A nanoparticle comprising cisplatin-linoleic acid and sn38-linoleic acid, method of preparation and use

Abstract

The present application belongs to the technical field of anti-tumor drug design, and particularly relates to a nanoparticle containing cisplatin-linoleic acid and SN38-linoleic acid, wherein the nanoparticle is formed by wrapping cisplatin-linoleic acid and SN38-linoleic acid with an amphiphilic polymer, and the amphiphilic polymer is distearoyl phosphatidyl ethanolamine-polyethylene glycol. By wrapping cisplatin-linoleic acid and SN38-linoleic acid prodrug molecules with the amphiphilic polymer, the water solubility of the drug molecules can be improved, and the in-vivo synchronous delivery and transportation of the cisplatin-linoleic acid and SN38-linoleic acid prodrug molecules can be realized, so that the synergistic anti-tumor effect can be achieved. In the mechanism, it is proved that the SN38-linoleic acid prodrug in the co-wrapped nanoparticle can not only cause DNA damage, but also inhibit the DNA damage repair protein Rad51, so as to increase the drug efficacy of the cisplatin-linoleic acid prodrug in drug-resistant tumors, and achieve the best tumor killing effect.

Description

Technical Field

[0001] This invention belongs to the field of antitumor drug design technology, specifically relating to a nanoparticle containing cisplatin-linoleic acid (Pt-LA2) and SN38-linoleic acid (SN38-LA), its preparation method, and its application. Background Technology

[0002] While surgery is an important treatment for NSCLC patients, most are diagnosed at an advanced stage with metastasis, making chemotherapy the primary treatment option. Cisplatin (also known as cis-dichlorodiammineplatinum, abbreviated as Pt) is currently the first-line chemotherapy drug for NSCLC patients in clinical practice. It primarily kills tumor cells by forming DNA-cisplatin adducts, and its therapeutic effect is relatively good. However, long-term use of cisplatin leads to drug resistance in lung cancer, and coupled with the drug's own toxic side effects, the treatment options for lung cancer patients with cisplatin are becoming increasingly limited. Therefore, cisplatin resistance in lung cancer and the safety of cisplatin remain key issues that urgently need to be addressed in clinical practice.

[0003] 7-Ethyl-10-hydroxycamptothecin is the active metabolite of irinotecan (CPT-11) in vivo. It primarily exerts its antitumor activity by inhibiting DNA topoisomerase I, leading to replication fork arrest and ultimately DNA double-strand breaks. Its tumor-killing mechanism differs from cisplatin, and no studies have reported cross-resistance to this drug in cisplatin-resistant tumors, thus demonstrating its potential against cisplatin resistance. Meanwhile, in preclinical studies, SN38 has shown more significant antitumor efficacy than CPT-11. However, SN38 itself has extremely poor water solubility and cannot yet be directly used clinically to replace its water-soluble prodrug, CPT-11. Summary of the Invention

[0004] This invention addresses the problems existing in the prior art by providing nanoparticles containing cisplatin-linoleic acid (Pt-LA2) and SN38-linoleic acid (SN38-LA), a preparation method, and applications. By encapsulating and linking cisplatin-linoleic acid and SN38-linoleic acid prodrug molecules with an amphiphilic polymer, the water solubility of the drug molecules can be improved, and the simultaneous in vivo delivery and transport of the prodrug molecules cisplatin-linoleic acid and SN38-linoleic acid can be achieved, thereby realizing a synergistic anti-tumor effect.

[0005] One object of the present invention is to provide nanoparticles comprising cisplatin-linoleic acid and SN38-linoleic acid, said nanoparticles being formed by encapsulating cisplatin-linoleic acid and SN38-linoleic acid with an amphiphilic polymer;

[0006] The amphiphilic polymer is distearate phosphatidylethanolamine-polyethylene glycol.

[0007] Pt-LA2 was obtained using the preparation method disclosed in Chinese Patent CN109021026B. The structural formula of Pt-LA2 is shown below:

[0008]

[0009] SN38-linoleic acid was obtained using the preparation method disclosed in Chinese Patent CN105315294B. The structural formula of SN38-linoleic acid is shown below:

[0010]

[0011] This invention combines cisplatin, a widely used antitumor drug for lung cancer treatment, with a linoleic acid conjugate of irinotecan's active product SN38. Co-loaded nanoparticles passively target the tumor site, exhibiting synchronized in vivo pharmacokinetic properties. Upon reaching the cell, the prodrug molecule is released, and SN38 induces DNA damage and inhibits DNA damage repair, significantly enhancing the efficacy of cisplatin and reversing drug resistance. Simultaneously, the co-assembly of these two drugs with an amphiphilic polymer to form nanoparticles significantly improves the water solubility of both drugs, enabling intravenous administration and reducing the hepatotoxicity and nephrotoxicity associated with rapid drug release.

[0012] Preferably, the amphiphilic polymer is distearylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG). 2k ).

[0013] Preferably, the total mass ratio of cisplatin-linoleic acid and SN38-linoleic acid to the amphiphilic polymer in the nanoparticles is (7-15):1.

[0014] Preferably, the molar ratio of cisplatin to SN38 in the nanoparticles is 1:10 to 10:1.

[0015] Preferably, the molar ratio of cisplatin to SN38 in the nanoparticles is 5:1.

[0016] Preferably, the average particle size of the nanoparticles is <200 nm.

[0017] Another objective of this invention is achieved through the following technical solution: a method for preparing the nanoparticles containing cisplatin-linoleic acid and SN38-linoleic acid, comprising the following steps:

[0018] Cisplatin-linoleic acid, SN38-linoleic acid, and an amphiphilic polymer were dissolved in an organic solvent and mixed evenly. The mixture was then added dropwise to an aqueous phase while being sonicated, followed by dialysis to remove the organic solvent, resulting in uniformly dispersed nanoparticles.

[0019] Preferably, the organic solvent is one or more of dimethyl sulfoxide, acetone, methanol, and ethanol.

[0020] Another objective of the present invention is achieved through the following technical solution: a pharmaceutical preparation comprising the nanoparticles containing cisplatin-linoleic acid and SN38-linoleic acid.

[0021] Preferably, the pharmaceutical preparation is one of tablets, granules, capsules, emulsions, or suspensions.

[0022] Another objective of the present invention is achieved through the following technical solution: the application of the nanoparticles containing cisplatin-linoleic acid and SN38-linoleic acid in an antitumor drug, wherein the antitumor drug comprises the nanoparticles containing cisplatin-linoleic acid and SN38-linoleic acid and pharmaceutical excipients.

[0023] This invention uses DSPE-PEG 2k Pt-LA2 and SN38-LA were non-covalently encapsulated into nanoparticles for in vivo delivery. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) 2k In the composition, DSPE is hydrophobic and PEG is hydrophilic, DSPE-PEG 2k In an aqueous phase, nanoparticles are assembled, with DSPE forming the core and PEG forming the crown. Upon intravenous injection, the DSPE-PEG combination has shown promising results. 2k The PEG crown in the nanoparticles protects them from phagocytosis by the reticuloendothelial system, endowing the drug with long-circulation properties in the bloodstream. Furthermore, encapsulating Pt-LA2 and SN38-LA into nanoparticles significantly improves their water solubility, enabling intravenous injection. In addition, the resulting nanoparticles allow for simultaneous delivery and slow release of the drug in vivo, avoiding hepatotoxicity and nephrotoxicity caused by rapid, large-volume drug release. They also passively accumulate at the tumor site through the high permeability and retention effect (EPR), thereby significantly reducing systemic toxicity while exerting higher antitumor activity.

[0024] The nanoparticles (Pt) containing cisplatin-linoleic acid prepared in this invention (IV) The average particle size of nanoparticles containing SN38-linoleic acid (SN38-NPs) and nanoparticles co-encapsulated by both (Cocktail-NPs) is less than 200 nm. The small particle size of the nanoparticles makes them easier to accumulate at the tumor site through the EPR effect, thus better exerting anti-tumor effects and reducing damage to normal tissues.

[0025] This invention verified through in vitro cytotoxicity experiments that the co-loaded nanoparticles of cisplatin-linoleic acid and SN38-linoleic acid (Cocktail-NPs) are more effective than Pt alone in cytotoxicity against cisplatin-resistant cells.(IV) Cisplatin-linoleic acid (CLA) or SN38-linoleic acid alone, and the combination of the two drugs significantly reduced the half-maximal inhibitory concentration (IC50) of tumor cells treated with the single drug and showed a good combination index (CI), indicating that CLA and SN38-linoleic acid exhibit a good synergistic effect in the co-loaded nanoparticle system. This was observed in cisplatin-acquired resistant lung cancer cells A549. cisR In this study, the molar ratio of cisplatin to SN38 of 5:1 showed the lowest IC50 and the best combination index (CI50).

[0026] This invention also provides evidence that co-loaded nanoparticles promote apoptosis in vitro, and that the combination therapy of nanoparticles co-loaded with cisplatin-linoleic acid and SN38-linoleic acid is superior to Pt alone in drug-resistant non-small cell lung cancer. (IV) -NPs significantly promote apoptosis. This indicates that the combined use of the two drugs has a synergistic effect in promoting apoptosis.

[0027] This invention demonstrates, through comet electrophoresis experiments, that treatment with co-loaded nanoparticles of cisplatin-linoleic acid and SN38-linoleic acid prodrugs induces widespread DNA damage in drug-resistant cells. Tail DNA content (%tail DNA) and tail moment (TM) are important indicators for measuring the degree of cellular DNA damage. Both of these indicators significantly increased in drug-resistant cells after treatment with SN38-NPs and co-loaded nanoparticles of cisplatin-linoleic acid and SN38-linoleic acid prodrugs, proving that the co-loaded nanoparticles induce DNA damage and are a direct cause of increased apoptosis.

[0028] This invention demonstrates, through Western blotting experiments, the mechanism by which prodrug-coated nanoparticles synergistically resist cisplatin resistance. Due to the presence of Rad51, a protein involved in cisplatin DNA damage repair, Pt... (IV) -NPs alone are not significantly effective against drug-resistant cells, and the accumulation of the DNA damage marker protein γH2AX within cells is not obvious. Drug-resistant cells are unable to repair the types of DNA damage caused. Therefore, SN38-NPs alone can lead to the accumulation of the intracellular damage marker protein γH2AX. At the same time, SN38-NPs can also inhibit the repair protein Rad51, thereby enhancing Pt (IV) The therapeutic effect of -NPs. Therefore, after treatment with prodrug co-loaded nanoparticles, the damage marker protein γH2AX in drug-resistant cells was most significantly upregulated. As a result, intracellular apoptosis-related proteins (including P53, p-P53, c-Caspase 3, and c-PARP) were all significantly upregulated, and drug-resistant cells were cleared.

[0029] This invention further provides in vivo evaluation of the efficacy of co-loaded nanoparticles. Results showed that, compared to the saline control group, the combination therapy of high-dose and low-dose co-loaded nanoparticles reduced tumor volume by 7.5-fold and 4.0-fold, respectively; correspondingly, the combination therapy of high-dose and low-dose clinical formulations reduced tumor volume by only 2.3-fold and 1.7-fold, respectively. This demonstrates that the combination therapy based on co-loaded nanoparticles is significantly more effective than existing clinical formulations. The in vivo test results provide strong evidence for the superiority of the nanoparticles containing cisplatin-linoleic acid and SN38-linoleic acid provided by this invention, and have broad application prospects.

[0030] The present invention has the following advantages over the prior art:

[0031] (1) The encapsulation material used in this invention is distearylphosphatidylethanolamine-polyethylene glycol, which is non-toxic, harmless, easily degradable in vivo, and highly biocompatible.

[0032] (2) The present invention uses distearylphosphatidylethanolamine-polyethylene glycol to encapsulate cisplatin-linoleic acid and SN38-linoleic acid, which improves the aqueous solubility of Pt-LA2 and SN38-LA, and provides the possibility that small-dose intravenous administration can also play an effective role in inhibiting tumors;

[0033] (3) The nanoparticles (co-supported nanoparticles) containing cisplatin-linoleic acid and SN38-linoleic acid prepared by the present invention have a particle size in the range of 200 nm. The small particle size makes it easy to passively accumulate in the tumor site and exert drug efficacy through the EPR effect at the tumor site.

[0034] (4) The distearate phosphatidylethanolamine-polyethylene glycol used in this invention is amphiphilic. Pt-LA2 and SN38-LA are located in the hydrophobic core of the nanoparticles and are protected by the PEG crown from being phagocytosed by the reticuloendothelial system, giving the drug long circulation properties in the blood circulation. The prodrug co-loaded nanoparticles can remain stable in PBS solution and PBS solution containing 20% ​​FBS.

[0035] (5) The co-loaded nanoparticles used in this invention can achieve the controllable synchronous delivery and release of two drugs, enabling drugs with different functions to work simultaneously in drug-resistant tumors. This drug delivery method is of great significance to the current research on anti-tumor drug delivery systems.

[0036] (6) This invention demonstrates that SN38-LA in co-loaded nanoparticles not only directly induces DNA damage in drug-resistant tumors, but also inhibits the DNA repair protein Rad51, thereby enhancing the efficacy of Pt-LA2. This provides a scientific explanation and basis for the clinical application of this therapy in drug-resistant tumors.

[0037] (7) Cisplatin-linoleic acid and SN38-linoleic acid drugs exhibit good synergistic effects in the co-loaded nanoparticle system, especially when the molar ratio of cisplatin to SN38 is 5:1, it shows the lowest IC50 and the best combination index (CI50).

[0038] (8) In vivo studies have shown that the combination therapy based on co-delivered nanoparticles has a significantly better anti-tumor effect than the current clinical formulations. Furthermore, this therapy has a minimal impact on body weight, indicating that it has few toxic side effects, good safety, and is more likely to enter clinical trials and obtain approval for marketing. Attached Figure Description

[0039] Figure 1 Cisplatin-linoleic acid, SN38-linoleic acid, and DSPE-PEG 2k The structural formula and a schematic diagram of the formation of nanoparticles containing cisplatin-linoleic acid and SN38-linoleic acid;

[0040] Figure 2 a is Pt (IV) Transmission electron microscopy (TEM) images of -NPs, SN38-NPs, and Cocktail-NPs nanoparticles. Figure 2 b is Pt (IV) Particle size distribution diagrams of three types of nanoparticles: -NPs, SN38-NPs, and Cocktail-NPs. Figure 2 c is Pt (IV) Zeta potential of three types of nanoparticles: -NPs, SN38-NPs, and Cocktail-NPs. Figure 2 d is Pt (IV) The stability of -NPs, SN38-NPs and Cocktail-NPs nanoparticles in PBS solution. Figure 2 e is Pt (IV) Stability of -NPs, SN38-NPs and Cocktail-NPs nanoparticles in PBS solution containing 20% ​​FBS;

[0041] Figure 3 a and b are Pt (IV) In vitro cytotoxicity of cisplatin-sensitive cells (a) and drug-resistant cells (b) to Cocktail-NPs, SN38-NPs and Cocktail-NPs nanoparticles in different molar ratios; Figure 3 c represents the IC50 values ​​of the two cell lines under various treatments; Figure 3 d is the combination index (CI50) of cisplatin and SN38 at different molar ratios in nanoparticles containing cisplatin-linoleic acid and SN38-linoleic acid in two cell lines;

[0042] Figure 4a and b are Pt (IV) The study and quantitative results of the effects of SN38-NPs and Cocktail-NPs nanoparticles on apoptosis in cisplatin-resistant non-small cell lung cancer. Figure 4 c and d represent the evaluation of Pt using comet electrophoresis, respectively. (IV) Research and quantitative results on DNA damage caused by -NPs, SN38-NPs and Cocktail-NPs nanoparticles. Figure 4 e is to study the synergistic mechanism of Cocktail-NPs nanoparticles against cisplatin resistance using protein immunoblotting;

[0043] Figure 5 a represents the curve showing the change in tumor size in nude mice after different administration methods. Figure 5 b represents the average inhibition rate of different drug treatments on tumors in nude mice. Figure 5 c represents the tumor proliferation status after different drug treatments. Figure 5 d is Figure 5 c. Statistical graph of tumor proliferation. Figure 5 e represents the change in body weight of the nude mice during the observation period. Detailed Implementation

[0044] The technical solutions of the present invention will be further described and illustrated below with reference to specific embodiments and accompanying drawings, but these descriptions do not constitute a limitation on the present invention. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commonly used in the art, and the methods used in the embodiments are all conventional methods in the art.

[0045] Example 1

[0046] In this embodiment, the nanoparticles containing cisplatin-linoleic acid and SN38-linoleic acid were prepared by precipitation method, as shown in the schematic diagram below. Figure 1 As shown, the specific steps are as follows:

[0047] Weigh 2.86 mg of Pt-LA2 containing 1 mg cisplatin and 1.67 mg of SN38-LA containing 1 mg SN38 using an electronic balance, and dissolve them separately in 500 μL of dimethyl sulfoxide. Then weigh 0.45 mg of DSPE-PEG. 2k Dissolve the above three substances in 500 μL of dimethyl sulfoxide, then mix them together. Add the mixture dropwise at 25°C to a beaker containing 9 mL of secondary ultrapure water, which is then placed in a water bath for sonication. After the addition is complete, stir evenly and stably for 10 min. Then, remove excess dimethyl sulfoxide through a dialysis bag (molecular weight cutoff 1 kDa), concentrate the solution using an ultrafiltration tube, and bring the final volume to 10 mL to obtain an aqueous solution of co-loaded drug nanoparticles containing Pt-LA2 and SN38-LA with an effective amount of 0.1 mg / mL, named Cocktail-NPs.

[0048] Example 2

[0049] The nanoparticles containing cisplatin-linoleic acid and SN38-linoleic acid in this embodiment were prepared by the following method:

[0050] Weigh 5.14 mg of Pt-LA2 containing 6 μmol cisplatin and 0.39 mg of SN38-LA containing 0.6 μmol SN38 using an electronic balance, and dissolve them separately in 500 μL of dimethyl sulfoxide. Then weigh 0.5 mg of DSPE-PEG. 2k Dissolve in 500 μL of dimethyl sulfoxide, then mix the above three solutions, and follow the same steps as in Example 1 to obtain an aqueous solution of nanoparticles containing Pt-LA2 and SN38-LA with a molar ratio of cisplatin to SN38 of 10:1.

[0051] Example 3

[0052] The nanoparticles containing cisplatin-linoleic acid and SN38-linoleic acid in this embodiment were prepared by the following method:

[0053] 4.72 mg of Pt-LA2 containing 5.5 μmol cisplatin and 0.72 mg of SN38-LA containing 1.1 μmol SN38 were weighed using an electronic balance and dissolved in 500 μL of dimethyl sulfoxide. The subsequent steps were the same as in Example 2, resulting in an aqueous solution of nanoparticles containing Pt-LA2 and SN38-LA with a cisplatin to SN38 molar ratio of 5:1.

[0054] Example 4

[0055] The nanoparticles containing cisplatin-linoleic acid and SN38-linoleic acid in this embodiment were prepared by the following method:

[0056] 3.77 mg of Pt-LA2 containing 4.4 μmol cisplatin and 1.44 mg of SN38-LA containing 2.2 μmol SN38 were weighed using an electronic balance and dissolved in 1000 μL of dimethyl sulfoxide. The subsequent steps were the same as in Example 2, to obtain an aqueous solution of nanoparticles containing Pt-LA2 and SN38-LA with a cisplatin to SN38 molar ratio of 2:1.

[0057] Example 5

[0058] The nanoparticles containing cisplatin-linoleic acid and SN38-linoleic acid in this embodiment were prepared by the following method:

[0059] 2.83 mg of Pt-LA2 containing 3.3 μmol cisplatin and 2.16 mg of SN38-LA containing 3.3 μmol SN38 were weighed using an electronic balance and dissolved in 1000 μL of dimethyl sulfoxide. The subsequent steps were the same as in Example 2, to obtain an aqueous solution of nanoparticles containing Pt-LA2 and SN38-LA with a cisplatin to SN38 molar ratio of 1:1.

[0060] Example 6

[0061] The nanoparticles containing cisplatin-linoleic acid and SN38-linoleic acid in this embodiment were prepared by the following method:

[0062] 1.89 mg of Pt-LA2 containing 2.2 μmol cisplatin and 2.88 mg of SN38-LA containing 4.4 μmol SN38 were weighed using an electronic balance and dissolved in 500 μL of dimethyl sulfoxide. The subsequent steps were the same as in Example 2, resulting in an aqueous solution of nanoparticles containing Pt-LA2 and SN38-LA with a cisplatin to SN38 molar ratio of 1:2.

[0063] Example 7

[0064] The nanoparticles containing cisplatin-linoleic acid and SN38-linoleic acid in this embodiment were prepared by the following method:

[0065] 0.94 mg of Pt-LA2 containing 1.1 μmol cisplatin and 3.60 mg of SN38-LA containing 5.5 μmol SN38 were weighed using an electronic balance and dissolved in 500 μL of dimethyl sulfoxide. The subsequent steps were the same as in Example 2, resulting in an aqueous solution of nanoparticles containing Pt-LA2 and SN38-LA with a cisplatin to SN38 molar ratio of 1:5.

[0066] Example 8

[0067] The nanoparticles containing cisplatin-linoleic acid and SN38-linoleic acid in this embodiment were prepared by the following method:

[0068] Weigh 0.51 mg of Pt-LA2 containing 0.6 μmol cisplatin and 3.93 mg of SN38-LA containing 6 μmol SN38 using an electronic balance, and dissolve them in 500 μL of dimethyl sulfoxide. The subsequent steps are the same as in Example 2, to obtain an aqueous solution of nanoparticles containing Pt-LA2 and SN38-LA with a cisplatin to SN38 molar ratio of 1:10.

[0069] Comparative Example 1

[0070] Comparative Example 1 consists of Pt-LA2 nanoparticles, which were prepared by the following method:

[0071] Weigh 2.86 mg of Pt-LA2 containing 1 mg cisplatin into 500 μL of dimethyl sulfoxide using an electronic balance, then weigh 0.29 mg of DSPE-PEG. 2k Dissolve the above three solutions in 500 μL of dimethyl sulfoxide (DMSO). Then, mix the three solutions together and add them dropwise to a beaker containing 9 mL of secondary ultrapure water, which is then placed in a water bath and sonicated. After the addition is complete, stir evenly and stably for 10 min. Subsequently, remove excess DMSO through a dialysis bag (molecular weight cutoff 1 kDa), concentrate the solution using an ultrafiltration tube, and bring the final volume to 10 mL to obtain a 0.1 mg / mL aqueous solution containing Pt-LA2 nanoparticles, named Pt. (IV) -NPs.

[0072] Comparative Example 2

[0073] Comparative Example 2 consists of nanoparticles containing SN38-LA, which were prepared by the following method:

[0074] Weigh 1.67 mg of SN38-LA containing 1 mg SN38 and dissolve it in 500 μL of dimethyl sulfoxide using an electronic balance. Then weigh 0.17 mg of DSPE-PEG. 2k Dissolve the above three substances in 500 μL of dimethyl sulfoxide, then mix them together. Add the mixture dropwise at 25°C to a beaker containing 9 mL of secondary ultrapure water, which is then placed in a water bath for sonication. After the addition is complete, stir evenly and stably for 10 min. Then, remove excess dimethyl sulfoxide through a dialysis bag (molecular weight cutoff 1 kDa), concentrate the solution using an ultrafiltration tube, and bring the final volume to 10 mL to obtain a 0.1 mg / mL aqueous solution containing SN38-LA nanoparticles, named SN38-NPs.

[0075] (1) Particle size and zeta potential of nanoparticles

[0076] The morphology of the particles was observed using transmission electron microscopy (TEM). Sample preparation: 2 mL of nanoparticles from Example 1 and Comparative Examples 1-2 were respectively added to ultrafiltration tubes and centrifuged at 2500 rpm for 6-12 minutes. After concentration to 400 μL, nanoparticles with a concentration of approximately 0.5 mg / mL were obtained. A drop of the 0.5 mg / mL nanoparticle solution was placed on a copper grid, negatively stained with 2% uranium acetate, and observed under a transmission electron microscope after air drying. The results are as follows: Figure 2 As shown in figure a. The particle size distribution and zeta potential of the nanoparticles were measured using a dynamic light scattering instrument. The particle size and its distribution (PDI) and zeta potential of the nanoparticles were measured at 25℃, the equilibration time was 120 seconds, the laser incident wavelength was 633nm, and the incident angle was 90°. Each sample was tested in triplicate, and the obtained particle size and potential are the average values ​​of the samples. Pt (IV)The transmission electron microscopy observation results and particle size analysis results of -NPs, SN38-NPs, and Cocktail-NPs are as follows: Figure 2 As shown in a and 2b, the Pt of the present invention can be seen. (IV) The co-loaded nanoparticles of -NPs, SN38-NPs, and Cocktail-NPs all have a particle size in the range of 200 nm. Their small particle size makes them easy to accumulate at the tumor site and exert drug effects through the EPR effect at the tumor site.

[0077] (2) Observation of the in vitro stability of nanoparticles

[0078] The prepared nanoparticles of Example 1 and Comparative Examples 1-2 were placed in 1 mL of PBS solution (pH 7.4, 0.01 M) or PBS solution containing 20% ​​FBS (pH 7.4, 0.01 M), respectively. The particle size changes of each nanoparticle were measured using a dynamic light scattering instrument at 0, 1, 2, 3, 4, 5, 6, and 7 days. The results are as follows. Figure 2 As shown in d and 2e, the nanoparticles of Example 1 and Comparative Examples 1-2 all exhibit good stability.

[0079] (3) Investigate the toxic effects of nanoparticles on non-small cell lung cancer cells.

[0080] The inhibitory effects of nanoparticles from Examples 2-8 and Comparative Examples 1-2 on the growth of cisplatin-sensitive and drug-resistant tumor cells were investigated using the CCK-8 assay, as detailed below:

[0081] Log-grown cisplatin-sensitive A549 cells and cisplatin-resistant A549 cells were collected. cisR Cells were digested with trypsin, centrifuged, and the supernatant was discarded. The cells were resuspended in 5 mL of culture medium, and cell counts were performed. The cell suspension density was adjusted based on the count results, and 3000 cells were seeded per well in 96-well plates. After overnight incubation, different concentrations of Pt were added to the cells. (IV) -NPs, SN38-NPs, and prodrugs co-loaded with nanoparticles at different concentrations and molar ratios (10:1, 5:1, 2:1, 1:1, 1:2, 1:5, or 1:10) were cultured at 37°C for 72 hours. Cell viability was assessed using the CCK-8 assay at the drug treatment endpoint. Cells were further cultured in CCK-8 reagent-containing medium at 37°C for 1-3 hours, and the absorbance of each well was measured at 450 nm using a microplate reader. Cell viability (%) was calculated as: Cell viability (%) = (At-Ab) / (Acon-Ab) × 100%, where At, Acon, and Ab are the absorbance of the treatment group, untreated control group, and blank well, respectively. The corresponding values ​​were imported into Graphpad Prism 8.0.2 software to fit cell survival curves and calculate the half-inhibitory concentration (IC50).50 The drug concentrations and corresponding inhibition rates (FA; 1 - cell viability / 100) of each treatment group were imported into Calculusyn software for analysis of the two-drug combination index. (IV) The synergistic effect of -NPs and SN38-NPs at different molar ratios was analyzed using the combination drug index (CI) at IC50. 50 The evaluation is performed using the following formula: CI 50 =C Pt-NPs / C50 Pt-NPs +C SN38-NPs / C50 SN38-NPs C50 Pt-NPs and C50 SN38-NPs The corresponding treatment in IC 50 The drug concentration below, while C Pt-NPs and C SN38-NPs These are the concentrations of the two drugs that produce the same inhibitory effect in cells when used in combination. For example... Figure 3 As shown, individual Pt can be observed. (IV) -NPs nanoparticles are not highly toxic to tumor cells, but the IC50 value is significantly reduced when used in combination with SN38-NPs nanoparticles. (IV) -NPs nanoparticles and SN38-NPs nanoparticles exhibit a significant synergistic effect; when mixed, Pt (IV) -NPs nanoparticles significantly enhance the killing ability of both drug-resistant and drug-resistant cells, especially when the molar ratio of cisplatin to SN38 is 5:1.

[0082] (4) To investigate the apoptosis-promoting effect, DNA damage induction, and synergistic mechanism of the two drugs in cisplatin-resistant non-small cell lung cancer by nanoparticle system.

[0083] Apoptosis experiment: Cisplatin-resistant cells were cultured at 3×10⁻⁶ cells per cell line 5 The wells were seeded at a density suitable for each well in a six-well plate. After overnight adhesion, 10 μM Pt was added. (IV) -NPs, 2μM SN38-NPs, and nanoparticles encapsulating Pt-LA2 and SN38-LA at equal concentrations were incubated under the same conditions for 24 hours. At the end of incubation, cells in each well were digested with trypsin, washed with PBS, and centrifuged to prepare cell pellets. Cells were then resuspended in 600μL of binding buffer containing Annexin V-FITC / PI staining solution and incubated at room temperature in the dark for 20 minutes to complete staining. Each sample was analyzed by flow cytometry. Figure 4 As shown in a and b, the co-loaded nanoparticles encapsulating Pt-LA2 and SN38-LA significantly increased the apoptosis rate of drug-resistant lung cancer cells.

[0084] Comet electrophoresis experiment: Drug-resistant cells were seeded in six-well plates using the same method. 10 μM t was added to each well. (IV) -NPs, 2μM SN38-NPs, and nanoparticles encapsulating Pt-LA2 and SN38-LA at equal concentrations were added and cultured for another 24 hours. Upon reaching the experimental endpoint, cells were digested with trypsin and washed with PBS to prepare a cell suspension with a density of 10⁻⁶. 6 Cells / mL. The agarose gel coating of cells was prepared in three steps: 1. 100 μL of microwave-dissolved 0.5% constant-melting-point agarose was spread onto the slide, covered with a coverslip, and placed at 4°C to solidify; 2. The coverslip was removed, 10 μL of the suspension was mixed with 90 μL of preheated 0.7% low-melting-point agarose, and dropped onto the first layer of agarose. The coverslip was then placed at 4°C to solidify; 3. The coverslip was removed, 100 μL of preheated 0.7% low-melting-point agarose was added, and the coverslip was placed at 4°C in a high-humidity environment for 30 minutes to solidify. The coverslip was removed, and the slide was immersed in lysis buffer at 4°C for 1 hour for lysis. After rinsing with PBS, the slide was placed in alkaline electrophoresis buffer at room temperature for 30-60 minutes to complete DNA unwinding. Electrophoresis was then performed in an electrophoresis tank at 25V for 20-40 minutes. Remove the slide, neutralize with 0.4 mM Tris-HCl buffer, and stain with 20 μL PI in the dark for 10 minutes. Finally, observe nuclear DNA and migrating DNA under a fluorescence microscope and take pictures. Analyze the tail moment (TM) and comet tail content (% DNA in tail) of each cell using Comet Assay Software Project (CASP, version 1.2.3). Figure 4 As shown in c and d, it can be observed that the co-loaded nanoparticles encapsulating Pt-LA2 and SN38-LA can significantly damage the DNA of drug-resistant lung cancer cells.

[0085] Protein immunoblotting assay: Drug-resistant cells were seeded in six-well plates using the same method, and 10 μM Pt was added. (IV)After treatment with 2 μM N38-NPs and nanoparticles of equal concentration encapsulating Pt-LA2 and SN38-LA for a specified time, the cell pellet was collected by trypsin digestion and centrifugation. Each sample was vortexed with 70 μL of lysis buffer containing protease and phosphatase inhibitors, incubated on ice for 20 minutes, and then centrifuged at 12000 rpm for 10 minutes to collect the supernatant. The total protein concentration of each sample was determined by the BCA method and standardized using 1× loading buffer. After denaturation by boiling in a metal bath, equal volumes of protein samples from each group were subjected to gel electrophoresis at a constant voltage of 80V until the leading edge reached the bottom of the gel. The protein was then transferred to a 25 μm polyvinylidene fluoride (PVDF) membrane using wet transfer. The membrane was blocked for 1 hour in TBST buffer containing 5% skim milk powder. The PVDF membrane was then placed in the appropriate primary antibody working solution and incubated overnight at 4°C. After washing three times with 1×TBST, the PVDF membrane was incubated at room temperature for 1 hour in the secondary antibody working solution. After washing three times with 1×TBST, the sample was scanned using an ECL chromogenic reagent in a chemiluminescence imaging system, and the results were saved. For example... Figure 4 As shown in e, after drug-resistant cells were treated with co-loaded nanoparticles containing Pt-LA2 and SN38-LA for different durations, the expression levels of DNA damage marker proteins in the cells significantly increased, while DNA damage repair proteins significantly decreased. As a result, the expression levels of apoptosis-related proteins in the cells significantly increased.

[0086] (5) Trial of prodrug co-loaded nanoparticles inhibiting the growth of cisplatin-resistant small cell lung cancer xenograft tumors

[0087] A549 cisR The antitumor effect of the prodrug co-loaded nanoparticles from Example 1 on a human cisplatin-resistant non-small cell lung cancer xenograft nude mouse model was evaluated. The tumors were evaluated when the subcutaneous tumor volume in the nude mice reached 100 mm². 3 Initially, mice were randomly assigned to five groups of six each, receiving saline, low-dose clinical-grade combination therapy (3 mg / kg cisplatin + 3 mg / kg irinotecan), high-dose clinical-grade combination therapy (5 mg / kg cisplatin + 5 mg / kg irinotecan), low-dose prodrug co-loaded nanoparticle therapy (3 mg / kg equivalent Pt-LA2 prodrug + 3 mg / kg equivalent SN38-LA prodrug), and high-dose prodrug co-loaded nanoparticle therapy (5 mg / kg equivalent Pt-LA2 prodrug + 5 mg / kg equivalent SN38-LA prodrug). Administration was via tail vein injection on days 0, 3, and 6. During treatment, subcutaneous tumor length and width were measured every three days to calculate tumor volume, and body weight changes were monitored to assess tumor-suppressive efficacy and toxicity. The tumor-suppressive effects of each drug are as follows: Figure 5As shown in the figure, Free combo (LD) represents the combination therapy of low-dose clinical formulation cisplatin and CPT-11, Free combo (HD) represents the combination therapy of high-dose clinical formulation cisplatin and CPT-11, NP combo (LD) represents low-dose nanoparticles containing cisplatin-linoleic acid and SN38-linoleic acid, and NP combo (HD) represents high-dose nanoparticles containing cisplatin-linoleic acid and SN38-linoleic acid; * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. As can be observed from the figure, compared with the control group saline, the combination therapy of low-dose and high-dose clinical cisplatin and irinotecan was not as effective as the therapy of co-loaded nanoparticles containing Pt-LA2 and SN38-LA at the same dose. Immunohistochemical results showed that the tumor proliferation rate after treatment with co-loaded nanoparticles was indeed much lower than that of the combination therapy of the same dose clinical dosage form and the control group. Moreover, the high- and low-dose co-loaded nanoparticles had little effect on body weight, indicating that the toxicity was low, which further proves the advantages of the present invention.

[0088] The specific embodiments described herein are merely illustrative of the spirit of the invention and do not limit the scope of protection of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. Use of nanoparticles comprising cisplatin-linoleic acid and SN38-linoleic acid for the preparation of an antitumor medicament, characterized in that, The nanoparticles are formed by wrapping cisplatin-linoleic acid and SN38-linoleic acid with amphiphilic polymers; the amphiphilic polymers are distearoyl phosphatidyl ethanolamine-polyethylene glycol; In the nanoparticles, the molar ratio of cisplatin and SN38 is 1:10-10:1; The antitumor drug comprises the nanoparticles; The tumor is cisplatin-resistant non-small cell lung cancer; The cisplatin-linoleic acid has a structural formula as shown in Formula I: Formula I; The SN38-linoleic acid has a structural formula as shown in Formula II: Formula II.

2. Use according to claim 1, characterized in that, The amphiphilic polymers are distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000.

3. Use according to claim 1, characterized in that, In the nanoparticles, the mass ratio of cisplatin-linoleic acid and SN38-linoleic acid to the amphiphilic polymers is (7-15):

1.

4. Use according to claim 1, characterized in that, In the nanoparticles, the molar ratio of cisplatin and SN38 is 5:

1.

5. The use according to claim 1, characterized in that, The average particle size of the nanoparticles is <200 nm.

6. The use according to claim 1, characterized in that, The antitumor drug comprises the nanoparticles and pharmaceutical excipients.

7. The use according to claim 1, characterized in that, The preparation method of the nanoparticles comprises the following steps: The cisplatin-linoleic acid, SN38-linoleic acid and amphiphilic polymers are dissolved in an organic solvent and mixed uniformly, then dropped into an aqueous phase under ultrasonic treatment, and then dialyzed to remove the organic solvent to obtain uniformly dispersed nanoparticles.

8. Use according to claim 7, characterized in that, The organic solvent is one or more of dimethyl sulfoxide, acetone, methanol and ethanol.

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