Preparation and application of poly gossypol supramolecular prodrug nano-micelle

Abstract

The invention discloses preparation and application of a supramolecular nano-micelle based on gossypol (GP). A preparation method comprises the following steps: (1) preparing a polymer PGP; (2) preparation of a polymer PGPP; and (3) preparing the PGPP supramolecular nano-micelle. The prepared micelle has the advantages of stable particle size, uniform dispersion, drug controlled release behavior, high drug loading capacity, low toxic and side effects, good biological safety and the like. The gossypol drug belonging to polyphenol has a certain anti-cancer effect but has toxic and side effects, and research shows that the toxic and side effects of gossypol on normal cells are reduced by modifying the aldehyde group on gossypol, and meanwhile, the gossypol drug has a certain anti-cancer effect but has toxic and side effects. The supramolecular nano-micelle is triggered by glutathione (GSH) and a pH microenvironment in cancer cells to realize double-response controllable release, so that gossypol is effectively enriched at a tumor site, the cancer cells are killed, and a new strategy and a systematic theoretical support are provided for development of a supramolecular prodrug delivery system.

Description

Technical Field

[0001] This invention relates to polymeric chemical drugs and some biological fields, specifically to a method for preparing and applying polystyrene supramolecular prodrug nanomicelles. Background Technology

[0002] Cancer remains one of the leading diseases threatening human life and health. According to data from the International Agency for Research on Cancer (IARC), a branch of the World Health Organization (WHO), approximately 19.29 million new cancer cases were diagnosed in 2020, with a male-to-female ratio of about 1.09:1. With the rapid increase in cancer cases, researchers worldwide are striving to find new technologies and strategies to improve cancer survival rates. Whether it's conventional treatments like surgical removal of lesions, chemotherapy, and radiotherapy, or high-impact therapies like immunotherapy and targeted therapy, these are all the result of continuous research, countless studies, and gradual improvements, achieving significant breakthroughs and innovations in the fight against cancer.

[0003] The introduction of nanotechnology into the biomedical field has given rise to nanomedicines. In recent years, researchers have shown strong interest in strategies for delivering anticancer drugs using supramolecular delivery systems based on non-covalent interactions and possessing stimulus-responsive functions. Significant progress has been made in improving drug delivery through the rational design and modification of anticancer drugs using supramolecular chemistry. Supramolecular nanomedicines possess specific, dynamically tunable non-covalent interactions, enabling them to respond to minute changes in the surrounding environment's pH, ionic strength, temperature, or solvent polarity. This provides innovative space for designing structure-property transition and microenvironment-responsive supramolecular nanomedicines, leading to the construction of stimulus-responsive, recognition-based supramolecular nanomedicines with advantages such as molecular-level structure, ease of preparation, controllable loading, intelligent release, high reproducibility, and multi-drug applicability.

[0004] Polyphenolic drugs have long been active in the field of anticancer research. Gossypol, a polyphenol found in the mallow plant cotton, has been shown to have certain anticancer effects and is a promising anticancer drug. However, due to its strong toxicity, it has rarely been used in anticancer research. Literature indicates that the toxicity of gossypol is mainly related to the aldehyde group in its molecular formula. Therefore, chemically modifying gossypol is a crucial step towards its potential use in the anticancer field.

[0005] This invention focuses on modifying gossypol drugs. A Schiff base bond is generated by the condensation reaction between the amino group on cystamine and the aldehyde group on gossypol, followed by mPEG-NHS end-capping to obtain a PGPP polymer prodrug. Leveraging the acidic microenvironment of tumor cells and their high glutathione (GSH) expression levels, this supramolecular prodrug nanomicelle exhibits precise and controllable drug release under dual-response conditions and high biocompatibility, thereby further reducing the toxicity of gossypol drugs and improving their utilization in cancer treatment. Summary of the Invention

[0006] The technical problem solved by this invention is to provide a method for preparing polygossypol supramolecular prodrug nanomicelles, which solves the problem of significant toxic side effects of gossypol and broadens the application of gossypol drugs in the field of anticancer. This study investigates the basic characterization, drug release, phagocytosis, and cytotoxicity of supramolecular prodrugs under acidic and high GSH environments in tumors, aiming to provide a method for preparing gossypol supramolecular prodrugs and their application in cancer treatment.

[0007] The specific technical solution of the present invention is as follows:

[0008] A method for preparing polystyrene supramolecular prodrug nanomicelles, characterized by comprising the following steps: (1) The entire reaction process was carried out in an argon atmosphere, with argon acting as a protective gas to prevent the presence of oxygen or hydrogen from affecting the reaction. A certain amount of gossypol (GP) and cystamine hydrochloride were added to an appropriate amount of DMSO and stirred until dissolved. Then, a certain amount of triethylamine (TEA) was incorporated into the completely dissolved reaction solution, and the reaction was carried out in an oil bath at 50°C for 24 h. After the reaction was completed, methanol was used as the dialysis solution, and the resulting reaction mixture was placed in a dialysis bag with a molecular weight of 3500 and dialyzed in methanol for about 24 h. The dialysis product was then removed, rotary evaporated, and vacuum dried for 24 h to obtain the polymer PGP. (2) A certain amount of polymer PGP obtained in the previous step and a certain amount of mPEG-NHS (5000) were completely dissolved in an appropriate amount of DMSO. The mixture was then reacted at room temperature for 24 h. After the reaction was completed, the reaction mixture was taken out and placed into a dialysis bag with a molecular weight of 10000. Methanol was used as the dialysis solution and dialyzed for 24 h. The methanol solution was evaporated and dried under vacuum for 24 h to obtain PGPP polymer. (3) Dissolve a certain amount of polymer PGPP in DMSO, and then slowly drop it into deionized water under rapid stirring with a pipette to obtain a mixed solution. Vortex for 24 h, and transfer the obtained solution to a dialysis bag with a molecular weight of 3500 for dialysis for about 24 h. The dialysis solution is deionized water, and PGPP supramolecular nanomicelles are obtained.

[0009] According to claim 1, the method for preparing polygossypol supramolecular prodrug nanomicelles is characterized in that: in step (1), the molar concentration of gossypol (GP) in the reaction is in the range of 1~2 mol·L⁻¹. -1 The molar ratio of gossypol (GP) to cystamine hydrochloride is 1:2, the molar concentration of cystamine hydrochloride ranges from 2 to 4 mol·L⁻¹, and the molar concentration of triethylamine (TEA) ranges from 0.1 to 0.2 mol·L⁻¹. -1 .

[0010] According to claim 1, the method for preparing polystyrene supramolecular prodrug nanomicelles is characterized in that: in step (2), the molar ratio of mPEG-NHS(5000) to PGP is 2:1, and the molar concentration range of mPEG-NHS(5000) is 2~4 mol·L⁻¹. -1 .

[0011] The method for preparing polystyrene supramolecular prodrug nanomicelles according to claim 1 is characterized in that: in step (3), the mass concentration of polymer PGPP dissolved in DMSO is in the range of 10~20 mg·mL. -1 The volume ratio of micelles to deionized water during dialysis is in the range of 1:200 to 1:300.

[0012] The main advantages of this invention are:

[0013] To address the significant cytotoxic side effects of gossypol on normal cells in antitumor research, gossypol was modified with cystamine to remove the toxic aldehyde group, and then capped with PEG to enhance its water solubility, greatly facilitating its intravenous administration. The delivery method was successfully optimized to improve the therapeutic efficacy of gossypol.

[0014] Under the acidic environment and high GSH expression levels of the tumor microenvironment, the Schiff base bond connecting gossypol and cystamine breaks due to low pH, while the disulfide bond (-SS-) in cystamine intelligently responds to GSH and breaks, thereby releasing gossypol and achieving precise and controllable release of the drug at the tumor site. The modified polygossypol forms supramolecular micelles. Cytotoxicity studies show that free gossypol is significantly toxic to normal cells, while micelles are almost non-toxic, demonstrating the stability of the prepared micelles in normal physiological environments and exhibiting low toxicity and high biocompatibility in normal tissues. In tumor cells, the micelles exhibit higher toxicity than free gossypol, indicating that micelles precisely release gossypol in the tumor environment, improving drug utilization. Attached Figure Description

[0016] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided:

[0017] Figure 1 This is a schematic diagram illustrating the synthesis of the polystyrene prodrug in Example 1.

[0018] Figure 2 This is an NMR diagram of the polymers PGP and PGPP used in Example 1.

[0019] Figure 3 The image shows the ultraviolet spectrum of the polystyrene supramolecular prodrug nanomicelles in Example 1.

[0020] Figure 4 The images show the transmission electron microscope (TEM) image and dynamic light scattering (DLS) particle size distribution map of the polystyrene supramolecular prodrug nanomicelles in Example 1. Figure 5 This is a schematic diagram of the in vitro drug release of polystyrene supramolecular prodrug nanomicelles in Example 1.

[0021] Figure 6 This is a comparison of the toxicity of polystyrene supramolecular prodrug nanomicelles to 4T1 cells and L929 fibroblasts in Example 1. Detailed Implementation

[0022] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0023] Example 1

[0024] A schematic diagram of the overall synthesis of a polystyrene prodrug is shown below. Figure 1 As shown, the main steps include: (1) The entire reaction process was carried out in an argon atmosphere, with argon acting as a protective gas to prevent the presence of oxygen or hydrogen from affecting the reaction. GP (1 mmol) and cystamine hydrochloride (2 mmol) were added to 1 mL of DMSO and stirred until dissolved. Then, triethylamine (TEA) (0.1 mmol) was incorporated into the completely dissolved reaction solution, and the reaction was carried out in an oil bath at 50 °C for 24 h. The reaction mixture was then placed in a dialysis bag with a molecular weight of 3500 and dialyzed in methanol for 24 h. The dialyzed reaction mixture was then removed and evaporated to dryness to obtain polymer PGP. (2) The PGP (1 mmol) and mPEG-NHS (5000) (2 mmol) prepared in the previous step were completely dissolved in 1 mL DMSO and reacted at room temperature for 24 h. The reaction mixture was then placed in a dialysis bag and dialyzed in methanol solution for 24 h. The methanol solution was evaporated to dryness to obtain PGPP polymer. (3) Weigh 10 mg of PGPP and completely dissolve it in 1 mL of DMSO. Add the PGPP solution to 9 mL of rapidly vortexed deionized water and vortex for 24 h. Dialyze the resulting solution in a dialysis bag (MWCO=3500 kDa) for 24 h to obtain PGPP supramolecular nanomicelles.

[0025] Figure 3 The ultraviolet spectrum effectively proves the successful synthesis of micelles. Figure 4 The particle size distribution and size of PGPP micelles, and the particle size distribution and size of PGPP decomposed under corresponding conditions. Figure 5 This is a schematic diagram of the in vitro release of PGPP nanomicelles, showing that the drug has a good release effect in an in vitro simulated tumor acidic environment and a high GSH expression environment. Figure 6 The image shows a comparison of the toxicity of PGPP micelles to 4T1 tumor cells, L929 (fibroblasts), and HUVEC (human umbilical vein endothelial cells). It can be seen that the micelles have almost no toxic side effects on normal cells, while having a significant killing effect on cancer cells, highlighting the biosafety and good tumor treatment effect of the micelles.

[0026] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing polystyrene supramolecular prodrug nanomicelles, characterized in that, Includes the following steps: (1) The entire reaction process was carried out in an argon atmosphere, with argon acting as a protective gas to prevent the presence of oxygen or hydrogen from affecting the reaction. A certain amount of gossypol (GP) and cystamine hydrochloride were added to an appropriate amount of dimethyl sulfoxide (DMSO) and stirred until dissolved. Then, a certain amount of triethylamine (TEA) was incorporated into the completely dissolved reaction solution, and the reaction was carried out in an oil bath at 50°C for 24 h. After the reaction was completed, methanol was used as the dialysate, and the resulting reaction mixture was transferred to a dialysis bag with a molecular weight of 3500 and dialyzed in methanol for 24 h. The dialyzed product was then removed, rotary evaporated, and vacuum dried for 24 h to obtain polymer PGP. (2) A certain amount of polymer PGP obtained in the previous step and a certain amount of mPEG-NHS (5000) were completely dissolved in an appropriate amount of DMSO. The mixture was then reacted at room temperature for 24 h. After the reaction was completed, the reaction mixture was taken out and placed into a dialysis bag with a molecular weight of 10000. Methanol was used as the dialysis solution and dialyzed for 24 h. The methanol solution was evaporated and dried under vacuum for 24 h to obtain PGPP polymer. (3) Dissolve a certain amount of polymer PGPP in DMSO, and then slowly drop it into deionized water under rapid stirring with a pipette to obtain a mixed solution. Vortex for 24 h, and transfer the obtained solution to a dialysis bag with a molecular weight of 3500 for dialysis for about 24 h. The dialysis solution is deionized water, and PGPP supramolecular nanomicelles are obtained.

2. The method for preparing polystyrene supramolecular prodrug nanomicelles according to claim 1, characterized in that: In step (1), the molar concentration of gossypol (GP) in the reaction is in the range of 1~2 mol·L⁻¹. -1 The molar ratio of gossypol (GP) to cystamine hydrochloride is 1:2, and the molar concentration of cystamine hydrochloride ranges from 2 to 4 mol·L⁻¹. -1 Furthermore, the molar concentration range of triethylamine (TEA) is 0.1–0.2 mol·L⁻¹. -1 .

3. The method for preparing polystyrene supramolecular prodrug nanomicelles according to claim 1, characterized in that: In step (2), the molar ratio of mPEG-NHS(5000) to PGP is 2:1, and the molar concentration range of mPEG-NHS(5000) is 2~4 mol·L⁻¹. -1 .

4. The method for preparing polystyrene supramolecular prodrug nanomicelles according to claim 1, characterized in that: In step (3), the mass concentration of polymer PGPP dissolved in DMSO is in the range of 10~20 mg·mL. -1 The volume ratio of micelles to deionized water during dialysis is in the range of 1:200 to 1:300.

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