Nano-drug based on strategy of removing cfDNA, ROS and Ca < 2 + > and promoting NO release as well as preparation method and application of nano-drug

Abstract

The invention provides a nano-drug based on cfDNA, ROS and Ca < 2 + > removal and NO release promotion strategies and a preparation method and application thereof, the preparation method comprises the following steps: weighing polyethyleneimine PEI and L-arginine in deionized water to prepare a template PEI-arg; adding a calcium ion chelating agent into the template, uniformly mixing, freezing and standing; a polyethyleneimine arginine template simulates biomineralization reaction catalysis and is doped with a calcium ion chelating agent to prepare the nano-drug PEI-arg (at) MON (at) BA. The bionic silicon dioxide nano-drug is successfully prepared by adopting a one-step method, and the nano-system removes free DNA through electrostatic adsorption, eliminates ROS through redox reaction, chelates excessive Ca < 2 + > by virtue of complexation, and continuously releases NO through arginine biotransformation. Benefited from the passive targeting characteristic of the liver, the nano scavenger is enriched to the liver after intravenous injection, so that the liver microcirculation can be remarkably improved, the oxidative stress level can be reduced, the inflammatory cascade reaction can be reduced, and the tissue pathological injury can be effectively relieved.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-drugs and drug delivery, and in particular to nano-drugs based on cfDNA, ROS and Ca 2+ clearance and promotion of NO release strategies, and their preparation methods and applications. Background Art

[0002] As the only effective treatment for end-stage liver disease, liver transplantation (LT) has been continuously developing since its clinical application in the 1960s. Thanks to the progress in the fields of liver pathophysiology, immunosuppressant research and development, surgical technology innovation, and improvement of intensive care level, the number of liver transplantation surgeries has been increasing at an annual growth rate of 20% from 2015 to 2021. During liver transplantation (including organ procurement, transportation, and transplantation), liver ischemia-reperfusion injury (IRI), which is closely related to the liver injury and inflammation network, is an important cause of abnormal liver function, organ failure, and patient death.

[0003] Liver IRI mainly consists of two stages. One is the ischemic injury stage, which causes cell damage due to insufficient energy supply. Insufficient blood flow in the ischemic area leads to limited oxygen and nutrient levels, triggering a hypoxic environment. In the hypoxic environment, intracellular adenosine triphosphate (ATP) begins to glycolyze, producing a large amount of pyruvate and lactate, and the pH values of the cytosol and mitochondria decrease. When ATP is depleted, the signal pathway network in the liver and parenchymal cells is activated, ultimately leading to cell death. The intracellular free calcium level also increases due to ATP depletion. ATP depletion activates phospholipase, protease, ATPase, and endonuclease, resulting in cell membrane damage, degradation of structural and membrane proteins, ATP loss, and deoxyribonucleic acid (DNA) damage, respectively. In addition, mitochondria are also damaged by oxidative stress and phospholipid decomposition through the phospholipase A2 pathway, undergoing a permeability transition, releasing cytochrome C into the cytoplasm, and triggering apoptosis. The second is the reperfusion stage. Reperfusion injury is mainly induced by reactive oxygen species (ROS) and severe inflammatory immune responses, which involve indirect and direct cytotoxic mechanisms. The main processes causing ROS and inflammatory immune responses include activation of innate immune cells, ROS production, secretion of chemokines and inflammatory cytokines, infiltration of circulating immune cells, and increased expression of adhesion molecules. The excessive source of ROS in liver IRI tissues is generated by dysfunctional mitochondria, activated Kupffer cells (KCs), or neutrophils and intracellular oxidases. In the hypoxic environment, cellular energy metabolism shifts to anaerobic respiration, which interrupts oxidative phosphorylation and accumulates reducing electron carrier molecules in mitochondria. With the sudden re-injection of oxygen into these cells during reperfusion, a large amount of ROS is produced under normal oxygen concentration conditions.

[0004] A large number of studies have been dedicated to exploring strategies for the prevention and treatment of hepatic IRI, including surgical means such as ischemic preconditioning (IPC) and ischemic postconditioning (IPostC), as well as drugs with antioxidant, energy metabolism improvement, and inflammatory response inhibition effects, such as verapamil, glutathione, trimetazidine, and ulinastatin. However, due to the practical difficulties of preconditioning operations and the limited efficacy of existing drugs, there is currently no clinically recognized specific therapy for IRI. In addition, most antioxidants, anti-inflammatory drugs, therapeutic gases, and biological immunosuppressants have problems such as low solubility, short half-life, insufficient activity, poor pharmacokinetics, non-specific distribution, and serious side effects, which limit their clinical application. In short, given that the pathophysiological process of IRI is caused by a cascade of multiple malignant factors, the intervention strategies targeting single factors of IRI and the existing drugs cannot target the liver, resulting in poor clinical efficacy of the existing methods.

[0005] Nanodrugs with component diversity and functional specificity are gradually bringing hope for the treatment of hepatic IRI. A large number of studies have shown that by changing the morphology and components of nanomaterials and performing multiple modifications or loading of response modules on them, the diagnosis and treatment effects of nanomaterials can be affected, including prolonging blood circulation time, enhancing drug-stimulus-responsive release, and achieving targeted delivery. With the development of materials science and cell biology, nanomaterials can also be combined with biological agents such as stem cells, biomembranes, exosomes, and microvesicles to alleviate local ROS and inflammatory responses. However, these complex nanostructures still face great problems in terms of assembly stability, cost control, large-scale production, and immunogenicity, bringing numerous challenges to their subsequent clinical translation. At the same time, mesoporous silica, as a material with a stable, uniform, and ordered structure, adjustable morphology and pore channels, simple preparation conditions, low preparation cost, and large-scale production, has extensive application value. As a drug delivery system, mesoporous silica has a large surface area and pore volume ratio, and at the same time has abundant silanol groups, which are easy to surface modify, enabling the drug-loading system to obtain more abundant functions. And mesoporous silica nanoparticles have good biosafety, physiological non-toxicity, and degradability; virus-like mesoporous silica nanoparticles with a 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] To sum up, aiming at the clinical problem of hepatic IRI treatment, a biomimetic silica nanodrug with multiple pathogenic factor clearance functions is designed. Starting from multiple pathogenic mechanisms of hepatic IRI, free DNA is cleared by electrostatic adsorption, ROS is eliminated through redox reactions, and excessive Ca 2+ is chelated by complexation. At the same time, NO is continuously released through arginine biotransformation, and multiple pathogenic pathways are blocked, which is expected to be used for the efficient treatment of hepatic IRI. Summary of the Invention

[0007] In view of this, the object of the present invention is to provide a nanodrug based on cfDNA, ROS and Ca 2+ clearance and promotion of NO release strategies, as well as its preparation method and application, so as to achieve synergistic and efficient treatment of liver IRI.

[0008] The technical solution of the present invention is as follows: First, the present invention provides a preparation method of a silica nanodrug PEI-arg@MON@BA based on the clearance of cell-free DNA (cfDNA), reactive oxygen species (ROS) and calcium ions (Ca 2+ ) and the promotion of nitric oxide (NO) release strategy; including:

[0009] 1) Prepare the biomimetic template PEI-arg: Weigh polyethyleneimine PEI and L-arginine L-arg, dissolve them in deionized water and let it stand to obtain the template PEI-arg;

[0010] 2) Add the calcium ion chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid BAPTA-AM to the above template, mix evenly, and freeze and stand at -20°C to obtain the drug-loaded template PEI-arg@BA;

[0011] 3) Prepare the inorganic / organic hybrid silicon source MON: Mix the inorganic silicon source TEOS and the organic silicon source BTES at room temperature to obtain the inorganic / organic hybrid silicon source MON;

[0012] 4) Mix the template PEI-arg@BA obtained in 2) with pre-cooled deionized water, add the inorganic / organic hybrid silicon source MON, stir for 24 h under ice-water bath conditions, centrifuge to obtain the precipitate, collect and dry, and then grind to obtain the nanodrug PEI-arg@MON@BA.

[0013] Second, the present invention also provides the nanodrug PEI-arg@MON@BA prepared by the above method.

[0014] Third, the present invention provides the application of the nanodrug PEI-arg@MON@BA prepared by the above method in inhibiting inflammation-related factors at the cellular level.

[0015] Fourth, the present invention provides the application of the nanodrug PEI-arg@MON@BA prepared by the above method in the treatment of liver ischemia-reperfusion injury and liver transplantation.

[0016] The present invention provides a method based on cfDNA, ROS and Ca 2+Nano-drugs for strategies of clearing and promoting NO release, their preparation methods and applications. The present invention successfully prepares a biomimetic silica nano-drug with multiple functions of clearing pathogenic factors by a one-step method. This nano-system clears free DNA by electrostatic adsorption, eliminates ROS through redox reactions, and chelates excessive Ca by complexation 2+ , and continuously releases NO through arginine biotransformation. Benefiting from the passive liver targeting property, the nano-scavenger is enriched in the liver after intravenous injection, which can significantly improve hepatic microcirculation, reduce the level of oxidative stress, reduce the inflammatory cascade reaction, and effectively relieve tissue pathological damage.

[0017] In the preparation method, the nano-drug PEI-arg@MON@BA is biomimetically synthesized under the catalysis of the biomimetic template PEI-arg. The conditions are simple, the yield is high, the particle size is uniform, and the morphology is good. The nano-drug clears free DNA by electrostatic adsorption, eliminates ROS through redox reactions, and chelates excessive Ca by complexation 2+ to clear the stimulating factors in the pathological environment, and continuously releases NO through arginine biotransformation to promote vasodilation. Benefiting from the passive liver targeting property, the nano-scavenger is enriched in the liver after intravenous injection, which can significantly improve hepatic microcirculation, reduce the level of oxidative stress, reduce the inflammatory cascade reaction, and effectively relieve tissue pathological damage. The nano-drug delivery system formed by loading the commonly used therapeutic drugs for clinical treatment with the nano-drug PEI-arg@MON@BA can not only improve the downstream symptoms (clear inflammatory factors and ROS, inhibit the inflammatory cascade reaction, reduce oxidative damage, improve calcium overload), but also relieve the upstream mechanisms (clear cfDNA, block the vicious cycle path, improve the immune state), and at the same time promote NO release, dilate blood vessels to improve hepatic ischemia, and achieve synergistic and efficient treatment of hepatic IRI, providing a reference for the design of nano-drugs and subsequent research on hepatic ischemia-reperfusion injury. Description of the Drawings

[0018] The drawings here are incorporated into the specification and constitute a part of this specification, showing the embodiments in line with the present invention, and are used together with the specification to explain the principles of the present invention.

[0019] 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 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.

[0020] Figure 1 Schematic diagram of the synthesis process of the nano-drug provided by the disclosed embodiment of the present invention;

[0021] Figure 2Appearance and morphology of each stage of the synthesis of the nano-drug provided by the disclosed embodiments of the present invention;

[0022] Figure 3 Morphology characterization of the 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-arg@MON@BA);

[0023] Figure 4 Infrared spectrum of the nano-drug provided by the disclosed embodiments of the present invention;

[0024] Figure 5 XPS elemental analysis spectra of the nano-drug provided by the disclosed embodiments of the present invention (A: XPS spectra of MONs series nano-drugs; B: XPS spectra of PEI-arg@MON@BA);

[0025] Figure 6 XRD analysis (A) and SAXD analysis (B) of the nano-drug provided by the disclosed embodiments of the present invention;

[0026] Figure 7 Thermogravimetric analysis (TGA) of the nano-drug provided by the disclosed embodiments of the present invention;

[0027] Figure 8 Nitrogen adsorption curve (A) and BJH pore size distribution curve (B) of the nano-drug provided by the disclosed embodiments of the present invention;

[0028] Figure 9 Zeta potential of the nano-drug solution provided by the disclosed embodiments of the present invention;

[0029] Figure 10 Clearance mechanism of three pathogenic factors and NO generation mechanism of the nano-drug provided by the disclosed embodiments of the present invention;

[0030] Figure 11 Degradation trend of the series of nano-drugs provided by the disclosed embodiments of the present invention after incubation in different media;

[0031] Figure 12 Time adsorption curves of the nano-drug PEI-arg@MON@BA provided by the disclosed embodiments of the present invention for cfDNA (A) and Ca 2+ (B);

[0032] Figure 13 Scavenging ability of the nano-drug PEI-arg@MON@BA provided by the disclosed embodiments of the present invention for total free radicals (A: Reaction formula; B: Color change; C: Scavenging percentage);

[0033] Figure 14Clearance time curve of the nano-drug PEI-arg@MON@BA for cfDNA provided by the disclosed embodiments of the present invention;

[0034] Figure 15 Free radical scavenging ability of the nano-drug PEI-arg@MON@BA provided by the disclosed embodiments of the present invention (A: ·O2-; B: ·OH; C: H2O2);

[0035] Figure 16 Free radical scavenging ability of the nano-drug PEI-arg@MON@BA provided by the disclosed embodiments of the present invention (A: ·O2-; B: ·OH; C: quantitative analysis);

[0036] Figure 17 Confocal laser scanning microscopy (CLSM) images (A) and semi-quantitative fluorescence analysis of cellular uptake on RAW 264.7 cells (B) provided by the disclosed embodiments of the present invention;

[0037] Figure 18 Staining images of NMS treated on RAW 264.7 and THP-1 cells after LPS treatment provided by the disclosed embodiments of the present invention, respectively indicating intracellular NO level, ROS level, Ca 2+ level, live and dead cell level (A: DAF-FMDA; B: DCFH-DA; C: Fluo-3; D: calcein-AM / PI);

[0038] Figure 19 Fluorescence images (A) and semi-quantitative fluorescence analysis (B) of RITC-labeled PEI-arg@MON@BA in the main organs and blood of rats provided by the disclosed embodiments of the present invention;

[0039] Figure 20 Distribution of PEI-arg@MON@BA in the main organs (A) and blood (B) of healthy rats and IRI rats detected by inductively coupled plasma mass spectrometry (ICP-MS) provided by the disclosed embodiments of the present invention;

[0040] Figure 21 CLSM images (A) and semi-quantitative fluorescence analysis (B) of RITC-labeled PEI-arg@MON@BA in the liver lobes of rats during IRI provided by the disclosed embodiments of the present invention;

[0041] Figure 22 In vitro release curve of PEI-arg@MON@BA in PBS pH 7.4 provided by the disclosed embodiments of the present invention;

[0042] Figure 23Plasma concentration-time curves (A) and main organ distributions (A: 2 hours after intravenous injection; B: 6 hours after intravenous injection; C: 24 hours after intravenous injection) of PEI-arg@MON@BA and the raw drug BA provided by the disclosed embodiments of the present invention;

[0043] Figure 24 HE staining images of the IRI rat model 6 hours after surgery provided by the disclosed embodiments of the present invention;

[0044] Figure 25 Ischemic regions, SUZUKI scores, and serum cfDNA levels (A) and biochemical index levels (B) of IRI rats after 60 minutes of ischemia and 6 hours of reperfusion provided by the disclosed embodiments of the present invention;

[0045] Figure 26 Serum inflammatory factor levels of IRI rats after 60 minutes of ischemia and 6 hours of reperfusion provided by the disclosed embodiments of the present invention;

[0046] Figure 27 Tunel (A) and DHE (B) staining images of the liver tissue of IRI rats provided by the disclosed embodiments of the present invention;

[0047] Figure 28 Therapeutic mechanisms and Western blot WB analysis of TLR9, MyD88, P65, and P-65 in the peritoneal lavage fluid of IRI rats provided by the disclosed embodiments of the present invention;

[0048] Figure 29 Immunohistochemical analysis of TLR9, TLR4, MyD88, NF-κB, TNF-α, and IL-1β on the liver tissue of IRI rats provided by the disclosed embodiments of the present invention;

[0049] Figure 30 Relevant detections were performed on the liver tissue of liver transplant rats 48 hours after surgery (A: HE staining image; B: DHE staining image; C: disease score and ischemic region statistics) provided by the disclosed embodiments of the present invention;

[0050] Figure 31 Statistical charts of serum biochemical index levels (A) and inflammatory factor levels (B) of liver transplant rats provided by the disclosed embodiments of the present invention; Detailed implementation manners

[0051] Here, exemplary embodiments will be described in detail, and examples thereof are shown in the accompanying drawings. When the following description refers to the accompanying drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments 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.

[0052] The present invention provides a construction method of a chiral silica drug and a 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 hepatic ischemia-reperfusion injury. It includes: preparing a polyethylenimine-arginine mixture chiral template; the polyethylenimine-arginine template catalyzes an organic-inorganic hybrid silicon source to prepare chiral nano-drug PEI-arg@MON@BA through a biomimetic mineralization reaction; five first-line and second-line therapeutic drugs are loaded into PEI-arg@MON@BA 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 scavenge 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 therapeutic effect of hepatic ischemia-reperfusion injury treatment, 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 hepatic ischemia-reperfusion injury.

[0053] Specifically, it includes the following:

[0054] 1. This embodiment provides a method for preparing a nano-drug based on cfDNA, ROS, and Ca 2+ scavenging and promoting NO release strategies, using a polyethylenimine-arginine (PEI-arg) biomimetic catalytic template to rapidly induce the aggregation and deposition of an inorganic-organic silicon source MON, including:

[0055] 1) Prepare the biomimetic template PEI-arg: Weigh polyethylenimine PEI and L-arginine L-arg, dissolve them in deionized water, and let it stand to obtain the chiral template PEI-arg;

[0056] 2) Add the calcium ion chelator 1,2-bis(ortho-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid BAPTA-AM to the above template, mix evenly, and freeze and let it stand at -20°C to obtain the drug-loaded template PEI-arg@BA;

[0057] 3) Preparation of inorganic / organic hybrid silicon source MON: Mix the inorganic silicon source TEOS and the organic silicon source BTES at room temperature to obtain the inorganic / organic hybrid silicon source MON;

[0058] 4) Take the template PEI-arg@BA described in 2), mix it with pre-cooled deionized water, then add the inorganic / organic hybrid silicon source mixture MON. After stirring for 24 h under ice-water bath conditions, centrifuge to obtain a precipitate, collect, dry, and grind it to obtain the nano-drug PEI-arg@MON@BA.

[0059] 5) Meanwhile, the nanomaterial PEI@Si prepared by removing arg, BTES, and BAPTA-AM according to the above steps; the nanomaterial PEI@MON prepared by removing BTES and BAPTA-AM; and the nanomaterial PEI-arg@MON prepared by removing BAPTA-AM are used as controls.

[0060] Specifically, based on cfDNA, ROS, and Ca 2+ A method for preparing a nano-drug based on the strategies of cfDNA, ROS, and Ca clearance and promoting NO release, including:

[0061] 1) Preparation of the biomimetic template PEI-arg: Weigh 0.316 g of polyethyleneimine PEI and 0.696 g of L-arginine L-arg, dissolve them in 1 mL of deionized water, and let it stand to obtain the chiral template PEI-arg;

[0062] 2) Add 5 mg of the calcium ion chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid BAPTA-AM to the above template, mix evenly, and freeze and let it stand at -20 °C to obtain the drug-loaded template PEI-arg@BA;

[0063] 3) Preparation of inorganic / organic hybrid silicon source MON: Mix 2.7 mL of the inorganic silicon source TEOS and 3.3 mL of the organic silicon source BTES at room temperature to obtain the inorganic / organic hybrid silicon source MON;

[0064] 4) Take the template PEI-arg@BA described in 2), mix it with 30 mL of pre-cooled deionized water, then add the inorganic / organic hybrid silicon source mixture MON. After stirring for 24 h under ice-water bath conditions, centrifuge to obtain a precipitate, collect, dry, and grind it to obtain the nano-drug PEI-arg@MON@BA.

[0065] Meanwhile, the nanomaterial PEI@Si prepared by removing arg, BTES, and BAPTA-AM according to the above steps; the nanomaterial PEI@MON prepared by removing BTES and BAPTA-AM; and the nanomaterial PEI-arg@MON prepared by removing BAPTA-AM are used as controls.

[0066] The synthesis mechanism of the above method is as follows Figure 1 shown, and the appearance at different stages during synthesis is as follows Figure 2 shown;

[0067] 2. Compare the nano-drugs with different structures prepared above, namely PEI@Si, PEI@MON, PEI-arg@MON, and PEI-arg@MON@BA, to determine the effects of each component on drug properties, biological fate, and therapeutic mechanisms.

[0068] The TEM, SEM, EDS-Mapping, and particle size distribution results of the series of nano-drugs are as follows Figure 3 shown. It can be clearly seen from the transmission electron microscope (TEM) and scanning electron microscope (SEM) images that all the series of samples show similar morphological characteristics, presenting an irregular flaky polymeric nano-scavenger morphology, with the average particle size of a single particle between 20 - 30 nm. The Si, C, O, N, and S elements are evenly distributed in the energy-dispersive spectroscopy (EDS) mapping images.

[0069] The infrared spectra of the series of nano-drugs are as follows Figure 4 shown. Verified by Fourier transform infrared spectroscopy (FTIR), in the infrared spectrum, the broad peak at 3450 cm-1 belongs to the stretching vibration of ν-Si-O-Si, 1076 cm-1 and 462 cm-1 correspond to the asymmetric stretching vibration and bending vibration δ-Si-O-Si of ν-Si-OH respectively, while the characteristic peak at 1623.2 cm-1 is related to the in-plane bending vibration of v-N-H. Finally, the characteristic peak at 1075.7 cm -1 is in line with the stretching vibration of v-C-S. These results are highly consistent with the changes in the binding energies of Si 2p and N1s in the X-ray photoelectron spectroscopy characterization, confirming the chemical bonding state of the amino-functionalized silica framework.

[0070] The XPS spectra of the series of nano-drugs are as follows Figure 5 shown. X-ray photoelectron spectroscopy (XPS) shows the elemental peaks of Si, C, O, N, and S. The contents of each element in PEI-arg@MON@BA are 18.09% (Si), 38.01% (C), 38.21% (O), 4.39% (N), and 1.30% (S) respectively, which also verifies the existence of the disulfide bond.

[0071] The XRD and SAXD analyses of the nano-drugs are as follows 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.

[0072] The thermogravimetric analysis of the nano-drugs is as follows Figure 7As shown, the mass loss rates of PEI@Si, PEI@MON, PEI-arg@MON, and PEI-arg@MON@BA in the range of 25 - 700 °C are 43.56%, 43.75%, 58.62%, and 64.28% in sequence. The series of nano scavengers show a weight loss characteristic with gradient change. Except for the degradation of the PEI organic template, the silica inorganic framework still exhibits excellent thermal stability at high temperatures. At the same time, the grafting of organic functional groups significantly improves the thermal decomposition activity of the material. Especially, due to the dual effects of arginine modification and BAPTA-AM loading, the thermal decomposition process of PEI-arg@MON@BA brings more mass loss, which indicates the successful grafting of arginine and the successful loading of BAPTA-AM.

[0073] The nitrogen adsorption curve and pore size distribution curve of the nano drug are as Figure 8 shown. The PEI@Si sample presents a typical H2-type hysteresis loop. With the gradual grafting of organic functional groups, the mesoporous channels are occupied, resulting in a continuous decrease in specific surface area and a synchronous reduction in the amplitude of the isotherm hysteresis loop. This phenomenon is consistent with the structural evolution law when organic components are loaded on mesoporous silica, confirming the regulation effect of functional modification on the mesoporous structure.

[0074] The surface and interface characteristics of the nano drug are as Figure 9 . With the loading of functional modules, the Zeta potential on the surface of the nano scavenger shows a downward trend: +36.42 mV for PEI@Si, which drops to +17.08 mV for PEI@MON modified by a single layer of organic matter, and further drops to +6.27 mV for PEI-Arg@MO grafted with arginine functionalization. Finally, the surface potential of the BA-loaded sample PEI-arg@MON@BA stabilizes at +5.60 mV. This change indicates the successful modification of the multiple nano scavengers. The decrease in surface potential is expected to reduce the non-specific interaction between the material and biomolecules, contributing to the improvement of biocompatibility.

[0075] The reaction principles of PEI-arg@MON@BA for scavenging cfDNA, ROS, Ca 2+ and generating NO are as Figure 10 shown. The nano system scavenges free DNA through electrostatic adsorption, eliminates ROS through redox reactions, chelates excessive Ca 2+ by means of complexation, and continuously releases NO through the biotransformation of arginine. After being placed at room temperature for half a year, the morphology and infrared spectrum of PEI-arg@MON@BA do not change significantly, proving that the material has good stability.

[0076] The in vitro degradation trend of the series of materials is as Figure 11As shown, the series of nanomaterials generally remained stable in the three simulated liquids and slowly degraded over time. After the initial degradation in the first 7 days, the degradation rate of the core layer slowed down. In addition, compared with the simulated body fluid environment, the degradation rates of the PEI@MON and PEI-arg@MON samples increased in the oxidative H2O2 and reductive GSH environments because the cross-linked BTES organosilane source on the silicon skeleton has redox-sensitive disulfide bonds. It can be oxidized to sulfone or sulfoxide in an oxidative environment, enhancing hydrophilicity and promoting the degradation of the material.

[0077] As Figure 12 shown, the ability of PEI-arg@MON@BA to scavenge pathogenic factors in vitro was studied experimentally. A series of nanomaterials were used as scavengers, and calf thymus DNA (ctDNA) was used as a model DNA, which was almost completely removed within 4 h. In addition, only the nanodrugs loaded with BA could effectively adsorb Ca 2+ .

[0078] The ability of PEI-arg@MON@BA to scavenge total free radicals is as Figure 13 shown. The antioxidant ability of PEI-arg@MON@BA 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-arg@MON@BA 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-arg@MON@BA consumed reactive oxygen species (ROS) in a concentration-dependent manner within a certain range.

[0079] Figure 14 It shows that the clearance rate of PEI-arg@MON@BA for cfDNA increased with increasing concentration, showing a concentration-dependent trend.

[0080] The ability of PEI-arg@MON@BA to scavenge individual free radicals was detected using a kit, and the results are as Figure 15 . The ability of PEI-arg@MON@BA to scavenge individual free radicals (including ·O2 - , ·OH, and H2O2) was quantitatively studied using a kit. It was found that PEI-arg@MON@BA decreased the levels of various free radicals in a concentration-dependent manner.

[0081] The ability of PEI-arg@MON@BA to scavenge ·O2 - and ·OH was analyzed by electron paramagnetic resonance (EPR), and the results are as Figure 16The qualitative evaluation of the scavenging of individual free radicals was carried out using an electron paramagnetic resonance (EPR) spectrometer. The results showed that after incubation with PEI-arg@MON@BA, the peak intensities of both free radicals decreased, further demonstrating the scavenging ability of PEI-arg@MON@BA for ·O2 - and ·OH.

[0082] 3. This embodiment provides the application of the series of nano-drugs prepared by the above method in inhibiting inflammation-related factors at the cellular level. Specifically, the ability of the nano-drug PEI-arg@MON@BA constructed by the method provided by the present invention to scavenge intracellular pathogenic factors in NMSs was evaluated. RAW 264.7 cells and THP-1 cells were used as the main research objects. LPS was added to induce an inflammatory environment in the cells, and then the difference before and after treatment was compared to evaluate the ability of PEI-arg@MON@BA to scavenge intracellular pathogenic factors. From this section onwards, for convenience of representation, G1, G2, G3, and G4 are used in the figures to represent PEI@Si, PEI@MON, PEI-arg@MON, and PEI-arg@MON@BA respectively.

[0083] The results of confocal laser scanning microscopy of the series of nano-drugs are as Figure 17 (A) shown. After incubating the RITC-labeled nano-drugs with RAW 264.7 cells for 4 hours and observing under a confocal microscope, PEI@Si and PEI@MON showed weak RITC fluorescence signals, while PEI-arg@MON and PEI-arg@MON@BA showed bright red fluorescence signals near or inside the cell nucleus after 4 hours of culture, indicating that they effectively crossed the cell membrane and achieved intracellular accumulation. At the same time, the semi-quantitative analysis results of the fluorescence intensity in (B) and flow cytometry also proved this point. The flow cytometry results further verified that the cell uptake rates of PEI@Si, PEI@MON, PEI-arg@MON, and PEI-arg@MON@BA were 19.41%, 58.9%, 625%, and 89.8% respectively.

[0084] Figure 18 These are different fluorescence staining images of NMSs treated on RAW 264.7 and THP-1 cells after LPS treatment. In RAW 264.7 and THP-1 cells, the DCFH-DA probe produced strong green fluorescence after LPS stimulation, indicating an increase in the intracellular ROS level (Figure B), which was consistent with the in vitro ROS scavenging results. After treatment with PEI-arg@MON and PEI-arg@MON@BA, ROS could be significantly eliminated, manifested as almost disappearance of the green fluorescence. With the assistance of Fluo-4 and DAF-FMDA probes, the PEI-arg@MON@BA treatment group also showed Ca 2+ signal reduction and NO signal enhancement confirmed its ability of Ca 2 + chelation and NO generation. Live / dead cell staining showed that almost no dead cells appeared when PEI-arg@MON@BA was added before and after LPS exposure, demonstrating that the material has both low cytotoxicity and significant cell protection effects.

[0085] 4. The embodiments provide the application of a series of nano-drugs prepared by the above method in passive targeting in rats. Specifically, the targeting, retention and biocompatibility of the series of nano-drugs constructed by the method provided by the present invention in vivo were evaluated. At the same time, the drug release and pharmacokinetics of the nano-material PEI-arg@MON@BA and the raw drug were investigated. Secondly, a rat liver ischemia-reperfusion injury model was established by physically blocking the hepatic portal triad structure of rats for 1 h, and a rat orthotopic liver transplantation model was constructed by the double-cuff method. Then, drug treatment was given, and the therapeutic effect of the nano-drug PEI-arg@MON@BA on liver ischemia-reperfusion injury was evaluated by different methods.

[0086] For the treatment protocol of the rat IRI model, the blood flow of 70% of the hepatic portal triad structure (including the hepatic artery, bile duct and portal vein) of the left and middle lobes of the liver was blocked with a microvascular clamp for 60 min, and then the vascular clamp was removed for reperfusion. During the modeling process, it could be observed that the color of the central and left lobes of the liver changed from bright red to reddish-brown (ischemic period), and returned to normal red after reperfusion (reperfusion period). In terms of treatment intervention, rats in the IRI model group were given PEI-arg@MON@BA via the tail vein twice, 24 h before surgery and 6 h after surgery, with a dosage of 20 mg / kg, and the control group was given the same volume of normal saline.

[0087] Fluorescence images of RITC-labeled PEI-arg@MON@BA in the major organs and blood of rats and quantitative fluorescence analysis are as Figure 19 shown. PEI-arg@MON@BA showed obvious aggregation in MPS organs (liver, spleen). After 24 h of intravenous injection, the near-infrared fluorescence intensity in the liver reached the peak value (5.77×10 11 ph / s), indicating the liver-targeting property of PEI-arg@MON@BA. For the in vivo imaging of the model group, PEI-arg@MON@BA also showed obvious liver enrichment. At the same time, due to the consumption of PEI-arg@MON@BA by the inflammatory environment caused by liver ischemia-reperfusion, the nano-scavenger continued to accumulate in the rats, laying a foundation for the subsequent construction of a drug treatment system.

[0088] The distribution of PEI-arg@MON@BA in the major organs and blood of healthy rats and IRI rats was detected by ICP-MS method as Figure 20As shown, the content of the PEI-arg@MON@BA nanocomposite system in the blood continuously decreased within 24 h after intravenous administration. From the combined organ distribution, it can be seen that at 24 h, it showed significant organ-selective accumulation characteristics: the targeted enrichment amounts in the liver and spleen were significantly more than those in other organs. Pharmacokinetic analysis showed that the blood content of this nanocarrier remained at a relatively low level and began to gradually decrease during the 7-day observation period, indicating that the nanomaterials were continuously cleared within 7 days.

[0089] The CLSM images of RITC-labeled PEI-arg@MON@BA in the rat liver lobe during IRI are as Figure 21 shown. The fluorescence intensity of PEI-arg@MON@BA could be clearly observed at all time points and effectively accumulated in both the normal liver lobe and the IRI lobe, further demonstrating the passive targeting effect of the nanomaterials in the liver.

[0090] As Figure 22 shown, the in vitro release curve of PEI-arg@MON@BA in PBS pH 7.4 was not much different from that of BA. Generally speaking, it could improve the dissolution efficiency of BA to a certain extent.

[0091] Figure 23 showed the plasma concentration-time curves of PEI-arg@MON@BA and the raw drug BA and the main organ distributions at different times. By observing the peak time in vivo of the BAPTA-AM and PEI-arg@MON@BA groups, it can be seen that the peak time of BAPTA-AM became slightly longer after being loaded into PEI-arg@MON@BA, but the maximum plasma drug concentration (Cmax) increased by 11.4%, which was consistent with the sustained-release characteristics of the nanocarrier in the in vitro release experiment. Although the relative bioavailability (AUC0-t) of the nanocarrier did not show a significant increase compared with the raw drug (Table 1), but mainly focused on the specific liver distribution of PEI-arg@MON@BA and the multiple clearance effects in the target organs. Compared with the raw drug, PEI-arg@MON@BA did not reduce the effect of the raw drug. By observing the main organ distributions, it can be seen that in the PEI-arg@MON@BA group, the accumulation of BA in all main organs was significantly increased at 2 h, especially in the liver, and the drug amount was 43.5% higher than that of BA. At 6 h and 24 h, the drug contents in the heart, spleen, kidney and brain of the rats in the BA group were higher. Compared with the BA group, PEI-arg@MON@BA was passively targeted and accumulated in the liver, always showing a higher drug content. <0,

[0092] Table 1 Pharmacokinetic parameters of the raw drug group and the carrier formulation group

[0093]

[0094] The HE staining images of the rat IRI model 6 hours after surgery showed that PEI-arg@MON@BA could significantly improve hepatic ischemia-reperfusion injury, as Figures 24 - 26 shown. It could be visually seen in the pathological sections that compared with the normal group, the hepatic tissue lesions of the IRI rats given normal saline were more severe. Individual hepatocytes showed vacuolar degeneration (indicated by yellow arrows), ballooning degeneration of hepatocytes (indicated by yellow hollow arrows), and partial necrosis of hepatocytes (indicated by black arrows). There was lymphocyte infiltration in the portal area (indicated by green arrows). Similar situations also occurred in the PEI@MON group. In the PEI-arg@MON and PEI-arg@MON@BA groups, the hepatic tissue lesions were mild, with only individual cells showing vacuolar degeneration in the cytoplasm and a small amount of lymphocyte infiltration in the portal area. There was no collapse of sinusoidal endothelial cells, no cell necrosis, and no cell apoptosis. According to the degree of inflammatory cell infiltration, the order of lesion severity was: Control>PEI-arg@MON@BA>PEI-arg@MON>PEI@MON>Normal. The detection of blood biochemical and inflammatory factor indicators showed that compared with the normal group, the ALT and AST levels in the control group increased from 60.25 U / L to 1121.51 U / L (ALT) and from 78.20 U / L to 1236.38 U / L (AST) respectively, indicating severe liver function injury. Under the intervention of a series of samples, whether it was indicators such as ALT, AST or inflammatory factors, they all showed a trend of decreasing in gradients. At the same time, we measured the content of cfDNA, and its trend was consistent with the above indicators. The above results showed that a series of nano scavengers could alleviate liver injury to varying degrees.

[0095] Next, Tunel and DHE staining were selected to further observe the results of ROS and apoptosis. As Figure 27 shown, compared with the control group, the ROS intensity in the hepatic tissue of the rats in the liver IRI model group was significantly enhanced. After the intervention of two nano scavengers, PEI-arg@MON and PEI-arg@MON@BA, the ROS fluorescence signal showed an obvious weakening trend. It could be seen through Tunel staining that the apoptosis rate of hepatocytes in the IRI model group was significantly increased, while after the intervention of nano scavengers, the apoptosis phenomenon of hepatocytes was significantly inhibited. These results suggested that NMSs might effectively reduce the programmed cell death of hepatocytes caused by IRI by regulating the oxidative stress pathway and the expression of apoptosis-related proteins.

[0096] The expression level of the TLRs family (especially TLR9) and the MyD88-dependent endosomal signaling cascade mediated by it can mediate inflammation. Combining the WB and immunohistochemical results, as Figure 28, as shown in Fig. 29, the protein expressions of TLR9, MyD88, and NF-κB (p65) in macrophages of rats with ischemia-reperfusion injury model were activated. However, after treatment with PEI-arg@MON@BA, this pro-inflammatory signaling pathway was significantly blocked, and the secretion of pro-inflammatory cytokines decreased, indicating that it effectively inhibited the inflammatory cascade mediated by the TLR9–MyD88–NF-κB signaling pathway. The above experimental results are highly consistent with the molecular mechanism research of the TLR9 signaling pathway: As an endosome-localized pattern recognition receptor, TLR9 activates NF-κB through the MyD88-dependent pathway after recognizing damage-associated molecular patterns (such as mtDNA, cfDNA), promoting the expression of pro-inflammatory factors such as TNF-α and IL-1β. The inhibitory effect of PEI-arg@MON@BA on the TLR9-MyD88-NF-κB pathway may involve the following mechanisms: ① Competitive binding to mtDNA / cfDNA to block its recognition by TLR9; ② Inhibiting the ROS-TLR9 co-activation effect through the antioxidant properties of the nanomaterials; ③ Regulating the phosphorylation or ubiquitination modification of downstream signaling molecules (such as TRAF6, IKK complex), thereby interfering with signal transduction.

[0097] In summary, PEI-arg@MON@BA effectively alleviates liver inflammatory response and shows a significant protective effect on hepatic ischemia-reperfusion injury (IRI).

[0098] Subsequently, its therapeutic effect in a rat liver transplantation model was investigated. The HE staining images and DHE staining images are as Figure 30 shown. HE staining showed that the livers of rats in the control group had severe lesions, manifested as punctate necrosis and lymphocyte infiltration in the perivenous area; while after treatment with PEI-arg@MON@BA, only individual cytoplasmic vacuolization of cells was seen in the liver, and no collapse, necrosis, or apoptosis of sinusoidal endothelial cells was observed. DHE staining showed that the livers of the control group showed significant red fluorescence, while the ROS signal in the treatment group almost disappeared. Further, blood samples of rats were collected at 6, 24, and 48 hours after surgery for biochemical index and inflammatory factor detection, as Figure 31 shown. After intervention with PEI-arg@MON@BA, the liver function indexes including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin (ALB), and total protein (TP) were significantly decreased; the levels of pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β, and IL-17A were significantly decreased, while the levels of anti-inflammatory factors IL-10 and TGF-β were increased compared with the control group, clarifying its protective effect on ischemia-reperfusion injury (IRI) during liver transplantation surgery.

[0099] In summary, the present invention uses a biomimetic silicification strategy to construct cfDNA, ROS, and Ca with the structure-directing and catalytic ability of the template PEI-arg under normal temperature and pressure.2+ Nanodrugs for clearance and promotion of NO release strategies. The novel nanodrug PEI-arg@MON@BA prepared in the present invention has an amorphous structure and a rich nanoporous structure on the surface. After intravenous injection, due to its passive liver targeting property, PEI-arg@MON@BA shows great potential in the development of treatment strategies for liver diseases. This material can not only clear pathogenic factors, reduce oxidative stress, improve microcirculation, and relieve inflammatory responses, but also ultimately significantly reduce liver injury caused by IRI. Transcriptomic analysis suggests that it may act through the TLR9–MyD88–NF-κB signaling pathway, and this mechanism has been quantitatively and qualitatively verified by immunohistochemistry (IHC) and Western blotting (WB) experiments. Given the complex pathophysiological process of IRI and the co-regulatory design of PEI-arg@MON@BA targeting multiple factors, it is believed that more pathways related to the alleviation of oxidative stress and microcirculation homeostasis may be discovered in the future. In addition, the protective effect of this material on liver IRI has been fully verified in a rat liver transplantation model. PEI-arg@MON@BA clears free DNA through electrostatic adsorption, eliminates ROS through redox reactions, chelates excessive Ca 2+ , and continuously releases NO through arginine biotransformation to relieve oxidative stress, inhibit the inflammatory cascade reaction, maintain immune homeostasis, block the vicious cycle of hepatic ischemia-reperfusion injury, and achieve efficient treatment of hepatic ischemia-reperfusion injury, providing a reference for the design of nanodrugs and subsequent research on hepatic ischemia-reperfusion injury.

[0100] 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 modifications can be made, and these changes and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Preparation method of nano-drug based on cfDNA, ROS and Ca 2+ for clearing and promoting NO release strategies, characterized in that, Including: 1) Weigh polyethyleneimine (PEI) and L-arginine (L-arg), dissolve them in deionized water, and let it stand still to obtain the chiral template PEI-arg; 2) Add 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) to the chiral template PEI-arg and mix evenly. Then freeze and let it stand still at -20 °C to obtain the drug-loaded template PEI-arg@BA; 3) Mix the inorganic silicon source TEOS and the organic silicon source BTES at room temperature to obtain the inorganic / organic hybrid silicon source MON; 4) Mix the drug-loaded template PEI-arg@BA obtained in 2) with pre-cooled deionized water, add the inorganic / organic hybrid silicon source mixture MON, stir for 24 h under an ice-water bath condition, centrifuge to obtain the precipitate, collect, dry, and grind it to obtain the nano-drug PEI-arg@MON@BA.

2. The nano-drug PEI-arg@MON@BA prepared by the method according to claim 1.

3. Use of the nano-drug PEI-arg@MON@BA prepared by the method according to claim 1, characterized in that, Application in inhibiting inflammation-related factors at the cellular level.

4. Use of the nano-drug PEI-arg@MON@BA prepared by the method according to claim 1, characterized in that, Application of the nano-drug PEI-arg@MON@BA in liver ischemia-reperfusion treatment and liver transplantation.

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