Melphalan targeted drug delivery system as well as preparation method and application thereof

Abstract

The invention provides a melphalan targeted drug delivery system and a preparation method and application thereof, and the preparation method comprises the following steps: S1, dissolving rhodamine B in anhydrous tetrahydrofuran at-5-0 DEG C, and then slowly adding sodium borohydride until all hydrogen is released; s2, adding iodine, reacting for 8-10 hours, and quenching the reaction with water to obtain a first mixture; s3, extracting the first mixture for multiple times to obtain a solvent layer, drying, filtering, evaporating, purifying, separating and concentrating to obtain a first compound; s4, dissolving the melphalan and the first compound in anhydrous dichloromethane; s5, adding dicyclohexylcarbodiimide and 4-dimethylaminopyridine, and carrying out an esterification reaction so as to obtain a second mixture; and S6, washing, drying, filtering and evaporating the second mixture, purifying and separating, and concentrating to obtain a second compound. The melphalan targeted drug delivery system prepared by the invention can target tumor cells and tissues, can reduce the toxicity of the melphalan targeted drug delivery system to normal cells and tissues, and avoids serious side effects in a cancer treatment process.

Description

Technical Field

[0001] The invention belongs to the field of medicines and relates to a melphalan targeted drug delivery system and a preparation method and application thereof. Background Art

[0002] Melphalan is a bifunctional alkylating agent with antitumor and cytotoxic effects. The main mechanism of melphalan's antitumor and cytotoxic effects is that the single and double strands of DNA are cross-linked through alkylation, thereby destroying the function and synthesis of DNA, thereby exerting an antitumor effect. This process is toxic to both cancer cells and normal cells, so melphalan has serious side effects in the treatment of cancer. In order to reduce the side effects of melphalan treatment, a system that can selectively deliver drugs in tumor cells or tissues is needed. Summary of the invention

[0003] The purpose of the present invention is to provide a melphalan targeted drug delivery system and a preparation method and application thereof, aiming to solve the problem of side effects of melphalan in the process of treating cancer.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention to achieve the above purpose is: using reduced rhodamine B as a melphalan carrier, and combining the two to obtain a melphalan drug delivery system (HRhod-Mel).

[0005] The specific steps include:

[0006] 1) Reduction of Rhodamine B

[0007] S1. At -5-0°C, dissolving rhodamine B in a solvent, wherein the mass volume ratio of the rhodamine B to the solvent is 1:(18-22) (g / mL), and then slowly adding sodium borohydride, wherein the mass ratio of the sodium borohydride to the rhodamine B is 19:(180-220), until all hydrogen is released;

[0008] S2. adding iodine, wherein the molar ratio of the iodine to the rhodamine B is (9-11):21, reacting for 8-10 hours, quenching the reaction with water, and obtaining a first mixture;

[0009] S3. extracting the first mixture multiple times to obtain a solvent layer, drying, filtering and evaporating, purifying and separating, and concentrating to obtain the first compound;

[0010] 2) Preparation of melphalan targeted drug delivery system

[0011] S4. dissolving melphalan and the first compound in anhydrous dichloromethane at a molar ratio of (0.9-1.1):1, wherein the volume ratio of the anhydrous dichloromethane to the step S1 is (0.9-1.1):2;

[0012] S5. adding dicyclohexylcarbodiimide and 4-dimethylaminopyridine, using dicyclohexylcarbodiimide as a dehydrating agent and 4-dimethylaminopyridine as a catalyst, wherein the molar ratio of the dicyclohexylcarbodiimide, the 4-dimethylaminopyridine and the melphalan is (2.7-3.3):(1.8-2.2):2, and performing an esterification reaction to obtain a second mixture;

[0013] S6. The second mixture is washed, dried, filtered, evaporated, purified, separated and then concentrated to obtain a second compound, which is the melphalan targeted drug delivery system to be synthesized.

[0014] Furthermore, the solvent used in step S1 is any one of tetrahydrofuran, methanol and ethanol.

[0015] Furthermore, the reaction after adding iodine in step S2 is carried out under stirring at a reaction temperature of -5-0°C.

[0016] Furthermore, the specific operation of dissolving in step S4 is stirring at 0-25° C. for 9-11 minutes.

[0017] Furthermore, the temperature of the esterification reaction in step S5 is 60-80° C., the reaction time is 22-26 hours, and the esterification reaction is carried out under stirring.

[0018] Furthermore, in step S3 and step S6, the drying is carried out by drying with anhydrous sodium sulfate.

[0019] Furthermore, step S3 and step S6 are purified and separated by passing through a silica gel chromatography column at normal pressure.

[0020] Furthermore, the eluent used for column separation at normal pressure in step S3 is a mixture of petroleum ether and ethyl acetate in a volume ratio of 12:1.

[0021] Furthermore, the eluent used for column separation at normal pressure in step S6 is a mixture of petroleum ether and ethyl acetate in a volume ratio of 25:1.

[0022] Furthermore, the step S3 is extracted three times with dichloromethane, and the volume ratio of the dichloromethane to the anhydrous tetrahydrofuran is (4.5-5.5):2 each time.

[0023] Furthermore, in step S6, the second mixture is washed with hydrochloric acid having a concentration of 0.45-0.55 mol / L and saturated sodium bicarbonate in sequence.

[0024] The present invention also provides a melphalan targeted drug delivery system, which is prepared by the above-mentioned preparation method.

[0025] The present invention also provides the use of the above-mentioned melphalan targeted drug delivery system in the preparation of drugs for breast cancer, ovarian cancer, chronic lymphocytic and granulocytic leukemia, malignant lymphoma or multiple myeloma.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention utilizes the good biocompatibility, high fluorescence quantum yield and the feature of spontaneously locating mitochondria of rhodamine B to develop a drug delivery system (HRhod-Mel) that can target tumor cells and tissues, emit red fluorescence in tumor cells and tissues, and release the anticancer drug melphalan under green light (λ=556nm) irradiation conditions. The system can reduce the toxicity of melphalan to normal cells and tissues and avoid the serious side effects of melphalan in the treatment of cancer (breast cancer, ovarian cancer, chronic lymphocytic and granulocytic leukemia, malignant lymphoma, multiple myeloma, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0029] Figure 1 is the H NMR spectrum of compound 1 (HRhod-OH);

[0030] Figure 2 is the NMR carbon spectrum of compound 1 (HRhod-OH);

[0031] Figure 3 This is the mass spectrometry characterization result of compound 1 (HRhod-OH);

[0032] Figure 4 is the H NMR spectrum of compound 2 (HRhod-Mel);

[0033] Figure 5 is the NMR carbon spectrum of compound 2 (HRhod-Mel);

[0034] Figure 6 This is the mass spectrometry characterization result of compound 2 (HRhod-Mel);

[0035] Figure 7 is the H NMR spectrum of compound 3 (Rhod-Mel);

[0036] Figure 8 is the NMR carbon spectrum of compound 3 (Rhod-Mel);

[0037] Fig. 9 This is the mass spectrometry characterization result of compound 3 (Rhod-Mel);

[0038] Fig.10 is the H NMR spectrum of compound 4 (Rhod-OH);

[0039] Fig.11 is the NMR carbon spectrum of compound 4 (Rhod-OH);

[0040] Fig.12 This is the mass spectrometry characterization result of compound 4 (Rhod-OH);

[0041] Fig.13 The fluorescence intensity change of compound 2 (HRhod-Mel) in the presence of different concentrations of hydrogen peroxide;

[0042] Fig.14 HPLC identification diagram of compound 2 (HRhod-Mel) in the presence of different types of ROS;

[0043] Fig.15 HPLC chart of the photolytic release of drug melphalan (Mel) and photolytic drug carrier Rhod-OH by compound 2 (HRhod-Mel) under green light λ=556nm irradiation;

[0044] Fig.16 HPLC spectra of compound 2 (HRhod-Mel) releasing drugs under green light irradiation and in dry air and phosphate buffer solution (pH=7.4);

[0045] Fig.17 is the distribution of compound 2 (HRhod-Mel) in cancer cell line MCF-7 cells;

[0046] Fig.18 The distribution of compound 2 (HRhod-Mel) in normal cell line MCF-10 cells;

[0047] Fig.19 The results of cell viability assays of HRhod-Mel, Rhod-OH and free drug Melphalan in MCF-7 cell lines in the dark;

[0048] Fig. 20 The results of cell activity determination of HRhod-Mel, Rhod-OH and free drug Melphalan in MCF-7 cell line after illumination;

[0049] Fig.21The results of cell viability assays of HRhod-Mel, Rhod-OH and free drug Melphalan in MCF-10A cell line in the dark;

[0050] Fig. 22 The results of cell activity assay of HRhod-Mel, Rhod-OH and free drug Melphalan in light-exposed MCF-10A cell line. DETAILED DESCRIPTION

[0051] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The present invention is specifically introduced below in conjunction with the specific embodiments.

[0052] The embodiment of the present invention provides a method for preparing a melphalan targeted drug delivery system, comprising the following steps:

[0053] 1) Reduction of Rhodamine B

[0054] S1. At -5-0°C (exothermic reaction, the temperature is as low as possible), preferably at 0°C, dissolving rhodamine B in a solvent, the mass volume ratio of the rhodamine B to the solvent is 1:(18-22) (g / mL), preferably 1:20 (g / mL), and then slowly adding sodium borohydride, the mass ratio of the sodium borohydride to the rhodamine B is 19:(180-220), preferably 19:200, until all the hydrogen is released (the reason for slowly adding sodium borohydride is that sodium borohydride reacts violently, and the reaction process is an exothermic reaction, so the reaction temperature is 0°C, and the hydrogen released is because sodium borohydride is decomposed during the reaction);

[0055] S2. adding iodine, wherein the molar ratio of the iodine to the rhodamine B is (9-11):21, preferably 10:21, and after reacting for 8-10 hours, quenching the reaction with water (quenching with water is to quench the excess sodium borohydride to terminate the reaction, and water should be added slowly dropwise during the addition process, and a large number of bubbles will be generated during the addition), to obtain a first mixture;

[0056] S3. extracting the first mixture multiple times to obtain a solvent layer, drying, filtering and evaporating, purifying and separating, and concentrating to obtain the first compound;

[0057] 2) Preparation of melphalan targeted drug delivery system

[0058] S4. dissolving melphalan and the first compound in anhydrous dichloromethane at a molar ratio of (0.9-1.1):1, preferably, dissolving melphalan and the first compound in anhydrous dichloromethane at a molar ratio of 1:1, wherein the volume ratio of the anhydrous dichloromethane to the step S1 is (0.9-1.1):2, preferably, 1:2;

[0059] S5. adding dicyclohexylcarbodiimide and 4-dimethylaminopyridine, using dicyclohexylcarbodiimide as a dehydrating agent and 4-dimethylaminopyridine as a catalyst, wherein the molar ratio of the dicyclohexylcarbodiimide, the 4-dimethylaminopyridine and the melphalan is (2.7-3.3):(1.8-2.2):2, preferably, the molar ratio of the dicyclohexylcarbodiimide, the 4-dimethylaminopyridine and the melphalan is 3:2:2, and performing an esterification reaction to obtain a second mixture;

[0060] S6. The second mixture is washed, dried, filtered, evaporated, purified, separated and then concentrated to obtain a second compound, which is the melphalan targeted drug delivery system to be synthesized.

[0061] Among them, melphalan is a bifunctional alkylating agent that has a good inhibitory effect on malignant tumors such as breast cancer. However, melphalan is toxic to both cancer cells and normal cells during the treatment of cancer. The dye rhodamine B itself has good biocompatibility and high fluorescence quantum yield, and can spontaneously locate the mitochondria of cancer cells with excessive reactive oxygen species (ROS). By combining melphalan and reduced rhodamine B through an esterification reaction, a drug delivery system for melphalan to treat cancer and rhodamine B to locate cancer cells was synthesized. It avoids the side effects of melphalan in the treatment of cancer and has the advantages of rhodamine B. It can release the anticancer drug melphalan under green light (λ=556nm). Therefore, the developed drug delivery system (HRhod-Mel) may play a role in reducing the side effects of melphalan in the treatment of cancer in the future.

[0062] Among them, the solvent in step S4 can only be dichloromethane. The reason why dichloromethane is selected as the solvent for the esterification reaction of 4-dimethylaminopyridine (DMAP) is that dichloromethane has anhydrous properties, mild reaction conditions and good solubility; secondly, the anhydrous nature of dichloromethane is crucial for the DMAP esterification reaction. The dehydrating agent 1,3-dicyclohexylcarbodiimide (DCC) in the reaction is converted into dicyclohexylurea after the reaction, and this process requires an anhydrous environment. If moisture is introduced during the reaction, it may cause the reaction to fail or produce unnecessary by-products. Therefore, the use of dichloromethane can effectively dry the reaction system and ensure the smooth progress of the reaction. In addition, the reaction conditions of dichloromethane are mild and suitable for most esterification reactions. It will not damage substrates that are sterically hindered or sensitive to acid.

[0063] Specifically, the solvent used in step S1 is any one of tetrahydrofuran, methanol and ethanol. Preferably, the solvent used in step S1 is tetrahydrofuran. Compared with methanol and ethanol, tetrahydrofuran is better and can improve the reduction effect of sodium borohydride. Secondly, tetrahydrofuran is stable to sodium borohydride and will not react with it.

[0064] Specifically, the reaction after adding iodine in step S2 is carried out under stirring at a reaction temperature of -5-0°C. After adding iodine, a large amount of bubbles will be released and it is an exothermic reaction with a violent reaction.

[0065] Specifically, the specific operation of dissolving in step S4 is stirring at 0-25°C for 9-11 minutes, preferably, stirring at 0°C for 10 minutes. This step is to activate the reaction substrate, and the temperature should not be too high at the beginning.

[0066] Specifically, the temperature of the esterification reaction in step S5 is 60-80°C, preferably 80°C. Increasing the temperature increases the reaction rate, but too high a temperature will also generate many by-products, which is not conducive to the reaction. The reaction time is 22-26h, preferably 24h, and the esterification reaction is carried out under stirring.

[0067] Specifically, the drying in step S3 and step S6 is carried out by drying with anhydrous sodium sulfate.

[0068] Specifically, step S3 and step S6 are performed on a silica gel chromatography column at normal pressure for purification and separation.

[0069] Specifically, the eluent used for the column pass at normal pressure in step S3 is a mixture of petroleum ether and ethyl acetate in a volume ratio of 12:1.

[0070] Specifically, the eluent used for column separation at normal pressure in step S6 is a mixture of petroleum ether and ethyl acetate in a volume ratio of 25:1.

[0071] Specifically, the step S3 is extracted three times with dichloromethane, and the volume ratio of the dichloromethane to the anhydrous tetrahydrofuran each time is (4.5-5.5):2, preferably 5:2.

[0072] Specifically, in step S6, the second mixture is washed with hydrochloric acid having a concentration of 0.45-0.55 mol / L and saturated sodium bicarbonate in sequence, preferably, with hydrochloric acid having a concentration of 0.5 mol / L.

[0073] The embodiment of the present invention also provides a melphalan targeted drug delivery system, which is prepared by the above-mentioned preparation method.

[0074] The embodiments of the present invention also provide the use of the above-mentioned melphalan targeted drug delivery system in the preparation of drugs for breast cancer, ovarian cancer, chronic lymphocytic and granulocytic leukemia, malignant lymphoma or multiple myeloma.

[0075] Example 1: Synthesis of Melphalan Targeted Drug Delivery System

[0076] This example synthesized a melphalan targeted drug delivery system (HRhod-Mel), and the specific process was as follows:

[0077] (1) Reduction of Rhodamine B: At 0°C, commercially available Rhodamine B (1 g, 2.1 mmol) was dissolved in anhydrous tetrahydrofuran (THF, 20 mL), and then sodium borohydride (NaBH4, 0.095 g, 2.5 mmol) was slowly added until all hydrogen was released, and iodine (0.266 g, 1.0 mmol) was added dropwise to the mixed solution. After stirring and heating for 9 hours, the reaction was quenched with water (15 mL), and the mixture was extracted with dichloromethane (50 mL×3). The organic phase (extracted dichloromethane layer) was dried over anhydrous sodium sulfate, filtered and evaporated, and the mixture was chromatographed on a silica gel column at normal pressure (eluent petroleum ether: ethyl acetate = 12:1), and concentrated in vacuo to obtain a purple oily compound 1 (HRhod-OH) with a yield of 67% (0.64 g, 1.6 mmol). The reaction process is as follows:

[0078]

[0079] (2) Esterification reaction: Melphalan (0.05 g, 0.16 mmol) and compound 1 (0.069 g, 0.16 mmol) were dissolved in 10 mL of anhydrous dichloromethane (DCM), stirred at 0°C for 10 minutes, and then dicyclohexylcarbodiimide (DCC, 0.047 g, 0.24 mmol) and 4-dimethylaminopyridine (DMAP, 0.019 g, 0.16 mmol) were added to the solution, and then heated to 80°C and stirred for 24 hours. After the reaction was completed, the mixture was washed with hydrochloric acid (0.5 mol / L) and saturated sodium bicarbonate, dried over anhydrous sodium sulfate, filtered and evaporated. The mixture was chromatographed at normal pressure on a silica gel column (eluent: petroleum ether: ethyl acetate = 25:1), and concentrated in vacuo to obtain a purple oily compound 2 (HRhod-Mel) with a yield of 70% (0.084 g, 0.12 mmol), which was the melphalan targeted drug delivery system (HRhod-Mel) to be synthesized. The reaction process is as follows:

[0080]

[0081] This example also synthesized compound 3 (Rhod-Mel) and compound 4 (Rhod-OH), and the specific process was as follows:

[0082] (3) In a 10 mL reaction bottle, compound 2 (0.05, 0.07 mmol) was dissolved in dichloromethane (DCM, 10 mL), and chlorobenzoquinone (0.027 g, 0.11 mmol) was added to the reaction bottle. The mixture in the reaction bottle was stirred at room temperature for 2 h, and then the mixture in the reaction bottle was washed with water and saturated brine in turn. The washed mixture was dried with anhydrous Na2SO4, and the solid anhydrous sodium sulfate was removed by filtration. The solvent after drying with anhydrous sodium sulfate was evaporated. The remaining solid was purified by silica gel column chromatography (V 二氯甲烷 :V 甲醇 =65:15) to purify the residue to obtain purple compound 3 (Rhod-Mel) with a yield of 83% (0.036 g, 0.05 mmol). The reaction process is as follows:

[0083]

[0084] (4) In a 10 mL single-mouth reaction bottle, compound 1 (HRhod-OH, 0.05 g, 0.06 mmol) was dissolved in 5 mL of dichloromethane, and tetrachlorobenzoquinone (Chioranil, 0.03 g, 0.12 mmol) was added to the reaction bottle. The mixture in the reaction bottle was stirred at room temperature for 2 h, and then the mixture in the reaction bottle was washed with saturated brine. The mixture solution after brine washing was dried over anhydrous Na2SO4, and the anhydrous Na2SO4 was removed by filtration. The solution in the mixture was removed by rotary evaporation. Then (V) was added to the remaining residue. 己烷 :V 三氯甲烷 =3:7) was recrystallized at -20°C to obtain green crystalline compound 4 (Rhod-OH) with a yield of 72% (0.034 g, 0.043 mmol). The reaction process is as follows:

[0085]

[0086] Example 2: In vitro reactive oxygen species (ROS) oxidation of melphalan targeted drug delivery system (HRhod-Mel)

[0087] To observe the fluorescence spectrum changes of compound 2 (HRhod-Mel) in the presence of mitochondrial ROS in tumor cells, the inventors used common reactive oxygen species produced by tumor cells in vitro, including hydrogen peroxide (H2O2), potassium superoxide (KO2), hydroxyl radical (·OH), hypochlorous acid (HOCl) and peroxynitrite (ONOO ﹣) oxidation. First, the inventors treated a solution of compound 2 (HRhod-Mel) (10 μM) in acetonitrile / PBS buffer (1:19) with different concentrations of hydrogen peroxide (H2O2) (0-100 μM) and monitored the reaction by fluorescence spectroscopy. It was observed that with the gradual addition of H2O2 (0, 20, 40, 60, 80, 100 μM), the fluorescence intensity at λ=560 nm gradually increased, and the solution changed from no fluorescence to red fluorescence ( Fig.13 ). This indicates that the state of compound 2 has changed, and the fluorescence intensity has increased, which suggests that it may produce compound 3 (Rhod-Mel). Compound 2 was treated with 100 μM of other reactive oxygen species, such as hydrogen peroxide (H2O2), potassium superoxide (KO2), hydroxyl radical (·OH), hypochlorous acid (HOCl), and peroxynitrite (ONOO ﹣ ). The products produced by the oxidation with the above different active oxygen species were identified by HPLC. Fig.14 ,exist Fig.14 It can be observed that the chromatographic peak of compound 2 (HRhod-Mel) disappears after being treated with the above five reactive oxygen species, and a new peak appears, which corresponds to the chromatographic peak of compound 3 (Rhod-Mel). This shows that when compound 2 (HRhod-Mel) is treated with 100 μM of five different reactive oxygen species, compound 2 (HRhod-Mel) is oxidized to compound 3 (Rhod-Mel) by these ROS. This proves that compound 2 (HRhod-Mel) can be oxidized to compound 3 (Rhod-Mel) by the reactive oxygen species commonly found in tumor cells.

[0088] Example 3: HPLC characterization of the release of anticancer drug melphalan by melphalan targeted drug delivery system (HRhod-Mel) under green light (λ=556 nm) in vitro

[0089] HPLC was used to study whether compound 3 (Rhod-Mel) could release the anticancer drug melphalan (Mel) under the irradiation of green light (λ = 556nm). Compound 3 (Rhod-Mel) was dissolved in a mixed solution of acetonitrile / water (8:1 (v / v)) and irradiated with a medium-pressure mercury lamp in the green light band (λ = 556nm) for 0min, 20min, 40min and 60min, and samples were taken at each time period for HPLC determination. The results are shown in Table 1. Fig.15The inventors found that after 20 minutes of irradiation, the HPLC graph showed that compound 3 decomposed into two substances. By comparing the HPLC graphs of compounds 1-4, the inventors found that the decomposed substances were compound 4 (Rhod-OH) and the anticancer drug melphalan (Mel). At 60 minutes, compound 3 (Rhod-Mel) was almost completely decomposed to produce compound 4 (Rhod-OH) and the anticancer drug melphalan (Mel). It proves that the synthesized drug delivery system can release the anticancer drug melphalan (Mel) under green light irradiation.

[0090] Example 4: Stability of Melphalan Targeted Drug Delivery System (HRhod-Mel) in Dry Air and Phosphate Buffer Solution (pH = 7.4)

[0091] To verify the stability of compound 2, the inventors compared the decomposition of the dry solid compound 2 after exposure to air for five days with and without adding any solution. The inventors used HPLC to detect the decomposition of compound 2 in the above two cases after five days of exposure. The results were compared with the HPLC chromatograms of compounds 2, 3 and 4. Fig.16 It was found that they were stable after being placed in phosphate buffer solution or dry air for five days, and were completely oxidized to compound 3 within 30 minutes after adding 100 μM hydrogen peroxide (H2O2). This shows that compound 2 is relatively stable in air and in phosphate buffer solution (pH=7.4), and compound 2 can be quickly oxidized to compound 3 when encountering active oxygen such as hydrogen peroxide.

[0092] Example 5: Uptake of Melphalan Targeted Drug Delivery System (HRhod-Mel) in Tumor Cells MCF-7 and Normal Cells MCF-10

[0093] In order to study whether compound 2 (HRhod-Mel) can achieve localization in the mitochondria of tumor cells, MCF-7 and MCF-10A cell lines (purchased from the American Type Culture Collection (ATCC, USA)) were used. The cells were cultured in a humidified environment at 37°C and 5% CO2 using DMEM medium supplemented with 10% (v / v) fetal bovine serum. Under the above conditions, compound 2 (HRhod-Mel) (200nM) and mitochondrial green fluorescent probe (Mito-Tracker Green, final concentration of 200nM) were incubated together for 4 hours. The distribution of compound 2 (HRhod-Mel) in cells in MCF-7 and MCF-10A cell lines was monitored by cofluor microscopy (FL). Imaging was performed using green and red wavelength channels, and the results are shown in Figure 2. Fig.17 and Fig.18The green channel in the figure shows the mitochondria stained with mitochondrial fluorescent probe dye, the red channel shows the mitochondria stained with compound 2 (HRhod-Mel), and the red and green channels are the superposition of the two stainings. Fig.17 It can be seen that compound 2 (HRhod-Mel) can emit red fluorescence in the mitochondria of cancer cell MCF-7. Fig.18 It can be seen that compound 2 (HRhod-Mel) does not emit obvious red fluorescence in normal cells MCF-10A, proving that compound 2 may be oxidized to compound 3 (Rhod-Mel) only in cancer cells. It proves that the synthesized drug delivery system can be localized in MCF-7 cells.

[0094] Example 6: Cytotoxicity experiments of compound 2 (HRhod-Mel), melphalan (Mel) and compound 4 (Rhod-OH)

[0095] Example 2 shows that compound 2 (HRhod-Mel) will be oxidized to compound 3 (Rhod-Mel) by excessive reactive oxygen species after entering cancer cells, and Example 3 shows that compound 3 will release anticancer drug melphalan (Mel) and photolysis product compound 4 (Rhod-OH) under green light irradiation. Therefore, the inventors used MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to study the in vitro cytotoxicity of HRhod-Mel, Rhod-OH and free drug melphalan (Mel). Cancer cell line MCF-7 and normal cell line MCF-10A were inoculated in 96-well plates containing Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) for 8 hours. The cells were incubated with HEPES buffer containing different concentrations (5-50 μM) of HRhod-Mel, Rhod-OH and free drug melphalan (Mel) at 37°C in 5% CO2 for 72 hours. The cytotoxicity was then measured using the MTT assay before and after light irradiation. Figures 19 to 22 From the MTT analysis data, it is obvious that HRhod-Mel has low toxicity to the cancer cell line MCF-7 and the normal cell line MCF-10A before irradiation. However, after irradiation, a significant increase in HRhod-Mel cytotoxicity was observed, and the photolysis product Rhod-OH was not observed to show obvious cytotoxicity. The cytotoxicity of the free drug melphalan (Mel) did not change significantly before and after irradiation.

[0096] The above description is only a description of the preferred embodiments of the present invention, and is not intended to limit the scope of the present invention. Any changes or modifications made by a person skilled in the art in the field of the present invention based on the above disclosure shall fall within the scope of protection of the claims.

Claims

1. A method for preparing a melphalan targeted drug delivery system, characterized in that: The following steps are involved: 1) Reduction of Rhodamine B S1. At -5-0°C, dissolving rhodamine B in a solvent, wherein the mass volume ratio of the rhodamine B to the solvent is 1:(18-22) (g / mL), and then slowly adding sodium borohydride, wherein the mass ratio of the sodium borohydride to the rhodamine B is 19:(180-220), until all hydrogen is released; S2. adding iodine, wherein the molar ratio of the iodine to the rhodamine B is (9-11):21, reacting for 8-10 hours, quenching the reaction with water, and obtaining a first mixture; S3. extracting the first mixture multiple times to obtain a solvent layer, drying, filtering and evaporating, purifying and separating, and concentrating to obtain the first compound; 2) Preparation of melphalan targeted drug delivery system S4. dissolving melphalan and the first compound in anhydrous dichloromethane at a molar ratio of (0.9-1.1):1, wherein the volume ratio of the anhydrous dichloromethane to the step S1 is (0.9-1.1):2; S5. adding dicyclohexylcarbodiimide and 4-dimethylaminopyridine, using dicyclohexylcarbodiimide as a dehydrating agent and 4-dimethylaminopyridine as a catalyst, wherein the molar ratio of the dicyclohexylcarbodiimide, the 4-dimethylaminopyridine and the melphalan is (2.7-3.3):(1.8-2.2):2, and performing an esterification reaction to obtain a second mixture; S6. The second mixture is washed, dried, filtered, evaporated, purified, separated and then concentrated to obtain a second compound, which is the melphalan targeted drug delivery system to be synthesized.

2. The preparation method according to claim 1, characterized in that: The solvent used in step S1 is any one of tetrahydrofuran, methanol and ethanol; And / or, the reaction after adding iodine in step S2 is carried out under stirring at a reaction temperature of -5-0°C.

3. The preparation method according to claim 1, characterized in that: The specific operation of dissolving in step S4 is stirring at 0-25° C. for 9-11 minutes; And / or, the temperature of the esterification reaction in step S5 is 60-80° C., the reaction time is 22-26 hours, and the esterification reaction is carried out under stirring.

4. The preparation method according to claim 1, characterized in that: The drying in step S3 and step S6 is carried out by drying with anhydrous sodium sulfate.

5. The preparation method according to claim 1, characterized in that: Step S3 and step S6 are performed on a silica gel chromatography column at normal pressure for purification and separation.

6. The preparation method according to claim 5, characterized in that: The eluent used for the column under normal pressure in step S3 is a mixture of petroleum ether and ethyl acetate in a volume ratio of 12:1; And / or, the eluent used for column passing at normal pressure in step S6 is a mixture of petroleum ether and ethyl acetate in a volume ratio of 25:

1.

7. The preparation method according to claim 1, characterized in that: The step S3 is extracted three times with dichloromethane, and each time the volume ratio of the dichloromethane to the anhydrous tetrahydrofuran is (4.5-5.5):

2.

8. The preparation method according to any one of claims 1 to 7, characterized in that: In step S6, the second mixture is washed with hydrochloric acid having a concentration of 0.45-0.55 mol / L and saturated sodium bicarbonate in sequence.

9. A melphalan targeted drug delivery system, characterized in that: The method is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the melphalan targeted drug delivery system according to claim 1 in the preparation of drugs for breast cancer, ovarian cancer, chronic lymphocytic and granulocytic leukemia, malignant lymphoma or multiple myeloma.

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