A matrix-regulated drug-semiflavanochrome conjugate and its preparation method and application

Abstract

The application discloses a kind of matrix regulatory drug-hemicyanine conjugates.The chemical structure of the conjugate is as shown in formula (I);Wherein, R1 is matrix regulatory drug, R2 is stimulus-responsive connecting molecule, R3 is C1~C18 alkyl or alkyl sulfonic acid.The application also discloses the preparation method of the conjugate and its application in antitumor drugs.The conjugate provided by the application has the characteristics of stimulus-responsive release at tumor site, so as to release matrix regulatory drug and hemicyanine;On the one hand, matrix regulatory drug can directly realize the functional regulation of tumor-related fibroblasts, on the other hand, hemicyanine can present mild photothermal characteristics under laser irradiation to realize the functional regulation of tumor-related fibroblasts, so as to solve the problem that clinical drugs are difficult to penetrate deeply in tumor site due to the dense extracellular matrix in tumor site.

Description

Technical Field

[0001] This invention belongs to the fields of biomedical materials and pharmaceutical technology, and more specifically, relates to a matrix-regulated drug-hemicyanine conjugate, its preparation method and application. Background Technology

[0002] The dense extracellular matrix (ECM) of solid tumor cells continuously accumulates as the tumor develops and progresses, creating an abnormal tumor biomechanical microenvironment that severely hinders drug delivery to the tumor site and its deep penetration within tumor tissue. Insufficient drug delivery and distribution within tumor tissue not only affects the efficacy of anti-tumor therapy but is also a significant cause of tumor drug resistance, recurrence, and metastasis. Therefore, regulating tumor biomechanical properties to promote drug delivery and improve the effectiveness of conventional anti-tumor therapy has become an increasingly important research focus in the field of oncology. It is evident that tumor matrix components are key factors in the formation of the tumor biomechanical microenvironment, and downregulating the synthesis of tumor matrix or decomposing existing matrix components within the tumor is an effective strategy for regulating tumor biomechanical properties.

[0003] In recent years, nanomedicine-mediated mild photothermal effects have shown that reducing tumor-associated fibroblasts can improve the tumor extracellular matrix, further reducing tumor solid stress and enhancing the deep penetration and anti-tumor efficacy of chemotherapeutic drugs. Meanwhile, many small molecule drugs, such as losartan, tranisterol, and pirfenidone, can regulate tumor mechanical properties by downregulating the synthesis of tumor matrix components or by directly breaking down matrix proteins within tumor tissue through proteolytic enzymes.

[0004] However, there is currently a lack of drugs that can achieve a dual strategy of mild photothermal combined with matrix-modulated drugs. If this is achieved, it will have significant research implications for improving the abnormal biomechanical microenvironment of tumors and thus enhancing the anti-tumor efficacy of clinical drugs. Summary of the Invention

[0005] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a method for preparing and applying a multifunctional matrix-regulated drug-hemicyanine conjugate. This conjugate, with single molecules capable of self-assembling into nanoparticles, accumulates at high concentrations in tumor sites after systemic administration. It responsively releases the matrix-regulated drug and hemicyanine dye through endogenous stimulation at the tumor site. The matrix-regulated drug directly improves tumor biomechanics, while the hemicyanine dye generates mild photothermal activity under laser irradiation to further improve tumor biomechanics. This combined effect effectively addresses the problem of insufficient drug accumulation at tumor sites due to dense extracellular matrix.

[0006] To achieve the above objectives, according to one aspect of the present invention, a matrix-regulated drug-hemicyanine conjugate is provided, the chemical structure of which is shown in formula (I):

[0007]

[0008] Among them, R1 is a matrix-regulating drug, R2 is a stimulus-response linker molecule, and R3 is a C1-C18 alkyl or alkyl sulfonic acid.

[0009] Preferably, the matrix-regulating drug is losartan, tranisterol, pirfenidone, or calcipotriol.

[0010] Preferably, the linker molecules of the stimulus response are ROS response bonds, GSH response bonds, enzyme response bonds, or pH response bonds.

[0011] Preferably, the coupling compound exists in the dispersion in the form of self-assembled nanoparticles.

[0012] As a further preferred embodiment, the concentration of the coupling agent in the dispersion is 2–20 mg / mL.

[0013] According to another aspect of the present invention, a method for preparing the above-mentioned coupling compound is provided, wherein the synthetic route of the coupling compound is as follows:

[0014]

[0015] As a further preferred embodiment, the compound of formula (IV) is synthesized as follows:

[0016]

[0017] As a further preferred embodiment, the compound of formula (Ⅲ) is synthesized as follows:

[0018]

[0019] Preferably, it also includes obtaining a nanoparticle dispersion of the compound of formula (Ⅰ) by nanoprecipitation.

[0020] According to another aspect of the invention, the use of the above-described conjugate in antitumor drugs is also provided.

[0021] In summary, compared with the prior art, the above-described technical solutions conceived in this invention, through improvements to the synthetic route, yield conjugates with matrix-regulated drugs and hemicyanine structures, thus possessing the following beneficial effects:

[0022] 1. This invention provides a matrix-regulating drug-hemicyanine conjugate, which has the characteristic of responsive release at the tumor site, thereby releasing the matrix-regulating drug and hemicyanine. On the one hand, the matrix-regulating drug can directly regulate the function of tumor-associated fibroblasts; on the other hand, hemicyanine exhibits mild photothermal properties under laser irradiation, thereby regulating the function of tumor-associated fibroblasts. It has been verified that this conjugate can achieve dual improvement of the tumor biomechanical microenvironment, thereby solving the problem that the dense extracellular matrix at the tumor site causes clinical drugs to accumulate poorly and have difficulty penetrating deep into the tumor.

[0023] 2. The matrix-regulated drug-hemicyanine conjugate of the present invention can promote the accumulation of antitumor drugs at the tumor site, thereby improving the antitumor effect. It has great clinical potential in regulating tumor mechanics to improve the problem of insufficient delivery and distribution of nanomedicines at the tumor site.

[0024] 3. This matrix-regulated drug-hemicyanine conjugate is preferably obtained by nanoprecipitation to obtain a single-molecule self-assembled nanodrug delivery system. The prepared nano-formulation has good stability and does not require other additional stabilizers for stabilization. The preparation process is simple.

[0025] 4. This conjugate exhibits good stability in water, stronger killing and functional regulation of tumor-associated fibroblasts, and tumor microenvironment-responsive characteristics. Attached Figure Description

[0026] Figure 1 The proton nuclear magnetic resonance spectrum of Example 1 of this invention;

[0027] Figure 2 This is the high-resolution mass spectrometry of Example 1 of the present invention;

[0028] Figure 3 Comparison of UV absorption spectra (A) and fluorescence recovery changes (B) of the nanoparticles prepared in Example 1 of the present invention under in vitro conditions to detect their ROS response capability.

[0029] Figure 4 The images shown are transmission electron microscope (TEM) images (A) and particle size distribution (B) of Embodiment 1 of the present invention.

[0030] Figure 5 The results of zeta potential measurement of nanoparticles prepared in Example 1 of this invention in different environments;

[0031] Figure 6a The particle size and polymer dispersibility index of the nanoparticles prepared in Example 1 of this invention in ultrapure water;

[0032] Figure 6bThe particle size and polymer dispersibility index of the nanoparticles prepared in Example 1 of this invention in physiological saline;

[0033] Figure 6c The particle size and polymer dispersibility index of the nanoparticles prepared in Example 1 of this invention in phosphate buffer solution;

[0034] Figure 7 The heating conditions of the nanoparticles prepared in Example 1 of the present invention (A) and the results of infrared thermal imaging under different heating conditions (B) are shown.

[0035] Figure 8 The toxic effects of the nanoparticles of Example 1 of the present invention on tumor-associated fibroblasts (CAFs), tumor-associated fibroblasts, and normal cells NIH 3T3 after 24 hours and 48 hours.

[0036] Figure 9 This demonstrates the selective killing effect of Embodiment 1 of the present invention under illumination conditions.

[0037] Figure 10 The images show the tumor volume-time curves (A) and tumor photographs (B) after dissection of mice treated with tumors in Example 1 of this invention and other drugs, respectively.

[0038] Figure 11 The results of the biosafety study of Example 1 of the present invention are shown in which (A) represents the number of white blood cells, (B) represents the number of red blood cells, (C) represents the number of platelets, and (D) represents the number of hemoglobin.

[0039] Figure 12 Tissue sections of the main organs (heart, liver, spleen, lung, and kidney) of mice after treatment according to Example 1 of this invention. Detailed Implementation

[0040] 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. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0041] This invention provides a matrix-regulated drug-hemicyanine conjugate, the chemical structure of which is shown in formula (Ⅰ):

[0042]

[0043] Among them, R1 is a class of drugs that regulate the matrix, such as losartan, tranister, pirfenidone, calcipotriol and their derivatives (which do not affect their drug activity).

[0044] R2 is a type of stimulus-response linker molecule, such as ROS response bond, GSH response bond, enzyme response bond, or pH response bond; where the enzyme in the enzyme response bond refers to cytoplasmic enzyme, mitochondrial enzyme, lysosome, nuclease, or protease.

[0045] R3 is selected from C1 to C18 alkyl or alkyl sulfonic acids, preferably C1 to C10 alkyl or alkyl sulfonic acids, and more preferably C3 to C6 alkyl or alkyl sulfonic acids.

[0046] This invention also provides a method for preparing the above-mentioned matrix-regulated drug-hemicyanine conjugate, comprising the following steps:

[0047] Step 1: Obtain compound (Ⅲ) according to the following steps;

[0048]

[0049] Step 2: Obtain compound (IV) according to the following steps;

[0050]

[0051] Step 3: Obtain compound (Ⅰ) according to the following steps;

[0052]

[0053] Step 4: Obtain a nanoparticle dispersion of compound (I) using the nanoprecipitation method; specifically, dissolve compound (I) in a neutral organic solvent, then add it dropwise to deionized water and stir, and finally dialyze it with deionized water to obtain a self-assembled nanoparticle dispersion, which is the matrix-regulated drug-hemicyanine conjugate nanoformulation.

[0054] In some embodiments, the neutral organic solvent is one or more selected from dimethyl sulfoxide, ethanol, methanol, and tetrahydrofuran. The deionized water under stirring conditions is stirred at a speed of not less than 400 rpm, preferably 400–2000 rpm. The concentration of the coupling agent in the coupling agent solution is 2–20 mg / mL, more preferably 5–10 mg / mL. The molecular weight cutoff of the dialysis bag is determined according to the molecular weight of the target coupling agent, typically 1 kDa–10 kDa, and dialysis is performed for a total of 6–12 hours, with two water changes during the process.

[0055] The matrix-regulated drug-hemicyanine conjugate nanoformation composed of compound (I) can enhance the antitumor efficacy of clinical drugs. The aforementioned sensitizing clinical drugs include, but are not limited to, doxil hydrochloride liposome injection, oxaliplatin, paclitaxel, docetaxel, gemcitabine, capecitabine, hydroxycamptothecin, pirarubicin, epirubicin, etc.; the aforementioned tumors include, but are not limited to, breast cancer, ovarian cancer, pancreatic cancer, liver cancer, colon cancer, or melanoma, etc.

[0056] The present invention provides a matrix-regulating drug—hemicyanine conjugate—which mainly exhibits good stability in water, stronger killing and functional regulation of tumor-associated fibroblasts, and tumor microenvironment-stimulated response characteristics.

[0057] The following is an example:

[0058] Example 1

[0059] In this embodiment, R1 is pirfenidone (PFD), R2 is a linker molecule TK with a ROS-stimulated response, and R3 is C4H8O3S-.

[0060] like Figure 1 As shown, the synthesis steps include:

[0061] S1. Preparation of hydroxylated pirfenidone (PFD-OH)

[0062]

[0063] The preparation route for PFD-OH, i.e., hydroxyl-modified matrix-regulating drugs PFD, is as shown above, and its specific steps are as follows:

[0064] S11. Weigh out pirfenidone (185.5 mg, 1.0 mmol) and N-bromosuccinimide (NBS) (178.6 mg, 1.0 mmol) and mix them in 7.0 mL of carbon tetrachloride (CCl4), then add azobisisobutyronitrile (20.0 mg, 0.12 mmol).

[0065] S12. Reflux the above reaction solution at 90°C while magnetically stirring for 2 hours, and monitor the reaction by thin-layer chromatography. Place the reaction system in an ice bath for 30 minutes to promote solid precipitation.

[0066] S13. The above reaction liquid was collected by vacuum filtration and dried by rotary evaporation to obtain a yellow viscous liquid product, brominated pirfenidone (PFD-Br).

[0067] S14. Add 10 ml of sodium hydroxide with an equivalent concentration of 4N, stir at 40°C for 4 hours, monitor the reaction by thin-layer chromatography, filter to obtain a yellow liquid, and then extract with dichloromethane and dichloromethane + water, wash with saturated sodium chloride, dry with anhydrous sodium sulfate, concentrate by rotary evaporation and then vacuum dry, with the vacuum drying temperature controlled at 20-50°C to obtain PFD-OH.

[0068] Preparation of S2. Sulfonate-substituted hemicyanine fluorescent drugs (SO3Cy)

[0069]

[0070] The preparation route of SO3 Cy is as shown above, and its specific steps are as follows:

[0071] Resorcinol (88 mg, 0.8 mmol) and K2CO3 (110 mg, 0.8 mmol) were dissolved in 1.5 mL of anhydrous N,N-dimethylformamide (DMF). The mixture was stirred for 10 min under N2 protection at room temperature. Then, 2-[2-[2-chloro-3-[2-[1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indole-2-yl]ethylene]-1-cyclohexen-1-yl]vinyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indole sodium salt IR783 (300 mg, 0.4 mmol) dissolved in 2 mL of DMF was added to the above reaction system. The reaction was carried out at 45 °C for 6 h under N2 protection. DMF was removed by rotary evaporation at 90℃ using an oil pump. After drying, the sample was extracted with CH2Cl2, washed with saturated brine, dried with anhydrous sodium sulfate, concentrated, mixed with silica gel, and purified by column chromatography using CH2Cl2:MeOH = 50:1, CH2Cl2:MeOH = 20:1, and CH2Cl2:MeOH = 5:1 as eluents, respectively, to obtain SO3 Cy.

[0072] S3. Preparation of thioketal bond TK-OH (i.e., the stimulus-responsive linker molecule modified with dihydroxyl groups)

[0073]

[0074] The preparation route of TK-OH is as shown above, and its specific steps are as follows:

[0075] S31. Thioglycolic acid (9.2 g, 100 mmol), acetone (3.54 g, 60 mmol), and 10 μL of trifluoroacetic acid (TFA) were mixed and stirred at room temperature for 5 h. The mixture was then placed in an ice bath to promote complete crystallization. After centrifugation, the solution was washed three times with n-hexane and water to give a white product in 82% yield.

[0076] Centrifuge at 32.9000 rpm for 5 min. Wash the product sequentially with petroleum ether, water, water, and n-hexane, then centrifuge again and vacuum dry to obtain the white product TK-COOH.

[0077] S33. Dissolve compound TK-COOH (1.5 g, 6.69 mmol) in 150 mL of anhydrous tetrahydrofuran (THF), stir in an ice bath, slowly add lithium aluminum hydride (1.52 g, 40.1 mmol), stir for 30 min, and then purge with N2 to prevent hydrogen accumulation.

[0078] S34. After removing the ice bath, stir at room temperature for 4 hours. Then, slowly add ice water (1 mL), 10% potassium hydroxide aqueous solution, and ice water dropwise under ice bath conditions to quench the lithium aluminum hydride in the reaction. After stirring for 5 minutes, remove the ice bath, add anhydrous magnesium sulfate (4.5 g, 37.4 mmol), stir at room temperature for 20 minutes, filter, and concentrate the filtrate by rotary evaporation at 45°C.

[0079] S35. Mix the sample with silica gel and purify it by column chromatography using petroleum ether:ethyl acetate = 1:1 as the eluent to obtain TK-OH.

[0080] S4. React TK-OH with di(p-nitrobenzene) carbonate (NPC) to generate NPC-TK-OH

[0081]

[0082] The preparation route of NPC-TK-OH is as shown above, and its specific steps are as follows:

[0083] TK-OH (300 mg, 1.5 mmol) was dissolved in 5 mL of anhydrous CH2Cl2, and 300 μL of N,N-diisopropylethylamine (DIPEA) was added. Under ice bath conditions, di(p-nitrobenzene) carbonate (NPC) dissolved in 4 mL of anhydrous CH2Cl2 was added. After the white fumes disappeared, the ice bath was removed, and the mixture was stirred at room temperature for 4 h under N2 protection. The post-processing was performed by CH2Cl2 extraction, washing the organic phase with saturated brine, drying with anhydrous sodium sulfate, concentrating, and separating and purifying the sample by column chromatography with petroleum ether:ethyl acetate = 3:1 as the eluent to obtain NPC-TK-OH.

[0084] S5. Prepare PFD-TK-OH by reacting NPC-TK-OH with PFD-OH.

[0085]

[0086] The preparation route of PFD-TK-OH is as shown above, and its specific steps are as follows:

[0087] The PFD-OH (434 mg, 2.16 mmol) obtained in step S1 was dissolved in 10 mL of anhydrous CH2Cl2, and 4-dimethylaminopyridine DMAP (330 mg, 2.7 mmol) was added. Under stirring at room temperature, the NPC-TK-OH (1 g, 2.7 mmol) obtained in step S4 was added, which was dissolved in 10 mL of anhydrous CH2Cl2. The mixture was stirred overnight at room temperature under N2 protection. The post-processing was performed by CH2Cl2 extraction, washing the organic phase with saturated brine, drying with anhydrous sodium sulfate, concentrating, and purifying the sample by column chromatography with petroleum ether:ethyl acetate = 1:5 as the eluent to obtain PFD-TK-OH.

[0088] S6. Preparation of Pirfenidone Thioacetate Hemicyanine (PFD-TK-Cy)

[0089] S61. Triphosgene was dissolved in 4 mL of anhydrous CH2Cl2 (145 mg, 0.5 mmol). The mixture was stirred in an ice bath under N2 protection. Then, PFD-TK-OH (100 mg, 0.24 mmol) dissolved in 10 mL of anhydrous CH2Cl2 was added, followed by 50 μL of DIPEA. The ice bath was removed, and the mixture was stirred at room temperature for 10 minutes. After the reaction was complete, the solvent and unreacted triphosgene were removed by rotary evaporation.

[0090] S62. Add 5 mL of anhydrous CH2Cl2 to the above system to reconstitute, slowly add SO3Cy (50 mg, 0.1 mmol) dissolved in 3 mL of anhydrous CH2Cl2, and then add 20 μL of LIPEA. Stir at room temperature for 5 hours under N2 protection, and monitor the reaction by thin-layer chromatography.

[0091] S63. Post-processing involved extraction with CH2Cl2, washing the organic phase with saturated brine, drying with anhydrous sodium sulfate, concentrating, and then separating and purifying the sample by column chromatography using CH2Cl2:MeOH = 20:1 and CH2Cl2:MeOH = 10:1 as eluents, respectively, to obtain the final product PFD-TK-Cy, the structure of which is shown below.

[0092]

[0093] Figure 1 and Figure 2 The 1H NMR spectrum and high-resolution mass spectra of the compound prepared in Example 1 are shown.

[0094] Preparation of S7.PFD-TK-Cy nanoparticles

[0095] The self-assembly of PFD-TK-Cy nanoparticles is spontaneous and prepared using a nanoprecipitation method. Specifically, 2 mg of PFD-TK-Cy was dissolved in 200 μl of DMSO to achieve a concentration of 10 mg / mL. This was then added dropwise to 2 mL of ultrapure water, resulting in a DMSO:water (volume ratio) of 1:10, and the mixture was stirred until homogeneous. The resulting mixture was transferred to a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed for 6 hours, with the water changed twice to remove the organic solvent. The resulting nanoparticle dispersion was a clear blue solution.

[0096] Characterization of PFD-TK-Cy nanoparticles in Verification Example 1

[0097] Figure 3 This paper demonstrates the detection of the ROS responsiveness of PFD-TK-Cy nanoparticles prepared in Example 1 under in vitro conditions. Hydrogen peroxide, as a type of ROS, modifies the SO3Cy backbone by converting the phenolic hydroxyl groups connected to TK bonds into phenolic ester bonds, weakening the intramolecular charge transfer (ICT) effect. This results in the fluorescence intensity of PFD-TK-Cy being much lower than that of SO3Cy. However, the intervention of ROS causes the TK bonds of PFD-TK-Cy to break, releasing SO3Cy, thus enhancing the ICT effect and gradually restoring the fluorescence. In the diagram, A shows a comparison of UV absorption spectra, and B shows the fluorescence recovery changes in response to reactive oxygen species in vitro (B). The results show that after co-incubation with hydrogen peroxide, the fluorescence of PFD-TK-Cy gradually increases with time, indicating that ROS causes the breakage of the TK bonds in PFD-TK-Cy, releasing SO3Cy, thus proving that PFD-TK-Cy has ROS-responsive characteristics.

[0098] Figure 4 and Figure 5 Transmission electron microscopy (TEM) images of the PFD-TK-Cy nanoparticles prepared in Example 1 are shown. Figure 4 A) Particle size diagram Figure 4 B) and the zeta potential measurements of nanoparticles in ultrapure water, physiological saline, and phosphate buffer solution, respectively. Figure 5 The results showed that the prepared PFD-TK-Cy nanoparticles were uniform in size with an average diameter of about 150 nm, and the surface charge of the PFD-TK-Cy nanoparticles was weakly neutral.

[0099] Figure 6 shows the PFD-TK-Cy nanoparticles prepared in Example 1 in ultrapure water (… Figure 6a ), physiological saline ( Figure 6b ), phosphate buffer solution ( Figure 6cThe changes in particle size and polymer dispersibility index (PDI) of the prepared PFD-TK-Cy nanoparticles were investigated. The results showed that the particle size of the prepared PFD-TK-Cy nanoparticles remained at around 150 nm within 7 days, and the PDI was less than 0.2, indicating that the nanoparticles had good stability.

[0100] Verification Example 2: Temperature rise of PFD-TK-Cy nanoparticles

[0101] Different concentrations of PFD-TK-Cy nanoparticle aqueous solutions were placed in 1.5 ml Ep tubes and irradiated with a laser with an excitation wavelength of 660 nm for 4 minutes. The solution temperature was recorded in real time using an infrared thermal imager. Figure 7 The results show the heating of the nanoparticles (A) and the results of infrared thermal imaging under different heating conditions (B). The results show that the simple aqueous solution did not heat up. The higher the concentration of PFD-TK-Cy nanoparticles, the more obvious the heating effect. The temperature of nanoparticles with a concentration of 50 micrograms per milliliter can be raised to 46 degrees Celsius in 4 minutes, which meets the conditions of mild photothermal heating.

[0102] Example 3: Effect of PFD-TK-Cy nanoparticles on cytotoxicity

[0103] The effect of PFD-TK-Cy nanoparticles prepared in Example 1 on three types of cytotoxicity was investigated. Specifically, 10,000 4T1 cells were seeded in 96-well plates and cultured in a 37°C, 5% CO2 constant temperature and normoxic incubator. After cell adhesion, the culture medium was aspirated, and 100 μL of RPMI 1640 culture medium containing different concentrations of PFD-TK-Cy nanoparticles was added to each well. After incubation for 24 h, cell viability was calculated using the MTT assay. Figure 8 The results showed that PFD-TK-Cy nanoparticles had a significantly higher killing effect on tumor-associated fibroblasts (CAFs) than on both tumor-associated fibroblasts and normal cells (NIH 3T3) at 24h and 48h.

[0104] Example 4: Validation of the selective killing effect of PFD-TK-Cy nanoparticles on tumor cells

[0105] Example 1 describes the killing effect of PFD-TK-Cy nanoparticles prepared under light irradiation on tumor cells and tumor-associated fibroblasts (CAFs). Specifically, 10,000 4T1 cells were seeded in 96-well plates and cultured in a 37°C, 5% CO2 incubator. After cell adhesion, the culture medium was aspirated, and 100 μL of RPMI 1640 medium containing different concentrations of PFD-TK-Cy nanoparticles was added to each well. The cells were incubated for 8 h, and each well was then irradiated with a 660 nm laser for 5 min, with the power adjusted to maintain the temperature between 42 and 43°C. After culturing for another 24 h, cell viability was calculated using the MTT assay. Figure 9 The results showed that under illumination, the killing effect of PFD-TK-Cy nanoparticles on CAFs was much higher than that of 4T1, indicating that PFD-TK-Cy nanoparticles have higher killing selectivity for CAFs.

[0106] Example 5: Investigation of the antitumor efficacy of PFD-TK-Cy nanoparticles in subcutaneous breast cancer tumors in mice.

[0107] This validation example used a mouse 4T1 subcutaneous breast cancer tumor model to investigate the antitumor effect of PFD-TK-Cy nanoparticles under both light and no-light conditions, and compared it with the marketed drug Doxil. The specific steps are as follows:

[0108] Six-week-old, 16–18g female BALB / c mice were subcutaneously inoculated on the right side with a suspension of approximately 1 × 10⁻⁶ mouse mammary gland 4T1 cells. 6 A mouse model of 4T1 subcutaneous breast cancer tumor was established using [number] cells. When the subcutaneous tumor volume was approximately 100 mm²... 3 Mice were randomly divided into 5 groups of 6 mice each: saline group, PFD-TK-Cy nanoparticle + light irradiation group, Doxil group, PFD-TK-Cy nanoparticle + Doxil group, and PFD-TK-Cy nanoparticle + light irradiation + Doxil group. During treatment, the dosage of PFD-TK-Cy nanoparticles was 8 mg / kg, and the dosage of Doxil was 2.5 mg / kg. The first day of administration was designated as Day 1. Doxil was administered at the same dosage on days 5 and 12. One hour after administration, the light irradiation group received light irradiation at 660 nm for 10 minutes, maintaining the tumor temperature between 42℃ and 43℃. Doxil was administered intravenously 24 hours after administration. From Day 1, mouse body weight and tumor volume were measured every other day, and a tumor volume-time curve was plotted. Mice were sacrificed on day 15, and subcutaneous tumors were dissected and photographed. Figure 10Content (A) shows the tumor volume-time curve in mice, and content (B) shows a photograph of the tumor after dissection. The results showed that the inhibitory effect of PFD-TK-Cy nanoparticles alone plus light irradiation on tumors was no different from that of the saline group alone. This indicates that the mild photothermal effect generated by PFD-TK-Cy nanoparticles does not kill tumors, but only alters the tumor's mechanical environment. The tumor inhibition rate was 54% in the Doxil group, 69% in the PFD-TK-Cy nanoparticle + Doxil group, and as high as 87% in the PFD-TK-Cy nanoparticle + light irradiation + Doxil group. This demonstrates that both PFD-TK-Cy nanoparticles and PFD-TK-Cy nanoparticles plus light irradiation can improve the abnormal mechanical microenvironment of the tumor, thereby promoting the accumulation of the marketed drug Doxil at the tumor site and enhancing the anti-tumor effect. This result further demonstrates the great clinical potential of the highly effective PFD-TK-Cy nanoparticles in regulating tumor mechanics to overcome the challenge of insufficient delivery and distribution of nanomedicines at the tumor site.

[0109] Verification Example 6: Biosafety of PFD-TK-Cy Nanoparticles

[0110] After the antitumor efficacy of the PFD-TK-Cy nanoparticles in mouse breast cancer subcutaneous tumors ended, the blood routine tests of the mice were performed to evaluate the safety of the single-drug group and the combination dosing regimen. Figure 11 The results of the biosafety study of PFD-TK-Cy nanoparticles are shown. In the figure, (A) represents the number of white blood cells, (B) represents the number of red blood cells, (C) represents the number of platelets, and (D) represents the number of hemoglobin. The results showed that the blood routine analysis of mice in the PFD-TK-Cy nanoparticle treatment group was within the normal range, indicating that PFD-TK-Cy nanoparticles have good biosafety.

[0111] After the antitumor efficacy of the PFD-TK-Cy nanoparticles in mouse subcutaneous breast cancer subcutaneous tumors was exhausted, the safety of tissue sections from the major organs (heart, liver, spleen, lung, and kidney) of mice was evaluated. Figure 12 The results showed that the nano-formulation prepared in Example 2 did not cause significant damage to the major organs (heart, liver, spleen, lungs, and kidneys) of mice, indicating that the nanoparticle dispersion prepared in this example did not cause significant toxic side effects on normal tissues.

[0112] Examples 2-18 were also prepared using the same steps as in Example 1, and their structures are shown in Table 1.

[0113] Table 1 Overall Structure of Examples 2-18

[0114]

[0115]

[0116]

[0117]

[0118] In addition to the matrix-regulating drugs and stimulus-responsive linkers already discussed in Example 1, the structures of the other matrix-regulating drugs and stimulus-responsive linkers are as follows:

[0119]

[0120]

[0121] (The carboxyl group in the original trastuzumab is modified to a hydroxyl group, which does not affect its drug activity.)

[0122]

[0123] It has been verified that Examples 2-18 also have similar properties to Example 1.

[0124] Those skilled in the art will readily understand that the above description is merely 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 scope of protection of the present invention.

Claims

1. A matrix modulating prodrug-hemicyanine conjugate, characterized in that, The chemical structure of the conjugate is shown in formula (I): The compound of formula (I) is 。 2. The conjugate of claim 1, wherein, The conjugate exists in the form of nanoparticles formed by self-assembly in the dispersion.

3. The conjugate of claim 2, wherein, The concentration of the conjugate in the dispersion is 2-20 mg / mL.

4. Process for the preparation of the conjugate according to any one of claims 1 to 3, characterized in that, The synthesis route of the conjugate is as follows: 。 5. The method of claim 4, wherein, The synthesis of the compound of formula (IV) is as follows: 。 6. The method of claim 5, wherein, The synthesis of the compound of formula (III) is as follows: 。 7. Use of the conjugate of any one of claims 1-3 in the preparation of an antitumor drug.

8. Use according to claim 7, wherein the compound is ###0002### The tumor is breast cancer, ovarian cancer, pancreatic cancer, liver cancer, colon cancer, or melanoma.

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