CN110859817A

CN110859817A - Nanoparticle drug delivery system and preparation method and application thereof

Info

Publication number: CN110859817A
Application number: CN201911182894.XA
Authority: CN
Inventors: 蔡林涛; 周海梅; 刘兰兰; 何华美; 梁锐晶
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2019-11-27
Filing date: 2019-11-27
Publication date: 2020-03-06
Also published as: WO2021104088A1

Abstract

The invention provides a nano drug-carrying system with simple preparation method, mature process, stable property and good biocompatibility, wherein nano particles are coupled to the surface of macrophages through maleic amide bonds, the tumor treatment is carried out by combining the immune regulation function of the macrophages and the function of the nano particles, so that the anti-tumor curative effect is improved, the problem that small drug molecules cannot be effectively enriched and targeted at tumor parts in the prior art is solved, and the nano drug-carrying system has important application prospects in the fields of nano medicine and tumor treatment.

Description

Nanoparticle drug delivery system and preparation method and application thereof

Technical Field

The invention belongs to the field of nano-medicine, and particularly relates to a drug delivery system with nano-particles loaded on the surfaces of macrophage cells, a preparation method of the drug delivery system and application of the drug delivery system in tumor immunotherapy.

Background

Tumor immunotherapy is known as the fourth tumor treatment method after surgery, chemotherapy, and radiotherapy. Unlike traditional treatment, tumor immunotherapy is a treatment method aiming at the immune system of human body rather than directly at tumor, and can enhance the anti-tumor immunity of the tumor microenvironment by exciting or mobilizing the immune system of the body, thereby controlling and killing tumor cells. In recent years, cellular immunotherapy has been attracting attention as a main tumor immunotherapy. Cellular immunotherapy, which is collectively referred to as adoptive immunotherapy, refers to the delivery of immune cells (both specific and non-specific) with anti-tumor activity to tumor patients to directly kill tumors or to stimulate an immune response in the body to kill tumor cells.

Macrophages are important components of the inherent immune response of an organism, are cell populations with plasticity and heterogeneity, maintain the steady state of normal tissues by removing abnormal cells, and play an important role in the non-specific immune function of the organism. Macrophages can exert a wide range of anti-tumor effects through multiple pathways and multiple steps. Bacterial cell wall components and cytokines can activate macrophages, and the activated macrophages can efficiently and specifically recognize and lyse tumor cells, including those resistant to cytotoxic drugs, but with little damage to normal cells. After the macrophages and the tumor cells are directly contacted for 1-3 days, some cytotoxic substances (such as tumor necrosis factors, nitric oxide, serine protease, lysosomal enzyme, active oxygen and the like) can be secreted and released, so that the combined tumor cells can be dissolved or killed, the process is slow, and the cells are required to be directly contacted. Macrophages can also kill tumor cells directly through antibody-dependent cytotoxicity. Activated macrophages can process and present tumor antigens, activate T cells, and stimulate the body to mount a specific immune response to tumor cells. In contrast to T cells, macrophage killing of tumor cells is independent of the immunogenicity, metastatic potential and sensitivity to drugs of the tumor cells. Therefore, when the specific T cells are difficult to exert effect in vivo, the killing effect of the activated macrophages is rarely resistant to most tumor cells, especially the metastatic tumor cells with the tumor antigens easy to generate variation. In addition, macrophages have tumor targeting properties and are widely used as tumor targeting vectors.

CN109893515A discloses a patent entitled "a macrophage drug-carrying microparticle preparation and a preparation method thereof", which indicates that the macrophage drug-carrying microparticle preparation comprises a cell vesicle and a drug small molecule active ingredient encapsulated in the cell vesicle, and the cell vesicle is released by mannose-modified macrophage apoptosis. The drug-loaded microparticles provided by the preparation method are beneficial to high enrichment in tumor tissues and easier to be absorbed by M2 type tumor-related macrophages, the reverse polarization effect of micromolecular drugs on M2 type tumor-related macrophages is improved, the tumor microenvironment is improved, and the killing effect on tumor cells is enhanced. CN104771764A discloses a patent entitled "a macrophage targeting vector system and its preparation", which indicates that the macrophage targeting vector is mannosylated protamine, and electropositive mannosylated protamine is loaded with electronegative nucleic acid to form a positively charged nanoparticle. Compared with the non-viral gene vector protamine, the mannosylated protamine has the functions of nuclear localization and macrophage targeting, and can improve the gene transfection mediating efficiency of the protamine in macrophages.

Although the prior art has a certain effect on the treatment of tumors by means of macrophage drug loading, macrophage targeting systems and the like, the prior art only takes the macrophages as carriers to deliver drugs to tumor sites to play a role, and does not utilize the functions of the macrophages, namely the natural immune cells. This not only increases the process and cost of preparation, but also wastes the action of macrophages. The research shows that the macrophage can play the specific immune function and tumor targeting property when serving as a carrier to transport the medicine, and the macrophage and the carrier play a role together, so that the anti-tumor curative effect is improved.

In recent years, the nano material has obvious advantages in the aspect of targeted drug delivery due to unique physicochemical properties, targeted modification and the like. Compared with the problems of poor biocompatibility and the like of artificial synthetic nano drug carriers, the utilization of cells or vesicles derived from the cells as drug carriers attracts wide attention. Therefore, by combining the nano-materials with cellular immunotherapy, a new idea for tumor treatment may be provided.

Disclosure of Invention

Aiming at the problems, the invention discloses a nano drug delivery system which has the advantages of simple preparation method, mature process, stable property and good biocompatibility, loads nano particles on the surface of macrophage, treats tumors in a combined manner by utilizing the immunoregulation function of the macrophage and the function of the nano particles, and has good application prospect in the fields of nano biomedicine, tumor cell treatment and the like.

The invention aims to provide a nano drug delivery system, which comprises macrophages and nano particles, wherein the nano particles have the loading, delivery and/or sustained release properties; the nanoparticle comprises a polymer selected from any one or more of polyesters, polyanhydrides, polyorthoesters, polyphosphazenes, polyhydroxy acids, polypropylmethacrylates, polyamides, polyamino acids, polyacetals, polyethers, polyurethanes, polymethacrylates, polyacrylates, polycyanoacrylates, polylactic acid (PLA), polyglycolic acid (PGA), Polycaprolactone (PCL), polyglutaractone, poly (lactide-co-glycolide) (PLG), polylactic acid-glycolic acid (PLGA), polyglycolic acid-polyethylene glycol (PLGA-PEG), poly (lactide-co-caprolactone) (PLC), poly (glycolide-co-caprolactone) (PGC), polycaprolactone-polyethylene glycol (PCL-PEG); the nanoparticles and/or macrophages have or are altered to have one or more functional groups that allow the nanoparticles to be loaded onto the surface of the macrophages.

Preferably, the functional group of the nanoparticle is a maleimide bond, and the nanoparticle is a nanoparticle coated with the maleimide bond synthesized by an ultrasonic method.

Preferably, the functional group of the macrophage is a thiol group, and the macrophage is treated with a thiol reducing agent before coupling to the nanoparticle; preferably, the thiol reducing agent is TCEP.

Preferably, the drug delivery nano-system further comprises an anti-tumor drug.

Preferably, the anti-tumor drug comprises an anti-tumor broad spectrum drug and/or an anti-tumor targeted drug.

Preferably, the anti-tumor broad spectrum medicine is selected from any one or more of camptothecin medicines, adriamycin medicines, taxol medicines or platinum medicines.

Preferably, the anti-tumor targeting agent is selected from any one or more of zertinib, nilotinib, imatinib, vismodegib, vemurafenib, temsirolimus, sunitinib, ceritinib, regorafenib, afatinib, tremitinib, pranatinib, bortezomib, pazopanib, axitinib, romidepsin, everolimus, ibrutinib, lenvatinib, dabrafenib, crizotinib, carfilzomib, osetinib, cabozantinib, cabertinib, gefitinib, vorinostat, vandetanib, eltanib, dinoselamemo, sonedgi, sorafenib, bosutinib, belita, olaparipatinib, aflibercept, lapatinib, dasatinib, palbociclib, or erlotinib.

Preferably, the composition further comprises a polypeptide material, the polypeptide comprising an antigen or an antibody.

Preferably, the antibody is selected from any one or more of adalimumab, cetuximab, ibritumomab tiuxetan, trastuzumab, nivolumab, darunavir anti-ramucirumab, nixituzumab, pembromumab, ofatumumab, bornatuzumab, bevacizumab, panitumumab, obint itumumab, benitumumab, dinumumab, tositumumab, erlotuzumab, trastuzumab, or rituximab.

Preferably, the tumor is selected from any one or more of basal cell carcinoma, squamous cell carcinoma, esophageal carcinoma, glioblastoma, bladder cancer, cervical cancer, breast cancer, lung cancer, liver cancer, stomach cancer, colon cancer, rectal cancer, nasopharyngeal cancer, pancreatic cancer, thyroid cancer, prostate cancer, leukemia, lymphoma, kidney tumor, sarcoma, and blastoma.

The invention also aims to provide a medicament containing the nano medicament-carrying system.

Preferably, the drug is an anti-tumor drug.

Preferably, the medicament is administered by injection.

Preferably, the administration by injection comprises any one or more of subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection, intra-lymph node injection, intratumoral injection or underfoot injection.

Preferably, the medicament further comprises medically or pharmaceutically acceptable auxiliary substances and/or excipients.

The invention also aims to provide the application of the nano drug delivery system or the drug in preparing an anti-tumor drug.

Another object of the present invention is to provide a method for preparing the nano drug delivery system, comprising the following steps:

(1) preparing the nanoparticles encapsulating the maleic amide bond;

(2) removing and inducing and culturing primary macrophages from the spinal cord;

(3) when the growth of macrophages was good, the macrophages were counted and transferred to a 1.5mL centrifuge tube, to which a TCEP solution diluted with phosphate buffer was added at 37 ℃ with 5% CO₂Co-incubation is carried out in an incubator, the centrifuge tube is turned upside down every 10 minutes, and then the centrifuge tube is washed for a plurality of times by phosphate buffer;

(4) and (3) transferring the nanoparticles obtained in the step (1) and the macrophages treated in the step (3) into a 15mL centrifuge tube, continuously dropwise adding a phosphate buffer solution until the total volume is 4mL, then mixing the phosphate buffer solution in a shaking table at 37 ℃, and finally centrifuging the mixed liquid to successfully load the nanoparticles on the surfaces of the macrophages.

Preferably, the preparation method of step (1) is to synthesize nanoparticles wrapping the maleic amide bond by an ultrasonic method.

Preferably, the number of the macrophages transferred into the 1.5mL centrifuge tube in the step (3) is 1X 10⁵-1×10⁸Preferably 1-5X 10⁶More preferably 2 × 10⁶And (4) respectively.

Preferably, the concentration of the TCEP solution in the step (3) is 1 mM.

Preferably, the incubation time in step (3) is 20 minutes.

Preferably, the mixing conditions of the shaker in step (4) are 120 revolutions for 20 minutes.

Preferably, the centrifugation in step (4) is performed at 1000 rpm for 4 minutes.

The invention provides a preparation method of a nano drug-carrying system loading nano particles on the surfaces of macrophages, which aims to solve the technical problem that small drug molecules cannot be effectively enriched and targeted at tumor sites in the prior art, and the nano drug-carrying system prepared by the invention is characterized in that ① the macrophages used for loading the nano particles are processed by TCEP and aim to expose sulfydryl on the surfaces of cells, a maleimide bond is arranged at the periphery of the nano particles with ② core-shell structures and is used for being combined with the sulfydryl on the surfaces of the macrophages, ③ the nano particles are loaded on the surfaces of the macrophages instead of entering the cells, the ④ macrophage loaded with the nano particles has better biocompatibility and biodegradability and can reduce the toxicity of drugs to organisms, the ⑤ nano particle-loaded macrophage has pH responsiveness and can realize the response control release of the drugs, and the ⑥ nano particle-loaded macrophages directly contact tumors after being targeted at the tumor sites and can be activated to release the drugs wrapped in the nano particles in situ and can play the immune regulation of the macrophages and improve the anti-tumor curative effect.

Drawings

Fig. 1 is a scanning electron microscope image of nanoparticle-loaded macrophages.

Fig. 2 is a confocal laser image of nanoparticle-loaded macrophages.

FIG. 3 is a graph showing the in vitro killing effect of macrophages loaded with nanoparticles on their surface on cancer cells, BMDM representing macrophages, MPIP representing macrophages loaded with nanoparticles.

Detailed Description

The present invention will be described in further detail with reference to specific examples below so that those skilled in the art can better understand the present invention and practice the present invention, but the examples are not intended to limit the present invention.

The experimental procedures used in the following examples are conventional unless otherwise specified. The materials, reagents and the like used are commercially available unless otherwise specified.

Example 1 preparation of macrophage cell surface-loaded with nanoparticles

① synthesizing nanometer particles wrapping the maleic amide bond by an ultrasonic method;

② primary macrophages were removed and induced at the spinal cord of mice;

③ when the macrophage growth state is good, count 2X 10⁶Left and right macrophages were added to a 1.5mL centrifuge tube 1mM TCEP solution (diluted with phosphate buffer) at 37 deg.C with 5% CO₂Co-incubating for 20 minutes in an incubator, turning the centrifuge tube upside down every 10 minutes, and washing for 3-4 times by using a phosphate buffer solution;

④ transferring the nanoparticles and the treated macrophages into a 15mL centrifuge tube, continuously dripping phosphate buffer solution until the total volume is 4mL, then putting the centrifuge tube into a shaking table at 37 ℃, rotating the shaker tube for 20 minutes at 120 ℃, finally putting the mixed liquid into a 1000-rotation shaker tube, and centrifuging the shaker tube for 4 minutes, namely successfully loading the particles on the surfaces of the macrophages.

Example 2 Performance and characterization of macrophage cells surface-loaded with nanoparticles

1. Scanning electron microscope image of macrophage with surface loaded with nano particles

Firstly, diluting a solution of macrophage with the surface loaded with nano particles by 10 times, preparing a sample according to a scanning electron microscope sample preparation method, and then observing under a scanning electron microscope. As a result, it was found that the nanoparticles were loaded on the surface of macrophages, as shown in fig. 1.

2. Laser confocal image of macrophage with surface loaded with nanoparticles

Firstly, diluting a solution of macrophages with nanoparticles loaded on the surface by 100 times, then dropwise adding the solution into a confocal small dish, and then placing the confocal small dish under a laser confocal microscope for observation. The results show that the nanoparticles are loaded on the macrophage surface, as shown in figure 2.

3. Detection of in-vitro killing effect of macrophage with surface loaded with nanoparticles on cancer cells

Breast carcinoma cells (4T1) were seeded in 96-well plates (10)⁴One/well), respectively, will contain macrophages in an amount of 10⁴Individual macrophages alone or macrophages surface-loaded with nanoparticles were added and allowed to co-incubate for 24 hours. Then, the survival rate of breast cancer cells was detected by a killing kit, and as a result, it was found that the tumor killing effect of macrophages with surface-loaded nanoparticles was much stronger than that of macrophages alone, as shown in fig. 3 (BMDM represents macrophages, MPIP represents macrophages with loaded nanoparticles).

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A nano drug delivery system comprising macrophages and nanoparticles, the nanoparticles having loading, delivery and/or sustained release properties; the nanoparticle comprises a polymer selected from any one or more of polyesters, polyanhydrides, polyorthoesters, polyphosphazenes, polyhydroxy acids, polypropylmethacrylates, polyamides, polyamino acids, polyacetals, polyethers, polyurethanes, polymethacrylates, polyacrylates, polycyanoacrylates, polylactic acid (PLA), polyglycolic acid (PGA), Polycaprolactone (PCL), polyglutaractone, poly (lactide-co-glycolide) (PLG), polylactic acid-glycolic acid (PLGA), polyglycolic acid-polyethylene glycol (PLGA-PEG), poly (lactide-co-caprolactone) (PLC), poly (glycolide-co-caprolactone) (PGC), polycaprolactone-polyethylene glycol (PCL-PEG); the nanoparticles and/or macrophages have or are altered to have one or more functional groups that allow the nanoparticles to be loaded onto the surface of the macrophages.

2. The nanoparticie delivery system of claim 1, wherein the functional group of the nanoparticle is a maleimide bond, and the nanoparticle is a nanoparticle coated with a maleimide bond synthesized by an ultrasonic method.

3. The nanoplatelet system of claim 1 wherein the functional group of the macrophage is a thiol group, and the macrophage is treated with a thiol reducing agent prior to coupling to the nanoparticle; preferably, the thiol reducing agent is TCEP.

4. The nanoparticie system of claim 1, the nanoparticles further comprising an anti-tumor drug and/or a polypeptide substance; the anti-tumor medicine comprises an anti-tumor broad-spectrum medicine and/or an anti-tumor targeted medicine; the polypeptide includes an antigen or an antibody.

5. The nano drug delivery system of claim 4, wherein the anti-tumor broad-spectrum drug is selected from any one or more of camptothecin drugs, adriamycin drugs, taxol drugs or platinum drugs.

6. The Nanotercint system of claim 4, wherein the antitumor targeting agent is selected from any one or more of Zebutinib, nilotinib, imatinib, vismodegib, Verofibrib, temsirolimus, sunitinib, Seritinib, regorafenib, Afatinib, trametinib, Prnatinib, Bortezomib, Pazopanib, acitinib, Romidepsin, Everolimus, Ibrutinib, Levatinib, Darafenib, crizotinib, Carfilzomib, Osertinib, Cabotinib, Cabinitinib, Gefitinib, Vorinostat, vandetanib, Identinib, Dinosapide, Sonedgi, Sorafenib, Bosutinib, Bellitastat, Olapatinib, Abiracil, Lapatinib, Pabesinib, or erlotinib.

7. The nanoparticulable system of claim 4, wherein the antibody is selected from any one or more of adalimumab, cetuximab, ibritumomab tiuxetan, trastuzumab, nivolumab, darunavailantimazerumab, nixituzumab, pembrolizumab, ofatumumab, bonatuzumab, bevacizumab, panitumumab, obint itumumab, benitumumab, dinumumab, tositumomab, erlotuzumab, trastuzumab, or rituximab.

8. A medicament containing a drug delivery nanosystem according to any of claims 1 to 7, which is an antineoplastic medicament.

9. The medicament of claim 8, which is administered by injection.

10. The medicament of claim 9, wherein the administration by injection comprises any one or more of subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection, intra-lymph node injection, intratumoral injection or underfoot injection.

11. Pharmaceutical according to any one of claims 8-10, further comprising pharmaceutically or pharmacologically acceptable auxiliary substances and/or excipients.

12. Use of the nanopharmaceutical delivery system of any of claims 1-7 or the medicament of any of claims 8-11 in the preparation of an anti-tumor medicament.

13. The Nanocarotemporal system according to any one of claims 4 to 7 or the medicament according to any one of claims 8 to 11 or the use according to claim 12, wherein the tumor is selected from any one or more of basal cell carcinoma, squamous cell carcinoma, esophageal carcinoma, glioblastoma, bladder carcinoma, cervical carcinoma, breast carcinoma, lung carcinoma, liver carcinoma, stomach carcinoma, colon carcinoma, rectal carcinoma, nasopharyngeal carcinoma, pancreatic carcinoma, thyroid carcinoma, prostate carcinoma, leukemia, lymphoma, renal tumor, sarcoma, blastoma.

14. A method of preparing a nano drug delivery system comprising the steps of:

(1) preparing the nanoparticle of any one of claims 1-7;

15. The method according to claim 14, wherein the step (1) is carried out by synthesizing nanoparticles coated with maleic amide bond by ultrasonic method.

16. The method of claim 14, wherein the number of macrophages transferred into the 1.5mL centrifuge tube in step (3) is 1 x 10⁵-1×10⁸Preferably 1-5X 10⁶More preferably 2 × 10⁶And (4) respectively.

17. The method according to claim 14, wherein the concentration of the TCEP solution in step (3) is 1 mM.

18. The method of claim 14, wherein the incubation in step (3) is for 20 minutes.

19. The method of claim 14, wherein said shaker mixing in step (4) is performed at 120 revolutions for 20 minutes.

20. The method of claim 14, wherein the centrifugation in step (4) is at 1000 rpm for 4 minutes.