Lipoic acid-choline ionic liquid solution as well as preparation method and application thereof

Abstract

The invention discloses a lipoic acid-choline ionic liquid solution as well as a preparation method and application thereof, and belongs to the technical field of tumor treatment, the lipoic acid-choline ionic liquid solution has a microwave heat effect, the microwave heat effect is positively correlated with the molar ratio of choline to lipoic acid, the concentration of the lipoic acid-choline ionic liquid solution is 2-25%, and the concentration of the lipoic acid-choline ionic liquid solution is 2-25%. The lipoic acid-choline ionic liquid solution consists of lipoic acid, choline and water / ethanol, wherein the molar ratio of the lipoic acid to the choline is (0.25-1): 1. According to the lipoic acid-choline ionic liquid solution and the preparation method and application thereof, the microwave ablation area is increased through the lipoic acid-choline ionic liquid solution, immunosuppression related to incomplete microwave ablation is obviously relieved, and the anti-tumor effect far better than that of independent microwave ablation is achieved; and tumor recurrence and metastasis are effectively inhibited.

Description

Technical Field

[0001] The invention relates to the technical field of tumor treatment, in particular to a lipoic acid-choline ionic liquid solution and a preparation method and application thereof. Background Art

[0002] Cancer poses a serious threat to human life and health, and current treatments are still difficult to completely cure cancer. Different from traditional treatments (surgery, chemotherapy, radiotherapy, etc.), local thermal therapy (LTT) may be a more promising anti-tumor treatment due to its advantages such as multiple anti-tumor mechanisms, systemic immune activation and minimal invasiveness. For example, microwave thermal ablation (MWA) effectively induces tumor tissue necrosis by generating high temperatures around the electrode needle within a few minutes. However, the area near the electrode needle of MWA may generate excessively high temperatures (>80°C), which can lead to water evaporation and carbonization of tissues, which in turn greatly hinders the diffusion of microwave sensitizers and microwave heat to distal areas (also called mass transfer / heat transfer). The temperature decreases rapidly with increasing distance from the electrode needle. The high heterogeneity and irregular shape of the tumor further complicate heat / mass transfer within the tumor, and large tumors with a diameter greater than 3.0 cm are often unable to be completely ablated. Tumor tissue near large blood vessels or bile ducts is also difficult to completely ablate. The residual tumor cells after incomplete ablation show a very aggressive phenotype, rapidly recur and metastasize, and lead to poor prognosis for patients. In addition, it has been reported that tumor recurrence and metastasis are highly correlated with the infiltration of heat-induced immunosuppressive cells such as myeloid suppressor cells (MDSCs) and regulatory T cells (Tregs). Therefore, how to improve the heat / mass transfer within the tumor tissue during LTT and improve the immunosuppressive microenvironment after thermal ablation to achieve specific and effective ablation of tumor tissue remains a huge challenge.

[0003] Ionic liquids (ILs) generally refer to organic salts that can melt at room temperature (usually with a melting point of <100°C) and are widely used in various industrial sectors. Recently, ILs used together with active pharmaceutical ingredients (APIs) have attracted increasing attention due to their diverse bioactivities and favorable properties (e.g., high conductivity and thermal / chemical stability). In cancer therapy, researchers have developed various antitumor API-ILs and ILs-dependent drug carriers by taking advantage of the various antitumor activities, drug solubility properties, and enhanced tissue permeability of ILs. For example, choline / geranyl ILs (LATTE) exhibited higher intratumoral diffusion by disrupting the intratumoral osmotic pressure balance and membrane permeability, thereby achieving better tumor ablation than other chemical ablative agents. Since the high ion content and polarizability are conducive to microwave thermosensitization, researchers have utilized classic [Bmim][PF6]-ILs to improve the antitumor efficacy of MWA. To avoid toxicity caused by ILs leakage, researchers strictly encapsulated them in biocompatible nanoparticles, which in turn may lead to poor heat / mass transfer in the tumor and incomplete MWA. Therefore, it is highly desirable to develop biocompatible, highly permeable ILs as microwave sensitizers to achieve complete tumor ablation.

[0004] Lipoic acid (LA) is an endogenous disulfide fatty acid that functions as a coenzyme factor in mitochondrial energy metabolism. Due to its powerful antioxidant effect, LA has been widely used as a dietary supplement and drug for a variety of diseases associated with high oxidative stress in more than 180 clinical studies. In addition, it has been reported that LA and its derivatives selectively kill various cancer cells through multiple anti-tumor effects (inducing apoptosis, inhibiting oncogenic signals, inhibiting cell cycle, anti-tumor metastasis, etc.) targeting mitochondria, while normal cells are hardly affected. Summary of the invention

[0005] The purpose of the present invention is to provide a lipoic acid-choline ionic liquid solution and a preparation method and application thereof. The lipoic acid-choline ionic liquid solution increases the ablation area of ​​microwave ablation, significantly alleviates the immunosuppression associated with incomplete microwave ablation, achieves an anti-tumor effect far better than microwave ablation alone, and effectively inhibits tumor recurrence and metastasis.

[0006] To achieve the above object, the present invention provides a thioctic acid-choline ionic liquid solution, which has a microwave thermal effect, and the microwave thermal effect is positively correlated with its choline / thioctic acid molar ratio. The concentration of the thioctic acid-choline ionic liquid solution is 2-25%, and the thioctic acid-choline ionic liquid solution is composed of thioctic acid, choline and water / ethanol, wherein the molar ratio of thioctic acid to choline is (0.25-1):1.

[0007] Preferably, the lipoic acid-choline ionic liquid solution LACH includes LACH4, LACH2, and LACH1; the molar ratio of lipoic acid to choline in LACH4 is 0.25:1, the molar ratio of lipoic acid to choline in LACH2 is 0.5:1, and the molar ratio of lipoic acid to choline in LACH1 is 1:1.

[0008] The method for preparing the above-mentioned lipoic acid-choline ionic liquid solution comprises the following steps:

[0009] Step 1, mixing lipoic acid and choline, dissolving them in water / ethanol and stirring to obtain a stirred product;

[0010] Step 2: placing the stirred product obtained in step 1 in a rotary evaporator for rotary evaporation, and then placing the solution obtained by rotary evaporation in a vacuum drying oven to obtain a yellow viscous or yellow elastic liquid;

[0011] Step 3: dissolving the yellow viscous or yellow elastic liquid obtained in step 2 in water to obtain a lipoic acid-choline ionic liquid solution.

[0012] Preferably, the volume of water / ethanol in step 1 is 10-50 mL, and the stirring time is 10-12 h.

[0013] Preferably, in step 2, the rotary evaporation temperature is 50-100° C., the temperature in the vacuum drying oven is 50-100° C., and the reaction time in the vacuum drying oven is 36-48 h.

[0014] The application of the above-mentioned lipoic acid-choline ionic liquid solution is to apply the above-mentioned lipoic acid-choline ionic liquid solution to the preparation of anti-tumor drugs.

[0015] Therefore, the present invention adopts the above-mentioned lipoic acid-choline ionic liquid solution and its preparation method and application, which has the following beneficial effects:

[0016] (1) Lipoic acid-choline ionic liquid solution LACH, as a multifunctional MW sensitizer and chemical ablation agent to enhance the antitumor efficacy of MWA.

[0017] (2) LACH is uniformly retained and diffused in the tumor, thereby generating high-performance heat / mass transfer in the tumor and achieving a larger ablation area than iMWA.

[0018] (3) LACH induces specific cytotoxicity to liver cancer cells, but only inhibits the proliferation of normal liver cells, thereby reducing the toxic side effects on normal cells.

[0019] (4) Due to the good heat / mass transfer and specific killing of liver cancer cells by LACH, LACH significantly alleviated the iMWA-related immunosuppression and achieved a much better antitumor efficacy than iMWA.

[0020] (5) LACH can be developed as a multifunctional sensitizer for clinical translation to achieve precise synergistic effects of chemical ablation and MWA.

[0021] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The present invention is a lipoic acid-choline ionic liquid solution and its preparation method and application examples LA, CH, LACH1, LACH2 and LACH4 1 H-NMR spectrum;

[0023] Figure 2 The present invention is a lipoic acid-choline ionic liquid solution and its preparation method and application example LACH1 1 H-NOESY spectrum;

[0024] Figure 3 It is a DOSY diagram of a lipoic acid-choline ionic liquid solution and a preparation method and application example LACH1 of the present invention;

[0025] Figure 4 The viscosity and conductivity of a lipoic acid-choline ionic liquid solution and its preparation method and application examples LACH1, LACH2 and LACH4 at 25°C, 37°C and 50°C of the present invention;

[0026] Figure 5 The present invention is a lipoic acid-choline ionic liquid solution and its preparation method and application example LACH2 1 H-NOESY spectrum;

[0027] Figure 6 It is a DOSY diagram of a lipoic acid-choline ionic liquid solution and a preparation method and application example LACH2 of the present invention;

[0028] Figure 7 The present invention is a lipoic acid-choline ionic liquid solution and its preparation method and application example LACH4 1 H-NOESY spectrum;

[0029] Figure 8 It is a DOSY diagram of a lipoic acid-choline ionic liquid solution and a preparation method and application example LACH4 of the present invention;

[0030] Fig. 9 This is an infrared thermal image of a lipoic acid-choline ionic liquid solution and its preparation method and application examples LACH1, LACH2 and LACH4 under microwave radiation;

[0031] Fig.10 The present invention is a lipoic acid-choline ionic liquid solution and its preparation method and application example. The dose-response curve after LACH1, LACH2 and LACH4 are co-incubated with HepG2 and LO2 cells for 48 hours respectively;

[0032] Fig.11 The present invention discloses a lipoic acid-choline ionic liquid solution and a preparation method and application example thereof. The dose-response curves after LACH1, LACH2 and LACH4 are co-incubated with Hepa1-6 and FL83b cells for 48 hours respectively;

[0033] Fig.12 It is a representative photo of the cell morphology changes after LACH1, LACH2 and LACH4 are co-incubated with different cells in a lipoic acid-choline ionic liquid solution and a preparation method and application example of the present invention;

[0034] Fig.13 It is an in vivo fluorescence image of mice at predetermined time points (6h, 12h and 24h) after different treatments of a lipoic acid-choline ionic liquid solution and its preparation method and application examples of the present invention;

[0035] Fig.14 The invention discloses a lipoic acid-choline ionic liquid solution and a preparation method thereof and application examples thereof for quantitative analysis of the fluorescence diffusion area of ​​tumors in vivo after different treatments;

[0036] Fig.15 The invention discloses a lipoic acid-choline ionic liquid solution and a preparation method thereof and application examples thereof for quantitative analysis of the fluorescence intensity of tumors in vivo after different treatments;

[0037] Fig.16 It is a fluorescence image (left), a real-time temperature image (middle) and an overlapping image of the fluorescence area and the ablation area (T>50°C) of a lipoic acid-choline ionic liquid solution and its preparation method and application examples after different treatments in vitro Hepa1-6 tumor tissues of the present invention;

[0038] Fig.17 It is a diffusion area diagram of ILACH in in vitro Hepa1-6 tumor tissue after different treatments of a lipoic acid-choline ionic liquid solution and its preparation method and application examples of the present invention;

[0039] Fig.18 It is a statistical diagram of the overlap of the diffusion area and the ablation area of ​​ILACH in Hepa1-6 tumor tissue in vitro after different treatments of a lipoic acid-choline ionic liquid solution and its preparation method and application examples of the present invention;

[0040] Fig.19This is a schematic diagram of animal experiment time of a lipoic acid-choline ionic liquid solution and its preparation method and application example of the present invention;

[0041] Fig. 20 The present invention is a lipoic acid-choline ionic liquid solution and its preparation method and application examples. The average tumor growth curve of subcutaneous Hepa1-6 tumors in mice after different treatments;

[0042] Fig.21 The present invention is a lipoic acid-choline ionic liquid solution and its preparation method and application example. After different treatments, the Treg (CD25 + Foxp3 + / CD3 + CD4 + T cells) and CD8 + / Treg ratio statistics;

[0043] Fig. 22 The present invention discloses a lipoic acid-choline ionic liquid solution and a preparation method and application examples thereof, and provides statistical percentages of MDSCs, PMN-MDSCs, and M1 and M2 tumor-related macrophages in tumor tissues after different treatments;

[0044] Fig.23 The invention discloses a lipoic acid-choline ionic liquid solution and a preparation method and application examples thereof, and statistical percentages of Tcm and Tem in tumor tissue after different treatments. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0046] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0047] The invention provides a thioctic acid-choline ionic liquid solution. The thioctic acid-choline ionic liquid solution has a microwave thermal effect, and the microwave thermal effect is positively correlated with the choline / thioctic acid molar ratio thereof. The concentration of the thioctic acid-choline ionic liquid solution is 2-25%. The thioctic acid-choline ionic liquid solution is composed of thioctic acid, choline and water / ethanol, wherein the molar ratio of thioctic acid to choline is (0.25-1):1.

[0048] The lipoic acid-choline ionic liquid solution LACH includes LACH4, LACH2, and LACH1; the molar ratio of lipoic acid to choline in LACH4 is 0.25:1, the molar ratio of lipoic acid to choline in LACH2 is 0.5:1, and the molar ratio of lipoic acid to choline in LACH1 is 1:1.

[0049] The method for preparing the above-mentioned lipoic acid-choline ionic liquid solution comprises the following steps:

[0050] Step 1: Mix lipoic acid and choline, dissolve in water / ethanol and stir to obtain a stirrer, the volume of water / ethanol is 10-50 mL, and the stirring time is 10-12 h.

[0051] Step 2: Place the stirred substance obtained in step 1 in a rotary evaporator for rotary evaporation, and then place the solution obtained by rotary evaporation in a vacuum drying oven to obtain a yellow viscous or yellow elastic liquid; the rotary evaporation temperature is 50-100° C., the temperature in the vacuum drying oven is 50-100° C., and the reaction time in the vacuum drying oven is 36-48 hours.

[0052] Step 3: dissolving the yellow viscous or yellow elastic liquid obtained in step 2 in water to obtain a lipoic acid-choline ionic liquid solution.

[0053] An application of a lipoic acid-choline ionic liquid solution is to apply the lipoic acid-choline ionic liquid solution to the preparation of an anti-tumor drug, wherein the tumor is preferably a liver tumor.

[0054] A multifunctional ionic liquid solution (LACH) was constructed by the ion metathesis reaction of lipoic acid (LA) and cationic choline (CH). LACH is used as a multifunctional tumor ablative agent to overcome tumor recurrence and metastasis caused by incomplete microwave ablation. LACH has the following three advantages: 1) The high tissue permeability generated by the reduced tumor stiffness promotes intratumor heat / mass transfer and achieves a more uniform microwave heat distribution in the tumor; 2) The high ionic conductivity helps LACH enhance the MW thermal effect and improve the antitumor efficacy of MWA; 3) Based on the difference in heat resistance between tumor cells and normal cells, the thermosensitization effect can enhance the specific cytotoxicity of LACH to tumor cells. Given that iMWA usually produces an immunosuppressive microenvironment, LACH-induced tumor cell immunogenic cell death (ICD) and relieved immunosuppression to improve the tumor immune microenvironment were studied. Finally, the tumor inhibitory effect of LACH in the mouse Hepa1-6 subcutaneous tumor model was evaluated. LACH can be used as a new multifunctional ablative agent to overcome the tumor recurrence and metastasis of iMWA.

[0055] Example 1

[0056] A synthesis of lipoic acid-choline ionic liquid solution LACH1 comprises the following steps:

[0057] In order to synthesize lipoic acid-choline ionic liquid solution LACH1, choline carbonate (CH, 0.097 mol) and lipoic acid (LA, 20 g, 0.097 mol) were dissolved in 20 mL of water / ethanol at room temperature, the reaction system was placed on a magnetic stirrer and stirred overnight until CO2 stopped generating, the mixture was rotary evaporated at 50° C. for 2 hours, and then dried in a vacuum drying oven at 60° C. for 48 hours to remove the residual water in the solution to obtain LACH1, and then the yellow transparent ionic liquid was dissolved in 10 mL of water (concentration of 2%-25%) and stirred to obtain the lipoic acid-choline ionic liquid solution LACH1 for use.

[0058] like Figure 1 As shown, 1 H NMR ( 1 H-NMR) proved the successful synthesis of LACH1. Among them, ag represents the characteristic peak of LA, and hl represents the characteristic peak of CH. The chemical shifts δ3.44ppm and δ3.58ppm are the characteristic peaks of the methylene (c) on CH in LACH and the methine (k) on the disulfide five-membered ring of LA, respectively. The peak area ratio is 2:1, so the molar ratio of lipoic acid and choline in the obtained LACH1 is 1:1.

[0059] like Figure 2 As shown, the NOESY spectrum results of LACH1 showed that LA and CH in LACH1 were in close contact (distance <0.5 nm) and there was an ion pair.

[0060] In order to further understand the diffusion-permeation characteristics of LACH, diffusion-ordered spectroscopy (DOSY) was used to calculate its self-diffusion coefficient. Figure 3 It can be seen that the self-diffusion coefficient of LACH1 is 5.55×10 -6 m 2 ·s -1 The diffusion of molecules is closely related to their physical properties (such as viscosity and conductivity). The viscosity and conductivity of LACH1 at different temperatures were further tested. The results showed that at 25°C, 37°C and 50°C, the higher the temperature, the higher the viscosity and conductivity of LACH1. Figure 4 shown.

[0061] Example 2

[0062] A synthesis of lipoic acid-choline ionic liquid solution LACH2 comprises the following steps:

[0063] In order to synthesize lipoic acid-choline ionic liquid solution LACH2, choline carbonate (CH, 0.194 mol) and lipoic acid (LA, 20 g, 0.097 mol) were dissolved in 20 mL of water / ethanol at room temperature, and the reaction system was placed on a magnetic stirrer and stirred overnight until CO2 stopped generating, and four yellow transparent ionic liquids were obtained. Rotary evaporation was performed at 50°C for 2 hours, and then the residual water in the solution was removed by drying at 60°C in a vacuum drying oven for 48 hours to obtain LACH2, and then the yellow transparent ionic liquid was dissolved in 10 mL of water and stirred to obtain lipoic acid-choline ionic liquid solution LACH2 for use.

[0064] like Figure 1 As shown, 1 H NMR ( 1 H-NMR) confirmed the successful synthesis of LACH2, and the molar ratio of lipoic acid to choline in the obtained LACH2 was 0.5:1.

[0065] The NOESY spectrum of LACH2 shows the interaction between the methyl protons on the CH nitrogen in LACH2 (H k -H h,i,j , H l -H h,i,j and H k -H l ) dominates, and there is no paired LACH2 ion pair in LACH2. It is a complex molecular network structure with a high degree of exchange between ions, such as Figure 5 shown.

[0066] like Figure 3 , Figure 6 and Figure 8 As shown in the DOSY spectrum, the self-diffusion coefficient of LACH2 is 5.62×10 -6 m 2 ·s -1 , the self-diffusion ability of LACH2 is stronger than that of LACH1 and LACH4. The results of viscosity and conductivity of LACH at different temperatures show that at 25℃, 37℃ and 50℃, the higher the temperature, the higher the viscosity and conductivity, such as Figure 4 shown.

[0067] Example 3

[0068] A synthesis of lipoic acid-choline ionic liquid solution LACH4 comprises the following steps:

[0069] In order to synthesize lipoic acid-choline ionic liquid solution LACH4, choline carbonate (CH, 0.388 mol) and lipoic acid (LA, 20 g, 0.097 mol) were dissolved in 20 mL of water / ethanol at room temperature, and the reaction system was placed on a magnetic stirrer and stirred overnight until CO2 stopped generating, and four yellow transparent ionic liquids were obtained. Rotary evaporation was performed at 50°C for 2 hours, and then the residual water in the solution was removed by drying at 60°C in a vacuum drying oven for 48 hours to obtain LACH4, and then the yellow transparent ionic liquid was dissolved in 10 mL of water and stirred to obtain lipoic acid-choline ionic liquid solution LACH4 for use.

[0070] like Figure 1 As shown, 1 H NMR ( 1 H-NMR) confirmed the successful synthesis of LACH4, and the molar ratio of lipoic acid to choline in the obtained LACH4 was 0.25:1.

[0071] The NOESY spectrum of LACH4 shows the interaction between the methyl protons on the CH nitrogen in LACH4 (H k -H h ,i,j , H l -H h,i,j , H k -H l and H m -H l ) dominates, and there is no paired LACH4 ion pair in LACH4. It is a complex molecular network structure with a high degree of exchange between ions, such as Figure 7 shown.

[0072] like Figure 3 , Figure 6 and Figure 8 As shown in Figure 2, the DOSY spectrum shows that the self-diffusion coefficient of LACH4 is 4.64×10 -6 m 2 ·s -1 , the self-diffusion ability of LACH4 is weaker than that of LACH2 and LACH1. The results of viscosity and conductivity of LACH4 at different temperatures show that at 25℃, 37℃ and 50℃, the higher the temperature, the higher the viscosity and conductivity. Figure 4 shown.

[0073] Example 4

[0074] The microwave thermal response characteristics of LACH1, LACH2 and LACH4 are characterized as follows:

[0075] Microwave electromagnetic fields can cause ionic liquids to rotate in a confined space and collide violently with each other, thereby enhancing the microwave thermal effect. Different LACH1, LACH2 and LACH4 were irradiated at a microwave power of 3W for 300s to study the microwave thermal curve of LACH over time. The results showed that LACH4 had the best microwave thermal effect (the ΔT of LACH1, LACH2 and LACH4 were 28.3±3.1, 45.2±2.7 and 52.0±2.7℃, respectively). With the increase of conductivity, the energy conversion efficiency of microwave electromagnetic energy to thermal energy of LACH4 is higher, such as Fig. 9 shown.

[0076] Example 5

[0077] The studies on LACH1, LACH2 and LACH4 inducing specific damage to various liver cancer cells are as follows:

[0078] First, the cytotoxicity of the three LACHs was compared by co-culturing LACH1, LACH2, and LACH4 with hepatoma cells and normal hepatocytes at concentrations ranging from 0.02 to 20 mM for 48 h. The half inhibitory concentrations (IC 50 s) are 0.62mM, 0.13mM and 1.70mM respectively, such as Fig.10 However, for LO2 cells (normal human hepatocytes), the IC 50 As high as 1.67mM, which is about 13 times that of HepG2 cells. Similarly, the IC of LACH2 for Hepa1-6 cells (mouse liver cancer cells) is 50 is 0.43 mM, while the IC 50 The latter is about 3.7 times of the former. Fig.11 As shown, the above results show that LACH has specific cytotoxicity to various liver cancer cells, especially LACH2 has the greatest difference in cytotoxicity between tumor cells and normal cells. Further study on the effect of LACH2 on the morphological structure of normal cells and tumor cells, Fig.12 The results showed that compared with the control group, after treatment with LA and LACH2, HepG2 and Hepa1-6 cells showed obvious apoptotic morphological characteristics, while the morphological characteristics of normal cells (FL83b and LO2) did not change significantly, only showing a decrease in number. The CH and PBS treatment groups had no significant effect on the morphological characteristics and number of normal cells and tumor cells, indicating that LACH2 can induce apoptosis of tumor cells through LA and only inhibit the proliferation of normal cells to achieve specific killing of tumor cells.

[0079] Example 6

[0080] The diffusion and retention balance of LACH1, LACH2, and LACH4 in the tumor was evaluated to expand the range of microwave ablation. The specific operation is as follows:

[0081] As a chemical ablative agent and microwave sensitizer, LACH needs to be retained in the tumor while being uniformly diffused throughout the tumor tissue. In order to evaluate the balance of LACH diffusion and retention in the tumor, LACH1, LACH2, and LACH4 (named ILACH1, ILACH2, and ILACH4, respectively) with ICG-COOH added, ethanol (a medical chemical ablative agent, named IEtOH), and IPBS (fluorescent dye ICG-COOH was added to all materials) were injected into the tumors of tumor-bearing mice, and the diffusion area and total fluorescence (Total radiant efficiency, TRE) of the drug in the tumor at 6h, 12h, and 24h after drug injection were detected using a small animal imaging system, as shown in Figure 2. Fig.13 After injection, the three ILACHs showed a larger diffusion area than IEtOH, indicating that their diffusion within the tumor was almost unaffected. Fig.14 However, the TREs of ILACH are much higher than those of IEtOH, as shown in Fig.15 This indicates that the three ILACHs are retained in the tumor, which is conducive to exerting a sustained anti-tumor effect. In addition, the retention of ILACH4 in the tumor is lower than that of ILACH1 and ILACH2, and the diffusion area of ​​ILACH2 in the tumor is much larger than that of ILACH1, so ILACH2 can achieve a better balance between diffusion and retention in the tumor.

[0082] Since LACH has the best diffusion and retention ability, its effect on the range of microwave ablation was further studied in two in vitro tumor models (mouse Hepa1-6 subcutaneous tumor tissue and human hepatocellular carcinoma tissue). Freshly excised Hepa1-6 tumors were treated with low-power MWA (3W, 5min) 24h after injection of LACH1, LACH2, and LACH4 (50μL). The tumor ablation area ( Fig.16 , refers to the area where T>50℃) highly overlaps with the fluorescence diffusion area ( Fig.17 The overlap between the MWA ablation area and the fluorescence diffusion area injected by ILACH2 [(2×overlap area / (ablation area+fluorescence diffusion area)] reached 84.4±13.45%, which was greater than 32.45±14.23% of ILACH1 and 71.13±6.36% of ILACH4. Fig.18 As shown, ILACH2 can achieve a better expansion of the microwave ablation area within the tumor.

[0083] Example 7

[0084] The synergistic antitumor efficacy of chemoablation of LACH1, LACH2, and LACH4 and MWA was evaluated as follows:

[0085] Two mouse subcutaneous liver cancer models (Hepa1-6) were used to study the synergistic antitumor efficacy of LACH chemical ablation and MWA. 3 After that, LACH (PBS and EtOH, as controls) were injected into the mice tumors, and MWA (3.0W) was performed for 5.0 min simultaneously. Fig.19 shown.

[0086] As a chemical ablative agent with specific killing ability for liver cancer cells, LACH1, LACH2 and LACH4 have stronger inhibitory effects on tumor growth than EtOH, such as Fig. 20 When LACH2 and MWA were used in combination, the volume of Hepa1-6 subcutaneous tumors increased from 323.53±78.46mm on day 0 to 40.5±10.2mm on day 1. 3 Reduced to 46.778±11.658mm at 4 days 3 , and only increased to about 232.468±56.56mm on 14 days 3 When LACH1 was used in combination with MWA, the volume of Hepa1-6 subcutaneous tumors increased to approximately 589.45±101.37mm on day 14. 3 When LACH4 was used in combination with MWA, the volume of Hepa1-6 subcutaneous tumor increased to about 498.46±189.46mm on the 14th day, which was smaller than the tumor volume of mice in the Control group, EtOH group, and MWA group. This indicates that LACH1, LACH2, and LACH4 synergistically inhibit tumor growth by expanding the MWA area and specifically killing tumor cells in combination with MWA, among which LACH2 has a better effect.

[0087] Example 8

[0088] Discussion on the immunosuppression-relieving properties of LACH2:

[0089] The immune microenvironment of subcutaneous Hepa1-6 tumors in mice after LACH2 treatment was investigated. Fig.21 As shown, the immune microenvironment iMWA induced a higher proportion of Tregs (CD25 + Foxp3 + / CD3 + CD4 + T cells) and significantly reduced CD8 +In addition, the content of MDSCs and PMN-MDSCs increased, and the ratio of M1 / M2 tumor-associated macrophages decreased, indicating that iMWA has a strong tumor immunosuppressive effect, such as Fig. 22 However, after LACH2 treatment of mice, these immunosuppressive cells (Tregs, MDSCs, and PMN-MDSCs) were significantly reduced, and the proportion of M1 / M2 tumor-associated macrophages was significantly increased, indicating that the iMWA-mediated immunosuppressive microenvironment was improved. In addition, after LACH2 treatment, the central memory T cells (Tcm, CD3 + CD8 + CD44 + CD62L + ) and effector memory T cells (Tem, CD3 + CD8 + CD44 + CD62L - ) were much higher than those in mice treated with iMWA, e.g. Fig.23 These results all indicate that LACH2 can induce a good anti-tumor immune response, greatly alleviate iMWA-mediated tumor immunosuppression, and help improve tumor recurrence and metastasis after iMWA.

[0090] Therefore, the present invention adopts the above-mentioned lipoic acid-choline ionic liquid solution and its preparation method and application. The lipoic acid-choline ionic liquid solution increases the ablation area of ​​microwave ablation, significantly alleviates the immunosuppression associated with incomplete microwave ablation, achieves an anti-tumor effect far better than microwave ablation alone, and effectively inhibits tumor recurrence and metastasis.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A thioctic acid-choline ionic liquid solution, wherein the thioctic acid-choline ionic liquid solution has a microwave thermal effect, and the microwave thermal effect is positively correlated with its choline / thioctic acid molar ratio, characterized in that: The concentration of the lipoic acid-choline ionic liquid solution is 2-25%, and the lipoic acid-choline ionic liquid solution consists of lipoic acid, choline and water / ethanol, wherein the molar ratio of lipoic acid to choline is (0.25-1):

1.

2. A lipoic acid-choline ionic liquid solution according to claim 1, characterized in that: The lipoic acid-choline ionic liquid solution LACH includes LACH4, LACH2, and LACH1; the molar ratio of lipoic acid to choline in LACH4 is 0.25:1, the molar ratio of lipoic acid to choline in LACH2 is 0.5:1, and the molar ratio of lipoic acid to choline in LACH1 is 1:

1.

3. A method for preparing a lipoic acid-choline ionic liquid solution as described in any one of claims 1-2, characterized in that: The following steps are involved: Step 1, mixing lipoic acid and choline, dissolving them in water / ethanol and stirring to obtain a stirred product; Step 2: placing the stirred product obtained in step 1 in a rotary evaporator for rotary evaporation, and then placing the solution obtained by rotary evaporation in a vacuum drying oven to obtain a yellow viscous or yellow elastic liquid; Step 3: dissolving the yellow viscous or yellow elastic liquid obtained in step 2 in water to obtain a lipoic acid-choline ionic liquid solution.

4. The method for preparing a lipoic acid-choline ionic liquid solution according to claim 3, characterized in that: In step 1, the volume of water / ethanol is 10-50 mL, and the stirring time is 10-12 h.

5. The method for preparing a lipoic acid-choline ionic liquid solution according to claim 3, characterized in that: In step 2, the rotary evaporation temperature is 50-100° C., the temperature in the vacuum drying oven is 50-100° C., and the reaction time in the vacuum drying oven is 36-48 hours.

6. An application of a lipoic acid-choline ionic liquid solution, characterized in that: The lipoic acid-choline ionic liquid solution described in claims 1-2 is used in the preparation of anti-tumor drugs.

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