A nano-molecular probe with targeting property and its preparation method and application

Abstract

The present application relates to the technical field of biological medicine, in particular to a kind of nano-molecular probe with targeting and its preparation method and application.The plasmid of immune checkpoint corresponding receptor of stable expression tumor surface negative regulation molecule is synthesized in the present application, which is prepared into targeted nano-molecular probe with cell membrane nanocapsule of enhancing T cell activity ligand and being marked with fluorescence, and melanin nanosphere of simultaneously loading therapeutic drug.The targeted nano-molecular probe provided in the present application can be long-circulated in vivo, reduce the dosage and administration frequency of chemotherapeutic drug.It also has the advantages of improving the drug resistance of tumor site to therapeutic drug, targeting tumor DNA damage and significantly enhancing the infiltration and activation of immune cells in tumor site.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a targeted nanomolecular probe, its preparation method, and its application. Background Technology

[0002] Pancreatic cancer is one of the most malignant solid tumors, and its early diagnosis is difficult. Most patients are diagnosed at an advanced stage, with an overall five-year survival rate of only about 10%. Treatment of pancreatic cancer involves many complex factors, such as high molecular weight heterogeneity, drug resistance, an immunosuppressive microenvironment, a dense, highly fibrotic stroma, and high hypoxia, all of which greatly complicate the treatment process. Currently, the main treatments for pancreatic cancer are surgical resection and adjuvant chemotherapy, but the surgical resection rate is low and the postoperative recurrence rate is high. Oxaliplatin, as a chemotherapy drug for pancreatic cancer, commonly causes gastrointestinal reactions, primarily nausea and vomiting, but may also manifest as diarrhea, nausea, vomiting, and loss of appetite. Another common side effect of oxaliplatin is neurotoxicity, specifically numbness in the hands and feet after administration, especially upon contact with cold water or exposure to cold stimuli.

[0003] Improving the treatment efficacy of pancreatic cancer treatment requires a deeper understanding of tumor biology, the discovery of therapeutic targets, and targeted therapy to address these targets. Nanomedicine shows great potential to overcome the barriers to pancreatic cancer treatment. Through the careful design and rational modification of nanomaterials, multifunctional intelligent nanosystems can be obtained. Compared with traditional drugs, nanomedicines have many advantages, such as passively targeting tumor tissue through enhanced penetration and retention (EPR) effects, improved circulation, high drug loading capacity, and even intrinsic diagnostic and therapeutic properties. This indicates that nanomedicines have enormous translational potential in pancreatic cancer treatment. This invention studies biological drug therapies that inhibit or kill pancreatic cancer, explores more possibilities for targeted biological drugs, and provides new drugs for the treatment of pancreatic cancer. Summary of the Invention

[0004] This invention addresses the shortcomings and defects of existing technologies by improving commonly used treatments for pancreatic cancer, providing a targeted nanomolecular probe, its preparation method, and its applications. This invention involves encapsulating tumor therapeutic drugs onto melanin nanospheres within TIGIT / CD40 nanovesicles (Ti40 NVs), enabling targeted delivery to tumor cells. This enhances the infiltration and activation of immune cells at the tumor site, promotes tumor cell apoptosis, inhibits tumor cell proliferation, and reduces the toxic side effects of the drugs.

[0005] The above-mentioned objective of this invention is achieved through the following technical solutions:

[0006] This invention provides a targeted nanomolecular probe comprising an immune checkpoint receptor that stably expresses negative regulatory molecules on the tumor surface, cell membrane nanovesicles that enhance T cell activity ligands, and melanin nanospheres loaded with therapeutic drugs.

[0007] Optionally, the drug loading in the melanin nanospheres is 0-30%, and the surface of the cell membrane nanovesicles is loaded with green fluorescent GFP or red fluorescent Mcherry.

[0008] The immune checkpoints mentioned correspond to receptors such as TIGIT, CD40, PD-1, CD226, CD96, or CD27.

[0009] The treatment drugs may be oxaliplatin, paclitaxel, gemcitabine, carboplatin, or doxorubicin.

[0010] This invention also provides a method for preparing a targeted nanomolecular probe, the method comprising:

[0011] (1) Cell lines were transfected with an immune checkpoint receptor plasmid that stably expresses negative regulatory molecules on the tumor surface and a lentiviral packaging plasmid in equal proportions. The cell lines were fluorescently labeled and cell membrane nanovesicles were obtained by lysis and extrusion filtration.

[0012] (2) Melanin nanospheres were prepared by grinding and then centrifugation, and then modified with PEG to obtain PEG-modified melanin nanospheres. The therapeutic drug was loaded into the PEG-modified melanin nanospheres.

[0013] (3) The cell membrane nanovesicles obtained in step (1) and the melanin nanospheres obtained in step (2) are mixed and added to the electroporation solution for electroporation to prepare a targeted nanomolecular probe.

[0014] Further, the lentiviral packaging plasmid described in step (1) is a 1:1 mixture of psPAX2 and pMD2G.

[0015] The fluorescent labeling in step (1) is green fluorescent GFP or red fluorescent Mcherry.

[0016] The immune checkpoint receptor plasmids mentioned in step (1) are one or more of TIGIT, CD40, CD226, CD96, and CD27.

[0017] The cell lines mentioned in step (1) are cell lines that enhance the activity of T cells, including HEK293T, PANC02 or KPC cells.

[0018] The loading of the therapeutic drug in step (2) is 0% to 30%.

[0019] The electroporation described in step (3) was performed using a Bio-Rad electroporator, a 0.4 cm electroporation cuvette, two electroporations at 300 mV, followed by 30 min recovery on ice, three washes with ice-cold PBS, centrifugation at 4 ℃ for 20 min, resuspending in ice-cold PBS, and storage at -80 ℃.

[0020] This invention also provides the application of the targeted nanomolecular probes described above in the preparation of tumor therapeutic drugs.

[0021] Furthermore, the tumor is pancreatic cancer; the treatment drugs are oxaliplatin, paclitaxel, gemcitabine, carboplatin, or doxorubicin.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] This invention synthesizes an immune checkpoint receptor plasmid that stably expresses a tumor surface negative regulatory molecule. This plasmid is then combined with cell membrane nanovesicles labeled with green fluorescent GFP and red fluorescent McCormick ligands to enhance T cell activity, and melanin nanospheres simultaneously encapsulating therapeutic drugs to prepare targeted nanomolecular probes. Overexpression of the immune checkpoint negative regulatory molecule TIGIT can recognize CD155, which is widely expressed on the tumor surface, while overexpression of CD40 can promote T lymphocyte infiltration at the tumor site. This allows for better targeted delivery of chemotherapeutic drugs to tumor tissue, achieving drug accumulation in tumor cells while simultaneously activating and enhancing the cytotoxic activity of CD8. + T cells. The targeted nanomolecular probes provided by this invention can circulate in vivo for extended periods, reducing the dosage and frequency of chemotherapy drugs. They also possess the advantages of improving tumor resistance to therapeutic drugs, targeting tumor DNA damage, and significantly enhancing the infiltration and activation of immune cells at the tumor site. Attached Figure Description

[0024] Figure 1 These are laser confocal microscopy comparison images of the HEK293T cell line;

[0025] Figure 2 This is a graph validating the efficiency of TI40 NVs in expressing GFP-TIGIT and Mcherry-CD40 in the cell membrane.

[0026] Figure 3 This is a comparison chart of vesicle morphology and dynamic light scattering analysis; Figure 3 In the diagram, A represents the morphology of Ti40 NVs, MNS, and MO@Ti40NVs vesicles, and B represents the particle size distribution range of Ti40 NVs, MNS, and MO@Ti40 NVs.

[0027] Figure 4This is a localization diagram of cell membrane nanovesicles on the surface of cell lines; in the diagram, A represents TIGIT NVs and CD155-HEK293T, B represents CD40 NVs and CD40L-HEK 293T, and C represents Ti40 NVs and PANC02 cells.

[0028] Figure 5 This is a targeting and aggregation map of vesicles in a subcutaneous pancreatic cancer tumor model after 6 hours; where A is the targeting and aggregation at the tumor site, and B is the aggregation in the tumor, heart, liver, spleen, lung and kidney tissues.

[0029] Figure 6 These are comparison images of tumor tissue and changes in tumor volume among the mice in each group.

[0030] Figure 7 This is a graph showing the expression of Ki67 in the tumor tissues of mice in each group;

[0031] Figure 8 Flow cytometry was used to analyze CD8+ in tumor sites of mice in different treatment groups. + A representative diagram of T cells. Implementation

[0032] This invention discloses a targeted nanomolecular probe, its preparation method, and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0033] This invention uses cell membrane nanovesicles overexpressing TIGIT and CD40 as an example to load oxaliplatin into nanomolecular probes for targeted therapy. Examples demonstrate that after intravenously injected nanomolecular probes reach the tumor site via blood circulation, they recognize and bind to the ligand CD155 on the tumor surface, achieving drug accumulation at the tumor site, disrupting the TIGIT / CD155 immune checkpoint inhibitory axis, and preventing the depletion of cytotoxic CD8+ T cells. Simultaneously, CD40 binds to CD40L on the surface of T cells, activating the activity of CD8+ T cells. When used in combination, these two methods can prevent immune escape by tumor cells, restore an effective anti-tumor immune response, enhance the infiltration and activation of immune cells at the tumor site, and achieve the goal of enhancing immunotherapy for pancreatic cancer. It should be noted that the therapeutic drugs loaded in this invention are merely exemplary and do not constitute a limitation on therapeutic drugs.

[0034] Those skilled in the art will understand that, unless otherwise specified below, the materials and operating methods used in this invention are well-known in the art, the experimental methods used are all conventional methods, and the materials and reagents used can be purchased from chemical reagent companies. The GFP-TIGIT plasmid, Mcherry-CD40 plasmid, psPAX2 plasmid, and pMD2G plasmid involved in this invention were all purchased from Guangzhou Jidan Biotechnology Co., Ltd.

[0035] Example 1: Construction of TIGIT / CD40 cell membrane nanovesicles

[0036] (1) Construction of stable HEK 293T cell lines overexpressing TIGIT / CD40: HEK293T cells were transfected with the immune checkpoint negative regulatory molecule receptors GFP-TIGIT and / or Mcherry-CD40 and lentiviral packaging plasmids psPAX2 and pMD2G at a ratio of 2:1:1 for 72 h to obtain viral packaging solution, which was used to infect HEK293T cells. After screening with 2 μg / ml puromycin for 72 h, HEK293T cell lines overexpressing GFP-TIGIT, Mcherry-CD40 and co-expressing GFP-TIGIT and Mcherry-CD40 (Ti40) were established. HEK293T cells expressing only GFP and Mcherry were prepared using the same method, except that the receptor plasmid was replaced with GFP or Mcherry.

[0037] (2) Characterization of HEK293T cell line: The HEK293T cell line established in step (1) was plated in 12-well plates with round glass slides and cultured overnight. It was stained with cell membrane dye WGA405 at a concentration of 5 μg / ml for 20 min in the dark. After washing twice with PBS, the supernatant was discarded and fixed with 4% paraformaldehyde for 10 min. The binding of vesicles to target cells was observed by laser confocal microscopy. Figure 1 This is a laser confocal microscopy comparison image of the HEK293T cell line; in the image, A shows HEK293T cells overexpressing the empty GFP vector (GFP-293T) and GFP-TIGIT (TIGIT-293T), B shows HEK293T cells overexpressing the empty Mcherry vector (Mcherry-293T) and Mcherry-CD40 (CD40-293T), and C shows simultaneous co-expression of GFP-TIGIT and Mcherry-CD40 (Ti40). Figure 1As shown, HEK293T cell lines overexpressing GFP-TIGIT, Mcherry-CD40, and simultaneously overexpressing GFP-TIGIT and Mcherry-CD40 (Ti40) have GFP with green fluorescence and Mcherry with red fluorescence on their blue fluorescent cell membrane surfaces, respectively. HEK293T cell lines expressing only GFP show green fluorescence, while HEK293T cell lines expressing only Mcherry show red fluorescence.

[0038] (3) Obtaining cell membrane vesicles: HEK293T cells overexpressing GFP-TIGIT, Mcherry-CD40 and co-expressing GFP-TIGIT and Mcherry-CD40 (Ti40) were collected. After rinsing with PBS, HM lysis buffer (42.79g sucrose, 2.385g 4-hydroxyethylpiperazine ethanesulfonic acid (Hepes), 3.7g ethylenediaminetetraacetic acid (EDTA), pH 7.4) was added for 10 minutes for lysis. The cells were then completely ruptured by grinding on ice with a grinding stick. Subsequently, the cells were centrifuged at 5000 r / min and 4℃ for 10 minutes. The lower layer of cell nuclei and cytoplasmic proteins were discarded, and the supernatant was collected. The supernatant was then centrifuged at 12000 r / min and 4℃ for 10 minutes. The lower precipitate (i.e., cell membrane) was resuspended in PBS and then filtered sequentially through 0.45 mm and 0.22 mm filter membranes to obtain PBS-resuspended cell membrane nanovesicles, which were designated as TIGIT NVs, CD40 NVs, and Ti40 NVs, respectively. Figure 2 This is a graph validating the efficiency of TI40 NVs in expressing GFP-TIGIT and Mcherry-CD40 on the cell membrane; in the graph, Na-K-ATPase is used as an internal reference protein for membrane proteins. Figure 2 As shown, GFP-TIGIT and Mcherry-CD40 can be stably expressed on the HEK293T cell membrane co-expressing GFP-TIGIT and Mcherry-CD40 (Ti40).

[0039] Example 2: Preparation of MO@Ti40 NVs

[0040] Synthesis of melanin nanospheres: 3.6 mL of ammonia, 24 mL of anhydrous ethanol, and 54 mL of deionized water were stirred at 30 °C and 500 rpm for 30 min to ensure homogeneity. During stirring, 6 mL of 50 mg / mL dopamine hydrochloride aqueous solution was slowly added. The reaction was carried out at a constant temperature for 18 h, followed by centrifugation at 9000 rpm for 15 min. The nanospheres were washed with deionized water and centrifuged again. This process was repeated three times. The resulting melanin nanospheres were collected, and 30 mL of deionized water was added to disperse them evenly, yielding a 2 mg / mL aqueous solution of melanin nanospheres. This solution was stored at 4 °C for later use.

[0041] PEG modification on the surface of melanin nanospheres: 1 mL of 5 mg / mL polyethylene glycol (PEG) aqueous solution was mixed with 5 mL of the melanin nanosphere aqueous solution obtained in step (1), and 0.5 mL of ammonia was added. The mixture was reacted in a shaker at room temperature for 6 h. The mixture was washed three times with deionized water to obtain polyethylene glycol-modified melanin nanospheres (MNS).

[0042] Loading oxaliplatin (OXA): 1 mg of oxaliplatin was added to 1 mL of 1 mg MNS aqueous solution. The mixture was shaken at 100 rpm for 12 h under light-protected conditions to mix thoroughly. After centrifugation at 9000 rpm for 15 min, oxaliplatin-loaded melanin nanospheres (MO) were obtained and washed twice with deionized water.

[0043] Preparation of MO@Ti40 NVs: 1 mg of Ti40 NVs vesicles prepared in Example 1 and 1 mg of MO were added to 1 mL of electroporation buffer (21% 25 mM potassium chloride, 1.15 mM potassium hydrogen phosphate at pH 7.2). The mixture was gently mixed at pH 10 and 4 °C. The mixture was electroporated twice at 300 mV using a Bio-Rad electroporator and a 0.4 cm electroporation cuvette. The mixture was then restored on ice for 30 min, washed three times with ice-cold PBS, centrifuged at 13,500 rpm and 4 °C for 20 min, and resuspended in ice-cold PBS for three times to obtain MO@Ti40 NVs. The mixture was stored at -80 °C.

[0044] The morphology of vesicles was observed using transmission electron microscopy, and the particle size distribution range of cell membrane vesicles was analyzed by dynamic light scattering. Figure 3 This is a comparison chart of vesicle morphology and dynamic light scattering analysis; Figure 3 In the diagram, A represents the morphology of Ti40 NVs, MNS, and MO@Ti40 NVs vesicles, and B represents the particle size distribution range of Ti40 NVs, MNS, and MO@Ti40 NVs; for example... Figure 3 As shown in Figure A, Ti40 NVs exhibit a spherical, complete cell membrane structure with a size of approximately 110 nm, and no obvious special structures are observed within the sphere. MNS exhibits a circular structure with a particle size less than 120 nm; MO@Ti40 NVs exhibit an elliptical, complete cell membrane structure with a longest diameter of approximately 140 nm. The particle size of Ti40 NVs is mainly distributed around 110 nm, the particle size of MNS is mainly distributed around 120 nm, and the particle size of MO@Ti40 NVs is mainly distributed around 140 nm.

[0045] Example 3: Targeting effect of MO@Ti40 NVs on pancreatic cancer cells and subcutaneous pancreatic cancer model

[0046] (1) Receptor-ligand binding between cell membrane nanovesicles and receptors on cells: TIGITNVs prepared in Example 1 were co-incubated with CD155-HEK 293T cells, and CD40 NVs were co-incubated with CD40L-HEK 293T cells. The experimental results were observed using a laser confocal microscope after 2 hours. TI40 NVs were co-incubated with PANC02 cells using the same method, and the experimental results were observed using a laser confocal microscope after 2 hours.

[0047] Figure 4 This is a localization diagram of cell membrane nanovesicles on the surface of cell lines; in the diagram, A represents TIGIT NVs and CD155-HEK293T, B represents CD40 NVs and CD40L-HEK 293T, and C represents Ti40 NVs and PANC02 cells. Figure 4 As shown, TIGIT NVs can be accurately localized on the surface of CD155-HEK293T cell membranes, and CD40 NVs can bind to CD40L on the cell surface via receptor-ligand binding, accurately localizing on the surface of CD40L-HEK293T cell membranes. PANC02 cells express CD155, and TIGIT NVs can be accurately localized on the surface of PANC02 cell membranes.

[0048] (2) Targeted aggregation of cell membrane nanovesicles at tumor sites: A subcutaneous tumor-bearing mouse model was constructed using PANC02, with three mice in each group. After the tumor-bearing model was established, the Free NVs, TIGIT NVs, CD40 NVs, and Ti40 NVs groups were injected into the mice via the tail vein (25 mg / kg). The prepared nanovesicles were injected into the mice, and the location of fluorescence enrichment was detected by in vivo imaging technology 6 hours later. It can be observed that the fluorescence distribution site was detected 6 hours after administration via tail vein injection. Fluorescence enrichment could be observed at the pancreatic tumor. Subcutaneous tumors, hearts, livers, spleens, lungs, and kidneys were removed from each mouse and detected using an immunofluorescence device. Figure 5 This is a targeting and aggregation map of vesicles in a subcutaneous pancreatic cancer tumor model after 6 hours; where A represents the targeting and aggregation at the tumor site, and B represents the aggregation in the tumor, heart, liver, spleen, lung, and kidney tissues. Figure 5 It is evident that Ti40 NVs target and accumulate at the tumor site, with most Ti40 NVs enriched in the tumor tissue and a small portion detectable in the liver, considering that the drug may be metabolized by the liver; while in the control group (FreeNVs group), fluorescence was found scattered in various organs.

[0049] Example 4: The therapeutic effect of MO@Ti40 NVs cell membrane nanovesicles encapsulating melanin-oxaliplatin on pancreatic cancer

[0050] In this embodiment, physiological saline (Saline) group, Free NVs group, TIGIT NVs group, CD40 NVs group, Ti40 NVs group, MNS group, OXA group, MO group, MO@Free NVs group, and MO@Ti40 NVs group were used as therapeutic drugs to treat a mouse model of pancreatic cancer by injection. The therapeutic effect of oxaliplatin melanin nanovesicles on pancreatic cancer was analyzed. Each group was divided into three replicates. The animal experiments were conducted with the approval of the Animal Experiment Ethics Committee of Jiangsu University. Specifically, 1×10 6 indivual PANC02 mouse pancreatic cancer cells were injected subcutaneously into C57BL / 6 mice. After successful tumor implantation, the weight and tumor size of the male C57BL / 6 mice were measured every other day. The weight of the male C57BL / 6 mice remained relatively stable at around 22-28 g during the treatment cycle. The tumor was allowed to grow to 50 mm. 3 Mice were divided into ten groups and treated with oxaliplatin (5 μg / kg) via tail vein injection, once every two days for a total of seven doses. After 14 days, the mice were sacrificed and their tumors, spleens, and lymph nodes were extracted and photographed.

[0051] Figure 6 These are comparative images of tumor tissue and changes in tumor volume in different groups of mice; in the figures, A is a line graph of tumor volume in tumor-bearing mice (n=5), and B is a comparative image of tumor volume in each group; Figure 6 As can be seen, the tumor growth rate in the MO@Ti40 NVs-loaded vesicle group was significantly lower than that in other groups, demonstrating a clear advantage of MO@Ti40 NVs in anti-tumor activity. Dissection of the tumors revealed that the mice treated with MO@Ti40 NVs had the smallest tumor volume.

[0052] Subsequently, the tumor tissue was paraffin-embedded, sectioned, and the expression of Ki67 was analyzed by immunohistochemical staining to determine the tumor proliferation. Figure 7 This is a graph showing the expression of Ki67 in the tumor tissues of mice in each group; by Figure 7 As shown, Ki67 expression in the MO@Ti40NVs group was significantly lower than in other groups, indicating a strong ability to inhibit tumor proliferation.

[0053] To observe whether MO@Ti40 NVs has enhanced CD8 + T cell function was assessed by shearing, digesting with collagenase I, and thoroughly grinding the collected tumor and spleen tissues. The resulting single-cell suspension was then filtered through a 100-mesh sieve. After incubation with APC-CD3, FITC-CD8, and Percp-IFNg flow cytometry antibodies, CD8 levels were measured. +Expression levels of Granzyme B, IFNγ, and TNFα in T cells. Figure 8 Flow cytometry was used to analyze CD8+ in tumor sites of mice in different treatment groups. + Representative diagrams of T cells; such as Figure 8 As shown, CD8 levels at the tumor site after MO@Ti40 NVs treatment + The number of T cells was significantly increased. This demonstrates that MO@Ti40 NVs can inhibit tumor growth and recruit and activate CD8+ at the tumor site. + T cells.

[0054] Based on all the experimental results, it can be concluded that MO@Ti40 NVs inhibit pancreatic cancer proliferation and promote CD8 in vivo. + T cell activation has a strong ability to inhibit tumors.

[0055] The foregoing description and demonstration of the basic principles, main features, and advantages of this invention are as follows. Those skilled in the art should understand that this 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 this invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A targeted nanomolecular probe, characterized in that, The probes include cell membrane nanovesicles that stably express TIGIT and CD40, and melanin nanospheres loaded with therapeutic drugs.

2. The targeted nanomolecular probe according to claim 1, characterized in that, The drug loading in the melanin nanospheres is 0-30%, and the surface of the cell membrane nanovesicles is loaded with green fluorescent GFP or red fluorescent Mcherry.

3. The targeted nanomolecular probe according to claim 1, characterized in that, The therapeutic drugs are selected from oxaliplatin, paclitaxel, gemcitabine, carboplatin, or doxorubicin.

4. A method for preparing a targeted nanomolecular probe, characterized in that, The preparation method includes: (1) The plasmids that stably express TIGIT and CD40 are mixed with lentiviral packaging plasmids in equal proportions and transfected into cell lines. Cell membrane nanovesicles are obtained by lysis, extrusion and filtration. The surface of the cell membrane nanovesicles is loaded with green fluorescent GFP or red fluorescent Mcherry. (2) Melanin nanospheres were prepared by grinding and then centrifugation, and then modified with PEG to obtain PEG-modified melanin nanospheres. The therapeutic drug was loaded into the PEG-modified melanin nanospheres. (3) The cell membrane nanovesicles obtained in step (1) and the melanin nanospheres obtained in step (2) are mixed and added to the electroporation solution for electroporation to prepare a targeted nanomolecular probe.

5. The preparation method according to claim 4, characterized in that, The lentivirus packaging plasmid mentioned in step (1) is a 1:1 mixture of psPAX2 and pMD2G.

6. The preparation method according to claim 4, characterized in that, The cell lines mentioned in step (1) are HEK293T, PANC02 or KPC cells.

7. The preparation method according to claim 4, characterized in that, The loading of the therapeutic drug in step (2) is 0% to 30%.

8. The preparation method according to claim 4, characterized in that, The electroporation described in step (3) was performed using a Bio-Rad electroporator, a 0.4 cm electroporation cuvette, two electroporations at 300 mV, followed by 30 min recovery on ice, three washes with ice-cold PBS, centrifugation at 4 ℃ for 20 min, resuspending in ice-cold PBS, and storage at -80 ℃.

9. The use of the targeted nanomolecular probe according to any one of claims 1-3 in the preparation of tumor therapeutic drugs.

10. The application according to claim 9, characterized in that, The tumor is pancreatic cancer; the treatment drugs are oxaliplatin, paclitaxel, gemcitabine, carboplatin, or doxorubicin.

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