Preparation method and application of an enzyme-catalyzed self-assembled multi-targeted Pt(IV) prodrug

Abstract

The present invention aims to provide a method for preparing and applying an enzyme-catalyzed self-assembled multi-targeted Pt(Ⅳ) prodrug, belonging to the field of antitumor drug technology. This invention employs a chemical coupling method to construct the enzyme-catalyzed self-assembled multi-targeted Pt(Ⅳ) prodrug TPP-FFK(p)Y-Pt(Ⅳ), which can form a supramolecular self-assembled structure with nanofibers in situ on the surface of cancer cells under the action of phosphatases, confining the drug within the tumor. Furthermore, the drug exhibits GSH-responsive release, consuming GSH while further reducing Pt(Ⅳ) to cisplatin (Pt(Ⅱ)), avoiding premature drug disintegration. The drug is delivered to the intracellular mitochondria via triphenylphosphine, causing mitochondrial dysfunction, reducing ATP production, and thus inhibiting drug efflux and DNA damage repair. This system exhibits good stability and biocompatibility, and shows significant inhibitory effects on cisplatin-resistant lung cancer cells.

Description

Technical Field

[0001] This invention belongs to the field of antitumor drug technology, specifically relating to a method for preparing and applying an enzyme-catalyzed self-assembled multi-targeted Pt(Ⅳ) prodrug. Background Technology

[0002] Cancer is a major public health problem that is the second leading cause of death worldwide, posing a continuous and severe threat to human society.

[0003] Chemotherapy is an essential treatment strategy for the vast majority of lung cancer patients, with four irreplaceable advantages: (1) broad-spectrum killing and comprehensive coverage. (2) synergistic with immunotherapy to achieve a "1+1>2" effect. (3) highly efficient and rapid, controlling acute conditions. (4) economically accessible and universally beneficial. Platinum-based drugs (including cisplatin, carboplatin, nedaplatin, lobaplatin, etc.) are the "main force" of chemotherapy for lung cancer. Cisplatin (CDDP) occupies an irreplaceable "cornerstone" position in the treatment of lung cancer and has obvious therapeutic uniqueness. However, there are also some thorny issues in the clinical application of CDDP, mainly manifested in the fact that after repeated intake, cancer cells have established a complex drug resistance mechanism, thereby reducing anti-cancer activity. There are three main reasons for cancer cells' resistance to CDDP: reduced drug intake and increased efflux, glutathione (GSH)-induced drug inactivation, and enhanced DNA damage repair capacity.

[0004] Tetravalent platinum (Pt(Ⅳ)) prodrugs are widely considered a core option for next-generation platinum-based drugs to overcome cisplatin resistance, improve efficacy, and reduce toxicity. First, Pt(Ⅳ) compounds are chemically inert, stable in the bloodstream, and do not readily react with non-target molecules such as plasma proteins, thus reducing systemic toxicity (especially nephrotoxicity and neurotoxicity). Second, upon entering tumor cells, Pt(Ⅳ) prodrugs are primarily reduced by intracellular reducing agents (such as GSH, ascorbic acid, and mitochondrial reductase), releasing active Pt(Ⅱ) (equivalent to CDDP). This reduction process is crucial for its efficacy and overcoming resistance. Besides reversing CDDP resistance itself, Pt(Ⅳ) can be combined with other anticancer drugs or components that reverse resistance pathways, or delivered to the lesion site in the form of nanomedicines. Therefore, in-situ self-assembled Pt(Ⅳ) prodrugs with high targeting, strong aggregation, and intelligent responsiveness will show enhanced antitumor and anti-resistance potential.

[0005] Nanomaterials possess enhanced permeability and retention effects (EPR), resulting in higher targeting and uptake rates in tumor tissues. Furthermore, enzyme-instructed self-assembly (EISA) is a bio-inspired strategy that utilizes enzyme-catalyzed reactions to regulate molecular self-assembly. Its core feature is the use of overexpressed enzymes in cells (such as alkaline phosphatase (ALP)) to trigger and regulate drug molecule self-assembly under specific conditions through enzymatic reactions, constructing functionalized nanostructures for targeted cancer cell therapy. In addition, mitochondria are the cell's "power source," synthesizing adenosine triphosphate (ATP) through oxidative phosphorylation. Mitochondrial-targeted therapy can directly cut off the energy supply to cancer cells, causing them to "starve" and die. Mitochondrial-targeted drugs act directly on the mitochondrial membrane, unaffected by drug efflux pumps, overcoming resistance to traditional chemotherapy. By increasing the permeability of the outer mitochondrial membrane, apoptosis is irreversibly initiated, more effectively eliminating cancer cells and reducing tumor recurrence. In summary, drugs that target tumors / cells / organelles in a cascade and are activated in an orderly manner within the tumor microenvironment cleverly integrate the above advantages. This approach is an effective way to overcome the obstacles of individual strategies by using a single drug system, efficiently avoid drug resistance, and improve the efficacy of anticancer treatment.

[0006] Recent reports on CDDP treatment for cancer have not covered the area of ​​using triple-targeted therapy in combination to overcome drug resistance and reduce side effects. Summary of the Invention

[0007] To address the above problems, the present invention aims to provide a method for preparing and applying an enzyme-catalyzed self-assembled multi-targeted Pt(Ⅳ) prodrug, wherein a uniformly morphologically homogeneous TPP-FFK(p)Y-Pt(Ⅳ) was prepared by chemical coupling.

[0008] The present invention adopts the following technical solution: A method for preparing an enzymatically self-assembled multi-targeted Pt(Ⅳ) prodrug includes the following steps: Synthesis of S1 and Pt(NH3)2(Cl)2(OH)2: A mixture of CDDP and hydrogen peroxide was placed in a 100 mL round-bottom flask and heated at 50 °C for 3 h in the dark with strong magnetic stirring. The mixture was cooled and reprecipitated overnight at 4 °C. The sample was then placed in a 50 mL centrifuge tube and centrifuged at 10,000 rpm for 10 min. The supernatant was discarded to obtain bright yellow Pt(NH3)2(Cl)2(OH)2 powder, referred to as Pt(OH)2 powder. The powder was washed successively with cold water, anhydrous ethanol, and diethyl ether, and centrifuged successively. The powder was then vacuum dried for 24 h. Synthesis of S2 and Pt(NH3)2(Cl)2(OOCCH2CH2COOH)2: Succinic anhydride was added to an anhydrous DMF suspension of Pt(OH)2 and reacted at 70℃ in the dark for 24 h. After removing DMF, the product was dissolved in a small amount of anhydrous methanol, and an appropriate amount of diethyl ether was added. The sample was placed in a 50 mL centrifuge tube and centrifuged at 10000 rpm for 10 min. The supernatant was discarded. The product was washed with anhydrous methanol and diethyl ether, centrifuged, and the supernatant was discarded. The product was then dried under vacuum for 60 h to obtain Pt(NH3)2(Cl)2(OOCCH2CH2COOH)2, abbreviated as Pt-COOH. Preparation of S3 and TPP-FFK(p)Y: Using 2-chlorotriphenylmethyl chloro resin as a carrier and various amino acids with N-Fmoc side chain protection as reactants, first, 2-chlorotriphenylmethyl chloro resin was soaked in DCM to swell, and the first amino acid Fmoc-D-Tyr(HPO3Bzl)-OH was added. After 2 h, the unreacted parts in the 2-chlorotriphenylmethyl chloro resin were quenched with a blocking liquid of DCM / MeOH / DIPEA with a volume ratio of 17 / 4 / 1. 200 mL of liquid with V(hexahydropyridine):V(DMF)=1:4 was added, and the reaction was carried out for 15 min to remove the Fmoc protecting group and carry out subsequent reactions with D-amino acids. TBTU was used as a condensing agent to couple subsequent amino acids protected by Fmoc. The amino acid coupling and deprotection steps were repeated until the condensation of the last amino acid TPP was completed. 200 mL of 82.5% TFA cleavage reagent was added, and the reaction was carried out for 3 h. After filtration, the filtrate was concentrated by rotary evaporation. 10 mL of ice-cold ether was added to the concentrate to precipitate the precipitate. The precipitate was centrifuged at 5000 rpm for 5 min, and the supernatant was discarded. The reaction was repeated three times. The precipitate was dried under vacuum to obtain TPP-{D-Phe}-{D-Phe}-{D-Lys}-{D-Tyr(H2PO3), i.e., crude TPP-FFK(p)Y. The crude TPP-FFK(p)Y was purified by reverse-phase chromatography and lyophilized to obtain TPP-FFK(p)Y. Synthesis of S4, TPP-FFK(p)Y-Pt(Ⅳ): Pt-COOH, NHS and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were dissolved in anhydrous dimethyl sulfoxide and stirred for 12 h to form the NHS active ester of Pt-COOH. TPP-FFK(p)Y was added, and DIPEA was added until the solution was weakly alkaline. The solution was stirred at room temperature for 24 h, transferred to a dialysis bag with MWCO = 2000 Da, and dialyzed with deionized water for 48 h. Finally, the solution was lyophilized to obtain TPP-FFK(p)Y-Pt(Ⅳ). S5. Preparation of TPP-FFKY-Pt(Ⅳ) HG: TPP-FFK(p)Y-Pt(Ⅳ) was added to PBS at pH = 7.4 until completely dissolved. The solution was treated with ALP and placed at 37℃ for 2 h. TPP-FFK(p)Y-Pt(Ⅳ) underwent dephosphorization under the catalysis of ALP to form a hydrogel-like self-assembled TPP-FFKY-Pt(Ⅳ) (Hydrogel), named TPP-FFKY-Pt(Ⅳ) HG.

[0009] Further, in S1, the amount of CDDP used is 1000-1200mg, and the amount of hydrogen peroxide used is 20-25mL.

[0010] Further, in S2, the amount of succinic anhydride is 800-1000 mg, the amount of Pt(OH)2 is 600-800 mg, the amount of DMF is 20 mL, the amount of anhydrous methanol is 5 mL, and the amount of diethyl ether is 20 mL.

[0011] Further, in S3, the amount of 2-chlorotriphenylmethyl chloride resin is 15-20 g, the amount of DCM is 200-500 mL, the amount of TBTU is 5-10 g, and the amount of TPP is 5-20 g.

[0012] Amino acids protected by N-Fmoc side chains include Fmoc-D-Tyr(HPO3Bzl)-OH in amounts of 8-12 g, Fmoc-D-Lys(Boc)-OH in amounts of 5-10 g, Fmoc-D-Phe-OH in amounts of 8-10 g, and Fmoc-D-Phe-OH in amounts of 8-10 g.

[0013] Further, in S4, the amount of Pt-COOH is 100-120 mg, the amount of NHS is 40-60 mg, the amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 60-80 mg, the amount of anhydrous dimethyl sulfoxide is 10-15 mL, and the amount of TPP-FFK(p)Y is 400-600 mg.

[0014] Further, in S5, the amount of TPP-FFK(p)Y-Pt(Ⅳ) is 1-2 mg, the amount of PBS with pH = 7.4 is 1-2 mL, and the amount of ALP is 1-5 U.

[0015] The present invention also provides the use of an enzyme-catalyzed self-assembled multi-targeted Pt(Ⅳ) prodrug as and / or in the preparation of antitumor drugs.

[0016] The principle of this invention is as follows: This invention uses a chemical coupling method to successfully construct an enzyme-catalyzed self-assembled nanomedicine system TPP-FFK(p)Y-Pt(Ⅳ), which delivers functional components to the mitochondria of tumor cells via triphenylphosphine (TPP). The Pt(Ⅳ) prodrug consists of four parts: (1) a Pt(Ⅳ) module, which is linked to the polypeptide backbone via succinic anhydride and responds to the high concentration of GSH in tumor cells, reducing and dissolving it into cytotoxic Pt(Ⅱ); (2) a phosphate group, which responds to phosphatase stimulation, changing the hydrophilicity and hydrophobicity of the molecule to achieve nano-self-assembly behavior; (3) a mitochondrial targeting group TPP, which enhances the mitochondrial enrichment of the drug; and (4) a polypeptide backbone, which endows the nanomedicine with good biocompatibility. TPP-FFK(p)Y-Pt(Ⅳ) has the following advantages: (1) It achieves triple targeting through ALP-responsive in-situ self-assembly, nanostructure regulation, and TPP targeting mitochondria, reducing side effects; (2) It responds to high concentrations of GSH in cancer cells, decomposes and releases Pt(Ⅱ), targets and attacks mitochondrial DNA, increases mitochondrial membrane permeability, interferes with energy metabolism, and induces apoptosis and mitochondrial autophagy death; (3) It effectively overcomes chemotherapy resistance mediated by high levels of GSH in tumor cells; (4) Its mitochondrial targeting avoids drug inactivation caused by nuclear DNA repair mechanisms. This multi-targeted and cascade-response in-situ self-assembled nanomedicine cleverly circumvents traditional drug resistance pathways by specifically interfering with mitochondrial function, and efficiently and synergistically kills cisplatin-resistant lung cancer cells. This design idea is a deepening and expansion of existing drug delivery theories and the understanding of tumor drug resistance mechanisms. By optimizing drug distribution, it concentrates drug efficacy on key core targets, reduces systemic toxicity, and ultimately improves the effective cure rate and overall quality of life of lung cancer patients.

[0017] Therefore, the Pt(Ⅳ) prodrug, which combines in-situ self-assembly of nanomedicines, cell targeting, and mitochondrial targeting, can effectively solve the existing problems of drug-resistant tumor treatment and the large side effects in current clinical treatments.

[0018] The beneficial effects of this invention are as follows: The preparation method of this invention is simple, and the synthesis conditions are green and pollution-free. The obtained TPP-FFK(p)Y-Pt(Ⅳ) can form supramolecular self-assembled structures with nanofiber structures in situ on the surface of cancer cells under the action of phosphatases, thus confining the drug within the tumor. Furthermore, the drug exhibits GSH-responsive release, consuming GSH while further reducing Pt(Ⅳ) to cisplatin (Pt(Ⅱ)), avoiding premature drug disintegration. The drug is delivered to the intracellular mitochondria via triphenylphosphine, causing mitochondrial dysfunction, reducing ATP production, and thereby inhibiting drug efflux and DNA damage repair.

[0019] The system of this invention has good stability and biocompatibility, and has a significant inhibitory effect on cisplatin-resistant lung cancer cells. It will promote the development of drug-resistant tumor treatment, explore new directions for personalized nanomedicine, and provide a possibility for overcoming drug resistance in clinical treatment. Attached Figure Description

[0020] Figure 1 The synthetic route of TPP-FFK(p)Y-Pt(Ⅳ) prepared in Example 1 of this invention and the release diagram of cisplatin under the action of GSH are shown.

[0021] Figure 2 The TPP-FFK(p)Y-Pt(Ⅳ) prepared in Example 1 of this invention 1 H NMR spectrum.

[0022] Figure 3 ESI-MS image of TPP-FFK(p)Y-Pt(Ⅳ) prepared in Example 1 of this invention.

[0023] Figure 4 Transmission electron microscope image of TPP-FFK(p)Y-Pt(Ⅳ) prepared in Example 1 of this invention.

[0024] Figure 5 Transmission electron microscope image of TPP-FFKY-Pt(Ⅳ) HG prepared in Example 1 of this invention.

[0025] Figure 6 The particle size distribution diagram is shown for TPP-FFKY-Pt(Ⅳ) HG prepared in Example 1 of this invention.

[0026] Figure 7 The cumulative release curves of Pt from TPP-FFKY-Pt(Ⅳ) HG prepared in Example 1 of this invention under different concentrations of GSH are shown.

[0027] Figure 8 Transmission electron microscope image of TPP-FFKY-Pt(Ⅳ) HG prepared in Example 1 of this invention after GSH response release.

[0028] Figure 9 The image shows the result of TPP-FFKY-Pt(Ⅳ) HG being generated on the cell surface after TPP-FFKY-Pt(Ⅳ) prepared in Example 1 of this invention was incubated with A549 / DDP cells for 48 h.

[0029] Figure 10 The figure shows the effect of TPP-FFK(p)Y-Pt(Ⅳ) prepared in Example 1 of this invention on the viability of A549, A549 / DDP and BEAS-2B cells.

[0030] Figure 11 The graph shows the changes in mitochondrial membrane potential of A549 / DDP cells in different experimental groups after drug treatment.

[0031] Figure 12 This diagram shows the changes in apoptosis in A549 / DDP cells from different experimental groups.

[0032] Figure 13 The diagram shows the cell cycle changes of A549 / DDP cells in different experimental groups.

[0033] Figure 14 The diagram shows the migration changes of A549 / DDP cells in different experimental groups.

[0034] Figure 15 The image shows actual photos of tumors after different drugs were administered to the tail vein of tumor-bearing mice.

[0035] Figure 16 This is a comparison of tumor volume after different drugs were administered to the tail vein of tumor-bearing mice.

[0036] Figure 17 TUNEL staining of tumors in tumor-bearing mice after treatment with different drugs in the tail vein.

[0037] Figure 18 HE staining images of tissues from tumor-bearing mice after treatment with different drugs in the tail vein. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1 A method for preparing an enzymatically self-assembled multi-targeted Pt(Ⅳ) prodrug includes the following steps: Synthesis of S1 and Pt(NH3)2(Cl)2(OH)2 (abbreviated as Pt(OH)2): A mixture of CDDP (1007.9 mg), hydrogen peroxide (30% w / v, 9 mL, H2O (22 mL)) was placed in a 100 mL round-bottom flask and heated at 50 °C for 3 h in the dark with strong magnetic stirring. The mixture was cooled and redeposited overnight at 4 °C. The sample was transferred to a 50 mL centrifuge tube and centrifuged at 10,000 rpm for 10 min, and the supernatant was discarded. A bright yellow Pt(OH)2 powder was obtained, which was washed successively with cold water, anhydrous ethanol, and diethyl ether, and centrifuged successively. The powder was then vacuum dried for 24 h.

[0040] Synthesis of S2 and Pt(NH3)2(Cl)2(OOCCH2CH2COOH)2 (abbreviated as Pt-COOH): Succinic anhydride (876.4 mg) was added to a suspension of Pt(OH)2 (730.3 mg) in anhydrous DMF (19 mL), and the mixture was reacted at 70 °C in the dark for 24 h. After removing the DMF, the product was dissolved in a small amount of anhydrous methanol (5 mL), and an appropriate amount of diethyl ether (20 mL) was added. The sample was then transferred to a 50 mL centrifuge tube and centrifuged at 10,000 rpm for 10 min. The supernatant was discarded. The product was washed with anhydrous methanol and diethyl ether, centrifuged, and the supernatant was discarded. The product was then vacuum dried for 60 h.

[0041] Preparation of S3 and TPP-FFK(p)Y: 2-chlorotriphenylmethyl chloride resin (18.18 g) was used as a carrier, and various amino acids with N-Fmoc side chain protection were used as reactants. First, the resin was soaked in 300 mL of DCM to swell, and the first amino acid, Fmoc-D-Tyr(HPO3Bzl)-OH (10.8 g), was added. After 2 h, unreacted sites in the resin were quenched with a blocking solution (17 / 4 / 1 DCM / MeOH / DIPEA). 200 mL of V(hexahydropyridine):V(DMF) = 1:4 was added, and the reaction was allowed to proceed for 15 min to remove the Fmoc protecting group, allowing for subsequent reactions with the D-amino acid. TBTU (9 g) was used as a condensing agent to couple subsequent Fmoc-protected amino acids (Fmoc-D-Lys(Boc)-OH (7.5 g), Fmoc-D-Phe-OH (8.3 g), Fmoc-D-Phe-OH (8.3 g)). The amino acid coupling and deprotection steps were repeated until the condensation of the last amino acid (TPP (10 g) was considered the last amino acid) was complete. 200 mL of cleavage reagent (82.5% TFA) was added, and the reaction was carried out for 3 h. After filtration, the filtrate was concentrated by rotary evaporation. 10 mL of ice-cold diethyl ether was added to the concentrate, and a precipitate was formed. The precipitate was centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and the reaction was repeated three times. The precipitate was then dried under vacuum to obtain crude TPP-{D-Phe}-{D-Phe}-{D-Lys}-{D-Tyr(H2PO3) (i.e., TPP-FFK(p)Y). The crude TPP-FFK(p)Y was purified using a reverse-phase chromatography column and then lyophilized to obtain TPP-FFK(p)Y.

[0042] Synthesis of S4, TPP-FFK(p)Y-Pt(Ⅳ): Pt-COOH (106.6 mg), NHS (51.8 mg), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (76.7 mg) were dissolved in 12 mL of anhydrous dimethyl sulfoxide and stirred for 12 h to form the NHS active ester of Pt-COOH. 456.5 mg of TPP-FFK(p)Y was added. DIPEA was added until the solution became weakly alkaline, and the mixture was stirred at room temperature for 24 h. The solution was transferred to a dialysis bag (MWCO = 2000 Da) and dialyzed with deionized water for 48 h. Finally, the solution was lyophilized to obtain 236.6 mg of TPP-FFK(p)Y-Pt(Ⅳ).

[0043] S5. Preparation of TPP-FFKY-Pt(Ⅳ) HG: TPP-FFK(p)Y-Pt(Ⅳ) (1 mg) was added to 1 mL of PBS at pH = 7.4 until completely dissolved, and the solution was treated with 2 U ALP. After being placed at 37℃ for 2 h, TPP-FFK(p)Y-Pt(Ⅳ) underwent a dephosphorization reaction catalyzed by ALP to form a hydrogel-like self-assembled TPP-FFKY-Pt(Ⅳ) (Hydrogel) (named TPP-FFKY-Pt(Ⅳ) HG).

[0044] Figure 1 The synthetic route of TPP-FFK(p)Y-Pt(Ⅳ) prepared in Example 1 of this invention and the release diagram of cisplatin under the action of GSH are shown in the figure. TPP-FFK(p)Y-Pt(Ⅳ) was synthesized by chemical coupling method and Pt(Ⅱ) was released under the action of GSH.

[0045] Figure 2 The TPP-FFK(p)Y-Pt(Ⅳ) prepared in Example 1 of this invention 1 The 1H NMR spectrum is shown in the figure. 1 H NMR (500 MHz, DMSO-) d 6 ) δ 8.37 (s, 2H), 8.22 (s, 2H), 7.98 (s, 2H), 7.87-7.86 (m,6H), 7.85-7.60 (m, 24H), 7.35-7.25 (m, 4H), 7.22-6.92 (m, 25H), 6.72-6.60 (m,5H,), 4.48-4.40 (m, 4H), 4.21-4.08 (m, 4H), 3.04-2.74 (m, 16H), 2.65-2.58 (m,2H), 2.42-2.36 (m, 6H), 2.28 (s, 8H), 1.55-1.44 (m, 8H), 1.32–1.17 (m, 8H), with characteristic hydrogen atoms that are different from those in Pt-COOH and TPP-FFK(p)Y.

[0046] Figure 3 The ESI-MS chromatogram of TPP-FFK(p)Y-Pt(Ⅳ) prepared in Example 1 of the present invention is shown in the figure. ESI-MS: 2527.7712, indicating the successful synthesis of TPP-FFK(p)Y-Pt(Ⅳ).

[0047] Figure 4The image shows a transmission electron microscope image of TPP-FFK(p)Y-Pt(Ⅳ) prepared in Example 1 of the present invention. As shown in the figure, TPP-FFK(p)Y-Pt(Ⅳ) appears as an irregular aggregate.

[0048] Figure 5 The image shows a transmission electron microscope image of TPP-FFKY-Pt(Ⅳ) HG prepared in Example 1 of the present invention. As shown in the figure, the nanofibers of TPP-FFKY-Pt(Ⅳ) HG are interwoven to form a dense network.

[0049] Figure 6 The image shows the particle size distribution of TPP-FFKY-Pt(Ⅳ) HG prepared in Example 1 of this invention. As shown in the image, the average hydrodynamic size of the TPP-FFKY-Pt(Ⅳ) HG nanofibers is 166 nm, and the PDI is 0.021.

[0050] Figure 7 The cumulative release curves of Pt for TPP-FFKY-Pt(Ⅳ) HG prepared in Example 1 of this invention under different concentrations of GSH are shown in the figure. TPP-FFKY-Pt(Ⅳ) HG showed concentration-dependent drug release in the presence of 10 mM and 10 μM GSH, and the cumulative release rates of Pt(Ⅱ) at 48 h were 79.6% and 15.2%, respectively.

[0051] Figure 8 The image shows a transmission electron microscope image of TPP-FFKY-Pt(Ⅳ) HG prepared in Example 1 of this invention after GSH response release. As shown in the figure, the nanofibers are dispersed into irregular individual particles.

[0052] Example 2 In this embodiment, the self-assembly behavior of the sample TPP-FFK(p)Y-Pt(Ⅳ) on the cell surface was verified, and the method is as follows: (1) Cell culture A549 / DDP cells were cultured in Ham's F-12K medium containing 10% fetal bovine serum and placed in a cell culture incubator at 37°C and 5% CO2.

[0053] (2) TPP-FFK(p)Y-Pt(Ⅳ) self-assembles on the cell surface A549 / DDP cells were loaded at 2.0... 10 6 / wells were seeded in 6 cm cell culture dishes, and incubated for 48 h with drug-free medium and medium containing 100 μM TPP-FFK(p)Y-Pt(IV).

[0054] Figure 9The figure shows the results of TPP-FFK(p)Y-Pt(Ⅳ) HG generation on the cell surface after incubating A549 / DDP cells with TPP-FFK(p)Y-Pt(Ⅳ) prepared in Example 1 of this invention for 48 h. As shown in the figure, the bottom of the culture dish in the TPP-FFK(p)Y-Pt(Ⅳ) treatment group was covered with a layer of obvious gel-like substance, while the bottom of the culture dish in the drug-free treatment group was relatively clean, indicating that TPP-FFK(p)Y-Pt(Ⅳ) underwent effective self-assembly on the surface of tumor cells.

[0055] Example 3 In this embodiment, the effect of sample TPP-FFK(p)Y-Pt(Ⅳ) from Example 1 on cell viability was determined using the following method: A549 / DDP, A549, and BEAS-2B cells were seeded in 96-well plates at a cell density of approximately 1.0 g / cm³. 10 4 Cells per well. After 24 h of incubation, the supernatant culture medium was discarded, the cells were washed once with PBS, and treated with a certain concentration of TPP-FFK(p)Y-Pt(Ⅳ) solution for 48 h. The original culture medium was replaced with fresh culture medium containing 10% CCK-8. After incubation for 20 min, the cells were placed in a microplate reader to measure the absorbance at a wavelength of 450 nm. The absorbance of each group was subtracted from the absorbance of the blank PBS solution. The absorbance of each well was divided by the absorbance of the control group to obtain the cell viability. Six parallel wells were set up for each group.

[0056] Figure 10 The figure shows the effect of TPP-FFK(p)Y-Pt(Ⅳ) prepared in Example 1 of this invention on the viability of A549, A549 / DDP and BEAS-2B cells. The figure also shows the effect of TPP-FFK(p)Y-Pt(Ⅳ) on the IC50 of A549 cells. 50 The value was 3.94 ± 0.22 μM, and the IC50 of A549 / DDP cells was... 50 The value was 5.46 ± 0.34 μM, while the cytotoxicity to BEAS-2B cells was mild, indicating that TPP-FFK(p)Y-Pt(Ⅳ) selectively targets cancer cells while having the lowest side effects on normal cells.

[0057] Example 4 In this embodiment, we evaluated the effect of TPP-FFK(p)Y-Pt(Ⅳ) on the cell membrane potential of A549 / DDP cells using the JC-1 experiment, and conducted parallel experiments with different drug treatment groups as controls, as follows: A549 / DDP cells were loaded at 6.0... 10 5Cells were seeded in 24-well plates with a crawling plate at the bottom. After incubation for 24 h, the supernatant was discarded, and the cells were washed once with PBS. PBS, cell culture medium containing 20 μM CDDP, 20 μM Pt-COOH, 40 μM TPP-FFK(p)Y, and 20 μM TPP-FFK(p)Y-Pt(Ⅳ) were added. After incubation for 48 h, the supernatant was discarded, and the cells were washed with pre-cooled PBS. Cells were stained with JC-1 using an enhanced mitochondrial membrane potential detection kit (JC-1). The crawling plate was removed, and green and red fluorescence signals were acquired using a confocal laser scanning microscope at excitation wavelengths of 490 nm and 525 nm.

[0058] Figure 11 The image shows the changes in mitochondrial membrane potential in A549 / DDP cells from different experimental groups after drug treatment. In normal mitochondria, a polarized state (high membrane potential) is observed: JC-1 aggregates in the mitochondrial matrix to form polymers (J-aggregates), emitting red fluorescence. In apoptotic cells, a depolarization process (decreased membrane potential) is observed: JC-1 is released from the mitochondria into the cytoplasm, existing as monomers and emitting green fluorescence. As shown in the image, compared with the PBS group, the red fluorescence of J-aggregates in A549 / DDP cells treated with TPP-FFK(p)Y-Pt(Ⅳ) was weakened, while the intensity of green fluorescence was significantly enhanced, indicating a decrease in mitochondrial membrane potential. This suggests that the active ingredient Pt(Ⅱ) released by TPP-FFK(p)Y-Pt(Ⅳ) can effectively depolarize mitochondria.

[0059] Example 5 To further investigate whether the toxic effects of TPP-FFK(p)Y-Pt(Ⅳ) on A549 / DDP cells are related to apoptosis, we examined changes in apoptosis in each group.

[0060] (1) Take A549 / DDP cells in the logarithmic growth phase cultured in a cell culture incubator (37℃, 5% CO2), digest them into single cells, and then seed them into 6-well plates at an appropriate density and continue to culture them in the incubator.

[0061] (2) The next day, when the cells reach a suitable density, the old culture medium is removed and a cell culture medium containing 20 μM CDDP, 20 μM Pt-COOH, 40 μM TPP-FFK(p)Y and 20 μM TPP-FFK(p)Y-Pt(Ⅳ) is added. The control group is given complete cell culture medium without drugs. The cells are then placed back in the cell culture incubator and cultured for another 48 h.

[0062] (3) Collect the old culture medium of cells from each treatment group in the 6-well plate, wash twice with cold PBS, collect the PBS wash solution, digest the cells with trypsin for 1-2 min, stop the digestion with the old culture medium and resuspend the cells, add the collected PBS wash solution, and centrifuge at 1000 rpm for 5 min using a 4℃ centrifuge.

[0063] (4) Collect the cell pellet, gently resuspend the cell pellet on ice with 100 μL Binding Buffer, add 5 μL Annexin V FITC and 10 μL PI staining solution, and stain in the dark for 30 min.

[0064] (5) After staining, transfer each group of cells to flow cytometry tubes and immediately perform flow cytometry analysis on changes in apoptosis.

[0065] Figure 12 The image shows the changes in apoptosis in A549 / DDP cells in different experimental groups. As shown, the apoptosis rate was only 1.92% in the untreated group, 3.85% in the CDDP-treated group, and 22.05% in the TPP-FFK(p)Y-Pt(IV)-treated group, which was 11.48 times that of the PBS group and 5.73 times that of the CDDP group. This indicates that TPP-FFK(p)Y-Pt(IV) can induce apoptosis in CDDP-resistant cells, demonstrating a more effective antitumor effect than free CDDP.

[0066] Example 6 To further investigate whether the toxic effects of TPP-FFK(p)Y-Pt(Ⅳ) on A549 / DDP cells are related to the cell cycle, we examined cell cycle changes in each group.

[0067] (1) Take A549 / DDP cells in the logarithmic growth phase cultured in a cell culture incubator (37℃, 5% CO2), digest them into single cells, and then seed them into 6-well plates at an appropriate density and continue to culture them in the incubator.

[0068] (2) The next day, when the cells reach a suitable density, the old culture medium is removed and a cell culture medium containing 20 μM CDDP, 20 μM Pt-COOH, 40 μM TPP-FFK(p)Y and 20 μM TPP-FFK(p)Y-Pt(Ⅳ) is added. The control group is given complete cell culture medium without drugs. The cells are then placed back in the cell culture incubator and cultured for another 48 h.

[0069] (3) Collect the old culture medium of cells from each treatment group in a 6-well plate, wash twice with cold PBS, collect the PBS wash buffer, digest the cells with trypsin for 1-2 min, stop the digestion with the old culture medium and resuspend the cells, add the collected PBS wash buffer, centrifuge at 1000 rpm for 5 min at 4℃, and discard the supernatant. Add 1 mL of 70% ethanol pre-chilled on ice, gently pipette to mix, and fix at 4℃ for 24 h. Centrifuge the fixed cells at 800 rpm for 3 min and carefully aspirate the supernatant. Add 1 mL of PBS pre-chilled on ice and resuspend the cells. Centrifuge again to precipitate the cells and carefully aspirate the supernatant.

[0070] (4) Collect the cell pellet. Add a staining detection solution to each sample, which is made by gently mixing 1 mL staining buffer, 20 μL L Nase A and 50 μL propidium iodide staining solution. Slowly and thoroughly resuspend the cell pellet and incubate at 37°C in the dark for 30 min.

[0071] (5) After staining, transfer each group of cells to flow cytometry tubes and immediately perform flow cytometry analysis on changes in apoptosis.

[0072] Figure 13 The image shows the cell cycle changes of A549 / DDP cells in different experimental groups. The S phase is the critical period for cancer cell replication and clonal expansion, and it is also the period when the cells are most sensitive to chemotherapy. As shown in the image, TPP-FFK(p)Y-Pt(Ⅳ) can arrest 31.7% of A549 / DDP cells in the S phase, which is significantly higher than that of CDDP-exposed cells.

[0073] Example 7 To further investigate the inhibitory effect of TPP-FFK(p)Y-Pt(Ⅳ) on A549 / DDP cell migration, we examined cell migration in each group.

[0074] (1) Take A549 / DDP cells in the logarithmic growth phase cultured in a cell culture incubator (37℃, 5% CO2), digest them into single cells, and then seed them into 6-well plates at an appropriate density and continue to culture them in the incubator.

[0075] (2) The next day, when the cells reach a suitable density, draw several parallel positioning lines on the back of the plate with a marker. Carefully remove the old culture medium with a pipette, slowly add preheated PBS along the well wall, gently shake and then discard, repeating twice. Using the tip of a 200 μL sterile pipette, hold it perpendicular to the bottom of the plate and draw a straight line steadily and at a uniform speed along the marked line on the back of the plate. Immediately after drawing the line, add preheated PBS gently along the well wall, gently shake the culture plate, and wash away the cell debris drawn off to make the edge of the line clear. Remove the PBS, add 5 μM CDDP, 20 μM Pt-COOH, 20 μM TPP-FFK(p)Y, and 1.5 μM TPP-FFK(p)Y-Pt(Ⅳ) prepared with cell basal medium (serum-free) to the experimental wells at a volume of 2 mL / well, and add serum-free cell basal medium to the control wells.

[0076] (3) Place the culture plate under an inverted microscope, locate the scratched area, and take a picture at the marked line. Put the culture plate back into the cell culture incubator to continue culturing. Take out the culture plate at 24 h, 48 h, and 72 h after scratching and take a picture at the previously recorded position.

[0077] Figure 14 The figure shows the migration of A549 / DDP cells in different experimental groups. The migration rate of cells treated with the drug TPP-FFK(p)Y-Pt(Ⅳ) was significantly slower. Except for the TPP-FFK(p)Y-Pt(Ⅳ) group, the cells in the other groups merged 72 h after scratching without obvious scratch boundaries. However, the cells in the TPP-FFK(p)Y-Pt(Ⅳ) group retained a clear scratch area, indicating that TPP-FFK(p)Y-Pt(Ⅳ) can more effectively inhibit the migration of tumor cells.

[0078] Example 8 To investigate the therapeutic effect of TPP-FFK(p)Y-Pt(Ⅳ) on A549 / DDP cell tumor-bearing mice, the following methods were used: (1) A549 / DDP cells were loaded with 1 10 7 One tumor per mouse was injected into the right back of BALB / c mice. After successful tumor modeling, the mice were randomly divided into 5 groups of 5 mice each and treated with the drug. The length (L) and width (W) of the tumor were measured using calipers, and the result was calculated using the formula V = 0.5 × L × W. 2 The volume of the tumor was estimated when the subcutaneous tumor in each group of BALB / c mice grew to a volume of 100 mm. 3The treatment experiment began around 10:00 AM. PBS, CDDP (3 mg / kg), Pt-COOH (5.3 mg / kg), TPP-FFK(p)Y (20.3 mg / kg), and TPP-FFK(p)Y-Pt(Ⅳ) (25.3 mg / kg) (with the same Pt ​​and TPP-FFK(p)Y concentrations) were administered via tail vein injection, once every 7 days for a total of 3 times.

[0079] (2) After the start of treatment, observe the tumor growth of BALB / c mice every other day, measure the length and width of the tumor, and weigh the mice. To ensure animal welfare, the tumor volume should not exceed 2000 mm. 3 In the pre-termination experiment, tumor-bearing BALB / c mice were euthanized, and organs and tumors were collected from BALB / c mice in each treatment group. Figure 15 These are photographs of tumors in the tail vein of tumor-bearing mice after treatment with different drugs. Figure 16 This image shows a comparison of tumor volume after different drugs were administered to the tail vein of tumor-bearing mice. As shown in the image, on day 21, the tumor volume of mice treated with PBS increased to approximately 1500 mm. 3 However, when treated with TPP-FFK(p)Y-Pt(Ⅳ) during the same period, the tumor volume was significantly suppressed to 274 mm. 3 The average tumor inhibition rate of TPP-FFK(p)Y-Pt(Ⅳ) was approximately 81.7%, significantly higher than that of CDDP (56.2%). These results indicate that TPP-FFK(p)Y-Pt(Ⅳ) possesses significant in vivo tumor growth inhibitory activity.

[0080] Example 9 1. TUNEL staining, the method is as follows: On day 21 post-treatment, mice were euthanized, tumors were removed, fixed in 4% paraformaldehyde for 24 h, dehydrated, and then embedded in paraffin for subsequent paraffin sectioning. The paraffin sections were dewaxed to water by immersion in xylene I for 10 min, xylene II for 10 min, 100% ethanol I for 5 min, 100% ethanol II for 5 min, 95% ethanol for 5 min, and 85% ethanol for 5 min. The sections were then washed three times with PBS and stained using the TUNEL apoptosis detection kit. After staining, the staining process involved hematoxylin counterstaining for 15 s, washing with water for 1 min, differentiation solution for 7 s, washing with water for 1 min, blueing solution for 15 s, washing with water for 1 min, 95% ethanol for 20 s, eosin for 12 s, 95% ethanol for 1 min, 100% ethanol for 1 min, 100% ethanol for 1 min, xylene for 1 min, and xylene for 1 min. Remove the sections from xylene, air dry them, mount them with neutral resin, dry them overnight in a 37°C oven, remove them and let them air dry in a cool place, and scan them with a high-throughput slide scanner.

[0081] 2. HE staining, the method is as follows: On day 21 post-treatment, mice were euthanized, and tissues were removed, fixed in 4% paraformaldehyde for 24 h, dehydrated, and then embedded in paraffin for subsequent paraffin sectioning. The paraffin sections were placed in xylene I for 10 min, xylene II for 10 min, 100% ethanol I for 5 min, 100% ethanol II for 5 min, 95% ethanol for 5 min, and 85% ethanol for 5 min for dewaxing to water, followed by 1 min of water washing, 2 min of hematoxylin, 1 min of water washing, 7 s of differentiation solution, 1 min of water washing, 15 s of blueing solution, 1 min of water washing, 20 s of 95% ethanol, 12 s of eosin, 1 min of 95% ethanol, 1 min of 100% ethanol, 1 min of 100% ethanol, 1 min of xylene, and 1 min of xylene. The sections were removed from xylene, air-dried, mounted with neutral resin, and dried overnight in a 37°C oven. They were then removed and air-dried in a cool place before being scanned using a high-throughput slide scanner.

[0082] Figure 17 TUNEL staining images of tumors in the tail vein of tumor-bearing mice after treatment with different drugs. Figure 18 HE staining images of tissues from tumor-bearing mice after treatment with different drugs in the tail vein. As shown in the images, compared with other groups, the TPP-FFK(p)Y-Pt(Ⅳ) group showed extensive and more apoptotic (red) cells. Mice treated with the TPP-FFK(p)Y-Pt(Ⅳ) group did not show obvious tissue damage compared with the PBS group, indicating that it has good tissue compatibility.

[0083] The above description only illustrates the preferred embodiments of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention, and all such changes should be included within the protection scope of the present invention.

Claims

1. A method for preparing an enzymatically self-assembled multi-targeted Pt(Ⅳ) prodrug, characterized in that: Includes the following steps: Synthesis of S1 and Pt(NH3)2(Cl)2(OH)2: A mixture of CDDP and hydrogen peroxide was placed in a 100 mL round-bottom flask and heated at 50 °C for 3 h in the dark with strong magnetic stirring. The mixture was cooled and reprecipitated overnight at 4 °C. The sample was then placed in a 50 mL centrifuge tube and centrifuged at 10,000 rpm for 10 min. The supernatant was discarded to obtain bright yellow Pt(NH3)2(Cl)2(OH)2 powder, referred to as Pt(OH)2 powder. The powder was washed successively with cold water, anhydrous ethanol, and diethyl ether, and centrifuged successively. The powder was then vacuum dried for 24 h. Synthesis of S2 and Pt(NH3)2(Cl)2(OOCCH2CH2COOH)2: Succinic anhydride was added to an anhydrous DMF suspension of Pt(OH)2 and reacted in the dark at 70 °C for 24 h. After removing DMF, the product was dissolved in a small amount of anhydrous methanol, and an appropriate amount of diethyl ether was added. The sample was placed in a 50 mL centrifuge tube and centrifuged at 10,000 rpm for 10 min. The supernatant was discarded. The product was washed with anhydrous methanol and diethyl ether, centrifuged, and the supernatant was discarded. The product was then dried under vacuum for 60 h to obtain Pt(NH3)2(Cl)2(OOCCH2CH2COOH)2, abbreviated as Pt-COOH. Preparation of S3 and TPP-FFK(p)Y: Using 2-chlorotriphenylmethyl chloro resin as a carrier and various amino acids with N-Fmoc side chain protection as reactants, first, 2-chlorotriphenylmethyl chloro resin was soaked in DCM to swell, and the first amino acid Fmoc-D-Tyr(HPO3Bzl)-OH was added. After 2 h, the unreacted parts in the 2-chlorotriphenylmethyl chloro resin were quenched with a blocking liquid of DCM / MeOH / DIPEA with a volume ratio of 17 / 4 / 1. 200 mL of liquid with V(hexahydropyridine):V(DMF)=1:4 was added, and the reaction was carried out for 15 min to remove the Fmoc protecting group and carry out subsequent reactions with D-amino acids. TBTU was used as a condensing agent to couple subsequent amino acids protected by Fmoc. The amino acid coupling and deprotection steps were repeated until the condensation of the last amino acid TPP was completed. 200 mL of 82.5% TFA cleavage reagent was added, and the reaction was carried out for 3 h. After filtration, the filtrate was concentrated by rotary evaporation. 10 mL of ice-cold ether was added to the concentrate to precipitate the precipitate. The precipitate was centrifuged at 5000 rpm for 5 min, and the supernatant was discarded. The reaction was repeated three times. The precipitate was dried under vacuum to obtain TPP-{D-Phe}-{D-Phe}-{D-Lys}-{D-Tyr(H2PO3), i.e., crude TPP-FFK(p)Y. The crude TPP-FFK(p)Y was purified by reverse-phase chromatography and lyophilized to obtain TPP-FFK(p)Y. Synthesis of S4, TPP-FFK(p)Y-Pt(Ⅳ): Pt-COOH, NHS and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were dissolved in anhydrous dimethyl sulfoxide and stirred for 12 h to form the NHS active ester of Pt-COOH. TPP-FFK(p)Y was added, and DIPEA was added until the solution was weakly alkaline. The solution was stirred at room temperature for 24 h, transferred to a dialysis bag with MWCO = 2000 Da, and dialyzed with deionized water for 48 h. Finally, the solution was lyophilized to obtain TPP-FFK(p)Y-Pt(Ⅳ). S5. Preparation of TPP-FFKY-Pt(Ⅳ) HG: TPP-FFK(p)Y-Pt(Ⅳ) was added to PBS at pH = 7.4 until completely dissolved. The solution was treated with ALP and placed at 37℃ for 2 h. TPP-FFK(p)Y-Pt(Ⅳ) underwent dephosphorization under the catalysis of ALP to form a hydrogel-like self-assembled body, TPP-FFKY-Pt(Ⅳ), named TPP-FFKY-Pt(Ⅳ) HG.

2. The method for preparing an enzymatically self-assembled multi-targeted Pt(Ⅳ) prodrug according to claim 1, characterized in that: In S1, the amount of CDDP used is 1000-1200mg, and the amount of hydrogen peroxide used is 20-25mL.

3. The method for preparing an enzymatically self-assembled multi-targeted Pt(Ⅳ) prodrug according to claim 1, characterized in that: In S2, the amount of succinic anhydride is 800-1000 mg, the amount of Pt(OH)2 is 600-800 mg, the amount of DMF is 20 mL, the amount of anhydrous methanol is 5 mL, and the amount of diethyl ether is 20 mL.

4. The method for preparing an enzymatically self-assembled multi-targeted Pt(Ⅳ) prodrug according to claim 1, characterized in that: In S3, the amount of 2-chlorotriphenylmethyl chloride resin is 15-20 g, the amount of DCM is 200-500 mL, the amount of TBTU is 5-10 g, and the amount of TPP is 5-20 g. Amino acids protected by N-Fmoc side chains include Fmoc-D-Tyr(HPO3Bzl)-OH in amounts of 8-12 g, Fmoc-D-Lys(Boc)-OH in amounts of 5-10 g, Fmoc-D-Phe-OH in amounts of 8-10 g, and Fmoc-D-Phe-OH in amounts of 8-10 g.

5. The method for preparing an enzymatically self-assembled multi-targeted Pt(Ⅳ) prodrug according to claim 1, characterized in that: In S4, the amount of Pt-COOH is 100-120 mg, the amount of NHS is 40-60 mg, the amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 60-80 mg, the amount of anhydrous dimethyl sulfoxide is 10-15 mL, and the amount of TPP-FFK(p)Y is 400-600 mg.

6. The method for preparing an enzymatically self-assembled multi-targeted Pt(Ⅳ) prodrug according to claim 1, characterized in that: In S5, the amount of TPP-FFK(p)Y-Pt(Ⅳ) used is 1-2 mg, the amount of PBS with pH = 7.4 used is 1-2 mL, and the amount of ALP used is 1-5 U.

7. The use of an enzymatically self-assembled multi-targeted Pt(Ⅳ) prodrug prepared by the preparation method as described in claim 1 in the preparation of antitumor drugs.

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