Preparation method and application of hybrid nanogel for enhancing sonodynamic therapy of tumor

Abstract

The application relates to a preparation method of a hybrid nanogel for strengthening tumor sonodynamic therapy, which adopts a hydrophilic polymer derivative to self-assemble and encapsulate an oxygen-deficient response drug in a manganese dioxide inorganic salt solution; the hydrophilic polymer derivative is obtained by modifying benzene boronic acid and a sonodynamic agent on a hydrophilic polymer material through a one-step method; the hydrophilic polymer material is selected from any one of starch, dextrin, hyaluronic acid, chitosan, dextran, gelatin or silk fibroin. The hydrophilic polymer derivative is constructed through one-step modification, and is self-assembled with manganese dioxide nano to form a hybrid nanogel, so that efficient sonodynamic therapy effect and inhibition of energy metabolism at a tumor site are realized; the oxygen-deficient response drug encapsulated by the nanogel can be specifically activated at an oxygen-deficient part of the tumor, so that a synergistic tumor sonodynamic therapy effect is realized.

Description

Technical Field

[0001] This invention relates to a method for preparing nanogels and their applications, specifically to a method for preparing hybrid nanogels for enhancing sonodynamic therapy of tumors and their applications. Background Technology

[0002] Sonodynamic therapy, with its advantages of high spatiotemporal accuracy, minimal invasiveness, and low systemic toxicity, is widely used in the treatment of malignant tumors. During photodynamic therapy, external laser irradiation activates photosensitizers at the tumor site, generating reactive oxygen species and upregulating intracellular oxidative stress. Due to the hydrophobicity of traditional sonodynamic agents, various carriers are needed to encapsulate them for in vivo delivery. However, traditional sonodynamic agents tend to accumulate in the hydrophobic regions of the carrier, leading to aggregation quenching and reduced sonodynamic efficiency. Furthermore, ultrasound therapy still lacks effective methods for treating hypoxic segments of tumors with insufficient oxygen supply. Therefore, optimizing sonodynamic tumor treatment strategies based on traditional sonodynamic agents is crucial to improving sonodynamic efficiency.

[0003] Tumor cells exhibit various abnormal physiological indicators compared to normal cells, making tumor therapy by regulating tumor cell metabolism possible. Tumor cells have higher concentrations of glutathione and adenosine triphosphate (ATP) than normal cells, and hypoxic regions exist within tumors due to abnormal vascular distribution and insufficient nutrient supply. These abnormal physiological characteristics of tumors also significantly reduce the effectiveness of single-method treatments. For example, the high levels of reactive oxygen species (ROS) induced by sonodynamic processes can effectively kill tumor cells, but the high concentration of glutathione within tumor cells neutralizes ROS, reducing the tumor-killing effect. Furthermore, the hypoxic microenvironment in tumors is largely due to the tumor's vigorous self-consumption, which also limits the therapeutic effect of sonodynamic therapy. In addition, the hypoxic microenvironment weakens the tumor-killing effect of amplified oxidative stress.

[0004] By utilizing starvation therapy to deplete glucose at the tumor site, we can reduce the tumor's own metabolism and enhance the efficacy of sonodynamic therapy. Furthermore, adenosine triphosphate (ATP), as the body's most direct energy source, has attracted our attention. The ATP-based energy supply mechanism of tumor cells is a restricted process, requiring rapid conversion between ATP and adenosine diphosphate (ADP). Therefore, restricting the free conversion between ATP and ADP at the tumor site may be an effective energy blocking strategy, which can enhance the therapeutic effect of sonodynamic therapy by reducing the tumor's own endogenous oxygen consumption. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing hybrid nanogels that enhance the sonodynamic therapy of tumors.

[0006] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A method for preparing a hybrid nanogel to enhance tumor sonodynamic therapy, wherein a hydrophilic polymer derivative is used to self-assemble and encapsulate a hypoxia-responsive drug in manganese dioxide in an inorganic salt solution; the hydrophilic polymer derivative is obtained by modifying phenylboronic acid and a sonosensitive agent onto a hydrophilic polymer material in one step; the hydrophilic polymer material is selected from any one of starch, dextrin, hyaluronic acid, chitosan, dextran, gelatin or silk fibroin.

[0007] The beneficial effects of this invention are: This invention constructs a hydrophilic polymer derivative through one-step modification and autonomously packages it with nano-manganese dioxide to form a hybrid nanogel, thereby achieving a highly efficient sonodynamic therapy effect and inhibiting the energy metabolism of the tumor site itself. The hypoxia-responsive drug loaded in the nanogel can be specifically activated at the hypoxic site of the tumor, thereby achieving a synergistic sonodynamic therapy effect on the tumor.

[0008] This invention utilizes the aggregation of manganese dioxide in inorganic salts. With the assistance of inorganic salts, manganese dioxide can be directionally loaded and self-assembled into the interior of hydrophilic polymer materials to form nanogels. The nanogels obtained through this directional loading and self-assembly have stable properties.

[0009] Based on the above technical solution, the present invention can be further improved as follows.

[0010] Furthermore, the molecular weight of the hydrophilic polymer material is 10,000-200,000; the degree of substitution of the sound-sensitizing agent on the main chain of the hydrophilic polymer material is 20-50 μg / mg, preferably 40 μg / mg; and the degree of substitution of phenylboronic acid on the main chain of the hydrophilic polymer material is 20-200 μg / mg.

[0011] The beneficial effects of adopting the above-mentioned further scheme are as follows: Since the acoustic sensitizer will cause aggregation quenching, the present invention can reduce its aggregation quenching by controlling the modification ratio of the acoustic sensitizer on the dextrin polymer to make it in a loose state. The acoustic sensitizer modification greater than 50 μg / mg will cause aggregation quenching, which will significantly affect the acoustic dynamic efficiency; the acoustic sensitizer with a substitution degree of less than 20 μg / mg cannot provide an effective hydrophobic region on the hydrophilic polymer material and cannot form a hybrid nanogel.

[0012] This invention utilizes phenylboronic acid and a sonication agent to modify a hydrophilic polymer material in a one-step process. This allows the phenylboronic acid exposed on the hydrogel surface to bind to sialic acid receptors overexpressed on the surface of tumor cells, thereby exerting a tumor-targeting effect. Internally, the phenylboronic acid can bind to ATP within tumor cells, reducing the tumor cells' own energy metabolism and providing more oxygen for sonication.

[0013] Furthermore, the hydrophilic polymer material is dextrin; the sonosensitive agent is protoporphyrin IX; and the phenylboronic acid is 4-carboxyphenylboronic acid.

[0014] Furthermore, the inorganic salt solution is a sodium dihydrogen phosphate and disodium hydrogen phosphate solution; the manganese dioxide surface is modified with any one of polyallylamine, polyethyleneimine or chitosan; the phenylboronic acid is any one of 4-aminophenylboronic acid, 3-aminophenylboronic acid, o-aminophenylboronic acid, 4-carboxyphenylboronic acid, 3-carboxyphenylboronic acid or 2-carboxyphenylboronic acid.

[0015] Furthermore, the surface-modified manganese dioxide has a positive potential and a size of 10-40 nanometers.

[0016] Furthermore, the hypoxia-responsive drug is one of telatrazamine, apatziquinone, evafosinate, banoanthraquinone, and tasotinib.

[0017] Furthermore, the method of modifying a hydrophilic polymer material with phenylboronic acid and a sound-sensitizing agent in one step involves dissolving the hydrophilic polymer material in dimethyl sulfoxide solvent, then adding dicyclohexylcarbodiimide and 4-dimethylaminopyridine to obtain a hydrophilic polymer material solution. The mixture of phenylboronic acid and the sound-sensitizing agent is stirred under nitrogen protection at 40-55°C to obtain a mixed solution, preferably 50°C. The mixed solution is then added to ice-cold diethyl ether, centrifuged, and a white precipitate is obtained. This precipitate is dissolved in deionized water, dialyzed using a dialysis bag, and then freeze-dried.

[0018] Furthermore, the selected dialysis bag has a molecular weight of 10,000 to 100,000, and the concentration of the dialysate is 0.01 mol.

[0019] Furthermore, the mass ratio of the hydrophilic polymer material to manganese dioxide is 100:0.5-10, preferably 100:1-6, and the concentration of the hypoxia-responsive drug is 100 μg / mL.

[0020] The present invention also provides the application of the hybrid nanogel obtained by the above-mentioned method for preparing hybrid nanogels for enhanced tumor sonodynamic therapy in the preparation of therapeutic drugs for tumors or hypoxic tumor-related diseases. Attached Figure Description

[0021] Figure 1 This is the 1H NMR spectrum of the hydrophilic polymer derivative dextrin-protoporphyrin IX-phenylboronic acid (Dex-PpIX-PBA) in this invention.

[0022] Figure 2 The infrared spectrum of dextrin-protoporphyrin IX-phenylboronic acid (Dex-PpIX-PBA), a hydrophilic polymeric derivative of this invention.

[0023] Figure 3The images show the UV absorption peaks of different concentrations of protoporphyrin IX in this invention, as well as its concentration-UV absorbance standard curve at 406 nm.

[0024] Figure 4 The images show the UV absorption peaks of phenylboronic acid at different concentrations in this invention, as well as the concentration-UV absorbance standard curve at 252 nm.

[0025] Figure 5 This describes the singlet oxygen generation of Dex-PpIX-PBA with different degrees of protoporphyrin IX substitution prepared in this invention.

[0026] Figure 6 The particle size distribution and transmission electron microscopy (scale bar = 200 nm) of the hybrid nanogel (DPP / TPZ@MnO2) prepared in this invention are shown.

[0027] Figure 7 The particle size distribution and transmission electron microscopy changes of the hybrid nanogel prepared in this invention after incubation with 10 mM GSH for 24 h are shown (scale bar = 100 nm).

[0028] Figure 8 The release curves of the hybrid nanogel prepared in this invention and terazamine loaded with 10 mM GSH after incubation for different times are shown.

[0029] Figure 9 This describes the singlet oxygen generation in the hybrid nanogel prepared in this invention.

[0030] Figure 10 The ROS generation of the hybrid nanogel prepared in this invention under normal oxygen (21% O2) conditions is shown.

[0031] Figure 11 The ROS generation of the hybrid nanogel prepared in this invention under hypoxic (1% O2) conditions is shown.

[0032] Figure 12 The hybrid nanogel prepared in this invention exhibits its ability to kill breast cancer cells under normal oxygen (21% O2) conditions.

[0033] Figure 13 The hybrid nanogel prepared in this invention exhibits its ability to kill breast cancer cells under hypoxic (1% O2) conditions. Detailed Implementation

[0034] The principles and features of the present invention are described below with reference to embodiments. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0035] Example 1

[0036] Synthesis of the hydrophilic polymeric derivative dextrin-protoporphyrin IX-phenylboronic acid (Dex-PpIX-PBA)

[0037] Step 1: Dissolve 1g of dextrin in 10mL of dimethyl sulfoxide solvent, then add 970mg of dicyclohexylcarbodiimide and 180mg of 4-dimethylaminopyridine.

[0038] Step 2: Add 150 mg of protoporphyrin IX and 600 mg of 4-carboxyphenylboronic acid to the solution obtained in Step 1, and stir the mixture at 50 °C under nitrogen protection for 24 hours.

[0039] Step 3: Add 100 mL of ice-cold diethyl ether to the mixed solution obtained in Step 2, and centrifuge the solvent at 5000 rpm for 10 minutes. Dissolve the obtained white precipitate in 10 mL of deionized water, dialyze the solution using a dialysis bag with a MWCO of 12 kDa, and then freeze-dry it to obtain the hydrophilic polymeric derivative dextrin-protoporphyrin IX-phenylboronic acid.

[0040] Figure 1 The figure shows the 1H NMR spectrum of the hydrophilic polymer derivative dextrin-protoporphyrin IX-phenylboronic acid (Dex-PpIX-PBA) in this invention. Compared with the 1H NMR spectrum of dextrin, the additional peaks in the 7.0 ppm to 8.0 ppm range of Dex-PpIX-PBA are attributed to the porphyrin ring (marked 1 in the figure), and the additional proton peak at 10.2 ppm is attributed to the benzene ring (marked 2 in the figure), proving the successful modification of protoporphyrin IX and phenylboronic acid.

[0041] Figure 2 This is the infrared spectrum of the hydrophilic polymeric derivative dextrin-protoporphyrin IX-phenylboronic acid (Dex-PpIX-PBA) in this invention. Compared with dextrin, Dex-PpIX-PBA exhibits a higher infrared spectrum at 1201 cm⁻¹. -1 And 677cm -1 The absorption peak at 1725 cm⁻¹ is attributed to the CN stretching and -CH₃- stretching of protoporphyrin IX. -1 The absorption peak at that point is attributed to the benzene ring stretching of phenylboronic acid, proving the successful modification of protoporphyrin IX and phenylboronic acid.

[0042] Figure 3 The standard curve of concentration-UV absorbance of protoporphyrin IX at 406 nm in this invention is shown. By substituting it into the curve, the degree of substitution of protoporphyrin IX, the sonosensitive agent in Dex-PpIX-PBA, is calculated to be 26 μg / mg.

[0043] Figure 4 The standard curve of phenylboronic acid concentration-UV absorbance at 252 nm in this invention is shown. By substituting it into the curve, the degree of substitution of phenylboronic acid in Dex-PpIX-PBA is calculated to be 34 μg / mg.

[0044] Figure 5 This paper presents the singlet oxygen generation of Dex-PpIX-PBA with different degrees of protoporphyrin IX substitution prepared in this invention. The singlet oxygen green fluorescent probe, Singlet Oxygen Sensor Green (SOSG), can selectively bind singlet oxygen to generate green fluorescence. By comparing the ratio of the fluorescence intensity (IA) of SOSG after 3 minutes of sonication (1W / cm², 1MHz) to its initial fluorescence intensity (I0), it can be seen that the singlet oxygen generation effect of Dex-PpIX-PBA increases with increasing protoporphyrin IX substitution (13 μg / mg to 47 μg / mg). However, the singlet oxygen generation capacity of Dex-PpIX-PBA with a high degree of protoporphyrin IX substitution decreases (62 μg / mg) due to aggregation quenching.

[0045] Example 2

[0046] Preparation of hybrid nanogels

[0047] Step 1: Mix 18 mL of 3.5 mg / mL potassium permanganate solution with 2 mL of 37.4 mg / mL polyallylamine solution and react at room temperature for 2 hours to obtain positively charged manganese dioxide nanoparticles.

[0048] Step 2: Dissolve 13 μg of manganese dioxide nanoparticles, 1 mg of Dex-PpIX-PBA and 100 μg of terazamine obtained in Step 1 into a mixed solution of 5 mL of deionized water and 5 mL of ethanol, and vortex the mixed solution for 1 minute.

[0049] Step 3: Dialyze the mixed solution from Step 2 using 10 mL of a solution containing 0.01 mol sodium dihydrogen phosphate and disodium hydrogen phosphate for 24 hours.

[0050] Step 4: Centrifuge the solution in the dialysis bag from Step 3 at 12000 rpm for 30 minutes to remove uncoated manganese dioxide. Use a 0.22 μm cellulose acetate membrane to remove uncoated Dex-PpIX-PBA. Then, use a cellulose ultrafiltration membrane with a molecular weight cutoff of 10000 to remove uncoated tirazamine.

[0051] Figure 6 The images show the particle size distribution and transmission electron microscopy morphology of the hybrid nanogel (DPP / TPZ@MnO2) prepared in this invention. It can be seen that the average particle size of the prepared hybrid nanogel is approximately 100 nanometers. This suitable nanosize not only facilitates intravenous injection in vivo but also enables it to effectively accumulate at the tumor site through the EPR effect (Enhanced Permeability and Retention Effect).

[0052] Figure 7 The figures show the particle size distribution and transmission electron microscopy changes of the hybrid nanogel prepared in this invention after incubation with 10 mM GSH for 24 h. As can be seen from the figures, the manganese dioxide component in the hybrid nanogel can react with the high concentration of glutathione at the tumor site, achieving rapid disintegration of the nanogel at the tumor site. In this state, the DPP in the nanogel system returns to a loose state, which can enhance the sonodynamic effect of the sonosensitive agent; at the same time, the encapsulated TPZ can achieve rapid release, exerting a synergistic anti-tumor effect.

[0053] Figure 8 The figures show the release curves of the hybrid nanogel prepared in this invention after incubation with 10 mM GSH for different times and encapsulated terazamine. As can be seen from the figures, the hybrid nanogel can react with high concentrations of glutathione, resulting in the rapid release of the encapsulated contents.

[0054] Example 3

[0055] Singlet oxygen generation in hybrid nanogels

[0056] Step 1: Using a singlet oxygen green fluorescent probe (SOSG) as the singlet oxygen detection probe, it was added to a nanogel containing 100 μg / ml protoporphyrin IX sonosensitive agent dissolved in isotonic phosphate buffer, and then sonicated (1 W / cm², 1 MHz) for 3 minutes. The fluorescence intensity change of SOSG was observed using a multi-functional microplate reader (Ex = 488 nm, Em = 525 nm).

[0057] Step 2: The reactive oxygen species (ROS) generation of the hybrid nanogel within mouse breast cancer cells (4T1) was investigated. 4T1 cells were seeded into 24-well plates and cultured under normoxic (21% oxygen) and hypoxic (1% oxygen) conditions. The nanogel containing 10 μg / ml protoporphyrin IX sonicator was incubated with 4T1 cells for 4 hours, followed by sonication (1 W / cm²). 2 The ROS levels in 4T1 cells were examined using a ROS probe at 1 MHz for 3 minutes.

[0058] Figure 9 The figure shows the singlet oxygen generation of the hybrid nanogel prepared in this invention. As can be seen from the figure, the nanogel can generate efficient singlet oxygen under ultrasonic conditions, and the acoustic dynamics of the nanogel, which disintegrates after reacting with a high concentration of GSH, is improved.

[0059] Figure 10 The diagram shows the ROS generation of the hybrid nanogel prepared in this invention under normal oxygen (21% O2) conditions. Compared with liposomes loaded with protoporphyrin IX sonosensitive agent, the nanogel exhibits highly efficient sonodynamic therapeutic effects under normal oxygen conditions.

[0060] Figure 11 The diagram shows the ROS generation of the hybrid nanogel prepared in this invention under hypoxic (1% O2) conditions. Compared with liposomes loaded with protoporphyrin IX sonosensitive agent, the nanogel exhibits highly efficient sonodynamic therapeutic effects under hypoxic conditions.

[0061] Example 4

[0062] Cytotoxicity detection of hybrid nanogels

[0063] Step 1: 4T1 cells were seeded into 96-well plates and then cultured under normoxic (21% oxygen) and hypoxic (1% oxygen) conditions. After the cell density increased to 60%-80%, the tumor cells were incubated with different concentrations of hybrid nanogels for 4 hours.

[0064] Step 2: Replace the nanogel with fresh culture medium and sonicate for 3 minutes (1W / cm2, 1MHz).

[0065] Step 3: After incubation for another 24 hours, add 20 μl of 5 mg / mL thiazolyl blue and incubate for another 4 hours. Then discard the supernatant and add 200 μl of DMSO. Observe the absorbance of cells at 570 nm using a multi-functional microplate reader. Cell viability = (Absorbance of drug-treated group - Absorbance of blank group) / (Absorbance of negative control group - Absorbance of blank group) * 100%.

[0066] Figure 12 The figure shows the killing effect of the hybrid nanogel prepared in this invention on breast cancer cells under normal oxygen (21% O2) conditions. As can be seen from the figure, the nanogel exhibits good sonodynamic tumor killing effect under normal oxygen conditions.

[0067] Figure 13 The figure shows the killing effect of the hybrid nanogel prepared in this invention on breast cancer cells under hypoxic (1% O2) conditions. As can be seen from the figure, the nanogel exhibits good sonodynamic tumor killing effect under hypoxic conditions.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a hybrid nanogel to enhance sonodynamic therapy for tumors, characterized in that, A hypoxia-responsive drug is encapsulated by self-assembly of a hydrophilic polymer derivative with manganese dioxide in an inorganic salt solution; the surface of the manganese dioxide is modified with any one of polyallylamine, polyethyleneimine or chitosan, and the surface-modified manganese dioxide has a positive potential. The hydrophilic polymer derivative is obtained by modifying a hydrophilic polymer material with phenylboronic acid and a sound-sensitizing agent in one step; the hydrophilic polymer material is dextrin, the sound-sensitizing agent is protoporphyrin IX, and the phenylboronic acid is 4-carboxyphenylboronic acid; the degree of substitution of the sound-sensitizing agent on the dextrin backbone is 20-50 μg / mg, and the degree of substitution of phenylboronic acid on the dextrin backbone is 20-200 μg / mg; The mass ratio of dextrin to manganese dioxide is 100:1 to 6.

2. The method for preparing a hybrid nanogel for enhancing tumor sonodynamic therapy according to claim 1, characterized in that, The inorganic salt solution is a solution of sodium dihydrogen phosphate and disodium hydrogen phosphate.

3. The method for preparing a hybrid nanogel for enhancing tumor sonodynamic therapy according to claim 1, characterized in that, The surface-modified manganese dioxide has a size of 10-40 nanometers.

4. The method for preparing a hybrid nanogel for enhancing tumor sonodynamic therapy according to claim 1, characterized in that, The hypoxia-responsive drug is one of telatazamine, apatziquinone, evaphosphatamide, banoanthraquinone, and tasotinib.

5. The method for preparing a hybrid nanogel for enhancing tumor sonodynamic therapy according to claim 1, characterized in that, The method of modifying a hydrophilic polymer material with phenylboronic acid and a sound-sensitizing agent in one step involves: dissolving the hydrophilic polymer material in dimethyl sulfoxide solvent, then adding dicyclohexylcarbodiimide and 4-dimethylaminopyridine to obtain a hydrophilic polymer material solution; stirring the mixture of phenylboronic acid and the sound-sensitizing agent under nitrogen protection at 40-55°C to obtain a mixed solution; adding the mixed solution to ice-cold diethyl ether, centrifuging to obtain a white precipitate, dissolving it in deionized water, dialyzing using a dialysis bag, and freeze-drying.

6. The method for preparing a hybrid nanogel for enhancing tumor sonodynamic therapy according to claim 5, characterized in that, The selected dialysis bag has a molecular weight of 10,000 to 100,000 and the dialysis solution concentration is 0.01 mol.

7. A method for preparing a hybrid nanogel for enhancing tumor sonodynamic therapy according to any one of claims 1 to 6, characterized in that, The concentration of the hypoxia-responsive drug is 100 μg / mL.

8. The use of a hybrid nanogel obtained by the method for preparing a hybrid nanogel for enhancing tumor sonodynamic therapy according to any one of claims 1 to 7 in the preparation of a therapeutic drug for hypoxic tumors.

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