Fibronectin-coated core-shell type polymer drug-loaded microsphere system as well as preparation method and application of fibronectin-coated core-shell type polymer drug-loaded microsphere system

Abstract

The invention relates to a fibronectin-coated core-shell type polymer drug-loaded microsphere system and a preparation method and application thereof.The preparation method comprises the steps that a drug is loaded in a hydrophobic cavity of an amphiphilic polymer to form drug-loaded micelles, and the drug-loaded micelles are packaged in polymer microspheres through a coaxial electronic injection technology to form core-shell type drug-loaded microspheres; and further physically adsorbing and wrapping fibronectin on the surfaces of the drug-loaded microspheres. The method is simple in operation condition, easy to operate and separate, and capable of effectively improving the bioavailability of the micromolecule drug and the protein, and cooperating the biological activity of the micromolecule drug and the protein in in-vitro and in-vivo lung injury models, thereby providing a new thought for constructing a safe, intelligent and efficient drug carrier.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and particularly relates to a fibronectin-coated core-shell polymer drug-loaded microsphere system and a preparation method and application thereof. Background Art

[0002] Acute lung injury is a clinical syndrome with an acute onset and rapid progression. It causes extensive inflammatory reactions in the lungs and seriously affects gas exchange, which may eventually lead to respiratory failure and threaten the patient's life safety. Due to the complex pathological process of acute lung injury, which involves the interaction of multiple inflammatory mediators and cytokines, traditional treatment methods often fail to achieve ideal therapeutic effects. In this context, it is particularly urgent to develop new treatment methods and means. Drug carrier technology provides new possibilities 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, thereby 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 duration of drug action in the lungs. This can not only enhance the efficacy of drugs, but also reduce the side effects of drugs on other normal tissues. In addition, drug carriers can also control the release rate of drugs, so that drugs can continue to play a therapeutic role in the lungs, thereby extending 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 potential risks to patients. This is especially important for patients with acute lung injury, because their lungs are already damaged and their tolerance to drugs is poor. In addition, the development of drug carrier technology can also promote scientific and technological progress and industrial development in related fields. With the continuous progress in 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, and help promote the improvement of medical standards and the quality of life of patients.

[0003] Microsphere drug carriers can achieve a variety of release modes, such as zero-order, first-order or second-order release, through different preparation methods to meet the release requirements 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 the drug. In addition, microspheres can protect drugs from environmental influences, reduce degradation, extend the shelf life, and improve drug stability and targeting in vivo, 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] As a drug carrier, PLGA has shown significant advantages in drug controlled release and targeted delivery, mainly due to its good biocompatibility, degradability, 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 the molecular weight and copolymer ratio, and the regulation process is complex and requires precise control. In addition, the drug delivery effect depends on the design optimization of specific diseases and drugs. Our research group has shown good results in drug loading and bioavailability by preparing fiber microspheres by electrospinning (Xiao et al. NanoToday. 2021, 38, 101123), (Zhang et al. Adv. Fiber Mater. 2022, 4, 807–819). Resveratrol (Res) has broad application prospects 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 the potential for 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 a major challenge in its application.

[0005] A search of relevant domestic and foreign literature and patents has not yet found any reports on the use of electrosprayed microspheres as a carrier for the co-delivery of Res and FN for the anti-inflammatory and antioxidant treatment of acute lung injury. Summary of the invention

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

[0007] The invention provides a protein-coated core-shell polymer drug-loaded microsphere composite material, wherein the composite material is a core-shell structure drug-loaded nano-microsphere with a surface coated with protein; wherein the core of the core-shell structure drug-loaded nano-microsphere is a drug-loaded nano-micelle, and the shell layer is a polymer material.

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

[0009] Furthermore, the composite material utilizes the hydrophobic cavity of the amphiphilic polymer to load the drug to form drug-loaded micelles, the drug-loaded micelles are encapsulated inside the polymer microspheres through coaxial electrospinning technology to form core-shell drug-loaded microspheres, and fibronectin is further physically adsorbed and wrapped on the surface of the drug-loaded microspheres.

[0010] The present invention provides a method for preparing a protein-coated core-shell polymer drug-loaded microsphere composite material, comprising:

[0011] (1) adding an amphiphilic polymer solution and a drug solution into water under ultrasonic conditions, stirring, dialyzing, filtering, and centrifuging to obtain drug-loaded nanomicelles;

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

[0013] (3) Co-extruding the core-shell drug-loaded microspheres of step (2) and the protein solution, and centrifuging and purifying to obtain a protein-coated core-shell polymer drug-loaded microsphere composite material.

[0014] Preferably, in step (1), the amphiphilic polymer is polyethylene glycol-polycaprolactone 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 to 1:5; the volume ratio of the solvent to water in the solution is 1:8 to 1:12. (The solvent is the sum of the solvents of the amphiphilic polymer solution and the drug solution)

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

[0017] Preferably, the solvent of the drug-loaded nanomicelle solution in step (2) is water, and further the water is ultrapure water; the mass percentage concentration of the drug-loaded nanomicelle 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 dissolution condition is stirring at room temperature for 1-4 hours.

[0018] Preferably, in the coaxial electrostatic process in step (2), 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; the PVA dissolution conditions are heating and stirring at 90° C. for 2 hours.

[0019] Furthermore, step (2) is specifically as follows: the drug-loaded nanomicelle solution is used as the core layer solution, and the polylactic acid-glycolic acid copolymer PLGA solution is used as the shell layer solution, which are respectively loaded into the core layer channel and the shell layer channel of the coaxial electrospinning device, and assembled together with the polyvinyl alcohol PVA solution as the receiving solution into a coaxial electrospinning device.

[0020] By adjusting the voltage, receiving distance and pushing rate of the shell-core solution of the coaxial electrospray device, core-shell drug-loaded microspheres RPG with internal micelles were obtained. Then, RPG with uniform size distribution was obtained by differential centrifugation, and ultrapure water was repeatedly eluted to remove the residual PVA and organic solvent on the surface.

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

[0022] Preferably, the coaxial electrospinning is followed by differential centrifugation; wherein the process parameters of the differential centrifugation are as follows: a centrifugation temperature of 4°C, 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 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 to 4 mg / mL; and the mass ratio of the core-shell drug-loaded microspheres to the protein is 1:2 to 1:6.

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

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

[0026] The present invention provides an application of the protein-coated core-shell polymer drug-loaded microsphere composite material in the preparation of anti-inflammatory and antioxidant drugs.

[0027] The present invention provides an application of the protein-coated core-shell polymer drug-loaded microsphere composite material in the preparation of a lung injury drug.

[0028] The present invention also provides a fibronectin-coated core-shell polymer drug-loaded microsphere composite material (system) for use in enhanced anti-inflammatory and antioxidant treatment of an acute lung injury mouse model.

[0029] The present invention uses the amphiphilic polymer PEG-PCL to encapsulate Res with anti-inflammatory and antioxidant activity in the hydrophobic cavity of micelles, and further encapsulates the drug-loaded micelles inside PLGA microspheres by electrospraying 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 co-extrusion to improve the intracellular delivery efficiency of the protein and realize the inflammatory cell targeting function of the drug-loaded microspheres, thereby achieving efficient anti-inflammatory and antioxidant treatment of acute lung injury.

[0030] The present invention 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 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 effects of the materials on intracellular ROS levels; flow cytometry is used to evaluate the effects of the materials on protein intracellular delivery; flow cytometry is used to evaluate the effects of the materials on macrophage transformation; enzyme-linked immunosorbent assay (ELISA) is used to evaluate the effects of the microsphere platform on anti-inflammatory and pro-inflammatory cytokine expression; and finally, an acute lung injury mouse model is established to verify the in vivo anti-inflammatory and antioxidant therapeutic effects of the drug-loaded microsphere system.

[0031] The present invention provides a fibronectin-coated core-shell polymer drug-loaded microsphere system and a preparation method and application thereof. The drug-loaded microsphere system has good biosafety, 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 a safe, intelligent and efficient drug carrier.

[0032] Beneficial Effects

[0033] (1) The reaction conditions of the present invention are simple, easy to operate and separate, and have good development prospects.

[0034] (2) The drug-loaded microsphere platform prepared by the present invention effectively improves the water solubility of Res and can release Res in the inflammatory microenvironment, providing a new idea for constructing a safe and efficient drug carrier.

[0035] (3) The drug-loaded microsphere platform prepared by the present invention can target alveolar macrophages through the surface-coated FN, on the one hand, clearing excessive reactive oxygen species in the lesion site and restoring the homeostasis of oxidative stress; on the other hand, the drug-loaded microsphere platform can synergize the anti-inflammatory activity of FN and Res, induce macrophages to polarize from M1 type to M2 type, and inhibit excessive immune activation in the lesion site. At the same time, the drug-loaded microsphere platform can also downregulate pro-inflammatory inflammatory factors by inhibiting the NF-κB signaling pathway, enhance the expression of anti-inflammatory inflammatory factors, and enhance the therapeutic effect.

[0036] (4) After the drug-loaded microsphere platform prepared by the present invention enters the mouse body through the tracheal spray drug delivery device, the microspheres can be retained in the mouse alveoli for a long time due to their large hydrodynamic diameter, achieving a long-term drug sustained release effect, and have potential clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the preparation and application of RPG@FN in the present invention;

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

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

[0040] Figure 4 Single-channel (a) and double-channel (b) confocal laser microscope images of the core-shell structure microsphere FPG with FITC fluorescence labeling prepared in Example 1, with a scale bar of 1 μm;

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

[0042] Figure 6 The SDS-PAGE electrophoresis diagram of RPG, RPG@FN and RPG@BSA prepared in Example 1;

[0043] Figure 7 The Res release kinetic curves of RPG@FN, RPG@BSA, and RPG prepared in Example 1 within 72 hours;

[0044] Figure 8 Schematic diagram of the degradation of RPG, RPG@BSA and RPG@FN prepared in Example 1 over time;

[0045] Fig. 9 This is a graph of cell viability after PEG-PCL / Res, Res, and RPG prepared in Example 1 were co-incubated with MH-S cells for 24 hours;

[0046] Fig.10 The flow cytometry graph (a) and the mean fluorescence intensity quantitative graph (b) of RPG@FN and FN prepared by the present invention in Example 1 after being co-incubated with MH-S cells for 12 hours; and the flow cytometry graph (c) and the mean fluorescence intensity quantitative graph (d) of RPG@FN+RGD and RPG@FN after being co-incubated with MH-S cells for 12 hours;

[0047] Fig.11 Flow cytometry analysis (a), relative fluorescence intensity quantification (b) and laser confocal microscopy image (c) of the expression level of reactive oxygen species in cells after RPG@FN, RPG@BSA, Res and FN prepared by the present invention were co-incubated with LPS-activated MH-S cells for 24 hours. The scale bar in the figure is 10 μm.

[0048] Fig.12 Flow cytometric analysis of CD86 and CD206 expression levels in MH-S cells activated by LPS after co-incubation of RPG, RPG@BSA and RPG@FN prepared by the present invention for 24 hours (a), percentage of M1 macrophages (b) and M2 macrophages (c), and (d) relative M2 / M1 macrophage ratio;

[0049] Fig.13 ELISA test results 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] Fig.14 This is the histological section analysis of the lung tissue of mice in each experimental group in Example 12; the gray arrows indicate alveolar wall congestion, the black arrows indicate inflammatory cells, and the scale bar in the figure is 50 μm. DETAILED DESCRIPTION

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

[0052] PLGA was purchased from Jinan Daigang Bioengineering Co., Ltd. PEG-PCL was purchased from Xi'an Ruixi Biotechnology Co., Ltd. Res was purchased from Shanghai Hongye Biotechnology Co., Ltd. Fibronectin was from Shanghai Xianlian Biotechnology 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 Saiolesen Biomedicine Co., Ltd. Reactive oxygen species (ROS) detection probe (DCFH-DA) was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Cell counting kit (CCK-8) was purchased from Shanghai Qihai Biotechnology Co., Ltd. ELISA kit was purchased from Shanghai Zhuocai Biotechnology Co., Ltd. BALB / c mice were purchased from Shanghai Slake Experimental Animal Center. Anti-CD80-PE and Anti-CD206-FITC were purchased from Thermo Fisher Scientific (Waltham, MA). Water with a resistivity higher than 18.2 MΩ.cm used in all experiments 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. The solution was then added dropwise to 5 mL ultrapure water under ultrasonic conditions and stirred for 24 h. After the reaction, DMSO was removed by dialysis in ultrapure water for 3 days using a dialysis bag with a molecular weight cutoff of 3000 Da. After the dialysis, the product was filtered through a microporous filter membrane with a pore size of 1 μm to remove the unloaded Res. Finally, the product was concentrated by centrifugation at 4500 rpm for 30 min to obtain PEG-PCL / Res.

[0055] (2) Dissolve 40 μg PLGA in 960 μL hexafluoroisopropanol, stir and mix for 2 h, and then load it into a 1 mL syringe set as the outer layer of the coaxial needle. At the same time, take the 40 μg PEG-PCL / Res prepared in step (1) and dissolve it in 960 μL ultrapure water, load it into a 1 mL syringe set as the inner layer of the coaxial needle, and place the prepared 1% PVA solution below as a receiving device. Adjust the high voltage power supply of the electrospray equipment to 8 kV, the receiving distance to 10 cm, the shell injection pump propulsion speed to 2 mL / h, the core injection pump propulsion speed to 1 mL / h, and place a magnetic stirring device below the receiving device to stir at a constant temperature of 750 rpm to prevent adhesion between microspheres. Collect the prepared microspheres RPG and solidify them at 4°C for 12 h. Then use differential centrifugation, first centrifuge at 200 g for 10 min, discard the precipitate, collect the supernatant, and centrifuge again at a speed of 500 g for 30 min, discard the supernatant, collect the precipitate and solidify it at 4°C for 12 h. After freeze drying in a freeze dryer, RPG with uniform particle size was obtained. Similarly, Res was replaced with FITC to prepare core-shell microspheres FPG with fluorescence labeling.

[0056] (3) Take 15 mL of fibronectin stock solution and place it in a 10000Da dialysis bag for 24 hours, change the water twice in the middle, freeze the dialyzed stock solution at -80°C for more than 12 hours, and then place it in a freeze dryer for freeze drying to obtain purified fibronectin FN.

[0057] (4) 4 mg of the purified FN in step (3) was dissolved in 1 mL of ultrapure water to obtain an FN solution, which was then mixed with 1 mg of RPG. The solution was extruded 12 times using an Avanti micro extruder, centrifuged at 10,000 rpm for 6 min to remove excess fibronectin FN, and the precipitate was collected to obtain RPG@FN.

[0058] (5) Using the same method as in step (3) and step (4), fibronectin was replaced with bovine serum albumin (BSA) to prepare the control material RPG@BSA.

[0059] Example 2

[0060] Take an appropriate amount of RPG@FN, disperse it evenly with ultrapure water, and ultrasonicate for 30 minutes to completely disperse it into a suspension to prepare an RPG@FN solution with a Res concentration of 4mM; and take an appropriate amount of Res and PEG-PCL / Res, RPG, RPG@BSA and RPG@FN prepared in Example 1, dilute them with water to prepare a solution with a Res concentration of 4mM for measuring the hydrated particle size and surface potential. The results are shown in Figure 2As shown, the hydrated particle size of Res alone is 120.3nm, the hydrated particle size of PEG-PCL / Res micelles is 310.2nm, and the hydrated particle size of RPG is 841.3±174nm. When BSA or FN is coated on the surface of drug-loaded microspheres RPG microspheres, the hydrated particle sizes of RPG@BSA and RPG@FN increase to 1668.6±196nm and 1684.4±124nm, respectively, and the surface potential is further reduced, which means that BSA or FN is successfully coated on the surface of the microspheres. In addition, the hydrated particle size of the drug-loaded microsphere system RPG@FN in various solutions (water, PBS, RPMI-1640 culture medium) remained almost unchanged within a week ( Figure 3 ), which proves that RPG@FN has good colloidal stability.

[0061] Example 3

[0062] In order to verify the core-shell structure of the prepared microsphere system, the Res encapsulated in PEG-PCL was replaced with FITC of the same concentration according to the steps of Example 1 to obtain fluorescently labeled microspheres FPG. The images were observed by laser confocal microscopy. Figure 4 As shown in Figure 2, the prepared RPG microspheres can completely encapsulate the micelles containing FITC inside the microspheres, indicating that RPG has obvious core-shell structural characteristics. In addition, the size and morphology of the prepared RPG@FN were characterized, as shown in Figure 2. Figure 5 As shown in the SEM images, RPG@FN microspheres are uniformly distributed spherical structures with an average size of approximately 1298±433nm.

[0063] Example 4

[0064] In order to further verify the successful loading of BSA or FN in RPG microspheres, RPG, RPG@FN, and RPG@BSA prepared in Example 1 were used for SDS-PAGE gel electrophoresis experiments. 20 μL of RPG, RPG@FN, RPG@BSA, FN, and BSA (protein concentration was 1 mg / mL) were taken and mixed with SDS loading buffer in a boiling water bath for 5 minutes. RPG, RPG@FN, RPG@BSA, purified BSA and FN, and standard protein marker (5 μL) were added to the corresponding channels of the gel in turn and run for 30 minutes at a voltage of 120 V. After the gel run, the gel was taken out and placed in a container filled with Coomassie Brilliant Blue for staining for 30 minutes. The staining solution was then replaced with the same volume of destaining solution and destained on a decolorizing shaker for 2 hours. Finally, the protein bands on the precast gel were recorded with a scanner. The results are shown in the figure. Figure 6As shown in the figure, RPG did not show obvious protein bands, while RPG@FN showed the same bands as FN, indicating the successful combination of FN and RPG microspheres; RPG@BSA also showed the same characteristic bands as BSA, verifying the successful combination of BSA and RPG microspheres. Combined with 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 were successfully prepared.

[0065] Example 5

[0066] In order to further analyze the drug sustained release performance of Res in the drug-loaded microsphere system, the 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 retention capacity of 3500Da). The dialysis bag was then 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 the liquid outside the dialysis bag was aspirated, and 1 mL of fresh buffer solution was added to the container. The absorbance of the removed liquid at 338 nm was measured to quantify the content of Res released. After the sustained release, the drug release curves of RPG, RPG@BSA and RPG@FN under different conditions were plotted. Figure 7 As shown in the figure, the cumulative release rate of RPG after 72 hours reached 74.3%, which has the effect of long-term sustained release of drugs, while the cumulative release rate of RPG@FN after 72 hours was 75.2%, proving that the coating of FN will not affect the overall release of Res from drug-loaded microspheres. This shows that the prepared drug-loaded microsphere system is expected to release the drug Res in the lesion microenvironment for a long time, achieving enhanced anti-inflammatory and antioxidant effects.

[0067] Example 6

[0068] The microspheres prepared in Example 1 were taken to characterize their biodegradable properties. First, a certain amount of RPG, RPG@BSA and RPG@FN were weighed and their initial weights (W0) were 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 oscillator at 37 ° C with an oscillation speed of 100 rpm. During the 8-week experimental period, the microspheres were taken out and dried every week 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) / W0×100%(1)

[0070] The results are as follows Figure 8As shown, over time, the degradation efficiency of RPG, RPG@BSA and RPG@FN reached 50% and above at the 8th week, proving that the prepared microsphere drug carriers have good biodegradability.

[0071] Example 7

[0072] In order to verify the effect of RPG@FN on cell viability, cells in the logarithmic growth phase were collected and cultured at 1×10 4 The cells were inoculated into a 96-well plate at a density of 10 cells per well, cultured at 5% CO2 and 37°C for 24 hours, and then replaced with fresh RPMI-1640 medium containing different concentrations of Res, PEG-PCL / Res or RPG (the concentrations of Res were 0, 5, 10, 15, 20, 25, 35, and 50 μg / mL) for 24 hours. The normal group was treated with PBS. The culture medium was then discarded, washed three times with PBS, and 100 μL of fresh serum-free DMEM culture medium containing 10% (v / v) CCK-8 solution was added to each well and incubated in an incubator for 3 hours. The absorbance of each well was tested at a wavelength of 450 nm using a multifunctional microplate reader. The cells treated with PBS were used as blank controls, and cell viability was recorded as 100%. Fig. 9 The experimental results showed that within the test concentration range, the cell viability of each group of materials was above 90%, which indicates that the prepared drug-loaded microsphere system has excellent cell compatibility.

[0073] Example 8

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

[0075] MH-S cells were used as a model to verify the effect of RPG in promoting FN intracellular delivery and the role of FN in targeting inflammatory macrophages. Cells in the logarithmic growth phase were collected and cultured at 1×10 5The cells were seeded at a density of 10 cells per well in a 12-well plate and cultured at 5% CO2 and 37°C for 24 hours. The culture medium was replaced with fresh serum-free RPMI-1640 medium containing FN, RPG or RPG@FN, RPG@FN+RGD and incubated for 4 hours (FN was labeled with Cy5.5 and the concentration of FN was 40 μg / mL). The normal group was treated with PBS. The culture medium was discarded and washed three times with PBS. 1 mL of trypsin was added to each well to digest the cells. The digestion was terminated and the cells were collected by centrifugation. 300 μL of PBS was added to the collected cells to resuspend the cell pellet, and the pellet was transferred to a flow tube. The fluorescence intensity of each group was detected by flow cytometry. The results are shown in Fig.10 As shown in ab, 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, providing the main guarantee for the biological function of FN. Among them, the experimental results of 10c-d showed that the fluorescence intensity of the RPG@FN group was higher than that of the RPG@FN+RGD group, verifying that FN has the effect of targeting inflammatory macrophages, and that the targeting is achieved through the interaction of RGD-α4β1 integrin.

[0076] Example 9

[0077] In order to verify the scavenging effect of RPG@FN microspheres complex on reactive oxygen species (ROS) in alveolar macrophages, MH-S cells in the logarithmic growth phase were collected and cultured at 1×10 5 The cells were seeded at a density of 10 cells per well in a 12-well plate and cultured at 5% CO2 and 37°C for 24 hours. The medium was replaced with RPMI-1640 medium containing LPS (at a concentration of 2 μg / mL) and incubated with the cells for 24 hours. PBS was added to the control group. Subsequently, the medium was replaced with fresh serum-free RPMI-1640 medium containing Res, FN, RPG@BSA, and RPG@FN (BSA or FN concentration of 20 μg / mL) and incubated for 4 hours. The old medium was discarded, and serum-containing RPMI-1640 medium was added to incubate the cells for 20 hours. After collecting the cells by trypsin digestion, the cell pellet was resuspended with 1 mL of PBS, and the ROS fluorescent probe DCFH-DA was added and incubated with the cells at 37°C for 20 minutes. After washing three times with PBS to remove the probes that were not bound to the cells, the cell pellet was resuspended with 300 μL of PBS and transferred to a flow tube. The effect of the nanomaterial on ROS clearance in macrophages was evaluated by flow cytometry. The results are shown in Fig.11As shown in ab, the relative fluorescence intensity of the Res and FN groups is lower than that of the LPS group, indicating that both can reduce intracellular ROS to a certain extent. Compared with the FN or Res group, RPG@FN showed a lower level of fluorescence intensity, which shows that RPG@FN can synergize the antioxidant activity of Res and FN in macrophages and most effectively reduce the level of intracellular ROS, while the RPG@BSA group can only play the role of Res, and its cell phagocytosis is significantly higher than that of free Res, so it has a higher ROS scavenging ability than free Res.

[0078] The ability of RPG@FN to remove intracellular ROS was further verified by laser confocal microscopy. MH-S cells in the logarithmic growth phase were collected and plated at 1×10 5 MH-S cells were seeded in a laser confocal microscopy culture dish at a density of 10 cells per well and incubated at 5% CO2 and 37°C for 24h. The culture medium was replaced with RPMI-1640 medium containing LPS (at a concentration of 2μg / mL) and incubated with the cells for 24h. PBS was added to the control group. Then, each well plate was replaced with fresh serum-free RPMI-1640 medium containing Res, FN, RPG@BSA, and RPG@FN (BSA or FN at a concentration of 20μg / mL) and incubated for 4h. The old culture medium was discarded and replaced with fresh culture medium containing serum and incubated with the cells for 20h. The culture medium was discarded, washed three times with PBS, 1mL DCFH-DA was added and incubated with the cells at 37°C for 20min, washed three times with PBS, 1mL 2.5% glutaraldehyde was added and fixed at room temperature for 15min, washed three times with PBS, 1mL LDAPI was added and incubated with the cells for 5min, washed three times with PBS, and 200μL PBS was added to observe the changes in cell fluorescence intensity under a laser confocal microscope. The results are as follows Fig.11 As shown in Figure 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 proved that the drug-loaded microsphere system had the best antioxidant performance. This is due to the fact that RPG@FN can effectively deliver Res and FN into macrophages, thereby improving the bioavailability of the two.

[0079] Example 10

[0080] In order to verify the effect of RPG@FN nanocomplex on macrophage polarization, MH-S cells in the logarithmic growth phase were collected and cultured at 1×10 5The cells were inoculated at a density of 10 cells per well in a 12-well plate and cultured at 5% CO2 and 37°C for 24 hours. The culture medium was discarded and replaced with RPMI-1640 culture medium containing LPS (at a concentration of 2 μg / mL) and incubated with the cells for 24 hours. PBS was added to the control group. Subsequently, fresh serum-free RPMI-1640 culture medium containing Res, FN, RPG@BSA, and RPG@FN (BSA or FN at a concentration of 20 μg / mL) was replaced and incubated for 4 hours. The old culture medium was discarded and serum-containing RPMI-1640 culture medium was added to incubate the cells for 20 hours. Subsequently, the culture medium was discarded, PBS was washed twice, and the cells were collected by trypsin digestion. The cell pellet was resuspended in 200 μL PBS, and Anti-CD206-FITC and Anti-CD86-PE antibodies were added to the cells and incubated for 30 minutes at 4°C in the dark. After washing three times with PBS to remove the antibodies not bound to the cells, the cell pellet was resuspended with 300 μL PBS and transferred to a flow cytometry tube. The effect of the nanomaterial on macrophage polarization was evaluated by detecting the changes in the expression levels of CD86 and CD206 in the cells. Fig.12 As shown in ad, Res and RPG@BSA can partially reduce the level of M1 macrophages induced by LPS. Although FN alone has difficulty entering the cells, FN still exhibits the ability to induce macrophages to polarize to M2, which may be because FN binds to macrophage surface receptors and participates in regulating 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 its M2 / M1 ratio was 2.6 times that of the RPG@BSA group, indicating that RPG@FN further enhances the ability to induce macrophages to polarize to M2 by synergistically utilizing the biological activities of FN and Res.

[0081] Embodiment 11

[0082] All animal experiments were conducted in strict accordance with the standards of the Experimental Animal Ethics Committee of Donghua University. Six-week-old male BALB / c mice were purchased from Shanghai Slake Experimental Animal Center. The animal model of acute lung injury was established by intratracheal aerosolization of LPS (5 mg / kg) 24 hours later, and the control group was intratracheally aerosolized 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 was 2 mg / kg) was administered to the lungs of mice in each group through a nebulizer. 24 hours after treatment, the lungs were perfused with PBS, and the alveolar lavage fluid was extracted and centrifuged at 4°C (4000 rpm, 5 min). The supernatant was transferred to a new centrifuge tube to obtain the alveolar lavage fluid, which was stored at -80°C. Then, ELISA was used to detect the expression levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and anti-inflammatory cytokines (IL-10) in bronchoalveolar lavage fluid. Fig.13 As shown in ad, compared with the control group, the pro-inflammatory cytokines in the bronchoalveolar lavage fluid of mice in the Res and FN treatment groups were reduced to a certain extent. Compared with the single Res and FN groups, 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 therapeutic effects on mice with acute lung injury by promoting the intracellular delivery of FN and improving the bioavailability of Res.

[0083] Example 12

[0084] 24 hours after treatment, one mouse was selected from each of the six experimental groups, and its lung tissue was taken and immersed in tissue fixative for 24 hours, and H&E staining was performed to analyze the degree of lung tissue damage. Fig.14 As shown in the figure, compared with the control group, the degree of alveolar wall congestion and inflammatory cell infiltration in mice treated with Res and FN were reduced, but there were still obvious pathological features of inflammatory cell infiltration, alveolar wall congestion and alveolar wall thickening, which indicated that the therapeutic effect of Res and FN alone on ALI mice was limited. In mice treated with RPG@FN, alveolar wall congestion, lung inflammatory infiltration and alveolar structure were restored to the level of normal mice, which was mainly attributed to the synergistic effect of anti-inflammatory and antioxidant activities of Res and FN mediated by the drug-loaded microsphere platform.

Claims

1. A protein-coated core-shell polymer drug-loaded microsphere composite material, characterized in that: The composite material is a core-shell structure drug-loaded nano-microsphere with a surface coated with protein; wherein the core of the core-shell structure drug-loaded nano-microsphere is a drug-loaded nano-micelle, and the shell is a polymer material.

2. A method for preparing a protein-coated core-shell polymer drug-loaded microsphere composite material, comprising: (1) adding an amphiphilic polymer solution and a drug solution into water under ultrasonic conditions, stirring, dialyzing, filtering, and centrifuging to obtain drug-loaded nanomicelles; (2) using the drug-loaded nanomicelle solution as the core layer solution and the polylactic acid-co-glycolic acid copolymer PLGA solution as the shell layer solution, and conducting coaxial electrospinning to obtain core-shell drug-loaded microspheres; (3) Co-extruding the core-shell drug-loaded microspheres of step (2) and the protein solution, and centrifuging to obtain a protein-coated core-shell polymer drug-loaded microsphere composite material.

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

12.

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

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

6. The preparation method according to claim 2, characterized in that: In the coaxial electrostatic process in step (2), polyvinyl alcohol (PVA) solution is used as the receiving solution, 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 spraying in step (2) is coaxial electrostatic spraying, and the process parameters include: adjusting the voltage to 6-12 kV; the receiving distance to 10-15 cm; the pushing rate of the shell solution to 1.5-2.5 mL / h; the pushing rate of the core solution to 0.5-1.5 mL / h; The coaxial electrospinning is followed by differential centrifugation; the process parameters of the differential centrifugation are as follows: the centrifugation temperature is 4°C, the centrifugation is first performed at 200-400g for 10-30min to remove the precipitate, and then the centrifugation is performed at 500g-600g for 10-30min to collect the precipitate.

8. The preparation method according to claim 2, characterized in that: The protein in step (3) is at least one of fibronectin FN and bovine serum albumin; the solvent of the protein solution is water; the concentration of the protein solution is 2 to 4 mg / mL; the mass ratio of the core-shell drug-loaded microspheres to the protein is 1:2 to 1:6; The extrusion in step (3) is performed by repeatedly extruding 10 to 15 times using an Avanti micro extruder with a filter membrane pore size of 5 to 7 μm; The centrifugal process parameters are centrifugation at 8000-10000 rpm for 6-8 minutes.

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

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

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