Sonosensitizer-silane conjugate and its preparation method and application

Abstract

The present application discloses a sonosensitizer-silane conjugate and its preparation method and application. The sonosensitizer-silane conjugate comprises a sonosensitizer, a water-soluble polymer, and an aminosilane; one end of the water-soluble polymer is connected to the sonosensitizer, and the other end is connected to the aminosilane. The conjugate is combined with the in situ silicification of tumor cells, which can improve the rigidity of tumor cells while giving the silicon layer excellent sonodynamic properties, alleviating the shortcomings of traditional sonodynamic therapy that promotes tumor escape and metastasis, and increasing the effect of T cells on target cells. By utilizing the synergistic effect of blocking mechanical immune checkpoints and sonodynamic immunotherapy, the body's own immune efficacy is greatly improved.

Description

Technical Field

[0001] The present invention relates to the field of tumor treatment, and in particular to a sonosensitizer-silane conjugate and a preparation method and application thereof. Background Art

[0002] Immune checkpoints are a series of molecules expressed on immune cells that regulate immune activation and play a key role in preventing autoimmunity (abnormal immune function / attack on normal cells). The underlying principle of immune checkpoint blockade therapy is to exploit the activation mechanism of immune T cells, blocking the "passivation" of immune cells by tumor cells, thereby maintaining normal immune system activity and preventing tumor cell escape. To date, the US Food and Drug Administration (FDA) has approved four types of immune checkpoint inhibitors for clinical use: cytotoxic T lymphocyte-associated protein-4 (CTLA-4); programmed cell death receptor-1 (PD-1); programmed cell death-ligand 1 (PD-L1); and lymphocyte activation gene-3 (LAG-3). These four types of immune checkpoint inhibitors have led to the development of numerous related drugs, but significant side effects (multi-organ toxicity), high treatment costs (40,000 to 350,000 yuan / year), and low immune response (only 10% to 30% of patients achieve an objective response) have driven researchers to continuously search for better immune checkpoint inhibitors and corresponding inhibitors. However, studies have shown that tumor cells can use cell softness to weaken the force of cytotoxic T cells at the immune synapse, thereby serving as a mechanical immune checkpoint to evade immune cell attack.

[0003] The prerequisite for blocking mechanical immune checkpoints is the infiltration of sufficient numbers of immune cells into the tumor tissue, that is, the transformation from a "cold tumor" to a "hot tumor" must first be completed. According to previous research reports, sonodynamic therapy can effectively accomplish this task. Sonodynamic therapy is a new non-invasive treatment method in which sonosensitizers effectively produce reactive oxygen species under the activation of low-intensity ultrasound. It can effectively induce immunogenic death of tumor cells, improve the immunogenicity of tumor tissue, and increase the infiltration of immune cells. However, traditional sonodynamic therapy has significant disadvantages: it damages the cytoskeletal system, further "softens" tumor cells, and increases their ability to escape through mechanical immune checkpoints; it also induces the production of apoptotic exosomes and loosens the tumor cell extracellular matrix, increasing serum exosome levels and promoting the occurrence of lung metastasis.

[0004] Therefore, obtaining better immune checkpoints and corresponding blockers, or improving existing sonodynamic therapy has become a technical problem that needs to be urgently solved by existing technologies. Summary of the Invention

[0005] The primary purpose of the present invention is to overcome the limitations of current immune checkpoint therapy and alleviate the shortcomings and deficiencies of existing sonodynamic therapy technologies, thereby providing a sonosensitizer-silane conjugate.

[0006] The second object of the present invention is to provide a method for preparing the sonosensitizer-silane conjugate.

[0007] The third object of the present invention is to provide the use of the above-mentioned sonosensitizer-silane conjugate in the preparation of tumor immunotherapy products.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A sonosensitizer-silane conjugate comprises a sonosensitizer, a water-soluble polymer and aminosilane; one end of the water-soluble polymer is connected to the sonosensitizer, and the other end is connected to the aminosilane.

[0010] Furthermore, the sonosensitizer includes at least one of hematoporphyrin, protoporphyrin (PpIX), hematoporphyrin monomethyl ether (HMME), verteporfin, dihydrochlorin e6 (Ce6) and dihydrochlorin e6-C15 monomethyl ester; and further, it is hematoporphyrin.

[0011] Furthermore, the water-soluble polymer includes at least one of PEG and PEG derivatives; further, the PEG derivative includes N-hydroxysuccinimide-PEG-carboxylic acid (HOOC-PEG-NHS).

[0012] Further, the aminosilane includes at least one of 3-aminopropyltriethoxysilane, 3-(2-aminoethylamino)propyltriethoxysilane, 3-aminopropyldimethoxymethylsilane, 3-aminopropyltrimethoxysilane, (3-aminopropyl)dimethylmethoxysilane, [3-(6-aminohexylamino)propyl]trimethoxysilane, N-[3-(trimethoxysilyl)propyl]ethylenediamine, and 1-[3-(trimethoxysilyl)propyl]urea, and further 3-aminopropyltriethoxysilane.

[0013] Furthermore, when the sonosensitizer is hematoporphyrin, the PEG derivative is N-hydroxysuccinimide-PEG-carboxylic acid, and the aminosilane is 3-aminopropyltriethoxysilane, the structure of the sonosensitizer-silane conjugate is shown in Formula I:

[0014]

[0015] The preparation method of the sonosensitizer-silane conjugate comprises the following steps: (1) dissolving the sonosensitizer and the water-soluble polymer in a solvent and mixing them uniformly, adding a catalyst 1, and reacting them under an inert atmosphere; purifying the reaction solution after the reaction by recrystallization, and collecting the solid;

[0016] (2) dissolving the solid collected in step (1) and catalyst 2 in a solvent, mixing them evenly, adding aminosilane, reacting under an inert atmosphere, purifying the reaction solution after the reaction by recrystallization, collecting the solid, and obtaining a sonosensitizer-silane conjugate.

[0017] Furthermore, the sonosensitizer described in step (1) includes at least one of hematoporphyrin, protoporphyrin (PpIX), hematoporphyrin monomethyl ether (HMME), verteporfin, dihydrochlorin e6 (Ce6) and dihydrochlorin e6-C15 monomethyl ester; further, it is hematoporphyrin.

[0018] Furthermore, the solvent in step (1) includes dimethyl sulfoxide.

[0019] Furthermore, the concentration of the sonosensitizer in step (1) in the solvent is 1-10 mM; further preferably 6 mM.

[0020] Furthermore, the water-soluble polymer in step (1) includes at least one of PEG and PEG derivatives; further, the PEG derivative includes N-hydroxysuccinimide-PEG-carboxylic acid (HOOC-PEG-NHS).

[0021] Furthermore, the concentration of the water-soluble polymer in the solvent in step (1) is 15-25 mM; further preferably 18 mM.

[0022] Furthermore, the catalyst 1 in step (1) comprises 4-dimethylaminopyridine (DMAP).

[0023] Furthermore, the molar ratio of the catalyst 1 and the sonosensitizer in step (1) is 3 to 4:1.

[0024] Furthermore, the molar ratio of the sonosensitizer to the water-soluble polymer in step (1) is 1:2-4; furthermore, 1:3.

[0025] Furthermore, the reaction temperature in step (1) is 10-60°C; further preferably 15-40°C.

[0026] Furthermore, the reaction time in step (1) is 12 to 72 hours; further preferably 24 to 60 hours; further preferably 24 hours.

[0027] Furthermore, the recrystallization in step (1) comprises the following steps: adding the reaction solution dropwise into diethyl ether.

[0028] Furthermore, the catalyst 2 in step (2) includes at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0029] Furthermore, the molar ratio of the catalyst 2 to the aminosilane in step (2) is 3 to 4:1.

[0030] Furthermore, the molar ratio of the sonosensitizer described in step (1) to the aminosilane described in step (2) is 1:3 to 7; further preferably 1:5.

[0031] Furthermore, the concentration of the aminosilane in step (2) in the reaction system is 20-40 mM; further preferably 30 mM.

[0032] Furthermore, the aminosilane described in step (2) includes at least one of 3-aminopropyltriethoxysilane, 3-(2-aminoethylamino)propyltriethoxysilane, 3-aminopropyldimethoxymethylsilane, 3-aminopropyltrimethoxysilane, (3-aminopropyl)dimethylmethoxysilane, [3-(6-aminohexylamino)propyl]trimethoxysilane, N-[3-(trimethoxysilyl)propyl]ethylenediamine, and 1-[3-(trimethoxysilyl)propyl]urea, and further 3-aminopropyltriethoxysilane.

[0033] Furthermore, the solvent in step (2) includes dimethyl sulfoxide.

[0034] Furthermore, the reaction temperature in step (2) is 10-60°C; further 30-50°C; further 30°C.

[0035] Furthermore, the reaction time in step (2) is 12 to 72 hours; further preferably 24 to 60 hours; further preferably 24 hours.

[0036] Furthermore, the recrystallization in step (2) comprises the following steps: adding the reaction solution dropwise into diethyl ether.

[0037] Application of the above-mentioned sonosensitizer-silane conjugate in the preparation of tumor treatment products.

[0038] Furthermore, the tumor includes breast cancer.

[0039] A tumor treatment product comprises a sonosensitizer-silane conjugate and an organosilicon coupling reagent.

[0040] Furthermore, the tumor treatment product includes a siliconized stock solution, and the preparation method of the siliconized stock solution includes the following steps:

[0041] Adjusting the pH of the culture medium to the pH of the solid tumor; adding the sonosensitizer-silane conjugate and the organosilicon coupling reagent to the culture medium, mixing them evenly, to obtain a siliconized stock solution;

[0042] Furthermore, the culture medium is serum-free 1640 cell culture medium;

[0043] Furthermore, the pH of the regulated culture medium is adjusted by adding a pH regulating solution; further still, the pH regulating solution comprises at least one of disodium hydrogen phosphate solution, sodium hydrogen phosphate solution, hydrochloric acid solution, sodium citrate, nitric acid solution, Tris-hydrochloric acid solution, sulfuric acid solution, and phosphoric acid solution, and the concentration of the pH regulating solution is 1 to 15 mol / L; further still, the pH regulating solution comprises at least one of potassium hydroxide solution, sodium hydroxide solution, and hydrochloric acid solution, and the concentration of the pH regulating solution is 1 to 6 mol / L;

[0044] Furthermore, the pH of the solid tumor is 6.5;

[0045] Furthermore, the organosilicon coupling agent includes at least one of tetraethyl orthosilicate (TEOS), methyl orthosilicate, 3-chloropropyltriethoxysilane, dimethyldimethoxysilane, dimethyldimethoxysilane, hexamethyldisiloxane, hexamethyldisilazane, 3-aminopropyltriethoxysilane, N-[3-(trimethoxysilyl)propyl]ethylenediamine, and isocyanatepropyltriethoxysilane;

[0046] Furthermore, the molar ratio of the sonosensitizer-silane conjugate to the organosilicon coupling reagent is 20-320:1-100; further 50-200:1-30; further 160:3.

[0047] Furthermore, the concentration of the sonosensitizer-silane conjugate is 20-320 μM; further 50-200 μM; further 160 μM.

[0048] Furthermore, the concentration of the organosilicon coupling reagent is 1-100 mM, further 1-30 mM, and further 3 mM.

[0049] A method for mineralizing tumor cells using the tumor treatment product comprises the following steps: injecting the tumor treatment product into the tumor cells and incubating for mineralization.

[0050] Furthermore, the tumor cells include 4T1 cells.

[0051] Furthermore, the ratio of the sonosensitizer-silane conjugate, the organosilicon coupling reagent and the tumor cells in step S2 in the tumor treatment product is 20-320 mmol: 1-100 mmol: 1-106 further to 50~200:1~30:10 5 ~10 6 further to 160:3:10 5 ~10 6 indivual.

[0052] Furthermore, the mineralization time is 5 to 180 minutes, and further 30 to 120 minutes.

[0053] The present invention synthesizes a sonosensitizer-silane conjugate; for example, when the sonosensitizer is hematoporphyrin and the silane is 3-aminopropyltriethoxysilane, the synthesis reaction formula is shown in Formula II:

[0054]

[0055] Based on this, the present invention proposes a strategy of combining mechanical immune checkpoint blockade with sonodynamic immunotherapy. Sonodynamic therapy induces immunogenic death of tumor cells, enhancing the immunogenicity of the tumor site. At the same time, a highly rigid amorphous silica exoskeleton generated on the cell membrane surface is used to increase the cortical hardness of target cells, thereby blocking mechanical immune checkpoints. Furthermore, this approach addresses potential drawbacks of traditional sonodynamic therapy, achieving a synergistic effect between mechanical immune checkpoint blockade and sonodynamic therapy. This method complements existing mechanical immune checkpoint blockade therapies, providing another dimension of thinking for sonodynamic immunotherapy and broadening the clinical application prospects for sonodynamic immunotherapy based on mechanical immune checkpoint blockade.

[0056] The present invention has the following advantages and effects compared to the prior art:

[0057] (1) The present invention aims to improve the rigidity of tumor cells through a biomimetic silicon layer, and the addition of hematoporphyrin-silane conjugate not only has no obvious interference with the rigidity of tumor cells, but also gives the silicon layer excellent acoustic dynamic properties.

[0058] (2) By increasing the hardness of target cells, the disadvantage of traditional sonodynamic therapy in promoting tumor escape and metastasis is alleviated, and the effect of T cells on target cells is increased. By utilizing the synergistic effect of blocking the mechanical immune checkpoints and sonodynamic immunotherapy, the body's own immune efficacy is greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is the hydrogen spectrum of hematoporphyrin-silane conjugate.

[0060] Figure 2 These are the infrared and ultraviolet spectra of hematoporphyrin-silane conjugates.

[0061] Figure 3This is a graph showing the results of testing the sonodynamic performance of the material using a single-wire oxygen probe SOSG.

[0062] Figure 4 This is a data graph showing that cell hardness increases with increasing silicon source concentration at the cellular level.

[0063] Figure 5 This is the result of testing the sonodynamic performance using DCFH-DA as a reactive oxygen species probe at the cellular level.

[0064] Figure 6 This is a data chart showing that biomimetic siliconization-assisted sonodynamic immunotherapy induces DC maturation at the cellular level.

[0065] Figure 7 This is an analysis of the effect of in situ silicification assisting tumor cells in regulating their own hardness to block mechanical immune checkpoints combined with sonodynamic therapy. DETAILED DESCRIPTION

[0066] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0067] Example 1

[0068] The synthesis of hematoporphyrin-silane conjugates comprises the following steps:

[0069] (1) Synthesis of intermediate product PEG-HP.

[0070] Dissolve N-hydroxysuccinimide-PEG-carboxylic acid (120 mg, 0.09 mmol) and hematoporphyrin dihydrochloride (17.6 mg, 0.03 mmol) in 5 mL of anhydrous dimethyl sulfoxide (DMSO) and stir at room temperature for 30 minutes. Then, add 4-dimethylaminopyridine (DMAP) (12.5 mg, 0.1 mmol) to the system, purge with nitrogen, and allow to react at room temperature for 24 hours. The reaction solution is then added dropwise to anhydrous ether and recrystallized to yield a reddish-brown solid.

[0071] (2) Synthesis of the target product APTES-PEG-HP.

[0072] The reddish-brown solid obtained above was dissolved in 5 mL of anhydrous dimethyl sulfoxide (DMSO) along with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) (20 mg) and N-hydroxysuccinimide (NHS) (20 mg). The mixture was purged with nitrogen and stirred at room temperature in the dark for 4 hours. 3-Aminopropyltriethoxysilane (35 μL, 0.15 mmol) was added to the reaction solution, which was then purged with nitrogen and allowed to react at room temperature for 24 hours. The reaction solution was then added dropwise to anhydrous ether and recrystallized to obtain the final product as a reddish-brown solid.

[0073] The hydrogen spectrum of the above hematoporphyrin-silane conjugate is as follows Figure 1 The above hematoporphyrin-silane conjugate was compared with the raw material hematoporphyrin by infrared and ultraviolet spectra. The results are as follows: Figure 2 As shown, compared with the raw material hematoporphyrin, the structure of the final product changes without affecting the energy absorption characteristics of the porphyrin ring.

[0074] Example 2

[0075] Material level acoustic dynamic performance test of hematoporphyrin-silane conjugate:

[0076] The hematoporphyrin-silane conjugate and the raw material hematoporphyrin were prepared into 5 μg / ml aqueous solutions respectively and mixed thoroughly.

[0077] Each time, 1 mL was taken into a 2 mL centrifuge tube for sonodynamic effect test.

[0078] The test parameter of the acoustic dynamic instrument is 1.5W / cm 2 , working time is 2 minutes, working interval is 1 minute, and coupling agent thickness is 2 cm.

[0079] Each group set 11 time nodes for testing, namely 0 minutes, 2 minutes, 4 minutes, 6 minutes, 8 minutes, 10 minutes, 12 minutes, 14 minutes, 16 minutes, 18 minutes and 20 minutes. The results are as follows: Figure 3 As shown in the figure, after 10 minutes of ultrasonic treatment, the reactive oxygen species production rate in the presence of hematoporphyrin-silane conjugate was 1.5 times that in the presence of hematoporphyrin, and 2.0 times that of the control group (no sonosensitizer), that is, the molecular modification of hematoporphyrin greatly improved its sonodynamic response effect.

[0080] Example 3

[0081] Specific experimental methods for improving the hardness of 4T1 cells by biomimetic silicification:

[0082] 4T1 cells were cultured at 10 5 The cells were seeded at a density of 100 μg / mL in a 6-cm culture dish and incubated in an incubator for 24 hours. 4T1 cells in the exponential growth phase were digested and dispersed into a single-cell suspension, washed three times with PBS, and then centrifuged.

[0083] The sonosensitizer-silane conjugate prepared in Example 1 and varying amounts of tetraethyl orthosilicate (TEOS) were added to serum-free 1640 cell culture medium and mixed to obtain siliconization stock solutions. Siliconization stock solutions with TEOS concentrations of 0 mM, 1 mM, 3 mM, 6 mM, and 9 mM were prepared (with a sonosensitizer-silane conjugate concentration of 160 μM).

[0084] 4T1 cells (approximately 2.5×10 6 The cells were washed three times with PBS solution, and then 3 mL of siliconization solution of different concentrations was added. The cells were placed in a cell culture incubator (CO2 concentration of 5%, temperature of 36.5°C) and mineralized for 30 minutes.

[0085] The mineralization solution was discarded, and the cells were washed with PBS three times and then 1 mL of PBS solution was added. The Young's modulus of the cells was tested using an atomic force microscope. The results were as follows: Figure 4 As shown in Figure 3, as the concentration of tetraethyl orthosilicate (TEOS) increases, the cortical hardness of mineralized 4T1 cells also increases.

[0086] Example 4

[0087] Specific protocol for testing sonodynamic performance using DCFH-DA as a reactive oxygen species probe at the cellular level:

[0088] 4T1 cells in the exponential growth phase were digested and dispersed into a single-cell suspension, washed three times with PBS, and then centrifuged.

[0089] Different amounts of the hematoporphyrin-silane conjugate prepared in Example 1 and TEOS were added to serum-free 1640 cell culture medium and mixed to obtain siliconization stock solutions. Siliconization stock solutions with concentrations of 0 μM, 40 μM, 80 μM, 120 μM, 160 μM, and 320 μM hematoporphyrin-silane conjugate (wherein the concentration of TEOS was 3 mM) were prepared.

[0090] Resuspend 4T1 cells in siliconization stock solution to a cell concentration of 10 5 / mL, mix well and place in a cell culture incubator (CO2 concentration of 5%, temperature of 36.5°C) for mineralization for 30 minutes.

[0091] After mineralization, centrifuge and discard the supernatant. Wash the cells three times with PBS solution and then incubate in DCFH-DA working solution for no more than 30 minutes.

[0092] After the injection, the cells were centrifuged, washed three times with PBS solution, resuspended with PBS solution, and subjected to sonodynamic therapy.

[0093] The parameters of the sonodynamic therapy device are set as follows: power is 1.5W / cm 2 , the interval time was 1 minute, and the ultrasound time was 2 minutes.

[0094] The following experimental groups were set up according to the sonosensitizer concentration gradient: 0μM, 40μM, 80μM, 120μM, 160μM, 320μM. The reactive oxygen species production in each group was quantitatively analyzed by fluorescence staining and fluorescence intensity. Figure 5As shown in the figure, when the concentration of sonosensitizer-silane reaches 160 μM, reactive oxygen species can be generated efficiently.

[0095] Example 5

[0096] These experiments demonstrate that organically doped hematoporphyrin within the silica layer can effectively function as a sonosensitizer. To investigate its ability to induce immunogenic cell death in vivo, further analysis is needed to determine whether the presence of the silica layer affects the release of damage-associated molecular patterns. The specific experimental steps are as follows:

[0097] 1. Preparation of Immature DCs (iDCs)

[0098] (1) Preparation of single-cell suspension of primary mouse bone marrow-derived dendritic cells (BMDCs).

[0099] All surgical instruments were sterilized prior to the experiment, and the experiment was conducted in a Class II biosafety cabinet using appropriate safety procedures. A healthy 8-week-old female BALB / c mouse was sacrificed by cervical dislocation and soaked in 75% ethanol for 2 minutes. The femur and tibia were surgically removed, and the surrounding muscle was removed using surgical scissors and forceps without damaging the bone tissue.

[0100] Place all bones to be used in a 6 cm Petri dish containing 70% ethanol and soak for 30 seconds. Use a scalpel blade to trim the ends of the tibia and femur. Use a syringe filled with 1 mL of 1640 medium (serum-free) to flush the bone marrow contents from one end of the bone into a 15 mL centrifuge tube. Repeat this three times.

[0101] Gently pipette the resulting mixture repeatedly through a 1 mL pipette tip to create a single-cell suspension. Filter through a 75 μm filter into a 15 mL centrifuge tube. Centrifuge at 300 g for 5 minutes at 4°C in a temperature-controlled centrifuge. Carefully remove the supernatant. Resuspend the cells in 1 mL of red blood cell lysis buffer and lyse at room temperature for 5 minutes to remove red blood cells.

[0102] Add 9 mL of 1640 complete medium and centrifuge at 300 g for 5 min at 4°C in a temperature-controlled centrifuge. Discard the supernatant and resuspend the cell suspension in 10 mL of 1640 complete medium. Filter the cell suspension through a 40 μm filter and measure the cell concentration.

[0103] (2) In vitro induction of bone marrow-derived dendritic cells.

[0104] The cell concentration was adjusted to 1×10 cells using 1640 complete medium containing 20 ng / mL GM-CSF and 40 ng / mL IL-4. 6 / mL. Use a 6-well plate for culture, add 3mL of cell suspension to each well, and culture normally in an incubator. This is marked as day 0. On the second day of culture, gently shake the culture plate, remove 1 / 2 of the culture medium in the well (which can be added to a new 6-well plate for continued culture), and add an equal amount of differentiation medium. This step aims to remove granulocytes and lymphocytes.

[0105] On the fourth day of adherence, DCs aggregated and adhered to the bottom of the plate, and by the sixth day, DC colonies could be observed. On the fifth day of culture, 3 mL of 1640 complete medium supplemented with 20 ng / mL GM-CSF and 40 ng / mL IL-4 was added to each well for continued culture, bringing the volume per well to 6 mL.

[0106] On day 6 of culture, gently aspirate the culture medium with a pipette to obtain loosely adherent BMDCs, which are considered immature DCs (iDCs). Add 3 mL of 1640 medium supplemented with 20 ng / mL GM-CSF and 10 ng / mL IL-4 to the 6-well plate and continue culturing. Centrifuge the BMDC suspension at 300 g for 5 minutes at 4°C in a temperature-controlled centrifuge. Discard the supernatant and resuspend the cells in 1640 complete medium until ready to use.

[0107] 2. Treatment Experiments

[0108] The experimental groups are as follows:

[0109] Control group: 4T1 cells in the exponential growth phase were digested and dispersed into single cell suspension, washed with PBS three times and centrifuged; 4T1 cells were mixed with serum-free 1640 cell culture medium (cell concentration after mixing was 10 5 Cells were then incubated in a cell culture incubator (5% CO2, 36.5°C) for 30 minutes. The cells were centrifuged, the supernatant discarded, and the cells were washed three times with PBS and resuspended in PBS. 2 mL of the cells were collected from each group and placed in a 5 mL centrifuge tube. No sonodynamic therapy was performed.

[0110] TEOS T-HP Si Group: 4T1 cells in the exponential growth phase were digested and dispersed into a single cell suspension, washed 3 times with PBS, and then centrifuged. The hematoporphyrin-silane conjugate prepared in Example 1 and TEOS were added to serum-free 1640 cell culture medium and mixed to obtain a siliconization stock solution; the siliconization stock solution contained 3mM tetraethyl orthosilicate (TEOS) and 160μM hematoporphyrin-silane conjugate. The 4T1 cells were resuspended in the siliconization stock solution so that the cell concentration in the suspension was 10 5Cells were mixed and placed in a cell culture incubator (5% CO2, 36.5°C) for 30 minutes. The cells were centrifuged, the supernatant discarded, and the cells were washed three times with PBS and resuspended in PBS. 2 mL of the cells were collected from each group and placed in a 5 mL centrifuge tube. No sonodynamic therapy was performed.

[0111] US group: 4T1 cells in the exponential growth phase were digested and dispersed into single cell suspension, washed with PBS three times and then centrifuged; 4T1 cells were mixed with serum-free 1640 cell culture medium (cell concentration after mixing was 10 5 Cells were then incubated in a cell culture incubator (5% CO2, 36.5°C) for 30 minutes. The cells were centrifuged, the supernatant discarded, and the cells were washed three times with PBS and resuspended in PBS. 2 mL of the suspension was transferred to each 5 mL centrifuge tube according to grouping and sonodynamic therapy was performed.

[0112] HP+US ​​group: 4T1 cells in the exponential growth phase were digested and dispersed into a single cell suspension, washed with PBS three times, and then centrifuged. The hematoporphyrin-silane conjugate prepared in Example 1 was added to serum-free 1640 cell culture medium and mixed to obtain a siliconization stock solution; the siliconization stock solution contained 160 μM hematoporphyrin-silane conjugate. 4T1 cells were resuspended in the siliconization stock solution so that the cell concentration in the suspension was 10 5 Cells were mixed and placed in a cell culture incubator (5% CO2, 36.5°C) for 30 minutes for mineralization. The cells were centrifuged, the supernatant discarded, and the cells were washed three times with PBS and resuspended in PBS. 2 mL of the cells were collected from each group and placed in a 5 mL centrifuge tube for sonodynamic therapy.

[0113] T-HPSi+US group: 4T1 cells in the exponential growth phase were digested and dispersed into a single cell suspension, washed with PBS three times, and then centrifuged. The hematoporphyrin-silane conjugate prepared in Example 1 and TEOS were added to serum-free 1640 cell culture medium and mixed to obtain a siliconization stock solution; the siliconization stock solution contained 3mM tetraethyl orthosilicate (TEOS) and 160μM hematoporphyrin-silane conjugate. The 4T1 cells were resuspended in the siliconization stock solution so that the cell concentration in the suspension was 10 5 Cells were mixed and placed in a cell culture incubator (5% CO2, 36.5°C) for 30 minutes for mineralization. The cells were centrifuged, the supernatant discarded, and the cells were washed three times with PBS and resuspended in PBS. 2 mL of the cells were collected from each group and placed in a 5 mL centrifuge tube for sonodynamic therapy.

[0114] The sonodynamic therapy is performed by using a sonodynamic therapy device at a power of 1.5 W / cm 2 Then, ultrasonicate for 1 minute, pause for 1 minute, and ultrasonicate for another 1 minute.

[0115] 3. Efficacy verification

[0116] After treatment, each group was incubated in the incubator for 30 minutes. Each group was fully mixed and 200 μL was taken from each group and placed in the upper chamber of the Transwell. 800 μL of the above-treated iDCs were added to the lower chamber of the Transwell. Each group was paralleled. Incubated in the incubator for 6 hours, DCs in the lower chamber were collected and mixed with pre-prepared CD11c + CD80 + and CD11c + CD86 + Antibody co-staining and detection by flow cytometry.

[0117] The experimental results are as follows Figure 6 As shown in the figure, the ability of different therapies to induce cell immunogenic death and promote DC maturation in vivo and thus enhance the body's immune function was judged by the maturation of DCs in each group. The results showed that the T-HPSi+US group had the best ability to promote DC maturation and enhance the body's immune function.

[0118] Example 6

[0119] Using a 4T1 bilateral tumor model, we investigated the efficacy of in situ silicification in assisting tumor cells in regulating their own stiffness to block mechanical immune checkpoints combined with sonodynamic therapy. The specific experimental steps are as follows:

[0120] Twenty-five healthy 6-week-old BALB / c mice were randomly divided into five groups, with five mice in each group. Orthotopic tumors were implanted on day -7, ectopic tumors were implanted on day -3, and the tumor volume was about 100 mm on day 0. 3 ), it is processed by group, and the specific groups are as follows:

[0121] Control group (Control): 50 μL of serum-free 1640 cell culture medium was injected into the orthotopic tumor using an automatic injection system and incubated in a normal growth environment for 2 hours.

[0122] T-HP Si Group: Prepare a silication stock solution by adding the hematoporphyrin-silane conjugate prepared in Example 1 and TEOS to serum-free 1640 cell culture medium and mixing thoroughly to obtain a silication stock solution containing 3 mM tetraethyl orthosilicate (TEOS) and 160 μM hematoporphyrin-silane conjugate. 50 μL of the mineralization solution was injected into the orthotopic tumor using an automated injection system. Mineralization proceeded for 2 hours in a normal growth environment.

[0123] In the US group, 50 μL of serum-free 1640 cell culture medium was injected into the orthotopic tumor using an automated injection system and incubated for 2 hours in a normal growth environment. The orthotopic tumor was then treated with sonodynamic therapy.

[0124] HP+US ​​group: Prepare the silication solution by adding the hematoporphyrin-silane conjugate prepared in Example 1 to serum-free 1640 cell culture medium and mix thoroughly to obtain the silication solution; the silication solution contains 160 μM of the hematoporphyrin-silane conjugate. 50 μL of the mineralization solution was injected into the in situ tumor using an automated injection system. Mineralization proceeded for 2 hours in a normal growth environment. The in situ tumor was then treated with sonodynamic therapy.

[0125] T-HPSi+US Group: Prepare the silication solution by adding the hematoporphyrin-silane conjugate prepared in Example 1 and TEOS to serum-free 1640 cell culture medium and mix thoroughly to obtain the silication solution; the silication solution contains 3 mM tetraethyl orthosilicate (TEOS) and 160 μM hematoporphyrin-silane conjugate. 50 μL of the mineralization solution was injected into the in situ tumor using an automated injection system. Mineralization proceeded for 2 hours in a normal growth environment. The in situ tumor was then treated with sonodynamic therapy.

[0126] The sonodynamic therapy is performed using a sonodynamic therapy device, the thickness of the coupling agent does not exceed 2 cm, and the power is 1.5 W / cm 2 The ultrasound was performed for a total of 5 minutes, with a 1-minute pause after every 1-minute ultrasound (i.e., the treatment process was a total of 9 minutes, with a total of 5 minutes of ultrasound and a total of 4 minutes of pause).

[0127] During the experiment, the length and width of the tumor were measured every other day with an electronic vernier caliper to calculate the tumor volume (0.5 × length × width). 2 ) and measure the weight of each mouse using an electronic scale. Also, inspect the mice daily for any abnormalities in appearance and any interference from non-experimental factors, such as infection with other pathogens. Mice were maintained in the same environmental conditions and fed the same diet until day 14, at which time each group of mice was sacrificed by removing their eyeballs and drawing blood.

[0128] The treatment effect of each experimental group can be seen from the bilateral tumor growth curve within 14 days. Figure 7 As shown, mechanical immune checkpoint blockade assisted sono-immunotherapy group (T-HP Si + US) has the best therapeutic effect: after 14 days of treatment, T-HP Si The tumor inhibition rate in the +US group reached 98.6% for orthotopic tumors and 97.4% for ectopic tumors. This study demonstrates that mechanoimmune checkpoint blockade combined with sonodynamic immunotherapy has achieved excellent therapeutic effects in solid tumor models, providing strong case support for mechanoimmune checkpoint research and opening up a new dimension for combined sonodynamic immunotherapy.

[0129] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A tumor treatment composition, characterized in that: Including sonosensitizer-silane conjugates and organosilicon coupling reagents, The structure of the sonosensitizer-silane conjugate is shown in Formula I: Formula I.

2. The tumor treatment composition according to claim 1, wherein The organosilicon coupling reagent includes at least one of tetraethyl orthosilicate, methyl orthosilicate, 3-chloropropyltriethoxysilane, dimethyldimethoxysilane, dimethyldimethoxysilane, hexamethyldisiloxane, hexamethyldisilazane, 3-aminopropyltriethoxysilane, N-[3-(trimethoxysilyl)propyl]ethylenediamine, and isocyanatepropyltriethoxysilane.

3. The method for preparing the sonosensitizer-silane conjugate in the tumor therapeutic composition according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) Hematoporphyrin and N-hydroxysuccinimide-PEG-carboxylic acid are dissolved in a solvent and mixed evenly, and catalyst 1 is added and reacted under an inert atmosphere; the reaction solution is purified by recrystallization and the solid is collected; (2) The solid collected in step (1) and catalyst 2 are dissolved in a solvent, mixed, 3-aminopropyltriethoxysilane is added, and the reaction is carried out under an inert atmosphere. The reaction solution after the reaction is purified by recrystallization, and the solid is collected to obtain a sonosensitizer-silane conjugate.

4. The preparation method according to claim 3, characterized in that The concentration of the hematoporphyrin in the solvent in step (1) is 1 to 10 mM; The concentration of N-hydroxysuccinimide-PEG-carboxylic acid in the solvent described in step (1) is 15 to 25 mM; The catalyst 1 described in step (1) includes 4-dimethylaminopyridine; The molar ratio of the catalyst 1 to hematoporphyrin in step (1) is 3 to 4:1; The molar ratio of hematoporphyrin to N-hydroxysuccinimide-PEG-carboxylic acid in step (1) is 1:2-4; The reaction temperature in step (1) is 10-60°C; The reaction time in step (1) is 12 to 72 hours.

5. The preparation method according to claim 3, characterized in that The catalyst 2 in step (2) comprises at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; The molar ratio of the hematoporphyrin described in step (1) to the 3-aminopropyltriethoxysilane described in step (2) is 1:3-7; The concentration of 3-aminopropyltriethoxysilane in the reaction system in step (2) is 20-40 mM; The reaction temperature in step (2) is 10-60°C; The reaction time in step (2) is 12 to 72 hours.

6. Use of the tumor therapeutic composition according to any one of claims 1 to 2 in the preparation of tumor therapeutic drugs.

7. The use according to claim 6, characterized in that The tumor treatment drug includes a siliconized stock solution, and the preparation method of the siliconized stock solution includes the following steps: The pH of the culture medium is adjusted to the pH of the solid tumor; the sonosensitizer-silane conjugate and the organosilicon coupling reagent in the tumor treatment composition according to any one of claims 1 to 2 are added to the culture medium, mixed, and a siliconized stock solution is obtained.

8. The use according to claim 7, characterized in that The organosilicon coupling reagent includes tetraethyl orthosilicate; The molar ratio of the sonosensitizer-silane conjugate to the organosilicon coupling reagent is 20-320:1-100; The concentration of the sonosensitizer-silane conjugate is 20 to 320 μM; The concentration of the organosilicon coupling reagent is 1-100 mM.

9. The use according to claim 6, characterized in that The use of the medicine includes the following steps: injecting the tumor treatment product into the tumor to incubate mineralization.

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