A drug nanoparticle targeting PD-L1, its preparation method, and its application.

Abstract

This application discloses a drug nanoparticle targeting PD-L1, its preparation method, and its application, belonging to the field of oncology drug technology. The drug nanoparticle includes a drug and a carrier loading the drug. The drug is atorvaquinone. The carrier is prepared by coupling a second intermediate product with a first intermediate product. The first intermediate product is obtained by reacting bovine serum albumin with a heterobifunctional cross-linking agent. The second intermediate product is generated by reducing a thiol-modified nucleic acid aptamer, where the nucleic acid aptamer is a PD-L1 aptamer, and the heterobifunctional cross-linking agent is Sulfo-SMCC. This application uses a PD-L1-targeting nucleic acid aptamer coupled with bovine serum albumin to encapsulate atorvaquinone, preparing a novel atorvaquinone nanoparticle-targeted drug. This improves the precise targeting effect of atorvaquinone, overcomes the drawbacks of poor water solubility and high dosage of atorvaquinone, and reduces its systemic toxicity.

Description

Technical Field

[0001] This application belongs to the field of tumor drug technology, and specifically relates to a drug nanoparticle targeting PD-L1, its preparation method, and its application. Background Technology

[0002] Esophageal cancer is the seventh leading cause of cancer-related death worldwide. Although its incidence and mortality rates have improved in recent years, the prognosis remains grim. Esophageal cancer mainly includes two subtypes: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC), with ESCC being particularly common in Asia. Currently, various treatment modalities are available for esophageal cancer, including chemotherapy, targeted therapy, and surgery. Despite advancements in treatment, the overall five-year survival rate for ESCC remains very low, less than 20%, highlighting the urgent need to develop more effective ESCC-specific targeted therapies to improve esophageal cancer treatment outcomes.

[0003] Atorvaquinone (ATO), a classic antimalarial drug, has shown remarkable potential for "drug repurposing" in the field of anti-tumor therapy in recent years. Its application is not directly as a potent cytotoxic agent, but primarily based on its unique mitochondrial mechanism of action. Atorvaquinone can reduce the oxygen consumption of solid tumors by inhibiting the activity of mitochondrial complex III, thereby reducing the oxygen consumption rate of tumor cells, increasing the oxygen partial pressure in the tumor microenvironment, reversing hypoxia, and thus exerting its anti-tumor effect. Tumor stem cells, to meet the demands of rapid proliferation, often rely heavily on oxidative phosphorylation for energy. This metabolic dependence makes them particularly sensitive to the effects of atorvaquinone. Atorvaquinone inhibits oxidative phosphorylation, cutting off its energy source, increasing reactive oxygen species levels, leading to mitochondrial dysfunction and oxidative stress, ultimately inducing apoptosis. Atorvaquinone can selectively inhibit the self-renewal and tumorigenesis ability of tumor stem cells, which is crucial for preventing recurrence and metastasis. However, atorvaquinone has extremely low solubility in water and poor targeting, resulting in poor bioavailability, low blood concentrations, high dosages, and high systemic toxicity.

[0004] Aptamers are short-chain, single-stranded oligonucleotides capable of forming secondary and tertiary structures, exhibiting high affinity and specificity for target targeting. Based on the excellent properties of nucleic acid aptamers, various aptamer-mediated drug delivery systems have been developed, including aptamer-drug conjugates (ApDCs), aptamer-siRNAs, and aptamer-functionalized nanoparticle systems, aiming to effectively treat cancer, reduce potential toxicity, and improve therapeutic efficacy. Programmed death-ligand 1 (PD-L1) is highly expressed in cancer cells and inhibits T cell activity by binding to programmed death protein 1 (PD-1), making PD-L1 a key target for cancer therapy. Based on the properties of nucleic acid aptamers, research on aptamer-mediated tumor therapy targeting PD-L1 has been extensive. In the treatment of uveal melanoma (UM), integrating the PD-L1 aptamer into the drug-loaded nucleic acid nanomedicine SNAMA significantly enhanced the drug's tumor targeting and immunomodulatory capabilities, thereby improving therapeutic efficacy and providing a new strategy for UM treatment. A lipid nanoparticle (Apt-LNP) coupled with an anti-PD-L1 DNA aptamer can precisely deliver PTEN-encoding mRNA to castration-resistant prostate cancer cells. Compared with lipid nanoparticles without targeting ligands, aptamer-mediated endocytosis enables Apt-LNP to achieve higher transfection efficiency in vitro, significantly upregulate PTEN and inhibit PI3K / AKT phosphorylation, reduce cell viability, block migration, and induce apoptosis. These studies demonstrate that PD-L1 aptamer-mediated tumor therapy exhibits significant targeting and therapeutic effects in the treatment of various cancers. Combining the superior properties of aptamers targeting PD-L1, nano-targeted drugs prepared based on PD-L1 aptamers may provide new insights for the precision treatment of esophageal cancer.

[0005] Nanoparticle drug delivery systems have attracted significant attention due to their unique advantages in cancer treatment. These advantages include improved drug solubility, enhanced drug distribution within tumor tissues, reduced cytotoxicity, and the ability to cross biological barriers. Targeted delivery using nanocarriers can improve drug accumulation at the target site, reduce toxicity to non-cancer cells, and decrease the dosage. Albumin, with its biodegradable, biocompatible, and non-toxic properties, has become an ideal material for developing nanoparticles to deliver anticancer drugs. Summary of the Invention

[0006] To address the aforementioned issues, this application provides a drug nanoparticle targeting PD-L1, its preparation method, and its application.

[0007] The first objective of this application is to provide a drug nanoparticle targeting PD-L1, comprising a drug and a carrier loading the drug, wherein the drug is atovaquone (ATO), and the carrier is prepared by coupling a second intermediate product with a first intermediate product. The first intermediate product is obtained by reacting bovine serum albumin with a heterobifunctional cross-linking agent, and the second intermediate product is generated by reducing a thiol-modified nucleic acid aptamer, wherein the nucleic acid aptamer is PD-L1aptamer with the sequence: 5'-ACGGGCCACATCAACTCATTGATAGACAATGCGTCCACTGCCCGTTTTTTTTTTT-3'; and the heterobifunctional cross-linking agent is Sulfo-SMCC.

[0008] In a specific embodiment of this application, the PD-L1-targeting drug nanoparticles (referred to as Apt-BSA@ATO) have a particle size of 175-190 nm, a PDI value of 0.037-0.097, a Zeta potential of -19.0 to (-17.6) mV, an encapsulation efficiency of 71-91%, and a drug loading rate of 5.9-7.7%.

[0009] The second objective of this application is to provide a method for preparing drug nanoparticles targeting PD-L1, comprising: Bovine serum albumin was reacted with a heterobifunctional cross-linking agent to obtain the first intermediate product; The thiol-modified nucleic acid aptamer undergoes a reduction reaction with a reducing agent to yield a second intermediate product; The first intermediate product is coupled with the second intermediate product to obtain the carrier; An ethanol solution of atovaquinone was added dropwise to a PBS solution containing a delivery carrier, stirred, and the precipitate was collected to obtain drug nanoparticles targeting PD-L1.

[0010] In a specific embodiment of this application, the mass ratio of bovine serum albumin to heterobifunctional cross-linking agent is 10:1-2.

[0011] In a specific embodiment of this application, the mass ratio of bovine serum albumin to the thiol-modified nucleic acid aptamer is 50000:43-45.

[0012] In a specific embodiment of this application, the reducing agent is a phosphine-based reducing agent.

[0013] In a specific embodiment of this application, the thiol-modified nucleic acid aptamer is 5'-SH-C6-ACGGGCCACATCAACTCATTGATAGACAATGCGTCCACTGCCCGTTTTTTTTTTT-3', which is obtained through operations well known to those skilled in the art. The steps for obtaining the thiol-modified nucleic acid aptamer will not be described again in this application.

[0014] In a specific embodiment of this application, the mass ratio of the thiol-modified nucleic acid aptamer to the phosphine reducing agent is 43-45:114660.

[0015] In a specific embodiment of this application, the reaction conditions between the first intermediate product and the second intermediate product are: shaking for 8-12 hours in an ice-water bath.

[0016] In a specific embodiment of this application, the heterobifunctional crosslinking agent is 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester, abbreviated as Sulfo-SMCC.

[0017] The third objective of this application is to provide the application of PD-L1-targeting drug nanoparticles in the preparation of drugs for the treatment of esophageal cancer.

[0018] Compared with the prior art, this application has the following advantages: This application discloses a drug nanoparticle targeting PD-L1, its preparation method, and its application. This application uses an aptamer targeting PD-L1 conjugated with bovine serum albumin to encapsulate atovaquinone, thereby preparing a novel atovaquinone nano-targeting drug, which improves the precise targeting effect of atovaquinone, overcomes the disadvantages of poor water solubility and high dosage of atovaquinone, and reduces its systemic toxic side effects.

[0019] This application provides new ideas for the development of novel anti-esophageal cancer drugs and new strategies for the clinical treatment of esophageal cancer, and has important theoretical significance and clinical translational value.

[0020] Other features and advantages of this application will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the application. The objectives and other advantages of this application may be realized and obtained by means of the structures pointed out in the description, claims and drawings. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The test results diagram according to Embodiment 2 of this application is shown; Figure 2 The following diagram shows the expression levels of PD-L1 in EC109 and KYSE520 cells detected by Western blotting according to an embodiment of this application. Figure 3 The test results according to embodiments 3-4 of this application are shown as follows: Figure 3 As shown; where, Figure 3 In Figure A, the fluorescence graph of the uptake capacity of different esophageal cancer cells for different concentrations of Cy5-Apt-BSA was detected by flow cytometry. The vertical axis, count, represents the number of cells, and the horizontal axis, fluorescence intensity, represents the fluorescence intensity. Figure 3 B is Figure 3 The mean fluorescence intensity (MFI) graph of quantitative EC109 cells and KYSE520 cells in China is shown. The vertical axis represents the mean fluorescence intensity, and the horizontal axis represents the concentration of quantitative EC109 cells and KYSE520 cells. Blank represents the control group. Figure 3 Image C shows a laser confocal microscopy image of esophageal cancer cells co-incubated with different concentrations of Cy5-Apt-BSA for 4 h, where 'merge' represents the merged channel of different fluorescence channels. Scale bar = 50 μm. P<0.001; Figure 4 The test results according to embodiments 5-6 of this application are shown as follows: Figure 4 As shown; Figure 4 Image A shows a fluorescence image obtained by flow cytometry analysis of the Cy5-Apt-BSA uptake pathway in esophageal cancer cells. Figure 4 Figure B is a bar graph of the average fluorescence intensity obtained from flow cytometry results (n=3); Figure 4 Image C in the middle is a laser confocal microscope image of esophageal cancer cells after co-incubation with Cy5-Apt-BSA and three inhibitors for 4 h. Figure 4 In this context, CAB refers to Cy5-Apt-BSA, with a scale bar of 50 μm. P<0.001; Figure 5 The test results according to Embodiment 11 of this application are shown as follows: Figure 5 As shown; Figure 5 Image A shows fluorescence distribution in EC109 tumor-bearing nude mice at different time intervals after tail vein injection of Cy5-Apt-BSA@ATO. Figure 5 Image B shows the fluorescence distribution of KYSE520 tumor-bearing nude mice after tail vein injection of Cy5-Apt-BSA@ATO at different time intervals; Figure 5 Image C shows the in vitro fluorescence images of the internal organs and tumors of tumor-bearing nude mice after the tail vein injection of Cy5-Apt-BSA@ATO. Figure 6 The test results according to Embodiment 12 of this application are shown as follows: Figure 6 As shown; Figure 6 In Figure A, the antiproliferative effect of bare ATO on EC109 cells at different concentrations was detected using CCK-8 assay. Figure 6 In Figure B, the anti-proliferative capacity of Apt-BSA@ATO nanomedicine at different concentrations was detected using CCK-8 assay. Figure 6 In the graph, the vertical axis represents relative cell viability, and the horizontal axis represents concentration. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0024] Example 1: Preparation of Apt-BSA monomer 1. Accurately weigh 10 mg of BSA and dissolve it in 2 mL of PBS solution to prepare a concentration of 5 mg / mL for later use.

[0025] 2. Accurately weigh 20 mg of Sulfo-SMCC and dissolve it in 2 mL of PBS solution to prepare a concentration of 10 mg / mL. Take 100 μL and add it to the above solution.

[0026] 3. Under ice bath conditions, the reaction was carried out on a shaker at 150 rpm for 4 h to allow the N-hydroxysuccinimide ester group of Sulfo-SMCC to form a stable amide bond with the amino group of BSA.

[0027] 4. After the reaction is complete, remove unreacted Sulfo-SMCC by ultrafiltration using a 30 kDa ultrafiltration tube (MWCO). Centrifuge at 8000 rpm for 15 min. After centrifugation, resuspend in 200 μL PBS solution.

[0028] 5. Accurately weigh 0.02293 g TCEP and dissolve it in 100 μL of ultrapure water. Take 5 μL of 5'-thiol-modified PD-L1 nucleic acid aptamer (sequence 5'-SH-C6-ACGGGCCACATCAACTCATTGATAGACAATGCGTCCACTGCCCGTTTTTTTTTTT-3') solution (100 μM) and dilute it in 750 μL of ultrapure water. Combine the two solutions and react them in an ice bath at 150 rpm for 30 min. Activate the thiol group in the aptamer with TCEP (thiol groups are easily oxidized to disulfide bonds, and TCEP can reduce them to the active state).

[0029] 6. After combining the solutions described in steps 4 and 5, incubate overnight in an ice bath at 150 rpm on a shaker. This allows the Sulfo-SMCC, with BSA attached to one end, to bind to the thiol group on the aptamer via a maleimide group at the other end, thereby binding the aptamer to BSA and obtaining Apt-BSA.

[0030] 7. The reaction product was ultrafiltered using a 30 kDa ultrafiltration tube (MWCO) to remove unreacted free thiol-modified PD-L1 aptamers. The mixture was centrifuged three times, 15 min each time, at 8000 rpm. After each centrifugation, the product was resuspended in 1 mL of PBS. After all three centrifugations, the product was resuspended in 1 mL of PBS.

[0031] 8. Store the final product at -80℃ for later use.

[0032] Example 2: Agarose gel electrophoresis verification of the successful preparation of Apt-BSA monomer Agarose gel electrophoresis was used to assess whether the PD-L1 aptamer successfully conjugated with bovine serum albumin. The specific steps are as follows: 1. Accurately weigh 0.5 g of agarose and dissolve it in 25 mL of TAE solution by heating. After slightly cooling, add Super Red nucleic acid dye, mix well, and pour into a gel casting chamber to solidify.

[0033] 2. On an ice box, mix appropriate amounts of free thiol-modified PD-L1 nucleic acid aptamer, free BSA, and Apt-BSA with the loading buffer and load the samples. Electrophore the samples at 120 V for 35 min.

[0034] 3. Observe the position of the DNA bands and take pictures using an ultraviolet imaging device (Tanon-1600 gel image).

[0035] Example 3: Flow cytometry to investigate the ability of tumor cells to take up Apt-BSA To determine the uptake capacity of esophageal cancer cells EC109 and KYSE520 for Apt-BSA, flow cytometry was used to analyze the uptake capacity of esophageal cancer cells EC109 and KYSE520 for Cy5-Apt-BSA. The specific steps are as follows: 1. Esophageal cancer cells in the logarithmic growth phase, specifically EC109 and KYSE520, were seeded in six-well plates (3 × 10⁻⁶ cells / well). 5 Cells per well were incubated for 24 h in 2 mL of RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin solution.

[0036] 2. Divide each type of cell into three groups. After discarding the old culture medium, add 2 mL of fresh complete cell culture medium (corresponding to the blank group), 2 mL of fresh complete cell culture medium containing 0.1 mg / mL Cy5-Apt-BSA, and 2 mL of fresh complete cell culture medium containing 0.5 mg / mL Cy5-Apt-BSA to the blank group. Incubate at 37℃ in a 5% CO2 incubator in the dark for 4 h.

[0037] 3. The cells were digested with trypsin, centrifuged, washed, and then resuspended in EP tubes with 1 mL of PBS solution.

[0038] 4. Filter the cells through a 300-mesh nylon mesh, collect them in flow cytometry tubes, and analyze them using a flow cytometer to determine the signal intensity of each group of cells.

[0039] Example 4: Investigating the ability of tumor cells to take up Apt-BSA using laser confocal microscopy 1. Esophageal cancer cells in the logarithmic growth phase, EC109 and KYSE520, were collected at a concentration of 3 × 10⁻⁶. 4 One cell per well was seeded into an eight-well chamber, 800 μL of complete cell culture medium was added, and the chamber was incubated in a cell culture incubator for 24 h.

[0040] 2. Discard the old culture medium and gently wash twice with sterile PBS solution. Add 500 μL of fresh cell complete culture medium, 500 μL of fresh cell complete culture medium containing 0.1 mg / mL Cy5-Apt-BSA, and 500 μL of fresh cell complete culture medium containing 0.5 mg / mL Cy5-Apt-BSA to each cell type, and incubate at 37°C in a 5% CO2 incubator in the dark for 4 h.

[0041] 3. Discard the solution, gently wash twice with 500 μL PBS solution, then fix with 500 μL 4% paraformaldehyde solution in the dark for 15 min, wash once with 500 μL PBS solution, discard the solution, add anti-fluorescence quencher containing DAPI to stain the cell nuclei, observe and photograph under a laser confocal microscope.

[0042] Example 5: Flow cytometry analysis of tumor cell uptake of Apt-BSA To investigate the uptake mechanism of Apt-BSA in esophageal cancer cells, different uptake pathway inhibitors, including chlorpromazine (CPZ, a clathrin pathway inhibitor), genistein (Gen, a caveolin pathway inhibitor), and amiloride (EIPA, a macropinocytosis pathway inhibitor), were used to block different endocytic pathways. Flow cytometry was then used to analyze the uptake capacity of esophageal cancer cells EC109 and KYSE520 for Cy5-Apt-BSA to explore its uptake mechanism. The specific steps are as follows: 1. Esophageal cancer cells in the logarithmic growth phase, specifically EC109 and KYSE520, were seeded in six-well plates (3 × 10⁻⁶ cells / well). 5 Add 2 mL of complete cell culture medium to each well (1 cell / well) and incubate in a cell culture incubator for 24 h.

[0043] 2. Each cell type was divided into Control, CPZ, Gen, and EIPA groups. After discarding the old culture medium, 2 mL of fresh culture medium containing 0.1 mg / mL Cy5-Apt-BSA was added to the Control group, and 2 mL of mixed fresh culture medium containing inhibitors (CPZ 30 μM, EIPA 80 μM, Gen 200 μM) and 0.1 mg / mL Cy5-Apt-BSA was added to the inhibitor groups. The cells were incubated at 37°C in a 5% CO2 incubator in the dark for 4 h.

[0044] 3. The cells were digested with trypsin, centrifuged, washed, and then resuspended in EP tubes with 1 mL of PBS solution.

[0045] 4. Filter the cells through a 300-mesh nylon mesh, collect them in flow cytometry tubes, and analyze them using a flow cytometer to determine the signal intensity of each group of cells.

[0046] Example 6: Investigating the uptake of Apt-BSA by tumor cells using laser confocal microscopy 1. Esophageal cancer cells in the logarithmic growth phase, EC109 and KYSE520, were collected at a concentration of 3 × 10⁻⁶. 4 One cell per well was seeded into an eight-well chamber, 800 μL of complete cell culture medium was added, and the chamber was incubated in a cell culture incubator for 24 h.

[0047] 2. Discard the old culture medium and gently wash twice with sterile PBS solution. Divide each cell type into four groups (CAB group, CPZ group, Gen group and EIPA group). Add 500 μL of fresh culture medium containing 0.1 mg / mL Cy5-Apt-BSA to the CAB group. Add 500 μL of fresh culture medium containing the inhibitor (CPZ 30 μM, EIPA 80 μM, Gen 200 μM) and 0.1 mg / mL Cy5-Apt-BSA to the inhibitor groups. Incubate at 37℃ in a 5% CO2 incubator in the dark for 4 h.

[0048] 3. Discard the solution, gently wash twice with 500 μL PBS solution, fix with 500 μL 4% paraformaldehyde solution in the dark for 15 min, wash once more with 500 μL PBS solution, discard the solution, add anti-fluorescence quencher containing DAPI to stain the cell nuclei, observe and photograph under a laser confocal microscope.

[0049] Example 7: Preparation of Apt-BSA@ATO Apt-BSA was prepared into a 5 mL solution with a concentration of 2 mg / mL using PBS. 5 mg of atovaquinone was accurately weighed and dissolved in 5 mL of anhydrous ethanol to prepare a 1 mg / mL atovaquinone ethanol solution. At room temperature and 600 rpm, 0.5 mL of the prepared atovaquinone solution was added dropwise to the prepared Apt-BSA solution (at a constant rate of 0.5 mL / min). After stirring, the liquid was centrifuged at 12000 rpm for 15 min, the supernatant was discarded, and the precipitated Apt-BSA@ATO nanoparticles were resuspended in 1 mL of PBS and stored at 4°C for later use.

[0050] Example 8: Determination of encapsulation efficiency of Apt-BSA@ATO by high performance liquid chromatography 1. Preparation of mobile phase and standard solution (prepare fresh for immediate use): The mobile phase is acetonitrile (A) and 0.05% phosphoric acid water (B). After filtration to remove impurities and ultrasonication to remove air bubbles, it is ready for use. To prepare the standard solution, accurately weigh 2 mg of atovaquinone and dissolve it in 2 mL of anhydrous ethanol to prepare 2 mL of atovaquinone ethanol solution with a concentration of 1 mg / mL. Then, measure 1 mL of each of these six concentration gradients and dilute them to 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, and 0.01 mg / mL. Filter 1 mL of each of these six concentration gradients through a 0.22 μm microporous membrane for later use.

[0051] 2. Preparation of sample solution: Take 500 μL of the Apt-BSA@ATO nanoparticle solution prepared above, add 1.5 mL of acetonitrile, vortex for 3 min to fully demulsify, then centrifuge at 12000 rpm for 10 min to remove Apt-BSA, and filter the supernatant through a microporous membrane with a pore size of 0.22 μm for later use.

[0052] 3. High-performance liquid chromatography (HPLC) program settings: An Amethyst C-18H column (lengh×id, 4.6×250 mm, 5 μm particle size) was selected. The column temperature was maintained at 40℃. The mobile phase was acetonitrile (A) and 0.05% phosphoric acid water (B). The program was set to A:B=70:30, and the flow rate was 1 mL / min. The sample injection volume was preset to 10 μL. The absorbance was detected at 251 nm, and the run time was 35 min (atorvaquinone showed a peak at approximately 24 min under these conditions).

[0053] 4. Data processing: A standard curve is prepared based on the peak area of ​​the standard. The concentration of atovaquinone in the sample is calculated from the peak area of ​​the sample according to the standard curve. Then, the encapsulation efficiency is calculated according to the encapsulation efficiency formula.

[0054] Encapsulation efficiency formula: Encapsulation efficiency (EE)% = (Amount of drug encapsulated / Total amount of drug applied) × 100% Example 9: Determination of drug loading rate of Apt-BSA@ATO nanoparticles Accurately weigh the lyophilized vials and record their gross weight. Pipette three 500 μL samples of Apt-BSA@ATO pure aqueous solution into each vial, freeze at -80℃, seal with plastic wrap, and place in a vacuum freeze dryer for 48 h until the solution is completely dried into powder. Then, accurately weigh the lyophilized vials and subtract their gross weight to obtain the powder mass. Calculate the drug loading rate using the formula: Drug Loading Rate (DL)% = (Total mass of drug in the system / Total mass of the drug-loaded system) × 100%.

[0055] Example 10: Determination of particle size, PDI, and Zeta potential of Apt-BSA@ATO nanoparticles The particle size, dispersion index (PDI), and zeta potential of Apt-BSA@ATO nanoparticles were measured using a Malvern particle size analyzer. 200 μL of the prepared Apt-BSA@ATO nanoparticle solution was diluted in 800 μL of PBS solution. The diluted solution was filtered through a microporous membrane, and then transferred to a four-way permeable cuvette, which was cleaned with lens paper. The particle size and PDI were measured after setting the program. The Apt-BSA@ATO nanoparticle solution was then transferred to a Malvern potential sample cell using the same method, and the zeta potential was measured after setting the program.

[0056] Example 11: In vivo imaging of small animals to investigate the distribution of Apt-BSA@ATO in vivo. Six-week-old nude mice were inoculated with 5 × 10⁵ esophageal cancer cells, KYSE520 and EC109, in the right axilla. 6 (1 cell / each), until the tumor grows to 300 mm 3 Up to 500 mm 3 At the time of injection, Cy5-Apt-BSA@ATO (ATO: 15 mg / kg) was administered via the tail vein. Nude mice were anesthetized with isoflurane gas. In vivo tumor enrichment of Cy5-Apt-BSA@ATO was imaged at 6 h, 24 h, 96 h, 120 h, 144 h, 168 h, 192 h, and 240 h after Cy5-Apt-BSA@ATO injection. After the experiment, the nude mice were sacrificed, and their internal organs and tumors were dissected for fluorescence enrichment imaging. Image analysis was performed using Living Image.

[0057] Example 12: Determination of the antiproliferative capacity of ATO and Apt-BSA@ATO by CCK-8 assay 1. EC109 esophageal cancer cells in the logarithmic growth phase were seeded into 96-well plates (4 × 10⁻⁶ cells / well). 3 Add 100 μL of complete cell culture medium to each cell (1 cell / well) and incubate in a 37°C, 5% CO2 cell culture incubator for 24 h.

[0058] 2. Set up three groups: blank group, control group, and experimental group. The blank group contains only culture medium (without cells) for background correction; the control group contains cells and culture medium, but no drug treatment; the experimental group contains cells and different concentrations of the test drug, and the outermost wells of the plate are filled with PBS solution to reduce evaporation during culture.

[0059] 3. After 24 h of adherent cell culture, discard the old culture medium. Add 100 μL of fresh cell complete culture medium to each well of the control group, and add 100 μL of Apt-BSA@ATO solution (diluted with cell complete culture medium to concentrations of 120 μM, 60 μM, 30 μM, 15 μM, 7.5 μM, and 3.75 μM) to each well of the experimental group respectively. Incubate at 37℃ in a 5% CO2 incubator for 48 h.

[0060] 4. Under light-protected conditions, add CCK-8 reagent to the complete cell culture medium, discard the old culture medium, and wash with PBS. Add 100 μL of complete cell culture medium containing CCK-8 to each well of the blank group, control group, and experimental group, and incubate in a 37℃, 5% CO2 incubator for 1.5 h in the dark.

[0061] 5. Measure the absorbance (OD value) of each well at a wavelength of 450 nm using an ELISA reader, calculate the average OD value of each group of replicates, and calculate the cell viability (%) according to the formula: Cell viability (%) = [(OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group)] × 100%.

[0062] The results of Example 2 are shown in the figure below. Figure 1 As shown. Figure 1 Agarose gel electrophoresis was used to evaluate the coupling of Apt-BSA. Lane 1 was the marker, lane 2 was the free thiol-modified PD-L1 aptamer monomer, lane 3 was Apt-BSA, and lane 4 was free BSA.

[0063] To evaluate the successful coupling of PD-L1 aptamer with BSA, agarose gel electrophoresis was used for verification. The difference in electrophoretic velocity caused by the difference in molecular weight was used to investigate whether Apt-BSA was successfully coupled. The electrophoresis results are as follows: Figure 1 As shown, the PD-L1 aptamer modified with free thiol (lane 2) exhibits the fastest electrophoretic speed due to its lower molecular weight. Apt-BSA (lane 3), due to its binding to BSA, has a higher molecular weight and therefore a slower electrophoretic speed than the PD-L1 aptamer modified with free thiol. Free BSA (lane 4), lacking aptamer modification, shows no band. These results indicate successful coupling between the PD-L1 aptamer and BSA.

[0064] Among them, the results of Western blotting analysis of the differential expression of PD-L1 protein in esophageal cancer cells are as follows: Figure 2As shown. Programmed death-ligand-1 (PD-L1) is a protein highly expressed on the surface of tumor cells and is closely related to poor prognosis in cancer. This application used Western blotting to investigate the expression level of PD-L1 protein in esophageal cancer cells EC109 and KYSE520. Figure 2 As shown, PD-L1 is expressed in both EC109 and KYSE520 esophageal cancer cells, with higher expression levels in KYSE520 cells. This result suggests that PD-L1 may serve as a potential target for the treatment of esophageal cancer.

[0065] The test results of Examples 3-4 are as follows: Figure 3 As shown.

[0066] To investigate the uptake of Apt-BSA by esophageal cancer cells, flow cytometry was used to evaluate the uptake capacity of different human esophageal cancer cells. Figure 3 China A and Figure 3 As shown in Figure B, compared with the blank control group, esophageal cancer cells EC109 and KYSE520 showed different intensities of fluorescence signals after co-incubation with different concentrations of Cy5-Apt-BSA. Furthermore, the fluorescence signal of KYSE520 was higher than that of EC109 at both concentrations, indicating that KYSE520 cells, with their relatively high PD-L1 expression level, had a stronger ability to take up Apt-BSA. Laser confocal microscopy was used to evaluate the uptake of Apt-BSA by esophageal cancer cells. Figure 3 As shown in Figure C, the red fluorescence signal of KYSE520 cells incubated with different concentrations of Cy5-Apt-BSA was stronger than that of EC109. Therefore, it can be inferred that Apt-BSA can interact with PD-L1 overexpressed on the surface of cancer cells and be specifically taken up by it.

[0067] The test results of Examples 5-6 are as follows: Figure 4 As shown.

[0068] This application employs three major uptake pathway inhibitors: chlorpromazine (CPZ), genistein (Gen), and amiloride (EIPA) to block clathrin-mediated endocytosis, caveolin-mediated endocytosis, and macropinocytosis-mediated endocytosis, respectively, thereby elucidating the uptake pathway of Apt-BSA by esophageal cancer cells. Figure 4As shown in Figure A, flow cytometry analysis revealed that for EC109 cells, the fluorescence signal in the Gen inhibitor group was significantly weakened, indicating reduced cellular uptake in the presence of Gen. In contrast, the fluorescence signal intensity in the CPZ and EIPA groups did not decrease, suggesting that EC109 cells primarily rely on caveolin-mediated endocytosis for Apt-BSA uptake. For KYSE520 cells, all three inhibitor groups showed weakened fluorescence signals, with the EIPA group exhibiting the most significant decrease. Therefore, macropinocytosis-mediated endocytosis is the main pathway for Apt-BSA uptake in KYSE520 cells, while clathrin and caveolin-mediated pathways represent potential uptake pathways. The average fluorescence intensity statistics from flow cytometry further illustrate this conclusion. Figure 4 (Middle B). The uptake of Apt-BSA by esophageal cancer cells was investigated using laser confocal microscopy, such as... Figure 4 As shown in Figure C, the red fluorescence intensity of the Gen group in EC109 cells was significantly reduced, indicating that Gen is its main uptake channel inhibitor. In contrast, the red fluorescence intensity of all three inhibitor groups in KYSE520 cells was weakened, with the EIPA group showing the darkest red fluorescence, indicating that EIPA is its main uptake channel inhibitor, while CPZ and Gen are potential uptake channel inhibitors. These experimental results are consistent with the conclusions obtained from flow cytometry.

[0069] The test results of Examples 8-10 are shown in Table 1.

[0070] Table 1

[0071] The particle size, polydispersity index (PDI), and zeta potential were characterized using a Malvern particle size analyzer. The experimental results are shown in Table 1. The measured particle size was 183.0 ± 6.3 nm, the PDI was 0.067 ± 0.030, and the zeta potential was -18.30 ± 0.66 mV. As shown in Table 1, the encapsulation efficiency of Apt-BSA@ATO was 81.1 ± 9.2%, and the drug loading rate was 6.80 ± 0.86%, indicating that the nanoparticles were successfully prepared.

[0072] The test results of Example 11 are as follows: Figure 5 As shown.

[0073] To investigate the in vivo targeting ability of Apt-BSA@ATO, this application administered Cy5-Apt-BSA@ATO via tail vein injection to nude mice inoculated with KYSE520 and EC109 cells, followed by in vivo imaging analysis of the small animals. Figure 5 China A, Figure 5As shown in Figure B, Cy5-Apt-BSA@ATO effectively accumulates in tumor tissue over time. After the experiment, major organs and tumor tissues from nude mice were removed for in vitro fluorescence imaging. Figure 5 As shown in Figure C, the fluorescence intensity in KYSE520 cell tumor tissue from nude mice with high PD-L1 expression was significantly higher than that in EC109 cell tumor tissue from nude mice. In vivo and ex vivo imaging analyses indicated that Cy5-Apt-BSA@ATO exhibited targeting ability and specifically targeted tumor tissues with high PD-L1 expression levels. This suggests that the drug has a targeting effect and is more suitable for in vivo application than traditional atorvaquinone monotherapy.

[0074] The test results of Example 12 are shown in the figure below. Figure 6 As shown. Figure 6 In Figure A, the antiproliferative capacity of bare ATO at different concentrations against EC109 cells was detected using CCK-8 assay. Figure 6 In Figure B, the anti-proliferative capacity of Apt-BSA@ATO nanomedicine at different concentrations was detected using CCK-8 assay.

[0075] The cell viability of EC109 cells was determined using the CCK-8 assay for both ATO-naïve and Apt-BSA@ATO nanomedicine. Figure 6 The results showed that Apt-BSA@ATO significantly inhibited the proliferation of EC109 esophageal cancer cells, with a half-maximal inhibitory concentration (IC50) of 100%. 50 The concentration was 7.1 ± 1.3 μM, compared to ATO's IC50. 50 =13.0±0.1 μM is lower, and Apt-BSA@ATO has stronger anti-proliferative ability at high concentrations, indicating that Apt-BSA@ATO has targeted anti-tumor activity and the potential to reduce ATO drug use.

[0076] Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A drug nanoparticle targeting PD-L1, characterized in that, The invention includes a drug and a carrier loaded with the drug, wherein the drug is atorvaquinone, and the carrier is prepared by coupling a second intermediate product and a first intermediate product. The first intermediate product is obtained by reacting bovine serum albumin with a heterobifunctional cross-linking agent, and the second intermediate product is generated by reducing a thiol-modified nucleic acid aptamer, wherein the nucleic acid aptamer is PD-L1 aptamer, and the heterobifunctional cross-linking agent is Sulfo-SMCC.

2. The drug nanoparticle targeting PD-L1 according to claim 1, characterized in that, The particle size of the drug nanoparticles targeting PD-L1 is 175-190 nm.

3. A method for preparing PD-L1-targeting drug nanoparticles according to claim 1 or 2, characterized in that, include: Bovine serum albumin was reacted with a heterobifunctional cross-linking agent to obtain the first intermediate product; The thiol-modified nucleic acid aptamer was reduced with a reducing agent to obtain the second intermediate product; The first intermediate product is coupled with the second intermediate product to obtain the carrier; An ethanol solution of atovaquinone was added dropwise to a PBS solution containing a delivery carrier, stirred, and the precipitate was collected to obtain drug nanoparticles targeting PD-L1.

4. The method for preparing PD-L1-targeting drug nanoparticles according to claim 3, characterized in that, The mass ratio of bovine serum albumin to heterobifunctional cross-linking agent is 10:1-2.

5. The method for preparing PD-L1-targeting drug nanoparticles according to claim 3, characterized in that, The mass ratio of bovine serum albumin to the thiol-modified nucleic acid aptamer is 50000:43-45.

6. The method for preparing PD-L1-targeting drug nanoparticles according to claim 3, characterized in that, The reducing agent is a phosphine-based reducing agent.

7. The method for preparing PD-L1-targeting drug nanoparticles according to claim 6, characterized in that, The mass ratio of the thiol-modified nucleic acid aptamer to the phosphine reducing agent is 43-45:114660.

8. The method for preparing PD-L1-targeting drug nanoparticles according to claim 3, characterized in that, The reaction conditions for the first intermediate product and the second intermediate product are: shaking for 8-12 hours in an ice-water bath.

9. The method for preparing PD-L1-targeting drug nanoparticles according to claim 6, characterized in that, The heterobifunctional crosslinking agent is 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (Sulfo-SMCC).

10. The use of a PD-L1-targeting drug nanoparticle according to any one of claims 1-2 in the preparation of a drug for treating esophageal cancer.

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