Ultrasonic activation type carrier-free nano-drug as well as preparation method and application thereof

Abstract

The invention discloses an ultrasonic activation type carrier-free nano-drug as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The nano-drug is formed by self-assembly of a sound-sensitive agent and a chemotherapy prodrug, the sound-sensitive agent is an organic small molecule capable of generating ROS through ultrasonic irradiation, and the chemotherapy prodrug is an ROS response type SN38 prodrug. Only after the carrier-free nano-drug provided by the invention is subjected to ultrasonic irradiation, the sound-sensitive agent can generate active oxygen, further activate the chemotherapeutic prodrug and release the active substance of the chemotherapeutic drug to induce tumor cell apoptosis, the tumor treatment effect is improved in an ultrasonic triggering manner, and toxic and side effects caused by non-specific distribution of the chemotherapeutic drug are avoided. The preparation method disclosed by the invention is simple and mild in reaction condition, and has great potential in the fields of preparation and application of tumor drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an ultrasound-activated carrier-free nano drug and a preparation method and application thereof. Background Art

[0002] Tumors are important diseases that affect human health. Current methods for tumor treatment include traditional methods such as surgery, radiotherapy, chemotherapy, and new treatment methods such as targeted therapy and immunotherapy. Although emerging cancer treatments such as immunotherapy (such as pembrolizumab and atezolizumab) have achieved exciting clinical success, conventional surgical resection, chemotherapy, and radiotherapy remain the first choice for the treatment of malignant tumors. Among them, chemotherapy shows better therapeutic efficacy for advanced solid tumors that cannot be treated with surgery and radiotherapy. However, chemotherapeutic drugs (such as doxorubicin, paclitaxel, and irinotecan) have serious side effects such as cardiotoxicity, hepatotoxicity, renal toxicity, neutropenia, and anemia due to their off-target biodistribution, which limits their clinical efficacy. Therefore, it is of great significance to improve the quality of life of cancer patients by developing ideal chemotherapeutic drug preparations and treatment strategies to reduce systemic toxicity and improve treatment efficacy.

[0003] At present, many chemotherapy preparations have been developed, including excipients modified with hydrophilic biocompatibility, active targeting ligands, and tumor-responsive prodrugs. However, these carrier delivery systems have problems such as low drug loading, complex preparation methods, and safety of the carrier itself. Based on this, carrier-free nanomedicines have emerged. This strategy is based on the nanostructure formed by the self-assembly of drug molecules, and its drug loading can reach 100%. Compared with nano drug delivery systems, carrier-free nanomedicines have simple preparation methods, high anti-cancer efficiency, and avoid the related toxicity and immunogenicity caused by carriers. At present, many carrier-free nanomedicines have entered clinical phase II trials, such as Theralus, Panzem-NCD, etc. (Multifunctional cargo-free nanomedicine for cancer therapy [J]. International Journal of Molecular Sciences, 2018, 19 (10): 2963.).

[0004] Although carrier-free nanomedicines have made certain progress in the field of tumor treatment, different drugs have different densities, hydrophobic properties, surface charges, etc., which will lead to different fates of nanoparticles based on different materials in tumor treatment. In the process of research, it is necessary to conduct special analysis of specific nanomaterials. In addition, the many problems faced in the in vivo delivery process affect its therapeutic effect, thereby limiting its clinical transformation. Specifically, carrier-free nanomedicines have no carriers and rely on non-covalent interactions to achieve self-assembly. Once dissociated, chemotherapeutic drugs are easily distributed in normal tissues and produce toxic side effects; in addition, tumors are a microenvironment with high heterogeneity and complexity, and some of its characteristic manifestations cannot achieve universal activation of prodrugs, resulting in poor drug treatment effects.

[0005] In order to solve the above problems, preparing a carrier-free nanodrug system with a simple structure and independent of the tumor microenvironment is the key to reducing the toxic and side effects of chemotherapy drugs and improving the therapeutic efficacy. Summary of the invention

[0006] The purpose of the present invention is to provide a controllable nano drug delivery system with a simple structure, high drug loading and independence from the tumor microenvironment, so as to achieve efficient tumor treatment.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The present invention provides an ultrasound-activated carrier-free nanomedicine, wherein the nanomedicine is formed by self-assembly of a sonosensitizer and a chemotherapeutic prodrug, wherein the sonosensitizer is an organic small molecule that can generate ROS upon ultrasound irradiation, and the chemotherapeutic prodrug is a ROS-responsive SN38 prodrug.

[0009] SN-38 is an active metabolite of the topoisomerase I inhibitor irinotecan (CPT11). The present invention uses ROS-responsive groups to modify SN-38 to prepare a chemotherapeutic prodrug, so that SN-38 is released under the action of reactive oxygen species (ROS) to exert a tumor cell-killing effect.

[0010] The mode of action of the ultrasound-activated carrier-free nanomedicine provided by the present invention is as follows: due to the enhanced permeability and retention (EPR) of tumor tissue, nanoparticles are easily accumulated in tumor tissue. Once the nanomedicine accumulates in the tumor and is internalized into tumor cells, after ultrasound irradiation, the sonosensitizer produces reactive oxygen and further activates the chemotherapeutic prodrug, releasing SN38 to induce tumor cell apoptosis. The ultrasound-triggered method improves the tumor treatment effect and further reduces the toxic side effects of chemotherapeutic drugs on normal tissues.

[0011] Furthermore, the ROS-responsive SN38 prodrug is prepared by modifying the phenolic hydroxyl site of SN38 with a borate group.

[0012] The present invention calculates the molecular binding energy of different sonosensitizers and chemotherapeutic prodrugs through molecular simulation, and analyzes the intermolecular π-π stacking, electrostatic and hydrophobic interactions based on the molecular simulation results, thereby guiding the preparation of nanomedicines formed by self-assembly of sonosensitizers and chemotherapeutic prodrugs. Studies have shown that porphyrin sonosensitizers, phthalocyanine sonosensitizers, xanthene sonosensitizers, phenothiazine sonosensitizers, and fluoroquinolone sonosensitizers can self-assemble with SN38 prodrugs modified by borate groups to form nanoparticles through π-π stacking, electrostatic and hydrophobic interactions. Other organic small molecule sonosensitizers that can self-assemble with SN38 prodrugs modified by borate groups to form nanoparticles are also suitable for the present invention.

[0013] Further, the sonosensitizer is hematoporphyrin, protoporphyrin IX, hematoporphyrin monomethyl ether, dihydrochlorin e6, sodium chlorin, zinc phthalocyanine, tetra-α-(3-carboxyphenoxy) phthalocyanine zinc, disulfonyl diphthalimide methyl phthalocyanine zinc, phthalocyanine, hematoporphyrin dimethyl ester, diphenyl porphyrin, tetraphenyl porphyrin, tetra-p-tolyl porphyrin, tetra-aldehyde phenyl porphyrin, tetra-(2 -naphthyl)porphyrin, tetraphenylporphyrin manganese, 5,10,15,20-tetrakis(4-fluorophenyl)-21H,23H-porphyrin, tetraphenylporphyrin tetrasulfonic acid hydrate, 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin, 5,15-(aminophenyl)-10,20-phenylporphyrin, 5,15-di(4-pyridyl)-10, 20-diphenylporphyrin, 5,10,15,20-tetrakis(1H-imidazole-4-yl)porphyrin, 5,10,15,20-tetrakis(N-methyl-4-pyridine)-21,23H-tetrachloroporphyrin, m-tetrakis(p-bromophenyl)porphyrin, fluorescein, erythrosine, rose bengal, rose bengal lactone, methylene blue, promethazine hydrochloride, dioxypromethazine hydrochloride, 1-(5,5-dioxyphenothiazine-10-yl)-N,N-dimethylpropane-2-amine hydrochloride, ciprofloxacin, gatifloxacin, sparfloxacin, levofloxacin, curcumin, rhamnol, artemether, artesunate, hydroxysafflor yellow A, hyperforin, 5-aminolevulinic acid, IR780, indocyanine green, acridine orange. The sonosensitizer applicable to the present invention is not limited thereto.

[0014] Furthermore, the ROS-responsive SN38 prodrug is a pinacol boronate-modified SN38 prodrug (PBSN38), and its structural formula is shown in formula (I):

[0015]

[0016] Preparation of PBSN38 and the mechanism of ROS activation Figure 1 shown.

[0017] The present invention also provides a method for preparing the ultrasound-activated carrier-free nanomedicine, which comprises: dissolving the sonosensitizer and the ROS-responsive SN38 prodrug in an organic solution, adding the solution dropwise into water to form nanoparticles, and then removing the organic solvent by dialysis to obtain the ultrasound-activated carrier-free nanomedicine.

[0018] Furthermore, the organic solvent may be but is not limited to dimethyl sulfoxide (DMSO).

[0019] Furthermore, the molar ratio of the sonosensitizer to the ROS-responsive SN38 prodrug is 5:1-1:5.

[0020] The present invention also provides the use of the ultrasound-activated carrier-free nanomedicine in the preparation of anti-tumor drugs. The ultrasound-activated carrier-free nanomedicine accumulates in tumor tissue and releases SN38 after ultrasound irradiation.

[0021] Specifically, the tumor is a solid tumor. After the nanomedicine accumulates in the tumor and is internalized into the tumor cells, it is combined with ultrasound irradiation to achieve the purpose of tumor treatment. The conditions of the ultrasound irradiation can be changed according to the physical condition of the subject to be treated, so as to achieve the activation of the sonosensitizer and then react to produce reactive oxygen.

[0022] Furthermore, the tumor is a solid tumor such as colorectal cancer, breast cancer, liver cancer, pancreatic cancer, etc.

[0023] The present invention has the following beneficial effects:

[0024] The present invention utilizes a sonosensitizer that can generate ROS and a ROS-responsive anti-tumor chemotherapy prodrug to self-assemble to form a carrier-free nano drug, significantly increasing the drug loading and avoiding the toxic and side effects caused by the introduction of nano carriers. Only after the carrier-free nano drug is irradiated with ultrasound, the sonosensitizer can generate reactive oxygen and further activate the chemotherapy prodrug, releasing the active substance of the chemotherapy drug to induce tumor cell apoptosis, improving the tumor treatment effect by ultrasound triggering, and avoiding the toxic and side effects caused by the non-specific distribution of the chemotherapy drug. The preparation method of the present invention is simple, the reaction conditions are mild, and it has great potential in the field of tumor drug preparation and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the preparation of PBSN38 and the principle of ROS activation.

[0026] Figure 2 For PBSN38 1 HNMR spectrum.

[0027] Figure 3 This is a visualization image of the molecular docking of the sonosensitizer and PBSN38 in Example 1.

[0028] Figure 4 Graph showing the particle size of the carrier-free nanomedicines in Examples 2-7.

[0029] Figure 5 This is a graph showing the generation of ROS after cells are incubated with nanomedicine under ultrasound in Example 2.

[0030] Figure 6 This is a graph showing the survival rate of cells after incubation with nanomedicine under ultrasound in Example 2.

[0031] Figure 7 This is the in vivo tumor inhibition effect of the nanomedicine in Example 2 in the mouse subcutaneous colorectal cancer tumor model under the action of ultrasound.

[0032] Figure 8 These are H&E staining images of the main organs (heart, liver, spleen, lung, and kidney) of Balb / C mice after nanomedicine treatment in Example 2.

[0033] Fig. 9 The results of ROS production (A) and survival rate (B) after cells were incubated with nanomedicine under ultrasound in Example 3.

[0034] Fig.10 The results of ROS production (A) and survival rate (B) after cells were incubated with nanomedicine under ultrasound in Example 4.

[0035] Fig.11 The results of ROS production (A) and survival rate (B) after cells were incubated with nanomedicine under ultrasound in Example 5 are shown.

[0036] Fig.12 The results of ROS production (A) and survival rate (B) after cells were incubated with nanomedicine under ultrasound in Example 6 are shown.

[0037] Fig.13 The results of ROS production (A) and survival rate (B) after cells were incubated with nanomedicine under ultrasound in Example 7 are shown. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0039] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0040] The chemotherapeutic prodrug used in the following examples is a ROS-responsive SN38 prodrug (PBSN38), and the synthesis process is as follows: First, 4-(hydroxymethyl)phenylboronic acid pinacol ester (7.37 g, 31.5 mmol) was dissolved in dry dichloromethane (DCM, 50 mL), and then carbonyl diimidazole (CDI, 10.20 g, 62.9 mmol) was added to a 100 mL flask and stirred for 30 minutes. The above mixture was diluted in ethyl acetate (200 mL), washed three times with water, and the obtained organic layer was washed twice with saturated brine, dried in anhydrous magnesium sulfate, and concentrated in vacuo to obtain CDI-activated phenylboronic acid pinacol ester as a pure white solid (7.9 g, yield of about 75.6%). Next, the above product (0.98 g, 3 mmol), SN38 (1.57 g, 4 mmol) and dimethylaminopyridine (DMAP, 0.3 mmol, 37 mg) were dissolved in anhydrous DCM (50 mL) and stirred at room temperature for 6 hours. The solvent was removed by vacuum evaporation, and the final product, PBSN38 (pale yellow solid, 1.81 g, yield about 70.2%), was obtained by chromatography (silica gel, DCM / ethyl acetate=3:2, v / v) and vacuum drying. 1 HNMR diagram Figure 2 .

[0041] Protoporphyrin IX, zinc phthalocyanine, methylene blue, rose bengal, gatifloxacin, and indocyanine green were purchased from the exploration platform website.

[0042] CT26 colorectal cancer cells were purchased from ATCC cell bank.

[0043] Example 1

[0044] In this example, the molecular binding energies of different sonosensitizers and chemotherapeutic prodrug PBSN38 were calculated by molecular simulation, and the intermolecular π-π stacking, electrostatic and hydrophobic interactions were analyzed based on the molecular simulation results, thereby guiding the preparation of self-assembled nanomedicines composed of sonosensitizers and chemotherapeutic prodrugs.

[0045] The sonosensitizers used in this embodiment are shown in Table 1, which are small organic molecules that can generate ROS after ultrasonic radiation. The molecular simulation calculation method is as follows:

[0046] The sdf files of each sonosensitizer and PBSN38 molecule were downloaded from the Pubchem database, and the molecular docking of sonosensitizer and PBSN38 was simulated and the molecular binding energy was calculated using the Hex8.0.0 molecular docking software. The results are shown in Table 1. The pdb files of the simulated complexes were visualized using Pymol software to analyze various interactions between molecules.

[0047] Table 1. Molecular binding energy data of various sonosensitizers and PBSN38 obtained by molecular simulation calculation

[0048]

[0049]

[0050] As can be seen from Table 1, the molecular binding energy of all sonosensitizers with PBSN38 is <0 kcal / mol, among which the binding energy of porphyrin sonosensitizers with PBSN38 is lower than that of other types of sonosensitizers, indicating that there is good binding force between the molecules. It is speculated that they can interact with each other to form a stable structure during the self-assembly process of nanoparticles. The molecular docking visualization image of protoporphyrin IX and PBSN38 is shown in Figure 1. Figure 3 As shown, the molecular docking results indicate that the two molecules self-assemble through π-π stacking, electrostatic and hydrophobic interactions.

[0051] Based on the above theoretical calculation results, and combined with the fact that sonosensitizers are cost-effective, easy to purchase from the market, have good sonodynamic effects, and are commonly used and effective as reported in the literature, we selected a small molecule from each category for subsequent nanomedicine preparation and characterization. Specifically, in subsequent experiments, protoporphyrin IX, zinc phthalocyanine, methylene blue, rose bengal, gatifloxacin, and indocyanine green were selected to self-assemble with PBSN38 for nanomedicine preparation.

[0052] Example 2

[0053] 1. Preparation method of protoporphyrin IX-PBSN38 carrier-free nanomedicine

[0054] Weigh protoporphyrin IX and PBSN38 and dissolve them in DMSO to prepare a 10mM raw material mother solution. Take 10μL protoporphyrin IX and 10μL PBSN38 mother solution and ultrasonically dissolve them into a homogeneous solution. Take the above solution and add it dropwise into 1mL deionized water during stirring, and then stir and react for 2 hours. After dialyzing the above solution (Mw: 3500Da) for 48 hours, the protoporphyrin IX-PBSN38 carrier-free nanomedicine was prepared.

[0055] 2. Particle size characterization of nanomedicine

[0056] 1 mL of the protoporphyrin IX-PBSN38 carrier-free nanodrug sample (0.1 mM) obtained after dialysis was taken and placed in a special sample pool of the particle size analyzer. A dynamic light scattering test was performed using the particle size analyzer to measure the hydration kinetic particle size and particle size distribution of the nanoparticles. The experiment was repeated three times and the average value was taken.

[0057] like Figure 4 As shown, the average particle size of the nano drug prepared in this example is about 100 nm.

[0058] 3. Verify the effect of nanomedicine on producing ROS and inducing tumor cell death at the cellular level

[0059] 40,000 CT26 colorectal cancer cells were inoculated in each well of a 24-well plate, and the above-mentioned drugs (SN38 concentration was 50 μM) were added. After incubation for 24 hours, ultrasonic irradiation (3 MHz, 1.5 W / cm 2 , 50% duty cycle, time 1 min). ROS signal was detected by cell ROS kit.

[0060] like Figure 5 As shown, after incubation with nanomedicine and ultrasound irradiation, the intracellular ROS fluorescence intensity increased by about 7 times, while no ROS signal was detected in cells without ultrasound irradiation.

[0061] 5000 CT26 colorectal cancer cells were inoculated in each well of a 96-well plate, and the above drugs (SN38 concentration was 50 μM) were added. After incubation for 24 hours, ultrasound irradiation (3 MHz, 1.5 W / cm 2 , 50% duty cycle, time 1 min). Cytotoxicity was detected by CCK8 kit.

[0062] like Figure 6 As shown, after incubation with nanomedicine and ultrasound irradiation, the cell survival rate was only 16%, indicating that a large number of tumor cells died.

[0063] The above results indicate that nanomaterials can produce a large amount of ROS and cause tumor cell death after ultrasound irradiation.

[0064] 4. Verify the tumor treatment effect of nanomedicine at the experimental animal level

[0065] CT26 tumor cells (500,000 tumor cells per mouse) were inoculated subcutaneously into Balb / C mice and the tumor volume reached 70 mm. 3 The mice were divided into 3 groups, 5 in each group, and injected with PBS, CPT11 and nanomaterials (SN38 dose of 4 mg / kg) through the tail vein. After 6 hours, they were irradiated with ultrasound (frequency 3 MHz, duty cycle 50%, intensity 1.5 W / cm 2 , time 10min), and the tumor volume of mice was measured every two days.

[0066] like Figure 7 As shown, compared with the control group (PBS and CPT11 group), the tumor growth of the nanomaterials in the experimental group was significantly inhibited, indicating that the nanomaterials can improve the tumor treatment effect by ultrasound triggering.

[0067] 5. Safety assessment of nanomedicine

[0068] The nanomaterials were injected into healthy ICR mice through the tail vein (SN38 dose was 4 mg / kg). Three days later, the mice were killed in accordance with animal ethics, and the main organs (heart, liver, spleen, lungs, and kidneys) were dissected and characterized for biosafety by H&E staining.

[0069] like Figure 8 As shown, after the mice were injected with nanomaterials via the tail vein, no obvious tissue damage was found in their major organs (heart, liver, spleen, lungs, and kidneys), proving the biosafety of nanomaterials to normal tissues.

[0070] Example 3

[0071] 1. Preparation method of zinc phthalocyanine-PBSN38 carrier-free nanomedicine

[0072] Weigh zinc phthalocyanine and PBSN38 respectively and dissolve them in DMSO to prepare a 10mM raw material mother solution. Take 50μL zinc phthalocyanine and 10μL PBSN38 mother solution and ultrasonically dissolve them into a homogeneous solution. Take the above solution and add it drop by drop into 1mL deionized water during stirring, and then stir and react for 2 hours. After dialyzing the above solution (Mw: 3500Da) for 48 hours, the zinc phthalocyanine-PBSN38 carrier-free nanomedicine is obtained.

[0073] 2. Particle size characterization of nanomedicine

[0074] The test method is the same as in Example 2, and the results are as follows Figure 4 As shown, the average particle size of the nano drug prepared in this embodiment is about 120 nm.

[0075] 3. Verify the effect of nanomedicine on producing ROS and inducing tumor cell death at the cellular level

[0076] The test method is the same as in Example 2, and the results are as follows Fig. 9 As shown, after incubation of nanomedicine and ultrasonic irradiation, the result graph of cell ROS production and the result graph of cell survival rate indicate that the nanomaterial can produce a large amount of ROS (fluorescence intensity increased by about 7 times) and cause tumor cell death (cell survival rate 17%) after ultrasonic irradiation.

[0077] Example 4

[0078] 1. Preparation method of methylene blue-PBSN38 carrier-free nanomedicine

[0079] Weigh methylene blue and PBSN38 separately and dissolve them in DMSO to prepare a 10mM raw material mother solution. Take 30μL of methylene blue and 10μL of PBSN38 mother solution and ultrasonically dissolve them into a homogeneous solution. Take the above solution and add it dropwise into 1mL of deionized water during stirring, and then stir and react for 2 hours. After dialyzing the above solution (Mw: 3500Da) for 48 hours, the methylene blue-PBSN38 carrier-free nanomedicine is obtained.

[0080] 2. Particle size characterization of nanomedicine

[0081] The test method is the same as in Example 2, and the results are as follows Figure 4 As shown, the average particle size of the nano drug prepared in this embodiment is about 150 nm.

[0082] 3. Verify the effect of nanomedicine on producing ROS and inducing tumor cell death at the cellular level

[0083] The test method is the same as in Example 2, and the results are as follows Fig.10 As shown, after incubation of nanomedicine and ultrasonic irradiation, the result graph of cell ROS production and cell survival rate shows that the nanomaterial can produce a large amount of ROS (fluorescence intensity increased by about 6 times) and cause tumor cell death (cell survival rate 15%) after ultrasonic irradiation.

[0084] Example 5

[0085] 1. Preparation method of Rose Bengal-PBSN38 carrier-free nanomedicine

[0086] Rose Bengal and PBSN38 were weighed and dissolved in DMSO to prepare a 10mM raw material mother solution. 10μL of Rose Bengal and 30μL of PBSN38 mother solution were ultrasonically dissolved into a homogeneous solution. The above solution was added dropwise into 1mL of deionized water during stirring, and then stirred for reaction for 2 hours. After the above solution was dialyzed (Mw: 3500Da) for 48 hours, Rose Bengal-PBSN38 carrier-free nanomedicine was obtained.

[0087] 2. Particle size characterization of nanomedicine

[0088] The test method is the same as in Example 2, and the results are as follows Figure 4 As shown, the average particle size of the nano drug prepared in this example is about 145 nm.

[0089] 3. Verify the effect of nanomedicine on producing ROS and inducing tumor cell death at the cellular level

[0090] The test method is the same as in Example 2, and the results are as follows Fig.11As shown, after incubation of nanomedicine and ultrasonic irradiation, the result graph of cell ROS production and the result graph of cell survival rate indicate that the nanomaterial can produce a large amount of ROS (fluorescence intensity increased by about 6 times) and cause tumor cell death (cell survival rate 13%) after ultrasonic irradiation.

[0091] Example 6

[0092] 1. Preparation method of gatifloxacin-PBSN38 carrier-free nanomedicine

[0093] Gatifloxacin and PBSN38 were weighed and dissolved in DMSO to obtain a 10mM raw material mother solution. 10μL of gatifloxacin and 50μL of PBSN38 mother solution were ultrasonically dissolved into a homogeneous solution. The above solution was added dropwise into 1mL of deionized water during stirring, and then stirred for 2 hours. After the above solution was dialyzed (Mw: 3500Da) for 48 hours, the gatifloxacin-PBSN38 carrier-free nanomedicine was obtained.

[0094] 2. Particle size characterization of nanomedicine

[0095] The test method is the same as in Example 2, and the results are as follows Figure 4 As shown, the average particle size of the nano drug prepared in this example is about 132 nm.

[0096] 3. Verify the effect of nanomedicine on producing ROS and inducing tumor cell death at the cellular level

[0097] The test method is the same as in Example 2, and the results are as follows Fig.12 As shown, after incubation of nanomedicine and ultrasonic irradiation, the result graph of cell ROS production and cell survival rate shows that the nanomaterial can produce a large amount of ROS (fluorescence intensity increased by about 3 times) and cause tumor cell death (cell survival rate 23%) after ultrasonic irradiation.

[0098] Example 7

[0099] 1. Preparation method of indocyanine green-PBSN38 carrier-free nanomedicine

[0100] Weigh indocyanine green and PBSN38 respectively and dissolve them in DMSO to prepare a 10mM raw material mother solution. Take 10μL indocyanine green and 10μL PBSN38 mother solution and ultrasonically dissolve them into a homogeneous solution. Take the above solution and add it dropwise into 3mL deionized water during stirring, and then stir and react for 2 hours. After dialyzing the above solution (Mw: 3500Da) for 48 hours, the indocyanine green-PBSN38 carrier-free nanomedicine is obtained.

[0101] 2. Particle size characterization of nanomedicine

[0102] The test method is the same as in Example 2, and the results are as follows Figure 4 As shown, the average particle size of the nano drug prepared in this example is about 125 nm.

[0103] 3. Verify the effect of nanomedicine on producing ROS and inducing tumor cell death at the cellular level

[0104] The test method is the same as in Example 2, and the results are as follows Fig.13 As shown, after incubation of nanomedicine and ultrasonic irradiation, the result graph of cell ROS production and the result graph of cell survival rate indicate that the nanomaterial can produce a large amount of ROS (fluorescence intensity increased by about 5 times) and cause tumor cell death (cell survival rate 20%) after ultrasonic irradiation.

[0105] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. An ultrasound-activated carrier-free nanomedicine, characterized in that: The nano drug is formed by self-assembly of a sonosensitizer and a chemotherapeutic prodrug, wherein the sonosensitizer is an organic small molecule that can generate ROS after ultrasonic irradiation, and the chemotherapeutic prodrug is a ROS-responsive SN38 prodrug.

2. The ultrasound-activated carrier-free nanomedicine according to claim 1, characterized in that: The sonosensitizer is a porphyrin sonosensitizer, a phthalocyanine sonosensitizer, a xanthene sonosensitizer, a phenothiazine sonosensitizer or a fluoroquinolone sonosensitizer; the ROS-responsive SN38 prodrug is prepared by modifying the SN38 phenolic hydroxyl site with a borate group.

3. The ultrasound-activated carrier-free nanomedicine according to claim 1, characterized in that: The sonosensitizer is hematoporphyrin, protoporphyrin IX, hematoporphyrin monomethyl ether, dihydrochlorin e6, sodium chlorin, zinc phthalocyanine, tetra-α-(3-carboxyphenoxy) phthalocyanine zinc, disulfonyl diphthalimide methyl phthalocyanine zinc, phthalocyanine, hematoporphyrin dimethyl ester, diphenyl porphyrin, tetraphenyl porphyrin, tetra-p-tolyl porphyrin, tetraaldehyde phenyl porphyrin, tetra(2-naphthyl) porphyrin, )porphyrin, tetraphenylporphyrin manganese, 5,10,15,20-tetrakis(4-fluorophenyl)-21H,23H-porphyrin, tetraphenylporphyrin tetrasulfonic acid hydrate, 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin, 5,15-(aminophenyl)-10,20-phenylporphyrin, 5,15-di(4-pyridyl)-10,20 -diphenylporphyrin, 5,10,15,20-tetrakis(1H-imidazol-4-yl)porphyrin, 5,10,15,20-tetrakis(N-methyl-4-pyridinyl)-21,23H-tetrachloroporphyrin, m-tetrakis(p-bromophenyl)porphyrin, fluorescein, erythrosine, rose bengal, rose bengal lactone, methylene blue, promethazine hydrochloride, dioxypromethazine hydrochloride, 1-(5,5-dioxyphenothiazin-10-yl)-N,N-dimethylpropan-2-amine hydrochloride, ciprofloxacin, gatifloxacin, sparfloxacin, levofloxacin, curcumin, rhamnol, artemether, artesunate, hydroxysafflor yellow A, hyperforin, 5-aminolevulinic acid, IR780, indocyanine green, acridine orange.

4. The ultrasound-activated carrier-free nanomedicine according to claim 1, characterized in that: The ROS-responsive SN38 prodrug is a SN38 prodrug modified with pinacol borate, and its structural formula is shown in formula (I):

5. The method for preparing the ultrasound-activated carrier-free nanomedicine according to any one of claims 1 to 4, characterized in that: include: The sonosensitizer and ROS-responsive SN38 prodrug are dissolved in an organic solution, added dropwise to water to form nanoparticles, and then the organic solvent is removed by dialysis to obtain the ultrasound-activated carrier-free nanoparticle.

6. The preparation method according to claim 5, characterized in that: The organic solvent is dimethyl sulfoxide.

7. The preparation method according to claim 5, characterized in that: The molar ratio of the sonosensitizer to the ROS-responsive SN38 prodrug is 5:1-1:

5.

8. Use of the ultrasound-activated carrier-free nanomedicine according to any one of claims 1 to 4 in the preparation of anti-tumor drugs, characterized in that: The ultrasound-activated carrier-free nanomedicine accumulates in tumor tissue and releases SN38 after ultrasound irradiation.

9. The use according to claim 8, characterized in that The tumor is colorectal cancer, breast cancer, liver cancer, or pancreatic cancer.

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