Quercetin-loaded TPP-PEG-PCL nano microcapsule as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of preparation of sepsis acute lung injury drugs, relates to a novel quercetin drug delivery system or dosage form, and particularly relates to a quercetin-loaded TPP-PEG-PCL nano microcapsule as well as a preparation method and application thereof. The preparation method comprises the following steps: by taking (3-carboxypropyl) triphenylphosphonium bromide, polyethylene glycol-b-polycaprolactone, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide as raw materials, preparing a TPP-PEG-PCL (Triphenyl Polyethylene Glycol-Polycaprolactone) nano-micelle; and then loading the quercetin on the TPP-PEG-PCL nano-micelle, so as to obtain the TPP-PEG-PCL nano-microcapsule loaded with the quercetin. The quercetin-loaded TPP-PEG-PCL nano-micelle obtained by the method disclosed by the invention is used as a quercetin drug delivery system to be applied to preparation of drugs for treating acute lung injury caused by sepsis.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of drugs for acute lung injury caused by sepsis, is a novel quercetin delivery system or dosage form, and specifically relates to a quercetin-loaded TPP-PEG-PCL nano-microcapsule and a preparation method and application thereof. Background Art

[0002] Sepsis is caused by severe infection, burns, trauma and other factors, and is an important cause of multiple organ dysfunction syndrome. Epidemiological surveys show that there are about 31.5 million sepsis patients and 5.3 million deaths from sepsis each year worldwide. The high incidence and mortality of sepsis pose a huge challenge to clinical diagnosis and treatment. The lung is the most vulnerable target organ of sepsis. The incidence of acute lung injury (AIL) secondary to sepsis is as high as 25% to 50%. If ALI patients cannot receive timely treatment, their mortality rate can be as high as 70%. Therefore, timely diagnosis and treatment are the key to saving patients' lives. In the clinical treatment of sepsis and its complications, the widespread use of antibiotics has led to bacterial resistance, increased the risk of adverse drug reactions, and increased the difficulty of treatment. Studies have shown that traditional Chinese medicine has unique advantages in the treatment of bacterial infectious diseases, and can block the progression of sepsis in many aspects, such as enhancing the body's nonspecific immunity and reducing lung damage caused by intestinal endotoxin immunity. Therefore, it is extremely urgent to find new drugs to treat ALI.

[0003] Quercetin (Q), chemical formula is C 15 H 10 O7 is a secondary metabolite widely found in plants in nature. It is present in various vegetables and fruits, such as raspberries, broccoli and apples, as well as foods such as tea, red wine, olive oil and propolis. Quercetin has a wide range of pharmacological effects, and has been widely studied in the fields of antioxidant, anti-inflammatory, antibacterial, anti-tumor and cardiovascular protection. However, due to its poor solubility and almost insoluble in water, its clinical application is greatly limited. In addition, Q has poor cell penetration ability and low bioavailability, and cannot reach the target tissue well to play its role in scavenging reactive oxygen. Therefore, it is urgent to construct and develop a new type of quercetin dosage form to solve the solubility, bioavailability and targeting problems of Q. Summary of the invention

[0004] The purpose of the present invention is to provide a quercetin-loaded TPP-PEG-PCL nanocapsule and a preparation method thereof and an application thereof in the preparation of a drug for treating acute lung injury caused by sepsis. The present invention proposes a new dosage form of quercetin, which has a better therapeutic effect after being applied to a drug for treating acute lung injury caused by sepsis, and has great prospects for promotion and application.

[0005] The implementation process of the present invention is as follows:

[0006] A method for preparing quercetin-loaded TPP-PEG-PCL nanomicelles comprises the following steps:

[0007] (1) dissolving (3-carboxypropyl) triphenylphosphonium bromide and polyethylene glycol-b-polycaprolactone in methanol, adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to the mixture, and performing an esterification reaction in a light-proof environment and under the protection of an inert gas. After the reaction is completed, removing the unreacted small molecule (3-carboxypropyl) triphenylphosphonium bromide, and freeze-drying to obtain TPP-PEG-PCL nanomicelles;

[0008] (2) Quercetin, TPP-PEG-PCL nanomicelles and methanol were mixed in a container, and a dry film was formed at the bottom of the container after rotary evaporation. After freeze-drying, PBS buffer was added and reacted in a water bath. After the reaction, Q@TPP-PEG-PCL nanomicelles were obtained by ultrasonic treatment, microporous membrane filtration and freeze-drying.

[0009] Furthermore, the reaction temperature of the esterification reaction in step (1) is room temperature, and the reaction time is 20 to 30 hours.

[0010] Furthermore, in step (1), unreacted small molecule (3-carboxypropyl)triphenylphosphonium bromide is removed using a dialysis bag.

[0011] Furthermore, in step (2), after mixing quercetin, TPP-PEG-PCL nanomicelles and methanol, dissolution is promoted by slight shaking.

[0012] Furthermore, in step (2), the rotary evaporation water temperature is set at 35-45°C.

[0013] Furthermore, the pH of the PBS buffer in step (2) is 7.4.

[0014] Furthermore, the water bath temperature of the water bath reaction in step (2) is 35 to 45° C., and the water bath reaction time is 10 to 20 min.

[0015] Furthermore, the pore size of the microporous filter membrane in step (2) is 0.2 μM.

[0016] The quercetin-loaded TPP-PEG-PCL nanomicelles obtained by the above method.

[0017] The quercetin-loaded TPP-PEG-PCL nanomicelles obtained by the above method are used as a quercetin drug delivery system in the preparation of a drug for treating acute lung injury caused by sepsis.

[0018] The design idea of ​​the preparation method of the quercetin-loaded TPP-PEG-PCL nanocapsules of the present invention is as follows:

[0019] We modified polyethylene glycol-b-polycaprolactone (PEG-PCL) with triphenylphosphine bromide (TPP) to form TPP-PEG-PCL polymers, and loaded quercetin (Q) into them to prepare quercetin-loaded TPP-PEG-PCL nanocapsules (Q@TPP-PEG-PCL), which are expected to deliver quercetin to the treatment of acute lung injury.

[0020] The application principle of the quercetin-loaded TPP-PEG-PCL nanocapsules of the present invention in the preparation of a drug for treating sepsis-induced acute lung injury is as follows:

[0021] The quercetin-loaded TPP-PEG-PCL nano-microcapsules of the present invention are easily soluble in water and have amphiphilicity. The periphery is a hydrophilic hook material, and the core is a hydrophobic polycaprolactone fragment that is polymerized into a cluster, which has enhanced permeability and a long retention time in inflammatory tissues. Due to the large vascular gaps in lung tissue, the quercetin-loaded TPP-PEG-PCL nano-microcapsules can stay in these gaps and passively accumulate in lung tissue for delivering the quercetin loaded on the micelles. In the process of delivering quercetin, the cell membrane and mitochondrial membrane in the lung tissue are negatively charged, and the surface of the quercetin-loaded TPP-PEG-PCL nano-microcapsules of the present invention is positively charged. Because of the attraction of positive and negative charges, the quercetin-loaded TPP-PEG-PCL nano-microcapsules break through the barriers of the cell membrane and mitochondrial membrane in the lung tissue, and finally target mitochondria to treat acute lung injury caused by sepsis.

[0022] Positive effects of the present invention:

[0023] (1) The quercetin-loaded TPP-PEG-PCL nano-microcapsules of the present invention solve the key problems of quercetin's hydrophobicity, low oral bioavailability (<2%) and low brain permeability. The quercetin-loaded TPP-PEG-PCL nano-microcapsules constructed by the present invention have good stability, can spontaneously assemble into micelles, have high encapsulation efficiency and drug loading, and good in vitro sustained release performance, thereby increasing the solubility and permeability of quercetin, improving bioavailability, and prolonging the half-life of the drug in the blood circulation.

[0024] (2) The quercetin-loaded TPP-PEG-PCL nanocapsules of the present invention achieve the "targeting" effect of mitochondrial targets. The quercetin-loaded TPP-PEG-PCL nanocapsules constructed by the present invention can be well localized in mitochondria after entering MEL-12 cells through fluorescence detection. This fully demonstrates that the quercetin-loaded TPP-PEG-PCL nanocapsules constructed by the present invention can achieve the effect of targeting mitochondria.

[0025] (3) The quercetin-loaded TPP-PEG-PCL nanocapsules constructed by the present invention can solve the core key problems of quercetin such as low solubility and low bioavailability, and can also achieve targeted enrichment of lung target organs and precise treatment of mitochondrial targets. This not only provides a new idea for the drug development of quercetin, but also opens up a new strategy for the treatment of acute lung injury in sepsis, with great potential for clinical and socioeconomic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Characterization diagram of Q@TPPPEG-PCL nanocapsules; (A) 1H-NMR spectrum of TPP-PEG-PCL nanocapsules; (B) Tyndall effect of Q@TPP-PEG-PCL; (C) Transmission electron microscopy image of Q@TPP-PEG-PCL;

[0027] Figure 2 This is the in vitro release curve of Q@TPP-PEG-PCL nanocapsules;

[0028] Figure 3 This is the pharmacokinetic parameter diagram of Q@TPP-PEG-PCL nanocapsules;

[0029] Figure 4 is the critical micelle concentration curve of Q@TPP-PEG-PCL nanocapsules;

[0030] Figure 5 The stability diagram of Q@TPP-PEG-PCL nanocapsules; (A) is the change of particle size over time, and (B) is the change of electromotive force over time;

[0031] Figure 6 Figure 2 is the mitochondrial targeting of Q@TPP-PEG-PCL nanocapsules;

[0032] Figure 7The in vivo effect diagram of Q@TPP-PEG-PCL nanocapsules in treating ALI; (AB) are w / d and MPO in the lungs of septic mice; (C) is the level of ROS; (DE) are MDA and SOD in the lungs; (F) is hematoxylin-eosin (HE) staining of representative lung tissue sections; (GI) are lung cytokines; all data are expressed as mean ± standard deviation; compared with the Ctr group: # P<0.05, ## P<0.01. Compared with LPS group: * P<0.05, ** P < 0.01;

[0033] Figure 8 The in vitro effect diagram of Q@TPP-PEG-PCL nanocapsules in treating ALI; (AB) is the ROS level; (CE) is the cytokine in MLE-12 cells; (FG) is the SOD and MDA levels; all data are expressed as mean ± standard deviation; compared with the Ctr group: # P<0.05, ## P<0.01. Compared with LPS group: * P<0.05, ** P<0.01. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with the embodiments.

[0035] Example 1 Preparation of Q@TPP-PEG-PCL Nanocapsules

[0036] The method for preparing quercetin-loaded TPP-PEG-PCL nanomicelles described in this embodiment comprises the following steps:

[0037] (1) Preparation of TPP-PEG-PCL nanomicelles

[0038] Accurately weigh 4.29 mg of (3-carboxypropyl) triphenylphosphine bromide (TPP) and 25 mg of polyethylene glycol-b-polycaprolactone (PEG-PCL), and then dissolve them in 50 ml of chromatographic methanol. Add 6.2 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 3.2 mg of N-hydroxysuccinimide (NHS) to the mixture to promote the esterification reaction between the carboxyl group of (3-carboxypropyl) triphenylphosphine bromide and the terminal amino group of PEG-PCL. In a light-proof environment, argon gas is introduced to protect the reaction. The reaction is carried out at room temperature for 24 hours, and then the unreacted small molecule TPP (relative molecular weight of 2000) is removed with a dialysis bag. The final product is freeze-dried to obtain TPP-PEG-PCL nano-micelles.

[0039] (2) Preparation of Q@TPP-PEG-PCL nanocapsules

[0040] Accurately weigh 10 mg of quercetin (Q) and 100 mg of TPP-PEG-PCL nanocapsules into a pear-shaped bottle, add 50 ml of chromatographic methanol, and shake slightly to promote full dissolution. Then install the pear-shaped bottle on a rotary evaporator, turn on the rotary evaporator, set the water temperature at 40°C, and form a uniformly distributed dry film at the bottom of the pear-shaped bottle. Then freeze-dry overnight to fully remove residual methanol, add an appropriate amount of PBS buffer with a pH of 7.4 (the amount of PBS buffer is larger than the amount of the dry film to meet the reaction), shake slightly in a 40°C water bath for 15 minutes to promote hydration, treat with ultrasound (power: 250 W, frequency: 40 KHz) for 15 minutes, filter through a 0.2 μM microporous filter membrane, and then freeze-dry for 24 hours to obtain Q@TPP-PEG-PCL nanocapsules.

[0041] Comparative Example: Preparation of Q@PEG-PCL Nanocapsules

[0042] Accurately weigh 10 mg of quercetin (Q) and 100 mg of polyethylene glycol-b-polycaprolactone (PEG-PCL) and put them into a pear-shaped bottle, add 50 ml of chromatographic methanol, and shake slightly to promote full dissolution. Then install the pear-shaped bottle on a rotary evaporator, turn on the rotary evaporator, set the water temperature at 40°C, and form a uniformly distributed dry film at the bottom of the pear-shaped bottle. Then freeze-dry overnight to fully remove residual methanol, add an appropriate amount of PBS buffer with a pH of 7.4, shake slightly in a 40°C water bath to promote hydration, treat with ultrasound (power: 250 W, frequency: 40 kHz) for 15 min, filter through a 0.2 μM microporous filter membrane, and then freeze-dry for 24 h to obtain Q@PEG-PCL nanocapsules.

[0043] The preparation process of PBS buffer in the above method is as follows: weigh 8g NaCl, 0.2g KCl, 0.24g KH2PO4, and 1.44g Na2HPO4 and put them into a clean beaker, add a small amount of ultrapure water and stir to dissolve, then dilute to 1L with ultrapure water and adjust the pH to 7.4.

[0044] 1. Characterization of Q@TPP-PEG-PCL nanocapsules

[0045] 1.1 Nuclear magnetic resonance, particle size distribution, zeta potential, Tyndall phenomenon and electron microscopy analysis

[0046] A small amount of Q@TPP-PEG-PCL nanocapsules were adsorbed, diluted with distilled water, and the zeta potential and the particle size of the micelles were measured using a Zetasizer nano ZSe nanoparticle potentiometer; the Tyndall phenomenon was detected from the side of the micelle solution irradiated with a laser pen. A small amount of Q@TPP-PEG-PCL nanocapsule solution was dropped on a 200-mesh copper grid, slightly dried with an ear bulb, and the morphology of the nanocapsules was observed using a transmission electron microscope.

[0047] The results showed that TPP-PEG-PCL micelles were verified by NMR, such as Figure 1 As shown in A, about δ7.5 is the triphenyl ring peak of TPP, and about δ3.5 and δ1.4 are the characteristic peaks of polymers PEG and PCL, respectively. The results show that TPP-PEG-PCL micelles have been successfully synthesized.

[0048] The average particle size of Q@TPP-PEG-PCL nanocapsules is (16.1±0.3) nm, and the zeta potential is (14.5±0.2) mV. When a laser pen is used to illuminate one side of the micelle solution, a bright beam of light can be seen, showing a good Tyndall effect ( Figure 1 B). Electron microscopy images show that Q@TPP-PEG-PCL nanocapsules are spherical and have regular particles ( Figure 1 C).

[0049] 1.2 Encapsulation efficiency and drug loading

[0050] 2mL of Q@TPP-PEG-PCL nano-microcapsule solution was placed in an ultrafiltration centrifuge tube (retaining the relative molecular weight of 2000), and the solution was centrifuged at low temperature and high speed for 5 minutes at a speed of 15000r / min. The Q encapsulated in the nano-microcapsule was retained in the ultrafiltration centrifuge tube (retaining the relative molecular weight of 2000), and 10 times the volume of methanol was added to destroy the structure of the nano-microcapsule. The mass (Mn) of Q carried in the nano-microcapsule was detected by high performance liquid chromatography, and there was free Q outside the centrifuge tube. The mass (Mo) of free Q outside the micelle was determined by the same method, and the encapsulation efficiency and drug loading rate of Q@TPP-PEG-PCL nano-microcapsule were calculated:

[0051] Encapsulation efficiency = Mn / (Mn+Mo)%.

[0052] Drug loading = Mn / M.

[0053] Mn: free Q amount; Mo: Q amount in micelles; M: Q@TPP-PEG-PCL nanocapsules.

[0054] The results showed that the encapsulation efficiency of Q was 84.7±0.6%. In terms of drug loading, the results showed that the drug loading of Q was 7.81±0.4%.

[0055] 1.3 In vitro release

[0056] 1 mg of Q and 1 mg of Q@PEG-PCL nanomicelles (containing 1 mg of Q) and Q@TPP-PEG-PCL nanomicelles (containing 1 mg of Q) were placed in a spectral laboratory dialysis bag (retained relative molecular weight of 2000), the two ends of the dialysis bag were sealed with plastic clips, and placed in 100 ml of PBS buffer solution with pH = 7.4, and 1.0 g of polysorbate 80 was added as a surfactant to ensure that 1 mg of TEMPO reached the sink condition during the release process. The water temperature was set at 37 ° C, and samples were taken with a mobile gun at the predetermined time points of 1, 2, 5, 8, 10, 12, 16, 18, 24, 48, 72 and 96 h, each time 0.5 ml of sample was drawn, and the same volume of PBS buffer (pH = 7.4) was supplemented to calculate the cumulative release rate of Q in the micelles.

[0057] The results are as follows Figure 2 As shown in the figure, Q was released rapidly in vitro, while Q@PEG-PCL nanomicelles (containing 1 mg Q) and Q@TPP-PEG-PCL (containing 1 mg Q) nanomicelles showed a slow release trend. The drug release rate of Q@TPP-PEG-PCL nanomicelles was the slowest, showing good sustained-release performance.

[0058] 1.4 Pharmacokinetics of Q@TPP-PEG-PCL

[0059] Twenty-four SD rats (half male and half female, weighing 200-250 g) were randomly divided into three groups (N=8): Q (10 mg / kg) group, Q@TPP-PEG-PCL (10 mg / kg) group, and Q@PEG-PCL (10 mg / kg) group. Blood was collected from the eye sockets at 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, and 12 hours after administration. 50 μL of plasma was added with 150 μL of methanol to precipitate protein, centrifuged for 5 minutes, and then centrifuged at 15000 r / min for 10 minutes. 200 μL of supernatant was drawn and dried with liquid nitrogen. High performance liquid chromatography-diode array detector (HPLC-DAD) was used to detect the content of Q in plasma samples, and the blood drug concentration at each time point was calculated.

[0060] like Figure 3 As shown, the results showed that Q@TPP-PEG-PCL nanocapsules significantly improved the bioavailability of Q, thereby prolonging the retention time of the drug.

[0061] 1.5 Critical micelle concentration (CMC)

[0062] Preparation of pyrene solution: 40 μl of pyrene was added to 40 ml of water.

[0063] The TPP-PEG-PCL nanocapsules were prepared into 1-10 μg / mL micellar solutions for detecting CMC, and then mixed with the pyrene solution prepared above, stirred at room temperature for more than 6 hours, and the emission spectrum of pyrene was detected by a fluorescence photometer. 394 and I 373 , with I 394 / I 373 The ratio of fluorescence intensity is the ordinate and the mass concentration of nanocapsules is the abscissa, and a curve is drawn.

[0064] like Figure 4 As shown in the figure, the ratio of pyrene fluorescence intensity I394 / I373 in the core of the micelle changes with the increase of the mass concentration of TPP-PEG-PCL nanocapsules, presenting an approximately S-shaped curve. The inflection point of the curve is exactly the CMC of TPP-PEG-PCL nanocapsules, which is about 5.6μg / mL, indicating that TPP-PEG-PCL can spontaneously assemble into micelles.

[0065] 1.6 Stability

[0066] The Q@TPP-PEG-PCL nanocapsules were placed in a refrigerator at 4°C for 60 days and taken out at 0, 10, 20, 30, 40, 50 and 60 days. The particle size and zeta potential of the nanocapsules were measured using the above method.

[0067] like Figure 5 As shown, compared with the initial results at 0d, the particle size and electromotive force of Q@TPP-PEG-PCL nanocapsules did not change significantly after refrigeration at 4°C for 60 days, and Q@TPP-PEG-PCL micelles were stable at low temperatures.

[0068] 1.7 Mitochondrial targeting

[0069] After incubating Q@TPP-PEG-PCL with MLE-12 cells for 1 and 3 h, the distribution of Q@TPP-PEG-PCL in MLE-12 cells was observed under laser confocal microscopy.

[0070] like Figure 6 As shown, after the blue fluorescent nanocapsules enter the MEL-12 cells, they can be well localized in the mitochondria, and the coincidence coefficient reflects the co-localization of the nanocapsules and mitochondria.

[0071] The therapeutic effect of 2Q@TPP-PEG-PCL on acute lung injury

[0072] 2.1 Establishment of ALI mouse model

[0073] The ALI model was established by intraperitoneal injection of lipopolysaccharide (LPS) 10 mg / kg in mice. The mice (control group) were given an equal amount of normal saline in the same way. The mice were divided into control group (Ctr), LPS (M), LPS+Q group (20 mg / kg), LPS+Q@TPP-PEG-PCL group (containing 20 mg / kg Q). After 6 hours of intervention, LPS (10 mg / kg) was intraperitoneal. Q@TPP-PEG-PCL, Q (both were administered orally).

[0074] 2.2 Establishment of in vitro model

[0075] MEL-12 cells were cultured at 1×10 6 The cells were seeded at a density of 100 μM / well in a 6-well plate for 24 hours, and then divided into the following groups: control group (Ctr), LPS (model, 50 μM) group, LPS+Q (5 mM / mL) group, LPS+Q@TPP-PEG-PCL (containing 5 mM / mLQ) group. The dose was set according to the drug loading of Q in Q@TPP-PEG-PCL. After 24 hours of treatment with Q@TPP-PEG-PCL and Q, the cells were co-incubated with 50 μM LPS for 24 hours, while the control group was treated with an equal amount of PBS buffer (pH = 7.4). The cell supernatant and cells were collected for subsequent detection.

[0076] 2.3 Measurement of lung wet / dry ratio (w / d)

[0077] After the experiment, the right lungs of the mice were removed. The trachea and esophagus were passively separated, the right lung lobe was obtained, and the wet weight was immediately measured. Subsequently, the lungs were dried at 60 °C for 72 hours to remove all moisture, and then the dried lungs were weighed to calculate the wet weight / dry weight ratio of the lungs.

[0078] 2.4 Mitochondrial ROS detection

[0079] The accumulation of ROS was measured using flow cytometry according to the protocol provided by the manufacturer (Thermos Fisher, Massachusetts, USA). Briefly, MEL-12 cells were collected and cultured at 2 × 10 5 After 24 hours of culture, CellROX was added to each well at a final concentration of 5 μM. TM Deep Red reagent, incubated for 30 min at 37° C. To measure ROS content, cells were washed, digested, resuspended in PBS buffer (pH=7.4) and analyzed by flow cytometry.

[0080] 2.5 MPO and cytokine detection

[0081] Serum, cell supernatant, and lung fluid were collected and tested for the measurement of MPO, SOD, MDA, IL-6, IL-1β, and TNF-α. The operation process was carried out strictly according to the instructions of the kit.

[0082] 2.6 Hematoxylin-eosin (HE) staining

[0083] The lungs were fixed with 4% formalin solution, embedded in paraffin, cut into 4 μm thick sections, and placed on glass slides for hematoxylin-eosin staining.

[0084] Statistical analysis

[0085] All data were expressed as mean ± standard deviation (SD), and one-way analysis of variance (ANOVA) and Tukey's multiple comparison test were performed using GraphPad prism 8 (GraphPad Software, USA). p < 0.05 was considered statistically significant. Design Expert 8.0.6.1 was used to analyze the results of the response surface experiment, and Origin 8.0 was used to analyze the results of the response surface experiment.

[0086] 3. Experimental study results of Q@TPP-PEG-PCL in the treatment of acute lung injury

[0087] 3.1 Experimental subjects are septic mice

[0088] (1) Effects of Q@TPP-PEG-PCL on water-to-humidity ratio, ROS, and MPO in septic mice

[0089] like Figure 7 As shown in AC, the w / d, ROS, and MPO levels of the model group were significantly increased compared with the control group, while Q and Q@TPP-PEG-PCL significantly reduced the w / d, ROS, and MPO levels compared with the model group, and the effect of Q@TPP-PEG-PCL was more obvious.

[0090] (2) Oxidative stress, lung histopathology, and lung inflammation in mice

[0091] like Figure 7 As shown in Figures DE, compared with the control group, the MDA level in the lung of the LPS group increased and the SOD level decreased. In addition, Q and Q@TPP-PEG-PCL also significantly reduced MDA and increased SOD in the lung, and the effect of Q@TPP-PEG-PCL was more obvious.

[0092] HE staining was used to observe the pathological changes of lung tissue. Figure 7F), Sepsis lung tissue pathology showed diffuse congestion, local atrophy, widening of alveolar cavity, and inflammatory cell infiltration in the lung tissue of mice in the model group, while Q and Q@TPP-PEG-PCL could significantly restore the above lung tissue pathological changes, and the effect of Q@TPP-PEG-PCL was more obvious.

[0093] Inflammation plays an important role in ALI. The levels of cytokines TNF-α, IL-1β, and IL-6 in the lung tissues of septic mice were higher than those in the control group ( Figure 7 GI), Q and Q@TPP-PEG-PCL significantly reduced the levels of cytokines TNF-α, IL-1β, and IL-6 in lung tissue, and the effect of Q@TPP-PEG-PCL was more obvious.

[0094] 3.2 Experimental subjects are MLE-12 cells

[0095] (1) Effects of Q@TPP-PEG-PCL on reactive oxygen species and cytokines in LPS-induced MLE-12 cells

[0096] like Figure 8 As shown in AE, compared with the control group, LPS can induce the release of cytokines and the production of ROS in the supernatant of MEL-12 cells. Q and Q@TPP-PEG-PCL significantly reduced cytokines and ROS, and the effect of Q@TPP-PEG-PCL was more obvious.

[0097] (2) Effect of Q@TPP-PEG-PCL on LPS-induced oxidative stress in MLE-12 cells

[0098] like Figure 8 As shown in FG, compared with the control group, LPS increased MDA and decreased SOD in MEL-12 cells, while Q and Q@TPP-PEG-PCL significantly reduced SOD elevated by MDA, and the effect of Q@TPP-PEG-PCL was more obvious.

[0099] Example 2

[0100] The steps and parameters are the same as those in Example 1, except that:

[0101] During the preparation of TPP-PEG-PCL nanomicelles, the esterification reaction time was 20 hours.

[0102] During the preparation of Q@TPP-PEG-PCL nanocapsules, the rotary evaporation water temperature was set at 35°C; the water bath temperature was 45°C, and the water bath reaction time was 10 min.

[0103] Example 3

[0104] The steps and parameters are the same as those in Example 1, except that:

[0105] During the preparation of TPP-PEG-PCL nanomicelles, the esterification reaction time was 30 hours.

[0106] During the preparation of Q@TPP-PEG-PCL nanocapsules, the rotary evaporation water temperature was set at 45°C; the water bath temperature was 35°C, and the water bath reaction time was 20 min.

[0107] The peripheral polyethylene glycol of PEG-PCL selected in the present invention has good water solubility, the core PCL is lipophilic, and the modification of PEG-PCL by TPP cation can overcome the barrier of high viscosity cell fluid and mediate the entry of drugs into mitochondria. The Q@TPP-PEG-PCL nano-microcapsules finally prepared make the nanoparticles with smaller volume into regular spheres with good stability and water solubility, which not only improves the bioavailability of Q, but also significantly improves the targeting of Q in mitochondria. Q@TPP-PEG-PCL nano-microcapsules can locate mitochondria well, and its compliance phenomenon is significantly better than that of PEG-PLC nano-microcapsules. Q@TPP-PEG-PCL nano-microcapsules deliver Q to mitochondria.

[0108] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and the specific implementation of the present invention cannot be limited to these descriptions. For ordinary technicians in the field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A method for preparing quercetin-loaded TPP-PEG-PCL nanomicelles, characterized in that: The steps include: (1) dissolving (3-carboxypropyl) triphenylphosphonium bromide and polyethylene glycol-b-polycaprolactone in methanol, adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to the mixture, and performing an esterification reaction in a light-proof environment and under the protection of an inert gas. After the reaction is completed, removing the unreacted small molecule (3-carboxypropyl) triphenylphosphonium bromide, and freeze-drying to obtain TPP-PEG-PCL nanomicelles; (2) Quercetin, TPP-PEG-PCL nanomicelles and methanol were mixed in a container, and a dry film was formed at the bottom of the container after rotary evaporation. After freeze-drying, PBS buffer was added and reacted in a water bath. After the reaction, Q@TPP-PEG-PCL nanomicelles were obtained by ultrasonic treatment, microporous membrane filtration and freeze-drying.

2. The method for preparing quercetin-loaded TPP-PEG-PCL nanomicelles according to claim 1, characterized in that: The reaction temperature of the esterification reaction in step (1) is room temperature, and the reaction time is 20 to 30 hours.

3. The method for preparing quercetin-loaded TPP-PEG-PCL nanomicelles according to claim 1, characterized in that: In step (1), a dialysis bag is used to remove unreacted small molecule (3-carboxypropyl)triphenylphosphonium bromide.

4. The method for preparing quercetin-loaded TPP-PEG-PCL nanomicelles according to claim 1, characterized in that: In step (2), quercetin, TPP-PEG-PCL nanomicelles and methanol are mixed and then gently shaken to promote dissolution.

5. The method for preparing quercetin-loaded TPP-PEG-PCL nanomicelles according to claim 1, characterized in that: In step (2), the rotary evaporation water temperature is set at 35-45°C.

6. The method for preparing quercetin-loaded TPP-PEG-PCL nanomicelles according to claim 1, characterized in that: The pH of the PBS buffer in step (2) is 7.

4.

7. The method for preparing quercetin-loaded TPP-PEG-PCL nanomicelles according to claim 1, characterized in that: The water bath temperature of the water bath reaction in step (2) is 35 to 45° C., and the water bath reaction time is 10 to 20 minutes.

8. The method for preparing quercetin-loaded TPP-PEG-PCL nanomicelles according to claim 1, characterized in that: The pore size of the microporous filter membrane in step (2) is 0.2 μM.

9. The quercetin-loaded TPP-PEG-PCL nanomicelles obtained by the method of claim 1.

10. Use of the quercetin-loaded TPP-PEG-PCL nanomicelles obtained by the method of claim 1 as a quercetin drug delivery system in the preparation of a drug for treating acute lung injury caused by sepsis.

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