A fibronectin-coated core-shell type polymer drug-loaded microsphere system and a preparation method and application thereof

Abstract

The application relates to a fibronectin-coated core-shell type polymer drug-loaded microsphere system and a preparation method and application thereof, and comprises the following steps: a hydrophobic cavity of an amphiphilic polymer is used to load a drug to form a drug-loaded micelle; the drug-loaded micelle is encapsulated in a polymer microsphere inside to form a core-shell type drug-loaded microsphere through a coaxial electrospinning technology; and fibronectin is further physically adsorbed and wrapped on the surface of the drug-loaded microsphere. The method has simple operation conditions, is easy to operate and separate, can effectively improve the bioavailability of small molecule drugs and proteins, can coordinate the bioactivity of the two in an in-vitro and in-vivo lung injury model, and provides a new idea for constructing a safe, intelligent and efficient drug carrier.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a fibronectin-coated core-shell type polymer drug-loaded microsphere system and a preparation method and application thereof. BACKGROUND

[0002] Acute lung injury is a clinical syndrome with rapid onset and rapid progression, which leads to extensive inflammatory response in the lungs and seriously affects gas exchange, and eventually can lead to respiratory failure, threatening the life safety of patients. Due to the complex pathological process of acute lung injury involving the interaction of multiple inflammatory mediators and cytokines, the traditional treatment method often has difficulty in achieving ideal curative effect. Under this background, it is particularly urgent to develop new treatment methods and means. Drug carrier technology provides a new possibility for the treatment of acute lung injury. Drug carriers can change the way drugs enter the human body and the characteristics of their distribution in the body, thus achieving precise delivery and efficient use of drugs. Specifically, drug carriers can deliver drugs directly to damaged lung tissues through specific targeting mechanisms, thereby increasing the concentration and action time of drugs in the lungs. This not only can enhance the efficacy of drugs, but also can reduce the side effects of drugs on other normal tissues. In addition, drug carriers can control the release rate of drugs, allowing them to continuously exert therapeutic effects in the lungs, thereby prolonging the effective period of drugs. In addition to improving the efficacy and safety of drugs, drug carriers can also be made of biocompatible, biodegradable and renewable materials, which can reduce the immunogenicity and toxicity of drug carriers and reduce the potential risks to patients. This is particularly important for patients with acute lung injury, as their lungs are already damaged and less tolerant to drugs. In addition, the development of drug carrier technology can also promote the technological progress and industrial development in related fields. With the continuous progress of materials science, nanotechnology, biotechnology and other fields, the design and preparation technology of drug carriers are also constantly improving and innovating. This will provide more options and possibilities for the treatment of acute lung injury, helping to improve the level of medical treatment and improve the quality of life of patients.

[0003] Microsphere drug carriers can achieve various release modes such as zero-order, first-order or second-order release through different preparation methods to meet the release needs of different drugs. By adjusting the material, size and structure of the microspheres, the microspheres can achieve rapid, delayed or sustained release, enhancing the bioavailability and efficacy of drugs. In addition, microspheres can protect drugs from environmental influences, reduce degradation, prolong the effective period, and improve drug stability and targeting in the body, reducing adverse reactions. Common preparation methods for microspheres include solvent evaporation, emulsion gelation and spray drying, which are convenient for controlling their size and release characteristics, and are suitable for large-scale production and long-term storage.

[0004] PLGA as a drug carrier, in drug controlled release and targeted delivery show significant advantages, mainly due to its good biocompatibility, biodegradable, low immunogenicity and low toxicity. These characteristics make it suitable for long-term drug release or targeted therapy. However, the degradation and release rate of PLGA is affected by molecular weight and copolymer ratio, the regulation process is complex, and needs to be accurately controlled. In addition, the drug delivery effect depends on the design optimization of specific diseases and drugs. Our group has prepared fiber microspheres by electrospinning, which has shown good results in drug loading and bioavailability (Xiao et al. NanoToday. 2021, 38, 101123), (Zhang et al. Adv. Fiber Mater. 2022, 4, 807-819). Resveratrol (Res) has a wide application prospect in the medical field due to its significant anti-inflammatory and antioxidant properties. Res can effectively reduce tissue damage and inflammatory response by scavenging free radicals and blocking the production of inflammatory mediators, showing potential in anti-aging, improving immunity, and treating arthritis, dermatitis and other diseases. However, the water insolubility of Res limits its administration route and bioavailability, which is the main challenge in its application.

[0005] After searching relevant literature and patents at home and abroad, no related reports have been found on the use of electrospun microspheres as Res and FN co-delivery carriers for anti-inflammatory and antioxidant therapy of acute lung injury. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a fibronectin-coated core-shell type polymer drug-loaded microsphere system and its preparation method and application, to fill the gap in the prior art.

[0007] The present application provides a protein-coated core-shell type polymer drug-loaded microsphere composite material, which is a protein-coated core-shell structure drug-loaded nanomicrosphere; wherein the core of the core-shell structure drug-loaded nanomicrosphere is a drug-loaded nanomicelle, and the shell layer is a polymer material.

[0008] The shell layer is a polylactic acid-glycolic acid copolymer PLGA.

[0009] Further, the composite material: uses the hydrophobic cavity of the amphiphilic polymer to load the drug to form a drug-loaded micelle, the drug-loaded micelle is encapsulated in the polymer microsphere inside to form a core-shell type drug-loaded microsphere by coaxial electrospinning technology, and further physically adsorbs fibronectin on the surface of the drug-loaded microsphere.

[0010] The present application provides a preparation method of a protein-coated core-shell type polymer drug-loaded microsphere composite material, comprising:

[0011] (1) adding the amphiphilic polymer solution and the drug solution into water under ultrasonic condition, stirring, dialysis, filtration, centrifugation, to obtain the drug-loaded nanomicelles;

[0012] (2) taking the drug-loaded nanomicelles solution as the core layer solution, and taking the polylactic acid-glycolic acid copolymer (PLGA) solution as the shell layer solution, to perform coaxial electrospinning, to obtain the core-shell drug-loaded microspheres;

[0013] (3) performing co-extrusion on the core-shell drug-loaded microspheres of step (2) and the protein solution, and performing centrifugal purification, to obtain the protein-coated core-shell polymer drug-loaded microsphere composite material.

[0014] Preferably, in step (1), the amphiphilic polymer is polyethylene glycol-poly (ε-caprolactone) (PEG-PCL); the drug is resveratrol (Res); the solvent of the amphiphilic polymer solution is dimethyl sulfoxide (DMSO); and the solvent of the drug solution is dimethyl sulfoxide (DMSO).

[0015] Preferably, in step (1), the mass ratio of the drug to the amphiphilic polymer is 1:1-1:5; and the volume ratio of the solvent of the solution to water is 1:8-1:12. (The solvent is the total solvent of the amphiphilic polymer solution and the drug solution)

[0016] Preferably, in step (1), the stirring is room temperature stirring for 20-30 h; the molecular weight cut-off of the dialysis bag in the dialysis is 8000-14000 Da; the filtration condition is that a microporous filter membrane with a pore size of 3-5 μm is used; and the centrifugal process parameter is centrifugation at 4000-5000 rpm for 10-30 min.

[0017] Preferably, in step (2), the solvent of the drug-loaded nanomicelles solution is water, and further, the water is ultrapure water; the mass percentage concentration of the drug-loaded nanomicelles solution is 4-6%; the solvent of the PLGA solution is hexafluoroisopropanol (HFIP); the mass percentage concentration of the PLGA solution is 4-6%, and the PLGA dissolving condition is room temperature stirring for 1-4 h.

[0018] Preferably, in step (2), in the coaxial electrospinning process, the polyvinyl alcohol (PVA) solution is used as the receiving solution, wherein the mass fraction of the PVA solution is 1-4%; the solvent of the PVA solution is water, and further, the water is ultrapure water; and the PVA dissolving condition is heating and stirring at 90 ℃ for 2 h.

[0019] Further, step (2) is specifically as follows: taking the drug-loaded nanomicelles solution as the core layer solution, and taking the polylactic acid-glycolic acid copolymer (PLGA) solution as the shell layer solution, and respectively loading into the core layer channel and the shell layer channel of the coaxial electrospinning device, and assembling into a coaxial electrospinning device together with the polyvinyl alcohol (PVA) solution as the receiving solution.

[0020] The core-shell type drug-loaded microspheres RPG loaded with the inner micelles are obtained by adjusting the voltage of the coaxial electrospinning device, the receiving distance and the pushing rate of the shell-core layer solution.

[0021] Preferably, the coaxial electrostatic in the step (2) is coaxial electrostatic spraying, and the process parameters include: the adjusting voltage is 6-12kV; the receiving distance is 10-15cm; the pushing rate of the shell layer solution is 1.5-2.5mL / h; and the pushing rate of the core layer solution is 0.5-1.5mL / h.

[0022] Preferably, the coaxial electrostatic spinning is followed by differential centrifugation, and the process parameters of the differential centrifugation include: the centrifugation temperature is 4℃, first centrifugation at 200g-400g for 10-30min to remove the precipitate, and then centrifugation at 500g-600g for 10-30min to collect the precipitate.

[0023] Preferably, the protein in the step (3) is at least one of fibronectin FN and bovine serum albumin; the solvent of the protein solution is water, and further, the water is ultrapure water; the concentration of the protein solution is 2-4mg / mL; and the mass ratio of the core-shell type drug-loaded microspheres to the protein is 1:2-1:6.

[0024] In the step (3), the core-shell type drug-loaded microsphere solution is co-extruded with fibronectin (FN) and bovine serum albumin (BSA) solution in a micro-extrusion device to coat FN or BSA on the surface of the core-shell type drug-loaded microspheres, and the core-shell type drug-loaded microspheres@FN or the core-shell type drug-loaded microspheres@BSA are obtained after centrifugal purification.

[0025] Preferably, the extrusion in the step (3) is repeated 10-15 times by using an Avanti micro-extruder with a filter membrane pore size of 5-7μm; and the centrifugation process parameters include centrifugation at 8000-10000rpm for 6-8min.

[0026] The application provides application of the protein-coated core-shell type high-molecular drug-loaded microsphere composite material in preparation of anti-inflammatory and antioxidant drugs.

[0027] The application provides application of the protein-coated core-shell type high-molecular drug-loaded microsphere composite material in preparation of lung injury drugs.

[0028] The application also provides application of the fibronectin-coated core-shell type high-molecular drug-loaded microsphere composite material (system) in enhanced anti-inflammatory and antioxidant treatment of an acute lung injury mouse model.

[0029] The application encapsulates Res with anti-inflammatory and antioxidant activity in the hydrophobic cavity of micelles by using amphiphilic polymer PEG-PCL, and further wraps the drug-loaded micelles in the PLGA microspheres by the way of electrospinning, so as to improve the drug release rate and bioavailability of Res. Finally, a layer of fibronectin is uniformly coated on the surface of the microspheres by the way of co-extrusion, so as to improve the intracellular delivery efficiency of the protein and realize the inflammation cell targeting function of the drug-loaded microspheres, and realize the efficient anti-inflammatory and antioxidant treatment of acute lung injury.

[0030] The application uses Zeta potential and dynamic light scattering analysis (DLS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), laser confocal microscopy (CLSM) and other means to characterize the physical and chemical properties of the prepared fibronectin-coated core-shell type polymer drug-loaded microsphere system. Then, the CCK-8 method is used to analyze and evaluate the cytotoxicity of RPG@FN and related control materials; flow cytometry and CLSM are used to evaluate the influence of the materials on the intracellular ROS level; flow cytometry is used to evaluate the influence of the materials on the intracellular delivery of proteins; flow cytometry is used to evaluate the influence of the materials on the transformation of macrophages; enzyme-linked immunosorbent assay (ELISA) is used to evaluate the influence of the microsphere platform on the expression of anti-inflammatory and pro-inflammatory cytokines; and finally, an acute lung injury mouse model is established to verify the in vivo anti-inflammatory and antioxidant treatment effect of the drug-loaded microsphere system.

[0031] The application provides a fibronectin-coated core-shell type polymer drug-loaded microsphere system, a preparation method and application thereof, the drug-loaded microsphere system has good biological safety, can effectively improve the bioavailability of small molecule drugs and proteins, can synergize the biological activities of the two in in vitro and in vivo models, and provides a new idea for constructing safe, intelligent and efficient drug carriers.

[0032] Advantages

[0033] (1) The application has simple reaction conditions, is easy to operate and separate, and has good development prospect.

[0034] (2) The prepared drug-loaded microsphere platform effectively improves the water solubility of Res and releases Res in the inflammatory microenvironment, and provides a new idea for constructing safe and efficient drug carriers.

[0035] (3) The drug-loaded microsphere platform prepared by the application can target alveolar macrophages by surface-coated FN, on the one hand, clear excessive active oxygen at the lesion site, and restore the oxidative stress homeostasis; on the other hand, the drug-loaded microsphere platform can synergize the anti-inflammatory activity of FN and Res to induce macrophages to polarize from M1 type to M2 type, and inhibit excessive immune activation at the lesion site. Meanwhile, the drug-loaded microsphere platform can also down-regulate pro-inflammatory inflammatory factors by inhibiting the NF-κB signaling pathway, enhance the expression of anti-inflammatory inflammatory factors, and enhance the treatment effect.

[0036] (4) The drug-loaded microsphere platform prepared by the application can enter the mouse body through a tracheal spray drug delivery device, and due to the large hydrodynamic diameter of the microspheres, the microspheres can be retained in the alveolar part of the mouse for a long time, realizing long-acting drug sustained release, and having potential clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 It is a schematic diagram for the preparation and application of RPG@FN in the application;

[0038] Figure 2 It is the hydrodynamic size (a) and surface potential (b) data of PEG-PCL / Res, RPG@BSA and RPG@FN prepared in Example 1;

[0039] Figure 3 It is a curve graph of the hydrodynamic diameter of RPG@FN prepared in Example 1 in water, PBS and 1640 culture medium changing with time;

[0040] Figure 4 It is a single-channel (a) and double-channel (b) confocal laser microscope picture of the core-shell structure microspheres FPG with FITC fluorescent label prepared in Example 1, and the scale is 1 μm;

[0041] Figure 5 It is an SEM image (a) and a particle size distribution histogram (b) of RPG@FN prepared in Example 1, and the scale in the graph is 10 μm;

[0042] Figure 6 It is an SDS-PAGE electrophoresis graph of RPG, RPG@FN and RPG@BSA prepared in Example 1;

[0043] Figure 7 It is a Res release kinetics curve of RPG@FN, RPG@BSA and RPG prepared in Example 1 within 72 h;

[0044] Figure 8 It is a degradation diagram of RPG, RPG@BSA and RPG@FN prepared in Example 1 with time;

[0045] Figure 9 Cell viability plots of PEG-PCL / Res, Res, RPG prepared in Example 1 after co-incubation with MH-S cells for 24 h;

[0046] Figure 10 Flow cytometry plots (a) and mean fluorescence intensity quantification plots (b) of RPG@FN and FN prepared in Example 1 of the present application after co-incubation with MH-S cells for 12 h, respectively; and flow cytometry plots (c) and mean fluorescence intensity quantification plots (d) of RPG@FN+RGD and RPG@FN after co-incubation with MH-S cells for 12 h, respectively;

[0047] Figure 11 Flow cytometry analysis plots (a), relative fluorescence intensity quantification plots (b) and laser confocal microscope images (c) of reactive oxygen species expression levels in cells after co-incubation of RPG@FN, RPG@BSA, Res and FN prepared in the present application with LPS-activated MH-S cells for 24 h, the scale bar in the images is 10 μm;

[0048] Figure 12 Flow cytometry analysis plots (a) of CD86, CD206 expression levels in cells, percentage content plots of M1 macrophages (b) and M2 macrophages (c) and (d) relative M2 / M1 macrophage ratio schematic diagram after co-incubation of RPG, RPG@BSA, RPG@FN prepared in the present application with LPS-activated MH-S cells for 24 h;

[0049] Figure 13 ELISA test results plots of interleukin (IL)-1β (a), IL-6 (b), IL-10 (c) and tumor necrosis factor (TNF)-α (d) in the bronchoalveolar lavage fluid of mice in each experimental group in Example 11;

[0050] Figure 14 Histological section analysis of lung tissues of mice in each experimental group in Example 12; gray arrows indicate alveolar wall congestion, black arrows indicate inflammatory cells, the scale bar in the images is 50 μm. DETAILED DESCRIPTION

[0051] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. Furthermore, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the content taught by the present application, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

[0052] PLGA was purchased from Jinan Daigang Biological Engineering Co., Ltd. PEG-PCL was purchased from Xi'an Rixi Biological Technology Co., Ltd. Res was purchased from Shanghai Hongye Biological Technology Co., Ltd. Fibronectin was from Shanghai Fibro Connect Biological Technology Co., Ltd. MH-S cells (mouse alveolar macrophage cell line) were from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. RPMI-1640 complete medium was purchased from Shanghai Sailaiolun Biological Medicine Co., Ltd. Reactive oxygen species (ROS) detection probe (DCFH-DA) was purchased from Shanghai Biyun Tian Biological Technology Co., Ltd. Cell counting kit (CCK-8) was purchased from Shanghai Qihai Biological Technology Co., Ltd. ELISA kit was purchased from Shanghai Zucai Biological Technology Co., Ltd. BALB / c mice were purchased from Shanghai Slike Experimental Animal Center. Anti-CD80-PE, Anti-CD206-FITC were purchased from Thermo Fisher Scientific (Waltham, MA). The water used in all experiments with a resistivity higher than 18.2 MΩ·cm was purified by a laboratory water purification system (Cascada I, PALL, Beijing, China).

[0053] Example 1

[0054] (1) 1 mg Res and 2.67 mg PEG-PCL were dissolved in 500 μL DMSO, respectively, and then the solution was added dropwise into 5 mL ultrapure water under ultrasonic conditions, and stirred for 24 h. After the reaction was completed, the DMSO was removed by dialysis in ultrapure water using a dialysis bag with a molecular weight cutoff of 3000 Da for 3 days, and after dialysis, the product was filtered through a microporous filter membrane with a pore size of 1 μm to remove the unloaded Res, and finally the product was concentrated by centrifugation at 4500 rpm for 30 min, to obtain PEG-PCL / Res.

[0055] (2) 40 μg PLGA was dissolved in 960 μL hexafluoroisopropanol, after stirring and mixing for 2 h, it was loaded into a 1 mL syringe with coaxial needle outer layer push. At the same time, 40 μg PEG-PCL / Res prepared in step (1) was dissolved in 960 μL ultrapure water, and it was loaded into a 1 mL syringe with coaxial needle inner layer push. The prepared 1% PVA solution was placed below as a receiving device. The high voltage power supply of the electrospinning device was adjusted to 8 kV, the receiving distance was 10 cm, the shell layer syringe pump push speed was 2 mL / h, the core layer syringe pump push speed was 1 mL / h, and a magnetic stirring device was placed below the receiving device to stir at 750 rpm to prevent adhesion between the microspheres. The prepared microspheres RPG were collected and solidified at 4°C for 12 h, then differential centrifugation was performed, first 200 g centrifugation for 10 min, the precipitate was discarded, the supernatant was collected, and then 500 g centrifugation for 30 min, the supernatant was discarded, and the precipitate was collected and solidified at 4°C for 12 h. After freeze-drying machine treatment, RPG with uniform particle size was obtained. Similarly, Res was replaced by FITC, and core-shell structure microspheres FPG with fluorescent labeling were prepared.

[0056] (3) 15 mL fibronectin stock solution was placed in a 10000 Da dialysis bag and dialyzed for 24 h, with water change twice in between. The dialyzed stock solution was frozen at -80°C for 12 h or more, and then placed in a freeze-drying machine to obtain purified fibronectin FN after freeze-drying.

[0057] (4) 4 mg of purified FN in step (3) was dissolved in 1 mL ultrapure water to obtain a FN solution, which was mixed with 1 mg RPG. The Avanti micro-extruder was used to extrude the solution 12 times, and centrifuged at 10000 rpm for 6 min to remove excess fibronectin FN. The precipitate was collected to obtain RPG@FN.

[0058] (5) In the same way as in steps (3) and (4), bovine serum albumin (BSA) was used instead of fibronectin to prepare the control material RPG@BSA.

[0059] Example 2

[0060] An appropriate amount of RPG@FN was dispersed uniformly with ultrapure water, and ultrasonic treatment was performed for 30 min to completely disperse it into a suspension, and a RPG@FN solution with a Res concentration of 4 mM was prepared. An appropriate amount of Res and PEG-PCL / Res, RPG, RPG@BSA and RPG@FN prepared in Example 1 were diluted with water to prepare a solution with a Res concentration of 4 mM for determination of hydration particle size and surface potential. The results are shown in Table 1. Figure 2The hydrodynamic size of Res alone was 120.3 nm, the hydrodynamic size of PEG-PCL / Res micelles was 310.2 nm, and the hydrodynamic size of RPG was 841.3 ± 174 nm. When the surface of the drug-loaded microspheres RPG was coated with BSA or FN, the hydrodynamic size of RPG@BSA and RPG@FN increased to 1668.6 ± 196 nm and 1684.4 ± 124 nm, respectively, and the surface potential further decreased, which meant that BSA or FN was successfully coated on the surface of the microspheres. In addition, the hydrodynamic size of the drug-loaded microsphere system RPG@FN remained almost unchanged in various solutions (water, PBS, RPMI-1640 medium) within a week Figure 3 ), which proved that RPG@FN had good colloidal stability.

[0061] Example 3

[0062] To verify the core-shell structure of the prepared microsphere system, FITC was used to replace Res of the same concentration in PEG-PCL according to the procedure of Example 1 to obtain fluorescently labeled microspheres FPG. The images observed by laser confocal microscopy are shown in Figure 4 As shown, the prepared microspheres RPG can completely encapsulate the micelles containing FITC inside the microspheres, indicating that RPG has obvious core-shell structure characteristics. In addition, the size and morphology of the prepared RPG@FN were characterized, and the SEM images are shown in Figure 5 As shown, the RPG@FN microspheres have a uniform distribution of spherical structure, and the average size is about 1298 ± 433 nm.

[0063] Example 4

[0064] To further verify the successful loading of BSA or FN in the RPG microspheres, the RPG, RPG@FN, and RPG@BSA prepared in Example 1 were subjected to SDS-PAGE gel electrophoresis experiment. 20 μL of RPG, RPG@FN, RPG@BSA, FN, BSA (protein concentration of 1 mg / mL) and SDS loading buffer were mixed in a boiling water bath for 5 min, and then RPG, RPG@FN, RPG@BSA, and purified BSA and FN and standard protein Marker (5 μL) were sequentially added to the corresponding wells of the gel and run at a voltage of 120 V for 30 min. After the gel was run, the gel was taken out and placed in a container containing Coomassie brilliant blue for staining for 30 min, and then the staining solution was replaced with the same volume of destaining solution, and the destaining was performed on a destaining shaker for 2 h. Finally, a scanner was used to record the protein bands on the precast gel. The results are shown in Figure 6As shown, RPG did not have obvious protein bands, while RPG@FN had bands identical to FN, indicating the successful combination of FN and RPG microspheres; RPG@BSA also had a characteristic band identical to BSA, verifying the successful combination of BSA and RPG microspheres. Combining the potential, hydrodynamic size and morphology analysis of RPG, RPG@FN and RPG@BSA, it can be proved that the core-shell structure drug-loaded microsphere platform RPG@FN and RPG@BSA loaded with Res and FN are successfully prepared.

[0065] Example 5

[0066] In order to further analyze the drug release performance of Res in the drug-loaded microsphere system, RPG, RPG@BSA and RPG@FN prepared in Example 1 were dispersed in 1 mL of PBS buffer (concentration of 1 mg / mL) and placed in a dialysis bag (molecular weight cut-off of 3500 Da), and then the dialysis bag was transferred to a centrifuge tube containing 9 mL of PBS buffer, and shaken in a constant temperature shaker at 37°C. At different time points, 1 mL of liquid outside the dialysis bag was taken, and 1 mL of fresh buffer solution was added to the container, and the absorbance value of the removed liquid at 338 nm was measured to quantify the content of released Res. After the end of the drug release, the drug release curves of RPG, RPG@BSA and RPG@FN under different conditions were drawn. As shown in Figure 7 As shown, the cumulative release rate of RPG reached 74.3% after 72h, which had the effect of long-acting drug release, and the cumulative release rate of RPG@FN was 75.2% after 72h, which proved that the coating of FN would not affect the overall release of Res of the drug-loaded microspheres. This indicates that the prepared drug-loaded microsphere system is expected to release drug Res in the lesion microenvironment for a long time, and achieve enhanced anti-inflammatory and antioxidant effects.

[0067] Example 6

[0068] The microspheres prepared in Example 1 were taken to characterize their biodegradability. First, a certain amount of RPG, RPG@BSA and RPG@FN was weighed, and the initial weight (W0) was recorded. Thereafter, the microspheres were transferred to a centrifuge tube, 10 mL of PBS (pH = 7.4 ± 0.1) was added, and placed in a constant temperature shaker at 37°C with a shaking speed of 100 rpm. Within 8 weeks of experimental time, the microspheres were taken out every week, dried and weighed again (W1) to calculate the mass loss of the microspheres over time. The weight loss rate (WL) was calculated according to formula (1):

[0069] WL(%) = (W0-W1) / W0x100% (1)

[0070] The results are shown in Figure 8As shown, the degradation efficiencies of RPG, RPG@BSA and RPG@FN were all above 50% at the 8th week, which proved that the prepared microspheres had good biodegradable properties.

[0071] Example 7

[0072] To verify the effect of RPG@FN on cell viability, cells in the logarithmic growth phase were collected and seeded in a 96-well plate at a density of 1 x 10 4 cells per well, and incubated in a 5% CO2, 37°C incubator for 24 h. Then, the cells were incubated in fresh RPMI-1640 medium containing different concentrations of Res, PEG-PCL / Res or RPG (the concentration of Res was 0, 5, 10, 15, 20, 25, 35 and 50 μg / mL, respectively) for 24 h, and the normal group was treated with PBS. Subsequently, the medium was discarded, and the cells were washed with PBS for 3 times. Then, 100 μL of fresh serum-free DMEM medium containing 10% (v / v) CCK-8 solution was added to each well, and the plate was incubated in the incubator for 3 h. The absorbance of each well was measured at a wavelength of 450 nm using a multifunctional enzyme labeler, and the cells treated with PBS were used as a blank control. The cell viability was recorded as 100%. Figure 9 The experimental results showed that the cell viability of each group of materials was above 90% in the tested concentration range, which indicated that the prepared drug-loaded microsphere system had excellent cell compatibility.

[0073] Example 8

[0074] FN is a macromolecular extracellular membrane protein widely present on the surface of various animal cells, and is the main non-collagenous glycoprotein in the extracellular matrix and basement membrane. It plays a central role in cell adhesion, and can regulate cell polarity, differentiation and growth, and is involved in wound repair, tissue regeneration and angiogenesis. In addition, FN can be used as a therapeutic protein to treat inflammatory diseases by inducing macrophage polarization and reducing the expression of inflammatory factors, but the low intracellular delivery efficiency hinders the exertion of the biological activity of FN, so microspheres can be used to improve the delivery efficiency of FN and exert its biological functions.

[0075] The MH-S cells were used as a model to verify the effect of RPG on promoting the intracellular delivery of FN and the role of FN in targeting inflammatory macrophages. Cells in the logarithmic growth phase were collected and seeded in a 96-well plate at a density of 1 x 10 5The density of 1 x 105cells per well was inoculated in a 12-well plate, and incubated at 37°C in 5% CO2for 24 h, and then replaced with fresh serum-free RPMI-1640 medium containing FN, RPG or RPG@FN, RPG@FN+RGD (FN was labeled with Cy5.5, and the concentration of FN was 40 μg / mL), and the normal group was treated with PBS. The medium was discarded, and the cells were washed with PBS for 3 times, 1 mL of trypsin was added to each well to digest the cells, and the digestion was terminated, and the cells were collected by centrifugation. The collected cells were resuspended in 300 μL of PBS, and transferred to a flow tube, and the fluorescence intensity of each group was detected by flow cytometry. The results are shown in Figure 10 As shown in a-b, the fluorescence intensity of the RPG@FN group was significantly higher than that of the free FN group, indicating that RPG can promote the delivery of FN in alveolar macrophages, and provide a major guarantee for the biological function of FN. The experimental results of 10c-d show that the fluorescence intensity of the RPG@FN group is higher than that of the RPG@FN+RGD group, which verifies that FN has the effect of targeting inflammatory macrophages, and the targeting is achieved through the interaction of RGD-α4β1 integrin.

[0076] Example 9

[0077] In order to verify the ROS scavenging effect of the RPG@FN microsphere complex in alveolar macrophages, the MH-S cells in the logarithmic growth phase were collected, and the density of 1 x 105cells per well was inoculated in a 12-well plate, and incubated at 37°C in 5% CO2for 24 h, and then replaced with fresh serum-free RPMI-1640 medium containing FN, RPG or RPG@FN, RPG@FN+RGD (FN was labeled with Cy5.5, and the concentration of FN was 40 μg / mL), and the normal group was treated with PBS. The medium was discarded, and the cells were washed with PBS for 3 times, 1 mL of trypsin was added to each well to digest the cells, and the digestion was terminated, and the cells were collected by centrifugation. The collected cells were resuspended in 300 μL of PBS, and transferred to a flow tube, and the fluorescence intensity of each group was detected by flow cytometry. The results are shown in 5 The density of 1 x 105cells per well was inoculated in a 12-well plate, and incubated at 37°C in 5% CO2for 24 h, and then replaced with fresh serum-free RPMI-1640 medium containing FN, RPG or RPG@FN, RPG@FN+RGD (FN was labeled with Cy5.5, and the concentration of FN was 40 μg / mL), and the normal group was treated with PBS. The medium was discarded, and the cells were washed with PBS for 3 times, 1 mL of trypsin was added to each well to digest the cells, and the digestion was terminated, and the cells were collected by centrifugation. The collected cells were resuspended in 300 μL of PBS, and transferred to a flow tube, and the fluorescence intensity of each group was detected by flow cytometry. The results are shown in Figure 11As shown in ab, the relative fluorescence intensity of the Res and FN groups was lower than that of the LPS group, indicating that both could reduce intracellular ROS to some extent. Compared with the FN or Res groups, RPG@FN exhibited a lower level of fluorescence intensity, indicating that RPG@FN can synergistically enhance the antioxidant activity of Res and FN in macrophages, most effectively reducing intracellular ROS levels. In contrast, the RPG@BSA group could only exert the effect of Res, and its phagocytic capacity was significantly higher than that of free Res, thus exhibiting a higher ROS scavenging capacity than free Res.

[0078] The ability of RPG@FN to scavenge intracellular ROS was further verified using laser confocal microscopy. MH-S cells in logarithmic growth phase were collected and cultured at 1×10⁻⁶ cells / cells. 5 MH-S cells were seeded in laser confocal microscopy plates at a density of 10 cells per well and incubated at 37°C with 5% CO2 for 24 h. The medium was then replaced with RPMI-1640 medium containing 2 μg / mL LPS and incubated for another 24 h. PBS was added to the control group. Next, each well was replaced with fresh serum-free RPMI-1640 medium containing Res, FN, RPG@BSA, and RPG@FN (BSA or FN concentration 20 μg / mL) and incubated for 4 h. The old medium was discarded, and the cells were incubated with fresh serum-containing medium for 20 h. The medium was discarded, and the cells were washed three times with PBS. 1 mL of DCFH-DA was added and the cells were co-incubated at 37°C for 20 min. The cells were washed three times with PBS, fixed with 1 mL of 2.5% glutaraldehyde at room temperature for 15 min, washed three times with PBS, and then co-incubated with 1 mL of LDAPI for 5 min. After washing three times with PBS, 200 μL of PBS was added, and the changes in cell fluorescence intensity were observed under a laser confocal microscope. The results are as follows Figure 11 As shown in c, compared with the LPS, Res, FN and RPG@BSA groups, the green fluorescence intensity of the RPG@FN group was significantly reduced, which also proves that the drug-loaded microsphere system has the best antioxidant performance. This is due to the fact that RPG@FN can effectively deliver Res and FN into macrophages and improve their bioavailability.

[0079] Example 10

[0080] To verify the effect of RPG@FN nanocomplex on macrophage polarization, MH-S cells in logarithmic growth phase were collected and subjected to polarization at a ratio of 1×10⁻⁶ cells / cells. 5The density of one cell per well was inoculated in a 12-well plate, and incubated at 37°C in 5% CO2 for 24 h. The culture medium was discarded and replaced with RPMI-1640 medium containing LPS (2 pg / mL) and incubated with cells for 24 h. The control group was added with PBS. Subsequently, fresh serum-free RPMI-1640 medium containing Res, FN, RPG@BSA, RPG@FN (the concentration of BSA or FN was 20 pg / mL) was added and incubated for 4 h. The old culture medium was discarded, and the cells were incubated with RPMI-1640 medium containing serum for 20 h. Subsequently, the culture medium was discarded, and the cells were washed twice with PBS, trypsinized, and collected. The cell pellet was resuspended with 200 pL PBS, and Anti-CD206-FITC and Anti-CD86-PE antibodies were added and incubated with the cells at 4°C in the dark for 30 min. After washing with PBS for three times to remove the unbound antibodies, the cell pellet was resuspended with 300 pL PBS and transferred to a flow tube. The effect of the nanomaterials on macrophage polarization was evaluated by detecting the expression level of CD86 and CD206 in the cells. As shown in FIGS. 10A-10D, Res and RPG@BSA can partially reduce the level of LPS-induced M1 macrophages. Although FN alone is difficult to enter the cell interior, FN still shows the ability to induce macrophage polarization to M2, which may be because FN binds to the macrophage surface receptor to regulate the behavior of macrophages. Importantly, the RPG@FN group showed the highest level of CD206 (40.0%) and the lowest level of CD86 (4.7%) expression, and the M2 / M1 ratio was 2.6 times that of the RPG@BSA group, indicating that RPG@FN further enhances the ability to induce macrophage polarization to M2 by synergistically utilizing the biological activity of FN and Res. Figure 12 a-d, Res and RPG@BSA can partially reduce the level of LPS-induced M1 macrophages. Although FN alone is difficult to enter the cell interior, FN still shows the ability to induce macrophage polarization to M2, which may be because FN binds to the macrophage surface receptor to regulate the behavior of macrophages. Importantly, the RPG@FN group showed the highest level of CD206 (40.0%) and the lowest level of CD86 (4.7%) expression, and the M2 / M1 ratio was 2.6 times that of the RPG@BSA group, indicating that RPG@FN further enhances the ability to induce macrophage polarization to M2 by synergistically utilizing the biological activity of FN and Res.

[0081] Example 11

[0082] All animal experiments were performed in strict accordance with the standard of the Experimental Animal Ethics Committee of Donghua University. Six-week-old male BALB / c mice were purchased from Shanghai Slac Laboratory Animal Center. The acute lung injury animal model was established by intratracheal nebulization of LPS (5 mg / kg) for 24 h, and the control group was intratracheally nebulized with an equal amount of PBS. The mice were randomly divided into 6 groups (PBS control group, LPS treatment group, Res treatment group, FN treatment group, RPG@FN treatment group, and RPG@BSA treatment group), with 7 mice in each group. 100 μL of PBS, Res, FN, or RPG@FN solution (FN or BSA concentration of 2 mg / kg) was administered to the lungs of mice in each group by a nebulization dosing device. At 24 h after treatment, the lungs were perfused with PBS, and the bronchoalveolar lavage fluid was extracted, centrifuged (4000 rpm, 5 min) at 4°C, and the supernatant was transferred to a new centrifuge tube to obtain the bronchoalveolar lavage fluid, which was stored at -80°C. The expression levels of pro-inflammatory cytokines (TNF-a, IL-6, IL-1β) and anti-inflammatory cytokines (IL-10) in the bronchoalveolar lavage fluid were detected by ELISA. As shown in FIGS. 1a-1d, compared with the control group, the pro-inflammatory cytokines in the bronchoalveolar lavage fluid of the Res and FN treatment groups were reduced to a certain extent. Compared with the Res and FN groups alone, the RPG@FN treatment group showed lower levels of pro-inflammatory cytokines and higher levels of anti-inflammatory cytokines, indicating that the RPG@FN group achieved enhanced anti-inflammatory treatment effect on acute lung injury mice by promoting the intracellular delivery of FN and improving the bioavailability of Res. Figure 13 a-d, the pro-inflammatory cytokines in the bronchoalveolar lavage fluid of the Res and FN treatment groups were reduced to a certain extent compared with the control group. Compared with the Res and FN groups alone, the RPG@FN treatment group showed lower levels of pro-inflammatory cytokines and higher levels of anti-inflammatory cytokines, indicating that the RPG@FN group achieved enhanced anti-inflammatory treatment effect on acute lung injury mice by promoting the intracellular delivery of FN and improving the bioavailability of Res.

[0083] Example 12

[0084] At 24 h after treatment, one mouse was selected from each of the 6 experimental groups, and the lung tissue was immersed in tissue fixative for 24 h for H&E staining to analyze the degree of lung tissue damage. The experimental results are shown in FIGS. 2a-2d. Figure 14 As shown in FIGS. 2a-2d, compared with the control group, the degree of lung alveolar wall congestion and inflammatory cell infiltration was reduced in mice treated with Res and FN, but there were still obvious pathological features of inflammatory cell infiltration, lung alveolar wall congestion, and lung alveolar wall thickening, indicating that the treatment effect of Res and FN alone on ALI mice was limited. After treatment with RPG@FN, the degree of lung alveolar wall congestion, lung inflammation infiltration, and lung alveolar structure recovery in mice returned to the level of normal mice, which was mainly due to the synergistic effect of Res and FN anti-inflammatory and antioxidant activities mediated by the drug-loaded microsphere platform.

Claims

1. A protein-coated core-shell type polymer drug-loaded microsphere composite material, characterized by, The composite material is a protein-coated core-shell structure drug-loaded nanomicrosphere; wherein the core of the core-shell structure drug-loaded nanomicrosphere is a drug-loaded nanomicelle, and the shell layer is a high polymer material; The drug is resveratrol Res; and the protein is fibronectin FN. The preparation method of the protein-coated core-shell type high polymer drug-loaded microsphere composite material comprises: (1) adding an amphiphilic polymer solution and a drug solution to water under ultrasonic conditions, stirring, dialysis, filtration, centrifugation, and obtaining drug-loaded nanomicelles; wherein the amphiphilic polymer is polyethylene glycol-poly-caprolactone PEG-PCL; (2) taking the drug-loaded nanomicelle solution as a core layer solution, taking a polylactic acid-glycolic acid copolymer PLGA solution as a shell layer solution, and performing coaxial electrostatic treatment to obtain core-shell type drug-loaded microspheres; (3) performing co-extrusion of the core-shell type drug-loaded microspheres of step (2) and a protein solution, and centrifuging to obtain the protein-coated core-shell type high polymer drug-loaded microsphere composite material.

2. A preparation method of a protein-coated core-shell type high polymer drug-loaded microsphere composite material, comprising: (1) adding an amphiphilic polymer solution and a drug solution to water under ultrasonic conditions, stirring, dialysis, filtration, centrifugation, and obtaining drug-loaded nanomicelles; wherein the drug is resveratrol Res; and wherein the amphiphilic polymer is polyethylene glycol-poly-caprolactone PEG-PCL; (2) taking the drug-loaded nanomicelle solution as a core layer solution, taking a polylactic acid-glycolic acid copolymer PLGA solution as a shell layer solution, and performing coaxial electrostatic treatment to obtain core-shell type drug-loaded microspheres; (3) performing co-extrusion of the core-shell type drug-loaded microspheres of step (2) and a protein solution, and centrifuging to obtain the protein-coated core-shell type high polymer drug-loaded microsphere composite material; wherein the protein is fibronectin FN.

3. The preparation method according to claim 2, characterized in that, In step (1), the solvent of the amphiphilic polymer solution is dimethyl sulfoxide DMSO; and the solvent of the drug solution is dimethyl sulfoxide DMSO. In step (1), the mass ratio of the drug and the amphiphilic polymer is 1:1-1:5; and the volume ratio of the solvent of the solution to water is 1:8-1:

12.

4. The preparation method according to claim 2, characterized in that, In step (1), the stirring is room temperature stirring for 20-30 h; the molecular weight cut-off of the dialysis bag in the dialysis is 8000-14000 Da; the filtration condition is that a microporous filter membrane with a pore size of 3-5 μm is used; and the centrifugation process parameter is centrifugation at 4000-5000 rpm for 10-30 min.

5. The preparation method according to claim 2, characterized in that, In step (2), the solvent of the drug-loaded nanomicelle solution is water; the mass percentage concentration of the drug-loaded nanomicelle solution is 4-6%; the solvent of the PLGA solution is hexafluoroisopropanol HFIP; and the mass percentage concentration of the PLGA solution is 4-6%.

6. The preparation method according to claim 2, characterized in that, In step (2), polyvinyl alcohol PVA solution is used as a receiving solution in the coaxial electrostatic treatment process, wherein the mass fraction of the PVA solution is 1-4%; and the solvent of the PVA solution is water.

7. The preparation method according to claim 2, characterized in that, The coaxial electrostatic in the step (2) is coaxial electrostatic spraying, and process parameters include: an adjusting voltage is 6-12 kV; a receiving distance is 10-15 cm; a pushing rate of the shell layer solution is 1.5-2.5 mL / h; and a pushing rate of the core layer solution is 0.5-1.5 mL / h; The coaxial electrostatic spinning is followed by differential centrifugation, wherein process parameters of the differential centrifugation are a centrifugation temperature of 4 ℃, first centrifugation at 200-400 g for 10-30 min to remove the precipitate, and then centrifugation at 500 g-600 g for 10-30 min to collect the precipitate.

8. The preparation method according to claim 2, characterized in that, The solvent of the protein solution in the step (3) is water; the concentration of the protein solution is 2-4 mg / mL; and the mass ratio of the core-shell type drug-loaded microspheres to the protein is 1:2-1:

6. The extrusion in the step (3) is repeated 10-15 times by using an Avanti micro-extruder with a filter membrane pore size of 5-7 μm; and centrifugation process parameters are centrifugation at 8000-10000 rpm for 6-8 min.

9. Use of the protein-coated core-shell type polymer drug-loaded microsphere composite material of claim 1 in preparation of anti-inflammatory and antioxidant drugs.

10. Use of the protein-coated core-shell type polymer drug-loaded microsphere composite material of claim 1 in preparation of drugs for acute lung injury.

Images