Targeted nanoformulation for treatment of melanoma and carriers and uses thereof

Abstract

The application discloses a drug targeting delivery carrier, a preparation method and application, and has the following structure: the carrier is non-toxic, has good biocompatibility, and is simple to prepare, and is favorable for penetrating tumor tissues. In addition, the carrier has high drug loading and encapsulation rates, and can effectively encapsulate hydrophobic antitumor drugs. The application also discloses a drug combination with combined anti-melanoma efficacy and application, and the drug combination comprises Afuresertib (ASB) and Celecoxib (CXB). A series of experiments on a cell level show that ASB has a strong inhibiting effect on tumor growth, and CXB can promote macrophage reprogramming, improve a tumor immunosuppressive microenvironment, and combined administration of Afuresertib and Celecoxib can reduce the risk of side effects caused by single drug action, and the drug combination can be applied to melanoma treatment.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to drug carriers, drug compositions, and their preparation and application, specifically to targeted nano-formulations for the treatment of melanoma, their carriers, and their applications. Background Technology

[0002] Melanoma is a type of skin cancer, and its incidence has increased significantly worldwide in recent years. Furthermore, malignant melanoma is the leading cause of death from skin cancer, accounting for over 90% of all skin cancer-related deaths. Malignant melanoma is a highly malignant tumor originating from melanocytes in the skin and other tissues, with an insidious onset, and it is more prevalent in people with light skin. Factors contributing to melanoma include genetic susceptibility, skin melanin deposition, and environmental factors such as ultraviolet radiation.

[0003] Early diagnosis of melanoma is crucial for its treatment, and drug resistance is a significant characteristic of melanoma. Currently, surgery, chemotherapy, and immunotherapy are the most common treatments. However, these methods are largely limited by advanced cancer diagnosis, drug off-target effects, systemic toxicity, and adverse drug reactions. While early-stage melanoma can generally be surgically removed, surgery often fails to achieve the desired results in malignant melanoma. Chemotherapy is currently the most commonly used cancer treatment, with drugs such as vemurafenib, dacarbazine, and paclitaxel used clinically. Chemotherapy drugs typically show good initial efficacy, but later, patients experience severe side effects and drug resistance. Furthermore, chemotherapy causes significant toxic side effects on the nervous system, kidneys, and liver, severely impacting patients' quality of life. To overcome the limitations of current melanoma treatments, new approaches are needed. The emergence of nanomedicine delivery systems can precisely target tumors, reducing side effects while increasing drug concentration at the tumor site. Nanoparticle drug delivery systems passively target tumor tissues through the EPR (Enhanced Permeability and Retention Effect) and other mechanisms, improving drug bioavailability through sustained drug release while preventing clearance by the reticuloendothelial system and reducing systemic toxicity. Furthermore, the intracellular environment of tumors is currently considered a decisive factor influencing cancer treatment. Of particular interest are nanoscale drug delivery systems using molecularly targeted ligands, such as antibodies, peptides, and fatty acids, applied to cancer therapy. These targeted and personalized nanoparticle drug delivery systems increase targeted drug accumulation in the tumor region based on the molecular characteristics and microenvironment of tumor cells, and can control drug release, thereby reducing side effects and further lowering the risk of treatment for patients. IL4RPep-1 is an IL4R-binding peptide that preferentially binds to the highly expressed IL4R receptor on the surface of melanoma cells in the tumor microenvironment. Therefore, in this invention, we enhance the active targeting capability of the drug delivery system by modifying the surface of the nanocarrier with IL4RPep-1.

[0004] Currently, traditional drug therapies are severely limited by their lack of therapeutic efficacy, high toxicity to healthy tissues, and non-specificity towards cancer cells. Furthermore, traditional chemotherapy drugs generally exhibit poor stability and solubility in the bloodstream, lacking selective delivery capabilities. Recent studies have found that single-drug therapy may show good efficacy in treating early-stage cancers, but the clinical efficacy decreases with prolonged treatment, possibly related to acquired multidrug resistance in tumor cells during treatment. The emergence of acquired multidrug resistance leads to the rapid clearance of large amounts of anticancer drugs (DOX, PTX, dabrafenib, etc.) from tumor cells, rendering low levels of the drug ineffective in killing tumor cells, ultimately leading to chemotherapy failure. This means patients must switch to other new drug treatments; however, a new drug may bring more potential side effects and higher treatment costs.

[0005] In recent years, studies have found that combination chemotherapy, which administers two or more drugs with similar or different pharmacological mechanisms simultaneously, is often a superior treatment strategy compared to monotherapy. ASB is a biologically bioavailable, orally available serine / threonine protein kinase Akt inhibitor. CXB, as a selective inhibitor of cyclooxygenase-2 (COX-2), is mainly used clinically as an anti-inflammatory and analgesic drug.

[0006] There are currently no reports of using a combination of ASB and CXB nanomedicine delivery systems to treat melanoma. Summary of the Invention

[0007] To address the aforementioned technical problems in the prior art, this invention provides targeted nano-formulations for the treatment of melanoma, their carriers, and their applications.

[0008] The first objective of this invention is to provide a simple and practical nanomedicine delivery carrier with biodegradability and targeted delivery, and to further verify the good anticancer effect and low toxicity of the nanodelivery system in in vitro cell experiments.

[0009] A nanomedicine delivery carrier, wherein the delivery carrier is IL4RPep-1-polylactic acid-glycolic acid copolymer-polyethylene glycol nanoparticles, and the chemical structure of the IL4RPep-1-polylactic acid-glycolic acid copolymer-polyethylene glycol nanoparticles is shown in Formula I:

[0010]

[0011] Wherein, IL4RP is IL4RPep-1, and n = 50, m = 40-70.

[0012] Furthermore, the molecular weight of polylactic acid-glycolic acid copolymer is 38,000-54,000 Da; the molecular weight of polyethylene glycol is 2,000-3,500 Da; and the amino acid sequence of IL4RPep-1 is CRKRLDRNC.

[0013] Furthermore, the particle size of the nanomedicine delivery carrier is 154.6-175.5 nm.

[0014] A second objective of this invention is to provide a method for preparing the aforementioned nanomedicine delivery carrier, the method comprising the following steps:

[0015] (1) Preparation of PLGA-NHS: Weigh 1-100 mg equivalent of PLGA and dissolve it in acetone, add EDC and NHS, and react at room temperature for 4-12 h to form activated PLGA-NHS.

[0016] (2) Preparation of PLGA-PEG polymer: 100 mg equivalent of PEG is added to the activated PLGA-NHS formed in step (1), and stirring is continued at room temperature for 12-48 h. The product is dialyzed and freeze-dried under vacuum to obtain PLGA-PEG polymer; preferably, the molecular cutoff of the dialyzed polymer is 4000 Da.

[0017] (3) Preparation of IL4RPep-1-PLGA-PEG triblock polymer: The PLGA-PEG polymer obtained in step (2) was prepared into a 10 mg / mL solution, and IL4RPep-1 was prepared into a 5 mg / mL solution. The PLGA-PEG polymer solution and the IL4RPep-1 solution were reacted at a volume ratio of 1:1. EDC and NHS were added at room temperature for catalysis. The reaction was carried out under light and stirred for 12-48 h. The product was dialyzed and freeze-dried under vacuum to obtain IL4RPep-1-PLGA-PEG triblock polymer. Preferably, the reaction temperature under light and stirred was 4 °C.

[0018] (4) Preparation of IPP NPs: 1-100 mg equivalent of IL4RPep-1-PLGA-PEG triblock polymer is added to acetone solution and fully dissolved. It is then added to a 1-20% (w / w) polyvinyl alcohol solution and sonicated at 4°C. After sonication, it is added to an equal volume of ddH2O and finally centrifuged to obtain IL4RPep-1-polylactic acid-glycolic acid copolymer-polyethylene glycol nanoparticles. Preferably, the sonication is performed at 4°C.

[0019] A third object of the present invention is to provide a pharmaceutical composition comprising the aforementioned nanomedicine delivery carrier, the pharmaceutical composition further comprising alflurotem and celecoxib, the nanomedicine delivery carrier encapsulating alflurotem and celecoxib.

[0020] Furthermore, the mass ratio of the nanomedicine delivery carrier, aflurtrol, and celecoxib is 5:1:1.

[0021] A fourth object of the present invention is to provide the use of the aforementioned nanomedicine delivery carrier or the aforementioned pharmaceutical composition in the preparation of a medicament for treating tumors.

[0022] Furthermore, the tumor is melanoma. The combined use of ASB and CXB, delivered via a nanomedicine delivery carrier, significantly inhibits the development of melanoma in vitro.

[0023] The technical solution of this invention has the following advantages over the prior art:

[0024] In this invention, a novel nanomedicine delivery system, IPP NPs, is prepared for the co-delivery of ASB and CXB to melanoma cells. Utilizing the pharmacological characteristics of ASB and CXB respectively, it simultaneously kills melanoma cells and activates cytotoxic CD8. + T lymphocyte infiltration and the promotion of tumor-associated M2 macrophage reprogramming into M1 macrophages, through synergistic effects, can reshape the tumor immune microenvironment while inhibiting melanoma growth, thereby enhancing anti-tumor activity and improving the pathological process of melanoma.

[0025] This invention is the first to utilize an IPP NPs drug delivery system to deliver ASB and CXB in combination for the treatment of melanoma. Specifically, the IPP NPs drug delivery system was first synthesized and characterized, and its in vitro release and uptake mechanisms were studied. Based on this, drug-loaded ASB & CXB IPP NPs were synthesized. On one hand, using human melanoma cells A375 and SK-MEL-28 as model cells, the in vitro antitumor efficacy of ASB was evaluated. CCK-8, TUNEL, and flow cytometry results showed that ASB exhibited a strong dose-dependent ability to promote tumor cell apoptosis in both types of human melanoma cells, and the drug-loaded NPs enhanced its antitumor activity. On the other hand, in this invention, human monocytic leukemia cells THP-1 were induced to become macrophages, and then stimulated with IL-4 and IL-13 to polarize them into the M2 type. At the molecular level, we found that CXB could induce the conversion of macrophages from M2 to M1 type, and the drug-loaded NPs promoted this reprogramming effect. In summary... This invention reveals that CXB can improve the tumor immunosuppressive microenvironment by inducing macrophage reprogramming. Furthermore, we comprehensively evaluated its safety at both the cellular and animal levels. The IPP NPs drug delivery system disclosed in this invention, which targets and co-delivers ASB and CXB, can effectively improve the therapeutic effect of melanoma.

[0026] This invention constructs an IPP NPs drug delivery system for synergistic drug administration and comprehensively evaluates its biosafety and tumor targeting. Leveraging the advantages of this nanomedicine delivery system, which combines active and passive targeting, we successfully delivered ASB and CXB together to melanoma tissue. Simultaneously, the in vitro efficacy of the combined effect of ASB and CXB was evaluated, showing that its efficacy was greater than that of either drug alone. This combination significantly inhibited melanoma growth, improved the tumor immunosuppressive microenvironment, and demonstrated strong anti-melanoma efficacy. Attached Figure Description

[0027] Figure 1 IPP 1 H NMR spectrum.

[0028] Figure 2 PLGA-PEG NPs ( Figure 2 A) and IPP NPs ( Figure 2 B) Transmission electron microscopy image.

[0029] Figure 3 PLGA-PEG NPs ( Figure 3 A) and IPP NPs ( Figure 3 B) Particle size and PdI value.

[0030] Figure 4 Transmission electron microscopy (TEM) images of IPP NPs in acidic media and zeta potentials of IPP NPs in different media; where A. TEM images of IPP NPs in acidic media, particle size and PdI value; B. zeta potentials of PLGA-PEG NPs and IPP NPs in different media.

[0031] Figure 5 Serological stability and hemolysis assays of IPP NPs; A. Stability of IPP NPs in PBS containing 10% FBS; B. Hemolytic assay of IPP NPs.

[0032] Figure 6 In vitro safety assessment of IPP NPs; A. Safety assessment of IPP NPs in mouse primary hepatocytes, n=3; B. Safety assessment of IPP NPs in A375 cells, n=3.

[0033] Figure 7 In vitro and in vivo safety assessment of IPP NPs; A. Changes in mouse body weight during 21 days of continuous administration of IPP NPs; B. Changes in liver weight in mice after administration of IPP NPs;

[0034] Figure 8Evaluation of liver and kidney function parameters in mice after IPP NPs administration: A. ALT, B. AST, C. BUN, D. CRE.

[0035] Figure 9 H&E staining sections of various organs and tissues of mice after IPP NPs administration, n=5.

[0036] Figure 10 IPP NPs encapsulation efficiency and drug loading rate; among them, A. ASB drug encapsulation efficiency and drug loading rate; B. CXB drug encapsulation efficiency and drug loading rate.

[0037] Figure 11 ASB & CXB IPP NPs were released in vitro; where A. ASB NPs were released in vitro; B. CXB NPs were released in vitro; n = 3.

[0038] Figure 12 In vitro cell uptake laser confocal images of IPP NPs.

[0039] Figure 13 IPP NPs were detected by in vitro cell uptake using flow cytometry; A. Flow cytometry detection of Rhodamine B / IPP NPs concentration and statistical graph in A375 cells; B. Flow cytometry detection of Rhodamine B / IPP NPs concentration and statistical graph in SK-MEL-28 cells.

[0040] Figure 14 Study on the cellular uptake mechanism of IPP NPs.

[0041] Figure 15 In vitro efficacy evaluation of combined administration of ASB and CXB; A. Combined administration to A375 cells; B. Combined administration to SK-MEL-28 cells.

[0042] Figure 16 Inhibitory effects of ASB NPs on melanoma cells; A. A375 cell survival rate; B. SK-MEL-28 cell survival rate.

[0043] Figure 17 TUNEL analysis was conducted to investigate the effects of different administration methods on apoptosis in A375 and SK-MEL-28 cells.

[0044] Figure 18 Flow cytometry was used to analyze the effects of different drug administration methods on apoptosis in A375 cells and to generate flow cytometry apoptosis statistics. A. Flow cytometry apoptosis graphs for different drug administration methods; B. Flow cytometry apoptosis statistics bar charts for different drug administration methods.

[0045] Figure 19Flow cytometry was used to analyze the effects of different drug administration methods on apoptosis in SK-MEL-28 cells and to generate flow cytometry apoptosis statistics. A. Flow cytometry apoptosis graphs for different drug administration methods; B. Flow cytometry apoptosis statistics bar charts for different drug administration methods.

[0046] Figure 20 IL4 and IL13 stimulation resulted in the expression of M2-related genes, including A. TGFβ; B. CD163; and C. IL10.

[0047] Figure 21 M1-related gene expression after CXB administration, including A. TNFα; B. CD86; C. CD80.

[0048] The IPP NPs mentioned in this invention refer to: IL4RPep-1-polylactic acid-glycolic acid copolymer-polyethylene glycol nanoparticles, with a chemical structure as shown in Formula I:

[0049] The ASB mentioned in this invention refers to: Alfluoceretide, whose chemical structure is shown in Formula II:

[0050] The CXB mentioned in this invention refers to celecoxib, whose chemical structure is shown in Formula III:

[0051] The ASB&CXB IPP NPs mentioned in this invention refer to: IL4RPep-1-polylactic acid-glycolic acid copolymer-polyethylene glycol nanoparticles simultaneously loaded with aflurtrol and celecoxib.

[0052] The PLGA mentioned in this invention refers to poly(lactic-co-glycolic acid) copolymer (PLGA).

[0053] In this invention, PEG refers to polyethylene glycol.

[0054] The IL4RPep-1 mentioned in this invention refers to an IL4R binding peptide with the amino acid sequence CRKRLDRNC.

[0055] The EDC mentioned in this invention refers to carbodiimide (EDC).

[0056] The NHS mentioned in this invention refers to N-hydroxysuccinimide (NHS).

[0057] In this invention, NPs refers to nanoparticles. Detailed Implementation

[0058] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies.

[0059] Example 1: Preparation of IL4RPepe-1-PLGA-PEG Nanodrug Delivery System

[0060] (1) Chemical synthesis route of IL4RPepe-1-PLGA-PEG

[0061]

[0062] (2) Preparation method of IL4RPepe-1-PLGA-PEG

[0063] 1) Preparation of PLGA-NHS: 10 mg of PLGA (manufacturer: Aladdin, specification: P134564-1g) was fully dissolved in 1 mL of acetone, and 5 mg of EDC and 5 mg of NHS were added as reaction catalysts. The mixture was stirred at room temperature for 4 h to form activated PLGA-NHS.

[0064] 2) Preparation of PLGA-PEG polymer: 10 mg of PEG (manufacturer: Aladdin, specification: N164051-100 mg) was added to the activated PLGA-NHS formed in step 1. The mixture was stirred at room temperature for 24 h. The resulting product was placed in a dialysis bag with a molecular weight cutoff of 4000 Da and dialyzed with deionized water for 3 days. After 3 days, the dialyzed product was removed and freeze-dried under vacuum to obtain the PLGA-PEG polymer. For subsequent experiments such as transmission electron microscopy, 10 mg of the PLGA-PEG triblock polymer was added to 1 mL of acetone solution and vortexed to fully form an emulsion. This emulsion was then added to a 1% polyvinyl alcohol (manufacturer: Aladdin, specification: P139532-500 g) solution and stirred until homogeneous, forming an water / oil emulsion. The solution was intermittently sonicated at 4 °C (150 W power for 30 s, followed by a 2 s pause after 3 s of sonication). After sonication, the reaction solution was added to an equal volume of ddH2O, vortexed, and then centrifuged at 12,000 rpm and 4°C for 15 minutes. After centrifugation, ddH2O was added to resuspend the solution, and the filtrate was then freeze-dried under vacuum to obtain a white powder, which is PLGA-PEG NPs.

[0065] 3) Preparation of IL4RPep-1-PLGA-PEG triblock polymer: The PLGA-PEG polymer obtained in step 2 was prepared into a 10 mg / mL solution, and IL4RPep-1 was prepared into a 5 mg / mL solution. The reaction was carried out at a 1:1 volume ratio. 5 mg EDC and 5 mg NHS were added as reaction catalysts at room temperature. The reaction was carried out under light and stirred at 4 °C for 48 h. The resulting product was placed in a dialysis bag with a molecular cutoff of 4000 Da and immersed in deionized water for dialysis for 3 days. After 3 days, the dialysis product was taken out and freeze-dried under vacuum to obtain IL4RPep-1-PLGA-PEG triblock polymer.

[0066] 4) Preparation of IPP NPs: 10 mg of IL4RPep-1-PLGA-PEG triblock polymer was added to 1 mL of acetone solution and vortexed to fully form an emulsion. This emulsion was then added to a 1% polyvinyl alcohol (manufacturer: Aladdin, specification: P139532-500g) solution and stirred until homogeneous, forming an water / oil emulsion. The solution was intermittently sonicated at 4℃ (150W power for 30s, followed by a 3s sonication pause for 2s). After sonication, the reaction solution was added to an equal volume of ddH2O and vortexed until homogeneous. The solution was then centrifuged at 12000 rpm at 4℃ for 15 min. After centrifugation, ddH2O was added to resuspend the solution, and the filtrate was then freeze-dried under vacuum to obtain a white powder, which is the IPP NPs.

[0067] Characterization of IPP NPs prepared in Example 2

[0068] IPP NPs 1 H-NMR spectrum ( Figure 1 The chemical shift peaks of the amino (-NH2) and thio (-SH) groups on IL4RPep-1 were 2.4 ppm and 1.4 ppm, respectively, and were successfully grafted onto PLGA-PEG. In addition, a chemical shift peak of the amino (-CH2) group on PLGA-PEG was also observed at 4.8 ppm. The experimental results prove that IL4RPep-1 was successfully attached to PLGA-PEG.

[0069] To further study the morphological structure of the prepared IPP NPs, such as... Figure 2 and 3 As described, transmission electron microscopy (TEM) images and Malvern particle size analyzer results show that IPP NPs have a smooth surface and a uniform spherical structure. IPP NPs are stable in neutral environments, with an average particle size of approximately 159.0 nm; however, in acidic environments, such as… Figure 4As shown, IPP NPs exhibit irregular spherical shapes and undergo breakage. Both PdI values ​​are less than 0.5, indicating good dispersion of the NPs in solution. After modification with IL4RPep-1, the zeta potential increases, with the zeta potential of IPP NPs rising from -2.02 mV to -1.11 mV. As pH decreases, the amount of negative charge carried by the NPs also decreases, indicating that the release of NPs increases under acidic conditions.

[0070] like Figure 5 As shown, serological stability results indicate that the particle size did not change significantly before and after incubation, suggesting that IPP NPs can stably exist in the bloodstream for a certain period of time. The biocompatibility of IPP NPs was evaluated using a hemolysis test. Compared with the positive control, erythrocytes in both the IPP NPs and negative control groups did not rupture or hemolyze, thus demonstrating good biocompatibility.

[0071] Example 3: In vitro and in vivo safety assessment of IPP NPs

[0072] To test the safety of IPP NPs, this study co-incubated mouse primary hepatocytes with different doses of IPP NPs, such as... Figure 6 As shown, IPP NPs did not exhibit significant cytotoxicity. Furthermore, IPP NPs did not significantly affect the survival rate of melanoma cells. To further evaluate the in vivo safety of IPP NPs, we divided C57BL6 / J mice into two groups. The experimental group received IPP NPs (25 mg / kg) via tail vein injection for 21 days, administered every three days. The control group received the same dose of PBS. Figure 7 As shown in A and B, compared with the PBS group, IPP NPs did not significantly alter mouse body weight and liver weight. We also measured serum parameters related to liver and kidney injury in the experimental and control groups, including liver injury markers ALT and AST, and kidney injury markers BUN and CRE, such as... Figure 8 The results showed no significant difference between the two groups. Furthermore, as... Figure 9 As shown, HE staining revealed no tissue lesions in any of the mouse tissues and organs after drug administration. In conclusion, IPP NPs exhibit good biocompatibility and can serve as an ideal nanomedicine delivery carrier.

[0073] Example 4 Preparation of ASB NPs and CXB NPs

[0074] The preparation methods for ASB NPs and CXB NPs are as follows: ASB or CXB is prepared into a 50 mg / mL solution using DMSO. 2 mg of the IPP NPs prepared in Example 1 is dispersed in 0.2 mL of acetone. 8 μL of the ASB or CXB solution is added dropwise to the above IPP NPs acetone solution. After the addition is complete, it is added to a 1% polyvinyl alcohol solution, stirred and mixed, and then sonicated for 30 s. After sonication, the reaction solution is added to an equal volume of ddH2O, vortexed and mixed. The solution is then centrifuged at 12000 rpm and 4 °C for 15 min. After centrifugation, ddH2O is added to resuspend the solution, and the filtrate is then freeze-dried under vacuum to obtain a white powder, which is the ASB NPs or CXB NPs.

[0075] Example 5: Encapsulation efficiency and drug loading rate of drug-loaded IPPs (NPs)

[0076] 0.04 mg, 0.08 mg, 0.2 mg, and 0.4 mg of ASB and CXB, respectively, were added to 0.2 mL of IPP triblock polymer (10 mg / mL). The resulting mixture was then transferred to a 1% polyvinyl alcohol solution, stirred thoroughly, and sonicated for 30 s. After sonication, the reaction solution was added to an equal volume of ddH₂O, vortexed, and centrifuged at 12000 rpm and 4 °C for 15 min. ASB₁, ASB₂, ​​ASB₃, ASB₄ and CXB₁, CXB₂, CXB₃, CXB₄ NPs solutions were prepared. The supernatant of each sample was taken, and the absorbance was measured by UV spectrophotometry (the characteristic absorption peak of ASB is 260 nm; the characteristic absorption peak of CXB is 250 nm) using ddH₂O as a blank control. Based on the pre-measured ASB and CXB standard curves, the drug loading content was calculated, and then the drug loading efficiency (LE) and encapsulation efficiency (EE) of the drug-loaded nanoparticles for the four samples were calculated respectively:

[0077]

[0078]

[0079] Where M0 is the total mass of ASB or CXB; M1 is the free mass in the supernatant of ASB or CXB; and Q0 is the unloaded nanomass of IPP NPs.

[0080] like Figure 10As shown in the experiment, increasing the mass ratio of ASB or CXB to the nanocarrier increased both the drug loading and encapsulation efficiency of ASB or CXB. The IPP NPs prepared in Example 1 showed an encapsulation efficiency of 28.8-79.9% for ASB and a drug loading efficiency of 0.6-16.0%; the IPP NPs showed an encapsulation efficiency of 6.6-85.1% for CXB and a drug loading efficiency of 0.13-17.0%.

[0081] Example 6 Drug Release from IPP NPs Co-loaded with ASB and CXB

[0082] Six parallel preparations were made, each consisting of 0.4 mg ASB and 0.4 mg CXB co-loaded onto 2 mg IPP NPs. One mL of each ASB and CXB nanocarrier was encapsulated in a dialysis bag with a molecular cutoff of 4000 Da, and then placed in release systems at pH 7.4 and pH 5.0, with three samples in each system. The mixture was incubated at 37°C for 100 rpm. The absorbance of free ASB and CXB in the release medium was measured at different time points using a UV spectrophotometer, and the free drug concentration was calculated based on their respective standard curves. The cumulative release amounts of ASB and CXB over different time periods were then plotted against the corresponding time points. The characteristic absorption peak of ASB was 260 nm; the characteristic absorption peak of CXB was 250 nm.

[0083] like Figure 11 As shown, the drug release behavior of IPP NPs is pH-sensitive under different pH conditions. Under normal physiological conditions (pH 7.4), the cumulative release of ASB and CXB is low and the release rate is slow, with cumulative release of 29.8% and 37.7% at 6 hours. However, under tumor acidic conditions (pH 5.0), the cumulative release of ASB and CXB is high, and the release rate is significantly accelerated, reaching 84.3% and 99% at 6 hours.

[0084] Example 7: In vitro cellular uptake and mechanism study of IPP NPs

[0085] In vitro cell uptake of unloaded IPP NPs: Human malignant melanoma cells A375 or SK-MEL-28 cells (2×10⁻⁶) were added. 5Cells (per well) were seeded into laser confocal dishes containing 1 mL of culture medium and cultured for 24 h. After removing the old culture medium and washing three times with PBS, cells were cultured in Rhodamine B / IPP NPs cell culture medium (140 μg / mL) at 37 °C for 0 h, 6 h, 12 h, and 24 h. After incubation, the old culture medium was discarded, cells were washed three times with PBS, fixed with 4% paraformaldehyde for 30 min, washed three times with PBS, and the nuclei were stained with DAPI. Finally, 200 μL of PBS was added to the laser confocal dish, and cell uptake was observed under LSCM.

[0086] Cell uptake efficiency: Human malignant melanoma cells A375 or SK-MEL-28 cells (6 × 10⁻⁶) were used to achieve the following results: 5 Cells were seeded into 6-well plates (cells / well) and cultured for 24 h. Cell culture medium containing Rhodamine B / IPP NPs (140 μg / mL) was added, and the cells were cultured at 37°C for 0 h, 6 h, 12 h, and 24 h, respectively. After incubation, cells were digested with EDTA-free trypsin, centrifuged (1500 rpm, 5 min) to collect cells, washed three times with pre-chilled PBS, and the uptake efficiency of A375 or SK-MEL-28 cells was detected by flow cytometry.

[0087] In vitro cellular uptake mechanism of IPP NPs: Human malignant melanoma cells A375 or SK-MEL-28 cells (2×10⁻⁶) were used. 5 Cells (per well) were seeded into laser confocal dishes containing 1 mL of culture medium and cultured for 24 h. Then, the cells were pre-incubated for 30 min in serum-free medium containing inhibitors (Filipin, 1 μg / mL; CPZ, 10 μg / mL; and EIPA, 10 μg / mL). After pre-incubation, Rhodamine B / IPP NPs cell culture medium (140 μg / mL) was added, and incubation continued for another 24 h. After incubation, the old culture medium was discarded, and the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 30 min, washed three times with PBS, and finally stained with DAPI. 200 μL of PBS was added to the laser confocal dish, and cell uptake in each inhibitor group was observed under LSCM.

[0088] like Figure 12 , 13As shown, no red fluorescence was observed in either cell type at the initial incubation time of 0 h. However, the intensity of intracellular red fluorescence gradually increased over time, and a strong red fluorescence signal appeared under the microscope after 24 h of incubation. Furthermore, the cell uptake efficiency of A375 and SK-MEL-28 cells was detected by flow cytometry. After 24 h, the cell uptake rates of A375 and SK-MEL-28 cells were 21.62% and 30.04%, respectively. In conclusion, the uptake of IPP NPs in human melanoma cells A375 and SK-MEL-28 cells is time-dependent.

[0089] To further analyze the mechanism of tumor cell uptake of IPP NPs, such as Figure 14 As shown, compared with the control group, the uptake of IPP NPs by A375 and SK-MEL-28 cells was significantly inhibited when EIPA (inhibits caveolin-mediated endocytosis) was applied, while the inhibitor treatment in other groups only slightly altered the uptake of IPP NPs by cells.

[0090] In summary, IPP NPs are mainly taken up by A375 and SK-MEL-28 cells by inhibiting caveolin-mediated endocytosis.

[0091] Example 8: In vitro efficacy evaluation of combined administration of ASB and CXB

[0092] A375 and SK-MEL-28 cells were seeded into 96-well plates (1×10⁻⁶ cells per well). 4 Cells / well were cultured for 24 h (5% CO2, 37℃). Then, different concentrations of drug combinations (ASB & CXB: 0 μM & 0 μM, 0 μM & 2.5 μM, 0 μM & 5 μM, 0 μM & 10 μM; 2.5 μM & 0 μM, 2.5 μM & 2.5 μM, 2.5 μM & 5 μM, 2.5 μM & 10 μM; 5 μM & 0 μM, 5 μM & 2.5 μM, 5 μM & 5 μM, 5 μM & 10 μM; 10 μM & 0 μM, 10 μM & 2.5 μM, 10 μM & 5 μM, 10 μM & 10 μM) were added and incubated in a cell culture incubator for 24 h. Finally, the cell viability of each drug-treated group was detected by CCK8 assay.

[0093] like Figure 15 As shown, the addition of different concentrations of CXB did not significantly affect the survival rate of A375 and SK-MEL-28 cells. The killing effect on A375 and SK-MEL-28 cells mainly depended on the action of ASB. In conclusion, the direct killing effect of CXB on tumor cells has been ruled out, thus ruling out the synergistic effect of ASB and CXB in killing tumor cells. CXB can only exist as an immunotherapy drug.

[0094] Example 9: Effect of ASB NPs on melanoma survival

[0095] A375 and SK-MEL-28 cells in logarithmic growth phase were seeded into 96-well plates (1×10⁻⁶ cells per well). 4 Cells / well were cultured for 24 h (5% CO2, 37℃). Then, different concentrations of drug combinations were added (ASB Free: 0 μM, 2.5 μM, 5 μM, 10 μM; ASBNPs: 0 μM, 2.5 μM, 5 μM, 10 μM), and the cells were incubated in a cell culture incubator for 24 h. Finally, the cell viability of each drug-treated group was detected by CCK8 assay.

[0096] like Figure 16 As shown, in A375 and SK-MEL-28 cells, cell viability gradually decreased with increasing ASB drug concentration. ASB NPs significantly enhanced the antitumor activity of ASB; at the same drug concentration, ASB NPs exhibited a higher lethality rate against A375 and SK-MEL-28 cells. The half-maximal inhibitory concentration (IC50) of ASB against A375 and SK-MEL-28 tumor cells was calculated. 50 The values ​​are 8.3 μM and 7.3 μM.

[0097] Example 10: In vitro antitumor efficacy evaluation of ASB & ASB NPs

[0098] TUNEL analysis: Human malignant melanoma cells A375 or SK-MEL-28 cells in logarithmic growth phase (2 × 10⁻⁶) were analyzed. 5 Cells (per well) were seeded into laser confocal dishes containing 1 mL of culture medium and cultured for 24 h. Then, based on the IC50 values ​​of the two cell types... 50 Free and nanomedicines were added to the culture medium, respectively, and incubated for 24 h. After incubation, cells were washed three times with PBS, fixed with 4% paraformaldehyde for 30 min, stained with a one-step TUNEL apoptosis detection kit, and finally stained with DAPI for cell nuclei. 200 μL of PBS was added to a laser confocal dish, and cell uptake in each inhibitor group was observed under LSCM. Flow cytometry analysis: Logarithmic growth phase human malignant melanoma cells A375 or SK-MEL-28 cells (6 × 10⁻⁶) were analyzed. 5 Cells (per well) were seeded into 6-well plates and cultured for 24 hours. Then, based on the IC50 values ​​of the two cell types... 50Free and nanomedicines were added to the culture medium separately, and incubation continued for 24 h. After incubation, cells were digested with EDTA-free trypsin, centrifuged (1000g, 5 min), and the cells were collected. 500 μL of binding buffer was added to resuspend the cells, followed by the addition of 5 μL of PI and 5 μL of Annexin V-FITC. The mixture was gently mixed and incubated in the dark for 10 min. The cells were then analyzed by flow cytometry.

[0099] like Figure 17 , 18 As shown in Figure 19, the cells in the untreated group were in good condition, exhibiting a normal spindle shape with a clear background; while the cells in the treated group were atrophied and rounded, with numerous apoptotic bodies appearing. ASB promoted tumor cell apoptosis in both types of melanoma cell lines, and ASB IPP NPs more significantly induced pyroptosis in tumor cells. Flow cytometry analysis revealed that ASB primarily induced early apoptosis in tumor cells, while ASB IPP NPs induced a transition from early apoptosis to late apoptosis.

[0100] Example 11 Evaluation of CXB & CXB NPs in vitro inducing macrophage reprogramming

[0101] Human monocytic leukemia THP-1 cells in logarithmic growth phase (3×10) 5 Cells (per well) were seeded into 12-well plates and cultured for 24 h with PMA (100 μg / mL) to induce adherence and macrophage transformation. IL4 (1 μg / mL) and IL13 (1 μg / mL) were then added and cultured for another 72 h to induce M2 macrophage transformation. The culture medium was then discarded, and CXB and CXBNPs (30 μM) were added and cultured for another 48 h. After culture, cells were lysed with TRIzol, and total RNA was extracted. Reverse transcription was performed using the Takara reverse transcription kit. Primers for M2 genes (IL10, CD163, TGFβ) and M1 genes (CD80, CD83, TNFα) were designed using Primer 3 software and synthesized by Beijing Qingke Biotechnology Co., Ltd. The reaction system was prepared according to the instructions of the Vazyme kit from Nanjing Novizan Biotechnology Co., Ltd., and amplification was performed using a qPCR instrument. The relative expression level of the target gene was calculated based on the Ct value.

[0102] like Figure 20 As shown, compared with the control group, stimulation with IL4 and IL13 significantly increased the mRNA expression of M2-related genes CD163, IL10, and TGFβ, increasing by 3.0, 2.3, and 1.2 times, respectively; while after further incubation with CXB and CXB NPs (30 μM) for 48 h, as shown... Figure 21As shown, the mRNA expression levels of M1-related genes CD80, CD83, and TNFα continued to increase, and the presence of NPs made the mRNA expression levels even more pronounced. In conclusion, CXB & CXB NPs have the ability to induce macrophage reprogramming in vitro.

Claims

1. A pharmaceutical composition for treating melanoma, characterized in that, The pharmaceutical composition includes a nanomedicine delivery carrier, and the pharmaceutical composition further includes alflurtrolide and celecoxib. The nanomedicine delivery carrier encapsulates alflurtrolide and celecoxib, and the mass ratio of the nanomedicine delivery carrier, alflurtrolide, and celecoxib is 5:1:

1. The nanomedicine delivery carrier is IL4RPep-1-polylactic acid-glycolic acid copolymer-polyethylene glycol nanoparticles, and the chemical structure of the IL4RPep-1-polylactic acid-glycolic acid copolymer-polyethylene glycol nanoparticles is shown in Formula I: Formula I Wherein, IL4RP is IL4RPep-1, n=50, m=40-70; The molecular weight of the polylactic acid-glycolic acid copolymer is 38,000-54,000 Da; the molecular weight of the polyethylene glycol is 2,000-3,500 Da; and the amino acid sequence of IL4RPep-1 is CRKRLDRNC.

2. The pharmaceutical composition according to claim 1, characterized in that, The particle size of the nanomedicine delivery carrier is 154.6-175.5 nm.

3. The pharmaceutical composition according to claim 1, characterized in that, The preparation method of the nanomedicine delivery carrier includes the following steps: (1) Preparation of PLGA-NHS: Weigh 1-100 mg equivalent of PLGA and dissolve it in acetone, add EDC and NHS, and react at room temperature for 4-12 h to form activated PLGA-NHS; (2) Preparation of PLGA-PEG polymer: 1-100 mg equivalent of PEG was added to the activated PLGA-NHS formed in step (1), and stirring was continued at room temperature for 12-48 h. The product was dialyzed and freeze-dried under vacuum to obtain PLGA-PEG polymer. (3) Preparation of IL4RPep-1-PLGA-PEG triblock polymer: The PLGA-PEG polymer obtained in step (2) was prepared into a 10 mg / mL solution, and IL4RPep-1 was prepared into a 5 mg / mL solution. The PLGA-PEG polymer solution and IL4RPep-1 solution were reacted at a volume ratio of 1:

1. EDC and NHS were added for catalysis. The reaction was carried out under light and stirred for 12-48 h. The product was dialyzed and freeze-dried under vacuum to obtain IL4RPep-1-PLGA-PEG triblock polymer. (4) Preparation of IPP NPs: 1-100 mg equivalent of IL4RPep-1-PLGA-PEG triblock polymer was added to acetone solution and dissolved completely. It was then added to a 1-20% polyvinyl alcohol solution and sonicated. After sonication, it was added to an equal volume of ddH2O and finally centrifuged to obtain IL4RPep-1-polylactic acid-glycolic acid copolymer-polyethylene glycol nanoparticles.

4. Use of the pharmaceutical composition of claim 1 in the preparation of a medicament for treating melanoma.

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