Activated methylene blue bonding medicine as well as preparation method and application thereof

Abstract

The invention relates to the technical field of small molecule bonding drugs, in particular to an activated type methylene blue bonding drug which comprises an adriamycin-methylene blue bonding drug DOX-MB and a 1-methyl-tryptophan-methylene blue bonding drug MT-MB. Two bonding drugs DOX-MB or MT-MB based on methylene blue are designed and synthesized, MT or DOX is bonded with methylene blue through urea bonds, and the synthesis method is simple and easy to implement; the two bonding drugs DOX-MB or MT-MB and the distearoyl phosphatidyl ethanolamine-polyethylene glycol are assembled in an aqueous solution to form the nano-drug, so that efficient loading of DOX or MT can be realized; the bonding drug DOX-MB or MT-MB has no cytotoxicity, and singlet oxygen generated by methylene blue under near-infrared laser irradiation or ultrasonic conditions can break urea bonds to release a parent drug, so that precise and controllable release and combined treatment of the drug are realized; the fluorescence of methylene blue in the bonding drug DOX-MB or MT-MB is quenched, and the methylene blue fluorescence released by urea bond breakage under near-infrared laser irradiation or ultrasonic conditions is recovered, so that the method can be used for real-time detection of drug release.

Description

Technical Field

[0001] The present invention relates to the technical field of small molecule bonded drugs, and in particular to an activated methylene blue bonded drug and a preparation method and application thereof. Background Art

[0002] Cancer is one of the most common and deadly diseases that threaten human life and health. Traditional methods of treating cancer include surgery, chemotherapy and radiotherapy. However, these treatments have strong toxic side effects, are prone to drug resistance, lack targeting of cancer cells, and are prone to recurrence. The treatment results are difficult to be satisfactory. Doxorubicin is a broad-spectrum chemotherapy drug belonging to the anthracycline class of drugs. It has strong anti-tumor effects and definite efficacy, but it usually causes toxic side effects such as hair loss, bone marrow suppression and cardiotoxicity. In particular, cardiotoxicity is the most serious side effect of this type of drug, which limits its clinical application.

[0003] Indoleamine-(2,3)-dioxygenase (IDO) is an immune checkpoint protein kinase that catalyzes tryptophan metabolism to produce kynurenine. When overexpressed in tumor cells, it can form an immunosuppressive microenvironment, causing inactivation or apoptosis of immune function cells, which is beneficial for tumor cells to escape the immune system and survive. Therefore, inhibiting the expression of IDO can block the kynurenine pathway, promote the proliferation of T cells, enhance anti-tumor immune response, and ultimately improve the therapeutic effect on tumors. 1-Methyltryptophan (MT) is an IDO inhibitor. MT has the advantages of traditional small molecule drugs, such as a defined and simple structure and easy manufacturing. However, it also has the common disadvantages of small molecule drugs, namely, rapid drug metabolism and short retention time in the body.

[0004] In order to solve the above problems of DOX and MT, the existing technology often prepares DOX or MT into nano drugs, so that it has a more suitable half-life, prolongs the circulation time of the drug in the body and is not easily metabolized; it can also achieve passive targeting by enhancing the permeability and retention effect (EPR), effectively increasing its selectivity for tumor cells and reducing its toxic side effects on normal cells. At present, the more successful clinical is liposomal doxorubicin, which uses liposomes as shells to encapsulate ordinary doxorubicin. After liposome loading, the drug is redistributed in the body, which significantly reduces the drug concentration in the heart, kidney, and gastrointestinal tissues, so it can reduce the toxic effects on these organs, especially reducing the toxicity of doxorubicin to the heart. However, the current doxorubicin nano drugs cannot achieve specific activation in tumor cells, and it is difficult to achieve precise treatment. On the other hand, MT has good hydrophilicity and cannot self-assemble to form nano drugs in the aqueous phase. It needs to be modified and combined with excipients to make nano drugs, which leads to low drug loading rate (<10%), so that the drug cannot reach an effective therapeutic concentration in tumor cells, and it is easy to induce cancer cells to develop drug resistance.

[0005] Therefore, in view of the problems of short circulation time, poor selectivity, serious toxic side effects and low drug loading rate of the above drugs in the body after using excipients, an activated methylene blue bonded drug is designed. The prepared methylene blue bonded drug is activated to release DOX or MT under near-infrared light irradiation or ultrasound conditions. Methylene blue can be used to detect the release of drugs in real time, realizing precise and controllable treatment of tumors. Summary of the invention

[0006] In order to overcome the problems of short drug circulation time in the body, poor selectivity, serious toxic side effects, and low drug loading rate after using excipients, an activated methylene blue bonded drug was designed to achieve precise and controllable treatment of tumors.

[0007] The technical scheme of the present invention is: an activated methylene blue bonded drug, comprising doxorubicin-methylene blue bonded drug DOX-MB and 1-methyl-tryptophan-methylene blue bonded drug MT-MB;

[0008] The compound structural formula is:

[0009]

[0010] R is selected from the following small molecules:

[0011]

[0012] The method for preparing an activated methylene blue bonded drug comprises the activated methylene blue bonded drug as described above, and the synthesis route is as follows:

[0013]

[0014] The following steps are involved:

[0015] Step 1: In a nitrogen or argon inert gas environment, in a water and dichloromethane mixed solvent (V 水： V 二氯甲烷 =2:1), under the catalysis of sodium carbonate, methylene blue and sodium dithionite react at 40°C for 1h, then cool the reaction system to 0°C, add triphosgene and react in an ice-water bath for 4h, after the reaction is completed, add ice water to quench the reaction, extract with dichloromethane, wash with brine, dry with anhydrous sodium sulfate, and separate by silica gel column chromatography to obtain the intermediate product MB-Cl;

[0016] Step 2: The MB-Cl obtained in step 1 is dissolved in DMF (20 mg / mL), triethylamine is added, and the molar ratio of triethylamine to MB-Cl is 3-5:1. DOX is dissolved in DMF (20 mg / mL), and the molar ratio of DOX to MB-Cl is 1-1.5:1. In a nitrogen or argon inert gas environment and an ice bath, the DMF solution of MB-Cl is added to the DMF solution of DOX, and the reaction is continued for 6 hours. After the reaction is completed, it is extracted with ethyl acetate, washed with deionized water, and the product is separated by silica gel column chromatography and dried in vacuo to obtain doxorubicin-methylene blue bonded drug DOX-MB;

[0017] Step 3: MB-Cl was dissolved in DMF (20 mg / mL), and triethylamine was added. The molar ratio of triethylamine to MB-Cl was 3-5:1. 1-Methyl-tryptophan (MT) was dissolved in methanol (20 mg / mL). The molar ratio of MT to MB-Cl was 1-1.5:1. Under ice bath conditions, the DMF solution of MB-Cl was added to the methanol solution of MT. The reaction was continued for 6 hours. After the reaction was completed, the product was separated by silica gel column chromatography and vacuum dried to obtain 1-methyl-tryptophan-methylene blue bonded drug MT-MB.

[0018] Preferably, in step 1, under the catalysis of sodium carbonate, the molar ratio of sodium carbonate to methylene blue is 4-6:1; methylene blue and sodium dithionite are reacted at 40°C for 1 hour, and the molar ratio of methylene blue to sodium dithionite is 1:3-7; and the molar ratio of methylene blue to triphosgene is 1.5-2:1.

[0019] A methylene blue bonded drug nanomedicine comprises the activated methylene blue bonded drug as described above and is obtained by assembling molecules of the methylene blue bonded drug.

[0020] The present invention also provides a method for preparing an activated methylene blue bonded drug, the steps of which are as follows:

[0021] S1: dissolving the methylene blue bonded drug and distearoylphosphatidylethanolamine-polyethylene glycol in an organic solvent to obtain solution A;

[0022] S2: Add solution A obtained in step S1 dropwise into distilled water, stir, dialyze and freeze-dry to obtain the solution.

[0023] Preferably, the mass ratio of the methylene blue bonded drug to distearoylphosphatidylethanolamine-polyethylene glycol in S1 is 100:0 to 10:90.

[0024] Preferably, the organic solvent in S1 is one of tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide, acetone and ethanol.

[0025] The activated methylene blue bonded drug as described above or the use of the activated methylene blue bonded drug in anti-tumor.

[0026] The invention has the following beneficial effects: two bonded drugs DOX-MB or MT-MB based on methylene blue are designed and synthesized, MT or DOX is bonded to methylene blue through a urea bond, and the synthesis method is simple and easy; the two bonded drugs DOX-MB or MT-MB are assembled with distearoylphosphatidylethanolamine-polyethylene glycol in an aqueous solution to form a nano drug, which can realize efficient loading of DOX or MT; the bonded drugs DOX-MB or MT-MB have no cytotoxicity, and the singlet oxygen generated by methylene blue under near-infrared laser irradiation or ultrasonic conditions can break the urea bond to release the parent drug, thereby reducing the toxic and side effects of the drug on normal tissues; the fluorescence of MB in the bonded drugs DOX-MB or MT-MB is quenched, and the fluorescence of MB released by breaking the urea bond under near-infrared laser irradiation or ultrasonic conditions is restored, which can be used for real-time detection of drug release. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The methylene blue intermediate (MB-Cl) of Example 1 of the present invention 1 HNMR spectrum;

[0028] Figure 2 is the DOX-MB of Example 1 of the present invention 1 HNMR spectrum;

[0029] Figure 3 MT-MB of Example 2 of the present invention 1 HNMR spectrum;

[0030] Figure 4 is the ultraviolet absorption curve of DOX-MB in Example 1 of the present invention;

[0031] Figure 5 is a high performance liquid chromatogram of DOX-MB of Example 1 of the present invention;

[0032] Figure 6 is a high performance liquid chromatogram of MT-MB of Example 2 of the present invention;

[0033] Figure 7 The ultraviolet absorption and fluorescence emission spectra of MT-MB of Example 2 of the present invention;

[0034] Figure 8 The fluorescence emission spectrum change curve of DOX-MB in Example 1 of the present invention;

[0035] Fig. 9 The method is to detect the generation of singlet oxygen by DOX-MB using an ultraviolet spectrophotometer under illumination conditions in Example 1 of the present invention;

[0036] Fig.10 The DOX-MB nanoformulation of Example 3 of the present invention was subjected to light (660nm, 400mW / cm 2 ) and the inhibition rate of 4T1 cells under non-light conditions;

[0037] Fig.11 The MB-MT nanoformulation of Example 4 of the present invention was subjected to ultrasound (1.5 W / cm 2 Live-dead staining microscopic photos of 4T1 cells under ultrasound (1MHz) and without ultrasound. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0039] Example 1: Synthesis of doxorubicin-methylene blue bonded drug (DOX-MB)

[0040] (1) Methylene blue (2.5 g, 7.82 mmol) and sodium carbonate (3.31 g, 31.23 mmol) were weighed and dissolved in 50 mL of deionized water under nitrogen atmosphere, and placed in an oil bath at 40°C. Sodium dithionite (6.54 g, 37.56 mmol) was dissolved in 25 mL of DCM and added to the system under nitrogen atmosphere, and stirred for reaction for 1 h. After cooling in an ice-water bath, a 20 mL DCM solution of triphosgene (1.39 g, 4.68 mmol) was added, and stirred for reaction for 4 h. After the reaction was completed, ice water was added to quench the reaction, and the mixture was extracted with DCM, washed with brine, dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography to obtain the intermediate product MB-Cl;

[0041] (2) MB-Cl (40 mg, 0.115 mmol) was dissolved in 3 mL DMF, and triethylamine (96 μL, 0.69 mmol) was added. DOX (80 mg, 0.138 mmol) was dissolved in 7 mL DMF. In an inert gas environment of nitrogen or argon, the DMF solution of DOX was added to the MB-Cl system. The reaction was carried out in an ice bath for 30 min and then the ice bath was removed. The reaction was carried out at room temperature for 6 h. After the reaction was completed, the mixture was extracted with water and ethyl acetate, washed with deionized water, and separated by silica gel column chromatography to obtain the target product DOX-MB.

[0042] The structures of the intermediate product MB-Cl and the bonded drug DOX-MB in Example 1 were determined by H NMR spectroscopy. The results are shown in the attached figure. Figure 1 and Figure 2 As shown, the solvents used in NMR are CDCl 3 and deuterated DMSO.

[0043] Example 2: Synthesis of 1-methyl-tryptophan-methylene blue (MT-MB)

[0044] (1) Methylene blue (2.5 g, 7.82 mmol) and sodium carbonate (3.31 g, 31.23 mmol) were weighed and dissolved in 50 mL of deionized water under nitrogen atmosphere, and placed in an oil bath at 40°C. Sodium dithionite (6.54 g, 37.56 mmol) was dissolved in 25 mL of DCM and added to the system under nitrogen atmosphere for stirring and reaction for 1 h. After cooling in an ice-water bath, a 20 mL DCM solution of triphosgene (1.39 g, 4.68 mmol) was added and stirred for reaction for 4 h. After the reaction was completed, ice water was added to quench the reaction, and the mixture was extracted with DCM, washed with brine, dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography to obtain the intermediate product MB-Cl;

[0045] (2) Under nitrogen environment, MB-Cl (348 mg, 1.0 mmol) and triethylamine (0.5 mL) were dissolved in 10 mL of dichloromethane and added to the reaction flask. MT (327.37 mg) was dissolved in 3 mL of dichloromethane and added to the reaction system. The mixture was stirred at room temperature for 4 h. After the reaction was completed, the solution was evaporated using a rotary evaporator and the crude product was purified by column chromatography to obtain the target product MT-MB.

[0046] The structure of the bonded drug in Example 2 was determined by hydrogen nuclear magnetic resonance spectroscopy. Figure 3 As shown, the solvent used in NMR is CDCl 3 .

[0047] Example 3: Preparation of DOX-MB nanomedicine

[0048] Weigh 2 mg of DOX-MB and dissolve it in 100 μL of dimethyl sulfoxide; weigh 8 mg of distearoylphosphatidylethanolamine-polyethylene glycol and dissolve it in 200 μL of dimethyl sulfoxide, mix the two solutions, slowly add them dropwise into magnetically stirred deionized water (5 mL), assemble them into uniform nanoparticles (DOX-MB nanoparticles) at room temperature, stir at room temperature for 6 hours, load them into a dialysis bag with a molecular weight cutoff of 1KD, dialyze with deionized water for 12 hours, and filter through a 0.45 μm aqueous membrane to obtain a DOX-MB bonded drug nanoparticle solution.

[0049] Example 4: Preparation of MT-MB nanomedicine

[0050] Weigh 5 mg of MT-MB and dissolve it with 100 μL of dimethyl sulfoxide; weigh 5 mg of distearoylphosphatidylethanolamine-polyethylene glycol and dissolve it with 200 μL of dimethyl sulfoxide, mix the two solutions, slowly add them dropwise into magnetically stirred deionized water (10 mL), assemble them into uniform nanoparticles (MT-MB nanoparticles) at room temperature, stir at room temperature for 6 hours, load them into a dialysis bag with a molecular weight cutoff of 1KD, dialyze with deionized water for 12 hours, and filter through a 0.45 μm aqueous membrane to obtain a MT-MB drug-bonded nanoparticle solution.

[0051] Example 5: UV absorption detection of DOX-MB bonded drug

[0052] The bonded drug DOX-MB prepared in Example 1 was prepared into a dimethyl sulfoxide solution (0.1 mg / mL), and dimethyl sulfoxide solutions of DOX and MB with corresponding concentrations were prepared, and UV absorption detection was performed respectively. Figure 4 As shown, Figure 4 A is the UV absorption curve of physical mixture of DMSO solutions with different concentrations of DOX and MB; Figure 4 B is the UV absorption curve of DOX-MB bonded drug with different concentrations; Figure 4 C is a mixture of small molecule DOX and MB. The UV absorption curves of DOX-MB bonded drug and polymer-encapsulated DOX-MB nanodrug are given by Figure 4 B shows that the ultraviolet absorption of MB in the bonded drug DOX-MB is significantly reduced, which verifies the bonding between DOX and MB. Figure 4 D is the standard curve of DOX-MB conjugate in dimethyl sulfoxide.

[0053] Example 6: Release of DOX-MB bonded drug under near-infrared light irradiation

[0054] The DOX-MB conjugate prepared in Example 1 was prepared into a 400 μM dimethyl sulfoxide solution and illuminated at 400 mW / cm 2 The samples were irradiated with 660nm laser for 0min, 2min, 4min, 6min, 8min and 10min respectively, and then HPLC and fluorescence detection were performed. The results are shown in Figure 5 As shown in the figure, after 10 minutes of laser irradiation, most of the bonded drug has been cleaved, releasing the original drug DOX.

[0055] Example 7: Release of MT-MB bonded drug under ultrasonic conditions

[0056] The MT-MB bonding agent prepared in Example 2 was prepared into a 400 μM dimethyl sulfoxide solution and ultrasonically treated (1.5 W / cm 2 , 1MHz) for 0min, 2min, 4min, 6min, and 8min, and then HPLC detection was performed. The results were as follows Figure 6 As shown, after 8 min of ultrasonic treatment, the bonded drug has been mostly cleaved, releasing the original drug MT.

[0057] Example 8: Detection of UV absorption and fluorescence of MT-MB bonded drug after ultrasonic treatment

[0058] The MT-MB bonding agent prepared in Example 2 was subjected to ultrasound (1.5 W / cm 2, 1MHz) were treated for 0min, 2min, 4min, 6min, and 8min, and then UV absorption and fluorescence detection were performed. The results are shown in Figure 7 As shown in Figure 2, after 8 min of ultrasonic treatment, the bonded drug has been mostly cleaved, and the ultraviolet absorption of methylene blue ( Figure 7 A) and fluorescence ( Figure 7 B) gradually recover.

[0059] Example 9: Fluorescence detection of DOX-MB bonded drug under illumination conditions

[0060] The DOX-MB bonded drug prepared in Example 1 was irradiated with light (660 nm, 400 mW / cm 2 ) were treated for 0min, 2min, 4min, 6min, 8min, and 10min, and then fluorescence detection was performed. The results are shown in Figure 8 As shown, after 10 min of light treatment, the fluorescence of methylene blue gradually recovered, proving that the bonded drug had been mostly cleaved to release the original drug MB.

[0061] Example 10: Detection of singlet oxygen produced by DOX-MB bonded drug under light irradiation

[0062] The DOX-MB bonded drug prepared in Example 1 was irradiated with light (660 nm, 400 mW / cm 2 ) were treated for 0min, 2min, 4min, 6min, and 8min, respectively, and 1,3-Diphenylisobenzofuran (DPBF) was used as a probe to detect the generated singlet oxygen. DPBF was oxidized to form endoperoxides after combining with singlet oxygen, and its absorption intensity at UV-visible light (416nm) decreased rapidly. Fig. 9 It can be seen that DOX-MB produces singlet oxygen under light conditions.

[0063] Example 9: In vitro cytotoxicity experiment of DOX-MB nanomedicine

[0064] The CCK8 method was used to investigate the cytotoxicity of the DOX-MB nanodrug prepared in Example 3 on mouse breast cancer cells (4T1). 4T1 cells in the logarithmic growth phase were taken and inoculated into a 96-well plate so that the number of cells in each well was 5×103. After culturing for 24 h (37° C.) to allow them to adhere to the wall, 100 μL of DOX, MB, DOX and MB blended drugs (DOX+MB), DOX-MB and DOX-MB nanodrug culture medium solution prepared in Example 3 were added respectively. Three replicate wells were set for each concentration. Cells treated with blank culture medium were used as controls. After the sample was added and cultured for 48 h (37° C.), the 96-well plate was taken out, the old culture medium was discarded, 100 μL of CCK8 solution (5 mg / mL) was added to each well, and the OD value of each well was measured at 450 nm using an enzyme reader. At the same time, the above experiment was illuminated (illumination conditions: 400 mW / cm 2 660nm laser) and non-illumination comparison, the results are as follows Fig.10 shown.

[0065] Example 10: Cell live-dead staining experiment of MT-MB bonded drug nanomedicine

[0066] In order to more intuitively observe the effect of the MT-MB nanomedicine prepared in Example 4 on the survival status of 4T1 cells, the cells after different treatment conditions were stained and fluorescently detected using the Calcein-AM / PI live / dead cell double staining kit. First, 4T1 cells were cultured at 2×10 5 The cells were inoculated at a density of 1.57 μg / well in each well of a 24-well cell culture plate. After the cells were cultured until they were completely attached to the wall, the original culture medium was aspirated and culture medium containing different drugs (PBS, MT, MB and MT-MB, where the concentration of MB was 5 μM, two wells for each drug) was added to each well. After culturing in a 37°C incubator for 6 h, the original drug-containing culture medium was aspirated and one well of the same drug group was subjected to ultrasonic treatment (1.5 W / cm 2 , 1MHz, 5min), and the other well was not treated. After that, the culture was continued for 2h, the culture medium was aspirated, and the cells were washed three times with PBS. 1mL of pre-prepared staining solution was added to each well. After staining at room temperature for 30min, the staining solution was removed, and 1mL of PBS was added to each well. The cell staining was observed under an inverted fluorescence microscope and photographed. The cell staining fluorescence photos are shown in Fig.11 As shown, red represents dead cells, and the bonded drug has strong cytotoxicity after ultrasonic treatment.

[0067] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge scope of those skilled in the art without departing from the spirit of the present invention.

Claims

1. An activated methylene blue bonding drug, characterized in that: Including doxorubicin-methylene blue bonded drug DOX-MB and 1-methyl-tryptophan-methylene blue bonded drug MT-MB; The compound structural formula is: R is selected from the following small molecules:

2. A method for preparing an activated methylene blue bonded drug, characterized in that The activated methylene blue bonded drug according to claim 1 has the following synthesis route: The following steps are involved: Step 1: In a nitrogen or argon inert gas environment, in a water and dichloromethane mixed solvent (V 水 :V 二氯甲烷 =2:1), under the catalysis of sodium carbonate, methylene blue and sodium dithionite react at 40°C for 1h, then cool the reaction system to 0°C, add triphosgene and react in an ice-water bath for 4h, after the reaction is completed, add ice water to quench the reaction, extract with dichloromethane, wash with brine, dry with anhydrous sodium sulfate, and separate by silica gel column chromatography to obtain the intermediate product MB-Cl; Step 2: The MB-Cl obtained in step 1 is dissolved in DMF (20 mg / mL), triethylamine is added, and the molar ratio of triethylamine to MB-Cl is 3-5:

1. Doxorubicin is dissolved in DMF (20 mg / mL), and the molar ratio of doxorubicin to MB-Cl is 1-1.5:

1. In a nitrogen or argon inert gas environment and an ice bath, the DMF solution of MB-Cl is added to the DMF solution of doxorubicin, and the reaction is carried out for 6 hours. After the reaction is completed, it is extracted with ethyl acetate, washed with deionized water, and the product is separated by silica gel column chromatography and dried in vacuo to obtain doxorubicin-methylene blue bonded drug DOX-MB; Step 3: MB-Cl was dissolved in DMF (20 mg / mL), and triethylamine was added. The molar ratio of triethylamine to MB-Cl was 3-5:

1. 1-Methyl-tryptophan (MT) was dissolved in methanol (20 mg / mL). The molar ratio of MT to MB-Cl was 1-1.5:

1. Under ice bath conditions, the DMF solution of MB-Cl was added to the methanol solution of MT. The reaction was continued for 6 hours. After the reaction was completed, the product was separated by silica gel column chromatography and vacuum dried to obtain 1-methyl-tryptophan-methylene blue bonded drug MT-MB.

3. The method for preparing an activated methylene blue bonded drug according to claim 2, characterized in that: In step 1, under the catalytic action of sodium carbonate, the molar ratio of sodium carbonate to methylene blue is 4-6:1; methylene blue and sodium dithionite are reacted at 40° C. for 1 hour, and the molar ratio of methylene blue to sodium dithionite is 1:3-7; and the molar ratio of methylene blue to triphosgene is 1.5-2:

1.

4. A methylene blue bonded drug nanomedicine, characterized in that It comprises the activated methylene blue bonded drug according to claim 1, which is obtained by assembling methylene blue bonded drug molecules.

5. The activated methylene blue bonded drug according to claim 4, characterized in that: The preparation steps are as follows: S1: dissolving the methylene blue bonded drug and distearoylphosphatidylethanolamine-polyethylene glycol in an organic solvent to obtain solution A; S2: Add solution A obtained in step S1 dropwise into distilled water, stir, dialyze and freeze-dry to obtain the solution.

6. The activated methylene blue bonded drug according to claim 5, characterized in that: The mass ratio of methylene blue bonded drug to distearoylphosphatidylethanolamine-polyethylene glycol in S1 is 100:0 to 10:

90.

7. The activated methylene blue bonded drug according to claim 5, characterized in that: The organic solvent in S1 is one of tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide, acetone and ethanol.

8. Use of the activated methylene blue bonded drug according to claim 1 or the activated methylene blue bonded drug according to claim 4 in anti-tumor treatment.

Images