Tissue ablation methods and materials

Abstract

The invention relates to methods and materials for tissue ablation. For example, methods are provided for tissue ablation using a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution). In some cases, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) may be used to ablate tumor tissue in a mammal suffering from cancer (e.g., to treat a mammal).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. patent application serial number 63 / 066,024, filed on August 14, 2020. The disclosure of the prior application is considered a part of (and incorporated by reference into) the disclosure of this application.

[0003] Statement Regarding Federal Funding

[0004] This invention was made with government support from the National Institutes of Health under Grant No. EP 024403. The government has certain rights in this invention. Technical Field

[0005] The present invention relates to methods and materials for tissue ablation. For example, compositions comprising one or more ionic liquids can be used to ablate one or more tissues in a mammal. In some cases, compositions comprising one or more ionic liquids can be used to ablate tumor tissue in a mammal suffering from cancer (e.g., to treat the mammal). Background Art

[0006] Cancer is a leading cause of morbidity and mortality, with an estimated 10 million deaths worldwide each year (World Cancer Report 2014, (World Health Organization, 2014)), and costs in the United States alone exceeding $200 billion annually (Mattiuzzi et al., J Epidemiol Glob Health 9:217-222 (2019)). Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Once diagnosed, the 5-year survival rates for regional and metastatic liver cancer are 9% and 3%, respectively. Summary of the Invention

[0007] Systemic chemotherapy is the cornerstone of cancer treatment. However, the development of new therapies for liver cancer is limited by its inability to achieve uniform delivery within the tumor, side effects on the non-cancerous liver, and systemic side effects. Despite significant efforts worldwide, systemic and local therapies (LRT), such as percutaneous microwave ablation or endovascular embolization, have not resulted in changes in overall survival for these patients.

[0008] Provided herein are methods and materials for tissue ablation. For example, provided herein are ionic liquids (e.g., compositions comprising one or more ionic liquids) and methods for tissue ablation using such ionic liquids. In some cases, compositions comprising one or more ionic liquids (e.g., compositions comprising a local active agent (LATTE) solution for tumor therapy and eradication) can be used to ablate tumor tissue in a mammal (e.g., to treat cancer). As described herein, compositions comprising LATTE solutions can be spread evenly in a circular manner while destroying tumor tissue. When compositions comprising LATTE solutions are administered together with chemotherapeutic agents such as doxorubicin, the chemotherapeutic agents can stay in the ablation zone for up to 28 days. Also as described herein, compositions comprising LATTE solutions can be used to ablate adipose tissue, cardiac tissue, blood clots, and deplete nucleated cells in the blood. Therefore, compositions comprising one or more ionic liquids (e.g., compositions comprising LATTE solutions) can be used as therapeutic agents (e.g., as anticancer agents) to ablate tissue (e.g., tumor tissue) in a mammal (e.g., a human).

[0009] In general, one aspect of the present application features a method for ablating at least a portion of a tissue in a mammal. The method may include or consist essentially of percutaneously injecting a composition comprising an ionic liquid into the tissue in the mammal, wherein the ionic liquid comprises: (a) a cationic component comprising a cation selected from the group consisting of choline, benzylpyridinium, benzyldimethyldodecylammonium, phosphonium, tetraalkylphosphonium, phenethylonium, imidazolium, pyridinium, piperidinium, quinolinium, morpholinium, quaternary phosphonium, and quaternary ammonium; and (b) an anionic component comprising a cation selected from the group consisting of geranate, bistrifluorocarbonimide, oleate, and bis(trifluoromethanesulfonate). , hexanoate, dodecyldimethylaminopropane sulfonate, N-lauryl sarcosinate, geraniolate, tetrafluoroborate, hexafluorophosphate, methyl sulfate, octyl sulfate, acesulfame potassium, halide, bis(trifluoromethylsulfonyl)amide, bis(trifluoromethyl)amide, dicyanamide, and anion of trifluoromethanesulfonate; wherein the composition can effectively produce an ablation zone in the tissue, and wherein the composition can effectively reduce the number of cells in the ablation zone. The mammal can be a human. The tissue can be adipose tissue, cardiac tissue, connective tissue, bone tissue, synovial tissue, abscess tissue or cyst. The percutaneous injection step can include guided injection. The composition can also include a contrast agent. The contrast agent can be indocyanine green, a radiopaque contrast agent, iohexol tantalum nanoparticles, tantalum microparticles, gold nanoparticles, gadolinium, indium 111 or microbubbles. The ablation zone can be about 0.1 cm to about 4 cm. The composition can be in the form of a hydrogel. The hydrogel can include a nanosilicate. The hydrogel can include about 1% (w / v) to about 10% (w / v) of the nanosilicate. The nanosilicate can include montmorillonite clay.

[0010] In another aspect, the invention features a method of treating a mammal suffering from cancer. The method can include or consist essentially of percutaneously injecting a composition comprising an ionic liquid into a tissue in the mammal, wherein the ionic liquid comprises: (a) a cationic component comprising a cation selected from the group consisting of choline, benzylpyridinium, benzyldimethyldodecylammonium, phosphonium, tetraalkylphosphonium, phenethylonium, imidazolium, pyridinium, piperidinium, quinolinium, morpholinium, quaternary phosphonium, and quaternary ammonium; and (b) an anionic component comprising a cation selected from the group consisting of geranate, bistrifluorocarbonimide, oleate, hexane, benzophenone ... The invention relates to a method for preparing an anion of a trifluoromethanesulfonate, a tetrafluoroborate, a hexafluorophosphate, a methylsulfate, an octylsulfate, an acesulfame potassium, a halide, a bis(trifluoromethylsulfonyl)amide, a bis(trifluoromethyl)amide, a dicyanamide, and a trifluoromethanesulfonate; wherein the composition can effectively produce an ablation zone in the tumor tissue, and wherein the composition can effectively reduce the number of cells in the ablation zone. The mammal can be a human. The cancer can be liver cancer, biliary tract cancer, pancreatic cancer, colorectal cancer, kidney cancer, ovarian cancer, breast cancer, prostate cancer, colon cancer, bladder cancer, lung cancer, thyroid cancer, melanoma, brain cancer, stomach cancer, cervical cancer, uterine cancer, skin cancer, synovial cancer, appendix cancer, or adrenal gland. When the cancer is liver cancer, the liver cancer can be hepatocellular carcinoma (HCC). When the cancer is biliary tract cancer, the biliary tract cancer can be cholangiocarcinoma. When the cancer is colorectal cancer, the colorectal cancer can be colorectal cancer liver metastasis (CRCLM). The composition can also include a chemotherapeutic agent and / or a radioactive agent. The chemotherapeutic agent can be doxorubicin, cisplatin, paclitaxel, olaparib, everolimus, mitomycin, atezolizumab, bevacizumab, cabozantinib-s-malate, ramucirumab, pembrolizumab, lenvatinib mesylate, sorafenib tosylate, nivolumab, pemmetinib, pembrolizumab, ramucirumab, regorafenib or pomacillin. The radioactive agent can be Y90. The method can effectively deliver the chemotherapeutic agent and / or radioactive agent to the ablation zone. The method can effectively maintain the chemotherapeutic agent and / or radioactive agent in the ablation zone for about 1 day to about 30 days. This method can effectively reduce the volume of the cancer by at least 2 times. The method can include determining that the mammal suffers from cancer. The composition can be in the form of a hydrogel. The hydrogel can include nanosilicates. The hydrogel may comprise from about 1% (w / v) to about 10% (w / v) nanosilicate.The nanosilicate may comprise montmorillonite clay.

[0011] In another aspect, the invention features a method for treating a mammal suffering from a disease or condition associated with fat accumulation. The method can include or consist essentially of percutaneously injecting a composition comprising an ionic liquid into adipose tissue in a mammal, wherein the ionic liquid comprises: (a) a cationic component comprising a cation selected from the group consisting of choline, benzylpyridinium, benzyldimethyldodecylammonium, phosphonium, tetraalkylphosphonium, phenethylonium, imidazolium, pyridinium, piperidinium, quinolinium, morpholinium, quaternary phosphonium, and quaternary ammonium; and (b) an anionic component comprising a cation selected from the group consisting of geranate, bistrifluorocarbonimide, oleate, hexane, benzophenone ... The invention further comprises a composition comprising an anion of a trifluoromethanesulfonate, a tetrafluoroborate, a hexafluorophosphate, a methylsulfate, an octylsulfate, an acesulfame potassium, a halide, a bis(trifluoromethylsulfonyl)amide, a bis(trifluoromethyl)amide, a dicyanamide, and a trifluoromethanesulfonate; wherein the composition is effective in producing an ablation zone within the adipose tissue, and wherein the composition is effective in reducing the number of adipocytes within the ablation zone. The mammal may be a human. The disease or condition associated with fat accumulation may be obesity, lipedema, a fat storage disorder, or a cancer with a fat-depositing tumor. The method may include determining that the mammal suffers from a disease or condition associated with fat accumulation. The composition may be in the form of a hydrogel. The hydrogel may include a nanosilicate. The hydrogel may include about 1% (w / v) to about 10% (w / v) of the nanosilicate. The nanosilicate may include montmorillonite clay.

[0012] In another aspect, the invention features a method of treating a mammal suffering from a cardiac disease or condition. The method can include or consist essentially of percutaneously injecting a composition comprising an ionic liquid into atrophic myocardium in the mammal, wherein the ionic liquid comprises: (a) a cationic component comprising a cation selected from the group consisting of choline, benzylpyridinium, benzyldimethyldodecylammonium, phosphonium, tetraalkylphosphonium, phenethylonium, imidazolium, pyridinium, piperidinium, quinolinium, morpholinium, quaternary phosphonium, and quaternary ammonium; and (b) an anionic component comprising a cation selected from the group consisting of geranate, bistrifluorocarbonimide, oleate, hexanoate, benzoic acid ... Salt, dodecyldimethylaminopropane sulfonate, N-lauryl sarcosinate, geraniolate, tetrafluoroborate, hexafluorophosphate, methyl sulfate, octyl sulfate, acesulfame potassium, halides, bis(trifluoromethylsulfonyl)amide, bis(trifluoromethyl)amide, dicyanamide, and anions of trifluoromethanesulfonate; wherein the composition can effectively produce an ablation zone in the atrophic myocardium, and wherein the composition can effectively reduce the number of atrophic myocardial cells in the ablation zone. The mammal can be a human. The cardiac disease or condition can be hypertrophic cardiomyopathy, arrhythmia, or atrial fibrillation lesions. The method can include determining that the mammal has a cardiac disease or condition. The composition can be in the form of a hydrogel. The hydrogel can include nanosilicates. The hydrogel can include about 1% (w / v) to about 10% (w / v) nanosilicates. The nanosilicates can include montmorillonite clay.

[0013] In another aspect, the invention features a method for treating a mammal having a disease or condition associated with blood clotting. The method may comprise or consist essentially of percutaneously injecting into a mammal a composition comprising an ionic liquid into the blood clot, wherein the ionic liquid comprises: (a) a cationic component comprising a cation selected from the group consisting of choline, benzylpyridinium, benzyldimethyldodecylammonium, phosphonium, tetraalkylphosphonium, phenethylonium, imidazolium, pyridinium, piperidinium, quinolinium, morpholinium, quaternary phosphonium, and quaternary ammonium; and (b) an anionic component comprising an anion selected from the group consisting of geranate, bistrifluoroformimide, oleate, hexanoate, dodecyldimethylaminopropanesulfonate, N-laurylsarcosinate, geraniolate, tetrafluoroborate, hexafluorophosphate, methylsulfate, octylsulfate, acesulfame K, a halide, bis(trifluoromethylsulfonyl)amide, bis(trifluoromethyl)amide, dicyanamide, and trifluoromethanesulfonate; wherein the composition is effective to reduce the size of the blood clot. The mammal can be a human. The disease or condition associated with blood clots can be acute deep vein thrombosis, chronic deep vein thrombosis, antiphospholipid syndrome, arteriosclerosis, atherosclerosis, or pulmonary embolism. The method can include determining that the mammal suffers from a disease or condition associated with blood clots. The composition can be in the form of a hydrogel. The hydrogel can include a nanosilicate. The hydrogel can include about 1% (w / v) to about 10% (w / v) of the nanosilicate. The nanosilicate can include montmorillonite clay.

[0014] In another aspect, the invention features a method for treating a mammal having an infected tissue. The method can include or consist essentially of percutaneously injecting a composition comprising an ionic liquid into the infected tissue in the mammal, wherein the ionic liquid comprises: (a) a cationic component comprising a cation selected from the group consisting of choline, benzylpyridinium, benzyldimethyldodecylammonium, phosphonium, tetraalkylphosphonium, phenethylonium, imidazolium, pyridinium, piperidinium, quinolinium, morpholinium, quaternary phosphonium, and quaternary ammonium; and (b) an anionic component comprising a cation selected from the group consisting of geranate, bistrifluorocarbonimide, oleate, hexane, benzophenone ... The invention relates to a method for treating a mammal comprising: administering to the mammal a composition comprising: a tetrafluoroborate, a hexafluorophosphate, a methylsulfate, an octylsulfate, an anion of a trifluoromethanesulfonate; a halide, a bis(trifluoromethylsulfonyl)amide, a bis(trifluoromethyl)amide, a dicyanamide, and a trifluoromethanesulfonate; wherein the composition is effective in producing an ablation zone within the infected tissue, and wherein the composition is effective in reducing the number of infected cells within the ablation zone. The mammal may be a human. The infected tissue may be at a wound site. The wound may be a diabetic wound or a surgical wound. The method may include determining that the mammal has infected tissue. The composition may be in the form of a hydrogel. The hydrogel may include a nanosilicate. The hydrogel may include about 1% (w / v) to about 10% (w / v) of the nanosilicate. The nanosilicate may include montmorillonite clay.

[0015] In another aspect, the invention features a method for delivering a therapeutic agent to a tissue in a mammal. The method can include or consist essentially of: percutaneously injecting a composition comprising (a) an ionic liquid and (b) a therapeutic agent into a tissue in the mammal at a depth of about 0.1 picometer (pm) to about 12 pm; wherein the ionic liquid comprises: (a) a cationic component comprising a cation selected from the group consisting of choline, benzylpyridinium, benzyldimethyldodecylammonium, phosphonium, tetraalkylphosphonium, phenethylonium, imidazolium, pyridinium, piperidinium, quinolinium, morpholinium, quaternary phosphonium, and quaternary ammonium; and (b) an anionic component comprising a cation selected from the group consisting of geranium, chrysogenum ... The invention relates to an ionic liquid comprising an anion of a tetrafluoroborate, a bistrifluoroformimide, an oleate, a hexanoate, a dodecyldimethylaminopropanesulfonate, a N-dodecylsarcosinate, a geraniolate, a tetrafluoroborate, a hexafluorophosphate, a methylsulfate, an octylsulfate, an acesulfame potassium, a halide, a bis(trifluoromethylsulfonyl)amide, a bis(trifluoromethyl)amide, a dicyanamide, and a trifluoromethanesulfonate; wherein the ionic liquid can effectively produce an ablation zone in the tissue, and wherein the method can effectively maintain the therapeutic agent in the ablation zone. The mammal can be a human. The tissue can be adipose tissue, cardiac tissue, connective tissue, bone tissue, synovial tissue, abscess tissue, or a cyst. The tissue can be tumor tissue. The therapeutic agent can be a chemotherapeutic agent, a radioactive agent, an antibody, an angiogenic factor, a therapeutic polypeptide, a nucleic acid encoding a therapeutic polypeptide, or an immunomodulator. The ablation zone can be about 0.1 cm to about 4 cm. The method can effectively maintain the therapeutic agent in the ablation zone for about 1 day to about 30 days. The percutaneous injection step can include guided injection. The composition may also include a contrast agent. The contrast agent may be indocyanine green, a radiopaque contrast agent, iohexol tantalum nanoparticles, tantalum microparticles, gold nanoparticles, gadolinium, indium 111 or microbubbles. The composition may be in the form of a hydrogel. The hydrogel may include a nanosilicate. The hydrogel may include from about 1% (w / v) to about 10% (w / v) of the nanosilicate. The nanosilicate may include montmorillonite clay.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar to or equivalent to those described herein can be employed in the practice of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of any conflict, the present specification, including definitions, will prevail. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0017] The accompanying drawings and the following description further illustrate one or more embodiments of the present invention in detail. Other features, objectives and advantages of the present invention will be apparent from the description, drawings and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A The penetration and ablation effects of LATTE after intrahepatic injection into the rat liver were evaluated using microcomputed tomography and ultrasound imaging. Figures 1A-1B Photographs of rat livers during and 24 hours after injection, showing the LATTE-treated areas. Figures 1C-1D Representative micro-CT images and illustration of 3D volume analysis showing that 25% LATTE concentration induced higher diffusion volume (*p=0.03, **p=0.004, n=5). Figure 1E 、 1F and 1 G. Micrographs of H&E-stained rat liver sections obtained 24 hours after intraparenchymal injection of 50%, 25%, or 6.25% LATTE, respectively, show necrotic areas (dashed lines, scale bar 200 μm). Figure 1H Morphometric analysis of the necrotic areas induced by 50%, 25% or 6.25% LATTE showed that the necrotic areas were larger at the 50% and 25% LATTE injection sites compared with the 6.25% LATTE injection sites (*p=0.048, n=5). Figure 1I 、 1J and 1K. Show photos of liver tumors in N1S1 rats immediately and 2 weeks after intratumoral injection of 25% LATTE, respectively. Gross examination showed tumor regression. Figure 1L and 1M Corresponding ultrasound scans of liver lesions in N1S1 rats obtained two weeks after direct intratumoral injection of saline or LATTE (yellow dashed line, scale bar, 0.5 cm). Figure 1N and 1O Corresponding graphs of N1S1 tumor volume and relative changes in lesion volume measured by ultrasound in saline or LATTE injected rats (**p=0.002). Figure 1P and 1Q 3D-rendered images of in vivo micro-CT scans of normal rat liver (black arrows) segmented from hypodense N1S1 tumor lesions two weeks after direct injection of saline or LATTE, respectively. Figure 1R Quantitative micro-CT analysis of N1S1 tumor volume showed a significant reduction in lesions two weeks after LATTE injection compared with saline (**p = 0.002). Figure 1S Linear regression plots of the corresponding tumor volumes determined by ultrasound (US) and micro-CT showed a linear relationship and a significant correlation between the two imaging modalities (Pearson correlation coefficient (r) = 0.96, p < 0.0001). Figure 1T and 1U NIRF scanning was performed on the livers of explanted N1S1 tumor-bearing rats two weeks after direct intratumoral injection of 25% LATTE or saline. Figure 1V Graph showing that tissue ICG diffusion was higher in LATTE-injected tumors than in saline (**p=0.002). Figure 1W Summary of fluorescence intensity in transverse liver sections showed significantly higher fluorescence intensity in LATTE-treated lesions (**p = 0.002). Data are presented as mean ± SEM, n = 6 per group. Statistical significance among the three groups was assessed using one-way analysis of variance and Tukey's post hoc test, while comparisons between two groups were calculated using the Mann-Whitney test.

[0019] Figures 2A-2D . LATTE viscosity and injection force test. Figure 2A Rheological study graphs evaluating the viscosity of 6.25, 25, 35, 50% or 100% LATTE concentrations following dilution in 0.9 w / v saline, showing concentration-dependent viscosity changes compared to pure LATTE (100%). Figure 2B A plot comparing the adhesion of 6.25%, 25% and 50% LATTE concentrations at a shear rate of 50 1 / s shows that the viscosity of 50% LATTE is significantly higher compared to 6.25% or 25% (n=3). Figure 2C Summary graph of the evaluation of the viscosity change over time for different LATTE concentrations measured 0, 10, or 20 days after dilution, showing the stability of each solution. Figure 2D Injection force testing of 25%, 35%, 50%, and 100% LATTE on a 1 mL syringe injected through a 21-gauge percutaneous needle demonstrated concentration-dependent injection force, with 25% LATTE generating similar force compared to 35% LATTE. Data represent mean ± SEM, with *p < 0.05, ***p < 0.0001, calculated using analysis of variance and Tukey's post hoc test (n = 3).

[0020] Figures 3A-3I To evaluate the ablation effect and drug retention of LATTE on N1S1 tumors. Figure 3A and 3B Representative photomicrographs of H&E-stained N1S1 tissue sections obtained two weeks after saline or LATTE injection, respectively, show decreased cellularity and architectural degradation in LATTE-treated tumors compared with saline. Figure 3C A graph summarizing the morphometric analysis of cross-sectional tumor lesions two weeks after treatment shows that LATTE-treated tumor lesions were significantly smaller compared with saline. Figure 3D and 3ERepresentative photomicrographs of PCNA-stained rat liver sections obtained from saline- or LATTE-treated N1S1 tumors, respectively. Figure 3F Summary of nuclei counts within each N1S1 tumor border for saline- or LATTE-injected tumors. Figure 3G and 3H Corresponding images of immunostained tissue sections show cleaved caspase-3 in saline- or LATTE-treated N1S1 tumors. Figure 3I Quantitative analysis of the mean proliferating (PCNA expression) or apoptotic (caspase-3 positivity) cell counts within the borders of N1S1 tumors. A graphical summary shows a significant decrease in proliferation and a parallel increase in apoptotic cells detected in LATTE-treated tumors compared to saline. Statistical significance was calculated using the Mann-Whitney test (** and ++ p < 0.01). Data are presented as mean ± SEM, n = 6 per group. Scale bar = 100 μm.

[0021] Figures 4A-4I Effects of intratumoral LATTE injection on T-lymphocyte and macrophage recruitment. Figure 4A and 4B Representative immunostaining identifying CD3 T-lymphocytes in N1S1 tumor tissue sections obtained two weeks after injection of saline or LATTE, respectively. Figure 4C Quantitative analysis of CD3-expressing cell counts in tumor tissue sections showed a higher infiltration of CD3+ cells within the borders of LATTE-injected tumors (**p-0.002, using unpaired Mann-Whitney test). Figure 4D and 4E Representative micrographs of CD68-expressing cells in saline- or LATTE-treated N1S1 tumors. Figure 4F Quantitative analysis of CD68-bearing cell counts in tumor tissue sections revealed higher infiltration in LATTE-injected tumors. Figure 4G and 4I Representative fluorescence micrographs of CD8-expressing lymphocytes in LATTE-treated tumors are shown. Data are presented as mean ± SEM, n = 6 per group. Scale bar, 100 μm.

[0022] Figures 5A-5D LATTE cytotoxicity and synergistic interactions with chemotherapy in human cancer cells. Figure 5A The summary of the cell viability dose-response fractions for LATTE treatment in human hepatocellular carcinoma (Hep-G2), cholangiocarcinoma (SNU-478), or pancreatic ductal adenocarcinoma (Panc-1) cells after 24 h of incubation using water-soluble tetrazolium-1 (WST-1) output curves yielded an effective dose (EC50) of 0.19%, 0.2%, or 0.3%, respectively. Figure 5B Figure 4. Dose-response curve of doxorubicin on the viability of HepG2 cells incubated with serially diluted doxorubicin concentrations ranging from 80 to 0.156 μM in growth medium, resulting in an EC50 of 1.63 μM 24 hours after treatment. Figures 5C-5D The synergistic matrix based on the Loewe model was generated at 24 and 48 h after incubation using selected concentrations close to the EC50 values ​​of each compound for all possible combinations of LATTE and doxorubicin mixtures (0.05%-0.78% for LATTE and 1.25-10 μM for doxorubicin) and revealed that the synergistic matrix was significantly different with 0.39% LATTE and 5 μM doxorubicin at 24 h ( Figure 5C ), 0.19% LATTE and 1.25 μM doxorubicin at 48 h ( Figure 5D ) Synergistic cytotoxic effects were observed on HepG2 cells. The results are representative of replicates of three independent experiments with n=12.

[0023] Figures 6A-6O Time-dependent evaluation of chemotherapeutic drug diffusion and retention after LATTE injection in rat liver. Sprague-Dawley rats were injected with a 50 μL volume of 25% LATTE and ICG into the lateral lobe, while the medial lobe was injected with 50 μg of doxorubicin and 50 μL of 25% LATTE and ICG. Survival time was 1, 7, or 28 days after injection (n=10). Figure 6A 、 6B and 6C. After necropsy, near-infrared fluorescence (NIRF) scanning showed ICG retention at both injection sites. Figure 6D 、 6E and 6F. Fluorescence scans show doxorubicin located in the middle of the same liver. Figure 6G Summary images of ICG diffusion / doxorubicin fluorescence intensity at each injection site show that diffusion peaked at 7 days (*p=0.015, **p=0.001). Figure 6H Quantitative analysis showed that the fluorescence intensity of doxorubicin was consistent at 1, 7, and 28 days after injection. Figures 6I-6O Representative photomicrographs of H&E-stained tissue sections obtained from LATTE-treated sites at 1, 7, and 28 days after injection showed maximal necrosis in the center of the treated area and less necrosis at the peripheral border at 1 and 7 days after injection, with evidence of tissue remodeling associated with fibrogenesis at 28 days after injection. Figure 6L 、 6M and 6N. 28 days after injection, corresponding Picrosirius red-stained tissue sections showed enhanced collagen staining in the LATTE-treated area (black arrows).

[0024] Figures 7A-7W LATTE image-guided injection of VX2 into rabbit or normal pig liver tumors. Figure 7A Photographs depict percutaneous ultrasound-guided injection into a VX2 tumor in the rabbit liver. Figure 7B Representative ultrasound images after injection of 25% LATTE mixture into VX2 tumors show the needle tracks (white arrows). Figure 7C Gross image of a VX2 tumor immediately after injection of a 25% LATTE and ICG mixture shows LATTE solution visible subcapsularly within the tumor lesion. Figure 7D and 7E Representative color Doppler ultrasound scans of rabbit livers before and after LATTE injection show flow in the hypoechoic VX2 tumor area. Figure 7F and 7G Separate laser speckle perfusion scans showed decreased perfusion after LATTE injection into VX2 tumors. Figure 7H Summary of laser speckle image analysis of VX2 tumor-bearing livers, showing a significant decrease in perfusion after LATTE injection. Figure 7I Maximum intensity projection micro-CT of an explanted rabbit liver 1 hour after LATTE injection demonstrated efficient distribution and retention of Exitron throughout the VX2 lesion. Figure 7J Volumetric micro-CT analysis of LATTE-injected VX2 tumors showed that an average of 150% of Exitron in four VX2 tumors diffused beyond the 1 mL LATTE injection volume. Figure 7K The images of cross-sectioned VX2 tumors are shown in gray (white dashed outline), NIRF images show ICG diffusion beyond the tumor area, and fluorescence images ( Figure 7I ) showed that doxorubicin was retained throughout the tumor. Figure 7M Image summary of ICG and doxorubicin diffusion showed a larger diffusion area compared to the tumor area. Figures 7N-7O H&E-stained sections obtained from untreated or treated VX2 tumors, respectively (scale bar 200 μm). Figure 7P Photographs and ultrasound images show percutaneous image-guided LATTE injection into a pig liver, with the US image showing advancement of the access needle within the liver parenchyma, the needle track (white arrow), and accumulated LATTE (yellow dashed outline) visible in the post-injection US image. Figure 7R NIRF scan of the transplanted pig liver lobe showed a strong ICG signal in the affected area. Figures 7S-7T NIRF and microscopy images of pig liver tissue sections obtained from the injection site showed strong signals corresponding to necrotic areas in the stained sections. Figure 7U Representative T1-weighted axial MR images show attenuation in the center of the affected area and enhancement due to LATTE spreading toward the periphery. Figure 7V MRI volume analysis showed that the diffusion volume increased 2.8-fold within 90 minutes after LATTE injection (p=0.038, n=4, using paired t-test). Figure 7W Subsequent NIRF scans of the injected pig livers showed ICG spreading throughout the affected areas.

[0025] Figures 8A-8G LATTE was injected ex vivo into explanted human cancer tissue. Figures 8A-8E . The images of resected solid tumors include; ( Figure 8A ) Colorectal cancer liver metastasis (CRCLM), ( Figure 8B ) hepatocellular carcinoma (HCC), ( Figure 8C )Choledochocarcinoma (CAA), ( Figure 8D )Breast cancer (BC) and ( Figure 8E ) Renal cancer. Each panel (from left to right) includes a photograph of tumor tissue, a NIRF scan obtained 10 minutes after LATTE injection, a NIRF scan obtained 24 hours after LATTE injection, and H&E-stained sections of ablated and nonablated tumors. Scale bar 200 μm. Figure 8F Summary images of fluorescence intensity assessed 10 minutes and 24 hours after LATTE injection showed a significant increase compared to baseline (BL). Figure 8G Figure 3 ICG diffusion area plots assessed 10 minutes and 24 hours after LATTE injection. Data represent mean ± SEM calculated using repeated-measures ANOVA, **p < 0.001, ***p < 0.0001 (n = 12).

[0026] Figure 9 Ablation of adipose tissue from the skin. Control skin: Arrows indicate subcutaneous fat. Treated skin: Pigs treated with LATTE liquid ablative agent showed a significant reduction in subcutaneous fat, as shown in H&E-stained (upper panel) and trichrome-stained (lower panel) histological sections.

[0027] Figure 10 A-10B. Ablation of fat from adipocytes. Figure 10 A. Fat from pigs. Figure 10 B. Fat removed from a pig is placed in a LATTE solution. After one hour, the fat in the adipocytes is depleted.

[0028] Figure 11 Imaging during ablation of adipose tissue. Porcine fat was treated with LATTE liquid ablative agent mixed with indocyanine green (ICG). 1cc of the mixture was injected. One hour after the LATTE liquid ablative agent spread, the distribution of ICG-enhanced fluorescence was visualized using a near-infrared fluorescence imager.

[0029] Figure 12A and 12B . The effects of ablative agents on human blood. Figure 12A A normal blood smear showing blood cells in a microscopic field. Figure 12BNo blood cells were detected in the blood samples treated with the LATTE ablative agent.

[0030] Figure 13 A-13C. Effects of ablative agents on human blood clots. Figure 13 A. Image of coagulated blood in a multiwell plate covered with saline. Figure 13 B. Image of coagulated blood covered with LATTE in multiple wells showing clot lysis. Figure 13 C. Images of blood clots formed in test tubes in a petri dish, showing complete dissolution after incubation with LATTE. These images demonstrate the ability of LATTE to dissolve blood clots; these data suggest that LATTE could be used to lyse or dissolve clots, potentially enabling the treatment of arterial and venous clots in conditions including stroke, myocardial infarction, deep vein thrombosis, and ischemic leg or organ disease.

[0031] Figures 14A-14B Effects of ablative agents on porcine cardiac tissue. Figure 14A In a live Yorkshire pig weighing 50-55 kg, a syringe attached to a 21-gauge, 7-cm needle was guided through the skin into the myocardium using handheld ultrasound guidance. At necropsy, 14A shows a near-infrared image of the pig myocardium after injection of LATTE mixed with indocyanine green, showing a strong signal. Figure 14B Myocardial staining histological images showed extensive myocardial ablation in the treatment area after direct percutaneous image-guided injection of 25% LATTE.

[0032] Figures 15A-15G Characterization of the mechanical properties of hydrogels containing LATTE (nanogels). Figure 15A Representative flow curves for hydrogels containing 3 wt%, 4.5 wt%, 6 wt% or 9 wt% nanosilicate (NS) showing shear thinning behavior. Figure 15B Graph showing the effect of increasing NS ratio on the storage modulus (G') produced by hydrogels containing 3 wt%, 4.5 wt%, 6 wt% or 9 wt% NS. Figure 15C The summary graph of the rheological results shows a concentration-dependent increase in G' in nanogels containing 1.25 wt% or 25 wt% IL compared to NS alone (e.g. Figure 15B ), while 50 wt% IL resulted in a much higher G′. Figure 15D and 15E As shown, representative flow curves and graphs of NS hydrogel, nanogel (NG), NG containing 0.25% mg / mL ICG (NG+ICG), 1.25 mg / mL doxorubicin (Dox) (NG+Dox), 1 mg / mL Nivo (Nivolumab) (NG+Nivo), or NG+Dox+Nivo. Figure 15FGraph showing the injection force generated by different nanogel formulations loaded into a 1 cc syringe and injected at 1 mL / min- 1 The injection rate was through a 110 cm2.8F microcatheter. Figure 15G Table showing examples of different NG formulations incorporating Dox, nivolumab (Nivo; PD-1 IgG), and iohexol contrast agents, added as single components or in combination, for different testing purposes. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison post hoc test (n = 6 for each test). ns; not significant, *p < 0.05, ***p < 0.0001.

[0033] Figures 16A-16F . The microstructural appearance of the nanogels was evaluated. Figure 16A An image showing an example of a nanogel containing 3 wt% NS, 25 wt% IL, 1 mg / mL Nivo, and 0.25 mg / mL ICG, loaded into a clinical-grade syringe equipped with a 21-gauge vascular puncture needle for direct intratumoral or intravascular injection. Figure 16B Images showing injection of the nanogel through the needle clearly demonstrate shear-thinning behavior by remaining coherent as it leaves the needle tip. Figures 16C-16F To demonstrate the effects of IL or Nivo on hydrogel appearance at the microscopic level, scanning electron microscopy images of nanosilicate hydrogels (NS), NS mixed with Nivo (NS+Nivo), nanogels alone (NG), or nanogels mixed with 1 mg / mL Nivo (NG+Nivo) revealed porous microstructures in the NS and NS+Nivo hydrogels, respectively. Compared with nanogels containing ionic liquids or NG+Nivo, the microtubular structures in the latter were less porous and sieve-like, suggesting that IL altered the interactions of the nanocomposites in the NS hydrogels, which may explain the changes in their mechanical properties. Scale bar, 200 μm.

[0034] Figures 17A-17J Effects of IL on coagulation and cell death. Figure 17A Stained blood smears prepared from porcine blood treated with IL at concentrations of 0% (control), 0.78 wt%, 1.56 wt%, and 3.12 wt% exhibited concentration-dependent changes in morphology, with decreased leukocyte counts beginning at 1.56% IL and evidence of complete hemolysis observed at 3.12 wt% IL. Scale bar, 100 μm. Figure 17B. Representative rheometer flow curves used to evaluate the effect of IL treatment on thrombus formation and modulus in pig blood aliquots treated with IL concentrations of 0% (control), 0.78%, 1.56%, and 3.12% wt%. Control and 0.78 wt% IL-treated pig blood showed consistent G' and G" modulus curves, while 1.56% IL-treated blood showed delayed clotting lag time and lower levels of G' and G" (modulus) compared to control blood (n=5). 3.12 wt% IL-treated blood failed to clot during the 30 minute test period (n=5). Figure 17C Quantitative analysis of the lag time of coagulation onset (t-lag) showed that the lag time increased slightly under 0.78% IL treatment and was significantly prolonged after 1.56 wt% IL treatment. Figure 17D The summary plot of the storage modulus (G') 30 min after treatment with 0 wt%, 0.78 wt%, 1.56 wt%, or 3.12 wt% IL showed that the storage modulus decreased by approximately 20% at 0.78 wt% IL, compared with a 95% decrease in the storage modulus at 1.56 wt% IL and a 100% decrease in the modulus at 3.12 wt% IL, indicating that IL has a concentration-dependent anticoagulant effect. Figure 17E Hemolysis assays performed in pig blood treated with 0 wt%, 0.78%, 1.56%, 3.12%, 6.25%, 12.5 wt%, and 25 wt% IL showed a significant increase in hemolysis (***p<.0001). Figures 17F-17J . Shows a complete blood count of fresh blood aliquots treated with increasing concentrations of 0.78 wt%, 1.56 wt% and 3.12 wt% IL, showing red blood cells (RBC, Figure 17F ) and white blood cells (WBC, Figure 17G ) counts decreased in a concentration-dependent manner, which was consistent with the Figure 17J ), and monocyte count ( Figure 17I ). However, lymphocyte counts in the same aliquots did not change compared to the control group ( Figure 17H ), indicating resistance to IL treatment at the tested concentrations (n=6). Blood count data are expressed as a percentage of the control value (0 wt% IL) from the same blood pool. Data are mean ± SEM. Statistical analysis was performed using one-way ANOVA with Tukey's post hoc test (*p<.05, **p<.01, ***p<.001, and ****p<.0001).

[0035] Figures 18A-18F To evaluate the effects of nanogels on drug diffusion and stability in vitro. Figure 18AFluorescence images showing radial diffusion of the naturally fluorescent anticancer drug Dox. Dox was added to NS hydrogels (control hydrogels without ionic liquid) or to nanogels containing 6.25%-IL or 25%-IL for 24 hours after equal aliquots were loaded into designated wells of 2% agarose cast in multiwell plates to assess radial diffusion at 37°C for 24 hours. Figure 18B Graphs showing continuous measurement of Dox fluorescence in NS+Dox, NG+Dox 6.25%-IL, or NG+Dox with 25%-IL revealed that the diffusion area in NG+Dox25%-IL was significantly enhanced compared with NG+Dox6.25%-IL and NS+Dox (n=4). Figure 18C and 18D . Showing the fluorescence of Dox incorporated into nanogels or NS hydrogels ( Figure 18C ) or ICG( Figure 18D ) of the continuous detection and measurement of fluorescence images and graphs, respectively, showing that Dox( Figure 18C ) or ICG( Figure 18D ) continued to increase over 56 days (n = 4). **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. Statistical differences were calculated using one-way ANOVA followed by Turkey's post hoc test. Data in all graphs are mean ± SEM.

[0036] Figures 19A-19D Effects of nanogels on HepG2 cell viability, Dox diffusion and release. Figure 19A Figure 4 shows the cumulative Dox release profiles from nanogels (NG) mixed with 0.25 mg / mL Dox and then incubated for 7 days under physiological (pH = 7.4) or acidic (pH = 5.0) conditions, demonstrating sustained release of doxorubicin (n = 3). Figure 19B The viability fraction of HepG2 cells 24 h after treatment with serially diluted NG extracts resulted in an IC of 0.14% at an IL concentration of 50 , indicating that the cytotoxic effect of NG was retained. Figure 19C HepG2 cells incubated with NG extract containing 0.25 mg / mL Dox (NG+Dox) showed enhanced cytotoxicity compared to nanosilicate extract containing a similar amount of Dox (NS+Dox), indicating a synergistic effect. NS alone did not show cytotoxic effects. Figure 19DGraphs showing the sterility of NG or NG+Dox after incubation in LB broth at 37°C for 24 hours or 2 months. LB broth inoculated with E. coli was used as a positive control (n = 6). ns: not significant, ***: p < 0.001, ****: p < 0.0001. Statistical differences were calculated using one-way ANOVA with Turkey's post hoc test. Data in all figures are mean ± SEM.

[0037] Figures 20A-20E Demonstration of percutaneous ultrasound-guided nanogel injection into tissue with enhanced detection using magnetic resonance imaging. Figure 20A The nanogel was encased in a latex sleeve and then placed in a human phantom for ultrasound scanning, which showed high echogenicity, indicating its compatibility and visibility with ultrasound. Figure 20B and 20C Representative in vivo ultrasound images during direct injection of nanogels into rat liver parenchyma demonstrate the feasibility of direct percutaneous injection into target tissues ( Figure 20B The arrow in the middle indicates the injection needle, Figure 20C The outline in the figure shows the hyperechoic nanogel after injection). Figure 20D and 20E Compared with human liver or aorta, the T1( Figure 20D ) and T2( Figure 20E ) Graphs of T-weighted MR images and corresponding transverse views, showing 2- to 3-fold enhancement on T1-weighted images and 4- to 8-fold enhancement on T2-weighted images (n = 6). *: p < 0.5, ***: p < 0.01 using ANOVA.

[0038] Figures 21A-21I The tissue ablation and drug diffusion and retention abilities of the nanogels were evaluated in rats. 3D rendering reconstructed micro-CT and ICG fluorescence analysis of rat livers showed tissue ablation at each injection site in rats that received 50 μL of NS, NG, or NG+Dox injections on days 1, 14, or 28, respectively. Figure 21A 、 Figure 21C and Figure 21E As shown in the 3D rendered image. Figure 21B , 21D and 21F. Representative near-infrared images of transplanted rat livers showing the area of ​​ICG-enhanced fluorescence at each injection site on day 1, 14, or 28 after NS, NG, or NG+Dox injection, and graphs showing the diffusion area 28 days after NG and NG+Dox injection compared with NS demonstrate that ICG diffusion and retention were higher in the presence of IL in the nanogel formulation (n = 5). Figures 21G-21I *: p < 0.5, ***: p < 0.01. Statistical analysis was performed using one-way ANOVA and Tukey's post hoc test. Figure 21A 、 21C Scale bars in Figures 2 and 21E are 0.5 cm.

[0039] Figure 22A and 22B Sterility and MRI visibility of nanogels. Figure 22A Diagram of T2-weighted MRI images of syringes loaded with NS or NS-IL hydrogel and the corresponding transverse views showing signal enhancement compared with the liver or aorta. Figure 22B Graphs showing sterility of NS-IL and NS-IL-Dox 24 hours and 2 months after culture.

[0040] Figures 23A-23D Liquid chromatography-tandem mass spectrometry (LC–MS / MS) analysis of Dox levels in rat plasma. Figure 23A 、 23B and 23C. The different ionization channels of Dox molecules showed the same retention time at 4.1 min. Figure 23D The LC-MS / MS quantitative analysis of Dox levels showed linearity (R 2 =0.99).

[0041] Figures 24A-24H Effects of intratumoral injection of nanogel on HCC tumors in N1S1 rats. Figures 24A-24D Representative US images of N1S1 tumors at baseline before intratumoral injection ( Figure 24A and 24B ), and intratumoral injection of control NS-Dox-PD-1 antibody hydrogel ( Figure 24A and 24C ) or nanogel (NS-Dox-IL-PD-1 antibody hydrogel) ( Figure 24B and 24D )-treated N1S1 tumors 2 weeks after treatment ( Figure 24C and 24D ). Scale bar, 0.5 cm. Figure 24E and 24F . Control ( Figure 24E ) or nanogel-treated tumors ( Figure 24F ) H&E stained histological sections. Figure 24G and 24H . Control ( Figure 24G ) or nanogel-treated N1S1 tumors ( Figure 24H ) Histological sections showed PD-1 antibody immunostaining (brown; black arrows). n=3.

[0042] Figures 25A-25E . To evaluate the effect of nanogel injection on inflammatory cell infiltration. Figure 25A and 25B The histological sections of rat liver tissues were used to analyze the expression of myeloperoxidase (MPO)-carrying inflammatory cells ( Figure 25A ) or CD3+ T lymphocytes ( Figure 25B ) for immunostaining. Figure 25C Graphs summarizing the ablation area in liver sections of rats on days 1, 14, and 28 after injection of NS, NG, or NG+Dox showed that the ablation area was significantly larger at the NG and NG+Dox injection sites compared with the NS injection sites on days 1 and 14. Figure 25D Figure 4 shows morphometric analysis of MPO-positive cells, demonstrating early MPO-positive cell recruitment on day 1, which gradually decreased on days 14 and 28, suggesting a transient acute proinflammatory response (n=4). Figure 25E Histological analysis of the number of immunostained CD3+ cells counted within each injection site revealed significantly higher CD3+ cells in the NG+Dox site compared with the NS or NG injection site, indicating greater T lymphocyte recruitment and higher CD3+ cell numbers at the NG+Dox injection site 28 days after injection (n = 4). Statistical analysis was performed using two-way ANOVA with post hoc tests. Ns, not significant *: p < 0.05, **: p < 0.01, ****: p < 0.0001. Scale bars in histological images A and B = 1 mm in stitched images, 75 μm in high-magnification images on the right.

[0043] Figures 26A-26L Ultrasound-guided intratumoral injection of nanogels into a mouse colorectal cancer model. Figure 26A Gross view of an immunocompetent C57BL6 mouse inoculated subcutaneously with MC38 colon adenocarcinoma cells (colorectal cancer cells) showing a tumor in the right lower flank (arrow in the image). Figure 26B Ultrasound images of an MC38 tumor obtained during direct intratumoral injection of the nanogel show a hyperechoic needle (dashed outline) within a hypoechoic tumor lesion. Figure 26C and 26D 49 days after nanogel injection, the nanogel-treated tumors showed a complete therapeutic response, leaving only a small scar on the mouse skin ( Figure 26C ), which can be seen on ultrasound ( Figure 26D ). Figure 26E and 26F Gross images and corresponding ultrasound images of MC38 tumor-bearing mice showed that the tumor had progressed to approximately 2 cm 21 days after saline injection. 2 (Compare, arrows in the general figure; Figure 26E and dotted outline of US; Figure 26F ). Scale bar, 5 mm. Figure 26G Survival curves of MC38 tumor-bearing mice after ultrasound-guided intratumoral injection of nanogel or control. p = 0.0003. Figure 26H Figure 4. MC38 tumor growth curves of individual tumors assessed by ultrasound in nanogel (black, n=7) or control (red, n=7) are shown, showing early tumor progression in the control group compared to persistently lower tumor volumes in nanogel-injected tumors, indicating tumor response to treatment. Scale bar = 5 mm. Figure 26I Mean changes in tumor volume measured before intratumoral injection of nanogel or control and at the end of survival period for each group. n = 7 mice per group. **p < 0.1. Statistical analysis was performed using an unpaired t-test. Figure 26J Histological sections of MC38 tumors 1 hour after nanogel injection showed tumor cell ablation in high-magnification images. Figure 26K Histological sections of MC38 tumors 48 days after intratumoral injection of the nanogel showed evidence of reduced lesion size, cell ablation, and fibrogenesis. Figure 26L Histological sections of untreated MC38 tumors from the control group showed significantly larger tumor areas and evidence of actively proliferating tumor cells.

[0044] Figures 27A-27J Effects of intratumoral injection of nanogels on the N1S1 rat hepatocellular carcinoma model. Figure 27A Representative US images of N1S1 tumor ablation two weeks after intratumoral injection of nanogel mixed with 1 mg / mL Nivo and 0.25 ng / mL anticancer drug Dox. Figure 27B Representative US images of N1S1 tumors 2 weeks after intratumoral injection with NS hydrogel mixed with Nivo (control) and doxorubicin without IL. Figure 27C and 27D Corresponding stained histological sections of N1S1 tumors two weeks after injection of nanogels or control hydrogels, respectively. Scale bar in US images, 0.5 cm. Figure 27G and 27H . Infrared-corrected image of N1S1 tumor section on gold slide, showing the effect of nanogel injection ( Figure 27E ) or control ( Figure 27F ) Silicate oxide (Si-O, a derivative of nanosilicate) distribution using reflectance mode after two weeks. Figure 27G and 27H . Figure 27E and 27F Corresponding fluorescence imaging of N1S1 tissue sections. Figure 27I and 27JHistological sections showed that a large amount of Nivo (brown staining, black arrows) was detected in the N1S1 tumor lesions two weeks after the injection of the nanogel containing Dox and Nivo ( Figure 27A ), whereas Nivo detection was reduced in tumors that received NS hydrogels containing Dox or Nivo ( Figure 27B ). n=3 per group.

[0045] Figures 28A-28D Intratumoral injection of the nanogel effectively delivered anticancer immunotherapy and enhanced T lymphocyte recruitment in the N1S1 rat liver cancer model. Figure 28A and 28B Histological sections showed that two weeks after injection of the nanogel containing Dox and Nivo, tissue ablation and abundant Nivo distribution were detected within the N1S1 tumor lesions (brown staining, black arrows). Figure 28C and 28D Two weeks after intratumoral injection of the nanogel, representative immunostained histological sections of N1S1 tumors showed abundant lymphocyte infiltration (CD3+ cells in brown) at the interface between the ablated tumor area and the adjacent liver, as shown in Figure 3. Figure 28C and higher magnification images Figure 28D shown. Figure 28D The high-magnification images in the Figure 28C The area marked with a black box (n=3).

[0046] Figures 29A-29G Image-guided intratumoral injection of nanogels in a rabbit VX2 liver cancer model. Figure 29A Ultrasound scan of a rabbit liver showing a VX2 tumor, outlined by a white dashed line. Figure 29B and 29C Image showing ultrasound-guided injection of the nanogel using a standard 21-gauge vascular puncture needle (tumor outlined, arrow indicates needle). Figure 29D One hour after injection of NG mixed with 1.25 mg / mL Dox and 1 mg / mL Nivo, images of transected VX2 tumors showed red Dox material distributed throughout the transected tumor (arrows). Figure 29E . Figure 29D Ex vivo fluorescence image of a mid-section VX2 tumor showing Dox fluorescence throughout the treatment area 1 hour after injection of NG+Dox+Nivo. Figure 29F and 29G Representative histological sections 1 hour after intratumoral injection of NG+Dox+Nivo, H&E stained sections ( Figure 29D ) is located in the ablation area of ​​the PD-1 antibody (Nivo) immunostained section ( Figure 29G ). Scale bar in ultrasound image ( Figure 29A and29C ), 1 cm. Scale bar in histological sections ( Figure 29F and 29G ), 200μm.

[0047] Figure 30 Representative normal rabbit liver treated with nanogel (NG) containing 1.25 mg / mL doxorubicin (Dox) and 1 mg / mL nivolumab (Nivo).

[0048] Figure 31 VX2 tumor treated with NG+Dox+Nivo. Images show ablation and diffusion of Dox.

[0049] Figures 32A-32G Intravascular chemoembolization and drug delivery in porcine renal arteries. Figure 32A and 32B Representative fluoroscopic images before and after renal artery embolization with 2-3 cc of NG containing 0.25 mg / mL ICCG and 20% iohexol contrast agent, providing imaging enhancement on both fluorescence and X-ray-based imaging platforms such as CT or fluoroscopy. Figure 32C Gross images of explanted and transected kidneys 1 hour after NG embolization. Figure 32D Near-infrared fluorescence images of porcine kidneys 1 hour after NG embolization showed diffuse fluorescence enhancement of ICG throughout the renal cortex and medulla. Figure 32E and 32F Gross views and near-infrared fluorescence imaging of harvested and transected porcine kidneys one hour after embolization with NS hydrogel containing 0.25 mg / mL and 20% iohexanol showed that the fluorescence enhancement of ICG was reduced and confined to the renal vascular network. Figure 32G Figure 1 shows the increase in ICG fluorescence intensity in a single cross-section of renal tissue obtained after NG+ICG embolization compared with NS+ICG embolization. ****p<0.0001, unpaired Student's t-test, n=4 per group. Data are reported as mean ± SEM. Scale bar = 10 mm.

[0050] Figures 33A-33D Histological evaluation of transarterial spread of immunotherapy after transcatheter embolization of nivolumab-containing nanogels. Figure 33A H&E-stained histological sections of renal artery branches in the renal cortex showed complete casting of the artery within one hour after embolization of the renal artery with nanogel, indicating that NG has the ability to reach smaller arterial branches. Figure 33B .exist Figure 33A High-magnification images of serial sections in Figure 3 show immunohistochemical detection of Nivo. Nivo was localized in and around the renal artery, suggesting transarterial drug delivery. Figure 33C and 33DImages of hematoxylin-stained histological sections show reduced nuclear staining in the arterial wall, indicating successful transarterial delivery of IL and ablation after NG embolization. Scale bar in AC = 150 μm. Figure 33D Scale bar in = 50 μm. DETAILED DESCRIPTION

[0051] Provided herein are methods and materials for tissue ablation. For example, provided herein are ionic liquids having a cationic component and an anionic component (e.g., compositions comprising one or more ionic liquids having a cationic component and an anionic component) and methods for tissue ablation using such ionic liquids. In some cases, compositions comprising one or more ionic liquids (e.g., compositions comprising a LATTE solution) can be used to ablate (e.g., scar and / or destroy) at least a portion of a tissue in a mammal. For example, compositions comprising one or more ionic liquids (e.g., compositions comprising a LATTE solution) can be used to ablate tumor tissue in a mammal (e.g., to treat a mammal with cancer). For example, compositions comprising one or more ionic liquids (e.g., compositions comprising a LATTE solution) can be used to ablate adipose tissue in a mammal (e.g., for treating a mammal with a disease or condition associated with fat accumulation). In some cases, compositions comprising one or more ionic liquids (e.g., compositions comprising a LATTE solution) can be used to ablate cardiac tissue in a mammal (e.g., for treating a mammal with a cardiac disease or condition). In some cases, a composition comprising one or more ionic liquids (eg, a composition comprising a LATTE solution) can be used to eliminate blood clots in a mammal (eg, to treat the mammal).

[0052] In some cases, the compositions provided herein (e.g., compositions including one or more ionic liquids such as LATTE solution) can be sterile.

[0053] In some cases, the compositions provided herein (e.g., compositions including one or more ionic liquids such as LATTE solutions) can be antimicrobial.

[0054] The composition comprising one or more ionic liquids can include any suitable ionic liquid. In some cases, the ionic liquid can be a eutectic ionic liquid (e.g., a deep eutectic solvent (DES)). For example, when the ionic liquid is a eutectic ionic liquid (e.g., a DES), the melting temperature of the eutectic ionic liquid can be lower than the melting point of the cationic component and lower than the melting point of the anionic component.

[0055] The ionic liquid can include any suitable cationic component. In some cases, the cationic component can be an organic cationic component. In some cases, the cationic component can be an inorganic cationic component. Examples of cations that can be included in the cationic component of the ionic liquid include, but are not limited to, choline (e.g., choline cation), benzylpyridinium, benzyldimethyldodecylammonium, phosphonium, tetraalkylphosphonium, phenethylonium, imidazolium, pyridinium, piperidinium, quinolinium, morpholinium, quaternary phosphonium, and quaternary ammonium. In some cases, the cationic component can be in the form of a salt (e.g., choline bicarbonate).

[0056] The ionic liquid may include any suitable anionic component. Examples of anions that may be included in the anionic component of the ionic liquid include, but are not limited to, geranate, bistrifluoroformimide, oleate, hexanoate, dodecyldimethylaminopropanesulfonate, N-lauryl sarcosinate, geraniolate, tetrafluoroborate, hexafluorophosphate, methylsulfate, octylsulfate, acesulfame K, halides, bis(trifluoromethylsulfonyl)amide, bis(trifluoromethyl)amide, dicyanamide, and trifluoromethanesulfonate.

[0057] When the ionic liquid is a eutectic ionic liquid (e.g., a DES), the eutectic ionic liquid may include one or more hydrogen bond donors. In some cases, the hydrogen bond donor may provide a hydroxyl group (e.g., an -OH group). In some cases, the hydrogen bond donor may provide an amine group (e.g., a secondary amino group such as an -NH group). The hydrogen bond donor may be any type of molecule (e.g., an alcohol, a fatty acid, and an amine). Examples of hydrogen bond donors that may be included in the eutectic ionic liquid include, but are not limited to, imidazolium and chloride ions.

[0058] In some cases, the ionic liquid can have a cationic component that includes choline and can have an anionic component that includes geranate.

[0059] In some cases, the ionic liquid can be as described elsewhere (see, e.g., U.S. Patent No. 10,449,254, e.g., at column 4, line 2 to column 6, line 52; and column 11, line 6 to column 14, line 66).

[0060] The ionic liquid can include any suitable ratio of cationic components to anionic components. In some cases, the ionic liquid can include from about 1:2 (cation:anion) to about 2:1 (cation:anion). For example, the ionic liquid can include a ratio of cationic components to anionic components of 1:1, 1:2, 2:1, 1:3, 3:1, 2:3, or 3:2.

[0061] In some cases, the ionic liquid (eg, LATTE solution) can be in the form of a solution (eg, an aqueous solution) or a suspension.

[0062] In some cases, the ionic liquid (e.g., LATTE solution) can be in the form of (e.g., can be incorporated into) a hydrogel (e.g., a shear-thinning hydrogel). For example, a shear-thinning hydrogel comprising an ionic liquid can include one or more nanosilicates, one or more gelatins, and an ionic liquid.

[0063] When the compositions provided herein (e.g., compositions comprising one or more ionic liquids such as LATTE solutions) are incorporated into a hydrogel, the compositions can be incorporated into any suitable hydrogel. Examples of hydrogels into which the compositions provided herein can be incorporated include, but are not limited to, nanosilicate hydrogels, tantalum microparticle hydrogels, gelatin-based hydrogels, alginate hydrogels, gelatin methacrylate hydrogels, extracellular matrix-based hydrogels, self-assembling peptide-based hydrogels, polyethylene glycol hydrogels, and chitosan hydrogels. In some cases, the hydrogels into which the compositions provided herein can be incorporated can be as described elsewhere (see, e.g., Altun et al., Adv. Mater., 32(52):e2005603 (2020); Hu et al., Adv. Mater., 32(33):e2002611 (2020); Albadawi et al., Adv. Sci., 8(1):2003327 (2020); and Avery et al., Sci. Transl. Med., 8(365):365ra156 (2016)).

[0064] In some cases, the compositions provided herein can be incorporated into a hydrogel comprising one or more nanosilicates. The hydrogel can comprise any suitable type of nanosilicate. In some cases, the hydrogel can comprise a single type of nanosilicate. In some cases, the hydrogel can comprise two or more (e.g., two, three, four or more) types of nanosilicates. In some cases, the nanosilicate can be a synthetic nanosilicate. Examples of nanosilicates that can be included in a hydrogel that can be used in a hydrogel composition provided herein include, but are not limited to, montmorillonite clays (e.g., synthetic montmorillonite clays, such as LAPONITE™, XLG, XLS and XL21).

[0065] When the compositions provided herein (e.g., compositions comprising one or more ionic liquids such as LATTE solutions) are incorporated into a hydrogel comprising one or more nanosilicates, the hydrogel can include any amount of nanosilicates. For example, the compositions provided herein (e.g., compositions comprising one or more ionic liquids such as LATTE solutions) can be incorporated into a hydrogel comprising from about 1% (w / v) to about 10% (w / v) of nanosilicates (e.g., from about 1% (w / v) to about 9% (w / v), from about 1% (w / v) to about 8% (w / v), from about 1% (w / v) to about 7% (w / v), from about 1% (w / v) to about 5% (w / v), from about 1% (w / v) to about 3% (w / v), from about 2% (w / v) to about 10% (w / v), from about 3% (w / v) to about 10% (w / v) , about 4% (w / v) to about 10% (w / v), about 5% (w / v) to about 10% (w / v), about 7% (w / v) to about 10% (w / v), about 9% (w / v) to about 10% (w / v), about 2% (w / v) to about 9% (w / v), about 3% (w / v) to about 8% (w / v), about 4% (w / v) to about 7% (w / v), about 5% (w / v) to about 6% (w / v), about 1% (w / v) to about 4% (w / v), about 3% (w / v) to about 7% (w / v), or about 5% (w / v) to about 8% (w / v) nanosilicates). In some cases, a composition provided herein (e.g., a composition comprising one or more ionic liquids such as a LATTE solution) can be incorporated into a hydrogel comprising about 3% (w / v) to about 9% (w / w) nanosilicate (e.g., synthetic montmorillonite clay).

[0066] When the compositions provided herein (e.g., compositions comprising one or more ionic liquids such as LATTE solutions) are incorporated into a hydrogel, the hydrogel compositions comprising one or more ionic liquids (e.g., LATTE solutions) can include any amount of the ionic liquid. In some cases, the hydrogel compositions can include from about 3% by weight (w / w or wt%) to about 50% (w / w) of the one or more ionic liquids (e.g., from about 3% by weight (w / w or wt%) to about 40% (w / w), from about 3% by weight (w / w or wt%) to about 30% (w / w), from about 3% by weight (w / w or wt%) to about 25% (w / w), from about 3% by weight (w / w or wt%) to about 20% (w / w) of the one or more ionic liquids. w), about 3 weight % (w / w or wt%) to about 15% (w / w), about 3 weight % (w / w or wt%) to about 10% (w / w), about 10 weight % (w / w or wt%) to about 50% (w / w), about 20 weight % (w / w or wt%) to about 50% (w / w), about 25 weight % (w / w or wt%) to about 50% (w / w), about 30 weight % (w / w or wt%) to about 50% (w / w), about 35 weight % (w / w or wt%) to about 50% (w / w), about 40 weight % (w / w or wt%) to about 50% (w / w), about 5 weight % (w / w or wt%) to about 40% (w / w), about 10 weight % (w / w or wt%) to about 30% (w / w), about 5 weight % (w / w or wt%) to about 15% (w / w), about 10 weight % (w / w or wt%) to about % (w / w) of the one or more ionic liquids. For example, a hydrogel including one or more ionic liquids (e.g., a LATTE solution) can include about 3.25% (w / w) of the one or more ionic liquids. For example, a hydrogel including one or more ionic liquids (e.g., a LATTE solution) can include about 12.5% ​​(w / w) of the one or more ionic liquids. For example, a hydrogel including one or more ionic liquids (e.g., a LATTE solution) can include about 25% (w / w) of the one or more ionic liquids. For example, a hydrogel including one or more ionic liquids (eg, a LATTE solution) can include about 50% (w / w) of the one or more ionic liquids.

[0067] When a composition provided herein (eg, a composition comprising one or more ionic liquids such as a LATTE solution) is incorporated into a hydrogel, the hydrogel can be a shear-thinning hydrogel composition. For example, the viscosity of a hydrogel into which the compositions provided herein can be incorporated can decrease at a shear rate of from about 0.1 1 / s to about 1000 1 / s (e.g., from about 0.1 1 / s to about 750 1 / s, from about 0.1 1 / s to about 500 1 / s, from about 0.1 1 / s to about 250 1 / s, from about 0.1 1 / s to about 100 1 / s, from about 0.1 1 / s to about 75 1 / s, from about 0.1 1 / s to about 50 1 / s, from about 0.1 1 / s to about 25 1 / s, from about 0.1 1 / s to about 10 1 / s, from about 10 1 / s to about 1000 1 / s, from about 50 1 / s to about 1000 1 / s, from about 100 1 / s to about 1000 1 / s, from about 250 1 / s to about 1000 1 / s, from about 500 1 / s to about 1000 1 / s, about 750 1 / s to about 1000 1 / s, about 10 1 / s to 800 1 / s, about 50 1 / s to 600 1 / s, about 100 1 / s to 500 1 / s, about 200 1 / s to 300 1 / s, about 100 1 / s to 300 1 / s, about 300 1 / s to 500 1 / s, about 400 1 / s to 600 1 / s, about 500 1 / s to about 700 1 / s, about 600 1 / s to about 800 1 / s, or about 700 1 / s to 900 1 / s).

[0068] In some cases, ionic liquids (eg, LATTE solutions) can be incorporated into microparticles or nanoparticles.

[0069] Any suitable method can be used to obtain an ionic liquid (such as a LATTE solution). In some cases, an ionic liquid can be synthesized by combining a cationic component and an anionic component in the presence of a solute. Examples of solutes that can be used to synthesize ionic liquids include, but are not limited to, acetone. When the ionic liquid is a eutectic ionic liquid (such as DES), a salt metathesis reaction of the cationic component and the anionic component can be used to synthesize a eutectic ionic liquid in the presence of a hydrogen bond donor. For example, a 1:1 molar ratio of choline bicarbonate and geranate can be used to synthesize a LATTE solution). In some cases, the ionic liquid can be synthesized as described in Example 1. In some cases, the ionic liquid can be synthesized as described elsewhere (see, for example, U.S. Patent No. 10,449,254, for example, column 11, line 6 to column 14, line 66). When the ionic liquid is incorporated into a hydrogel, a hydrogel comprising an ionic liquid can be synthesized as described in Example 5. In some cases, hydrogels including ionic liquids can be synthesized as described elsewhere (see, e.g., Albadawi et al., Adv. Sci., 8(1):2003327 (2020); and Avery et al., Sci. Transl. Med., 8(365):365ra156 (2016)).

[0070] A composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can include any amount of the ionic liquid. In some cases, a composition comprising one or more ionic liquids can include from about 6 weight percent (wt%) to about 100 wt% (e.g., from about 6 wt% to about 100 wt%, from about 10 wt% to about 100 wt%, from about 20 wt% to about 100 wt%, from about 30 wt% to about 100 wt%, from about 40 wt% to about 100 wt%, from about 50 wt% to about 100 wt%, from about 60 wt% to about 100 wt%, from about 70 wt% to about 100 wt%, from about 80 wt% to about 100 wt%, from about 90 wt% to about 100 wt%, from about 6 wt% to about 90 wt%, from about 6 wt% to about 80 wt%, from about 6 wt% to about 70 wt%, from about 6 wt% to about % to about 60 wt %, about 6 wt % to about 50 wt %, about 6 wt % to about 40 wt %, about 6 wt % to about 30 wt %, about 6 wt % to about 20 wt %, about 6 wt % to about 10 wt %, about 10 wt % to about 90 wt %, about 20 wt % to about 80 wt %, about 30 wt % to about 70 wt %, about 40 wt % to about 60 wt %, about 10 wt % to about 30 wt %, about 20 wt % to about 40 wt %, about 30 wt % to about 50 wt %, about 40 wt % to about 60 wt %, about 50 wt % to about 70 wt %, about 60 wt % to about 80 wt %, or about 70 wt % to about 90 wt %) of one or more ionic liquids. For example, a composition including one or more ionic liquids (e.g., a composition including a LATTE solution) can include about 6.25 wt % of one or more ionic liquids. For example, a composition including one or more ionic liquids (e.g., a composition including a LATTE solution) can include about 25 wt % of one or more ionic liquids. For example, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can include about 35 wt% of the one or more ionic liquids. For example, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can include about 50 wt% of the one or more ionic liquids. For example, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can include 100 wt% of the one or more ionic liquids.

[0071] In some cases, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can include one or more therapeutic agents. For example, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be used to deliver one or more therapeutic agents to the ablation zone created by the composition. Examples of therapeutic agents that may be included in a composition including one or more ionic liquids (e.g., a composition including a LATTE solution) include, but are not limited to, chemotherapeutics (e.g., doxorubicin, cisplatin, and paclitaxel), radioactive agents, antibodies (e.g., antibodies targeting specific cell types (e.g., cancer cells), angiogenic factors (e.g., factors that can inhibit angiogenesis or factors that can stimulate angiogenesis), therapeutic polypeptides, nucleic acids encoding therapeutic polypeptides (e.g., vectors such as viral vectors or expression plasmids encoding therapeutic polypeptides), immunomodulators (e.g., factors that can enhance immune responses or factors that can inhibit immune responses, including but not limited to immune checkpoint inhibitors (e.g., anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies) and immunostimulants (e.g., interleukins and interferons)), hormones, antibiotics, and blood thinners (e.g., lovenox, coumarin, and FactorXA inhibitors). In some cases, therapeutic agents can be coupled to nanoparticles. In some cases, therapeutic agents can be contained within nanoparticles.

[0072] When a composition comprising one or more ionic liquids (eg, a composition comprising a LATTE solution) includes one or more therapeutic agents, the composition can include any amount of the therapeutic agent. In some cases, the compositions provided herein (e.g., compositions comprising one or more ionic liquids, such as LATTE solutions) can include from about 50 μg of the therapeutic agent per ml of composition (μg / mL) to about 2000 μg / mL of the therapeutic agent (e.g., from about 50 μg / mL to about 1500 μg / mL, from about 50 μg / mL to about 1000 μg / mL, from about 50 μg / mL to about 700 μg / mL, from about 50 μg / mL to about 500 μg / mL, from about 50 μg / mL to about 300 μg / mL, from about 50 μg / mL to about 200 μg / mL, from about 50 μg / mL to about 100 μg / mL, from about 100 μg / mL to about 2000 μg / mL, from about 200 μg / mL to about 2000 μg / mL, from about 300 μg / mL to about 2000 μg / mL, from about 500 μg / mL to about 2000 μg / mL , about 700 μg / mL to about 2000 μg / mL, about 1000 μg / mL to about 2000 μg / mL, about 1200 μg / mL to about 2000 μg / mL, about 1500 μg / mL to about 2000 μg / mL, about 100 μg / mL to about 1500 μg / mL, about 200 μg / mL to about 1200 μg / mL, about 400 μg / mL to about 1000 μg / mL,

[0014] In some embodiments, the present invention provides a composition comprising an ionic liquid such as LATTE solution, for example, a composition comprising one or more ionic liquids such as LATTE solution, for example, a therapeutic agent (e.g., doxorubicin) at a concentration of about 1 mg / mL to about 2 mg / mL (e.g., 1.25 mg / mL).

[0073] In some cases, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can include one or more contrast agents. In some cases, the contrast agent can be a radiopaque contrast agent. In some cases, the contrast agent can be an earth metal-based contrast agent. In some cases, the contrast agent can be compatible with magnetic resonance imaging. In some cases, the contrast agent can be compatible with nuclear imaging. In some cases, the contrast agent can be compatible with ultrasound imaging. In some cases, the contrast agent can be compatible with fluorescence imaging. Examples of contrast agents that can be included in a composition comprising one or more ionic liquids include, but are not limited to, indocyanine green, ExiTron, TM 、 Iohexol tantalum nanoparticles, tantalum microparticles, gold nanoparticles, gadolinium, indium 111 , iodine and microbubbles.

[0074] When a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) comprises one or more contrast agents, the composition can include any amount of the contrast agent. In some cases, a composition provided herein (e.g., a composition comprising one or more ionic liquids such as a LATTE solution) can include from about 10% (w / w) to about 30% (w / w) of a contrast agent (e.g., from about 10% (w / w) to about 25% (w / w), from about 10% (w / w) to about 20% (w / w), from about 10% (w / w) to about 15% (w / w), from about 15% (w / w) to about 30% (w / w) of a contrast agent. w), about 20% (w / w) to about 30% (w / w), about 25% (w / w) to about 30% (w / w), about 12% (w / w) to about 27% (w / w), about 15% (w / w) to about 25% (w / w), about 18% (w / w) to about 22% (w / w), about 15% (w / w) to about 20% (w / w), or about 20% (w / w) to about 25% (w / w) contrast agent. For example, the compositions provided herein contained in a hydrogel (e.g., a composition containing one or more ionic liquids such as a LATTE solution) can contain about 20% (w / w) of a contrast agent.

[0075] When a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) includes other agents (e.g., one or more therapeutic agents, such as one or more therapeutic agents coupled to or contained within nanoparticles, or a contrast agent), the other agents can be of any suitable size. In some cases, the size of the other agents included in a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be up to about 110 nm (e.g., across the longest dimension). For example, the other agents included in the composition include one or more ionic liquids (e.g., a composition comprising a LATTE solution) that can be from about 1 nm to about 110 nm (e.g., from about 1 nm to about 100 nm, from about 1 nm to about 90 nm, from about 1 nm to about 80 nm, from about 1 nm to about 70 nm, from about 1 nm to about 60 nm, from about 1 nm to about 50 nm, from about 1 nm to about 40 nm, from about 1 nm to about 30 nm, from about 10 nm to about 110 nm, from about 20 nm to about 110 nm, from about 30 nm to about 110 nm, from about 40 nm to about 110 nm m, about 50 nm to about 110 nm, about 60 nm to about 110 nm, about 70 nm to about 110 nm, about 80 nm to about 110 nm, about 90 nm to about 110 nm, about 10 nm to about 100 nm, about 20 nm to about 90 nm, about 30 nm to about 80 nm, about 40 nm to about 70 nm, about 10 nm to about 40 nm, about 20 nm to about 50 nm, about 30 nm to about 60 nm, about 40 nm to about 70 nm, about 50 nm to about 80 nm, about 60 nm to about 90 nm, or about 70 nm to about 100 nm).

[0076] When a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) includes one or more therapeutic agents and / or one or more contrast agents, the one or more therapeutic agents and / or contrast agents can reside within the ablation zone for up to about 28 days (e.g., up to about 30 days, up to about 1 month, up to about 6 weeks, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, or up to about 6 months). In some cases, when a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) includes one or more therapeutic agents, the therapeutic agents and / or contrast agents can remain within the ablation zone for about 1 day to about 30 days (e.g., about 1 day to about 30 days, about 1 day to about 28 days, about 1 day to about 25 days, about 1 day to about 22 days, about 1 day to about 20 days, about 1 day to about 15 days, about 1 day to about 12 days, about 1 day to about 10 days, about 1 day to about 8 days). days, about 1 day to about 5 days, about 5 days to about 30 days, about 7 days to about 30 days, about 10 days to about 30 days, about 12 days to about 30 days, about 15 days to about 30 days, about 18 days to about 30 days, about 20 days to about 30 days, about 22 days to about 30 days, about 25 days to about 30 days, about 27 days to about 30 days, about 5 days to about 10 days, about 10 days to about 15 days, about 15 days to about 20 days, about 20 days to about 25 days, or about 25 days to about 30 days).

[0077] In some cases, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can include one or more other components. In some cases, the other component can be a stabilizer. Examples of other components that can be included in the hydrogel compositions provided herein include, but are not limited to, polysorbates, surfactants, organic solvents (e.g., dimethyl sulfoxide (DMSO)), and detergents (e.g., sodium dodecyl sulfate (SDS)).

[0078] A composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered to a mammal (e.g., a human) by any suitable route. In some cases, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered to a mammal (e.g., a human) by percutaneous injection; for example, intramuscular injection, subcutaneous injection, intratumoral injection, intraparenchymal injection, intradermal injection, intrathecal injection, intracathecal injection, intravascular injection, intraosseous injection, and intraarticular injection. For example, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered to a mammal (e.g., a human) by direct percutaneous injection into the tissue to be ablated. For example, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered to a mammal (e.g., a human) by percutaneous injection within a distance of about 0.1 pm to about 12 pm (e.g., about 0.1 pm to about 10 pm, about 0.1 pm to about 9 pm, about 0.1 pm to about 7 pm, about 0.1 pm to about 5 pm, about 0.1 pm to about 3 pm, about 0.5 pm to about 12 pm, about 3 pm to about 12 pm, about 5 pm to about 12 pm, about 8 pm to about 12 pm, about 10 pm to about 12 pm, about 0.5 pm to about 10 pm, about 2 pm to about 8 pm, about 3 pm to about 7 pm, about 4 pm to about 6 pm, about 1 pm to about 4 pm, about 2 pm to about 5 pm, about 3 pm to about 6 pm, about 4 pm to about 7 pm, about 5 pm to about 8 pm, about 6 pm to about 9 pm, about 7 pm to about 10 pm, or about 8 pm to about 11 pm) from the tissue to be ablated. In some cases, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered to a mammal (e.g., a human) without the need for any anesthesia (e.g., without the need for general anesthesia). In some cases, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered to a mammal (e.g., a human) using guided injection (e.g., using ultrasound guidance). In some cases, when a composition is administered to a mammal (e.g., a human) by transdermal injection, a single injection can be used to administer the composition. In some cases, when a composition is administered to a mammal (e.g., a human) by transdermal injection, two or more (e.g., two, three, four, five, or more) injections can be used to administer the composition. For example, multi-hole injection or multi-angle injection can be used to inject a mammal two or more (e.g., two, three, four, five, or more) times. In some cases, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered to a mammal (e.g., a human) by topical administration; for example, spraying onto a tissue.

[0079] Compositions comprising one or more ionic liquids (e.g., compositions comprising LATTE solutions) can be used to ablate at least a portion of one or more tissues in any suitable mammal. As described herein, examples of mammals in which one or more tissues can be ablated include, but are not limited to, humans, non-human primates such as monkeys, horses, bovine species, porcine species, dogs, cats, horses, cows, pigs, sheep, mice, rabbits, and rats.

[0080] Compositions comprising one or more ionic liquids (e.g., compositions comprising LATTE solutions) can be used to ablate at least a portion of any type of tissue. Examples of tissues that can be ablated using ionic liquids (e.g., compositions comprising LATTE solutions) include, but are not limited to, adipose tissue, cardiac tissue, connective tissue (e.g., blood), bone tissue, synovial tissue, abscess tissue, and cysts. In some cases, the tissue can be tumor tissue (e.g., which can include one or more cancer cells). The tumor tissue can be either benign or malignant.

[0081] In some cases, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be used to create an ablation zone in a mammal (e.g., a human). For example, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be diffused in a circular manner (e.g., can diffuse from an administration site) to create an ablation zone in a mammal (e.g., a human). The ablation zone can be of any suitable size. In some cases, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can create an ablation zone of up to about 5 cm (e.g., across the longest dimension) in a mammal. For example, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can produce an ablation zone in a mammal having a diameter of about 0.1 cm to about 5 cm (e.g., about 0.1 cm to about 4.5 cm, about 0.1 cm to about 4 cm, about 0.1 cm to about 3.5 cm, about 0.1 cm to about 3 cm, about 0.1 cm to about 2.5 cm, about 0.1 cm to about 2 cm, about 0.1 cm to about 1.5 cm, about 0.1 cm to about 1 cm, about 0.1 cm to about 0.5 cm, about 0.5 cm to about 4 cm, about 1 cm to about 4 cm, about 1.5 cm to about 4 cm, about 2 cm to about 4 cm, about 2.5 cm to about 4 cm, about 2.8 cm to about 4 cm, about 3 cm to about 4 cm, about 3.2 cm to about 4 cm, about 3.5 cm to about 4 cm, about 0.5 cm to about 3.8 cm, about 1 cm to about 3.5 cm, about 2 cm to about 3 cm, or about 1 cm to about 2 cm).

[0082] In some cases, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be used to reduce the number of cells within the ablation zone. For example, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered to a mammal (e.g., a human) to reduce the number of cells within the ablation zone produced by the composition by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

[0083] In some cases, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be used to induce an inflammatory response within the ablation zone created by the composition. For example, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered to a mammal (e.g., a human) to recruit T cells (e.g., activated T cells) to the ablation zone created by the composition.

[0084] In some cases, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be used to treat a mammal (e.g., a human) having a disease or condition that would benefit from ablation of at least a portion of tissue in the mammal. For example, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be used to ablate tumor tissue in a mammal having cancer (e.g., treating the mammal). In some cases, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be used to ablate adipose tissue in a mammal having a disease or condition associated with fat accumulation (e.g., treating the mammal). In some cases, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be used to ablate cardiac tissue in a mammal having a cardiac disease or condition (e.g., treating the mammal). In some cases, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be used to ablate blood clots in a mammal (e.g., treating the mammal). In some cases, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be used to ablate one or more infected tissues in a mammal (e.g., treating the mammal).

[0085] As described herein, when treating a mammal (e.g., a human) having cancer (e.g., by administering a composition comprising one or more ionic liquids (e.g., LATTE solution)), the composition can be effective in reducing the size of the cancer in the mammal (e.g., reducing the number of cancer cells in the mammal and / or reducing the volume of one or more tumors in the mammal). For example, as described herein, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having cancer) to reduce the size of the cancer by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some cases, as described herein, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having cancer) to reduce the size of the cancer by at least 2-fold (e.g., by 2-fold, 3-fold, 4-fold, 5-fold, or more).

[0086] In some cases, when treating a mammal (e.g., a human) having a cancer described herein (e.g., by administering a composition comprising one or more ionic liquids (e.g., LATTE solution)), the composition can effectively promote the entry of one or more T cells (e.g., activated T cells) into a tumor in the mammal (e.g., increasing the number of one or more T cells in the tumor). For example, as described herein, a composition comprising one or more ionic liquids (e.g., a composition comprising LATTE solution) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human suffering from cancer) to increase the number of one or more T cells in the tumor in the mammal by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. Following administration of a composition comprising one or more ionic liquids (e.g., a composition comprising LATTE solution), examples of T cells that can be increased in a tumor include, but are not limited to, CD4+ T cells, CD8+ T cells, and natural killer T cells.

[0087] In some cases, as described herein, when treating a mammal (e.g., a human) having cancer (e.g., by administering a composition comprising one or more ionic liquids (e.g., LATTE solution)), the composition can be effective in inducing an inflammatory response within the ablation zone formed by the composition. For example, as described herein, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered to a mammal (e.g., a human) (e.g., a human having cancer) to a locally accessible cancerous lesion (e.g., a locally accessible metastatic lesion, such as a lesion on the skin or peritoneal surface) to recruit T cells (e.g., activated T cells) to treat the locally accessible cancerous lesion. For example, as described herein, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered to a mammal (e.g., a human) (e.g., a human having cancer) to a locally accessible cancerous lesion (e.g., a locally accessible metastatic lesion, such as a lesion on the skin or peritoneal surface) to recruit T cells (e.g., activated T cells) to treat otherwise inaccessible cancerous lesions in the mammal.

[0088] As described herein, when treating a mammal (e.g., a human) having cancer (e.g., by administering a composition comprising one or more ionic liquids (e.g., LATTE solution)), the cancer can be any type of cancer. In some cases, the cancer can include one or more solid tumors. For example, the cancer can include one or more fat-rich solid tumors. In some cases, the cancer can be a blood cancer. In some cases, the cancer can be a primary cancer. In some cases, the cancer can be a metastatic cancer. In some cases, the cancer can be a cancer that escapes and / or does not respond to chemotherapy (e.g., a chemotherapy-resistant cancer). Examples of cancers that can be treated as described herein (e.g., using a composition comprising one or more ionic liquids, such as a LATTE solution) include, but are not limited to, liver cancer (e.g., HCC), biliary tract cancer (e.g., bile duct cancer), pancreatic cancer (e.g., pancreatic adenocarcinoma), colorectal cancer (e.g., colorectal cancer liver metastasis (CRCLM)), kidney cancer, ovarian cancer, breast cancer, prostate cancer, colon cancer, bladder cancer, lung cancer, thyroid cancer, melanoma, brain cancer, stomach cancer, cervical cancer, uterine cancer, skin cancer, synovial cancer, appendix cancer, adrenal cancer, sarcoma, and lymphoma.

[0089] In some cases, the methods of treating a mammal having cancer as described herein (e.g., by administering a composition comprising one or more ionic liquids (e.g., LATTE solution)) can also include identifying the mammal as having cancer. Methods for identifying the mammal as having cancer include, but are not limited to, physical examination, laboratory tests (e.g., blood and / or urine), biopsy, imaging tests (e.g., X-ray, PET / CT, MRI, and / or ultrasound), nuclear medicine scans (e.g., bone scans), endoscopy, and / or genetic testing.

[0090] In some cases, when a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) is used to treat a mammal (e.g., a human) having cancer, the composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can include the ionic liquid as the sole active agent for treating the cancer.

[0091] In some cases, when a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) is used to treat a mammal (e.g., a human) suffering from cancer, the composition comprising one or more ionic liquids may include one or more (e.g., one, two, three, four, five or more) other therapeutic agents for treating cancer. In some cases, the therapeutic agent for treating cancer may be a chemotherapeutic agent. In some cases, the therapeutic agent for treating cancer may be a radioactive agent. In some cases, the therapeutic agent for treating cancer may be an immunotherapeutic agent (e.g., an immune checkpoint inhibitor, such as an anti-PD-1 antibody and / or an anti-PD-L1 antibody). In some cases, the therapeutic agent for treating cancer may be a stimulator of an interferon (IFN) gene (STING) agonist. Examples of therapeutic agents for treating cancer that can be administered to a mammal having cancer with a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) include, but are not limited to, doxorubicin, cisplatin, paclitaxel, olaparib, everolimus, mitomycin, radioisotopes (e.g., yttrium Y-90, lutetium-177, actinium, fluorine-18, gallium-67, krypton-81m, rubidium-82, nitrogen-13, technetium-99m, indium-111, iodine-123, xenon-133, and thallium-201), atezolizumab, bevacizumab, cabozantinib-s-malate, ramucirumab, pembrolizumab, lenvatinib mesylate, sorafenib tosylate, nivolumab, pemmetinib, pembrolizumab, ramucirumab, regorafenib, and abemaciclib. In some cases, one or more other therapeutic agents can be administered together with a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution). In some cases, one or more other therapeutic agents can be administered independently of a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution). When one or more other therapeutic agents are administered independently of a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution), the composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered first, followed by administration of the one or more other therapeutic agents, or vice versa.

[0092] In some cases, a method for treating a mammal (e.g., a human) having cancer as described herein (e.g., by administering a composition comprising one or more ionic liquids, such as a LATTE solution) can also include subjecting the mammal to one or more (e.g., one, two, three, four, five, or more) other treatments (e.g., therapeutic interventions) effective for treating cancer. Examples of other treatments that can be used to treat cancer as described herein include, but are not limited to, radiation therapy, surgery, percutaneous tumor ablation, transcatheter embolization, and cancer immunotherapy. In some cases, one or more other treatments effective for treating cancer can be administered concurrently with the administration of a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution). In some cases, one or more other treatments effective for treating cancer can be administered before and / or after the administration of a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution).

[0093] When treating a mammal (e.g., a human) having a disease or condition associated with fat accumulation as described herein (e.g., by administering a composition comprising one or more ionic liquids, such as a LATTE solution), the composition can be effective in reducing the number of fat cells in the mammal. For example, as described herein, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human suffering from cancer) to reduce the number of fat cells in the mammal by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

[0094] When treating a mammal (e.g., a human) having a disease or condition associated with fat accumulation as described herein (e.g., by administering a composition comprising one or more ionic liquids, such as LATTE solution), the disease or condition associated with fat accumulation can be any disease or condition associated with fat accumulation. Examples of diseases and conditions associated with fat accumulation that can be treated as described herein (e.g., with a composition comprising one or more ionic liquids, such as LATTE solution) include, but are not limited to, overweight (e.g., obesity), lipedema, fat storage disorders (e.g., glycogen storage disorders), and cancer characterized by fat-depositing tumors.

[0095] In some cases, a method of treating a mammal (e.g., a human) having a disease or condition associated with fat accumulation as described herein (e.g., by administering a composition comprising one or more ionic liquids such as a LATTE solution) can further comprise identifying that the mammal has a disease or condition associated with fat accumulation. Examples of methods for identifying that a mammal has a disease or condition associated with fat accumulation include, but are not limited to, physical examination, laboratory tests (e.g., blood and / or urine), CT imaging, and / or MRI.

[0096] In some cases, when a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) is used to treat a mammal (e.g., a human) suffering from a disease or condition associated with fat accumulation, the composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) may include the ionic liquid as the sole active agent for treating the disease or condition associated with fat accumulation.

[0097] In some cases, when a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) is used to treat a mammal (e.g., a human) suffering from a disease or condition associated with fat accumulation, the composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) may include one or more (e.g., one, two, three, four, five or more) other therapeutic agents for treating a disease or condition associated with fat accumulation. Examples of therapeutic agents for treating a disease or condition associated with fat accumulation that can be administered together with a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) include, but are not limited to, orlistat, phentermine, topiramate, bupropion, naltrexone, liraglutide, and combinations thereof. In some cases, one or more other therapeutic agents can be administered together with a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution). In some cases, one or more other therapeutic agents can be administered independently of a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution). When one or more other therapeutic agents are administered separately from a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution), the composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered first and the one or more other therapeutic agents can be administered secondarily, or vice versa.

[0098] In some cases, a method for treating a mammal (e.g., a human) having a disease or condition associated with fat accumulation as described herein (e.g., by administering a composition comprising one or more ionic liquids, such as a LATTE solution) may also include subjecting the mammal to one or more (e.g., one, two, three, four, five, or more) other treatments (e.g., therapeutic interventions) effective for treating the disease or condition associated with fat accumulation. As described herein, examples of other treatments that can be used to treat a disease or condition associated with fat accumulation include, but are not limited to, dietary changes (e.g., dietary changes that reduce calories), increased activity levels, endoscopic surgery for weight loss, bariatric surgery, vagal nerve blockade, and left gastric artery embolization. In some cases, one or more other treatments effective for treating a disease or condition associated with fat accumulation may be administered concurrently with the administration of a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution). In some cases, one or more other treatments effective for treating a disease or condition associated with fat accumulation may be administered before and / or after the administration of a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution).

[0099] When treating a mammal (e.g., a human) suffering from a cardiac disease or condition as described herein (e.g., by administering a composition comprising one or more ionic liquids, such as a LATTE solution), the composition can effectively reduce the amount of cardiac tissue in the mammal (e.g., reduce the number of atrophic cardiomyocytes and / or hypertrophic cardiomyocytes in the myocardium). For example, as described herein, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human suffering from cancer) to reduce the number of atrophic cardiomyocytes in the mammal by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

[0100] When treating a mammal (e.g., a human) having a cardiac disease or condition as described herein (e.g., by administering a composition comprising one or more ionic liquids, such as LATTE solution), the cardiac disease or condition can be any cardiac disease or condition. Examples of cardiac diseases and conditions that can be treated as described herein (e.g., with a composition comprising one or more ionic liquids, such as LATTE solution) include, but are not limited to, hypertrophic cardiomyopathy, arrhythmias, and atrial fibrillation lesions.

[0101] In some cases, a method of treating a mammal (e.g., a human) having a cardiac disease or condition described herein (e.g., by administering a composition comprising one or more ionic liquids such as a LATTE solution) can also include identifying the mammal as having the cardiac disease or condition. Examples of methods for identifying a mammal as having a disease or condition associated with fat accumulation include, but are not limited to, a physical examination and / or laboratory tests (e.g., blood and / or urine).

[0102] In some cases, when a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) is used to treat a mammal (e.g., a human) having a cardiac disease or condition, the composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) may include the ionic liquid as the sole active agent for treating the cardiac disease or condition.

[0103] In some cases, when a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) is used to treat a mammal (e.g., a human) suffering from a heart disease or condition, the composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) may include one or more (e.g., one, two, three, four, five or more) other therapeutic agents for treating the heart disease or condition. In some cases, the therapeutic agent for treating the heart disease or condition may be an anticoagulant. In some cases, the therapeutic agent for treating the heart disease or condition may be an ACE inhibitor. In some cases, the therapeutic agent for treating the heart disease or condition may be a beta blocker. In some cases, the therapeutic agent for treating the heart disease or condition may be a calcium channel blocker. In some cases, the therapeutic agent for treating the heart disease or condition may be a cholesterol-lowering drug. Examples of therapeutic agents for treating cardiac disease or illness that can be administered to a mammal with cardiac disease or illness together with a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) include, but are not limited to, captopril, enalapril, fosinopril, lisinopril, perindopril, quinapril, ramipril, tranepril, and combinations thereof. In some cases, one or more other therapeutic agents can be administered together with a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution). In some cases, one or more other therapeutic agents can be administered independently of a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution). When one or more other therapeutic agents are administered independently of a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution), a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered first, and one or more other therapeutic agents can be administered secondary, or vice versa.

[0104] In some cases, a method for treating a mammal (e.g., a human) having a cardiac disease or condition as described herein (e.g., by administering a composition comprising one or more ionic liquids, such as a LATTE solution) can also include subjecting the mammal to one or more (e.g., one, two, three, four, five, or more) other treatments (e.g., therapeutic interventions) effective for treating the cardiac disease or condition. Examples of other treatments that can be used to treat a cardiac disease or condition as described herein include, but are not limited to, dietary changes (e.g., dietary changes that reduce calories) and increased activity levels. In some cases, one or more other treatments effective for treating the cardiac disease or condition can be administered concurrently with the administration of a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution). In some cases, one or more other treatments effective for treating the cardiac disease or condition can be administered before and / or after the administration of a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution).

[0105] When treating a mammal (e.g., a human) having one or more blood clots as described herein (e.g., by administering a composition comprising one or more ionic liquids, such as a LATTE solution), the composition can be effective in reducing the size of the blood clots in the mammal (e.g., reducing the number of blood clots in the mammal and / or the volume of one or more blood clots in the mammal). For example, a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered as described herein to a mammal (e.g., a human) in need thereof (e.g., a human having one or more blood clots) to reduce the size of the blood clot by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

[0106] When a mammal (e.g., a human) having one or more blood clots as described herein is treated (e.g., by administering a composition comprising one or more ionic liquids, such as a LATTE solution), the mammal may have a disease or condition associated with one or more blood clots. Examples of diseases and conditions associated with one or more blood clots that can be treated as described herein (e.g., with a composition comprising one or more ionic liquids, such as a LATTE solution) include, but are not limited to, deep vein thrombosis (e.g., acute deep vein thrombosis and chronic deep vein thrombosis), antiphospholipid syndrome, arteriosclerosis, atherosclerosis, embolism (e.g., pulmonary embolism), stroke, and arterial thrombosis.

[0107] In some cases, a method of treating a mammal having one or more blood clots as described herein (e.g., by administering a composition comprising one or more ionic liquids such as a LATTE solution) can also include identifying the mammal as having one or more blood clots. Examples of methods for identifying a mammal as having one or more blood clots include, but are not limited to, a physical examination and / or imaging tests (e.g., venography and / or MRI).

[0108] In some cases, when a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) is used to treat a mammal (e.g., a human) having one or more blood clots, the composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can include the ionic liquid as the sole active agent for treating the blood clot.

[0109] In some cases, when a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) is used to treat a mammal (e.g., a human) with one or more blood clots, the composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) may include one or more (e.g., one, two, three, four, five or more) other therapeutic agents for treating blood clots. In some cases, the therapeutic agent for treating blood clots may be an anticoagulant. In some cases, the therapeutic agent for treating blood clots may be a thrombolytic. Examples of therapeutic agents for treating blood clots that can be administered to a mammal with cancer together with a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) include but are not limited to heparin, warfarin, dabigatran, apixaban, and rivaroxaban. In some cases, one or more other therapeutic agents may be administered together with a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution). In some cases, one or more other therapeutic agents may be administered independently of a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution). When one or more other therapeutic agents are administered separately from a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution), the composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered first and the one or more other therapeutic agents can be administered secondarily, or vice versa.

[0110] In some cases, a method for treating a mammal (e.g., a human) having one or more blood clots as described herein (e.g., by administering a composition comprising one or more ionic liquids, such as a LATTE solution) can further comprise subjecting the mammal to one or more (e.g., one, two, three, four, five, or more) other treatments (e.g., therapeutic interventions) effective for treating the one or more blood clots. Examples of other treatments that can be used to treat one or more blood clots as described herein include, but are not limited to, thrombectomy, thrombolytic therapy, and an inferior vena cava filter. In some cases, one or more other treatments effective for treating the one or more blood clots can be administered concurrently with the administration of a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution). In some cases, one or more other treatments effective for treating the one or more blood clots can be administered before and / or after the administration of a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution).

[0111] When treating a mammal (e.g., a human) having one or more infected tissues as described herein (e.g., by administering a composition comprising one or more ionic liquids, such as LATTE solution), the composition can effectively reduce the number of infected cells in the mammal. For example, as described herein, a composition comprising one or more ionic liquids (e.g., a composition comprising LATTE solution) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having cancer) to reduce the number of infected cells in the mammal by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

[0112] When treating a mammal (e.g., a human) having one or more infected tissues described herein (e.g., by administering a composition comprising one or more ionic liquids such as LATTE solution), the infected tissue can be any type of tissue. In some cases, the infected tissue may be located at a wound (e.g., a diabetic wound or a surgical wound). Examples of tissues that can be infected and can be treated as described herein (e.g., with a composition comprising one or more ionic liquids such as LATTE solution) include, but are not limited to, skin, abscess cavities, and enterocutaneous fistulas.

[0113] In some cases, a method of treating a mammal (e.g., a human) having one or more infected tissues described herein (e.g., by administering a composition comprising one or more ionic liquids such as a LATTE solution) can also include identifying that the mammal has infected tissue. Examples of methods for identifying that a mammal has infected tissue include, but are not limited to, physical examination and / or laboratory tests (e.g., blood and / or urine).

[0114] In some cases, when a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) is used to treat a mammal (e.g., a human) having one or more infected tissues, the composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE fluid) may include the ionic liquid as the sole active agent for treating the infected tissue.

[0115] In some cases, when a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) is used to treat a mammal (e.g., a human) having one or more infected tissues, the composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) may include one or more (e.g., one, two, three, four, five, or more) other therapeutic agents for treating the infected tissues. Examples of therapeutic agents for treating infected tissues include, but are not limited to, antibiotics, antifungal drugs, and combinations thereof, which can be administered to a mammal having one or more infected tissues together with a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution). In some cases, one or more other therapeutic agents can be administered together with a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution). In some cases, one or more other therapeutic agents can be administered independently of a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution). When one or more other therapeutic agents are administered independently of a composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution), the composition comprising one or more ionic liquids (e.g., a composition comprising a LATTE solution) can be administered first, followed by the one or more other therapeutic agents, or vice versa. The present invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0116] Example

[0117] Example 1: Percutaneous liquid ablative agents for tumor therapy and drug delivery

[0118] A co-crystal ionic liquid formulation was developed that can be used to ablate liver tumor tissue while delivering the drug uniformly throughout the ablation zone. A choline-geranylgeranate (CAGE) ionic liquid formulation, referred to herein as LATTE, was prepared and tested for needle-based delivery in normal rat, rabbit, and porcine liver tissue; diffusion capacity was monitored by micro-CT and real-time MRI imaging, and drug loading capacity was monitored by infrared imaging. Subsequently, ablation capability was demonstrated in a rat liver tumor model, a rabbit VX2 liver tumor model, and ultimately in 12 ex vivo human tumors.

[0119] LATTE was synthesized using salt metathesis of choline bicarbonate and geranic acid in a 1:1 molar ratio to produce a deep eutectic ionic liquid, as reported elsewhere (Banerjee et al., Adv Healthc Mater 6:1601411 (2017)). Briefly, pure choline and geranic acid ionic liquids were first prepared using salt metathesis. To this end, one equivalent of pure geranic acid (Sigma-Aldrich, St. Louis, MO) was recrystallized five times in acetone at -70°C in a 500 mL round-bottom flask and added to one equivalent volume of choline bicarbonate (80 wt% solution, Sigma-Aldrich, St. Louis, MO). The mixture was stirred at room temperature until CO2 evolution ceased. Residual HO was removed by rotary evaporation at 60°C for 2 hours and drying in a vacuum oven at 60°C for 96 hours. Various LATTE mixtures were prepared by mixing pure LATTE (100%) with 0.25 mg / mL indocyanine green in saline (ICG, Sigma-Aldrich, St. Louis, MO) in predetermined proportions. For example, a 6.25% LATTE solution was prepared by mixing 6.25% by weight of pure LATTE and 93.75% by weight of ICG solution. Using this method, 25%-LATTE, 50%-LATTE, and 100%-LATTE (pure) were also prepared for characterization. The livers of healthy rats received three subcapsular injections of 50%, 25%, or 6.25% LATTE solutions containing equal amounts of indocyanine green (ICG) and the radiopaque nanoparticle contrast agent Exitron (Figures 1a, b). The addition of contrast agents allowed tracking during and after the intervention via standard computed tomography (CT) imaging and, in the case of Exitron, demonstrated the ability of LATTE to co-transport nanoparticles (i.e., 110 nm) throughout the ablation zone.

[0120] After injection, LATTE rapidly diffused within the liver parenchyma, producing a well-defined ablation zone that remained visible for 24 hours without disrupting the liver capsule, thus demonstrating an immediate local effect (Figure 1b). 3D-reconstructed and segmented in vivo microCT images showed uniform enhancement at each treatment site, while saline containing a similar amount of Exitron showed no enhancement (Figure 1c). High-resolution microCT analysis showed that 25% LATTE had a larger diffusion volume compared to 6.25% or 50% (Figure 1d); this may be a result of the lower viscosity of 25% LATTE compared to 50% LATTE, as liquid viscosity directly affects its penetration and diffusion distribution within the tissue (Figures 2a-c). In addition, 25% LATTE demonstrated a comfortable manual injection force to facilitate percutaneous needle-based intratumoral delivery (Figure 2d). Histological evaluation of treated tissue sections showed that the ablation areas caused by 50%-LATTE and 25%-LATTE were similar and significantly larger than the size of the ablated liver parenchyma receiving 6.25%-LATTE (Figures 1e-h). The ablation zone exhibited necrosis associated with loss of cell nuclei, scattered red blood cell aggregation, and granulocyte infiltration, which abruptly transitioned to surrounding interstitial edema and cellular swelling. These data suggest that LATTE has a unique ability to transport and retain lytic molecules while exerting a potent tissue-destructive effect throughout the treated area. Based on the favorable mechanical, micro-CT, and histological appearance, 25%-LATTE was used in subsequent experiments.

[0121] Next, in vivo experiments were conducted in immunocompetent rats bearing highly malignant orthotropic N1S1 liver tumors using LATTE. LATTE was injected percutaneously, and evaluations also included CT imaging to mimic the clinical situation. Gross examination showed that intratumoral injection of 25%-LATTE resulted in significant tumor ablation at two weeks (Figures 1i-k). Grayscale US images showed that tumor masses were significantly larger in the saline-injected group compared to tumors that were five times smaller in the group receiving LATTE (Figures 1l-o). Following intravenous injection of Exitron contrast agent, these tumors were also subjected to high-resolution in vivo microCT imaging to accurately segment the liver tumors (Figures 1p-q). Analysis of the microCT images produced results similar to those from ultrasound, showing that LATTE-treated animals had significantly smaller tumor volumes two weeks after treatment (Figure 1r). A linear relationship was established between tumor volumes assessed by ultrasound and micro-CT, confirming the consistency and reproducibility of these measurements (Figure 1s). Tissue harvested from LATTE-injected tumors revealed a uniformly distributed, intense fluorescent signal from ICG extending to the tumor margins, whereas no ICG signal was detected in significantly larger tumors in the saline group (Figures 1t-u). Quantitative analysis of ICG diffusion and fluorescence intensity in the LATTE-treated group yielded a 28-fold larger diffusion area and significantly higher fluorescence intensity compared to the saline control group (Figures 1v-w). These data demonstrate that LATTE can successfully ablate highly malignant N1S1 liver tumors and evenly distribute and retain the co-administered ICG. Micro-CT images evaluated by a board-certified radiologist demonstrated that LATTE-treated tumors were characterized as completely responsive based solely on size criteria. These tumors were also harvested and evaluated histologically at two weeks. LATTE-treated N1S1 tumors demonstrated complete tumor necrosis with absent nuclear staining, whereas saline-treated tumors demonstrated hypercellularity, loss of normal tissue architecture, and significant N1S1 tumor cell infiltration (Figures 3a-b). Morphometric analysis revealed a significant reduction in tumor size and cell count in LATTE-injected tumors (Figures 3c-d). Quantitative analysis of proliferating (Figure 3e-f) or apoptotic cells (Figure 3g-h) showed that the number of proliferating cells in the LATTE group was significantly reduced and the number of apoptotic cells increased 24-fold compared with the control saline group (Figure 3l). In addition, LATTE-treated samples showed a 16-fold increase in macrophages expressing CD68 (Figure 4a-c) and enhanced CD3+ T lymphocyte infiltration within the treated tumor margins (Figure 4d-f). Immunostaining identified many CD3+ cells as CD8+ subsets (Figure 2g-i). These data indicate that LATTE treatment can lead to significant tumor destruction while effectively inhibiting cell proliferation and stimulating a strong immune response in the tumor area, suggesting that LATTE may enhance immunotherapy.Furthermore, analysis of serum samples collected two weeks after treatment revealed normal liver and kidney function, with no evidence of systemic effects or damage to the noncancerous liver, indicating that the treatment was safe (Table 1).

[0122] Table 1. Rat Serum Blood Chemistry: Serum levels of alkaline phosphatase (ALP) and alanine aminotransferase (ALT), creatinine (Cre), blood urea nitrogen (BUN), and glucose (Glu) were measured in serum aliquots using a DRI-CHEM 4000 analyzer. C-reactive protein (CRP) was measured using a quantitative ELISA. Changes in ALP and ALT are commonly used as indicators of altered liver function, while elevated creatinine levels indicate renal dysfunction. Results showed slight differences in ALP and BUN levels compared to controls, but all serum values ​​were within the normal range for healthy Sprague-Dawley rats (n = 6).

[0123] LATTE brine comparison P-value Normal range ALP U / L *211±29 *199±42 227±15 *p＝01 0-260 ALT U / L 57±13 51±10 48±10 0.4 10-190 CRE mg / dL 0.3±0.04 0.3±0.05 0.2±0 0.2 0.5-1.6 CRP ng / mL 459±38 472±39 425±58 0.76 300-600 BUN mg / dL **25±2.4 **25±4.2 18±1.5 **0.003 20-26 GLU mg / dL 268±75 220±54 196±17 0.2 190-280 Total protein g / dL 5.2±0.3 5.5±0.6 5.7±0.3 0.28 5-7

[0124] To determine the synergistic effects and concentrations of chemotherapeutic agents such as doxorubicin co-administered with LATTE, cell culture experiments were performed. To test whether LATTE exerts cytotoxic effects on human cancer cells, patient-derived cholangiocarcinoma, pancreatic adenocarcinoma cell lines, and HepG2 liver cancer cells were incubated with serial dilutions of LATTE (25-0.048% w / v) for 24 or 48 hours, and concentration-dependent cytotoxicity dose-response curves were obtained. Viability scores showed that an effective concentration (EC) of 0.18-0.3% that caused 50% cell death was 100%. 50 5a), indicating that LATTE exerts a strong cytotoxic effect on cancer cells even at very low concentrations. To examine whether dual treatment with LATTE and chemotherapy can enhance the anticancer effect, the viability score EC 50 Hep-G2 cells were incubated with LATTE and doxorubicin at selected concentrations and extensive synergistic analysis was performed (Figure 5a-b). Synergy plots showed a significant enhancement of synergistic cytotoxicity, with maximum effects observed at 24 and 48 hours for LATTE 0.19-0.39% and doxorubicin 2.5-5μM, respectively (Figure 5c-d). This data suggests that LATTE can not only induce cancer cell death but also maintain the function of chemotherapeutic drugs such as doxorubicin, resulting in synergistic effects that maximize tumor response and potentially extend the therapeutic range.

[0125] Next, doxorubicin and ICG were dissolved in 25%-LATTE and then injected into rat livers. NIRF imaging and histopathology of explanted rat livers at 3, 7, and 28 days after injection showed that doxorubicin was persistently retained throughout the affected area for up to 28 days (Figure 6a-h). This novel ability to retain chemotherapy within the ablation zone is highly desirable, suggesting that it may help prevent tumor recurrence, which often occurs in the surrounding area of ​​the ablation in a clinical setting.

[0126] Under US guidance, 25% LATTE was mixed with 184 μM doxorubicin and injected into rabbit liver tumors. The rabbit VX2 liver tumor model was used because it is a commonly used cancer model for testing preclinical interventional technologies. A 21-gauge vascular puncture needle was used to inject 1 mL of 25% LATTE containing doxorubicin, ICG, and exitron into the syringe into the tumor (Figures 7a-b). One hour after injection, the rabbit liver was exposed and examined, showing subcapsular accumulation of the LATTE mixture, similar to what was observed in rat N1S1 tumor injection (Figure 7c, dotted outline). In vivo ultrasound Doppler imaging (Figures 7d, e) and laser speckle perfusion scanning (Figures 7f-h) showed a significant decrease in perfusion in the tumor after LATTE injection. Micro-CT imaging using Exitron contrast imaging showed that the contrast agent was evenly distributed throughout the tumor and demonstrated that LATTE has the unique ability to deliver nanoparticles measuring 110 nm throughout the tumor tissue (Figures 7i-j). Furthermore, consistent with the micro-CT findings, NIRF imaging of explanted rabbit liver VX2 lesions demonstrated ICG (Figure 7k) and doxorubicin (Figure 7l) retention throughout the tumor lesion, with spread beyond the tumor margin, similar to the retention observed in rat N1S1 tumors (Figure 1m). Histological evaluation of LATTE-injected VX2 tumors revealed consistent tissue ablation and extensive necrosis compared with saline-injected tumors (Figures 7n and 7o). Similar to the rabbit experiments, a feasibility study of transdermal ablation was also conducted in pigs to demonstrate the practicality of image-guided LATTE delivery and the consistency of liver parenchymal spread and ablation. Figure 7p shows the injection of 2 mL of 25% LATTE mixed with ICG; Figure 7q demonstrates the echogenic appearance of the needle and LATTE within the liver tissue, allowing real-time tracking. Post-mortem, near-infrared fluorescence scanning of the explanted liver lobe revealed a strong fluorescence signal corresponding to the LATTE injection site (Figure 7r). Before hematoxylin and eosin staining, the injection site was harvested and histological sections were imaged with a fluorescence scanner. This localized the fluorescent area to the ablation zone (Figure 7s), indicating that the LATTE mixture penetrated and helped retain ICG throughout the ablation zone. Histological examination showed complete destruction of tissue architecture, interstitial edema, and loss of nuclear staining, consistent with rat and rabbit tissue (Figure 7t). This data suggests that transdermal LATTE injection is possible in pigs and produces similar ablation effects, enabling ablation zones up to 4 cm in length.

[0127] Because intrahepatic injection of LATTE results in rapid diffusion and significant volumetric tissue destruction, real-time magnetic resonance imaging (MRI) was used in a clinical interventional MRI suite to examine the temporal changes in the volume of the ablation zone in porcine livers. T1-weighted MR imaging sequences demonstrated a 2.8-fold increase in the circumferential spread of LATTE within 90 minutes of injection (Figure 7u-w). These data identified an early period of rapid diffusion of LATTE within seconds of intraparenchymal injection, followed by a period of gradual diffusion over 90 minutes.

[0128] Finally, to show whether LATTE can ablate human tumors, 12 consecutive freshly resected human tumors were collected and stored in RPMI medium; within 1 hour of resection, the ex vivo tumors were treated with 25% LATTE mixed with ICG. Tumor tissues were photographed and NIRF images were acquired 10 minutes after injection, followed by incubation at 37°C in a humidified tissue culture chamber in RPMI medium for 24 hours. At 24 hours, fluorescence scans were repeated, and the tissues were processed for histological evaluation. Fluorescence intensity and diffusion area were calculated using similar parameters for all tissues. Representative images of different types of human tumor tissue and their corresponding fluorescence scans at 10 minutes and 24 hours are shown in Figure 8. Analysis of the results showed that LATTE significantly diffused throughout the tumor tissue, peaking at 24 hours, with significant tissue destruction, consistent with results obtained from rat, rabbit, and pig liver (Figure 8). LATTE is capable of ablating a variety of highly malignant human tumors.

[0129] In summary, LATTE represents a new family of LRTs that can be easily delivered under US guidance. While LATTE is highly effective in ablating tissue, it can also be used as a drug carrier in free form or within nanoparticles for chemotherapy delivery, with retention within the ablation zone for up to 28 days. LATTE also induces a significant immune response at the tumor margin and may be an effective approach to activate T cells and improve solid tumor responses when combined with immunotherapy drugs. LATTE treatment has the potential to improve survival outcomes in HCC by enabling more patients to transition to liver transplantation.

[0130] Materials and methods

[0131] LATTE synthesis and formulation

[0132] Pure choline and geranate ionic liquids were prepared for the first time using a salt metathesis method. To this end, one equivalent of pure geranic acid (Sigma-Aldrich, St. Louis, MO) was recrystallized five times in acetone at -70°C in a 500 mL round-bottom flask and added to one equivalent of choline bicarbonate (80 wt% solution, Sigma-Aldrich, St. Louis, MO). The mixture was stirred at room temperature until CO2 evolution ceased. Residual H2O was removed by rotary evaporation at 60°C for 2 hours and drying in a vacuum oven at 60°C for 96 hours.

[0133] Various LATTE mixtures were prepared by mixing pure LATTE (100%) with 0.25 mg / mL indocyanine green (ICG, Sigma-Aldrich, St. Louis, MO) in saline at predetermined ratios. For example, a 6.25% LATTE solution was prepared by mixing 6.25% by weight of pure LATTE with 93.75% by weight of ICG solution. Using this method, 25% LATTE, 50% LATTE, and 100% LATTE (pure) were also prepared for characterization.

[0134] The viscosity was measured using an Anton Paar MCR 302 rheometer using a 1° aluminum plate with a diameter of 25 mm. The gap between the top and bottom plates was maintained at 0.048 mm. Before the experiment, the LATTE was placed on the bottom plate and equilibrated at 25 °C for 10 minutes. For each LATTE formulation, the viscosity was measured at 10 and 1000 s. -1 Flow curves were performed at shear rates between 10 and 20 days. Viscosity tests were performed in triplicate. In addition, the viscosity of each variant of LATTE was measured on day 0, day 10, and day 20 to assess its stability.

[0135] The syringeability of LATTE was tested using a mechanical tester (Instron, Model 5942). The injection force generated by different LATTE formulations loaded into a 1 mL syringe (Becton Dickinson, Franklin Lakes, NJ) at a flow rate of 10 μL / s through a 7 cm 21-gauge needle (COOK Medical, Bloomington, IN) was recorded. Each test was repeated five times.

[0136] Intraparenchymal injection of LATTE in normal rats

[0137] Sprague-Dawley rats were injected intrahepatically with the LATTE mixture by laparotomy. Anesthetized rats were placed in the supine position on a warming platform. Abdominal hair was removed with an electric razor, and the skin was scrubbed and disinfected with three alternating applications of povidone-iodine and 70% alcohol. After preparing and draping the abdomen in a standard surgical manner, a vertical subxiphoid mini-laparotomy incision was made with a No. 15 blade, and the peritoneum was exposed through the avascular linea alba using blunt dissection. The peritoneal wall was carefully divided with a retractor to visualize the liver. Care was taken to avoid tearing the liver capsule; the exposed organ was kept moist with wet gauze. Using a cotton-tipped applicator moistened with sterile saline, blunt forceps were used to expose and locate the left lower liver lobe. Two injections of 100 μl of the LATTE mixture were made in each lobe at 1 cm intervals using a 28-gauge needle syringe. The medial injection site received 100 μL of 25% LATTE and 65 μg of indocyanine green (ICG, Sigma-Aldrich) in normal saline, while the lateral injection site received 100 μL of 25% v / v LATTE, 65 μg of ICG, and 100 μg of doxorubicin in normal saline. After the injection was completed, the subcutaneous tissue was re-sutured in a continuous manner with 5-0 Velcro sutures, and the final dermis was approximated with 5-0 Velcro subcutaneous sutures. Subgroups of rats survived 1, 3, 7, or 28 days after injection. At the end of the survival period, the explanted liver was fixed and ex vivo fluorescence imaging was performed to detect ICG and doxorubicin at the two injection sites, and the diffusion area and fluorescence intensity were calculated based on the mean radiant efficiency.

[0138] Evaluation of tumor response to LATTE treatment in the N1S1 rat hepatocellular carcinoma model

[0139] All procedures were approved by the Institutional Animal Care and Use Committee and performed in accordance with institutional guidelines. Eighteen male Sprague-Dawley rats (Envigo, CA) initially weighing 300-325 g were used to induce hepatocellular carcinoma. N1S1 rat hepatoma cells (ATCC, CRL-1604, Manassas, VA) were cultured in Iscove's modified Dulbecco's medium (IMDM, ATCC, Manassos, VA) supplemented with 10% heat-inactivated calf serum (SH30072.03HI, HyClone, UT). N1S1 cells were plated at 75 cm 2 The cells were maintained in suspension in culture flasks at 37°C and 5% CO2. Before the tumor inoculation procedure, N1S1 cell viability was documented to be greater than 95% using trypan blue exclusion. To prepare cells for inoculation, N1S1 cell aliquots were rinsed and suspended in plain IMDM (without antibiotics or serum), with 2 x 10 cells in a 100 μL fresh aliquot obtained for each inoculation. 6cells. Under isoflurane anesthesia, the rat liver was exposed by upper midline laparotomy, and then N1S1 cells were subcapsularly inoculated into the left liver lobe. Gauze was gently pressed to stop bleeding to prevent cell reflux. The abdominal incision was sutured using interrupted Vicoli sutures (for abdominal muscles) and subcutaneous sutures (for skin), and then coated with Vetbond tissue adhesive (3M, St.Paul, MN) and recovered from anesthesia. In three groups, 0.5 cm 3 Rats with N1S1 tumor lesions were injected intratumorally with an ionic liquid mixture and measured using ultrasound. Intratumoral injections consisted of the following mixtures: 25% (w / v) LATTE, saline, or 100% ethanol. All solutions contained 0.25 mg / mL indocyanine green (ICG, Sigma-Aldrich, St. Louis, MO) dissolved in saline. Following intratumoral injection, treated rats were allowed to survive for 2 weeks, and tumor volume was recorded using ultrasound. Transcutaneous ultrasound was used to assess the progression and treatment response of N1S1 tumors. Selected focal depth, gain, and tissue harmonics settings were optimized during baseline initial imaging acquisitions, and the same parameters were applied during acquisitions following intratumoral injection of the LATTE mixture. Serial ultrasound examinations of rat livers were performed after N1S1 inoculation to confirm tumor formation and assess tumor volume. Rats were anesthetized using a gas mixture of 2-3% isoflurane in 100% oxygen via a nose cone. During imaging, the temperature was maintained at 37°C on an electronically controlled warming platform, and the rats were secured in a supine position. The abdominal area was shaved and prepared with depilatory cream (Nair, Church & Dwight Co.INC). Initially, abdominal ultrasound was performed using an ACUSON S2000 system (Siemens Inc., Germany) and a multi-frequency linear transducer (9L4, 9.0 MHz) to depict the tumor mass boundary in grayscale (B mode). The transducer was positioned to obtain a 2D scan through the subcostal window to record the liver tumor and measure the lesion diameter. The maximum diameter of the echo based on the superior-inferior (SI), lateral-medial (LM), and anterior-posterior (AP) planes of the tumor lesion was measured. The tumor volume was calculated as follows: V = (4 / 3) × π × (1 / 2) SI × (1 / 2) LM × 1 / 2 AP. Color Doppler images were also collected to detect the distribution of tumor blood flow per week.

[0140] Tumor volume measurement using microcomputed tomography

[0141] After direct injection using SkyScan-1276 (Bruker, Kontich, Belgium), micro-computed tomography (μ CT) was performed in rats or ex vivo to fixed liver tissue explanted from rats or rabbits. In order to show normal liver parenchyma on CT imaging, rats were injected with 400 μL alkaline earth metal-based nanoparticle suspension Exitronnano 12000 (Miltenyi Biotec, Auburn, CA) two hours before micro-CT scanning. Rats were anesthetized using 2-3% isoflurane inhaled at a constant flow rate of 2L / min in 100% O2 and placed on a box equipped with a heating system, continuous gas exchange and integrated real-time motion detection camera. Fixed rat tissue was placed in a humid plastic chamber and fixed using foam beads. In vivo micro-CT scans of the upper abdominal region were acquired after flat-field correction using a 0.25 aluminum filter and the following parameters: 85 kVp, 200 μA, 275 ms exposure, 20 μm pixel size, and 360° rotation with a 0.6° rotation step size, while the fixed liver tissue scanning protocol consisted of the following: 40 kVp, 200 μA, 288 ms exposure, 20 μm pixel size, and 360° rotation with a 0.4° rotation step size, with 2-frame averaging without using a filter. 3D image stacks were reconstructed using NRecon software and the InstaRecon CBR server (version: 1.7.4.6, Bruker, Kontich, Belgium), with subsequent adjustments for random motion, beam hardening correction, and ring artifact reduction and smoothing. Rendered 3D volumes were rendered using CTVox software (version: 3.3.0r1383, Kontich, Belgium). The stack was virtually rotated and axially oriented using Data Viewer software (Bruker, Kontich, Belgium) to generate transverse projections of the selected volume of interest (VOI). 3D morphometric analysis of the segmented VOI was performed using CTAn software (version: 1.18.8.0, Bruker, Belgium) to measure tumor volumes in vivo and ex vivo. Segmentation was performed by applying a global thresholding procedure followed by a series of morphological operations to separate highly opaque healthy liver from low-density tumor volumes. Tumor volume data are expressed in cubic centimeters as mean ± SEM (* indicates p < 0.05, **p indicates < 0.01, n = 6 per group).

[0142] Fluorescence imaging of explanted rat liver

[0143] To evaluate the differences in the diffusion and retention of ICG or doxorubicin after intrahepatic injection, ex vivo spectral fluorescence imaging of fixed liver tissue was performed using the IVIS200 system (PerkinElmer, Inc., USA). A cross-section of each tumor lesion was cut using a scalpel and positioned within the imaging system. Cross-sectional images were obtained after near-infrared irradiation with an excitation wavelength of 750 nm, while fluorescence emission was obtained at 850 nm to observe indocyanine green (ICG); doxorubicin was detected at an excitation wavelength of 460 nm and an emission wavelength of 560 nm. Bright-field photographs were also obtained for each imaging sequence. All fluorescence images were acquired using a 1-second exposure time (aperture (f / stop) = 2) and displayed using the same scale of fluorescence intensity. Fluorescence intensity was quantified in the region of interest (ROI) of each tissue. All images were acquired using the same illumination settings (lamp voltage, filter, f / stop (aperture value), field of view, distribution), and fluorescence emission intensity was normalized to the number of photons per square centimeter per second per steradian (p / s / cm2) in quantitative analysis. 2 / sr).

[0144] In vitro evaluation of LATTE cytotoxicity and synergistic effects with chemotherapy

[0145] HepG2 human hepatoma cell line (ATCC CRL10741; American Type Culture Collection, Manassas, VA, USA) was cultured at 75 cm 2 The cells were cultured in flasks using a growth medium consisting of low-glucose Dulbecco's modified Eagle's medium (DMEM, ThermoFisher) and 10% heat-inactivated bovine serum, supplemented with 100 IU penicillin and 10 μg / mL streptomycin (Thermo Fisher Scientific). The cells were incubated in a 5% CO2 incubator at 37°C until confluence; they were then detached using 0.05% trypsin-EDTA solution (Millipore Sigma) and plated at 5 × 10 3 Cells were seeded at a constant density / well in 96-well replica plates for 24 hours. The anticancer activity of LATTE against HepG2 cells was determined using the WST-1 reagent (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium) as an indicator of viability / cytotoxicity. The WST-1 viability assay is based on the reduction of the tetrazolium salt WST-1 to insoluble formazan via electron transport across the plasma membrane of living cells. Dye, thereby changing the optical density of the culture medium. After the 24-hour inoculation period, the growth medium was replaced with 200 μL of fresh growth medium containing serial dilutions of LATTE or doxorubicin in designated replicate wells. The cells were incubated for 24 or 48 hours after treatment. At the end of the incubation period, the culture medium was discarded, the wells were rinsed three times with DPBS solution, and then 100 μL of growth medium and 10 μL of freshly prepared WST-1 reagent were added to each well, followed by incubation in a 5% CO2 incubator at 37°C for 2 hours. After the incubation period, the optical density percentage was measured at a wavelength of 450 nm using a microplate reader (SpectraMax iD5, Molecular Devices, San Jose, California). Cell viability was calculated relative to the control wells that received growth medium alone. Viability was calculated as follows: Viability (%) = (1-OD 处理 / OD 对照 ) x 100%.

[0146] The viability score dose-response curves for LATTE or doxorubicin were used to calculate the concentration that caused cytotoxicity in 50% of HepG2 cells at 24 or 48 hours (EC50) to determine the synergistic effect score of the LATTE and doxorubicin treatment combination in vitro using statistical software (Prizm software, version 7, GraphPad, San Diego, CA). To establish the cytotoxic synergy of the combination of LATTE and doxorubicin at low concentrations, HepG2 cells were incubated with the single agents using 4-5 steps spanning the EC50 of LATTE or doxorubicin, respectively. 50 concentrations. Subsequently, all possible combinations of paired treatments were generated to produce a 4x5 matrix for each individually tested drug; (C) Pairwise concentration values ​​were interpolated from the fitted Hill curves of each single treatment dose-response curve using the Loewe model and the Loewe synergy score calculated and plotted using Combefit software. The graphical output consists of the following: single agent dose-response data and their fits, the combined dose-response, and a graphical mapping of the synergistic effect distribution on the dose-response surface. All reported values ​​are the average of three replicates, with 8 wells per dose level per study. A plot of the logarithmic concentration of each drug versus the viability score was generated for each study.

[0147] Induction of VX2 hepatocarcinoma in rabbits

[0148] An aliquot of preserved VX2 tumor tissue slurry obtained from anesthetized donor rabbits stored in liquid nitrogen was freshly thawed and suspended in 1 mL of DMEM tissue culture medium and then injected into the calf muscle of donor New Zealand female white rabbits using a 16-gauge needle. After tumor growth, muscle tissue containing VX2 tumor lesions was harvested, placed in ice-cold DMEM, and then cut into 1 mm 3Two freshly harvested and minced tumor tissue fragments were surgically implanted into a small incision in the medial lobe of the left liver using aseptic technique through upper abdominal exposure. The liver incision was gently compressed for three minutes using absorbable gelatin sponge (Ethicon, Inc., Summerville, NJ) to control bleeding. Ultrasound-confirmed VX2 tumor-bearing rabbits were injected with 2 mL of LATTE formulation under ultrasound guidance or after surgical exposure. One hour after injection, ultrasound imaging was repeated, and the rabbits were then euthanized and the livers were subsequently harvested and fixed.

[0149] Ultrasound-guided percutaneous injection of LATTE mixture into the liver of rabbits and pigs

[0150] Euthanized pigs or rabbits were injected with LATTE solution under ultrasound guidance. A mixture of 2 mL of 25% LATTE and 0.25 mg / mL indocyanine green (ICG) was loaded into a syringe (Becton Dickenson, Franklin Lakes, NJ) and placed on a 7 cm 21-gauge puncture needle (COOK Medical, Bloomington, IN). Ultrasound examination was performed using a high-frequency transducer (9-MHz multi-frequency linear probe, ACUSON S2000, Siemens, Germany) to visualize the left liver parenchyma. The location of the entry site was marked on the skin, and a small incision was made in the center of the marked line using a surgical blade. The puncture needle was passed through the skin incision in front of the transducer parallel to the long axis of the transducer until the desired location was reached, and then the LATTE mixture was slowly injected over one minute. Ten minutes after injection, liver tissue was collected and near-infrared fluorescence imaging (NIRF) (IVIS200, PerkinElmer, Inc. Waltham, MA) was performed, followed by fixation and histological evaluation.

[0151] Magnetic resonance imaging after subcapsular injection of LATTE solution in pig liver

[0152] After subcutaneous injection of 2 mL of a solution (including a 25% LATTE aqueous solution containing 0.25 mg / mL indocyanine green) with a 21-gauge vascular puncture needle, MR imaging of the explanted pig liver was performed. Injection images were collected at 0 and 90 minutes after injection. The pig liver was scanned using a 3TMAGNETOM Skyra MRI (Siemens Healthcare, Erlangen, Germany), which has an 18-channel front coil and a 32-channel posterior spine coil. The following MR scans were performed: coronal T2 single-shot fast spin echo (HASTE), FOV 300×300 mm, resolution 448×310, slice thickness 1.3 mm, TR 800 ms, TE 120 ms, BW 620 Hz / Px, 4NEX, scan time 3:15 minutes; this is for anatomical reference. A high-resolution coronal 3D T1 fast gradient echo volume scan (MPRAGE) with a FOV of 320 × 320 mm, a resolution of 512 × 512, a slice thickness of 0.6 mm, a TR of 1350 ms, a TE of 2.34 ms, a flip angle of 9 degrees, a TI of 900 ms, a bandwidth of 390 Hz / Px, and a scan time of 6 minutes achieved a 3D volume resolution of 0.6 × 0.6 × 0.6 mm; this sequence was performed to visualize the liver parenchyma. A high-resolution coronal 3D T2 fast spin echo volume scan (SPACE) with a FOV of 300 × 300 mm, a resolution of 320 × 320, a slice thickness of 0.9 mm, a TR of 1700 ms, a TE of 105 ms, a flip angle of 135 degrees, a bandwidth of 600 Hz / Px, a NEX of 1.4, and a scan time of 9:24 minutes achieved a 3D volume resolution of 0.9 × 0.9 × 0.9 mm; this sequence was performed to visualize blood vessels. Coronal T2 cine rapid steady-state free precession (TRUFI) scans were acquired with a FOV of 380 × 380 mm, a resolution of 256 × 256 pixels, a slice thickness of 5 mm, a TR of 630 ms, a TE of 11.77 ms, a flip angle of 60°, a bandwidth of 1300 Hz / px, a scan time of 3:09 minutes, and a temporal resolution of approximately 1.5 frames / second. Scans were performed at 0 and 90 minutes after injection. Segmentation and volume calculations were performed using Materialise3-Matic and Mimics 3D image processing software (Materialise, Belgium).

[0153] Human cancer tissue processing

[0154] Explanted human cancer tissue was collected after surgical resection and stored in RPMI medium. A 25-gauge needle was used to inject a 25% LATTE mixture into the core of the tumor mass. Near-infrared fluorescence imaging (NIRF) was performed 5 minutes after injection. The treated tissue was incubated in a humidified chamber for 24 hours, partially immersed in tissue culture medium. NIRF was repeated 24 hours after LATTE injection. The tissue was cross-sectioned to reveal the ablation area, fixed in 10% buffered formalin, embedded in paraffin, and then sectioned and stained with hematoxylin and eosin for microscopic evaluation.

[0155] Histopathology and immunohistochemistry

[0156] At autopsy, liver tissue was collected and fixed in 10% buffered formaldehyde and transected axially to expose the core of the treated area. Each liver was scanned for fluorescence before and after transsection, and the tissue was subsequently embedded in paraffin and serially sectioned to generate 4 μm thick sections. Serial sections were stained with hematoxylin and eosin (H&E) to observe tissue morphology and cell infiltration, or immunohistochemistry (IHC) staining was performed. To identify actively proliferating cells and cells undergoing apoptosis, tissue sections were incubated with a 1:250 dilution of proliferating cell nuclear antigen-specific rabbit IgG (PCNA, AB13847, Abcam) or an IgG that recognizes cleaved caspase 3 (1:250, AB13847 Abcam, MA). Rat anti-mouse CD3-IgG3, κ (1:20, 550295, BD Pharmingen) was used to visualize naive T cells; polyclonal rabbit anti-CD68 IgG (1:250, AB125212, Abcam) was used to identify local monocytes and macrophages; those used to characterize neutrophil-granulocyte infiltration included rabbit monoclonal anti-myeloperoxidase IgG (MPO, 1:250, AB208670, Abcam.). The sections were then incubated with goat anti-rabbit horseradish peroxidase-conjugated IgG HL (1:300, AB97051, Abcam) secondary antibody at room temperature for 30 minutes. Specific proteins were detected using 3,3′-diaminobenzidine (DAB, Dako) reagent and counterstained with hematoxylin. The sections were dehydrated and coverslipped using Richard-Allen loading medium (ThermoFisher Scientific). An EVOS FL automated microscope was used to obtain mosaic digital micrographs. Appropriate thresholds and particle sizes were set to count the number of positive cells per field of view, and limited concentric radial measurements of each cross-sectional area were compared for all tumor samples. Data are expressed as the mean number of positive cells per cubic millimeter.

[0157] Statistical analysis

[0158] All results were analyzed using Prism software, version 7 (GraphPad, San Diego, CA) to assess statistical differences between groups. Data are reported as mean ± standard error of the mean (SEM) or percentage (%), as appropriate for categorical variables. Differences between two groups were compared using the Mann–Whitney test (U test), or continuous variables were analyzed between three groups using analysis of variance (ANOVA) for comparisons between different treatments. Linear regression plots of in vivo measurements of tumor size determined by ultrasound (US) or micro-CT imaging were compared by Pearson's factor to analyze the correlation of values ​​obtained using the two imaging modalities. A p value of ≤0.05 for each comparison was considered statistically significant.

[0159] Taken together, these results suggest that LATTE compositions can be used alone or in combination with chemotherapeutic drugs to ablate tumor tissue for the treatment of cancer.

[0160] Example 2: Effect of ablative agents on fat

[0161] To demonstrate the ability of LATTE to ablate adipose tissue, a 25% LATTE mixture was injected subcutaneously in pigs.

[0162] Skin tissue collected for histological evaluation showed complete ablation of adipocytes (fat cells) in the treated area ( Figure 9 ) and demonstrated that LATTE was able to dissolve subcutaneous adipose tissue without a significant inflammatory response. Additional experiments were performed on explanted adipose tissue obtained from the inguinal region of pigs in culture dishes immersed in saline ( Figure 10 ). The explanted adipose tissue was then incubated with 25% LATTE and showed complete adipocyte lysis within 10-15 minutes of incubation. LATTE mixed with indocyanine green was injected into the adipose tissue and showed significantly enhanced fluorescence in the ablation area using near-infrared imaging ( Figure 11 ).

[0163] Taken together, these results suggest that LATTE compositions can be used to ablate adipose tissue for the treatment of obesity.

[0164] Example 3: Effects of ablative agents on blood

[0165] To demonstrate the ability of LATTE to lyse cells in human blood, whole blood was placed in a slide chamber after dilution with saline and then observed and photographed under a bright field microscope ( Figure 12A After incubation with 25% LATTE solution, all blood cells could not be detected in the slide showing intact cells ( Figure 12B). In another independent experiment, coagulation was induced in non-coagulating subjects. A blood volume of 4 mL of citrated blood was loaded into a polypropylene tube and mixed with 400 μL of 0.2 M calcium chloride (CaCl2) for 10 seconds. 100 μL aliquots were deposited in multiple wells of a 96-well plate, and the remaining blood volume remained in the test tube. Coagulation was initiated by incubating the samples at 37°C for 10 minutes. After coagulation was complete, 100 μL of 25% LATTE was overlaid on the coagulated blood in the wells, while the control wells were overlaid with a similar volume of saline, as shown in FIG. Figure 13 A and 13B. In addition, the blood clot formed in the tube was moved to a culture dish and then immersed in 25% LATTE, as shown in Figure 13 As shown in C, it leads to thrombolysis.

[0166] LATTE causes blood clots to dissolve in real time. It could be a potential treatment for both acute and chronic deep vein thrombosis.

[0167] Together, these results suggest that LATTE compositions may be used to ablate blood clots to treat diseases and conditions associated with blood clots.

[0168] Example 4: Effects of ablative agents on cardiac tissue

[0169] Under ultrasound guidance, the septum of pig heart tissue received an injection of 1cc of LATTE mixture and ICG. One hour later, the myocardium was removed for near-infrared imaging (NIRF) and histological examination. NIRF imaging showed localized enhanced fluorescence in the myocardium at the injection site ( Figure 14A Histological sections of the injection site showed significant ablation of myocardial fibers limited to the treated area ( Figure 14B ).

[0170] Taken together, these results suggest that LATTE compositions may be used to ablate cardiac tissue to treat cardiac diseases and disorders.

[0171] Example 5: Nanogel Formulation for Effective Tissue Ablation and Drug Delivery

[0172] This embodiment describes the synthesis and characterization of ionic liquids (ILs) based on choline. Selected ILs are used to prepare hydrogels containing LATTE (nanogel formulations) and doxorubicin (Dox), immune checkpoint inhibitors (ICIs) and / or imaging agents. The properties (such as viscosity, elastic modulus, injectability and sterility), cytotoxicity characteristics and drug release kinetics of nanogel materials are evaluated in vitro. Tissue ablation, drug diffusion / delivery and retention are also evaluated in vivo in normal rat liver.

[0173] method

[0174] Nanogel preparation and characterization

[0175] Stock solutions of pure CAGE ILs were prepared using salt metathesis as described elsewhere (see, e.g., Zakrewsky et al., Adv. Healthc. Mater., 5(11):1282-9 (2016); Banerjee et al., Adv. Healthc. Mater., 6(15)(2017)). Briefly, geranic acid (Sigma Aldrich, St. Louis, MO) was recrystallized five times in acetone at -70°C in a 500 mL round-bottom flask and a molar equivalent of choline bicarbonate (80 wt% solution, Sigma Aldrich, St. Louis, MO) was added. The mixture was stirred at room temperature until CO2 evolution ceased. Residual HO was removed by rotary evaporation at 60°C for 2 hours and drying in a vacuum oven at 60°C for 96 hours. After incubation at 4°C or 65°C, the long-term stability of the ILs was verified using NMR. Any IL that did not meet the optimal physical properties or long-term stability was excluded. To prepare the nanogels, use XLG (BYK) was prepared by physical mixing in ice-cold water. Nanosilicate (NS) hydrogels were prepared from powders as described elsewhere (see, e.g., Albadawi et al., Adv. Sci., 2020; 8(1):2003327 (2020); Avery et al., Sci. Transl. Med., 8(365):365ra156 (2016)). Aliquots of freshly prepared NS hydrogels were mixed with a constant weight ratio of Dox, ICI, or ExiTron nano12000 (Miltenyi Biotec, Germany) and efficiently mixed using a SpeedMixer (DAC-150.1, FlackTek Inc). Various nanogel formulations were prepared by combining aliquots of NS hydrogels containing Dox or species-specific ICI with the relevant amount of pure IL (100%) at predetermined weight ratios to produce nanogels with a range of IL concentrations (6.25, 12.5, 25, and 50 wt% IL). Anti-PD1 (LSBio, anti-rabbit PD1, LS-C55247) and anti-PDL1 (Biorbyt, anti-rabbit PDL1, orb228661) were used as ICI candidates.

[0176] The physical properties of each nanogel formulation were analyzed to determine viscosity and syringeability (rheometer and injection force testing), molecular integrity, conductivity, and density.

[0177] Nanogel viscosity variables of NS (2-6 wt%) were tested to produce nanogels with ease of syringeability (rheology) and stability after injection.

[0178] Drug release kinetics from nanogels

[0179] To understand the interactions between anticancer drugs and NS and IL, as well as possible interactions between anticancer drugs, the release kinetics of synergistic IL / Dox / ICI-loaded nanogel formulations were compared with the release profiles of Dox-loaded and ICI-loaded hydrogels prepared individually. Dox and ICI were loaded onto NS hydrogels in the same manner (NS was prepared first, followed by the addition of Dox or ICI). To evaluate the release profiles, nanogels were synthesized, and 200 mg aliquots of the different formulations, along with IL, Dox, or ICI, were dispensed into transwell inserts fitted with polyethylene terephthalate filters and incubated at 37°C for 30 days. The release kinetics of Dox or / and ICI were continuously analyzed at different time points using fluorescence intensity from Dox- or ICI-specific ELISAs and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Furthermore, to assess the chemical structure stability and functionality of IL, ICI, and Dox, the samples were analyzed using FTIR and nuclear magnetic resonance (NMR). Each formulation was also subjected to zeta potential analysis (Malvern Panalytical) to analyze any changes in surface charge, corresponding to possible interactions and their effects on the release profile. In addition, injection force (Instron), viscosity, storage / loss modulus (rheometer), and injectability testing (Instron) were analyzed as described elsewhere (Albadawi et al., Adv. Sci., 2020; 8(1): 2003327(2020); Avery et al., Sci. Transl. Med., 8(365): 365ra156(2016)).

[0180] Determining nanogel cytotoxicity and synergy with chemotherapy

[0181] The cytotoxic effects of the selected nanogel formulations were evaluated in different human liver cancer cell lines, including human hepatocellular carcinoma (CRL10741 American Type Culture Collection, Manassas, VA) and cholangiocarcinoma (SNU-478). In addition, the selected nanogel formulations were tested in animal cancer cell lines, including: rat hepatocellular carcinoma cell line N1S1 (ATCC, CRL-1604) and mouse colorectal adenocarcinoma cell line MC38. Both cell lines were used to establish cancer models for in vivo experiments. The viability scores (IC 50) were measured after incubation with nanogel extracts, which were compared to cells treated with the single components using a WST-1 assay (Cayman Chemicals, Ann Arbor, MI) and a microplate reader (SpectraMax iD5, Molecular Devices, San Jose, CA), as described elsewhere (Albadawi et al., Sci. Transl. Med., 13(580)(2021)). To assess the effects of nanogels on cancer cell energy metabolism, steady-state levels of ATP, NAD / NADH ratio, and lactate were measured using a chemiluminescent assay (Promega, Madison, WI), as described elsewhere (Albadawi et al., Sci. Transl. Med., 13(580)(2021)). To determine the effects of nanogels on Dox intracellular uptake and nuclear localization, confocal microscopy was performed on cells grown on microscope slide chambers. To quantify the uptake of Dox in cells, nuclear, or mitochondrial compartments, cells were isolated and suspended in 0.5 mL ethanol / 0.3 N HCl. The amount of Dox retained was determined using a fluorescence plate reader and extrapolated from the calibration curve. In order to evaluate the ICI-loaded nanogels in vitro, HepG2 cells were also used to test the in vitro immunogenicity. Primary isolated rabbit or human splenocytes (Zen-Bio) were pre-activated for 2-4 days with well plates coated with ICI-loaded nanogels at different concentrations to stimulate anti-tumor sensitivity. Activated splenocytes were then co-cultured with HepG2 at a ratio of 1:20 and 1:40 in commercially available ELISPOT modules (MABTECH, IgG (#3865-2H), IFN-γ (#33321-2H), TNF-α (#3511-2H)) for 48 hours.

[0182] Rheological testing

[0183] Rheological evaluation of the nanogels or Ns hydrogels was performed using an Anton Paar MCR 302 rheometer (Anton Paar USA Inc., Torrance, CA). All measurements were performed using a 25 mm diameter sandblasted aluminum upper plate and aluminum lower plate with a 500 μm gap between them. Flow curves and amplitude sweeps (10 rad s) were obtained at 25 °C and 37 °C. -1 For the tests at 37°C, a solvent trap was used and filled to the brim with water to provide a moist environment. For each experiment, data were obtained in at least three replicates.

[0184] Injectability

[0185] The injectability of NS hydrogel or NG through clinical catheters was studied using a mechanical tester (Instron, Norwood, MA). Bluehill software version 3 (Instron, Norwood, MA, US) was used to record the force required for NG or NS gel (loaded into a 1cc BD syringe) to pass through a 2.8F, 110cm catheter (Terumo Medical Corporation, Somerset, NJ) at a flow rate of 1mL / min. Subsequently, the injection force for each sample was obtained.

[0186] Scanning electron microscopy (SEM)

[0187] After freezing at -80 ° C and then lyophilizing (Labconco, 0.120 mbar and -50 ° C), the microstructure of the nanogels or NS hydrogels was observed using a scanning electron microscope (JCM-6000 Plus). All prepared samples were then sputter-coated with 7 nm gold / palladium (Leica EMACE200) and imaged using SEM.

[0188] Fluorescence imaging of nanogels in vitro

[0189] In vitro spectral fluorescence imaging was performed on 250 μL aliquots of nanogel or NS hydrocolloid containing 1.25 mg / mL Dox or 0.25 mg / mL ICG, which were loaded into 96-well plates to evaluate the difference in doxorubicin or ICG after intratumoral injection of nanogel or NS hydrocolloid using the IVIS200 system (PerkinElmer Inc., Waltham, MA). Fluorescence images of doxorubicin were obtained using an excitation wavelength of 460 nm and an emission wavelength of 560 nm. ICG was visualized using near-infrared irradiation with an excitation wavelength of 750 nm and an emission wavelength of 850 nm. The same set of 1 -s Fluorescence images were collected from different experimental samples using an exposure time (f-stop = 2) and displayed using the same scale in each group. Each plate was incubated in a humidified chamber at 37°C and imaged continuously for 56 days. The fluorescence intensity in the wells was quantified using the radiance value in the region of interest and normalized to the number of photons per square centimeter per second per steradian (p / s / cm 2 / sr), and the fluorescence enhancement area in each sample was calculated after applying a normalized threshold.

[0190] Cytotoxicity

[0191] Cytotoxicity of the human hepatoma cell line HepG2 (CRL10741, American Type Culture Collection, Manassas, VA) was assessed after incubation with serially diluted nanogel extracts under growth conditions consisting of Dulbecco's modified Eagle's medium (DMEM, ThermoFisher Scientific, Waltham, MA) and 10% heat-inactivated bovine serum supplemented with 100 IU penicillin and 10 μg / mL streptomycin (Thermo Fisher Scientific, Waltham, MA). Cells were seeded at a density of 5,000 cells per well in 96-well replicate plates for 24 hours. After 24 hours of seeding, the medium was replaced with 200 μL of fresh growth medium containing serially diluted nanogel extracts in designated replicate wells and incubated for 24 hours. At the end of the incubation period, the medium was removed, and the wells were rinsed three times with Dulbecco's modified phosphate-buffered saline (DPBS, Sigma-Aldrich, Saint-Louis, MO), followed by the addition of 100 μL of growth medium. The cytotoxicity of the nanogels was determined by adding 10 μL of freshly prepared water-soluble 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium reagent (WST-1, Cayman Chemicals, Ann Arbor, MI) solution to each well and then incubating in a 5% CO2 humidified incubator at 37°C for 2 hours. The output of WST-1 was assessed by measuring the optical density at a wavelength of 450 nm using a microplate reader (SpectraMax iD5, Molecular Devices, San Jose, CA). Cell viability was calculated relative to control wells that received an aliquot of growth medium alone. Viability was calculated as follows: Viability (%) = (1 - OD 处理 / OD 对照 ) x 100%. The concentration that induces cytotoxicity in 50% of HepG2 cells at 24 h (IC) was calculated using Prism software version 8 (GraphPad, San Diego, CA) using a viability fraction dose-response plot. 50 ).

[0192] Assess sterility

[0193] The sterility of NS or NG was tested using Escherichia coli (E. coli) according to established protocols with minor modifications. 7 CFU mL -1 10 mL of E. coli suspension was added on top of 1 mL of gel to achieve 10 8A final concentration of CFU / mL of gel was used as a positive control. A sample of pain Luria-Bertani (LB) broth was used as a negative control. All groups were incubated at 37°C in a shaker incubator at 180 rpm for 24 hours. The optical density of the suspension was measured at 600 nm using a microplate reader. Each suspension was measured three times, and each test was performed three times independently.

[0194] result

[0195] The mechanical properties of NG were characterized. NS was used to provide NG with shear-thinning and drug-loading properties, allowing it to be injected directly into tumor lesions via a needle or injected into tumor-feeding vessels via a catheter to cause embolization. Initial experiments were conducted to evaluate the shear-thinning properties of hydrogels containing different proportions of NS. Hydrogels containing 3wt%, 4.5wt%, 6wt% or 9wt% NS exhibited shear-thinning behavior ( Figure 15A The effect of increasing the NS ratio on the storage modulus (G′) of hydrogels containing 3 wt%, 4.5 wt%, 6 wt% or 9 wt% NS is shown in Figure 2. Figure 15B As shown. 3wt% NS provided sufficient storage modulus with the least amount of solid material in the hydrogel formulation and was therefore chosen for further characterization. To evaluate the effect of adding different concentrations of ionic liquid (IL) to the hydrogel formulation, NG containing 3wt% NS and 6.25wt%, 12.5wt%, 25wt% or 50wt% IL were prepared and their mechanical properties were tested by rheometer. Rheological results showed a concentration-dependent increase in G' in nanogels containing 1.25wt% or 25wt% IL compared to NS alone ( Figure 15C ,and Figure 15B Since 25 wt% IL was demonstrated to be effective in tissue ablation in previous examples, NG formulations containing 25 wt% IL were generated for further testing.

[0196] Next, the researchers investigated the effects of adding other ingredients, such as anticancer drugs, on the mechanical properties of NG. NG containing 3 wt% NS and 25 wt% IL was mixed with 1.25 mg / mL of the anticancer drug doxorubicin, 1 mg / mL of the immunotherapeutic agent nivolumab (an anti-PD-1 antibody), or 0.25 mg / mL of the near-infrared fluorescent agent ICG, which has been used to track drug delivery in in vivo studies. Compared with NS, the G′ of the different NG formulations increased significantly ( Figure 15D Compared with using NG alone, the added components have little effect on viscosity and G' value ( Figure 15E ).

[0197] To verify whether NG can be conveniently injected through an angiographic catheter, injection force tests were performed. The injection forces generated by different NG formulations in a 1cc syringe and injected through a 110cm 2.8F microcatheter at an injection rate of 1mL / min showed that the injection was comfortable with an average hand, and the addition of these components to NG had no effect on the injection force values ​​( Figure 15F (G) Exemplary NG formulations containing doxorubicin, nivolumab, or iohexol Figure 15G shown.

[0198] The microstructural appearance of NG was evaluated. NG containing 3 wt% NS, 25 wt% IL, 1 mg / mL nivolumab, or 0.25 mg / mL ICG was loaded into a clinical-grade syringe equipped with a 21-gauge vascular puncture needle for direct intratumoral or intravascular injection ( Figure 16A Images showing NG injected through a needle show that it maintains consistency as it leaves the needle tip and exhibits clear shear-thinning behavior ( Figure 16B ). Figures 16A to 16F Microscopic and SEM images of NS, NS mixed with Nivo (NS+Nivo), NG alone, and NG mixed with 1 mg / mL Nivo are shown, respectively, and reveal the porous microstructures in the NS and NS+Nivo hydrogels compared to the less porous and network-like microstructures in the nanogels containing ionic liquid or NG+Nivo. These data suggest that IL alters the interactions of the nanocomposites in the NS hydrogels.

[0199] The effects of IL on blood coagulation and cell death were evaluated in vitro. To evaluate the effect of IL on cell death at the edge of the ablation zone, in particular the effect of IL on inflammatory cells, blood treated with low concentrations of IL was used to simulate the concentration along the ablation edge. To analyze the effects of ionic liquids on blood coagulation, erythrocyte hemolysis, and immune cells, blood smears were analyzed after incubation of pig blood aliquots with different concentrations of IL, including rheology, hemolysis analysis, and complete blood counts. Stained blood smears prepared from pig blood treated with IL showed concentration-dependent morphological changes, decreased white blood cell counts, and evidence of complete hemolysis ( Figure 17A Control and 0.78 wt% IL-treated pig blood showed consistent G' and G" modulus profiles, whereas 1.56% IL-treated blood showed delayed clotting lag time and lower G' and G" (modulus) levels compared to control blood ( Figure 17B ). 3.12 wt% IL treated blood failed to clot during the 30 min test period ( Figure 17BSince 3.12 wt% IL did not induce coagulation, higher concentrations were not tested by rheometer. Quantitative analysis of the lag time to the onset of coagulation (t-lag) showed a slight increase in the lag time at 0.78% IL treatment and a significant increase in the lag time at 1.56 wt% IL treatment ( Figure 17C The storage modulus (G′) 30 minutes after IL treatment showed that the storage modulus decreased by about 20% at 0.78% IL, compared with a decrease of 95% at 1.56wt% IL and a decrease of 100wt% at 3.12wt% IL. Figure 17D ), indicating that IL has a concentration-dependent anticoagulant effect. The hemolysis test of pig blood treated with IL showed a significant increase in hemolysis ( Figure 17E A complete blood count was performed on aliquots of fresh blood treated with increasing concentrations of IL, and red blood cells (RBC, Figure 17F ) and white blood cells (WBC, Figure 17G ) decreased in a concentration-dependent manner, and the total count of granulocytes ( Figure 17J ) and monocytes ( Figure 17I ) counts were paralleled by a similar decrease in the lymphocyte counts. Lymphocyte counts in the same aliquots did not change compared to the control group ( Figure 17H ), indicating resistance to IL treatment at the tested concentrations.

[0200] The effect of NG on drug diffusion and stability was evaluated in vitro. (A) Fluorescence images show the radiative diffusion of the naturally fluorescent anticancer drug. Doxorubicin was added to NS hydrogels (control hydrogels without ionic liquid) or to nanogels containing 6.25%-IL or 25%-IL. Equal aliquots were loaded into the center of designated wells of 2% agarose cast in multiwell plates. Fluorescence images were acquired over a 24-hour period to assess radiative diffusion ( Figure 18A A consistently larger Dox diffusion area was observed in NG+Dox with 25 wt %-IL compared to NG+Dox with 6.25 wt %-IL ( Figure 18B ). The radiation diffusion in the NS+Dox-loaded wells was limited throughout the 24-hour test period ( Figure 18B ). Showing the fluorescence of Dox incorporated into NG or NS hydrogels ( Figure 18C ) or ICG( Figure 18D ) showed that Dox and ICG were continuously enhanced over 56 days, compared with the decreased detection in NS hydrogels. These data indicate that ILs mediate the diffusion of co-administered drugs and enhance their stability.

[0201] The drug release kinetics, cytotoxicity, and sterility of NG were evaluated in vitro. The effects of the nanogel on HepG2 cell viability, Dox diffusion, and release were observed. Cumulative release of doxorubicin was observed in NG mixed with 0.25 mg / mL Dox after incubation for 7 days under physiological (pH = 7.4) or acidic (pH = 5.0) conditions, indicating sustained release of doxorubicin. Figure 19A ). HepG2 cells were treated with serially diluted NG extracts and the viability fraction was observed 24 h after treatment, resulting in an IC of 0.14% for the IL concentration. 50 , indicating that the cytotoxic effect of NG was retained ( Figure 19B The cytotoxicity of HepG2 cells was also enhanced after treatment with NG extract containing 0.25 mg / mL Dox (NG+Dox) compared with NS extract containing a similar amount of doxorubicin (NS+Dox), indicating a synergistic effect ( Figure 19C NS alone did not show cytotoxic effects ( Figure 19C NG and NG+Dox remained sterile after 24 hours or 2 months of culture in LB broth ( Figure 19D LB broth inoculated with E. coli was used as a positive control ( Figure 19D ).

[0202] These data collectively suggest that IL-containing NS hydrogels can be used to induce cancer cell ablation, maintain and enhance the function of anticancer drugs such as Dox, and synergistically maximize the anticancer response.

[0203] Example 6: Drug distribution ability of nanogel after injection

[0204] This example describes the ablation and drug distribution capabilities of the nanogels following injection into the rat liver.

[0205] method

[0206] Nanogel preparation

[0207] Nanogels were prepared according to the method described in Example 5.

[0208] Experimental design

[0209] In 6 groups of Sprague-Dawley rats (Charles River, 192 rats, 10-12 weeks, 250-300 grams, 1:1 male and female), normal rat livers were injected with nanogel formulations. The rats in each experimental group received injections of nanogel formulations and were compared with rats that received intraparenchymal injections of NS-IL, NS-Dox and / or NS-ICI. Assuming a standard deviation of 30-40%, a 90% confidence level and a 0.05α level, 8 rats were used for each data point to achieve statistical significance based on efficacy analysis (p<0.05). Briefly, anesthetized rats were placed in a supine position on a warming platform. After standard surgical preparation, the liver was exposed by laparotomy and three intraparenchymal injections of 50 μL of nanogel formulation, NS-IL, NS-Dox or NS-ICI were performed on the lower left liver lobe using a 27-gauge needle.

[0210] Result Analysis

[0211] To assess ExiTron diffusion, in vivo microcomputed tomography (microCT) was performed immediately after injection to obtain baseline volumes, as described elsewhere (Albadawi et al., Sci. Transl. Med., 13(580)(2021)). Subsequently, a subgroup of rats underwent microCT follow-up at 3, 7, 14, and 28 days after injection and were subsequently euthanized. At autopsy, the rat liver was explanted and transected into two parts at the midline through each injection site for ex vivo fluorescence imaging to calculate the Dox diffusion area and fluorescence intensity at each injection site. Subsequently, the tissue was incubated with triphenyltetrazolium chloride (TTC, Sigma) reagent to assess tissue viability or processed for histological evaluation. To calculate the ablation area, freshly harvested tissue was incubated in 1% TTC solution to visually distinguish between surviving and dead / dying areas. The ablation area and volume were calculated using the total ablation area and apoptosis versus necrosis area in the transverse and longitudinal planes as described elsewhere (Albadawi et al., Sci. Transl. Med., 13(580)(2021); Bhonsle et al., J. Vasc. Interv. Radiol., 27(12):1913-22e2(2016); Siddiqui et al., HPB (Oxford), 2016; 18(9):726-34(2016); Stadlbauer et al., Histol. Histopathol., 31(1):115-29(2016)). After fixation, serial sections were prepared and analyzed to confirm the ablation area, assess vascular integrity, and evaluate biliary architecture. The degree of apoptosis was assessed in sections immunostained for cleaved caspase-3 IgG and scored across the entire treatment area using ImageJ IHC profiler software. In addition, the local inflammatory response, including T lymphocytes (CD3 + 、CD4 + 、CD8 + ), NK cell infiltration, and quantitative analysis of myeloid cell infiltration were performed. Systemic responses in blood samples were compared using complete blood counts and assessment of markers of liver function (ALT, AST, GGT, LD, ALP, and bilirubin), renal function (creatinine and urea nitrogen), and cardiac injury (CK, troponin, C-reactive protein, and lipids). In addition, serum samples were analyzed for cytokine and chemokine levels (Eve technology). Tissue specimens of the brain, lung, kidney, liver, and spleen were collected for histological analysis to rule out any abnormalities.

[0212] result

[0213] Image-guided intrahepatic injection was used to deliver 50 μL of different hydrogel formulations, all containing 0.25 mg / mL ICG, into normal rat livers.

[0214] In vivo ultrasound imaging during direct injection of the nanogel into the liver parenchyma demonstrated the feasibility of direct percutaneous injection into the target tissue ( Figures 20A-20B Neither Dox nor Nivo affected the signal enhancement on MRI ( Figure 20D and 20E ). These data suggest that NG can be used for real-time image-guided injection using ultrasound or MRI.

[0215] 3D-reconstructed micro-CT and ICG fluorescence analysis of rat livers showed tissue ablation at each injection site in rats receiving NS, NG, or NG+Dox ( Figure 21A 、 21C and 21E). NG+Dox induced higher ablation volumes at all time points, which was associated with a time-dependent decrease in ablation volume at day 28 after injection ( Figure 21A 、 21C and 21E), reflecting the normal healing of the ablation area. Near-infrared imaging of explanted rat livers showed that after NS, NG, or NG+Dox injection, the ICG-enhanced fluorescence area at each injection site ( Figure 21B 、 21D and 21F). Compared with NS, a larger ICG diffusion area was observed 28 days after injection of NG and NG+Dox ( Figure 21B 、 21D and 21F), indicating that the diffusion and retention of ICG were higher in the presence of IL in the nanogel formulation. After injection of NG+Dox, Dox enhanced the fluorescence, diffusion area, and fluorescence intensity (mean radiance) ( Figure 21G –21I), indicating enhanced diffusion and long-term retention of chemotherapy.

[0216] To evaluate the imaging characteristics of multiple imaging modalities, the visibility of NS or NS-IL hydrogels loaded into syringes was evaluated on magnetic resonance imaging (MRI), computed tomography (CT), and ultrasound (US). Figure 22A As shown. It is well known that IL has the property of neutralizing a variety of pathogens. To verify whether the antibacterial properties of IL were maintained when NS hydrogel was mixed with IL or Dox, the sterility test showed that no bacterial growth was detected after 1 day and 2 months of incubation at 37 °C ( Figure 22B ).

[0217] These data suggest that the combination of IL and Dox in NS hydrogels can synergistically expand the therapeutic boundary and broaden the drug distribution area.

[0218] Example 7: Time to progression and overall survival in rodent models of solid tumors

[0219] This example describes the in vivo tumor response, ablation efficacy, imaging characteristics, drug distribution and retention, immune response, and survival rate in animal cancer models injected with nanogels.

[0220] method

[0221] Nanogel ablation efficacy, drug distribution, immune response, and survival rate

[0222] The N1S1 rat hepatocellular carcinoma model was induced in 160 Sprague-Dawley rats (Envigo, CA) weighing 300-325 g (male and female) as described elsewhere (Albadawi et al., Sci. Transl. Med., 2021; 13(580)(2021)). Rat Novikoff hepatoma (N1S1) cells obtained from ATCC (CRL-1604, Manassas, VA) were propagated in Iscove's modified Dulbecco's medium (IMDM) containing 10% fetal bovine serum. The rat liver was exposed by midline laparotomy under anesthesia, and 1×10 cells were inoculated subcutaneously in the left lower liver lobe with a 25-gauge syringe needle in a volume of 100 μL. 6 N1S1 cell suspension. After recovery, serial ultrasound imaging was performed using an ultrahigh-frequency transducer (Vevo-3100, FUJIFILM) to confirm tumor formation, delineate tumor mass boundaries in grayscale (B-mode) to calculate volume, and assess vascularity in color mode. Four groups of rats bearing N1S1 tumors with a diameter of 0.5 cm measured by ultrasound were randomly divided into four groups and received intratumoral injections of nanogels (selected from target 1.4), NS-IL, NS-Dox, and NS-ICS hydrogels, all of which contained an equal amount of ExiTron Nano 12000 contrast agent (Miltenyi Biotec). The injection volume was calculated based on 1.25 times the ultrasound tumor volume according to the following formula v = 4 / 3π[r + 0.5] 3 Time-dependent studies were performed by euthanizing subgroups of rats on days 3, 7, 14, 28, or 42.

[0223] Tumor burden, Dox and ICI distribution and retention, and host response were evaluated and compared in rats bearing N1S1 liver cancer injected with nanogels, NS-IL, NS-Dox, and NS-ICIs hydrogels. All rats survived for predetermined time points of 3, 7, 14, 28, or 42 days, and tumor volume and vascularity were serially recorded twice weekly using ultrasound. To provide 3D rendering and tumor volume measurements and tissue architecture on a microscopic scale, in vivo micro-CT analysis was performed using a SkyScan-1276 system (Bruker, Kontich, Belgium). Before autopsy, laser speckle angiography (LASCA, Perimed) was performed on exposed livers directly after euthanasia. Ex vivo fluorescence imaging of rat livers was performed at the midline to calculate the area of ​​Dox diffusion, and the fluorescence intensity of Dox within each tumor core was measured using an IVIS200 system (PerkinElmer, Inc., Waltham, MA). Subsequently, the tumor tissue was fixed or frozen and stained with H&E to evaluate tumor morphology, calculate the ablation area, and use specific pathological staining of active proliferation markers (Ki-67) and metastatic potential marker cytokeratin-19 (which is associated with poor prognosis in HCC patients after liver resection or ablation) to examine whether there are residual viable tumor cells in the tumor periphery of the surgical margin of the treated tumor. As shown in Figure 23, samples from different areas within and around the tumor lesion were analyzed using LC-MS / MS. Representative chromatograms of rat plasma spiked with 0.8 μM Dox in three ionization channels; all samples showed a consistent retention time at 4.1 minutes, confirming the molecular structure of Dox ( Figures 23A-23C Quantitative analysis of Dox levels in rat plasma showed a linear relationship between the measured concentrations (r 2 =0.99, Figure 23D ). Immunostaining for PD-1 or PD-L1 antibodies was performed to assess the tissue distribution and retention of ICI (Figure 24). The following in situ criteria were used to evaluate / confirm the ablation and drug distribution effects: 1) cellular uptake of Dox (quantified using LC-MS / MS); 2) the average transport distance of red fluorescent Dox in segmented tumor margins (measured using confocal fluorescence microscopy); 3) the number of actively proliferating cells in digitized microscopic fields obtained from the tumor core, margin, and peritumoral areas (measured using apoptosis rate (based on TUNEL and cleaved caspase-3 immunostaining) and proliferation rate (based on cytokeratin-19, Ki-67, and PCNA immunostaining); and 4) ICI distribution (measured by immunostaining for anti-PD-1 and anti-PD-L1). In addition, serially cut tissue sections were immunostained and the infiltrating lymphocyte lineages (CD3 + 、CD4 + 、CD8 +The local immune response was elucidated by measuring the number of inflammatory cells (eg, NK cells, leukocytes, and myeloid inflammatory cells) and granulocytes, monocytes, and macrophages. Blood chemistry of the blood samples obtained at each endpoint was analyzed as in Example 6.

[0224] Effects of intratumoral nanogel injection on survival and host immune response

[0225] The immunocompetent mouse model of MC38 colorectal cancer was used to test the ablation effect of the selected nanogel formulations. MC38 colorectal cancer cells (approximately 1 x 10 6 The cells were subcutaneously inoculated into the right lower flank of 10-12 week old C57BL6 mice (200 mice). The tumor volume was about 200 mm 3 The mice were randomly divided into three groups and injected directly into the tumor with 250 μL of nanogel, while the control group of mice was injected with NS-IL, NS-Dox or NS-ICS. The ICIs evaluated included mouse-specific PD-1 and PD-L1 inhibitors, anti-mPD-1-mIgG1e3 InvivoFit TM , and anti-PD-L1-mIgG1e3 InvivoFit TM Monoclonal antibodies (InvivoGen, San Diego, CA) were used to assess binding using specific ELISAs containing serially diluted nanogel extracts of anti-PD-1 and anti-PD-L1. These antibodies were obtained sterile, endotoxin-free, preservative-free, and lyophilized and mixed to achieve a concentration of 1 mg / mL. Following survival, subsets of mice were euthanized on days 1, 3, 7, 14, and 28 after intratumoral injection to assess immune responses and survival.

[0226] The effect of intratumoral nanogel injection on tumor response and animal survival was assessed by serially measuring tumor volume twice weekly using ultrasound after treatment, as shown in FIG26 .

[0227] Mouse colorectal cancer tumor model

[0228] Twelve-week-old female C57BL6 / J mice (n=14, Jackson Laboratory) were housed in a diurnal animal housing facility with a 12-hour light / dark cycle and free access to food. Mice were anesthetized by continuous inhalation of isoflurane. 2×10 α-Hydroxyproline suspended in 0.1 mL of Hanks' balanced salt solution was injected subcutaneously in the right flank. 6 MC38 colorectal cancer cells. Ultrasound was used to measure the vertical diameter of the tumor, and the tumor volume was calculated using the formula: 0.523 × (length × width × depth). When the tumor volume reached 150 mm 3At 14 days, tumor-bearing mice were randomly divided into two groups and received intratumoral injection of normal saline (control group, n=7) or nanogel (n=7). The injection volume was calculated based on 1.25 times the tumor volume. Tumor volume was continuously assessed using ultrasound twice a week. The mouse survival criterion was based on reaching the maximum allowed tumor volume of 2000 mm 3 Tumor progression was considered on days before the end of the study, or when the tumor developed severe ulceration, and individual mice were counted as dead according to the Institutional Animal Care and Use Committee. Tumor volume was used to calculate log-rank survival using Prism software to compare survival between the two groups. Mice were euthanized at the endpoint, and tumors were harvested for histological examination.

[0229] Establishment of N1S1 rat hepatocellular carcinoma model

[0230] Male Sprague-Dawley rats weighing 300 to 325 g (Envigo, CA) were used to induce N1S1 HCC. N1S1 rat hepatoma cells (CRL-1604, American Type Culture Collection, Manassas, VA) were cultured in Iscove's modified Dulbecco's medium supplemented with 10% heat-inactivated calf serum (HyClone, UT). To prepare cells for plating, an aliquot of N1S1 cells was rinsed and suspended in plain Iscove's modified Dulbecco's medium to generate 2 × 10 cells in 100 μL aliquots in a 1 ml syringe. 6 cells. The rat liver was exposed by upper midline laparotomy under anesthesia, and then N1S1 cells were inoculated into the left lower liver lobe under the capsule using a 25-gauge syringe needle. Gently press with gauze to achieve hemostasis and prevent cell reflux. Subsequently, the subcutaneous tissue and dermis were re-sutured with 5-0 Velcro sutures (Ethicon, Somerville, NJ). After recovery, serial ultrasound imaging was performed using the ACUSON S2000 system (Germany GmbH) and a multi-frequency linear transducer (9L4, 9.0 MHz) to confirm tumor formation and assess tumor volume, with the boundaries of the tumor mass depicted in grayscale (B mode). The two groups measured with US carried approximately 0.15 cm 3 Rats with N1S1 tumor lesions received intratumoral injections of NG or NS hydrogels containing 0.25% ICG and 1.25 mg / mL doxorubicin. The injection volume was calculated based on delivering 125% of the calculated tumor volume on US, as previously described for human chemical ablation procedures using ethanol injection. Following intratumoral injection, treated rats were allowed to survive for 2 weeks, and tumor volume was recorded using US.

[0231] In vitro and ex vivo fluorescence imaging

[0232] After intratumoral injection of NG or NS hydrogel using the IVIS200 system (PerkinElmer Inc., Waltham, MA), the tissue was subjected to in vitro spectral fluorescence imaging to evaluate the differences in doxorubicin or ICG. Cross-sectional fluorescence images of doxorubicin were obtained using an excitation wavelength of 460 nm and an emission wavelength of 560 nm. ICG was visualized using near-infrared irradiation with an excitation wavelength of 750 nm and an emission wavelength of 850 nm. Fluorescence images in different experimental samples were collected using the same setting of 1 s exposure time (aperture value = 2) and displayed using the same scale in each group. Fluorescence intensity was quantified using the radiance value in the region of interest and normalized to the number of photons per square centimeter per steradian per second (p / s / cm 2 / sr), and the fluorescence enhancement area in each sample was calculated after applying a normalized threshold.

[0233] Less than 32mm 3 Tumors larger than 2000 mm were considered to have completely regressed, while those larger than 2000 mm 3 Tumors were considered to be progressive, and animals were euthanized. Based on survival results, survival rates and tumor volumes were compared and calculated using statistical software (GraphPad Prism). At necropsy, tumor tissues were resected and randomized for fluorescence imaging and histological analysis, or for evaluation of host immune responses, which was performed after generating single-cell suspensions from each tissue, labeling, and analyzing tissues using a Helios mass spectrometer system (Fluidigm) and a Hyperion imaging module for antibody profiling.

[0234] result

[0235] These results demonstrate the feasibility of intratumoral injection of a nanogel formulation containing 3 wt% NS, 25 wt% IL, 250 mg / mL Dox, and 1 mg / mL anti-PD-1 antibody into rat N1S1 tumors, compared to intratumoral injection of a control hydrogel containing the same concentrations of NS, IL, Dox, and anti-PD-1 antibody without IL ( FIG24 ). Ultrasound examinations of the rat livers at baseline and 2 weeks after injection showed significantly smaller tumors in the nanogel-injected tumors compared to the larger tumors in the control hydrogel-treated tumors ( FIG24 ). Figures 24A-24D Histological evaluation of N1S1 tissues harvested at 2 weeks revealed extensive areas of necrosis, little nuclear staining, and loss of normal tissue architecture compared to hypercellularity, indicative of active proliferation, when the tumors were injected with the nanogel ( Figures 24E-24F Immunostaining with anti-PD-1 antibodies showed that ICI antibodies were evenly distributed throughout the ablated tumors 2 weeks after nanogel injection, compared with reduced antibody detection in control tumors ( Figures 24G-24HThese results suggest that ILs can be used for tumor ablation and enable the distribution and long-term retention of ICIs in tumors.

[0236] The effect of nanogel injection on inflammatory cell infiltration was evaluated. Histological sections of rat liver tissue showed the presence of myeloperoxidase (MPO)-carrying inflammatory cells ( Figure 25A ) or CD3+ T lymphocytes ( Figure 25B ) were immunostained. The ablation areas of rat liver sections on days 1, 14, and 28 after NS, NG, or NG+Dox injection showed that the ablation areas at the NG and NG+Dox injection sites were significantly larger than those at the NS injection sites on days 1 and 14 ( Figure 25C Compared with NS, the ablation area measured at the injection site of NG+Dox on day 28 was larger. Morphometric analysis of MPO-positive cells showed early recruitment of MPO-positive cells on day 1, which gradually decreased on days 14 and 28 ( Figure 25D ), indicating a transient acute proinflammatory response. Histological analysis of the number of immunostained CD3+ cells counted within each injection site showed that CD3+ cells were significantly higher in the NG+Dox site compared with the NS or NG injection site, indicating that T lymphocyte recruitment and CD3+ cell numbers were higher in the NG+Dox injection site up to 28 days after injection ( Figure 25E ).

[0237] The nanogel was inoculated into a mouse colorectal cancer model using ultrasound-guided intratumoral injection. MC38 colon adenocarcinoma cells were subcutaneously inoculated to form tumors in the right lower flank of immunocompetent C57BL6 mice ( Figure 26A Tumor volume was calculated by measuring the length, width, and depth of the tumor on ultrasound imaging, and tumor growth was monitored twice a week. Ultrasound imaging of MC38 tumors obtained during direct intratumoral injection of nanogels showed a hyperechoic needle within a hypoechoic tumor lesion ( Figure 26B ; dotted outline). 49 days after nanogel injection, the nanogel-treated tumors showed a complete therapeutic response, leaving a small scar on the mouse skin ( Figure 26C ), visible on ultrasound ( Figure 26D ). General picture of MC38 tumor-bearing mice ( Figure 26E ) and the corresponding ultrasound image ( Figure 26F ) showed that 21 days after saline injection, the tumor had progressed to approximately 2 cm 2 Mouse survival was based on reaching the maximum permissible tumor volume of 2000 mm 3 Before 1 hour, or when the tumor developed severe ulceration, the tumor was considered to have progressed and the individual mouse was counted as dead. Figure 26G Tumor growth curves of individual tumors assessed by ultrasound showed earlier tumor progression in the control group ( Figure 26H ), indicating that the tumor responded to treatment. The mean change in tumor volume was measured before intratumoral injection of nanogel or control and at the end of the survival period of each group ( Figure 26I The mean tumor volume of the control group showed a significant increase in tumor volume, indicating continued tumor progression compared to the MC38 tumors that received nanogel injections, which showed no change in tumor volume. Histological sections showed tumor cell ablation 1 hour after nanogel injection ( Figure 26J ) and showed evidence of lesion size reduction, cell ablation, and fibrogenesis 48 days after intratumoral nanogel injection ( Figure 26K Histological sections of untreated MC38 tumors in the control group showed significantly larger tumor areas and evidence of actively proliferating tumor cells ( Figure 26L ).

[0238] Experiments were conducted to see if NG could also help deliver nivolumab. NG was mixed with Nivo to see if it could also evenly distribute, retain, and deliver the drug throughout the tumor. Figures 27A-27J ). The results showed that NG has the ability to ablate tumors and deliver chemotherapy and immunotherapy uniformly throughout the ablation zone.

[0239] To evaluate the recruitment of T lymphocytes after intratumoral injection of nanogels containing anticancer immunotherapy ( Figure 28A –28D). These images demonstrate high Nivo levels, tumor cell death, and high CD3 levels within the ablation zone, indicating that the nanogel containing anticancer immunotherapeutic agents can achieve immunotherapy of solid tumors.

[0240] Example 8: Image-guided intratumoral injection of nanogel

[0241] This example describes the use of intratumoral nanogel injection to treat tumors.

[0242] method

[0243] Construction of a rabbit VX2 hepatocellular carcinoma model

[0244] New Zealand white rabbits (Charles River; 2.5-3.0 kg; male; 120 rabbits (male and female)) were used to construct the VX2 hepatocarcinoma model as described elsewhere (Albadawi et al., Sci. Transl. Med., 13(580)(2021)). Power analysis showed that the effective sample size for ANOVA (f = 10; mean SD = 2, mean = 1-5) was approximately 88% power with an α of 0.05 and 8 animals per data point per group. Briefly, cryopreserved VX2 slurry was expanded in vivo after intramuscular injection into the rabbit thigh muscle. The formed muscle tumor was then aseptically isolated and minced and stored in cold DMEM. VX2 hepatocarcinoma was induced by freshly implanting a muscle tumor excised from a donor rabbit into the deep recess of the left lobule of the recipient rabbit. Tumor size was monitored twice a week by ultrasound. Once the VX2 tumor reached a size of 1 cm 3 Rabbits were randomly divided into three treatment groups and injected intratumorally with 1.25 mL of nanogel, NS alone, or ethanol under ultrasound guidance. The V was calculated based on the following formula: V = 4 / 3π[r + 0.5] 3 To simulate the clinical approach, intratumoral injection was performed using a standard 21-gauge needle. The needle was inserted into the tumor, and equal parts of nanogel, NS, or ethanol were slowly injected to make the tumor tissue echogenic while the needle was slowly retracted to the proximal edge of the tumor.

[0245] Result Analysis

[0246] Serial US, endpoint angiography, CT imaging, histopathology, blood values, and molecular profiling data were used to assess: 1) technical success of intratumoral injection; 2) ablation efficacy, local tumor progression, and vascularity; 3) drug distribution and long-term retention of ICI and Dox; 4) survival; and 5) rule out potential complications. Tumor burden was serially assessed using ultrasound to compare volume changes. A subgroup of 10 rabbits underwent contrast-enhanced angiography and laser speckle scanning before euthanasia 1, 3, 7, 21, and 28 days after intratumoral injection to assess tumor vascularity. At necropsy, explanted VX2 tumor-bearing livers underwent gross examination, ex vivo microCT, and fluorescence imaging to measure segmented 3D tumor size, Dox fluorescence intensity, and distribution area. Whole blood samples were collected for complete blood count (CBC) and organ function markers (i.e., LFTs, BUN / Cr). The in situ standard assessments used to evaluate / confirm the efficacy of ablation and drug distribution as described in Example 7 were as follows: 1) Dox chemotherapy levels were assessed using LC-MS / MS; 2) Dox transport distance was measured at six segmented tumor edges using confocal fluorescence microscopy; 3) immunostaining was used to assess apoptosis (TUNEL and caspase-3), proliferation rate (Ki-67 and PCNA), and local immune cell infiltration (lymphocytes, macrophages, and granulocytes) in three different areas within the tumor boundary (tumor core, edge) and the peritumoral area; and 4) immunostaining was used to assess ICI distribution (PD-1 and PD-L1).

[0247] Chemical embolization of porcine renal artery by nanogel

[0248] Healthy Yorkshire pigs weighing 48 to 55 kg (S&S Farms, Brentwood, CA) were acclimated for at least 4 days under standard housing conditions and appropriate temperature. 5 mg kg -1 Tiritamine-zorazepam (Telazol, Zoetis), 2 mg mL -1 Xylazine and 0.02 mg kg -1Pigs were anesthetized with glucopyranose. The pigs were then placed in the supine position and intubated on an X-ray compatible operating table (Pannomed Aeron, DRE, KY). After intubation, anesthesia was maintained with inhaled 1.5-3% isoflurane. During the procedure, electrocardiogram, transcutaneous oxyhemoglobin saturation (SpO2), end-tidal CO2 concentration, inspired oxygen fraction, and core temperature were monitored. Percutaneous access to the carotid artery was performed using ultrasound guidance (ACUSON S2000, Siemens) and fluoroscopy (OECElite C-Arm, GE Healthcare Systems, Chicago, IL). The puncture needle and guidewire were replaced with a 5 French catheter (Cook Medical). The thoracoscopic catheter was anesthetized using a GT slide wire (Terumo Medical) and contrast-enhanced fluoroscopy (350 mg / mL) was used. -1 Omnipaque, GE Healthcare, MA) was used to advance the catheter tip into the kidney. Intravenous contrast medium (350 mg / mL) was used. -1 Omnipaque, GE HealthCare, MA) performed renal artery angiography under real-time fluoroscopic guidance. A syringe filled with nanogel or NS embolic agent was directly connected to the catheter using a Luer lock, and 2 mL of nanogel or NS hydrogel was delivered to the renal artery through the catheter. Digital subtraction angiography was used to evaluate the radiopacity and vascular patency of the nanogel or NS hydrogel. Angiography was performed repeatedly to check the embolization effect. Pigs were killed 1 hour after embolization (non-survival group; n=4) or 1 week after embolization (survival group; n=4). Angiography was repeated before euthanasia to confirm embolism. At autopsy, the explanted embolized kidneys were explanted for fluorescence imaging and histological examination.

[0249] Statistical analysis

[0250] Animal survival was analyzed using the Kaplan–Meier method and the log-rank test to compare survival rates between groups. Statistical analysis was performed using two-way repeated-measures analysis of variance (ANOVA) to assess tumor response using serial ultrasound. Kruskal–Wallis analysis was used to test the difference in total ablation, apoptosis, and necrosis areas between groups, and the Wilcoxon signed-rank test was used for paired tests. Ablation area, volume, and apoptosis versus necrotic area / volume are expressed as mean ± SEM, as well as median, minimum, and maximum values. Statistical analysis was performed using Prism software. A p value < 0.05 was considered statistically significant.

[0251] result

[0252] No survival experiments were performed to demonstrate the feasibility of ultrasound-guided intratumoral injection of nanogels. 25 wt%-IL was mixed with 1.25 mg / mL Dox and 1 mg / mL anti-PD-1 antibody in nanogels and injected into VX2 tumors using a 21-gauge standard puncture needle ( Figures 29A-29C One hour after injection, the animals were euthanized and liver tissue was collected for fluorescence imaging. Gross examination and fluorescence imaging of cross-sectioned VX2 tumors showed that Dox fluorescence was evenly distributed and covered the ablation area ( Figure 29D and 29E H&E histological sections showed extensive tumor ablation 1 hour after injection ( Figure 29F Furthermore, immunostaining with PD-1 antibodies revealed extensive areas of positive staining confined to areas of complete tissue ablation ( Figure 29G These results indicate that the nanogel can induce rapid tumor ablation and evenly distribute Dox and ICI throughout the treatment area.

[0253] Example 9: Nanogel Formulation for Effective Tissue Ablation and Drug Delivery

[0254] In a large animal model, we investigated whether NG and its components delivered via catheters into the arteries of organs such as the kidneys would cross the vessel wall to achieve parenchymal delivery. As shown by digital subtraction angiography, 1 hour after embolization, there was a complete absence of flow from the renal artery to the kidney (white arrows) ( Figure 32A –32C), indicating successful embolization. One hour after embolization, diffuse fluorescence enhancement of ICG was observed throughout the renal cortex and medulla ( Figure 32D One hour after embolization with NS hydrogel containing 0.25 mg / mL ICG and 20% iodine hexanol, the ICG fluorescence enhancement was observed to be limited to the renal vascular network ( Figure 32E and 32F These results suggest that NG can be used to achieve temporal vascular embolization and drug delivery (e.g., ICG) into the parenchyma of highly vascular organs such as the kidney. Furthermore, this delivery is not instantaneous—the ICG is retained, indicating sustained delivery of the drug / therapy.

[0255] We also examined whether the components contained in NG could be transported through the blood vessel wall. H&E staining of the renal artery branches in the renal cortex region showed that within one hour after embolization of the renal artery with nanogel, the artery was completely cast, indicating that NG had the ability to reach smaller arterial branches ( Figure 33A Immunohistochemical analysis of nivolumab showed that Nivo was localized in and around the renal artery, suggesting that it was administered through the artery ( Figure 33B A decrease in nuclear staining of the arterial wall was observed, indicating successful transarterial delivery of IL and ablation after NG embolization ( Figure 33C and 33DThese results demonstrate the ability to deliver the drug continuously through the arterial wall. Furthermore, these results indicate that NG embolization is capable of ablating all layers of the vessel wall.

[0256] Other implementations

[0257] It should be understood that although the invention has been described in conjunction with specific embodiments, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and improvements are within the scope of the claims.

Claims

1. Use of a composition comprising an ionic liquid for preparing a percutaneously injectable medicament for ablating at least a portion of a tissue in a mammal by injecting the medicament into the tissue in the mammal. The ionic liquid comprises: (a) a cationic component comprising a cation selected from the group consisting of choline, benzylpyridinium, benzyldimethyldodecylammonium, phosphonium, tetraalkylphosphonium, phenethylonium, imidazolium, pyridinium, piperidinium, quinolinium, morpholinium, quaternary phosphonium, and quaternary ammonium; and (b) an anionic component comprising an anion selected from the group consisting of geranate, bistrifluoroformimide, oleate, hexanoate, dodecyldimethylaminopropanesulfonate, N-laurylsarcosinate, geranilate, tetrafluoroborate, hexafluorophosphate, methylsulfate, octylsulfate, acesulfame K, a halide, bis(trifluoromethylsulfonyl)amide, bis(trifluoromethyl)amide, dicyanamide, and trifluoromethanesulfonate; The drug is free of other additional therapeutic agents and is effective to create an ablation zone in the tissue, and wherein the drug is effective to reduce the number of cells in the ablation zone.

2. The use according to claim 1, wherein The mammal is a human.

3. The method of claim 1, wherein the tissue is selected from the group consisting of adipose tissue, cardiac tissue, connective tissue, bone tissue, synovial tissue, abscess tissue, and cyst.

4. The method of claim 1, wherein the injection is a guide injection.

5. The use according to claim 4, wherein the medicament further comprises a contrast agent.

6. The method according to claim 5, wherein the contrast agent is selected from the group consisting of indocyanine green, radioactive dense contrast agent, iohexol, tantalum nanoparticles, tantalum microparticles, gold nanoparticles, gadolinium, indium 111 or microbubbles.

7. The use of claim 1, wherein the ablation zone is about 0.1 cm to about 4 cm.

8. Use of a composition comprising an ionic liquid for preparing a transdermal injectable medicament for treating a mammal suffering from cancer by injecting the medicament into a tumor tissue in the mammal. The ionic liquid comprises: (a) a cationic component comprising a cation selected from the group consisting of choline, benzylpyridinium, benzyldimethyldodecylammonium, phosphonium, tetraalkylphosphonium, phenethylonium, imidazolium, pyridinium, piperidinium, quinolinium, morpholinium, quaternary phosphonium, and quaternary ammonium; and (b) an anionic component comprising an anion selected from the group consisting of geranate, bistrifluoroformimide, oleate, hexanoate, dodecyldimethylaminopropanesulfonate, N-laurylsarcosinate, geranilate, tetrafluoroborate, hexafluorophosphate, methylsulfate, octylsulfate, acesulfame K, a halide, bis(trifluoromethylsulfonyl)amide, bis(trifluoromethyl)amide, dicyanamide, and trifluoromethanesulfonate; The composition does not contain any additional therapeutic agent and is effective in generating an ablation zone within the tumor tissue, and wherein the drug is effective in reducing the number of cancer cells within the ablation zone.

9. The use according to claim 8, wherein The mammal is a human.

10. The method of claim 8, wherein the cancer is selected from the group consisting of liver cancer, biliary tract cancer, pancreatic cancer, colorectal cancer, kidney cancer, ovarian cancer, breast cancer, prostate cancer, colon cancer, bladder cancer, lung cancer, thyroid cancer, melanoma, brain cancer, stomach cancer, cervical cancer, uterine cancer, skin cancer, synovial cancer, appendix cancer, and adrenal cancer.

Images