CN115957188A - Application of miplatin liposomes in anti-drug resistant tumors - Google Patents

Abstract

The invention belongs to the field of medicines, and relates to application of miriplatin liposome in drug resistance. The invention discovers that the miboplatin liposome can reverse the tumor cell natural and acquired drug resistance to the tumor drug. 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.

Description

Application of miplatin liposomes in anti-drug resistant tumors

technical field

The invention belongs to the field of medicine, and relates to the application of miplatin liposome in anti-drug resistance.

Background technique

Drug therapy plays an important role in anti-tumor treatment, but cancer patients often develop drug resistance (including natural and acquired) to tumor drugs after a period of medication, which makes many patients insensitive to tumor drugs and limits the treatment of tumors .

Cancer stem cells are a kind of highly tumorigenic and multidirectionally differentiated cell population existing in tumor tissues, and are the main cause of tumor drug resistance, recurrence and metastasis. It is not sensitive to general tumor drugs, and often survives conventional anti-tumor drug treatment, and can return to the proliferation cycle under certain conditions, leading to tumor recurrence. The combination of treatment targeting tumor stem cells and conventional chemotherapy drugs is the key to completely curing tumors and improving the prognosis of patients. So far, no clear anti-tumor stem cell drugs have been reported.

Platinum drugs have always played an important role in antitumor, but their toxicity limits their application. Oxaliplatin, as a third-generation platinum drug with gradually improved performance, has always played an important role in colorectal cancer and pancreatic cancer. In the 2019 edition of the US National Comprehensive Cancer Guidelines, it is listed as the first-line drug for pancreatic cancer in the "FOLFIRINOX" combination. The "FOLFOX" treatment plan in which oxaliplatin is used in combination is also used as the first-line treatment for colorectal cancer, and has become the gold standard of chemotherapy for advanced colorectal cancer, showing significant therapeutic effects in clinical use. Its non-specific distribution in vivo leads to toxicity in different tissues, most notably neurotoxicity and myelosuppressive toxicity.

Miplatin is an upgraded drug based on oxaliplatin. The anti-tumor active intermediate formed after metabolism is the same as the anti-tumor active ingredient of oxaliplatin. It has a broad anti-tumor spectrum, strong anti-tumor activity, and low adverse reactions. . However, it has poor water solubility and cannot be administered systemically. It can only be administered through the hepatic artery after dissolving in poppy seed oil, which is expensive and not easy to obtain. The limitation of the way of administration and the difficulty in obtaining the solvent limit the treatment of other tumors and the route of administration of miplatin. So far, there is no report on the antitumor activity of miplatin through systemic administration against pancreatic cancer and colorectal cancer.

In tumor therapy, drug combination is a very important treatment method. Combination drugs are often based on different anti-tumor mechanisms, synergistically blocking different pro-proliferation pathways in tumor cells to play a synergistic role, and the anti-tumor effect is obvious, especially for advanced third-line tumor treatment. Therefore, new platinum-based drugs with novel and unique anti-tumor mechanisms, especially new platinum-based drugs with strong anti-tumor activity and low toxicity, will play an important role in expanding the scope of combined use of platinum-based drugs and other drugs in combination with anti-tumor therapy. The mechanism of action of platinum-based drugs is to target DNA to exert anti-tumor effects.

The preparation of miplatin liposomes has been authorized Chinese patent (patent number: ZL 2015 1 0070080.2; patent name: miplatin liposomes and preparation method). Miplatin liposomes are prepared from miplatin, phospholipids, cholesterol and water.

Miriplatin (Miriplatin) is a fat-soluble platinum metal complex. It is a new type of platinum (II) anti-tumor drug developed and marketed by Sumitomo Pharmaceutical Co., Ltd., code-named SM-11355. Available in monohydrate form, it is used in the treatment of liver cancer. The chemical name of miplatinum is: cis-[(1R,2R)-1,2-cyclohexanediamine-N,N'] ditetradecanoyloxyplatinum, and the English chemical name is (SP-4-2) -[(1R,2R)-1,2-Cyclohexanediamine-kappaN,kappaN'])bis(myristato-kappa O)platinum(II), the chemical formula of its monohydrate is: C34H68N2O4Pt H2O, molecular weight: 782.01, CAS registration No.: 141977-79-9.

The present invention finds that the miplatin liposome can reverse the natural and acquired drug resistance of tumor cells to tumor drugs. It was further found that it has a significant killing effect on tumor stem cells at the root of tumor recurrence, inhibits metastasis and tumor recurrence, and provides a good direction for the thorough treatment of tumors, benefiting more cancer patients, and has important clinical application value.

Miplatin liposome is a new type of nano-medicine prepared on the basis of Miplatin. It has a large drug loading capacity and is completely soluble in water. It can be administered intravenously and systemically, expanding the range of tumor treatment. Its anti-tumor activity is significantly stronger than miplatin raw material drug and opalplatin, and has a passive targeting effect, which makes the drug have the characteristics of attenuating toxicity and increasing efficiency. It is expected to become a new nano-platinum drug for tumor treatment and benefit more patients. It has important clinical application value.

The invention discovers that the miplatin liposome has a novel and unique action mechanism different from other anticancer drugs, and can expand the combined application with other drugs with different action mechanisms from the miplatin liposome. Especially for cancer patients with few choices of drugs in the advanced stage, it is of good value for saving the lives of patients.

Contents of the invention

The invention solves the problem that many patients are insensitive to the tumor drug due to drug resistance after taking the existing tumor drug for a period of time and limits the treatment of the tumor.

The purpose of the present invention is to provide the application of miplatin liposome in the preparation of anti-drug resistant tumor drugs.

Among them, tumors include: lung cancer, breast cancer, prostate cancer, skin cancer, nasal cancer, oral cancer, pancreatic cancer, and colorectal cancer. Preferably, the tumor is pancreatic cancer, colorectal cancer.

Among them, human colorectal cancer cell lines: human rectal cancer cells HCT8 and HT29;

Among them, human pancreatic cancer cells: AsPC-1, BxPC-3, MIA-PaCa-2, PANC-1, SW1990, SU.86.86.

Among them, human oxali-resistant colorectal cancer cell line HCT8L; human cisplatin-resistant tumor cell line PATU8988/DDP.

Among them, miplatin liposomes reversed the resistance (acquired drug resistance) of colorectal cancer cell HCT8 to oxaliplatin; miplatin liposomes reversed the natural resistance of pancreatic cancer cells MIA-PaCa-2 and PANC-1 to gemcitabine. Drug resistance; miplatin liposomes reversed drug resistance of oxali-resistant colorectal cancer cell HCT8L; and cisplatin-resistant tumor cell PATU8988/DDP.

Among them, miplatin liposomes have a killing effect on 3D-tumor stem cell spheres cultured from colorectal cancer HCT8 cells.

Among them, miplatin liposome inhibits the ability of soft agar colony formation of colorectal cancer tumor cells.

Among them, miplatin liposomes increased the expression of mesothelin markers ZO1 and E-cadherin, while down-regulating the expression of epithelin marker proteins N-cadherin, Snail, and Slug, indicating that miplatin liposomes inhibited EMT transformation, that is, inhibited tumor cell metastasis.

Among them, miplatin liposomes mainly enter cancer cells through the endocytic pathway mediated by caveolin, and the macropinocytosis pathway plays an auxiliary role in the entry of miplatin liposomes.

Among them, miplatin liposomes caused a large number of endoplasmic reticulum and mitochondria vacuolization in tumor cells, and it was observed that the vacuolated mitochondria were contained in autophagic vacuoles, suggesting that autophagy occurred in mitochondria.

Among them, miplatin liposomes enter the mitochondria of tumor cells, causing the anti-tumor mechanism of autophagy and endoplasmic reticulum damage caused by mitochondrial DNA replication disorders, so as to exert anti-tumor effects.

Another object of the present invention is to provide the anticancer activity of miplatin liposomes in different tumors.

Miplatin liposomal pair reverses natural resistance of pancreatic cancer to the first-line drug gemcitabine.

Using gemcitabine, a commercially available first-line drug for pancreatic cancer, as a positive control drug, 6 pancreatic cancer cell lines (the IC50 of 6 pancreatic cancer cell lines at 24 hours were all greater than 75 μM, and the IC50 of 3 lines of pancreatic cancer cells at 48 hours were greater than 75 μM) and MTT method were used to evaluate the 24-hour and MTT method. 48-hour miplatin liposome reverses natural resistance to gemcitabine in pancreatic cancer in vitro.

Miplatin liposomes reverse acquired resistance to the first-line drug oxaliplatin in colorectal cancer.

The SRB method was used to evaluate the role of miplatin liposomes in reversing the acquired resistance of colorectal cancer against 2 colon cancer cell lines with acquired resistance to oxaliplatin. That is, first, oxaliplatin was used to continuously treat colorectal cancer HCT8 tumor cells and HT29 tumor cells. After 48 hours of oxaliplatin treatment, the supernatant containing the drug was removed and replaced with normal medium, so that the surviving tumor cells continued to proliferate for 72 hours, that is, Obtain oxaliplatin-resistant cells. The oxaliplatin-resistant cells in the well plate were treated with miplatin liposomes and oxaliplatin for 48 hours, the state of the cells was observed under a light microscope, and the cells were fixed and stained by the SRB method.

Miplatin liposomes reversed drug resistance in established tumor drug-resistant cell lines (acquired drug resistance).

Using the established human colorectal cancer oxaliplatin-resistant tumor cell lines HCT8L, PATU8988/DDP, the MTT method was used to evaluate the role of miplatin liposomes in reversing the acquired drug resistance of tumor cell lines. Oxaliplatin and miplatin liposomes were used to treat human-derived colorectal cancer tumor oxaliplatin-resistant cell line HCT8L, and cisplatin and miplatin liposomes were used to treat human-derived pancreatic cancer cisplatin-resistant cell line PATU8988 /DDP, after 48 hours, the state of the cells was observed under a light microscope, and the cell viability was detected by the MTT method.

Antitumor activity of miplatin liposomes against xenograft tumors in nude mice.

Human pancreatic cancer APSC-1 cells were inoculated subcutaneously in nude mice BALB/c Nude mice. When the tumor volume reached 100 cubic centimeters, miplatin liposomes, first-line pancreatic cancer drugs gemcitabine and oxaliplatin were administered through the tail vein, and the tumors were detected. Proliferation curves and mouse survival time were monitored for body weight changes.

Antitumor activity of miplatin liposomes in mice with primary colorectal carcinoma.

Another object of the present invention is to provide miplatin liposomes with strong anti-tumor stem cells (drug-resistant cells).

In this experiment, an experiment reflecting the characteristics of tumor stem cells was used to observe whether the tumor stem cells were inhibited after miplatin liposome treatment.

Whether the ability to form spheres is reduced: 3D-tumor stem cell spheres were cultured in the sphere formation medium, and the tumor stem cell spheres were treated with miplatin liposomes, and the killing effect of the drug on the tumor stem cell spheres was observed.

Whether the ability to form a tumor is reduced: Treat tumor cells with miplatin liposomes, observe the ability of tumor cells to form clones in soft agar after drug treatment, and detect whether miplatin liposomes can inhibit tumor stem cells from forming tumor tissues through self-cloning.

Reflecting the characteristics of stem cells, whether epithelial-mesenchymal transition (EMT) is reduced: Epithelial-mesenchymal transition (EMT) refers to the transformation of epithelial-derived tumor cells to mesenchymal cells, acquiring the characteristics of mesenchymal cells, and then metastasizing to distant places Important biological behavior, closely related to tumor metastasis and invasion, is also one of the important characteristics of tumor stem cells. Western blot was used to detect changes in the expression levels of EMT-related molecular marker proteins to reflect whether EMT was reduced.

Another object of the present invention is to provide the anti-tumor mechanism of miplatin liposome.

Study on the pathway of miplatin liposomes into cells

The endocytosis of nanoparticles by cells is mainly achieved by pinocytosis, which can be subdivided into four endocytic pathways: clathrin-mediated, caveolin-mediated, macropinocytosis, clathrin- and caveolin-independent pathways . Affected by the properties of nanoparticles and cell types, nanoparticles finally enter cells through different pathways.

Different pathways of endocytosis inhibitors were selected to act on cells, and the uptake of fluorescent liposomes by cells and the accumulation of platinum in cells were used as indicators to investigate the effects of different treatments on miplatin liposomes entering cells, so as to determine the The cellular entry pathway of platinum liposomes.

Study on the intracellular transport process of miplatin liposomes in cells

According to the conventional liposome entry pathway and orientation, we first observed its orientation in endosomes and lysosomes. By marking cancer cell endosomes and lysosomes, observing their co-localization with miplatin liposomes, judging the transport process of miplatin liposomes in cancer cells, and providing an experimental basis for explaining the mechanism of miplatin liposomes.

Miplatin liposomes cause mitophagy by targeting mitochondrial DNA.

Whether the expression of mitophagy protein markers BNIP3 and BNIP3L/Nix increased was detected.

Laser co-scanning technology was used to detect whether the autophagy protein marker LC3B co-localized with mitochondria to reflect whether autophagy occurred in mitochondria.

qRT-PCR method was used to detect the changes of mitochondrial DNA (mtDNA) copy number.

Study on the damage of miplatin liposomes to the endoplasmic reticulum of pancreatic cancer cells

After miplatin liposomes treated pancreatic cancer cells, Western Blot was used to detect the changes of endoplasmic reticulum stress response-related protein markers BiP, eIF2α and their phosphorylated forms, in order to explain the damage of endoplasmic reticulum by miplatin liposomes. The mechanism provides an experimental basis.

Wherein, the medicament of the present invention contains acceptable auxiliary materials and other active ingredients that play a compatible and synergistic role.

Wherein, the medicine of the present invention is prepared into any pharmaceutical dosage form, including: capsules, tablets, powders, liquors, suspensions, emulsions, syrups, aerosols, or injections.

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

The present invention studies the activity of reversing tumor drug resistance of miplatin liposome. Using natural gemcitabine-resistant pancreatic cancer cell lines (the first-line drug for pancreatic cancer), as well as two acquired oxaliplatin-resistant colorectal cancer cell lines and other established drug-resistant cell lines, the activity of miplatin liposomes was detected, and the results It shows that miplatin liposome can overcome the natural drug resistance of pancreatic cancer to its first-line drug gemcitabine at 24 hours and 48 hours, and also overcome the acquired drug resistance of drug-resistant tumor cell lines. This discovery is beneficial to many patients who have natural and acquired resistance to first-line tumor drugs in clinical practice, especially for patients who have been treated multiple times in the middle and late stages, and will have beneficial therapeutic effects. At the same time, liposome preparations have extremely low drug toxicity, making miplatin liposomes a new type of platinum-based liposome drug that can overcome drug resistance in the future.

The invention discovers a new mechanism of platinum drugs for treating tumors. Although miplatin liposomes still belong to platinum drugs, its anti-tumor mechanism is different from the DNA targeting mechanism of traditional platinum drugs and is a new mechanism. The mechanism is that, through endosome and lysosome transport and processing, it targets mitochondrial DNA and endoplasmic reticulum, reduces mitochondrial DNA replication, leads to decreased synthesis of various mitochondrial key functional proteins, and triggers mitophagy.

Combination drugs based on different anti-tumor mechanisms are the basis for combination therapy of tumor drugs. The combined application of drugs with different mechanisms can synergistically block the proliferation of tumor cells from different pathways, and has obvious anticancer effects. It is often used in tumor treatment, especially in the treatment of advanced tumors. Therefore, the discovery of drugs with novel and unique mechanisms, especially drugs with strong anti-tumor activity and weak toxicity, will play an important role in expanding the scope of combined use of platinum-based drugs and other drugs in combination, and in anti-tumor effects of combined drugs.

Description of drawings

Figure 1A flow chart of oxaliplatin-acquired drug-resistant cell culture

Figure 1B Miplatin liposomes can overcome tumor cell resistance to oxaliplatin-SRB quantification

Figure 1C Miplatin liposomes can overcome tumor cell resistance to oxaliplatin-microscopic observation

Figure 2A Miplatin liposomes can overcome the acquired drug resistance of tumor drug-resistant cell lines HCT8L, PATU8988/DDP-MTT quantification

Figure 2B Miplatin liposomes can overcome the acquired drug resistance of tumor drug-resistant cell lines HCT8L, PATU8988/DDP-microscopic observation

Figure 3A Miplatin liposomes can kill 3D-tumor stem cell spheres-12 hours

Figure 3B Miplatin liposomes can kill 3D-tumor stem cell spheres-24 hours

Figure 4. Miplatin liposomes inhibit the ability of tumor cell soft agar colony formation

Figure 5. Miplatin liposome inhibits epithelial-mesenchymal transition (EMT) of tumor cells

Figure 6. Effect of cell entry inhibitors on fluorescence intensity

Figure 7. Effect of cell entry inhibitors on platinum content in cells

Figure 8. Miplatin liposomes enter endosomes and lysosomes after entering cells, the scale bar is 20 μm

Figure 9. Transmission electron microscope image of AsPC-1 cells

Figure 10A. Colocalization of mitochondria and LC3B in AsPC-1 cells after treatment with miplatin liposomes

Figure 10B. Colocalization of mitochondria and LC3B in MIA-PaCa-2 cells treated with miplatin liposomes

Figure 11. The expression levels of LC3B, BNIP3 and BNIP3L in cells increased after treatment with miplatin liposomes

Figure 12. The expression level of LC3B in the mitochondria of cells increased after treatment with miplatin liposomes

Detailed ways

The present invention will be further illustrated by the following specific examples, but not as a limitation of the present invention.

All colorectal cancer cell lines of the present invention: human colorectal adenocarcinoma cells HCT8 and HT29 were purchased from the National Experimental 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 Experimental Cell Resource Sharing Platform and ATCC Cell Bank (Rockville, MD, USA). Experimental example 1: Miplatin liposome reverses the natural drug resistance of pancreatic cancer

The invention adopts the MTT (tetramethylazolazolium salt) method to detect the anti-drug-resistant cell activity of miplatin liposome (abbreviated as LMPt hereinafter). Six pancreatic cancer cell lines were cultured in a complete medium containing 10% fetal bovine serum, cultured in a constant temperature incubator at 37°C and 5% CO2, and the cells in the logarithmic growth phase were digested, counted, and inoculated in a 96-well plate In , after 24 hours of culture, when the cells were completely attached to the wall, they were treated with miplatin liposomal miplatin and oxaliplatin respectively, and a concentration gradient was set. Proliferation was detected after 24 hours and 48 hours, and IC50 was calculated by SigmaPlot. And set gemcitabine, oxaliplatin and other drugs as controls.

The IC50 results of miplatin liposome, gemcitabine and oxaliplatin, the first-line drugs for pancreatic cancer, on pancreatic cancer cells are shown in Table 1, and the results show that the six pancreatic cancer tumor cells are resistant to oxaliplatin and gemcitabine within 24 hours , miplatin liposomes have strong killing activity on pancreatic cancer tumor cells at 24 hours and 48 hours, and its activity is much higher than that of the control oxaliplatin and gemcitabine. drug resistance.

Table 1 Anti-tumor activity IC50 statistics of Miplatin liposomes on pancreatic cancer cells in vitro

Test Example 2: Miplatin liposomes reverse colorectal cancer's resistance to oxaliplatin (acquired drug resistance). The first-line drug for colorectal cancer is oxaliplatin, and resistance to oxaliplatin seriously hinders colorectal cancer. cancer treatment. Based on this, we established drug-resistant cells resistant to oxaliplatin to test whether miplatin liposomes can acquire drug resistance. Colorectal cancer cells HCT8 and HT29 were treated with oxaliplatin for 48 hours, the drug-containing supernatant was removed, and the drug-resistant surviving cells continued to proliferate for 72 hours to obtain oxaliplatin-resistant cells.

Thereafter, miplatin liposomes were added, and oxaliplatin and a blank control group were set at the same time. After 48 hours, the state of the cells was observed under the microscope, and the cells were fixed and stained by the SRB method. Experimental results found that, compared with the control group, 80% oxaliplatin-resistant tumor cells can be killed by miplatin liposomes, which proves that miplatin liposomes can overcome tumor cell resistance to oxaliplatin (see figure 1).

Test Example 3: Miplatin liposome reverses the drug resistance of established drug-resistant tumor cell lines (acquired drug resistance)

The resistance of tumor cells to common chemotherapeutic drugs is a serious challenge in clinical tumor treatment. In this experiment, we tested whether miplatin liposomes have killing activity against drug-resistant tumor cell lines and reversed their acquired drug resistance. HCT8L used in the experiment is a human-derived colorectal cancer oxaliplatin-resistant tumor cell line, and PATU8988/DDP is a human-derived pancreatic cancer cisplatin-resistant tumor cell line.

Miplatin liposomes and oxaliplatin were used to treat HCT8L tumor cell lines respectively, and an untreated control was set. After 48 hours of drug treatment, the state of the cells was observed under the microscope, and the proliferation activity of the cells was detected by the MTT method. PATU8988/DDP tumor cell lines were treated with miplatin liposomes and cisplatin, respectively, and an untreated control was set. The test method is the same as HTCTL. The test results found that HCT8L and PATU8988/DDP were resistant to oxaliplatin and cisplatin, respectively, and miplatin liposomes had a significant killing effect on them, which could reverse their acquired drug resistance (see Figure 2).

The existence of tumor stem cells is the main reason why tumor cells are resistant to common chemotherapy drugs and cannot be completely killed during treatment. Therefore, targeting and killing these cells is the key to tumor treatment.

We used the following experiments reflecting tumor stemness to detect the killing activity of miplatin lipids on tumor stem cells.

Test Example 4: Miplatin liposomes have strong killing activity on tumor stem cells

4.1 M platinum liposomes can kill 3D-tumor stem cell spheres

Colorectal cancer HCT8 cells in the logarithmic growth phase were used for 3D-tumor stem cell sphere culture. After the cells formed spheres, miplatin liposomes and oxaliplatin were added to treat the tumor stem cell spheres, and the state of the tumor stem cell spheres were observed and counted after 12 hours and 24 hours of drug treatment. The experimental results found that compared with the non-administration group, the tumor stem cell spheres in the miplatin liposome treatment group were almost completely killed, while about 50% of the oxaliplatin group still survived (see Figure 3). Experiments have shown that miplatin liposomes have a killing effect on 3D-tumor stem cell spheres cultured from colorectal cancer HCT8 cells.

4.2 M Platinum liposomes inhibit the ability of tumor cell soft agar colony formation

The ability of tumor cells to form colonies in soft agar is often used to reflect the stemness characteristics of tumor cells. We treated HCT8 cells with miplatin liposomes and oxaliplatin respectively, and performed soft agar colony formation experiments on the treated tumor cells. The colony formation rate in soft agar was only 10%-20%, while the colony formation rate of HCT8 cells in the oxaliplatin-treated group was about 70% (see Figure 4). Experiments have shown that miplatin liposomes inhibit the colony formation ability of colorectal cancer tumor cells in soft agar.

4.3 M Platinum liposome inhibits epithelial-mesenchymal transition (EMT) of tumor cells

Epithelial-mesenchymal transition (EMT) refers to the transformation of epithelial-derived tumor cells into mesenchymal cells, becoming an important biological behavior with the characteristics of mesenchymal cells, and then acquiring distant metastasis, which is closely related to tumor metastasis and invasion, and also One of the important characteristics of cancer stem cells. In order to verify whether miplatin liposomes can inhibit this tumor stem cell characteristic, we treated HCT8 cells and HT29 cells with miplatin liposomes, and detected the expression levels of EMT-related proteins.

The experimental results show that miplatin liposomes can increase the expression of mesothelin markers ZO1 and E-cadherin, while down-regulating the expression of epithelin marker proteins N-cadherin, Snail, and Slug, indicating that miplatin liposomes inhibit EMT transformation , which inhibited tumor cell metastasis (see Figure 5)

Experimental Example 5: Study on the Cell Entry Pathway of Miplatin Liposomes

Nanomedicine mainly enters cells through the endocytosis pathway. Specifically, the endocytosis pathway is mainly divided into: clathrin-mediated endocytosis pathway, caveolin-mediated endocytosis pathway, macropinocytosis pathway, and independent The clathrin and caveolin pathway. To monitor the miplatin liposome entry pathway, we first prepared fluorescent miplatin lipids. Miplatin liposomes use the same method as described in Chinese patent ZL 2015 1 0070080.2, Miplatin liposomes and preparation methods, adding fluorescent substances to the lipid material for preparing Miplatin liposomes to prepare fluorescently labeled Miplatin Liposome (hereinafter referred to as LMPt-DiI).

Human pancreatic cancer cells AsPC-1, BxPC-3, MIA-PaCa-2 and colorectal cancer cells were cultured to the logarithmic phase, seeded in 6-well plates, 20× ¹⁰⁴ cells/well, after the cells adhered to the wall, treated with inhibitors After 2 hours of treatment, the cells were treated with an inhibitor of the nanoparticle entry pathway, and the pathway of the miplatin liposomes into the tumor cells was determined by fluorescence microscopy and platinum content determination. The results (Fig. 6-7) showed that miplatin liposomes mainly entered into cancer cells through the endocytic pathway mediated by caveolin, and the macropinocytosis pathway played an auxiliary role in the entry of miplatin liposomes.

Experimental Example 6: Transport of Miplatin Liposomes in Tumor Cells

After the nanomedicine is endocytized by the cell, the cytoplasm wraps the nanoparticle to form a pit, then continues to sink, and finally breaks away from the plasma membrane and enters the cytoplasm to form an endosome. Early endosomes mature into late endosomes, which eventually fuse with lysosomes. The endocytosed nanomedicine will be degraded under the action of low pH environment (endosome/lysosome) and lysosomal enzyme system, which is conducive to the release of the coated drug.

In order to further observe the translocation of miplatin liposomes, we used fluorescently labeled miplatin liposomes and endosome/lysosome system to detect the translocation of miplatin liposomes in cells.

Inoculate pancreatic cancer cells and colorectal cancer cells in glass-bottom 96-well plates. After the cells adhere to the wall, replace the medium in the corresponding wells with the medium containing fluorescent reagents, and continue to incubate the cells at 37°C and 5% CO2 for 24 hours. , to ensure that organelles in tumor cells are fluorescently labeled. Then add LMPt-DiI to each well, discard the culture medium at 30 min, 1, and 6 h respectively, wash the cells with PBS, wash away the fluorescent liposomes adhering to the cell surface, add fresh culture medium to the wells, and use Fluorescence microscopy was used to observe the co-localization of LMPt-DiI and organelles.

The co-localization of LMPt-DiI with early endosomes, late endosomes and lysosomes is shown in Figure 8. The results showed that pancreatic cancer cells and colorectal cancer cells obtained similar results, that is, co-localization of LMPt-DiI with early endosomes and late endosomes was observed when LMPt-DiI was co-incubated with cells for 1 h; Body co-determined. The above results indicated that miplatin liposomes entered the cells and then first entered the endosomes and then transported to the lysosomes.

Experimental Example 7: Mechanism of Miplatin Liposome Killing Tumor

7.1 Transmission electron microscopy observation of the damage of miplatin lipids to tumor cells

In order to further confirm the damage of miplatin to the cells after entering the cells, we further observed the loss of organelles by electron microscopy.

AsPC-1 was cultured in vitro to the logarithmic phase and seeded in 6-well plates at a seeding density of 20×10 ⁴ cells/well, with 2 mL of medium per well, and then continued to culture in a cell culture incubator for 24 hours. Dilute miplatin liposomes with medium to 30 μM, and when cells adhere to the wall, replace the medium in the corresponding well with the medium containing miplatin liposomes, and set the wells without miplatin liposomes as a negative control. Discard the medium after continuing to incubate for 24 hours, wash the cells with PBS, wash away the miplatin liposomes adhering to the cell surface, nitrate the cells with 0.25% trypsin and collect them in Ep tubes, centrifuge (800×g, 5min) to obtain Cell pellet. Cells were prefixed in 2.5% glutaraldehyde followed by fixation in 1% osmium tetroxide and 1.5% potassium ferrocyanide for 1 h. Cells were dehydrated with 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 resin. The embedded sample was cut into 60nm thick slices using an ultramicrotome and fixed on a copper grid. Sections were observed using a JEM-1400Plus transmission electron microscope system (JEOL, Japan) (120 kV voltage).

The results showed that miplatin liposomes could cause a large number of vacuolization of endoplasmic reticulum and mitochondria, and it was observed that the vacuolated mitochondria were included in the autophagic vesicles, suggesting that autophagy occurred in mitochondria (Figure 9).

7.2M Platinum liposome-targeted organelles

Miplatin liposomes enter the next step of target organelles after being processed by inclusion bodies and lysosomes containing a large number of degrading enzymes to exert their anti-tumor activity. Electron micrographs suggested that miplatin paper liposomes may have damaged the endoplasmic reticulum and mitochondria and played a role, so we extracted cell organelles to detect the platinum content to determine the direction of platinum. Take the cells treated with miplatin liposomes, extract the nucleus, endoplasmic reticulum, mitochondria and other organelles to detect platinum content, the results show that platinum is mainly concentrated in the endoplasmic reticulum and mitochondria, which is the main content of miplatin liposomes target organelles.

7.3M platinum liposomes induce mitophagy

According to the aggregation of miplatin liposomes in mitochondria and the results of electron microscopy, it was suggested that mitochondria were targeted for autophagy, so we further detected mitochondria to confirm that mitochondria were involved in autophagy

In order to maintain the normal state of cell activity, damaged or unnecessary mitochondria need to be removed in time. Cells selectively clear damaged mitochondria primarily through the autophagic mechanism, a process known as mitophagy. When mitophagy occurs, the mitophagy receptor is first activated, and by binding to the key autophagy protein LC3, LC3 is recruited to the damaged mitochondria, so that the damaged mitochondria are wrapped by autophagosomes. Autophagosomes, which wrap mitochondria, fuse with lysosomes, and damaged mitochondria are released into the lysosomal cavity for final degradation. The occurrence of mitophagy can be proved by detecting mitophagy markers and verifying the co-localization of mitochondria and autophagy signals.

7.3.1 Mitochondria and trigger autophagy marker LC3B

The co-localization of mitochondria and autophagy marker LC3B in rice liposome-treated cells was judged by immunofluorescence. The results showed that the LC3B signal in the cytoplasm co-localized with the mitochondria, indicating that LC3B was recruited into the mitochondria and mitophagy occurred (Fig. 10).

7.3.2 Detection of mitophagy markers

After the cells were treated with miplatin liposomes, the whole cell lysate was taken for western blot detection, and the non-administered group was used as a negative control. The results showed that the key intracellular autophagy protein LC3B, the mitochondrial autophagy receptor BNIP3, and the mitochondrial autophagy receptor The expression of BNIP3L/Nix increased (Figure 11), suggesting the occurrence of mitophagy. Mitochondria were extracted afterwards, the miplatin liposome treatment group was the experimental group, and the non-administration group was used as the negative control group. Western blot was used to detect the LC3B in the mitochondria. The results showed that with the increase of the administration concentration, the enriched LC3B increased (Figure 12), indicating that LC3B produced by autophagy is enriched into mitochondria.

The above results all proved that miplatin liposomes induced the occurrence of mitophagy.

Claims (9)

1. The application of the miboplatin liposome in preparing the drug-resistant tumor medicament, wherein the tumor is pancreatic cancer.

2. The use of claim 1, wherein the human pancreatic cancer cell: aspC-1, bxPC-3, MIA-PaCa-2, PANC-1, SW1990, SU.86.86.

3. The use according to claim 1, characterized in that the human cisplatin-resistant pancreatic cancer cell line PATU8988/DDP.

4. The use as claimed in claim 1, characterized in that the miriplatin liposomes reverse the natural gemcitabine resistance of pancreatic cancer cells MIA-PaCa-2 and PANC-1; the miboplatin liposome reverses the drug resistance of cisplatin-resistant pancreatic cancer cells PATU8988/DDP.

5. 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.

6. The use as claimed in claim 1, wherein the miboplatin liposomes enter the cancer cells primarily through caveolin-mediated endocytosis, and the macropinocytosis pathway assists in the entry of the miboplatin liposomes.

7. 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.

8. 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.

9. 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.

Images