CN110859817A - Nanoparticle drug-carrying system, preparation method and application thereof - Google Patents

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-carrying system, preparation method and application thereof

technical field

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

Background technique

Tumor immunotherapy is known as the fourth tumor treatment method after surgery, chemotherapy and radiotherapy. Different from traditional treatment methods, tumor immunotherapy is a treatment method that targets the human immune system rather than directly targeting the tumor. By stimulating or mobilizing the body's immune system, it enhances the anti-tumor immunity of the tumor microenvironment, thereby controlling and killing tumor cells. . In recent years, cellular immunotherapy has received extensive attention as the main tumor immunotherapy. Cellular immunotherapy, its full name is adoptive immune cell therapy, is to transport immune cells (specific and non-specific) with anti-tumor activity to tumor patients to directly kill tumors or stimulate the body's immune response to kill tumor cells.

Macrophages are an important component of the body's innate immune response. They are a type of plastic and heterogeneous cell population. They maintain the homeostasis of normal tissues by removing abnormal cells and play an important role in the body's non-specific immune function. 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 activated macrophages can efficiently and exclusively recognize and lyse tumor cells, including those that are resistant to cytotoxic drugs, but damage normal cells very rarely. few. Macrophages and tumor cells can secrete and release some cytotoxic substances (such as tumor necrosis factor, nitric oxide, serine proteases, lysosomal enzymes, reactive oxygen species, etc.) after direct contact with tumor cells for 1 to 3 days, which can lead to the lysis of the bound tumor cells. or apoptosis, a process that is slow and requires direct contact between cells. Macrophages can also directly kill tumor cells 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. Compared with T cells, tumor cell killing by macrophages is independent of tumor cell immunogenicity, metastatic potential, and drug sensitivity. Therefore, most tumor cells, especially those metastatic tumor cells that are prone to mutation of tumor antigens, are rarely resistant to the killing effect of activated macrophages when specific T cells are difficult to exert their effects in vivo. In addition, macrophages have tumor-targeting properties and are widely used as tumor-targeting carriers.

CN109893515A published a patent titled "A macrophage drug-loaded microparticle preparation and its preparation method", the patent pointed out that the macrophage drug-loaded microparticle preparation includes cell vesicles and small drug molecules encapsulated in the cell vesicles Active ingredient, cell vesicles are released from apoptosis of mannose-modified macrophages. They believe that the drug-loaded microparticles provided by it are beneficial to be highly enriched in tumor tissue and more easily taken up by M2 tumor-associated macrophages, improve the depolarization effect of small molecule drugs on M2 tumor-associated macrophages, and improve tumors. The microenvironment enhances the killing of tumor cells. CN104771764A published a patent titled "A macrophage targeting carrier system and its preparation", pointing out that the macrophage targeting carrier is mannosylated protamine, and the positively charged mannosylated protamine is loaded with negatively charged nucleic acid to form a kind of positively charged nanoparticles. Compared with the non-viral gene carrier protamine, mannosylated protamine has nuclear localization function and macrophage targeting, which can improve the gene transfection mediation efficiency of protamine in macrophages.

The above-mentioned prior art through macrophage drug loading, macrophage targeting system, etc., has a certain effect on the treatment of tumors, but these methods only use macrophages as a carrier to deliver the drug to the tumor site. The function of the innate immune cell itself, macrophages, is not utilized. This not only increases the process and cost of preparation preparation, but also wastes the role of macrophages. Our study found that macrophages can also play their own unique immune function and tumor targeting while acting as a carrier to transport drugs. The two work together to improve the anti-tumor efficacy.

In recent years, nanomaterials have obvious advantages in targeted drug delivery due to their unique physicochemical properties and targeted modification. Compared with the poor biocompatibility of synthetic nano-drug carriers, the use of cells or cell-derived vesicles as drug carriers has attracted widespread attention. Therefore, by combining nanomaterials and cellular immunotherapy, it may provide new ideas for tumor treatment.

SUMMARY OF THE INVENTION

In view of the above problems, the present invention discloses a nano-drug loading system with simple preparation method, mature technology, stable properties and good biocompatibility, which loads nanoparticles on the surface of macrophages, and uses the immune system of macrophages themselves The combination of modulating function and nanoparticle function to treat tumors has good application prospects in the fields of nanobiomedicine and tumor cell therapy.

One object of the present invention is to provide a nano-drug delivery system, including macrophages and nanoparticles, the nanoparticles have the properties of loading, delivery and/or sustained release; the nanoparticles include polymers, and the polymers Selected from polyester, polyanhydride, polyorthoester, polyphosphazene, polyphosphate, polyhydroxy acid, polypropyl fumarate, polyamide, polyamino acid, polyacetal, polyether, polyurethane, polymethyl Acrylates, Polyacrylates, Polycyanoacrylates, Polylactic Acid (PLA), Polyglycolic Acid (PGA), Polycaprolactone (PCL), Polyvalerolactone, Poly(lactide-co-glycolide) ) (PLG), polylactic acid-glycolic acid (PLGA), polyglycolic acid-polylactic acid-polyethylene glycol (PLGA-PEG), poly(lactide-co-caprolactone) (PLC), poly(ethylene glycol) any one or more of lactide-co-caprolactone) (PGC), polycaprolactone-polyethylene glycol (PCL-PEG); the nanoparticles and/or macrophages have or are altered to Having one or more functional groups enables the nanoparticles to be loaded onto the surface of the macrophages.

Preferably, the functional group of the nanoparticle is a maleamide bond, and the nanoparticle is synthesized by a method of ultrasonication and wraps the maleamide bond.

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

Preferably, the nano-drug loading system further includes anti-tumor drugs.

Preferably, the anti-tumor drugs include broad-spectrum anti-tumor drugs and/or targeted anti-tumor drugs.

Preferably, the anti-tumor broad-spectrum drug is selected from any one or more of camptothecin-based drugs, doxorubicin-based drugs, paclitaxel-based drugs or platinum-based drugs.

Preferably, the anti-tumor targeted drug is selected from zanubrutinib, nilotinib, imatinib, vemodagi, vemurafenib, temsirolimus, sunitinib, ceritinib Afatinib, regorafenib, afatinib, trametinib, ponatinib, bortezomib, pazopanib, axitinib, romidepsin, everolimus, ibrutinib , lenvatinib, dabrafenib, crizotinib, carfilzomib, ostinib, cabozantinib, cabitinib, gefitinib, vorinostat, vandetanib , alectinib, denosumab, sonidegi, sorafenib, bosutinib, belistat, olaparib, aflibercept, lapatinib, dasatinib, Any one or more of palbociclib, panobinostat, or erlotinib.

Preferably, the composition further includes a polypeptide substance, and the polypeptide includes an antigen or an antibody.

Preferably, the antibody is selected from the group consisting of adalimumab, cetuximab, tiimumab, trastuzumab, nivolumab, daratumumab, ramucirumab, nexacitab Pembrolizumab, pembrolizumab, pembrolizumab, ofatumumab, blinatumumab, bevacizumab, panitumumab, obinutuzumab, brentuximab , any one or more of denutuzumab, tositumumab, elotuzumab, trastuzumab, or rituximab.

Preferably, the tumor is selected from the group consisting of basal cell carcinoma, squamous cell carcinoma, esophageal cancer, malignant glioma, bladder cancer, cervical cancer, breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, nasopharyngeal cancer, Any one or more of pancreatic cancer, thyroid cancer, prostate cancer, leukemia, lymphoma, kidney tumor, sarcoma, blastoma.

Another object of the present invention is to provide a drug containing the nano-drug delivery system.

Preferably, the drug is an antitumor drug.

Preferably, the drug is administered by injection.

Preferably, the injection administration includes any one or more of subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection, intralymphatic injection, intratumoral injection or foot injection.

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

Another object of the present invention is to provide a nano-drug loading system or an application of the drug in the preparation of anti-tumor drugs.

Preferably, the tumor is selected from the group consisting of basal cell carcinoma, squamous cell carcinoma, esophageal cancer, malignant glioma, bladder cancer, cervical cancer, breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, nasopharyngeal cancer, Any one or more of pancreatic cancer, thyroid cancer, prostate cancer, leukemia, lymphoma, kidney tumor, sarcoma, blastoma.

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

(1) prepare the described nanoparticle that wraps maleamide bond;

(2) Take out and induce and culture primary macrophages from the spinal cord;

(3) When the macrophages grow well, count the macrophages, transfer them into a 1.5mL centrifuge tube, add TCEP solution diluted with phosphate buffer, and co-incubate them in a 37°C, 5% _CO2 incubator. During the period, after turning the centrifuge tube upside down every 10 minutes, wash it several times with phosphate buffer;

(4) Transfer the nanoparticles obtained in step (1) and the macrophages treated in step (3) into a 15 mL centrifuge tube, continue to add phosphate buffer solution dropwise to a total volume of 4 mL, and then put them into a shaker at 37°C. The bed is mixed, and the mixed liquid is finally centrifuged, that is, the nanoparticles are successfully loaded on the surface of macrophages.

Preferably, the preparation method of the step (1) is to synthesize nanoparticles encapsulated with maleamide bonds by ultrasonic method.

Preferably, the number of the macrophages transferred into the 1.5mL centrifuge tube in the step (3) is 1×10 ⁵ -1×10 ⁸ , preferably 1-5×10 ⁶ , more preferably 2 x 10 ⁶ pcs.

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

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

Preferably, in the step (4), the shaker mixing condition is 120 rpm for 20 minutes.

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

The present invention provides a preparation method of a nano-drug-carrying system loaded with nanoparticles on the surface of macrophages. The technical problem to be solved is to solve the problem that small drug molecules in the prior art cannot be effectively enriched and targeted at tumor sites. question. The characteristics of the nano-drug-loading system prepared by the invention are as follows: ① the macrophages used to load the nanoparticles must be treated with TCEP, in order to expose the thiol groups on the cell surface; ② the core-shell structured nanoparticles have horses on the periphery of the nano-particles. The lymide bond is used to combine with the sulfhydryl group on the surface of macrophages; ③ the nanoparticles are loaded on the surface of macrophages, rather than entering the interior of the cells; ④ the macrophages loaded with nanoparticles have good biocompatibility and biological properties. Degradability can reduce the toxicity of drugs to the body; ⑤ Macrophages loaded with nanoparticles are pH responsive and can achieve controlled release of drugs in response; ⑥ After macrophages loaded with nanoparticles are targeted to the tumor site, they interact with the tumor. Direct contact can not only activate the release of drugs encapsulated in nanoparticles in situ, but also exert the immune regulation of macrophages and improve the anti-tumor efficacy.

Description of drawings

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

Figure 2 is a confocal image of nanoparticle-loaded macrophages.

Figure 3 shows the in vitro killing effect of nanoparticle-loaded macrophages on cancer cells. BMDM represents macrophages, and MPIP represents nanoparticle-loaded macrophages.

Detailed ways

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

The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified.

Example 1 Preparation of surface-loaded nanoparticle macrophages

①Synthesize nanoparticles encapsulating maleamide bonds by ultrasonic method;

②Take out and induce primary macrophages from the spinal cord of mice;

③When the macrophages grow well, count about 2×10 ⁶ macrophages in a 1.5mL centrifuge tube, add 1mM TCEP solution (diluted with phosphate buffer), and incubate at 37°C, 5% CO ₂ . Incubate in the box for 20 minutes, invert the centrifuge tube upside down every 10 minutes, and then wash with phosphate buffer 3-4 times;

④ Transfer the nanoparticles and the treated macrophages into a 15mL centrifuge tube, continue to add dropwise phosphate buffer to a total volume of 4mL, then put them into a shaker at 37°C, 120 rpm for 20 minutes, and finally put the mixed liquid into the tube. After centrifugation at 1000 rpm for 4 minutes, the particles were successfully loaded on the surface of macrophages.

Example 2 Properties and characterization of surface-loaded nanoparticle macrophages

1. SEM images of surface-loaded nanoparticle macrophages

First, the solution of macrophages loaded with nanoparticles on the surface was diluted 10 times and then prepared according to the scanning electron microscope sample preparation method, and then observed under the scanning electron microscope. It was found that the nanoparticles were loaded on the surface of macrophages, as shown in Figure 1.

2. Confocal images of surface-loaded nanoparticle macrophages

First, the solution of macrophages loaded with nanoparticles on the surface was diluted 100 times and then dropped into a confocal small dish, and then the confocal small dish was placed under a laser confocal microscope for observation. The results showed that the nanoparticles were loaded on the surface of macrophages, as shown in Figure 2.

3. In vitro killing effect of surface-loaded nanoparticle macrophages on cancer cells

Breast cancer cells (4T1) were seeded in 96-well plates (10 ⁴ cells/well), and macrophages containing 10 ⁴ macrophages alone or nanoparticle-loaded macrophages were added to them, respectively. Let them incubate for 24 hours. Then the survival rate of breast cancer cells was detected by a killing kit, and it was found that the surface-loaded nanoparticle macrophages had a much stronger killing effect on tumors than single macrophages, as shown in Figure 3 (BMDM stands for macrophages, MPIP represents nanoparticle-loaded macrophages).

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. range.

Claims (20)

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;

(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.

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.

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