Preparation and application of mesoporous polydopamine drug delivery system with multiple antioxidant functions

Abstract

The invention discloses preparation and application of a mesoporous polydopamine drug delivery system with multiple antioxidant functions, and belongs to the technical field of medicines.The drug delivery system is characterized in that plant exosomes with antioxidant and anti-inflammatory performance wrap mesoporous polydopamine to serve as a carrier, and the carrier with antioxidant and anti-inflammatory functions is loaded through an adsorption equilibrium method. A nano drug delivery system of a drug having an anti-inflammatory effect; the constructed nano drug delivery system has multiple antioxidant properties, and can regulate the phenotype of macrophages, increase the release of anti-inflammatory factors and promote the apoptosis of hepatic stellate cells.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a mesoporous polydopamine carrier with multiple antioxidant properties, its preparation method, and the application of the mesoporous polydopamine carrier in a drug delivery system. Background Technology

[0002] Reactive oxygen species (ROS) and reactive nitrogen species (RNS), or free radicals, are highly reactive molecules produced as metabolic byproducts in biological systems and also serve as signaling molecules. ROS are substances produced by aerobic organisms during normal physiological activities. These substances generally refer to oxygen-containing free radicals and non-free radical derivatives with strong redox properties, including superoxide anion radicals (O2·kJ·m ... - Hydrogen peroxide (H₂O₂), hydroxyl radicals (·OH), etc. RNS is another class of reactive intermediates, including nitric oxide (·NO), peroxynitrite (·NO₂), etc. - Furthermore, RNS and ROS molecules can interact, establishing molecular networks between RNS and ROS signaling pathways. Their interactions play a crucial role in regulating stress responses. An antioxidant system exists within organisms, composed of non-enzymatic antioxidant molecules and natural antioxidant enzymes, maintaining a balance of reactive oxygen species (ROS). However, with age or when the body is damaged, the rate of ROS production increases to the point where it can overwhelm intracellular levels of non-enzymatic (e.g., glutathione, α-tocopherol, ascorbic acid, carotenoids) and enzymatic (e.g., catalase, superoxide dismutase, and glutathione peroxidase) antioxidants. This imbalance between ROS and antioxidant levels leads to cellular damage and health problems, resulting in oxidative stress. When oxidative stress occurs, cellular structures are disrupted, including oxidative damage to DNA, lipids, proteins, and more. Reactive oxygen species and nitrogenous substances (RONS) also produce aging substances, disrupting genetic factors and contributing to most diseases and aging. Therefore, regulating the level of RONS in the body can effectively prevent and treat diseases related to oxidative stress, such as sepsis, neurodegenerative diseases, brain injury, and cardiovascular diseases.

[0003] The liver, as the largest metabolic and detoxification organ in the human body, is irreplaceable in its importance. Due to the dramatic increase in unhealthy lifestyles among modern people, it is estimated that more than 2 million people die from liver-related diseases each year. Liver fibrosis refers to the diffuse, excessive deposition and abnormal distribution of extracellular matrix (ECM) components such as collagen, glycoproteins, and proteoglycans in the liver during the development of chronic liver disease. It is a pathological repair response of the liver to chronic damage and a key step in the progression of various chronic liver diseases to cirrhosis, significantly affecting the prognosis of chronic liver diseases. Further development of liver fibrosis can lead to structural disorder of the liver, nodular regeneration of hepatocytes, and the formation of pseudolobular structures, i.e., cirrhosis. Liver fibrosis is histologically reversible; with active treatment during this period, it can still be reversed. Otherwise, if liver fibrosis progresses to the cirrhosis stage, reversal becomes very difficult, and the prognosis is relatively poor.

[0004] Liver fibrosis is a complex multicellular pathophysiological process with many causes, including irregular lifestyle, alcohol, medications, and infections. Despite the different causes, such as alcoholic liver injury, viral liver injury, non-alcoholic fatty liver disease, and cholestatic liver injury, hepatocyte death is a common early trigger for liver fibrosis. Hepatocytes are the innate epithelial cells of the liver, accounting for approximately 80% of all liver cells and performing most liver-related functions. Previously, it was thought that hepatocytes did not directly participate in collagen fiber production; however, many recent studies have shown that once hepatocytes transform into fibroblasts through epithelial-mesenchymal transition, they play a crucial role in the occurrence and development of liver fibrosis. When the liver is subjected to exogenous or endogenous inflammatory damage, damaged hepatocytes exhibit a burst of apoptotic cytosomes, reactive oxygen species (ROS), and damage-associated molecular patterns, stimulating intrahepatic macrophages and hematopoietic stem cells. Activated macrophages, by releasing large amounts of inflammatory factors and oxidation-related mediators, stimulate resting hepatic stellate cells (HSCs) to differentiate into activated myofibroblasts, the main ECM-synthesizing cells. This disrupts the balance between ECM synthesis and degradation, leading to massive ECM deposition and proliferation, resulting in liver fibrosis. Furthermore, activated HSCs also promote the recruitment of macrophages from the bone marrow to increase the already large number of macrophages, further exacerbating inflammation and fibrosis. The integration of these pathological behaviors coordinates the occurrence, development, and progression of liver fibrosis, with persistent inflammation and ROS-mediated oxidative stress being two major fibrotic factors.

[0005] Developing effective anti-fibrotic therapies has been challenging to date. Despite numerous promising preclinical studies, results often fail to translate into clinical trials for human disease; therefore, treatment options for liver fibrosis are currently under active investigation. Summary of the Invention

[0006] The purpose of this invention is to construct a mesoporous polydopamine nanoparticle drug delivery system encapsulated in exosomes, possessing both multiple antioxidant and biocompatibility properties. This drug delivery system utilizes the unique structural advantages of the mesoporous framework of mesoporous polydopamine (MPDA), which not only increases drug loading and stability but also exhibits excellent antioxidant activity. Encapsulation with plant exosomes (GENs) containing multifunctional proteins further enhances the biocompatibility of the carrier and endows it with dual antioxidant properties. MP@GE possesses antioxidant, anti-inflammatory, and immunomodulatory effects, capable of scavenging various free radicals, polarizing macrophages to the M2 anti-inflammatory phenotype, inhibiting the release of pro-inflammatory factors, and increasing the release of anti-inflammatory factors. The drugs encapsulated in the drug-loaded nanoparticles are small molecule drugs with antioxidant and anti-inflammatory effects, including curcumin, tripterygium wilfordii, and quercetin; the plant exosomes are derived from one of ginger, carrot, grapefruit, and grape.

[0007] This invention encapsulates mesoporous polydopamine (MPDA) with plant exosome nanoparticles (GENs) possessing antioxidant and anti-inflammatory properties as a carrier, and loads curcumin (Cur) using an adsorption equilibrium method, resulting in C / MP@GE. The constructed C / MP@GE nanosystem exhibits multiple antioxidant properties, can regulate macrophage phenotype, increase the release of anti-inflammatory factors, and promote HSC apoptosis, thereby achieving the goal of anti-liver fibrosis.

[0008] The technical solution adopted in this invention is as follows:

[0009] (1) Preparation of MPDA: Using Pluronic F127 and m-trimethylbenzene (TMB) as template agents, the nanoparticles were dispersed in a mixed solution of distilled water and anhydrous ethanol. The pH of the reaction solution was adjusted with ammonia water, and dopamine hydrochloride solution was added dropwise. The prepared nanoparticles were dispersed in a mixed solvent of acetone and ethanol to remove the template.

[0010] (2) Extraction and purification of exosomes: Exosomes were crudely extracted from plant juices by gradient centrifugation, and then purified by ultra-high speed centrifugation and sucrose gradient centrifugation.

[0011] (3) Exosome-coated mesoporous polydopamine: Low-power ultrasound disrupts the intact membrane structure of exosomes, which are then co-incubated with mesoporous polydopamine.

[0012] In the above preparation method, wherein:

[0013] Preferably, in step (1), the system pH is 8-10, the amount of dopamine hydrochloride added is 150-300 mg, the reaction time is 4-12 h, the reaction temperature is 25-30 °C, and the volume ratio of acetone to ethanol is 1:2.

[0014] Preferably, in step (2), the centrifugation gradient is 3000-5000×g for 30 min, 5000-9000×g for 30 min, and 9000-12000×g for 30 min, the ultra-high speed centrifugation speed is 150000×g, and the centrifugation time is 45-90 min; the sucrose concentration gradient is 5%-15%, 15%-30%, 30%-45%, and 50%-60% respectively; and the plant juice is ginger juice.

[0015] Preferably, in step (3), the incubation temperature of exosomes and mesoporous polydopamine is 25-40°C, and the incubation time is 1-6 hours.

[0016] The specific preparation steps are as follows:

[0017] First, F127 and TMB were dispersed in a mixed solution of water and ethanol. The solution was ultrasonically mixed in a water bath until it was milky white. The mixture was stirred in a 30°C water bath. 150-300 mg of DA was weighed, dissolved in 2 mL of distilled water, and added dropwise to a conical flask. Finally, ammonia was added dropwise to adjust the pH to 8-10. The reaction was allowed to proceed for 4-12 hours. After centrifugation, the precipitate was collected and washed alternately with distilled water and anhydrous ethanol. The precipitate was then dispersed in a mixed solution of ethanol and acetone and ultrasonically removed using a probe. The carrier MPDA was obtained.

[0018] Take fresh ginger, wash it, peel it, cut it into pieces, crush it with a homogenizer, filter it with gauze to remove the residue. The collected liquid was centrifuged using a gradient centrifugation method (3000-5000×g for 30 min, 5000-9000×g for 30 min, and 9000-12000×g for 30 min). The supernatant was collected and allowed to stand at 4℃ for 6-12 h. The supernatant was then collected and centrifuged at 150000×g for 45-90 min. The precipitate was collected, resuspended with an appropriate amount of PBS, and added to a centrifuge tube for later use. Sucrose solutions with mass concentrations of 5%-15%, 15%-30%, 30%-45%, and 50%-60% were prepared and sequentially added to the centrifuge tube through the sample layer. The solutions were centrifuged at 150000×g for 60-90 min. The liquid between the 15%-30% and 30%-45% sucrose solution layers was collected, diluted with PBS, and centrifuged again at 150000×g for 30-60 min. The precipitate was collected to obtain GENs. The precipitate was resuspended in an appropriate amount of PBS, filtered through a 0.22 μm microporous membrane, and stored at -80°C for later use.

[0019] Weigh 3 mg MPDA into a 10 mL centrifuge tube, add 3 mL of pH 7.4 PBS, and sonicate to disperse evenly. Add 1-4 mL of GENs, mix well, and place the mixture in an ice-water bath. Use a cell disruptor to sonicate and disrupt the cell membrane. After sonication, incubate at 25-40℃ for 1-6 h, centrifuge (4℃, 8000-11000 rpm, 5-15 min), and collect the precipitate to obtain MP@GE.

[0020] This invention uses the above-mentioned preparation method to prepare exosome-coated mesoporous polydopamine nanoparticles. The nanoparticles are polydopamine nanoparticles coated with exosomes. The nanoparticles not only have a spherical mesoporous morphology that can efficiently load drugs, but also have stable free radical scavenging ability, anti-inflammatory ability and excellent immunomodulatory ability, which can carry out efficient anti-liver fibrosis treatment.

[0021] The GENs-coated mesoporous polydopamine nanocarrier system constructed in this invention not only enhances the stability of the nanocarrier and effectively improves the solubility of poorly soluble drugs, thereby increasing their bioavailability, but more importantly, it endows the nanocarrier with the ability to target macrophages and reduce toxic side effects.

[0022] Therefore, this invention also provides the application of the aforementioned functionalized hollow mesoporous polydopamine nanoparticles as a drug delivery system combining anti-inflammatory, antioxidant, and immunomodulatory effects. The drug is selected from curcumin, tripterygium wilfordii, quercetin, and other drugs with anti-inflammatory and antioxidant properties.

[0023] The ratio of drug loading to carrier is between 2:1 and 1:3 by mass.

[0024] Using Cur as a model drug:

[0025] First, Cur is loaded into the MPDA prepared in step (1), and then the exosome-coated carrier containing the drug (Cur / MPDA@GENs, abbreviated as C / MP@GE) is prepared according to the following steps.

[0026] The specific steps for loading Cur into the carrier are as follows:

[0027] Disperse 2-10 mg MPDA uniformly in 2-10 mL of ethanol, add 1-30 mg Cur to the carrier suspension and disperse evenly, sonicate in a water bath for 20-40 min, then magnetically stir at 50 °C for 4 h, and collect the product by centrifugation. Subsequently, disperse the product in PBS buffer, add purified GENs and incubate for 1-6 h, collect the precipitate by centrifugation to obtain the drug-loaded system C / MP@GE.

[0028] The drug delivery system constructed in this invention enables the drug to be highly dispersed at the nanoscale within the mesoporous channels of the carrier, allowing the drug to exist in an amorphous state (see attached figure). Figure 2 .

[0029] The antioxidant capacity of C / MP@GE at the cellular level was investigated. Mouse monocytes / macrophages (RAW264.7) were induced with LPS. The intracellular ROS clearance after treatment is shown in the appendix. Figure 8 .

[0030] The beneficial effects of this invention are:

[0031] This invention utilizes MPDA as a drug carrier. As a mussel-like material, MPDA exhibits excellent biocompatibility, and its unique mesoporous structure, with its internal pore structure and outer mesoporous walls providing confinement, enables efficient drug loading and stable dispersion. Furthermore, the prepared carrier not only possesses good biodegradability but also exhibits excellent free radical scavenging capabilities, effectively treating problems caused by oxidative stress in vivo.

[0032] The drug delivery system prepared in this invention can scavenge various free radicals, regulate macrophage phenotype, reduce the release of pro-inflammatory factors while increasing the release of anti-inflammatory factors, and promote hepatic stellate cell apoptosis, thereby exerting an anti-hepatic fibrosis effect.

[0033] This invention introduces plant exosomes, which are rich in bioactive lipids, mRNA, and proteins. Exosomes act as extracellular messengers, stimulating intercellular communication and biological defense against pathological diseases. Exosomes are not only safe, biocompatible, and biodegradable, with no negative impact on intestinal barrier function or other organ toxicity, but also possess inherent biological properties such as antioxidant, anti-inflammatory, and regenerative activities. Encapsulation with exosomes can further improve the biocompatibility of the carrier and endow the carrier with multiple antioxidant capabilities.

[0034] This invention uses Curcumin as a model drug, which possesses excellent antioxidant and anti-inflammatory effects. Curcumin is rich in phenolic hydroxyl groups, enabling it to transfer electrons from two methoxyphenol groups or readily donate H atoms to conduct redox reactions. Furthermore, curcumin also contains various other functional groups, such as β-diketones and several π electrons with prominent electron transfer capabilities. Attached Figure Description

[0035] Figure 1 The particle size distribution and zeta potential of MPDA prepared in Example 1 and C / MP prepared in Example 2 are shown, where A is the particle size and B is the zeta potential.

[0036] Figure 2Differential calorimetric scanning spectroscopy (DCS) spectra of the MDA prepared in Example 1, the active pharmaceutical ingredient Cur, the C / MP prepared in Example 2, and the physical mixture of the active pharmaceutical ingredient and the blank carrier.

[0037] Figure 3 The particle size and particle concentration of the GENs prepared in Example 4.

[0038] Figure 4 The images show TEM images of MPDA prepared in Example 1 and MP@GE prepared in Example 5, where A represents MPDA and B represents MP@GE.

[0039] Figure 5 The nitrogen adsorption-desorption isotherms and pore size distributions of MPDA prepared in Example 1 and MP@GE prepared in Example 5 are shown in Figure A. Figure B shows the pore size distribution.

[0040] Figure 6 This study validates the DPPH and ABTS free radical scavenging abilities of MPDA prepared in Example 1, the active pharmaceutical ingredient Cur, C / MP prepared in Example 2, GENs extracted in Example 4, and C / MP@GE prepared in Example 5. Figure A shows the DPPH scavenging ability validation, and Figure B shows the ABTS scavenging ability validation.

[0041] Figure 7 Figure 5 shows the in vitro release curves of the active pharmaceutical ingredient Cur and the prepared drug delivery system (C / MP@GE) in Example 5. Figure A shows the release under pH 7.4 PBS conditions, and Figure B shows the release under pH 6.5 PBS conditions.

[0042] Figure 8 The ability of the active pharmaceutical ingredient Cur, C / MP prepared in Example 2, and MP@GE and C / MP@GE prepared in Example 5 to scavenge ROS at the cellular level was verified.

[0043] Figure 9 The effects of the active pharmaceutical ingredient Cur, C / MP prepared in Example 2, and MP@GE and C / MP@GE prepared in Example 5 on the production of inflammation-related factors by cells are shown. Where A represents the concentration of released TNF-α, B represents the concentration of released IL-6, and C represents the concentration of released IL-10.

[0044] Figure 10 The effects of factors released from RAW264.7 cells after treatment with the active pharmaceutical ingredient Cur, C / MP prepared in Example 2, and MP@GE and C / MP@GE prepared in Example 5 on HSC cells.

[0045] Figure 11The values ​​represent the AST and ALT levels in the serum of each group in Example 13. Where A represents the AST level and B represents the ALT level.

[0046] Figure 12 The content of Hyp in the liver tissue of each group in Example 13. Detailed Implementation

[0047] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by this invention.

[0048] Example 1

[0049] MPDA preparation

[0050] 15 mL of distilled water and 15 mL of anhydrous ethanol were added sequentially to a 50 mL Erlenmeyer flask, and the solvents were sonicated in a water bath until homogeneous. 300 mg of F127 was added to the Erlenmeyer flask and sonicated in a water bath until completely dissolved. Then, 0.48 mL of TMB was added, and the mixture was sonicated until the TMB was uniformly dispersed and the solution was milky white. The mixture was stirred in a 30°C water bath. 150 mg of DA was weighed, dissolved in 2 mL of distilled water, and added dropwise to the Erlenmeyer flask. Finally, 1.2 mL of ammonia was added dropwise, and the reaction was allowed to proceed for 4 hours. The precipitate was collected by centrifugation and washed three times alternately with distilled water and anhydrous ethanol. The precipitate was then dispersed in a mixed solution of ethanol and acetone (2:1 volume ratio) and sonicated to remove the template. The final carrier, MPDA, was obtained.

[0051] Example 2

[0052] Preparation of C / MP

[0053] Take 2 mg of MPDA prepared in Example 1, disperse it in 2 mL of anhydrous ethanol, add 2 mg of curcumin, sonicate for 30 min, and continue stirring in a 50 °C water bath for 4 h. The product is obtained by centrifugation, which is C / MP.

[0054] Example 3

[0055] Basic representation

[0056] Take 1 mg each of MPDA and C / MP prepared in Examples 1-2, disperse them in water, and use a Nano ZS90 potentiodynamic instrument to measure the particle size and potential changes of polydopamine nanoparticles before and after drug loading. The results are shown in the appendix. Figure 1Differential scanning calorimetry (DSC) was performed on C / MP prepared in Example 2, the active pharmaceutical ingredient Cur, the blank carrier MPDA prepared in Example 1, and a physical mixture of the active pharmaceutical ingredient and the blank carrier to examine the change in drug crystallinity after drug loading. Detailed results are shown in the appendix. Figure 2 .

[0057] Example 4

[0058] Extraction and purification of ginger exosomes

[0059] Fresh ginger roots were washed, peeled, and chopped. The mixture was then homogenized and filtered through gauze to remove residue. The collected liquid was centrifuged at 3000×g for 30 min, 5000×g for 30 min, and 10000×g for 30 min, respectively. The supernatant was collected and allowed to stand at 4℃ for 12 h. The supernatant was then collected and centrifuged at 150000×g for 1 h. Sucrose solutions with concentrations of 8%, 30%, 45%, and 60% were prepared and sequentially added to centrifuge tubes through the sample layer. The solutions were centrifuged at 150000×g for 1 h. The liquid between the 30% and 45% sucrose solution layers was collected, diluted with PBS, and centrifuged again at 150000×g for 1 h. The precipitate was collected to obtain GENs. The precipitate was resuspended in an appropriate amount of PBS, filtered through a 0.22 μm microporous membrane, and stored at -80℃.

[0060] Example 5

[0061] Preparation of C / MP@GE (MP@GE)

[0062] Take 3 mg of C / MP prepared in Example 2 or MPDA prepared in Example 1, add 3 mL of pH 7.4 PBS, and sonicate to disperse evenly. Add 2 mL of GENs extracted in Example 4, mix well, and place the mixture in an ice-water bath. Use a cell disruptor to sonicate the cell membrane (2 kHz, 15% power, 3 min × 4, 2 min interval). After sonication, incubate at 37°C for 2 h, centrifuge, collect the precipitate, and obtain C / MP@GE and MP@GE, which are then lyophilized for later use.

[0063] Example 6

[0064] Basic representation

[0065] Take 200 μL of GENs extracted in Example 4, dilute the GENs with PBS at pH 7.4 at a ratio of 1:10, and determine the particle concentration of GENs using a Nanosight nanoparticle size analyzer. The results are shown in the appendix. Figure 3 As shown. Figure 4TEM images of MPDA prepared in Example 1 and MP@GE prepared in Example 5 are shown. The images reveal that MPDA exhibits a clear pore structure, while the presence of genans (GENs) on the MPDA surface obscures the pores, confirming the GENs coating the MPDA surface. 30 mg of the MPDA support prepared in Example 1 and 30 mg of MP@GE prepared in Example 5 were used to determine their pore size distribution curves and nitrogen adsorption-desorption isotherms. The results are attached. Figure 5 As shown, after GENs coating, the pores are blocked, the MPDA specific surface area decreases, and the pore volume decreases.

[0066] Example 7

[0067] DPPH scavenging capability verification

[0068] Cur, GENs (Example 4), MPDA (Example 1), C / MP (Example 2), and C / MP@GE (Example 5) were prepared at different concentrations (each system contained the same amount of Cur, MPDA, and GENs). 1 mL of sample was added to each system and placed in a centrifuge tube. 1 mL of DPPH reaction solution was then added, followed by 1 mL of anhydrous ethanol. The reaction solution was mixed thoroughly and reacted in the dark for 25 min. The absorbance was measured at 515 nm. Anhydrous ethanol solution was used as a negative control instead of the sample solution, and ethanol was used as a blank control instead of the DPPH reaction solution. The scavenging rate was used to represent the DPPH free radical scavenging activity. The results are attached. Figure 6 As shown in (A), Cur, GENs and MPDA all have DPPH scavenging activity, and MPDA has better scavenging ability under the same concentration conditions. After MPDA is loaded with Cur, the scavenging ability is further enhanced, while C / MP@GE shows better DPPH scavenging ability. The scavenging ability is concentration-dependent.

[0069] Example 8

[0070] ABTS clearance capability verification

[0071] Cur, GENs (Example 4), MPDA (Example 1), C / MP (Example 2), and C / MP@GE (Example 5) were prepared at different concentrations (each system contained the same amount of Cur, MPDA, and GENs). 1 mL of sample was added to each system and placed in a centrifuge tube. 1 mL of ABTS reaction solution was then added, followed by 1 mL of anhydrous ethanol. The mixture was thoroughly mixed and reacted in the dark for 10 min. The absorbance at 734 nm was then measured. Anhydrous ethanol was used as a negative control instead of the sample solvent, and ethanol was used as a blank control instead of the ABTS reaction solution. The scavenging rate was used to represent the ABTS free radical scavenging activity. The results are attached. Figure 6As shown in (B), Cur, GENs, and MPDA all have ABTS scavenging activity. The scavenging ability is further enhanced after Cur is loaded onto MPDA. After GENs are coated, C / MP@GE shows better ABTS scavenging ability. The scavenging ability is concentration-dependent.

[0072] Example 9

[0073] In vitro release study of drug delivery system

[0074] Two mL solutions of Cur and C / MP@GE containing the same amount of Cur were placed in separate dialysis bags and sealed. The bags were then dialyzed with pH 7.4 PBS and pH 6.5 PBS (both containing 1% Tween 80 as a solubilizer), respectively. The bags were placed in a constant-temperature shaker (37°C) and shaken in the dark. The absorbance of the supernatant at 425 nm was measured at specific time points (0.5, 1, 2, 4, 6, 10, 12, 24, 48, 72 h and 120 h). After sampling, an equal volume of the original dispersion medium was added to the original sample.

[0075] like Figure 7 As shown, free Cur can be released in pH 7.4 PBS and pH 6.5 PBS containing 1% Tween 80, with release rates reaching 60.60% and 59.07% respectively after 120 h. However, the drug-loaded system C / MP@GE releases more slowly due to the strong adsorption capacity of MPDA and the coating of GENs. Figure 7 As shown in (A), the cumulative drug release of C / MP@GE in pH 7.4 PBS over 120 h was 32.79%, while... Figure 7 As shown in (B), the release was 29.48% in pH 6.5 PBS, slightly lower than in pH 7.4 PBS. This is contrary to the expected result of higher drug release under the weakly acidic conditions of liver fibrosis, possibly because Cur is more soluble in pH 7.4 PBS than in pH 6.5 PBS, thus leading to a higher release in vitro in pH 7.4 PBS.

[0076] Example 10

[0077] Intracellular ROS scavenging capacity assay

[0078] RAW264.7 cells were seeded in 24-well plates and cultured for 12 h before the culture medium was discarded. The experimental groups were set as follows: (1) blank control group (no drugs and LPS), (2) LPS group (LPS induction only), (3) Cur group, (4) C / MP group, (5) MP@GE group, and (6) C / MP@GE group. The Cur, MPDA and GENs contained in each system were the same, and all contained Cur 5 μg / mL. LPS (final concentration 0.25 μg / mL) was added to the LPS group and the experimental group, and the same volume of DMEM medium was added to the blank control group. After culturing for 2 h, the solution in the wells was aspirated. The experimental group was added to the solutions prepared by the culture medium of each nanosystem, and the blank control group and the model group were added to the same volume of DMEM medium. They were incubated together for 24 h. After washing cells with PBS, they were incubated with 2,7-dichlorofluorescein diacetate (DCFH-DA) solution (20 μM, 10 μL) for 30 min. Cells were washed three times with PBS. The fluorescence of 2,7-dichlorofluorescein (DCF) was observed using a CLSM analyzer. The results are shown in the appendix. Figure 8 LPS treatment produced significant fluorescence signals, indicating an increase in ROS levels in RAW264.7 cells. However, the fluorescence intensity decreased to varying degrees after treatment with different drugs. The fluorescence intensity of the LPS-treated group decreased most significantly after C / MP@GE treatment, suggesting that C / MP@GE has a stronger scavenging effect on ROS compared to other groups.

[0079] Example 11

[0080] Detection of cellular inflammatory factors

[0081] RAW264.7 was seeded into 24-well plates. After LPS induction for 2 hours, Cur, C / MP, MP@GE, and C / MP@GE (with equal final concentrations of Cur, MPDA, and GENs in each group) were added to the corresponding wells. After incubation for 24 hours, the plates were centrifuged at 3000 rpm for 15 minutes, and the supernatant was collected. Following the ELISA kit instructions, the changes in the concentrations of TNF-α, IL-6, and IL-10 in the supernatant of each group were calculated based on the standard curve. The relevant results are shown in the appendix. Figure 9 After LPS-induced macrophages were treated with a drug delivery system, the secretion levels of TNF-α, IL-6, and IL-10 changed significantly, with the C / MP@GE group showing the most significant changes. After drug administration, the levels of pro-inflammatory factors TNF-α and IL-6 decreased significantly by 98.3-fold and 1.99-fold, respectively, and the release of anti-inflammatory factor IL-10 was significantly increased to 39.49 pg / mL.

[0082] Example 12

[0083] The impact of RAW264.7 released substances on HSC

[0084] Cell crawling slides were added to 24-well plates, and HSC cells were seeded on the slides and incubated overnight. The supernatant from RAW264.7 obtained in Example 12 was used to prepare HSC culture medium at a 1:1 ratio with fresh HSC culture medium. This medium was then used to incubate HSC cells for another 24 hours. After washing with PBS, 250 μL of Calcein AM fluorescent dye was added to each well for staining for 30 minutes. After washing again, 250 μL of PI fluorescent dye was added for further staining. After washing with PBS, the viable / dead cell status was observed using a CLSM assay; the results are shown in the appendix. Figure 10 Following LPS induction, the factors released by RAW264.7 had little effect on HSCs, and the HSCs remained viable. However, red fluorescence began to appear in all treatment groups, indicating cell death. The C / MP@GE group showed almost no green fluorescence, indicating that the cells were essentially dead. This suggests that the factors released by RAW264.7 after C / MP@GE treatment can lead to HSC death.

[0085] Example 13

[0086] In vivo anti-liver fibrosis effect

[0087] SPF-grade healthy male C57BL / 6 mice were randomly divided into 6 groups (n=5 per group):

[0088] (1) Blank control group: Olive oil was injected intraperitoneally at 2 mL / kg for the first two weeks and at 3 mL / kg for the next four weeks, for a total of 6 weeks;

[0089] (2) Model group: 20% CCl4 was injected intraperitoneally at 2 mL / kg for the first two weeks, and 20% CCl4 was injected intraperitoneally at 3 mL / kg for the next four weeks, for a total of 6 weeks;

[0090] (3) Cur group: 20% CCl4 was injected intraperitoneally at 2 mL / kg for the first two weeks, and 20% CCl4 was injected intraperitoneally at 3 mL / kg for the next four weeks for a total of 6 weeks. At the same time, starting from the fourth week, 2 mg / kg Cur was injected intravenously via the tail vein.

[0091] (4) C / MP group: For the first two weeks, 20% CCl4 was injected intraperitoneally at 2 mL / kg, and for the next four weeks, 20% CCl4 was injected intraperitoneally at 3 mL / kg for a total of 6 weeks. At the same time, starting from the fourth week, C / MP containing 8 mg / kg was injected via the tail vein.

[0092] (5) MP@GE group: For the first two weeks, 20% CCl4 was injected intraperitoneally at 2 mL / kg, and for the next four weeks, 20% CCl4 was injected intraperitoneally at 3 mL / kg for a total of 6 weeks. At the same time, starting from the fourth week, MP@GE containing 10 mg / kg was injected via the tail vein.

[0093] (6) C / MP@GE group: 20% CCl4 was injected intraperitoneally at 2 mL / kg for the first two weeks, and 20% CCl4 was injected intraperitoneally at 3 mL / kg for the next four weeks, for a total of 6 weeks. At the same time, starting from the fourth week, C / MP@GE containing 12 mg / kg was injected intravenously via the tail vein.

[0094] After the last administration, mice were fasted for 12 hours but allowed free access to water. Anesthesia was administered intraperitoneally to the mice. Their whiskers were trimmed with surgical scissors, and their eyeballs were compressed to protrude. The eyeballs were then quickly removed with forceps. Blood was collected in 1.5 mL sterile centrifuge tubes. After coagulation, the blood was separated and centrifuged (4℃, 3000 rpm, 15 min). The supernatant serum was collected in centrifuge tubes and labeled for subsequent analysis. Mice were euthanized by cervical dislocation, and the livers were removed and photographed. The removed livers were routinely dewaxed and stained according to the Masson staining kit instructions. The results were observed under a microscope. Figure 11 As shown in the figure. After CCl4 induction, compared with the control group, blue collagen fibers were visible in the liver tissue sections of the model group mice, mainly concentrated around the portal area, extending outward from the portal area to form pseudolobular structures, indicating increased collagen deposition in the liver. The liver surface of the mice was rough and contained vacuoles. The serum alanine aminotransferase (AST) and aspartate aminotransferase (ALT) levels were detected according to the kit instructions, and the results are as follows. Figure 12 As shown in the figure, the AST and ALT levels in the model group were higher than those in the control group, indicating liver inflammation and the successful establishment of the CCl4-induced liver fibrosis model. Subsequent treatment with different drug groups resulted in varying degrees of decrease in AST and ALT, with the most significant decrease observed in the C / MP@GE group, demonstrating that C / MP@GE can effectively alleviate liver fibrosis.

[0095] The above description is only the best specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the invention.

Claims

1. A mesoporous polydopamine drug delivery system with multiple antioxidant functions, characterized in that, The medicine delivery system is a nano medicine delivery system which uses plant exosome with anti-oxidation and anti-inflammatory performance as a carrier, and uses adsorption equilibrium method to load medicine with anti-oxidation and anti-inflammatory performance; the constructed nano medicine delivery system has multiple anti-oxidation and anti-inflammatory performances.

2. The mesoporous polydopamine drug delivery system with multiple antioxidant functions according to claim 1, characterized in that, The plant exosome is derived from one of ginger, carrot, grapefruit and grape. 3.The mesoporous polydopamine drug delivery system with multiple antioxidant functions of claim 1, wherein, The medicine with anti-oxidation and anti-inflammatory performance includes curcumin, triptolide and quercetin.

4. The method for preparing the mesoporous polydopamine drug delivery system with multiple antioxidant functions according to any one of claims 1-3, characterized in that, The method comprises the following steps: (1) preparation of mesoporous polydopamine: pluronics F127 and m-xylene are used as a template agent, which is dispersed into a mixed solution of distilled water and anhydrous ethanol, and then ammonia water is used to adjust the pH of the reaction solution, and hydrochloric acid dopamine solution is added dropwise, and the prepared nanoparticles are dispersed into a mixed solvent of acetone and ethanol, and the template is removed; (2) extraction and purification of exosomes: gradient centrifugation is used to extract exosomes from plant juice, and then ultrahigh-speed centrifugation and sucrose gradient centrifugation are used to purify the exosomes; (3) exosome-coated mesoporous polydopamine: low-power ultrasonic is used to destroy the complete membrane structure of the exosomes, and then the exosomes are incubated with mesoporous polydopamine.

5. The preparation method according to claim 4, characterized in that, In step (1), the pH of the system is 8-10, the amount of hydrochloric acid dopamine added is 150-300 mg, the reaction time is 4-12 h, the reaction temperature is 25-30 DEG C, and the volume ratio of acetone to ethanol is 1:

2.

6. The preparation method according to claim 4, characterized in that, In step (2), the centrifugal gradient is 3000-5000xg for 30 min, 5000-9000xg for 30 min and 9000-12000xg for 30 min; the ultrahigh-speed centrifugal speed is 150000xg, and the centrifugal time is 45-90 min; the sucrose concentration gradient is 5%-15%, 15%-30%, 30%-45% and 50%-60% respectively; and the plant juice is ginger juice.

7. The preparation method according to claim 4, characterized in that, In step (3), the incubation temperature of the exosomes with mesoporous polydopamine is 25-40 DEG C, and the incubation time is 1-6 h.

8. The mesoporous polydopamine medicine delivery system with multiple anti-oxidation functions according to any one of claims 1-3 is used as an anti-oxidation, anti-inflammatory and immune regulation combined medicine delivery system.

9. Use according to claim 8, characterized in that, The method comprises the following steps: (1) 2-10 mg of mesoporous polydopamine is uniformly dispersed in 2-10 mL of ethanol, 1-30 mg of curcumin is added to the carrier suspension and uniformly dispersed, ultrasonic is performed for 20-40 min in a water bath, and then magnetic stirring is performed at 50 DEG C for 4 h, and the product is collected by centrifugation; (2) the exosome-coated mesoporous polydopamine medicine delivery system is prepared according to steps (2) and (3) of claim 4.

10. The use of the mesoporous polydopamine drug delivery system with multiple antioxidant functions according to any one of claims 1-3, characterized in that, The mesoporous polydopamine medicine delivery system with multiple anti-oxidation functions can scavenge multiple free radicals, regulate macrophage phenotype, reduce the release of pro-inflammatory factors while increasing the release of anti-inflammatory factors, and promote the apoptosis of hepatic stellate cells, and is used for preparing an anti-hepatic fibrosis medicine.

Images