IRE electrical sensitization nanometer material capable of regulating and controlling tissue dielectric constant

Abstract

The invention relates to an IRE electrical sensitization nanometer material capable of regulating and controlling a tissue dielectric constant, a manganese carbonate nanometer material is manganese carbonate nanometer particles, the manganese carbonate nanometer particles are of a rhombohedral structure, and the average particle size is about 100-200 nm. According to the invention, the IRE ablation efficiency and tumor selectivity are obviously improved, and the residue and recurrence risk is reduced; economically, expensive equipment and complex processes are not needed, and the cost is controllable; in society, prognosis of patients is improved, life quality is improved, and safe and efficient treatment of solid tumors such as liver cancer is promoted.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to an IRE electrical sensitizing nanomaterial with adjustable tissue dielectric constant. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide, especially in its middle and late stages, where the prognosis is extremely poor due to the lack of effective treatments. my country has a high incidence of chronic liver disease, and liver cancer has become a serious threat to national health. Current clinical treatments include surgical resection, liver transplantation, local ablation (such as radiofrequency ablation, microwave ablation, and irreversible electroporation), and systemic therapy. Although local treatments can achieve tumor control to some extent, each has its limitations. For example, traditional thermal ablation is susceptible to the "heat sink effect," leading to incomplete ablation of tumors near large blood vessels; transarterial chemoembolization and radiotherapy often involve systemic toxic side effects due to the chemotherapy drugs and radiation.

[0003] Irreversible electroporation (IRE) is a non-thermal ablation method that has been increasingly applied in clinical practice in recent years. This technology uses a short-duration high-voltage pulsed electric field to create irreversible nanopores in the cell membrane, leading to cell death. It can ablate tumors while preserving key anatomical structures such as blood vessels and bile ducts, thus having a unique advantage in the treatment of tumors near blood vessels.

[0004] However, existing IRE technology still has the following shortcomings:

[0005] Non-specific damage: All tissues within the ablation area will be affected by the high-frequency electrical pulse, which may cause damage to the function of normal tissues.

[0006] Uneven electric field distribution: Differences in conductivity and dielectric constant between tumors and surrounding normal tissues lead to uneven electric field distribution between electrodes. In some areas, the electric field strength is insufficient to reach the perforation threshold, resulting in incomplete ablation or even recurrence. Furthermore, increasing the voltage and pulse intensity may introduce risks such as bile duct injury and thrombosis.

[0007] Limitations of Electrosensitization Strategies: Current electrosensitization research largely focuses on the introduction of conductive nanomaterials (such as carbon-based materials and metal nanoparticles) to increase tissue conductivity and lower the perforation threshold. While these methods improve the efficiency of IRE to some extent, they lack tumor specificity and pose long-term biocompatibility risks. More importantly, these methods primarily rely on increasing tissue conductivity to enhance current conduction, but they struggle to alter voltage distribution between different tissues, thus failing to significantly improve the selective electroporation efficiency in tumor areas.

[0008] Under the pulsed conditions commonly used in IRE (typical pulse frequency band of kHz–MHz), biological tissue as a whole exhibits a predominantly capacitive electrical response. Based on this, the IRE ablation region can be approximated as a composite capacitor composed of three dielectric components: normal tissue, tumor tissue, and normal tissue again. For ease of voltage distribution analysis, a series capacitance model can be used. In this model, the total voltage is constant; when the effective dielectric constant of the tumor tissue decreases, its equivalent capacitance decreases, and the corresponding voltage distribution increases, thus helping to enhance the electroporation efficiency of the tumor region. However, an effective means to controllably regulate the dielectric properties of tumors in vivo is still lacking.

[0009] In summary, existing IRE technology still has significant shortcomings in terms of electric field distribution control and electrical sensitization, and there is an urgent need for a new, safe, controllable approach that can achieve dielectric modulation locally in tumors to solve this problem. Summary of the Invention

[0010] To address the shortcomings of existing technologies, the technical problem this invention aims to solve is to provide an IRE (Intense Respiratory Electrochemical) nanomaterial with adjustable tissue dielectric constant. This invention aims to address the following key issues existing in current IRE technology for tumor treatment: 1. Insufficient tumor-selective voltage distribution: In heterogeneous tissue structures of "normal-tumor-normal," the voltage distribution in the tumor region is insufficient, making it difficult to achieve sufficient electroporation under a fixed global voltage. How to enhance the voltage share and effective electric field in the tumor region without increasing the overall electric field strength is a core problem that urgently needs to be solved. 2. Uncontrollable dielectric properties: Existing technologies lack means to controllably adjust the tumor dielectric constant in vivo, making it difficult to achieve voltage zoning optimization at the macroscopic level. 3. Limited microscopic perforation efficiency: The local perforation threshold of the cell membrane is high, and existing methods cannot form electric field hotspots at the subcellular scale, leading to incomplete ablation. 4. Limited clinical translation: Currently, there is a lack of a simple, safe, and directly compatible electrosensitization strategy with IRE devices and parameters.

[0011] This invention is based on an electrosensitization strategy using manganese carbonate (MnCO3). By injecting MnCO3 nanoparticles into the tumor, CO2 is released through decomposition in the acidic microenvironment of the tumor, thereby reducing the dielectric constant of the tumor tissue and decreasing its equivalent capacitance. Under the three-capacitor series model, a higher voltage is shared, which significantly enhances the efficiency of irreversible electroporation (IRE) ablation in the tumor region.

[0012] This invention provides a manganese carbonate nanomaterial, wherein the manganese carbonate nanomaterial is manganese carbonate nanoparticles, wherein the manganese carbonate nanoparticles have a rhombohedral structure and an average particle size of about 100-200 nm.

[0013] Manganese carbonate nanomaterials decompose and release CO2 bubbles in an acidic tumor microenvironment. The release of CO2 can reduce the dielectric constant of tumor tissue, thereby decreasing Cr and increasing V, which enhances the electroporation effect without increasing the global electric field strength.

[0014] This invention provides a method for preparing manganese carbonate nanomaterials, comprising:

[0015] (1) Mix hexadecyltrimethylammonium bromide (CTAB), cyclohexane, and pentanol, then add an aqueous solution of manganese source, mix well, and stir to obtain microemulsion A;

[0016] (2) Mix hexadecyltrimethylammonium bromide (CTAB), cyclohexane, and pentanol, then add water, NaHCO3, and NH4HCO3, and stir to obtain microemulsion B;

[0017] (3) Mix microemulsion A and microemulsion B, stir and react, purify, and obtain manganese carbonate nanomaterials.

[0018] In step (1), the ratio of CTAB, cyclohexane, pentanol, and manganese source aqueous solution is 0.5-0.8 g: 18-25 mL: 0.8-1.2 mL: 0.5-0.8 mL; the manganese source is MnCl2·4H2O.

[0019] In step (1), the ratio of manganese source to water in the aqueous solution of manganese source is 3.0-3.5 mmol: 0.5-0.8 mL.

[0020] In step (2), the ratio of CTAB, cyclohexane, pentanol, water, NaHCO3 and NH4HCO3 is 2.4-3.0 g: 70-90 mL: 3.5-4.5 mL: 2.4-3.0 mL: 6.0-6.5 mmol: 0.3-0.35 mmol.

[0021] In steps (1) and (2), the reaction is stirred for 1-2 hours.

[0022] In step (3), the reaction is stirred for 0.5-1.5 hours.

[0023] In step (3), the purified product is collected by centrifugation, washed 2-3 times with anhydrous ethanol, and then washed 2-3 times with deionized water to remove residual reactants.

[0024] The manganese carbonate nanomaterials from step (3) can be used directly in experiments or freeze-dried for later use.

[0025] This invention provides an application of manganese carbonate nanomaterials as an IRE (Inductively Coupled Receptor) electrosensitizer.

[0026] This invention proposes an electrosensitization strategy based on manganese carbonate (MnCO3) nanomaterials. By injecting MnCO3 nanoparticles into the tumor, CO2 is decomposed and released in the acidic microenvironment of the tumor, thereby reducing the dielectric constant of the tumor tissue and decreasing its equivalent capacitance. Under the three-capacitor series model, a higher voltage is shared, which significantly enhances the efficiency of irreversible electroporation (IRE) ablation in the tumor region.

[0027] This invention provides an application of manganese carbonate nanomaterials in the preparation of antitumor drugs, wherein the manganese carbonate nanomaterials are used in combination with IRE.

[0028] This invention provides the application of manganese carbonate nanomaterials in the preparation of anti-hepatocellular carcinoma drugs, wherein the manganese carbonate nanomaterials are used in combination with IRE.

[0029] Administration: Inject once every 3 days, for a total of 3 times. The dosage of MnCO3 nanomaterials is 3-5 mg / kg. To ensure sufficient decomposition and CO2 release within the tumor tissue, irreversible electroporation (IRE) treatment is performed 8-10 minutes after injection. The IRE ablation parameters are as follows: using a BTX ECM 830 pulse generator (Harvard Bioscience), voltage 1000 V, number of pulses 30, single pulse width 100 μs, and repeating the treatment 3 times for each tumor site.

[0030] Beneficial effects

[0031] 1. Voltage distribution optimization

[0032] Manganese carbonate (MnCO3) decomposes in the acidic tumor microenvironment, releasing CO2, which lowers the local dielectric constant and reduces the equivalent capacitance of the tumor region, thus achieving higher voltage sharing in a three-capacitor series model. This significantly enhances the electroporation efficiency in the tumor region without increasing the global electric field strength, avoiding additional damage to surrounding normal tissues.

[0033] 2. Improved micro-perforation efficiency

[0034] CO2 bubbles form a gas-liquid interface on the cell membrane surface, leading to local current concentration and an electric field "hot spot effect," which promotes rapid cell membrane destruction, thereby improving the thoroughness and efficiency of IRE ablation.

[0035] 3. Improved security

[0036] MnCO3 nanoparticles decompose and release CO2 in the acidic tumor microenvironment. This CO2 can be selectively accumulated in the tumor region through in-situ injection or by relying on the EPR (Enhanced Permeability and Retention) effect, achieving a spatially confined mode of action. This reduces systemic exposure and off-target effects, lowers the risk of systemic toxicity, and improves treatment safety. Furthermore, the Mn released from the decomposition of MnCO3... 2＋ Ions are known to activate the cGAS–STING pathway, and therefore are expected to synergize with IRE ablation-induced immunogenic cell death, thereby further enhancing the immune activation effect.

[0037] 4. Advantages in clinical application

[0038] This method is directly compatible with existing IRE equipment and parameters, requiring no additional modifications. The manganese carbonate preparation process is simple, the raw materials are readily available, and it possesses feasibility for large-scale production and promising prospects for clinical application. Furthermore, MnCO3 nanoparticles can be applied to the IRE process via intratumoral injection, which is simple to operate and highly compatible with interventional therapy procedures, facilitating clinical implementation. Meanwhile, Mn... 2＋ The immune activation effect and MRI imaging enhancement provide added value to this invention while improving the efficacy of ablation. Therefore, this invention combines the advantages of electrical sensitization, clinical feasibility, and multifunctionality, and has significant clinical implications.

[0039] In terms of overall effectiveness, technically it significantly improves IRE ablation efficiency and tumor selectivity, reducing the risk of residual disease and recurrence; economically, it eliminates the need for expensive equipment and complex processes, making costs controllable; and socially, it helps improve patient prognosis, enhances quality of life, and promotes safe and efficient treatment of solid tumors such as liver cancer. Attached Figure Description

[0040] Figure 1 Characterization diagram of morphology and elemental distribution of manganese carbonate (MnCO3) nanoparticles;

[0041] Figure 2 A comparison of the gases released by manganese carbonate (MnCO3) nanoparticles under different pH conditions;

[0042] Figure 3 A comparison of electrochemical impedance spectroscopy (Nyquist plot) of ex vivo tissues under conditions with and without bubbles;

[0043] Figure 4 A comparison graph of pulse voltages under conditions with and without bubbles;

[0044] Figure 5To simulate the electric field and current density distribution in the extracellular environment in the presence of gas;

[0045] Figure 6 Follow-up results of tumor growth curves in mice of different treatment groups;

[0046] Figure 7 The survival rate of mice in different treatment groups was followed up.

[0047] Figure 8 The curves show the changes in body weight of mice in different treatment groups during follow-up. Detailed Implementation

[0048] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0049] Example 1

[0050] (I) Material Preparation

[0051] Manganese carbonate (MnCO3) nanoparticles were prepared using a reverse microemulsion method. The specific steps are as follows:

[0052] Dissolve 0.667 g of hexadecyltrimethylammonium bromide (CTAB) in a mixture of 20 mL cyclohexane and 1 mL pentanol, then add 0.667 mL of deionized water (containing 3.33 mmol MnCl2·4H2O) to obtain solution A;

[0053] 2.668 g CTAB was dispersed in a mixture of 80 mL cyclohexane and 4 mL pentanol, and 2.668 mL of deionized water (containing 6.33 mmol NaHCO3 and 0.33 mmol NH4HCO3) was added to obtain solution B;

[0054] Solution A and solution B were stirred separately for 2 h and then mixed. Stirring was continued for 1 h to induce the precipitation of MnCO3 nanoparticles.

[0055] The product was collected by centrifugation, washed three times with anhydrous ethanol, then washed twice with deionized water, and finally freeze-dried for storage.

[0056] The morphology and elemental distribution of the prepared nanoparticles were characterized by transmission electron microscopy (TEM) and energy dispersive spectroscopy (EDS).

[0057] (ii) Gas release performance

[0058] MnCO3 nanoparticles were tested under different pH conditions. The solution state was observed and photographed in environments with pH=7.4 and pH=6.5.

[0059] (III) External electrical verification

[0060] CO2 gas was artificially introduced into isolated porcine liver tissue to construct an electrode model for electrical detection.

[0061] Two-electrode model: Electrochemical impedance spectroscopy (EIS) was used to test changes in tissue dielectric constant and equivalent capacitance;

[0062] Four-electrode model: Comparison of pulse voltage amplitude and distribution when detecting the presence or absence of bubbles.

[0063] In addition, a single-cell model was established for finite element numerical simulation. Bubbles were introduced into the extracellular environment to calculate the distribution of electric field and current density, which was used to evaluate the effect of bubbles on the concentration effect of electric field in the cell membrane neighborhood.

[0064] (iv) Animal experiments

[0065] Establish an H22 subcutaneous hepatocellular carcinoma (HCC) mouse model. H22 cells (1×10⁻⁶) were used... 6 Each tumor (containing 100 μL of PBS suspension) was subcutaneously inoculated into the right axilla of BALB / c mice. The inoculation continued until the tumor volume reached approximately 150 mm. 3 Mice were randomly divided into six groups (n=5): PBS control group, MnCl2 group, MnCO3 group, PBS+IRE group, MnCl2+IRE group, and MnCO3+IRE group.

[0066] The administration method was intratumoral injection, once every 3 days, for a total of 3 times. The dosage of MnCO3 was 5 mg / kg, and the dosage of MnCl2·4H2O was 215.2 μg (equivalent to 8.61 mg / kg).

[0067] IRE treatment was performed 10 min after injection, with the following parameters: a BTX ECM 830 pulse generator (Harvard Bioscience) was used, with an output voltage of 1000 V, 30 pulses, and a single pulse width of 100 μs. Each tumor site was treated for 3 rounds.

[0068] During the experimental period, mouse body weight and tumor volume (V = 1 / 2 × length × width) were monitored periodically. 2 Monitoring was conducted every 3–4 days, and tumor weighing, histological examination, and survival follow-up were performed at the study endpoint.

[0069] Manganese carbonate (MnCO3) nanoparticles were prepared in this example. Transmission electron microscopy (TEM) and energy dispersive spectroscopy (EDS) results showed that the prepared MnCO3 nanoparticles exhibited a regular rhombohedral structure with an average particle size of approximately 100–200 nm, and that Mn, C, and O elements were uniformly distributed, confirming the composition and structure of the material. MnCO3 can decompose and release carbon dioxide (CO2) under acidic conditions (e.g., ...). Figure 1 As shown), this provides a foundation for subsequent electrical control (such as...). Figure 2 (As shown).

[0070] To verify the electrosensitizing effect of CO2 gas in tissues, CO2 was artificially introduced into isolated porcine liver tissue, and two-electrode and four-electrode models were constructed for detection. In the two-electrode model, electrochemical impedance spectroscopy (EIS) revealed that the effective dielectric constant of the tissue decreased and the equivalent capacitance decreased after CO2 introduction (e.g., ...). Figure 3 As shown); in the four-electrode model, the detection results show that the local pulse voltage amplitude increases when bubbles are present, indicating that CO2 can change the current distribution and affect the local electric field strength (e.g. Figure 4 (As shown). Furthermore, a finite element numerical model was established to simulate and analyze the extracellular environment in the presence of bubbles. The results show that both the electric field and current density exhibit concentration phenomena in the vicinity of the bubbles (e.g., ...). Figure 5 As shown in the figure, this further illustrates that the presence of CO2 can affect the local electric field distribution and provide favorable conditions for the electroporation process.

[0071] like Figure 3 As shown, the electrochemical impedance spectroscopy (Nyquist plot) of ex vivo tissue under conditions with and without bubbles is compared. The tissue impedance characteristics were detected using a two-electrode model, where the horizontal axis represents the real part of the impedance and the vertical axis represents the imaginary part. The square curve represents the bubble-free condition, and the dotted curve represents the condition after the introduction of CO2 bubbles. It can be observed that the vertical axis value of the curve increases overall under the bubble condition. Combined with the analysis of the equivalent circuit model, this indicates that the presence of CO2 bubbles affects the tissue's equivalent capacitance, thereby reducing its effective dielectric constant and altering the tissue's electrical response characteristics.

[0072] like Figure 4 As shown, the pulse voltage is compared under conditions with and without CO2 bubbles. The voltage distribution with and without CO2 bubbles was detected in a four-electrode model. The results show that the local pulse voltage amplitude increases after the introduction of bubbles, producing a stronger electric field effect compared to the bubble-free condition, suggesting that CO2 bubbles can change the tissue electric field distribution and enhance the local electroporation effect.

[0073] like Figure 5The diagram illustrates the distribution of electric field and current density in the extracellular environment under simulated gas conditions. The left figure is a schematic diagram of the cell structure under an applied electric field, including the cytoplasm, cell membrane, and external environment, with gas bubbles distributed in the region adjacent to the cell membrane. The right figure shows the distribution of the electric field (E-field) and current density (Current density). The results indicate that electric field concentration and current density enhancement occur at the bubble-membrane interface, which is conducive to cell membrane breakdown.

[0074] Animal experiments showed that tumor growth in the MnCO3+ IRE group was significantly inhibited, with almost no recurrence during the observation period, and tumor volume and weight were significantly lower than those in the control group. Figure 6-7 Survival curves showed that the survival time of this group of mice was significantly prolonged. Figure 8 Meanwhile, there was no significant difference in body weight changes among the groups of mice. Figure 8 This indicates that the method has good security.

[0075] like Figure 6 The following table shows the follow-up results of tumor growth curves in mice under different treatment groups. In the established H22 subcutaneous hepatocellular carcinoma model, mice were randomly divided into PBS control group, MnCl2 group, MnCO3 group, PBS+IRE group, MnCl2+IRE group, and MnCO3+IRE group (n=5). After treatment with predetermined drug administration and IRE parameters, the tumor volume of mice was measured periodically. The results showed that tumor growth in the MnCO3+IRE group was significantly inhibited, with a better tumor-suppressing effect than that in the MnCl2+IRE group, and almost no obvious recurrence was observed throughout the observation period; while the tumor volume in the control group and other treatment groups showed a continuous increasing trend.

[0076] like Figure 7 As shown, the survival rate follow-up results of mice in different treatment groups were observed in the H22 subcutaneous hepatocellular carcinoma model. Mice were randomly divided into six groups (n=5): PBS, MnCl2, MnCO3, IRE+PBS, IRE+MnCl2, and IRE+MnCO3. After treatment with predetermined drug administration and IRE parameters, the mice in each group were followed up and their survival status was recorded. The results showed that the survival rate of mice in the IRE+MnCO3 group was significantly higher than that of the other groups, suggesting that this treatment can achieve better results in prolonging survival time.

[0077] like Figure 8As shown, the weight change curves of mice in different treatment groups were observed during the follow-up experiment in the H22 subcutaneous hepatocellular carcinoma model experiment. Mice were randomly divided into six groups (n=5): PBS, MnCl2, MnCO3, IRE+PBS, IRE+MnCl2, and IRE+MnCO3. Mouse weight was monitored regularly during the observation period after drug administration and IRE treatment. The results showed that the weight of mice in each group gradually increased, and no significant differences were observed between groups, indicating that the treatment described in this invention has good biosafety.

Claims

1. A manganese carbonate nanomaterial, characterized in that, The manganese carbonate nanomaterial is manganese carbonate nanoparticles, wherein the manganese carbonate nanoparticles have a rhombohedral structure and an average particle size of about 100-200 nm.

2. A method for preparing manganese carbonate nanomaterials, comprising: (1) Mix hexadecyltrimethylammonium bromide (CTAB), cyclohexane, and pentanol, then add an aqueous solution of manganese source, mix well, and stir to obtain microemulsion A; (2) Mix hexadecyltrimethylammonium bromide (CTAB), cyclohexane, and pentanol, then add water, NaHCO3, and NH4HCO3, and stir to obtain microemulsion B; (3) Mix microemulsion A and microemulsion B, stir and react, purify, and obtain manganese carbonate nanomaterials.

3. The preparation method according to claim 2, characterized in that, In step (1), the ratio of the aqueous solution of CTAB, cyclohexane, pentanol, and manganese source is 0.5-0.8 g: 18-25 mL: 0.8-1.2 mL: 0.5-0.8 mL; the manganese source is MnCl2·4H2O.

4. The preparation method according to claim 2, characterized in that, In step (1), the ratio of manganese source to water in the aqueous solution of manganese source is 3.0-3.5 mmol: 0.5-0.8 mL.

5. The preparation method according to claim 2, characterized in that, In step (2), the ratio of CTAB, cyclohexane, pentanol, water, NaHCO3 and NH4HCO3 is 2.3-3.0 g: 70-90 mL: 3.5-4.5 mL: 2.4-3.0 mL: 6.0-6.5 mmol: 0.3-0.35 mmol.

6. The preparation method according to claim 2, characterized in that, In steps (1) and (2), the reaction is stirred for 1-2 hours.

7. The preparation method according to claim 2, characterized in that, In step (3), the reaction is stirred for 0.5-1.5 hours.

8. Application of a manganese carbonate nanomaterial as an IRE electrical sensitizer.

9. The application of a manganese carbonate nanomaterial in the preparation of antitumor drugs, characterized in that, The manganese carbonate nanomaterials are used in conjunction with IRE.

10. The application of a manganese carbonate nanomaterial in the preparation of an anti-hepatocellular carcinoma drug, characterized in that, The manganese carbonate nanomaterials are used in conjunction with IRE.

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