Multi-responsive traceable targeted multi-drug co-delivery nanomicelles, preparation method and application thereof

Abstract

The application relates to the technical field of medicines, and particularly discloses a multi-response traceable targeted multi-drug co-delivery nanomicelle as well as a preparation method and application thereof. The nanomicelle is formed by synergistic assembly of a molecule A and a molecule B through electrostatic and hydrophobic interactions; the molecule A is mPEG-targeting ligand-pH sensitive bond-cationic hydrophobic chain, and the molecule B is an anionic hydrophilic segment P-disulfide bond-0831A-hydrophobic tail. The pH sensitive bond and the disulfide bond are introduced to realize dual-response cascade release of a tumor microenvironment pH / oxidation-reduction, and the problem of inaccurate single pH response release is solved; active targeting is realized through the targeting ligand, and the problem of low passive targeting enrichment efficiency is solved. The nanomicelle has good stability and high bioavailability, can significantly enhance the antitumor efficacy and reduce the systemic toxicity, and has a wide application prospect in the preparation of antitumor drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to a multi-responsive traceable targeted multi-drug co-delivery nanomicelle, its preparation method, and its application. Background Technology

[0002] Cancer is a major disease that seriously threatens human health, and chemotherapy remains one of the main methods for treating malignant tumors in clinical practice. However, traditional chemotherapy drugs generally have drawbacks such as poor water solubility, low bioavailability, and significant toxic side effects, which limit their effectiveness in clinical application.

[0003] Microtubule inhibitors, by interfering with microtubule homeostasis, induce cell cycle arrest and apoptosis in tumor cells, demonstrating good activity in the treatment of solid tumors. SKLB060 is a novel microtubule depolymerizing agent developed by Sichuan University. To further improve its water solubility and bioavailability, its amino acid derivative 0831A (SKLB060-methionine) was subsequently developed. Studies have shown that 0831A retains the excellent antitumor activity of SKLB060, but its intravenous administration still exhibits significant irritation and systemic toxicity, limiting its clinical application.

[0004] To overcome the aforementioned problems, nanomedicine delivery systems have emerged. Polymer micelles, due to their controllable particle size, structural stability, and ability to prolong drug circulation time, have become a research hotspot for antitumor drug delivery. In particular, by utilizing the differences between the microenvironments of tumor tissue and normal tissue, microenvironment-responsive smart micelles can be designed to achieve selective drug release at the target site. For example, due to rapid proliferation leading to local hypoxia and enhanced glycolysis, the extracellular pH of tumor tissue (6.5–6.8) is significantly lower than that of normal tissue (pH 7.4). Therefore, pH-sensitive micelles have been extensively studied. Currently reported pH-sensitive micelles mostly use acid-hydrolyzable chemical bonds (such as hydrazone bonds, acetal bonds, orthoester bonds, etc.) to link the drug to the polymer backbone, or are formed through the self-assembly of pH-sensitive block copolymers. For example, a search revealed that Chinese patent CN103071159A discloses a pH-sensitive doxorubicin prodrug micelle, which links doxorubicin to a polyethylene glycol-polyaspartic acid block copolymer via hydrazone bonds, enabling rapid drug release under acidic conditions. However, existing pH-sensitive micelles still face the following problems in practical applications:

[0005] (1) Insufficient precision and controllability of drug release. The single pH response mechanism relies on the slightly acidic environment of tumor tissue, but the pH value of the tumor microenvironment varies from person to person and changes dynamically. In addition, there may be slight acidity in local areas of the blood circulation, which may lead to premature drug leakage. More importantly, after the drug enters the tumor cells, it needs to be released rapidly into the cell to fully exert its efficacy. The single pH response is difficult to meet this "extracellular trigger, intracellular burst" delivery requirement.

[0006] (2) Limited tumor targeting ability. Existing micelles mostly rely on passive targeting through the EPR effect and lack the ability to actively recognize tumor cells, resulting in low drug enrichment efficiency at the tumor site. Some drugs will still be distributed to normal tissues, producing toxic side effects.

[0007] (3) The structure and function are relatively simple. Existing micelle structures are relatively fixed, making it difficult to flexibly adjust the physicochemical properties such as particle size and surface charge according to treatment needs; at the same time, most micelles can only deliver a single drug, making it difficult to achieve synergistic effects of chemotherapy and immunotherapy.

[0008] Therefore, how to construct a novel nanodelivery system that can achieve precise and controllable release, active targeted enrichment, tunable structure, and synergistic therapeutic potential has become a technical challenge that urgently needs to be solved in this field.

[0009] To address these issues, this application proposes a multi-responsive traceable targeted multi-drug co-delivery nanomicelle, its preparation method, and its application. Summary of the Invention

[0010] The purpose of this invention is to solve the technical problems mentioned in the background art above, and to provide a multi-responsive traceable targeted multi-drug co-delivery nanomicelle, its preparation method and application, so as to achieve precise and controllable release and active targeted enrichment of drugs at tumor sites, and have the potential for structurally tunable and synergistic therapy.

[0011] The above-mentioned objective of the present invention is achieved as follows:

[0012] One aspect of the present invention provides a multi-responsive traceable targeted multi-drug co-delivery nanomicelle, wherein the targeted multi-drug co-delivery nanomicelle is formed by the synergistic assembly of molecules A and B through electrostatic and hydrophobic interactions;

[0013] The molecule A has the structure shown in formula (Ⅰ):

[0014]

[0015] (I)

[0016] Wherein, mPEG is monomethoxy polyethylene glycol with a number average molecular weight of 1000-5000; L1 is a linker bond selected from amide bonds or ester bonds; T is a tumor-targeting ligand; L2 is a pH-sensitive linker arm; and H is a cationic hydrophobic segment.

[0017] The molecule B has the structure shown in formula (II):

[0018]

[0019] (II)

[0020] Wherein, P is the anionic hydrophilic segment; SS is the disulfide bond; 0831A is the antitumor drug SKLB060-methionine; L3 is the linking bond, selected from ester or amide bonds; and H' is the hydrophobic tail.

[0021] Furthermore, the tumor-targeting ligand T is selected from RGD peptide, folic acid, hyaluronic acid, or galactose;

[0022] The pH-sensitive linker L2 is selected from acetal bonds, orthoester bonds, or vinyl ether bonds;

[0023] The cation-containing hydrophobic segment H is a C12-C18 alkyl chain containing a quaternary ammonium salt;

[0024] The anionic hydrophilic segment P is selected from hyaluronic acid oligosaccharide, polyglutamic acid, or polyaspartic acid; the hydrophobic tail H' is selected from cholesterol, vitamin E, or C16-C18 fatty acids.

[0025] Furthermore, in molecule A, the number-average molecular weight of mPEG is 2000; T is RGD peptide or folic acid; L2 is an acetal bond; H is a C16 alkyl chain containing quaternary ammonium salt; in molecule B, P is hyaluronic acid oligosaccharide with a molecular weight of 1000-3000; and H' is cholesterol or vitamin E.

[0026] Furthermore, the molar ratio of molecule A to molecule B is 1:1 to 3:1.

[0027] Furthermore, its core also contains a therapeutic agent; the therapeutic agent is selected from at least one of immunomodulators, photosensitizers, anti-angiogenic drugs, or diagnostic probes.

[0028] Furthermore, the immunomodulator is NLG919, indomod, or ralsimod; the photosensitizer is dihydroporphyrin e6 or indocyanine green; the anti-angiogenic drug is apatinib or sunitinib; and the diagnostic probe is a near-infrared fluorescent probe Cy5.5, DiR, or superparamagnetic iron oxide nanoparticles.

[0029] The present invention also provides a method for preparing multi-responsive, traceable, targeted multi-drug co-delivery nanomicelles, comprising the following steps:

[0030] S1. Synthesize molecules A and B respectively;

[0031] S2. Dissolve molecules A and B in an organic solvent at a predetermined molar ratio to obtain a mixed solution;

[0032] S3. Under stirring, the mixed solution obtained in step S2 is added dropwise to a buffer solution with pH 6.0 to 7.5, and spontaneously assembles to form nanomicelles.

[0033] S4. Remove the organic solvent by dialysis or ultrafiltration to obtain the nanomicelle solution.

[0034] Furthermore, the organic solvent mentioned in step S2 is selected from dimethyl sulfoxide, N,N-dimethylformamide, or acetonitrile;

[0035] The buffer solution mentioned in step S3 is a phosphate buffer or an acetate buffer, and the assembly temperature is 20–37°C.

[0036] The present invention also provides a pharmaceutical composition comprising the above-described nanomicelles and a pharmaceutically acceptable carrier.

[0037] The present invention also provides the use of nanomicelles or the above-described pharmaceutical composition in the preparation of a medicament for treating cancer; said cancer being colorectal cancer, breast cancer, lung cancer, liver cancer, or ovarian cancer.

[0038] The difficulty and significance of the technical problem solved by this invention lie in:

[0039] This technical solution overcomes long-standing problems in existing pH-sensitive nanodelivery systems (inaccurate drug release due to a single response mechanism, low tumor enrichment efficiency due to passive targeting, and difficulty in achieving synergistic therapy due to a single structure and function). Its technical challenges lie in multiple aspects, including the synergistic design of multiple response mechanisms, precise matching of binary molecules, chemical synthesis of multifunctional linkers, and biological verification of cascade release effects. It requires overcoming technical challenges such as the stability and response sequence control of different sensitive bonds in the complex in vivo environment, the electrostatic and hydrophobic balance of anions and cations, and the compatibility and release coordination of multiple drug co-loading. The solution of this invention... This study proposes a novel cascade release mechanism of "extracellular pH triggering and intracellular GSH burst" and develops a "multivalent synergistic" binary molecule co-assembly strategy, providing a new paradigm for the design of intelligent drug delivery systems. Clinically, it is significant because it can precisely deliver the anti-tumor drug 0831A to the tumor site through active targeting, while simultaneously encapsulating immunomodulators to achieve chemotherapy-immunotherapy combination therapy, significantly enhancing anti-tumor efficacy and greatly reducing systemic toxicity. By encapsulating diagnostic probes, it enables the visualization and tracking of the treatment process, providing technical support for personalized precision medicine. Its industrial value lies in the controllable preparation process and tunable structure, possessing good potential for clinical translation.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] 1. The present invention cleverly combines a pH-sensitive linker (acetal bond) and a redox-sensitive linker (disulfide bond) to achieve a "cascade release" mechanism. Both types of links are stable in the bloodstream (pH 7.4). Upon reaching the tumor tissue, the slightly acidic environment (pH 6.5-6.8) first triggers the acetal bond to break, causing molecule A to dissociate from molecule B and expose the disulfide bond. Subsequently, it is cleaved by the high concentration of glutathione in the tumor cells, rapidly releasing 0831A. This dual response mechanism of "extracellular triggering and intracellular burst" significantly improves the accuracy and controllability of drug release and reduces systemic exposure.

[0042] 2. In the present invention, the targeting ligand (such as RGD or folic acid) introduced in molecule A can specifically recognize the receptor overexpressed on the surface of tumor cells, mediate the active endocytosis of nanomicelles, and increase the cell uptake rate by 2 to 3 times compared with the non-targeted system, significantly enhancing the enrichment efficiency of the drug at the tumor site.

[0043] 3. The present invention employs a binary molecular co-assembly strategy. By adjusting the molar ratio of molecule A to molecule B (1:1 to 3:1), the particle size (80 to 180 nm), surface charge (-10 to +15 mV), and drug loading (5% to 15%) of the nanomicelles can be precisely controlled to meet the needs of different tumor treatments. At the same time, the hydrophobic space of the core can flexibly embed a second therapeutic agent to achieve chemotherapy-immunotherapy combination therapy. By embedding diagnostic probes, real-time tracking at the in vivo level can be achieved to monitor the in vivo distribution and tumor enrichment of the nanomicelles, providing a basis for personalized treatment.

[0044] 4. In this invention, through multiple interactions of electrostatics and hydrophobicity between two molecules, the critical micelle concentration (CMC) of the nanomicelles is as low as 2-4 μg / mL, exhibiting extremely high stability in blood. Pharmacokinetic studies show that its blood circulation half-life is 8-10 times longer than that of the 0831A active pharmaceutical ingredient in the prior art, significantly improving bioavailability. Animal experiments show that the nanomicelles of this invention can effectively inhibit the growth of CT26 colon cancer xenografts, with a tumor inhibition rate of over 80%, and there is no significant decrease in mouse body weight, indicating good safety. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the synthetic routes of molecules A and B in this invention;

[0046] Figure 2 This is a schematic diagram of the formation process and "cascade release" mechanism of nanomicelles in this invention;

[0047] Figure 3 This is a particle size distribution diagram (dynamic light scattering) of the RGD targeted nanomicelles prepared in Example 1 of this invention.

[0048] Figure 4 This is a transmission electron microscope image (scale bar 100 nm) of the nanomicelles prepared in Example 1 of this invention.

[0049] Figure 5 These are the in vitro release curves of the nanomicelles prepared in Example 1 of this invention under different conditions (pH 7.4, pH 6.5, pH 6.5+10 mM GSH).

[0050] Figure 6 This is the result of the in vitro cytotoxicity experiment of CT26 cells by the nanomicelles containing NLG919 and DiR prepared in Example 2 of this invention;

[0051] Figure 7 This is a live fluorescence imaging image of the DiR-encapsulated nanomicelles prepared in Example 2 of this invention in CT26 tumor-bearing mice.

[0052] Figure 8 This is the tumor inhibition curve of the NLG919-encapsulated nanomicelles prepared in Example 2 of this invention in CT26 tumor-bearing mice;

[0053] Figure 9 This is the curve showing the change in body weight of mice after treatment with the nanomicelles prepared in Example 2 of this invention. Detailed Implementation

[0054] 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.

[0055] Terminology Explanation: mPEG: Monomethoxy polyethylene glycol; RGD: Arginine-glycine-aspartic acid tripeptide; EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; DMAP: 4-dimethylaminopyridine; DMSO: Dimethyl sulfoxide; PBS: Phosphate buffer; GSH: Glutathione; HPLC: High performance liquid chromatography; PDI: Polymer dispersity index; TEM: Transmission electron microscopy; DiR: Near-infrared fluorescent probe; NLG919: Immunomodulator.

[0056] This invention provides a multi-responsive traceable targeted multi-drug co-delivery nanomicelle, its preparation method, and its application. The aim is to construct a nanomicelle that integrates multi-responsive cascade release, active targeted enrichment, multi-drug synergistic therapy, and in vivo visualization and tracking, so as to precisely solve the problems of uncontrollable release, poor targeting, and single function in existing anti-tumor drug delivery systems, thereby significantly improving efficacy and reducing systemic toxicity.

[0057] The implementation of the present invention will be described in detail below with reference to specific embodiments.

[0058] Example 1: Preparation and characterization of RGD-targeted multi-responsive nanomicelles

[0059] 1. Synthesis of molecule A (mPEG2000-RGD-acetal bond-C16 quaternary ammonium salt):

[0060] Step 1: Preparation of mPEG2000-COOH

[0061] 10 g (5 mmol) of mPEG2000 was dissolved in 50 mL of anhydrous dichloromethane. Succinic anhydride (1.0 g, 10 mmol), DMAP (0.12 g, 1 mmol), and triethylamine (1.4 mL, 10 mmol) were added, and the mixture was stirred at room temperature for 24 h. The reaction solution was washed with dilute hydrochloric acid, dried over anhydrous sodium sulfate, filtered, concentrated by rotary evaporation, precipitated in ice-cold diethyl ether, filtered under vacuum, and dried under vacuum to give a white solid mPEG2000-COOH (9.2 g, 88% yield).

[0062] Step 2: Synthesis of mPEG2000-RGD

[0063] mPEG2000-COOH (5 g, 2.5 mmol) was dissolved in anhydrous dichloromethane (30 mL), and EDCI (0.96 g, 5 mmol) and N-hydroxysuccinimide (0.58 g, 5 mmol) were added. The mixture was activated in an ice bath for 2 h. Then, RGD peptide (1.3 g, 2.5 mmol) and triethylamine (0.35 mL, 2.5 mmol) were added, and the reaction was carried out at room temperature for 24 h. The reaction solution was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, precipitated with diethyl ether, and dried under vacuum to obtain mPEG2000-RGD (4.5 g, 76% yield). The number average molecular weight was approximately 2600 as determined by GPC.

[0064] Step 3: Synthesis of the linker arm containing the acetal bond

[0065] A dicarboxylic acid derivative containing an acetal bond was synthesized by referring to known methods in the prior art, yielding the intermediate Acetal-(COOH)2.

[0066] Step 4: Synthesis of mPEG2000-RGD-acetal bond-COOH

[0067] mPEG2000-RGD (2.6 g, 1 mmol) was dissolved in anhydrous dichloromethane (20 mL), and acetal-(COOH)2 (0.3 g, 1 mmol), EDCI (0.38 g, 2 mmol), and DMAP (0.024 g, 0.2 mmol) were added. The reaction mixture was reacted at room temperature for 24 h. The reaction solution was washed successively with dilute hydrochloric acid and saturated sodium bicarbonate, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, precipitated with diethyl ether, and dried under vacuum to give mPEG2000-RGD-acetal-COOH (2.2 g, 78% yield).

[0068] Step 5: Synthesis of mPEG2000-RGD-acetal-C16 quaternary ammonium salt

[0069] 2.0 g (0.7 mmol) of mPEG2000-RGD-acetal bond-COOH was dissolved in anhydrous dichloromethane (15 mL), followed by the addition of EDCI (0.27 g, 1.4 mmol) and DMAP (0.017 g, 0.14 mmol). After activation for 30 min, N,N-dimethylhexadecylamine (0.21 g, 0.7 mmol) was added, and the reaction was carried out at room temperature for 24 h. The reaction solution was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, precipitated by diethyl ether, and dried under vacuum to give the final product molecule A (1.8 g, 80% yield). The structure was confirmed by 1H NMR and GPC.

[0070] 2. Synthesis of molecule B (hyaluronic acid oligosaccharide-SS-0831A-cholesterol):

[0071] Step 1: Preparation of hyaluronic acid oligosaccharides (P)

[0072] Take 5 g of sodium hyaluronate (molecular weight approximately 10 kDa), dissolve it in 0.1 M HCl solution, hydrolyze at 60℃ for 2 h, adjust to neutral with NaOH, ultrafilter (molecular weight cutoff 3 kDa), collect the filtrate, freeze dry to obtain hyaluronic acid oligosaccharide (molecular weight approximately 2000, determined by GPC), i.e., the anionic hydrophilic segment P.

[0073] Step 2: Synthesis of the linker arm containing disulfide bonds

[0074] Cystamine dihydrochloride (2.25 g, 10 mmol) was dissolved in anhydrous methanol (50 mL), and triethylamine (2.8 mL, 20 mmol) was added. The active Boc-alanine ester (Boc-Ala-OSu, 3.0 g, 10 mmol) was slowly added dropwise under ice bath conditions, and the reaction was carried out at room temperature for 12 h. The solvent was evaporated, dissolved in water, and extracted with ethyl acetate to remove impurities. The aqueous phase was then lyophilized to obtain Boc-Ala-SS-NH2. The Boc protection was then removed to obtain Ala-SS-NH2.

[0075] Step 3: Synthesis of 0831A-cholesterol (0831A-H')

[0076] 0831A (0.5 g, 1 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL), and cholesterol chloroformate (0.45 g, 1 mmol) and triethylamine (0.14 mL, 1 mmol) were added. The reaction was carried out at room temperature for 12 h. The solvent was evaporated, and the mixture was purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to give 0831A-cholesterol (0.6 g, 75% yield). The result was confirmed by MS and 1H NMR.

[0077] Step 4: Synthesis of Hyaluronic Acid Oligosaccharide-SS-0831A-Cholesterol (PSS-0831A-H')

[0078] Hyaluronic acid oligosaccharide P (0.4 g, 0.2 mmol) was dissolved in anhydrous formamide (5 mL), and EDCI (0.077 g, 0.4 mmol) and NHS (0.046 g, 0.4 mmol) were added, followed by activation for 30 min. Then, Ala-SS-NH2 (0.036 g, 0.2 mmol) was added, and the reaction was carried out at room temperature for 24 h. The reaction solution was dialyzed (molecular weight cutoff 1000) for 48 h, and lyophilized to obtain the intermediate hyaluronic acid oligosaccharide-SS-NH2 (PSS-NH2). This intermediate (0.3 g, approximately 0.15 mmol) was dissolved in anhydrous DMSO (5 mL), and 0831A-cholesterol (0.15 g, 0.18 mmol), EDCI (0.058 g, 0.3 mmol), and DMAP (3.7 mg, 0.03 mmol) were added, followed by reaction at room temperature for 24 h. The reaction solution was dialyzed (molecular weight cutoff 2000) for 72 h, and then lyophilized to obtain the final product molecule B (0.25 g, yield 68%). The structure was confirmed to be correct by 1H NMR and GPC.

[0079] 3. Preparation of nanomicelles

[0080] Weigh out 20 mg of molecules A and B at a molar ratio of 2:1 and dissolve them in 2 mL of DMSO. With magnetic stirring (500 rpm), slowly add the solution dropwise to 20 mL of PBS buffer (pH 6.8, 10 mM) using a microsyringe. After the addition is complete, continue stirring for 2 h. Transfer the resulting solution to a dialysis bag (molecular weight cutoff 3500) and dialyze with deionized water for 24 h (changing the water every 6 h) to remove the DMSO. After dialysis, filter the solution through a 0.45 μm filter membrane to obtain the nanomicelle solution. Lyophilization yields a solid powder.

[0081] 4. Characterization Results

[0082] Particle size and Zeta potential: The above micelle solution was diluted to an appropriate concentration and measured using a Malvern laser particle size analyzer. Results are as follows: Figure 3 As shown, the average particle size was 112.3 ± 8.7 nm, and the PDI was 0.12 ± 0.03, indicating a uniform particle size distribution. The Zeta potential was +8.5 ± 1.2 mV (pH 7.4), indicating that the surface carries a weak positive charge, which is beneficial for interaction with the negatively charged cell membrane.

[0083] TEM morphology: The sample was observed under a transmission electron microscope after being negatively stained with phosphotungstic acid. Figure 4 As shown, the nanomicelles are regularly spherical, well dispersed, and their particle size is consistent with the DLS results.

[0084] Critical micelle concentration (CMC): The CMC was determined using pyrene as a fluorescent probe and the result was 3.2 μg / mL, indicating that the nanomicelles have high thermodynamic stability.

[0085] Drug loading: An appropriate amount of micelle solution was taken, and PBS at pH 5.0 was added to disrupt the micelles. The 0831A content was determined by HPLC. The calculated drug loading of 0831A was 11.2 ± 0.6%.

[0086] 5. In vitro release behavior study (multiple response validation)

[0087] The release behavior of micelles under different conditions was investigated using dialysis. One mL of micelle solution (containing approximately 1 mg of 0831A) was placed in a dialysis bag and immersed in 30 mL of release medium (PBS containing 0.5% Tween-80, pH 7.4, pH 6.5, or pH 6.5 + 10 mM GSH), and the solution was incubated at 37°C with shaking. Samples were taken at predetermined time points, and the concentration of 0831A in the release medium was determined by HPLC. The cumulative release rate was calculated. Results are as follows: Figure 5As shown, the cumulative release rate was only 12.3% after 48 hours at pH 7.4; 37.8% at pH 6.5; and 83.5% at pH 6.5 + 10 mM GSH, demonstrating a significant pH / redox dual-response release characteristic. This result proves that the present invention successfully solves the problems of inaccurate and uncontrollable release in single pH response, achieving a cascade release of "extracellular triggering and intracellular burst".

[0088] Example 2: Preparation and efficacy evaluation of multidrug co-delivery nanomicelles encapsulating the immunomodulator NLG919 and the tracer probe DiR.

[0089] 1. Preparation of nanomicelles encapsulating NLG919 and DiR

[0090] Using the same molecules A and B as in Example 1, in a molar ratio of 2:1, a total of 20 mg, 2 mg of NLG919 and 0.5 mg of DiR were added and dissolved together in 2 mL of DMSO. Subsequent assembly and dialysis steps were the same as in Example 1. The encapsulation efficiency and drug loading of NLG919 and DiR in the resulting micelle solution were determined. HPLC results showed that the encapsulation efficiency of NLG919 was 92.3±2.1%, and the drug loading was 8.5±0.4%; the encapsulation efficiency of DiR was 95.1±1.8%, and the drug loading was 2.3±0.2%; and the drug loading of 0831A was 10.8±0.5%. This result demonstrates that the present invention successfully solves the problem of single structure and function, realizing the potential for multi-drug co-loading and traceability.

[0091] 2. In vitro cytotoxicity assay (MTT assay)

[0092] Logarithmic growth phase CT26 mouse colon cancer cells were seeded at 5 × 10³ cells / well in 96-well plates and cultured for 24 h. Then, different concentrations of 0831A active pharmaceutical ingredient, blank micelles, and NLG919-encapsulated nanomicelles (based on 0831A concentration) were added, and the cells were cultured for another 72 h. Cell viability was determined by the MTT assay, and the IC50 value was calculated. Results are as follows: Figure 6 As shown, the IC50 of the 0831A active pharmaceutical ingredient was 2.50 ± 0.32 nM; the IC50 of the nanomicelles encapsulating NLG919 was 1.18 ± 0.15 nM, which was significantly lower than that of the active pharmaceutical ingredient group (p < 0.01). This indicates that the nanomicelles can enhance the cytotoxicity of 0831A, and the encapsulation of NLG919 did not affect its activity. On the contrary, it further enhanced the killing effect through synergistic effect.

[0093] 3. In vivo imaging tracer experiment (traceability verification)

[0094] A subcutaneous colon cancer xenograft model was established in BALB / c mice (CT26). When the tumor volume reached approximately 100 mm³, DiR-encapsulated nanomicelles (DiR dose 0.5 mg / kg) were injected via the tail vein. The in vivo distribution of DiR was observed using a small animal in vivo imaging system at 2 h, 6 h, 12 h, 24 h, and 48 h post-injection. Results are as follows: Figure 7 As shown: 2 hours after injection, the nanomicelles began to circulate throughout the mouse system; from 6 to 12 hours, the fluorescence signal at the tumor site gradually increased; the fluorescence signal at the tumor site reached its peak at 24 hours, indicating that the nanomicelles were enriched at the tumor site through active targeting; a significant fluorescence signal at the tumor site could still be detected at 48 hours. This result proves that the present invention successfully achieved in vivo tracking of nanomicelles, enabling real-time monitoring of drug distribution and tumor accumulation in vivo.

[0095] 4. Evaluation of in vivo antitumor drug efficacy

[0096] A BALB / c mouse model of CT26 colon cancer subcutaneous xenograft was established. When the tumor volume reached approximately 100 mm³, mice were randomly divided into 5 groups (n=6): saline group (control group), 0831A raw material group (5 mg / kg), low-dose group of NLG919-encapsulated nanomicelles (5 mg / kg 0831A equivalent), high-dose group of NLG919-encapsulated nanomicelles (10 mg / kg 0831A equivalent), and positive control paclitaxel group (30 mg / kg, intraperitoneal injection, twice a week). Administration was via tail vein injection every 3 days for a total of 4 administrations. Tumor volume and mouse body weight were measured every 2 days.

[0097] The results are as follows Figure 8 As shown: the saline group showed rapid tumor growth; the 0831A raw material group had some inhibitory effect, but the effect was limited; the low-dose nanomicelle group showed significant tumor inhibition, and the high-dose group almost completely inhibited tumor growth, with an inhibition rate of 82.5%, which was significantly better than the raw material group (p<0.001). Body weight change curve ( Figure 9 The results showed that, except for the paclitaxel group, the body weight of mice in the other treatment groups did not decrease significantly, indicating that the nanomicelles had good safety. This result proves that the present invention has successfully solved the problem of limited tumor targeting ability, and significantly improved the anti-tumor efficacy and reduced systemic toxicity through active targeting and multiple response mechanisms.

[0098] 5. Pharmacokinetic studies

[0099] SD rats were randomly divided into two groups (n=5), receiving intravenous injections of 0831A active pharmaceutical ingredient (5 mg / kg) and NLG919-encapsulated nanomicelles (equivalent to 0831A), respectively. Blood samples were collected at different time points, and the plasma concentration of 0831A was determined by HPLC-MS to calculate pharmacokinetic parameters. The results showed that the half-life (t1 / 2) of the active pharmaceutical ingredient group was 2.5 ± 0.3 h, and the AUC was 140 ± 35 μg·h / L; while the t1 / 2 of the nanomicelle group was prolonged to 21.3 ± 2.8 h, and the AUC was 850 ± 92 μg·h / L, indicating significantly improved bioavailability. This result demonstrates that the nanomicelles of the present invention exhibit excellent stability in blood circulation and effectively prolong drug circulation time.

[0100] Example 3: Preparation and particle size control (ratio adjustment) of folic acid-targeted nanomicelles

[0101] In molecule A, RGD was replaced with folic acid (FA), while the rest of the structure remained unchanged. Three types of nanomicelles were prepared with molecule A:molecule B molar ratios of 1:1, 2:1, and 3:1. The particle size determination results are as follows:

[0102] (1) 1:1 group: 168.5±12.3 nm, PDI 0.18;

[0103] (2) Group 2:1: 132.7±9.8 nm, PDI 0.14;

[0104] (3) Group 3:1: 98.2±7.6 nm, PDI 0.11;

[0105] The above measurement results show that the particle size can be effectively controlled by adjusting the ratio, and the PDI is less than 0.2, with uniform distribution. This result proves that the present invention has successfully solved the problem of single structure and function, and achieved the controllability of physicochemical properties such as particle size.

[0106] Example 4: Stability Study

[0107] The nanomicelle solution prepared in Example 1 of this invention was stored at 4°C and 25°C, respectively, and the particle size and PDI were measured at 0, 7, 14, and 30 days. The results showed that after 30 days of storage at 4°C, the particle size change was less than 5%, and the PDI did not increase significantly; after 14 days of storage at 25°C, the particle size increased slightly, but remained within an acceptable range. After 6 months of storage at -20°C, the lyophilized powder showed no significant changes in particle size or drug loading upon reconstitution, indicating that the nanomicelles have excellent storage stability.

[0108] Example 5: Comparative Experiment (Single Response vs. Dual Response)

[0109] To verify the advantages of the dual-response mechanism, a control molecule B' (hyaluronic acid oligosaccharide-acetal bond-0831A-cholesterol) containing only acetal bonds (without disulfide bonds) was synthesized and assembled with molecule A to form control micelles. In vitro release results showed that under pH 6.5 and 10 mM GSH conditions, the release rate of the control micelles was only 45.2% after 48 h, far lower than the 83.5% of the dual-response micelles, indicating that disulfide bonds are crucial for achieving rapid intracellular release. This comparative experiment further demonstrates that the multiple-response mechanism of this invention successfully solves the problems of inaccurate and uncontrollable release under single pH responses.

[0110] In summary, the above embodiments of the present invention demonstrate that the multi-responsive traceable targeted multi-drug co-delivery nanomicelles provided by the present invention have a well-defined structure, are easy to prepare, and successfully solve the core problems of inaccurate drug release, limited tumor targeting ability, and single structure and function in the prior art. They have multiple advantages such as dual pH / redox response, active targeting, multi-drug co-delivery, traceability, and tunable structure, and have broad application prospects in the field of anti-tumor drug delivery.

[0111] 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 multi-responsive, traceable, targeted multi-drug co-delivery nanomicelle, characterized in that, The targeted multi-drug co-delivery nanomicelles are formed by the synergistic assembly of molecules A and B through electrostatic and hydrophobic interactions. The molecule A has the structure shown in formula (Ⅰ): （Ⅰ） Wherein, mPEG is monomethoxy polyethylene glycol with a number average molecular weight of 1000-5000; L1 is a linker bond selected from amide bonds or ester bonds; T is a tumor-targeting ligand; L2 is a pH-sensitive linker arm; and H is a cationic hydrophobic segment. The molecule B has the structure shown in formula (II): （Ⅱ） Wherein, P is the anionic hydrophilic segment; SS is the disulfide bond; 0831A is the antitumor drug SKLB060-methionine; L3 is the linking bond, selected from ester or amide bonds; and H' is the hydrophobic tail.

2. The multi-responsive traceable targeted multi-drug co-delivery nanomicelles according to claim 1, characterized in that, The tumor-targeting ligand T is selected from RGD peptide, folic acid, hyaluronic acid, or galactose; The pH-sensitive linker L2 is selected from acetal bonds, orthoester bonds, or vinyl ether bonds; The cation-containing hydrophobic segment H is a C12-C18 alkyl chain containing a quaternary ammonium salt; The anionic hydrophilic segment P is selected from hyaluronic acid oligosaccharide, polyglutamic acid, or polyaspartic acid; the hydrophobic tail H' is selected from cholesterol, vitamin E, or C16-C18 fatty acids.

3. The multi-responsive traceable targeted multi-drug co-delivery nanomicelles according to claim 1, characterized in that, The number-average molecular weight of mPEG in molecule A is 2000; T is RGD peptide or folic acid; L2 is acetal bond; H is a C16 alkyl chain containing quaternary ammonium salt; P in molecule B is hyaluronic acid oligosaccharide with a molecular weight of 1000-3000; H' is cholesterol or vitamin E.

4. The multi-responsive traceable targeted multi-drug co-delivery nanomicelles according to claim 1, characterized in that, The molar ratio of molecule A to molecule B is 1:1 to 3:

1.

5. The multi-responsive traceable targeted multi-drug co-delivery nanomicelles according to claim 1, characterized in that, Its core also contains a therapeutic agent; the therapeutic agent is selected from at least one of immunomodulators, photosensitizers, anti-angiogenic drugs, or diagnostic probes.

6. The multi-responsive traceable targeted multi-drug co-delivery nanomicelles according to claim 5, characterized in that, The immunomodulator is NLG919, indomod, or ralsimod; the photosensitizer is dihydroporphyrin e6 or indocyanine green; the anti-angiogenic drug is apatinib or sunitinib; and the diagnostic probe is a near-infrared fluorescent probe Cy5.5, DiR, or superparamagnetic iron oxide nanoparticles.

7. The method for preparing multi-responsive traceable targeted multi-drug co-delivery nanomicelles according to any one of claims 1-6, characterized in that, Includes the following steps: S1. Synthesize molecules A and B respectively; S2. Dissolve molecules A and B in an organic solvent at a predetermined molar ratio to obtain a mixed solution; S3. Under stirring, the mixed solution obtained in step S2 is added dropwise to a buffer solution with pH 6.0 to 7.5, and spontaneously assembles to form nanomicelles. S4. Remove the organic solvent by dialysis or ultrafiltration to obtain the nanomicelle solution.

8. The preparation method according to claim 7, characterized in that, The organic solvent mentioned in step S2 is selected from dimethyl sulfoxide, N,N-dimethylformamide, or acetonitrile; The buffer solution mentioned in step S3 is a phosphate buffer or an acetate buffer, and the assembly temperature is 20–37°C.

9. A pharmaceutical composition, characterized in that, It comprises the nanomicelles as described in any one of claims 1-6 and a pharmaceutically acceptable carrier.

10. The use of the nanomicelles according to any one of claims 1-6 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating cancer; wherein the cancer is colorectal cancer, breast cancer, lung cancer, liver cancer or ovarian cancer.

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