A nano-drug preparation targeting colorectal cancer and a preparation method thereof

Abstract

This invention belongs to the field of pharmaceutical formulation technology, and particularly relates to a nanomedicine formulation targeting colorectal cancer and its preparation method. The preparation method of the nanomedicine formulation targeting colorectal cancer of this invention includes the following steps: S1. Dissolving modified silk fibroin in deionized water to obtain a modified silk fibroin solution, adding doxorubicin to it to obtain an doxorubicin-modified silk fibroin solution for later use; S2. Dissolving DSPE-PEG-RGD, camptothecin, and naringin derivative into a mixed solution to obtain a drug mixture; S3. Adding the drug mixture dropwise to the doxorubicin-modified silk fibroin solution and freezing, thawing, centrifuging to collect the obtained nanoparticles, washing the nanoparticles, resuspending them in deionized water, ultrasonically dispersing, and freeze-drying to obtain the nanomedicine formulation targeting colorectal cancer. The pharmaceutical formulation of this invention can co-load multiple drugs, possesses targeting function, and has a good inhibitory effect on colorectal cancer cell lines.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, and particularly relates to a nanomedicine formulation targeting colorectal cancer and its preparation method. Background Technology

[0002] Colorectal cancer is one of the leading causes of cancer-related deaths worldwide, posing a serious threat to human health. Currently, clinical treatment primarily relies on surgery, supplemented by chemotherapy, radiotherapy, and targeted therapy. Chemotherapy is a crucial treatment for advanced or metastatic colorectal cancer. Doxorubicin, a broad-spectrum anthracycline antitumor antibiotic, exerts its potent antitumor effects through multiple mechanisms, including DNA double-strand insertion, inhibition of topoisomerase II, and the generation of reactive oxygen species. Camptothecin and its derivatives are specific inhibitors of topoisomerase I, causing single-strand breaks in DNA and blocking DNA replication and transcription; they also exhibit significant activity against various solid tumors. However, these two traditional chemotherapy drugs face many serious challenges in clinical application: First, they have poor water solubility (especially camptothecin), are widely distributed in the body, and are difficult to achieve effective therapeutic concentrations at the tumor site; second, they lack tumor targeting and produce serious toxic side effects on normal tissues and organs (such as the heart, bone marrow, and gastrointestinal tract), which limits their clinical application dosage and efficacy; third, tumor cells are prone to multidrug resistance, leading to treatment failure; fourth, although their mechanisms of action are different, the efficacy of single-drug therapy is limited, and simple combination therapy often fails to achieve the ideal synergistic effect due to differences in pharmacokinetics and the superposition of toxicities.

[0003] To overcome the aforementioned bottlenecks, nanomedicine delivery systems (NDDS) have emerged and become a research hotspot in the field of cancer therapy. Among numerous nanocarrier materials, biogenic proteins, especially silk fibroin, have attracted widespread attention due to their excellent biocompatibility, degradability, good mechanical properties, ease of chemical modification, and wide availability. While silk fibroin itself possesses a certain drug-carrying capacity, its unmodified form still suffers from limitations in drug loading, insufficient affinity for hydrophobic drugs, and a lack of targeting ability when used as a drug carrier. On the other hand, from the perspective of drug combination, in addition to classic chemotherapeutic drugs, searching for natural products or their derivatives that can enhance drug efficacy and reduce toxicity is an important research direction. Naringin is a natural furanone compound. However, how to incorporate such hydrophobic derivatives with hydrophilic chemotherapeutic drugs into a stable and efficient nanodelivery platform and verify their combined anti-tumor effect has not yet been systematically reported.

[0004] Based on this, constructing a novel nanodelivery system capable of co-loading multiple drugs and possessing targeting capabilities is of great significance for the treatment of colorectal cancer and reducing systemic toxicity. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, one objective of this invention is to provide a method for preparing a nanomedicine formulation targeting colorectal cancer. This formulation, through ingenious molecular design, covalently introduces a specific chemical group (2-(benzoazo)cyano) with π-π stacking interaction and hydrophobic interface-providing capabilities into the silk fibroin side chain. This not only achieves stable binding with doxorubicin but also provides a high-affinity loading interface for camptothecin and naringin derivatives. Furthermore, the network structure is strengthened through calcium ion-induced β-sheet cross-linking, greatly enhancing the stability and leakage prevention of the carrier. Finally, it is co-assembled with DSPE-PEG-RGD to form core-shell structured or mixed micelle-like nanoparticles, achieving targeted uptake of colorectal cancer cells and providing a novel solution for highly efficient and low-toxicity combined chemotherapy for colorectal cancer.

[0006] The second objective of this invention is to provide a nanomedicine formulation that targets colorectal cancer.

[0007] The above objectives are achieved using the following technical solution: A method for preparing a nanomedicine formulation targeting colorectal cancer, the method comprising the following steps: S1. Dissolve the modified silk fibroin in deionized water to obtain a modified silk fibroin solution, add doxorubicin to it to obtain a doxorubicin-modified silk fibroin solution for later use; S2. Dissolve DSPE-PEG-RGD, camptothecin, and naringin derivative into a mixed solution to obtain the drug mixture; S3. The drug mixture obtained in step S2 is added dropwise to the doxorubicin-modified silk fibroin solution in step S1 and frozen. After thawing, the nanoparticles are collected by centrifugation. The nanoparticles are washed, resuspended in deionized water, ultrasonically dispersed, and freeze-dried to obtain a nanomedicine preparation targeting colorectal cancer. The chemical structural formula of the terpineone derivative is as follows: .

[0008] Furthermore, the preparation of the naringin derivative includes the following steps: (1) Under a nitrogen atmosphere, naringin, mercaptoacetic acid and sodium ascorbate were added to methanol, stirred evenly, and then tris(2,2'-bipyridine) ruthenium dichloride and trichlorobromomethane were added. The mixture was reacted at room temperature for 6-10 h under light irradiation. After purification, naringin-mercaptoacetic acid was obtained. (2) Under a nitrogen atmosphere, cyperione-mercaptoacetic acid was added to tetrahydrofuran and stirred until homogeneous. Thionyl chloride was added and reacted at 70°C for 3-5 h. The reaction solution was concentrated to obtain a concentrate. The concentrate was dissolved in a solvent to form a solution. 4,4-dithiodiphenylamine and diisopropylethylamine were added to tetrahydrofuran and stirred until homogeneous. The solution was then added dropwise to the solution and reacted at 0-3°C for 1-3 h.

[0009] Further, in step (1), the molar ratio of naringin, thioglycolic acid, sodium ascorbate, tris(2,2'-bipyridine) ruthenium dichloride and trichlorobromomethane is 10 mmol: 30-50 mmol: 0.03-0.05 mmol: 0.01-0.03 mmol: 0.05-0.08 mmol; the volume ratio of naringin to methanol is 10 mmol: 25 mL.

[0010] Further, in step (2), the molar ratio of naringin-mercaptoacetic acid, thionyl chloride, 4,4-dithiodiphenylamine, and diisopropylethylamine is 5 mmol: 12-16 mmol: 2 mmol: 10-20 mmol; when naringin-mercaptoacetic acid is added to tetrahydrofuran, the ratio of naringin-mercaptoacetic acid to tetrahydrofuran is 5 mmol: 20 mL; the ratio of the concentrate to the solvent is 5 mmol: 30 mL, and the solvent is tetrahydrofuran; when 4,4-dithiodiphenylamine and diisopropylethylamine are added to tetrahydrofuran, the ratio of 4,4-dithiodiphenylamine to tetrahydrofuran is 5 mmol: 20 mL.

[0011] Furthermore, the preparation of the modified silk fibroin includes the following steps: a. Add silkworm cocoons to sodium carbonate solution and reflux for 30 minutes, then dry to obtain degummed silk; add the degummed silk to a mixed solution and dissolve at 65-75℃ for 3-6 hours; dialyze and freeze-dry the reaction solution to obtain silk fibroin. b. Add silk fibroin, ethyl 2-(benzoazo)cyanoacetate, and triethylamine to anhydrous N,N-dimethylformamide and react at 60-80°C for 6-10 hours; concentrate the reaction solution, dialyze, and freeze-dry to obtain the final product.

[0012] Further, in step a, the ratio of silkworm cocoon to sodium carbonate solution is 10g:80-100mL, and the concentration of sodium carbonate solution is 0.02-0.03mol / L; the ratio of degummed silk to mixed solution is 10g:80-100mL, and the mixed solution is obtained by mixing lithium chloride solution and ethanol solution at a volume ratio of 9:1, the concentration of lithium chloride solution is 9mol / L, and the volume concentration of ethanol solution is 10%.

[0013] Further, in step b, the ratio of silk fibroin, ethyl 2-(benzoazo)cyanoacetate, triethylamine, and anhydrous N,N-dimethylformamide is 1g:0.3-0.5g:0.5-0.8g:20mL.

[0014] Further, the concentration of the modified silk fibroin solution in step S1 is 5 mg / mL, and the concentration of doxorubicin in the modified silk fibroin solution is 0.1-0.2 mg / mL; the mass ratio of DSPE-PEG-RGD, camptothecin, and naringin derivative in step S2 is 1:(0.1-0.3):(0.3-0.5); the concentration of DSPE-PEG-RGD in the mixed solution is 10-15 mg / mL, and the mixed solution is prepared by mixing ethanol, acetone, and methanol in a volume ratio of 1:1:1.

[0015] Further, in step S3, the volume ratio of the drug mixture and the doxorubicin-modified silk fibroin solution is 1:5; the freezing temperature is -15~-20℃, and the freezing time is 12-16h.

[0016] A nanomedicine formulation targeting colorectal cancer was prepared using the above-described preparation method.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention provides a novel application of doxorubicin, camptothecin, and naringin derivatives in the preparation of drugs for treating colorectal cancer. Research results show that the combined use of doxorubicin, camptothecin, and naringin derivatives significantly inhibits the proliferation of colorectal cancer cell lines. The naringin derivative is prepared by reacting the double bond of naringin with the sulfhydryl group of thioglycolic acid to obtain intermediate 1, which is then reacted with the amino group of 4,4-dithiodiphenylamine. The combined use of doxorubicin, camptothecin, and naringin derivatives can significantly inhibit tumor growth and prolong the survival time of mice.

[0018] 2. This invention provides a nanomedicine formulation targeting colorectal cancer. The invention synthesizes modified silk fibroin by reacting some of its amino groups with the active ester group of ethyl 2-(benzoazo)cyanoacetate, introducing a 2-(benzoazo)cyano group into the amide group. This forms an aqueous phase with doxorubicin. Camptothecin, naringin derivatives, and DSPE-PEG-RGD form an oil phase. The two phases are then prepared by solvent evaporation to obtain the nanomedicine formulation targeting colorectal cancer. This formulation exhibits excellent encapsulation efficiency and improves the overall targeting of the drug for colorectal cancer. The 2-(benzoazo)cyano group introduced into the modified silk fibroin side chain can form π-π stacking with doxorubicin, while simultaneously providing a high-affinity interface for the hydrophobic drug, achieving synergistic loading. This group is covalently anchored to the protein amino group, supplemented by a calcium ion-induced β-sheet cross-linking network, significantly inhibiting drug leakage during preparation and cycling, thereby improving the encapsulation efficiency. Furthermore, after the modified silk fibroin is co-assembled with DSPE-PEG-RGD, the particle size is reduced and the surface negative charge is increased, which reduces plasma protein adsorption, prolongs systemic circulation time, and enhances the passive penetration ability in the colorectal mucus layer, providing a favorable condition for DSPE-PEG-RGD-mediated targeting. Attached Figure Description

[0019] Figure 1 The infrared spectrum of the modified silk fibroin obtained in Example 1 of this invention; Figure 2 This is a graph showing the uptake results of the drug formulation of the present invention on SW620 cells and HT29 cells. Detailed Implementation

[0020] The present invention will now be further described with reference to the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all reagents or instruments used are conventional products obtained through commercial channels.

[0021] Example 1 A method for preparing a nanomedicine formulation targeting colorectal cancer includes the following steps: S1. Dissolve the modified silk fibroin in deionized water to prepare a 5 mg / mL modified silk fibroin solution. Add doxorubicin to the solution to obtain a doxorubicin-modified silk fibroin solution. The concentration of doxorubicin in the modified silk fibroin solution is 0.15 mg / mL. S2. Dissolve DSPE-PEG-RGD (distearylphosphatidylethanolamine-polyethylene glycol-arabinoglobulin, PEG molecular weight 2000 Daltons), camptothecin, and naringin derivative in a mixed solution (prepared from ethanol, acetone, and methanol in a volume ratio of 1:1:1), wherein the mass ratio of DSPE-PEG-RGD, camptothecin, and naringin derivative is 1:0.2:0.4; the concentration of DSPE-PEG-RGD in the mixed solution is 12 mg / mL; thus, the drug mixture is obtained. S3. The drug mixture obtained in step S2 is added dropwise to the doxorubicin-modified silk fibroin solution in step S1, wherein the volume ratio of the drug mixture to the doxorubicin-modified silk fibroin solution is 1:5; then the mixture is frozen at -20°C for 14 hours, and after thawing, the nanoparticles are collected by centrifugation, then the nanoparticles are resuspended in deionized water, ultrasonically dispersed, and freeze-dried to obtain a nanomedicine formulation targeting colorectal cancer. The preparation of the naringin derivative includes the following steps: (1) Under a nitrogen atmosphere, naringin, thioglycolic acid and sodium ascorbate were added to methanol and stirred until homogeneous. Then, tris(2,2'-bipyridine) ruthenium chloride and trichlorobromomethane were added. The molar ratio of naringin, thioglycolic acid, sodium ascorbate, tris(2,2'-bipyridine) ruthenium chloride and trichlorobromomethane was 10 mmol: 40 mmol: 0.04 mmol: 0.02 mmol: 0.07 mmol. The volume ratio of naringin to methanol was 10 mmol: 25 mL. The reaction was carried out at room temperature for 8 h under blue LED (2W) illumination. The methanol was removed by concentration and purified by silica gel column chromatography (hexane / ethyl acetate volume ratio of 4:1) to obtain naringin-thioglycolic acid. 1 H NMR (C 17 H 26 O3, 400 MHz, DMSO): δ 12.14 (s, 1H), 5.88 (s, 1H), 2.98-2.73(m, 2H), 2.43-2.33 (m, 4H), 1.98-1.65 (m, 5H), 1.25-0.88 (m, 13H); HRMS(ESI+): [M+H] + The calculation yields 279.19, and the solution is 279.20. (2) Under a nitrogen atmosphere, terpinene-mercaptoacetic acid was added to tetrahydrofuran and stirred until homogeneous. Then, thionyl chloride was added. The ratio of terpinene-mercaptoacetic acid, tetrahydrofuran, and thionyl chloride was 5 mmol: 20 mL: 14 mmol. The mixture was then reacted at 70 °C for 4 h. The reaction solution was concentrated to remove the solvent and unreacted thionyl chloride, and a concentrate was obtained. The concentrate was dissolved in tetrahydrofuran to form a solution (the ratio of concentrate to tetrahydrofuran was 5 mmol: 30 mL). 4,4-Dithiodiphenylamine and diisopropyl... Ethylamine was added to tetrahydrofuran, with the ratio of terpineone-mercaptoacetic acid, 4,4-dithiodiphenylamine, diisopropylethylamine, and tetrahydrofuran being 5 mmol: 2 mmol: 15 mmol: 20 mL. After stirring thoroughly, the solution was added dropwise to the mixture, and the reaction was carried out at 0 °C for 2 h. The reaction solution was quenched with water, and the mixture was extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (hexane / ethyl acetate volume ratio of 3:1) to obtain the final product. 1 H NMR (C 46 H 60 N2O4S2, 400 MHz, DMSO): δ 10.04 (s, 2H), 8.09 (d, 4H), 7.79 (d, 4H), 5.88 (s, 2H), 2.98-2.73 (m, 4H), 2.43-2.33 (m, 4H), 2.10-1.65 (m, 14H), 1.25-0.88 (m, 26H); HRMS(ESI+): [M] Calculated value is 768.40, found value is 768.40.

[0022] The preparation of the modified silk fibroin includes the following steps: a. Add silkworm cocoons to a 0.025M sodium carbonate solution and reflux for 30 minutes. The ratio of silkworm cocoons to sodium carbonate solution is 10g:100mL. Dry the cocoons to obtain degummed silk. Prepare a mixed solution by mixing 9M lithium chloride solution and 10v / v% ethanol solution at a volume ratio of 9:1. Add the degummed silk to the mixed solution at a volume ratio of 10g:100mL. Dissolve the mixture at 70℃ for 5 hours. Pour the reaction solution into a dialysis bag with a capacity cutoff of 10000Da for dialysis and freeze-drying to obtain silk fibroin. b. Add silk fibroin, ethyl 2-(benzoazo)cyanoacetate, and triethylamine to anhydrous N,N-dimethylformamide, wherein the ratio of silk fibroin, ethyl 2-(benzoazo)cyanoacetate, triethylamine, and anhydrous N,N-dimethylformamide is 1 g: 0.4 g: 0.6 g: 20 mL; react at 70 °C for 8 h; concentrate the reaction solution to obtain a crude product, dissolve the crude product in deionized water at room temperature, and then place it in a dialysis bag with a capacity cutoff of 14000 Da for dialysis and freeze-drying to obtain modified silk fibroin.

[0023] A nanomedicine formulation targeting colorectal cancer was prepared using the method described above.

[0024] Example 2 A method for preparing a nanomedicine formulation targeting colorectal cancer includes the following steps: S1. Dissolve the modified silk fibroin in deionized water to prepare a 5 mg / mL modified silk fibroin solution. Add doxorubicin to the solution to obtain a doxorubicin-modified silk fibroin solution. The concentration of doxorubicin in the modified silk fibroin solution is 0.1 mg / mL. S2. Dissolve DSPE-PEG-RGD (molecular weight 2000 Daltons), camptothecin, and naringin derivative in a mixed solution (prepared from ethanol, acetone, and methanol in a volume ratio of 1:1:1), wherein the mass ratio of DSPE-PEG-RGD, camptothecin, and naringin derivative is 1:0.1:0.3; the concentration of DSPE-PEG-RGD in the mixed solution is 10 mg / mL; thus obtaining the drug mixture. S3. The drug mixture obtained in step S2 is added dropwise to the doxorubicin-modified silk fibroin solution in step S1, wherein the volume ratio of the drug mixture to the doxorubicin-modified silk fibroin solution is 1:5; then the mixture is frozen at -20°C for 12 hours, and after thawing, the nanoparticles are collected by centrifugation, then the nanoparticles are resuspended in deionized water, ultrasonically dispersed, and freeze-dried to obtain a nanomedicine formulation targeting colorectal cancer. The preparation of the naringin derivative includes the following steps: (1) Under a nitrogen atmosphere, terpineone, thioglycolic acid, and sodium ascorbate were added to methanol and stirred until homogeneous. Then, tris(2,2'-bipyridine)ruthenium chloride and trichlorobromomethane were added. The molar ratio of terpineone, thioglycolic acid, sodium ascorbate, tris(2,2'-bipyridine)ruthenium chloride, and trichlorobromomethane was 10 mmol: 30 mmol: 0.03 mmol: 0.01 mmol: 0.05 mmol. The volume ratio of terpineone to methanol was 10 mmol: 25 mL. The reaction was carried out at room temperature for 6 h under blue LED (2W) illumination. The methanol was removed by concentration, and the mixture was purified by silica gel column chromatography (hexane / ethyl acetate volume ratio of 4:1) to obtain terpineone-thioglycolic acid. 1 H NMR and HRMS were consistent with those in Example 1; (2) Under a nitrogen atmosphere, terpineone-mercaptoacetic acid was added to tetrahydrofuran and stirred until homogeneous. Then, thionyl chloride was added. The ratio of terpineone-mercaptoacetic acid, tetrahydrofuran, and thionyl chloride was 5 mmol: 20 mL: 12 mmol. The mixture was then reacted at 70 °C for 3 h. The reaction solution was concentrated to remove the solvent and unreacted thionyl chloride, and a concentrate was obtained. The concentrate was dissolved in tetrahydrofuran to form a solution (the ratio of concentrate to tetrahydrofuran was 5 mmol: 30 mL). 4,4-Dithiodiphenylamine and diisopropyl... Ethylamine was added to tetrahydrofuran, with the ratio of naringin-mercaptoacetic acid, 4,4-dithiodiphenylamine, diisopropylethylamine, and tetrahydrofuran being 5 mmol:2 mmol:10 mmol:20 mL. After stirring thoroughly, the solution was added dropwise, and the reaction was carried out at 3°C ​​for 1 h. The reaction solution was quenched with water, and the mixture was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (hexane / ethyl acetate volume ratio of 3:1) to obtain the naringin derivative. 1 H NMR and HRMS were consistent with those in Example 1.

[0025] The preparation of the modified silk fibroin includes the following steps: a. Add silkworm cocoons to a 0.02M sodium carbonate solution and reflux for 30 minutes. The ratio of silkworm cocoons to sodium carbonate solution is 10g:80mL. Dry the cocoons to obtain degummed silk. Prepare a mixed solution by mixing 9M lithium chloride solution and 10v / v% ethanol solution at a volume ratio of 9:1. Add the degummed silk to the mixed solution at a volume ratio of 10g:80mL. Dissolve the mixture at 65℃ for 6 hours. Pour the reaction solution into a dialysis bag with a capacity cutoff of 10000Da for dialysis and freeze-drying to obtain silk fibroin. b. Add silk fibroin, ethyl 2-(benzoazo)cyanoacetate, and triethylamine to anhydrous N,N-dimethylformamide, wherein the ratio of silk fibroin, ethyl 2-(benzoazo)cyanoacetate, triethylamine, and anhydrous N,N-dimethylformamide is 1 g: 0.3 g: 0.5 g: 20 mL; react at 60 °C for 10 h; concentrate the reaction solution to obtain a crude product, dissolve the crude product in deionized water at room temperature, and then place it in a dialysis bag with a capacity cutoff of 14000 Da for dialysis and freeze-drying to obtain modified silk fibroin.

[0026] A nanomedicine formulation targeting colorectal cancer was prepared using the method described above.

[0027] Example 3 A method for preparing a nanomedicine formulation targeting colorectal cancer includes the following steps: S1. Dissolve the modified silk fibroin in deionized water to prepare a 5 mg / mL modified silk fibroin solution. Add doxorubicin to the solution to obtain a doxorubicin-modified silk fibroin solution. The concentration of doxorubicin in the modified silk fibroin solution is 0.2 mg / mL. Set aside for later use. S2. Dissolve DSPE-PEG-RGD (molecular weight 2000 Daltons), camptothecin, and naringin derivative in a mixed solution (prepared from ethanol, acetone, and methanol in a volume ratio of 1:1:1), wherein the mass ratio of DSPE-PEG-RGD, camptothecin, and naringin derivative is 1:0.3:0.5; the concentration of DSPE-PEG-RGD in the mixed solution is 15 mg / mL; thus obtaining the drug mixture. S3. The drug mixture obtained in step S2 is added dropwise to the doxorubicin-modified silk fibroin solution in step S1, wherein the volume ratio of the drug mixture to the doxorubicin-modified silk fibroin solution is 1:5; then the mixture is frozen at -15°C for 16 hours, and after thawing, the nanoparticles are collected by centrifugation, then the nanoparticles are resuspended in deionized water, ultrasonically dispersed, and freeze-dried to obtain a nanomedicine formulation targeting colorectal cancer. The preparation of the naringin derivative includes the following steps: (1) Under a nitrogen atmosphere, terpinene, thioglycolic acid, and sodium ascorbate were added to methanol and stirred until homogeneous. Then, tris(2,2'-bipyridine)ruthenium chloride and trichlorobromomethane were added. The molar ratio of terpinene, thioglycolic acid, sodium ascorbate, tris(2,2'-bipyridine)ruthenium chloride, and trichlorobromomethane was 10 mmol: 50 mmol: 0.05 mmol: 0.03 mmol: 0.08 mmol. The volume ratio of terpinene to methanol was 10 mmol: 25 mL. The reaction was carried out at room temperature for 10 h under blue LED (2W) illumination. The methanol was removed by concentration, and the mixture was purified by silica gel column chromatography (hexane / ethyl acetate volume ratio of 4:1) to obtain terpinene-thioglycolic acid. 1 H NMR and HRMS were consistent with those in Example 1; (2) Under a nitrogen atmosphere, terpineone-mercaptoacetic acid was added to tetrahydrofuran and stirred until homogeneous. Then, thionyl chloride was added. The ratio of terpineone-mercaptoacetic acid, tetrahydrofuran, and thionyl chloride was 5 mmol: 20 mL: 16 mmol. The mixture was then reacted at 70 °C for 5 h. The reaction solution was concentrated to remove the solvent and unreacted thionyl chloride, and a concentrate was obtained. The concentrate was dissolved in tetrahydrofuran to form a solution (the ratio of concentrate to tetrahydrofuran was 5 mmol: 30 mL). 4,4-Dithiodiphenylamine and diisopropyl... Ethylamine was added to tetrahydrofuran, with the ratio of naringin-mercaptoacetic acid, 4,4-dithiodiphenylamine, diisopropylethylamine, and tetrahydrofuran being 5 mmol:2 mmol:20 mmol:20 mL. After stirring thoroughly, the solution was added dropwise, and the reaction was carried out at 0 °C for 3 h. The reaction solution was quenched with water, and the mixture was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (hexane / ethyl acetate volume ratio of 3:1) to obtain the naringin derivative. 1 H NMR and HRMS were consistent with those in Example 1.

[0028] The preparation of the modified silk fibroin includes the following steps: a. Add silkworm cocoons to a 0.03M sodium carbonate solution and reflux for 30 minutes. The ratio of silkworm cocoons to sodium carbonate solution is 10g:100mL. Dry to obtain degummed silk. Prepare a mixed solution by mixing 9M lithium chloride solution and 10v / v% ethanol solution at a volume ratio of 9:1. Add the degummed silk to the mixed solution at a volume ratio of 10g:100mL. Dissolve at 75℃ for 3 hours. Pour the reaction solution into a dialysis bag with a capacity cutoff of 10000Da for dialysis and freeze-dry to obtain silk fibroin. b. Add silk fibroin, ethyl 2-(benzoazo)cyanoacetate, and triethylamine to anhydrous N,N-dimethylformamide, wherein the ratio of silk fibroin, ethyl 2-(benzoazo)cyanoacetate, triethylamine, and anhydrous N,N-dimethylformamide is 1 g: 0.5 g: 0.8 g: 20 mL; react at 80 °C for 6 h; concentrate the reaction solution to obtain a crude product, dissolve the crude product in deionized water at room temperature, and then place it in a dialysis bag with a capacity cutoff of 14000 Da for dialysis and freeze-drying to obtain modified silk fibroin.

[0029] A nanomedicine formulation targeting colorectal cancer was prepared using the method described above.

[0030] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the modified silk fibroin is replaced with silk fibroin; otherwise, they are the same as in Example 1.

[0031] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the naringin derivative is replaced with naringin; otherwise, they are the same as in Example 1.

[0032] Experimental Example 1 The modified silk fibroin obtained in Example 1 of this invention was characterized by infrared spectroscopy, and the results are as follows: Figure 1 As shown.

[0033] Depend on Figure 1 It can be known that 2235cm -1 Stretching vibrations attributed to the cyano group; 1730 cm⁻¹ -1 Stretching vibrations attributable to the carbonyl group of the ester; 1485 cm⁻¹ -1 The stretching vibration is attributed to the benzoazo group; the above information indicates that ethyl 2-(benzoazo)cyanoacetate was successfully grafted onto silk fibroin.

[0034] Experimental Example 2 To investigate the encapsulation efficiency of the drug formulations obtained in Examples 1-3 and Comparative Examples 1-2, the following tests were conducted. During the preparation of the nano-formulations in Examples 1-3 and Comparative Examples 1-2, the supernatant after centrifugation following thawing in step S3 was collected, filtered through a 0.45 μm filter membrane, and the mass of free doxorubicin, camptothecin, and naringin derivatives in the supernatant was determined.

[0035] In the preparation of nano-formulations in Examples 1-3 and Comparative Examples 1-2, the products obtained after thawing in step S3 (without centrifugation) were added to 75% ethanol and sonicated for 10 min to demulsify, releasing the encapsulated drugs. The mixture was then filtered through a 0.45 μm filter membrane, and the masses of doxorubicin, camptothecin, and naringin derivatives in the drug formulation system were determined. The encapsulation efficiency was calculated using the following formula: Encapsulation efficiency (%) = (Total mass of drug in the drug formulation system - Mass of free drug in the supernatant) / Total mass of drug in the drug formulation system × 100%. The results are shown in Table 1.

[0036] Table 1 As shown in Table 1, the encapsulation efficiency of Examples 1-3 of this invention is superior to that of Comparative Examples 1-2. The encapsulation efficiency of Comparative Example 1 is significantly lower, indicating that the modified silk fibroin, through the introduction of specific groups and the construction of a cross-linked network, is the basis for achieving high encapsulation efficiency and high stability. The above experiments demonstrate that the pharmaceutical formulation of this invention has excellent encapsulation efficiency, laying a solid foundation for subsequent in vivo antitumor experiments.

[0037] Experimental Example 3 Cellular uptake assay: To verify the targeting ability of the drug formulations obtained in Examples 1-3 and Comparative Examples 1-2 against colorectal cancer cells, the following tests were performed: Human colorectal adenocarcinoma cell lines SW620 and HT29 were selected as positive target cells, and were administered at a rate of 2 × 10⁻⁶ cells / cells. 5 Cells were seeded at a density of 100 cells / well in 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum for 24 h. Fluorescein isothiocyanate (FITC) was added to each well (FITC group), and nanomedicine formulations from Examples 1-3 and Comparative Examples 1-2 containing FITC were added to each well (experimental group). After incubation at 37°C for 4 h, each group was washed and fixed with PBS, and the uptake of drug carriers by cells was observed using a laser scanning confocal microscope. Results are shown in [Figure number missing]. Figure 2 .

[0038] Figure 2 The fluorescence intensity data visually reflects the uptake of different nanomedicine formulations by colorectal cancer cells. For example... Figure 2 As shown, the pharmaceutical formulation of the present invention can recognize and bind to colorectal cancer cells, achieving efficient co-delivery of doxorubicin, camptothecin, and naringin derivative drugs, thereby improving the drug uptake capacity of tumor cells.

[0039] Test Example 4 To investigate the inhibitory activity of the drug formulations obtained in Examples 1-3 and Comparative Examples 1-2 against colorectal cancer cells, the following experiments were conducted: Logarithmic growth phase SW620 cells and HT29 cells were selected and adjusted to a size of 1×10⁻⁶. 4Cell suspension of 1 / mL was seeded into 96-well plates and cultured overnight at 37°C until cell attachment. The culture medium was then replaced with complete medium containing the drug formulations of Examples 1-3 and Comparative Examples 1-2 (final drug concentration 20 μg / mL). A blank group (medium only) and a cell control group (no drug) were also set up. After 72 hours of treatment, the original medium was removed, and MTT solution was added to each well. The cells were incubated at 37°C for 4 hours, the supernatant was discarded, DMSO was added, and the cells were incubated at 37°C for 10 minutes. The absorbance was measured using a microplate reader. Cell viability was calculated as: Cell viability = (Experimental group absorbance - Blank group absorbance) / (Control group absorbance - Blank group absorbance). The results are shown in Table 2.

[0040] Table 2 Table 2 shows the in vitro killing effects of different drug formulations on two human colorectal cancer cell lines (SW620 and HT29). Lower cell viability indicates a stronger anti-proliferative effect. As shown in Table 2, the cell viability of Examples 1-3 was lower than that of Comparative Examples 1 and 2. Comparative Example 2 showed the worst inhibitory effect on the colorectal cancer cell lines, indicating that the combined use of naringin derivatives with doxorubicin and camptothecin can enhance the inhibitory effect on colorectal cancer cells.

[0041] Experimental Example 5 To evaluate the in vivo antitumor activity of the drug formulations obtained in Examples 1-3 and Comparative Examples 1-2, a colon cancer orthotopic xenograft model was established using 6-8 week old SPF-grade male BALB / c nude mice. Before the experiment, the mice were fasted for 12 hours, and 0.1 mL of SW620 cell suspension (density 2×10⁻⁶) was added. 7 (number of tumor cells / mL) were inoculated into the serosa of the colon of nude mice. The tumors grew to a volume of 100 mm. 3 Subsequently, nude mice were randomly divided into 6 groups of 6 mice each. The experimental groups (Examples 1-3 and Comparative Examples 1-2) were administered the corresponding drug formulations by gavage (e.g., Example 1 group was administered the drug formulation obtained in Example 1 by gavage at a dose of 20 mg / kg). The blank control group was given an equal volume of physiological saline. All groups were administered the drug once every 2 days for 21 consecutive days. After the last administration, the nude mice were euthanized by cervical dislocation, the tumor tissue was completely dissected, and the tumor volume was measured and weighed. The results are shown in Table 3.

[0042] Table 3 Table 3 shows that, compared with the blank control group, all treatment groups exhibited a certain degree of tumor growth inhibition, but the inhibitory effects varied. Among them, the tumor volume in Examples 1-3 was the smallest, followed by Comparative Example 1, and then Comparative Example 2. This difference mainly stems from the modification of key components in the nanomedicine formulation and the resulting changes in physicochemical properties and pharmacodynamics. Comparative Example 1 replaced modified silk fibroin with unmodified silk fibroin. The lack of the 2-(benzoazo)cyano group in unmodified silk fibroin led to reduced affinity at the drug loading interface, decreased drug loading efficiency and stability, thus resulting in a decrease in the antitumor activity of the drug formulation in Comparative Example 1. Comparative Example 2 replaced naringin derivative with naringin. Using unmodified naringin may result in lower activity, and the synergistic effect of naringin derivative with doxorubicin and camptothecin is weakened, leading to a decrease in the overall antitumor effect.

[0043] In summary, the pharmaceutical formulation of the present invention can deliver doxorubicin, camptothecin, and naringin derivatives to the tumor site more efficiently and stably, thereby exerting a synergistic effect in inhibiting tumor growth.

[0044] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a nanomedicine formulation targeting colorectal cancer, characterized in that, The preparation method includes the following steps: S1. Dissolve the modified silk fibroin in deionized water to obtain a modified silk fibroin solution, add doxorubicin to it to obtain a doxorubicin-modified silk fibroin solution for later use; S2. Dissolve DSPE-PEG-RGD, camptothecin, and naringin derivative into a mixed solution to obtain the drug mixture; S3. The drug mixture obtained in step S2 is added dropwise to the doxorubicin-modified silk fibroin solution in step S1 and frozen. After thawing, the nanoparticles are collected by centrifugation. The nanoparticles are washed, resuspended in deionized water, ultrasonically dispersed, and dried to obtain a nanomedicine preparation targeting colorectal cancer. The chemical structural formula of the terpineone derivative is as follows: .

2. The method for preparing the nanomedicine formulation targeting colorectal cancer according to claim 1, characterized in that, The preparation of the naringin derivative includes the following steps: (1) Under a nitrogen atmosphere, naringin, mercaptoacetic acid and sodium ascorbate were added to methanol, stirred evenly, and then tris(2,2'-bipyridine) ruthenium dichloride and trichlorobromomethane were added. The mixture was reacted at room temperature for 6-10 h under light irradiation. After purification, naringin-mercaptoacetic acid was obtained. (2) Under a nitrogen atmosphere, cyperione-mercaptoacetic acid was added to tetrahydrofuran and stirred until homogeneous. Thionyl chloride was added and reacted at 70°C for 3-5 h. The reaction solution was concentrated to obtain a concentrate. The concentrate was dissolved in a solvent to form a solution. 4,4-dithiodiphenylamine and diisopropylethylamine were added to tetrahydrofuran and stirred until homogeneous. The solution was then added dropwise to the solution and reacted at 0-3°C for 1-3 h.

3. The method for preparing the nanomedicine formulation targeting colorectal cancer according to claim 2, characterized in that, In step (1), the molar ratio of naringin, thioglycolic acid, sodium ascorbate, tris(2,2'-bipyridine) ruthenium dichloride and trichlorobromomethane is 10 mmol: 30-50 mmol: 0.03-0.05 mmol: 0.01-0.03 mmol: 0.05-0.08 mmol; the volume ratio of naringin to methanol is 10 mmol: 25 mL.

4. The method for preparing the nanomedicine formulation targeting colorectal cancer according to claim 2, characterized in that, In step (2), the molar ratio of naringin-mercaptoacetic acid, thionyl chloride, 4,4-dithiodiphenylamine, and diisopropylethylamine is 5 mmol: 12-16 mmol: 2 mmol: 10-20 mmol; when naringin-mercaptoacetic acid is added to tetrahydrofuran, the ratio of naringin-mercaptoacetic acid to tetrahydrofuran is 5 mmol: 20 mL; the ratio of the concentrate to the solvent is 5 mmol: 30 mL, and the solvent is tetrahydrofuran; when 4,4-dithiodiphenylamine and diisopropylethylamine are added to tetrahydrofuran, the ratio of 4,4-dithiodiphenylamine to tetrahydrofuran is 5 mmol: 20 mL.

5. The method for preparing the nanomedicine formulation targeting colorectal cancer according to claim 1, characterized in that, The preparation of the modified silk fibroin includes the following steps: a. Add silkworm cocoons to sodium carbonate solution and reflux for 30 minutes, then dry to obtain degummed silk; add the degummed silk to a mixed solution and dissolve at 65-75℃ for 3-6 hours; dialyze and freeze-dry the reaction solution to obtain silk fibroin. b. Add silk fibroin, ethyl 2-(benzoazo)cyanoacetate, and triethylamine to anhydrous N,N-dimethylformamide and react at 60-80°C for 6-10 hours; concentrate the reaction solution, dialyze, and freeze-dry to obtain the final product.

6. The method for preparing the nanomedicine formulation targeting colorectal cancer according to claim 5, characterized in that, In step a, the ratio of silkworm cocoon to sodium carbonate solution is 10g:80-100mL, and the concentration of sodium carbonate solution is 0.02-0.03mol / L; the ratio of degummed silk to mixed solution is 10g:80-100mL, and the mixed solution is obtained by mixing lithium chloride solution and ethanol solution at a volume ratio of 9:1, the concentration of lithium chloride solution is 9mol / L, and the volume concentration of ethanol solution is 10%.

7. The method for preparing the nanomedicine formulation targeting colorectal cancer according to claim 5, characterized in that, The ratio of silk fibroin, ethyl 2-(benzoazo)cyanoacetate, triethylamine, and anhydrous N,N-dimethylformamide in step b is 1g:0.3-0.5g:0.5-0.8g:20mL.

8. The method for preparing the nanomedicine formulation targeting colorectal cancer according to claim 1, characterized in that, In step S1, the concentration of the modified silk fibroin solution is 5 mg / mL, and the concentration of doxorubicin in the modified silk fibroin solution is 0.1-0.2 mg / mL; in step S2, the mass ratio of DSPE-PEG-RGD, camptothecin, and naringin derivative is 1:(0.1-0.3):(0.3-0.5); the concentration of DSPE-PEG-RGD in the mixed solution is 10-15 mg / mL, and the mixed solution is prepared by mixing ethanol, acetone, and methanol in a volume ratio of 1:1:

1.

9. The method for preparing the nanomedicine formulation targeting colorectal cancer according to claim 1, characterized in that, In step S3, the volume ratio of the drug mixture and the doxorubicin-modified silk fibroin solution is 1:5; the freezing temperature is -15~-20℃, and the freezing time is 12-16h.

10. A nanomedicine formulation targeting colorectal cancer, characterized in that, It is prepared by the preparation method according to any one of claims 1-9.

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