Dual-targeting / drug-loaded tumor reduction-sensitive nanocarrier and application thereof

Abstract

The application discloses a kind of dual targeting / drug-loaded reduction-sensitive nano-carrier, and the micelle is formed by the micelle of block polymer by solvent evaporation method, and the multi-block polymer is Tel-PEG-ss-PCL, the hydrophilic end of which is PEG with targeting group Tel, and the hydrophobic end is PCL, which is a core-shell structure, the core is the hydrophobic end PCL, and the shell is the hydrophilic block with targeting group Tel.The nano-carrier selects the polymer micelle with hydrophilic end polyethylene glycol and hydrophobic end polycaprolactone as drug carrier to improve the problem of poor water solubility of drug;Tel, an angiotensin II type receptor (AT1R) ligand, is used to simultaneously target CAFs and tumor cells overexpressing AT1R, kill tumor cells and CAFs, and double drug loading to overcome drug resistance;Meanwhile, disulfide bond is used to respond to high concentration glutathione (GSH) in tumor cells to achieve precise control release of drug in cells.The nano-carrier realizes the treatment effect of solubilization of poorly soluble drug, tumor and CAFs dual targeting and reversal of drug resistance.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and nanomedicine, and in particular, it relates to a dual-targeting, low-toxicity, multifunctional responsive nanocarrier. Background Technology

[0002] Breast cancer is a globally recognized public health issue that seriously threatens the physical and mental health of women worldwide. Similar to breast cancer in humans, dogs, as companion animals, are exposed to the same carcinogenic factors due to living in the same environment as humans. Canine breast cancer is also the most common malignant tumor in female dogs, and it is the second most diagnosed cancer in dogs, with an average incidence rate as high as 25% to 42%. In unspayed female dogs, the incidence rate can even reach 50% to 70%, which is three times the malignancy rate of human breast tumors. Moreover, this rate increases with age.

[0003] Similar to human breast cancer, canine breast cancer has an extremely high incidence and a low cure rate. Drug resistance is one of the main reasons for treatment failure. Acquired resistance, which is the primary type of chemotherapy resistance, leads to decreased efficacy of subsequent drug administration. Dual-drug therapy is believed to reduce the decrease in sensitivity caused by acquired resistance. Furthermore, tumor-associated stromal cells (CAFs), such as CAFs, promote tumor cell survival through various mechanisms, including secreting alanine as a nutrient supply for the tricarboxylic acid cycle of tumor cells. This leads to decreased chemosensitivity and promotes tumor growth and metastasis. Therefore, developing nanocarriers that dual-target both CAFs and tumor cells and dual-load chemotherapeutic drugs is of great significance in overcoming drug resistance.

[0004] This invention addresses the problem of tumor drug resistance in current clinical practice by synthesizing and preparing a dual-targeting / drug-loaded tumor-reducing sensitive nanocarrier for the treatment of breast cancer, particularly canine breast cancer. Summary of the Invention

[0005] The purpose of this invention is to provide a dual-targeting / drug-loaded reductive nanocarrier with therapeutic potential to address the problem of drug resistance in breast tumors in current clinical practice.

[0006] Based on existing research, this invention creatively synthesizes an unreported long polymer chain. First, polymer micelles with hydrophilic polyethylene glycol and hydrophobic polycaprolactone are selected as drug carriers to improve the problem of poor drug water solubility. Then, the angiotensin II receptor (AT1R) ligand telmisartan (Tel) is used to simultaneously target CAFs overexpressing AT1R and tumor cells, killing both tumor cells and CAFs to overcome CAF-induced drug resistance. Simultaneously, disulfide bonds are used to respond to the high concentration of glutathione (GSH) within tumor cells to achieve precise controlled drug release within the cells. This material achieves therapeutic effects including solubilization of poorly soluble drugs, dual targeting of tumors and CAFs, and reversal of CAF-induced drug resistance in tumor cells. The material simultaneously achieves good biocompatibility and tumor targeting, exhibiting high targeting and killing effects on tumor cells. Furthermore, this invention verifies its antitumor effects at both cellular and animal levels, both in vitro and in vivo, aiming to provide a theoretical basis for the treatment of canine mammary cancer and offer new insights for the treatment of human mammary cancer.

[0007] To achieve this solution, the technical solution of the present invention is: a targeted ligand, a reduction-responsive carrier with dual targeting of tumor tissue, the structural formula of which is shown in the formula below. As shown ( Figure 1 ):

[0008] Where n≥2, m≥2;

[0009] Mode The compound is Tel-PEG-ss-PCL (TPSP), a block polymer, wherein n≥2, m≥2, and more preferably m is in the range of 30-70 and n is in the range of 50-80. TPSP powder and the drug are dissolved together in tetrahydrofuran (THF), and the TPSP is added to the THF solution under high-speed stirring. After the addition is complete, the stirring speed is slowed down. After the THF has completely evaporated, a drug-loaded micelle solution is obtained. This solution is transferred entirely to a dialysis bag (molecular weight cutoff of 1000), dialyzed with distilled water for 48 hours to remove unloaded drug, and then freeze-dried to obtain a dual-targeting / drug-loaded tumor-reducing sensitive nanocarrier. The TPSP micelles have a particle size distribution ranging from 100-140 nm, are relatively uniformly dispersed, and have a near-spherical structure. The micelles have a core-shell structure, with a hydrophilic end containing PEG with a targeting group Tel and a hydrophobic end containing PCL. This allows for targeting of tumor cells and CAFs, and rapid response to high concentrations of glutathione (GSH) within tumor cells to achieve precise and controlled drug release within the cells.

[0010] This invention creatively utilizes the high affinity of the AT1R ligand telmisartan to tumor-associated fibroblasts and tumor cells overexpressing AT1R to achieve targeted polymer micelles. After the drug enters the tumor cells, the carrier can use disulfide bonds to respond to the high concentration of GSH in the tumor cells to achieve precise controlled release of the drug within the cells, thereby enhancing the therapeutic effect of chemotherapy drugs.

[0011] This invention provides a carrier that can encapsulate chemotherapy drugs such as DOX and natural antitumor compounds such as aconitine linoleate within a nanocarrier. It is particularly suitable for drugs with poor water solubility and significant side effects. The carrier encapsulates the antitumor drug within the hydrophobic core of the polymer, increasing the drug's water solubility and bioavailability. When the drug is in the bloodstream, it is less prone to leakage and exhibits good targeting of tumor cells, thus reducing the toxic side effects of antitumor drugs.

[0012] The preparation method of the dual-targeting, low-toxicity tumor microenvironment-responsive nanocarrier includes the following steps:

[0013] 1) Synthesis of monoprotected 1,3-propanediamine (BOC-NH-NH2)

[0014] Dissolve 10-100 parts of 1,3-propanediamine in methanol. Under ice bath conditions, add 1-10 parts of a methanol solution of Boc₂O dropwise and react at room temperature for 12-36 h. After the reaction is complete, concentrate under reduced pressure to recover the solvent. Dissolve the product in ethyl acetate, recover the solvent under reduced pressure, add NaH₂PO₄ solution to the residue to adjust the pH to 5, and extract with petroleum ether:ethyl acetate = 1:2 to remove the double-protected product. After extraction, separate the aqueous phase, add NaOH solution to adjust the pH to 12, and add ethyl acetate to extract the monoprotected 1,3-propanediamine. Figure 2 ).

[0015] 2) Synthesis of monoprotected propylenediamine-telmisartan (Tel-NH-BOC)

[0016] Take 1-5 parts of telmisartan, 3-15 parts of EDCI, 1-6 parts of NHS, and an appropriate amount of triethylamine, dissolve them in dichloromethane, and add 1-5 parts of BOC-propylenediamine dichloromethane solution under ice bath conditions. Slowly raise the temperature to 30-50 °C and react for 24-60 h. After the reaction is complete, separate the organic phase and recover the solvent under reduced pressure. The product is purified by rapid silica gel chromatography and dried under vacuum to obtain monoprotected propylenediamine-telmisartan (… Figure 3 ).

[0017] 3) Synthesis of monoprotected cystamine (BOC-NH-ss-NH2)

[0018] Dissolve 1-5 parts of cystamine dihydrochloride and 3-15 parts of triethylamine in methanol. Under ice bath conditions, add 1-5 parts of a methanol solution of BOC anhydride dropwise and react at room temperature for 12-36 h. After the reaction is complete, recover the solvent under reduced pressure. Dissolve the product in ethyl acetate, recover the solvent, add NaH2PO4 solution to the residue to adjust the pH to 5, and extract with petroleum ether:ethyl acetate = 1:1 to remove the double-protected product. Collect the aqueous phase, add NaOH solution to adjust the pH to 12, extract with ethyl acetate, recover the solvent under reduced pressure, and dry under vacuum to obtain monoprotected cystamine. Figure 4 ).

[0019] 4) Synthesis of monoprotected cystamine-polycaprolactone (BOC-ss-NH2-PCL)

[0020] Take 1-5 parts of BOC-ss-NH2, 30-300 parts of ε-caprolactone, stannous octoate as catalyst, and react at 100-150℃ for 3-10 h under nitrogen protection. After the reaction is terminated, add a small amount of dichloromethane to dissolve the product into a paste, then add it dropwise to a large amount of ice-cold ethanol to precipitate it. Filter and dry to obtain monoprotected cystamine-polycaprolactone. Figure 5 ).

[0021] Where n≥2.

[0022] 5) Synthesis of bilaterally carboxylated polyethylene glycol (COOH-PEG-COOH)

[0023] 1-5 parts of PEG 2000 Dissolve 4-20 parts of succinic anhydride in dry dichloromethane and reflux at 50-80°C. After the reaction is complete, recover the organic phase under reduced pressure, dissolve the product in a small amount of dichloromethane until it becomes viscous, precipitate it by adding it dropwise to a large amount of ice-cold diethyl ether, filter and dry to obtain double-sided carboxylated polyethylene glycol (PEG). Figure 6 ).

[0024] Where m≥2.

[0025] 6) Synthesis of polyethylene glycol-cystamine-caprolactone (COOH-PEG-SS-PCL, PSP)

[0026] Take 1-5 parts of Boc-ss-NH-PCL and dissolve it in DCM:TFA (v:v, 10:1). Stir the reaction at room temperature until Boc is completely removed. Recover the solvent under reduced pressure. Dissolve the residue in dichloromethane and add an appropriate amount of triethylamine. Simultaneously, dissolve 1-5 parts of COOH-PEG-COOH, 1-5 parts of EDCI, and 1-5 parts of NHS in dry dichloromethane and add an appropriate amount of triethylamine. Mix the two solutions in an ice bath and react at 25-40 °C. After the reaction is complete, separate the organic phase, recover the organic solvent under reduced pressure, dissolve it in a small amount of dichloromethane until it becomes viscous, and precipitate it by adding it dropwise to a large amount of ice-cold ethanol. Repeat the dissolution and precipitation until COOH-PEG-COOH is completely removed. Dry under vacuum to obtain polyethylene glycol-cystamine-caprolactone (PSP). Figure 7 ).

[0027] Where n≥2, m≥2.

[0028] 7) Synthesis of Tel-PEG-ss-PCL

[0029] Take 1-5 parts of COOH-PEG-SS-PCL, 3-15 parts of EDCI, 10-15 parts of NHS, and an appropriate amount of triethylamine, and dissolve them in dichloromethane for later use. Separately, take 1-5 parts of Tel-NH2-BOC and dissolve it in DCM:TFA = (v:v, 10:1). React at room temperature until Boc is completely removed to expose the amino group. After recovering the organic solvent under reduced pressure, redissolve it in dichloromethane. Add this to the above COOH-PEG-SS-PCL solution, heat to 20-40 °C, and react. After the reaction is complete, separate the organic phase, recover the solvent under reduced pressure, and dissolve it in a small amount of dichloromethane until it becomes viscous. Precipitate this by adding it dropwise to a large amount of ice-cold ethanol, filter, and vacuum dry to obtain the polymer Tel-PEG-SS-PCL (TPSP) (e.g., ...). Figure 1 ).

[0030] As a further improvement to the above technical solution, the preferred conditions for the synthesis step are as follows:

[0031] The feeding ratio of 1,3-propanediamine to Boc2O in step 1) is 10:1, the reaction temperature is 25℃, and the reaction time is 24h.

[0032] In step 2), the feed ratio of telmisartan:EDCI:NHS:Boc is 1:3:1:1, the amount of triethylamine added is 1%, the reaction temperature is 30 °C, and the reaction time is 48 h.

[0033] In step 3), the ratio of cystamine dihydrochloride, triethylamine, and BOC anhydride is 1:3:1, the reaction temperature is 25°C, and the reaction time is 24 hours.

[0034] In step 4), the ratio of BOC-ss-NH2:ε-caprolactone is 1:65, the amount of stannous octoate added is 0.5% of the reaction system, the reaction temperature is 140 ℃, and the reaction time is 6 h.

[0035] PEG in step 5) 2000 The ratio of succinic anhydride to reactants was 1:5, the reaction temperature was 50 °C, and the reaction time was 24 h.

[0036] In step 6), the ratio of Boc-ss-NH-PCL:COOH-PEG-COOH:EDCI:Tel-NH2-BOC is 1:3:1:1, the amount of triethylamine added is 1%, the reaction temperature is 30 ℃, and the reaction time is 48 h.

[0037] In step 7), the ratio of COOH-PEG-SS-PCL:EDCI:EDCI:NHS is 1:3:1:1, the amount of triethylamine added is 1%, the reaction temperature is 30 ℃, and the reaction time is 72 h.

[0038] The synthesis route for Tel-PEG-ss-PCL is as follows: Figure 8 :

[0039] Working mechanism of the present invention:

[0040] Drugs are formulated into micelles using the block polymer Tel-PEG-ss-PCL, with a hydrophobic drug encapsulated within the micelles to form a hydrophobic core. Once the drug-loaded micelles enter the tumor, the high affinity of the AT1R ligand telmisartan linked to the PEG end for AT1R-overexpressing cancer cells (CAFs) and tumor cells enables targeted delivery and enhances the endocytosis of the nanocarrier by tumor cells and CAFs. After entering tumor cells, the drug-loaded nanocarrier responds to the high concentration of GSH within the tumor cells via disulfide bonds, achieving precise and controlled drug release within the cells and enhancing the therapeutic effect of anti-tumor drugs.

[0041] Beneficial effects:

[0042] This anti-tumor dual-targeting nanocarrier achieves solubilization of poorly soluble drugs; it dual-loads drugs, simultaneously targeting both tumor cells and CAFs, reversing drug resistance in tumor cells; the drug delivery system has responsive release capability, achieving stimulus-response release under reducing conditions, and exhibits good targeting and killing effects at both cellular and animal levels.

[0043] This patent utilizes fewer synthesis steps and milder reaction conditions to prepare long polymer chains, which can be easily used to prepare micelles with a simple preparation process. The prepared polymer micelles exhibit good tumor targeting and tumor microenvironment responsiveness for precise release control. Attached Figure Description

[0044] Figure 1 The structure of the dual-targeting polymer Tel-PEG-ss-PCL.

[0045] Figure 2 Single protection of the 1,3-propanediamine structure.

[0046] Figure 3 Single-protected propylenediamine-telmisartan structure.

[0047] Figure 4 Single-protected cystamine structure.

[0048] Figure 5 Single-protected cystamine-polycaprolactone structure.

[0049] Figure 6 Bilaterally carboxylated polyethylene glycol structure.

[0050] Figure 7 Polyethylene glycol-cystamine-caprolactone structure.

[0051] Figure 8 Tel-PEG-ss-PCL synthesis route diagram.

[0052] Figure 9 polyethylene glycol-cystamine-caprolactone 1 H NMR (CDCl3).

[0053] Figure 10 Tel-PEG-ss-PCL 1 H NMR (CDCl3).

[0054] Figure 11 Infrared spectra of synthetic materials and their intermediates.

[0055] Figure 12 UV spectra of synthetic materials and intermediates.

[0056] Figure 13 GPC spectra of synthetic materials and intermediates.

[0057] Figure 14 Polymer molecular weight and distribution.

[0058] Figure 15 Transmission electron microscopy (TEM) results of drug-loaded micelles.

[0059] Figure 16 Particle size variation of drug-loaded micelles DOX+L29@TPSP in PBS and DMEM (10% FBS).

[0060] Figure 17In vitro cumulative release curves of DOX+L29@TPSP micelles: a) DOX cumulative release curve; b) L29 cumulative release curve.

[0061] Figure 18 Semi-quantitative fluorescence analysis of DOX and CAF cells cultured with different DOX-loaded micelles for 1 h.

[0062] Figure 19 Semi-quantitative fluorescence analysis of DOX and different DOX-loaded micelles after 1 h of CMT-7364 cell culture.

[0063] Figure 20 Semi-quantitative fluorescence analysis of DOX and different DOX-loaded micelles after 1 h of culture with ADR / MCF-7 cells.

[0064] Figure 21 Cytotoxicity of dual-drug micelles DOX+L29@PSP and DOX+L29@TPSP against a) NIH / 3T3 cells and b) CAFs.

[0065] Figure 22 Before co-culture, the IC50 values ​​of naked drug single / drug and dual-drug micelles on CMT-7364 cells and ADR / MCF-7 cells were compared. 50 Synergistic Index (CI) with drugs.

[0066] Figure 23 After co-culture, the IC50 values ​​of naked drug single / drug and dual-drug micelles on CMT-7364 cells and ADR / MCF-7 cells were compared. 50 Synergistic Index (CI) with drugs.

[0067] Figure 24 Cytotoxicity of naked dual-drug DOX+L29 and dual-drug micelles DOX+L29@PSP and DOX+L29@TPSP against a) CMT-7364 cells and b) ADR / MCF-7 cells.

[0068] Figure 25 Changes in tumor volume in mice in each treatment group over 21 days.

[0069] Figure 26 The weight of the tumors excised from the body after dissection on day 21 in each treatment group.

[0070] Figure 27 Tumor growth inhibition rate (TGI) in mice of each treatment group.

[0071] Figure 28 Changes in body weight of mice in each treatment group from 0 to 21 days. Specific implementation plan:

[0072] To make the objectives, technical solutions, and advantages of the invention clearer, the following embodiments provide a more detailed description of the invention. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0073] Example 1:

[0074] 1) Synthesis of monoprotected 1,3-propanediamine

[0075] Ten parts of 1,3-propanediamine were dissolved in methanol and added dropwise to a methanol solution of 1 part of Boc2O under ice bath conditions. The reaction was carried out at 25°C for 24 h. After the reaction was completed, the solvent was recovered by concentration under reduced pressure. The product was dissolved in ethyl acetate, and the solvent was recovered under reduced pressure. NaH2PO4 solution was added to the residue to adjust the pH to 5, and the double-protected product was removed by extraction with petroleum ether:ethyl acetate = 1:2. After extraction, the aqueous phase was separated, and NaOH solution was added to adjust the pH to 12. Ethyl acetate was then added to extract the monoprotected 1,3-propanediamine.

[0076] 2) Synthesis of monoprotected propylenediamine-telmisartan (Tel-NH-BOC)

[0077] One part telmisartan, three parts EDCI, one part NHS, and 1% triethylamine were dissolved in dichloromethane. One part BOC-propylenediamine dichloromethane solution was added under ice bath conditions, and the mixture was slowly heated to 30 °C and reacted for 48 h. After the reaction was complete, the organic phase was separated, and the solvent was recovered under reduced pressure. The product was purified by rapid silica gel chromatography and dried under vacuum to obtain monoprotected propylenediamine-telmisartan.

[0078] 3) Synthesis of monoprotected cystamine (BOC-NH-ss-NH2)

[0079] One part of cystamine dihydrochloride and three parts of triethylamine were dissolved in methanol. Under ice bath conditions, one part of BOC anhydride methanol solution was added dropwise, and the reaction was carried out at 25 °C for 24 h. After the reaction was completed, the solvent was recovered under reduced pressure. The product was dissolved in ethyl acetate, and the solvent was recovered. NaH2PO4 solution was added to the residue to adjust the pH to 5, and the product was extracted with petroleum ether:ethyl acetate = 1:1 to remove the double-protected product. The aqueous phase was collected, and NaOH solution was added to adjust the pH to 12. Ethyl acetate was added for extraction, and the solvent was recovered under reduced pressure. The product was then dried under vacuum to obtain monoprotected cystamine.

[0080] 4) Synthesis of monoprotected cystamine-polycaprolactone (BOC-ss-NH2-PCL)

[0081] Take 1 part BOC-ss-NH2, 65 parts ε-caprolactone, and 0.5% stannous octanoate, and react at 140 °C for 6 h under nitrogen protection. After the reaction is terminated, add a small amount of dichloromethane to dissolve the product into a paste, then add it dropwise into a large amount of ice-cold ethanol to precipitate it. Filter and dry to obtain monoprotected cystamine-polycaprolactone.

[0082] 5) Synthesis of bilaterally carboxylated polyethylene glycol (COOH-PEG-COOH)

[0083] 1 part PEG 2000 Five parts of succinic anhydride were dissolved in dry dichloromethane and refluxed at 50°C for 24 h. After the reaction was complete, the organic phase was recovered under reduced pressure, and the product was dissolved in a small amount of dichloromethane until it became viscous. This product was then added dropwise to a large amount of ice-cold diethyl ether to precipitate the product. The precipitate was filtered and dried to obtain double-sided carboxylated polyethylene glycol.

[0084] 6) Synthesis of polyethylene glycol-cystamine-caprolactone (COOH-PEG-SS-PCL, PSP)

[0085] One part of Boc-ss-NH-PCL was dissolved in DCM:TFA (v:v, 10:1) and stirred at room temperature until Boc was completely removed. The solvent was recovered under reduced pressure, and the residue was dissolved in dichloromethane with the addition of a suitable amount of triethylamine. Simultaneously, three parts of COOH-PEG-COOH, one part of EDCI, and one part of NHS were dissolved in dry dichloromethane with the addition of 1% triethylamine. The two solutions were mixed in an ice bath and reacted at 30°C for 48 hours. After the reaction was completed, the organic phase was separated, the organic solvent was recovered under reduced pressure, and dissolved in a small amount of dichloromethane until it became viscous. This precipitate was then added dropwise to a large amount of ice-cold ethanol. The process of dissolving and precipitating was repeated until COOH-PEG-COOH was completely removed. The product was then dried under vacuum to obtain polyethylene glycol-cystamine-caprolactone (PSP).

[0086] 7) Synthesis of Tel-PEG-ss-PCL

[0087] Take 1 part COOH-PEG-SS-PCL, 3 parts EDCI, 1 part NHS, and 1% triethylamine, and dissolve them in dichloromethane for later use. Separately, take 1 part Tel-NH2-BOC and dissolve it in DCM:TFA = (v:v, 10:1). React at room temperature until Boc is completely removed to expose the amino group. After recovering the organic solvent under reduced pressure, redissolve it in dichloromethane. Add this to the above COOH-PEG-SS-PCL solution, heat to 30 °C, and react for 72 h. After the reaction is complete, separate the organic phase, recover the solvent under reduced pressure, and dissolve it in a small amount of dichloromethane until it becomes viscous. Precipitate this by adding it dropwise to a large amount of ice-cold ethanol, filter, and vacuum dry to obtain the polymer Tel-PEG-SS-PCL (TPSP), with the structural formula shown below. Figure 1 As shown.

[0088] In this embodiment, m=50 and n=68. The size and ratio of m and n affect the solubility of the polymer and the drug loading capacity of the polymer micelles. In this embodiment, the ratio of hydrophilic to hydrophobic ends is approximately 1:3.

[0089] 8) Characterization of synthetic materials

[0090] a. 1H NMR spectral analysis of synthetic materials and intermediates

[0091] Polyethylene glycol-cystamine-caprolactone

[0092] Figure 9 1H NMR spectrum of polyethylene glycol-cystamine-caprolactone (COOH-PEG-ss-PCL)

[0093] 1 H NMR (600 MHz, CDCl3): δ 4.24(m, H a ), δ4.06(t, J= 6.6 Hz, H j ), δ3.64(m, H b H c ), δ3.56 (m, H l ), δ3.52 (m, H f ), δ2.82 (m, H g ), δ2.62 (m, H e, H d ), δ2.30 (t, J = 7.2 Hz, H h ), δ1.64 (m, H k ), δ1.39 (m, H i The presence of proton peaks around the carboxylated PEG and disulfide bonds indicates the successful synthesis of COOH-PEG-ss-PCL.

[0094] Tel-PEG-ss-PCL

[0095] Figure 10 The NMR spectrum of Tel-PEG-ss-PCL is 1H NMR.

[0096] 1 H NMR (600 MHz, CDCl3): δ 7.60-6.50 (m, Tel-Ar-H), δ4.06(t, J= 6.6Hz, H j ), δ3.65 (m, H c ), δ2.31 (t, J= 7.2 Hz, H h ), δ1.65 (m, Hk ), δ1.39 (m, H i Chemical shifts of δ4.06, δ2.31, δ1.65, and δ1.39 represent the peaks of PCL, while δ3.65 represents the peak of PEG. The presence of hydrogen proton peaks on Tel in the low-field range of δ (7.60-6.50) confirms the successful connection of Tel.

[0097] b. Infrared Spectrum Analysis of Synthetic Materials and Intermediates

[0098] The infrared spectra of COOH-PEG-COOH, Boc-NH-ss-NH-PCL, and Tel-PEG-ss-PCL are as follows: Figure 11 As shown, the OH stretching vibration peak in the COOH group of carboxylated PEG is at 3500 cm⁻¹. -1 The CH2 stretching vibration peak is at 2865 cm⁻¹. -1 The carbonyl stretching vibration peak is at 1725 cm⁻¹. -1 The COC stretching vibration peak is at 1107 cm⁻¹. -1 .

[0099] In Boc-NH-ss-PCL, an NH stretching vibration peak was observed at 3300 cm⁻¹. -1 The carbonyl stretching vibration peak is at 1725 cm⁻¹. -1 amide peak at 1541 cm⁻¹ -1 The characteristic CH stretching vibration peak of PCL is 2945 cm⁻¹. -1 2865 cm -1 And CS peak 732 cm -1 .

[0100] Tel-PEG-ss-PCL exhibits characteristic peaks from both of the aforementioned intermediates, including the OH stretching vibration peak in COOH at 3400 cm⁻¹. -1 The CH2 stretching vibration peak is at 2945 cm⁻¹. -1 2865 cm -1 The carbonyl stretching vibration peak is at 1725 cm⁻¹. -1 amide peak at 1541 cm⁻¹ -1 And CS peak 732 cm -1 Because Tel constitutes a small fraction of the molecule, its aromatic skeleton signal was not observed.

[0101] c. UV spectrum analysis of synthetic materials and intermediates

[0102] Figure 12The UV absorption spectra of polymers PEG-ss-PCL and Tel-PEG-ss-PCL are shown. The 250nm-340nm band is the characteristic UV absorption peak of the benzene ring in the Tel structure, which also proves that Tel was successfully attached to the polymer material.

[0103] d. GPC spectrum analysis of synthetic materials and intermediates

[0104] GPC test results are as follows Figure 13-14 The data shows that the number-average molecular weight (Mn) of Tel-PEG-ss-PCL is 4799, and the weight-average molecular weight (Mw) is 8566.

[0105] Example 2:

[0106] Preparation of micelles using synthetic carrier materials

[0107] Micelles were prepared using a solvent evaporation method. First, 10 mg of PEG-ss-PCL or Tel-PEG-ss-PCL copolymer powder was weighed and completely dissolved in 5 mL of tetrahydrofuran (THF) as the oil phase. Then, the mixture was added dropwise to 10 mL of distilled water using a disposable syringe while stirring at high speed. After the THF had completely evaporated, blank micelles PSP and TPSP were obtained. Furthermore, the preparation process of doxorubicin (DOX) and aconitine linoleate (L29)-loaded dual-drug micelles was similar to that of the blank micelles. The difference was that doxorubicin hydrochloride (DOX·HCl) was first dissolved in the THF solution, then a few drops of triethylamine were added, and the mixture was stirred in the dark for 30 min. Subsequently, the supernatant was collected, and L29 and the copolymer were added. After dissolving, the mixture was added to the THF solution while stirring at high speed. After the addition was complete, the stirring speed was slowed down. After the THF had completely evaporated, the drug-loaded micelle solution was obtained. All of them were transferred to dialysis bags (molecular weight cutoff of 1000), dialyzed with distilled water for 48 hours to remove unloaded DOX and L29, and then freeze-dried to obtain drug-loaded micelles DOX+L29@PSP and DOX+L29@TPSP.

[0108] Figure 15Micelles prepared by solvent evaporation were measured using a nanolaser particle size analyzer. The prepared micelles exhibited suitable size and uniformity, and could accumulate around tumor tissue through the EPR effect. After grafting the Tel group, the drug-loaded micelle potential increased from -34.83 mV to -23.47 mV. This is because the Tel group neutralizes the negative charge at the PEG terminus. Furthermore, the particle size of both micelles increased to some extent after loading both drugs, indicating successful drug loading. The particle size of the DOX+L29@PSP micelles was approximately 130 nm, and that of the DOX+L29@TPSP micelles was approximately 140 nm. The particle size distribution was good, and the micelles were visually spherical, consistent with the DLS data.

[0109] HPLC analysis showed that the drug loading of DOX in PSP and TPSP micelles was 1.12 ± 0.01% and 1.05 ± 0.01%, respectively, with corresponding encapsulation efficiencies of 9.7 ± 0.07% and 9.15 ± 0.05%. Meanwhile, the drug loading of L29 in PSP and TPSP micelles was 12.1 ± 0.18% and 13.93 ± 0.04%, respectively, with corresponding encapsulation efficiencies of 75 ± 1.09% and 86.36 ± 0.27%.

[0110] To investigate the stability of drug-loaded micelles, DOX+L29@TPSP micelles were placed in PBS and DMEM containing 10% FBS. DLS analysis at four time points (0 h, 24 h, 48 h, and 72 h) revealed no significant changes in particle size, which remained around 140 nm, indicating that the drug-loaded micelles DOX+L29@TPSP maintain good stability. Figure 16 ).

[0111] To determine the in vitro release of the two drugs, their release curves were tested at pH 7.4 10 μM GSH (simulating the normal intravascular blood environment) and pH 6.8 10 μM GSH and pH 6.8 10 mM GSH (simulating the intracellular and extracellular microenvironment of tumor cells), respectively. Figure 17 The sulfhydryl group of glutathione attacks the disulfide bond, causing it to break and releasing the drug. Figure 17 As can be seen, when the glutathione concentration is high (10 mM), the drug-loaded micelles exhibit a significant burst release within the first 12 hours. PWith a concentration <0.01%, the cumulative release over 70% was achieved within 48 hours. Meanwhile, at lower glutathione concentrations (10 μM), the cumulative release of the drug-loaded micelles was only 21.1% and 16.6%, respectively, indicating that the drug-loaded micelles do not prematurely release the drug during in vivo circulation and remain relatively stable in the bloodstream. In conclusion, the DOX+L29@TPSP drug-loaded micelles exhibit sensitive reduction responsiveness and can achieve rapid release in tumor cells.

[0112] Example 3: Cellular uptake experiment of drug-loaded micelles

[0113] To evaluate the cellular uptake of drug-loaded nanomicelles, fluorescent drug DOX was encapsulated in different materials (concentration 10 μg / mL) and reacted with tumor-associated fibroblasts (CAFs), canine breast cancer cells (CMT-7364), and doxorubicin-resistant human breast cancer cell line (ADR / MCF-7) for 1 h. The results were then evaluated using fluorescence microscopy and flow cytometry, and the fluorescence intensity of DOX was analyzed using ImageJ fluorescence semi-quantitative analysis and flow cytometry results. Figure 18 (19,20). The results showed that the targeted material group (DOX@TPSP) achieved high uptake in all three cell types. In CAFs, CMT-7364 and ADR / MCF-7 cells, the uptake capacity of the DOX@TPSP group was increased by 1.5 times, 0.84 times and 0.67 times compared with the DOX@PSP group, respectively. It can be seen that the Tel targeting group can increase the uptake of micelles by cells, especially tumor-associated fibroblasts, which proves that the synthesized polymer micelles have a good ability to target tumor-associated fibroblasts.

[0114] Example 4: Investigation of Drug Synergistic Index and In Vitro Cytotoxicity Assay of Drug-Loaded Micelles

[0115] The MTS assay was used to investigate the cytotoxicity of DOX+L29@PSP and DOX+L29@TPSP against NIH / 3T3 cells and CAFs. Figure 21 As shown in (a), when NIH / 3T3 cells were not stimulated to form CAFs, there was no significant difference in cytotoxicity between the DOX+L29@PSP and DOX+L29@TPSP groups, and the survival rate of NIH / 3T3 cells still exceeded 60% when the DOX concentration was 2 μM and the L29 concentration was 16 μM; Figure 21As shown in (b), when NIH / 3T3 cells were stimulated to form CAFs, the overall survival rate of CAFs was lower than that of NIH / 3T3 cells. Furthermore, the study found that the cell survival rate of the DOX+L29@TPSP group was also lower than that of the DOX+L29@PSP group. When the DOX concentration was 2 μM and the L29 concentration was 16 μM, the cytotoxicity difference between the DOX+L29@PSP and DOX+L29@TPSP groups was significant. P <0.05), the above results indicate that NIH / 3T3 cells stimulated to become CAFs have increased drug sensitivity, especially the DOX+L29@TPSP group, which also suggests that CAFs may have better uptake capacity for the DOX+L29@TPSP group.

[0116] To investigate whether the combined use of DOX and L29 could produce better cytotoxicity against tumor cells, the cytotoxicity of DOX alone and in combination with L29 at different concentrations in vitro against CMT-7364 and ADR / MCF-7 cells was examined, and the CI values ​​were calculated. Figure 22 The study results show that DOX and L29 are co-encapsulated in micelles, and their respective ICs... 50 Compared to the bare-label group, there was a significant decrease in all cases:

[0117] For CMT-7364 cells, the IC50 of DOX in the DOX+L29@PSP group was [missing information]. 50 The concentration of L29 in the DOX+L29@TPSP group decreased from 4.34 μM to 1.32 μM, and in the PSP group it decreased to 1.09 μM; the IC50 of L29 in the DOX+L29@PSP group was reduced. 50 The concentration decreased from 15.23 μM to 9.21 μM, and in the DOX+L29@TPSP group it decreased to 8.78 μM.

[0118] For ADR / MCF-7 cells, the IC50 of DOX in the DOX+L29@PSP group was [missing information]. 50 The concentration of L29 in the DOX+L29@TPSP group decreased from 86.44 μM to 1.33 μM; the concentration of L29 in the DOX+L29@PSP group decreased to 0.92 μM. 50 The concentration decreased from 14.30 μM to 9.34 μM, and in the DOX+L29@TPSP group it decreased to 7.34 μM.

[0119] Example 5: Cytotoxicity assay after co-culturing tumor cells with tumor-associated fibroblasts (CAFs)

[0120] This study first co-cultured tumor-associated fibroblasts (CAFs) with two types of tumor cells, and then tested whether the two types of tumor cells would develop resistance to DOX and L29 after co-culture. Figure 23The results showed that co-culturing two types of tumor cell lines with tumor-associated fibroblasts (CAFs) resulted in different IC50 values ​​for the two drugs. 50 All showed some improvement; after co-culture, the IC50 of CMT-7364 cells against DOX was increased. 50 The concentration was 14.81 μM, which is the IC50 value before co-culture. 50 3.41 times the value; for L29 IC 50 The concentration was 21.53 μM, which is the IC50 value before co-culture. 50 The value was 1.41 times higher. Meanwhile, the IC50 of ADR / MCF-7 cells against DOX was... 50 The concentration was 96.59 μM, which is the IC50 value before co-culture. 50 1.12 times the value; for L29 IC 50 The concentration was 53.16 μM, which is the IC50 value before co-culture. 50 The value was 3.72 times higher, suggesting that tumor-associated fibroblasts induced drug resistance in tumor cells.

[0121] Next, the cytotoxicity of naked dual-drug DOX+L29, dual-drug micelle DOX+L29@PSP, and DOX+L29@TPSP against CMT-7364 and ADR / MCF-7 cells co-cultured with tumor-associated fibroblasts (CAFs) was tested. Figure 23 , Figure 24 The results showed that the naked dual-drug group significantly reduced the IC50 of both drugs on both cell types. 50 ( P The CI value (<0.05) also indicates that the combination of the two drugs has a synergistic effect. It is worth noting that the IC50 of the targeted group with the two drugs... 50 The values ​​were all lower than those of the non-targeted group and the naked double-drug group, with the lowest CI values. This suggests that the targeted drugs have stronger targeting ability to the upper tumor-associated fibroblasts (CAFs), and can better kill or alter the characteristics of CAFs, indirectly promoting tumor cell apoptosis and reducing the drug resistance induced by CAFs in tumor cells.

[0122] Example 6: In vivo antitumor study of drug-loaded micelles

[0123] A mixed tumor model was established by inoculating canine breast cancer cells CMT-7364 and tumor-associated fibroblasts (CAFs) into the abdominal mammary pads of SPF-grade female Nod-scid mice to simulate a tumor-resistant environment.

[0124] After inoculation, monitor tumor volume until it reaches 50 mm. 3 At that time, tumor-bearing mice were randomly divided into 5 groups, defined as treatment day 0. The treatment dose for each group was DOX 0.4 mg / kg + L29 5 mg / kg. Figure 25The results of tumor volume measurement over 21 days are shown. The tumor volumes of mice in the TPSP, DOX+L29, DOX+L29@PSP, and DOX+L29@TPSP groups were 96.63%, 68.23%, 47.33%, and 24.71% of those in the saline group, respectively. Figure 26 The results of tumor weight measurement over 21 days are shown. The tumor weights of mice in the TPSP, DOX+L29, DOX+L29@PSP, and DOX+L29@TPSP groups were 102.53%, 71.01%, 45.86%, and 19.49% of those in the saline group, respectively. Figure 27 The tumor inhibition rate (TGI) of each treatment group was shown. The TGIs for the TPSP, DOX+L29, DOX+L29@PSP, and DOX+L29@TPSP groups were 3.57%, 31.76%, 53.25%, and 76.70%, respectively, with the DOX+L29@TPSP group showing the best therapeutic effect. Figure 28 The study showed the changes in body weight of mice in each group during the 21-day treatment period. Due to the toxic side effects of DOX, the body weight of mice in the naked DOX+L29 group was significantly lower than that of other groups. However, the body weight of mice in the DOX+L29@PSP group and the DOX+L29@TPSP group did not change significantly compared with the control group, which also suggests the safety of micelle therapy.

[0125] Comparison of H&E staining results of organs from different groups of mice, combined with changes in mouse body weight, revealed that the bare drug DOX+L29 is toxic to the heart and kidneys. Neutrophil accumulation was observed in the heart of the DOX+L29 group, and glomerular swelling, indistinct glomerular structure, and neutrophil aggregation were observed in the kidneys. No significant lesions were observed in the organs of the other groups and the drug-loaded micelle group, indicating that micelle encapsulation can reduce the toxicity of the bare drug to the body and improve in vivo safety.

[0126] Analysis of tumor tissue sections from mice in each group using H&E, Ki67, and TUNEL staining revealed that the cells in the saline and TPSP groups were dense, structurally intact, and had clearly defined nuclei and cytoplasm. In the treatment groups, tumor cells showed varying degrees of necrosis, with nuclei exhibiting condensation, fragmentation, and dissolution, and cell outlines becoming blurred. Furthermore, Ki67 and TUNEL staining results showed that the DOX+L29@TPSP group had the lowest positive proliferation signal and the highest positive apoptosis signal compared to the other groups, indicating that the DOX+L29@TPSP group had a better anti-tumor effect.

Claims

1. A dual-targeting / drug-loaded tumor-reducing sensitive nanocarrier, characterized in that, The nanocarrier is a micelle formed by solvent evaporation of a block polymer. The block polymer is Tel-PEG-ss-PCL, with a hydrophilic end of PEG containing the targeting group telmisartan (Tel) and a hydrophobic end of PCL, forming a core-shell structure. The core is hydrophobic PCL, and the shell is a hydrophilic block containing the targeting group Tel. Its preparation steps include: 1) Synthesis of monoprotected 1,3-propanediamine Dissolve 10-100 parts of 1,3-propanediamine in methanol. Under ice bath conditions, add 1-10 parts of Boc2O methanol solution dropwise. React at room temperature for 12-36 h. After the reaction is complete, concentrate under reduced pressure to recover the solvent. Dissolve the product in ethyl acetate and recover the solvent under reduced pressure. Add NaH2PO4 solution to the residue to adjust the pH to 5. Extract with a 1:2 ratio of petroleum ether:ethyl acetate to remove the double-protected product. After extraction, separate the aqueous phase. Add NaOH solution to adjust the pH to 12. Add ethyl acetate to extract the monoprotected 1,3-propanediamine shown in Formula I, i.e., BOC-propanediamine. Equation I 2) Synthesis of monoprotected propylenediamine-telmisartan Take 1-5 parts of telmisartan, 3-15 parts of EDCI, 1-6 parts of NHS and an appropriate amount of triethylamine, dissolve them in dichloromethane, add 1-5 parts of BOC-propylenediamine dichloromethane solution under ice bath, slowly heat to 30-50 ℃ and react for 24-60 h. After the reaction is complete, separate the organic phase and recover the solvent under reduced pressure. The product is purified by rapid silica gel chromatography and dried under vacuum to obtain the monoprotected propylenediamine-telmisartan Tel-NH2-BOC shown in Formula II. Formula II 3) Synthesis of monoprotected cystamine Dissolve 1-5 parts of cystamine dihydrochloride and 3-15 parts of triethylamine in methanol. Under ice bath conditions, add 1-5 parts of BOC anhydride methanol solution dropwise. React at room temperature for 12-36 h. After the reaction is complete, recover the solvent under reduced pressure. Dissolve the product in ethyl acetate and recover the solvent. Add NaH2PO4 solution to the residue to adjust the pH to 5 and extract with a 1:1 ratio of petroleum ether:ethyl acetate to remove the double-protected product. Collect the aqueous phase, add NaOH solution to adjust the pH to 12, extract with ethyl acetate, recover the solvent under reduced pressure, and dry under vacuum to obtain the monoprotected cystamine BOC-ss-NH2 shown in Formula III. Formula III 4) Synthesis of monoprotected cystamine-polycaprolactone Take 1-5 parts of BOC-ss-NH2, 30-300 parts of ε-caprolactone, catalyst amount of stannous octoate, and react at 100-150 °C for 3-10 h under nitrogen protection. After the reaction is terminated, add a small amount of dichloromethane to dissolve the product into a paste, then drop it into a large amount of ice-cold ethanol to precipitate it. Filter and dry to obtain the monoprotected cystamine-polycaprolactone Boc-ss-NH-PCL shown in Formula IV. Formula IV Where n≥2; 5) Synthesis of bilaterally carboxylated polyethylene glycol 1-5 parts of PEG 2000 Dissolve 4-20 parts of succinic anhydride in dry dichloromethane and reflux at 50-80℃. After the reaction is complete, recover the organic phase under reduced pressure. Dissolve the product in a small amount of dichloromethane until it becomes viscous. Add it dropwise to a large amount of ice-cold diethyl ether to precipitate. Filter and dry to obtain the bilateral carboxylated polyethylene glycol COOH-PEG-COOH shown in Formula V. Formula V Where m≥2; 6) Synthesis of polyethylene glycol-cystamine-caprolactone Take 1-5 parts of Boc-SS-NH-PCL and dissolve it in a 10:1 DCM:TFA solution. Stir the mixture at room temperature until Boc is completely removed. Recover the solvent under reduced pressure. Dissolve the residue in dichloromethane and add an appropriate amount of triethylamine. Simultaneously, dissolve 1-5 parts of COOH-PEG-COOH, 1-5 parts of EDCI, and 1-5 parts of NHS in dry dichloromethane and add an appropriate amount of triethylamine. Mix the two solutions in an ice bath and react at 25-40 °C. After the reaction is complete, separate the organic phase, recover the organic solvent under reduced pressure, dissolve it in a small amount of dichloromethane until it becomes viscous, and precipitate it by adding it dropwise to a large amount of ice-cold ethanol. Repeat the dissolution and precipitation process until COOH-PEG-COOH is completely removed. Dry under vacuum to obtain polyethylene glycol-cystamine-caprolactone COOH-PEG-SS-PCL as shown in Formula VI. Formula VI Where n≥2, m≥2; 7) Synthesis of Tel-PEG-ss-PCL Take 1-5 parts of COOH-PEG-SS-PCL, 3-15 parts of EDCI, 10-15 parts of NHS, and an appropriate amount of triethylamine, and dissolve them in dichloromethane to obtain a COOH-PEG-SS-PCL solution for later use. Separately, take 1-5 parts of Tel-NH2-BOC and dissolve it in a 10:1 mixture of DCM:TFA. React at room temperature until Boc is completely removed to expose the amino group. After recovering the organic solvent under reduced pressure, redissolve it in dichloromethane. Add this to the above COOH-PEG-SS-PCL solution and heat to 20-40 °C to react. After the reaction is complete, separate the organic phase, recover the solvent under reduced pressure, and dissolve it in a small amount of dichloromethane until it becomes viscous. Add this to a large amount of ice-cold ethanol to precipitate the precipitate, filter, and vacuum dry to obtain the polymer Tel-PEG-SS-PCL shown in Formula VII. Equation VII Where n≥2, m≥2.

2. The nanocarrier according to claim 1, characterized in that, In the block polymer, the repeating unit m is in the range of 30-70 or n is in the range of 50-80.

3. The method for preparing the dual-targeting / drug-loaded tumor-reducing sensitive nanocarrier according to claim 1, characterized in that, Step 1) involves feeding 10 parts of 1,3-propanediamine and 1 part of Boc2O at a reaction temperature of 25°C for 24 hours. Step 2) involves feeding 1 part of telmisartan, 3 parts of EDCI, 1 part of NHS, and 1 part of Boc2O, with triethylamine added at 1%, at a reaction temperature of 30°C for 48 hours. Step 3) involves feeding 1 part of cystamine dihydrochloride, 3 parts of triethylamine, and 1 part of BOC anhydride at a reaction temperature of 25°C for 24 hours. Step 4) involves feeding 1 part of BOC-ss-NH2O. 2和65份 ε-caprolactone was added, stannous octoate was added at 5‰ of the reaction system, the reaction temperature was 140 ℃, and the reaction time was 6 h; 1 part of PEG in step 5) 2000 In step 6), 5 parts of succinic anhydride were added, the reaction temperature was 50 ℃, and the reaction time was 24 h; in step 7), 1 part of Boc-ss-NH-PCL, 3 parts of COOH-PEG-COOH, 1 part of EDCI, and 1 part of Tel-NH2-BOC were added, the triethylamine content was 1%, the reaction temperature was 30 ℃, and the reaction time was 48 h; in step 8), 1 part of COOH-PEG-SS-PCL, 3 parts of EDCI, and 1 part of NHS were added, the triethylamine content was 1%, the reaction temperature was 30 ℃, and the reaction time was 72 h.

4. The use of the nanocarrier according to any one of claims 1-2 in the preparation of drugs for treating breast cancer.

5. The application according to claim 4, characterized in that, The drug mentioned is for treating breast cancer in dogs.

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