A method for preparing acid-sensitive environment-responsive PEGylated self-assembled micelles and its application
By using an acid-sensitive environment-responsive PEGylated self-assembled micelles, the problems of poor water solubility of SKLB060 and premature drug release during in vivo circulation of nanocarriers were solved, achieving tumor-targeted delivery and controlled release of 0831A, improving anti-tumor efficacy and reducing systemic toxicity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- THE WEST CHINA SECOND UNIV HOSPITAL OF SICHUAN
- Filing Date
- 2026-03-01
- Publication Date
- 2026-06-02
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Figure CN122123975A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to a method for preparing and applying acid-sensitive environment-responsive PEGylated self-assembled micelles. Background Technology
[0002] Cancer is a polygenic and multicellular disease characterized by uncontrolled cell growth stimulated by environmental factors, resulting in a significantly poor clinical prognosis. Microtubules (MTs) are effective intracellular anti-tumor targets. Tubulin inhibitors exert cytotoxic effects on cells by affecting microtubule function, demonstrating good activity in the treatment of solid tumors and hematological malignancies.
[0003] SKLB060, synthesized by the research group of Professor Chen Lijuan at the State Key Laboratory of Biotherapy, Sichuan University, is a microtubule depolymerizing agent with excellent anti-tumor effects. However, its poor water solubility makes intravenous administration difficult. To improve the water solubility and bioavailability of SKLB060, a series of SKLB060 amino acid derivatives were synthesized, and SKLB060-methionine (0831A) was selected as the optimal one. Previous studies have shown that 0831A retains the good anti-tumor cell proliferation activity of SKLB060. Although 0831A improves the solubility of SKLB060 to some extent, significant irritation and toxicity reactions still occurred during initial intravenous administration.
[0004] Nanocarriers are now widely used for drug delivery. However, after entering the body, the drug delivery system may experience premature drug release during circulation, leading to insufficient drug release after accumulation in the target tissue. Currently, the most common method is to modify the surface of nanocarriers or drugs with flexible backbone water-soluble polymers such as polyethylene glycol (PEG), so that the drug carrier is not destroyed during blood circulation and avoids phagocytosis of the drug and drug carrier by the mononuclear phagocytic system (MPS). Simultaneously, to achieve selective drug release at tumor sites, tumor microenvironment-responsive nanocarriers are designed, utilizing the pH difference between the tumor and normal tissue microenvironments to design acid-sensitive micelles. It has been reported that the pH of normal tissues and blood is between 7.4 and 7.5, while the pH of the interstitial fluid in tumor tissues decreases to between 6.5 and 6.7 due to ischemia and abnormal extracellular acidification. pH-sensitive polymer micelles can improve the shortcomings of antitumor drugs, such as poor water solubility and short half-life, enabling selective drug release at tumor sites, reducing systemic toxicity, and exhibiting better antitumor activity. The basic method for preparing acid-sensitive micelles is to directly covalently attach drug molecules to the surface of existing nanocarriers using acid-labile chemical bonds, or to construct new nanocarriers. Acid-hydrolyzable chemical bonds include hydrazone bonds, cis-acetyl bonds, and ether bonds. Through covalent bonding, controlled release of anticancer drugs can be achieved through physical encapsulation or adsorption.
[0005] To reduce the in vivo toxicity of 0831A, it is necessary to prepare acid-responsive PEGylated nanomicelles for the tumor microenvironment and construct a polymeric micelle drug delivery system based on acid-sensitive chemical bonds. Existing technologies have attempted to construct acid-sensitive micelles using hydrazone bonds. For example, Chinese patent document CN118304437A discloses a pH-responsive acid-sensitive micelle, which involves first preparing mPEG-β-CD, then preparing 0831A-Hyd-AD, and finally utilizing the host-guest inclusion interaction between β-cyclodextrin and adamantane to non-covalently assemble 0831A-Hyd-AD and mPEG-β-CD into micelles. Although this technical solution utilizes the acid-sensitive properties of acylhydrazone bonds, the system relies on the non-covalent interaction between cyclodextrin and adamantane to achieve the final micelle assembly and stabilization. This multi-component non-covalent assembly system has the following potential problems: (1) The strength and stability of host-guest interactions may be affected by the complex biological environment in the body (such as protein competitive binding), and there is a risk that micelles may dissociate prematurely or become structurally unstable during cycling. (2) The preparation process involves multiple steps, including the synthesis of mPEG-β-CD, the synthesis of 0831A-Hyd-AD, and subsequent physical assembly. The process route is relatively long and there are many quality control points. (3) The final structure of micelles is driven and maintained by non-covalent forces, and its structural definition, batch-to-batch uniformity and long-term physical stability are challenged.
[0006] Therefore, in order to meet the in vivo delivery requirements of 0831A, there is an urgent need to develop a new acid-sensitive PEGylated micelle system with a simpler and more defined structure, easier preparation, and good stability in in vivo circulation, so as to more effectively achieve tumor-targeted delivery and controlled release of 0831A, while enhancing anti-tumor efficacy and significantly reducing its systemic toxicity.
[0007] To address these issues, this application proposes a method for preparing acid-sensitive environment-responsive PEGylated self-assembled micelles and its application. Summary of the Invention
[0008] The purpose of this invention is to solve the technical problems mentioned in the background art above, and to provide a method for preparing acid-sensitive environment-responsive PEGylated self-assembled micelles and their application. These micelles are formed by the self-assembly of simple amphiphilic prodrug molecules, can remain stable at physiological pH, and specifically release active drugs rapidly in the slightly acidic environment of tumors, thereby effectively reducing the systemic toxicity of 0831A and improving its anti-tumor efficacy.
[0009] The above-mentioned objective of the present invention is achieved as follows: One aspect of this invention provides a method for preparing acid-sensitive, environment-responsive PEGylated self-assembled micelles. The micelles are formed by the self-assembly of a 0831A-Hyd-mPEG conjugate in an aqueous solution. The 0831A-Hyd-mPEG conjugate is a product prepared via the following reaction pathway: First, monomethoxy polyethylene glycol is reacted with p-aldehyde benzoic acid to generate an intermediate product mPEG-CHO with an aldehyde group at the end; then, the mPEG-CHO is reacted with 0831A, so that the aldehyde group at the end of mPEG-CHO forms an acylhydrazone bond with the amino group of 0831A, thus obtaining the 0831A-Hyd-mPEG conjugate, which has the following structure (I): (I) Wherein, mPEG is monomethoxy polyethylene glycol with a number average molecular weight of 1000, 2000 or 5000, and 0831A is SKLB060-methionine; The preparation method includes the following steps: (1) Preparation of 0831A-Hyd-mPEG conjugate; (2) The 0831A-Hyd-mPEG conjugate obtained in step (1) is self-assembled in an aqueous solution to form micelles by thin film hydration or solvent evaporation.
[0010] Furthermore, in step (1), the number-average molecular weight of the mPEG is 2000.
[0011] Furthermore, in step (1), the reaction of mPEG with p-aldehyde benzoic acid is carried out in the presence of the dehydrating agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and the catalyst 4-dimethylaminopyridine; the reaction of mPEG-CHO with 0831A is carried out under conditions that provide an alkaline environment.
[0012] Furthermore, in step (2), the self-assembly method is a solvent evaporation method, specifically: the 0831A-Hyd-mPEG conjugate is dissolved in dichloromethane, the resulting solution is added dropwise to stirred deionized water, and the organic solvent is removed after mixing to obtain a micelle solution.
[0013] The present invention also provides an acid-sensitive environment-responsive PEGylated self-assembled micelles, which are prepared by the above-described preparation method.
[0014] Furthermore, the micelles are formed by the self-assembly of a conjugate formed by linking mPEG with 0831A via acylhydrazone bonds, which has a number average molecular weight of 2000.
[0015] Furthermore, the average particle size of the micelles is 157.1 ± 14.73 nm, and the polymer dispersibility index is 0.141 ± 0.082.
[0016] Furthermore, the critical micelle concentration is 9.6 μg / mL; The micelles achieved a cumulative drug release rate of 69.46±2.13% in a pH 5.0 buffer solution over 48 hours, and a cumulative drug release rate of 22.94±0.67% in a pH 7.4 buffer solution over 48 hours.
[0017] The present invention also provides a pharmaceutical composition comprising the above-described acid-sensitive, environment-responsive PEGylated self-assembled micelles and a pharmaceutically acceptable carrier.
[0018] The present invention also provides the use of acid-sensitive environment-responsive PEGylated self-assembled micelles or the above-described pharmaceutical compositions in the preparation of a medicament for treating cancer, wherein the cancer is colorectal cancer.
[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention abandons the complex non-covalent assembly mode of the prior art that relies on the host-guest interaction of cyclodextrin-adamantane, and designs a simple covalent reaction route to synthesize target micelles. The route is as follows: monomethoxy polyethylene glycol (mPEG) first reacts with p-aldehyde benzoic acid to form an ester bond, and the generated intermediate mPEG-CHO then forms an acylhydrazone bond with drug 0831A through its amino group, thereby directly obtaining a structurally well-defined "mPEG-acylhydrazone bond-0831A" amphiphilic single conjugate molecule. This molecule can self-assemble into micelles in water by being connected only by covalent bonds, which simplifies the composition of micelles and improves the structural clarity and batch-to-batch reproducibility.
[0020] 2. The preparation method of the present invention is simple and does not require the synthesis of multi-component host / guest molecules and subsequent physical mixing and assembly. Its core process only includes the aldehyde modification of mPEG, the acylhydrazone bond connection with 0831A, and the self-assembly of the final conjugate. The process route is clear and shortened, which is more conducive to quality control and large-scale production.
[0021] 3. The micelles obtained by the present invention have suitable nanoparticle size (approximately 157 nm) and low PDI, uniform distribution, and excellent stability and pH responsiveness. In vitro release experiments have demonstrated that they have significant acid-sensitive drug release characteristics, with a high drug release rate (69.46±2.13% after 48 hours) under simulated tumor microenvironment conditions at pH 5.0, while the release is slow under physiological conditions at pH 7.4 (22.94±0.67% after 48 hours), effectively avoiding premature drug leakage. Serum stability experiments show that the micelles have good stability in the blood environment.
[0022] 4. The micelles obtained by the present invention have significant in vitro and in vivo efficacy and safety. The micelles maintain the anti-tumor cell activity of 0831A and can promote cellular uptake. In the CT26 colon cancer-bearing mouse model, compared with the 0831A raw material, the micelles of the present invention can significantly inhibit tumor growth, while greatly reducing the systemic toxicity of the drug and keeping the animal weight stable. Pharmacokinetic studies show that the micelles can significantly prolong the in vivo circulation time of 0831A and improve its bioavailability.
[0023] 5. The micelles prepared by the present invention can be successfully prepared into lyophilized powder, which has good storage stability under suitable conditions and is convenient for clinical storage and transportation. Attached Figure Description
[0024] Figure 1 This is the chemical reaction formula of mPEG-0831A in the embodiments of the present invention; Figure 2 Here are the chemical structural formula and 1H-NMR spectrum of the intermediate product mPEG-CHO in this embodiment of the invention; Figure 3 Here are the chemical structural formula and 1H-NMR spectrum of 0831A-Hyd-mPEG in this embodiment of the invention; Figure 4 These are nanomicelle particle size diagrams from embodiments of the present invention (emulsion evaporation method (A), thin film hydration method (B)). Figure 5 This is a micelle size distribution diagram of 0831A-Hyd-mPEG n (n=1000 / 2000 / 5000) in an embodiment of the present invention; Figure 6 This is the critical micelle concentration of 0831A-Hyd-mPEG2000 micelles in the embodiments of the present invention; Figure 7 This is a TEM image of the 0831A-Hyd-mPEG micelles after reconstitution in an embodiment of the present invention (scale bar is 100 nm). Figure 8 This is a one-month stability test of micelle 0831A in this embodiment of the invention; Figure 9 This is a graph showing the absorbance change of 0831A-Hyd-mPEG micelles in serum in an embodiment of the present invention; Figure 10 This is an in vitro release simulation of 0831A micelles in 5% TW-80 solutions at pH 5.0 and pH 7.4 in the embodiments of the present invention (the results are average values ± SD values). Figure 11 This is the one-month stability test result of the 0831A-Hyd-mPEG lyophilized powder in the embodiments of the present invention; Figure 12 The figures show the in vitro cytotoxicity test results of 0831A active pharmaceutical ingredient and 0831A micelles in the embodiments of the present invention (all results are average values ± SD values). Figure 13 This is the flow cytometry result of CT26 cells taking up coumarin-6 in an embodiment of the present invention; Figure 14 The total uptake of 0831A by CT26 cells was detected by HPLC in this embodiment of the invention (all results are average ± SD values). Figure 15 The following are flow cytometry results of CT26 cell apoptosis and cell cycle arrest induced by 0831A active pharmaceutical ingredient and 0831A-Hyd-mPEG micelles in this embodiment of the invention: (A) In vitro apoptosis experiment results of 0831A active pharmaceutical ingredient and 0831A micelles; (B) In vitro cell cycle arrest experiment results of 0831A active pharmaceutical ingredient and 0831A micelles; (C) Statistical analysis of scratch assay; data are presented as mean ± SD, ***, P < 0.1 compared with the control group; (D) Statistical analysis of tube formation assay, **, P < 0.01 compared with the control group). Figure 16 In this embodiment of the invention, 0831A micelles inhibit the proliferation activity of HUVEC cells; Figure 17 This is an example of the scratch and tube formation experiments of 0831A micelles on HUVEC cells in this invention ((A) scratch results of 0831A micelles; (B) tube formation results of 0831A micelles; (C) statistics of the scratch experiment; data are presented as mean ± SD, ***, P < 0.1 compared with the control group; (D) statistics of the tube formation experiment, **, P < 0.01 compared with the control group). Detailed Implementation
[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0026] The implementation of the present invention will be described in detail below with reference to specific embodiments.
[0027] This invention provides a method for preparing acid-sensitive environment-responsive PEGylated self-assembled micelles and their application. Specifically, it describes a method for preparing PEGylated self-assembled micelles based on acylhydrazone bonds and exhibiting pH responsiveness to the tumor microenvironment, and the application of these micelles in antitumor drug delivery. The specific implementation of this invention is described below.
[0028] Example 1: Synthesis and characterization of 0831A-Hyd-mPEG with different molecular weights 1. Experimental Materials 1.1 Instruments Gel permeation chromatograph: RID-20 differential refractive index detector, Shimadzu Corporation, Japan.
[0029] 1.2 Reagents Monomethyl ether polyethylene glycol 1000 / 2000 / 5000 (mPEG) 1K / 2k / 5K Tokyo Chemical Industry Co., Ltd. Dichloromethane: Analytical grade, Chengdu Kelong Chemical Reagent Factory; Tetrahydrofuran (THF): Analytical grade, Chengdu Kelong Chemical Reagent Factory; Polystyrene standards: Polymer laboratories; 0831A: Prepared by the research group of Chen Lijuan, State Key Laboratory of Biotherapy; Toluene: Analytical grade, Chengdu Kelong Chemical Reagent Factory; p-Aldehydebenzoic acid: analytical grade, Chengdu Kelong Chemical Reagent Factory; Acetone: Analytical grade, Tianjin Fuyu Fine Chemical Co., Ltd.; Dichloromethane: Analytical grade, Chengdu Kelong Chemical Reagent Factory.
[0030] 2. Experimental Methods 2.1 Acid-sensitive design Monomethoxy polyethylene glycol (mPEG) (Mn = 1000, 2000, 5000) was modified with p-aldehyde benzoic acid to impart an aldehyde active group; the aldehyde group then formed an acid-sensitive acylhydrazone bond with the amino group of 0831A; the synthetic routes for mPEG-CHO and 0831A-Hyd-mPEG are shown below. Figure 1 .
[0031] 2.2 Synthesis of 0831A-Hyd-mPEG Dissolve p-aldehyde benzoic acid in 40 mL of dichloromethane (R=1.2), add triethylamine dropwise until clear, and then add mPEG sequentially. 1000 / 2000 / 5000 The reaction mixture was prepared using EDCI (R=2) as a dehydrating agent and DMAP (R=0.2) as a catalyst, with TEA (R=0.15) providing an alkaline environment. The reaction was carried out at room temperature for 48 h under N2 protection. The reaction solution was completely evaporated by rotary evaporation, the mixture was dissolved in deionized water and placed in a dialysis bag. After dialysis for 72 h, the mixture was extracted with dichloromethane and dried under reduced pressure to obtain the intermediate product. The intermediate product was dissolved in toluene and subjected to dehydration at 120 °C. The reactant ratio was 1:1. Purification was performed using a silica gel column with dichloromethane to remove toluene:methanol in a 10:1 ratio. The reaction formula for mPEG-Hyd-0831A is shown below. Figure 1 .
[0032] 2.3 Characterization of 0831A-Hyd-mPEG 2.3.1. Detection by proton nuclear magnetic resonance (NMR) spectrum The 1H NMR spectra of intermediate products mPEG-CHO and 0831A-Hyd-mPEG ( 1 Characterization was performed using a Bruker AV-11 400 NMR spectrometer (H-NMR). CDCl3 was selected as the solvent, and tetramethylsilane (TMS) was used as the internal standard.
[0033] 2.3.2 Gel permeation chromatography GPC measurements typically determine relative molecular weights, using methods such as monodisperse standard calibration, progressive differential calibration, and universal calibration. This sample underwent a universal calibration method to determine the polymer molecular weight distribution.
[0034] Take an appropriate amount of the sample to be tested, dissolve and dilute it with THF to prepare a solution containing approximately 5 mg in 1 mL, shake, and use this as the test solution. Take 5 reference standards (of known molecular weight) and prepare 1 mL of 5 mg reference standard solution using the same method. Perform molecular exclusion chromatography (General Chapter 0514, Chinese Pharmacopoeia 2015), using a lipophilic gel chromatography column, tetrahydrofuran, 45 ℃, 1 mL / min, and a differential refractive index detector at 45 ℃. Inject 20 μL of the above reference standard solution into the liquid chromatograph, record the chromatograms, and calculate the regression equation using GPC software. Take 20 μL of the test solution and determine it using the same method. Calculate the weight-average molecular weight Mw, number-average molecular weight Mn, and polydispersity index D (Mw / Mn) of the test sample using the universal correction method. The basic instrument operation procedure is as follows: column rinsing with aqueous phase, column packing with mobile phase, RID removal of light refraction signal (approximately 20 min), and final injection.
[0035] Chromatographic conditions were as follows: High-performance liquid chromatograph (HPLC) with a Shimadzu RID-20 differential refractive index detector; GPC column: Waters Styragel HR4 THF gel chromatography column; mobile phase: chromatographic grade THF (TEDIA, USA); flow rate: 1.0 mL / min; column temperature: 35 ℃; polystyrene standard: 0.5% THF solution; method: a standard curve was prepared using narrow-distribution polystyrene, and relative correction method was used.
[0036] 3. Experimental Results 3.1 Synthesis and Characterization of 0831A-Hyd-mPEG Conjugate Monomethoxy polyethylene glycol (mPEG) of different molecular weights (Mn = 1000, 2000, 5000) was condensed with p-aldehyde benzoic acid to obtain mPEG-CHO with a terminal aldehyde group. Then, 0831A reacted with mPEG-CHO of different molecular weights. The amino group at the terminal of 0831A reacted with the aldehyde group at the terminal of mPEG-CHO to form an acid-sensitive amide bond, thus yielding 0831A-Hyd-mPEG of different molecular weights. n (n=1000, 2000, 5000). Take 0831A-Hyd-mPEG 2000 Using the data results as an example, a graph was plotted, and the successful synthesis of the polymer was confirmed by using nuclear magnetic resonance hydrogen spectroscopy.
[0037] Characterization of mPEG-CHO: mPEG was esterified with p-aldehyde benzoic acid to obtain mPEG-CHO. The 1H NMR spectrum of the intermediate mPEG-CHO is shown below. Figure 2 As shown, the chemical structure is consistent with the 1H-NMR spectrum, and the positions of all peaks are marked. The spectrum shows a peak at δ=3.80 (c') representing the polyethylene glycol methylene (-CH2) group, a peak at δ=8.00~8.25 ppm (a') representing the characteristic peak of p-aldehyde benzoic acid, and a characteristic peak of the aldehyde group at δ=10.20 ppm (b'). This indicates the successful synthesis of mPEG-CHO.
[0038] Characterization of 0831A-Hyd-mPEG: 0831A reacts with mPEG-CHO to obtain 0831A-Hyd-mPEG. 1 See the H-NMR spectrum and diagram. Figure 3 The 1H NMR spectrum of 0831A-Hyd-mPEG showed characteristic peaks for p-aldehyde benzoic acid at δ = 8.00–8.25 ppm (c'), a polyethylene glycol methylene (-CH2) peak at δ = 3.50 ppm (b'), characteristic peaks for the benzene ring of 0831A at δ = 6.50–6.89 ppm (d'), and a hydrogen peak at δ = 10.11 ppm (a'), which was attributed to the H resonance of the Schiff base group. These results indicate the successful synthesis of the 0831A-Hyd-mPEG conjugate.
[0039] 3.2 Gel permeation chromatography results 0831A-Hyd-mPEG was characterized by gel permeation chromatography (GPC). nThe molecular weights of the prodrug micelles (n=1000, 2000, 5000) are shown in Table 1. The weight-average molecular weights of the 0831A-Hyd-mPEG conjugates were measured to be 3500 kDa, 5410 kDa, and 9489 kDa, respectively. Under different standards and detection methods, the elution time was always half the time path, and the molecular weight distribution of the three different molecular weight 0831-Hyd-mPEG conjugates was relatively concentrated. n (Da)a represents the number-average molecular weight of the polymer material obtained by the GPC method, M w (Da)a represents the weight-average molecular weight of the polymer material obtained by GPC. The Mw / Mn ratios of the three conjugates with different molecular weights are similar, indicating that three 0831-Hyd-mPEG conjugates with uniform molecular weight distribution were successfully prepared.
[0040] Table 1. GPC data results for mPEG-Hyd-0831A block copolymer Example 2: Preparation and stability study of 0831A-Hyd-mPEG micelles 1. Experimental Materials As described in the above embodiments.
[0041] 1.1 Instruments High-resolution transmission electron microscopy: HRTEM, Tecnai G2 F20 S-TWIN; Nanopotential particle size analyzer: Malvern Z3600, Malvern Ltd., UK; Filter: SLGP033RB Millipore 0.22 μm, Merck, Germany.
[0042] Multifunctional microplate reader: SpectraMax M5, Molecular Devices, USA.
[0043] 1.2 Reagents Disodium hydrogen phosphate: analytical grade, Chengdu Kelong Chemical Reagent Co., Ltd.; Sodium dihydrogen phosphate: analytical grade, Chengdu Kelong Chemical Reagent Co., Ltd.; Lithium dodecyl hydroxystearate (PEG) (HS15): BASF, Germany Methanol (MeOH): Analytical grade, Chengdu Kelong Chemical Reagent Factory; Physiological saline: purchased from Kelun Pharmaceutical Co., Ltd.; PBS: Purchased from Wuhan Boster Biological Engineering Co., Ltd. Fetal Bovine Serum (FBS): Thermo Scientific Hyclone, USA; 96-well plate: Costar 3599, Corning Corporation, USA; 5% Glucose Injection: Sichuan Kelun Pharmaceutical Co., Ltd.
[0044] 2. Experimental Methods 2.1 Investigation of preparation methods and molecular weight screening of 0831A-Hyd-mPEG micelles Thin film hydration method: Weigh 0831A-Hyd-mPEG 2000 The conjugate, with a target concentration of 1 mg / mL 0831A, was dissolved in an appropriate amount of dichloromethane at room temperature. The solvent was transferred to a round-bottom flask and rotary evaporated at 40 °C in a water bath until the organic solvent was completely removed. Deionized water was then added, and the mixture was manually shaken until the membrane was completely dissolved. The solution was observed to be a clear, transparent, pale yellow liquid. The solution was filtered through a 0.22 μm sterile filter to obtain the 0831A-Hyd-mPEG micelle solution. The particle size and polymer dispersibility index (PDI) of the micelles were measured using a particle size analyzer to examine the micelle size and dispersion uniformity.
[0045] Solvent evaporation method: Weigh 0831A-Hyd-mPEG 2000 The prodrug, with a target concentration of 1 mg / mL 0831A, was prepared by dissolving 0831A-Hyd-mPEG in an appropriate amount of dichloromethane. 2000 The conjugate was dissolved and added dropwise to deionized water stirred at 1000 rpm. After the oil and aqueous phases were fully mixed, and the oil phase was uniformly distributed in the aqueous phase as small particles, forming an emulsion, the mixture was rotary evaporated at 40 °C in a water bath until the organic solvent was completely removed. Deionized water was then added, and the mixture was manually shaken until the film was completely dissolved, yielding a yellow, transparent solution. This solution was filtered through a 0.22 μm filter and brought to a target concentration of 1 mg / mL. The micelle size and polymer dispersibility index (PDI) were measured using a particle size analyzer to assess the micelle size and dispersion uniformity.
[0046] By comparing the physical parameters and stability of the micelle solutions obtained by the two methods, the optimal method for micelle preparation is selected.
[0047] 2.4 Determination of drug loading in 0831A-Hyd-mPEG micelles Free 0831A in 0831A-Hyd-mPEG micelles was separated by filtration. 100 μL of the micelle solution was accurately measured and added to 400 μL of pH 5.0 phosphate buffer. The mixture was vortexed for 30 min, then diluted with 500 μL of methanol and vortexed for another 30 min. The mixture was centrifuged at 13000 rpm and 4 °C for 3 min. The supernatant was then used for HPLC analysis to determine the 0831A content.
[0048] 2.5. Detection of critical micelle concentration of 0831A-Hyd-mPEG Take 14 volumetric flasks (10 mL each), and transfer 80 μL of pyrene-acetone solution to each flask. After the acetone has evaporated naturally, transfer different volumes of 0831A-Hyd-mPEG micelle stock solution to the pyrene-containing flasks. Dilute to the mark with deionized water to obtain solution concentrations of 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 mg / mL in each flask. Place the polymer solutions in a 25 °C water bath in the dark for 24 h. Plot the I338 / I334 ratio against the logarithmic concentration of the polymer and calculate the CMC of the block polymer, i.e., the concentration at the inflection point of the curve.
[0049] 2.6 Transmission electron microscopy characterization of 0831A-Hyd-mPEG micelles The morphological characteristics of 0831A-Hyd-mPEG micelles were studied using high-resolution transmission electron microscopy (HRTEM, Tecnai G2 F20 S-TWIN). The micelle solution was diluted with distilled water and placed on a copper grid for air drying at room temperature. The samples were negatively stained with phosphotungstic acid, dried at room temperature, and then analyzed.
[0050] 2.7. Serum stability test of 0831A-Hyd-mPEG micelles The method is described in Section 3.2 of Chapter 2. 0831A micelles and a blank micelle solution (150 μL, 0.1 mg / mL) were mixed with an equal volume of FBS in a 96-well plate, with 5% glucose used as a negative control. After incubation at 37 °C for 72 hours, the absorbance at 415 nm was measured using a microplate reader at time intervals of 0 h, 4 h, 8 h, and 24 h, and the corresponding turbidity for each group at different time points was calculated.
[0051] 2.8 Study on pH sensitivity of 0831A-Hyd-mPEG micelles The in vitro release of micelles was detected using dialysis. The method is described in Chapter 2, Section 2.8. The specific responsiveness of 0831A-Hyd-mPEG micelles under acidic conditions was verified by examining their release curves under neutral and acidic environments. One mL of 0831A-Hyd-mPEG micelles (2 mg / mL) was placed in a dialysis bag (molecular weight cutoff 3.5 kDa), and the release behavior was observed at time intervals of 0.5, 1, 2, 4, 8, 12, 24, and 48 h under neutral and acidic conditions. The concentration of 0831A in the dialysate was determined by HPLC. All studies were repeated three times, and data are expressed as mean ± SD.
[0052] 2.9 Stability Study of 0831A-Hyd-mPEG Micelles and Lyophilized Powder 2.9.1 Investigation of the freeze-drying standard curve Considering that the pre-freezing temperature should generally be at least 15 °C below the eutectic point, and that the final drying temperature is directly related to the final moisture content, we set the pre-freezing temperature to -40 °C and selected a 64-hour freeze-drying curve for observation, examining the appearance of the freeze-dried powder. The freeze-drying curves are shown in Table 2. Table 2 Freeze-drying process curves 2.9.2 Stability Study of Freeze-Dried Powder To investigate the stability of the lyophilized powder, an influencing factor experiment and a one-month stability experiment were conducted, and the effect of different pH conditions on micelle stability was also investigated.
[0053] Influencing factors and experimental conditions: High temperature: 40 ℃; High humidity: 25 ℃, 90%±5%; Light: 4500 lx±500 lx; Observation time: 0, 1, 5, 10 days. One-month stability test conditions: Room temperature, refrigeration 2-8 ℃, freezing -20 ℃; Observation time: 0, 5, 10, 30 days. Two parallel lyophilized powder samples were taken from each group, for a total of six bottles. Each bottle of formulation was dissolved in 1 mL of deionized water. 100 μL of micelle solution was accurately measured, and 400 μL of pH 5.0 phosphate buffer was added. The mixture was vortexed for 30 min, diluted with 500 μL of methanol, and vortexed for 30 min. The mixture was centrifuged at 13000 rpm and 4 ℃ for 3 min. The supernatant was used for HPLC determination of the 0831A content.
[0054] 3. Experimental Results 3.1 Preparation and molecular weight screening of 0831A-Hyd-mPEG micelles With 0831A-Hyd-mPEG 2000 For example, the nanomicelles prepared are shown in Table 3 and Figure 4The results showed that the micelles prepared by both methods had good stability and no obvious precipitation occurred within 1 h. However, the polymer dispersibility index (PDI) of the thin film hydration method was higher and the particle size distribution of the micelles was not concentrated. Therefore, the solvent evaporation method was selected as the subsequent micelle preparation method.
[0055] Table 3 Optimal Results of Preparation Method 0831A-Hyd-mPEG was prepared using a solvent evaporation method. n Micelles (n=1000, 2000, 5000) were measured, and their stability and dispersibility were observed by detecting the average particle size (Z-Average) and polymer dispersibility index (PDI). The particle size and dispersibility coefficient of 0831A-Hyd-mPEG with different molecular weights are shown in Table 4. Figure 5 Theoretically, the smaller the PDI, the better the dispersibility of the micelles. Comparing 0831A-Hyd-mPEG micelles with different block ratios, the results show that when n=2000, the average particle size of the micelles is 157.1±14.73 nm, and the polymer dispersion index (PDI) of this micelle is 0.141±0.082. The PDI is smaller than that of the other two groups. This can be seen from the particle size distribution diagram of 0831A-Hyd-mPEG. 1000 The micelles exhibit a relatively concentrated particle size distribution, but show a distinct tail peak with a broad peak shape. 0831A-Hyd-mPEG 5000 The micelles have a particle size of 472.9 ± 100.3 nm and a PDI of 0.423 ± 0.173. The micelles are relatively large and have a non-concentrated particle size distribution. Therefore, compared to 0831A-Hyd-mPEG micelles with molecular weights of 1000 and 5000, the micelles exhibit a larger size and less concentrated particle size distribution. 2000 The micelles exhibited the most concentrated particle size distribution and showed no obvious tail peaks, indicating superior stability compared to the other two. Therefore, 0831A-Hyd-mPEG was prioritized for subsequent experiments. 2000 (Hereafter referred to as 0831A-Hyd-mPEG).
[0056] Table 4. Average particle size and PDI of mPEG-bound 0831A self-assembled micelles of different fragments 3.2 Characterization of 0831A-Hyd-mPEG micelles After 0831A and mPEG are bound by acylhydrazone bonds, 0831A is the hydrophobic end and mPEG is the hydrophilic end. The amphiphilic 0831A-Hyd-mPEG conjugate can self-assemble into micelles in aqueous solution. When 0831A-Hyd-mPEG comes into contact with water, the mPEG segment spontaneously becomes hydrophilic, forming self-assembled micelles with 0831A as the core.
[0057] To verify the self-assembly behavior occurring in the aqueous solution of 0831A-Hyd-mPEG micelles, we used pyrene as a probe to study the micelle self-assembly process. The results of the critical micelle concentration determination are shown below. Figure 6 The critical micelle concentration (CMC) of 0831A-Hyd-mPEG micelles was determined to be 9.6 μg / mL by calculating the inflection point in the figure. Therefore, when the concentration of 0831A-Hyd-mPEG conjugate is greater than this value, it can be prepared into nano micelles through nano-formulation.
[0058] TEM image of 0831A-Hyd-mPEG micelle lyophilized powder after reconstitution is shown below. Figure 7 As shown, the 0831A-Hyd-mPEG micelle nanoparticles are regularly spherical with a narrow particle size range. After reconstitution of the lyophilized micelle powder, there was no aggregation or adhesion between the micelles, indicating good stability of the lyophilized powder. The nanoparticles were dispersed uniformly and exhibited good dispersibility after reconstitution. TEM results were consistent with dynamic light scattering results, further confirming the successful preparation of 0831A-Hyd-mPEG micelles. The drug loading of the successfully prepared 0831A-Hyd-mPEG micelles was 10.20 ± 0.48%, demonstrating that 0831A-Hyd-mPEG micelles have good drug loading capacity.
[0059] 3.3 Stability Study of 0831A-Hyd-mPEG Micelles The one-month stability results of 0831A-Hyd-mPEG micelles are shown in the figure. Figure 8 It can be seen that the micelles exhibit poor stability when stored at room temperature. The content of 0831A in the micelles decreases over time, and a small amount of precipitation occurs. After one month, the 0831A content in the micelles is 87.83±0.80%. The micelles show relatively better stability when stored at -4 ℃. No significant precipitation occurs during the one-month storage period, and the final 0831A content in the micelles is 94.15±0.44%. The results indicate that the micelle solution has better stability when stored at -20 ℃. After one month, the 0831A content in the micelles is 98.27±0.13%, which is better than storage at room temperature and refrigeration at 2-4 ℃.
[0060] 3.4. Serum stability study of 0831A-Hyd-mPEG micelles To investigate the serum stability of 0831A-Hyd-mPEG micelles, the micelles were co-incubated with fetal bovine serum, and the changes in solution turbidity after the micelles interacted with proteins in the serum were observed. Results are shown below. Figure 9The absorbance curves of the 0831A-Hyd-mPEG conjugate group and the micelle group showed similar changes over time. Compared with the blank control 5% glucose, the absorbance was higher, indicating that the stability of the prodrug group and the micelle group in serum was similar, and both showed trace precipitation, resulting in higher absorbance values than the blank control group. This suggests that the acylhydrazone bond in the prodrug has good stability in serum and is not easily hydrolyzed by serum enzymes.
[0061] 3.5. Investigation of the in vitro release behavior of 0831A-Hyd-mPEG micelles The release behavior of 0831A in 083A-Hyd-mPEG micelles was investigated under simulated physiological conditions (pH 7.4 PBS, 37°C) and tumor environment conditions (pH 5.0 PBS, 37°C). Figure 10 As shown, the 0831A active pharmaceutical ingredient (API) was rapidly released from PBS solutions at different pH values at similar rates, exhibiting a linear relationship, which gradually slowed down over time. In PBS solution at pH 5.0, the micelle group released approximately 40% of 0831A after 24 hours, subsequently showing a release curve consistent with the API group. At pH 7.4, release was quite slow, with a burst release of about 10% in the first 10 hours, followed by only 15% drug release after 48 hours. This may be because after the formation of micelles from 0831A-Hyd-mPEG, a small amount of free 0831A API remained adsorbed on the micelle surface, and after 12 hours, there was virtually no 0831A release. This indicates that at pH 7.4, the 0831A micelles release only a small amount of 0831A API, and the cumulative release of API did not increase over time, suggesting that micelles may be more stable in neutral or slightly alkaline environments.
[0062] Based on the above experimental results, only a small amount of 0831A is released from the 0831Hyd-mPEG micelles during systemic circulation, thus reducing the drug's exposure time in the medium. When the micelles reach the tumor site, the acylhydrazone bonds break in the slightly acidic environment inside the tumor, and the micelles release 0831A relatively quickly, exerting their therapeutic effect at the tumor site.
[0063] 3.6. Results of stability study of 0831A-Hyd-mPEG micelles and lyophilized powder 3.6.1 Results of freeze-drying curve investigation The appearance of the freeze-dried powder was observed, and the presence of shrinkage, collapse, stratification, and porosity were used as indicators to evaluate its appearance. A moisture analyzer was used to determine the moisture content to evaluate the applicability of the freeze-drying curve. As shown in Table 5, the freeze-dried powder exhibited good appearance in the 64-h freeze-drying curve, without shrinkage or collapse. The moisture content determination results indicate that the freeze-drying curve is suitable for the freeze-drying of 0831A-Hyd-mPEG micelles; therefore, this curve will be used for subsequent freeze-drying processes.
[0064] Table 5 Results of the freeze-drying curve investigation 3.6.2 Influencing Factors Experiment The results of the influencing factors are shown in Table 6. The experimental results show that the lyophilized powder did not undergo significant degradation under high temperature, high humidity, and high light conditions. This demonstrates that the micelles after lyophilization have good stability, and strong light and high temperature have little impact on the lyophilized powder. Therefore, after packaging the lyophilized powder, it was refrigerated at 4°C to ensure that the drug content in the micelles would not change significantly in a dry environment.
[0065] Table 6. Results of Experiment 0831A on Drug Purity Affected by Factors Influencing Lyophilized Powder 3.6.3. Stability test of lyophilized powder The stability test results are shown in Figure 11 As shown, the drug stability of the lyophilized powder was tested under different storage conditions for one month and the drug stability of its reconstituted solution for 8 hours. The conditions were: drying at room temperature in the dark, drying under cold storage in the dark, and drying by freezing in the dark. The experimental results show that the lyophilized powder has poor stability at room temperature, while the lyophilized powder has the best stability under freezing conditions. Therefore, the prepared lyophilized powder was stored at -20 ℃.
[0066] Example 3: In vitro antitumor activity study of 0831A-Hyd-mPEG micelles 1. Experimental Materials 1.1 Instruments Low-temperature refrigerated centrifuge: Thermo Heraeus Fresco 17, Thermo Scientific; Cell Culture CO2 Incubator, ESCO; Micropipettes (2.5 μL, 10 μL, 100 μL, 200 μL, 1 ml): Eppendorf GmbH, Germany Low-speed centrifuges: Varifuge 3.0; Megafuge 1.0, Heraeus Six-hole plate: Costar 3599, Corning Corporation, USA; 24-well plate: Costar 3599, Corning Corporation, USA; Clean bench: Biological Safety Cabinets, Class II, NUAIR, SANYO, Japan; Constant temperature water bath: PolyScience 9505 Vortex Mixer: Type 16700 Mixer, Thermolyne Flow cytometer: FASC420 (USA) Variable speed horizontal shaker: Kylin Bell Lab Instruments Orthogonal research-grade microscope: Olympus BX51TRF Ordinary optical microscopes: CHS, Olympus; Inverted fluorescence microscope: Zeiss Axiovert 200 Cell counting chamber: Nanjing Jiancheng Bioengineering Co., Ltd.; 1.2 Reagents Methanol: chromatographic grade, Fisher Scientific; Formic acid: Fluka Analytical, 98% purity; Dimethyl sulfoxide (DMSO): purchased from Sigma-Aldrich, USA; 4',6-Diamidinyl-2-phenylindole (DAPI): Beyotime Corporation Coumarin-6: Shanghai Anaiji Chemical Co., Ltd., purity > 99%; Trypsin: GIBCO.BRL, USA; Binding Buffer: Biovision, Inc. (USA) Annexin V-FITC: Biovision, a US company; Propidium iodide (PI): Merck GmbH, Germany; BD Matrigel™ Basement Membrane Matirx: Corning Incorporated, USA; Triton-X100: Chengdu Kelong Chemical Reagent Factory 1.3 Cells 4T1, A2780s, A2780T, and CT26 cell lines were all purchased from the American Type Culture Collection (ATCC) and preserved by the State Key Laboratory of Biotherapy, West China Hospital, Sichuan University. A2780s and A2780T cells were cultured in RPMI 1640 medium (containing 10% FBS, 10 IU / mL penicillin, and 10 μg / mL streptomycin) and passaged in a 37°C incubator containing 5% CO2. 4T1 and CT26 cell lines were cultured in DMEM medium (containing 10% FBS, 10 IU / mL penicillin, and 10 μg / mL streptomycin) and passaged in a 37°C incubator containing 5% CO2. 2. Experimental Methods 2.1. Investigation of the in vitro antitumor effect of 0831Hyd-mPEG micelles The cytotoxicity of 0831A active pharmaceutical ingredient, 0831A-Hyd-mPEG conjugate, and 0831A-Hyd-mPEG self-assembled micelles on A2780T, HCT116, CT26, and A549 cell lines was evaluated using a cell growth inhibition assay. The methods are described in Chapter 2, Section 4, 2.1. In this experiment, the concentration gradients of 0831A were 3.125, 6.25, 12.5, 25, 50, 100, and 200 nmol / L, a total of seven concentrations. The drug was diluted with culture medium, and each concentration was tested in triplicate. A blank micelle control without the drug was also included. All cells were incubated in a constant temperature incubator for 72 h.
[0067] 2.2 Investigation of 0831A-Hyd-mPEG micelle uptake behavior The quantitative uptake of 0831A-Hyd-mPEG micelles by CT26 cells was detected by flow cytometry. Since 0831A itself lacks fluorescence capable of penetrating biological cells or tissues, coumarin-6 was selected as a small molecule substitute for 0831A. The quantitative uptake of coumarin-6 encapsulated in 0831A-Hyd-mPEG micelles by CT26 cells was detected by flow cytometry. Coumarin-6 / 0831A-Hyd-mPEG nanomicelles were prepared using an emulsion evaporation method. The high-intensity fluorescence of coumarin-6 itself was used to simulate the uptake of 0831A-Hyd-mPEG nanomicelles by tumor cells. 5 × 10⁶ cells were added to each well of a six-well plate. 4Cells were incubated overnight with a 2 mL / cell suspension. The growth medium was replaced with fresh medium containing free coumarin and coumarin micelles (coumarin concentrations: 100 ng / mL and 200 ng / mL). Free coumarin was dissolved in DMSO as a negative control. Cells were collected after 3 hours of incubation. After centrifugation at 1500 rpm, the supernatant was discarded, and the cells were washed three times with PBS. Intracellular fluorescence levels were detected by flow cytometry.
[0068] Quantitative uptake of 0831A from 0831A-Hyd-mPEG micelles by cells was detected by HPLC. High-performance liquid chromatography was also used to detect cellular uptake of 0831A. Cells were cultured at 5 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 100 cells / well in 24-well plates and in 1 mL of growth medium. After 24 hours of culture, 1 μL of 2 mg / mL 0831A active pharmaceutical ingredient and 0831A micelles were added to the corresponding wells for incubation. After 1 h of incubation, the cells were washed three times with PBS and digested with trypsin. Cells were lysed with 1% Triton lysis buffer and mixed with 100 μL of methanol. The mixture was centrifuged to obtain the supernatant, and the concentration of 0831A in the mixture was determined by HPLC.
[0069] 2.3 Detection of Cellular Apoptosis in 0831A-Hyd-mPEG Micelles Collect CT26 cells from passages 3 to 7, at a ratio of 2 × 10⁻⁶. 5 Seeds were seeded at a density of 2 mL / well in 6-well plates and incubated overnight in a 5% CO2 incubator at 37°C. Once cells adhered, the culture medium in the wells was discarded, and 2 mL of fresh culture medium containing the prescribed drug concentration was added to each well. An equal volume of fresh culture medium without the drug was added to the solvent control wells. After 24 or 48 hours of drug treatment, EDTA-free trypsin was added to the 6-well plates in a clean bench to digest the cells. The cells were then centrifuged at 1500 rpm for 3 min and washed twice with PBS.
[0070] Apoptosis assay: The concentrations of 0831A were set at 1, 3, and 6 nmol / L. The treatment groups were 0831A raw material, 0831-Hyd-mPEG conjugate, and 0831A-Hyd-mPEG micelles, respectively. Cells were collected 48 h after treatment and stained using the Annexin V-FITC / PI kit. Each flow cytometry tube was mixed with 500 μL Binding Buffer, 5 μL Annexin V, and 5 μL PI, and incubated at room temperature in the dark for 10 min. Flow cytometry was used to detect cell apoptosis and assess the degree of apoptosis induced in CT26 cells in different groups.
[0071] Cell cycle assays: The concentration gradients of 0831A were 1, 3, and 6 nmol / L. The treatment groups were 0831A raw material, 0831A-Hyd-mPEG conjugate, and 0831A-Hyd-mPEG micelles, respectively. Cells were collected 24 h after treatment, the supernatant was discarded, and 500 μL of pre-chilled 75% ethanol was added to centrifuge tubes for fixation overnight at 4°C. After washing twice with PBS, the cells were centrifuged at 1000 rpm and the supernatant was discarded. 500 μL of LPI staining solution was added to each tube, and the cells were treated in the dark for 10 min. Flow cytometry was then used to detect cell cycle arrest.
[0072] 2.4, 0831A-Hyd-mPEG micelle inhibition of cell migration experiment HUVEC cells passaged to the third to seventh generation were digested with trypsin, detached by pipetting, and centrifuged. The cells were then separated at a concentration of 1×10⁻⁶. 6 Cells were seeded at a concentration of 2 cells / mL in 6-well plates. When the cells nearly reached confluence, the medium was replaced with serum-free medium and incubated for 6 hours. Cells were scratched using a sterile medium-sized pipette tip. Floating cells were washed away with sterile PBS, and the medium was replaced with DMEM complete medium for further culture. Simultaneously, 0831A, 0831A-Hyd-mPEG conjugate, and 0831A-Hyd-mPEG micelles were added at 1 and 3 nM respectively. The 6-well plates with the added drugs were immediately photographed under a microscope and designated as the 0h group. After 24 hours of treatment with the three drugs, the cells were fixed with 4% paraformaldehyde and photographed under a microscope. Three different fields of view were randomly selected from each group to count the number of migrating cells.
[0073] 2.5. Experiment on the inhibition of angiogenesis by 0831A-Hyd-mPEG micelles Thaw Matrigel at 4 °C until it is liquid, and pre-cool the pipette tips, 96-well plates, and EP tubes used in the experiment. Add Matrigel liquid at 50 μL / well to the 96-well plate, and incubate the 96-well plate in a CO2 incubator at 37 °C for 45 min to solidify the Matrigel. Collect HUVEC cells from passages 3 to 7, and divide the HUVEC cells into cells at a rate of 2-4 × 10⁶ cells / well. 4 Cells were seeded at a concentration of 500 μL / well in 96-well culture plates. Then, 0831A and 0831A-Hyd-mPEG micelles were added to the wells at concentrations of 1 and 3 nM, respectively. The cells were then transferred to an incubator and incubated for 6-8 hours under the influence of the drugs. The cells were fixed with 4% paraformaldehyde, and the cell aggregation into tubes was compared and photographed under an inverted microscope. The number of the formed cavity structures was counted.
[0074] 3. Experimental Results 3.1 Evaluation of the anti-tumor cell proliferation activity of 0831A-Hyd-mPEG micelles The growth inhibition rate of 0831A-Hyd-mPEG micelles against different tumor cell lines was determined using the MTT assay to investigate the in vitro antitumor activity of these micelles. 0831A active pharmaceutical ingredient and the 0831A-Hyd-mPEG conjugate were used as controls.
[0075] Figure 12 The study investigated the survival rates of four cell types after treatment with three drug groups—0831A raw material, 0831A-Hyd-mPEG conjugate, and D0831A-Hyd-mPEG micelles—for 48 hours. The results showed that the inhibitory effects of all three drugs on cell cell survival were dose-dependent; that is, the survival rate of tumor cells decreased with increasing 0831A concentration.
[0076] The 0831A active pharmaceutical ingredient group, the 0831A-Hyd-mPEG conjugate group, and the 0831A-Hyd-mPEG micelle group were used in four types of cell IC50. 50 The values are shown in Table 7.
[0077] Table 7 IC50 in four tumor cell types in the treatment groups 50 Among them, the 0831A-Hyd-mPEG micelle group showed the most significant inhibitory effect on cell growth, IC50. 50 The concentration was 5.39 μg / mL, lower than that of the 0831A active pharmaceutical ingredient group and the 0831A-Hyd-mPEG conjugate group. Therefore, the acid-sensitive self-assembled micelles 0831A-Hyd-mPEG micelles prepared in this experiment can enhance the cytotoxicity of 0831A. When the micelles are in the acidic environment of tumor cells, the acylhydrazone bonds break, releasing 0831A, thus allowing the 0831A-Hyd-mPEG micelles to retain the good cytotoxicity of 0831A against tumor cells.
[0078] 3.2 Results of in vitro cellular uptake experiments using 0831A-Hyd-mPEG micelles The antitumor cell activity assay results showed that the IC50 of 0831A-Hyd-mPEG micelles was [missing information]. 50 The uptake of 0831A-Hyd-mPEG micelles by CT26 cells was investigated because the uptake was lower than that of the 0831A API group. Flow cytometry was used to analyze the uptake of 0831A-Hyd-mPEG micelles by CT26 cells. The uptake was compared by calculating the fluorescence intensity of free coumarin and the ratio of coumarin to 0831A-Hyd-mPEG micelles. After incubating CT26 cells with micelles for 3 hours, the fluorescence intensity was detected by flow cytometry. The results are as follows: Figure 13As shown, the average fluorescence intensities of CT26 cells in the free coumarin group and the coumarin / 0831A-Hyd-mPEG micelles were 5600 and 12000, respectively. The results indicate that the average intracellular fluorescence intensity of CT26 cells after incubation with coumarin / 0831A-Hyd-mPEG was twice that of the free coumarin group. This result demonstrates that encapsulating coumarin in 0831A-Hyd-mPEG micelles significantly increased cellular uptake of coumarin, further proving that 0831A-Hyd-mPEG micelles can significantly enhance cellular uptake of 0831A, thereby improving the anti-tumor cell activity of 0831A.
[0079] To further investigate the uptake of 0831A from 0831A-Hyd-mPEG micelles by cells, HPLC was used to detect the quantitative uptake of 0831A by cells. The uptake of 0831A by CT26 cells after 1 hour of incubation is shown in the figure. Figure 14 The total amount of 0831A in the cell lysate treated with the 0831A active pharmaceutical ingredient was 0.47 ± 0.07 μg, while that in the 0831A-Hyd-mPEG micelle group was 0.84 ± 0.20 μg. The 0831A content in the 0831A-Hyd-mPEG micelle group was nearly twice that of the free active pharmaceutical ingredient group, consistent with the results obtained by cell flow cytometry. This further indicates that the 0831A-Hyd-mPEG micelles enhance the uptake of 0831A by cells.
[0080] 3.3 Apoptosis cycle experiment results of 0831A-Hyd-mPEG micelles Microtubule inhibitors possess biological functions such as inducing tumor cell differentiation, causing G2 / M phase arrest, and promoting apoptosis, providing a basis for further research. The abilities of 0831A, 0831A-Hyd-mPEG conjugates, and 0831A-Hyd-mPEG micelles to induce tumor cell apoptosis and cell cycle arrest were investigated. The effects of 0831A-Hyd-mPEG micelles on inducing tumor cell differentiation and cell cycle arrest were also examined. Results are as follows: Figure 15 As shown in (A), after treating cells with 1 nM and 3 nM concentrations for 48 h, the cell death of CT26 cells was analyzed by flow cytometry using Annexin V & PI double staining. The three groups of CT26 cells showed a clear concentration gradient trend in apoptosis after drug treatment. Apoptosis in all three drug-treated groups was observed to be concentration-dependent. At a drug concentration of 3 nM, the proportion of apoptotic cells reached 38.7% in the 0831A raw material group, 38.5% in the 0831A-Hyd-mPEG conjugate group, and 26.7% in the 0831A-Hyd-mPEG micelle group. This indicates that the micelle group retained the ability of 0831A raw material to induce tumor cell apoptosis.
[0081] To further investigate the ability of the three drug-treated groups to induce cell cycle arrest in CT26 cells, CT26 cells were treated with predetermined concentrations (1 and 3 nM) of 0831A, 0831A-Hyd-mPEG conjugate, and 0831A-Hyd-mPEG micelles for 24 h, and cell cycle was analyzed by flow cytometry. The experimental results are as follows: Figure 15 As shown in (B), the results indicate that both 0831A active pharmaceutical ingredient (API) and 0831A-Hyd-mPEG micelles can induce significant G2 / M phase arrest after 24 hours of treatment. In the control group, 13.53% of CT26 cells were in the G2 / M phase. After 24 hours of incubation with 1 nmol / L and 3 nmol / L of 0831A API, the percentage of cells in the G2 / M phase was 56.35% and 65.56%, respectively. After incubation with the 0831A-Hyd-mPEG conjugate at concentrations of 1 nmol / L and 3 nmol / L, the percentage of cells in the G2 / M phase was 15.79% and 45.12%, respectively. The percentages of 0831A-Hyd-mPEG micelles at the same concentrations were 18.29% and 29.42%, respectively. These results indicate that 0831A-Hyd-mPEG micelles retain the common properties of microtubule inhibitors, arresting the cell cycle in the G2 / M phase. However, the proportion of cells arrested in the G2 / M phase was significantly lower than that in the 0831A active pharmaceutical ingredient group. This may be because the micelles did not fully release the active pharmaceutical ingredient within 24 hours, resulting in a weaker cell cycle arrest effect in the micelle group compared to the active pharmaceutical ingredient group.
[0082] 3.4 Results of 0831A-Hyd-mPEG micelles inhibiting cell migration and tube formation Colchicine-binding microtubule inhibitors typically disrupt tumor angiogenesis. These compounds possess a dual mechanism of action, killing both tumor cells and tumor-associated vascular endothelial cells. The effects of microtubule inhibitors on the microtubule system cause morphological changes in endothelial cells. The in vitro anti-angiogenic activity of 0831A-Hyd-mPEG micelles was investigated using HUVEC cells. First, the antiproliferative activity of 0831A-Hyd-mPEG micelles against HUVEC cells was examined, and the results are as follows: Figure 16 As shown. The IC50 of 0831A active pharmaceutical ingredient was calculated. 50 The value is 8.75 nM, IC of micelles 50 The value is 9.774 nM, indicating that the 0831A-Hyd-mPEG micelles retain the ability of 0831A to disrupt tumor vascular-associated networks.
[0083] Subsequent scratch and tube formation experiments were performed using HUVEC cells. The results are as follows: Figure 17As shown in (A), in the scratch assay, HUVEC cells migrated 24 h after scratching, exhibiting stronger migration activity compared to the initial scratch solvent control group. The 0831A-Hyd-mPEG micelle group dose-dependently inhibited HUVEC cell migration. When the drug concentration was 6 nM equivalent of 0831A, the 0831A-Hyd-mPEG micelles essentially completely inhibited HUVEC migration.
[0084] The results of the tube forming experiment are as follows Figure 17 As shown in (B), after 8 h of treatment, cells in the solvent control group assembled into tubular structures, while the 0831A micelle group dose-dependently inhibited HUVEC cell tubulation. When the drug concentration was 6 nM equivalent of 0831A, the morphology of HUVEC cells was nearly completely fragmented. The above vascular-related experiments demonstrate that the 0831A-Hyd-mPEG micelles retain the activity of 0831A as a microtubule inhibitor and have an anti-angiogenic effect.
[0085] In summary, through the above embodiments of the present invention, the solution of the present invention successfully solves the key problem of "significant toxicity after intravenous administration" mentioned in the background art of 0831A; furthermore, the solution of the present invention, through a simple "prodrug self-assembly" strategy, significantly enhances the in vivo anti-tumor efficacy while greatly reducing systemic toxicity, achieving a dual improvement in efficacy and safety.
[0086] The present invention designs and synthesizes a well-defined "mPEG-acylhydrazone-0831A" amphiphilic prodrug, utilizing its self-assembly behavior to construct an acid-sensitive environment-responsive micelle. Furthermore, it possesses advantages such as simple structure, convenient preparation, and good stability, and exhibits multiple biological advantages including significant pH-responsive drug release, enhanced cellular uptake, improved pharmacokinetic behavior, superior in vivo antitumor efficacy, and significantly reduced systemic toxicity. In particular, compared to existing technologies, the present invention solves the core bottleneck (toxicity problem) in the clinical application of 0831A more effectively with simpler technical means, while significantly improving efficacy.
[0087] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for preparing acid-sensitive environment-responsive PEGylated self-assembled micelles, characterized in that, The micelles were formed by the self-assembly of the 0831A-Hyd-mPEG conjugate in an aqueous solution, and the 0831A-Hyd-mPEG conjugate was a product prepared according to the following reaction pathway: First, monomethoxy polyethylene glycol is reacted with p-aldehyde benzoic acid to generate an intermediate product mPEG-CHO with an aldehyde group at the end; then, the mPEG-CHO is reacted with 0831A, so that the aldehyde group at the end of mPEG-CHO forms an acylhydrazone bond with the amino group of 0831A, thus obtaining the 0831A-Hyd-mPEG conjugate, which has the following structure (I): （I） Wherein, mPEG is monomethoxy polyethylene glycol with a number average molecular weight of 1000, 2000 or 5000, and 0831A is SKLB060-methionine; The preparation method includes the following steps: (1) Preparation of 0831A-Hyd-mPEG conjugate; (2) The 0831A-Hyd-mPEG conjugate obtained in step (1) is self-assembled in an aqueous solution to form micelles by thin film hydration or solvent evaporation.
2. The preparation method according to claim 1, characterized in that, In step (1), the number average molecular weight of the mPEG is 2000.
3. The preparation method according to claim 1, characterized in that, In step (1), the reaction of mPEG with p-aldehyde benzoic acid is carried out in the presence of the dehydrating agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and the catalyst 4-dimethylaminopyridine; the reaction of mPEG-CHO with 0831A is carried out under alkaline conditions.
4. The preparation method according to claim 1, characterized in that, In step (2), the self-assembly method is the solvent evaporation method, specifically: the 0831A-Hyd-mPEG conjugate is dissolved in dichloromethane, the resulting solution is added dropwise to stirred deionized water, and the organic solvent is removed after mixing to obtain a micelle solution.
5. A type of acid-sensitive environment-responsive PEGylated self-assembled micelles, characterized in that, It is prepared by the preparation method according to any one of claims 1-4.
6. The micelles according to claim 5, characterized in that, The micelles are formed by the self-assembly of a conjugate consisting of mPEG with a number average molecular weight of 2000 and 0831A linked by acylhydrazone bonds.
7. The micelles according to claim 5, characterized in that, The micelles have an average particle size of 157.1 ± 14.73 nm and a polymer dispersibility index of 0.141 ± 0.
082.
8. The micelles according to claim 5, characterized in that, The critical micelle concentration of the micelles is 9.6 μg / mL; The micelles achieved a cumulative drug release rate of 69.46±2.13% in a pH 5.0 buffer solution over 48 hours, and a cumulative drug release rate of 22.94±0.67% in a pH 7.4 buffer solution over 48 hours.
9. A pharmaceutical composition, characterized in that, It comprises the acid-sensitive environment-responsive PEGylated self-assembled micelles as described in any one of claims 5-8 and a pharmaceutically acceptable carrier.
10. The use of the acid-sensitive environment-responsive PEGylated self-assembled micelles according to any one of claims 5-8 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating cancer; wherein the cancer is colorectal cancer.