Enhanced sonodynamic nano-drug based on aldehyde dehydrogenase inhibitor as well as preparation method and application of enhanced sonodynamic nano-drug

Abstract

The invention discloses an enhanced sonodynamic nano-drug based on an aldehyde dehydrogenase inhibitor as well as a preparation method and application thereof, the nano-drug comprises two small organic molecules, and the two small organic molecules respectively have a formula 1 (BTz-IC-X, X = H, F or Cl, according to the compound disclosed by the invention, ROS is generated under an ultrasonic condition through an organic molecule BTz-IC-X and is used for sonodynamic therapy of tumors, and the generation of the ROS is enhanced through carrying of an aldehyde dehydrogenase inhibitor KS100, so that the sonodynamic therapy effect is enhanced; the invention provides a nano-drug which has an ultrasonic excitation sonodynamic therapy sonosensitizer and carries an aldehyde dehydrogenase inhibitor to enhance sonodynamic therapy, and efficient sonodynamic therapy of in-situ pancreatic cancer is achieved through ROS generated in the sonodynamic process. The preparation method of the nano-drug is mild in synthesis condition and simple in route, two small organic molecules are directly mixed with a surfactant, and an enhanced sonodynamic system is synthesized through ultrasound.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomedicines, and in particular relates to an enhanced sonodynamic nanomedicine based on an aldehyde dehydrogenase inhibitor, a preparation method and applications thereof. Background Art

[0002] Cancer remains a major global public health problem and the second leading cause of death worldwide. Sonodynamic therapy (SDT), primarily derived from photodynamic therapy (PDT), has emerged as a novel non-invasive cancer treatment. Low-intensity ultrasound (LIUS) is used as the energy source, replacing the laser used in PDT, to induce the production of reactive oxygen species (ROS), thereby damaging cancer cells and tissues and achieving the goal of tumor treatment. Compared to destructive X-rays, shallow tissue-penetrating light, and complex magnetic fields, SDT has become an attractive treatment method due to its penetration depth of up to 10 cm. It overcomes the major drawbacks of PDT, such as shallow penetration, collateral damage to adjacent tissues, and phototoxicity. The therapeutic efficacy of SDT depends largely on the properties of the sonosensitizer. However, existing sonosensitizers suffer from low ROS production under ultrasound, poor stability, and poor biocompatibility. Therefore, the development of novel sonosensitizers with high sonodynamic efficiency, excellent stability, and good biocompatibility for the treatment of cancer and other related diseases is crucial.

[0003] Aldehyde dehydrogenases (ALDHs) are enzymes that oxidize endogenous and exogenous aldehydes. ALDHs protect cells from oxidative stress and ROS and trigger lipid peroxidation and the accumulation of toxic aldehydes. Aldehydes form protein adducts via non-enzymatic covalent bonds with lysine, cysteine, and histidine residues and damage cells by increasing ROS production and lipid peroxidation. Overexpression of ALDH isoforms has been associated with progression, drug resistance, and poor prognosis in many cancers, including breast cancer, esophageal cancer, Ewing sarcoma, and head and neck squamous cell carcinoma.

[0004] Biomedical imaging plays a key role in the early detection and diagnosis of cancer. Although scientists have developed a variety of imaging techniques, such as magnetic resonance imaging and optical imaging, each method has certain limitations. Although magnetic resonance imaging has excellent tissue penetration and spatial resolution, it lacks sensitivity when using T1 contrast agents for molecular imaging, and T2 contrast agents are difficult to distinguish from tissues with similar magnetic resonance signals (such as bones and lungs) during imaging. On the other hand, optical imaging (near-infrared zone 1) is limited by the penetration depth of tissues. Therefore, the development of a near-infrared zone 2 fluorescent imaging contrast agent is crucial for imaging deep tumors and guiding tumor treatment. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide an enhanced sonodynamic nanomedicine based on aldehyde dehydrogenase inhibitors, a preparation method and its application, which realizes the generation of ROS under ultrasonic conditions for tumor sonodynamic therapy through the organic molecule BTz-IC-X, and enhances the generation of ROS by carrying the aldehyde dehydrogenase inhibitor KS100, thereby improving the sonodynamic therapy effect.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides an enhanced sonodynamic nanomedicine based on an aldehyde dehydrogenase inhibitor. The nanomedicine comprises two organic small molecules, each having a structure shown in Formula 1 and Formula 2, respectively:

[0008]

[0009] Furthermore, under ultrasonic excitation, the organic small molecule BTz-IC-X can generate a variety of ROS, such as hydroxyl radicals (·OH) and singlet oxygen ( 1 O2).

[0010] Furthermore, the organic small molecule BTz-IC-X can perform near-infrared second-region fluorescence imaging with the excitation wavelength in the near-infrared region.

[0011] Furthermore, the organic small molecule KS100 is an aldehyde dehydrogenase inhibitor, which can inhibit the activity of aldehyde dehydrogenase, promote the accumulation of aldehydes (such as acetaldehyde, malondialdehyde, etc.), enhance the production of ROS and lipid peroxidation, and improve the effect of sonodynamic therapy. The mechanism of action is as follows Figure 4 shown.

[0012] Furthermore, the nanomedicine also includes a surfactant to enhance the dispersibility of the nanomedicine in an aqueous solution.

[0013] Furthermore, the surfactant includes distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG 2k) and acid-responsive polymer (PEG-PDA).

[0014] The present invention also provides a method for preparing the enhanced sonodynamic nanomedicine based on the aldehyde dehydrogenase inhibitor, comprising the following steps:

[0015] (1) Synthesis of BTz-IC-X, KS100, and PEG-PDA;

[0016] (2) BTz-IC-X, KS100, DSPE-mPEG 2k and PEG-PDA in a predetermined ratio were added to THF, and then H2O was added, ultrasonic treatment was performed, and excess THF was removed by suspension evaporation, and ultrafiltration washing was performed to obtain the nanodrug BTZ-IC-X@KS00@PEG-PDANPs.

[0017] Furthermore, in step (2), the suspension temperature is set to 40-50°C, the ultrafiltration centrifuge speed is 4000-5000 rpm, the centrifugation time is 3-8 minutes, washing is performed 2-4 times, and the nanomedicine is stored in a refrigerator at 0-6°C for later use.

[0018] The present invention also provides applications of the enhanced sonodynamic nanomedicine based on the aldehyde dehydrogenase inhibitor, which can be used as a sonosensitizer for sonodynamic therapy and a near-infrared second-zone fluorescence imaging contrast agent.

[0019] Specifically, the nanomedicine can be used as a sonodynamic therapy agent for in situ pancreatic cancer and near-infrared second-zone fluorescence imaging of in situ pancreatic cancer.

[0020] The acetaldehyde dehydrogenase inhibitor KS100 can cause the accumulation of toxic aldehydes, increase ROS activity and lipid peroxidation, and effectively inhibit ALDH1A1, ALDH2 and ALDH3A1, leading to increased cell apoptosis and cell cycle G2 / M arrest, and has a killing effect on tumor cells.

[0021] The technical solution of the present invention has the following beneficial effects:

[0022] The present invention provides a nanomedicine that has both an ultrasound-excited sonodynamic therapy sonosensitizer and an aldehyde dehydrogenase inhibitor to enhance sonodynamic therapy, and guides the treatment of in situ pancreatic cancer through near-infrared second-zone fluorescence imaging.

[0023] The present invention provides a nanomedicine that has both an ultrasound-excited sonodynamic therapy sonosensitizer and an aldehyde dehydrogenase inhibitor to enhance sonodynamic therapy, and achieves efficient in situ pancreatic cancer treatment through the sonodynamic process.

[0024] The present invention provides a nanomedicine for enhancing sonodynamic therapy by using an aldehyde dehydrogenase inhibitor, and enhances sonodynamic therapy for in situ pancreatic cancer by using an aldehyde dehydrogenase inhibitor.

[0025] The preparation method of the nano drug of the present invention has mild synthesis conditions and a simple route. Small organic molecules are directly mixed with surfactants, and an enhanced sonodynamic system is synthesized through ultrasound. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the synthesis of the organic small molecule BTz-IC-X (X = H, F, Cl, Br).

[0027] Figure 2 Schematic diagram of the synthesis of the organic small molecule KS100.

[0028] Figure 3 Schematic diagram of the synthesis of acid-responsive polymer PEG-PDA.

[0029] Figure 4 Schematic diagram of the principle of enhanced sonodynamic nanomedicine based on aldehyde dehydrogenase inhibitors.

[0030] Figure 5 This is the UV image of BTz-IC-H@KS100@PEG-PDANPs prepared in Example 2.

[0031] Figure 6 TEM image of BTZ-IC-H@KS100@PEG-PDA NPs obtained in Example 2.

[0032] Figure 7 This is the particle size diagram of BTZ-IC-H@KS100@PEG-PDA NPs obtained in Example 2.

[0033] Figure 8 This is the surface Zeta potential diagram of BTZ-IC-H@KS100@PEG-PDANPs obtained in Example 3.

[0034] Figure 9 This is the cytotoxicity graph obtained in Example 4.

[0035] Figure 10 These are the in vivo near-infrared two-zone fluorescence imaging images of the subcutaneous tumor obtained in Example 5 at different times.

[0036] Figure 11 This is the subcutaneous tumor growth curve obtained in Example 5.

[0037] Figure 12 This is a tumor growth curve of in situ pancreatic cancer obtained in Example 5. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further illustrated below through specific experimental methods.

[0039] The experimental methods described in the following examples are conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.

[0040] Example 1 Synthesis of BTz-IC-X, KS100 and Acid-responsive Polymer PEG-PDA

[0041] Preparation of BTz-IC-X: Under nitrogen, a solution of compound 1 (10 mL) and a solution of 1,2-dichloroethane in DMF (10 mL) were mixed in a 100 mL three-necked flask and cooled to 0°C in an ice bath. POCl₃ (0.5 mL, 5.36 mmol) was slowly added dropwise, and the reaction was maintained at 0°C for 2 h. The temperature was then raised to 90°C and the reaction continued for 20 h. After completion of the reaction, the mixture was poured into 100 mL of ice water, the pH was adjusted to neutral with saturated NaOH solution, and the mixture was extracted with DCM. After drying over anhydrous Na₂CO₃, the mixture was concentrated and purified by column chromatography (DCM / PE = 1:1) to afford the yellow compound 2. CHCl₃ (30 mL), pyridine (0.1 g, 0.5 mmol), compound 2 (0.15 g, 0.13 mmol), and IC-X (0.5 mmol) were added sequentially to a 100 mL three-necked flask and the reaction was incubated at 65°C under nitrogen for 20 h. After cooling, the reaction solution was poured into 200 mL of methanol to precipitate, and the dark blue solid was collected by filtration. The target product BTz-IC-X (X = H, F, Cl, Br) was obtained after separation and purification by silica gel column chromatography (PE / DCM = 1:1) and vacuum drying (synthesis diagram shown in FIG. Figure 1 ).

[0042] Preparation of KS100: 500mg of 4,6-dibromoindigo (1.6mmol) was dissolved in 10mL of ethyl acetate, 1.76mmol of potassium carbonate (K2CO3) was added, and the mixture was stirred at room temperature for 1 hour. Subsequently, 1690mg of 1,4-di(bromomethyl)benzene (6.4mmol) was added and the reaction was allowed to proceed for 12 hours. The reaction solution was poured into ice water, extracted with ethyl acetate, and purified by silica gel column chromatography to obtain oily red crystals (compound 3). Compound 3 (300mg, 1.02mmol) and thiourea (47mg, 1.02mmol) were dissolved in 25mL of ethanol and refluxed at 90°C for 12 hours. The solvent was removed by rotary evaporation, and the solid was precipitated by adding ethyl acetate after redissolving in ethanol. The final product KS100 was obtained by washing with ethyl acetate several times (the synthesis diagram is shown in the figure). Figure 2 ).

[0043] Synthesis of acid-responsive polymer PEG-PDA: 44 mg NHS-CTA (0.117 mmol) was dissolved in 20 mL of anhydrous dichloromethane (DCM), and PEG was added dropwise at constant pressure. 113-NH2 (500 mg, 0.1 mmol) in DCM solution. After reacting at room temperature for 12 hours, excess ether was added to precipitate the product, and the pink solid PEG-CTA was obtained by vacuum drying. Using the RAFT polymerization method, PEG-CTA (50 mg, 0.01 mmol), diisopropylaminoethyl methacrylate (PDA, 256 mg, 1.2 mmol), and AIBN (0.41 mg, 0.0025 mmol) were dissolved in 2 mL of 1,4-dioxane. After three liquid nitrogen freeze-thaw cycles for deoxygenation, the reaction was carried out at 70 ° C for 48 hours. The reaction solution was dialyzed against pure water (molecular weight cutoff 5000 Da) and lyophilized to obtain PEG-PDA (synthesis diagram as shown in the figure). Figure 3 ).

[0044] Example 2 Synthesis and property verification based on BTz-IC-H@KS100@PEG-PDANPs

[0045] The present invention is based on the synthesis and property research of BTz-IC-H@KS100@PEG-PDANPs. The specific synthesis method is as follows:

[0046] (1) Preparation of BTz-IC-H@KS100@PEG-PDA NPs and control BTz-IC-H@PEG-PDA NPs (where X = H):

[0047] Preparation of BTz-IC-H@KS100@PEG-PDA NPs: Directly synthesized by nano-coprecipitation method ( Figure 4 ), first prepare 1 mL of THF mother solution containing BTz-IC-H (100 μg), aldehyde dehydrogenase inhibitor KS100 (100 μg), distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG 2k) (0.4 mg) and acid-responsive polymer PEG-PDA (0.2 mg), quickly inject 9 mL of H2O into the prepared tetrahydrofuran solution, and sonicate for 10 minutes; after removing excess THF by rotary evaporation, set the suspension temperature to 45°C, centrifuge (4500 rpm, 5 minutes), wash three times with ultrapure water, concentrate, calibrate the concentration with BTz-IC-H (the concentration of BTz-IC-H is 1 mg / mL), and store in a refrigerator at 4°C for use.

[0048] Preparation of control nanoparticles BTz-IC-H@PEG-PDA: First, prepare 1 mL of THF mother solution containing BTz-IC-H (100 μg), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG 2k) (0.4 mg) and acid-responsive polymer PEG-PDA (0.2 mg), quickly inject 9 mL of H2O into the prepared tetrahydrofuran solution, and sonicate for 10 minutes; after removing excess THF by rotary evaporation, set the suspension temperature to 45°C, centrifuge (4500 rpm, 5 minutes), wash three times with ultrapure water, concentrate, calibrate the concentration with BTz-IC-H (the concentration of BTz-IC-H is 1 mg / mL), and store in a refrigerator at 4°C for later use.

[0049] (2) Study on the properties of BTz-IC-H@KS100@PEG-PDA NPs: The obtained BTz-IC-H@KS100@PEG-PDA NPs were diluted to a certain extent, and then the UV spectra were tested by UV instruments ( Figure 5 ).

[0050] (3) Study on the particle size and surface potential properties of BTz-IC-H@KS100@PEG-PDA NPs: The mass concentration of the nanoparticles obtained in step (1) was determined by ultraviolet absorption, and the particle size of the nanoparticles was measured by transmission electron microscopy (TEM). Figure 6 ) and the surface Zeta potential of the nanoparticles ( Figure 8 ).

[0051] from Figure 5 It can be seen that the ultraviolet spectrum of molecules is between 600-900 nanometers.

[0052] Figure 6 TEM image of BTz-IC-H@KS100@PEG-PDANPs obtained in Example 2. Figure 6 It can be seen that the particle size of the synthesized particles is about 50-60 nm.

[0053] Figure 7 This is the DLS graph of BTz-IC-H@KS100@PEG-PDANPs obtained in Example 2. Figure 7 It can be seen that the particle size of the synthesized particles is about 40 nm, which is consistent with the results obtained by TEM.

[0054] Example 3 Synthesis and property verification based on BTz-IC-H@KS100@PEG-PDANPs

[0055] The embodiment of the present invention is based on the property study of BTz-IC-H@KS100@PEG-PDA NPs, and the specific synthesis steps are the same as step (1) of Example 2.

[0056] Figure 8 The surface zeta potential of the BTz-IC-H@KS100@PEG-PDA NPs obtained in Example 3 before and after acid response was plotted. The BTz-IC-H@KS100@PEG-PDA NPs were incubated with PBS at pH 7.4 and 5.4 for a specified time, followed by sonication for 5 minutes. The surface zeta potential after incubation at different pH conditions was measured using dynamic light scattering. As shown in Figure 8, the surface zeta potential of the BTz-IC-H@KS100@PEG-PDA NPs was -8 mV when incubated with PBS at pH 7.4, and +2.8 mV when incubated with PBS at pH 5.4.

[0057] Example 4 Cytotoxicity analysis based on BTz-IC-H@KS100@PEG-PDANPs

[0058] a) Toxicity Analysis: To evaluate the sonodynamic effect of the nanomedicine prepared in Example 2 at the cellular level, 4T1 cancer cells were seeded in 96-well plates and treated with different concentrations of BTz-IC-H@KS100@PEG-PDA NPs after they adhered to the plate. Two treatment groups were set up in the experiment: BTz-IC-H@KS100@PEG-PDA NPs and BTz-IC-H@KS100@PEG-PDA NPs. After the cells were incubated for a further 12 hours, cell viability was finally measured using the MTT assay. This protocol effectively analyzes the sonodynamic therapeutic effect of the material by comparing the differences between the two groups.

[0059] Figure 9 The toxicity analysis results of different concentrations of nanomedicine on cancer cells after different treatments are shown. Figure 9 It can be seen that BTz-IC-H@KS100@PEG-PDA NPs treatment has a good killing effect on cancer cells 4T1. In addition, we also performed cytotoxicity analysis on mouse pancreatic cancer cells Pan02 cells, and the results obtained were consistent with those of 4T1 cells.

[0060] From the above analysis, it can be seen that the nanomedicine prepared in Example 2 can be taken up by cancer cells, generate ROS under the action of ultrasound to kill tumors, and achieve sonodynamic therapy of tumors.

[0061] Example 5 Inhibition of tumor growth based on BTz-IC-H@KS100@PEG-PDA NPs nanoparticle system

[0062] For subcutaneous tumors, near-infrared two-zone imaging (0, 2, 8, 24 h) was performed at different times after injection of BTz-IC-H@KS100@PEG-PDA NPs to determine the optimal enrichment time. Figure 10 It can be seen that tumor enrichment reaches a maximum at 24 hours.

[0063] The animal experiments were divided into 5 groups (blank, BTz-IC-H@KS100@PEG-PDA NPs, ultrasound, BTz-IC-H@KS100@PEG-PDA NPs+ultrasound, BTz-IC-H@PEG-PDA NPs+ultrasound), and the cells were injected into the tail vein (1.5 mg / mL). Ultrasound treatment was performed at 24 h, and the tumor size was recorded every 1 day starting from day 0 for a total of 14 days.

[0064] Figure 11 is the tumor growth curve, from Figure 11 It can be seen that the nanosystem has a better effect in inhibiting tumor growth.

[0065] For orthotopic pancreatic cancer in mice, the animals were divided into two groups (blank and BTz-IC-H@KS100@PEG-PDA NPs + ultrasound). The mice were injected via the tail vein (1.5 mg / mL) and irradiated with ultrasound at the 12th hour. Within 24 hours after sonodynamic therapy, the mice were euthanized, and the pancreatic organs and pancreatic tumors were removed for H&E staining. As shown in Figure 12, in the BTz-IC-H@KS100@PEG-PDA NPs + ultrasound group, tumor cells showed significant nuclear shrinkage and nuclear-cytoplasmic separation, indicating a significant therapeutic effect on the tumor.

[0066] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. An enhanced sonodynamic nanomedicine based on aldehyde dehydrogenase inhibitor, characterized in that: The nanomedicine includes two organic small molecules, and the two organic small molecules have structures shown in Formula 1 (BTz-IC-X, X=H, F, Cl, Br) and Formula 2 (KS100):

2. The enhanced sonodynamic nanomedicine based on aldehyde dehydrogenase inhibitor according to claim 1, characterized in that: The nano medicine also includes a surfactant.

3. The enhanced sonodynamic nanomedicine based on aldehyde dehydrogenase inhibitor according to claim 2, characterized in that: The surfactants include DSPE-mPEG (2k) and PEG-PDA.

4. The method for preparing the enhanced sonodynamic nanomedicine based on aldehyde dehydrogenase inhibitor according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Synthesis of BTz-IC-X, KS100, and PEG-PDA; (2) BTz-IC-X, KS100, DSPE-mPEG 2k and PEG-PDA in a predetermined ratio were added to THF, and then H2O was added, ultrasonic treatment was performed, and excess THF was removed by suspension evaporation, and ultrafiltration washing was performed to obtain the nanodrug BTZ-IC-X@KS00@PEG-PDANPs.

5. The method for preparing the enhanced sonodynamic nanomedicine based on aldehyde dehydrogenase inhibitor according to claim 4, characterized in that: In step (2), the suspension temperature is set to 40-50°C, the ultrafiltration centrifuge speed is 4000-5000 rpm, the centrifugation time is 3-8 minutes, washing is performed 2-4 times, and the nanomedicine is stored in a refrigerator at 0-6°C for later use.

6. The use of the enhanced sonodynamic nanomedicine based on aldehyde dehydrogenase inhibitor according to any one of claims 1 to 3, characterized in that: It can be used as a sonosensitizer for sonodynamic therapy and a contrast agent for near-infrared zone II fluorescence imaging.

7. The use of the enhanced sonodynamic nanomedicine based on aldehyde dehydrogenase inhibitor according to claim 6, characterized in that: The nanomedicine can be used for enhanced sonodynamic therapy of in situ pancreatic cancer and near-infrared second-zone fluorescence imaging of in situ pancreatic cancer.

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