Active oxygen response drug release lipid nanoparticles, and preparation method and application thereof

Abstract

The application discloses a kind of active oxygen response drug release lipid nanoparticles and its preparation method and application, belong to the technical field of pharmaceutical preparation, the lipid nanoparticles include functional drug, lipid material and enteric coating material;The functional drug is anti-inflammatory drug or intestinal barrier regulator, the anti-inflammatory drug includes dexamethasone acetate, 5-amino salicylate or sulfasalazine, and the intestinal barrier regulator is PI3K inhibitor;The lipid material is the alpha-tocopherol and monoglyceride of thiole ketone bond bonding, obtained by the reaction of alpha-tocopherol, double carboxyl crosslinking monomer containing thiole ketone bond and monoglyceride;The lipid nanoparticles have active oxygen response drug release and colon sustained-release performance, can promote intestinal barrier repair, reduce inflammatory response and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical preparations, and particularly relates to a reactive oxygen species (ROS)-responsive drug release lipid nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] Inflammatory Bowel Disease (IBD) is a chronic intermittent inflammatory disease that cannot be cured. During the relapse period, the symptoms vary from mild to severe and can disappear or decrease during the remission period. Clinically, it is manifested as Ulcerative Colitis (UC) and Crohn's Disease (CD). The pathological features of UC are mainly diffuse mucosal inflammation extending from the rectum to the proximal end to varying degrees, mainly involving the superficial colonic mucosa, while CD involves the entire gastrointestinal tract and can affect any part of the gastrointestinal tract from the mouth to the anus. The most commonly affected parts are the terminal ileum, colon and perianal region. In contrast to UC, the disease process of CD is transmural, with the possibility of penetrating disease, fistula, ulcer, abscess and stricture formation. Currently, IBD has developed into a rapidly rising worldwide disease, which needs to be solved urgently.

[0003] The etiology of IBD is complex and unknown, and is related to various pathogenic factors, mainly including genetic factors and environmental factors, such as IBD-susceptible genes NOD2, intestinal flora imbalance, immune response imbalance, intestinal barrier damage, etc. Until recently, inducing clinical response and maintaining clinical remission have been the main goals of clinical practice and clinical trials. Since IBD patients need to receive long-term treatment, the quality of life of patients is poor, and the treatment cost is high. Therefore, it is necessary to further study the pathological process of IBD to explore new treatment strategies. In the field of drug delivery, due to rapid metabolism and serious off-target side effects of oral and systemic administration, effective carriers are urgently needed to achieve targeted drug delivery to improve bioavailability and reduce systemic side effects.

[0004] In the prior art, the treatment of IBD mostly focuses on inhibiting excessive immune response to reduce inflammation. Key research shows that intestinal barrier damage precedes IBD, in which barrier dysfunction promotes the entry of intestinal contents, triggering the activation of innate and adaptive immune cells. During the disease process, the barrier function is continuously disrupted, the mucosal immune system is disordered, leading to progressive tissue damage, and eventually leading to clinical IBD, which leads IBD to be included in the category of "barrier organ diseases". Intestinal epithelial cells and their tight junctions are key components of the intestinal epithelial barrier, and tight junctions mainly have three functions: (1) adhesion, maintaining tissue integrity; (2) forming a barrier, controlling the passage of ions, water, molecules, cells and pathogens through the epithelial layer; (3) signal transduction, receiving and transmitting signals that affect cell behavior and tissue function; It is the only dynamic barrier with selective function in the intestinal barrier. Barrier function plays a key role in maintaining intestinal epithelial homeostasis, and the destruction of the intestinal barrier will lead to increased intestinal permeability, causing pathological consequences such as infection and inflammation.

[0005] Regulating tight junction proteins to restore intestinal epithelial barrier function is promising to maintain long-term remission and prevent recurrence of IBD, and is a potential treatment. Claudins are the main components of the structure and function of tight junctions, and the intestinal barrier of IBD patients is generally damaged, and the dysregulation of tight junctions can be observed, for example, the expression of Claudin-1 is observed to be reduced and the expression of Claudin-2 is observed to be increased in a mouse model of chronic colitis. Previous studies have shown that the PI3K / Akt signaling pathway is involved in the regulation of tight junction proteins such as Claudin-1 and Claudin-2.

[0006] The Chinese patent document with the publication number CN103315959A discloses an oral colon-targeted preparation for treating inflammatory bowel disease, which takes 5-aminosalicylic acid as a model drug, takes pH-sensitive Eudragit S100 as a coating material, and is prepared into nanoparticles of 5-aminosalicylic acid coated by Eudragit S100, with a particle size of 70-400 nm. The prepared nanoparticles can protect the 1, 5-aminosalicylic acid drug from being destroyed by gastrointestinal juice until reaching the colon region with pH>7. The Chinese patent document with the publication number CN108014093A discloses a nano-preparation for treating inflammatory bowel disease, which is composed of corn protein nanoparticles and E. coli outer membrane vesicles coated outside the corn protein nanoparticles. The corn protein nanoparticles are wrapped with an antibacterial drug, which is an antibacterial peptide. The invention can quickly and successfully locate in host cells through bacterial outer membrane vesicles, and release the adhesive biological macromolecules and antibacterial components wrapped in the bacterial outer membrane vesicles, repair the mucus layer, inhibit the growth of pathogenic bacteria, and rebuild the balance of intestinal microecosystem. However, the above-mentioned prior art treats inflammatory bowel disease by anti-inflammatory or antibacterial methods, but does not repair the damaged intestinal barrier, resulting in continuous entry of intestinal cavity contents and continuous disorder of the mucosal immune system, and the disease cannot be fundamentally improved.

[0007] Therefore, it is necessary to develop a pharmaceutical preparation capable of promoting intestinal barrier repair, reducing inflammatory response, and having good treatment effect on inflammatory bowel disease. SUMMARY

[0008] The present application provides a reactive oxygen species-responsive drug release lipid nanoparticle, which uses reactive oxygen species-responsive lipid material and enteric coating material to load anti-inflammatory drugs or intestinal barrier modulators, has the performance of reactive oxygen species-responsive drug release and colon sustained release, and can promote intestinal barrier repair and reduce inflammatory response.

[0009] The specific technical solutions adopted are as follows:

[0010] A reactive oxygen species-responsive drug release lipid nanoparticle, comprising a functional drug, a lipid material and an enteric coating material; the functional drug is an anti-inflammatory drug or an intestinal barrier modulator, the anti-inflammatory drug comprises dexamethasone acetate, 5-aminosalicylate or sulfasalazine, and the intestinal barrier modulator is a PI3K inhibitor; the lipid material is an α-tocopherol and a monoglyceride bonded by a thioether bond, which is obtained by reacting α-tocopherol, a double-carboxyl crosslinking monomer containing a thioether bond and a monoglyceride;

[0011] The drug loading capacity of the reactive oxygen species-responsive drug release lipid nanoparticle is 4wt%-20wt%, and the enteric coating material increases the weight of the lipid material by 10wt%-100wt%.

[0012] The alpha-tocopherol and monoglyceride bonded by thioacetone bond in the active oxygen response drug release lipid nanoparticles of the application have active oxygen response, the enteric coating material has a colon targeting drug release function, can better cope with the change of active oxygen level in pathological environment such as inflammation, realize the targeted treatment of drugs at the colon inflammation site, improve the drug efficacy and reduce the side effects.

[0013] Preferably, the enteric coating material is polymethacrylate. Polymethacrylate is an enteric coating widely used in oral preparations, which can protect the lipid nanoparticles from gastric acid damage, has a colon targeting drug release function, and can realize the targeted treatment of drugs at the colon inflammation site.

[0014] Further preferably, the enteric coating material is Eudragit L100, which is an anionic polymer that can make the surface of the lipid nanoparticles negatively charged. Studies have shown that there are a large number of positively charged proteins in the inflamed colon, and the adsorption of positive and negative charges improves the adhesion and residence time of the lipid nanoparticles at the target site, enhances the colon targeting ability of the carrier, and thus increases the local drug concentration at the lesion site.

[0015] The PI3K inhibitor includes PI3K inhibitor LY294002, quercetin, buparlisib or alpelisib.

[0016] The monoglyceride includes but is not limited to glycerol monostearate, glycerol monooleate or glycerol monoacetate, etc.

[0017] Preferably, the preparation method of the alpha-tocopherol and monoglyceride bonded by thioacetone bond is as follows:

[0018] S01. Acetone and 3-mercapto propionic acid are reacted under the action of a catalyst to obtain a mixed solution, the product is precipitated by ice water bath, and after washing and drying, a double-carboxyl crosslinking monomer containing thioacetone bond is obtained;

[0019] S02. The carboxyl group at one end of the double-carboxyl crosslinking monomer containing thioacetone bond is activated by an activating agent, alpha-tocopherol is added, and the first esterification reaction occurs under the action of a catalyst. The carboxyl group at the other end of the double-carboxyl crosslinking monomer containing thioacetone bond is activated by an activating agent, monoglyceride is added, and the second esterification reaction is carried out under the action of a catalyst. After the reaction is completed, the product is precipitated by ice water bath, and the precipitate is obtained by centrifugation. After washing, purification and freeze-drying of the precipitate, the alpha-tocopherol and monoglyceride bonded by thioacetone bond are obtained.

[0020] Further preferably, in step S01, the catalyst is trifluoroacetic acid, and the reaction conditions are room temperature and 8-24 h.

[0021] Further preferably, in step S02, the activating agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide, the catalyst is 4-dimethylaminopyridine, and the molar ratio of the double carboxyl crosslinking monomer containing a thioacetone bond to the two activating agents is 0.5-2:1:1. By controlling the feeding ratio, the carboxyl group at one end of the double carboxyl crosslinking monomer containing a thioacetone bond is activated first, and then the carboxyl group at the other end is activated.

[0022] Preferably, the particle size of the active oxygen-responsive drug release lipid nanoparticles is 100-200 nm, and the encapsulation efficiency is greater than or equal to 90%.

[0023] The application also provides a preparation method of the active oxygen-responsive drug release lipid nanoparticles, comprising the following steps:

[0024] S11. Dissolve the functional drug, alpha-tocopherol bonded with a thioacetone bond, and monoglyceride in an organic solvent to obtain an organic phase, wherein the organic solvent is selected from dimethyl sulfoxide, methanol, ethanol, or N,N-dimethylformamide;

[0025] S12. Inject the organic phase of step S11 into an aqueous phase under heating, and stir in a water bath to obtain a mixed solution; then slowly drop a coating solution into the mixed solution, stir, and centrifuge to obtain the active oxygen-responsive drug release lipid nanoparticles; the coating solution is an organic solution of an enteric coating material.

[0026] Preferably, the mass ratio of the functional drug, alpha-tocopherol bonded with a thioacetone bond, monoglyceride, and enteric coating material is 1:4-20:0.4-20. Within this range, the prepared product lipid nanoparticles have good drug loading and encapsulation efficiency.

[0027] The application also provides a use of the active oxygen-responsive drug release lipid nanoparticles in the preparation of a medicine, wherein the medicine is used for treating inflammatory bowel disease.

[0028] Preferably, the medicine comprises two kinds of the active oxygen-responsive drug release lipid nanoparticles, one kind of active oxygen-responsive drug release lipid nanoparticles loaded with an anti-inflammatory drug, and one kind of active oxygen-responsive drug release lipid nanoparticles loaded with an intestinal barrier regulator.

[0029] Experiments prove that the active oxygen responsive drug release lipid nanoparticles have active oxygen responsive drug release and colon sustained release performance, when the lipid nanoparticles loaded with anti-inflammatory drugs and the lipid nanoparticles loaded with intestinal barrier regulator are used in combination, can promote down-regulation of intestinal barrier repair related signal pathway, up-regulation of tight junction barrier protein expression level, down-regulation of tight junction channel protein, recovery of barrier function, reduction of colon tissue injury, repair of intestinal barrier, reduction of expression of inflammatory factors, inhibition of inflammation, reduction of the level of IBD recurrence biomarker fecal calprotectin, promotion of intestinal barrier repair, reduction of inflammatory response, and is beneficial to the treatment of inflammatory bowel disease.

[0030] Further preferably, the drug administration mode is oral administration. Oral administration has higher patient compliance, is convenient and non-invasive, and is more suitable for the treatment of chronic diseases such as inflammatory bowel disease.

[0031] Compared with the prior art, the present application has the following beneficial effects:

[0032] (1) The present application designs an oral lipid nano drug delivery system with active oxygen responsiveness, which delivers anti-inflammatory drugs or intestinal barrier regulators, can respond to changes in active oxygen levels in pathological environments such as inflammation to release drugs, has colon sustained release performance, can improve drug efficacy and reduce side effects, promote intestinal barrier repair, maintain intestinal epithelial homeostasis, reduce inflammation while preventing further progression of the disease course, and improve treatment effect.

[0033] (2) When the product lipid nanoparticles loaded with anti-inflammatory drugs and the product lipid nanoparticles loaded with intestinal barrier regulators are used in combination to treat inflammatory bowel disease, they can repair the intestinal barrier while treating inflammation, close the door to invasion of external antigens, change the internal inflammatory environment from "hot" to "cold", restore intestinal homeostasis, and achieve prevention, treatment and prevention of recurrence of IBD. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 It is a synthesis route diagram of alpha-tocopherol and monoglyceride with thioacetone bond in Example 1.

[0035] Figure 2 It is a nuclear magnetic resonance spectrum of the double carboxyl crosslinking monomer containing thioacetone bond.

[0036] Figure 3 It is a nuclear magnetic resonance spectrum of alpha-tocopherol and monoglyceride with thioacetone bond.

[0037] Figure 4 It is the characterization results of four kinds of lipid nanoparticles in Table 3 of Example 2, wherein A is a transmission electron microscope image, B is a particle size and dispersion coefficient analysis chart, and C is a potential analysis chart.

[0038] Figure 5A is the morphological change diagram of Dex / LNPs in different concentrations of H2O2 in Example 3, B is the particle size change diagram of Dex / LNPs in different concentrations of H2O2, C is the in vitro release curve diagram of Dex / LNPs and L100 / Dex / LNPs in artificial gastric juice and artificial intestinal juice containing different concentrations of H2O2.

[0039] Figure 6 A is the fluorescence diagram, B is the statistical diagram.

[0040] Figure 7 A is the effect of different administration groups on the transmembrane resistance of Caco-2 cell monolayer in Example 5, B is the transmembrane permeation amount of FITC-Dextran measured by different administration groups, C-E are the TNF-α, IL-6, IL-1β contents of inflammatory cells in different administration groups.

[0041] Figure 8 A is the Western-blot band of tight junction protein regulated by different administration groups in Example 6, B is the immunofluorescence staining diagram of Claudin-1 and Claudin-2, C is the semi-quantitative analysis result of p-Akt protein content, D is the semi-quantitative analysis result of Claudin-1 protein content, E is the semi-quantitative analysis result of Claudin-2 protein content.

[0042] Figure 9 It is the distribution diagram of lipid nanoparticles in mice in Example 7 within 24 hours.

[0043] Figure 10 It is the disease activity index statistical diagram of lipid nanoparticles in model mice in Example 8.

[0044] Figure 11 It is the colon length of lipid nanoparticles in model mice in Example 8.

[0045] Figure 12 It is the colon H&E staining section diagram of lipid nanoparticles in model mice in Example 8.

[0046] Figure 13 It is the content statistical diagram of colon tissue inflammatory factors TNF-α, IL-6, IL-1β and mouse fecal calprotectin of lipid nanoparticles in model mice in Example 8, wherein A is TNF-α, B is IL-6, C is IL-1β, and D is mouse fecal calprotectin.

[0047] In the figure, *, **, ***, **** represent p values less than 0.05, 0.01, 0.001, 0.0001 respectively, and ns represents no significant difference between groups. DETAILED DESCRIPTION

[0048] The application will be further clarified by the following examples, which should not be construed as limiting the scope of the application.

[0049] Example 1: Synthesis of thioactone-linked a-tocopherol and monoglyceride

[0050] The synthesis route of thioactone-linked a-tocopherol and monoglyceride is shown in Figure 1 .

[0051] Precisely weigh 0.5 g of acetone and 0.457 g of 3-mercapto propionic acid into a round-bottom flask, add 200 μL of TFA (trifluoroacetic acid), stir at room temperature for 12 h under nitrogen protection, pour the mixture into an ice water bath to precipitate the product, wash with cold water and cold hexane for 3 times respectively, and vacuum dry to obtain white powder, which is TK crosslinking agent (double carboxyl crosslinking monomer containing thioactone bond). Deuterated chloroform is used as solvent for nuclear magnetic resonance structure confirmation, and the results are shown in Figure 2 .

[0052] Precisely weigh 252 mg of TK crosslinking agent and 192 mg of EDC (1-ethyl-(3- dimethylaminopropyl) carbonyldiimide) into 10 mL of DMF, activate the carboxyl group at one end of the TK crosslinking agent by stirring at 45°C water bath for 30 min, add 430 mg of a-T (a-tocopherol) and 122 mg of DMAP (4-dimethylaminopyridine), and perform the first step esterification reaction by stirring at 45°C water bath for 12 h under nitrogen protection. After the reaction is completed, add 192 mg of EDC, stir at 45°C water bath for 30 min to activate the carboxyl group at the other end of the TK crosslinking agent, then add 359 mg of monostearic acid glyceride (MG) and 122 mg of DMAP, and perform the second step esterification reaction by stirring at 45°C water bath for 12 h under nitrogen protection. After the reaction is completed, pour the mixture into a 100 mL ice water bath to precipitate the product, centrifuge at a speed of 10,000 rpm for 10 min to obtain a precipitate, wash the precipitate with dilute hydrochloric acid and ultrapure water to remove impurities, and further purify with a silica gel column using dichloromethane:methanol (25:1, v:v) as eluent. After freeze-drying, the final purified product, thioactone-linked a-tocopherol and monostearic acid glyceride (denoted as a-T-TK-MG), is obtained. Deuterated chloroform is used as solvent for nuclear magnetic resonance structure confirmation, and the results are shown in Figure 3 .

[0053] Example 2: Preparation and characterization of reactive oxygen species-responsive drug release liposomes

[0054] Precisely weigh 10 mg of α-T-TK-MG prepared in Example 1 and different mass of dexamethasone acetate and PI3K inhibitor LY294002 into 1 mL of anhydrous ethanol to obtain an organic phase, inject the organic phase into 10 mL of water, stir for 5 min at 60°C water bath, stir at room temperature until cooling, respectively obtain a mixed solution containing Dex / LNPs (dexamethasone acetate-loaded nanoparticles) and a mixed solution containing LY / LNPs (LY294002-loaded nanoparticles).

[0055] Precisely weigh different mass of Eudragit L100 into 1 mL of anhydrous ethanol to form a coating solution, slowly drop the coating solution into the above mixed solution respectively, stir for 1 h at 40°C water bath, to obtain lipid nanoparticles L100 / Dex / LNPs and L100 / LY / LNPs.

[0056] Test the micro-morphology, particle size and potential of Dex / LNPs, LY / LNPs, L100 / Dex / LNPs and L100 / LY / LNPs, determine the drug loading and encapsulation efficiency by high performance liquid chromatography, and the results are shown in Tables 1-3 and A-C in Figure 4

[0057] Table 1 Particle size, drug loading and encapsulation efficiency of drug-loaded lipid nanoparticles with different drug doses

[0058]

[0059] Table 2 Particle size, drug loading and encapsulation efficiency of drug-loaded lipid nanoparticles with different coating weight gains

[0060]

[0061] Table 3 Particle size, drug loading and encapsulation efficiency of four kinds of best ratio drug-loaded lipid nanoparticles

[0062]

[0063] Example 3: In vitro release study of lipid nanoparticles

[0064] ​Dex as the test object, artificial gastric juice and artificial intestinal juice containing 0.35% SDS (sodium dodecyl sulfonate) as the release medium, to ensure that the release of the drug meets the conditions of the leakage groove, and to measure the saturation solubility of Dex in the release medium. Prepare a drug content of 1 mg / mL of L100 / Dex / LNPs and Dex / LNPs nanoparticle dispersion in Table 3, take 1 mL into the dialysis bag (MWCO = 3.5 kDa), and place it in artificial gastric juice in a 37°C constant temperature water bath oscillator (100 rpm). After 2 h, place the dialysis bag in artificial intestinal juice containing different concentrations of H2O2 (0, 1, 10 mM), and take samples in turn at 0, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72 h and supplement fresh release medium. The sample is filtered through a 0.22 μm microporous filter, the Dex content is determined by HPLC, and three groups in parallel are used as the free Dex control group. The results are shown in Figure 5 A-C of FIG. 1, Figure 5 A of FIG. 1 shows that due to the presence of active oxygen response chemical bonds, the structure of the lipid nanoparticles is destroyed in the H2O2 solution, Figure 5 B of FIG. 1 proves that the lipid nanoparticles aggregate and the particle size increases due to the structural damage, Figure 5 C of FIG. 1 embodies that the drug release rate is proportional to the concentration of H2O2; all the above results show that the lipid nanoparticles prepared by bonding α-tocopherol and monoglyceride through thioketone bond have active oxygen responsiveness.

[0065] Example 4: Antioxidant ability of lipid nanoparticles

[0066] Take Caco-2 cells in good growth condition, inoculate into 96-well plates at a density of 1×10 4 LPS (lipopolysaccharide, 10 μg / mL), α-T-TK-MG LNPs (100 μg / mL), and MG SLNs (100 μg / mL) were used to treat cells for 24 h, then the culture solution was removed, 50 μL of active oxygen (ROS) fluorescent probe DCFH-DA (20 μmol / L) was added to fully cover the cells, and the cells were incubated in a 37°C cell incubator for 20 min. The cells were washed with HBSS solution three times to completely remove the DCFH-DA that did not enter the cells. The fluorescence absorbance of the cells was detected by a fluorescence enzyme marker, and the results are shown in Figure 6 A and B of FIG. 2, due to the presence of antioxidant α-tocopherol and thioketone that consumes ROS, which endows the carrier with antioxidant ability, α-T-TK-MG LNPs can significantly reduce the fluorescence intensity of DCF in cells, i.e., the level of ROS.

[0067] Example 5: In vitro pharmacodynamic evaluation of drug-loaded lipid nanoparticles

[0068] Caco-2 cells were inoculated into 96-well plates at a density of 2×105 / mL were seeded in the upper chamber of Transwell with 0.4 μm pore size and cultured for 21 days to form Caco-2 cell monolayer model. The cell monolayer was washed with HBSS solution for three times, and then the upper chamber of the cell monolayer was treated with LPS solution, LPS solution and mixed solution of Dex and LY294002, LPS solution and Dex / LNPs, LPS solution and LY / LNPs, LPS solution and mixed solution of Dex / LNPs and LY / LNPs (LPS final concentration was 10 μg / mL, Dex final concentration was 10 μg / mL, LY final concentration was 10 μg / mL, Dex:LY294002 = 1:1). The resistance values at 0, 3, 6, 9, 12 h were determined in sequence by using trans-epithelial electrical resistance meter. After 12 h, the drug solution was discarded, and the cell monolayer was washed with HBSS solution for three times. 1.5 mL of fresh HBSS solution was added to the lower chamber of Transwell, and 2 mg / mL FITC-Dextran 4-containing HBSS solution was added to the upper chamber. The transmembrane transport was carried out at 37°C for 2 h. After the end of the transport, the transport medium in the lower chamber was collected and the content of FITC-Dextran 4 in the lower chamber was determined by using a fluorescence spectrophotometer.

[0069] Caco-2 cells were seeded in a six-well plate at a density of 1 x 10 5 / mL. When the cell confluence reached 70%-80%, the cells were treated with LPS solution, LPS solution and mixed solution of Dex and LY294002, LPS solution and Dex / LNPs, LPS solution and LY / LNPs, and LPS solution and mixed solution of Dex / LNPs and LY / LNPs. The cells were cultured in an incubator for 24 h. After 24 h, the supernatant was collected, and the levels of inflammatory factors TNF-α, IL-6 and IL-1β released by the inflammatory stimulated Caco-2 cells were detected according to the instructions of the Elisa kit.

[0070] The results are shown in A-E of FIG. 6, wherein A and B show that the synergistic treatment of the two drug-loaded nanoparticles can protect the barrier function of the cell monolayer, and C-E show that the synergistic treatment of the two drug-loaded nanoparticles can achieve good anti-inflammatory effect. The combination of the two advantages can amplify the efficacy. Figure 7

[0071] Example 6: In vitro pharmacodynamic mechanism of drug-loaded lipid nanoparticles

[0072] ​The treated cells in Example 5 were washed with pre-cooled PBS for 2 times, 3 min each time, 100 μL of cell lysis solution was added to each well, and the cells were lysed on ice for 30 min, the well plate was blown every 10 min, the lysed cell suspension was transferred to a centrifuge tube and frozen and centrifuged (13000 rpm, 10 min, 4°C), the supernatant was taken, and the protein was quantified by a BCA kit, and the expression levels of Claudin-1 and Claudin-2, which are tight junction proteins, were detected by Western blotting for qualitative and semi-quantitative analysis.

[0073] Caco-2 cells were seeded in a 24-well plate containing a round glass slide at a density of 1 x 10 4 / mL, and when the cell confluence reached 70%-80%, drug treatment was performed, and the groups were as follows: LPS solution, mixed solution of LPS solution and Dex and LY294002, LPS solution and Dex / LNPs, LPS solution and LY / LNPs, and mixed solution of LPS solution and Dex / LNPs and LY / LNPs, and the plate was incubated in an incubator for 24 h. After the drug treatment was completed, the culture solution was discarded, and the cells were washed with PBS three times. An appropriate amount of 4% paraformaldehyde was added to each well for fixation at room temperature for 20 min, and then the cells were washed with PBS three times. The cell monolayer was blocked with 5% BSA for 30 min. After the blocking was completed, the cells were incubated with Claudin-1 and Claudin-2 primary antibodies (1:200, dissolved in 5% BSA) at 4°C overnight. After the incubation of the primary antibodies was completed, the cells were washed with PBS three times, 10 min each time, and then the cells were incubated with fluorescent secondary antibodies (1:200, dissolved in 5% BSA) at room temperature for 1 h. After the incubation of the secondary antibodies was completed, the cells were washed with PBS three times in the dark, and then the cells were stained with a nuclear staining reagent Hoechst 33342 (10 μg / mL) for 10 min, and then the cells were washed with cold PBS three times. The round glass slide in the 24-well plate was taken out, inverted on a pre-dropped glycerol-loaded glass slide, and mounted. The expression of Claudin-1 and Claudin-2, which are tight junction proteins, was observed under an inverted laser confocal microscope.

[0074] The results are shown in A-E of FIG. 7, which show that the synergistic treatment of the two drug-loaded nanoparticles protects the intestinal barrier by inhibiting the PI3K / Akt signaling pathway and regulating Claudin-1 and Claudin-2, which are tight junction proteins playing an important role in the intestinal barrier. Figure 8

[0075] Example 7: In vivo distribution of drug-loaded lipid nanoparticles

[0076] ​C57BL / 6 mice were used as model animals, and a 3% (w / v) dextran sodium sulfate (DSS) solution was prepared. The C57BL / 6 mice were allowed to freely drink the 3% DSS solution for one week to obtain a colonitis animal model. 10 mg of α-T-TK-MG was precisely weighed and dissolved in 0.5 mL of anhydrous ethanol. Then, 40 μL of a near-infrared dye DiR / DMSO solution (5 mg / mL) was added, and the mixture was dissolved by stirring in a 60°C water bath. DiR / LNPs were prepared by the solvent diffusion method, and L100 / DiR / LNPs were prepared by the double solvent diffusion method (the steps were similar to those in Example 2).

[0077] The normal mice and the colonitis mice were randomly divided into two groups (n=3) respectively, and were fasted for 12 h before the experiment. The first group of normal mice was administered with 0.5 mL of DiR / LNPs by gavage, and the second group of normal mice was administered with 0.5 mL of L100 / DiR / LNPs by gavage. The first group of colonitis mice was administered with 0.5 mL of DiR / LNPs by gavage, and the second group of colonitis mice was administered with 0.5 mL of L100 / DiR / LNPs by gavage. The mice were sequentially anesthetized with chloral hydrate (4%, w / v) at 2, 6, 12, and 24 h, and the fluorescence signal distribution of the lipid nanoparticles in vivo was observed by using the live imaging technology.

[0078] Figure 9 It was shown that the accumulation of the enteric-coated nanoparticles in the colonitis mice was higher than that in the healthy mice, and the accumulation of the coated nanoparticles was higher than that of the uncoated nanoparticles under the same healthy condition, indicating that the coated nanoparticles could well target the colon of the colonitis mice and had a strong retention capacity.

[0079] Example 8: In vivo pharmacodynamic evaluation of drug-loaded lipid nanoparticles

[0080] C57BL / 6 mice were randomly divided into 6 groups (n=6): Control group (drinking normal water), PBS group (drinking 3% DSS solution), mixed solution of L100 / Dex / LNPs and L100 / LY / LNPs, L100 / Dex / LNPs group, L100 / LY / LNPs group and mixed solution of Dex and LY294002 free drug. The drug dosage was 0.1 mg / kg. The administration of the drug was used to treat colitis mice in a timely manner. During treatment, changes in the Disease Activity Index (DAI) of mice were recorded. The DAI was calculated by combining three factors: percentage weight loss (less than 1 = 0, 1-5 = 1, 5-10 = 2, 10-15 = 3, greater than 15 = 4), stool consistency (normal = 0, loose stool = 2, diarrhea = 4), and fecal bleeding (normal = 0, occult blood = 2, overt bleeding = 4). The total score of these three factors was divided by 3 to obtain the DAI value. That is, DAI = (Body Mass Index + Stool Shape + Bleeding) / 3. After treatment, mice were euthanized by cervical dislocation. The colon was harvested, its length measured, and the contents carefully rinsed with saline. The colon was fixed with 4% paraformaldehyde, and a portion of the colon was embedded in paraffin. Sections were then stained with H&E for observation and analysis. Proteins were extracted from another portion of the colon, and the levels of inflammatory factors TNF-α, IL-6, and IL-1β were measured using ELISA. Mouse feces were collected, proteins were extracted, and the content of fecal calprotectin was determined using the ELISA method.

[0081] The results are as follows Figures 10-13 As shown, Figure 10 This indicates that the synergistic treatment with the two drug-loaded nanoparticles can minimize the disease activity index. Figure 11 This indicates that the synergistic treatment with the two drug-loaded nanoparticles best inhibits colonic atrophy. Figure 12 This indicates that the synergistic treatment with the two drug-loaded nanoparticles can inhibit epithelial destruction, crypt structural abnormalities, goblet cell depletion, and immune cell infiltration, thereby reducing colonic tissue damage. Figure 13 The AC in the figure indicates that the synergistic treatment of the two drug-loaded coated nanoparticles can achieve an anti-inflammatory effect. Figure 13 The figure D represents the statistical graph of fecal calprotectin in mice, a biomarker for inflammatory bowel disease relapse, indicating that synergistic treatment with two drug-coated nanoparticles can reduce the likelihood of relapse.

[0082] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A lipid nanoparticle for reactive oxygen species-responsive drug release, characterized in that, The product comprises functional drugs, lipid materials, and enteric-coated materials. The functional drugs are anti-inflammatory drugs or intestinal barrier modulators. The anti-inflammatory drugs include dexamethasone acetate, 5-aminosalicylate, or sulfasalazine. The intestinal barrier modulators are PI3K inhibitors such as LY294002 or quercetin. The lipid materials are α-tocopherol and monoglycerides linked by thioketone bonds, obtained by reacting α-tocopherol, a dicarboxylic acid crosslinking monomer containing thioketone bonds, and a monoglyceride, as shown in the following structural formula: The drug loading of the reactive oxygen species responsive drug-releasing lipid nanoparticles is 4wt% to 20wt%, and the weight gain of the enteric coating material is 10wt% to 100wt% of the lipid material. The preparation method of the aforementioned reactive oxygen species responsive drug-releasing lipid nanoparticles includes the following steps: S11. Dissolve the functional drug and the α-tocopherol and monoglyceride bonded by the thioketone in an organic solvent to obtain an organic phase, wherein the organic solvent is selected from dimethyl sulfoxide, methanol, ethanol or N,N-dimethylformamide. S12. The organic phase from step S11 is injected into the aqueous phase under heating conditions and stirred in a water bath to obtain a mixture; then the coating solution is slowly added dropwise to the mixture, stirred, and centrifuged to obtain the lipid nanoparticles with reactive oxygen species responsive drug release. The coating solution is an organic solution of enteric coating materials.

2. The lipid nanoparticles for reactive oxygen species-responsive drug release according to claim 1, characterized in that, The enteric coating material is polymethyl methacrylate.

3. The lipid nanoparticles for reactive oxygen species-responsive drug release according to claim 1, characterized in that, The method for preparing the thioketone-bonded α-tocopherol and monoglyceride includes the following steps: SO1. Acetone and 3-mercaptopropionic acid react under the action of a catalyst to obtain a mixture. The mixture is then subjected to an ice-water bath to precipitate the product. After washing and drying, a dicarboxyl crosslinked monomer containing a thioketone bond is obtained. S02. The carboxyl group at one end of the dicarboxyl crosslinked monomer containing a thioketone bond is activated by an activator, α-tocopherol is added, and the first esterification reaction occurs under the action of a catalyst. Then, the carboxyl group at the other end of the dicarboxyl crosslinked monomer containing a thioketone bond is activated by an activator, and monoglyceride is added, and the second esterification reaction occurs under the action of a catalyst. After the reaction is completed, the product is precipitated in an ice-water bath, centrifuged to obtain a precipitate, and the precipitate is washed, purified, and freeze-dried to obtain the thioketone bonded α-tocopherol and monoglyceride.

4. The lipid nanoparticles for reactive oxygen species-responsive drug release according to claim 3, characterized in that, In step S01, the catalyst is trifluoroacetic acid; and / or, in step S02, the activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide, the catalyst is 4-dimethylaminopyridine, and the molar ratio of the dicarboxylic acid crosslinking monomer containing thioketone bonds to the two activators is 0.5 to 2:1:

1.

5. The method for preparing lipid nanoparticles with reactive oxygen species responsive drug release according to any one of claims 1-4, characterized in that, Includes the following steps: S11. Dissolve the functional drug and the α-tocopherol and monoglyceride bonded by the thioketone in an organic solvent to obtain an organic phase, wherein the organic solvent is selected from dimethyl sulfoxide, methanol, ethanol or N,N-dimethylformamide. S12. The organic phase from step S11 is injected into the aqueous phase under heating conditions and stirred in a water bath to obtain a mixture; then the coating solution is slowly added dropwise to the mixture, stirred, and centrifuged to obtain the lipid nanoparticles with reactive oxygen species responsive drug release. The coating solution is an organic solution of enteric coating materials.

6. The method for preparing lipid nanoparticles with reactive oxygen species-responsive drug release according to claim 5, characterized in that, The mass ratio of the functional drug, thioketone-bonded α-tocopherol, monoglyceride, and enteric coating material is 1:4-20:0.4-20.

7. The application of the reactive oxygen species-responsive drug-releasing lipid nanoparticles according to any one of claims 1-4 in the preparation of drugs, characterized in that, The aforementioned drug is used to treat inflammatory bowel disease.

8. The application of the reactive oxygen species-responsive drug-releasing lipid nanoparticles according to claim 7 in the preparation of drugs, characterized in that, The drug comprises two types of reactive oxygen species (ROS) responsive drug-releasing lipid nanoparticles, one ROS responsive drug-releasing lipid nanoparticle loaded with an anti-inflammatory drug, and one ROS responsive drug-releasing lipid nanoparticle loaded with an intestinal barrier regulator.

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