Lyta-c-gem complex for enhancing anti-tumor effect of gemcitabine and application thereof

Abstract

The application discloses a LYTAG-Gem compound for enhancing the anti-tumor effect of gemcitabine and application thereof, relates to the technical field of biological medicine, and particularly relates to a gemcitabine (Gem) targeted delivery system based on a lysosome targeting chimera (LYTAC) and application thereof in enhancing the anti-tumor process. 2+ The system is assembled from heavy chain ferritin, Ni 2+ , NTA-PEG5000-DBCO and a targeting ligand TPP-1-N3 in a specific mass percentage, can efficiently load Gem and form a nano compound with a particle size of about 59-79 nm, the system targets tumor cells through heavy chain ferritin, and realizes site-specific release of Gem in cells by means of an endocytosis-lysosome pathway mediated by the LYTAC structure, in-vivo pharmacodynamic experiments show that the LYTAC-Gem compound can significantly inhibit the tumor growth of a KPC pancreatic cancer mouse model, molecular mechanism research further reveals that the LYTAC-Gem compound can down-regulate the expression of PD-L1 protein in tumor tissues, and it is indicated that the LYTAC-Gem compound has the potential to activate an anti-tumor immune response, and the application provides a novel targeted delivery strategy for overcoming the toxic side effects and tumor drug resistance of gemcitabine.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to a LYTAC-Gem complex for enhancing the antitumor effect of gemcitabine and its application. Background Technology

[0002] Gemcitabine is a first-line chemotherapy drug for the treatment of pancreatic cancer, non-small cell lung cancer, and breast cancer. However, its clinical application is severely limited: First, gemcitabine is a hydrophilic drug that is difficult to cross the cell membrane and needs to rely on the nucleoside transporter (hENT1) on the cell membrane to enter the cell; second, tumor cells often develop drug resistance by downregulating hENT1 expression or reducing deoxycytidine kinase (dCK) activity.

[0003] Lysosome-targeting chimeras (LYTACs) are an emerging targeted protein degradation technology. They are typically bifunctional molecules, with one end specifically binding to a protein of interest (POI) on the cell membrane via an antibody or small molecule ligand, and the other end linked to a cleavable linker (Lysosome-Targeting Receptor, LTR). The lysosome enters the cell as a complex via clathrin and is ultimately transported to the lysosome. In the acidic environment of the lysosome and under the action of abundant hydrolytic enzymes, the linker cleaves, achieving site-specific release.

[0004] LYTAC is primarily used to induce the degradation of cell membrane or extracellular proteins, but research on its application in the efficient and low-toxicity delivery of traditional chemotherapy drugs is still limited. Combining gemcitabine with LYTAC technology could theoretically achieve: 1) Targeted delivery: Utilizing the targeting properties of LYTAC, gemcitabine could be enriched in tumor tissues, reducing exposure to normal tissues; 2) Overcoming drug resistance: Delivery via the lysosomal pathway could potentially circumvent drug resistance mediated by cell membrane drug efflux pumps; 3) Synergistic effect: The specific receptor-ligand endocytosis and lysosomal release process mediated by LYTAC could disrupt tumor cell homeostasis, synergizing with the DNA-damaging effects of gemcitabine to enhance the killing effect.

[0005] Therefore, developing a gemcitabine targeted delivery system based on LYTAC is of great significance for overcoming the existing shortcomings of gemcitabine and improving the efficacy of tumor treatment. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing gemcitabine chemotherapy, such as difficulty in penetrating cell membranes and easy drug resistance, and to provide a delivery system based on LYTAC technology that can specifically target tumor cells and significantly enhance the efficacy of gemcitabine, as well as its application.

[0007] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a LYTAC-Gem complex for enhancing the antitumor efficacy of gemcitabine, comprising the following steps: The targeted portion first binds to gemcitabine, via Ni 2+ The function of NTA-PEG5000-DBCO and TPP-1-N3 to form targeted lysosomes; wherein the targeted portion can specifically bind to the targeted receptor on the surface of tumor cells.

[0008] Preferably, the LYTAC-Gem complex exhibits significant targeted accumulation properties.

[0009] On the other hand, the present invention provides a method for preparing a LYTAC-Gem complex, the specific steps of which are as follows: (1) Take 1 mL of ferritin solution with a concentration of 1 mg / mL, and mix Gem and heavy chain ferritin at a mass ratio of 1:10 continuously by stirring. (2) The fully reacted solution was transferred to an ultrafiltration tube to remove unreacted impurities, and the particle size was measured to be 59-79 nm. (3) Prepare 0.23 mg / mL Ni 2+ The reaction was carried out with 50 mg / mL NTA-PEG5000-DBCO and 20 mg / mL TPP-1-N3, and stirred at 4 °C for 12 h. (4) Transfer the fully reacted solution to an ultrafiltration tube to remove unreacted impurities and collect the ultrafiltration solution to obtain the LYTAC-Gem complex.

[0010] Preferably, the ultrafiltration tube is selected with a specification of 30 kDa.

[0011] Preferably, the LYTAC-Gem is delivered to tumor cells in the form of a complex.

[0012] In a third aspect, the present invention provides the application of the LYTAC-Gem complex for enhancing the antitumor efficacy of gemcitabine. (1) Model establishment: KPC mouse pancreatic cancer cells were inoculated into the thigh of C57BL / 6 mice; (2) Grouping and administration: When the tumor volume is approximately 100 mm 3 Mice were randomly divided into 8 groups: PBS group, free Gem group, PD-1 group, Fer group, Fer / Gem group, Fer / Gem+PD-1 group, LYTAC group, and LYTAC / Gem group. Administered via tail vein once daily for one week. The equivalent dose of Gem was 1 mg / kg. (3) Tumor diameter measurement and growth curve: The long diameter (a) and short diameter (b) of the tumor were measured every two days using vernier calipers, and the result was calculated using the formula V = 0.5 × a × b. 2 Calculate the tumor volume and plot the tumor growth curve.

[0013] (4) Study on the mechanism of LYTAC-Gem-induced tumor cell apoptosis: Tumor tissue was taken, fixed with 4% paraformaldehyde, and frozen sectioned. The sections were placed in citrate buffer, microwaved to boil to repair the antigen, and then naturally cooled to room temperature. The sections were washed three times with PBST for 5 minutes each time. 5% BSA or goat serum was added, and the sections were blocked at room temperature for 1 hour to block non-specific binding. The blocking solution was discarded, and diluted primary antibody was added. The sections were incubated overnight in a humidified chamber at 4°C. The sections were washed three times with PBST. Fluorescently labeled secondary antibody was added, and the sections were incubated at room temperature in the dark for 1 hour. The sections were washed three times with PBST, and mounting medium was added. The sections were then air-dried and mounted. Beneficial effects The LYTAC-Gem complex of this invention significantly inhibits tumor growth in a KPC pancreatic cancer mouse model. Further molecular mechanism studies revealed that it downregulates PD-L1 protein expression in tumor tissue, suggesting its potential to activate anti-tumor immune responses. This invention provides a novel targeted delivery strategy to overcome the toxicity and tumor resistance of gemcitabine. Attached Figure Description

[0014] Figure 1 This is the result of FTH-loaded Gem particle size distribution; Figure 2 These are the results of tumor growth rate curves in KPC pancreatic cancer mice from different treatment groups; Figure 3 The results show the immunofluorescence of PD-L1 in the tumors of KPC pancreatic cancer mice in each group. Detailed Implementation

[0015] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0016] The present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0017] Example 1: Synthesis of LYTAC-Gem Take 1 mL of 1 mg / mL ferritin solution, mix Gem and heavy chain ferritin at a mass ratio of 1:1 with continuous stirring, transfer the fully reacted solution to an ultrafiltration tube (30 kDa) to remove unreacted impurities, and determine the particle size to be 59-79 nm. Figure 1,join in , and The reaction was continued at 4 °C with stirring for 12 h. The fully reacted solution was transferred to an ultrafiltration tube (30 kDa) to remove unreacted impurities, and the ultrafiltration solution was collected to obtain the LYTAC-Gem complex.

[0018] Example 2 In vivo pharmacodynamic evaluation of LYTAC-Gem in a mouse KPC pancreatic cancer model Model establishment: KPC mouse pancreatic cancer cells were injected into the thigh of C57BL / 6 mice.

[0019] Grouping and administration: When the tumor volume is approximately 100 mm 3 Mice were randomly divided into 8 groups: PBS group, free Gem group, PD-1 group, Fer group, Fer / Gem group, Fer / Gem+PD-1 group, LYTAC group, and LYTAC / Gem group. The drugs were administered via tail vein once daily for one week. The equivalent dose of Gem was 1 mg / kg.

[0020] Tumor diameter measurement and growth curve: The major diameter (a) and minor diameter (b) of the tumor were measured every two days using vernier calipers, according to the formula V = 0.5 × a × b. 2 Calculate tumor volume and plot tumor growth curves, such as Figure 2 As shown.

[0021] Example 3 Mechanism study of LYTAC-Gem-induced tumor cell apoptosis Tumor tissue was collected, fixed with 4% paraformaldehyde, and frozen sectioned. Sections were placed in citrate buffer and microwaved to boil to retrieval antigens, then allowed to cool naturally to room temperature. Washed three times with PBST for 5 minutes each time. 5% BSA or goat serum was added, and the sections were blocked at room temperature for 1 hour to inhibit non-specific binding. The blocking solution was discarded, and diluted primary antibody was added, followed by incubation overnight at 4°C in a humidified chamber. The sections were washed three times with PBST. Fluorescently labeled secondary antibody was added, and the sections were incubated at room temperature in the dark for 1 hour. The sections were washed three times with PBST, mounted with mounting medium, air-dried, and then the samples were observed and photographed using a fluorescence microscope to confirm the specificity of the PD-L1 signal. Fluorescence counts were performed using ImageJ, and the number of PD-L1-positive cells was divided by the number of DAPI-positive cells to obtain the PD-L1-positive cell rate.

[0022] The results are as follows Figure 3 As shown, compared with the PBS group, the PD-L1 index decreased to varying degrees in each group. Compared with the LYTAC group, the PD-L1 content in the LYTAC / Gem group was lower, indicating that the anti-tumor effect of immune cells was activated.

[0023] Pancreatic cancer treatment has been found to easily trigger immune-related adverse events. Lysosomal targeting chimeras (LYTAC) bind to lysosomal targeting receptors and immune checkpoint proteins (such as PD-L1), internalizing the target protein (PD-L1) into the cell and degrading it to block the pathway. The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A LYTAC-Gem complex for enhancing the antitumor efficacy of gemcitabine, characterized in that, Includes the following steps: The targeted portion first binds to gemcitabine, via Ni 2+ The function of NTA-PEG5000-DBCO and TPP-1-N3 to form targeted lysosomes; wherein the targeted portion can specifically bind to the targeted receptor on the surface of tumor cells.

2. The LYTAC-Gem complex for enhancing the antitumor efficacy of gemcitabine according to claim 1, characterized in that, The LYTAC-Gem complex exhibits significant targeted accumulation properties.

3. The method for preparing the LYTAC-Gem complex according to any one of claims 1-2, characterized in that, The specific steps are as follows: (1) Take 1 mL of ferritin solution with a concentration of 1 mg / mL, and mix Gem and heavy chain ferritin at a mass ratio of 1:10 continuously by stirring. (2) The fully reacted solution was transferred to an ultrafiltration tube to remove unreacted impurities, and the particle size was measured to be 59-79 nm. (3) Prepare 0.23 mg / mL Ni 2+ The reaction was carried out with 50 mg / mL NTA-PEG5000-DBCO and 20 mg / mL TPP-1-N3, and stirred at 4°C for 12 h. (4) Transfer the fully reacted solution to an ultrafiltration tube to remove unreacted impurities and collect the ultrafiltration solution to obtain the LYTAC-Gem complex.

4. The method for manufacturing the LYTAC-Gem complex for enhancing the antitumor effect of gemcitabine according to claim 3, characterized in that, The ultrafiltration tube is selected to be 30 kDa.

5. The application of the LYTAC-Gem complex according to claim 3, characterized in that, The LYTAC-Gem is delivered to tumor cells in the form of a complex.

6. The application of the LYTAC-Gem complex according to claims 1-2, characterized in that, (1) Model establishment: KPC mouse pancreatic cancer cells were inoculated into the thigh of C57BL / 6 mice; (2) Grouping and administration: When the tumor volume is approximately 100 mm 3 Mice were randomly divided into 8 groups: PBS group, free Gem group, PD-1 group, Fer group, Fer / Gem group, Fer / Gem+PD-1 group, LYTAC group, and LYTAC / Gem group. The drugs were administered via tail vein once a day for one week. The equivalent dose of Gem was 1 mg / kg. (3) Tumor diameter measurement and growth curve: The long diameter (a) and short diameter (b) of the tumor were measured every two days using vernier calipers, and the result was calculated using the formula V = 0.5 × a × b. 2 Calculate tumor volume and plot tumor growth curve. (4) Study on the mechanism of LYTAC-Gem inducing tumor cell apoptosis.

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