Inflammation-targeted drug-loaded liposome as well as preparation method and application thereof

Abstract

The invention provides an inflammation-targeted drug-loaded liposome as well as a preparation method and application thereof, and belongs to the technical field of medicines. The inflammation-targeted drug-loaded liposome provided by the invention comprises a phospholipid membrane, macrophage membrane protein loaded on the phospholipid membrane, and a CCR2 inhibitor and a chemotherapeutic drug encapsulated in the phospholipid membrane. The inflammation-targeted drug-loaded liposome disclosed by the invention is beneficial to specifically delivering a drug to a tumor site treated by a nano-knife, and the loaded CCR2 inhibitor and chemotherapeutic drug such as gemcitabine can improve the drug resistance of chemotherapeutic drug treatment while inhibiting the recruitment of macrophages (TAMs), so that the effect of achieving double effects is achieved, and the inflammation-targeted drug-loaded liposome has a good application prospect. The recurrence of pancreatic tumor after nano-knife treatment can be effectively inhibited, and the method is a promising combined administration strategy after nano-knife treatment.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an inflammation-targeted drug-loaded liposome and a preparation method and application thereof. Background Art

[0002] Pancreatic cancer is a malignant tumor with a high mortality rate and extremely rapid progression. Most pancreatic cancer patients have already developed distant metastases at the time of diagnosis, and only about 20% of patients are eligible for surgical treatment. For patients with advanced progressive pancreatic cancer who cannot be removed by surgery, the current first-line treatment options include gemcitabine combined with albumin-bound paclitaxel chemotherapy, FOLFIRINOX chemotherapy (the drugs used are fluorouracil, calcium folinate, irinotecan and oxaliplatin) and stereotactic radiotherapy, which can moderately prolong survival. However, the efficacy of the above methods is still limited for pancreatic cancer patients, especially those with advanced pancreatic cancer. Therefore, new treatment strategies are urgently needed to improve patients' survival and quality of life, among which nanoknife percutaneous ablation therapy has attracted much attention.

[0003] Nanoknife is a non-thermal ablation technology under image guidance. It has the advantages of not damaging the surrounding normal tissue structure and multi-electrode mode. It can improve the quality of life of patients and prolong their survival. However, there is still a bottleneck problem of incomplete ablation leading to tumor recurrence after Nanoknife treatment, and its mechanism is still unclear. Regarding the problem of tumor recurrence after Nanoknife treatment, the current conventional treatment methods are secondary chemotherapy (such as modified FOLFIRINOX or gemcitabine combination regimen) or local radiotherapy, which have limited efficacy, high tumor resistance, and significant treatment-related toxic side effects. In addition, some patients cannot tolerate further active treatment due to tumor invasion of blood vessels or adjacent organs, deterioration of systemic condition, etc., resulting in limited treatment options. Summary of the invention

[0004] The purpose of the present invention is to provide an inflammation-targeted drug-loaded liposome and a preparation method and application thereof. The inflammation-targeted drug-loaded liposome provided by the present invention can effectively inhibit the recurrence of pancreatic tumors after nanoknife treatment, thereby improving the limitations of existing ablation technology.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides an inflammation-targeted drug-loaded liposome, comprising a phospholipid membrane, a macrophage membrane protein loaded on the phospholipid membrane, and a CCR2 inhibitor and a chemotherapeutic drug encapsulated in the phospholipid membrane.

[0007] Preferably, the raw materials for preparing the phospholipid membrane include the following components in molar proportions:

[0008] 2.8 to 3.2 parts of dipalmitoylphosphatidylcholine, 2.8 to 3.2 parts of 1,2-stearoyl-sn-glycero-3-phosphocholine, 0.8 to 1.2 parts of 1,2-stearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol) 2000], and 0.8 to 1.2 parts of cholesterol.

[0009] Preferably, the chemotherapeutic drug is a chemotherapeutic drug used to treat pancreatic cancer.

[0010] Preferably, the drug loading amount of CCR2 inhibitor in the inflammation-targeted drug-loaded liposome is 6.1-6.5wt%, and the drug loading amount of chemotherapy drug is 2.7-3.1wt%; the content of macrophage membrane protein in the inflammation-targeted drug-loaded liposome is 0.25-0.31wt%.

[0011] Preferably, the particle size of the inflammation-targeted drug-loaded liposome is 150 to 200 nm, and the Zeta potential is -30 to -40 mV.

[0012] The present invention provides a method for preparing the inflammation-targeted drug-loaded liposomes described in the above technical solution, comprising the following steps:

[0013] Mixing a CCR2 inhibitor, a chemotherapeutic drug, a raw material for preparing a phospholipid membrane, and an organic solvent, and removing the organic solvent from the resulting mixed liquid to obtain a precursor material;

[0014] Mixing the precursor material with a hydration liquid to perform a hydration treatment to obtain a hydration treatment liquid;

[0015] The hydration treatment liquid is mixed with macrophage membrane protein for loading treatment to obtain the inflammation-targeted drug-loaded liposome.

[0016] Preferably, the mass ratio of the raw material for preparing the phospholipid membrane to the CCR2 inhibitor is 45-55:4-6, and the mass ratio of the CCR2 inhibitor, the chemotherapy drug and the macrophage membrane protein is 4-6:4-6:0.13-0.2.

[0017] Preferably, the hydration liquid is water or PBS solution; the temperature of the hydration treatment is 45-55° C., and the time is 50-70 min.

[0018] The present invention provides the use of the inflammation-targeted drug-loaded liposomes described in the above technical solution or the inflammation-targeted drug-loaded liposomes prepared by the preparation method described in the above technical solution in the preparation of anti-macrophage migration reagents.

[0019] The present invention provides the use of the inflammation-targeted drug-loaded liposomes described in the above technical solution or the inflammation-targeted drug-loaded liposomes prepared by the preparation method described in the above technical solution in the preparation of a drug for inhibiting the recurrence of pancreatic tumors after nanoknife treatment.

[0020] The present invention provides an inflammation-targeted drug-loaded liposome, comprising a phospholipid membrane, a macrophage membrane protein loaded on the phospholipid membrane, and a CCR2 inhibitor and a chemotherapeutic drug encapsulated in the phospholipid membrane. The inflammation-targeted drug-loaded liposome in the present invention is conducive to the specific delivery of drugs to the pancreatic tumor site after nanoknife treatment, wherein the loaded CCR2 inhibitor and chemotherapeutic drug (such as gemcitabine) can improve the drug resistance of only using chemotherapeutic drugs while inhibiting the recruitment of macrophages (TAMs), thus achieving the effect of killing two birds with one stone. The inflammation-targeted drug-loaded liposome provided by the present invention can effectively inhibit the recurrence of pancreatic tumors after nanoknife treatment, thereby improving the limitations of existing ablation technology. Therefore, the present invention combines nanoknife with inflammation-targeted drug-loaded liposome, which is a very promising combined drug administration strategy after nanoknife treatment.

[0021] At the same time, the present invention provides a method for preparing the inflammation-targeted drug-loaded liposome, which is simple to operate and has good biocompatibility of raw materials, and is conducive to popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Transmission electron microscopy (TEM) image, DLS particle size distribution image and Zeta potential image of the inflammation-targeted drug-loaded liposomes prepared in Example 1;

[0023] Figure 2 This is a graph showing the drug loading and encapsulation efficiency test results of the inflammation-targeted drug-loaded liposomes prepared in Example 1;

[0024] Figure 3 This is a diagram showing the effect of the inflammation-targeted drug-loaded liposomes prepared in Example 1 on anti-macrophage migration;

[0025] Figure 4 This is a diagram showing the therapeutic effects of pancreatic tumors in the control group and the treatment group;

[0026] Figure 5 In vivo imaging of fluorescently labeled conventional liposomes and non-drug-loaded inflammation-targeted liposomes. DETAILED DESCRIPTION

[0027] The invention provides an inflammation-targeted drug-loaded liposome, comprising a phospholipid membrane, a macrophage membrane protein loaded on the phospholipid membrane, and a CCR2 inhibitor and a chemotherapeutic drug encapsulated in the phospholipid membrane.

[0028] Nanoknife therapy can temporarily relieve immunosuppression and promote anti-tumor immunity, but the intensity of anti-tumor immunity is not enough to cause tumor regression, so Nanoknife therapy needs to be combined with other treatment methods to improve the treatment effect. In the tumor microenvironment (TME) of pancreatic cancer, various inflammatory and immunomodulatory signals coexist, leading to dysregulated tumor suppressor responses. Tumor-associated macrophages (TAMs) are relevant targets in immuno-oncology, and their abundance is usually associated with treatment resistance, metastasis, and poor survival. They directly promote tumor progression and inhibit the anti-tumor effect of T cells. Single-cell RNA sequencing (scRNA-seq) studies of tumors have shown that TAMs are highly heterogeneous, including subpopulations with different individual development, functions, and therapeutic potential. Among them, the chemokine ligand 2 (CCR2)-positive TAMs subpopulation plays an important role in mobilizing TAMs from the bone marrow to infiltrate tumors. Inhibition of CCR2 can effectively reduce the infiltration of immunosuppressive TAMs. The liposome delivery carrier targeting the inflammatory environment has the advantages of good biocompatibility and prolonged half-life of the drug in vivo. The present invention uses it as a drug delivery platform, and by integrating macrophage membrane proteins into the phospholipid membrane, it can effectively target the tumor inflammatory environment after nanoknife ablation and reduce systemic toxicity. The inflammation-targeted drug-loaded liposome provided by the present invention is described in detail below.

[0029] The inflammation-targeted drug-loaded liposome of the present invention includes a phospholipid membrane, and the phospholipid membrane is specifically a phospholipid bilayer membrane. As an embodiment of the present invention, the raw materials for preparing the phospholipid membrane include the following components in molar proportions: 2.8 to 3.2 parts of dipalmitoylphosphatidylcholine, 2.8 to 3.2 parts of 1,2-stearoyl-sn-glycerol-3-phosphocholine, 0.8 to 1.2 parts of 1,2-stearoyl-sn-glycerol-3-phosphoethanolamine-N-[amino (polyethylene glycol) 2000], and 0.8 to 1.2 parts of cholesterol; specifically, the dipalmitoylphosphatidylcholine can be 3 parts, the 1,2-stearoyl-sn-glycerol-3-phosphocholine can be 3 parts, the 1,2-stearoyl-sn-glycerol-3-phosphoethanolamine-N-[amino (polyethylene glycol) 2000] can be 1 part, and the cholesterol can be 1 part.

[0030] The inflammation-targeted drug-loaded liposomes of the present invention include macrophage membrane proteins loaded on the phospholipid membrane. As an embodiment of the present invention, the content of macrophage membrane proteins in the inflammation-targeted drug-loaded liposomes can be 0.25-0.31wt%, specifically 0.28wt%. The introduction of macrophage membrane proteins into the inflammation-targeted drug-loaded liposomes of the present invention can give the liposomes macrophage-like properties, which is beneficial for actively identifying and targeting the inflammatory site after nanoknife treatment. In the embodiment of the present invention, the drug loading of macrophage membrane proteins is limited to the above range, which can ensure the stability of the inflammation-targeted drug-loaded liposomes and is beneficial for enhancing the efficiency of drug delivery.

[0031] The inflammation-targeted drug-loaded liposomes of the present invention include a CCR2 inhibitor and a chemotherapeutic drug encapsulated in the phospholipid membrane. As an embodiment of the present invention, the chemotherapeutic drug may include gemcitabine. As an embodiment of the present invention, the drug loading of the CCR2 inhibitor in the inflammation-targeted drug-loaded liposomes may be 6.1-6.5wt%, specifically 6.3wt%; the drug loading of the chemotherapeutic drug may be 2.7-3.1wt%, specifically 2.9wt%. In the embodiment of the present invention, the drug loading of the CCR2 inhibitor and the chemotherapeutic drug is limited to the above range, which can reduce the leakage of the drug in the blood circulation and is conducive to improving the biosafety of the drug.

[0032] As an embodiment of the present invention, the particle size of the inflammation-targeted drug-loaded liposome may be 150 to 200 nm, and the Zeta potential may be -30 to -40 mV.

[0033] The present invention provides a method for preparing the inflammation-targeted drug-loaded liposomes described in the above technical solution, comprising the following steps:

[0034] Mixing a CCR2 inhibitor, a chemotherapeutic drug, a raw material for preparing a phospholipid membrane, and an organic solvent, and removing the organic solvent from the resulting mixed liquid to obtain a precursor material;

[0035] Mixing the precursor material with a hydration liquid to perform a hydration treatment to obtain a hydration treatment liquid;

[0036] The hydration treatment liquid is mixed with macrophage membrane protein for loading treatment to obtain the inflammation-targeted drug-loaded liposome.

[0037] In the present invention, unless otherwise specified, the raw materials used are commercially available products well known to those skilled in the art.

[0038] The present invention mixes CCR2 inhibitor, chemotherapy drug, raw material for preparing phospholipid membrane and organic solvent, removes organic solvent from the obtained mixed liquid, and obtains precursor material. As one embodiment of the present invention, the mass ratio of the raw material for preparing phospholipid membrane to CCR2 inhibitor can be 45-55:4-6, specifically 50:5; the mass ratio of CCR2 inhibitor to chemotherapy drug can be 4-6:4-6, specifically 5:5. As one embodiment of the present invention, the organic solvent can be a chloroform-methanol mixed solvent, and the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent can be 2.5-3.5:1, specifically 3:1. As one embodiment of the present invention, the method for removing the organic solvent from the mixed liquid can be rotary evaporation, and the conditions of the rotary evaporation include: the temperature can be 43-47°C, specifically 45°C; the rotation speed can be 80-120rpm, specifically 100rpm; the pressure can be 80-120mbar, specifically 100mbar; the time can be 50-70min, specifically 1h. In the embodiment of the present invention, the CCR2 inhibitor, the chemotherapy drug and the raw materials for preparing the phospholipid membrane are uniformly dispersed in the organic solvent. After the organic solvent is removed, a solid film attached to the inner wall of the container is formed to obtain a precursor material.

[0039] After obtaining the precursor material, the present invention mixes the precursor material with a hydration liquid for hydration treatment to obtain a hydrated treatment liquid. As an embodiment of the present invention, the hydration liquid can be water or a PBS solution, and a PBS solution is specifically used in the embodiment; the dosage ratio of the precursor material to the hydration liquid can be 60 mg: 3-7 mL, specifically 60 mg: 5 mL. As an embodiment of the present invention, the temperature of the hydration treatment can be 45-55°C, specifically 50°C; the time can be 50-70 min, specifically 1 h. In the present invention, during the hydration treatment, since the raw materials for preparing the phospholipid membrane are hydrophobic, the phospholipid molecules will be dispersed and self-assembled to form a phospholipid bilayer structure, and the CCR2 inhibitor and the chemotherapeutic drugs will be evenly dispersed or encapsulated in the phospholipid bilayer structure.

[0040] After obtaining the hydration treatment liquid, the present invention mixes the hydration treatment liquid with macrophage membrane protein for loading treatment to obtain the inflammation-targeted drug-loaded liposome. As one embodiment of the present invention, the mass ratio of the chemotherapeutic drug to the macrophage membrane protein can be 4 to 6: 0.13 to 0.2, specifically 5: 0.17. As one embodiment of the present invention, the loading treatment may include: extruding the mixture obtained after mixing the hydration treatment liquid, macrophage membrane protein and chemotherapeutic drug using a cellulose acetate membrane, and the extrusion treatment may include sequentially performing a first extrusion treatment and a second extrusion treatment; the pore size of the cellulose acetate membrane used for the first extrusion treatment may be 450nm, and the number of the first extrusion treatment may be 10 to 20 times, and further may be 10 to 15 times; the pore size of the cellulose acetate membrane used for the second extrusion treatment may be 200nm, and the number of the second extrusion treatment may be 10 to 20 times, and further may be 10 to 15 times. As an embodiment of the present invention, the loading treatment preferably further includes: dialyzing the obtained feed liquid after the loading treatment to obtain the inflammation-targeted drug-loaded liposome; the dialysate used for the dialysis can be a PBS solution; the molecular weight cutoff of the dialysis membrane used for the second dialysis can be 500 to 2000 kDa, specifically 1000 kDa; the dialysis time can be 20 to 28 hours, specifically 24 hours. In the present invention, during the loading treatment, macrophage membrane proteins are integrated into the surface of the drug-loaded liposomes, and then free macrophage membrane proteins and chemotherapeutic drugs can be removed by dialysis.

[0041] The present invention provides the use of the inflammation-targeted drug-loaded liposomes described in the above technical solution or the inflammation-targeted drug-loaded liposomes prepared by the preparation method described in the above technical solution in the preparation of anti-macrophage migration reagents. As an embodiment of the present invention, the anti-macrophage migration reagent includes a functional component and a pharmaceutically acceptable excipient, and the functional component is the inflammation-targeted drug-loaded liposome; the content of the functional component in the anti-macrophage migration reagent can be 0.1-10wt%, for example, it can be 1wt%, 2wt%, 3wt%, 5wt%, 7wt% or 10wt%; the present invention does not specifically limit the types of the pharmaceutically acceptable excipients, and the pharmaceutically acceptable excipients familiar to those skilled in the art can be used; the present invention does not specifically limit the dosage form of the anti-macrophage migration reagent, and the dosage form familiar to those skilled in the art can be used, for example, it can be an injection.

[0042] The present invention provides the use of the inflammation-targeted drug-loaded liposomes described in the above technical solution or the inflammation-targeted drug-loaded liposomes prepared by the preparation method described in the above technical solution in the preparation of a drug for inhibiting the recurrence of pancreatic tumors after nanoknife treatment. As an embodiment of the present invention, the drug for inhibiting the recurrence of pancreatic tumors after nanoknife treatment comprises a functional component and a pharmaceutically acceptable excipient, and the functional component is the inflammation-targeted drug-loaded liposome; the content of the functional component in the drug for inhibiting the recurrence of pancreatic tumors after nanoknife treatment can be 0.1-10wt%, for example, 1wt%, 2wt%, 3wt%, 5wt%, 7wt% or 10wt%; the present invention has no special restrictions on the types of the pharmaceutically acceptable excipients, and pharmaceutically acceptable excipients familiar to those skilled in the art can be used. As an embodiment of the present invention, the present invention has no special restrictions on the dosage form of the drug for inhibiting the recurrence of pancreatic tumors after nanoknife treatment, and the dosage form familiar to those skilled in the art can be used, for example, an injection; the drug for inhibiting the recurrence of pancreatic tumors after nanoknife treatment can be administered by intravenous injection or intratumoral injection, and the dosage can be 4-20 mg / kg.

[0043] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Example 1

[0045] The preparation of inflammation-targeted drug-loaded liposomes comprises the following steps:

[0046] (1) Dipalmitoylphosphatidylcholine (DPPC), 1,2-stearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-stearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol) 2000] (DSPE-PEG-2000) and cholesterol are mixed in a molar ratio of 3:3:1:1 to obtain a mixed raw material; 50 mg of the mixed raw material, 5 mg of a CCR2 inhibitor (PF-4136309) and 5 mg of a chemotherapeutic drug (gemcitabine) are dissolved in 8 mL of a chloroform-methanol mixed solvent (the volume ratio of chloroform to methanol is 3:1), and a rotary evaporator is used to perform rotary evaporation for 1 hour at a temperature of 45° C., a rotation speed of 100 rpm and a pressure of 100 mbar to remove the solvent to obtain a lipid film;

[0047] (2) 5 mL of PBS solution was mixed with the lipid film, and hydrated at 50° C. for 60 min to obtain a liposome suspension, and then 0.17 mg of macrophage membrane protein was added to the obtained liposome suspension. The obtained mixture was extruded 10 times using a cellulose acetate membrane with a pore size of 450 nm, and then extruded 10 times using a cellulose acetate membrane with a pore size of 200 nm. The mixture was then dialyzed in a PBS solution using a dialysis membrane with a molecular weight cutoff of 1000 kDa for 24 h to remove free gemcitabine and free macrophage membrane protein, and finally freeze-dried to obtain inflammation-targeted drug-loaded liposomes.

[0048] Figure 1 The transmission electron microscope (TEM) image, DLS particle size distribution image and Zeta potential image of the inflammation-targeted drug-loaded liposomes prepared in Example 1, wherein a is the TEM image, b is the DLS particle size distribution image, and c is the Zeta potential image; the results show that the morphology of the inflammation-targeted drug-loaded liposomes is a perfect circle, the particle size is between 150 and 200 nm, and the Zeta potential is between -30 and -40 mV.

[0049] Test Example 1

[0050] The encapsulation efficiency and drug loading of CCR2 inhibitor (PF-4136309) and gemcitabine (GEM) in the inflammation-targeted drug-loaded liposomes prepared in Example 1 were determined as follows:

[0051] (1) The inflammation-targeted drug-loaded liposome suspension (concentration of 10 mg / mL, the dispersant used was PBS solution) was dissolved in 20 volumes of methanol, treated with a cell ultrasonic disruptor for 10 min, and then the concentration of gemcitabine was measured at a wavelength of 269 nm using a UV-visible spectrophotometer;

[0052] (2) The concentration of the CCR2 inhibitor was determined at 254 nm using high performance liquid chromatography (HPLC), wherein the HPLC conditions were as follows: Shimadzu Shimpack GIST C18 chromatographic column (4.6×250 mm, 5 μm); the mobile phases included mobile phase A and mobile phase B, wherein the mobile phase A was a 0.1% by volume phosphoric acid aqueous solution, and the mobile phase B was 100% acetonitrile; the flow rate was 1.5 mL / min, and the injection volume was 5 μL;

[0053] Figure 2 The drug loading and encapsulation efficiency test results of the inflammation-targeted drug-loaded liposomes prepared in Example 1 are shown in Figure a, where a is the encapsulation efficiency test result and b is the drug loading test result. The results show that the encapsulation efficiency of the CCR2 inhibitor and gemcitabine are 75.4% and 34.8%, respectively, and the drug loading is 6.3wt% and 2.9wt%, respectively.

[0054] Test Example 2

[0055] The in vitro anti-macrophage migration effect of the inflammation-targeted drug-loaded liposomes prepared in Example 1 was measured as follows:

[0056] Using a 24-well Transwell co-culture chamber, 200 μL containing 1×10 5 The serum-free culture medium of macrophages was placed in the upper chamber, and the inflammation-targeted drug-loaded liposome suspension with a final concentration of 50 μg / mL was added to the upper chamber, and 600 μL of tumor cell conditioned medium (pancreatic tumor KPC cells had been cultured in advance for 48 hours) was added to the lower chamber as a chemotactic attractant. After 12 hours of co-culture, the cells in the upper chamber were removed, and the macrophages that migrated to the lower chamber were fixed with 4% paraformaldehyde and stained with 1% amethyst, and then the number of migrated cells was counted; the group not treated with inflammation-targeted drug-loaded liposomes was used as the control group.

[0057] Figure 3 The effect diagram of the inflammation-targeted drug-loaded liposomes prepared in Example 1 on anti-macrophage migration, wherein a is a schematic diagram of the Transwell migration experiment, b is a representative diagram of the migrating cells of the control group, and c is a representative diagram of the migrating cells of the inflammation-targeted drug-loaded liposomes treatment group. The results show that the number of migrating cells in the inflammation-targeted drug-loaded liposome group is significantly less than that in the control group, indicating that the inflammation-targeted drug-loaded liposomes can effectively resist the migration of macrophages in vitro.

[0058] Test Example 3

[0059] The effect of the inflammation-targeted drug-loaded liposomes prepared in Example 1 on inhibiting the growth of recurrent pancreatic tumors after Nanoknife treatment was determined as follows:

[0060] (1) First, an orthotopic recurrent pancreatic tumor model was established. When the tumor size reached 7-9 mm (about 9 days after tumor implantation), nanoknife surgery was performed, with the double needles inserted perpendicular to the long axis of the tumor to ablate 80% of the tumor. The treatment parameters were as follows: double needle interval of 5 mm, voltage of 1000 V, pulse width of 100 μs, frequency of 1 Hz, and number of repeated pulses of 99.

[0061] (2) The mice were randomly divided into a control group (PBS) and a treatment group (inflammation-targeted drug-loaded liposomes). The treatment was carried out on the 10th, 12th, 14th, 16th, 18th and 20th days after tumor implantation. The therapeutic drugs were injected into the mice through the tail vein. At the designated time points (9th, 10th, 14th, 18th, 22nd and 27th days), the size of the orthotopic pancreatic tumor was monitored using the bioluminescence imaging technology of the IVIS system, and the survival rate of the mice was recorded and the Kaplan-Meier (KM) survival curve was plotted.

[0062] Figure 4 The figures are the treatment effect diagrams of pancreatic tumors in the control group and the treatment group, where a is the appearance of the pancreatic tumor, b is the comparison result diagram of the tumor weight, and c is the survival analysis diagram; the results show that the pancreatic tumor weight in the treatment group was significantly lower than that in the control group, the survival rate of the mice in the treatment group was 70% on the 50th day, while the survival rate of the mice in the control group was only 10%, indicating that the treatment of inflammation-targeted drug-loaded liposomes and nanoknife treatment has a significant synergistic effect.

[0063] Test Example 4

[0064] The preparation of conventional liposomes comprises the following steps:

[0065] (1) Dipalmitoylphosphatidylcholine (DPPC), 1,2-stearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-stearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol) 2000] (DSPE-PEG-2000) and cholesterol are mixed in a molar ratio of 3:3:1:1 to obtain a mixed raw material; 50 mg of the mixed raw material and 1 mg of DSPE-PEG-2000-Cy5.5 are dissolved in 8 mL of a chloroform-methanol mixed solvent (the volume ratio of chloroform to methanol is 3:1), and a rotary evaporator is used to perform rotary evaporation for 1 hour at a temperature of 45° C., a rotation speed of 100 rpm and a pressure of 100 mbar to remove the solvent to obtain a lipid film;

[0066] (2) 5 mL of PBS solution was mixed with the lipid film, and hydrated at 50° C. for 60 min to obtain a liposome suspension. The obtained liposome suspension was extruded 10 times through a cellulose acetate membrane with a pore size of 450 nm, and then extruded 10 times through a cellulose acetate membrane with a pore size of 200 nm to obtain conventional liposomes labeled with Cy5.5 fluorescence.

[0067] The method of Example 1 was followed, except that the CCR2 inhibitor and the chemotherapeutic drug were omitted to obtain drug-free inflammation-targeted liposomes.

[0068] The conventional liposome suspension (concentration of 10 mg / mL, dispersant used is PBS solution) and the non-drug-loaded inflammation-targeted liposome suspension (concentration of 10 mg / mL, dispersant used is PBS solution) were injected into the pancreatic cancer tumor-bearing mice of two groups after nanoknife treatment through the tail vein, respectively, with an injection volume of 100 μL, and then the fluorescence distribution in the mice was monitored at the designated time points (0h, 2h, 4h, 8h, 24h, 48h) using the chemiluminescence imaging technology of the IVIS system.

[0069] Figure 5These are in vivo images of fluorescently labeled conventional liposomes and non-drug-loaded inflammation-targeted liposomes, where "MML" represents non-drug-loaded inflammation-targeted liposomes and "Liposome" represents conventional liposomes. The results show that compared with conventional liposomes, the non-drug-loaded inflammation-targeted liposomes have a stronger targeting effect on pancreatic tumors in mice after nanoknife treatment.

[0070] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An inflammation-targeted drug-loaded liposome, comprising a phospholipid membrane, a macrophage membrane protein loaded on the phospholipid membrane, and a CCR2 inhibitor and a chemotherapeutic drug encapsulated in the phospholipid membrane.

2. The inflammation-targeted drug-loaded liposome according to claim 1, characterized in that: The raw materials for preparing the phospholipid membrane include the following components in molar proportions: 2.8 to 3.2 parts of dipalmitoylphosphatidylcholine, 2.8 to 3.2 parts of 1,2-stearoyl-sn-glycero-3-phosphocholine, 0.8 to 1.2 parts of 1,2-stearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol) 2000], and 0.8 to 1.2 parts of cholesterol.

3. The inflammation-targeted drug-loaded liposome according to claim 1, characterized in that: The chemotherapeutic drug is a chemotherapeutic drug used to treat pancreatic cancer.

4. The inflammation-targeted drug-loaded liposome according to any one of claims 1 to 3, characterized in that: The drug loading amount of CCR2 inhibitor in the inflammation-targeted drug-loaded liposome is 6.1-6.5wt%, and the drug loading amount of chemotherapy drug is 2.7-3.1wt%; the content of macrophage membrane protein in the inflammation-targeted drug-loaded liposome is 0.25-0.31wt%.

5. The inflammation-targeted drug-loaded liposome according to claim 1 or 2, characterized in that: The particle size of the inflammation-targeted drug-loaded liposome is 150 to 200 nm, and the Zeta potential is -30 to -40 mV.

6. The method for preparing the inflammation-targeted drug-loaded liposome according to any one of claims 1 to 5, comprising the following steps: Mixing a CCR2 inhibitor, a chemotherapeutic drug, a raw material for preparing a phospholipid membrane, and an organic solvent, and removing the organic solvent from the resulting mixed liquid to obtain a precursor material; Mixing the precursor material with a hydration liquid to perform a hydration treatment to obtain a hydration treatment liquid; The hydration treatment liquid is mixed with macrophage membrane protein for loading treatment to obtain the inflammation-targeted drug-loaded liposome.

7. The preparation method according to claim 6, characterized in that: The mass ratio of the raw materials for preparing the phospholipid membrane to the CCR2 inhibitor is 45-55:4-6, and the mass ratio of the CCR2 inhibitor, the chemotherapy drug and the macrophage membrane protein is 4-6:4-6:0.13-0.

2.

8. The preparation method according to claim 6, characterized in that: The hydration liquid is water or PBS solution; the temperature of the hydration treatment is 45-55° C., and the time is 50-70 minutes.

9. Use of the inflammation-targeted drug-loaded liposomes according to any one of claims 1 to 5 or the inflammation-targeted drug-loaded liposomes prepared by the preparation method according to any one of claims 6 to 8 in the preparation of anti-macrophage migration reagents.

10. Use of the inflammation-targeted drug-loaded liposomes according to any one of claims 1 to 5 or the inflammation-targeted drug-loaded liposomes prepared by the preparation method according to any one of claims 6 to 8 in the preparation of a drug for inhibiting the recurrence of pancreatic tumors after nanoknife treatment.

Images