Preparation method and application of nano-composite delivery system carrying cells

Abstract

The invention discloses a preparation method and application of a nano-composite delivery system carrying cells, and the preparation method comprises the following steps: 1, culturing the cells to a logarithmic phase, and preparing a single-cell suspension when the fusion degree is 90%; 2, preparing a mixed liquid from a drug and a polymer material, performing ultrasonic treatment to obtain a primary emulsion, adding the primary emulsion into the liquid containing the emulsifier, performing ultrasonic treatment to obtain a multiple emulsion, volatilizing a solvent, performing high-speed centrifugation for 3-5 times, performing water washing for 3-5 times, and performing freeze drying to obtain the polymer nano delivery system; and 3, incubating the cells and the polymer nano-delivery system together for a certain time to obtain the nano-composite delivery system carrying the cells. The method is simple, and the effect of cells and the effect of biological materials are combined together. In-vitro experiments prove that the nano-composite delivery system carrying the cells is successfully constructed and has good biocompatibility and good migration ability. Animal experiments prove that the nano-composite delivery system carrying the cells has a better disease treatment effect compared with free drugs.

Description

Technical Field

[0001] The invention relates to a preparation method and application of a cell-carrying nanocomposite delivery system, and belongs to the fields of cell biology and nanomedicine. Background Art

[0002] Cardiovascular diseases seriously endanger human health and are considered the main cause of high morbidity and mortality worldwide. The 2022 China Cardiovascular Disease Report shows that there are currently 330 million cardiovascular disease patients, and the morbidity and mortality rates are still on the rise. The burden of cardiovascular diseases in China is increasing and has become a major public health issue.

[0003] In clinical practice, in addition to surgery and interventional therapy, drug therapy is also a common means of treating cardiovascular diseases. The drugs used for treatment are roughly divided into five categories: vasodilators, calcium channel blockers or calcium antagonists, lipid-lowering drugs, antiplatelet drugs, thrombolytic drugs, and anticoagulants. Unfortunately, due to their nonspecific distribution, adverse reactions, low bioavailability, slow onset, and severe side effects, common oral drugs are limited to the treatment of early-stage diseases.

[0004] Bionic targeted nano-delivery systems provide a new perspective for the treatment of cardiovascular diseases. These nano-delivery systems can target lesions and thus improve inflammation. The current research is limited in that it only uses cell membranes for biomimetic purposes, without truly taking advantage of cell regulation and its own chemotaxis and homing advantages; and only cell membranes can only have short-term effects and are easily engulfed and metabolized by other cells.

[0005] Recently, the binding of foreign substances to the body's own cell proteins and other substances for "hitchhiking" delivery has become a hot topic in bionic therapy research. By using nano-delivery systems to carry them to cells, cells can be targeted to lesions through the effects of chemokines and other factors, while protecting nanoparticles from being metabolized by the reticulum system. This bionic hitchhiking strategy will open up new prospects for biocompatible nanosystems in disease treatment, especially cardiovascular diseases, and future clinical applications. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for preparing a cell-carrying nanocomposite delivery system.

[0007] The second object of the present invention is to provide an application of a cell-carrying nanocomposite delivery system.

[0008] The technical solution of the present invention is summarized as follows: 1. A preparation method and application of a cell-carrying nanocomposite delivery system, characterized in that it comprises the following steps: (1) The cells were cultured in a 37°C 5% CO2 incubator until the logarithmic phase. When the confluence reached 90%, single-cell suspension was prepared by cell scraping or trypsin digestion. (2) Dissolve 10-100 mg of the drug and 100-500 mg of the polymer material in an appropriate solvent, and ultrasonicate the mixture for 5-10 min at a shear rate of 10%-30% using an ultrasonic probe under ice bath conditions to obtain colostrum. Add 10-30 mL of an emulsifier aqueous solution to a beaker, and then add the colostrum obtained in step (2), and ultrasonicate the mixture for 10-20 min at a shear rate of 10%-30% using an ultrasonic probe under ice bath conditions. Volatilize the solvent, wash with distilled water 3-6 times under high-speed centrifugation conditions, and freeze-dry to obtain a nano delivery system; (3) incubating the cells and the polymer nano-delivery system in a 37°C 5% CO2 incubator for a certain period of time to obtain a nano-composite delivery system carrying cells; (4) Apply this prepared cell-loaded nanocomposite delivery system to the treatment of cardiovascular diseases and other diseases in animals.

[0009] 2. The cells are preferably primary or cell line type monocytes, macrophages, neutrophils, endothelial cells, cardiomyocytes or smooth muscle cells.

[0010] 3. The polymer is preferably polyester, polyalkyl cyanoacrylate (PBCA, PiBCA), amphiphilic block copolymer (Mal-PEG, mPEG), dendrimer (PAMAM), polylactic acid (PLA), polycaprolactone (PCL), polyethylene glycol (PEG), polylactic acid glycolic acid (PLGA), chitosan, etc.

[0011] 4. The drugs are various water-soluble or fat-soluble drugs for cardiovascular diseases and other diseases.

[0012] 5. The animals include mice, rats, rabbits, dogs, monkeys and other small and large animals.

[0013] 6. Cardiovascular diseases and other diseases include atherosclerosis, myocardial infarction, myocardial ischemia-reperfusion injury, arteritis, hemangioma and other diseases.

[0014] 7. A method for preparing a cell-carrying nanocomposite delivery system is obtained.

[0015] Advantages of the present invention: The preparation method of the cell-carrying nanocomposite delivery system of the present invention is simple and low in cost. Compared with the prior art, the present invention can improve the bioavailability of drugs, reduce drug side effects, protect drugs and nano delivery systems from being removed, and has the advantages of cell secretion factors, cell chemotaxis, etc. A cell-carrying nanocomposite delivery system has better disease treatment effects than free drugs at the animal experimental level. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A composite delivery system formed by nanoparticles carrying monocytes.

[0017] Figure 2 The fluorescence staining results are shown when nanoparticles were incubated with monocytes for different time periods.

[0018] Figure 3 Representative pictures of cell death and viability fluorescence staining. Green fluorescence indicates viable cells, and red fluorescence indicates dead cells.

[0019] Figure 4 This is a representative picture of the mouse aorta stained with Oil Red. The red dots are lipid droplets.

[0020] Figure 5 In vivo imaging of fluorescence distribution in the mouse heart and aorta.

[0021] In each figure, M represents the monocyte group; PP represents the polymer material-PLGA-PEG blank nanoparticles; ATV represents free atorvastatin; M-PPA represents monocytes loaded with PLGA-PEG-ATV nanodelivery system; Model represents the atherosclerosis positive control group. Example

[0022] A preparation method and application of a PLGA nanocomposite delivery system carrying macrophage RAW264.7, characterized in that it comprises the following steps: (1) RAW264.7 macrophages were cultured in a 37°C 5% CO2 incubator until the logarithmic phase. When the confluence reached 90%, single-cell suspension was prepared by cell scraping; (2) Dissolve 10 mg tPA and 150 mg PLGA polymer material in an appropriate solvent, and ultrasonicate for 5 min with an ultrasonic probe at a shear rate of 20% under ice bath conditions to obtain colostrum. Add 25 mL of 1% PVA emulsifier aqueous solution to a beaker, and then add the colostrum obtained in step (2), and ultrasonicate for 10 min with an ultrasonic probe at a shear rate of 20% under ice bath conditions. Volatilize the solvent, wash with distilled water 4 times under high-speed centrifugation conditions, and freeze-dry to obtain a nano delivery system; (3) RAW264.7 and the polymer nanodelivery system were co-incubated in a 37°C 5% CO2 incubator for 6 h to obtain a cell-carrying nanocomposite delivery system; (4) The prepared cell-loaded nanocomposite delivery system was applied to treat myocardial infarction in rats. Example

[0023] A preparation method and application of a PLGA-PEG nanocomposite delivery system carrying monocytes, characterized by comprising the following steps: (1) RAW264.7 macrophages were cultured in a 37°C 5% CO2 incubator until the logarithmic phase. When the confluence reached 90%, single-cell suspension was prepared by cell scraping. (2) 8 mg of atorvastatin and 100 mg of PLGA-PEG polymer material were dissolved in an appropriate solvent, and colostrum was obtained by ultrasonication with an ultrasonic probe at a shear rate of 25% for 3 min under ice bath conditions. 20 mL of 1% PVA emulsifier aqueous solution was added to a beaker, and then the colostrum obtained in step (2) was added, and ultrasonication with an ultrasonic probe at a shear rate of 25% for 9 min under ice bath conditions was performed. The solvent was evaporated, and the mixture was washed with distilled water 5 times under high-speed centrifugation conditions, and freeze-dried to obtain a nano delivery system; (3) The monocytes were co-incubated with the PLGA-PEG polymer nano-delivery system in a 37°C 5% CO2 incubator for 12 h to obtain a cell-loaded nanocomposite delivery system; (4) The prepared cell-loaded nanocomposite delivery system was applied to the treatment of atherosclerosis in mice. Example

[0024] The monocytes were co-incubated with the green fluorescent PLGA-PEG polymer nano-delivery system in a 37°C 5% CO2 incubator for 3 h, 6 h, 12 h, and 24 h to obtain the cell-carrying nanocomposite delivery system, which was observed under a confocal microscope. It was found that the fluorescence content of PLGA-PEG was similar at 3 h and 6 h, but reached a peak at 12 h, and remained undegraded at 24 h, proving that monocytes and the PLGA-PEG polymer nano-delivery system can form a carrier and can be maintained for a period of time. Example

[0025] Mouse monocytes were extracted and diluted to a concentration of 5 × 10 5 / mL, incubated in a 12-well plate. PLGA-PEG nanoparticles were incubated with monocytes for 48 h. The activity of monocytes was tested using the Calcein-AM-PI live cell-dead cell double staining kit. The dyes in the kit were diluted to obtain a working solution concentration of 2 μM for Calcein-AM and a working solution concentration of 1 μM for PI. After washing the cells three times with PBS, the dye solution at the working concentration was added and incubated at 37°C for 15 min. Under an inverted fluorescence microscope, green fluorescence excited at 488 nm represented live cells and red fluorescence excited at 545 nm represented dead cells. After incubation with monocytes for 48 h, the live and dead staining results showed that the cells in each group had good activity and almost no dead cells. The results showed that the nanoparticles had good biocompatibility. Monocytes almost died after engulfing the preparation and maintained good activity. In this way, a live cell carrier was successfully constructed. Example

[0026] APOE− / − mice (6-8 weeks old, male, 20 g) were fed a high-fat diet for 12 weeks to form a disease model. The mice were perfused with saline to wash out the blood, then perfused with paraformaldehyde, and the aorta was dissected and fixed with paraformaldehyde. The aorta was longitudinally sectioned and stained with oil red. The results showed that the oil red stained plaque area in the M-PPA group was significantly lower than that in the control group and the free ATV group. Example

[0027] APOE− / − mice (6-8 weeks old, male, 20 g) were fed a high-fat diet for 12 weeks to form a disease model. The mice were intraperitoneally injected with fluorescent drugs and fluorescent nanoparticles at a dose of 0.2 mL (20 mg / mL nanoparticles). Then, in vivo imaging was used to observe and quantify the fluorescence distribution of Cou-6 in the representative organ, the aorta. The results showed that the aggregation of the M-PPA group at the lesion site was significantly higher than that of the free drug group.

[0028] In summary, the preparation method of the cell-carrying nanocomposite delivery system of the present invention is simple and low-cost. Compared with the prior art, the present invention can improve the bioavailability of drugs, increase the retention time in lesions, reduce drug side effects, protect drugs and nanodelivery systems from being removed, and actively home to target organs with advantages such as cell secretion factors and cell chemotaxis. A cell-carrying nanocomposite delivery system has better biocompatibility than free drugs at the animal experimental level and has better disease treatment effects.

Claims

1. A preparation method and application of a cell-carrying nanocomposite delivery system, characterized in that The steps include: (1) The cells were cultured in a 37°C 5% CO2 incubator until the logarithmic phase. When the confluence reached 90%, single-cell suspension was prepared by cell scraping or trypsin digestion. (2) Dissolve 10-100 mg of the drug and 100-500 mg of the polymer material in an appropriate solvent, and ultrasonicate the mixture for 5-10 min at a shear rate of 10%-30% using an ultrasonic probe under ice bath conditions to obtain colostrum. Add 10-30 mL of an emulsifier aqueous solution to a beaker, and then add the colostrum obtained in step (2), and ultrasonicate the mixture for 10-20 min at a shear rate of 10%-30% using an ultrasonic probe under ice bath conditions. Volatilize the solvent, wash with distilled water 3-6 times under high-speed centrifugation conditions, and freeze-dry to obtain a nano delivery system; (3) incubating the cells and the polymer nano-delivery system in a 37°C 5% CO2 incubator for a certain period of time to obtain a nano-composite delivery system carrying cells; (4) Apply this prepared cell-loaded nanocomposite delivery system to the treatment of cardiovascular diseases and other diseases in animals.

2. The preparation method and application according to claim 1, characterized in that The cells are primary or cell line type monocytes, macrophages, neutrophils, endothelial cells, cardiomyocytes or smooth muscle cells.

3. The preparation method and application according to claim 1, characterized in that The polymers include polyesters, polyalkyl cyanoacrylates (PBCA, PiBCA), amphiphilic block copolymers (Mal-PEG, mPEG), dendrimers (PAMAM), polylactic acid (PLA), polycaprolactone (PCL), polyethylene glycol (PEG), polylactic acid glycolic acid (PLGA), chitosan, etc.

4. The preparation method and application according to claim 1, characterized in that The drugs are various water-soluble or fat-soluble drugs for cardiovascular diseases and other diseases.

5. The preparation method and use according to claim 1, characterized in that The animals include small animals such as mice, rats, rabbits, dogs, monkeys, and large animals.

6. The preparation method and use according to claim 1, characterized in that Cardiovascular diseases and other diseases include atherosclerosis, myocardial infarction, myocardial ischemia-reperfusion injury, arteritis, hemangioma and other diseases.

7. A method for preparing a cell-carrying nanocomposite delivery system prepared by the preparation method of any one of claims 1 to 6.

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