阅读说明：本技术 米铂脂质体在抗耐药性肿瘤的应用 (Application of miboplatin liposome in drug-resistant tumor resistance ) 是由 赵午莉 邵荣光 夏桂民 王梦燕 王晓葳 邱雨涵 于 2021-06-17 设计创作，主要内容包括：本发明属于药物领域,涉及米铂脂质体在抗耐药性中的应用。本发明发现米铂脂质体能够逆转,肿瘤细胞对肿瘤药物的天然和获得性耐药。进一步发现其对肿瘤复发根源的肿瘤干细胞具有显著杀伤作用,抑制转移和肿瘤复发,对肿瘤的彻底治疗提供了一个很好的方向,造益于更多肿瘤患者,具有重要的临床应用价值。(The invention belongs to the field of medicines, and relates to application of a miboplatin liposome in drug resistance. The invention discovers that the miboplatin liposome can reverse, and the tumor cells have natural and acquired drug resistance to tumor drugs. Further, the tumor stem cells which are the root of the tumor recurrence have obvious killing effect, the metastasis and the tumor recurrence are inhibited, a good direction is provided for the thorough treatment of the tumor, more tumor patients are benefited, and the clinical application value is important.)

1. Application of miboplatin liposome in preparing anti-drug-resistant tumor medicine is provided.

2. The use of claim 1, wherein the tumor comprises: pancreatic cancer, and carcinoma of large intestine.

3. The use of claim 1, wherein the large intestine cancer cell line: human colorectal cancer cells HCT8 and HT 29; human pancreatic cancer cells: AsPC-1, BxPC-3, MIA-PaCa-2, PANC-1, SW1990, SU.86.86;

4. the use according to claim 1, wherein the human Oselli resistant colon cancer cell line HCT 8L; human cisplatin-resistant pancreatic cancer cell line PATU 8988/DDP.

5. The use of claim 1, wherein the miriplatin liposomes reverse oxaliplatin resistance (acquired resistance) of colorectal cancer cells HCT 8; the miboplatin liposome reverses the natural resistance of pancreatic cancer cells MIA-PaCa-2 and PANC-1 to gemcitabine; the miboplatin liposome reverses the cells HCT8L of the Osxal-resistant colorectal cancer; and resistance to PATU8988/DDP in cis-platin-resistant pancreatic cancer cells.

6. The use as claimed in claim 1, wherein the miboplatin liposome has a killing effect on 3D-tumor stem cell spheres cultured from HCT8 cells of the large intestine cancer.

7. The use of claim 1, wherein the miriplatin liposomes inhibit the ability of colon cancer cells to form soft agar colonies.

8. The use of claim 1, wherein the miboplatin liposome increases the expression of the mesothelin marker ZO1, E-cadherin and simultaneously reduces the expression level of the epithelin marker N-cadherin, Snail, Slug, indicating that the miboplatin liposome inhibits EMT transformation, i.e. inhibits tumor cell metastasis.

9. The use of claim 1, wherein the miboplatin liposome enters the cancer cell primarily through a caveolin-mediated endocytosis pathway, and wherein the macropinocytosis pathway assists in the entry of the miboplatin liposome.

10. The use of claim 1, wherein the miriplatin liposome causes massive vacuolization of endoplasmic reticulum and mitochondria within tumor cells, and vacuolization of mitochondria is observed to be contained within the autophagy vesicle, suggesting that autophagy occurs in the mitochondria.

11. The use of claim 1, wherein the miriplatin liposome enters the mitochondria of tumor cells to exert an antitumor effect by inducing an antitumor mechanism of autophagy and endoplasmic reticulum damage caused by mitochondrial DNA replication dysfunction.

12. Use according to claim 1, characterized in that: the medicine contains acceptable auxiliary materials and other effective components which play a compatible and synergistic effect.

Technical Field

The invention belongs to the field of medicines, and relates to application of a miboplatin liposome in drug resistance.

Background

The drug therapy plays an important role in the anti-tumor therapy, but after a tumor patient takes the drug for a period of time, the drug resistance (including natural and acquired) to the tumor drug appears, so that many patients are not sensitive to the tumor drug, and the tumor therapy is limited.

Tumor stem cells are a group of cells that are present in tumor tissues, have high tumorigenicity and are divergently differentiated, and are the main causes of tumor drug resistance, recurrence and metastasis. Is not sensitive to common tumor drugs, often survives after the treatment of the conventional anti-tumor drugs, and can return to the proliferation cycle again under certain conditions, resulting in tumor recurrence. The combination of the treatment aiming at the tumor stem cells and the conventional chemotherapy drugs is the key for thoroughly curing the tumor and improving the prognosis of patients. At present, no specific anti-tumor stem cell medicine is reported.

Platinum drugs have been shown to play an important role in anti-tumor therapy, but toxicity limits their use. Oxaliplatin, as a third-generation platinum-based drug with gradually improved properties, has an important role in colorectal cancer and pancreatic cancer. In the us national comprehensive cancer guideline of 2019 edition, it is listed as the first line of pancreatic cancer in the "FOLFIRINOX" combination. Oxaliplatin is involved in the 'FOLFOX' treatment scheme used in compatibility and also serves as a first-line treatment scheme for colorectal cancer treatment, becomes the gold standard of chemotherapy for advanced colorectal cancer, and shows a remarkable treatment effect in clinical use. The non-specificity of the in vivo distribution of the compound causes the toxicity of different tissues, and the toxicity is more obvious to be neurotoxicity and myelosuppressive toxicity.

The miboplatin is an oxaliplatin-based upgraded medicine, and an antitumor active intermediate formed after metabolism is the same as the antitumor active ingredient of the oxaliplatin, so that the miboplatin has the advantages of wide antitumor spectrum, strong antitumor activity and low adverse reaction. However, the water solubility is poor, the systemic administration cannot be carried out, and only the hepatic artery administration can be carried out after the poppy seed oil which is expensive and not easy to obtain is dissolved. The limitation of the administration mode and the difficulty of solvent acquisition limit the treatment of other tumors and the administration route of the miboplatin. There is no report that miboplatin can be used for resisting the antitumor activity of pancreatic cancer and colorectal cancer through systemic administration.

In the treatment of tumors, the combination is a very important treatment method. The combined medicine is usually based on different anti-tumor mechanisms, and is used for synergistically blocking different proliferation promoting channels in tumor cells to play a role in synergism, the anti-tumor effect is obvious, and particularly for the treatment of advanced three-line tumors. Therefore, the novel platinum drug with novel and unique anti-tumor mechanism, especially strong anti-tumor activity and low toxicity, has important effect on expanding the combined drug range of the platinum drug and other drugs and resisting tumor treatment. The action mechanism of the platinum drugs is to target DNA to play an anti-tumor role.

The preparation of miboplatin liposome has been granted Chinese patent (patent No. ZL 201510070080.2; patent name: miboplatin liposome and preparation method). The miboplatin liposome is prepared from miboplatin, phospholipid, cholesterol and water.

Miriplatin (Miriplatin) is a fat-soluble platinum metal complex, is a novel platinum (II) antitumor drug developed and marketed by sumitomo pharmaceutical corporation, and has a code number of SM-11355, trade name: is clinically provided in the form of monohydrate and is used for treating liver cancer. The chemical name of miboplatin is: cis- [ (1R,2R) -1, 2-Cyclohexanediamine-N, N '] bistetradecanoyloxyplatinum, with the chemical name (SP-4-2) - [ (1R,2R) -1, 2-cyclohexadiomine-kappaN, kappaN' ]) bis (myristato-kappao) platinum (i), monohydrate of the formula: C34H68N2O4Pt · H2O, molecular weight: 782.01, CAS registry number: 141977-79-9.

The invention discovers that the miboplatin liposome can reverse, and the tumor cells have natural and acquired drug resistance to tumor drugs. Further, the tumor stem cells which are the root of the tumor recurrence have obvious killing effect, the metastasis and the tumor recurrence are inhibited, a good direction is provided for the thorough treatment of the tumor, more tumor patients are benefited, and the clinical application value is important.

The miboplatin liposome is a novel nano-drug prepared on the basis of miboplatin, has large drug-loading rate, is completely dissolved in water, can be administered through veins, and enlarges the treatment range of tumor types. The antitumor activity of the compound is obviously higher than that of a miboplatin bulk drug and olplatinum, and the compound has a passive targeting effect, so that the compound has the characteristics of toxicity reduction and synergy, is expected to become a novel nano platinum drug for tumor treatment, benefits more patients and has important clinical application value.

The invention finds that the miboplatin liposome is novel and unique in action mechanism different from other anti-cancer drugs, and can be combined with other drugs with different action mechanisms from the miboplatin liposome. Especially, the tumor patients with few medicines can be selected in the late stage, and the method has good value for saving the lives of the patients.

Disclosure of Invention

The invention solves the problem that the existing tumor medicament has drug resistance after being taken for a period of time, so that a plurality of patients are not sensitive to the tumor medicament and the treatment of tumors is limited.

The invention aims to provide application of a miboplatin liposome in preparation of an anti-drug-resistant tumor medicament.

Wherein the tumor comprises: lung cancer, breast cancer, prostate cancer, skin cancer, nasal cancer, oral cancer, pancreatic cancer, and carcinoma of large intestine. Preferably, the tumor is pancreatic cancer or colorectal cancer.

Wherein, the human large intestine cancer cell line: human rectal cancer cells HCT8 and HT 29;

wherein, the human pancreatic cancer cell: AsPC-1, BxPC-3, MIA-PaCa-2, PANC-1, SW1990, SU.86.86.

Wherein, the human Oselli resistant colon cancer cell strain HCT 8L; human cisplatin-resistant tumor cell line PATU 8988/DDP.

Wherein, the miboplatin liposome reverses the drug resistance (acquired drug resistance) of the colorectal cancer cells HCT8 to oxaliplatin; the miboplatin liposome reverses the natural resistance of pancreatic cancer cells MIA-PaCa-2 and PANC-1 to gemcitabine; the miboplatin liposome reverses the cells HCT8L of the Osxal-resistant colorectal cancer; and resistance to the cisplatin-resistant tumor cells PATU 8988/DDP.

Wherein, the miboplatin liposome has killing effect on 3D-tumor stem cell balls cultured by the HCT8 cells of the colorectal cancer.

Wherein, the miboplatin liposome inhibits the soft agar clone forming ability of colorectal cancer tumor cells.

The expression of a mesothelin marker ZO1, E-cadherin is improved by the miboplatin liposome, and the expression level of an epithelin marker protein N-cadherin, Snail, Slug is reduced at the same time, so that the miboplatin liposome can inhibit EMT transformation, namely tumor cell metastasis.

The miboplatin liposome mainly enters cancer cells through a caveolin-mediated endocytosis pathway, and meanwhile, a macropinocytosis pathway has an auxiliary effect on entry of the miboplatin liposome.

Among them, the miriplatin liposome caused vacuolation of a large number of endoplasmic reticulum and mitochondria in tumor cells, and it was observed that the vacuolated mitochondria were contained in autophagy vesicles, suggesting that autophagy occurred in mitochondria.

Wherein, the miboplatin liposome enters the mitochondria of tumor cells to cause autophagy and endoplasmic reticulum damage caused by mitochondrial DNA replication disorder, thereby playing the role of anti-tumor.

Another objective of the invention is to provide the anticancer activity of the miboplatin liposome in different tumors.

The miboplatin liposome pair reversed the natural resistance of pancreatic cancer to its linear gemcitabine.

The 24-hour and 48-hour miboplatin liposome in vitro reversal of natural gemcitabine resistance of pancreatic cancer was evaluated by using a commercial first-line drug gemcitabine for pancreatic cancer as a positive control drug, 6 pancreatic cancer cells (IC 50 of 6 pancreatic cancer cells is more than 75 mu M at 24 hours, and IC50 of 3 pancreatic cancer cells is more than 75 mu M at 48 hours) and a MTT method.

The miboplatin liposome reverses the acquired drug resistance of colorectal cancer to its linear oxaliplatin.

For the acquired oxaliplatin resistance to 2 colorectal cancer cells, the SRB method is adopted to evaluate the effect of the miboplatin liposome on reversing the acquired drug resistance pair of colorectal cancer. Firstly, oxaliplatin is adopted to continuously treat HCT8 tumor cells and HT29 tumor cells of colorectal cancer, and after oxaliplatin is treated for 48 hours, the supernatant containing the medicament is removed and replaced by a normal culture medium, so that the tumor cells which still survive continue to proliferate for 72 hours, and the oxaliplatin-resistant cells are obtained. Oxaliplatin-resistant cells in the well plate were treated with the miriplatin liposome and oxaliplatin for 48 hours, the cell state was observed under a light microscope, and the cells were fixed and stained by the SRB method.

The miplatin liposomes reversed the drug resistance of established tumor drug resistant cell lines (acquired drug resistance).

The established human colorectal cancer oxaliplatin-resistant tumor cell strains HCT8L and PATU8988/DDP are adopted, and the MTT method is adopted to evaluate the acquired drug resistance effect of the miboplatin liposome reversal tumor cell strain. Oxaliplatin and miriplatin liposome are respectively used for treating an oxaliplatin cell line HCT8L of a human colorectal cancer tumor, cisplatin and miriplatin liposome treatment drugs are used for treating a cisplatin-resistant cell line PATU8988/DDP of a human pancreatic cancer, the cell state is observed under a light mirror after 48 hours, and the MTT method is adopted for detecting the cell activity.

The antitumor activity of the miboplatin liposome on nude mouse xenograft tumor.

Inoculating human pancreatic cancer APSC-1 cells subcutaneously in a Nude mouse BALB/c Nude mouse, and after the tumor volume reaches 100 cubic centimeters, administering a miboplatin liposome, a first-line pancreatic cancer drug gemcitabine and oxaliplatin in a tail vein, detecting a tumor proliferation curve and the survival time of the mouse, and monitoring the weight change.

The anti-tumor activity of the miboplatin liposome primary colorectal cancer mouse.

Another objective of the invention is to provide a miboplatin liposome with strong antitumor stem cells (drug-resistant cells).

The experiment adopts an experiment reflecting the characteristics of the tumor stem cells to observe whether the tumor stem cells are inhibited after the miboplatin liposome is treated.

Whether the balling-up ability is reduced: culturing the 3D-tumor stem cell ball in a balling culture medium, treating the tumor stem cell ball with the miboplatin liposome, and observing the killing effect of the medicament on the tumor stem cell ball.

Whether the tumorigenic capacity is reduced: the miboplatin liposome is used for treating the tumor cells, the clonality of the tumor cells after the drug treatment in soft agar is observed, and whether the miboplatin liposome can inhibit the tumor stem cells from forming tumor tissues through self-cloning is detected.

Whether epithelial-to-mesenchymal transition (EMT) is reduced, reflecting the characteristics of stem cells: epithelial Mesenchymal Transition (EMT) refers to the transition from epithelial-derived tumor cells to mesenchymal cells, and the characteristics of mesenchymal cell pairs are obtained, so that the important biological behavior of distant metastasis is closely related to tumor metastasis and invasion, and is also one of the important characteristics of tumor stem cells. And detecting the change of the expression level of the EMT related molecular marker protein by using Western blot to reflect whether the EMT is reduced.

It is another object of the present invention to provide an anti-tumor mechanism of the miriplatin liposomes.

Study of mitoplatin Liposome entry route

Endocytosis of the nanoparticle by the cell is mainly realized through endocytosis and can be subdivided into 4 endocytosis ways: clathrin-mediated, caveolin-mediated, macropinocytosis, pathways independent of clathrin and caveolin. Influenced by the nature of the nanoparticle and the cell type, the nanoparticle finally enters cells through different ways.

Selecting endocytosis inhibitors in different ways to act on cells, taking the uptake of the cells to the fluorescent liposome and the accumulation amount of platinum elements in the cells as indexes, and investigating the influence of different treatments on the entry of the miboplatin liposome, thereby determining the entry way of the miboplatin liposome.

Study of intracellular transport process of miboplatin liposome in cells

According to the conventional liposome cell entry route and trend, we firstly observe the trend of the liposome in endosomes and lysosomes. The co-localization of the cancer cell endosome and the lysosome is observed by marking the cancer cell endosome and the lysosome, and the transport process of the miriplatin liposome in the cancer cell is judged, so that an experimental basis is provided for explaining the action mechanism of the miriplatin liposome.

The miboplatin liposome pair elicits mitophagy by targeting mitochondrial DNA.

Detecting whether expression of the mitophagemid markers BNIP3 and BNIP3L/Nix is increased.

And detecting whether the autophagy protein marker LC3B is co-localized with mitochondria by using a laser co-scanning technology to reflect whether the mitochondria generate autophagy.

The qRT-PCR method detects the copy number change of mitochondrial DNA (mtDNA).

Research on damage of miboplatin liposome to endoplasmic reticulum of pancreatic cancer cells

After the miboplatin liposome treats pancreatic cancer cells, the change conditions of endoplasmic reticulum stress response related protein markers BiP, eIF2 alpha and phosphorylation forms thereof are detected by using a Western Blot method, and an experimental basis is provided for explaining the mechanism of the miboplatin liposome damaging the endoplasmic reticulum.

The medicine comprises acceptable auxiliary materials and other effective components which play a compatible and synergistic effect.

Wherein, the medicine of the invention is prepared into any pharmaceutical dosage form, including: capsule, tablet, powder, medicated wine, suspension, emulsion, syrup, aerosol, or injection.

Compared with the existing antitumor drugs, the invention also has the following beneficial effects:

the invention researches the activity of the miboplatin liposome for reversing tumor drug resistance. The activity of the miboplatin liposome is detected by adopting a pancreatic cancer cell line with natural gemcitabine resistance (first-line medicament of pancreatic cancer), 2 large intestine cancer cell lines with acquired oxaliplatin resistance and other established medicament-resistant cell lines, and the result shows that the miboplatin liposome can overcome the natural medicament resistance of pancreatic cancer to first-line medicament gemcitabine within 24 hours and 48 hours and overcome the acquired medicament resistance of medicament-resistant tumor cell lines. This finding is beneficial in many patients who have natural and acquired resistance to first-line drugs in tumors in the clinic, especially in patients who have multiple treatments at the middle and late stages, and can produce beneficial therapeutic effects. Meanwhile, due to the characteristic of extremely low toxicity of liposome preparation drugs, the miboplatin liposome can possibly become a novel platinum liposome drug for overcoming drug resistance clinically in the future.

The invention discovers a new mechanism for treating tumors by using platinum drugs. Although the miboplatin liposome still belongs to the platinum group drugs, the anti-tumor mechanism of the miboplatin liposome is different from the targeted DNA mechanism of the traditional platinum group drugs, and the miboplatin liposome is a new mechanism. The mechanism is that after transportation and treatment of endosomes and lysosomes, mitochondrial DNA and endoplasmic reticulum are targeted, and mitochondrial DNA replication is reduced, so that synthesis of various mitochondrial key functional proteins is reduced, and mitochondrial autophagy is triggered.

The combination based on different anti-tumor mechanisms is the basis of the combination therapy of tumor drugs. The combined application of different mechanisms of medicines can synergistically block the proliferation of tumor cells from different passages, has obvious anticancer effect, and is often used for treating tumors, particularly late-stage tumors. Therefore, the discovery of the drugs with a novel and unique mechanism, especially the drugs with strong antitumor activity and weak toxicity, has important effects on enlarging the range of the combined medication of platinum drugs and other drugs and resisting tumors of the combined medication.

Drawings

FIG. 1, the miboplatin liposome can overcome the oxaliplatin resistance of tumor cells

FIG. 1A flow chart of cell culture for oxaliplatin acquired resistance

FIG. 1B shows that the miboplatin liposome can overcome the oxaliplatin resistance-SRB quantification of tumor cells

FIG. 1C shows that the miboplatin liposome can overcome the oxaliplatin resistance of tumor cells under the observation of an under-mirror

FIG. 2 shows that miboplatin liposome can overcome the acquired drug resistance of tumor drug-resistant cell lines HCT8L, PATU8988/DDP

FIG. 2A the acquired drug resistance-MTT quantitation of the platinum liposomes overcoming the tumor resistance cell lines HCT8L, PATU8988/DDP

FIG. 2B shows that the miboplatin liposome can overcome the acquired drug resistance of the tumor drug-resistant cell lines HCT8L, PATU8988/DDP

FIG. 3 shows that the miboplatin liposome can kill 3D-tumor stem cell balls

FIG. 3A shows that the miboplatin liposome can kill 3D-tumor stem cell balls for-12 hours

FIG. 3B shows that the platinum liposome can kill 3D-tumor stem cell balls for 24 hours

FIG. 4 shows that the miboplatin liposome can inhibit the formation of tumor cell soft agar clone

FIG. 5 shows that miboplatin liposome inhibits Epithelial Mesenchymal Transition (EMT) of tumor cells

FIG. 6 Effect of entry inhibitors on fluorescence intensity

FIG. 7 Effect of cellular entry inhibitors on cellular platinum content

FIG. 8, the mitoplatin liposomes entered the endosome and lysosome after entry, scale 20 μm

FIG. 9, transmission electron micrograph of AsPC-1 cells

FIG. 10 shows the co-localization of cellular mitochondria and LC3B after miboplatin liposome treatment

FIG. 10A, AsPC-1 Co-localization of cell mitochondria to LC3B after treatment of cells with miboplatin liposomes

FIG. 10B, MIA Co-localization of cell mitochondria to LC3B after PaCa-2 cell miboplatin liposomes treatment

FIG. 11 shows that the expression levels of intracellular LC3B, BNIP3 and BNIP3L were increased after miboplatin liposome treatment

FIG. 12 shows that the expression level of LC3B in mitochondria of cells was increased after miboplatin liposome treatment

Detailed Description

The present invention is further illustrated by the following specific examples, which are not to be construed as limiting the invention thereto.

All large intestine cancer cell lines of the invention: human large intestine adenocarcinoma cells HCT8 and HT29 were both purchased from the national laboratory cell resource sharing platform. Human pancreatic cancer cells: AsPC-1, BxPC-3, MIA-PaCa-2, PANC-1, SW1990, SU.86.86 were purchased from national laboratory cell resources sharing, et al, and ATCC cell banks (Rockville, Md., USA).

Test example 1: natural drug resistance of miboplatin liposome reverse pancreatic cancer

The invention adopts MTT (tetramethyl azodicarbonamide blue salt) method to detect the activity of the miboplatin liposome (abbreviated as LMPt hereinafter) in vitro drug-resistant cells. Culturing 6 pancreatic cancer cells in a complete culture medium containing 10% fetal calf serum, culturing in a cell constant temperature incubator containing 5% CO2 at 37 ℃, digesting and counting the cells in a logarithmic growth phase, inoculating the cells in a 96-well plate, culturing for 24 hours until the cells are completely attached to the wall, respectively administering and treating by using miboplatin liposome miboplatin and oxaliplatin, and setting an administration concentration gradient. Proliferation was detected after 24 hours and 48 hours, respectively, and SigmaPlot calculated IC 50. And gemcitabine, oxaliplatin and other drugs were set as controls.

The IC50 results of the platinum liposome, the first-line gemcitabine for pancreatic cancer and the oxaliplatin for pancreatic cancer cells are shown in Table 1, the results show that the 6 pancreatic cancer tumor cells are resistant to the oxaliplatin and the gemcitabine within 24 hours, the platinum liposome has strong killing activity for the pancreatic cancer tumor cells within 24 hours and 48 hours, the activity of the platinum liposome is far higher than that of the control oxaliplatin and the gemcitabine, and the platinum liposome has strong resistance for reversing natural resistance to the gemcitabine.

TABLE 1 statistics of in vitro antitumor Activity of platinum liposomes on pancreatic cancer cells IC50

Test example 2: miriplatin liposomes reverse oxaliplatin resistance to colorectal cancer (acquired resistance)

First-line administration of colorectal cancer is oxaliplatin, and the treatment of colorectal cancer is seriously hindered by the drug resistance of oxaliplatin. Based on the above, the oxaliplatin-resistant cells are established to detect whether the miplatin liposome can obtain the sex reversal drug resistance. Treatment of colorectal cancer cells with oxaliplatin HCT8 and HT 2948 hours, withdrawal of the drug-containing supernatant and continued proliferation of drug-resistant surviving cells for 72 hours to obtain oxaliplatin-resistant cells.

Thereafter, the miriplatin liposomes were added while oxaliplatin and the blank control group were set. After 48 hours, the state of the cells was observed under a mirror, and the cells were fixed and stained by the SRB method. The experimental result shows that 80% of oxaliplatin-resistant tumor cells can be killed by the miplatin liposome compared with the control group, and the miplatin liposome is proved to overcome the oxaliplatin resistance phenomenon of the tumor cells (see figure 1).

Test example 3: miriplatin liposome reversal of drug resistance (acquired resistance) of established drug-resistant tumor cell lines

The generation of drug resistance of tumor cells to common chemotherapeutic drugs is a serious challenge for clinical tumor treatment, and in the experiment, whether the miboplatin liposome has killing activity to drug-resistant tumor cell strains or not is detected, so that the acquired drug resistance is reversed. HCT8L used in the test is a constructed human colorectal cancer-resistant oxaliplatin tumor cell line, and PATU8988/DDP is a human pancreatic cancer-resistant cis-platinum tumor cell line.

The miplatin liposome and oxaliplatin were treated separately for HCT8L tumor cell line and an untreated control was set. After 48 hours of drug treatment, the cell state was observed under the mirror, and the proliferation activity of the cells was examined by the MTT method. PATU8988/DDP tumor cell lines were treated with miboplatin liposomes and cisplatin, respectively, and untreated controls were set. The test method is the same as HTCTL. The test result shows that HCT8L and PATU8988/DDP are resistant to oxaliplatin and cisplatin respectively, and the miboplatin liposome has obvious killing effect on the oxaliplatin and the cisplatin, and can reverse the acquired resistance (see figure 2).

The existence of tumor stem cells is the main reason that tumor cells are resistant to common chemotherapeutic drugs and cannot be completely killed in treatment, so that the targeting and killing of the cells are the key of tumor treatment.

We used the following experiment to reflect tumor dryness to test the killing activity of miboplatin lipid on tumor stem cells.

Test example 4: the miboplatin liposome has strong killing activity on tumor stem cells

4.1 platinum liposome capable of killing 3D-tumor stem cell ball

3D-tumor stem cell pellet culture was performed on colon cancer HCT8 cells in logarithmic growth phase. After the cells are pelleted, the miboplatin liposome and the oxaliplatin are respectively added to treat the tumor stem cell pellets, and the tumor stem cell pellets are respectively observed and counted after the drug treatment for 12 hours and 24 hours. The experimental results show that compared with the non-administration group, the miplatin liposome-treated group almost completely killed the tumor stem cell balls, and the oxaliplatin group still survived about 50% (see figure 3). Experiments prove that the miboplatin liposome has killing effect on 3D-tumor stem cell balls cultured by the HCT8 cells of the colorectal cancer.

4.2 ability of platinum liposome to inhibit tumor cell soft agar clone formation

The ability of tumor cells to form clones in soft agar is often used to reflect the sternness characteristics of tumor cells. The experiment results of soft agar colony formation experiments on HCT8 cells treated by the miriplatin liposome and the oxaliplatin respectively show that the colony formation rate of the miriplatin liposome-treated HCT8 cells in soft agar is only 10-20% and the colony formation rate of the oxaliplatin-treated HCT8 cells is about 70% by taking an untreated group as a control (see figure 4). Experiments prove that the miboplatin liposome can inhibit the soft agar clone forming ability of colorectal cancer tumor cells.

4.3 Mi Pt liposome inhibiting Epithelial Mesenchymal Transition (EMT) of tumor cell

Epithelial Mesenchymal Transition (EMT) refers to the transition from epithelial-derived tumor cells to mesenchymal cells, and has the characteristics of mesenchymal cells, so that important biological behaviors of distant metastasis are obtained, closely related to tumor metastasis and invasion, and also one of the important characteristics of tumor stem cells. To verify whether the miplatin liposome can inhibit the characteristics of the tumor stem cells, HCT8 cells and HT29 cells were treated with the miplatin liposome, and the expression level of EMT-related proteins was measured.

The experimental result shows that the miboplatin liposome can improve the expression of the mesothelin marker ZO1, E-cadherin and simultaneously reduce the expression level of the epithelial protein marker N-cadherin, Snail and Slug, and shows that the miboplatin liposome inhibits EMT transformation, namely inhibits tumor cell metastasis (see figure 5)

Experimental example 5: study of mitoplatin Liposome entry route

The nano-drug mainly enters cells through an endocytosis path, and specifically, the endocytosis path is mainly divided into: clathrin-mediated endocytosis pathway, caveolin-mediated endocytosis pathway, megalocytosis pathway, and clathrin-and caveolin-independent pathway. To monitor the platinum liposome entry pathway, we used first to prepare fluorescent platinum lipids. The miboplatin liposome adopts the same method as that described in Chinese patent ZL 201510070080.2, the miboplatin liposome and the preparation method, and fluorescent substances are added into lipid materials for preparing the miboplatin liposome to prepare the fluorescence-labeled miboplatin liposome (hereinafter referred to as LMPt-DiI).

Culturing human pancreatic cancer cells AspC-1, BxPC-3, MIA-PaCa-2 and colorectal cancer cells to logarithmic phase, inoculating to 6-well plate, 20 × 10⁴And (3) treating the cells for 2 hours by using an inhibitor after the cells are attached to the wall, then treating the cells by using the inhibitor aiming at the nanoparticle cell entry way, and judging the way of the miboplatin liposome entering the tumor cells by adopting a fluorescence microscope and platinum content measurement. The results (fig. 6-7) show that the miboplatin liposome enters cancer cells mainly through the caveolin-mediated endocytosis pathway, and the macropinocytic pathway has an auxiliary effect on the entry of the miboplatin liposome.

Experimental example 6: transport of miriplatin liposomes in tumor cells

After the nano-drug is endocytosed by cells, the cytoplasm wraps the nano-particles to form a small nest, and then the small nest continuously caves, finally the nano-drug is separated from the plasma membrane and enters the cytoplasm to form an endosome. Early endosomes mature into late endosomes, and finally fuse with lysosomes. The endocytosed nano-drug can be degraded under the action of a low pH environment (endosome/lysosome) and a lysosome enzyme system, and the release of the coated drug is facilitated.

In order to further observe the transport of the miplatin liposome, the miplatin liposome and a cell endosome/lysosome system are fluorescently labeled, and the transport of the miplatin liposome in the cell is detected.

The pancreatic cancer cells and the colorectal cancer cells are inoculated in a 96-well glass-bottom plate, after the cells adhere to the wall, a culture medium containing a fluorescent reagent is used for replacing the culture medium in the corresponding well, and the cells are continuously incubated for 24 hours at 37 ℃ under the condition of 5% CO2, so that the organelles in the tumor cells are fluorescently labeled. And then adding LMPt-DiI into each hole, discarding the culture medium within 30min, 1 and 6h respectively, washing the cells with PBS, washing off the fluorescent liposome adhered to the surfaces of the cells, adding a fresh culture medium into the holes, and observing the co-localization condition of the LMPt-DiI and the organelles by using a fluorescence microscope.

The co-localization of LMPt-DiI with early endosomes, late endosomes, lysosomes is shown in FIG. 8. The results show that the pancreatic cancer cells and the colorectal cancer cells have similar results, namely LMPt-DiI and the cells are incubated for 1h to observe that LMPt-DiI is co-localized with early endosomes and late endosomes; co-incubation for 6h was observed to co-localize LMPt-DiI with lysosomes. The above results indicate that the miboplatin liposome enters the cell, enters the endosome and then is transported to the lysosome.

Experimental example 7: mechanism for killing tumor by miboplatin liposome

7.1 Transmission Electron microscopy of Damage to tumor cells by Miplatin lipids

In order to further confirm the damage of the miboplatin to cells after entering the cells, we further observed the loss condition of organelles by adopting an electron microscope.

AsPC-1 was cultured in vitro to log phase and seeded in 6-well plates at a seeding density of 20X 10⁴2mL of medium per well, after which incubation in the cell incubator was continued for 24 h. And (3) diluting the miboplatin liposome to 30 mu M by using the culture medium, replacing the culture medium in the corresponding hole by using the culture medium containing the miboplatin liposome after the cells are attached to the wall, and setting the hole without adding the miboplatin liposome as a negative control. After further incubation for 24h, the medium was discarded, the cells were washed with PBS, the miboplatin liposomes adhered to the cell surface were washed off, the cells were nitrified with 0.25% pancreatin and collected in Ep tubes, and centrifuged (800 Xg, 5min) to obtain cell pellets. Cells were pre-fixed in 2.5% glutaraldehyde, followed by 1% osmium tetroxide and 1.5% potassium ferrocyanide for 1 h. The cells were dehydrated using increasing concentrations of ethanol solutions (50%, 60%, 70%, 80%, 90% and 100%), stained with 2% uranyl acetate containing 70% ethanol at room temperature, and then embedded in epoxy. The embedded samples were cut into sections 60nm thick using an ultra microtome and mounted on a copper mesh. The sections were observed (120kV voltage) using a JEM-1400Plus transmission electron microscope system (JEOL, Japan).

The results showed that the miboplatin liposome caused massive vacuolization of endoplasmic reticulum and mitochondria, and that vacuolized mitochondria were contained in the autophagy vesicle, suggesting that autophagy occurred in mitochondria (fig. 9).

7.2 Miplatin Liposome targeted organelles

After entering cells, the miboplatin liposome is treated by an inclusion body and lysosomes containing a large amount of degradation enzyme, enters a next target organelle and exerts the anti-tumor activity of the mitoplatin liposome. An electron microscope image indicates that the miboplatin paper liposome can damage endoplasmic reticulum and mitochondria to play a role, so that cell organelles are extracted to detect the content of platinum so as to judge the trend of the platinum. The cell treated by the platinum paper liposome is taken, the cell nucleus, the endoplasmic reticulum, the mitochondria and other organelles are respectively extracted to detect the platinum content, and the result shows that the platinum is mainly concentrated in the endoplasmic reticulum and the mitochondria and is the main target organelle of the platinum paper liposome.

7.3 m platinum liposomes cause mitophagy

According to the aggregation of the miboplatin liposome in the mitochondria and the result of an electron microscope, the fact that the mitochondria are targeted to autophagy is suggested, so that the mitochondria are further detected to determine that the mitochondria autophagy occurs

Damaged or unwanted mitochondria need to be cleared in time to maintain normal cellular activity. Cells selectively clear damaged mitochondria primarily through autophagy mechanisms, a process known as mitophagy. Upon occurrence of mitophagy, the mitophagy receptor is first activated, recruiting LC3 to the injured mitochondria by binding to autophagy key protein LC3, and encapsulating the injured mitochondria with autophagosomes. Autophagosomes encapsulating mitochondria fuse with lysosomes and damaged mitochondria are released into the lysosomal lumen and eventually degraded. The occurrence of the mitophagy can be proved by detecting the mitophagy marker and verifying the co-localization of the mitophagy and the autophagy signal.

7.3.1 mitochondria and marker LC3B for initiating autophagy

The co-localization of mitochondria and autophagy marker LC3B in rice liposome-treated cells was determined by immunofluorescence. The results indicate that the intracytoplasmic LC3B signal co-localizes with mitochondria, indicating that LC3B is recruited into mitochondria and that mitophagy occurs (fig. 10).

7.3.2 detection of mitochondrial autophagy markers

After the cells were treated with the platinum liposome, the cell lysate was subjected to western blot analysis, and the non-administered group was used as a negative control, and as a result, it was found that the expression levels of intracellular autophagy key protein LC3B, mitochondrial autophagy receptor BNIP3, and mitochondrial autophagy receptor BNIP3L/Nix were increased (fig. 11), suggesting the occurrence of mitochondrial autophagy. Mitochondria were extracted, the miboplatin liposome-treated group was the experimental group, the non-administered group was the negative control group, and the mitochondrial LC3B was detected by western blot, which indicated that as the administration concentration increased, the amount of LC3B enriched in mitochondria increased (fig. 12), indicating that LC3B produced by autophagy was enriched in mitochondria.

The above results all demonstrate that mitoplatin liposomes induced the development of mitochondrial autophagy.