Pancreatic cancer targeting and electric field responsive nano-micelle as well as preparation method and application thereof

Abstract

The invention provides a pancreatic cancer targeting and electric field responsive nano-micelle as well as a preparation method and application thereof, the pancreatic cancer targeting and electric field responsive nano-micelle is subjected to blood circulation after intravenous injection, and is specifically bound to a pancreatic cancer focus part through surface modified pancreatic cancer targeting peptide and P-lectin1 highly expressed on the surface of pancreatic cancer cells. The electroresponsive group ferrocene in the nano-micelle has hydrophobicity in a reduction state, and forms a part of the nano-material. After ablation of a pulsed electric field, the nano-material is oxidized to have hydrophilicity, so that the structure of the nano-material is broken, and the release of the immunologic adjuvant CpG ODN is realized. TAA and DAMP are released by cell death induced by pulsed electric field ablation, and APC maturation, M1 macrophage polarization, proinflammatory cytokine secretion and CTL activation are promoted. The synergistic effect maintains immunostimulation and prolongs the transient immunomodulatory effect of pulsed electric field ablation. The nano-micelle disclosed by the invention has a wide application prospect in improvement of a pancreatic cancer pulsed electric field ablation curative effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a pancreatic cancer-targeting, electric field-responsive nanomicelle, its preparation method, and its application. Background Technology

[0002] Pancreatic ductal adenocarcinoma (PDAC) remains the fourth leading cause of cancer-related deaths worldwide, with a 5-year survival rate of less than 7%. Pulsed electric field ablation (PFA) is a minimally invasive ablation therapy that uses transient high-voltage microsecond pulsed electric fields (μPEF) to trigger irreversible cell membrane permeability, leading to cancer cell death. Compared to other local ablation techniques, PFA has significant and unique advantages: it effectively preserves important structures such as the vascular system, pancreatic duct, and nerves, eliminates the interference of heat sink effects, and safely and precisely ablates tumor tissue located near critical structures. Recent preclinical studies have shown that PFA ablation can induce anti-tumor immunity.

[0003] However, the tumor microenvironment (TME) of PDAC exhibits an immune "cold" characteristic, specifically manifested as insufficient immune cell infiltration, defective antigen presentation, and the continuous accumulation of immunosuppressive molecules. This TME drives tumor cells to achieve immune escape, leading to rapid tumor recurrence after PFA treatment. Immune reactivation is crucial for enhancing anti-tumor efficacy, and immune adjuvants are a promising strategy. Among various immune adjuvants, cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs) are widely considered among the most potent stimulants. They activate Toll-like receptor 9 (TLR9)-mediated signaling pathways, leading to the upregulation of pro-inflammatory cytokines and chemokines. Furthermore, CpG ODNs promote the maturation of antigen-presenting cells (APCs) and enhance Th1 immune responses. Therefore, combining PFA with immunotherapy (electroimmunotherapy) may yield durable efficacy and broader applicability, including limiting tumor metastasis. However, the clinical application of CpGODNs is limited by several challenges, including easy degradation by nucleases, poor biodistribution, and systemic toxicity, which seriously impair their immunostimulatory efficacy.

[0004] To address these limitations, nanomedicine, with its unique advantages, has developed a promising strategic framework for the synergistic delivery of multiple therapeutic agents. This strategy not only cleverly circumvents various biological barriers but also significantly enhances the efficiency and precision of targeted delivery of therapeutic agents to pathological sites. Researchers have developed various responsive nanomaterials, such as nanoliposomes and micelles sensitive to tumor microenvironment (TME) (e.g., pH), light, or temperature, which partially enhance intratumoral drug accumulation. However, endogenous stimuli present in both the tumor microenvironment and normal physiological environments can lead to the unintended release of drugs from healthy tissues, thereby impairing the specificity of TME-responsive nanomaterials. Furthermore, due to the poor tissue penetration depth of light, photoresponsive nanomaterials are more suitable for superficial tumors than deep tumors like PDAC. During PFA treatment, the electric field can cover the pancreatic cancer tumor area, and as an external energy source, the electric field can effectively serve as a stimulus to activate drug release. Currently, there is no nanomicelle material that can simultaneously address both pancreatic cancer targeting and PFA electric field responsiveness while being safe and easy to prepare.

[0005] The relevant references are as follows: 1. Tempero, MA, Malafa, MP, Al-Hawary, M., Behrman, SW, Benson, AB, Cardin, DB, Chiorean, EG, Chung, V., Czito, B., Del Chiaro, M., etal. (2021). Pancreatic Adenocarcinoma, Version 2.2021, NCCN Clinical Practice Guidelines in Oncology. Journal of the National Comprehensive Cancer Network: JNCCN 19 , 439-457. doi:10.6004 / jnccn.2021.0017. 2. Burbach, B.J., O'Flanagan, S.D., Shao, Q., Young, K.M., Slaughter, J.R., Rollins, M.R., Street, T.J.L., Granger, V.E., Beura, L.K., Azarin, S.M., et al. (2021). Irreversible electroporation augments checkpoint immunotherapy in prostate cancer and promotes tumor antigen-specific tissue-resident memory CD8+ T cells. Nature communications 12 , 3862. doi:10.1038 / s41467-021-24132-6. 3. Zhu, Y.S., Tang, K., and Lv, J. (2021). Peptide-drug conjugate-based novel molecular drug delivery system in cancer. Trends in pharmacological sciences 42 , 857-869. doi:10.1016 / j.tips.2021.07.001. 4. Fatima, M., Almalki, W.H., Khan, T., Sahebkar, A., and Kesharwani, P. (2024). Harnessing the Power of Stimuli-Responsive Nanoparticles as an Effective Therapeutic Drug Delivery System. Advanced materials (Deerfield Beach, Fla.) 36 , e2312939. doi:10.1002 / adma.202312939。 Summary of the Invention

[0006] To address the aforementioned problems, this invention provides pancreatic cancer-targeting, electric field-responsive nanomicelles, their preparation method, and applications. These nanomicelles can solve problems such as difficulties in drug delivery and penetration for deep tumors like pancreatic cancer, and the lack of synergistically responsive nanomaterials during pulsed electric field ablation.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides pancreatic cancer-targeting, electric field-responsive nanomicelles, wherein the nanomicelles are composed of the following components by mass percentage: PLA2K-PEG2K 75%-92%, KTLLPTP 5%-10%, Fc 1%-5%, CpGODN 1%-5%, and OA-SPIO 1%-5%.

[0008] Furthermore, the nanomicelles have a particle size of 140~160nm and a dispersibility index of 0.10~0.15.

[0009] In a second aspect, the present invention provides a method for preparing the nanomicelles described in the first aspect, comprising the following steps: S1. Weigh PLGA2K-PEG2K-NHS, KTLLPTP and triethylamine and dissolve them in 3 mL DMF. The mass ratio of PLGA2K-PEG2K-NHS:KTLLPTP:triethylamine is 1:1.1:3. After stirring the reaction at room temperature for 12 h, dialyze the solution in pure water for 24 h. Collect the dialysate and freeze-dry it to obtain PLA2K-PEG2K-KTLLPTP. S2. Weigh out PEG2K-PLA2K, DCC, ferrocene formate, and DMAP in a mass ratio of 10:3:6:5, dissolve them in 20 mL of dichloromethane (CH2Cl2), stir magnetically for 24 h at room temperature, dialyze in pure water for 24 h, collect the dialysate and freeze-dry to obtain PLA2K-PEG2K-Fc; S3. Dissolve the PLA2K-PEG2K-KTLLPTP, PLA2K-PEG2K, PLA2K-PEG2K-Mal obtained in step S1 and the PLA2K-PEG2K-Fc obtained in step S2 in 2 mL of a mixture at a mass ratio of (5-10):(0-5):(1-5):(1-5), and then add 40-200 μL of OA-Fe3O4 with a concentration of 5 mg / mL. S4. Sonicate the mixture from step S3 for 1 min, slowly adding 2 mL of water during the sonication process to obtain an emulsion; S5. Add 0.2-1 mg of CpG ODN-SH to the emulsion obtained in step S4, add 0.5 M NaHCO3 solution dropwise to adjust the pH of the liquid to 7.5-8.0, stir for 4 h, dialyze in pure water for 48 h, collect the dialysate, and freeze-dry to obtain nanomicelles.

[0010] Furthermore, the molecular weight cutoff of the dialysis bag used in the dialysis process described in steps S1 and S2 is 2000 Da.

[0011] Furthermore, the mixture in step S3 is prepared by mixing tetrahydrofuran and dimethyl sulfoxide in a volume ratio of 9:1.

[0012] Furthermore, in step S5, the molecular weight cutoff of the dialysis bag during the dialysis process is 7000 Da.

[0013] Thirdly, the present invention provides the application of the nanomicelles described in the first aspect in the preparation of pancreatic cancer targeted pulsed electric field ablation drugs.

[0014] The present invention has the following beneficial effects: 1. The pancreatic cancer-targeting, electric field-responsive nanomicelles provided by this invention are easy to synthesize, have good electric field response performance, good pancreatic cancer targeting performance, uniform particle size, good stability, and have magnetic resonance T2-enhanced imaging capability.

[0015] 2. The nanomicelles prepared in this invention utilize the targeting effect of pancreatic cancer targeting peptides to solve the problem of difficult drug delivery and penetration in deep tumors.

[0016] 3. The nanomicelles prepared by this invention utilize the electric field influence function of the electroresponsive group ferrocene to solve the problem of controllable release of nanomaterials during pulsed electric field ablation.

[0017] 4. The nanomicelles prepared in this invention utilize nanomaterials for delivery, which solves the problem of easy degradation of the immune adjuvant CpG ODN in vivo.

[0018] 5. The nanomicelle pulsed electric field ablation combined with an immune adjuvant prepared in this invention addresses the immunosuppressive microenvironment problem in pancreatic cancer. Attached Figure Description

[0019] Figure 1 A simplified diagram of the nanomicelle synthesis process; Figure 2 The electric field response capability, particle size, and imaging characterization of nanomicelles are shown in Figure A, which is a scanning electron microscope image of nanomicelles before and after the action of a pulsed electric field; Figure B is the hydrated particle size of nanomicelles; Figure C is the imaging capability of nanomicelles with different concentrations of magnetic resonance T1 and T2 sequences; and Figure D is a statistical graph of the imaging capability of nanomicelles with magnetic resonance T1 and T2 sequences. Figure 3 To illustrate the killing effect of nanomicelles combined with pulsed electric field ablation on pancreatic cancer tumor cells, Figure A shows the CCK-8 assay used to detect the killing effect of nanomicelles combined with pulsed electric field ablation on pancreatic cancer tumor cells, and Figure B shows the flow cytometry analysis used to detect the effect of nanomicelles combined with pulsed electric field ablation on apoptosis of pancreatic cancer tumor cells. Figure 4 The ability of nanomicelles to target and bind to orthotopic pancreatic cancer in mice; Figure 5 To enhance the efficacy of pulsed electric field ablation of pancreatic cancer in mice using nanomicelles, Figure A shows actual images of pancreatic cancer tumor size in mice from different treatment groups, Figure B shows a statistical chart of pancreatic cancer tumor size in mice from different treatment groups, and Figure C shows survival curves of mice from different treatment groups. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0022] Unless otherwise specified, all materials and reagents used in the following examples are commercially available products. Among them, oleic acid-modified superparamagnetic nano-Fe3O4-SO4, polylactic acid-polyethylene glycol-N-hydroxysuccinimide PLA2K-PEG2K-NHS, polylactic acid-polyethylene glycol PLA2K-PEG2K, and polylactic acid-polyethylene glycol-maleimide PLA2K-PEG2K-Mal were purchased from Xi'an Ruixi Biotechnology Co., Ltd., thiol-modified cytosine-phosphate-guanine oligodeoxynucleotide CpG ODN-SH was purchased from Guangzhou Ruibo Biotechnology Co., Ltd., pancreatic cancer targeting peptide KTLLPTP was purchased from Shanghai Qiangyao Biotechnology Co., Ltd., and mouse pancreatic cancer cells KPC were obtained from the Zhejiang Provincial Key Laboratory of Pancreatic Disease Research.

[0023] Example 1 The detailed preparation method of pancreatic cancer-targeting peptide KTLLPTP modification, cytosine-phosphate-guanine oligodeoxynucleotide CpG ODN loading, ferrocene Fc integration, and superparamagnetic iron oxide nanoparticle SPIO encapsulation nanomicelles (PFSCs) is described below, with the specific process as follows: Figure 1 As shown: First, 100 mg of PLGA2K-PEG2K-NHS was weighed and dissolved in 3 ml of N,N-dimethylformamide (DMF). Based on the molar amount of PLGA2K-PEG2K-NHS, 1.1 equivalents of KTLLPTP peptide and 3.0 equivalents of triethylamine were added and dissolved completely. The reaction was carried out under magnetic stirring at room temperature for 12 h. The reaction solution was then transferred to a dialysis bag (molecular weight cutoff 2000 Da) and dialyzed in pure water for 24 h. The dialysate was collected and freeze-dried to obtain the pancreatic cancer-targeting peptide-modified block copolymer PLA2K-PEG2K-KTLLPTP.

[0024] PEG2K-PLA2K 100mg, N,N'-dicyclohexylcarbodiimide (DCC) 30mg, ferrocene formate 60mg, and 4-(dimethylamino)pyridine (DMAP) 50mg were dissolved in 20 mL of dichloromethane (CH2Cl2). The reaction mixture was magnetically stirred at room temperature for 24 h. The reaction solution was transferred to a dialysis bag (molecular weight cutoff 2000 Da) and dialyzed in pure water for 24 h. The dialysate was collected and freeze-dried to obtain the ferrocene-modified block copolymer PLA2K-PEG2K-Fc.

[0025] PLA2K-PEG2K-KTLLPTP (5.0 mg), PLA2K-PEG2K (5.0 mg), PLA2K-PEG2K-Mal (5.0 mg), and PLA-PEG-Fc (5 mg) were dissolved in a 5:5:5:5 ratio with OA-SPIO (40 μL, 5 mg / mL) in a 2 mL mixture containing tetrahydrofuran and dimethyl sulfoxide at a volume ratio of 9:1. Then, while sonicating (power: 60 W, time: 60 s), 2 mL of pure water was slowly added dropwise to obtain a brownish-yellow emulsion. 0.2 mg of thiol-modified cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN-SH) was added to a brownish-yellow emulsion. The pH of the solution was adjusted to 7.5–8.0 by dropwise addition of 0.5 M sodium bicarbonate (NaHCO3). The mixture was magnetically stirred for 4 h, and then dialyzed in pure water for 48 h. The molecular weight cutoff of the dialysis bag was 7000 Da. The liquid in the dialysis bag was lyophilized to obtain the final PFSC nanomicelles.

[0026] Example 2 The preparation methods for PLA2K-PEG2K-KTLLPTP and PLA2K-PEG2K-Fc are as described in Example 1. PLA2K-PEG2K-KTLLPTP (10.0 mg), PLA2K-PEG2K (5.0 mg), PLA2K-PEG2K-Mal (5.0 mg), and PLA-PEG-Fc (5 mg) were dissolved in a 10:5:5:5 ratio with OA-SPIO (40 μL, 5 mg / mL) in a mixture (2 mL) containing tetrahydrofuran and dimethyl sulfoxide at a volume ratio of 9:1. Then, while sonicating (power: 60 W, time: 60 s), 2 mL of pure water was slowly added dropwise to obtain a brownish-yellow emulsion. 0.2 mg of thiol-modified cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN-SH) was added to a brownish-yellow emulsion. The pH of the solution was adjusted to 7.5–8.0 by dropwise addition of 0.5 M sodium bicarbonate (NaHCO3). The mixture was magnetically stirred for 4 h, and then dialyzed in pure water for 48 h. The molecular weight cutoff of the dialysis bag was 7000 Da. The liquid in the dialysis bag was lyophilized to obtain the final PFSC nanomicelles.

[0027] Example 3 The electric field response capability, particle size, and imaging characterization of nanomicelles.

[0028] The PFSC nanomicelles prepared in Example 1 were characterized. The particle size of the nanomicelles was determined to be 145.20 nm by dynamic laser scattering (DLS), and the dispersion index (PDI) was 0.14. Figure 2 B). A 200 μg / ml PSFC solution was subjected to both no treatment and PFA treatment (electric field strength: 1000 V / cm, spacing: 0.4 cm, pulse number: 30, pulse width: 100 μs). Transmission electron microscopy revealed morphological changes in the nanomicelles after electric field stimulation, with the nanomicelles changing from round shapes to fragments and then aggregating. Figure 2 A). In addition, PSFC solutions with concentrations of 0 mg / ml (pure water), 0.2, 0.4, 0.6, 0.8, and 1.0 mg / ml were placed in EP tubes for magnetic resonance imaging (MRI). Test parameters: repetition time (TR) 400 ms, echo time (TE) 20 ms, resolution 156 mm × 156 mm (…). Figure 2 C). It can be observed that it differs significantly from the H2O signal in T2-weighted imaging, indicating potential for enhanced imaging. Figure 2 D).

[0029] Example 4 The killing effect of nanomicelles combined with pulsed electric field ablation on pancreatic cancer tumor cells.

[0030] The killing effect of the nanomicelles prepared in Example 1 was verified in mouse pancreatic cancer cells (KPC). Mouse pancreatic cancer cells (KPC) were seeded in 10 cm culture dishes and cultured at 37°C in a 5% CO2 incubator for 24 h until the cells reached 80-90% confluence. The culture medium was 10% fetal bovine serum high-glucose DMEM. The PFSC nanomicelles prepared according to method 1 were dissolved in the same medium to a concentration of 200 μg / ml. The experiment was divided into four groups: a blank control group (Control), a nanomicelle group (PSFC), a PFA treatment group (PFA alone), and a combined treatment group (PSFC+PFA). Cells were cultured in 5 × 10⁵ wells. 4 Cells were seeded into 96-well plates, and 200 μl of control medium and PFSC-containing micelle medium were added to each well for 12 h. After PFA treatment according to the group, the cells were incubated statically for 4 h, followed by 10 μL of CCK-8 working solution added to each well and incubated in the dark for 1 h. The absorbance (OD) value was measured at 450 nm using a microplate reader. Relative cell viability (%) = (OD detection – OD blank) / (OD negative – OD blank) × 100%. Five replicates were used for each group. The experimental results are as follows. Figure 3 As shown in Figure A, limited tumor cell killing ability was observed with PSFC alone in the CCK-8 assay. PFA alone induced more significant cytotoxicity, reducing cell viability to 64.56 ± 4.06%. Notably, the combination regimen showed a synergistic effect: PSFC + PFA treatment further reduced cell viability to 45.36 ± 6.65%.

[0031] The experiment was divided into four groups: a blank control group (Control), a nanomicelle group (PSFC), a PFA-only treatment group, and a combined treatment group (PSFC + PFA). Each well was prepared with 5 × 10⁻⁶ cells / well. 5 Cells were seeded into 6-well plates, and 200 μg / ml PSFC was added to the culture medium according to the group. The plates were incubated at 37°C with 5% CO2 for 24 h. After PFA treatment according to the group, the cells were incubated statically for 4 h, then digested with trypsin to form a cell suspension. Annexin V / PI staining was added according to the manufacturer's instructions before incubation and analysis. Flow cytometry analysis of Annexin V / PI staining confirmed the induction of apoptosis (…). Figure 3 B). The proportion of apoptotic cells increased significantly to 51.37±9.55% in the PFA group, 72.70±0.44% in the PSFC+PFA group, and only 12.87±1.29% in the control group, verifying the pro-apoptotic effect of these treatments.

[0032] Example 5 Nanomicelles' ability to target and bind to orthotopic pancreatic cancer in mice.

[0033] An orthotopic PDAC model was established in C57BL / 6J mice via a multi-step surgical procedure. Male mice aged 6 to 8 weeks were anesthetized with isoflurane. The abdominal cavity was opened through a 1.5 cm vertical incision in the abdominal wall to expose the organs. The pancreatic tail was carefully everted with a sterile swab to minimize mechanical damage, and 25 μL of PDAC cell suspension (containing 2 × 10⁶ cells / μL of serum-free medium) was injected using a 30G needle. 6 (KPC cells). The abdominal muscles and skin were sutured and disinfected with iodine swabs. Mice were cultured for 12 days, and 200 μL of CY5.5 fluorescently labeled PSFC nanomicelles were injected into the mice via tail vein injection. The biodistribution of PSFCs was measured using an IVIS small animal in vivo imaging system at 2h, 4h, 8h, 12h, and 24h. The results are as follows: Figure 4 As shown in the figure, fluorescence imaging indicates that PSFCs preferentially accumulate in in situ PDAC tumors. The fluorescence intensity of the tumor tissue in the PSFC group remained strong after 24 hours, indicating enhanced accumulation of in situ PDAC tumors due to targeted ligand modification.

[0034] Example 6 The therapeutic effect of pulsed electric field ablation on pancreatic cancer in mice enhanced by nanomicelles.

[0035] An orthotopic PDAC model was established in C57BL / 6J mice via a multi-step surgical procedure. Male mice aged 6 to 8 weeks were anesthetized with isoflurane. The abdominal cavity was opened through a 1.5 cm vertical incision in the abdominal wall to expose the organs. The pancreatic tail was carefully everted with a sterile swab to minimize mechanical damage, and 25 μL of PDAC cell suspension (containing 2 × 10⁶ cells / μL of serum-free medium) was injected using a 30G needle. 6 (KPC cells). The abdominal muscles and skin were sutured and disinfected with iodine swabs. After culturing the mice for 12 days, they were divided into the following groups for treatment, with 5 mice in each group: (1) PBS control group, which was controlled by injecting PBS into the tail vein; (2) PFA pulsed electric field ablation group, which was treated with pulsed electric field ablation 6 hours after injecting PBS into the tail vein; (3) PSFC+PFA nanomicelle combined pulsed electric field ablation group, which was treated with pulsed electric field ablation 6 hours after injecting PSFC into the tail vein. The mice were dissected and the tumor size was observed 14 days after treatment.

[0036] The results are as follows Figure 5 A shows that tumor growth was significantly inhibited in the PSFC + PFA group compared to PBS control and PFA-treated mice. Figure 5 B showed that the tumor weight in the combined group was significantly smaller than that in the control group or the group that used PFA alone.

[0037] A mouse model of KPC orthotopic pancreatic cancer was established using the same method described above. Mice were divided into groups of seven, as described above. After culturing for 12 days, mice were treated using the same method. Survival curves were observed and recorded from day 35 after treatment. Survival analysis results are as follows: Figure 5 The results showed that PSFC+PFA treatment had the highest survival benefit, with sustained tumor control and significantly improved long-term survival compared to the two control groups. At day 35 post-treatment, the experimental group with PSFC+PFA still maintained a survival rate of over 60%, highlighting the therapeutic advantages of the combination approach.

[0038] The above description is merely a specific embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A pancreatic cancer-targeting, electric field-responsive nanomicelle, characterized in that, The nanomicelles are composed of the following components by mass percentage: PLA2K-PEG2K 75%-92%, KTLLPTP 5%-10%, Fc 1%-5%, CpG ODN 1%-5%, and OA-SPIO 1%-5%.

2. The nanomicelles as described in claim 1, characterized in that, The nanomicelles have a particle size of 140~160nm and a dispersibility index of 0.10~0.

15.

3. A method for preparing nanomicelles according to any one of claims 1-2, characterized in that, Includes the following steps: S1. Weigh PLGA2K-PEG2K-NHS, KTLLPTP and triethylamine and dissolve them in 3 mL DMF. The mass ratio of PLGA2K-PEG2K-NHS:KTLLPTP:triethylamine is 1:1.1:

3. After stirring the reaction at room temperature for 12 h, dialyze the solution in pure water for 24 h. Collect the dialysate and freeze-dry it to obtain PLA2K-PEG2K-KTLLPTP. S2. Weigh out PEG2K-PLA2K, DCC, ferrocene formate, and DMAP in a mass ratio of 10:3:6:5, dissolve them in 20 mL of dichloromethane (CH2Cl2), stir magnetically for 24 h at room temperature, dialyze in pure water for 24 h, collect the dialysate and freeze-dry to obtain PLA2K-PEG2K-Fc; S3. Dissolve the PLA2K-PEG2K-KTLLPTP, PLA2K-PEG2K, PLA2K-PEG2K-Mal obtained in step S1 and the PLA2K-PEG2K-Fc obtained in step S2 in 2 mL of a mixture at a mass ratio of (5-10):(0-5):(1-5):(1-5), and then add 40-200 μL of OA-Fe3O4 with a concentration of 5 mg / mL. S4. Sonicate the mixture from step S3 for 1 min, slowly adding 2 mL of water during the sonication process to obtain an emulsion; S5. Add 0.2-1 mg of CpG ODN-SH to the emulsion obtained in step S4, add 0.5 M NaHCO3 solution dropwise to adjust the pH of the liquid to 7.5-8.0, stir for 4 h, dialyze in pure water for 48 h, collect the dialysate, and freeze-dry to obtain nanomicelles.

4. The preparation method according to claim 3, characterized in that, The molecular weight cutoff of the dialysis bag used in the dialysis process described in steps S1 and S2 is 2000 Da.

5. The preparation method according to claim 3, characterized in that, The mixture in step S3 is prepared by mixing tetrahydrofuran and dimethyl sulfoxide in a volume ratio of 9:

1.

6. The preparation method according to claim 3, characterized in that, In step S5, the molecular weight cutoff of the dialysis bag during the dialysis process is 7000 Da.

7. The use of the nanomicelles according to any one of claims 1-2 in the preparation of a drug for pancreatic cancer targeted pulsed electric field ablation.

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