Micromolecule prodrug with glutathione responsiveness and self-assembled nanoparticles thereof

Abstract

The invention discloses a small molecule prodrug with glutathione responsiveness and a self-assembled nanoparticle thereof, and the structure of the small molecule prodrug with glutathione responsiveness is as shown in formula I; the preparation method of the self-assembled nanoparticles comprises the following steps: dissolving the glutathione-responsive micromolecule prodrug in a mixed solution of dimethyl sulfoxide and ethanol, slowly dropwise adding the mixture into water in an ice bath and in a stirring state, and continuously stirring for 1 hour to prepare the self-assembled nanoparticles with the glutathione-responsive micromolecule prodrug. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of small molecule nano prodrugs, and particularly relates to a glutathione-responsive small molecule prodrug and its self-assembled nanoparticles. Background Art

[0002] The tumor microenvironment (TME) refers to the internal and external environment in which tumors occur, grow, and metastasize, including the structure, function, and metabolism of the tissue where the tumor is located, as well as a complex system formed by the interaction with the internal environment (nucleus and cytoplasm) of the tumor cells themselves.

[0003] Glutathione (GSH) is a sulfur-containing tripeptide composed of L-glutamic acid, cysteine, and glycine, and its concentration in tumor cells is higher than that in plasma and the extracellular matrix. This is mainly because tumor cells need GSH to relieve the effects of endogenous oxidative stress. In addition, some cellular and acellular components in the TME (such as tumor-associated fibroblasts) may secrete some factors that induce GSH synthesis, which will also lead to an increase in the GSH content in the TME. GSH can promote the proliferation and metastasis of tumor cells by regulating the cell signal transduction pathway and inhibiting apoptosis. Therefore, the characteristic of high GSH concentration in the TME is closely related to tumor growth, metabolism, metastasis, and response to treatment, and regulating it is expected to provide new ideas and methods for tumor treatment.

[0004] Nano prodrugs are a new type of drug delivery system formed by directly binding drugs to nano carriers through covalent bonds. These nanoparticles combine the activity of the drug and the targeting of the nano carrier, and can stably exist in the body and accurately reach the lesion site. The release mechanism of nano prodrugs usually depends on specific conditions in the tumor microenvironment, such as changes in pH value. Currently, some studies have pointed out that drugs can be released by responding to glutathione for tumor treatment, but in existing studies, polymers or high-molecular substances are introduced, which will lead to poor dispersibility of the nanoparticles and increased synthesis difficulty. Summary of the Invention

[0005] In order to solve the problems proposed in the above background art, the purpose of the present invention is to provide a glutathione-responsive small molecule prodrug and its self-assembled nanoparticles. The glutathione-responsive small molecule prodrug of the present invention involves small molecule synthesis, without adding any polymers or high-molecular substances, and can directly self-assemble into nanoparticles, thereby releasing the anti-tumor drug 10-hydroxycamptothecin in a responsive manner, providing new ideas and methods for the treatment of other types of tumors.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows: On the one hand, the present invention provides a glutathione-responsive small molecule prodrug, the structure of which is shown in Formula I,

[0007]

[0008] On the other hand, the present invention provides a preparation method of the above-mentioned glutathione-responsive small molecule prodrug, and the preparation method is as follows:

[0009] The above-mentioned glutathione-responsive small molecule prodrug is prepared by reacting 10-hydroxycamptothecin with 3,3'-dithiobis(propionic acid).

[0010] Furthermore, the preparation method of the above-mentioned glutathione-responsive small molecule prodrug includes the following steps:

[0011] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) is dissolved in a solvent to obtain a 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution;

[0012] 10-Hydroxycamptothecin and 3,3'-dithiobis(propionic acid) are mixed and dissolved in a solvent, then pyridine is added, and the mixture is cooled to 0-5°C; subsequently, under the condition of introducing nitrogen and stirring vigorously, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is slowly added dropwise, and stirred at 20-28°C for 20-24 h to prepare the glutathione-responsive small molecule prodrug.

[0013] Furthermore, the solvent is anhydrous dichloromethane.

[0014] Furthermore, the molar ratio of 10-hydroxycamptothecin, 3,3'-dithiobis(propionic acid), pyridine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.5:0.3:0.025:0.6.

[0015] On the other hand, the present invention provides a self-assembled nanoparticle of the above-mentioned glutathione-responsive small molecule prodrug, which is prepared by the following method:

[0016] Dissolve the glutathione-responsive small molecule prodrug in a mixed solution of dimethyl sulfoxide (DMSO) and ethanol. Slowly drip it into water under an ice bath with stirring, and continue stirring for 1 h to prepare self-assembled nanoparticles of the glutathione-responsive small molecule prodrug. Compared with using ethanol and DMSO alone, using a mixed solution of DMSO and ethanol can significantly reduce the particle size of the self-assembled nanoparticles. The ice bath condition is mainly used to control the reaction kinetics and maintain molecular stability through low temperature, reduce molecular thermal motion, make the self-assembly process slow and controllable, and make the self-assembled nanoparticles more stable and regular during the formation process. If the stirring time is too short, self-assembly cannot occur; if it is too long, it is unnecessary.

[0017] Further, the volume percentage of DMSO in the mixed solution of DMSO and ethanol is 40 - 80%.

[0018] On the other hand, the present invention provides an application of the above-mentioned glutathione-responsive small molecule prodrug or the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug described above in the preparation of anti-tumor drugs.

[0019] Further, the tumors include retinoblastoma, retinal pigment epithelioma, neuroblastoma, intraocular lymphoma, choroidal melanoma.

[0020] On the other hand, the present invention provides an anti-tumor drug composition, which contains the above-mentioned glutathione-responsive small molecule prodrug or the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug described above as an active substance.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] By introducing the sensitive group of disulfide bond and connecting it with 10-hydroxycamptothecin (HCPT) through chemical synthesis, the whole process only involves chemical reactions between small molecules, and GSH responsiveness is achieved through chemical bonds. The small molecule prodrug prepared by the present invention can self-assemble into nanoparticles in aqueous solution. It is a small molecule with a small particle size and good dispersibility, enabling it to release 10-hydroxycamptothecin in a responsive manner in the presence of high GSH, thereby achieving the anti-tumor effect. The responsive release of the drug improves the bioavailability of the drug, thereby reducing the dosage and cost of the drug, and providing new ideas and methods for the treatment of other types of tumors.

[0023] The glutathione-responsive small molecule prodrug of the present invention and its self-assembled nanoparticles have simple operations and preparation processes, do not require special equipment, and are easy to achieve batch production and application promotion. Description of the Drawings

[0024] Figure 1 1H NMR spectra of the glutathione-responsive small molecule prodrug HCPT-SS-HCPT, 10-hydroxycamptothecin, and 3,3'-dithiodipropionic acid prepared in Example 1 of the present invention;

[0025] Figure 2 13C NMR spectrum of the glutathione-responsive small molecule prodrug HCPT-SS-HCPT prepared in Example 1 of the present invention;

[0026] Figure 3 IR spectra of the glutathione-responsive small molecule prodrug HCPT-SS-HCPT, 10-hydroxycamptothecin, and 3,3'-dithiodipropionic acid prepared in Example 1 of the present invention;

[0027] Figure 4 UV absorption spectra of the glutathione-responsive small molecule prodrug HCPT-SS-HCPT, 3,3'-dithiodipropionic acid, 10-hydroxycamptothecin (HCPT), and methanol prepared in Example 1 of the present invention;

[0028] Figure 5 High-resolution mass spectrum of the glutathione-responsive small molecule prodrug HCPT-SS-HCPT prepared in Example 1 of the present invention;

[0029] Figure 6 SEM image of the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug prepared in Example 2 of the present invention;

[0030] Figure 7 Particle size diagrams of the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug prepared in Examples 2-3 and Comparative Examples 1-2 of the present invention;

[0031] Figure 8 Particle size diagrams of the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug prepared in Example 2 of the present invention and the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug after adding GSH;

[0032] Figure 9 Zeta potential diagrams of the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug prepared in Example 2 of the present invention and the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug after adding GSH;

[0033] Figure 10 Release curve of 10-hydroxycamptothecin in the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug prepared in Example 2 of the present invention under GSH conditions. Detailed implementation manners

[0034] The following further illustrates the glutathione-responsive small molecule prodrug and its self-assembled nanoparticles provided by the present invention through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as a limitation on the protection scope of the present invention. Those skilled in the art can still fall within the protection scope of the invention when making some non-essential improvements and adjustments to the present invention based on the above-mentioned invention content and implementing them specifically.

[0035] Example 1

[0036] Preparation of glutathione-responsive small molecule prodrug (HCPT-SS-HCPT) :

[0037] Dissolve 115.1 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) in 2 mL of anhydrous dichloromethane to obtain a 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution.

[0038] Mix 182.2 mg of 10-hydroxycamptothecin and 63.1 mg of 3,3'-dithiodipropionic acid and dissolve them in 10 mL of anhydrous dichloromethane, then add 2 mL of pyridine and cool it to 0 °C. Subsequently, under the condition of introducing argon and stirring vigorously, slowly dropwise add the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and stir at 25 °C for 24 h to obtain a crude product.

[0039] Put the crude product into a separatory funnel and extract it three times with saturated sodium bicarbonate aqueous solution, 1N hydrochloric acid solution and saturated sodium chloride solution, and wash it. Then collect the dichloromethane layer, rotary evaporate and dry it, and use a dichloromethane / methanol system for column chromatography separation at a ratio of 15:1 to obtain the purified glutathione-responsive small molecule prodrug.

[0040]

[0041] The 1H NMR spectra of the glutathione-responsive small molecule prodrug HCPT-SS-HCPT, 10-hydroxycamptothecin, and 3,3'-dithiodipropionic acid prepared in Example 1 are as Figure 1 shown. Since the glutathione-responsive small molecule prodrug prepared in Example 1 is an axisymmetric compound from the structural formula, only one side with the S-S as the axis of symmetry needs to be looked at. From Figure 1 it can be seen that at the peak positions of 3.0 - 3.3 ppm at points a and b in the figure, there are two triplets, which are the methylene peaks in the disulfide bond (the two peaks coincide in the figure), indicating the successful connection of the disulfide bond with 10-hydroxycamptothecin. The remaining peak positions can all correspond one by one to the 10-hydroxycamptothecin raw material, proving the successful synthesis of the product, and the NMR spectrum is clear without obvious impurities, proving that the product is relatively pure.

[0042] The carbon-13 NMR spectrum of the glutathione-responsive small molecule prodrug HCPT-SS-HCPT prepared in Example 1 is as follows Figure 2 shown. Only the positions of C atoms can be shown in the carbon-13 NMR spectrum. From Figure 2 it can be seen that the attribution of each C can be found. By comparing with the carbon spectrum positions of the 10-hydroxycamptothecin raw material in the literature, it can be found that at points a and b, that is, at 33.10 ppm and 34.23 ppm, there are two additional C atoms, that is, the C in the methylene group. The remaining peak positions are the same as those of the raw material 10-hydroxycamptothecin, which can prove the successful synthesis of the product and there are no other impurities.

[0043] The infrared spectra of the glutathione-responsive small molecule prodrug HCPT-SS-HCPT, 10-hydroxycamptothecin, and 3,3'-dithiodipropionic acid prepared in Example 1 were tested using a Fourier transform infrared spectrometer. The results are as follows Figure 3 shown. From Figure 3 it can be seen that there are obvious waveform changes at the positions pointed by the arrows in the figure. The two peaks appearing between the wave numbers 2850 - 2960 are the absorption peaks of the methylene group. The peak at 3224.4 disappears when compared with the peak at the same position of 10-hydroxycamptothecin, indicating the disappearance of the -OH on the benzene ring. The near-infrared spectrum also shows the successful synthesis of the product.

[0044] A small amount of 3,3'-dithiodipropionic acid, 10-hydroxycamptothecin (HCPT), and the glutathione-responsive small molecule prodrug HCPT-SS-HCPT prepared in Example 1 were respectively dissolved in an appropriate amount of methanol. The absorbance values were measured using a UV spectrophotometer, and methanol was used as a blank control to obtain the UV absorption spectra of 3,3'-dithiodipropionic acid, HCPT, HCPT-SS-HCPT, and methanol, as follows Figure 4 shown. From Figure 4 it can be seen that 3,3'-dithiodipropionic acid has a maximum absorption peak at a wavelength of 247 nm, HCPT has a maximum absorption peak at a wavelength of 384 nm, and HCPT-S-S-HCPT has maximum absorption peaks near 247 nm and 384 nm. This can prove that HCPT has been successfully connected to both ends of the disulfide bond.

[0045] The high-resolution mass spectrum of the glutathione-responsive small molecule prodrug HCPT-SS-HCPT prepared in Example 1 is as follows Figure 5 shown. First, the synthetic product was simulated in ChemDraw to obtain its standard molecular weight. After processing by high-resolution mass spectrometry and performing + hydrogen ion treatment on the product, the corresponding molecular weight, that is, 903.320020, can be found in Figure 5 , which is in line with the theoretical situation of the product, proving that the molecular weight of the product is consistent with the expectation and also proving the successful synthesis of the product.

[0046] Example 2

[0047] Preparation of Self-Assembled Nanoparticles with Glutathione-Responsive Small Molecule Prodrug:

[0048] Dissolve 4 mg of the glutathione-responsive small molecule prodrug prepared in Example 1 in a mixed solution of DMSO and ethanol (0.4 mL DMSO and 0.6 mL ethanol). Load the dissolved liquid into a syringe and slowly drip the liquid into 4 mL of ultrapure water under ice bath and stirring conditions. Continue stirring for 1 h until self-assembled into nanoparticles. After 1 h, dialyze in ultrapure water for 48 h to remove impurities such as DMSO, and the final self-assembled nanoparticle solution with glutathione-responsive small molecule prodrug is obtained.

[0049] Example 3

[0050] Preparation of Self-Assembled Nanoparticles with Glutathione-Responsive Small Molecule Prodrug:

[0051] Dissolve 4 mg of the glutathione-responsive small molecule prodrug prepared in Example 1 in a mixed solution of DMSO and ethanol (0.8 mL DMSO and 0.2 mL ethanol). Load the dissolved liquid into a syringe and slowly drip the liquid into 4 mL of ultrapure water under ice bath and stirring conditions. Continue stirring for 1 h until self-assembled into nanoparticles. After 1 h, dialyze in ultrapure water for 48 h to remove impurities such as DMSO, and the final self-assembled nanoparticle solution with glutathione-responsive small molecule prodrug is obtained.

[0052] Comparative Example 1

[0053] Preparation of Self-Assembled Nanoparticles with Glutathione-Responsive Small Molecule Prodrug:

[0054] Dissolve 4 mg of the glutathione-responsive small molecule prodrug prepared in Example 1 in a mixed solution of DMSO and ethanol (1 mL DMSO and 0 mL ethanol). Load the dissolved liquid into a syringe and slowly drip the liquid into 4 mL of ultrapure water under ice bath and stirring conditions. Continue stirring for 1 h until self-assembled into nanoparticles. After 1 h, dialyze in ultrapure water for 48 h to remove impurities such as DMSO, and the final self-assembled nanoparticle solution with glutathione-responsive small molecule prodrug is obtained.

[0055] Comparative Example 2

[0056] Preparation of Self-Assembled Nanoparticles with Glutathione-Responsive Small Molecule Prodrug:

[0057] Dissolve 4 mg of the glutathione-responsive small molecule prodrug prepared in Example 1 in a mixed solution of DMSO and ethanol (0 mL of DMSO and 1 mL of ethanol). Load the dissolved liquid into a syringe, and slowly drip the liquid into 4 mL of ultrapure water under an ice bath and stirring conditions. Continue stirring for 1 h until it self-assembles into nanoparticles. After 1 h, dialyze in ultrapure water for 48 h to remove impurities such as DMSO, and thus obtain the final self-assembled nanoparticle solution of the glutathione-responsive small molecule prodrug.

[0058] Perform electron microscopy scanning on the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug prepared in Example 2. The results are as Figure 6 shown. It can be seen from Figure 6 that the self-assembled nanoparticles present an oblate shape and are relatively regular.

[0059] Measure the particle sizes of the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug prepared in Examples 2-3 and Comparative Examples 1-2 by a dynamic light scattering particle size analyzer. The results are as Figure 7 shown. It can be seen from Figure 7 that the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug prepared with 0.4 mL of DMSO and 0.6 mL of ethanol, and 0.8 mL of DMSO and 0.2 mL of ethanol have smaller particle sizes.

[0060] Measure the particle sizes of the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug prepared in Example 2 and the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug after adding GSH by a dynamic light scattering particle size analyzer. The results are as Figure 8 shown. It can be seen from Figure 8 that the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug prepared in Example 2 have smaller particle sizes without glutathione stimulation. After adding glutathione, due to the specific response of the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug to glutathione, aggregation may occur during the reduction process of the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug. The molecules after reduction with GSH may re-aggregate into larger aggregates through hydrogen bonds, hydrophobic interactions or electrostatic interactions, and this aggregation phenomenon will lead to an increase in particle size.

[0061] Measure the zeta potentials of the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug prepared in Example 2 and the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug after adding GSH by a dynamic light scattering particle size analyzer. The results are as Figure 9 shown. It can be seen from Figure 9It can be seen that the potential value of the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug prepared in Example 2 is in the negative potential range. Since the molecule contains groups such as hydroxyl and carboxyl groups, these groups are prone to dissociation in aqueous solution to generate negative charges, and the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug may form a relatively stable system through electrostatic interactions. After the addition of GSH, the disulfide bond is broken, and after the self-assembled nanoparticles are disintegrated, the redistribution of molecules or fragments may reduce these interactions, resulting in a decrease in the absolute value of the potential.

[0062] The in vitro release within 48 h confirmed the responsive release of 10-hydroxycamptothecin by the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug prepared in Example 2 under the condition of GSH. Four completely identical solutions of the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug were placed in dialysis bags and divided into four groups: PBS, 0.1 mM GSH, 1 mM GSH, and 10 mM GSH. Each group was further set with three parallel samples, for a total of 12 groups. They were respectively put into 45 mL of medium (PBS, 0.1 mmol / L GSH, 1 mmol / L GSH, 10 mmol / L GSH) for in vitro release experiments. Samples were taken at 1 min, 10 min, 30 min, 2 h, 6 h, 12 h, 24 h, and 48 h to measure their OD values. According to the standard curve (first, the ultraviolet absorption peak position of 10-hydroxycamptothecin was measured at 384 nm with a UV-visible spectrophotometer, and then self-assembled nanoparticle solutions with different gradient concentrations of the glutathione-responsive small molecule prodrug were prepared. Then, the absorbance was measured with a UV-visible spectrophotometer respectively, and the absorbance values of these solutions at 384 nm were recorded. The standard curve was simulated with software to obtain a linear equation), the cumulative release amount was calculated. The results are as Figure 10 shown. As Figure 10 can be seen, the drug in the self-assembled nanoparticles was hardly released in PBS; with the increase of the GSH concentration, the release amount of 10-hydroxycamptothecin in the self-assembled nanoparticles increased significantly, and the release rate accelerated, demonstrating the responsiveness of the self-assembled nanoparticles in the presence of GSH. The concentration of GSH affects drug release and is positively correlated with the GSH concentration.

[0063] The above description is only the specific implementation manner of the present invention, not all of the implementation manners. Any equivalent transformation of the technical solution of the present invention by those of ordinary skill in the art through reading the specification of the present invention is covered by the claims of the present invention.

Claims

1. A glutathione-responsive small molecule prodrug, the structure of which is shown in Formula I, 2. The preparation method of the glutathione-responsive small molecule prodrug according to claim 1, characterized in that, The preparation method is as follows: The glutathione-responsive small molecule prodrug is prepared by reacting 10-hydroxycamptothecin with 3,3'-dithiobispropionic acid.

3. The preparation method of the glutathione-responsive small molecule prodrug according to claim 2, characterized in that, It includes the following steps: Dissolve 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in a solvent to obtain a 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution; Mix 10-hydroxycamptothecin and 3,3'-dithiobispropionic acid and dissolve them in a solvent, then add pyridine and cool it to 0-5°C; subsequently, under the condition of introducing nitrogen and stirring vigorously, slowly dropwise add the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, and stir at 20-28°C for 20-24 h to prepare the glutathione-responsive small molecule prodrug.

4. The preparation method of the glutathione-responsive small molecule prodrug according to claim 3, characterized in that, The molar ratio of 10-hydroxycamptothecin, 3,3'-dithiobispropionic acid, pyridine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.5:0.3:0.025:0.

6.

5. The self-assembled nanoparticles with glutathione-responsive small molecule prodrugs according to claim 1, characterized in that, It is prepared by the following method: Dissolve the glutathione-responsive small molecule prodrug in a mixed solution of dimethyl sulfoxide and ethanol, and slowly drop it into water under ice bath and stirring conditions, and continue to stir for 1 h to prepare self-assembled nanoparticles of the glutathione-responsive small molecule prodrug.

6. The self-assembled nanoparticles with glutathione-responsive small molecule prodrugs according to claim 5, characterized in that The volume percentage of dimethyl sulfoxide in the mixed solution of dimethyl sulfoxide and ethanol is 40-80%.

7. Use of the glutathione-responsive small molecule prodrug according to claim 1 or the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug according to any one of claims 5-6 in the preparation of anti-tumor drugs.

8. The application according to claim 7, characterized in that The tumors include retinoblastoma, retinal pigment epithelioma, neuroblastoma, intraocular lymphoma, and choroidal melanoma.

9. An anti-tumor drug composition, characterized in that, It contains the glutathione-responsive small molecule prodrug according to claim 1 or the self-assembled nanoparticles of the glutathione-responsive small molecule prodrug according to any one of claims 5-6 as the active substance.

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