A sonosensitizer for sonodynamic therapy, its preparation method and application in anti-tumor

Abstract

The application discloses a kind of for sonodynamic therapy acoustic sensitizer and its preparation method and application in antitumor, belong to medical technology field.The application solves the problem that existing methylene blue is easy to inactivate.The application synthesizes a kind of N-carboxyl cyclic anhydride small molecule containing methylene blue, further utilizes its ring-opening polymerization to obtain amphiphilic block copolymer, the block copolymer has good biocompatibility and biodegradability, and its assembly into nanoparticle in water, through SDT effect produces reactive oxygen species, can kill cancer cell, i.e.the application synthesizes environmentally friendly SDT preparation.Further, the urea bond of methylene blue connection in the block copolymer molecule synthesized by the application is a kind of ultrasonic response group, only under the action of ultrasound, MB is released, therefore it plays the selective protection effect to MB.In addition, the content of MB in unit mass in the obtained nanoparticle is higher, improves the anticancer effect mediated by SDT.

Description

Technical Field

[0001] This invention relates to a sonosensitive agent for sonodynamic therapy, its preparation method, and its application in anti-tumor therapy, belonging to the field of pharmaceutical technology. Background Technology

[0002] Sonodynamic therapy (SDT) is a novel anti-tumor treatment that utilizes ultrasound waves to penetrate deep into tissues and activate the antitumor effects of certain sonosensitive agents (such as hematoporphyrin and methylene blue). It is a treatment option following chemotherapy, radiotherapy, photodynamic therapy, and immunotherapy. However, conventional sonosensitive agents have certain drawbacks, such as short half-lives, phototoxicity, and damage to normal tissues. These issues inevitably affect the industrialization of novel sonosensitive agents and the clinical application of SDT.

[0003] Methylene blue (MB), as a water-soluble photosensitizer and sonosensitive agent, has been widely used in photodynamic therapy (PDT). It exhibits high singlet oxygen content within the human therapeutic window (600-900 nm). 1 O2 yield has made it a highly valuable photosensitizer. However, under in vitro conditions, pure methylene blue (MB) decomposes under visible light irradiation; in vivo, it is easily reduced to white methylene blue (LMB) by myocardial flavin enzyme, losing its photodynamic activity. Therefore, there is an urgent need to develop a method that can protect MB, reactivate it upon reaching cancer tissue, and exert its SDT or PDT effects for anti-cancer purposes. Summary of the Invention

[0004] To address the problem of easy inactivation of existing methylene blue, this invention provides a sonosensitive agent for sonodynamic therapy, its preparation method, and its application in antitumor therapy.

[0005] The technical solution of the present invention:

[0006] One objective of this invention is to provide a sound-sensitizing agent, which is a nanoparticle self-assembled from L-polylysine-methylene blue, the structural formula of which is as follows:

[0007]

[0008] In the formula, n is an integer.

[0009] A second objective of this invention is to provide a method for preparing the aforementioned sound-sensitive agent, the method comprising the following steps:

[0010] (1) Synthesis of L-lysine-methylene blue-N-carboxyl ring anhydride;

[0011] (2) L-polylysine-methylene blue was obtained by ring-opening polymerization of L-lysine-methylene blue-N-carboxylic anhydride initiated by amino polyethylene glycol monomethyl ether 5K.

[0012] (3) L-polylysine-methylene blue is self-assembled into nanoparticles, and the resulting nanoparticles are the sound-sensing agent.

[0013] Further specifying, the operation process of (1) is as follows:

[0014] Anhydrous tetrahydrofuran was heated in an oil bath, and Lys-MB and triphosgene were added under nitrogen purging. The mixture was stirred until the solution was clear and transparent, and stirring was continued for 4 hours. Heating was stopped and the nitrogen flow rate was increased. After 15 minutes, the mixture was precipitated in frozen n-hexane and stored at -20°C for 2 hours. After filtration, the crude product was dissolved in frozen ethyl acetate and washed with saturated sodium bicarbonate solution. The organic phase was dried with anhydrous magnesium sulfate, filtered through a G4 sintered glass filter to remove magnesium sulfate, and then transferred to an ammonia flask. The mixture was then dried by connecting a cold trap oil pump. Finally, using anhydrous tetrahydrofuran / n-hexane as raw material, L-lysine-methylene blue-N-carboxylated intracyclic anhydride was obtained by filtration, rotary evaporation, and recrystallization.

[0015] Furthermore, the ratio of anhydrous tetrahydrofuran, Lys-MB, and triphosgene was specified as 200 mL: 6.56 mmol: 39.33 mmol.

[0016] Furthermore, the oil bath temperature is specified as 45°C.

[0017] To further define the synthesis process of Lys-MB, it is as follows: Boc-Lys-OH, NaOH, THF and water are mixed, and the mixture is added dropwise to a solution containing MBCl and THF under ice bath conditions. After reacting for 12 hours, THF is removed by rotary evaporation. The pH is adjusted to 1 using hydrochloric acid, and the mixture is extracted multiple times with DCM. Then, DCM is removed by rotary evaporation. A mixed solvent of TFA and DCM is added, and after reacting for 6 hours, the mixture is precipitated in a mixed solvent of diethyl ether and petroleum ether. The precipitate is dried to obtain a dark blue solid, which is Lys-MB.

[0018] Furthermore, the ratio of Boc-Lys-OH, NaOH, THF and water is 40.19 mmol: 40.00 mmol: 60 mL: 30 mL.

[0019] To further specify, the solution containing MBCl and THF contains 20.12 mmol of MBCl and 90 mL of THF.

[0020] To further specify, THF is removed by rotary evaporation at 40°C.

[0021] Furthermore, the number of DCM extractions is limited to 10.

[0022] To further specify, DCM is removed by rotary evaporation at 33°C.

[0023] Furthermore, the TFA and DCM mixed solvents are prepared in a volume ratio of 1:1.

[0024] Furthermore, the mixture of diethyl ether and petroleum ether is prepared in a volume ratio of 1:1.

[0025] To further define the synthesis process of MBCl, the steps are as follows: dissolve methylene blue in water, add dichloromethane and dichloromethane, heat and stir the reaction under a nitrogen atmosphere, add sodium dithionite solution, continue the reaction until the solution turns yellow, cool in an ice-water bath, then add dichloromethane containing triphosgene, stir the reaction for 1.5 h, pour the reaction solution into ice water under stirring, then extract the mixture with dichloromethane, finally wash with saturated NaCl solution, dry with anhydrous magnesium sulfate, evaporate using a rotary evaporator, and obtain white solid MBCl by column chromatography.

[0026] Furthermore, the ratio of methylene blue, water, dichloromethane, and dichloromethane is specified as 31.26 mmol: 100 mL: 50 mL: 125.01 mmol.

[0027] To further specify, the sodium dithionite solution contains 125.09 mmol of sodium dithionite and 150 mL of water.

[0028] To further specify, the triphosgene-containing dichloromethane contains 18.77 mmol of triphosgene and 50 mL of dichloromethane.

[0029] Furthermore, the column chromatography solvent is a mixture of petroleum ether and dichloromethane in a volume ratio of 2:3.

[0030] Further specifying, (2) the operation process is as follows: under a nitrogen atmosphere, L-lysine-methylene blue-N-carboxylic acid anhydride, DMF and amino polyethylene glycol monomethyl ether 5K are mixed and reacted at room temperature in the dark for 48 hours. The reaction solution is then transferred to a dialysis bag with a molecular weight cutoff of 3500, dialyzed with water and then freeze-dried to obtain L-polylysine-methylene blue.

[0031] Furthermore, the ratio of L-lysine-methylene blue-N-carboxylic acid anhydride, DMF, and aminopolyethylene glycol monomethyl ether 5K is 2.00 mmol: 15 mL: 0.02 mmol.

[0032] Further specifying, (3) the operation process is as follows: weigh 10mg L-polylysine-methylene blue, dissolve it in 2mL DMF, add it dropwise to 20mL deionized water while stirring, stir for 2-4h to make it self-assemble into nanoparticles, then dialyze with deionized water for 24h to remove DMF from the solution, and make up to 25mL, which is the sound-sensitive agent.

[0033] A third objective of this invention is to provide an application of the aforementioned sonosensitive agent in sonodynamic therapy.

[0034] The fourth objective of this invention is to provide an application of the above-mentioned sound-sensitive agent in the preparation of pharmaceuticals.

[0035] A fifth objective of the present invention is to provide an antitumor pharmaceutical composition comprising the aforementioned sonosensitive agent and an anticancer drug and / or an immune agonist loaded with the sonosensitive agent.

[0036] Further specifying, the immune agonist is retcimod R848.

[0037] Beneficial effects:

[0038] This invention synthesizes a small molecule of N-carboxylic acid anhydride containing methylene blue, and further utilizes its ring-opening polymerization to obtain an amphiphilic block copolymer PEG-PMB. This block copolymer PEG-PMB exhibits good biocompatibility and biodegradability, and the nanoparticles PMB-NPs assembled in water generate reactive oxygen species (ROS) through SDT (sound-dependent catalytic degradation), which can kill cancer cells. Thus, this invention synthesizes an environmentally friendly SDT formulation. Furthermore, the urea bond connecting the methylene blue to the synthesized PEG-PMB molecule is a sonic responsive group; MB is only released under ultrasound (US) irradiation, thus providing selective protection against MB. In addition, the obtained nanoparticles PMB-NPs have a high MB content per unit mass, enhancing the SDT-mediated anticancer effect. Moreover, this sonic sensitizer, when combined with anticancer drugs and / or immune agonists, can achieve specific and rapid drug release at the tumor site, achieving the goal of synergistic efficacy and reduced toxicity in anticancer treatment. Attached Figure Description

[0039] Figure 1 Lys-MB-NCA prepared in Example 1 1 H NMR spectrum;

[0040] Figure 2 The ESI-MS spectrum of Lys-MB-NCA prepared in Example 1;

[0041] Figure 3 FITR spectra of Lys-MB and Lys-MB-NCA prepared in Example 1;

[0042] Figure 4 The PEG-PMB prepared in Example 1 1 H NMR spectrum;

[0043] Figure 5 A comparison of GPC test results between PEG-PMB and mPEG5K-NH2 prepared in Example 1;

[0044] Figure 6 The image shows the dynamic light scattering (DLS) test results of the PMB-NPs prepared in Example 1 before and after ultrasound.

[0045] Figure 7 TEM images of PMB-NPs before ultrasound prepared in Example 1;

[0046] Figure 8 TEM images of PMB-NPs after ultrasound preparation in Example 1;

[0047] Figure 9 Comparison of UV absorption spectra of PMB-NPs prepared in Example 1 at 0 min, 10 min, 20 min, 30 min, 40 min and 50 min of ultrasound;

[0048] Figure 10 The PMB-NPs prepared in Example 1 were detected after ultrasonication for 0 min, 10 min, 20 min, 30 min, 40 min, and 50 min. 1 Fluorescence spectrum of O2 generation;

[0049] Figure 11 MTT assay of PMB-NPs prepared for Example 1 after 24 hours of treatment with 4T1 cells under ultrasound and non-ultrasound conditions.

[0050] Figure 12 The image shows the dynamic light scattering (DLS) test results of PMB@R848-NPs prepared in Example 1 before and after ultrasound.

[0051] Figure 13 TEM images of PMB@R848-NPs before ultrasound, prepared in Example 1;

[0052] Figure 14 TEM images of PMB@R848-NPs after ultrasound preparation in Example 1;

[0053] Figure 15 Fluorescence spectra of the PMB@R848-NPs prepared in Example 1, under physiological conditions (pH 7.4) and in a tumor microenvironment (pH 5.0), showing the ultrasound-responsive release of R848.

[0054] Figure 16 The image shows the MTT assay results of mouse breast cancer cells (4T1) before and after ultrasound testing of PMB@R848-NPs prepared in Example 1. Detailed Implementation

[0055] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0056] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0057] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials, reagents, methods, and instruments used are conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art. Furthermore, all solid and liquid reagents used are of analytical grade.

[0059] Example 1

[0060] Step 1: Synthesis of Methylene Blue Chloride (MBCl)

[0061] Methylene blue (10.00 g, 31.26 mmol, 1.0 eq) was dissolved in 100 mL of water, and dichloromethane (50 mL) and Na₂CO₃ (13.25 g, 125.01 mmol, 4.0 eq) were added. The mixture was stirred at 40 °C under a nitrogen atmosphere. Sodium dithionite (21.78 g, 125.09 mmol, 4.0 eq) was dissolved in 150 mL of water and added directly to the solution using a syringe. The mixture was stirred at 40 °C under a nitrogen atmosphere. After the solution turned yellow, it was cooled in an ice-water bath. Triphosgene (5.57 g, 18.77 mmol, 0.6 eq) was added dropwise to 50 mL of dichloromethane, and the mixture was stirred for another 1.5 h. Then, while stirring, the solution was poured into 300 mL of ice water, and the mixture was extracted with 5 × 100 mL of dichloromethane. Finally, the sample was washed with saturated NaCl solution, dried over anhydrous magnesium sulfate, evaporated using a rotary evaporator, and subjected to column chromatography (petroleum ether / dichloromethane 2 / 3, vol) to obtain a white solid MBCl with a yield of 7.00 g and a yield of 64.52%.

[0062] Step 2: Synthesis of L-Lysine-Methylene Blue (Lys-MB)

[0063] Boc-Lys-OH (9.90 g, 40.19 mmol, 2.0 eq) and NaOH (1.60 g, 40.00 mmol, 2.0 eq) were dissolved in 60 mL of THF and 30 mL of water. The mixed solution was added dropwise to a round-bottom flask containing MBCl (7.00 g, 20.12 mmol, 1.0 eq) and 90 mL of THF in an ice bath and reacted for 12 h. THF was removed by rotary evaporation at 40 °C, and the pH was adjusted to 1 with hydrochloric acid. The mixture was extracted 10 times with 30 mL of DCM. DCM was removed by rotary evaporation at 33 °C, and 80 mL of mixed solvent (TFA / DCM = 1 / 1, vol) was added to the flask. The reaction was continued for 6 h, and the precipitate was then settled in 800 mL of mixed solvent (diethyl ether / petroleum ether = 1 / 1, vol). The precipitate was dried to obtain Lys-MB as a dark blue solid, with a yield of 7.20 g (78.18%).

[0064] Step 3: Synthesis of L-lysine-methylene blue-N-carboxylic acid anhydride (Lys-MB-NCA)

[0065] The three-necked flask was heated three times. 200 mL of anhydrous tetrahydrofuran was added, and N2 was bubbled through one end of a glass tube at approximately 2-3 bubbles per second. The flask was placed in an oil bath and heated to 45 °C. Lys-MB (3.00 g, 6.56 mmol, 1.0 eq) was added, followed by triphosgene (11.68 g, 39.33 mmol, 6.0 eq). The reaction mixture was stirred until the solution became clear and transparent, and stirring continued for 4 hours. Heating was stopped, the oil bath was removed, the stopper was removed, and nitrogen was purged at a high flow rate for 15 minutes. The mixture was then precipitated in 1 L of frozen n-hexane and stored at -20 °C for 2 hours. Finally, the crude product was obtained by filtration, dissolved again in 200 mL of frozen ethyl acetate, and washed three times with saturated sodium bicarbonate solution. The organic phase was dried overnight with anhydrous magnesium sulfate, then filtered through a G4 sintered glass funnel to remove the magnesium sulfate, transferred to a heated ammonia flask, and dried using a cold trap oil pump. Using anhydrous tetrahydrofuran / n-hexane as raw material, L-lysine-methylene blue-N-carboxylated intracyclic anhydride (Lys-MB-NCA) was obtained by filtration, rotary evaporation, and recrystallization, with a yield of 1.12 g and a yield of 35.33%.

[0066] The obtained product Lys-MB-NCA was characterized by 1H NMR spectroscopy (300M), and the results are as follows: Figure 1 As shown, by Figure 1 As can be seen, both chemical shift and integral data demonstrate that this method successfully synthesized the L-lysine-methylene blue-N-carboxyl ring anhydride with the following structural formula.

[0067]

[0068] Further ESI mass spectrometry analysis of the product Lys-MB-NCA yielded the following results:Figure 2 As shown, by Figure 2 As can be seen, the theoretical mass-to-charge ratio (m / z) is 483.19, while the actual measured values ​​are 484.19 and 506.18, further proving the successful synthesis of L-lysine-methylene blue-N-carboxylic acid anhydride (Lys-MB-NCA).

[0069] Further infrared spectral characterization of the products Lys-MB-NCA and Lys-MB yielded the following results: Figure 3 As shown, by Figure 3 It can be seen that the 1120 cm⁻¹ of Lys-MB after the reaction -1 The characteristic peaks of the α-carbon and amino groups disappeared after the reaction, and were replaced by a peak at 1850 cm⁻¹. -1 and 1780 cm -1 The stretching vibration peaks of the two carbonyl groups in the intracyclic anhydride were observed. This further verified the successful synthesis of L-lysine-methylene blue-N-carboxyl intracyclic anhydride (Lys-MB-NCA).

[0070] Step 4: Synthesis of L-Polylysine-Methylene Blue (PEG-PMB)

[0071] The synthesis route is as follows:

[0072]

[0073] The specific synthesis process was as follows: A round-bottom flask was heated three times, and nitrogen was purged three times. Under nitrogen atmosphere, L-lysine-methylene blue-N-carboxylic acid anhydride (0.96 g, 2.00 mmol, 100 eq) and 15 mL of anhydrous DMF were added, followed by mPEG5K-NH2 (0.10 g, 0.02 mmol, 1.0 eq). The reaction was carried out at room temperature in the dark for 48 h. The resulting reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 and dialyzed with water for 72 h, changing the water every 2 h. Finally, after lyophilization, L-polylysine-methylene blue (PEG-PMB) was obtained, with a yield of 0.81 g and a yield of 82.57%.

[0074] The obtained product PEG-PMB was characterized by 1H NMR spectroscopy (300M), and the results are as follows: Figure 4 As shown, by Figure 4 As can be seen, both chemical shift and integral can prove that this method successfully synthesized L-polylysine-methylene blue (PEG-PMB).

[0075] Further gel permeation chromatography (GPC) of the DMF mobile phase for the products PEG-PMB and mPEG5K-NH2 is as follows: Figure 5 As shown, by Figure 5It can be seen that the number average molecular weight of mPEG5K-NH2 is (11900, PDI=1.05), and the number average molecular weight of PEG-PMB is (31500, PDI=1.22). The peak time, molecular weight and distribution prove the successful polymerization of L-polylysine-methylene blue.

[0076] Step 4: Synthesis of L-polylysine-methylene blue nanoparticles (PMB-NPs)

[0077] L-polylysine-methylene blue nanoparticles (PMB-NPs) were prepared using a conventional dialysis method. The specific procedure was as follows: 10 mg of PEG-PMB was weighed and dissolved in 2 mL of DMF. While stirring, the solution was added dropwise to 20 mL of deionized water. The mixture was stirred for 3 h to allow the nanoparticles to self-assemble. Then, the solution was dialyzed with deionized water for 24 h to remove the DMF. The volume was adjusted to 25 mL to obtain PMB-NPs.

[0078] The obtained product PMB-NPs were analyzed before and after ultrasonication (ultrasonication conditions: 1MHz, 1.0W / cm). 2 The results of dynamic light scattering (DLS) tests (50% duty cycle, continuous ultrasound for 5 min) are as follows: Figure 6 As shown, by Figure 6 It can be seen that the diameter of the aqueous phase of PMB-NPs is (143.6 nm), and the diameter of the aqueous phase after sonication becomes (188.4 nm). This may be because sonication makes PMB-NPs more loose, which proves that the assembly of L-polylysine-methylene blue nanoparticles (PMB-NPs) was successful.

[0079] Further analysis of L-polylysine-methylene blue nanoparticles (PMB-NPs) before and after ultrasounding (ultrasounding conditions: 1MHz, 1.0W / cm). 2 Transmission electron microscopy (TEM) images (50% duty cycle, continuous sonication for 5 min) are shown below. Figure 7 and 8 As shown, by Figure 7 It can be seen that PMB-NPs are more compact and more uniform in size. (From...) Figure 8 It can be seen that PMB-NPs are more dispersed after ultrasounding, and L-polylysine-methylene blue nanoparticles (PMB-NPs) are spherical before and after ultrasounding, further proving the successful assembly of PMB-NPs.

[0080] Further testing of L-polylysine-methylene blue nanoparticles (PMB-NPs) at ultrasonication times of 0 min, 10 min, 20 min, 30 min, 40 min, and 50 min (ultrasonication conditions: 1 MHz, 1.0 W / cm²) was conducted. 2 The ultraviolet absorption spectrum (50% duty cycle) is as follows: Figure 9 As shown, byFigure 9 It can be seen that as the ultrasound time increases, the absorption peak of MB at 664 nm gradually increases, reaching its maximum value after 50 min of ultrasound, which proves that L-polylysine-methylene blue nanoparticles (PMB-NPs) can effectively release MB after ultrasound.

[0081] Further processing of L-polylysine-methylene blue nanoparticles (PMB-NPs) followed by sonication for 0 min, 10 min, 20 min, 30 min, 40 min, and 50 min (ultrasonic conditions: 1 MHz, 1.0 W / cm²) 2 (50% duty cycle), detected by 9,10-anthratridimyl-bis(methylene)dicarboxylic acid (ABDA). 1 The fluorescence spectrum of O2 generation is shown in the figure below. Figure 10 As shown, by Figure 10 It can be seen that the ABDA peak gradually decreases with increasing ultrasound time. This is because MB detaches from PMB-NPs after ultrasound, and MB produces [a new peak] as ultrasound continues. 1 O2, ABDA was 1 O2 oxidation caused a decrease in peak value, proving that PMB-NPs ultrasound can release MB and produce 1 O2.

[0082] L-Polylysine-Methylene Blue Nanoparticles (PMB-NPs) before and after ultrasound (ultrasound conditions: 1MHz, 1.0W / cm²) 2 The MTT assay was used to detect mouse breast cancer cells (4T1) at a 50% duty cycle and continuous sonication for 5 min. The results are as follows: Figure 11 As shown, by Figure 11 It can be seen that the PMB-NPs group had no inhibitory effect on 4T1 cells under the same dose of MB; however, the PMB-NPs+US group showed a significant inhibitory effect. This is because PMB-NPs produced a large amount of [unclear - possibly related to ultrasound treatment]. 1 O2, 1 O2, as a type of ROS, can damage DNA in the cell nucleus, leading to apoptosis of tumor cells. This demonstrates that the synthesized L-polylysine-methylene blue nanoparticles (PMB-NPs) exhibit an ultrasonic response.

[0083] Step 5: Synthesis of the antitumor drug composition (PMB@R848-NPs)

[0084] Using L-polylysine-methylene blue nanoparticles (PMB-NPs) as a carrier, R848 was physically loaded onto PMB-NPs using a conventional dialysis method. The specific procedure was as follows: 19.48 mg of PMB-NPs and 4.9 mg of R848 (the drug loading was designed to be 20%) were weighed and dissolved in 4 mL of DMF. While stirring, the solution was added dropwise to 30 mL of deionized water. The mixture was stirred for 3 h to allow it to self-assemble into nanoparticles. Then, the solution was dialyzed with deionized water for 24 h to remove the DMF. The volume was then adjusted to 50 mL to obtain PMB@R848-NPs.

[0085] The product PMB@R848-NPs was obtained before and after ultrasonication (ultrasonication conditions: 1MHz, 1.0W / cm). 2 The results of dynamic light scattering (DLS) tests (50% duty cycle, continuous ultrasound for 5 min) are as follows: Figure 12 As shown, by Figure 12 It can be seen that the aqueous phase diameter of PMB@R848-NPs is (93.4 nm), which is smaller than that of PMB-NPs. This is due to the effect of shear force. After the PEG-PMB shell encapsulates the hydrophobic R848, the entire nanoparticle becomes more compact. After sonication, the aqueous phase diameter becomes (172.1 nm), which may be due to the sonication causing the NPs to become more loose. The above proves that the assembly of R848 nanoparticles supported by L-polylysine-methylene blue is successful.

[0086] Further ultrasound examination of PMB@R848-NPs before and after (ultrasound conditions: 1MHz, 1.0W / cm²). 2 Transmission electron microscopy (TEM) images (50% duty cycle, continuous sonication for 5 min) are shown below. Figure 13 and 14 As shown, comparison Figure 13 and Figure 14 It can be seen that PMB@R848-NPs were more compact and uniform in size before ultrasound. After ultrasound, PMB@R848-NPs+US were more dispersed, and both PMB@R848-NPs before and after ultrasound were spherical, further proving that L-polylysine-methylene blue supported R848 nanoparticles were successfully assembled.

[0087] Further fluorescence spectra of PMB@R848-NPs releasing R848 in response to ultrasound under physiological conditions (pH 7.4) and tumor microenvironment (pH 5.0) are shown below. Figure 15 As shown, by Figure 15 It can be seen that PMB@R848-NPs release more R848 under acidic conditions, which is caused by protonation. Meanwhile, the sonication conditions after sonication were: 1 MHz, 1.0 W / cm². 2(50% duty cycle, continuous ultrasound for 5 minutes) PMB@R848-NPs are more dispersed, which can increase the release of R848, demonstrating the specificity of PMB@R848-NPs in ultrasound therapy in the tumor environment.

[0088] Further ultrasound examination of PMB@R848-NPs before and after (ultrasound conditions: 1MHz, 1.0W / cm²). 2 The MTT assay was used to detect mouse breast cancer cells (4T1) at a 50% duty cycle and continuous sonication for 5 min. The results are as follows: Figure 16 As shown, by Figure 16 It can be seen that the PMB@R848-NPs group had no inhibitory effect on 4T1 cells under the same dose of MB; however, the PMB@R848-NPs+US group showed a significant inhibitory effect. This is because PMB NPs produced a large number of cells after ultrasound. 1 O2 can damage DNA in the cell nucleus, leading to tumor cell apoptosis. This demonstrates that we have successfully synthesized a novel ultrasound-responsive L-polylysine-methylene blue-supported R848 nanoparticle. This reduces the need for high-dose R848 use, avoiding side effects, while achieving specific and rapid release at the tumor site, thus achieving the goal of synergistic efficacy and reduced toxicity in anti-cancer treatment.

[0089] Comparative Example 1

[0090] The difference between this comparative example and Example 1 is step three; the remaining steps one, two, and four are the same as in Example 1. Specifically, step three of this comparative example is as follows:

[0091] Lys-MB (0.042 g, 0.92 mmol, 1.0 eq), triphosgene (0.82 g, 2.76 mmol, 6.0 eq), and 100 mL of anhydrous THF were added to a round-bottom flask filled with N2 and stirred at 40 °C for 3 h. The mixture was precipitated in 1 L of frozen petroleum ether and stored at -20 °C for 2 h. The crude product was obtained by filtration and redissolved in 100 mL of frozen ethyl acetate. The product was washed three times with ice water and twice with saturated sodium bicarbonate solution. The organic solvent was collected and dried overnight with anhydrous magnesium sulfate. After filtration, rotary evaporation, and recrystallization, Lys-MB-NCA was given as a dark blue oily product with a yield of 85 mg (19.32%).

[0092] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A sound-sensitizing agent, characterized in that, This acoustic sensitizer is composed of L-polylysine-methylene blue nanoparticles assembled by self-assembly. The structural formula of the L-polylysine-methylene blue is as follows: In the formula, n is an integer.

2. A method for preparing the acoustic sensor according to claim 1, characterized in that, include: (1) Synthesis of L-lysine-methylene blue-N-carboxyl ring anhydride; (2) L-polylysine-methylene blue was obtained by ring-opening polymerization of L-lysine-methylene blue-N-carboxylic acid anhydride initiated by amino polyethylene glycol monomethyl ether 5K. (3) L-polylysine-methylene blue is self-assembled into nanoparticles, and the resulting nanoparticles are the sound-sensing agent.

3. The method for preparing the sound-sensitive agent according to claim 2, characterized in that, (1) The operation process is as follows: Anhydrous tetrahydrofuran was heated in an oil bath, and Lys-MB and triphosgene were added under nitrogen purging. The mixture was stirred until the solution was clear and transparent, and stirring was continued for 4 hours. Heating was stopped and the nitrogen flow rate was increased. After 15 minutes, the mixture was precipitated in frozen n-hexane and stored at -20°C for 2 hours. After filtration, the crude product was dissolved in frozen ethyl acetate and washed with saturated sodium bicarbonate solution. The organic phase was dried with anhydrous magnesium sulfate, filtered through a G4 sintered glass filter to remove magnesium sulfate, and then transferred to an ammonia flask. The mixture was then dried by connecting a cold trap oil pump. Finally, using anhydrous tetrahydrofuran / n-hexane as raw material, L-lysine-methylene blue-N-carboxylated intracyclic anhydride was obtained by filtration, rotary evaporation, and recrystallization.

4. The method for preparing the sound-sensitive agent according to claim 3, characterized in that, The synthesis process of Lys-MB is as follows: Boc-Lys-OH, NaOH, THF and water are mixed. The mixture is added dropwise to a solution containing MBCl and THF under ice bath conditions. After reacting for 12 hours, THF is removed by rotary evaporation. The pH is adjusted to 1 with hydrochloric acid. The mixture is extracted multiple times with DCM. Then, DCM is removed by rotary evaporation. A mixed solvent of TFA and DCM is added. After reacting for 6 hours, the mixture precipitates into a mixed solvent of diethyl ether and petroleum ether. The precipitate is dried to obtain a dark blue solid, which is Lys-MB.

5. The method for preparing the sound-sensitive agent according to claim 4, characterized in that, The synthesis process of MBCl is as follows: Methylene blue is dissolved in water, dichloromethane and sodium dithionite are added, and the mixture is heated and stirred under a nitrogen atmosphere. Sodium dithionite solution is added, and the reaction continues until the solution turns yellow. The mixture is then cooled in an ice-water bath, and dichloromethane containing triphosgene is added. After stirring for 1.5 hours, the reaction solution is poured into ice water under stirring. The resulting mixture is then extracted with dichloromethane, washed with saturated NaCl solution, dried over anhydrous magnesium sulfate, evaporated using a rotary evaporator, and purified by column chromatography to obtain white solid MBCl.

6. The method for preparing the sound-sensitive agent according to claim 2, characterized in that, (2) The operation process is as follows: Under a nitrogen atmosphere, L-lysine-methylene blue-N-carboxylic acid anhydride, DMF and amino polyethylene glycol monomethyl ether 5K are mixed and reacted at room temperature in the dark for 48 hours. The reaction solution is then transferred to a dialysis bag with a molecular weight cutoff of 3500, dialyzed with water and then freeze-dried to obtain L-polylysine-methylene blue.

7. The method for preparing the sound-sensitive agent according to claim 2, characterized in that, (3) The operation process is as follows: Weigh 10mg of L-polylysine-methylene blue, dissolve it in 2mL of DMF, and add it dropwise to 20mL of deionized water while stirring. Stir for 2-4h to allow it to self-assemble into nanoparticles. Then dialyze with deionized water for 24h to remove DMF from the solution and make up to 25mL to obtain the sound-sensitive agent.

8. The use of the acoustic sensitizer of claim 1 in the preparation of an anti-breast cancer drug.

9. An antitumor drug composition, characterized in that, Includes the acoustic sensitizer as described in claim 1 and anticancer drugs and / or immune agonists loaded with the acoustic sensitizer.

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