Responsive micelles for loading hydrophobic positively charged drugs and methods of making the same

Abstract

The application discloses a kind of response type micelles for loading hydrophobic positively charged drug and preparation method thereof, it is related to the field of biomedicine and nanomaterials.This carrier is based on polyethylene glycol and introduces stimulus-responsive disulfide bond and crosslinkable group, and the efficient loading and control release of drug are carried out.Through multi-step organic synthesis "ABA" type triblock structure polymer, four hydroxyl PEG is prepared first, and then coupled and photocrosslinking with undecanethiol undecanoic acid and sulfonated molecule in turn through disulfide bond at four hydroxyl ends, and finally obtain amphiphilic polymer with triblock structure.The polymer can efficiently load hydrophobic drug using its hydrophobic micro area, and promote the loading of positively charged drug by means of its hydrophobic end connected sulfonic group negative group, and realize good water solubility and long cycle by means of the hydrophilicity of PEG segment;The disulfide bond contained therein can be broken under high-concentration glutathione environment in tumor cells, to realize the intelligent control release of drug.

Description

Technical Field

[0001] This invention relates to the fields of functional materials technology and biomedical materials technology, specifically to a responsive micelle for loading hydrophobic positively charged drugs and its preparation method. Background Technology

[0002] Cancer, as a malignant disease that seriously threatens patients' lives and health, has always been a focus of scientific research and clinical attention in terms of innovation and optimization of treatment plans. Traditional chemotherapy involves a long treatment cycle, with patients taking oral chemotherapy drugs for extended periods, posing risks such as systemic drug exposure leading to thromboembolism and endometrial cancer. Therefore, developing a novel treatment strategy that can enhance efficacy while reducing systemic toxicity has become an urgent need in the field of cancer treatment. Many anti-tumor drugs have shown great therapeutic potential due to their broad anti-tumor activity, such as the multi-target receptor tyrosine kinase inhibitor sunitinib. However, the clinical application and efficacy improvement of many anti-tumor drugs face fundamental challenges due to their inherent physicochemical properties. For example, their extremely poor water solubility makes it difficult to form an effective formulation in vivo, resulting in significantly low oral bioavailability. This deficiency not only limits the effective concentration of the drug in systemic circulation but also leads to a severe shortage of the amount of drug that can be targeted to the tumor lesion area, failing to reach and maintain the critical concentration required for treatment within the tumor tissue, thus directly weakening its expected anti-tumor effect. Even if the drug reaches the tumor site, the inability of it to be released from the carrier to exert its therapeutic effect is also a major problem. Therefore, overcoming existing delivery bottlenecks for anti-tumor drugs is key to unlocking their clinical value. Current research focuses on developing novel drug delivery systems capable of efficiently loading anti-tumor drugs and achieving specific drug release at the tumor site. An ideal system should fundamentally improve drug solubility and in vivo stability, thereby significantly enhancing drug bioavailability within the lesion. The ultimate goal is to greatly enhance anti-tumor efficacy while minimizing systemic toxicity to normal tissues, providing cancer patients with a safer and more effective new strategy for precision treatment. This invention utilizes a polymer based on PEG and MUA, a safe and stable nanomaterial for drug delivery that is simple to operate and inexpensive. Summary of the Invention

[0003] The purpose of this invention is to provide a responsive micelle for loading hydrophobic positively charged drugs and its preparation method. This invention solves the problems of poor water solubility, low bioavailability, high systemic toxicity, insufficient tumor targeting and drug release efficiency of hydrophobic positively charged antitumor drugs (such as sunitinib), and the difficulty of efficiently loading such drugs with existing carriers.

[0004] This invention provides a method for preparing responsive micelles loaded with hydrophobic positively charged drugs, comprising the following steps:

[0005] Step 1: Using polyethylene glycol (PEG) as a starting material, react with epichlorohydrin to prepare polyethylene glycol with diepoxy end-modified.

[0006] Step 2: The diepoxy-terminated polyethylene glycol is subjected to a ring-opening reaction in an alkaline aqueous solution to obtain tetrahydroxy-terminated polyethylene glycol.

[0007] Step 3: React the tetrahydroxy-terminated polyethylene glycol with CDI to activate its terminal hydroxyl groups and obtain CDI-activated tetrahydroxy polyethylene glycol.

[0008] Step 4: CDI-activated tetrahydroxy polyethylene glycol is reacted with cystamine dihydrochloride in the presence of triethylamine to introduce a linker arm containing a disulfide bond, thereby obtaining disulfide-linked tetrahydroxy polyethylene glycol.

[0009] Step 5: After activating undecylmercaptoundecanoic acid (MUA), it is reacted with tetrahydroxy polyethylene glycol with disulfide bonds obtained in step 4 to obtain undecylmercaptoundecanoic acid-grafted disulfide bond polyethylene glycol.

[0010] Step 6: The disulfide-bonded polyethylene glycol grafted with undecylmercaptoundecanoic acid obtained in Step 5, the photoinitiator phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide (BAPO), and sulfonated butene glycol are mixed and, under 365 nm ultraviolet light irradiation, the negatively charged hydrophobic segments are connected to both ends of the polymer through a mercapto-olefin click chemical reaction to obtain an ABA-type triblock functionalized polymer.

[0011] Step 7: Dissolve the ABA-type triblock functionalized polymer obtained in step 6 in dimethyl sulfoxide, place the solution in a dialysis bag and dialyze for 48 h to remove unencapsulated sunitinib and solvent, centrifuge the resulting solution at 4000 rpm for 5 min, and take the supernatant to prepare responsive micelles for loading hydrophobic positively charged drugs.

[0012] The present invention also provides a responsive micelle for loading hydrophobic positively charged drugs, wherein the micelle is an ABA-type triblock functionalized polymer micelle composed of a hydrophilic end, a hydrophobic end and functionalized groups;

[0013] The hydrophilic end is polyethylene glycol (PEG), which serves as the central segment (B segment) of the triblock.

[0014] The hydrophobic end is undecylmercaptoundecanoic acid (MUA) modified with sulfonated groups, serving as the two end segments (A segment) of the triblock.

[0015] The hydrophilic end (section B) and the hydrophobic end (section A) are connected by a responsive disulfide bond bridge;

[0016] The functionalized groups include sulfonic acid groups and disulfide bond groups introduced through modification of ABA-type triblock polymers.

[0017] The purpose of this invention is to provide a multifunctional polymer micelle with GSH-responsive release capability for efficiently loading and delivering hydrophobic drugs.

[0018] Another object of the present invention is to provide a method for preparing the polymer micelles that has a clear preparation process, mild conditions, and low cost.

[0019] This invention cleverly integrates the hydrophilicity of PEG, the drug-carrying capacity of its hydrophobic segments, the intelligent responsiveness of its disulfide bonds, and the tunable hydrophobic end charge to promote drug loading by designing a novel "ABA"-type triblock polymer. This polymer can self-assemble in aqueous solution to form core-shell micelles, where the hydrophobic core efficiently encapsulates hydrophobic drugs, while the hydrophilic shell provides biocompatibility and prolongs in vivo circulation time. In specific microenvironments rich in GSH, such as tumors, the disulfide bonds of the bridging segments can break rapidly, achieving intelligent controlled drug release.

[0020] The present invention relates to functionalized polymer micelles for drug delivery, which are formed by the self-assembly of functionalized polymers in aqueous solution, wherein the core of the polymer micelles is loaded with a hydrophobic, positively charged therapeutic drug.

[0021] The functionalized polymer has a “ABA” type triblock structure with PEG as the hydrophilic end and modified MUA hydrophobic segments at both ends of the hydrophilic end. The A segment is MUA modified with sulfonated groups, and the B segment is hydrophilic PEG. The A and B segments are connected by a responsive disulfide bond bridge.

[0022] The preparation method of the functionalized polymer is based on organic synthesis reactions involving ring opening, substitution, and addition, and finally forms micelles through self-assembly via dialysis.

[0023] The functionalized polymer micelles can be tuned to adjust the molecular weight of PEG to form stable micelles suitable for drugs with different steric hindrances.

[0024] The functionalized polymer micelles can be adjusted to carry a certain amount of negative charge, allowing for the formation of stable micelles suitable for drugs with different amounts of positive charge.

[0025] The polymer concentration used in the preparation of the functionalized polymer micelles can be adjusted according to the required micelle size and drug loading, typically in the range of 1-5 mg / mL.

[0026] The polymer micelles of this invention for drug delivery possess a unique "ABA" triblock structure and multiple functional modifications. The hydrophilic and hydrophobic segments are connected by disulfide bonds, enabling specific responsive drug release in high GSH concentration environments. Its hydrophilic shell effectively prevents rapid clearance by the reticuloendothelial system, prolonging blood circulation time. The hydrophobic core and tunable negatively charged surface provide great flexibility for loading drugs with different properties. Furthermore, this system significantly increases the apparent water solubility of hydrophobic drugs, improving their bioavailability.

[0027] Experimental results show that the GSH-responsive polymeric micelles (P2SM) prepared in this invention achieve high loading and high encapsulation of hydrophobic, positively charged drugs (such as Sun). The P2SM@Sun micelles prepared by dialysis exhibit high EE and DL values ​​of 49.21% and 3.08%, respectively. The generated P2SM@Sun micelles have uniform particle size, ranging from 105 nm to 150 nm, and can utilize the high permeability and retention effect (EPR effect) of solid tumor tissue to achieve passive targeting and enrichment of drugs at the tumor site, and can release drugs in response to a high GSH environment. The P2SM polymeric micelles of this invention can significantly improve the water solubility of hydrophobic drugs and reduce their toxic side effects, thereby effectively improving drug bioavailability.

[0028] The functionalized polymer has an "ABA" type triblock structure, and its preparation method involves ring-opening reaction, substitution reaction and click chemistry.

[0029] The functionalized polymer with GSH-responsive release has a drug release mechanism derived from disulfide bonds in the linker arm.

[0030] The hydrophobic end charge of the polymer micelles can be adjusted by the number of introduced sulfonic acid groups to meet the loading requirements of different drugs.

[0031] During the preparation of the polymer micelles, the concentration of the "ABA" polymer is preferably 1-5 mg / mL.

[0032] The beneficial effects of this application, based on the above technical solutions, are as follows:

[0033] The polymer micelles of this invention for drug delivery possess advantages such as simple operation, low cost, good biocompatibility, wide drug loading range, and intelligent release, exhibiting very high clinical application potential. The micelles of this invention are particularly suitable for delivering chemotherapeutic drugs with poor water solubility and high systemic toxicity, such as for the treatment of solid tumors like breast cancer and melanoma. This invention significantly improves the therapeutic index by enhancing drug solubility, prolonging in vivo circulation time, and achieving lesion-specific drug release. The micelles prepared by this invention are used to deliver drugs to tumor sites and achieve controlled release at the tumor site. The preparation process is free of toxic and harmful substances, the reaction process is mild, the final product is easy to purify, environmentally friendly, and exhibits good stability. Attached Figure Description

[0034] Figure 1 This diagram illustrates the preparation method of the responsive polymer micelles for loading hydrophobic, positively charged drugs according to the present invention.

[0035] Figure 2 The quantitative data of EE of the responsive polymer micelles prepared in Examples 1-10 of this invention for loading hydrophobic, positively charged drugs after loading Sun under different feed ratios and solvent conditions are shown in the figure.

[0036] Figure 3 The quantitative data of DL after loading Sun with different feed ratios and solvent conditions are shown in Examples 1-10 of this invention for responsive polymer micelles used to load hydrophobic, positively charged drugs.

[0037] Figure 4 The quantitative data graphs show the particle size of the responsive polymer micelles prepared in Examples 8 and 11-14 of this invention for loading hydrophobic, positively charged drugs, and loaded with Sun under different molecular weight PEG conditions.

[0038] Figure 5 Quantitative data graphs of the zeta potentials of responsive polymer micelles prepared in Examples 8 and 11-14 of this invention for loading hydrophobic, positively charged drugs, loaded with Sun under different molecular weight PEG conditions.

[0039] Figure 6 The quantitative data of EE of the responsive polymer micelles prepared in Examples 11-14 of this invention for loading hydrophobic, positively charged drugs after loading Sun under different molecular weight PEG conditions are shown in the figure.

[0040] Figure 7 The quantitative data of DL of the responsive polymer micelles prepared in Examples 11-14 of this invention for loading hydrophobic, positively charged drugs after loading Sun under different molecular weight PEG conditions are shown in the figure.

[0041] Figure 8The quantitative data graph shows the ability of P2SM@Sun to release Sun within 24 h under different concentrations of GSH in the presence of responsive polymer micelles prepared for loading hydrophobic, positively charged drugs in Example 15 of this invention.

[0042] Figure 9 The image shows a microneedle image taken with a camera after the responsive polymer micelles prepared in Example 16 of this invention for loading hydrophobic, positively charged drugs were applied to the preparation of microneedles. Detailed Implementation

[0043] To fully illustrate the preparation concept of this invention, the process of this solution is described below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be interpreted or construed as limiting the protection of this invention.

[0044] Appendix Figure 1 The present invention illustrates the preparation method and operation process of responsive micelles for loading hydrophobic positively charged drugs. See the appendix for the following examples. Figure 1 :

[0045] Example 1

[0046] (1) MW2000 PEG (P2EG) was placed in a Schlenk flask, melted at 68 °C, dried under vacuum for 8 h, and then purged with argon for 5 min. Tetrahydrofuran (THF) was added to dissolve it. NaH was added under argon purging. The solution was stirred overnight at room temperature. Then epichlorohydrin was added and the reaction was stirred for 20 h. After the reaction was complete, the reaction mixture was filtered to remove excess NaH and salt. The filtered solution was concentrated and precipitated in diethyl ether. The precipitate was dried under vacuum to obtain the polymer Diepoxy P2EG (PEG with diepoxy end-modified).

[0047] (2) The polymer (Diepoxy P2EG) obtained in step (1) was stirred in a Schlenk flask with a mixture of 0.1 M NaOH aqueous solution at 60 °C for 10 h. Then hydrochloric acid was added to neutralize the solution. The water was evaporated under vacuum, and dichloromethane (DCM) and anhydrous sodium were added to the mixture sequentially for drying, followed by filtration. The DCM was evaporated under vacuum to obtain tetrahydroxy-terminated polyethylene glycol (polymer Tetrahydroxyl PEG, Tet P2EG), which was a white solid.

[0048] (3) The polymer (Tet P2EG) obtained in step (2) was placed in a Schlenk flask and dried under vacuum for 8 h, then purged with argon for 5 min. DCM (dichloromethane) was added to Tet P2EG to dissolve it. In addition, N,N'-carbonyldiimidazole (CDI) was dissolved in DCM, and the CDI solution was slowly added to the Tet P2EG solution at room temperature. The resulting mixture was stirred under a nitrogen atmosphere for 36 h. After the reaction was completed, saturated sodium chloride aqueous solution was added for extraction, and the organic layer was dried with sodium sulfate. The collected solution was used to evaporate DCM under vacuum to obtain CDI-activated tetrahydroxy polyethylene glycol (polymer Tet P2EG-CDI), which was a pale yellow solid powder.

[0049] (4) The polymer (Tet P2EG-CDI) obtained in step (3) was placed in a Schlenk flask and dried under vacuum for 8 h, then purged with argon for 5 min. Then, DMSO was added to Tet P2EG-CDI to dissolve it. In addition, cystamine dihydrochloride was dissolved in DMSO. The cystamine dihydrochloride solution and triethylamine were slowly added to the Tet P2EG-CDI solution. The resulting mixture was stirred at room temperature for 36 h. The reacted mixture was dialyzed and lyophilized to obtain disulfide-linked tetrahydroxy polyethylene glycol (polymer Tet P2EG-SS).

[0050] (5) Butene glycol was placed in a Schlenk flask and purged with argon for 5 min. Dimethylformamide (DMF) was added to dissolve it. Sulfur trioxide pyridine was dissolved in DMF and slowly added dropwise to the butene glycol solution through a constant pressure funnel. The resulting mixture was allowed to react at room temperature for 3 days. After the reaction was complete, the reaction was placed in an ice bath and an appropriate amount of water was slowly added to quench the reaction. The pH of the reaction solution was then adjusted to 8 with 5 mol / L NaOH solution, and the water was removed by rotary evaporation. 100 mL of methanol was added, and the solution was dried with anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the methanol and DMF were removed by rotary evaporation to obtain sulfonated butene glycol (3-butene-1,2-diol-Sul).

[0051] (6) Dissolve MUA, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) in DMSO, add the solution to a Schlenk flask under argon purging, and stir at room temperature for 18 h. Then dissolve the polymer (Tet P2EG-SS) obtained in step (4) in DMSO (dimethyl sulfoxide) and add it to the reaction solution, and continue to react at room temperature for 2 days. After the reaction is completed, dialyze the reaction solution and freeze-dry it to obtain undecylmercaptoundecanoic acid-grafted disulfide polyethylene glycol (polymer Tet P2EG-SS-MUA).

[0052] (7) The polymer (Tet P2EG-SS-MUA) obtained in step (6), the product (3-butene-1,2-diol-Sul) obtained in step (6), and BAPO (phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide) were added to an ep tube, DMF was added to dissolve them, and the mixture was irradiated under ultraviolet light at a wavelength of 365 nm for 2 h. After the reaction was completed, the reaction solution was dialyzed, centrifuged, and the supernatant was lyophilized to obtain the ABA-type triblock functionalized polymer (polymer Tet P2EG-SS-MUA-Sul, P2SM).

[0053] (8) The standard curve of Sun was determined using ultraviolet spectrophotometry. 1 mg of Sun was accurately weighed into a methanol solution to prepare a Sun standard solution. Six centrifuge tubes were used to prepare solutions with concentrations (C) of 1.25, 2.5, 5, 10, 20, and 40 ug / mL, respectively. The absorbance (A) at 425 nm was measured to establish the standard curve for Sun, which was A = 0.0614C + 7E - 0.5. Sun refers to sunitinib.

[0054] (9) To prepare P2SM@Sun micelles (responsive drug-loaded micelles formed by loading sunitinib onto a P2SM polymer carrier), GSH-sensitive P2SM@Sun micelles were prepared by dialysis. 5 mg of P2SM polymer and 1 mg of Sun were dissolved in 5 mL of formamide, and the mixture was placed in a dialysis bag and dialyzed for 48 h to remove unencapsulated Sun and solvent. The resulting solution was then centrifuged at 4000 rpm for 5 min, and the supernatant was collected to obtain the P2SM@Sun micelles.

[0055] (10) Dilute the P2SM@Sun micelles obtained in step (9) by dissolving them in formamide by 5 times in methanol to demulsify them. Then add 3 mL to a quartz dish and measure its absorbance using a UV spectrophotometer. Calculate its concentration using the Sun standard curve determined in step (8). Then, obtain the EE and DL of Sun in the P2SM@Sun micelles using the calculation formula. The quantitative data are as follows: Figure 2 and Figure 3 As shown. The formula for calculating EE (encapsulation efficiency) is m1 / m2*100%, and the formula for calculating DL (drug loading) is m1 / (m2+m3)*100%, where m1 is the drug loading mass of the carrier, m2 is the total drug loading mass, and m3 is the total carrier mass.

[0056] Experimental results show that the P2SM micelle delivery system constructed in this invention exhibits excellent loading performance for the hydrophobic, positively charged model drug Sun. P2SM@Sun micelles prepared by dialysis, with a feed ratio of 5 mg P2SM to 1 mg Sun and formamide as the solvent, achieved EE and DL of 13.42% and 2.24%, respectively, indicating that this carrier has a clear and efficient loading capacity for the target drug. The polymer micelles constructed in this invention, as a functionally defined and reliable drug delivery platform, demonstrate significant advantages in loading and controlled release of hydrophobic and positively charged drugs, providing solid experimental evidence for their further development and application in the field of disease treatment.

[0057] Example 2

[0058] Steps (1) to (8) are the same as in Example 1.

[0059] (9) To prepare P2SM@Sun micelles, GSH-sensitive P2SM@Sun micelles were prepared by dialysis. 10 mg of P2SM polymer and 1 mg of Sun were dissolved in 5 mL of formamide, and the mixture was placed in a dialysis bag and dialyzed for 48 h to remove unencapsulated Sun and solvent. The resulting solution was then centrifuged at 4000 rpm for 5 min, and the supernatant was collected to obtain P2SM@Sun micelles.

[0060] (10) Dilute the P2SM@Sun micelles obtained in step (9) by dissolving them in formamide by 5 times in methanol to demulsify them. Then add 3 mL to a quartz dish and measure its absorbance using a UV spectrophotometer. Calculate its concentration using the Sun standard curve determined in step (8). Then, obtain the EE and DL of Sun in the P2SM@Sun micelles using the calculation formula. The quantitative data are as follows: Figure 2 and Figure 3 As shown. The formula for calculating EE is m1 / m2*100%, and the formula for calculating DL is m1 / (m2+m3)*100%, where m1 is the drug-loaded mass of the carrier, m2 is the total mass of the drug, and m3 is the total mass of the carrier.

[0061] Experimental results show that the P2SM micelle delivery system constructed in this invention exhibits excellent loading performance for the hydrophobic, positively charged model drug Sun. P2SM@Sun micelles prepared by dialysis, with a feed ratio of 10 mg P2SM to 1 mg Sun and formamide as the solvent, achieved EE and DL of 18.32% and 1.67%, respectively, indicating that this carrier has a clear and efficient loading capacity for the target drug. The polymer micelles constructed in this invention, as a functionally defined and reliable drug delivery platform, demonstrate significant advantages in loading and controlled release of hydrophobic and positively charged drugs, providing a solid experimental basis for their further development and application in the field of disease treatment.

[0062] Example 3

[0063] Steps (1) to (8) are the same as in Example 1.

[0064] (9) To prepare P2SM@Sun micelles, GSH-sensitive P2SM@Sun micelles were prepared by dialysis. 15 mg of P2SM polymer and 1 mg of Sun were dissolved in 5 mL of formamide, and the mixture was placed in a dialysis bag and dialyzed for 48 h to remove unencapsulated Sun and solvent. The resulting solution was then centrifuged at 4000 rpm for 5 min, and the supernatant was collected to obtain P2SM@Sun micelles.

[0065] (10) Dilute the P2SM@Sun micelles obtained in step (9) by dissolving them in formamide by 5 times in methanol to demulsify them. Then add 3 mL to a quartz dish and measure its absorbance using a UV spectrophotometer. Calculate its concentration using the Sun standard curve determined in step (8). Then, obtain the EE and DL of Sun in the P2SM@Sun micelles using the calculation formula. The quantitative data are as follows: Figure 2 and Figure 3 As shown. The formula for calculating EE is m1 / m2*100%, and the formula for calculating DL is m1 / (m2+m3)*100%, where m1 is the drug-loaded mass of the carrier, m2 is the total mass of the drug, and m3 is the total mass of the carrier.

[0066] Experimental results show that the P2SM micelle delivery system constructed in this invention exhibits excellent loading performance for the hydrophobic, positively charged model drug Sun. P2SM@Sun micelles prepared by dialysis, with a feed ratio of 15 mg P2SM to 1 mg Sun and formamide as the solvent, achieved EE and DL of 22.58% and 1.41%, respectively, indicating that this carrier has a clear and efficient loading capacity for the target drug. The polymer micelles constructed in this invention, as a functionally defined and reliable drug delivery platform, demonstrate significant advantages in loading and controlled release of hydrophobic and positively charged drugs, providing solid experimental evidence for their further development and application in the field of disease treatment.

[0067] Example 4

[0068] Steps (1) to (8) are the same as in Example 1.

[0069] (9) To prepare P2SM@Sun micelles, GSH-sensitive P2SM@Sun micelles were prepared by dialysis. 20 mg of P2SM polymer and 1 mg of Sun were dissolved in 5 mL of formamide, and the mixture was placed in a dialysis bag and dialyzed for 48 h to remove unencapsulated Sun and solvent. The resulting solution was then centrifuged at 4000 rpm for 5 min, and the supernatant was collected to obtain P2SM@Sun micelles.

[0070] (10) Dilute the P2SM@Sun micelles obtained in step (9) by dissolving them in formamide by 5 times in methanol to demulsify them. Then add 3 mL to a quartz dish and measure its absorbance using a UV spectrophotometer. Calculate its concentration using the Sun standard curve determined in step (8). Then, obtain the EE and DL of Sun in the P2SM@Sun micelles using the calculation formula. The quantitative data are as follows: Figure 2 and Figure 3 As shown. The formula for calculating EE is m1 / m2*100%, and the formula for calculating DL is m1 / (m2+m3)*100%, where m1 is the drug-loaded mass of the carrier, m2 is the total mass of the drug, and m3 is the total mass of the carrier.

[0071] Experimental results show that the P2SM micelle delivery system constructed in this invention exhibits excellent loading performance for the hydrophobic, positively charged model drug Sun. P2SM@Sun micelles prepared by dialysis, with a feed ratio of 20 mg P2SM to 1 mg Sun and formamide as the solvent, achieved EE and DL of 34.71% and 1.65%, respectively, indicating that this carrier has a clear and efficient loading capacity for the target drug. The polymer micelles constructed in this invention, as a functionally defined and reliable drug delivery platform, demonstrate significant advantages in loading and controlled release of hydrophobic and positively charged drugs, providing solid experimental evidence for their further development and application in the field of disease treatment.

[0072] Example 5

[0073] Steps (1) to (8) are the same as in Example 1.

[0074] (9) To prepare P2SM@Sun micelles, GSH-sensitive P2SM@Sun micelles were prepared by dialysis. 25 mg of P2SM polymer and 1 mg of Sun were dissolved in 5 mL of formamide, and the mixture was placed in a dialysis bag and dialyzed for 48 h to remove unencapsulated Sun and solvent. The resulting solution was then centrifuged at 4000 rpm for 5 min, and the supernatant was collected to obtain P2SM@Sun micelles.

[0075] (10) Dilute the P2SM@Sun micelles obtained in step (9) by dissolving them in formamide by 5 times in methanol to demulsify them. Then add 3 mL to a quartz dish and measure its absorbance using a UV spectrophotometer. Calculate its concentration using the Sun standard curve determined in step (8). Then, obtain the EE and DL of Sun in the P2SM@Sun micelles using the calculation formula. The quantitative data are as follows: Figure 2 and Figure 3 As shown. The formula for calculating EE is m1 / m2*100%, and the formula for calculating DL is m1 / (m2+m3)*100%, where m1 is the drug-loaded mass of the carrier, m2 is the total mass of the drug, and m3 is the total mass of the carrier.

[0076] Experimental results show that the P2SM micelle delivery system constructed in this invention exhibits excellent loading performance for the hydrophobic, positively charged model drug Sun. P2SM@Sun micelles prepared by dialysis, with a feed ratio of 25 mg P2SM to 1 mg Sun and formamide as the solvent, achieved EE and DL of 35.92% and 1.38%, respectively, indicating that this carrier has a clear and efficient loading capacity for the target drug. The polymer micelles constructed in this invention, as a functionally defined and reliable drug delivery platform, demonstrate significant advantages in loading and controlled release of hydrophobic and positively charged drugs, providing a solid experimental basis for their further development and application in the field of disease treatment.

[0077] Example 6

[0078] Steps (1) to (8) are the same as in Example 1.

[0079] (9) To prepare P2SM@Sun micelles, GSH-sensitive P2SM@Sun micelles were prepared by dialysis. 5 mg of P2SM polymer and 1 mg of Sun were dissolved in 5 mL of DMSO, and the mixture was dialyzed in a dialysis bag for 48 h to remove unencapsulated Sun and solvent. The resulting solution was then centrifuged at 4000 rpm for 5 min, and the supernatant was collected to obtain P2SM@Sun micelles.

[0080] (10) Dilute the P2SM@Sun micelles obtained in step (9) by dissolving them in DMSO by 5 times in methanol to demulsify them. Then add 3 mL to a quartz dish and measure its absorbance using a UV spectrophotometer. Calculate its concentration using the Sun standard curve determined in step (8). Then, obtain the EE and DL of Sun in the P2SM@Sun micelles using the calculation formula. The quantitative data are as follows: Figure 2 and Figure 3 As shown. The formula for calculating EE is m1 / m2*100%, and the formula for calculating DL is m1 / (m2+m3)*100%, where m1 is the drug-loaded mass of the carrier, m2 is the total mass of the drug, and m3 is the total mass of the carrier.

[0081] Experimental results show that the P2SM micelle delivery system constructed in this invention exhibits excellent loading performance for the hydrophobic, positively charged model drug Sun. P2SM@Sun micelles prepared by dialysis, with a feed ratio of 5 mg P2SM to 1 mg Sun and DMSO as the solvent, achieved EE and DL of 15.30% and 2.55%, respectively, indicating that this carrier has a clear and efficient loading capacity for the target drug. The polymer micelles constructed in this invention, as a functionally defined and reliable drug delivery platform, demonstrate significant advantages in loading and controlled release of hydrophobic and positively charged drugs, providing solid experimental evidence for their further development and application in the field of disease treatment.

[0082] Example 7

[0083] Steps (1) to (8) are the same as in Example 1.

[0084] (9) To prepare P2SM@Sun micelles, GSH-sensitive P2SM@Sun micelles were prepared by dialysis. 10 mg of P2SM polymer and 1 mg of Sun were dissolved in 5 mL of DMSO / formamide, and the mixture was placed in a dialysis bag and dialyzed for 48 h to remove unencapsulated Sun and solvent. The resulting solution was then centrifuged at 4000 rpm for 5 min, and the supernatant was collected to obtain P2SM@Sun micelles.

[0085] (10) Dilute the P2SM@Sun micelles obtained in step (9) by dissolving them in DMSO by 5 times in methanol to demulsify them. Then add 3 mL to a quartz dish and measure its absorbance using a UV spectrophotometer. Calculate its concentration using the Sun standard curve determined in step (8). Then, obtain the EE and DL of Sun in the P2SM@Sun micelles using the calculation formula. The quantitative data are as follows: Figure 2 and Figure 3 As shown. The formula for calculating EE is m1 / m2*100%, and the formula for calculating DL is m1 / (m2+m3)*100%, where m1 is the drug-loaded mass of the carrier, m2 is the total mass of the drug, and m3 is the total mass of the carrier.

[0086] Experimental results show that the P2SM micelle delivery system constructed in this invention exhibits excellent loading performance for the hydrophobic, positively charged model drug Sun. P2SM@Sun micelles prepared by dialysis, with a feed ratio of 10 mg P2SM to 1 mg Sun and DMSO as the solvent, achieved EE and DL of 30.90% and 2.81%, respectively, indicating that this carrier has a clear and efficient loading capacity for the target drug. The polymer micelles constructed in this invention, as a functionally defined and reliable drug delivery platform, demonstrate significant advantages in loading and controlled release of hydrophobic and positively charged drugs, providing solid experimental evidence for their further development and application in the field of disease treatment.

[0087] Example 8

[0088] Steps (1) to (8) are the same as in Example 1.

[0089] (9) To prepare P2SM@Sun micelles, GSH-sensitive P2SM@Sun micelles were prepared by dialysis. 15 mg of P2SM polymer and 1 mg of Sun were dissolved in 5 mL of DMSO, and the mixture was placed in a dialysis bag and dialyzed for 48 h to remove unencapsulated Sun and solvent. The resulting solution was then centrifuged at 4000 rpm for 5 min, and the supernatant was collected to obtain P2SM@Sun micelles.

[0090] (10) Dilute the P2SM@Sun micelles obtained in step (9) by dissolving them in DMSO by 5 times in methanol to demulsify them. Then add 3 mL to a quartz dish and measure its absorbance using a UV spectrophotometer. Calculate its concentration using the Sun standard curve determined in step (8). Then, obtain the EE and DL of Sun in the P2SM@Sun micelles using the calculation formula. The quantitative data are as follows: Figure 2 and Figure 3 As shown. The formula for calculating EE is m1 / m2*100%, and the formula for calculating DL is m1 / (m2+m3)*100%, where m1 is the drug-loaded mass of the carrier, m2 is the total mass of the drug, and m3 is the total mass of the carrier.

[0091] (11) The P2SM@Sun micelle solution obtained in step (9) was subjected to dynamic light scattering (DLS) analysis using a Malvern Zeta Nanosizer to determine its particle size and Zeta potential. The quantitative data are as follows: Figure 4 and Figure 5 As shown.

[0092] Experimental results show that the P2SM micelle delivery system constructed in this invention exhibits excellent loading performance for the hydrophobic, positively charged model drug Sun. P2SM@Sun micelles prepared by dialysis, under conditions of a feed ratio of 15 mg P2SM to 1 mg Sun and DMSO as solvent, achieved EE and DL of 49.21% and 3.08%, respectively, indicating that this carrier has a clear and efficient loading capacity for the target drug. Furthermore, P2SM… @ Sun micelles, with a particle size of 105 nm and a zeta potential of -20.7 mV, can utilize the EPR effect of solid tumor tissue to achieve passive targeting and enrichment of drugs at the tumor site. The polymer micelles constructed in this invention serve as a functionally defined and reliable drug delivery platform, exhibiting significant advantages in loading and controlled release of both hydrophobic and positively charged drugs, providing a solid experimental basis for their further development and application in disease treatment.

[0093] Example 9

[0094] Steps (1) to (8) are the same as in Example 1.

[0095] (9) To prepare P2SM@Sun micelles, GSH-sensitive P2SM@Sun micelles were prepared by dialysis. 20 mg of P2SM polymer and 1 mg of Sun were dissolved in 5 mL of DMSO, and the mixture was placed in a dialysis bag and dialyzed for 48 h to remove unencapsulated Sun and solvent. The resulting solution was then centrifuged at 4000 rpm for 5 min, and the supernatant was collected to obtain P2SM@Sun micelles.

[0096] (10) Dilute the P2SM@Sun micelles obtained in step (9) by dissolving them in DMSO by 5 times in methanol to demulsify them. Then add 3 mL to a quartz dish and measure its absorbance using a UV spectrophotometer. Calculate its concentration using the Sun standard curve determined in step (8). Then, obtain the EE and DL of Sun in the P2SM@Sun micelles using the calculation formula. The quantitative data are as follows: Figure 2 and Figure 3 As shown. The formula for calculating EE is m1 / m2*100%, and the formula for calculating DL is m1 / (m2+m3)*100%, where m1 is the drug-loaded mass of the carrier, m2 is the total mass of the drug, and m3 is the total mass of the carrier.

[0097] Experimental results show that the P2SM micelle delivery system constructed in this invention exhibits excellent loading performance for the hydrophobic, positively charged model drug Sun. P2SM@Sun micelles prepared by dialysis, with a feed ratio of 20 mg P2SM to 1 mg Sun and DMSO as the solvent, achieved EE and DL of 31.92% and 1.52%, respectively, indicating that this carrier has a clear and efficient loading capacity for the target drug. The polymer micelles constructed in this invention, as a functionally defined and reliable drug delivery platform, demonstrate significant advantages in loading and controlled release of hydrophobic and positively charged drugs, providing solid experimental evidence for their further development and application in the field of disease treatment.

[0098] Example 10

[0099] Steps (1) to (8) are the same as in Example 1.

[0100] (9) To prepare P2SM@Sun micelles, GSH-sensitive P2SM@Sun micelles were prepared by dialysis. 25 mg of P2SM polymer and 1 mg of Sun were dissolved in 5 mL of DMSO / formamide, and the mixture was placed in a dialysis bag and dialyzed for 48 h to remove unencapsulated Sun and solvent. The resulting solution was then centrifuged at 4000 rpm for 5 min, and the supernatant was collected to obtain P2SM@Sun micelles.

[0101] (10) Dilute the P2SM@Sun micelles obtained in step (9) by dissolving them in DMSO by 5 times in methanol to demulsify them. Then add 3 mL to a quartz dish and measure its absorbance using a UV spectrophotometer. Calculate its concentration using the Sun standard curve determined in step (8). Then, obtain the EE and DL of Sun in the P2SM@Sun micelles using the calculation formula. The quantitative data are as follows: Figure 2 and Figure 3 As shown. The formula for calculating EE is m1 / m2*100%, and the formula for calculating DL is m1 / (m2+m3)*100%, where m1 is the drug-loaded mass of the carrier, m2 is the total mass of the drug, and m3 is the total mass of the carrier.

[0102] Experimental results show that the P2SM micelle delivery system constructed in this invention exhibits excellent loading performance for the hydrophobic, positively charged model drug Sun. P2SM@Sun micelles prepared by dialysis, with a feed ratio of 25 mg P2SM to 1 mg Sun and DMSO as the solvent, achieved EE and DL of 32.23% and 1.24%, respectively, indicating that this carrier has a clear and efficient loading capacity for the target drug. The polymer micelles constructed in this invention, as a functionally defined and reliable drug delivery platform, demonstrate significant advantages in loading and controlled release of hydrophobic and positively charged drugs, providing a solid experimental basis for their further development and application in the field of disease treatment.

[0103] Example 11

[0104] Steps (1) to (4) are the same as in Example 1.

[0105] (5) Dissolve MUA, EDC, and NHS in DMSO, add the solution to a Schlenk flask under argon purging, and stir at room temperature for 18 h. Then dissolve the polymer (Tet P2EG-SS) obtained in step (4) in DMSO and add it to the reaction solution, and continue the reaction at room temperature for 2 days. After the reaction is completed, dialyze the reaction solution and freeze-dry it to obtain the polymer Tet P2EG-SS-MUA.

[0106] (6) Add the polymer (Tet P2EG-SS-MUA) obtained in step (6), along with BAPO and butene glycol, to an EP tube, add DMF to dissolve it, and irradiate under ultraviolet light at a wavelength of 365 nm for 2 h. After the reaction is complete, dialyze the reaction solution, centrifuge, and freeze-dry the supernatant to obtain the polymer Tet P2EG-SS-MUA (P2SM). Un ).

[0107] (7) Determine the standard curve of Sun using ultraviolet spectrophotometry. Accurately weigh 1 mg of Sun into methanol solution to prepare a Sun standard solution. Take six centrifuge tubes and take 3 mL of the liquid from the Sun standard solution in sequence to prepare solutions with concentrations (C) of 1.25, 2.5, 5, 10, 20, and 40 ug / mL, respectively. Measure the absorbance (A) at 425 nm to establish the standard curve of Sun, which is A = 0.0614 C + 7E - 0.5.

[0108] (8) In order to prepare P2SM Un @Sun micelles, preparation of GSH-sensitive P2SM by dialysis Un @Sun micelles. P2SM Un15 mg of polymer and 1 mg of Sun were dissolved in 5 mL of DMSO, and the mixture was dialyzed for 48 h in a dialysis bag to remove unencapsulated Sun and solvent. The resulting solution was then centrifuged at 4000 rpm for 5 min, and the supernatant was collected to obtain P2SM. Un @Sun micelles

[0109] (9) Take the P2SM obtained in step (8) Un @Sun micelles were diluted 5 times in methanol for demulsification. Then, 3 mL was added to a quartz dish, and the absorbance was measured using a UV spectrophotometer. The concentration was calculated using the Sun standard curve determined in step (8). The P2SM concentration was then obtained using the calculation formula. Un EE and DL of Sun in @Sun micelles, quantitative data as follows: Figure 6 and Figure 7 As shown. The formula for calculating EE is m1 / m2*100%, and the formula for calculating DL is m1 / (m2+m3)*100%, where m1 is the drug-loaded mass of the carrier, m2 is the total mass of the drug, and m3 is the total mass of the carrier.

[0110] (10) Take the P2SM obtained in step (8) Un @Sun's micelle solution was analyzed by DLS using a Malvern Zeta Nanosizer to determine its particle size and Zeta potential. Quantitative data are as follows: Figure 4 and Figure 5 As shown.

[0111] Experimental results show that the P2SM prepared by the method of this invention... Un Micelles were successfully loaded with the hydrophobic, positively charged drug Sun. P2SM was prepared using a dialysis method. Un@ Sun micelles, in polymer P2SM Un P2SM was prepared by dialysis under the conditions of 15 mg feed, 1 mg Sun, and dissolution in DMSO. Un @Sun micelles had EE and DL of 18.29% and 1.14%, respectively. Further comparison revealed that, under the same preparation conditions, P2SM without sulfonic acid modification at the hydrophobic end... Un @Sun micelles exhibited low EE and DL; however, P2SM@Sun micelles with negatively charged sulfonic acid groups introduced at the hydrophobic end (Example 8) showed higher EE and DL. These results demonstrate that by attaching negatively charged sulfonic acid groups to the hydrophobic end of the micelles, their loading efficiency for hydrophobic and positively charged drugs can be significantly enhanced, thus validating the application potential of P2SM micelles in this type of drug delivery. Furthermore, P2SM... Un@Sun micelles, with a particle size of 152 nm and a zeta potential of -14.2 mV, can utilize the EPR effect of solid tumor tissue to achieve passive targeting and enrichment of drugs at the tumor site. The polymer micelles constructed in this invention serve as a functionally defined and reliable drug delivery platform, exhibiting significant advantages in loading and controlled release of both hydrophobic and positively charged drugs, providing solid experimental evidence for their further development and application in disease treatment.

[0112] Example 12

[0113] (1) MW1000 PEG (P1EG) was placed in a Schlenk flask, melted at 68 °C, dried under vacuum for 8 h, and then purged with argon for 5 min. THF was added to dissolve it. NaH was added under argon purging. The solution was stirred overnight at room temperature. Then epichlorohydrin was added and the reaction was stirred for 20 h. After the reaction was complete, the reaction mixture was filtered to remove excess NaH and salt. The filtered solution was concentrated and precipitated in diethyl ether. The precipitate was dried under vacuum to obtain the polymer DiepoxyP1EG.

[0114] (2) The polymer (Diepoxy P1EG) obtained in step (1) was mixed with a 0.1 M NaOH aqueous solution in a Schlenk flask and stirred at 60 °C for 10 h. Then hydrochloric acid was added to neutralize the solution. The water was evaporated under vacuum, and DCM and anhydrous sodium were added to the mixture in sequence for drying, followed by filtration. The DCM was evaporated under vacuum to obtain the polymer TetrahydroxylPEG (Tet P1EG), which was a white solid.

[0115] (3) The polymer (Tet P1EG) obtained in step (2) was placed in a Schlenk flask and dried under vacuum for 8 h, then purged with argon for 5 min. DCM was added to Tet P1EG to dissolve it. Meanwhile, CDI was dissolved in DCM, and the CDI solution was slowly added to the Tet P1EG solution at room temperature. The resulting mixture was stirred under a nitrogen atmosphere for 36 h. After the reaction was complete, saturated sodium chloride aqueous solution was added for extraction, and the organic layer was dried with sodium sulfate. The collected solution was used to evaporate DCM under vacuum to obtain the polymer Tet P1EG-CDI, which was a pale yellow solid powder.

[0116] (4) The polymer (Tet P1EG-CDI) obtained in step (3) was placed in a Schlenk flask and dried under vacuum for 8 h, then purged with argon for 5 min. Then, DMSO was added to Tet P1EG-CDI to dissolve it. In addition, cystamine dihydrochloride was dissolved in DMSO. The cystamine dihydrochloride solution and triethylamine were slowly added to the Tet P1EG-CDI solution. The resulting mixture was stirred at room temperature for 36 h. The reacted mixture was dialyzed and lyophilized to obtain the polymer Tet P1EG-SS.

[0117] (5) Butene glycol was placed in a Schlenk flask and purged with argon for 5 min. DMF was added to dissolve it. Sulfur trioxide pyridine was dissolved in DMF and slowly added dropwise to the butene glycol solution through a constant pressure funnel. The resulting mixture was allowed to react at room temperature for 3 days. After the reaction was complete, the reaction was placed in an ice bath and an appropriate amount of water was slowly added to quench the reaction. The pH of the reaction solution was then adjusted to 8 with 5 mol / L NaOH solution, and the water was removed by rotary evaporation. 100 mL of methanol was added, and the solution was dried with anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the methanol and DMF were removed by rotary evaporation to obtain sulfonated butene glycol (3-butene-1,2-diol-Sul).

[0118] (6) Dissolve MUA, EDC, and NHS in DMSO, add the solution to a Schlenk flask under argon purging, and stir at room temperature for 18 h. Then dissolve the polymer (Tet P1EG-SS) obtained in step (4) in DMSO and add it to the reaction solution, and continue the reaction at room temperature for 2 days. After the reaction is completed, dialyze the reaction solution and freeze-dry it to obtain the polymer Tet P1EG-SS-MUA.

[0119] (7) The polymer (Tet P1EG-SS-MUA) obtained in step (6) and the product (3-butene-1,2-diol-Sul) obtained in step (6) and BAPO were added to an ep tube, DMF was added to dissolve them, and the mixture was irradiated under ultraviolet light at a wavelength of 365 nm for 2 h. After the reaction was completed, the reaction solution was dialyzed, centrifuged, and the supernatant was lyophilized to obtain the polymer Tet P1EG-SS-MUA-Sul (P1SM).

[0120] (8) Determine the standard curve of Sun using ultraviolet spectrophotometry. Accurately weigh 1 mg of Sun into methanol solution to prepare a Sun standard solution. Take six centrifuge tubes and take 3 mL of the liquid from the Sun standard solution in sequence to prepare solutions with concentrations (C) of 1.25, 2.5, 5, 10, 20, and 40 ug / mL, respectively. Measure the absorbance (A) at 425 nm to establish the standard curve of Sun, which is A = 0.0614 C + 7E - 0.5.

[0121] (9) To prepare P1SM@Sun micelles, GSH-sensitive P1SM@Sun micelles were prepared by dialysis. 15 mg of P1SM polymer and 1 mg of Sun were dissolved in 5 mL of DMSO, and the mixture was placed in a dialysis bag and dialyzed for 48 h to remove unencapsulated Sun and solvent. The resulting solution was then centrifuged at 4000 rpm for 5 min, and the supernatant was collected to obtain P1SM@Sun micelles.

[0122] (10) The P1SM@Sun micelles obtained in step (9) by dissolving in DMSO were diluted 5 times in methanol for demulsification. Then, 3 mL was added to a quartz dish, and its absorbance was measured by a UV spectrophotometer. The concentration was calculated using the Sun standard curve determined in step (8). The EE and DL of Sun in the P1SM@Sun micelles were then obtained by calculation formulas, and the quantitative data are as follows: Figure 6 and Figure 7 As shown. The formula for calculating EE is m1 / m2*100%, and the formula for calculating DL is m1 / (m2+m3)*100%, where m1 is the drug-loaded mass of the carrier, m2 is the total mass of the drug, and m3 is the total mass of the carrier.

[0123] (11) The P1SM@Sun micelle solution obtained in step (9) was subjected to DLS analysis using a Malvern Zeta Nanosizer to determine its particle size and Zeta potential. The quantitative data are as follows: Figure 4 and Figure 5 As shown.

[0124] Experimental results show that the P1SM micelles prepared by the method of this invention can efficiently load the hydrophobic, positively charged drug Sun. P1SM@Sun micelles prepared by dialysis, with a feed ratio of 15 mg P1SM and 1 mg Sun, and DMSO as the solvent, exhibited excellent drug loading performance, with EE and DL reaching 39.04% and 2.44%, respectively. Further comparison revealed that, under the same preparation conditions, compared with the P2SM@Sun micelles in Example 8, the loading capacity of polymers prepared using different PEG molecular weights fluctuated to some extent for Sun. This was mainly due to the different steric hindrance of hydrophobic microdomains formed by the difference in the ratio of hydrophilic to hydrophobic chains in the polymer, and the different degrees of fit between these regions and the drug molecules. However, all micelles achieved stable encapsulation of the drug, demonstrating the good process robustness of this carrier system. In addition, P1SM... @ Sun micelles, with a particle size of 152 nm and a zeta potential of -12.6 mV, can utilize the EPR effect of solid tumor tissue to achieve passive targeting and accumulation of drugs at the tumor site. These results clearly demonstrate that by rationally controlling the molecular structure of polymer micelles, their loading efficiency for hydrophobic and positively charged drugs can be significantly improved. The micelle delivery platform constructed in this invention exhibits significant advantages in loading and controlled release of poorly soluble, positively charged drugs, providing a reliable experimental basis for their further development in the field of disease treatment.

[0125] Example 13

[0126] (1) MW3500 PEG (P3EG) was placed in a Schlenk flask, melted at 68 °C, dried under vacuum for 8 h, and then purged with argon for 5 min. THF was added to dissolve it. NaH was added under argon purging. The solution was stirred overnight at room temperature. Then epichlorohydrin was added and the reaction was stirred for 20 h. After the reaction was complete, the reaction mixture was filtered to remove excess NaH and salt. The filtered solution was concentrated and precipitated in diethyl ether. The precipitate was dried under vacuum to obtain the polymer DiepoxyP3EG.

[0127] (2) The polymer (Diepoxy P3EG) obtained in step (1) was mixed with a 0.1 M NaOH aqueous solution in a Schlenk flask and stirred at 60 °C for 10 h. Then hydrochloric acid was added to neutralize the solution. The water was evaporated under vacuum, and DCM and anhydrous sodium were added to the mixture in sequence for drying, followed by filtration. The DCM was evaporated under vacuum to obtain the polymer TetrahydroxylPEG (Tet P3EG), which was a white solid.

[0128] (3) The polymer (Tet P3EG) obtained in step (2) was placed in a Schlenk flask and dried under vacuum for 8 h, then purged with argon for 5 min. DCM was added to Tet P1EG to dissolve it. In addition, CDI was dissolved in DCM and the CDI solution was slowly added to the Tet P3EG solution at room temperature. The resulting mixture was stirred under a nitrogen atmosphere for 36 h. After the reaction was complete, saturated sodium chloride aqueous solution was added for extraction, and the organic layer was dried with sodium sulfate. The collected solution was used to evaporate DCM under vacuum to obtain the polymer Tet P3EG-CDI, which was a pale yellow solid powder.

[0129] (4) The polymer (Tet P3EG-CDI) obtained in step (3) was placed in a Schlenk flask and dried under vacuum for 8 h, then purged with argon for 5 min. Then, DMSO was added to Tet P3EG-CDI to dissolve it. In addition, cystamine dihydrochloride was dissolved in DMSO. The cystamine dihydrochloride solution and triethylamine were slowly added to the Tet P3EG-CDI solution. The resulting mixture was stirred at room temperature for 36 h. The reacted mixture was dialyzed and lyophilized to obtain the polymer Tet P3EG-SS.

[0130] (5) Butene glycol was placed in a Schlenk flask and purged with argon for 5 min. DMF was added to dissolve it. Sulfur trioxide pyridine was dissolved in DMF and slowly added dropwise to the butene glycol solution through a constant pressure funnel. The resulting mixture was allowed to react at room temperature for 3 days. After the reaction was complete, the reaction was placed in an ice bath and an appropriate amount of water was slowly added to quench the reaction. The pH of the reaction solution was then adjusted to 8 with 5 mol / L NaOH solution, and the water was removed by rotary evaporation. 100 mL of methanol was added, and the solution was dried with anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the methanol and DMF were removed by rotary evaporation to obtain sulfonated butene glycol (3-butene-1,2-diol-Sul).

[0131] (6) Dissolve MUA, EDC, and NHS in DMSO, add the solution to a Schlenk flask under argon purging, and stir at room temperature for 18 h. Then dissolve the polymer (Tet P3EG-SS) obtained in step (4) in DMSO and add it to the reaction solution, and continue the reaction at room temperature for 2 days. After the reaction is complete, dialyze the reaction solution and freeze-dry it to obtain the polymer Tet P3EG-SS-MUA.

[0132] (7) The polymer (Tet P3EG-SS-MUA) obtained in step (6) and the product (3-butene-1,2-diol-Sul) obtained in step (6) and BAPO were added to an ep tube, DMF was added to dissolve them, and the mixture was irradiated under ultraviolet light at a wavelength of 365 nm for 2 h. After the reaction was completed, the reaction solution was dialyzed, centrifuged, and the supernatant was lyophilized to obtain the polymer Tet P3EG-SS-MUA-Sul (P3SM).

[0133] (8) Determine the standard curve of Sun using ultraviolet spectrophotometry. Accurately weigh 1 mg of Sun into methanol solution to prepare a Sun standard solution. Take six centrifuge tubes and take 3 mL of the liquid from the Sun standard solution in sequence to prepare solutions with concentrations (C) of 1.25, 2.5, 5, 10, 20, and 40 ug / mL, respectively. Measure the absorbance (A) at 425 nm to establish the standard curve of Sun, which is A = 0.0614 C + 7E - 0.5.

[0134] (9) To prepare P3SM@Sun micelles, GSH-sensitive P3SM@Sun micelles were prepared by dialysis. 15 mg of P3SM polymer and 1 mg of Sun were dissolved in 5 mL of DMSO, and the mixture was placed in a dialysis bag and dialyzed for 48 h to remove unencapsulated Sun and solvent. The resulting solution was then centrifuged at 4000 rpm for 5 min, and the supernatant was collected to obtain P3SM@Sun micelles.

[0135] (10) The P3SM@Sun micelles obtained in step (9) by dissolving in DMSO were diluted 5 times in methanol for demulsification. Then, 3 mL was added to a quartz dish, and its absorbance was measured using a UV spectrophotometer. The concentration was calculated using the Sun standard curve determined in step (8). The EE and DL of Sun in the P3SM@Sun micelles were then obtained by calculation formulas, and the quantitative data are as follows: Figure 6 and Figure 7 As shown. The formula for calculating EE is m1 / m2*100%, and the formula for calculating DL is m1 / (m2+m3)*100%, where m1 is the drug-loaded mass of the carrier, m2 is the total mass of the drug, and m3 is the total mass of the carrier.

[0136] (11) The P1SM@Sun micelle solution obtained in step (9) was subjected to DLS analysis using a Malvern Zeta Nanosizer to determine its particle size and Zeta potential. The quantitative data are as follows: Figure 4 and Figure 5 As shown.

[0137] Experimental results show that the P3SM micelles prepared by the method of this invention can efficiently load the hydrophobic, positively charged drug Sun. P3SM@Sun micelles prepared by dialysis, with a feed ratio of 15 mg P3SM and 1 mg Sun, and using DMSO as solvent, exhibited excellent drug loading performance, with EE and DL reaching 45.92% and 2.87%, respectively. Further comparison revealed that, under the same preparation conditions, compared with the P3SM@Sun micelles in Example 8, the loading capacity of polymers prepared using different PEG molecular weights fluctuated to some extent for Sun. This was mainly due to the different steric hindrance of hydrophobic microdomains formed by the difference in the ratio of hydrophilic to hydrophobic chains in the polymer, and the different degrees of fit between these regions and the drug molecules. However, all micelles achieved stable encapsulation of the drug, demonstrating the good process robustness of this carrier system. In addition, P3SM... @ Sun micelles, with a particle size of 147 nm and a zeta potential of -14.1 mV, can utilize the EPR effect of solid tumor tissue to achieve passive targeting and accumulation of drugs at the tumor site. These results clearly demonstrate that by rationally controlling the molecular structure of polymer micelles, their loading efficiency for hydrophobic and positively charged drugs can be significantly improved. The micelle delivery platform constructed in this invention exhibits significant advantages in loading and controlled release of poorly soluble, positively charged drugs, providing a reliable experimental basis for their further development in the field of disease treatment.

[0138] Example 14

[0139] (1) MW6000 PEG (P6EG) was placed in a Schlenk flask, melted at 68 °C, dried under vacuum for 8 h, and then purged with argon for 5 min. THF was added to dissolve it. NaH was added under argon purging. The solution was stirred overnight at room temperature. Then epichlorohydrin was added and the reaction was stirred for 20 h. After the reaction was complete, the reaction mixture was filtered to remove excess NaH and salt. The filtered solution was concentrated and precipitated in diethyl ether. The precipitate was dried under vacuum to obtain the polymer DiepoxyP6EG.

[0140] (2) The polymer (Diepoxy P6EG) obtained in step (1) was mixed with a 0.1 M NaOH aqueous solution in a Schlenk flask and stirred at 60 °C for 10 h. Then hydrochloric acid was added to neutralize the solution. The water was evaporated under vacuum, and DCM and anhydrous sodium were added to the mixture in sequence for drying, followed by filtration. The DCM was evaporated under vacuum to obtain the polymer TetrahydroxylPEG (Tet P6EG), which was a white solid.

[0141] (3) The polymer (Tet P6EG) obtained in step (2) was placed in a Schlenk flask and dried under vacuum for 8 h, then purged with argon for 5 min. DCM was added to Tet P6EG to dissolve it. Meanwhile, CDI was dissolved in DCM and the CDI solution was slowly added to the Tet P6EG solution at room temperature. The resulting mixture was stirred under a nitrogen atmosphere for 36 h. After the reaction was complete, saturated sodium chloride aqueous solution was added for extraction, and the organic layer was dried with sodium sulfate. The collected solution was used to evaporate DCM under vacuum to obtain the polymer Tet P6EG-CDI, which was a pale yellow solid powder.

[0142] (4) The polymer (Tet P6EG-CDI) obtained in step (3) was placed in a Schlenk flask and dried under vacuum for 8 h, then purged with argon for 5 min. Then, DMSO was added to Tet P6EG-CDI to dissolve it. In addition, cystamine dihydrochloride was dissolved in DMSO. The cystamine dihydrochloride solution and triethylamine were slowly added to the Tet P6EG-CDI solution. The resulting mixture was stirred at room temperature for 36 h. The reacted mixture was dialyzed and lyophilized to obtain the polymer Tet P6EG-SS.

[0143] (5) Butene glycol was placed in a Schlenk flask and purged with argon for 5 min. DMF was added to dissolve it. Sulfur trioxide pyridine was dissolved in DMF and slowly added dropwise to the butene glycol solution through a constant pressure funnel. The resulting mixture was allowed to react at room temperature for 3 days. After the reaction was complete, the reaction was placed in an ice bath and an appropriate amount of water was slowly added to quench the reaction. The pH of the reaction solution was then adjusted to 8 with 5 mol / L NaOH solution, and the water was removed by rotary evaporation. 100 mL of methanol was added, and the solution was dried with anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the methanol and DMF were removed by rotary evaporation to obtain sulfonated butene glycol (3-butene-1,2-diol-Sul).

[0144] (6) Dissolve MUA, EDC, and NHS in DMSO, add the solution to a Schlenk flask under argon purging, and stir at room temperature for 18 h. Then dissolve the polymer (Tet P6EG-SS) obtained in step (4) in DMSO and add it to the reaction solution, and continue the reaction at room temperature for 2 days. After the reaction is complete, dialyze the reaction solution and freeze-dry it to obtain the polymer Tet P6EG-SS-MUA.

[0145] (7) The polymer (Tet P6EG-SS-MUA) obtained in step (6) and the product (3-butene-1,2-diol-Sul) obtained in step (6) and BAPO were added to an EP tube, DMF was added to dissolve them, and the mixture was irradiated under ultraviolet light at a wavelength of 365 nm for 2 h. After the reaction was completed, the reaction solution was dialyzed, centrifuged, and the supernatant was lyophilized to obtain the polymer Tet P1EG-SS-MUA-Sul (P6SM).

[0146] (8) Determine the standard curve of Sun using ultraviolet spectrophotometry. Accurately weigh 1 mg of Sun into methanol solution to prepare a Sun standard solution. Take six centrifuge tubes and take 3 mL of the liquid from the Sun standard solution in sequence to prepare solutions with concentrations (C) of 1.25, 2.5, 5, 10, 20, and 40 ug / mL, respectively. Measure the absorbance (A) at 425 nm to establish the standard curve of Sun, which is A = 0.0614 C + 7E - 0.5.

[0147] (9) To prepare P6SM@Sun micelles, GSH-sensitive P6SM@Sun micelles were prepared by dialysis. 15 mg of P6SM polymer and 1 mg of Sun were dissolved in 5 mL of DMSO, and the mixture was placed in a dialysis bag and dialyzed for 48 h to remove unencapsulated Sun and solvent. The resulting solution was then centrifuged at 4000 rpm for 5 min, and the supernatant was collected to obtain P6SM@Sun micelles.

[0148] (10) The P6SM@Sun micelles obtained in step (9) by dissolving in DMSO were diluted 5 times in methanol for demulsification. Then, 3 mL was added to a quartz dish, and its absorbance was measured by a UV spectrophotometer. The concentration was calculated using the Sun standard curve determined in step (8). The EE and DL of Sun in the P6SM@Sun micelles were then obtained by calculation formulas, and the quantitative data are as follows: Figure 6 and Figure 7 As shown. The formula for calculating EE is m1 / m2*100%, and the formula for calculating DL is m1 / (m2+m3)*100%, where m1 is the drug-loaded mass of the carrier, m2 is the total mass of the drug, and m3 is the total mass of the carrier.

[0149] (11) The P6SM@Sun micelle solution obtained in step (9) was subjected to DLS analysis using a Malvern Zeta Nanosizer to determine its particle size and Zeta potential. The quantitative data are as follows: Figure 4 and Figure 5 As shown.

[0150] Experimental results show that the P3SM micelles prepared by the method of this invention can efficiently load the hydrophobic, positively charged drug Sun. P3SM@Sun micelles prepared by dialysis, with a feed ratio of 15 mg P3SM and 1 mg Sun, and using DMSO as solvent, exhibited excellent drug loading performance, with EE and DL reaching 30.00% and 1.87%, respectively. Further comparison revealed that, under the same preparation conditions, compared with the P3SM@Sun micelles in Example 8, the loading capacity of polymers prepared using different PEG molecular weights fluctuated to some extent for Sun. This was mainly due to the different steric hindrance of hydrophobic microdomains formed by the difference in the ratio of hydrophilic to hydrophobic chains in the polymer, and the different degrees of fit between these regions and the drug molecules. However, all micelles achieved stable encapsulation of the drug, demonstrating the good process robustness of this carrier system. In addition, P3SM... @ Sun micelles, with a particle size of 150 nm and a zeta potential of -8.3 mV, can utilize the EPR effect of solid tumor tissue to achieve passive targeting and accumulation of drugs at the tumor site. These results clearly demonstrate that by rationally controlling the molecular structure of polymer micelles, their loading efficiency for hydrophobic and positively charged drugs can be significantly improved. The micelle delivery platform constructed in this invention exhibits significant advantages in loading and controlled release of poorly soluble, positively charged drugs, providing a reliable experimental basis for their further development in the field of disease treatment.

[0151] Example 15

[0152] Steps (1) to (9) are the same as in Example 8.

[0153] (10) The P2SM@Sun micelle solution obtained in step (9) was used to study the release of Sun from P2SM@Sun micelles in the presence of GSH by dialysis. In short, 1 mL of 74 ug / mL P2SM@Sun micelles was placed in a dialysis bag (MWCO 3.5 kDa) and clamped, then immersed in 45 mL of 0.1 M PBS buffer (phosphate buffer) containing 1% (w / v) Tween 80 and no GSH. The experiment was performed in a shaker at 37 °C at 100 rpm. 1 mL aliquots of culture medium were taken at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h, and the same volume of fresh culture medium was added. The release of Sun from the micelles was measured by UV spectrophotometry, and the quantitative data are as follows: Figure 8 As shown. The formula for cumulative release (Er) is: Where Ve is the replacement volume of PBS, Vo is the total volume of the release medium, Ci is the concentration of the release solution at the i-th replacement sampling, m is the total mass of the drug carried by the micelles, and n is the number of PBS replacements.

[0154] (11) As control group 1, the release of Sun from the P2SM@Sun micelles obtained in step (9) was studied by dialysis in the presence of GSH. In short, 1 mL of 74 ug / mL P2SM@Sun micelles was placed in a dialysis bag (MWCO 3.5 kDa) and clamped, then immersed in 45 mL of 0.1 MPBS buffer solution containing 1% (w / v) Tween 80 and 5 mM GSH. The experiment was conducted at 37 °C in a shaker at 100 rpm. 1 mL aliquots of culture medium were taken at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h, and the same volume of fresh culture medium was added. The release of Sun from the micelles was measured by UV spectrophotometry, and the quantitative data are as follows: Figure 8 As shown. The formula for cumulative release (Er) is: Where Ve is the replacement volume of PBS, Vo is the total volume of the release medium, Ci is the concentration of the release solution at the i-th replacement sampling, m is the total mass of the drug carried by the micelles, and n is the number of PBS replacements.

[0155] (12) As control group 2, the release of Sun from the P2SM@Sun micelles obtained in step (9) was studied by dialysis in the presence of GSH. In short, 1 mL of 74 ug / mL P2SM@Sun micelles was placed in a dialysis bag (MWCO 3.5 kDa) and clamped, then immersed in 45 mL of 0.1 MPBS buffer solution containing 1% (w / v) Tween 80 and 10 mM GSH. The experiment was conducted at 37 °C in a shaker at 100 rpm. 1 mL aliquots of culture medium were taken at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h, and the same volume of fresh culture medium was added. The release of Sun from the micelles was measured by UV spectrophotometry, and the quantitative data are as follows: Figure 8 As shown. The formula for cumulative release (Er) is: Where Ve is the replacement volume of PBS, Vo is the total volume of the release medium, Ci is the concentration of the release solution at the i-th replacement sampling, m is the total mass of the drug carried by the micelles, and n is the number of PBS replacements.

[0156] Experimental results show that the P2SM micelles prepared by the method of this invention can efficiently load the hydrophobic, positively charged drug Sun. P2SM@Sun prepared by dialysis releases the drug in the presence of high GSH. The results show that the release of Sun differs significantly under different GSH concentrations, with cumulative release percentages of 18.05% (0 mM GSH), 53.32% (5 mM GSH), and 87.67% (10 mM GSH) after 24 h, respectively. The sustained drug release in the presence of high GSH indicates that Sun is rapidly released after entering the tumor, thus quickly exerting its therapeutic effect. These results fully demonstrate that polymer micelles, after being adequately loaded with hydrophobic and positively charged drugs, can rapidly release the drug in a high GSH environment. The micelle delivery platform constructed in this invention exhibits significant advantages in loading and controlled release of poorly soluble, positively charged drugs, providing a reliable experimental basis for its further development in the field of disease treatment.

[0157] Example 16

[0158] Steps (1) to (9) are the same as in Example 8.

[0159] (10) Take the P2SM@Sun micelle solution obtained in step (9), add hyaluronic acid to the concentrated micelle solution to prepare a mixed solution, wherein the concentration of Sun is 250 μg / mL and the concentration of hyaluronic acid is 35 mg / mL, add it to the microneedle mold, centrifuge at 4000 rpm for 3 min, repeat this process three times, add hyaluronic acid solution (300 mg / mL), and take PVP solution (300 mg / mL) to use as a base. Dry at room temperature for 12 h, and peel off after drying to obtain P2SM@Sun microneedles.

[0160] (11) Take the P2SM@Sun microneedles obtained in step (9) and use a camera to photograph the morphology of the microneedles as shown in the image. Figure 9 As shown. Additionally, to determine the Sun-loaded content in the microneedle tips, all tips were scraped off with a scalpel, dissolved in water, diluted with methanol, and the Sun content was determined using a UV spectrophotometer.

[0161] Experimental results show that the P2SM micelles prepared using the method of this invention can efficiently load hydrophobic, positively charged drugs (Sun). P2SM@Sun, prepared by dialysis, was applied to microneedle loading for local drug delivery. The Sun content in the P2SM@Sun microneedles prepared by this invention was measured to be 43 μg. When this drug delivery system was applied, after the hyaluronic acid in the microneedle tip at the tumor site dissolved, P2SM@Sun reached the tumor site and released Sun in response to the high GSH environment of the tumor microenvironment, thereby exerting its therapeutic effect. These results fully demonstrate that polymer micelles, after being adequately loaded with hydrophobic and positively charged drugs, can rapidly release drugs in a high GSH (glutathione) environment and can be applied to microneedles for local tumor treatment. The micelle delivery platform constructed by this invention exhibits significant advantages in loading and controlled release of poorly soluble, positively charged drugs, providing a reliable experimental basis for its further development in the field of disease treatment.

Claims

1. A method for preparing responsive micelles for loading hydrophobic positively charged drugs, characterized in that, Includes the following steps: Step 1: Using polyethylene glycol as a starting material, react with epichlorohydrin to prepare polyethylene glycol with diepoxy end-modified. Step 2: The diepoxy-terminated polyethylene glycol is subjected to a ring-opening reaction in an alkaline aqueous solution to obtain tetrahydroxy-terminated polyethylene glycol. Step 3: React the tetrahydroxy-terminated polyethylene glycol with CDI to activate its terminal hydroxyl groups and obtain CDI-activated tetrahydroxy polyethylene glycol. Step 4: CDI-activated tetrahydroxy polyethylene glycol is reacted with cystamine dihydrochloride in the presence of triethylamine to introduce a linker arm containing a disulfide bond, thereby obtaining disulfide-linked tetrahydroxy polyethylene glycol. Step 5: After activating undecylmercaptoundecanoic acid, react it with tetrahydroxy polyethylene glycol with disulfide bonds obtained in step 4 to obtain disulfide bonded polyethylene glycol grafted with undecylmercaptoundecanoic acid. Step 6: Mix the disulfide bond polyethylene glycol grafted with undecylmercaptoundecanoic acid obtained in step 5, the photoinitiator, and sulfonated butene glycol. Under 365 nm ultraviolet light irradiation, the negatively charged hydrophobic segments are connected to both ends of the polymer through a mercapto-olefin click chemical reaction to obtain an ABA-type triblock functionalized polymer. Step 7: Dissolve the ABA-type triblock functionalized polymer obtained in step 6 in dimethyl sulfoxide, place the solution in a dialysis bag and dialyze for 48 h to remove unencapsulated sunitinib and solvent, centrifuge the resulting solution at 4000 rpm for 5 min, and take the supernatant to prepare responsive micelles for loading hydrophobic positively charged drugs.

2. The preparation method according to claim 1, characterized in that, The preparation steps of sulfonated butenyl glycol in step 6 are as follows: Sulfonated butenyl glycol was prepared by reacting butenyl glycol with a sulfur trioxide pyridine complex.

3. The preparation method according to claim 1, characterized in that, The photoinitiator is phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide.

4. A responsive micelle loaded with a hydrophobic positively charged drug, prepared by the method of claim 1.

5. The micelles according to claim 4, characterized in that, The molecular weight range of the hydrophilic polyethylene glycol central segment in the micelles is 1000 to 6000 Da.

6. The micelles according to claim 4, characterized in that, The micelles have a zeta potential ranging from -6.25 mV to -20.7 mV.

7. The micelles according to claim 4, characterized in that, The hydration dynamic diameter of the micelles ranges from 105 nm to 150 nm.

8. The micelles according to claim 4, characterized in that, The encapsulation efficiency of the micelles for the drug ranges from 13.42% to 49.21%.

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