Combined medicine for resisting tumor growth and metastasis and application thereof

Abstract

The invention discloses a combined medicine capable of effectively inhibiting tumor growth and metastasis. The combined drug for resisting tumor growth and metastasis comprises a Rho / ROCK inhibitor and a mitochondrial injury drug, wherein the mitochondrial injury drug is a chemotherapeutic drug coupled with a mitochondrial targeting ligand. The Rhoo / ROCK inhibitor destroys a cytoskeleton structure and blocks cytoskeleton-dependent mitochondrial transfer by inhibiting an Rhoo / ROCK signal channel, and the mitochondrial injury drug can selectively deliver a chemotherapeutic drug to mitochondria to cause mitochondrial injury. Compared with a single drug, the combined drug enhances damage to cytoskeletons and mitochondria, cuts off dynamic energy supply of the mitochondria to the cytoskeletons, makes up for the deficiency of tumor growth inhibition of the Rho / ROCK inhibitor, and has a good curative effect on tumor growth and metastasis resistance.

Description

Technical Field

[0001] The present invention relates to a combined drug and its application in treating tumor growth and metastasis, and specifically to the preparation and application of a combined drug of a Rho / ROCK inhibitor and a mitochondrial damage drug, belonging to the field of medical technology. Background Art

[0002] The malignant growth and distant metastasis of breast cancer in situ tumors are the main causes of poor prognosis and death. The metastatic cascade of tumors follows the order of initiation, circulation and colonization. Controlling the root of tumors and blocking the shedding of tumor cells from the primary site and their circulation in the blood are the main focuses of cancer treatment.

[0003] The cytoskeleton is the "weapon" of active attack and the "armor" of passive defense of cells. It reshapes its structure to dominate tumor metastasis by activating the Rho / ROCK signaling pathway. On the one hand, as a "weapon" of active attack of tumor cells, the cytoskeleton can promote cells to form angiogenic mimicry for deep oxygen and nutrient uptake in tumors, and promote tumor cells to enter the blood circulation through angiogenic mimicry; at the same time, after the cytoskeleton is remodeled, the epithelial-mesenchymal transition process is initiated, accelerating cell migration and invasion. On the other hand, as the "armor" of passive defense of tumor cells, the cytoskeleton can form tunnel nanotubes, provide a dynamic energy source for cytoskeleton activities, and avoid endogenous apoptosis caused by mitochondrial damage; in the process of detaching from the original tumor and distant metastasis, it can arm cells with a hard protective shield to resist the damage of external mechanical pressure and escape anoikis. Mitochondria are the "batteries" of cells. When cells are in a highly energy-consuming state due to migration, the cytoskeleton can form tunnel nanotubes to dynamically increase the energy source and ensure that migration is carried out quickly and efficiently.

[0004] Currently, Rho / ROCK inhibitors have been used in the treatment of tumor metastasis, but they have little effect on the proliferation of in situ tumor cells, which is not conducive to the long-term treatment of tumor metastasis. Since the root of tumor metastasis still exists, it can still cause metastasis in the later stage of tumor growth. Whether the combined use of Rho / ROCK inhibitors and mitochondrial damage drugs can make up for this defect, inhibit the proliferation, initiation and circulation of tumor cells, and exert a powerful effect of killing tumors and inhibiting metastasis has not been reported. Summary of the invention

[0005] In order to solve the problem that Rho / ROCK inhibitors have poor tumor killing efficacy and are prone to tumor metastasis, the present invention creatively combines Rho / ROCK inhibitors with mitochondrial damage drugs. While playing a good anti-metastatic role, Rho / ROCK inhibitors inhibit mitochondrial transfer through the cytoskeleton, cut off the energy supply of cytoskeleton activities, and cause cytoskeleton collapse and mitochondrial damage. Compared with a single drug, the combined drug achieves continuous inhibition of the entire process of tumor metastasis and significantly enhances the anti-tumor growth and metastasis effects.

[0006] One of the purposes of the present invention is to make up for the shortcomings of Rho / ROCK inhibitors in the treatment of tumor metastasis. By using mitochondrial damaging drugs in combination, the dynamic energy source of the cytoskeleton is cut off, the cytoskeleton network disorder is promoted, the mitochondrial damage is enhanced, and the anti-tumor growth and metastasis efficacy is improved.

[0007] Another object of the present invention is to provide a combined drug of a Rho / ROCK inhibitor and a mitochondrial damaging drug for use in preventing tumor growth and metastasis.

[0008] The purpose of the present invention is achieved through the following technical solution: a combined drug for preventing tumor growth and metastasis and its application, comprising a Rho / ROCK inhibitor and a mitochondrial damage drug as active ingredients. The mitochondrial damage drug is a chemotherapy drug coupled with a mitochondrial targeting ligand.

[0009] The combined drug for inhibiting tumor growth and metastasis may also contain at least one pharmaceutically acceptable carrier.

[0010] The combined drug for preventing tumor growth and metastasis can be further prepared into various forms such as injections, and drugs of various dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0011] The Rho / ROCK inhibitor is at least one of fasudil, hydroxyfasudil, besudil, rosudil, Y-27632, and Y-33075, preferably fasudil.

[0012] The mitochondrial targeting ligand is at least one of the small molecule triphenylphosphine, (E)-4-(2-(1 hydrogen-indol-3-yl)vinyl)-1-methylpyridine-1-salt (F16), guanidine / biguanide, glycyrrhetinic acid, or polypeptide sequences CFxrFxKFxrFxK, MLSLRQSIRFFK, HD-Arg-Dmt-Lys-Phe-NH2 (SS-31), preferably triphenylphosphine.

[0013] Specifically, the mitochondrial targeting ligand carries the coupled chemotherapeutic drug to the mitochondria through electrostatic interaction or receptor-ligand binding with the negatively charged mitochondrial inner membrane, thereby achieving mitochondrial damage.

[0014] The chemotherapy drug is at least one of camptothecin, doxorubicin, paclitaxel and lonidamine, preferably camptothecin.

[0015] The tumors include breast cancer, melanoma, liver cancer, lymphoma, lung cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, esophageal cancer, gastric cancer and prostate cancer.

[0016] The present invention is to simultaneously act the Rho / ROCK inhibitor and the mitochondrial damage drug on the tumor, or first treat with the Rho / ROCK inhibitor and then administer the mitochondrial damage drug, or first treat with the mitochondrial damage drug and then administer the Rho / ROCK inhibitor.

[0017] The combined use of the Rho / ROCK inhibitor and the mitochondrial damaging drug of the present invention can damage the cytoskeleton and mitochondria at the same time, effectively cut off the dynamic energy supply of mitochondria to the cytoskeleton, amplify the metastasis inhibition effect of the Rho / ROCK inhibitor, and significantly inhibit tumor growth and metastasis.

[0018] Compared with the prior art, the present invention has the following advantages and effects:

[0019] 1. The present invention provides a combination drug of a Rho / ROCK inhibitor and a mitochondrial damaging drug to achieve all-round inhibition of tumor growth and metastasis, involving the use of a Rho / ROCK inhibitor and a mitochondrial damaging drug.

[0020] 2. The present invention can destroy the cytoskeleton structure and damage mitochondria, cut off the dynamic energy supply of mitochondria to the cytoskeleton, amplify cell apoptosis, inhibit tumor cell proliferation in situ, and the initiation and circulation process of tumor metastasis.

[0021] 3. Compared with a single Rho / ROCK inhibitor, the combined drug provided by the present invention can enhance the anti-metastatic efficacy of the Rho / ROCK inhibitor and make up for its deficiency in killing tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Represents liposome active targeting peptide DSPE-PEG 2000 -Synthesis and characterization of KC27.

[0023] Figure 2 Diagram showing the synthesis and characterization of a mitochondrial damaging drug (triphenylphosphine-modified camptothecin).

[0024] Figure 3 A diagram showing the preparation and characterization of liposomes encapsulating Rho / ROCK inhibitors and mitochondrial damage drugs.

[0025] Figure 4 A diagram showing the effect of drug-loaded liposomes on the cytoskeleton structure.

[0026] Figure 5 A diagram showing the effect of drug-loaded liposomes on mitochondrial functional impairment.

[0027] Figure 6 A graph showing the effect of drug-loaded liposomes on the proliferation and metastasis of triple-negative breast cancer 4T1 cells.

[0028] Figure 7 Graphs showing the effects of drug-loaded liposomes on the inhibition of angiogenic mimicry in vitro and in vivo.

[0029] Figure 8 A graph showing the effect of drug-loaded liposomes on triple-negative breast cancer 4T1 cells in blood circulation.

[0030] Fig. 9 Graph showing the effect of drug-loaded liposomes on the inhibition of tunneling nanotube-dependent mitochondrial translocation.

[0031] Fig.10 A diagram showing the anti-tumor growth and metastasis therapeutic effects of drug-loaded liposomes in 4T1 orthotopic breast cancer model mice. DETAILED DESCRIPTION

[0032] The present invention will be described in further detail below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the embodiments of the present invention are not limited to the scope of the embodiments described. Moreover, those skilled in the art may perform equivalent substitutions, combinations, improvements or modifications to the present invention based on the description of the present invention, but these will all be included within the scope of the present invention.

[0033] Various commonly used chemical reagents used in the examples are all commercially available products.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0035] Example 1 DSPE-PEG 2000 -Synthesis and characterization of KC27.

[0036] The KC27 peptide (KE5NE4PLGLAGR9C) was combined with DSPE-PEG 2000 -Mal was dissolved in PBS (pH 7.4) at a molar ratio of 1.2:1 and stirred at 25°C for 48 h under argon protection. The reaction solution was dialyzed for 24 h (dialysis bag molecular weight 3500 Da) to remove unreacted KC27 peptide and DSPE-PEG 2000 -Mal. The dialysate was freeze-dried and stored at -20°C. The successful synthesis of the product was verified by hydrogen nuclear magnetic resonance spectroscopy.

[0037] Figure 1 The nuclear magnetic resonance spectroscopy results of B showed that the product had characteristic peaks of guanidine and peptide bond hydrogen between the displacement values ​​of 7-8, while the reactant DSPE-PEG 2000 The characteristic peak of the maleimide group of -Mal (shift value 8) disappeared, proving that KC27 was successfully connected to DSPE-PEG 2000 -Mal.

[0038] Example 2 Synthesis and characterization of triphenylphosphine-modified camptothecin (TPP-CPT).

[0039] Camptothecin (0.2mmol), triphenylphosphine (0.4mmol), (1-ethyl-3(3-dimethylpropylamine)carbodiimide) (1.2mmol), and 4-dimethylaminopyridine (0.8mmol) were dissolved in dichloromethane and reacted at room temperature in the dark for 24h. The reaction solution was extracted with saturated ammonium chloride solution and purified by silica gel column. Gradient elution was performed with dichloromethane and methanol. The product was obtained by rotary evaporation. The product structure was verified by mass spectrometry and nuclear magnetic resonance hydrogen spectrum, and the product purity was tested by high performance liquid chromatography (detection wavelength was 358nm, and the mobile phase was acetonitrile (containing 0.1% trifluoroacetic acid): water (containing 0.1% trifluoroacetic acid) = 45:55, v / v).

[0040] Figure 2 The nuclear magnetic resonance hydrogen spectrum and mass spectrometry results of BC showed that the characteristic peaks and molecular weight were consistent with the target product, proving that TPP-CPT was successfully synthesized. Figure 2 The HPLC chromatogram of D showed that the product was a single peak without any obvious impurities.

[0041] Example 3 Preparation and characterization of liposomes encapsulating Rho / ROCK inhibitors and mitochondrial damage drugs.

[0042] Liposomes loaded with Rho / ROCK inhibitors (KF Csk @LIP) was prepared by the ammonium sulfate gradient method, distearoylphosphatidylcholine (DSPC), cholesterol, DSPE-PEG 2000 -Ome and DSPE-PEG 2000 -KC27 was dissolved in a chloroform-methanol mixed solvent at a molar ratio of 65:33.24:1.71:0.085, and the organic solvent was removed by rotary evaporation to obtain a uniformly dispersed film. It was hydrated with 2 mL of ammonium sulfate aqueous solution (50 mM, pH 3.0), and probe ultrasound (120 W, 8 min) was used to obtain blank liposomes. Free ammonium sulfate was removed by G-50 dextran gel column, and 3 mg of fasudil (F Csk ), incubated at 60°C for 30 min. Mito@LIP) was obtained by thin film dispersion method, TPP-CPT (T Mito ), DSPC, cholesterol, DSPE-PEG 2000 -Ome and DSPE-PEG 2000 -KC27 was dissolved in a chloroform-methanol mixed solvent at a molar ratio of 61.28:30.64:6.38:1.61:0.08, and the organic solvent was removed by rotary evaporation to obtain a uniformly dispersed film, which was hydrated with 2mL RO water and subjected to probe ultrasound (120W, 8min). The above two liposomes were passed through a G-50 dextran gel column to remove the unencapsulated drugs and obtain pure liposomes. The particle size, polydispersity index PDI and potential of the liposomes were measured by a particle size analyzer, the morphology of the liposomes was observed by transmission electron microscopy, and the encapsulation efficiency of the two drugs (λF Csk =275nm,λT Mito =365nm).

[0043] Figure 3 AB showed that both liposomes had a round double-layer membrane structure, with a particle size of about 151 nm, a polydispersity coefficient of <0.3, a uniform distribution, potentials of -18.8 mV and -39.4 mV respectively, and encapsulation rates of more than 47%.

[0044] Example 4 Effects of drug-loaded liposomes on cytoskeleton structure.

[0045] (1) Observe the structure of microfilaments and microtubules using laser confocal microscopy.

[0046] 4T1 cells were seeded at a density of 10,000 cells / well in a 12-well plate pre-covered with a cover glass and placed in an incubator (5% CO2, 37°C) for overnight culture. Csk @LIP, KT Mito @LIP or Com(KF Csk @LIP and KT Mito @LIP physical mixture, F Csk :T Mito =2:1, T Mito =5 μg / mL). After 2 h, the old medium was removed, the cells were washed once with PBS, fixed with 4% paraformaldehyde for 15 min, washed three times with PBS, permeabilized with 0.1% Triton for 15 min, washed three times with PBS, stained with rhodamine-labeled phalloidin and microtubule antibody, washed three times with PBS, stained with DAPI for 3 min, washed three times with PBS, and then sealed and observed under a laser confocal microscope.

[0047] (2) Western Blot was used to investigate the expression of intermediate filament proteins.

[0048] 4T1 cells were cultured at 1×10 5Each well was inoculated into a 6-well plate and placed in an incubator (5% CO2, 37°C) for 24 h. Csk @LIP, KT Mito @LIP or Com (same as above, T Mito =3μg / mL). After 24h, the old culture medium was removed, and the cells were washed with PBS 2-3 times. The total cell protein was extracted with RIPA lysis buffer in an ice bath, and transferred to an EP tube. The probe was ultrasonicated (on 5s, off 5s, 2min) until there was a slight opalescence. The precipitate was removed by centrifugation (10000rpm, 10min), and the supernatant was collected for BCA quantification. 5×SDS-PAGE loading buffer was added and boiled at 100℃ for 10min. 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used for separation. The protein loading amount was unified to 30μg, and the membrane was transferred to a 0.45μm PVDF membrane, blocked with skim milk for 2h, incubated with primary antibodies for vimentin and internal reference protein (GAPDH) at 4℃ overnight, washed 3 times with TBST, incubated with secondary antibodies at 37℃ for 1h, washed 3 times with TBST, and developed on a gel imaging system.

[0049] like Figure 4 AC shows KF loaded with Rho / ROCK inhibitor Csk @LIP has a significant inhibitory effect on the polymerization of actin and tubulin and the expression of intermediate filament proteins, while the combined use of Rho / ROCK inhibitors and mitochondrial damage drugs maximizes the destruction of the cytoskeleton structure, thereby weakening the cell's active offensive and passive defensive capabilities.

[0050] Example 5: Damage to mitochondrial function caused by drug-loaded liposomes.

[0051] (1) Detection of intracellular reactive oxygen species (ROS).

[0052] 4T1 cells were cultured at 5 × 10 4 Each well was inoculated into a 12-well plate and placed in an incubator (5% CO2, 37°C) for 24 h. Csk @LIP, KT Mito @LIP or Com (same as above, T Mito =10 μg / mL). After 12 h, the old medium was removed, the cells were washed 2-3 times with PBS, the cells were digested, the reactive oxygen probe DCFH-DA was added, the cells were incubated at 37°C for 20 min, centrifuged (3000 rpm, 5 min), washed 3 times with PBS, and the cell fluorescence intensity was detected by flow cytometry.

[0053] (2) Mitochondrial membrane potential measurement.

[0054] 4T1 cells were cultured at 5 × 10 4Each well was inoculated into a 12-well plate and placed in an incubator (5% CO2, 37°C) for 24 h. Csk @LIP, KT Mito @LIP or Com (same as above, T Mito =10 μg / mL). After 12 hours, the old medium was removed, the cells were washed 2-3 times with PBS, digested, stained with mitochondrial membrane potential detection kit, and detected by flow cytometry. The ratio of JC-1 aggregates to monomers reflects the membrane potential. The higher the ratio, the higher the membrane potential.

[0055] (3) ATP level detection.

[0056] 4T1 cells were cultured at 5 × 10 3 Each well was inoculated into a 96-well plate and placed in an incubator (5% CO2, 37°C) for 24 h. Csk @LIP, KT Mito @LIP or Com (same as above, T Mito =10 μg / mL). After 12 h, the old medium was removed, and the cells were washed 2-3 times with PBS. The ATP level was determined using an ATP detection kit, and the bioluminescence intensity was detected using a multifunctional microplate reader. The cell number was corrected using BCA.

[0057] Figure 5 AC showed that destroying the cytoskeleton could reduce mitochondrial membrane potential and ATP level to a certain extent, while KT Mito @LIP can directly damage mitochondria, which is manifested by a significant increase in ROS content and a significant decrease in mitochondrial membrane potential and ATP levels. The combined use of the two can greatly increase the degree of mitochondrial damage and cut off the power source of the cytoskeleton.

[0058] Example 6 Drug-loaded liposomes inhibit the proliferation and metastasis of triple-negative breast cancer 4T1 cells.

[0059] (1) The killing effect of drug-loaded liposomes on 4T1 cells.

[0060] 4T1 cells were cultured at 5 × 10 3 Each well was inoculated into a 96-well plate and placed in an incubator (5% CO2, 37°C) for 24 h. Mito , F Csk , KF Csk @LIP, KT Mito @LIP. After 24 hours, the old culture medium was removed, MTT solution (0.5 mg / mL) was added and incubated for 4 hours, dimethyl sulfoxide (DMSO) was used to dissolve the generated formazan crystals, and the cells were shaken on a shaker for 20 minutes. The UV absorption at 490 nm was detected using a multifunctional microplate reader.

[0061] 4T1 cells were cultured at 2 × 10 3 Each well was inoculated into a 96-well plate pre-coated with agarose. When the tumor spheres grew to a diameter of 100 μm, KF Csk @LIP, KT Mito @LIP or Com (same as above, T Mito =3 μg / mL), and the size of tumor spheres was monitored daily using an optical microscope to draw a growth curve.

[0062] (2) The inhibitory effect of drug-loaded liposomes on the migration and invasion of 4T1 cells.

[0063] For cell migration, cells were plated at 1 × 10 5 100 μL / well were inoculated in the transwell chamber, 600 μL serum-free culture medium was added to the lower chamber, and the cells were placed in an incubator (5% CO2, 37°C) and cultured for 8 h until the cells adhered to the wall. The old culture medium was removed and KF Csk @LIP, KT Mito @LIP or Com (same as above, T Mito =1μg / mL), put in an incubator (5% CO2, 37°C) and culture for 24h, aspirate the upper chamber solution of the transwell, wash 3 times with PBS, fix with 4% paraformaldehyde for 15min, wash 3 times with PBS, stain with crystal violet (1mg / mL) for 15min, wash 5 times with PBS, gently wipe the upper chamber cells with a cotton swab, observe the cells migrating to the lower chamber under an optical microscope and take pictures. Dissolve the crystal violet with 33% glacial acetic acid solution, and use a multifunctional microplate reader to detect the ultraviolet absorption at 570nm.

[0064] For cell invasion, cells were plated at 1 × 10 5 100 μL / well were inoculated into a transwell chamber pre-coated with matrix gel, 600 μL serum-free culture medium was added to the lower chamber, and the cells were placed in an incubator (5% CO2, 37°C) for 8 h until the cells adhered to the wall. The old culture medium was removed and KF Csk @LIP, KT Mito @LIP or Com (same as above, T Mito =1μg / mL), put into incubator (5% CO2, 37℃) for 24h, aspirate the upper chamber solution of transwell, wash 3 times with PBS, fix with 4% paraformaldehyde for 15min, wash 5 times with PBS, stain with crystal violet (1mg / mL) for 15min, wash 3 times with PBS, gently wipe the upper chamber cells with cotton swab, observe the cells migrated to the lower chamber under optical microscope and take pictures. Dissolve crystal violet with 33% glacial acetic acid solution, and use multifunctional microplate reader to detect ultraviolet absorption at 570nm.

[0065] The results are as follows Figure 6AC shows KF loaded with Rho / ROCK inhibitor Csk @LIP IC 50 The value is higher than that of KT Mito @LIP, the effect of inhibiting the growth of tumor spheres is not as good as that of mitochondrial damage drugs, which reveals the weakness of Rho / ROCK inhibitors in killing in situ tumor cells. The combination drug group can quickly ablate tumor spheres, showing a strong ability to inhibit the proliferation of tumor cells in situ.

[0066] Figure 6 DE shows that KF loaded with Rho / ROCK inhibitor Csk @LIP has a good effect in anti-cell migration and invasion, but after combined use with mitochondrial damaging drugs, the cell migration and invasion rates were significantly reduced, proving that cutting off the dynamic energy supply of mitochondria can indeed enhance the anti-metastasis effect and reduce the ability of cells to actively attack, thereby killing the in situ tumor and inhibiting the initial stage of tumor metastasis.

[0067] Example 7 Inhibitory effect of drug-loaded liposomes on angiogenic mimicry in vivo and in vitro.

[0068] (1) Drug-loaded liposomes inhibit the formation of vasculogenic mimicry in vitro.

[0069] 4T1 cells were cultured at 2 × 10 4 Each well was inoculated in a 96-well plate pre-coated with matrix gel, and KF Csk @LIP, KT Mito @LIP or Com (same as above, T Mito =10 μg / mL), and cultured in an incubator (5% CO2, 37°C) for 4 h. The tubule formation was observed under an optical microscope, and the number of meshes, nodes and other indicators were analyzed using Image J.

[0070] (2) Drug-loaded liposomes inhibit the formation of vasculogenic mimicry in vivo.

[0071] 4T1 cells (1×10 5 100 μL PBS) was inoculated into the third pair of mammary gland pads on the left side of female BALB / c mice as a breast cancer model. 3 The mice were randomly divided into groups and KF was administered intravenously Csk @LIP, KT Mito @LIP or Com(T Mito : F Csk =1:2,T Mito=3mg / kg), administered once every two days, for a total of 3 times. Half of the mice were killed on the 6th and 8th days after administration, and tumor tissues were taken, homogenized with PBS, centrifuged at 10000rpm for 10min, and the supernatant was taken. The levels of glucose, cholesterol, and protein were detected using a biochemical kit; at the same time, tumor tissues were double-stained with PAS and CD31 to observe the formation of angiogenic mimicry tubules, and PAS positive / CD31 negative indicated angiogenic mimicry.

[0072] Figure 7 The in vitro results of AB showed that KF loaded with Rho / ROCK inhibitor Csk @LIP can effectively reduce the formation of vasculogenic mimicry, and the combined drug group can almost destroy the tubular structure; in vivo results ( Figure 7 CE) also showed that angiogenic mimicry can supply nutrients as a blood vessel. After destroying the cytoskeleton, the formation of angiogenic mimicry is reduced, and the supply of nutrients dependent on this pathway is also significantly reduced, thereby blocking the "active attack" of tumor cells to the blood circulation through angiogenic mimicry.

[0073] Example 8 Drug-loaded liposomes inhibit the survival of 4T1 cells in blood circulation.

[0074] (1) Anoikis detection.

[0075] 4T1 cells were cultured at 5 × 10 4 Each well was inoculated in a non-adhesive 12-well plate and KF Csk @LIP, KT Mito @LIP or Com (same as above, T Mito =1μg / mL), placed in an incubator (5% CO2, 37°C) and cultured for 24h, and the suspended cells were collected by centrifugation (3000rpm, 5min). The cells were stained with AnnexinV-FITC apoptosis kit, washed 3 times with PBS, and the apoptosis ratio was recorded by flow cytometry; live / dead cells were stained with Calcein-AM / PI double staining kit, washed 3 times with PBS, and the cell suspension was dropped onto a glass slide, sealed and observed under a confocal microscope; WB was used to detect anoikis-related proteins FAK, p-FAK, Integrin-β1, Bcl-2, Caveolin-1, and the method was the same as in Example 4 (2).

[0076] (2) In vitro simulation experiment of circulating tumor cells (CTC).

[0077] Digest 4T1-EGFP cells and take 1.5×10 6 Cells and KF Csk @LIP, KT Mito @LIP or Com (same as above, T Mito=5 μg / mL) and circulated in a circulation pump for 2 h (50% fetal bovine serum, 37°C, 15 mL / min). The proportion of surviving 4T1-EGFP cells was detected by flow cytometry.

[0078] Figure 8 AC showed that when cells are in an unanchored state, destroying the cytoskeleton can greatly reduce the passive defense ability of cells. Combined with KT Mito @LIP can further aggravate the anoikis of cells, which is manifested by an increase in the proportion of anoikis and the upregulation of anoikis-related protein expression. At the same time, when cells are in a cycling state, the destruction of the cytoskeleton and mitochondria can significantly reduce the cell survival rate ( Figure 8 D), proving that cells rely heavily on cytoskeleton defense and mitochondrial energy supply when migrating in the blood circulation. The above results show that combined drugs can effectively inhibit the circulation stage of tumor cell metastasis.

[0079] Example 9 Drug-loaded liposomes inhibit tunneling nanotube-dependent mitochondrial translocation.

[0080] (1) The inhibitory effect of drug-loaded liposomes on mitochondrial translocation in adherent cells.

[0081] For qualitative observation under confocal microscopy, 4T1 cells were plated at 1 × 10 4 The cells were seeded at a density of 100 / well in a 12-well plate covered with a cover glass and placed in an incubator (5% CO2, 37°C) for overnight culture. Melatonin (5 μg / mL) was pre-incubated for 2 h, and KF Csk @LIP, KT Mito @LIP or Com(T Mito =5 μg / mL). After 2 h, the old medium was removed, the cells were washed once with PBS, incubated with mitochondrial deep red fluorescent probe (250 mM) for 30 min, washed 3 times with PBS, fixed with 4% paraformaldehyde for 15 min, washed 3 times with PBS, permeabilized with 0.1% Triton for 15 min, washed 3 times with PBS, incubated with rhodamine-labeled phalloidin for 20 min, washed 3 times with PBS, stained with DAPI for 3 min, washed 3 times with PBS, and sealed. The transfer of mitochondria in tunneling nanotubes was observed under a laser confocal microscope.

[0082] For flow cytometry quantitative analysis, 4T1-EGFP cells were plated at 5 × 10 4 Each well was inoculated into a 6-well plate and placed in an incubator (5% CO2, 37°C) for 24 h. Csk @LIP, KT Mito @LIP or Com(T Mito=5μg / mL), and the cells were digested 4 hours later as mitochondrial recipient cells. 4T1 cells were digested, incubated with mitochondrial deep red fluorescent probe (250mM) for 30 minutes, washed 3 times with PBS, and used as mitochondrial donor cells. The two cells were mixed and cultured for 12 hours, and mitochondrial transfer was detected by flow cytometry. (2) Inhibitory effect of drug-loaded liposomes on mitochondrial transfer of circulating tumor cells.

[0083] 4T1-EGFP cells were cultured at 5×10 4 Each well was inoculated into a 6-well plate and placed in an incubator (5% CO2, 37°C) for 24 h. Csk @LIP, KT Mito @LIP or Com(T Mito =5μg / mL), and the cells were digested after 4 hours as mitochondrial recipient cells. 4T1 cells were digested, incubated with mitochondrial deep red fluorescent probe (250mM) for 30 minutes, washed 3 times with PBS, and used as mitochondrial donor cells. The two cells were mixed and circulated in a circulation pump for 2 hours (50% fetal bovine serum, 37°C, 15mL / min), and mitochondrial transfer was detected using a flow cytometer.

[0084] Fig. 9 AD shows KF Csk @LIP can effectively reduce mitochondrial transfer in static adherent cells and dynamic circulating tumor cells, proving that the dynamic energy acquisition of the cytoskeleton depends on the tunnel nanotubes formed by itself. Destroying this structure cuts off the energy supply, thereby blocking the active attack and passive defense of the cell. Example 10 Anti-tumor growth and metastasis effect of drug-loaded liposomes in 4T1 orthotopic breast cancer model mice.

[0085] 4T1 cells (1×10 5 100 μL PBS) was inoculated into the third pair of mammary gland pads on the left side of female BALB / c mice as a breast cancer model. 3 The mice were randomly divided into groups and KF was administered intravenously Csk @LIP, KT Mito @LIP or Com(T Mito : F Csk =1:2,T Mito =3 mg / kg), administered once every 3 days for a total of 6 times. The weight and tumor volume of mice were monitored every two days (V = 0.5 × a × b 2, a is the tumor length, b is the tumor width, measured with a vernier caliper). On the 18th day after administration, the mice were killed, the tumors and organs were removed, part of the tumor tissue was homogenized, and WB samples were prepared according to Example 4 (2) to examine the expression of epithelial-mesenchymal transition proteins E-cadherin and vimentin. After the lungs were washed, they were fixed in Bouin's stain, and the number of lung metastatic nodules was counted.

[0086] The results are as follows Fig.10 AE shows KF loaded with Rho / ROCK inhibitor Csk @LIP has a significant effect in blocking the epithelial-mesenchymal transition process and inhibiting lung metastasis, but its killing effect on in situ tumors is not as good as KT containing mitochondrial damage drugs. Mito @LIP, tumor inhibition rate is lower than KT Mito @LIP is more than 20%, but the combined drug group can significantly increase the tumor inhibition rate to more than 80%, and the number of lung metastatic nodules is only 1-2, which proves that on the basis of destroying the cytoskeleton, by damaging mitochondria and cutting off the dynamic energy supply, it can indeed reduce the cell's active attack and passive defense capabilities, effectively kill in situ tumor cells, and inhibit the initiation and circulation stages of tumor metastasis.

Claims

1. A combined drug for inhibiting tumor growth and metastasis, characterized in that: The combined drug comprises a Rho / ROCK inhibitor and a mitochondrial damage drug, and the combined drug is used for anti-tumor growth and metastasis treatment.

2. The anti-tumor growth and metastasis combined drug according to claim 1, characterized in that: The Rho / ROCK inhibitor is at least one of fasudil, hydroxyfasudil, besudil, rosudil, Y-27632, and Y-33075.

3. The anti-tumor growth and metastasis combined drug according to claim 1, characterized in that: The mitochondrial targeting ligand is at least one of small molecule triphenylphosphine, (E)-4-(2-(1 hydrogen-indol-3-yl)vinyl)-1-methylpyridine-1-salt (F16), guanidine / biguanide, glycyrrhetinic acid, or polypeptide sequences CFxrFxKFxrFxK, MLSLRQSIRFFK, HD-Arg-Dmt-Lys-Phe-NH2 (SS-31).

4. The combined anti-tumor growth and metastasis drug according to claim 1, characterized in that: The chemotherapy drug is at least one of camptothecin, doxorubicin, paclitaxel and lonidamine.

5. The combined drug for inhibiting tumor growth and metastasis according to claim 1, characterized in that: Rho / ROCK inhibitors and mitochondrial damage drugs are used as active ingredients.

6. The combined drug for preventing tumor growth and metastasis according to claims 2-4, characterized in that: The Rho / ROCK inhibitor and the mitochondrial damage drug are prepared into liposomes respectively, and the weight of the active components carried by the liposomes accounts for 0.1%-90% of the total weight of the liposomes.

7. The combined drug for inhibiting tumor growth and metastasis according to claim 1, characterized in that: The tumors include breast cancer, melanoma, liver cancer, lymphoma, lung cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, esophageal cancer, gastric cancer and prostate cancer.

8. The liposome according to claim 6, characterized in that: The liposomes are further prepared into various dosage forms such as injections, and drugs in various dosage forms can be prepared according to conventional methods in the pharmaceutical field.

9. The combined drug for inhibiting tumor growth and metastasis according to claim 1 may further contain at least one pharmaceutically acceptable carrier.

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