A dual-targeted nano-drug delivery system combining anti-glutamine metabolism and sensitized photodynamic therapy, its preparation and application

Abstract

The present invention belongs to the technical field of nano-drug formulations, and more specifically, relates to a dual-targeted nano-drug delivery system that combines anti-glutamine metabolism and sensitized photodynamic therapy, its preparation and application. It comprises three components, namely an anti-glutamine metabolism drug, a photosensitizer, and a targeting unit-hydroxyalkyl starch macromolecular conjugate. The anti-glutamine metabolism drug is a derivative of lonidamine, and experiments have proven that it can inhibit glutamine metabolism in tumor cells, thereby reducing the excessive glutathione in tumor stem cells. Moreover, experiments have also shown that the anti-glutamine metabolism drug and the photosensitizer of the present invention can act synergistically to enhance the killing effect of photodynamic therapy on tumor stem cells. By combining anti-glutamine metabolism and photodynamic therapy, the elimination of tumor stem cells is achieved, and the growth and metastasis of tumors are effectively inhibited.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano-drug formulations, and more specifically, relates to a dual-targeted nano-drug delivery system that combines anti-glutamine metabolism and sensitizes photodynamic therapy, its preparation and application. Background Art

[0002] Cancer stem cells are a small population of cells with stem cell properties in tumors, having high self-renewal, self-replication, and multi-directional differentiation potential, and being mainly responsible for the occurrence, development, recurrence, metastasis, and drug resistance of tumors. Existing studies have shown that one of the main reasons for cancer stem cell drug resistance is the high intracellular reduction potential (for example: elevated glutathione levels).

[0003] Photodynamic therapy (PDT) relies on the explosive growth of reactive oxygen species (ROS) under light irradiation by photosensitizers to achieve precise treatment of the lesion site. However, the excessive glutathione in cancer stem cells can rapidly scavenge ROS, reducing the efficacy of PDT. In addition, the insufficient enrichment concentration of photosensitizers at the tumor site also limits the efficacy of PDT. There is an urgent need to develop a drug delivery system for targeting tumors, disrupting the redox balance of cancer stem cells, sensitizing PDT, and eliminating cancer stem cells. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a dual-targeted nano-drug delivery system that combines anti-glutamine metabolism and sensitizes photodynamic therapy, its preparation and application, so as to solve the technical problems in the prior art that cancer stem cells are insensitive to photodynamic therapy and the insufficient enrichment concentration of photosensitizers at the tumor site affects the effect of photodynamic therapy.

[0005] To achieve the above purpose, the present invention provides a dual-targeted nano-drug delivery system that combines anti-glutamine metabolism and sensitizes photodynamic therapy, which comprises an anti-glutamine metabolism drug, a photosensitizer, and a targeting unit-hydroxyalkyl starch macromolecular conjugate. Among them, the anti-glutamine metabolism drug has the structure shown in formula (I):

[0006]

[0007] In formula (I), R is a phenyl group, or a phenyl group substituted by one or more substituents selected from halogen, C1-C4 alkyl, nitro, and trifluoromethyl; the photosensitizer is a cyanine or porphyrin photosensitizer; the targeting unit-hydroxyalkyl starch macromolecular conjugate is a macromolecular compound obtained by conjugating a molecule targeting tumors with hydroxyalkyl starch.

[0008] Preferably, the photosensitizer is an unsaturated fatty acid-semi-cyanine conjugate, which has the structure shown in formula (II):

[0009]

[0010] Among them, R 1 is an alkyl group with 1 - 18 carbon atoms or an alkyl sulfonic acid with 1 - 18 carbon atoms; R 2 is an unsaturated fatty chain, and the carbon number of the unsaturated fatty chain is 6 - 30.

[0011] Further preferably, the unsaturated fatty chain is derived from an unsaturated fatty acid, and the chemical formula of the unsaturated fatty acid is C n H 2n-1 COOH, C n H 2n-3 COOH, C n H 2n-5 COOH, C n H 2n-7 COOH or C n H 2n-9 COOH, where n is an integer from 8 - 29, and preferably n is an integer from 14 - 25.

[0012] Preferably, the molecule targeting the tumor is folic acid, galactose or RGD peptide, and the grafting rate of the targeting unit in the targeting unit - hydroxyalkyl starch macromolecular conjugate is less than or equal to 10%.

[0013] Preferably, the mass ratio of the anti - glutamine metabolism drug, photosensitizer and the targeting unit - hydroxyalkyl starch macromolecular conjugate in the nano - drug delivery system is (10 - 40):(10 - 30):(30 - 80).

[0014] According to another aspect of the present invention, a preparation method of the nano - drug delivery system is provided, including the following steps:

[0015] (1) Dissolve the anti - glutamine metabolism drug and the photosensitizer in a benign solvent to obtain a mixed solution;

[0016] (2) Drop the mixed solution into a poor solvent, and after dialysis, obtain a self - assembled nanoparticle dispersion of the anti - glutamine metabolism drug and the photosensitizer;

[0017] (3) Mix and stir the self - assembled nanoparticle dispersion obtained in step (2) with the targeting unit - hydroxyalkyl starch macromolecular conjugate in water, so that the targeting unit - hydroxyalkyl starch macromolecular conjugate is modified on the surface of the self - assembled nanoparticles, and obtain the dual - targeting nano - drug delivery system for combined anti - glutamine metabolism and PDT sensitization.

[0018] Preferably, the benign solvent in step (1) is one or more of dimethyl sulfoxide, tetrahydrofuran, methanol, ethanol, dichloromethane, acetonitrile, and the mass ratio of the anti - glutamine metabolism drug to the photosensitizer is 0.5 - 2:1;

[0019] The poor solvent described in step (2) is deionized water;

[0020] In the folic acid - hydroxyalkyl starch conjugate described in step (3), the grafting rate of folic acid is less than or equal to 10%. The mass ratio of the targeting unit - hydroxyalkyl starch conjugate to the total mass of the anti - glutamine metabolism drug and the photosensitizer is 0.5 - 2:1; the hydroxyalkyl starch is hydroxymethyl starch, hydroxyethyl starch, hydroxypropyl starch or hydroxybutyl starch, and is further preferably hydroxyethyl starch. The average molecular weight of the selected hydroxyalkyl starch is 40 - 200 kDa, and the molar substitution degree of the hydroxyalkyl group is 0.4 - 0.5.

[0021] According to another aspect of the present invention, there is provided an application of the described nano - drug delivery system in the preparation of a drug for treating and / or preventing tumors.

[0022] According to another aspect of the present invention, there is provided an application of the described nano - drug delivery system in the preparation of a drug for killing tumor stem cells.

[0023] Generally speaking, compared with the prior art through the above - mentioned technical solution conceived by the present invention, the following

[0024] Advantages are obtained:

[0025] (1) The present invention provides a dual - targeting nano - drug delivery system that combines anti - glutamine metabolism and sensitizes PDT, which contains three components, namely an anti - glutamine metabolism drug, a photosensitizer, and a targeting unit - hydroxyalkyl starch macromolecular conjugate. The anti - glutamine metabolism drug is a derivative of lonidamine. Experiments have proved that it can inhibit glutamine metabolism in tumor cells, thereby reducing the excessive glutathione in tumor stem cells. Moreover, experiments have also proved that the anti - glutamine metabolism drug and the photosensitizer can act synergistically to sensitize the killing of tumor stem cells by PDT.

[0026] (2) For the dual - targeting nano - drug delivery system that combines anti - glutamine metabolism and sensitizes PDT provided by the present invention, the modification of the targeting unit - hydroxyalkyl starch macromolecular conjugate not only stabilizes the nano - preparation, but also enables active targeting mediated by the targeting unit, increasing the enrichment of the nano - drug at the tumor site.

[0027] (3) For the dual - targeting nano - drug delivery system that combines anti - glutamine metabolism and sensitizes PDT provided by the present invention, this preparation can achieve active targeting led by the targeting unit and mitochondrial targeting mediated by the positively charged drug. The dual targeting increases the enrichment of the drug in the tumor.

[0028] (4) In the preferred embodiment of the present invention, the intelligent nano - preparation provided is named COHF NPs, which includes three components: the anti - glutamine metabolism drug HYL001, the photosensitizer CyOA, and the folic acid - hydroxyethyl starch conjugate (FA - HES). The surface - modified FA - HES can not only stabilize the nano - preparation but also achieve folic - acid - mediated active targeting; HYL001 inhibits the synthesis of glutathione by inhibiting glutamine metabolism, sensitizes PDT, and its positive charge mediates mitochondrial targeting; CyOA is a semi - cyanine photosensitizer, and the modification of unsaturated fatty chains promotes the self - assembly of the drug, ensuring a super - high drug - loading capacity of the nano - preparation.

[0029] (5) The present invention eradicates cancer stem cells by disrupting the redox balance of cancer stem cells and sensitizing PDT, which has a positive significance for tumor treatment. By combining anti - glutamine metabolism and photodynamic therapy, the elimination of cancer stem cells is achieved, and the growth and metastasis of tumors are effectively inhibited. The present invention fills the blank of using glutamine - metabolism - inhibition - sensitized PDT to inhibit cancer stem cells, providing a new effective strategy and technology for tumor treatment. Brief Description of the Drawings

[0030] Figure 1 Schematic flow chart of the synthesis method of the anti - glutamine metabolism drug HYL001;

[0031] Figure 2 Transmission electron micrograph of Example 1 (COHF NPs) of the present invention;

[0032] Figure 3 Transmission electron micrograph of Comparative Example 1 (COLF NPs) of the present invention;

[0033] Figure 4 Stability test of Example 1 (COHF NPs) of the present invention;

[0034] Figure 5 Quantitative ROS generation in vitro of Example 1 (COHF NPs) and Comparative Example 1 (COLF NPs) of the present invention;

[0035] Figure 6 Intracellular co - localization map of Example 1 (COHF NPs) and Comparative Example 1 (COLF NPs) of the present invention;

[0036] Figure 7 Band diagram of the effect of Example 1 (COHF NPs) and Comparative Example 1 (COLF NPs) of the present invention on GLS level;

[0037] Figure 8 Effect of Example 1 (COHF NPs) and Comparative Example 1 (COLF NPs) of the present invention on intracellular glutathione level;

[0038] Figure 9 Effect of Example 1 (COHF NPs) and Comparative Example 1 (COLF NPs) of the present invention on intracellular ROS levels;

[0039] Figure 10 Dose-cell activity graph of Example 1 (COHF NPs) and Comparative Example 1 (COLF NPs) of the present invention on 4T1 cells;

[0040] Figure 11 Dose-cell activity graph of Example 1 (COHF NPs) and Comparative Example 1 (COLF NPs) of the present invention on breast cancer stem cells;

[0041] Figure 12 In vivo tissue distribution map of Example 1 (COHF NPs) and Comparative Example 1 (COLF NPs) of the present invention, where content a is the in vivo imaging pictures of mice in different groups at different times, content b is the fluorescence quantification of tumor sites in different groups under in vivo imaging; content c is the ex vivo imaging pictures of various organs of mice; content d is the fluorescence quantification graph of each ex vivo organ;

[0042] Figure 13 In vivo pharmacodynamic graph of Example 1 (COHF NPs) and Comparative Example 1 (COLF NPs) of the present invention, where content a is the tumor volume-time curve, content b is the ex vivo photograph of the lung, and content c is the hematoxylin-eosin staining graph of the lung section. Detailed implementation manners

[0043] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0044] A dual-targeted nano-drug delivery system combining anti-glutamine metabolism and sensitizing PDT provided by the present invention comprises three components, namely an anti-glutamine metabolism drug, a photosensitizer and a targeting unit-hydroxyalkyl starch macromolecular conjugate. Among them, the anti-glutamine metabolism drug is a derivative of lonidamine and has the structure shown in formula (I):

[0045]

[0046] Among them, R is a phenyl group, or a phenyl group substituted by one or more substituents selected from halogen, C1-C4 alkyl, nitro and trifluoromethyl; the photosensitizer is a cyanine or porphyrin photosensitizer; the targeting unit-hydroxyalkyl starch macromolecular conjugate is a macromolecular compound obtained by conjugating a molecule targeting tumors with hydroxyalkyl starch.

[0047] The anti-glutamine metabolism drug of the present invention is a lonidamine derivative. In some embodiments, the preparation method of the lonidamine derivative includes the following steps:

[0048] (1) Etherify 2-hydroxy-5-methylisophthalic alcohol with a benzyl halide compound to obtain a bridged intermediate with a substituted phenolic hydroxyl group;

[0049] (2) Esterify the bridged intermediate described in step (1) with lonidamine to obtain a lonidamine-linker conjugate;

[0050] (3) React the conjugate described in step (2) with 4-carboxytriphenylphosphonium bromide to obtain a mitochondria-targeted lonidamine derivative.

[0051] In some embodiments, the benzyl halide compound described in step (1) is benzyl bromide or benzyl bromide substituted on the benzene ring by one or more of C1-C4 alkyl, nitro, and trifluoromethyl.

[0052] As a preferred embodiment, R in formula (I) is an unsubstituted benzene ring, and the structural formula of the anti-glutamine metabolism drug is as shown in formula (IV), named HYL001.

[0053]

[0054] In some embodiments, the synthesis method of HYL001 includes one-step etherification and two-step esterification reactions.

[0055] In some embodiments of the present invention, the photosensitizer is an unsaturated fatty acid-semi-cyanine conjugate, which has a structure as shown in formula (II):

[0056]

[0057] Wherein, R 1 is selected from alkyl groups with 1 to 18 carbon atoms and alkyl sulfonic acids with 1 to 18 carbon atoms; R 2 is an unsaturated fatty chain corresponding to the unsaturated fatty acid, and the carbon number of the unsaturated fatty chain is 6 to 30.

[0058] In a preferred embodiment, in formula (II), R 1 is selected from one of alkyl groups with 1 to 10 carbon atoms and alkyl sulfonic acids with 1 to 10 carbon atoms; more preferably one of alkyl groups with 1 to 6 carbon atoms and alkyl sulfonic acids with 1 to 6 carbon atoms.

[0059] In some embodiments, the chemical formula of the unsaturated fatty acid is C n H 2n-1 COOH, C n H 2n-3 COOH, C n H2n- 5 COOH, C n H 2n-7 COOH or C n H 2n-9 COOH, where n is an integer from 8 to 29, preferably an integer from 14 to 25. The unsaturated fatty acids include but are not limited to oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA).

[0060] In some embodiments, the unsaturated fatty acid-semi-cyanine conjugate is obtained by an esterification reaction of an unsaturated fatty acid with a semi-cyanine containing a phenolic hydroxyl group.

[0061] The targeting unit-hydroxyalkyl starch macromolecular conjugate of the present invention is a macromolecular compound in which a tumor-targeting molecule and hydroxyalkyl starch are conjugated through various chemical bonds such as ester bonds. The tumor-targeting molecules of the present invention include but are not limited to folic acid, galactose, or RGD peptide. The grafting rate of the targeting unit in the targeting unit-hydroxyalkyl starch macromolecular conjugate is less than or equal to 10%. The hydroxyalkyl starch is hydroxyethylmethyl starch, hydroxyethyl starch, hydroxypropyl starch, or hydroxybutyl starch, preferably hydroxyethyl starch. The selected hydroxyalkyl starch has an average molecular weight of 40-200 kDa and a molar substitution degree of hydroxyalkyl of 0.4-0.5. Taking folic acid as an example, the folic acid-hydroxyalkyl starch macromolecular conjugate is a macromolecular compound obtained by conjugating folic acid and hydroxyalkyl starch through an ester bond. In some embodiments, folic acid-hydroxyethyl starch has a structural schematic diagram as shown in formula (III):

[0062]

[0063] In some embodiments, the mass ratio of the anti-glutamine metabolism drug, photosensitizer, and the targeting unit-hydroxyalkyl starch macromolecular conjugate in the nano-drug delivery system is (10-40):(10-30):(30-80).

[0064] The present invention also provides a preparation method of the nano-drug delivery system described above, including the following steps:

[0065] (1) Dissolve the anti-glutamine metabolism drug and the photosensitizer in a benign solvent to obtain a mixed solution;

[0066] (2) Drop the mixed solution into a poor solvent, and after dialysis, obtain a self-assembled nanoparticle dispersion of the anti-glutamine metabolism drug and the photosensitizer;

[0067] (3) Disperse the self-assembled nanoparticle dispersion obtained in step (2) and the targeting unit-hydroxyalkyl starch macromolecular conjugate in water and mix and stir them to modify the targeting unit-hydroxyalkyl starch macromolecular conjugate on the surface of the self-assembled nanoparticles, thereby obtaining the dual-targeted nano-drug delivery system for combined anti-glutamine metabolism and PDT sensitization.

[0068] In some embodiments, the benign solvent in step (1) is one or more of dimethyl sulfoxide, tetrahydrofuran, methanol, ethanol, dichloromethane, and acetonitrile, and the mass ratio of the anti-glutamine metabolism drug to the photosensitizer is 0.5-2:1; the poor solvent in step (2) is deionized water; in step (2), the mixed solution is added dropwise to the poor solvent and fully mixed by stirring or ultrasonic treatment; dialysis is carried out in a dialysis bag, and the molecular weight cut-off of the dialysis bag is 1 kDa-14 kDa, and dialysis is carried out for 12-36 h in total. In step (3), the grafting rate of folic acid in the targeting unit-hydroxyalkyl starch conjugate is less than or equal to 10%, and the mass ratio of the total mass of the anti-glutamine metabolism drug and the photosensitizer to the mass of the targeting unit-hydroxyalkyl starch conjugate is 1:0.5-2; the hydroxyalkyl starch is hydroxymethyl starch, hydroxyethyl starch, hydroxypropyl starch, or hydroxybutyl starch, preferably hydroxyethyl starch. The selected hydroxyalkyl starch has an average molecular weight of 40-200 kDa and a molar substitution degree of hydroxyalkyl of 0.4-0.5.

[0069] The nano-drug delivery system provided by the present invention can be used to prepare drugs for treating and / or preventing tumors, drugs for killing tumor stem cells, especially drugs for treating and / or preventing tumors by photodynamic therapy, and drugs for killing tumor stem cells.

[0070] The following are examples:

[0071] The nano-drug delivery system provided in the following examples of the present invention contains three components, the anti-glutamine metabolism drug HYL001, the unsaturated fatty chain-modified hemicyanine photosensitizer CyOA, and the folic acid-hydroxyethyl starch conjugate FA-HES, wherein:

[0072] (1) The structural formula of the anti-glutamine metabolism drug HYL001 is as follows:

[0073]

[0074] The synthesis method of HYL001 is as Figure 1 shown and includes the following steps:

[0075] S1: Add 2-hydroxy-5-methylisophthalic dimethanol (1.7 g, 10 mmol), potassium carbonate (2.1 g, 15 mmol), and benzyl bromide (1.7 g, 10 mmol) into 10 mL of N’,N’-dimethylformamide solvent. After stirring at 80 °C for 6 hours, stop the reaction. The post-treatment is carried out by extraction with ethyl acetate, washing with saturated brine, drying with anhydrous sodium sulfate, concentration, mixing with silica gel, and column chromatography separation and purification using petroleum ether:ethyl acetate = 1:1 as the eluent to obtain intermediate 3.

[0076] S2: In a round-bottom flask, dissolve intermediate 3 (645.8 mg, 2.5 mmol) in 10 mL of dichloromethane, and dropwise add a mixed solution of lonidamine (642.3 mg, 2 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (479.3 mg, 2.5 mmol), and 4-dimethylaminopyridine (24.3 mg, 0.2 mmol) in 20 mL of dichloromethane. Stir the reaction at room temperature for 3 hours. After the reaction is completed, the post-treatment is carried out by extraction with dichloromethane, washing the organic phase with saturated brine, drying with anhydrous sodium sulfate, and concentration. The concentrate is purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1) to obtain intermediate 4.

[0077] S3: Similar to the synthesis method of intermediate 4, HYL001 is obtained by the esterification reaction of intermediate 4 and 4-carboxytriphenylphosphonium bromide. The specific steps are as follows: In a round-bottom flask, dissolve 4-carboxytriphenylphosphonium bromide (443.3 mg, 1.0 mmol) in 10 mL of dichloromethane, and dropwise add a mixed solution of intermediate 4 (561.5 mg, 1.0 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (191.7 mg, 1.0 mmol), and 4-dimethylaminopyridine (24.3 mg, 0.2 mmol) in 20 mL of dichloromethane. Stir the reaction at room temperature for 4 hours. After the reaction is completed, the post-treatment is carried out by extraction with dichloromethane, washing the organic phase with saturated brine, drying with anhydrous sodium sulfate, and concentration. The concentrate is purified by silica gel column chromatography (methanol:dichloromethane = 1:20 → 1:10) to obtain a white foamy solid powder. NMR and mass spectrometry data: 1 HNMR(600MHz,CDCl 3) δ 8.17 (d, J = 8.2 Hz, 1H), 7.85–7.72 (m, 9H), 7.68–7.62 (m, 6H), 7.43–7.26 (m, 10H), 7.15 (d, J = 1.8 Hz, 1H), 7.02–6.98 (m, 1H), 6.67–6.64 (m, 1H), 5.73 (s, 2H), 5.51 (s, 2H), 5.09 (s, 2H), 4.97 (s, 2H), 2.44 (t, J = 6.8 Hz, 2H), 2.29 (s, 3H), 2.04–1.93 (m, 4H), 1.75–1.65 (m, 2H). 31 P (600 MHz, CDCl 3 ) δ 24.44. HRMS (ESI): m / z calcd for C 54 H 48 BrCl 2 N 2 O 5 P[M - Br] + 905.2672, found 905.26628. It indicates that HYL001 was successfully prepared.

[0078] (2) The structural formula of the semi - cyanine photosensitizer CyOA modified with unsaturated fatty chains is as follows:

[0079]

[0080] The synthesis steps of CyOA are as follows:

[0081] S1: Dissolve resorcinol (220 mg, 2 mmol) and K 2 CO 3 (276 mg, 2 mmol) in 5 mL of anhydrous N’,N’ - dimethylformamide, stir at room temperature for 30 min, then add the solution of IR780 (667 mg, 1 mmol) dissolved in 2 mL of N’,N’ - dimethylformamide to the above reaction system. Under N 2 protection, react at 60 °C for 5 h. The post - treatment is carried out by extraction with dichloromethane, washing with saturated brine, drying with anhydrous sodium sulfate, concentration, mixing with silica gel, and column chromatography separation and purification using dichloromethane: methanol = 10:1 as the eluent to obtain the intermediate CyOH.

[0082] S2: In a round-bottom flask, dissolve CyOH (431 mg, 0.8 mmol) in 10 mL of dichloromethane, and add dropwise a mixed solution of oleic acid (282 mg, 1 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (192 mg, 1 mmol) and 4-dimethylaminopyridine (24.3 mg, 0.2 mmol) in 10 mL of dichloromethane. The reaction is stirred at room temperature for 8 h. After the reaction is completed, the post-treatment is carried out by extraction with dichloromethane. The organic phase is washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The concentrate is purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to obtain a dark blue viscous oily substance (yield: 71%). NMR and mass spectrometry data: 1 H NMR(600MHz,CDCl 3 )δ8.62(d,J=15.0Hz,1H),7.52–7.48(m,3H),7.44–7.36(m,2H),7.11(s,1H),7.07–7.03(m,1H),7.00–6.96(m,1H),6.87(d,J=15.0Hz,1H),5.37–5.32(m,2H),4.75–4.61(m,2H),2.88–2.82(m,2H),2.76–2.70(m,2H),2.61(t,J=7.6Hz,2H),2.10–1.92(m,10H),1.79(s,6H),1.31–1.22(m,21H),1.12–1.06(m,3H),0.88–0.85(m,3H).HRMS(ESI):m / z calcd for C 46 H 62 NO 3 + [M] + 676.4724,found 676.47155。

[0083] (3) Folic acid-hydroxyethyl starch conjugate FA-HES, the structural schematic is as follows:

[0084]

[0085] In the following examples, FA-HES refers to a macromolecular compound obtained by coupling hydroxyethyl starch with a molecular weight of 130KDa and folic acid through an ester bond. The specific preparation method is as follows: Dissolve folic acid (20 mmol, 8.828 mg) and dicyclohexylcarbodiimide (40 mmol, 8.253 mg) in 10 mL of dimethyl sulfoxide, add 4-dimethylaminopyridine (40 mmol, 4.887 mg), and react at 50 °C for 30 min. Dissolve hydroxyethyl starch (1 mmol, 130 mg) in 5 mL of dimethyl sulfoxide, add it to the above reaction system, and continue to react at 50 °C for 48 h. After the reaction is completed, add the reaction solution to 100 mL of ethanol for precipitation, centrifuge at 8000 rpm for 10 min, dissolve the precipitate in deionized water, place it in a dialysis bag (cut-off molecular weight: 8000 Da), dialyze with ultrapure water for two days, and then freeze-dry to obtain FA-HES.

[0086] Example 1

[0087] Preparation and characterization of COHF NPs. Among them, "CO" represents the unsaturated fatty chain-modified hemicyanine photosensitizer CyOA, "H" represents the anti-glutamine metabolism drug HYL001, and "F" represents FA-HES. The preparation method specifically includes the following two steps:

[0088] (1) Add 1 mmol of HYL001 and 1 mmol of photosensitizer CyOA dissolved in 1 mL of methanol dropwise to 10 mL of ultrapure water. After ultrasonic mixing, use a dialysis bag with a cut-off molecular weight of 3500 Da and dialyze for a total of 12 hours to obtain a dispersion of self-assembled nanoparticles of pure drugs of HYL001 and photosensitizer CyOA, named COH NPs;

[0089] (2) Mix and stir the dispersion of self-assembled nanoparticles of pure drugs COH NPs obtained in step (1) with 15 mg of folic acid-hydroxyethyl starch conjugate FA-HES dissolved in 2 mL of water to obtain folic acid-hydroxyethyl starch-stabilized nano-drug COHF NPs co-loaded with the anti-glutamine metabolism drug HYL001 and photosensitizer CyOA.

[0090] Figure 2 The transmission electron micrograph of the nano-drug COHF NPs prepared in Example 1 shows that the particle size distribution of COHF NPs is uniform, and the average diameter is about 80 nm.

[0091] Comparative Example 1

[0092] Preparation and characterization of COLF NPs. Among them, "CO" represents the unsaturated fatty chain-modified hemicyanine photosensitizer CyOA, "L" represents lonidamine, the precursor molecule for synthesizing HYL001, and "F" represents FA-HES. The preparation method specifically includes the following two steps:

[0093] (1) 1 mmol lonidamine and 1 mmol photosensitizer CyOA dissolved in 1 mL of methanol were added dropwise to 10 mL of ultrapure water. After ultrasonic mixing, dialysis was carried out for 12 hours using a dialysis bag with a molecular weight cut-off of 3500 Da to obtain a dispersion of the pure drug self-assembled nanoparticles of lonidamine and photosensitizer CyOA;

[0094] (2) The self-assembled nanoparticle dispersion obtained in step (1) was mixed and stirred with 15 mg of folic acid-hydroxyethyl starch conjugate FA-HES dissolved in 2 mL of water to obtain folic acid-hydroxyethyl starch-stabilized nanodrugs COLF NPs co-loaded with lonidamine and photosensitizer CyOA.

[0095] Figure 3 Figure for the transmission electron microscopy of the nanodrug COLF NPs prepared in Comparative Example 1. It can be seen that the average diameter of COHF NPs is about 100 nm.

[0096] Example 2

[0097] Stability test of Example 1 (COHF NPs)

[0098] The stability of COHF NPs was detected by dynamic light scattering. To investigate the effect of FA-HES modification on the stability of nanoparticles, we compared the changes in the particle size of nanoparticles before and after FA-HES modification. Specifically, the COHNPs and COHF NPs prepared in Example 1 were diluted 10-fold with phosphate buffer (pH = 7.4). At different time points, 1 mL of COH NPs and COHF NPs dispersed in phosphate buffer was taken and their particle sizes were detected by the instrument.

[0099] Figure 4 Contents a and b are the stabilities of COH NPs and COHF NPs respectively. It can be seen that after 24 h of placement, COHNPs significantly aggregated and the particle size increased; while the particle size of COHF NPs did not change significantly after 6 days of placement. Therefore, the modification of FA-HES can improve the stability of the self-assembly of HYL001 and CyOA pure drug nanoparticles.

[0100] Example 3

[0101] Quantitative graph of ROS generation in vitro of Example 1 (COHF NPs) and Comparative Example 1 (COLF NPs).

[0102] The absorbance value of 1,3-diphenylisobenzofuran (DPBF) is negatively correlated with the amount of ROS. Therefore, we used this reagent to quantify the in vitro ROS generation ability of the COHF NPs and COLF NPs prepared in Example 1 and Comparative Example 1. Specifically, 1 mg of DPBF was dissolved in 1 mL of ethanol to obtain a stock solution. Then, 120 μL of the stock solution was added to the COHF NPs or COLF NPs solution to make the total volume 4 mL, and the concentration of the photosensitizer CyOA in the reaction system was 2.5 μM. Under the laser irradiation at 660 nm (200 mW / cm 2 ), at each designed time point, 100 μL of the above mixed solution was taken out and mixed with 100 μL of ethanol, and then analyzed by the instrument to record its absorbance at 406 nm.

[0103] Figure 5 As the DPBF absorbance value-time change curve, it can be seen that there is no significant difference in the ability of COHF NPs and COLF NPs to generate ROS in vitro.

[0104] Example 5

[0105] Intracellular co-localization maps of Example 1 (COHF NPs) and Comparative Example 1 (COLF NPs).

[0106] Due to the negative potential of the inner mitochondrial membrane, cationic compounds can be efficiently enriched in mitochondria against the concentration gradient. We used a confocal microscope to investigate the intracellular location of the photosensitizer CyOA in Example 1 (COHF NPs) and Comparative Example 1 (COLF NPs). Specifically, 200,000 4T1 cells were seeded in a 20-mm confocal dish and incubated overnight. After the cells adhered, they were incubated with COHF NPs or COLF NPs for 8 h, with the final concentration of CyOA being 1 μM. Then, the uninternalized nanodrugs were washed away with phosphate buffer (pH = 7.4). After that, the mitochondria were labeled with a mitochondrial probe, and then the intracellular location of the nanoparticles was observed by the instrument.

[0107] Figure 6 are the co-localization pictures of COHF NPs and COLF NPs with mitochondria and lysosomes in cells. It can be obtained from the figure that the photosensitizer CyOA of both types of nanoparticles is polydispersed in mitochondria.

[0108] Example 5

[0109] Effects of Example 1 (COHF NPs) and Comparative Example 1 (COLF NPs) on glutaminase (GLS) in 4T1 cells.

[0110] The level of GLS after treatment with each group was measured by Western blotting. CTR represents no treatment; COHFNPs represents dilution treatment with COHF NPs (CyOA: 1 μM) without light irradiation; COLF NPs+L represents dilution treatment with COLF NPs (CyOA: 1 μM) and laser irradiation at 200 mW / cm 2 660 nm for 4 min; COHF NPs+L represents dilution treatment with COHF NPs (CyOA: 1 μM) and laser irradiation at 200 mW / cm 2 660 nm for 4 min. The specific process of Western blotting includes: preparation of 10% SDS-PAGE separation, extraction of total protein after treatment with each group, loading, electrophoresis (voltage 80 V), membrane transfer, incubation with primary antibody and secondary antibody, and development.

[0111] As Figure 7 shown, whether with or without light irradiation, treatment with COHF NPs can reduce the level of GLS; while treatment with COLF NPs (CyOA: 1 μM) has little effect on the level of GLS. It shows that HYL001 can reduce the expression of GLS in tumor cells, but the nano-drug prepared by replacing HYL001 with its precursor molecule lonidamine cannot reduce the level of GLS.

[0112] Example 6

[0113] Effect of Example 1 (COHF NPs) and Comparative Example 1 (COLF NPs) on intracellular glutathione in mitochondria of 4T1 cells.

[0114] 4T1 tumor cells (about 1×10 6 cells) after treatment with each group for 4 h were collected. CTR represents no treatment; COHFNPs represents treatment with COHF NPs (CyOA: 1 μM) without light irradiation; COLF NPs+L represents treatment with COLF NPs (CyOA: 1 μM) and laser irradiation at 200 mW / cm 2 660 nm for 4 min; COHF NPs+L represents treatment with COHF NPs (CyOA: 1 μM) and laser irradiation at 200 mW / cm 2 660 nm for 4 min. Mitochondria were obtained by differential centrifugation, and the content of mitochondrial glutathione was measured by 5,5'-dithiobis(2-nitrobenzoic acid).

[0115] As Figure 8As shown, in the absence of light, treatment with COHF NPs can reduce the glutathione level, which may be due to the decrease in GLS, the inhibition of glutamine metabolism, and thus the obstruction of glutathione synthesis; treatment with COLF NPs+L can also reduce the intracellular glutathione level, which may be due to the oxidation of some reducing substances by the intracellular burst of ROS; treatment with COHF NPs+L can reduce the mitochondrial GSH content to the greatest extent, by about 70%, which may be due to the dual effects of the inhibition of glutamine metabolism and the burst of ROS.

[0116] Example 7

[0117] Detection of the ability of Example 1 (COHF NPs) and Comparative Example 1 (COLF NPs) to generate intracellular ROS in 4T1 cells.

[0118] 2',7'-Dichlorodihydrofluorescein diacetate (DCFDA), which has cell membrane permeability and no fluorescence itself. Once it enters the cell, it is hydrolyzed by cell esterase to generate 2',7'-dichlorodihydrofluorescein and further oxidized to generate strongly fluorescent 2',7'-dichlorofluorescein (DHF), which is a common ROS indicator. Experimental procedure: First, 500,000 4T1 cells were seeded in a 6-well plate. After the cells adhered, they were incubated with COHF NPs (CyOA: 1 μM) or COLF NPs (CyOA: 1 μM) for 4 hours respectively. Then, 10 μM DCFDA was added and incubated for 30 min. The unentered drugs were removed by washing with phosphate buffer. Then, each well in the light group was irradiated with a 200 mW / cm 2 laser at 660 nm for 4 min. Flow cytometry was used to quantify the fluorescence intensity of each group.

[0119] Figure 9 Content a is the flow cytometry map of ROS detection for each group, and content b is the quantification of the fluorescence intensity. It can be obtained from the figure that: contrary to the trend of intracellular glutathione level, the intracellular ROS level can be increased after treatment in each group, and the increase amplitude in the COHF NPs+L group is the largest, which is 25 times that of the CTR. And under the same concentration of CyOA, the ability of the COHF NPs+L group to generate ROS is stronger than that of the COLF NPs+L group, indicating that the inhibition of glutamine metabolism contributes to the accumulation of ROS generated by PDT.

[0120] Example 8

[0121] Detection of the cytotoxicity of Example 1 (COHF NPs) and Comparative Example 1 (COLF NPs) to 4T1 cells.

[0122] 10,000 4T1 cells were inoculated in a 96-well plate and incubated at 37 °C and 5% CO 2Cultured in a constant temperature and normal oxygen incubator. After the cells adhered to the wall, the culture medium was aspirated, and 100 μL of RPMI 1640 culture medium containing different concentrations of COHF NPs and COLF NPs was added respectively, and incubated for 6 h. Each well was irradiated with a laser of 200 mW / cm 2 at 660 nm for 4 min. After continuous culture for 24 h, the MTT method was used to calculate the cell survival rate.

[0123] Figure 10 Histogram of the survival rate of -4T1 cells at different doses. It can be obtained from the figure that the half-maximal inhibitory concentration (IC 50 ) of the COHF NPs+L group is the smallest, about 0.9 μM. Under the same concentration of CyOA, the IC 50 of the COHF NPs+L group is less than that of the COLF NPs+L group, indicating that there is a synergistic effect between HYL001 and CyOA. Therefore, inhibiting glutamine metabolism can enhance the sensitivity of PDT.

[0124] Example 9

[0125] Detection of the toxicity of Example 1 (COHF NPs) and Comparative Example 1 (COLF NPs) to breast cancer stem cells.

[0126] The formation of tumor spheres in a low-attachment culture plate is the gold standard for the culture of tumor stem cells. First, mouse breast cancer single cells 4T1 were cultured in an ultra-low attachment culture dish using serum-free DMEM / F12 medium, supplemented with B-27 supplement, 2.05 mM glutamine, 20 ng / mL epidermal growth factor, 10 ng / mL basic fibroblast growth factor, 4 μg / mL insulin, and 0.4% low endotoxin bovine serum albumin. After 5 days, the spheres were harvested, dissociated into single cells with trypsin, and then re-cultured in a 96-well ultra-low attachment plate with the above medium. 100 μL of RPMI 1640 culture medium containing different concentrations of COHF NPs and COLF NPs was added respectively, and incubated for 6 h. The light irradiation group was irradiated with a laser of 200 mW / cm 2 at 660 nm for 4 min. After continuous culture for 24 h, the CCK8 method was used to calculate the cell survival rate.

[0127] Figure 11 Histogram of the survival rate of breast cancer stem cells at different doses. It can be obtained from the figure that the half-maximal inhibitory concentration (IC 50 ) of the COHF NPs+L group is the smallest, about 0.4 μM. Under the same concentration of CyOA, the IC 50 of the COHF NPs+L group is less than that of the COLF NPs+L group (0.7 μM), indicating that there is a synergistic effect between HYL001 and CyOA. Therefore, inhibiting glutamine metabolism can enhance the killing effect of PDT on tumor stem cells.

[0128] Example 10

[0129] In vivo tissue distribution of Example 1 (COHF NPs) and Comparative Example 1 (COLF NPs).

[0130] To highlight the tumor-targeting effect of FA-HES, we introduced CO NPs self-assembled only with the photosensitizer CyOA as a control to compare the differences in tumor enrichment concentration between it and COLF NPs and COHF NPs. Specifically, at 6 weeks of age, female BALB / c mice weighing 15 - 17 g were subcutaneously inoculated with 1×10 6 cells of mouse breast cancer 4T1 cell suspension to establish a subcutaneous tumor mouse model of mouse breast cancer 4T1. When the tumor reached approximately 200 mm 3 , the tumor-bearing mice were randomly divided into 3 groups with 3 mice in each group. CONPs, COLF NPs, and COHF NPs were administered via the tail vein, and the dosage was calculated as 3 mg / kg based on CyOA. The mice were anesthetized before administration and at 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h after administration, and fluorescence imaging was performed on the mice using a small animal in vivo imager. To further study the distribution behavior of the nano-drug in various organs in vivo, the mice were sacrificed at 48 h after administration, and the heart, liver, spleen, lung, kidney, and tumor were removed, and fluorescence imaging was performed using a small animal in vivo imager.

[0131] The results are as Figure 12 shown in Content a, Content b, Content c, and Content d. Compared with CO NPs, COLF NPs and COHF NPs have a longer half-life. The ex vivo tissue imaging pictures and semi-quantitative data after 48 h also show that after FA-HES modification, the drug enrichment amounts at the tumor sites in the COLF NPs and COHF NPs groups are significantly increased, which are 1.6 and 1.8 times that of the CO NPs group, respectively. Therefore, FA-HES improves the tumor-targeting property of the nano-drug.

[0132] Example 11

[0133] Antitumor efficacy investigation of Example 1 (COHF NPs) and Comparative Example 1 (COLF NPs) in orthotopic tumors of mouse breast cancer stem cells.

[0134] The culture method of breast cancer stem cells refers to Example 8. Female BALB / c mice at 6 weeks of age and weighing 16 - 18 g were inoculated with mouse breast cancer stem cells in the left breast pad of the fourth pair of mammary glands on the abdomen, approximately 5×10 4 cells. When the volume of the orthotopic tumor was approximately 80 mm 3At that time, the mice were randomly divided into 3 groups with 9 mice in each group, namely the saline group (Saline), the COLF NPs under light (COLF NPs+L), and the COHF NPs group (COHF NPs+L). The administration dose was calculated as 6 mg / kg based on CyOA. Record the administration time on the first day as day 1, and then administer the drug at the above dose on days 3, 5, 7, and 9 respectively. After 12 h of drug administration, the light group was irradiated. Irradiation parameters: 200 mW / cm 2 , 660 nm, 10 min. Since day 1, the body weight and in-situ tumor volume of the mice were measured once a day, and the tumor volume-time curve was plotted. On day 13, 6 mice were sacrificed, the subcutaneous tumors were dissected and photographed. For the remaining 3 mice in each group, after being sacrificed on day 22, the lung tissues were dissected to observe the lung metastasis situation.

[0135] Figure 13 Content a is the tumor volume-time curve of the mice, content b is the photo after fixation in Bouin's fixative of the dissected lung tissue, and content c is the hematoxylin-eosin staining of the lung section. It can be seen that COHF NPs+L can significantly inhibit the growth of tumor stem cells, and the tumor inhibition rate reaches 78%, while the tumor inhibition rate of COLF NPs+L is 51%. The results show that HYL001 can enhance the photodynamic efficacy of CyOA. The number of lung nodules and the section observation also show that the COHF NPs+L group has better anti-tumor metastasis ability. Therefore, these results further prove the synergistic effect between HYL001 and the photodynamic effect of CyOA, and inhibiting glutamine metabolism can enhance the killing of PDT on tumor stem cells and inhibit their metastasis.

[0136] It is easy for those skilled in the art to understand that the above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A dual-targeted nano-drug delivery system for combined anti-glutamine metabolism and sensitized photodynamic therapy, characterized in that, it comprises an anti-glutamine metabolism drug, a photosensitizer and a targeting unit-hydroxyalkyl starch macromolecular conjugate, wherein the anti-glutamine metabolism drug has a structure shown in formula (I): In formula (I), R is a phenyl group, or a phenyl group substituted by one or more substituents selected from halogen, C1-C4 alkyl, nitro and trifluoromethyl; The photosensitizer is an unsaturated fatty acid-semi-cyanine conjugate, which has a structure shown in formula (II): Among them, R 1 is an alkyl group with 1 to 18 carbon atoms or an alkyl sulfonic acid with 1 to 18 carbon atoms; R 2 is an unsaturated fatty chain, and the unsaturated fatty chain has 6 to 30 carbon atoms; The targeting unit-hydroxyalkyl starch macromolecular conjugate is a macromolecular compound obtained by conjugating a tumor-targeting molecule with hydroxyalkyl starch; the tumor-targeting molecule is folic acid; the mass ratio of the anti-glutamine metabolism drug to the photosensitizer in this nano-drug delivery system is (10-40):(10-30).

2. The nano-drug delivery system according to claim 1, characterized in that, The unsaturated fatty chain is derived from unsaturated fatty acids, and the chemical formula of the unsaturated fatty acid is C n H 2n-1 COOH, C n H 2n-3 COOH, C n H 2n-5 COOH, C n H 2n-7 COOH or C n H 2n-9 COOH, where n is an integer from 8 to 29.

3. The nano-drug delivery system according to claim 2, characterized in that, The unsaturated fatty acid-semi-cyanine conjugate is obtained by an esterification reaction of the unsaturated fatty acid with a semi-cyanine containing a phenolic hydroxyl group.

4. The nano-drug delivery system according to claim 1, characterized in that, The grafting rate of the targeting unit in the targeting unit-hydroxyalkyl starch macromolecular conjugate is less than or equal to 10%.

5. The nano-drug delivery system according to claim 1, characterized in that, The mass ratio of the anti-glutamine metabolism drug, the photosensitizer to the targeting unit-hydroxyalkyl starch macromolecular conjugate in this nano-drug delivery system is (10-40):(10-30):(30-80).

6. A preparation method of the nano-drug delivery system according to any one of claims 1 to 5, characterized in that, it comprises the following steps: (1) Dissolve the anti-glutamine metabolism drug and the photosensitizer in a benign solvent to obtain a mixed solution; (2) Drop the mixed solution into a poor solvent, and after dialysis, obtain a self-assembled nanoparticle dispersion of the anti-glutamine metabolism drug and the photosensitizer; (3) Mix and stir the self-assembled nanoparticle dispersion obtained in step (2) with the targeting unit-hydroxyalkyl starch macromolecular conjugate in water, so that the targeting unit-hydroxyalkyl starch macromolecular conjugate is modified on the surface of the self-assembled nanoparticles to obtain the dual-targeted nano-drug delivery system for combined anti-glutamine metabolism and sensitized PDT.

7. The preparation method according to claim 6, characterized in that, The benign solvent in step (1) is one or more of dimethyl sulfoxide, tetrahydrofuran, methanol, ethanol, dichloromethane, acetonitrile, and the mass ratio of the anti-glutamine metabolism drug to the photosensitizer is 0.5-2:1; The poor solvent in step (2) is deionized water; The mass ratio of the targeting unit-hydroxyalkyl starch conjugate to the total mass of the anti-glutamine metabolism drug and the photosensitizer in step (3) is 0.5-2:1; the hydroxyalkyl starch is hydroxymethyl starch, hydroxyethyl starch, hydroxypropyl starch or hydroxybutyl starch, and the selected hydroxyalkyl starch has an average molecular weight of 40-200 kDa and a molar substitution degree of hydroxyalkyl of 0.4-0.

5.

8. Use of the nano-drug delivery system according to any one of claims 1 to 5 in the preparation of a drug for treating and / or preventing tumors.

9. Use of the nano-drug delivery system according to any one of claims 1 to 5 in the preparation of a drug for killing cancer stem cells.

Images