Light cage molecule for double-response drug release, preparation method and application

Abstract

The invention discloses a light cage molecule for double-response drug release and a preparation method and application thereof.According to the light cage molecule, a light cage constructed based on a Sanger reagent is combined with tumor oxidation reduction activation for the first time, a double-drug release system is constructed, the light reaction is rapid, methylene blue can be completely released through photolysis within about 60 s, and the light cage molecule has the advantages of being simple in preparation method, low in cost and the like. And the 10-hydroxycamptothecine is slowly released in the presence of GSH, especially in the presence of high-concentration GSH, so that the 10-hydroxycamptothecine has high selectivity. Light cage molecule for double-response drug release, provided by the invention, has a structure as shown in a formula Sanger-MB-HCPT: # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a dual-responsive molecule, a preparation method and an application thereof, and more particularly to a photocaged molecule for dual-responsive drug release, a preparation method and an application thereof. Background Art

[0002] As a class of "molecular switch" systems with photoreactive properties, photocaged compounds have shown important research value in the fields of chemical biology, precision medicine, and functional materials in recent years. Such compounds construct an intelligent system capable of precisely controlling molecular release by molecular-level coupling of a photosensitive group with an active substance (such as a drug, a fluorescent molecule, a biological macromolecule, etc.) through a chemical bond. Its core function is to utilize a light source with a specific wavelength to trigger a controllable photoreaction of the photosensitive group, thereby realizing the precise and controllable release of active molecules in both the time and space dimensions. The light-controlled release system also faces many challenges: the limited tissue penetration depth of light, the low quantum yield of some systems, the need for the photosensitive molecule to be stable in the physiological environment and the degradation products to be non-toxic, and the high synthesis and purification costs of complex photocaged compounds.

[0003] The concentration of glutathione (GSH) in cancer cells can reach 4-10 times that of normal cells. The disulfide bond (S-S) dynamic covalent bond system designed based on this characteristic exhibits a unique "molecular switch" function: it remains stable in the normal physiological environment, but can be reduced and cleaved into mercapto groups (-SH) in the high-GSH tumor microenvironment, thereby triggering the release of targeted drugs. This "intelligent" reaction mechanism realizes the selective activation of chemotherapeutic drugs. However, the fluctuations in the GSH concentration in the microenvironment caused by tumor heterogeneity may still induce multi-drug resistance (MDR). Therefore, it is necessary to develop a photocaged molecule for dual-responsive drug release, a preparation method and an application thereof. Summary of the Invention

[0004] The technical problem to be solved by the present invention is: overcoming the deficiencies existing in the prior art, providing a photocaged molecule for dual-responsive drug release, which for the first time combines a photocage constructed based on Sanger reagent with tumor redox activation to construct a dual-drug release system, has a rapid light response, can photolyze and completely release methylene blue in about 60 s, and slowly releases 10-hydroxycamptothecin in the presence of GSH, especially under high-concentration GSH conditions, with high selectivity.

[0005] The present invention also provides a preparation method of the photocaged molecule for dual-responsive drug release and the application of the photocaged molecule in rapidly releasing drug 1 (methylene blue) under light irradiation conditions and slowly releasing drug 2 (10-hydroxycamptothecin) under high-concentration GSH conditions.

[0006] The technical concept and principle of the present invention are as follows: The photocaged molecule for dual-responsive drug release can rapidly photolyze under ultraviolet light to produce 690 nm fluorescence, and the disulfide bond can be cleaved under the high concentration of glutathione in the tumor microenvironment to produce 560 nm fluorescence. The inventors of the present invention first combined the photocage constructed based on Sanger reagent with tumor redox activation to construct a dual-drug release system, thereby solving the problems existing in the prior art.

[0007] The technical solution of the present invention for solving its technical problems is as follows:

[0008] The photocaged molecule for dual-responsive drug release of the present invention has a structure shown in the formula Sanger-MB-HCPT:

[0009]

[0010] The preparation method of the above-mentioned photocaged molecule for dual-responsive drug release of the present invention includes the following steps:

[0011] Dissolve 2-(2-((((4-nitrophenoxy)carbonyl)oxy)ethyl)disulfanyl)ethyl-4-(((2-(3,7-bis(dimethylamino)-10H-phenothiazin-10-yl)-2-oxoethyl)(2,4-dinitrophenyl)amino)methyl)benzoate and 10-hydroxycamptothecin in a solvent, add a triethylamine solution, and stir and react in the dark at 70-90 °C under a nitrogen atmosphere for more than 12 hours. After the reaction, purify and vacuum dry to obtain the photocaged molecule for dual-responsive drug release, abbreviated as Sanger-MB-HCPT.

[0012] A further technical solution of the preparation method of the above-mentioned photocaged molecule for dual-responsive drug release of the present invention is that the reaction is carried out at a temperature of 75-85 °C, the reaction needs to be carried out in the dark, and the solvent is an acetonitrile solution; the mass ratio of 2-(2-((((4-nitrophenoxy)carbonyl)oxy)ethyl)disulfanyl)ethyl-4-(((2-(3,7-bis(dimethylamino)-10H-phenothiazin-10-yl)-2-oxoethyl)(2,4-dinitrophenyl)amino)methyl)benzoate: 10-hydroxycamptothecin: triethylamine is 4.66:2.34:1. The reaction temperature in the present invention is preferably 75-85 °C.

[0013] A further technical solution of the preparation method of the above-mentioned photocaged molecule for dual-responsive drug release of the present invention can also be that the purification step after the reaction is: subject the crude product to vacuum filtration, and further purify the crude product on a silica gel column with dichloromethane / methanol to obtain Sanger-MB-HCPT.

[0014] Application of the above-mentioned photocage molecule for dual-responsive drug release in the rapid release of methylene blue under light illumination and the slow release of 10-hydroxycamptothecin in the presence of GSH.

[0015] For the above application of the present invention, a further technical solution is that in the presence of GSH, the slow release of 10-hydroxycamptothecin is such that the higher the GSH concentration, the faster the release of 10-hydroxycamptothecin. A further technical solution is that the GSH concentration is not less than 500 μM.

[0016] The preparation reaction equation of the photocage molecule for dual-responsive drug release of the present invention is as follows:

[0017]

[0018] The present invention has the following beneficial effects:

[0019] 1) The photocage molecule for dual-responsive drug release of the present invention has almost no fluorescence in a buffer solution with pH = 7.4, releases near-infrared fluorescence at 690 nm after ultraviolet light illumination, and simultaneously releases fluorescence at 560 nm in a high-concentration glutathione solution.

[0020] 2) The photocage molecule for dual-responsive drug release of the present invention combines, for the first time, a photocage constructed based on Sanger's reagent with redox activation to construct a dual-drug release system.

[0021] 3) The photocage molecule for dual-responsive drug release of the present invention reacts rapidly under ultraviolet light illumination, and strongly reacts only with glutathione containing multiple sulfhydryl groups, partially reacts with cysteine, and has no reaction with other common ions, amino acids, and enzymes. It has good selectivity and specificity, and has appropriate fluorescence emission wavelengths (560 nm and 690 nm).

[0022] 4) The preparation method of the photocage molecule for dual-responsive drug release of the present invention is simple and easy to implement, and is easy to scale up production. Description of the Drawings

[0023] Figure 1 1H-NMR spectrum of the photocage molecule for dual-responsive drug release prepared in Example 1 of the present invention. 1 1H-NMR spectrum.

[0024] Figure 2 Fluorescence spectrum at 690 nm of the photocage molecule for dual-responsive drug release of the present invention after ultraviolet light illumination for different times.

[0025] Figure 3 Fluorescence spectrum of the photocage molecule for dual-responsive drug release of the present invention reacting with various analytes.

[0026] Figure 4Bar chart of fluorescence intensity of the photocaged molecule for dual-responsive drug release of the present invention reacting with various analytes.

[0027] Figure 5 Increment graph of fluorescence of the photocaged molecule for dual-responsive drug release of the present invention reacting with glutathione solutions of different concentrations. Detailed implementation manners

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and in conjunction with embodiments, but the present invention is not limited to the given examples.

[0029] Example 1 Preparation of the photocaged molecule for dual-responsive drug release

[0030] Dissolve 100 mg of Sanger-MB-Cl and 50.18 mg of 10-hydroxycamptothecin in 10 ml of acetonitrile solution, add 21.44 mg of triethylamine solution, and stir in the dark at 80 °C for 12 hours under a nitrogen atmosphere; after the reaction is completed, first concentrate the reaction solution under reduced pressure, then purify the crude product by silica gel column chromatography (dichloromethane / methanol), and finally dry it under vacuum to obtain 48 mg of yellow solid, which is the pure product of the photocaged molecule for dual-responsive drug release, Sanger-MB-HCPT( 1 The 1H-NMR spectrum is shown in Figure 1 ).

[0031] Example 2 Response of the fluorescence intensity of the photocaged molecule for dual-responsive drug release to changes over time under ultraviolet light irradiation

[0032] Accurately weigh 5.84 mg of solid Sanger-MB-HCPT and dissolve it in 5 mL of DMSO solution to prepare a 1 mM stock solution.

[0033] Ultraviolet light irradiation test: Add 30 μL of the above mother liquor to 10 mM PBS buffer solution (pH 7.4) to make the final concentration of the photocaged molecule 10 μM, and then detect the fluorescence intensity of the photocaged molecule at 690 nm under ultraviolet light irradiation for different times. The emission slit width is 10 nm, and the emission wavelength is set at 660 nm. The obtained results are as Figure 2 shown.

[0034] The experimental results show that the photocaged molecule prepared in Example 1 itself has no fluorescence emission at 690 nm, and the fluorescence of methylene blue is quenched. However, as the ultraviolet light irradiation time prolongs, the near-infrared fluorescence intensity of the photocaged molecule Sanger-MB-HCPT at 690 nm gradually increases and reaches the maximum value at about 50 - 60 seconds, proving that as the ultraviolet light irradiation time prolongs, methylene blue is gradually released and reaches the peak at 50 - 60 seconds.

[0035] Spectral properties of the photocaged molecule that releases drugs with dual responses reacting with various analytes in Example 3

[0036] Add 30 μL of the mother liquor in Example 2 into 10 mM PBS buffer solution (pH 7.4), and then add various analytes respectively: (1) blank, (2) Na + , (3) K + , (4) phenylalanine (Phe), (5) glutamic acid (Glu), (6) tyrosine (Tyr), (7) valine (Val), (8) urease, (9) glucose oxidase (GOD), (10) lysozyme, (11) esterase, (12) pepsin, (13) trypsin, (14) lysine, (15) cysteine, (16) glutathione (GSH). Make the final concentration of the analyte 5 mM and the final concentration of the probe 10 μM. After incubation for 3 hours, test its fluorescence spectrum. The emission slit width is 10 nm. The obtained results are as shown in Figure 3 and Figure 4 .

[0037] The above results show that:

[0038] (1) The photocaged molecule prepared in Example 1 itself has a little fluorescence at 560 nm. Under the incubation of glutathione, the disulfide bond in the probe is broken to release 10-hydroxycamptothecin, and the fluorescence intensity gradually increases.

[0039] (2) The photocaged molecule prepared in Example 1 has high selectivity and specificity for glutathione, and can distinguish from other interfering substances under the above conditions, and successfully releases 10-hydroxycamptothecin.

[0040] Spectral properties of the reaction product of the photocaged molecule that releases drugs with dual responses and glutathione in Example 4

[0041] Add 30 μL of the mother liquor of the photocaged molecule in Example 2 into 10 mM PBS buffer solution (pH 7.4), and then add different equivalents of glutathione. Make the final concentration of the photocaged molecule 10 μM, and the final concentrations of glutathione are 10 μM, 25 μM, 50 μM, 100 μM, 200 μM, 250 μM, 500 μM, 1 mM, 2.5 mM, 5 mM respectively. After adding the glutathione solution and incubating for 3 hours, measure its fluorescence spectrum. The emission slit width is 10 nm. The obtained increment diagram of fluorescence intensity is shown in Figure 5 . The experimental results show that the fluorescence intensity at 560 nm after the reaction increases with the increase of glutathione concentration.

Claims

1. A photocaged molecule for dual-responsive drug release, characterized in that, The described photocage molecule has a structure shown by the formula Sanger-MB-HCPT:

2. A method for preparing a photocaged molecule for dual-responsive drug release as described in claim 1, characterized in that, Including the following steps: Dissolve 2-(2-((((4-nitrophenoxy)carbonyl)oxy)ethyl)disulfanyl)ethyl-4-(((2-(3,7-bis(dimethylamino)-10H-phenothiazin-10-yl)-2-oxoethyl)(2,4-dinitrophenyl)amino)methyl)benzoate and 10-hydroxycamptothecin in a solvent, add a triethylamine solution, stir and react in the dark at 70 - 90 °C under a nitrogen atmosphere for more than 12 hours. After the reaction, purify and vacuum dry to obtain the photocage molecule that releases drugs with dual responses, abbreviated as Sanger-MB-HCPT.

3. The preparation method of the photocage molecule for dual-responsive drug release according to claim 2, wherein, The reaction is carried out at a temperature of 75 - 85 °C, the reaction needs to be carried out in the dark, and the solvent is an acetonitrile solution; the mass ratio of 2-(2-((((4-nitrophenoxy)carbonyl)oxy)ethyl)disulfanyl)ethyl-4-(((2-(3,7-bis(dimethylamino)-10H-phenothiazin-10-yl)-2-oxoethyl)(2,4-dinitrophenyl)amino)methyl)benzoate : 10-hydroxycamptothecin : triethylamine is 4.66 : 2.34 :

1.

4. The preparation method of the photocage molecule for dual-responsive drug release according to claim 2, wherein, The purification step after the reaction is as follows: Filter the crude product under reduced pressure, and further purify the crude product on a silica gel column with dichloromethane / methanol to obtain Sanger-MB-HCPT.

5. Use of a photocage molecule that releases drugs with dual responses as described in claim 1 for rapidly releasing methylene blue under light irradiation conditions and slowly releasing 10-hydroxycamptothecin in the presence of GSH.

6. The application according to claim 5, wherein For the slow release of 10-hydroxycamptothecin in the presence of GSH, the higher the GSH concentration, the faster the release of 10-hydroxycamptothecin.

7. The application according to claim 6, wherein The GSH concentration is not less than 500 μM.

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