Plumbagin isomer PLB-1 as well as composition and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a plumbagin isomer PLB-1 as well as a composition and application of the plumbagin isomer PLB-1. The plumbagin isomer PLB-1 and the composition are deeply researched through molecular biology, transcriptome sequencing and animal experiment related experimental means, the specific regulation and control mechanism of the plumbagin isomer to an NQO1 / HO-1 pathway and the specific synergistic effect of HAS-NPs (at) PLB-1 (at) PD-L1 to plumbagin, and the specific regulation and control mechanism of the plumbagin isomer PLB-1 (at) PD-L1 and the specific regulation and control mechanism of the a leading bioinformatics method is combined, a new regulatory pathway of the PLB-1 is explored, and the action effect of the PLB-1 is further improved by taking an albumin nano material as a drug carrier. A new treatment mode is provided for the lung cancer, meanwhile, a new way is provided for application of PLB-1, and breakthrough of fundamental research on treatment of the lung cancer by PLB is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a plumbagolin isomer PLB-1, its composition, and its applications. Background Technology

[0002] Lung cancer is one of the leading causes of cancer death worldwide, with persistently high incidence and mortality rates. According to data released by the International Agency for Research on Cancer (IARC) of the World Health Organization, the incidence and mortality rates of lung cancer are as high as 11.4% and 18.0%, respectively, posing a serious threat to human health. Currently, treatment methods for lung cancer mainly include surgery, radiotherapy and chemotherapy, targeted therapy, and immunotherapy. However, these treatments all have certain limitations.

[0003] Surgical treatment is primarily suitable for early-stage lung cancer patients, but most lung cancer patients are diagnosed at an intermediate or advanced stage, missing the optimal window for surgery. While radiotherapy and chemotherapy can kill tumor cells, they also damage normal cells, leading to severe side effects. Targeted therapy, although initially showing good responses, is prone to developing resistance, making sustained benefit difficult. Immunotherapy, especially immune checkpoint inhibitors (ICIs) such as PD-1 / PD-L1, has achieved significant efficacy in lung cancer treatment, but its response rate in advanced non-small cell lung cancer (NSCLC) patients remains limited, at only about 20%.

[0004] Plumbagozeylanica (5-hydroxy-2-methyl-1,4-naphthoquinone, PLB) is a natural compound extracted from the roots of the medicinal plant Plumbagozeylanica. It possesses a wide range of biological activities, including anticancer, antimicrobial, anti-inflammatory, and anti-atherosclerotic effects. Recent studies have found that PLB exhibits significant anticancer activity against various cancer cell lines, with its anticancer mechanisms involving apoptosis, autophagy, cell cycle arrest, anti-angiogenesis, anti-invasion, and anti-metastasis. However, the effective concentration of PLB for inhibiting various tumors is relatively high, and its poor water solubility, easy degradation, and rapid in vivo clearance limit its clinical application.

[0005] To overcome the limitations of plasma albumin (PLB), researchers have begun exploring the use of nanotechnology to improve its pharmacokinetic properties. Nanoparticle drug carriers can control drug release rates, prolong the drug's half-life in vivo, reduce toxic side effects, and possess targeting capabilities. Human serum albumin (HSA), as a natural plasma protein, has good biocompatibility, stability, and drug-loading capacity, and is widely used in drug delivery systems. Loading PLB onto albumin nanoparticles can not only improve the water solubility and stability of PLB but also achieve targeted drug delivery, thereby enhancing its anti-tumor efficacy.

[0006] Furthermore, immunotherapy is playing an increasingly important role in lung cancer treatment. Immune checkpoint inhibitors, by blocking immunosuppressive signals, activate the body's own immune system to attack tumor cells, achieving significant therapeutic effects. Combining immunotherapy with nanomedicine delivery systems holds promise for synergistic effects, further improving survival benefits for lung cancer patients.

[0007] Based on the above background, this invention proposes a novel anticancer drug composition utilizing albumin nanoparticles to load the pistachioamine isomer PLB-1 and the immune checkpoint inhibitor PD-L1 monoclonal antibody. This composition aims to improve the pharmacokinetic properties of PLB-1 through nanotechnology, enhance its antitumor effect, and achieve synergistic effects when combined with immunotherapy, potentially providing a novel, highly effective, and low-toxicity treatment option for lung cancer patients. Summary of the Invention

[0008] The purpose of this invention is to provide a plumbagoside isomer PLB-1, its composition, and its application, which provides a new treatment for lung cancer and also provides a new approach for the application of PLB-1, achieving a breakthrough in the basic research of PLB for lung cancer treatment.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] This invention provides a leptin isomer PLB-1, the chemical structural formula of which is:

[0011]

[0012] This invention also provides a method for synthesizing the leptin isomer PLB-1, comprising the following steps:

[0013] (1) Mix 1,5-dihydroxynaphthalene, acetic anhydride and pyridine and stir to react, then add water, stir and cool to 0 degrees under ice water bath, filter and dry to obtain compound 1;

[0014] (2) After dissolving compound 1 in acetic acid, NBS solution was added dropwise. After the reaction was carried out, the mixture was cooled to room temperature, extracted, washed with water, dried and concentrated to obtain compound 2.

[0015] (3) Compound 2 was dispersed in ethanol, sulfuric acid aqueous solution was added, the mixture was heated under reflux and cooled to room temperature, and then extracted, washed with water, dried and concentrated to obtain compound 3;

[0016] (4) Compound 3, cyclopropylboronic acid, potassium phosphate and tricyclohexylphosphine were mixed in toluene solution, palladium acetate was added under nitrogen protection, the reaction was heated and then cooled to room temperature, and after extraction, washing with water, drying and concentration, the plumbagoside isomer PLB-1 was obtained.

[0017] The present invention also provides a composition loaded with the plumbagoside isomer PLB-1, the composition being prepared by loading plumbagoside isomer PLB-1 and PD-L1 monoclonal antibody onto albumin nanoparticles HAS-NPs.

[0018] The present invention also provides a method for preparing a composition, comprising the following steps:

[0019] (1) Albumin nanoparticles were synthesized by solvent removal method and cross-linked with glutaraldehyde to further stabilize the nanoparticles.

[0020] (2) Loading the hydrophobic drug PBL1 to synthesize HAS-NPs@PBL1 nanoparticles;

[0021] (3) PDL1-PLL is generated through coupling between primary PDL1 amine and PLL aldehyde;

[0022] (4) PDL1-PLL is sequentially coated onto the surface of HSA-NPs@PBL1 by electrostatic interaction, and then washed three times with distilled water to obtain the composition.

[0023] The present invention also provides a medicament for treating lung cancer, the medicament comprising the pampasodilator isomer PLB-1 or the composition thereof.

[0024] Furthermore, the plumbagoside isomer PLB-1 or the composition thereof is the sole active ingredient.

[0025] The present invention also provides the use of the plumbagoside isomer PLB-1 or the composition thereof in the preparation of products for the prevention and treatment of lung cancer.

[0026] Furthermore, the prevention and treatment of lung cancer involves promoting ferroptosis in lung cancer patients through NQO1 / / HO-1 to enhance immunotherapy and thus prevent and treat lung cancer.

[0027] Furthermore, when the product acts on lung cancer cells, it upregulates HMOX1, downregulates NQO1, and downregulates CD81.

[0028] Beneficial effects:

[0029] Previous studies have confirmed the effects of plumbagoside on lung cancer, but the effects of the plumbagoside isomer PLB-1 on lung cancer have not yet been reported, and the specific molecular mechanism remains unclear. This invention also marks the first time that albumin nanomaterials have been used as drug carriers to achieve dual-drug, targeted release of the plumbagoside isomer and the immune checkpoint inhibitor PD-L1 monoclonal antibody. This invention utilizes molecular biology, transcriptome sequencing, and animal experiments to investigate the specific regulatory mechanism of the plumbagoside isomer on the NQO1 / HO-1 pathway and the specific synergistic effect of HAS-NPs@PLB-1@PD-L1 on plumbagoside. Furthermore, it combines cutting-edge bioinformatics methods to discover new regulatory pathways for PLB-1 and further enhances the efficacy of PLB-1 using albumin nanomaterials as drug carriers. This provides a new treatment approach for lung cancer and also offers a new avenue for the application of PLB-1, representing a breakthrough in basic research on PLB therapy for lung cancer. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a schematic diagram of the PLB-1 drug synthesis.

[0032] Figure 2 A schematic diagram illustrating the construction process of HAS-NPs@PLB-1@PD-L1;

[0033] Figure 3 Characterization diagrams of the HSA-NPs@PBL1@PD-L1 composite material prepared in Example 1 of this invention are shown, where A) transmission electron microscope images of HAS, HSA-NPs@PBL1 and HSA-NPs@PBL1@PD-L1 nanoparticles; B) measurement of the Zeta potential of HAS, HSA-NPs@PBL1 and HSA-NPs@PBL1@PD-L1 nanoparticles; CE) DLS measurement of HAS, HSA-NPs@PBL1 and HSA-NPs@PBL1@PD-L1 nanoparticles in water; FH) Fourier transform infrared spectroscopy detection of the infrared absorption peaks of HSA-NPs@PBL1 and HSA-NPs@PBL1@PD-L1.

[0034] Figure 4The figure shows the effect of PLB-1 on the proliferation of four lung cancer cell lines: A549, H460, H1650, and H1292. In the figure, A represents the effect of PLB-1 on A549; B represents the effect of PLB-1 on H460; C represents the effect of PLB-1 on H1650; and D represents the effect of PLB-1 on H1292.

[0035] Figure 5 The figure shows the results of PLB-1's inhibitory effect on apoptosis and invasion of two lung cancer cell lines, H1650 and A549.

[0036] Figure 6 Figure showing transcriptomic analysis results for PLB-1 treatment;

[0037] Figure 7 The figure shows the effect of HSA-NPs@PLB1@PDL1 on lung cancer. In this figure, A represents the morphological characteristics of terminal tumors, and B represents the tumor growth kinetics. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0042] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0043] Example 1

[0044] 1. Synthesis of PLB-1 (PTC20436) drug

[0045] (1) Synthesis of Naphthalene-1,5-diyl:

[0046] 1,5-Dihydroxynaphthalene (20 g, 125 mmol), acetic anhydride (100 mL), and pyridine (100 mL) were mixed together and stirred at room temperature for 12 hours. After the starting materials had reacted completely, water (2 L) was added, and the mixture was cooled to 0°C with stirring in an ice-water bath. The mixture was filtered and dried to obtain a brown solid product (28 g, 91% yield).

[0047] (2) Synthesis of 6-Bromo-5,8-dioxo-5,8-dihydronaphthalen-1-yl:

[0048] Compound 1 (20 g, 81.9 mmol) was dissolved in 330 mL of hot acetic acid, and then a solution of NBS (65.6 g, 368.5 mmol) in 330 mL of acetic acid and 330 mL of water was added dropwise while maintaining the temperature at 65°C. After the addition was complete, the reaction was continued for 45 minutes. The mixture was cooled to room temperature, and 600 mL of water was added. The mixture was extracted twice with 700 mL of chloroform. The combined organic phases were washed with 500 mL of brine, dried, and concentrated. The crude product was recrystallized from ethanol to obtain an orange solid, compound 2 (15 g, 62% yield).

[0049] (3) Synthesis of 2-Bromo-5-hydroxynaphthalene-1,4-dione:

[0050] Compound 2 (10 g, 33.9 mmol) was dispersed in 200 mL of ethanol, and 102.5 mL of 3N sulfuric acid aqueous solution was added. The mixture was heated under reflux for 2 hours. After cooling to room temperature, it was diluted with 250 mL of water and extracted twice with 200 mL of chloroform. The combined organic phases were washed with 200 mL of saturated brine, dried, and concentrated to dryness. The crude product was purified by column chromatography to obtain an orange solid compound 3 (5.8 g, 67% yield).

[0051] (3) Synthesis of 2-Cyclopropyl-5-hydroxynaphthalene-1,4-dione (PTC20436):

[0052] Compound 3 (3.9 g, 15.4 mmol), cyclopropylboronic acid (1.76 g, 20.5 mmol), potassium phosphate (11.74 g, 55.3 mmol), and tricyclohexylphosphine (443 mg, 1.58 mmol) were mixed in 80 mL of toluene and 4 mL of water. Palladium acetate (193 mg, 0.79 mmol) was added under nitrogen protection, and the mixture was heated to 100 °C for 3 hours. After cooling to room temperature, 100 mL of water was added, and the mixture was extracted with 100 mL of ethyl acetate. The organic phase was washed with 50 mL of brine, dried, and concentrated to dryness. The residue was purified by column chromatography, and the resulting solid was further purified by preparative HPLC to give an orange solid, compound PTC20436 (825 mg, 25% yield).

[0053] The schematic diagram of the PLB-1 drug synthesis is shown below. Figure 1 As shown, the purity of the drug, as determined by Bruker nuclear magnetic resonance (NMR) spectrometry, was 98.78%.

[0054] 2. Drug Loading and Identification

[0055] Albumin nanoparticles (HSA-NPs) were synthesized using a solvent-free method and cross-linked with glutaraldehyde to further stabilize the nanoparticles. Hydrophobic drug PBL1 was loaded to synthesize HAS-NPs@PBL1 nanoparticles. PDL1-PLL was generated through coupling between primary PDL1 amine and PLL aldehyde. PDL1-PLL was sequentially coated onto the surface of HSA-NPs@PBL1 via electrostatic interactions. The surface was washed three times with distilled water to remove excess PDL1-PLL, yielding HSA-NPs@PLB1@PDL1. (A schematic diagram of the HAS-NPs@PLB-1@PD-L1 construction process is shown below.) Figure 2 (As shown)

[0056] The specific steps for synthesizing HSA-NPs@PLB1@PDL1 in this embodiment are as follows:

[0057] (1) Synthesis of HSA-NPs

[0058] In short, first, weigh 100 mg of BSA and dissolve it in 10 mL of deionized water, then adjust the pH of the solution to 9.0. Next, slowly add approximately 35 mL of anhydrous ethanol dropwise to the albumin solution at a rate of 1.0 mL / min until the solution exhibits an opalescent appearance, with the magnetic stirrer set to 250 rpm / min. After stirring for 20 minutes, add 1 mL of 0.25% glutaraldehyde to the solution to crosslink the albumin, and then continue the reaction at room temperature with stirring for 16 hours. After the reaction is complete, remove the ethanol from the suspension using a vacuum rotary dryer at 35°C and concentrate it to obtain a high-concentration HSA-NPs solution.

[0059] (2) Synthesis of HSA-NPs@PBL1

[0060] First, 10 mg of HSA-NPs were dispersed in a phosphate buffer solution at pH 9. Then, under magnetic stirring, 100 μL of DMSO-soluble PBL-1 (10 mg / mL concentration) was added dropwise to the above HSA-NPs solution. After stirring for 30 minutes, 50 μL of 25% glutaraldehyde solution was added to initiate further cross-linking between albumins. After reacting for 6 hours, the mixture was centrifuged at 1200 rpm for 5 minutes to remove potential aggregates. Finally, free PB-L1 and other aggregates in the mixture were removed using a dextran gel column G50 to obtain purified drug-loaded HSA-NPs@PBL-1.

[0061] (3) Synthesis of PD-L-PLL.

[0062] Dry PLL-acetal (NanoSoft Polymers, Winston-Salem, NS, USA) was dissolved in 10 mL of phosphate-buffered saline (PBS: pH 4.5) at a concentration of 0.3 mg / mL, converting the acetal to an aldehyde. PD-L1 (100 μL, 500 mg / 10 mL) was coupled to a PLL-aldehyde (mass ratio 1:5) to generate PD-L1-PLL via coupling between the primary PD-L1 amine and the PLL-aldehyde. Excess product was removed using a Sephadex PD10 column (GE Healthcare Life Sciences, Marburg, MA). The product was collected by freeze-drying, and its chemical structure was analyzed by FT-RI spectroscopy.

[0063] (4) PDL1-PLL sequentially coats the phase-locked loop onto the surface of HSA-NPs@PBL-1 through electrostatic interaction. Excess PD-L1-PLL is removed by washing with distilled water.

[0064] Transmission electron microscope (TEM, LIBRA200CS, Carl Zeiss, Germany) images ( Figure 3 As shown in Figure A), the original HSA-NPs exhibit a spherical structure. After modification with PBL1 to form HSA-NPs@PBL1, and further assembly with PD-L1 to form HSA-NPs@PBL1@PD-L1, the nanoparticles still maintain a good spherical morphology.

[0065] The Z-Average hydrodynamic diameter, measured by dynamic light scattering (DLS, Nano ZS90 Zetasizer, Malvern Instruments Co., UK), further confirms the size variation of the nanoparticles. Figure 3(C, D, E). The Z-Average diameters of HSA-NPs, HSA-NPs@PBL1, and HSA-NPs@PBL1@PD-L1 are 155.4±1.2 nm, 158.4±0.8 nm, and 159.8±1.5 nm, respectively. The particle size gradually increases from HSA-NPs to HSA-NPs@PBL1 and then to HSA-NPs@PBL1@PD-L1, indicating that PBL11 and PD-L1 were successfully modified or assembled onto the surface of HSA-NPs.

[0066] The surface charge of the nanoparticles was determined using a zeta potential meter (NanoZS90Zetasizer, Malvern Instruments Co., UK). Figure 3 (B) The surface potential of HSA-NPs was (3.2±0.1) mV. After modification with PBL1, the Zeta potential of HSA-NPs@PBL1 decreased significantly to (-25.9±0.1) mV, indicating that the negatively charged PBL1 successfully bound to the surface of HSA-NPs. Subsequent modification with PD-L1 caused the potential to rise back to -2.3±0.2 mV. This significant reversal of potential confirmed the sequential assembly of PBL1 and PD-L1 on the nanoparticle surface.

[0067] Fourier transform infrared spectroscopy (FTIR, model 6300, Bio-Rad Ltd., USA) was used to analyze changes in the chemical groups of nanoparticles. Figure 3 (G). By comparing the infrared spectra of HSA-NPs, HSA-NPs@PBL1, and HSA-NPs@PBL1@PD-L1, shifts in characteristic absorption peaks or the appearance of new peaks can be observed. Figure 3 In the G (HSA-NPs@PBL1) sample, the length is 1246.94cm. -1 The presence of a characteristic COC peak at the ester bond directly confirms the introduction of PBL1; the amide I band remains at 1634 cm⁻¹. -1 The interaction force is primarily physical adsorption. Figure 3 In H(HSA-NPs@PBL1@PD-L1), the amide I band blue shifts to 1637.15 cm⁻¹. -1 (+3.04cm -1 The red shift of ) and amide III bands to 1241.55 cm⁻¹ -1 (-5.39cm -1 This indicates that PD-L1 interacts with proteins through hydrogen bond remodeling. (1246.94cm) -1 Peak shift further supports changes in the surface microenvironment.

[0068] 3. Detection of the inhibitory efficacy of PLB-1 against lung cancer cells

[0069] Lung cancer cells were digested and prepared into a cell suspension for cell counting. Based on the cell count, 8 × 10³ cells were seeded per well, with five replicates per well. Cells were incubated at 37°C for approximately 8 hours to allow for cell adhesion. PLB-1 and PBS were added as controls. At specified time points, μl of DMEM complete medium containing 10% CCK-8 was added to each well, and the cells were incubated for another 4 hours. The absorbance at 450 nm was measured, and a curve was plotted. The IC50 values ​​of pampasodilator (PLB-1) in lung cancer cell lines A549, H1650, H460, and H292 were determined using CCK8 assay.

[0070] Using CCK8 assay, the 24-hour IC50 values ​​of the plumbagolide isomer (PLB-1) in A549, H460, H1650, and H1292 cells were all less than 1 μM. The IC50 was used as the experimental concentration to assess the drug's inhibitory efficacy against lung cancer cells. The results showed that PLB-1 alone was only significantly effective against lung cancer cells within 24 hours, and was almost ineffective after 48 hours. Figure 4 (As shown in A, B, C, and D). Therefore, the search for sustained-release nanomaterials is of great scientific significance.

[0071] 4. Effects of PLB-1 on lung cancer cell proliferation, invasion, and migration

[0072] Lung cancer cells were seeded uniformly at a density of 30% in six-well plates 24 hours in advance. The medium was changed the next day: 1.5 ml of DMEM high-glucose medium was used, replacing the original cell culture conditions. PLB-1 and PBS were added as controls. The transfection promoter and transfection fragment were mixed and allowed to stand for 20 minutes before being added to the corresponding six-well plates. Cells were cultured for another 72 hours. After 72 hours, cells were collected for transwell experiments. 2 hours in advance, the matrix gel (BD, USA) was dissolved at 4°C and the pipette tip was pre-cooled. A 96-well plate was removed, and the chambers were placed in the 96-well plate. 150 μl of matrix gel was added to each well, and the plate was incubated at 37°C to solidify. 30,000 cells were transferred to the corresponding chambers at a volume of 50 μl (transfection method as before), and the plates were cultured for another 24 hours. The chambers were removed, washed twice with PBS, and fixed with 4% paraformaldehyde for 15 minutes. After washing twice with PBS, crystal violet staining (Beyotime, China) was performed. Wash with PBS, let it dry slightly, scrape off a thin film from the bottom of the chamber with a blade, mount it on a glass slide, observe and photograph it under a microscope.

[0073] Flow cytometry analysis revealed that the apoptosis rate of PLB-1 was significantly higher than that of the control group (P<0.01). Figure 5 (As shown in Figures A and B). The EDU and invasion experiments were used for detection, and the results are as follows. Figure 5As shown, PLB-1 significantly inhibited the proliferation, invasion, and migration of lung cancer H1650 and A549 cells.

[0074] 5. Transcriptome sequencing analysis of changes in the expression of related signaling pathways after PLB-1 treatment of lung cancer cells.

[0075] Lung cancer cells A549 and PC-9 were treated with PLB-1 for 24 hours before transcriptomics sequencing. For pretreatment, an appropriate amount of sample was taken according to extraction requirements, and RNA was extracted using the phenol / chloroform method. The extracted total RNA was diluted at a specific ratio before concentration and integrity testing. Purity was determined using a NanoDrop 2000 & 8000 micro-spectrophotometer; concentration and integrity were determined using an Agilent 2100 Bioanalyzer and an Agilent RNA 6000 NanoKit.

[0076] After treating lung cancer cells A549 and PC-9 with PLB-1, transcriptome analysis was performed. Heatmaps and KEEG pathway enrichment analysis revealed significant changes in the ferroptosis pathway in both cell lines. Intersection analysis indicated the presence of HMOX1 (upregulated), NQO1 (downregulated), and CD81 (downregulated) in both cell groups. Figure 6 Transcriptomic analysis revealed significant changes in the ferroptosis pathway after PLB-1 treatment. PLB-1 was analyzed in two lung cancer cell lines, A549 and PC-9, after 24 hours.

[0077] 6. The impact of HSA-NPs@PLB1@PDL1 on lung cancer

[0078] 6.1 Animal Model Establishment and Dosing Regimen

[0079] Six-week-old male nude mice (strain: BALB / cnude) were used for Lewis lung cancer cell xenograft experiments.

[0080] 6.2 Tumor Model Establishment:

[0081] (1) Lewis lung cancer cells in the logarithmic growth phase were injected with 1×10 6 Cells / cell density were resuspended in 1 mL of sterile phosphate-buffered saline (PBS, pH 7.4).

[0082] (2) Use a 26G needle to subcutaneously inject cell suspension into the scapular region on the back of mice, with an injection volume of 100 μL per mouse.

[0083] 6.3 Grouping and Dosing:

[0084] When the transplanted tumor grows to 50±5mm 3At 7-10 days post-injection, tumor-bearing mice were randomly divided into 6 groups (n=6) and administered the drug via intraperitoneal injection every 3 days according to the protocol shown in Table 1 below, for 3 weeks:

[0085] Table 1 Dosing Regimen

[0086]

[0087] Note: All drugs / nanoparticles were prepared using PBS as a solvent, and the control group was injected with an equal volume of PBS.

[0088] 6.4 Endpoint Indicator Analysis:

[0089] Tumor monitoring: Measure the tumor's long diameter (L) and short diameter (W) every 3 days using calipers, according to the formula... Calculate the volume.

[0090] Sample collection: 24 hours after the last administration, euthanasia was performed by cervical dislocation, and the tumor tissue was completely removed and weighed (accurate to 0.1 mg).

[0091] 6.5 Experimental Results

[0092] Morphological characteristics of end-stage tumors, such as Figure 7 As shown in A, by Figure 7 As shown in section A, the differences in volume and morphology of the six groups of tumor specimens can be observed using the scale bar (77-90 units):

[0093] ① Volume ranking: The tumors in the PBS group were the largest, exhibiting typical expansive growth, with smooth surfaces and a brownish-red color; the HSA nanoparticle group and the PD-L1 monotherapy group (approximately 84 units) were slightly smaller, but their morphology was similar to that of the PBS group; the combination therapy group (HAS@PLB-1@PD-L1) had the smallest tumors, with a volume only about 1 / 3 that of the PBS group.

[0094] ② Characteristic pathological changes: Tumors in the free PLB-1 group and HAS@PLB group are light pink with local wrinkling on the surface; tumors in the HAS@PLB-1@PD-L1 group are grayish-white with obvious depression on the surface (suggesting local necrosis or fibrosis).

[0095] Tumor growth kinetics results as follows Figure 7 As shown in B, by Figure 7 As shown in the B-cell curve, tumor volume increased in all treatment groups over time (weeks 1 to 5), but the HAS@PLB-1@PD-L1 combination therapy group exhibited a significant tumor-suppressing effect. At week 3, the tumor volume (mean ± SEM: approximately 350 mmHg) in the combination therapy group (HAS@PLB-1@PD-L1) was approximately 350 mmHg. 3 The result was significantly lower than that of the PD-L1 monotherapy group (approximately 620 mm). 3 ), PLB-1 single-drug group (approximately 600 mm)3 ) and HAS@PLB group (approximately 450mm) 3 (*p<0.01). At week 5, the tumor volume in the combined treatment group was 510±45mm. 3 This is only equivalent to the PBS control group (1860±120 mm). 3 The tumor inhibition rate was 72.6%, with a tumor inhibition rate of 27.4%. Its efficacy was significantly superior to that of free PLB-1 monotherapy (1420±100mm). 3 (tumor inhibition rate 23.7%); PD-L1 monotherapy (1450±95mm) 3 (tumor inhibition rate 22.0%); HAS@PLB (980±70mm) 3 (Tumor inhibition rate 47.3%).

[0096] 6.6 Conclusion

[0097] The HAS@PLB-1@PD-L1 nanocomposite significantly inhibited the growth of Lewis lung cancer xenografts (tumor inhibition rate of 72.6%) and induced tumor necrosis-like morphological changes by synergistically enhancing drug delivery and immunomodulation. Its efficacy was significantly superior to single-therapy (free drugs or single-loaded nanoparticles), providing experimental evidence for targeted-immunotherapy combination therapy for lung cancer.

[0098] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A leucanthin isomer PLB-1, characterized in that, The chemical structural formula of the plumbagoside isomer PLB-1 is as follows: 。 2. The method for synthesizing the leptin isomer PLB-1 as described in claim 1, characterized in that, Includes the following steps: (1) Mix 1,5-dihydroxynaphthalene, acetic anhydride and pyridine and stir to react, then add water, stir and cool to 0 degrees under ice water bath, filter and dry to obtain compound 1; (2) After dissolving compound 1 in acetic acid, NBS solution was added dropwise, followed by reaction. After cooling to room temperature, compound 2 was obtained by extraction, washing with water, drying and concentration. (3) Compound 2 was dispersed in ethanol, sulfuric acid aqueous solution was added, the mixture was heated to reflux and then cooled to room temperature. After extraction, washing with water, drying and concentration, compound 3 was obtained. (4) Compound 3, cyclopropylboronic acid, potassium phosphate and tricyclohexylphosphine were mixed in toluene solution, palladium acetate was added under nitrogen protection, the reaction was heated and then cooled to room temperature, and after extraction, washing with water, drying and concentration, the plumbagoside isomer PLB-1 was obtained.

3. A composition loaded with the leptin isomer PLB-1 as described in claim 1, characterized in that, The composition was prepared by loading albumin nanoparticles HAS-NPs with plumbagoside isomer PLB-1 and PD-L1 monoclonal antibody.

4. The method for preparing the composition according to claim 3, characterized in that, Includes the following steps: (1) Albumin nanoparticles were synthesized by solvent removal method and cross-linked with glutaraldehyde to further stabilize the nanoparticles; (2) Loading the hydrophobic drug PBL1 to synthesize HAS-NPs@PBL1 nanoparticles; (3) PDL1-PLL is generated through coupling between primary PDL1 amine and PLL aldehyde; (4) PDL1-PLL is sequentially coated onto the surface of HSA-NPs@PBL1 by electrostatic interaction, and then washed three times with distilled water to obtain the composition.

5. A drug for treating lung cancer, characterized in that, The drug comprises the plumbagolin isomer PLB-1 as described in claim 1 or the composition as described in claim 4.

6. The drug as described in claim 5, characterized in that, The plumbagoside isomer PLB-1 or the composition thereof is the sole active ingredient.

7. The use of the plumbagoside isomer PLB-1 as described in claim 1 or the composition as described in claim 4 in the preparation of products for the prevention and treatment of lung cancer.

8. The application as described in claim 7, characterized in that, The proposed prevention and treatment of lung cancer involves promoting ferroptosis in lung cancer patients through NQO1 / HO-1 to enhance immunotherapy and thus prevent and treat lung cancer.

9. The application as described in claim 7, characterized in that, When the product acts on lung cancer cells, it upregulates HMOX1, downregulates NQO1, and downregulates CD81.

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