Gemcitabine nano-particles, anti-tumor combined medicine and application of gemcitabine nano-particles and anti-tumor combined medicine

Abstract

The invention relates to the technical field of biological pharmacy, in particular to a gemcitabine nano-particle, an anti-tumor combined medicine and application, and the gemcitabine nano-particle is composed of a gemcitabine core and a cell membrane covering the gemcitabine core; the cell membrane is derived from a CT26-PD-1 cell which is subjected to over-expression of PD-1 after genetic engineering modification; the particle size of the gemcitabine nano-particles ranges from 150 nm to 200 nm. The gemcitabine is coated by a PD-1 overexpressed tumor cell membrane, the enrichment capacity of the gemcitabine in tumors is enhanced by virtue of the interaction of PD-1 and PD-L1, and the biotoxicity and short blood half-life period of the gemcitabine are improved; by combining with a CD73 inhibitor AB680, precise targeted delivery and immune microenvironment regulation and control on the colorectal cancer are realized, and an efficient and low-toxicity innovative scheme is provided for tumor treatment.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to gemcitabine nanoparticles, anti-tumor combination drugs, and their applications. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive system, the third most common cancer worldwide, and the second leading cause of cancer-related deaths, posing a significant challenge to public health. CRC patients are often diagnosed at an advanced stage due to the insidious nature of early symptoms. Although surgery, chemotherapy, and radiotherapy are currently the main treatments for advanced CRC, clinical efficacy still faces multiple challenges. While surgical resection combined with chemotherapy remains the primary treatment for advanced CRC, the efficacy of traditional chemotherapy drugs is significantly limited due to systemic toxicity, drug resistance, and the immunosuppressive properties of the tumor microenvironment (TME). Gemcitabine (GEM) is commonly used to treat advanced colorectal cancer, pancreatic cancer, and lung cancer, but its short half-life, non-specific distribution, and immune escape caused by PD-L1 upregulation further exacerbate the risk of treatment failure, making it difficult to overcome the bottleneck of the "efficacy-toxicity" balance. How to overcome the immunosuppressive barrier, reverse drug resistance, and achieve precise targeted therapy has become a core challenge that urgently needs to be addressed in the field of CRC. Summary of the Invention

[0003] To address the above problems, this invention provides gemcitabine nanoparticles, a combination antitumor drug, and its application.

[0004] This invention is achieved through the following technical solution: A gemcitabine nanoparticle consists of a gemcitabine core and a cell membrane covering it; the cell membrane is derived from CT26-PD-1 cells that have been genetically engineered to overexpress PD-1; the gemcitabine nanoparticle has a particle size of 150 nm to 200 nm.

[0005] Preferably, the specific steps include: By genetically engineering CT26 cells, a cell line overexpressing PD-1, CT26-PD-1, was constructed. CT26-PD-1 cell membranes were extracted and gemcitabine was encapsulated in the CT26-PD-1 cell membranes to form gemcitabine nanoparticles.

[0006] An antitumor combination drug, wherein the antitumor combination drug uses gemcitabine nanoparticles as described in claim 1 and a CD73 inhibitor as active ingredients; the mass ratio of the gemcitabine nanoparticles to the CD73 inhibitor is 5-10:1.

[0007] Preferably, the CD73 inhibitor is AB680.

[0008] Preferably, the antitumor combination drug can be formulated into a clinically acceptable formulation together with or separately from pharmaceutically acceptable excipients.

[0009] Preferably, the pharmaceutically acceptable carrier is physiological saline.

[0010] Preferably, the formulation is a liquid formulation.

[0011] The application of the aforementioned combination of tumor drugs in the preparation of reagents for the prevention and treatment of colorectal cancer.

[0012] Compared with the prior art, the present invention has the following beneficial effects: This invention provides gemcitabine nanoparticles, consisting of a gemcitabine core and an outer cell membrane; the cell membrane is derived from CT26-PD-1 cells overexpressing PD-1 through genetic engineering; the gemcitabine nanoparticles have a particle size of 150nm~200nm. CM@GEM NPs actively target tumor cells highly expressing PD-L1 via the PD-1 / PD-L1 axis, improving the efficiency of intratumoral drug accumulation.

[0013] This invention constructs a biomimetic nanodelivery system (CM@GEM NPs) based on active targeting of the PD-1 / PD-L1 axis. Experimental results demonstrate that GEM treatment can upregulate PD-L1 expression levels in colorectal cancer cells. This invention enhances gemcitabine accumulation in tumors by coating tumor cells overexpressing PD-1 with gemcitabine (GEM), leveraging the interaction between PD-1 and PD-L1, thus improving gemcitabine's biotoxicity and shortening its blood half-life. Combined with the CD73 inhibitor AB680, it blocks the adenosine metabolic pathway, reverses the immunosuppressive microenvironment, and drives macrophage M1 polarization and CD8+. + T-cell infiltration breaks the "cold tumor" dilemma of CRC. This strategy, through the synergistic effect of targeted delivery and immune regulation, not only overcomes gemcitabine resistance and induces durable anti-tumor immune memory, but also reveals the key mechanism of the CD73-adenosine pathway in chemotherapy resistance, providing a new idea with theoretical basis and clinical translational value for precision treatment of CRC. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention 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 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.

[0015] Figure 1 These are morphological features and property characterization diagrams of the CM@GEM of this invention; Figure 1 In the diagram, A shows the immunofluorescence staining results of PD-L1 in CT26 cells after different drug treatments; B shows the statistical analysis of PD-L1 fluorescence intensity; C shows the PD-L1 fluorescence intensity of CT26 cells after different drug treatments. CD274 The results of mRNA transcription levels of CM@GEM NPs are shown in Figure 1 (control group, AB680 alone treatment group, GEM alone treatment group, and AB680 and GEM combined treatment group); Figure 2 shows the verification results of PD-1 overexpression efficiency after lentiviral transfection; Figure 3 shows the morphology of CM@GEM NPs under transmission electron microscopy, scale bar = 100 nm; Figure 4 shows the particle size distribution of empty CM NPs and CM@GEM NPs; Figure 5 shows the zeta potential distribution of empty CM NPs and CM@GEM NPs; Figure 6 shows the SDS-PAGE analysis results of CT26 cell lysate, purified cell membrane, and CM@GEM NPs; Figure 7 shows the detection results of PD-1 protein expression on the surface of CM@GEM NPs; Figure 8 shows the release curve of CM@GEM NPs at different pH values; Figure 9 shows the particle size change of CM@GEM NPs over 5 days; Figure 10 shows the change of PDI of CM@GEM NPs over 5 days; Figure 11 shows the flow cytometry detection of CM@GEM endocytosis in CT26 cells. The efficiency results of NPs are shown in the figure; N is the statistical graph corresponding to M; O is the result of laser confocal microscopy detection of CT26 cells phagocytizing CM@GEM NPs.

[0016] Figure 2 This is a hemolysis test image of the CM@GEM of this invention; Figure 2 In the diagram, A represents the results of the hemolysis test; B represents the statistical chart corresponding to A.

[0017] Figure 3 This is a diagram illustrating the in vitro tumor-killing effect of CM@GEM according to the present invention. Figure 3 In the diagram, A is the growth curve of CT26 cells after different treatments within 48 hours monitored by the live cell workstation; B is the relative viability of CT26 cells after different treatments; C is the proliferation status of CT26 cells after different treatments; D is the detection result of the expression levels of CD73 and γ-H2AX in CT26 cells after different treatments; E is the statistical graph corresponding to D; and F is the statistical graph corresponding to D.

[0018] Figure 4 This is a biosafety test image of the CM@GEM of this invention.

[0019] Figure 5 This is a targeting detection map of the CM@GEM of the present invention; Figure 5In the diagram, A represents the results of the in vivo targeting detection experiment of nanoparticles; B is a magnified view of the corresponding part in A.

[0020] Figure 6 This is a graph illustrating the in vivo tumor suppression effect of CM@GEM from this invention. Figure 6 In the diagram, A represents the animal experiment model; B represents the overall growth curve of subcutaneous tumors in mice of each group; C represents the growth curve of subcutaneous tumors in mice in the control group (injected with physiological saline); D represents the growth curve of subcutaneous tumors in mice in the AB680 treatment group (n=6); E represents the growth curve of subcutaneous tumors in mice in the GEM treatment group (n=6); F represents the growth curve of subcutaneous tumors in mice in the AB680+GEM combined treatment group (n=6); G represents the growth curve of subcutaneous tumors in mice in the CM@GEM treatment group (n=6); H represents the growth curve of subcutaneous tumors in mice in the AB680+CM@GEM treatment group (n=6); I represents photographs of ex vivo tumors in mice of each group (n=6); J represents the statistical results of subcutaneous tumor weight in mice of each group (n=6); and K represents the results of weight changes in mice of each group during the treatment period (n=6).

[0021] Figure 7 This is a graph showing the results of verifying the killing effect of the present invention in mice after different treatments; Figure 7 In the diagram, A represents the stained pathological sections of mouse tumors after different treatments; B represents the statistical graph corresponding to A; and C represents the statistical graph corresponding to A.

[0022] Figure 8 This invention describes the infiltration of immune cells into the tumor of mice after different treatments. Figure 8 In the diagram, A is an immunohistochemical staining image of mouse tumors; B is a statistical graph corresponding to A; and C is a statistical graph corresponding to A. Detailed Implementation

[0023] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0024] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0025] The inventive concept of this invention is as follows: Colorectal cancer, one of the most common malignant tumors worldwide, often presents with subtle early symptoms, leading to approximately 65% ​​of patients being diagnosed at an advanced stage, thus losing the opportunity for radical surgery. Gemcitabine (GEM), a nucleoside analogue chemotherapy drug, is widely used in the treatment of solid tumors, but its inherent limitations—short plasma half-life, dose-dependent toxicity, and acquired resistance—severely restrict its clinical efficacy.

[0026] Based on this, the present invention innovatively constructs PD-1 overexpression engineered cell membrane nanoparticles CM@GEM, uses a nano-drug delivery system to prolong the GEM cycle time and reduce toxicity, and utilizes the phenomenon of GEM inducing colorectal cancer cells to overexpress PD-L1. Through the binding effect of PD-1 / PD-L1, it enhances tumor-specific accumulation and kills tumor cells, improves the biotoxicity of gemcitabine and has a shorter blood half-life.

[0027] Furthermore, this invention provides an anti-tumor combination drug that combines nanoparticles CM@GEM with the CD73 small molecule inhibitor AB680 to block the adenosine-mediated immunosuppressive pathway activated by the release of intracellular ATP due to tumor cell death. This synergistically overcomes chemotherapy resistance in colorectal cancer and reshapes the anti-tumor immune response, providing a new strategy for the comprehensive treatment of colorectal cancer.

[0028] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0029] The GEM of this invention was purchased from MCE Company, item number HY-B0003; AB680 was purchased from MCE Company, item number HY-125286.

[0030] Example 1 To investigate the effects of different drug treatments on PD-L1 expression in cells, CT26 mouse colorectal cancer cells in logarithmic growth phase were divided into a control group, an AB680-only treatment group, a GEM-only treatment group, and a combined AB680 and GEM treatment group for in vitro culture, immunofluorescence staining, and quantitative real-time fluorescence PCR (qPCR) experiments.

[0031] The control group consisted of cells treated with complete culture medium containing 0.1% DMSO by volume.

[0032] The AB680-only treatment group consisted of cells treated with complete culture medium containing 200 nM AB680.

[0033] The GEM-only treatment group consisted of cells treated with complete culture medium containing 100 nM gemcitabine.

[0034] The AB680 and GEM combined treatment group consisted of cells treated with a complete culture medium containing 200 nM AB680 and 100 nM GEM.

[0035] Cells from all groups were collected for further testing 24 hours after processing.

[0036] Immunofluorescence staining results as follows Figure 1 As shown in A~B, compared with the control group, the immunofluorescence intensity of PD-L1 protein was significantly enhanced in CT26 cells treated with GEM and in AB680 combined with GEM.

[0037] Furthermore, the qPCR experimental results Figure 1 As shown in Figure C, the GEM-only treatment group and the AB680 + GEM combined treatment group significantly upregulated PD-L1 (CD274) expression in CT26 cells at the mRNA level. The control group and the AB680-only treatment group had no significant effect on PD-L1 expression.

[0038] Example 2: Preparation method of CM@GEM NPs (1) Construction of CT26-PD-1 cell line overexpressing PD-1 CT26 cells were seeded into culture dishes. When the cells reached approximately 20% confluence, the medium was replaced with fresh medium, and the transfection reagent HitransGA was added. Control virus and PD-1 overexpression virus were added separately at an MOI of 50 and mixed well. The cells were incubated in a CO2 incubator for 16 hours, then the medium was replaced with complete medium, and the cells were cultured further. Approximately 48 hours post-infection, the medium was replaced with fresh medium, and puromycin was added. After 48 hours of selection, all untransfected cells died, and the remaining cells were all positive. The puromycin concentration was reduced to a maintenance concentration, and infected cells were screened again. Cells with correct identification results were seeded. The identification results are shown below. Figure 1 As shown in D, this indicates that a stable CT26 cell line overexpressing PD-1 was successfully constructed through lentiviral transfection, denoted as CT26-PD-1.

[0039] (2) Extraction of CT26 cell membrane CT26-PD-1 cells were digested and collected, resuspended in PBS, and washed three times by centrifugation at 500g for 5 minutes. The washed cell pellet was resuspended in 10 mL of hypotonic buffer containing 20 mM Tris-HCl, 10 mM KCl, 2 mM MgCl2, and one EDTA-free mini protease inhibitor tablet per 10 mL solution, and lysed overnight. The lysed cell solution was transferred to a Dounce homogenizer and homogenized 20 times on ice. The homogenate was centrifuged at 3500g for 5 minutes at 4°C to remove cell debris. The supernatant was then ultracentrifuged at 100,000g for 1 hour at 4°C. The resulting pellet was the purified cell membrane.

[0040] (3) GEM load The purified cell membrane (20 mg, based on protein concentration) was mixed with 1 mL of 67 μM GEM solution and incubated at 37 °C with shaking for 30 minutes. The mixture of cell membrane and GEM was then sonicated for 5 minutes (80 W, 5 seconds sonication / 5 seconds interval). The sonicated solution was squeezed through a 400 nm polycarbonate membrane 20 times to ensure uniform nanoparticle size. Free GEM was removed by ultracentrifugation at 100,000 g for 1 hour at 4 °C. The nanoparticles were washed three times with PBS, with each centrifugation under the same conditions, to obtain gemcitabine nanoparticles, denoted as CM@GEM NPs, or CM@GEM for short.

[0041] Example 3: Characterization of CM@GEM NPs 1. Experimental Methods (1) Flow cytometry detection of nanoparticle endocytosis Empty cell membrane nanoparticles were extracted. The extracted nanoparticles were labeled with Did dye and co-incubated with CT26 cells. Cells were collected at 0.5 h, 1 h, 2 h, 3 h, and 4 h, digested with EDTA-free trypsin, centrifuged at 500 g for 5 min at 4 °C, and washed three times with pre-cooled PBS to remove free or adherent nanoparticles. The washed cells were resuspended in 500 μl PBS and analyzed by flow cytometry within 1 hour to extract empty cell membrane nanoparticles, denoted as CM NPs, or CM for short.

[0042] (2) Detection of nanoparticle endocytosis by confocal microscopy CT26 cells were seeded into 6-well plates with pre-coated slides, and CFDA-SE dye was added to label the cells. Empty cell membrane nanoparticles extracted using RhB were labeled. When the CT26 cells reached approximately 50% confluence, the fluorescently labeled nanoparticles were added, and after co-incubation for 4 hours, the cells were washed three times with PBS to remove free or adherent nanoparticles from the cell surface.

[0043] (3) Hemolysis test Add 10 IU / mL of heparin sodium to centrifuge tubes and collect blood from the orbital region of mice. Mix the blood with the heparin sodium thoroughly. Centrifuge at 1000 rpm for 5 minutes, remove serum, add 1 mL of PBS, and wash the blood cells three times, centrifuging under the same conditions each time, until the supernatant is clear and colorless. Centrifuge to remove PBS, and aliquot the blood cells into EP tubes, 20 μL per tube. Using the CM@GEM NPs prepared in Example 1, resuspend the particles in physiological saline to prepare drug solutions of 50 nM, 125 nM, 250 nM, 500 nM, and 1000 nM according to the GEM equivalent concentration. Add these solutions to the blood cells and mix gently. A negative control group (physiological saline) and a positive control group (deionized water) are also set up for control. Incubate at 37°C for 1 hour. Centrifuge at 1000 rpm for 5 minutes at room temperature, observe hemolysis, and record the results by photography. Collect 200 μL of supernatant and measure its absorbance at 540 nm using an ELISA reader.

[0044] The hemolysis rate is calculated using the formula: Hemolysis rate = (Absorbance of experimental group - Absorbance of negative control group) / (Absorbance of positive control group - Absorbance of negative control group) × 100%.

[0045] 2. Experimental Results Transmission electron microscopy (TEM) images such as Figure 1 The E data shows that CM@GEM NPs exhibit a typical spherical or near-spherical core-shell structure with uniform distribution.

[0046] Dynamic light scattering (DLS) detection, such as Figure 1 As shown in F~G, the hydrated particle size of CM@GEM NPs is approximately 180 nm, consistent with the particle size of unloaded cell membrane nanoparticles (CM NPs), while the zeta potentials of both are negative, consistent with the typical characteristics of cell membrane vesicles. The slight positive shift in the zeta potential of the nanoparticles after GEM loading indicates successful GEM encapsulation.

[0047] Figure 1 H in the figure represents the lysate of CT26 cells overexpressing PD-1 (corresponding to the control in the figure). The purified cell membrane of CT26 cells overexpressing PD-1 (corresponding to CM-PD1OE in the figure) and the SDS-PAGE analysis of CM@GEM show that most of the proteins on the cell membrane surface were preserved during the synthesis process.

[0048] Figure 1In the figure, I represents the detection of PD-1 protein expression levels on the surface of nanoparticles constructed using normal CT26 cell membranes and CT26 cell membranes overexpressing PD-1, respectively (CT26@GEM is nanoparticle constructed using normal CT26 cell membranes, and CM@GEM is nanoparticle constructed using CT26 cell membranes overexpressing PD-1). The method for constructing nanoparticles using normal CT26 cell membranes is the same as the method for constructing nanoparticles using CT26 cell membranes overexpressing PD-1 in Example 2.

[0049] In vitro drug release curves at different pH values ​​are as follows: Figure 1 As shown in J, CM@GEM releases GEM faster in an acidic environment.

[0050] Particle size and polydispersity index (PDI) monitoring results are as follows: Figure 1 As shown in K~L, CM@GEM did not undergo significant changes in particle size and PDI after being stored in PBS for 5 days, demonstrating that the nano-formulation has good colloidal stability and can meet the requirements for in vivo delivery.

[0051] The results of the hemolysis test are as follows Figure 2 As shown in A~B, this indicates that even at high concentrations, CM@GEM does not cause a significant hemolytic effect.

[0052] The experimental results of this embodiment fully demonstrate that the present invention has successfully prepared biomimetic nanoparticles CM@GEM NPs with uniform particle size, good stability, pH-responsive drug release characteristics, and surface display of PD-1 protein. These nanoparticles can be efficiently internalized by tumor cells and exhibit good biocompatibility.

[0053] Example 4: Validation of the in vitro tumor-killing effect of AB680 combined with CM@GEM 1. Experimental Methods (1) Cell proliferation experiment CT26 cells in logarithmic growth phase were collected and seeded at a density of 3000 cells per well into 100 μL of complete culture medium per well of a 96-well plate. The edge wells were sealed with 1×PBS to prevent moisture evaporation. The plate was incubated at 37°C with 5% CO2 for 24 hours until the cells were fully adherent. After cell adhesion, the culture medium was discarded, and fresh complete culture medium containing the following treatment drugs was added:

[0054] Control group: Complete culture medium containing 0.1% DMSO (by volume).

[0055] AB680 monotherapy group: complete culture medium containing 200 nM AB680.

[0056] GEM-only treatment group: complete culture medium containing 100 nM gemcitabine (GEM).

[0057] AB680+GEM co-treatment group: complete culture medium containing 200 nM AB680 and 100 nM GEM.

[0058] CM@GEM treatment group: CM@GEM suspension containing GEM equivalent concentration of 100 nM.

[0059] AB680+CM@GEM co-treatment group: containing 200 nM AB680 and CM@GEM suspension with an equivalent concentration of 100 nM GEM.

[0060] The treated cells were placed in a live cell workstation and continuously monitored for 48 hours. The cell confluence at each time point was automatically recorded to plot growth curves and calculate the relative viability and proliferation status of each group of cells.

[0061] (2) Western Blot To investigate the effects of different treatments on DNA damage and the expression of the immunosuppressive molecule CD73, cells from each group were collected 24 hours after drug treatment. Total protein was extracted using RIPA lysis buffer and quantified using the BCA method. Equal volumes of protein samples were subjected to SDS-PAGE electrophoresis and transferred to PVDF membranes. After blocking with 5% skim milk, the membranes were incubated overnight at 4°C with primary antibodies against anti-CD73 (12231-1-AP, Proteintech), anti-γ-H2AX (GB111841-100, Servicebio), and anti-β-actin (AF7018, Affinity), respectively. The next day, after washing with PBST, the membranes were incubated with the corresponding horseradish peroxidase-labeled secondary antibody at room temperature for 1 hour. Finally, the target protein bands were detected using an imaging system with ECL chemiluminescence imaging solution.

[0062] 2. Experimental Results like Figure 3 The experimental results showed that after CT26 cells were treated with 0.1% DMSO (control group), AB680 alone, GEM alone, AB680+GEM combined treatment, CM@GEM treatment, and AB680+CM@GEM combined treatment in vitro, CM@GEM had similar tumor inhibitory effects and genomic toxicity to free GEM at equivalent concentrations in vitro, while the tumor inhibitory effect of AB680 by promoting immune cell infiltration could not be observed in the in vitro environment.

[0063] Example 5: Verification of the in vivo tumor-killing effect of AB680 combined with CM@GEM 1. Experimental Methods (1) Construction of mouse subcutaneous tumor model Eight-week-old female BALB / c mice weighing 18g were randomly selected and acclimatized for one week. CT26 cells were collected from the digestive tract according to the method described above, and the cell concentration was adjusted to 5×10⁻⁶ cells using PBS. 6 100 μL of CT26 cell suspension was injected into the groin of mice after disinfection with an alcohol swab, and the needle was slowly withdrawn to prevent the cell suspension from flowing out.

[0064] (2) Biosafety testing of CM@GEM To assess the in vivo safety of CM@GEM, tumors were cultured at a size of approximately 75 mm. 3 Subsequently, tumor-bearing mice were randomly divided into three groups (n=3): ① saline control group (tail vein injection, injection volume 100 μl), ② empty CM NPs group (tail vein injection, dose consistent with CM@GEM group membrane protein level, injection volume 100 μl), ③ CM@GEM group (tail vein injection, GEM equivalent dose 20 mg / kg, injection volume 100 μl). Administered the medication every three days for four consecutive days. On the third day after the last administration, the mice were euthanized, and major organs such as the heart, liver, spleen, lungs, and kidneys were harvested. These organs were fixed in 4% paraformaldehyde (by weight / volume), embedded in paraffin, stained with H&E, and observed under a light microscope for histopathological changes.

[0065] (3) Small animal live imaging To verify the tumor targeting ability of CM@GEM, a subcutaneous tumor model of CT26 was established, with the tumor volume reaching approximately 100 mm. 3 The experiment began at that time. The tumor volume was calculated according to V = 1 / 2ab. 2 Calculations were performed (a and b represent the length and width of the tumor, respectively). The length and width of the tumor were measured using calipers. Unloaded CM NPs nanoparticles and CM@GEM nanoparticles were synthesized and labeled with the fluorescent dye Rhodamine B (RhB). Tumor-bearing mice were injected intravenously with saline, free Rhodamine B (RhB), RhB-labeled unloaded cell membrane nanoparticles CM NPs, or RhB-labeled CM@GEM, respectively. The RhB dose for each group was 25 mg / kg. The distribution of fluorescence signals in the mice was detected at 1, 2, 4, 8, and 12 hours using a broadband small animal imaging system. After the final in vivo observation, the mice were euthanized, and the major organs (heart, liver, spleen, lung, and kidney) and subcutaneous tumors were harvested. The fluorescence of the isolated organs was observed using a broadband small animal imaging system to semi-quantitatively analyze the distribution of nanoparticles in each tissue.

[0066] (4) Evaluation of combined in vivo efficacy To systematically evaluate the combined antitumor effect of AB680 and CM@GEM, tumors with a volume of 75 mm were tested. 3 At that time, tumor-bearing mice were randomly divided into 6 groups (n=6) and subjected to the following intervention: The study included: control group (tail vein and peritumoral injection of 100 μL and 50 μL of normal saline, respectively); AB680 monotherapy group (peritumoral injection of AB680, 7.5 mg / kg, dissolved in 50 μL of normal saline); GEM monotherapy group (tail vein injection of free GEM, 50 mg / kg, dissolved in 100 μL of normal saline); AB680+GEM combination group (peritumoral injection of AB680 7.5 mg / kg, dissolved in 50 μL of normal saline + tail vein injection of GEM 50 mg / kg, dissolved in 100 μL of normal saline); CM@GEM monotherapy group (tail vein injection of CM@GEM, GEM equivalent dose 50 mg / kg, dissolved in 100 μL of normal saline); and AB680+CM@GEM combination group (peritumoral injection of AB680 7.5 mg / kg, dissolved in 50 μL of normal saline + tail vein injection of CM@GEM, GEM equivalent dose 50 mg / kg, dissolved in 100 μL of normal saline).

[0067] All interventions were performed every 3 days, for a total of 4 times. The long and short diameters of the tumor were measured every 3 days using calipers, and calculated using the formula V = 1 / 2 × a × b. 2 Tumor volume was calculated and mouse weight was recorded. On the third day after the last administration, all mice were euthanized, tumors were dissected, weighed, and photographed. Part of the tumor tissue was fixed with 4% paraformaldehyde (w / v) for subsequent histological analysis (H&E, TUNEL, Ki67, immunohistochemistry), while the other part was cryopreserved for other molecular biological assays.

[0068] 5. Experimental Results (1) CM@GEM has good in vivo biosafety. like Figure 4 As shown, compared with the saline control group, the heart, liver, spleen, lungs, kidneys and other important organs of mice in the empty CM NPs group and CM@GEM group had intact tissue structures and no pathological damage such as inflammatory infiltration, necrosis or fibrosis, which confirms that cell membrane nanoparticles have good biocompatibility in vivo.

[0069] (2) CM@GEM can be efficiently enriched at the tumor site. like Figure 5In vivo and in vitro imaging results showed that, compared with the free RhB group, the RhB-labeled CM@GEM group exhibited a stronger and more persistent fluorescence signal at the tumor site. This indicates that nanoparticles can effectively prolong the circulation time of drugs in vivo and significantly improve the enrichment efficiency in tumor tissues by utilizing the active targeting effect between the PD-1 protein on their surface and the PD-L1 highly expressed by tumor cells.

[0070] (3) AB680 and CM@GEM together showed synergistic anti-tumor efficacy. like Figure 6 As shown, the experimental results indicate that, compared with the control group, the tumor growth inhibition rates of the GEM, AB680+GEM, CM@GEM, and AB680+CM@GEM treatment groups reached 43.2%, 85.9%, 78.3%, and 93.9%, respectively. Specifically, the tumor inhibition rate of the AB680+GEM treatment group was 32.7% higher than that of the GEM-only treatment group; the inhibition rate of the CM@GEM treatment group was 42.7% higher than that of the GEM treatment group; and the inhibition rate of the AB680+CM@GEM treatment group was 15.6% higher than that of the CM@GEM treatment group and 8% higher than that of the AB680+GEM treatment group. This demonstrates that the combination of CM@GEM and AB680 has a significant inhibitory effect on colorectal cancer. This synergistic effect of "1+1>2" (the combined group's effect is superior to either single-drug group or the simple sum of two single-drug groups) proves that the precision chemotherapy achieved by CM@GEM and the immune microenvironment regulation mediated by AB680 are two complementary and mutually reinforcing anti-tumor pathways. Furthermore, throughout the entire treatment period, the body weight of mice in all groups remained stable without significant decline, further confirming that the combined treatment regimen has a good safety window.

[0071] (4) Combination therapy exerts a synergistic effect by promoting apoptosis, inhibiting proliferation, and remodeling the immune microenvironment. Tumor cell level analysis such as Figure 7 As shown in the results, the number of TUNEL-positive cells in the tumor tissue of the AB680+CM@GEM treatment group was significantly increased, indicating that the intervention of nanoparticles combined with AB680 led to damage and increase of tumor cells. Furthermore, the level of tumor cell proliferation within the tumor was assessed by Ki67 immunofluorescence staining. The results showed that the Ki67 fluorescence intensity in the tumor tissue of the AB680+CM@GEM treatment group was significantly reduced, indicating a significant decrease in tumor proliferation capacity.

[0072] Immunohistochemical staining results as follows Figure 8 As shown, this indicates that intervention with nanoparticles combined with AB680 leads to intratumoral CD8... + Increased T cell infiltration and decreased Treg cell infiltration indicate that the tumor microenvironment has been remodeled.

[0073] In summary, this invention demonstrates that the combination therapy of CM@GEM NPs and AB680 represents a highly effective and low-toxicity innovative anti-tumor strategy. Its mechanism of action is a complete synergistic cycle: CM@GEM NPs actively and precisely deliver GEM to the tumor site, efficiently killing tumor cells and potentially inducing immunogenic cell death; simultaneously, AB680, by blocking the CD73-adenosine pathway, reverses the immunosuppressive microenvironment that chemotherapy may exacerbate, promoting the infiltration and function of cytotoxic T cells. Ultimately, this achieves a powerful synergy between chemotherapy and immunotherapy, providing a new solution for the treatment of colorectal cancer.

[0074] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0075] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. 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 scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. A gemcitabine nanoparticle, characterized in that, It consists of a gemcitabine core and a cell membrane covering it; the cell membrane is derived from CT26-PD-1 cells that have been genetically engineered to overexpress PD-1; the gemcitabine nanoparticles have a particle size of 150nm~200nm.

2. The method for preparing gemcitabine nanoparticles as described in claim 1, characterized in that, Specifically, the steps include the following: By genetically engineering CT26 cells, a cell line overexpressing PD-1, CT26-PD-1, was constructed. CT26-PD-1 cell membranes were extracted and gemcitabine was encapsulated in the CT26-PD-1 cell membranes to form gemcitabine nanoparticles.

3. A combination antitumor drug, characterized in that, The antitumor combination drug uses gemcitabine nanoparticles and CD73 inhibitor as active ingredients as described in claim 1; the mass ratio of gemcitabine nanoparticles to CD73 inhibitor is 5~10:

1.

4. The antitumor combination drug as described in claim 3, characterized in that, The CD73 inhibitor is AB680.

5. The antitumor combination drug as described in claim 3, characterized in that, The aforementioned antitumor combination drug can be formulated into a clinically acceptable formulation together with or separately from pharmaceutically acceptable excipients.

6. The antitumor combination drug as described in claim 5, characterized in that, The pharmaceutically acceptable carrier is physiological saline.

7. The antitumor combination drug as described in claim 3, characterized in that, The formulation is a liquid formulation.

8. The use of the combination drug for tumors as described in any one of claims 1 to 7 in the preparation of a reagent for the prevention and treatment of colorectal cancer.

Images