Chiral nano-drug based on LPS, cfDNA and ROS removal strategy, preparation method of chiral nano-drug, construction method of delivery system and application of chiral nano-drug

Abstract

The invention provides a chiral nano-drug based on LPS, cfDNA and ROS removal strategies, a preparation method of the chiral nano-drug, a construction method of a delivery system and application of the chiral nano-drug, and the preparation method comprises the following steps: weighing PEI (polyethyleneimine) and L / D-tartaric acid L / D-TA, dissolving the PEI and the L / D-TA in deionized water, and standing to prepare a chiral template PEI-L / D-TA; the polyethyleneimine tartaric acid template catalyzes an organic-inorganic mixed silicon source through a simulated biomineralization reaction to prepare a chiral nano-drug PEI-L / D-TA (at) MON; the PEI-L / D-TA (at) MON is loaded with five therapeutic drugs through an in-situ synthesis method so as to play a synergistic therapeutic effect. The nano-drug with LPS, cfDNA and ROS removal capability is successfully prepared by adopting a one-step method, so that the nano-drug can remove pathogens and a plurality of pathogenic factors such as LPS, cfDNA and ROS through physical adsorption, electrostatic interaction and reduction reaction, the inflammatory cascade reaction is inhibited, the nano-drug shows superiority in the aspect of treatment effect on inflammatory bowel diseases, and the nano-drug has a good application prospect. The application potential in the field of nano medicine is huge, and the material can be used as a drug carrier for loading various clinical drugs to achieve a synergistic treatment effect.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-drugs and drug delivery, and particularly relates to a chiral nano-drug based on LPS, cfDNA and ROS scavenging strategies, a preparation method thereof, a construction method of a delivery system, and an application. Background Art

[0002] Inflammatory bowel disease (IBD) is a disease characterized by chronic intestinal inflammation involving the ileum, rectum, and colon. Among them, Crohn's disease (CD) and ulcerative colitis (UC) are two main types of IBD. An epidemiological statistic points out that the incidence rate of IBD is gradually increasing globally and the incidence rate in some parts of Asia is still rising, which deserves wide attention. The pathogenesis of IBD is the result of the interaction of factors such as genetics, environment, intestinal flora, and immune response. The imbalance of intestinal flora accelerates the progression of intestinal inflammation, and the intestinal barrier formed by intestinal epithelial cells and the innate immune system is also damaged. The reason why the disease is difficult to cure lies in the complex pathogenesis: various external stimuli such as the endotoxin lipopolysaccharide (LPS) secreted by pathogens and the free DNA (cfDNA) in the intestine trigger an abnormal immune response of the body to produce a large number of inflammatory factors, and the reactive oxygen species (ROS) generated by the recruited immune cells working will cause the apoptosis of intestinal cells and produce new inflammatory factors to damage the intestine, resulting in the release of cfDNA and the further displacement of LPS to the intestinal mucosa. These important pathogenic factors and pathological molecules are interconnected and form a vicious cycle, causing the disease to develop continuously. Clinically, simple symptomatic treatment methods such as antidiarrheal, immunomodulatory, antibacterial and anti-inflammatory cannot achieve a cure for the multi-pathogenic causes, and are restricted by the non-specific distribution of drugs, etc. The remission rate in actual treatment does not exceed 30-60%. If a drug delivery system that specifically locates the lesion can be constructed to simultaneously remove multiple upstream pathogenic factors, block the vicious cycle path, and inhibit the inflammatory cascade reaction in the development process of IBD, it will become an effective strategy for the treatment of IBD.

[0003] Clinically, symptomatic treatment is generally carried out for IBD. For example, salicylic acid drugs such as Mesalazine (Mes) inhibit the synthesis of prostaglandins and play an anti-inflammatory role; glucocorticoids such as Dexamethasone (Dex) stabilize cell and lysosomal membranes and improve the state of ulcerative colitis; immunosuppressants such as Tacrolimus (Tac) reduce abnormal immune activation and decrease the sensitivity of the intestine to various stimulatory factors; antibiotics such as Berberine (Ber) and Norfloxacin (Nor) can treat infectious abdominal pain and diarrhea caused by pathogenic bacteria. The clinical thinking of the above treatment regimens mainly targets single factors or specific symptoms in the pathogenic pathway of IBD, lacking the regulation of upstream stimulatory factors and the vicious chain of mutual causation in the disease, leaving a hidden danger for disease recurrence. In addition, from the perspective of the inherent properties of drugs, the above drugs are all BCS class II-IV drugs and biologic macromolecule drugs. Defects in chemical properties such as the crystal form, solubility, and permeability of the drugs themselves lead to problems with drugability and dissolution, affecting drug absorption; the colon-retention ability of drug preparations is insufficient, and they are rapidly cleared by intestinal mucus, making it difficult to achieve the ideal local drug concentration.

[0004] In recent years, nanomedicine has shown great potential in disease treatment. The first-generation drug delivery system for IBD encapsulates drugs in polymers such as liposomes and solid lipid nanoparticles to prepare drug-loaded nanoparticles, which play the function of drug carriers, improve drug stability, and enhance solubility. The second-generation nanomedicine is based on stimulus-responsive and targeted preparations on this basis, including delivery systems based on the physiological characteristics of the colon, pH or reactive oxygen species stimulus-responsive drug release systems based on the inflammatory microenvironment, and targeted systems for mannose receptors, etc. The third-generation nanomedicine uses active biological or microbial matrices such as cells, bacteria, biofilms, and exosomes to escape immune recognition in the body through sophisticated "camouflage" to achieve the purpose of delivering drugs or improving the immune and microbial environment. The latest generation of nanomedicine uses nanomaterials themselves as drugs, targeting multiple targets and pathways in disease occurrence, and can exert functions in a drug-free environment, and can treat IBD alone or in combination with other drugs. It is also the current hot spot and frontier in the treatment of inflammatory diseases using nanomedical means.

[0005] Mesoporous silica has a stable structure, adjustable morphology and pore channels, uniform and ordered structure, simple preparation conditions, low preparation cost, and can be mass-produced. As a drug delivery system, it has a large surface area and pore volume ratio, and is rich in silanol groups, which are easy to modify on the surface, enabling the drug-loading system to obtain more abundant functions. Moreover, mesoporous silica nanoparticles have good biosafety, physiological non-toxicity, and degradability; virus mesoporous silica nanoparticles with uniform structure can enter living cells in large numbers and show an uptake level significantly higher than that of solid silica nanoparticles and conventional mesoporous silica nanoparticles;

[0006] In summary, to construct a nano-drug based on mesoporous silica, targeting the colonic lesion environment, with the ability to remove stimulating factors such as cfDNA, reactive oxygen species, and LPS in the pathological environment, so as to further improve the efficiency, intelligence, and targeting of the drug, and it is expected to achieve efficient treatment of IBD. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a nano-drug based on LPS, cfDNA, and ROS scavenging strategies, its preparation method, construction method of the delivery system, and application to achieve synergistic and efficient treatment of IBD.

[0008] The technical solution of the present invention is as follows: First, the present invention provides a preparation method of a chiral silica nano-drug PEI-L / D-TA@MON based on lipopolysaccharide (LPS), extracellular free DNA (cfDNA), and reactive oxygen species (ROS) scavenging strategies; including:

[0009] 1) Preparation of the biomimetic chiral template PEI-L / D-TA: Weigh polyethyleneimine PEI and L / D-tartaric acid L / D-TA, dissolve them in deionized water, and let it stand to obtain the chiral template PEI-L / D-TA;

[0010] 2) Preparation of the inorganic / organic hybrid silicon source MON: Mix the inorganic silicon source TMOS, the organic silicon source BTES, and absolute ethanol at room temperature to obtain the inorganic / organic hybrid silicon source MON;

[0011] 3) Obtain the aqueous phase of the chiral template PEI-L / D-TA, that is, the PEI-L / D-TA template solution, add it to an EP tube, and add the inorganic / organic silicon source mixture MON to the EP tube. After mixing, add the silane coupling agent APTES, quickly shake it until it gels, dry it, and grind it to obtain the chiral nano-drug PEI-L / D-TA@MON.

[0012] Second, the present invention also provides the chiral nano-drug PEI-L / D-TA@MON prepared by the above method.

[0013] Third aspect, the present invention provides the application of the chiral nano-drug PEI-L / D-TA@MON prepared by the method, and the application of the chiral nano-drug PEI-L / D-TA@MON in the treatment of inflammatory bowel disease.

[0014] Fourth aspect, the present invention provides a method for constructing a chiral nano-drug delivery system Mes / Ber / Dex / Nor / Tac-PEI-L-TA@MON using the chiral nano-drug

[0015] PEI-L / D-TA@MON prepared by the method. Using mesalazine (Mes), berberine (Ber), dexamethasone (Dex), norfloxacin (Nor), and tacrolimus (Tac) as model drugs and PEI-L-TA@MON as a carrier, drug loading is carried out by the in-situ drug loading method to obtain the chiral nano-drug delivery system Mes / Ber / Dex / Nor / Tac-PEI-L-TA@MON.

[0016] It includes: respectively dissolving Mes / Ber / Dex / Nor / Tac in water or ethanol according to their dissolution properties to prepare Mes / Ber / Dex / Nor / Tac drug solutions; respectively loading the Mes / Ber / Dex / Nor / Tac drug solutions into the pores of the chiral nano-drug PEI-L / D-TA@MON by in-situ loading to correspondingly obtain the chiral nano-drug delivery system Mes / Ber / Dex / Nor / Tac-PEI-L-TA@MON.

[0017] Fifth aspect, the present invention provides the application of the above-prepared chiral nano-drug delivery system

[0018] Mes / Ber / Dex / Nor / Tac-PEI-L-TA@MON, and the application of the chiral nano-drug delivery system Mes / Ber / Dex / Nor / Tac-PEI-L-TA@MON in the treatment of inflammatory bowel disease.

[0019] The present invention provides a nano-drug based on LPS, cfDNA and ROS scavenging strategies, its preparation method, a construction method of a delivery system, and applications. In the preparation method, the chiral nano-drug PEI-L- / D-TA@MON is biomimetically and rapidly synthesized under the catalysis of the biomimetic chiral template PEI-L / D-TA, with simple conditions, high yield and uniform particle size, and good morphology. In the chiral nano-drug, polyethyleneimine, MON and mesoporous silica can respectively scavenge stimulatory factors such as cfDNA, reactive oxygen species and LPS in the pathological environment through electrostatic interaction, redox reaction and adsorption in situ. After oral administration, the chiral nano-drug is efficiently retained in the intestine mediated by the adhesion of mesoporous silica and tartaric acid chirality, and specifically locates at the positively charged lesion site through the negative charge of carboxyl to exert a therapeutic effect. It can not only improve downstream symptoms (scavenging inflammatory factors and ROS, inhibiting the inflammatory cascade reaction, reducing oxidative damage), but also alleviate upstream mechanisms (scavenging cfDNA and LPS, blocking the vicious cycle path, improving the immune state). In addition, PEI-L- / D-TA@MON can act as a drug carrier to achieve in-situ and efficient loading of various therapeutic drugs, which is beneficial to improving the drug release state and enhancing the targeting property, and realizing the synergistic and efficient treatment of IBD, providing a reference for nano-drug design and subsequent research on inflammatory bowel disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings herein are incorporated into the specification and constitute a part of this specification, showing embodiments consistent with the present invention, and are used together with the specification to explain the principles of the present invention.

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

[0022] Figure 1 Schematic diagram of the synthesis process of the chiral nano-drug provided by the disclosed embodiments of the present invention;

[0023] Figure 2 Appearance and morphology of each stage of the synthesis of the chiral nano-drug provided by the disclosed embodiments of the present invention;

[0024] Figure 3 Morphology characterization of the chiral nano-drug provided by the disclosed embodiments of the present invention (A: TEM image; B: SEM image; C: Particle size distribution in SEM; D: EDS mapping image of PEI-L-TA@MON);

[0025] Figure 4 Infrared spectrum diagram of the chiral nano-drug provided by the disclosed embodiments of the present invention;

[0026] Figure 5 XPS elemental analysis spectra of the chiral nanodrugs provided by the disclosed embodiments of the present invention (A: XPS spectra of the MONs series of nanodrugs; B: XPS spectra of PEI-L-TA@MON);

[0027] Figure 6 XRD analysis (A) and SAXD analysis (B) of the chiral nanodrugs provided by the disclosed embodiments of the present invention;

[0028] Figure 7 Thermogravimetric analysis (TGA) of the chiral nanodrugs provided by the disclosed embodiments of the present invention;

[0029] Figure 8 Circular dichroism spectrum of the chiral nanodrugs provided by the disclosed embodiments of the present invention;

[0030] Figure 9 Nitrogen adsorption curve (A) and BJH pore size distribution curve (B) of the chiral nanodrugs provided by the disclosed embodiments of the present invention;

[0031] Figure 10 Instantaneous contact angle images (A) and instantaneous contact angles (B) of the chiral nanodrugs provided by the disclosed embodiments of the present invention;

[0032] Figure 11 Zeta potential (A) of the agglutinated liquid of the chiral nanodrugs and Zeta potential (B) of the MONs series of nanodrugs provided by the disclosed embodiments of the present invention;

[0033] Figure 12 Fluorescence stability of RITC-labeled PEI-L / D-TA@MON of the chiral nanodrugs provided by the disclosed embodiments of the present invention in different media;

[0034] Figure 13 Oil-water partition images (A) and oil-water partition coefficients (B) of the chiral nanodrugs provided by the disclosed embodiments of the present invention;

[0035] Figure 14 Clearance mechanisms of three pathogenic factors of the chiral nanodrugs provided by the disclosed embodiments of the present invention;

[0036] Figure 15 PEI-L-TA@MON after incubation of the chiral nanodrugs provided by the disclosed embodiments of the present invention with H2O2 (A: infrared spectrum; B: XPS spectrum; C: TEM);

[0037] Figure 16 Contact angle of MONs after incubation of the chiral nanodrugs provided by the disclosed embodiments of the present invention with H2O2;

[0038] Figure 17Degradation trend of the chiral nanomedicine PEI-L / D-TA@MON provided by the disclosed embodiments of the present invention after incubation in different media;

[0039] Figure 18 Color (A) of the chiral nanomedicine provided by the disclosed embodiments of the present invention before and after adsorption by RhB and EB solutions; Time adsorption curves of PEI-L / D-TA@MON for RhB and EB;

[0040] Figure 19 Time adsorption curves of the chiral nanomedicine PEI-L / D-TA@MON provided by the disclosed embodiments of the present invention for cfDNA (A) and LPS (B);

[0041] Figure 20 Free radical scavenging ability of the chiral nanomedicine PEI-L-TA@MON provided by the disclosed embodiments of the present invention (A: reaction formula; B: color change; C: scavenging percentage);

[0042] Figure 21 Time scavenging curve of the chiral nanomedicine PEI-L-TA@MON provided by the disclosed embodiments of the present invention for DPPH;

[0043] Figure 22 Catalytic activity of hydrogen peroxide of the chiral nanomedicine PEI-L-TA@MON provided by the disclosed embodiments of the present invention;

[0044] Figure 23 Free radical scavenging ability of the chiral nanomedicine PEI-L-TA@MON provided by the disclosed embodiments of the present invention (A: ·O2-; B: ·OH; C: H2O2);

[0045] Figure 24 Free radical scavenging ability of the chiral nanomedicine PEI-L-TA@MON provided by the disclosed embodiments of the present invention (A: ·O2-; B: ·OH; C: quantitative analysis);

[0046] Figure 25 Diffusion images (A) and diffusion efficiencies (B) of the RITC-labeled nanomedicine provided by the disclosed embodiments of the present invention in different media;

[0047] Figure 26 Movement trajectory diagrams of the chiral nanomedicine provided by the disclosed embodiments of the present invention in different media;

[0048] Figure 27 Logarithmic distributions of the mean square displacement MSD (A) and the effective diffusion coefficient (Deff) of the chiral nanomedicine provided by the disclosed embodiments of the present invention (B);

[0049] Figure 283D CLSM images (A), fluorescence intensity (B), and depth (C) of chiral nanodrugs penetrating the colon segments of humans, healthy rats, and DSS model rats provided by the disclosed embodiments of the present invention;

[0050] Figure 29 Intestinal adhesion ability scheme (A) of chiral nanodrugs and appearance of the eluent (B) provided by the disclosed embodiments of the present invention;

[0051] Figure 30 Elution time curve (A), fluorescence intensity (B), intestinal segment appearance, and fluorescence images (C) of chiral nanodrugs provided by the disclosed embodiments of the present invention;

[0052] Figure 31 CLSM images of chiral nanodrugs retained on the intestinal mucosa provided by the disclosed embodiments of the present invention;

[0053] Figure 32 Fluorescence intensity and penetration depth in the CLSM images of chiral nanodrugs retained on the intestinal mucosa provided by the disclosed embodiments of the present invention;

[0054] Figure 33 CLSM images of the permeability of chiral nanodrugs provided by the disclosed embodiments of the present invention (A: incubated with the human ex vivo intestine for 1.5 h; B: mouse colon 2 h after oral administration; C: DSS model mouse colon 2 h after oral administration);

[0055] Figure 34 Fluorescence images of nanodrugs labeled with RITC in the GIT provided by the disclosed embodiments of the present invention;

[0056] Figure 35 Fluorescence intensity and penetration depth of nanodrugs penetrating the intestinal mucosa provided by the disclosed embodiments of the present invention (A); fluorescence intensity of the GIT and major organs after oral administration (B);

[0057] Figure 36 Blood compatibility images (A) and hemolysis rate (B) of chiral nanodrugs provided by the disclosed embodiments of the present invention;

[0058] Figure 37 Body weight (A) and viscerosomatic ratio (B) of chiral nanodrugs after oral administration provided by the disclosed embodiments of the present invention;

[0059] Figure 38Blood routine after oral administration of the chiral nanodrug provided by the disclosed embodiments of the present invention (A: Lymphocyte percentage Lymph%; B: Lymphocyte count Lymph#; C: Hematocrit HCT; D: Mean corpuscular volume MCV; E: Mean corpuscular hemoglobin concentration MCHC; F: Mean corpuscular hemoglobin MCH; G: Neutrophil percentage Gran%; H: Neutrophil count Gran#; I: White blood cell count WBC; J: Platelet count PLT; K: Platelet volume distribution width PDW; L: Plateletcrit PCT; M: Mean platelet volume MPV; N: Monocyte percentage Mon%; O: Red blood cell volume distribution width RDW%; P: Monocyte count Mon#; Q: Hemoglobin HGB; R: Red blood cell count RBC);

[0060] Figure 39 Blood biochemistry after oral administration of the chiral nanodrug provided by the disclosed embodiments of the present invention (A: Creatinine CREA-M; B: Aspartate aminotransferase AST; C: Albumin ALB; D: Uric acid UA; E: Total bilirubin TBIL-Z; F: Alanine aminotransferase ALT; G: Alkaline phosphatase ALP; H: Total protein TP; I: Urea UREA);

[0061] Figure 40 HE staining of major organs after oral administration of the MONs series of carriers provided by the disclosed embodiments of the present invention;

[0062] Figure 41 HE staining of the gastrointestinal tract (GIT) after oral administration of the MONs series of carriers provided by the disclosed embodiments of the present invention;

[0063] Figure 42 Double staining of ZO-1 and Occduin in the colon after oral administration of the MONs series of carriers provided by the disclosed embodiments of the present invention;

[0064] Figure 43 HE staining of the chiral nanodrug after incubation with the MONs series of carriers in ex vivo human intestinal tissues provided by the disclosed embodiments of the present invention;

[0065] Figure 44 Weight change (A), DAI score (B), colon length (C), cfDNA level (D) of mice administered with the chiral nanodrug for preventive effect within 10 days;

[0066] Figure 45 Colon image of mice administered with the chiral nanodrug for preventive effect on the 10th day provided by the disclosed embodiments of the present invention;

[0067] Figure 46 HE staining and histological scoring of the colon of mice administered with the chiral nanodrug for preventive effect provided by the disclosed embodiments of the present invention;

[0068] Figure 47 Levels of inflammatory factors in the colon of mice administered with chiral nanodrugs for preventive effect provided by the disclosed embodiments of the present invention;

[0069] Figure 48 Immunofluorescence staining of the colon of mice administered with chiral nanodrugs for preventive effect provided by the disclosed embodiments of the present invention (A: Reactive oxygen species ROS; B: Apoptosis TUNEL; C: Double staining of LY6G and caspase3; D: Double staining of CD86 and CD206);

[0070] Figure 49 Immunofluorescence staining of the colon of mice administered with chiral nanodrugs for preventive effect provided by the disclosed embodiments of the present invention (A: Double staining of ZO-1 and OCCLUDIN; B: Double staining of CD31 and F480; C: Double staining of MPO and NF-κB; D: Double staining of CD4 and FoxP3);

[0071] Figure 50 Statistical results of immunofluorescence staining of the colon of mice administered with chiral nanodrugs for preventive effect provided by the disclosed embodiments of the present invention;

[0072] Figure 51 Immunohistochemistry (TLR4 / 9, MyD88, NF-κB, TNF-α and IL-1β) of the colon of mice administered with chiral nanodrugs for preventive effect provided by the disclosed embodiments of the present invention;

[0073] Figure 52 Immunohistochemical statistics of the colon of mice administered with chiral nanodrugs for preventive effect provided by the disclosed embodiments of the present invention;

[0074] Figure 53 Cluster heat map (A), KEGG enrichment histogram (B), Venn diagram (C), KEGG enrichment scatter plot (D) and volcano plot (E) of DEGs in the colon tissue of DSS model mice after treatment with chiral nanodrugs provided by the disclosed embodiments of the present invention;

[0075] Figure 54 Changes in body weight (A), DAI score (B), colon length (C), cfDNA level (D) of mice administered with chiral nanodrugs for therapeutic effect provided by the disclosed embodiments of the present invention within 10 days;

[0076] Figure 55 Colon image of mice administered with chiral nanodrugs for therapeutic effect provided by the disclosed embodiments of the present invention on the 10th day;

[0077] Figure 56HE staining and histological scoring of the colon of mice administered with chiral nanodrugs for therapeutic effects provided by the disclosed embodiments of the present invention;

[0078] Figure 57 Levels of inflammatory factors in the colon of mice administered with chiral nanodrugs for therapeutic effects provided by the disclosed embodiments of the present invention;

[0079] Figure 58 16S rRNA flora analysis of the feces of mice administered with chiral nanodrugs for therapeutic effects provided by the disclosed embodiments of the present invention: heatmap of species abundances at the phylum level (A); heatmap of species abundances at the genus level (B); percentage of species abundances at the phylum level (C); percentage of species abundances at the genus level (D); PCoA analysis based on ASV's Weighted Unifrac distance (E); box plot of inter-group differences in the observed species index based on ASV (F);

[0080] Figure 59 Drug delivery system design of the nano-drug PEI-L-TA@MON provided by the disclosed embodiments of the present invention (A: Schematic diagram of the synthesis route of the nano-drug delivery system Drug / PEI-L / D-TA@MON; B: Morphology of the nano-drug delivery system Drug / PEI-L / D-TA@MON);

[0081] Figure 60 Characterization of the nano-drug PEI-L-TA@MON drug delivery system provided by the disclosed embodiments of the present invention (A: FT-IR spectrum of the nano-drug delivery system Drug / PEI-L-TA@MON; B: XRD pattern of the nano-drug delivery system Drug / PEI-L-TA@MON);

[0082] Figure 61 Evaluation of the in vitro drug release ability of the nano-drug PEI-L-TA@MON drug delivery system provided by the disclosed embodiments of the present invention (A: Line graph of in vitro drug release of Mes-PEI-L-TA@MON; B: Line graph of in vitro drug release of Ber-PEI-L-TA@MON; C: Line graph of in vitro drug release of Dex-PEI-L-TA@MON; D: Line graph of in vitro drug release of Nor-PEI-L-TA@MON; E: Line graph of in vitro drug release of Tac-PEI-L-TA@MON);

[0083] Figure 62Investigation of the in vitro cumulative absorption amount of the nano-drug PEI-L-TA@MON drug delivery system provided by the disclosed embodiments of the present invention (A: Schematic diagram of the everted gut sac experiment procedure; B: Line graph of the cumulative drug absorption amount of Mes-PEI-L-TA@MON; C: Line graph of the cumulative drug absorption amount of Ber-PEI-L-TA@MON; D: Line graph of the cumulative drug absorption amount of Dex-PEI-L-TA@MON; E: Line graph of the cumulative drug absorption amount of Nor-PEI-L-TA@MON; F: Line graph of the cumulative drug absorption amount of Tac-PEI-L-TA@MON);

[0084] Figure 63 In vivo anti-inflammatory effect of the nano-drug PEI-L-TA@MON drug delivery system provided by the disclosed embodiments of the present invention (A: Schematic diagram of the construction of the DSS mouse model; B: H&E stained histological sections of different treatment groups in the DSS mouse model; C: Statistical analysis of histological scores). Detailed implementation manners

[0085] Here, the exemplary embodiments will be described in detail, and the examples are shown in the drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The implementation manners described in the following exemplary embodiments do not represent all implementation manners consistent with the present invention. On the contrary, they are merely examples of systems consistent with some aspects of the present invention as detailed in the appended claims.

[0086] The present invention provides a construction method of a chiral silica drug and a chiral nano-drug delivery system for loading common therapeutic drugs for clinical treatment based on lipopolysaccharide (LPS), extracellular free DNA (cfDNA), and reactive oxygen species (ROS) scavenging strategies, and a therapeutic application for inflammatory bowel disease. It includes: preparing a polyethyleneimine-tartaric acid mixture chiral template; the polyethyleneimine tartaric acid template catalyzes an organic-inorganic hybrid silicon source through a biomimetic mineralization reaction to prepare the chiral nano-drug PEI-L / D-TA@MON; five first-line and second-line therapeutic drugs are loaded into PEI-L / D-TA@MON by an in-situ synthesis method to exert a synergistic therapeutic effect. In addition, the present invention successfully prepares a nano-drug with the ability to scavenge LPS, cfDNA, and ROS by a one-step method, enabling it to clear pathogens and various pathogenic factors such as LPS, cfDNA, and ROS through physical adsorption, electrostatic interaction, and reduction reaction, inhibit the inflammatory cascade reaction, show superiority in the treatment effect of inflammatory bowel disease, have great application potential in the field of nanomedicine, and can be used as a drug carrier to load a variety of clinical drugs to exert a synergistic therapeutic effect, providing a reference for the design of nano-drugs for inflammatory bowel disease.

[0087] Specifically, it includes the following:

[0088] 1. This implementation provides a preparation method of chiral nanodrugs based on LPS, cfDNA, and ROS scavenging strategies, which uses a polyethyleneimine-tartaric acid (PEI-L / D-TA) biomimetic catalytic template to rapidly induce the aggregation and deposition of inorganic-organic silicon source MON, including:

[0089] 1) Preparation of the biomimetic chiral template PEI-L / D-TA: Weigh polyethyleneimine PEI and L / D-tartaric acid L / D-TA, dissolve them in deionized water, and let it stand still to obtain the chiral template PEI-L / D-TA.

[0090] 2) Preparation of the inorganic / organic hybrid silicon source MON: Mix the inorganic silicon source TMOS, the organic silicon source BTES, and absolute ethanol at room temperature to obtain the inorganic / organic hybrid silicon source MON.

[0091] 3) Obtain the aqueous phase of the chiral template PEI-L / D-TA, namely the PEI-L / D-TA template solution, add it to an EP tube, and then add the inorganic / organic silicon source mixture MON to the EP tube. After mixing, add the silane coupling agent APTES, quickly shake it until it gels, and then grind it after drying to obtain the chiral nanodrug PEI-L / D-TA@MON.

[0092] Preferably, the preparation method of the chiral nanodrug based on LPS, cfDNA, and ROS scavenging strategies includes:

[0093] 1) Preparation of the biomimetic chiral template PEI-L / D-TA: Weigh 0.4 g of PEI and 0.38 g of L / D-tartaric acid, dissolve them in 40 ml of deionized water, and let it stand still for 24 h to obtain a 10 mg / ml chiral template agglutination solution PEI-L / D-TA.

[0094] At the same time, prepare an achiral PEI template without tartaric acid as a control according to this step.

[0095] 2) Preparation of the inorganic / organic hybrid silicon source MON: Mix 0.6 ml of TMOS, 0.6 ml of BTES, and 1 ml of absolute ethanol at room temperature and normal pressure.

[0096] 3) Preparation of the chiral nanodrug PEI-L / D-TA@MON: Take 1 ml of the PEI-L / D-TA template solution and add it to a 5 ml EP tube. Then add 2.2 mL of the mixture (0.6 ml of TMOS, 0.6 ml of BTES, and 1 ml of absolute ethanol mixed), mix well, immediately add 0.1 mL of APTES, quickly shake it until it gels, and record the gelling time. After opening the lid and drying and grinding, PEI-L / D-TA@MON is obtained.

[0097] The synthesis mechanism of the above method is as Figure 1As shown, the appearance at different stages of the synthesis is as Figure 2 shown;

[0098] 2. Compare and study the nano-drugs PEI-L-TA@MON and PEI-D-TA@MON with different chiral structures prepared above. At the same time, compare PEI-L-TA@SiO2 and PEI-D-TA@SiO2 without organosilicon source and PEI@SiO2 without chiral structure to determine the effects of each component on drug properties, biological fate, and therapeutic mechanism.

[0099] The TEM, SEM, EDS-Mapping, and particle size distribution results of the series of chiral nano-drugs are as Figure 3 shown. It can be clearly seen in the transmission electron microscope (TEM) and scanning electron microscope (SEM) images that all the nano-drugs form irregularly densely aggregated nanoparticles (10 - 20 nm), with abundant nanopores distributed around the surface. The Si, C, O, N, and S elements are evenly distributed in the energy-dispersive spectroscopy (EDS) mapping images.

[0100] The infrared spectra of the series of chiral nano-drugs are as Figure 4 shown. Verified by Fourier transform infrared spectroscopy (FTIR), absorption bands of ν-NH (3265.7 cm -1 ) and δ-N-H (1635.1 cm -1 ) can be observed in the spectrum of PEI. However, in the spectra of PEI-L / D-TA, the band of ν-NH disappears and is replaced by the band of v-OH (3403.8 cm -1 ). At the same time, there are also absorption bands of v-CONH (3157.4 cm -1 ), v-C=O (1726.2 cm -1 , belonging to the carboxyl group), and ν-C=O (1600.6 cm -1 ). PEI-L / D-TA@MON has characteristic absorptions at 3355.9, 1102.9, 458.1, 1622.5, 1080.2, and 557.5 cm -1 in the FTIR spectrum, which belong to ν-Si-O-Si, ν-Si-OH, δ-Si-O-Si, v-N-H, v-C-S, and v-S-S bonds respectively. It shows that the carboxyl group of chiral tartaric acid binds to the amino group of PEI in the form of an amide bond, and BTES as an organosilicon source containing disulfide bonds is also successfully modified.

[0101] The XPS spectra of the series of chiral nano-drugs are as Figure 5As shown, X-ray photoelectron spectroscopy (XPS) shows the elemental peaks of Si, C, O, N, and S. The contents of each element in PEI-L-TA@MON are 15.6% (Si), 47.6% (C), 28.8% (O), 1.8% (N), and 6.3% (S), respectively, which also verifies the existence of the tetrasulfide bond.

[0102] XRD and SAXD analyses of the nano-drugs are as Figure 6 shown. Small-angle X-ray diffraction (SAXD) analysis indicates the mesoporous structure of these nano-drugs, while X-ray diffraction (XRD) studies confirm their amorphous nature.

[0103] Thermogravimetric analysis of the nano-drugs is as Figure 7 shown. Due to the cross-linking of the organosilicon source, PEI-L / D-TA@MON shows more weight loss (54.8%) compared with PEI@SiO2 (14.0%) and PEI-L / D-TA@SiO2 (18.6%).

[0104] Circular dichroism spectroscopy analysis of the nano-drugs is as Figure 8 shown. The chirality of L / D-TA shows negative and positive Cotton effects, which are derived to PEI-L / D-TA and are also manifested in PEI-L / D-TA@SiO2 and PEI-L / D-TA@MON.

[0105] Nitrogen adsorption curves and pore size distribution curves of the nano-drugs are as Figure 9 shown. Through nitrogen adsorption / desorption tests, the mesoporous structure of PEI-L / D-TA@MON was studied. Among them, different type-IV isothermal adsorption / desorption isotherms were observed for PEI@SiO2, PEI-L-TA@SiO2, and PEI-D-TA@SiO2.

[0106] Surface and interface properties of the nano-drugs are as Figure 10 and Figure 11 shown. After modification with L / D-TA, the Zeta potential of the PEI agglomerate decreased from 18.0 to 13.5 and 10.7 mV, indicating that positively charged PEI binds to negatively charged carboxyl groups. In summary, compared with PEI@SiO2, L / D-TA modification decreased the Zeta potential, and BTES cross-linking improved the surface hydrophobicity.

[0107] Fluorescence stability and oil-water partition coefficient of the nano-drugs are as Figure 12 and Figure 13 shown. Oil-water partition tests also verified that after adding the organosilicon source BTES containing tetrasulfide bonds, the nano-drugs became more hydrophobic.

[0108] The reaction principle of PEI-L-TA@MON for clearing cfDNA, ROS, and LPS is asFigure 14 shown. The characterization of PEI-L-TA@MON after incubation with H2O2 is as Figure 15 and Figure 16 shown. As Figure 14 , PEI-L-TA@MON scavenges cfDNA, ROS, and LPS through electrostatic interaction, redox reaction, and adsorption, respectively. After incubation of PEI-L-TA@MON in an oxidative environment, the absorption band of disulfide bonds around 557.0 cm -1 disappeared, and absorption bands of sulfoxide (ν-S=O, 1027.3 cm -1 ) and sulfone (ν-S=O, 1350.1 cm -1 ) appeared in the FTIR spectrum. Meanwhile, sulfur changed from divalent to 4-6 valence in the XPS spectrum, the morphology under transmission electron microscopy became loose, and the contact angle of PEI-L / D-TA@MON with H2O2 also decreased.

[0109] The in vitro degradation trend of MONs is as Figure 17 shown. PEI-L / D-TA@MON scavenged ROS in an oxidative environment, and the disulfide bonds were oxidized into sulfoxide or sulfone with good hydrophilicity, thus showing a relatively high degradation rate. At the same time, the degradation of PEI-L / D-TA@MON in simulated intestinal fluid was slightly faster than that in simulated gastric fluid and simulated body fluid.

[0110] The adsorption results of PEI-L-TA@MON for RhB and EB are as Figure 18 shown. The adsorption results of PEI-L-TA@MON for cfDNA and LPS are as Figure 19 shown. The adsorption ability of PEI-L-TA@MON was first verified by the fading after incubation with rhodamine B (RhB) and eosin blue (EB). Then, we experimentally studied the ability of PEI-L / D-TA@MON to scavenge pathogenic factors in vitro. Using PEI@SiO2 and PEI-L-TA@MON as scavengers and calf thymus DNA (ctDNA) as a model DNA, it was almost completely removed within 4 h. In addition, all the nanodrugs could effectively adsorb LPS in vitro.

[0111] The scavenging ability of PEI-L-TA@MON for total free radicals is as Figure 20 and Figure 21As shown. The antioxidant capacity of PEI-L / D-TA@MON was evaluated by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) tests. As the concentration of PEI-L-TA@MON increased, the colors of the solutions in the ABTS and DPPH experiments changed from blue-green to colorless and from purple to colorless, respectively, indicating that PEI-L-TA@MON consumed ROS in a concentration-dependent manner within a certain range. Figure 21 It shows that the scavenging rate of PEI-L-TA@MON on DPPH increased with time and finally stabilized.

[0112] The TMB color reaction was used to verify the catalytic activity of PEI-L-TA@MON on hydrogen peroxide, as Figure 22 shown. Hydrogen peroxide can oxidize TMB to generate oxidized TMB, which has a characteristic absorption peak at 652 nm and changes from colorless to blue at the same time. The catalytic effect of PEI-L-TA@MON on hydrogen peroxide can promote this reaction. As Figure 22 can be seen, since PEI-L / D-TA@MON contains tetrasulfide bonds and can react with hydrogen peroxide, promoting the TMB color reaction, the color deepens.

[0113] The scavenging ability of PEI-L-TA@MON on individual free radicals was detected using a kit, and the results are as Figure 23 . The scavenging ability of PEI-L / D-TA@MON on individual free radicals (including ·O2 - , ·OH and H2O2) was quantitatively studied using a kit. The results showed that PEI-L-TA@MON decreased the levels of various free radicals in a concentration-dependent manner.

[0114] The scavenging ability of PEI-L-TA@MON on ·O2 - and ·OH was analyzed by EPR, and the results are as Figure 24 . The qualitative evaluation of the scavenging of individual free radicals was carried out using an electron paramagnetic resonance (EPR) spectrometer. The results showed that after co-incubation with PEI-L-TA@MON, the peak intensities of both free radicals decreased, further demonstrating the scavenging ability of PEI-L-TA@MON on ·O2 - and ·OH.

[0115] 3. This embodiment provides the chiral nano-drug PEI-L / D-TA@MON prepared by the above method, and the application of the chiral nano-drug PEI-L / D-TA@MON in the treatment of inflammatory bowel disease. Specifically, the targeting, retention and biocompatibility of the chiral nano-drug PEI-L / D-TA@MON constructed by the method provided by the present invention in the intestine were evaluated. A mouse model of inflammatory bowel disease induced by DSS was used, and the preventive and therapeutic administration methods were used to evaluate the therapeutic effect of the chiral nano-drug PEI-L / D-TA@MON on inflammatory bowel disease.

[0116] The results of the intestinal mucus penetration ability of MONs are as Figure 25 shown. The RITC-labeled nano-drug was placed on the top layer of a 2-cm-thick medium and allowed to freely diffuse for 24 h. Under human and rat mucus and simulated physiological conditions, PEI-L-TA@SiO2 showed a neutral charged surface and the highest preferential conformational penetration efficiency (except for the HEC medium, where particle size and shape are the determining factors), while PEI@SiO2 was restricted to the upper layer of the medium due to its positive surface charge. In a simulated pathological environment containing hydrogen peroxide, the ROS-reactive PEI-L-TA@MON produced the best mucus permeability, which was 1.7 times, 1.2 times, 1.3 times and 1.2 times higher than that of PEI@SiO2, PEI-L-TA-@SiO2, PEI-D-TA-@SiO2 and PEI-D-TA@MON, respectively.

[0117] The results of the multi-particle analysis of the movement of nano-drugs in intestinal mucus are as Figure 26 and Figure 27 shown. The trajectories, diffusion rates and diffusion efficiencies of the particles were analyzed by multi-particle tracking technology. The positively charged PEI@SiO2 was easily captured by the negatively charged mucus and showed a restricted and irregular Brownian motion trajectory within 10 min. In contrast, PEI-L / D-TA-@SiO2 and PEI-L / D-TA@MON moved more freely, faster and over a longer distance. PEI-L-TA@MON had the highest mean squared displacement (MSD) and relative frequency in both rat and human mucus, which were 2.9 - 8.7 times that of other nano-drugs within 10 min. Long-term mucosal inflammation may lead to abnormal mucus secretion, and the mucus of DSS rats will be thinner, and these manifestations will be more obvious.

[0118] The results of the penetration of nano-drugs along the Z-axis direction in the intestine are as Figure 28As shown. Consistent with the in vitro mucus penetration trend, we found that the red fluorescence signals of PEI-L-TA@SiO2 and PEI-L-TA@MON directly observed in the z-stack mode were significantly stronger than those of the competitors, indicating chiral recognition. In the intestine of IBD model rats, the penetration depths of PEI@SiO2, PEI-L-TA-@SiO2, PEI-D-TA-@SiO2, PEI-L-TA@MON, and PEI-D-TA@MON were 56.0, 100.0, 77.3, 143.7, and 91.7 μm, respectively, and PEI-L-TA@MON had an advantage in penetration in the z-axis direction.

[0119] The results of the adhesion ability of the nanodrugs to the intestinal mucosa are shown in Figure 29 , Figure 30 , Figure 31 and Figure 32 As shown. Regarding the superior mucus coverage and permeability of the nanodrugs, we studied the adhesion ability of the nanodrugs to the intestinal mucosa by the elution method. Under the washing of simulated intestinal fluid (SIF), the elution amounts of PEI@SiO2, PEI-L-TA-@SiO2, PEI-D-TA--@SEI-L-TA, and PEI-D-TA@MON at 1 min were 71.8%, 80.3%, 77.0%, 85.3%, and 79.7%, respectively, and at 5 min were 26.0%, 50.6%, 47.4%, 66.7%, and 49.6%, respectively. After elution, more PEI-L-TA@MON remained on the mucosa, followed by PEI-L-TA@SiO2, and strong fluorescence intensity was also detected on the mucosa, indicating significant chiral recognition and stereomatching between the chiral nanostructures and the inherent chiral topology of the biological surface. Similar phenomena were also found on the intestinal mucosa of humans and IBD rats, and the adhesion rates of PEI-L-TA@MON were 3.0, 1.1, 1.6, 1.2 times higher than those of other nanodrugs and 3.1, 1.1, 1.5, 1.3 times higher, respectively. Then the intestinal mucosa was fixed and evaluated using CLSM. The results showed that in the inflammatory state, due to weakened mucus and mucosal damage, PEI-L-TA@MON infiltrated deeper into the intestinal mucosa of IBD rats. Generally speaking, the penetration depths of PEI-L-TA@MON and PEI-D-TA@MON in the IBD intestinal mucosa were 1.1 times and 1.2 times higher than those of the healthy mucosa, respectively, and highly accumulated at the lesion sites with obvious mucosal damage and villous atrophy.

[0120] The intestinal uptake and retention ability of the nanodrugs are shown in Figure 33As shown below. To ensure the optimal application of PEI-L / D-TA@MON in the treatment of intestinal diseases, it was investigated whether improving the bioadhesion of the nanomedicine would enhance the retention and penetration ability at the lesion site. Mice were orally administered RITC-labeled nanomedicine for 2 h, and the colon was excised for CLSM observation. In the cross-section of the colon of healthy mice, PEI@SiO2 mainly aggregated in the center of the intestinal lumen rather than entering the villi, while PEI-L-TA@MON penetrated deep along the intestinal wall, and PEI-L-TA@MON could also penetrate the in vitro intestinal tube of the human body. According to the trend of mucus penetration and mucosal adhesion, the nanomedicine effectively accumulated and penetrated into the villi of the inflamed colon, indicating its good targeting ability. Subsequently, we detected the GIT retention and biodistribution of the nanomedicine by an in vivo imaging system. It was observed from the fluorescence images that the RITC-labeled nanomedicine was found in the small intestine of healthy mice, distributed in the large intestine at 2-6 h, and then excreted. In contrast, due to the increased intestinal permeability, the accumulation of the nanomedicine in the colon of IBD mice increased. After 4 h of administration, the fluorescence intensity of PEI-L-TA@MON in the colon part of acute ulcerative colitis mice was significantly higher than that of other groups, and the retention time at the colon site was also longer, and there were still more nanoparticles distributed at the colon site at 24 h. In addition, all the nanomedicines mainly aggregated in the GIT, did not enter the blood circulation, and were hardly distributed in the main organs, belonging to the concept of targeted therapy, eliminating potential safety hazards.

[0121] The blood compatibility images and hemolysis rates are shown as Figure 36 below. PEI-L / D-TA@MON showed good blood compatibility in the concentration range of 0-800 μg / mL.

[0122] The body weight and viscera-body ratio after oral administration of the MONs series carriers are shown as Figure 37 below. The blood routine after oral administration of the MONs series carriers is shown as Figure 38 below. The blood biochemistry after oral administration of the MONs series carriers is shown as Figure 39 below. The HE staining of the main organs after oral administration of the MONs series carriers is shown as Figure 40 below. The HE staining of the gastrointestinal tract (GIT) after oral administration of the MONs series carriers is shown as Figure 41 below. The double staining of ZO-1 and Occduin in the colon after oral administration of the MONs series carriers is shown as Figure 42 below. The HE staining of the in vitro human intestinal tissue after incubation with the MONs series carriers is shown as Figure 43As shown below. Subsequently, we conducted a biocompatibility study on mice by orally administering PEI-L / D-TA@MON. Whole blood and serum were collected for routine blood and blood biochemical tests, and the main organs and GIT were removed for histopathological examination. The results showed that the chiral nanodrugs had good biocompatibility, with no deaths or significant weight loss, and the routine blood and blood biochemical indexes were all within the reference range. HE staining of the hearts, livers, spleens, lungs, kidneys, stomachs, small intestines, and colons of mice confirmed no histopathological abnormalities or injuries, indicating that the toxicity or irritation of these nanodrugs was negligible. In addition, exposure of PEI-L / D-TA@MON to the human intestinal mucosa did not cause any irritation.

[0123] Mice not treated with DSS showed normal behavior and stable body weight, while the control group of DSS model mice had severe weight loss and clinical symptoms of diarrhea and bloody stools. The symptoms improved significantly after treatment with the nanodrugs. As Figure 44 shown, the weight loss in the PEI@SiO2, PEI-L-TA-@SiO2, PEI-D-TA-@SiO2, PEI-L-TA@MON, and PEI-D-TA@MON treatment groups decreased from 100% to 77.2%, 84.7%, 77.8%, 89.8%, and 81.7% respectively, and the DAI scores decreased from 4 to 4, 3.3, 3.8, 2.1, and 3.1 respectively.

[0124] The colon length was measured, and it was observed that the colon length decreased significantly after DSS stimulation. As Figure 45 shown, the average colon lengths of mice in the Normal, Control, PEI@SiO2, PEI-L-TA-@SiO2, PEI-D-TA-@SiO2, PEI-L-TA@MON, and PEI-D-TA@MON groups were 7.3, 4.0, 4.5, 5.5, 5.1, 6.2, and 5.6 cm respectively, indicating that the colon injury was alleviated after treatment.

[0125] Colon staining of the Control group showed typical histological abnormalities of IBD, as Figure 46 shown, including local mucosal ulcers, epithelial cell necrosis, extensive disappearance of crypts, and infiltration of a large number of inflammatory cells. Nanodrugs with two (PEI@SiO2) or three (PEI-L / D-TA-@SiO2) functional moieties partially alleviated the injury. PEI-L-TA@MON could protect mice from DSS injury, with the mucosal structure being basically normal, the crypts being regular, a small number of inflammatory cells, no lymphoid follicle formation, and the histological score decreasing from 3.7 to 0.7, indicating its efficacy against colitis.

[0126] In addition, after treatment with nano-drugs, the serum cfDNA level was also significantly reduced. Enzyme-linked immunosorbent assay (ELISA) was used to measure the inflammatory cytokines in colon tissues. The results were as Figure 47 shown that PEI-L-TA@MON treatment could restore intestinal homeostasis, reduce the levels of pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6, IL-17, MCP-1, IFN-γ, and improve the anti-inflammatory cytokines (IL-4 and IL-10).

[0127] DHE staining indicated a sharp increase in ROS levels in murine colitis and obvious red fluorescence signals. As Figure 48 shown, PEI-L-TA@MON treatment reduced the ROS signal, showing superior antioxidant effects. Meanwhile, the apoptosis of intestinal epithelial cells induced by colitis injury in the PEI-L-TA@MON group was significantly alleviated. The expressions of tight junction proteins ZO-1 and Occludin in the colon tissues of colitis mice were significantly downregulated, and the expressions of these two proteins basically returned to normal levels after PEI-L-TA@MON treatment, and the intestinal barrier was restored. The IF images of F4 / 80 and CD31 showed the activation of monocytes and macrophages and a decrease in the number of endothelial cells in IBD model mice. CD86 and CD206 staining was used to understand the role of macrophages in guiding host inflammation and immune processes. For colitis mice, the number of M1-type macrophages increased in the Control group, while after treatment with nano-drugs, the macrophages began to shift to the M2 type.

[0128] Next, Ly6G and MPO were selected as neutrophil markers, and obvious neutrophil infiltration was found in murine colon tissues. As Figure 49 shown, this indicated that PEI-L-TA@MON could inhibit neutrophil migration and promote the transformation of macrophage phenotype from inflammatory M1 phenotype to another activated M2 phenotype with anti-inflammatory effects. Meanwhile, both apoptosis related to inflammation and the activation of NF-κB signaling pathway increased. The activities of MPO, Ly6G, caspase-3, and NF-κB in the PEI-L-TA@MON group were similar to those of the untreated normal group and were significantly lower than those of other colitis groups, indicating that the inflammatory cascade reactions related to macrophages and neutrophils were blocked. From the IF images of CD4 and FoxP3, a small number of Treg cells were found after treatment with PEI@SiO2 and PEI-L / D-TA@SiO2, and a large number of Treg cells were found in the PEI-L-TA@MON group, further suggesting the subsidence of the inflammatory state.

[0129] Transcriptomic analysis of murine colon tissues was performed to reveal the therapeutic effects of PEI-L-TA@MON at the gene level. As Figure 53As shown, compared with the Control group, PEI-L-TA@MON downregulated the expression of Marco, Fpr1, Fpr2, F10, IL1a, Acod1, Mmp12, Cxcl2, IL18, IL10, IL1r2, Ctla4, and Ptx3 genes and upregulated the expression of Pkp1, Ccnb1, Ccnb2, Cdc20, Pif1, Nccrp1, Melk, Gp2, Brca1, and Lrr1 genes. The heatmap of differential gene clusters showed that PEI-L-TA@MON treatment significantly altered the different gene levels in cellular processes, environmental information processing, and human diseases. KEGG analysis highlighted the differential genes in the P53, TNF-α, NF-κB, T cell receptor signaling pathways, and cytokine-cytokine receptor interactions, which were highly correlated with the inflammatory cascade and the secretion of pro-inflammatory cytokines related to the progression of IBD. In addition, IHC images strongly confirmed the high expression of TLR4 and TLR9 in the control group mice, which was induced by LPS and cfDNA enriched in the inflamed tissues. In the PEI-L-TA@MON group, the activation of immune cells was inhibited by reducing the TLR4 / TLR9-MyD88-NF-κB signaling pathway induced by LPS and cfDNA.

[0130] Mice were given DSS to induce colitis, received nanomedicine treatment from day 5 to day 11, and were sacrificed on day 14. The results were as Figure 54 shown. In the DSS model group mice, typical symptoms of IBD appeared, including weight loss (19.2%), mucous bloody stools (DAI = 4), partial death, and shortening of the colon length (from 6.9 to 3.4 cm), while the treatment with PEI-L-TA@MON effectively rescued the IBD symptoms of the mice, and the clinical manifestations were alleviated.

[0131] Histopathological examination of the colon tissues of IBD mice showed severe intestinal wall ulcers, epithelial cell necrosis, and inflammatory cell infiltration. After the colon tissues of mice were treated with PEI-L-TA@MON, a complete mucosal structure was observed, as Figure 55 and Figure 56 illustrated its protective effect on IBD.

[0132] Higher levels of cfDNA and pro-inflammatory cytokines were found in DSS model mice, such as Figure 57 shown, and were significantly reduced after treatment with PEI-L-TA@MON.

[0133] Feces of mice in each group were collected, and the composition and diversity of the bacterial flora in the feces of mice were detected by 16S rRNA gene sequencing. AsFigure 58 As shown, at the phylum level, DSS significantly increased the abundance of Proteobacteria and decreased the abundance of Bacteroidota, and these results were improved after PEI-L-TA@MON intervention. The analysis results of Chao1 α-diversity showed that the richness of the intestinal flora in colitis mice decreased significantly, indicating a significant decrease in species diversity and composition. PEI-L-TA@MON could effectively regulate the intestinal microbiota of mice in the DSS model group, increase the diversity of the intestinal flora in colitis mice, and regulate the characteristics of the intestinal flora to change from a dysregulated state to a balanced state.

[0134] 4. A method for constructing a chiral nano-drug delivery system Mes / Ber / Dex / Nor / Tac-PEI-L-TA@MON using the chiral nano-drug PEI-L / D-TA@MON prepared by the above method, comprising: respectively dissolving Mes / Ber / Dex / Nor / Tac in water or ethanol according to their dissolution properties to prepare Mes / Ber / Dex / Nor / Tac drug solutions; respectively loading the Mes / Ber / Dex / Nor / Tac drug solutions into the pores of the chiral nano-drug PEI-L / D-TA@MON by in-situ loading to obtain the chiral nano-drug delivery system Mes / Ber / Dex / Nor / Tac-PEI-L-TA@MON.

[0135] The chiral nano-drug delivery system Mes / Ber / Dex / Nor / Tac-PEI-L-TA@MON is applied in the treatment of inflammatory bowel disease.

[0136] The nano-drug delivery system of PEI-L-TA@MON is designed as Figure 59 shown. Although the nano-drug PEI-L-TA@MON has a certain effect on alleviating IBD to some extent, due to its mesoporous structure, it can be used as a drug-loading site. Considering the feasibility of combination therapy in clinical practice, the first- or second-line clinical drugs mesalazine (Mes), berberine (Ber), dexamethasone (Dex), norfloxacin (Nor), and tacrolimus (Tac) are selected for loading respectively. As shown in the data in Table 1, the correlation coefficients of the linear standard curves of Mes, Ber, Dex, Nor, and Tac in simulated intestinal fluid are all above 0.99, indicating a good linear relationship between the concentrations of these five raw materials and the ultraviolet absorbance. And the relative standard deviations of intra-day and inter-day precision are both less than 2%, meeting the requirements of in vitro analytical methodology. Subsequently, according to their solubility, these five raw materials are respectively pre-dissolved in the aqueous phase or organic phase, and by the in-situ drug-loading method ( Figure 59A) The nano-drug delivery systems Mes / PEI-L-TA@MON, Ber / PEI-L-TA@MON, Dex / PEI-L-TA@MON, Nor / PEI-L-TA@MON, and Tac / PEI-L-TA@MON were respectively formed within a few seconds Figure 59 B)

[0137] Table 1 Standard curves and precision of the raw materials Mes, Ber, Dex, Nor, and Tac in simulated intestinal fluid

[0138]

[0139]

[0140] The characterization of the nano-drug delivery system PEI-L-TA@MON is as follows Figure 60 shown. Due to the limited degree of drug dispersion and the existence of some drugs in crystal form, if not all drugs are loaded into the pores of the nano-drugs, it will lead to a decrease in the dissolution rate and affect the therapeutic effect. We performed FT-IR and XRD analyses on the five nano-drug delivery systems Mes / PEI-L-TA@MON, Ber / PEI-L-TA@MON, Dex / PEI-L-TA@MON, Nor / PEI-L-TA@MON, and Tac / PEI-L-TA@MON prepared by the in-situ drug loading method. As shown in the figure, the characteristic peaks of the groups of the raw materials Mes, Ber, Dex, Nor, and Tac did not appear in the nano-drug delivery system Figure 60 A), indicating that these five drugs were all loaded into the nano-drugs respectively. And the XRD results Figure 60 B) showed that the appearance of diffraction peaks in the spectrum of the raw material indicated that the drug existed in crystal form. However, the diffraction peaks of the crystal were not detected in the nano-drug delivery system, indicating that the drug might be encapsulated in the carrier in an amorphous form. These results suggest that the nano-drug delivery system has a good degree of dispersion, laying a foundation for subsequent drug release, drug transport, and therapeutic effect evaluation.

[0141] The evaluation of the in vitro drug release ability of the nano-drug delivery system PEI-L-TA@MON is as follows Figure 61As shown. The drug release rate is crucial for the efficacy and safety of drugs. When drugs are loaded into the pores of the carrier, due to the steric hindrance in the carrier pores, the directional arrangement between drug molecules can be blocked, resulting in the existence of drug molecules in an amorphous state. In addition, the loose porous structure of the nano-drug PEI-L-TA@MON increases its specific surface area, thereby expanding the contact area between drug molecules and the solution. The drug release rates of drugs Mes, Ber, Dex, Nor, and Tac before and after being loaded into PEI-L-TA@MON were measured by the USPII paddle method with simulated intestinal fluid as the release medium. As Figure 61 can be seen, the release characteristics of the drugs are gradually decreasing and then tending to be stable, while the drug release rate of the nano-drug delivery system shows the characteristics of gradually increasing and then tending to be stable. This result also indicates that the nano-drug delivery system has a sustained-release effect and can serve as a continuous drug storage depot.

[0142] The in vitro cumulative absorption amount of the nano-drug PEI-L-TA@MON drug delivery system was investigated as Figure 62 shown. The drug transport ability of the nano-drug delivery system not only affects the targeting of drugs but also directly relates to the bioavailability of drugs. Previous studies by the research group have demonstrated that PEI-L / D-TA@MON can effectively stereomatch with the mucosa after oral administration and anchor on the positively charged damaged intestine to target the lesion. In this study, the everted gut sac experiment was used to further measure the cumulative absorption amount of drugs in the isolated intestinal segments, which can predict the drug transport ability of the nano-drug PEI-L-TA@MON drug delivery system. By measuring the intestinal cumulative absorption amounts of Mes, Ber, Dex, Nor, and Tac before and after being loaded into the nano-drug PEI-L-TA@MON respectively ( Figure 62 A-E), it can be seen that the cumulative absorption amount of the nano-drug PEI-L-TA@MON drug delivery system is significantly improved compared with the cumulative absorption amount of only using the drug (P<0.001), indicating that the nano-drug PEI-L-TA@MON drug delivery system can effectively transport drugs into the intestine. The above results also suggest that the nano-drug PEI-L-TA@MON drug delivery system can achieve intestinal retention and further confirm its potential as a drug storage depot for continuous and targeted drug delivery.

[0143] The in vivo anti-inflammatory effect of the nano-drug PEI-L-TA@MON drug delivery system was as Figure 63 shown. H&E staining of the colon tissues of different treatment groups was performed ( Figure 63 B) and histological scoring was carried out ( Figure 63C) It was found that ulcer formation, massive infiltration of inflammatory cells, expansion of crypt area, necrosis of a small part of intestinal mucosa, and disappearance of goblet cells were visible in the sections of the Control group after DSS treatment. Although the tissues treated with only the therapeutic drugs Mes, Ber, Dex, Nor, and Tac had certain recovery, ulcer formation, infiltration of inflammatory cells, mucosal atrophy, and disappearance of goblet cells still existed. Moreover, the relevant histological scores decreased slightly from 3.7 points to 2.3 points, 3.0 points, 3.3 points, 2.7 points, and 2.7 points respectively compared with the Control group. However, in the tissue sections treated with the nano-drug delivery systems Mes / PEI-L-TA@MON, Ber / PEI-L-TA@MON, Dex / PEI-L-TA@MON, Nor / PEI-L-TA@MON, and Tac / PEI-L-TA@MON, the recovery of the colon tissue was shown to be good, with few or almost no ulcers, disappearance of crypts and goblet cells. The histological scores were significantly lower than those of the treatment groups without using or using only the therapeutic drugs. This result was mainly due to the antioxidant and anti-inflammatory activities of the nano-drugs themselves and the synergistic effect of their good drug loading and release behaviors with the drugs. The nano-drug delivery system significantly improved the damaged colon tissue and alleviated inflammation more than using clinical drugs alone, showing a good therapeutic effect.

[0144] In summary, the present invention uses a biomimetic silicification strategy to construct chiral silica nano-drugs with the ability to scavenge LPS, cfDNA, and ROS under normal temperature and pressure by utilizing the structure-directing and catalytic ability of the chiral template PEI-L / D-TA. After oral administration, the negatively charged PEI-L-TA@MON stereomatches with the mucosa, realizes long-term intestinal retention, and anchors on the positively charged inflammatory sites to target the lesions. PEI-L-TA@MON scavenges LPS, ROS, and cfDNA respectively through adsorption, redox reaction, and electrostatic interaction, alleviates oxidative stress, inhibits the inflammatory cascade reaction, maintains immune homeostasis, blocks the vicious cycle of IBD, and achieves efficient treatment of IBD. The novel nano-drug PEI-L-TA@MON prepared in this study has an amorphous structure and a rich nanoporous structure on the surface. Taking PEI-L-TA@MON as a drug carrier, the nano-drugs loaded with the drugs Mes, Ber, Dex, Nor, and Tac not only improve the drug release ability and intestinal retention ability, are beneficial to improving the drug release state and enhancing the targeting property, but also achieve synergistic and efficient treatment of IBD, providing a reference for nano-drug design and subsequent research on inflammatory bowel disease.

[0145] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and deformations can be made, and these changes and deformations should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing chiral nanomedicine based on LPS, cfDNA and ROS scavenging strategy, characterized in that: include: 1) Preparation of biomimetic chiral template PEI-L / D-TA: polyethyleneimine PEI and L / D-tartaric acid L / D-TA were weighed, dissolved in deionized water and allowed to stand to obtain a chiral template PEI-L / D-TA; 2) Preparation of inorganic / organic hybrid silicon source MON: Mixing inorganic silicon source TMOS, organic silicon source BTES and anhydrous ethanol at room temperature to obtain inorganic / organic hybrid silicon source MON; 3) obtaining the aqueous phase of the chiral template PEI-L / D-TA, i.e., the PEI-L / D-TA template liquid, and adding it to the EP tube; adding the inorganic / organic silicon source mixed liquid MON to the EP tube; adding the silane coupling agent APTES after mixing; and quickly shaking until gelling; drying and grinding to obtain the chiral nano drug PEI-L / D-TA@MON.

2. The chiral nanomedicine PEI-L / D-TA@MON prepared by the method as claimed in claim 1.

3. The use of the chiral nano drug PEI-L / D-TA@MON prepared by the method according to claim 1, characterized in that: Application of chiral nanodrug PEI-L / D-TA@MON in the treatment of inflammatory bowel disease.

4. A method for constructing a chiral nanodrug delivery system Mes / Ber / Dex / Nor / Tac-PEI-L-TA@MON using the chiral nanodrug PEI-L / D-TA@MON prepared by the method of claim 1, characterized in that: include: According to the solubility properties of Mes / Ber / Dex / Nor / Tac, Mes / Ber / Dex / Nor / Tac were respectively dissolved in water or ethanol in advance to prepare Mes / Ber / Dex / Nor / Tac drug solutions; the Mes / Ber / Dex / Nor / Tac drug solutions were respectively introduced into the pores of the chiral nanodrug PEI-L / D-TA@MON through in situ loading to obtain the chiral nanodrug delivery system Mes / Ber / Dex / Nor / Tac-PEI-L-TA@MON.

5. Application of the chiral nano drug delivery system Mes / Ber / Dex / Nor / Tac-PEI-L-TA@MON prepared according to claim 4, characterized in that: Application of the chiral nanodrug delivery system Mes / Ber / Dex / Nor / Tac-PEI-L-TA@MON in the treatment of inflammatory bowel disease.

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