19 It can be seen that the weight of mice in each group increased steadily, and there was no significant difference in weight gain among the groups, which means that the TIPGA nanotherapeutic platform has good biosafety.

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

[0092] The Apt-M sequence is:

[0093] NH 2 -(CH 2 ) 6 -GCAGTTGATCCTTTGGATACCCTGGGTTTTTTTTTT;

[0094] The Apt-C sequence is:

[0095] NH2 -(CH 2 ) 6 -ATTGCACTTACTATATTGCACTTACTATATTGCAC;

[0096] The Apt-Mf sequence is:

[0097] NH 2 -(CH 2 ) 6 -GCAGTTGATCCTTTGGATACCCTGGGTTTTTTTTTT-FAM;

[0098] The Apt-Cf sequence is:

[0099] NH 2 -(CH 2 ) 6 -ATTGCACTTACTATATTGCACTTACTATATTGCAC-FAM.

[0100] The contents not described in detail in the specification of the present invention belong to the prior art known to the professional and technical personnel in the field. Although the illustrative specific embodiments of the present invention are described above to facilitate the understanding of the present invention by the technical personnel in the field, it should be clear that the present invention is not limited to the scope of the specific embodiments. For the ordinary technical personnel in the field, as long as various changes are within the spirit and scope of the present invention defined and determined by the attached claims, these changes are obvious, and all inventions and creations using the concept of the present invention are protected.

Claims

1. A multifunctional tumor-targeted cryotherapy nanoplatform, characterized in that: Using two-dimensional Ti3C2 nanomaterials as carriers, indocyanine green, polydopamine and gambogic acid were used in sequence for surface modification, and then coupled with nucleic acid aptamers that can recognize specific cell surface proteins.

2. The multifunctional tumor-targeted cryotherapy nanoplatform according to claim 1, characterized in that: The average size of the two-dimensional Ti3C2 nanomaterial is 100 nm, and the average thickness is 5.0 nm.

3. The multifunctional tumor-targeted cryotherapy nanoplatform according to claim 1, characterized in that: The indocyanine green is used as a photosensitizer and loaded onto the two-dimensional Ti3C2 nanomaterial using electrostatic interaction.

4. The multifunctional tumor-targeted cryotherapy nanoplatform according to claim 1, characterized in that: The nucleic acid aptamer is coupled to the surface of the two-dimensional Ti3C2 nanomaterial by covalent bonding.

5. A method for preparing the multifunctional tumor-targeted cryotherapeutic nanoplatform according to claim 1, characterized in that: The following steps are involved: Step 1, ICG loading: Ti3C2 nanosheets are added to an aqueous solution of indocyanine green, and the precipitate is 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 then Ti3C2 / ICG / PDA nanosheets were collected by centrifugation; Step 3, loading GA: first activate gambogic acid with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, react the activated gambogic acid with mPEG-SS-NH2 to obtain PEG-SS-GA, and then add PEG-SS-GA to Ti3C2 / ICG / PDA nanosheets to prepare Ti3C2 / ICG / PDA / GA nanosheets; Step 4, coupling of Apt-M: disperse the Ti3C2 / ICG / PDA / GA nanosheets in Tris-HCl buffer containing nucleic acid aptamers and stir to react to obtain the Ti3C2 / ICG / PDA / GA / Apt-M nanoplatform.

6. The method for preparing the multifunctional tumor-targeted cryotherapeutic nanoplatform according to claim 5, characterized in that: The preparation method of the two-dimensional Ti3C2 material is as follows: Ti3AlC2 powder is mixed with HF aqueous solution, stirred for reaction at room temperature, centrifuged and precipitated with deionized water, and dispersed in tetrapropylammonium hydroxide aqueous solution, stirred for reaction, centrifuged and precipitated with deionized water to obtain a two-dimensional Ti3C2 nanomaterial.

7. Use of the multifunctional tumor-targeted cryotherapeutic nanoplatform according to claim 1 in the preparation of tumor-targeted therapeutic drugs.

Images