Active oxygen response type self-luminous nitric oxide controlled-release nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses active oxygen response type self-luminous nitric oxide controlled-release nanoparticles as well as a preparation method and application thereof. The nanoparticles are obtained by grafting an NO donor and a multi-amino polymer, then carrying out a luminol amidation reaction with carboxyl modified cerium dioxide for grafting to obtain a copolymer, and modifying the outer edge of the copolymer with hyaluronic acid, according to the nano-particles disclosed by the invention, when an NO donor is exposed to relatively low energy irradiation and within a visible light wavelength range (400-450 nm) with low cytotoxicity, accurate controlled release of NO in time and space along with the change of light intensity can be realized, and unnecessary damage of redundant toxicity to normal tissues is avoided; under the endogenous ROS visible light triggering mechanism, the precise control release of NO is realized; the endogenous light-triggered release system disclosed by the invention not only improves the solubility and the targeting property of NO, but also greatly improves the accuracy of hepatic fibrosis treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to a nitric oxide controlled release nanoparticle, in particular to a reactive oxygen species responsive self-luminescent nitric oxide controlled release nanoparticle, and also relates to a preparation method of the above nanoparticle and application of the nanoparticle in preparation of an anti-fibrosis, inflammation and tumor or immune disease drug. BACKGROUND

[0002] Liver fibrosis reflects the reversible stage of the development of various chronic liver diseases to cirrhosis to some extent. The pathological state is characterized by an imbalance between tissue damage and repair, with fibrogenesis in the liver exceeding its degradation rate. The excessive deposition of ECM forms severe fibrous scars. Damage to the liver structure further worsens and transforms into cirrhosis and even liver cancer. In the normal liver microenvironment, non-parenchymal hepatic stellate cells are located in the hepatic sinusoidal space, and HSCs exhibit normal resting state. When the liver is damaged, the accumulation of hypoxia and hydrogen peroxide (H2O2) forms a pro-fibrotic liver microenvironment, involving fibrogenesis and chronic stimulation of hepatic stellate cells (HSCs), inducing HSC activation and proliferation and further increasing ECM deposition, while up-regulating hypoxia-inducible factor (HIF) and transforming growth factor beta 1 (TGF-β1). Therefore, activated HSCs are called the "executors" of liver fibrosis, and inhibition of HSC activation is the key to anti-fibrosis therapy, but due to the complex regulation of HSC activation pathways, the therapeutic effect of a single drug on fibrosis often fails to meet expectations. At the same time, in the pathological state of the liver microenvironment, the physical barrier of activated HSCs seriously hinders the efficiency of drug delivery. At this time, the hepatic sinusoidal endothelial cells serve as the first layer of barrier, accompanied by the formation of basement membrane and the closure of particle passage windows. ECM serves as the second layer of barrier, forming a dense spatial barrier, further hindering the internalization of nanoparticles. Considering the above obstacles, restoring the portal structure of LSECs, degrading ECM deposition barriers and targeting HSCs are key steps to improve the effect of anti-fibrosis.

[0003] Studies have shown that LSECs are regulated by the nitric oxide-cyclic guanosine monophosphate cyclase (NO-sGC) pathway. At the same time, ECM is regulated by matrix metalloproteinases, and NO can induce the expression of MMPs under ROS conditions to produce activity and degrade type I collagen. However, under fibrotic conditions, LSECs dysfunction leads to reduced eNOS activity and reduced self-generated NO production, and many studies have focused on collagenase-modified nanoparticles to degrade deposited ECM or deliver gene drugs to block ECM biosynthesis, thereby promoting the penetration of nanoparticles into HSCs. In order to solve the problem of inactivation of collagenase or gene drugs during delivery, existing technologies often use exogenous NO donors to target delivery to the body, break through the double barrier and improve the efficiency of drug delivery.

[0004] Nitric oxide (NO) as an important gas molecule plays a significant role in the regulation of hematopoietic stem cells, which mainly manifests as inhibiting vasoconstriction, cell proliferation and migration, and promoting apoptosis [6]. NO can react with reactive oxygen species (ROS) to generate peroxynitrite, thereby weakening the activation of nuclear factor-kappa B (NF-κB) signaling pathway and inhibiting the expression of fibrosis and inflammation-related genes. In addition, NO can also down-regulate the expression of transforming growth factor-beta (TGF-β) and weaken the activity of its downstream signaling pathway. Studies have shown that NO is also involved in repairing the fenestration structure of damaged liver sinus endothelial cells (LSECs), promoting the penetration of nanoparticles into the liver sinusoidal barrier and into the perisinusoidal tissue. At the same time, NO activates matrix metalloproteinases (MMPs), destroys the extracellular matrix (ECM) barrier of the perisinusoidal space, and promotes the internalization of nanoparticles by hepatic stellate cells (HSCs). However, NO is a double-edged sword, and its concentration directly affects the treatment effect. Its solubility in water is relatively low, and the local concentration is difficult to accurately control. The stability and controlled release of single administration are poor, and it has high reactivity, short half-life and lack of targeting ability.

[0005] The NO controllable switch designed in the prior art, such as pH, enzyme and external light source responsive release, wherein the external light source as a clean energy can control the release of NO in time and space, but the external light responsive NO donor material currently mainly reacts to ultraviolet (UV) or near ultraviolet. Due to the unnecessary light toxicity and poor tissue penetration, it is harmful to biomedical applications. Although long-wavelength sensitive near-infrared responsive NO donors can be developed, the extended covalent structure usually leads to reduced water solubility and reduced NO loading content. In the process of near-infrared up-conversion, energy loss occurs, resulting in low NO release efficiency. Moreover, the external high-energy light source will simultaneously affect the target area and the non-target area in controlling the release of NO, lacks specificity, causes tissue damage, and the flexibility in time and space is difficult to control. Therefore, it is particularly important to develop a prodrug capable of realizing the precise control of the release of NO at the target site under the irradiation of endogenous visible light, improving the targeting and bioavailability of NO, and reducing side effects. SUMMARY

[0006] The purpose of the present application is to provide an active oxygen responsive self-luminescent nitric oxide controlled release nanoparticle, and also to provide a preparation method of the nanoparticle and its application in the preparation of anti-fibrosis, inflammation and tumor or immune disease drugs.

[0007] Technical solution: The active oxygen responsive self-luminescent nitric oxide controlled release nanoparticle of the present application is grafted with a NO donor and a polyamino polymer, and then grafted with carboxyl modified cerium dioxide luminol amide reaction to obtain a copolymer, and the outer edge of the copolymer is modified with hyaluronic acid.

[0008] The carboxyl-modified cerium dioxide-luminol is modified by coating the cerium dioxide with a polycarboxylic compound, and then coupled with luminol.

[0009] The polycarboxylic compound is citric acid or oleic acid, and the mass ratio of the carboxyl-modified cerium dioxide to luminol is 1:1 to 1:3.

[0010] The polyamino polymer is polyethyleneimine, amino PEG or a derivative thereof, the NO donor is a nitrate ester or nitrite ester NO donor with a carboxyl group at the end, and the grafting molar ratio of the NO donor to the polyamino polymer is 1:1 to 1:5.

[0011] The outer edge is modified by electrostatic adsorption or chemical coupling for connection, and the mass ratio of hyaluronic acid to copolymer is 2:1 to 1:1.

[0012] The preparation method of the active oxygen-responsive self-luminous nitric oxide controlled-release nanoparticle comprises the following steps:

[0013] (1) A polyamino polymer is used as a graft copolymer, and an activated carboxyl NO donor is dissolved in a solvent according to a molar ratio, and a polyamino polymer-NO copolymer is synthesized by preparing a polyamino polymer;

[0014] (2) The polyamino polymer-NO copolymer and the carboxyl-modified cerium dioxide-luminol are respectively dissolved in a solvent, and a polyamino polymer-NO-cerium dioxide-luminol is prepared by amidation after activation of the carboxyl group.

[0015] (3) The polyamino polymer-NO-cerium dioxide-luminol and hyaluronic acid are dissolved, stirred and reacted under room temperature and light shielding conditions, and then freeze-dried to obtain the active oxygen-responsive self-luminous nitric oxide controlled-release nanoparticle.

[0016] In step (1), the molecular weight of the polyamino polymer is 0.6-40 KDa, in step (2), the molecular weight of the hyaluronic acid is 1-40 KDa, in step (1) or (2), the solvent includes dichloromethane, trichloromethane or tetrahydrofuran, and in step (3), the solvent is deionized water.

[0017] The active oxygen-responsive self-luminous nitric oxide controlled-release nanoparticle can also be applied in the preparation of anti-fibrosis, inflammation and tumor or immune disease drugs.

[0018] The active oxygen-responsive self-luminous nitric oxide controlled-release nanoparticle as a prodrug triggers the release of NO at the lesion site in response to endogenous ROS self-luminous and auxiliary in situ treatment imaging evaluation.

[0019] Invention principle: the active oxygen response type self-luminous one nitrogen monoxide controlled release nanoparticle of the application is grafted by NO donor and polyamino polymer, and then grafted by carboxyl modified cerium dioxide lumino amidation reaction to obtain copolymer, and the outer edge of the copolymer is modified by hyaluronic acid. Among them, the problem of agglomeration and biocompatibility defects of unmodified particles is solved by carboxyl modified cerium dioxide (CeO2NP), at the same time, active sites are provided for lumino (Lum) grafting, and Lum is covalently grafted on the surface of carboxyl modified CeO2NP by amidation reaction, which overcomes the limitations of poor solubility, short light duration and weak intensity when Lum is used alone; after the polyamino polymer is grafted with the NO donor, the amino group is further amidated and combined with the carboxyl group of CeO2NP and the amino group of Lum, forming a stable conjugate structure, which not only improves the stability of the NO donor (reduces spontaneous decomposition), but also enhances the overall biocompatibility with the help of the polymer skeleton; the outer edge modified hyaluronic acid (HA) targets the surface CD44 receptor of hepatic stellate cells (HSC), which solves the problem of lack of nanoparticle targeting.

[0020] The synergistic effect of each component of the nanoparticle prodrug of the application produces unexpected technical effects: the enzyme-like activity of carboxyl modified CeO2NP catalyzes the decomposition of excess reactive oxygen species (ROS) in the lesion area, provides a continuous light substrate for Lum, and generates 400-425nm blue light in situ, which precisely regulates the release of NO by the NO donor, forming a closed-loop mechanism of "ROS response-self-luminous-NO controlled release", without the need for exogenous light intervention, avoiding the phototoxicity and equipment dependence of traditional light control systems; covalent assembly and HA modification synergistically improve the biocompatibility of the nanoparticles. More importantly, the system can dynamically adjust the amount of NO release according to the ROS concentration, accurately expand the NO treatment window, and avoid the problems of too high concentration (toxic substances with ROS) or too low concentration (treatment failure), providing an innovative solution for precise treatment of liver fibrosis.

[0021] Beneficial effects: compared with the prior art, the present application has the following remarkable advantages: (1) the active oxygen response type self-luminous nitric oxide controlled release nanoparticle of the present application triggers the lumi-CeO2 system to produce 400-450 nm visible light by means of endogenous ROS, so that the NO donor is released in the visible light range with low energy and low cytotoxicity, the amount of NO release is linearly related to the light intensity, the release rate can be dynamically regulated by the ROS level of the lesion, the spatial and temporal precise control is realized, the accumulation of the nanoparticle in the activated hepatic stellate cells is enhanced by more than 2 times, the accumulation of the nanoparticle in the liver cells is effectively reduced, the activity of the liver cells is maintained at more than 80%, so that the release of NO for treating liver fibrosis is realized while the toxic damage of excess NO to normal tissues is effectively avoided; (2) in terms of physical and chemical properties and targeting, the solubility of the NO donor in neutral aqueous solution is improved by 3 times through the synergistic effect of multi-amino polymer grafting and carboxyl modified CeO2; at the same time, the NO release intensity of HA-NO-CE-LU in the high H2O2 model cell is 2 times that of the low H2O2 model cell; the system realizes the on-demand release of NO in the fibrosis core area through the closed-loop linkage of "ROS-light-NO release", and effectively treats liver fibrosis. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 For the in vitro characterization results of HA-NO-CE-LU in Example 3 of the present application: A is the change of the composite graft particle size; B is the particle size frequency distribution and electron microscopy (TEM) characterization of HA-NO-CE-LU; C is the composite potential characterization; D is the infrared spectrum; E is the thermal gravimetric (TG) analysis characterization;

[0023] Figure 2 For the characterization results of the enzyme activity of the HA-NO-CE-LU nanoparticle in Example 4 of the present application: A is the XRD result graph of cerium dioxide (CE) and cerium dioxide lumi (CE-LU); B is the XPS result graph of cerium dioxide and cerium dioxide lumi;

[0024] Figure 3 For the NO-triggered spectrum verification and self-luminous quantification of HA-NO-CE-LU in Example 5 of the present application: A is the ultraviolet spectrum of NO and PEI-NO; B is the fluorescence spectrum range of HA-NO-CE-LU, CE-LU and LU; C is the fluorescence quantification under different pH conditions; D is the luminescence graph under the simulated in vitro inflammatory environment determined by IVIS;

[0025] Figure 4 For the in vitro NO release verification graph of HA-NO-CE-LU under different pH and H2O2 in Example 6 of the present application;

[0026] Figure 5Figure of the inhibitory effect of HA-NO-CE-LU on HSCs in the model group and the normal group in Example 7 of the present application, and figure for normal AML12 toxicity detection;

[0027] Figure 6 Figure of the targeting effect of the HA-modified nanoparticle with or without a target head after the HSCs were treated with HA-NO-CE-LU in the model group and the normal group in Example 8 of the present application;

[0028] Figure 7 Figure of the NO release effect of the nanoparticle in the cell under the condition of HA-NO-CE-LU with or without a target head and different model concentrations after the HSCs were treated with HA-NO-CE-LU in the model group and the normal group in Example 9 of the present application;

[0029] Figure 8 Figure of the self-luminous NO controlled release of HA-NO-CE-LU in vivo in Example 10 of the present application, wherein A is a model staining schematic diagram, B is the luminous intensity of the nanoparticle observed by IVIS imaging at different modeling periods, C is the hydrogen peroxide accumulation amount, and D is the NO release fluorescence intensity comparison of the nanoparticle without a target P-NO-CE-LU and HA modification;

[0030] Figure 9 Figure of the effect evaluation of HA-NO-CE-LU in vivo against liver fibrosis in Example 11 of the present application, wherein A is the modeling and treatment process of fibrotic mice, and B is a hematoxylin and eosin (H&E) staining, a picro-sirius red staining, and a Masson's trichrome staining treatment schematic diagram. DETAILED DESCRIPTION

[0031] The technical solutions of the present application are further described below in combination with examples. The test materials used in the examples can be purchased through a conventional route.

[0032] Example 1

[0033] The synthesis of the carboxyl-modified cerium dioxide luminol coordination polymer includes the following steps:

[0034] (1) Synthesis of CeO2-COOH, the synthesis route and process are as follows:

[0035] A commercial CeO2-NPs suspension was prepared in a 200 mL water:ethanol solution. An equivalent citric acid solution (pH = 7-8) was prepared in another 200 mL water:ethanol solution. The two solutions were mixed under continuous stirring and reflux conditions for 3 h. Then the mixture was incubated in an oven for 24 hours to cover the carboxyl groups on the nanoparticles. Finally, the CeO2-COOH was collected from the mixture by centrifugation.

[0036]

[0037] (2) Synthesis of CeO2-Luminol, synthesis route and process as follows:

[0038] CeO2-COOH and luminol were added to chloroform solvent according to the mass ratio of 1:1, first add CeO2-COOH 1g, add 500mg of EDC.HCL / NHS room temperature activation reaction for half an hour, then add 1g luminol room temperature reaction 12H. After the reaction, the reaction liquid was rotary evaporated under reduced pressure. CeO2-Luminol nanoparticles were obtained.

[0039]

[0040] Example 2

[0041] The active oxygen response type self-luminous nitric oxide controlled release nanoparticles of the application comprise the following steps:

[0042] (1) Synthesis of nitrate NO donor, synthesis route and process as follows:

[0043] Take ethyl bromoacetate 2g, silver nitrate 4g, dissolve in acetonitrile, reflux, reaction avoid light overnight. Filter out silver bromide, rotary evaporation to remove acetonitrile, add 100ml DCM, see white precipitate, room temperature for 2H, filter again, take the filtrate rotary evaporation, get yellow oil product.

[0044]

[0045] Synthesis of polyethyleneimine-nitrate NO donor polymer, synthesis route and process as follows:

[0046] Take polyethyleneimine 10k 100mg, according to the grafting rate of 20% of the amino group unit on polyethyleneimine, take nitrate NO donor 56mg, add EDC 178mg, NHS 107mg dissolved in 10ml DCM to activate nitrate NO donor for 30min, add PEI 10K , room temperature overnight reaction in dark, after the reaction is completed, rotary evaporation under reduced pressure.

[0047]

[0048] (2) Synthesis of polyethyleneimine-nitrate NO donor-cerium dioxide luminol polymer, synthesis route and process as follows:

[0049] According to the grafting rate of 30% of the amino group unit on PEI 10K -NO, take CeO2-Luminol 30mg, EDC / NHS 30mg each, 18mg dissolved in 10ml DMSO, activate for half an hour, then add PEI10K - NO 11 mg, continue to react in the dark for 24 H. After the reaction is completed, take 3.5 K dialysis bag deionized water dialysis 2 Day, freeze-drying.

[0050]

[0051] (3) Synthesis of active oxygen response type self-luminous nitric oxide controlled-release nanoparticles, the synthesis route and method are as follows:

[0052] Take polyethyleneimine-nitrate ester NO donor-cerium dioxide luminol 10 mg, dissolve HA 10 mg in deionized water, and use electrostatic adsorption principle for compounding, react for 12 h, freeze-drying to obtain the final product, recorded as HA-NO-CE-LU.

[0053] Example 3

[0054] In vitro characterization of HA-NO-CE-LU:

[0055] Take about 2 mg of the solid powder of HA-NO-CE-LU prepared in Example 2 and disperse it in 2 ml of deionized water, add 1 ml of the dispersion to a particle size dish, and add another 1 ml of the dispersion to a potential dish, set the detection temperature to 25℃, and use a dynamic laser scattering particle size instrument (Litesizer 500) to measure the particle size and Zeta potential of the nanoparticles prepared under the above conditions. Transmission electron microscopy (FEI Philips Tecnai 20) is used to observe the morphology, and Zeta potential is used to evaluate the dispersion stability. Thermogravimetric analyzer (TG209 F3 Tarsus) and infrared Fourier spectroscopy are used to characterize and detect the surface polymer modification.

[0056] The size of the prepared nanoparticles is observed by transmission electron microscopy (TEM) to be between 100-200 nm, and further detected by DLS to have a hydrodynamic diameter of 153 nm ( Figure 1 A and B), the average size of the nanoparticles measured by DLS is within the nanometer range observed by TEM, indicating successful preparation. In addition, Zeta potential analysis and thermogravimetric analysis (TGA) analysis further confirm the structure of HA-NO-CE-LU nanoparticles. Zeta potential ( Figure 1 C) shows a stepwise change in charge from CeO2 to Luminol modification, PEI grafting to HA coating, confirming successful layer-by-layer assembly. Infrared spectroscopy clearly shows that the characteristic absorption bands at 1560, 1550 and 1350 cm -1 indicate that luminol and nitroacetic acid NO donor as the core functional group are stably present in all nanosystem materials, ensuring their chemical properties ( Figure 1 D) The grafting and compounding process is characterized by thermogravimetry (TGA), which further confirms the success of each step of graftingFigure 1 Example 4

[0057] Example 4

[0058] Characterization of HA-NO-CE-LU Nanoparticle Enzyme-like Activity:

[0059] The crystal structure of CeO2-based nanoparticles was analyzed by X-ray diffraction (XRD) analysis, Figure 2 Example 2A) showed that CE-LU exhibited diffraction peaks at 2Θ = 28.5°, 33.1°, 47.5°, 56.3°, which were consistent with the (111), (220), (311), (400) crystal planes of CeO2, indicating that the polymer modification did not destroy the original crystal structure of CeO2. X-ray photoelectron spectroscopy (XPS) analysis of the Ce 3d orbital signal showed that the Ce 3d orbital signal of CE-LU was similar to that of pure CeO2 Figure 2 Example 2B) and luminol-modified CeO2 Figure 2 Example 2C) compared with Figure 2 Example 2C) showed that the Ce(III) / Ce(IV) ratio in the modified nanoparticles was 31.43%:68.57%, which had a slight increase, ensuring its antioxidant function (clearing excess ROS at the fibrosis lesion site) and enhancing the chemiluminescence-triggered NO release.

[0060] Example 5

[0061] In vitro luminescence characterization of HA-NO-CE-LU:

[0062] To explore whether the photons generated by ceria-enhanced luminol could trigger the photolysis of NO donors and o-nitrobenzyl derivatives, NO monomers and PEI-NO were dissolved in deionized water and ultrasonically dispersed for 10 min to prepare 1 mM deionized water stock solutions. The ultraviolet-visible spectrophotometer (1 cm quartz cuvette) was used to scan the ultraviolet-visible absorption spectrum of the sample in the wavelength range of 200-800 nm Figure 3 Example 3A). Luminol, 100 μM, CeO2-Luminol, 100 μM, and HA-NO-CE-LU, 100 μM were dissolved in phosphate buffer (PBS, pH 7.4), and the fluorescence spectrophotometer was used to scan the fluorescence emission spectrum in the range of 400-600 nm at the excitation wavelength λ_ex = 350 nm (slit width: 5 nm, scanning speed: 200 nm / min) Figure 3 Example 3B).

[0063] To quantify the effect of pH on the in vitro luminescence intensity of the nanoparticles (HA-NO-CE-LU), a microplate reader with luminescence detection function was used to measure the luminescence intensity of 96-well plates with the same concentration of free luminol, CE-LU and HA-NO-CE-LU. Different pH environments were prepared by 1x PBS buffer (pH 6.4, 7.4, 8.4), and 1 mL of PBS solution corresponding to the pH was added to each well with the drug solution. After mixing, the luminescence intensity was immediately detected Figure 3 In vitro experiment C). At the same time, in order to detect the luminescence intensity in the simulated in vitro fibrosis weak acidic condition, 100 μL of 50 mM H2O2 and 100 μL of the same concentration of free luminol, CeO2-Luminol (CE-LU) and HA-NO-CE-LU were added to the 24-well plate, and the luminescence image was recorded by IVIS Spectrum. Figure 3 In vitro experiment D)

[0064] The experiment verified the strong chemiluminescence ability of HA-NO-CE-LU in the simulated fibrosis environment in vitro. The results showed that the nanoparticles exhibited strong blue luminescence at the longest ≈425 nm, and the fluorescence spectrum was similar to that of luminol. The spectral range overlapped with the ultraviolet-visible absorption spectrum range of the nitric acid NO donor dissolved in aqueous solution and PEI-NO, and the overlapping region had moderate absorbance. This indicated that the chemiluminescence of the nanoparticles might induce the photolysis of NO molecules to release.

[0065] Example 6

[0066] In vitro NO release of HA-NO-CE-LU:

[0067] Incubation was carried out in phosphate buffer solution (PBS, 10 mM) containing different pH values and H2O2, and the specific systems were as follows: (1) PBS, pH 6.4; (2) PBS, pH 6.4, containing 10, 20, 30, 40, 50 mM H2O2;

[0068] (3) PBS, pH 7.4; (4) PBS, pH 7.4, containing 50 mM H2O2; (5) PBS, pH 8.4; (6) PBS, pH 8.4, containing 50 mM H2O2. 1.0 mL of HA-NO-CE-LU solution (1 mg / mL) was added to a dialysis bag with a molecular weight cutoff (MWCO) of 3500 Da, and placed in 20 mL of the corresponding medium system. The bags were incubated at 37°C on a shaker. 2 mL of release medium was collected at preset time points, and an equal volume of fresh PB buffer under the same conditions was immediately added after sampling. 1 mL of release medium was used to detect the NO content in the release medium using the Griess reagent method. The absorbance value of the corresponding medium was detected using a microplate reader at a wavelength of UV = 540 nm. The NO concentration was calculated based on the standard curve of sodium nitrite solution. The results are as follows: Figure 4 Displays A and B

[0069] To simulate the difference in NO release caused by ROS changes in a fibrotic microenvironment, 100 μL of the 10-50 mM release medium from system (2) was taken, and an equal volume of fresh medium was immediately added after sampling. The NO release was detected by the Griess method at UV=540 nm using an ELISA reader.

[0070] In vitro NO release experiments verified that HA-NO-CE-LU releases NO via chemiluminescence triggered by H2O2. The released NO was quantitatively detected using the Griess method. Results are as follows: Figure 4 As shown in Figure A, NO release peaks at pH 6.4 because the chemiluminescence intensity is highest under this condition, achieving the light energy required for NO release and enabling photo-triggered on-demand NO release, which is positively correlated with light intensity. Meanwhile, in systems with different H₂O₂ concentrations at pH 6.4 (… Figure 4 In the case of NO (B), the release amount increases with increasing H2O2 concentration. The release kinetic curve shows that 76% of the total NO is released within 12 hours. Figure 4 (C)

[0071] Therefore, HA-NO-CE-LU can generate strong fluorescence signals under conditions that simulate the fibrotic microenvironment in vitro, and the photons generated thereby can realize the on-demand and controllable release of NO.

[0072] Example 7

[0073] The MTT assay was used to determine the cytotoxicity of nanomedicines and conventional photosensitized S-NO (irradiated with exogenous light) on resting and H2O2-activated HSC-T6 cells and normal AML12 cells. After treatment, the optical density was measured at 490 nm using a Bio-Rad microplate reader.

[0074] At the same concentration of material, activated HSCs are more sensitive to drug toxicity and cytotoxicity is significantly increased. The release of NO triggered by in situ chemiluminescence can be attributed to the accumulation of H2O2 in activated HSCs, while the cytotoxicity of nanoparticles is significantly reduced without release in normal HSCs Figure 5 A), while the inhibitory effect of S-NO on the model group is significantly lower than that of HA-NO-CE-LU Figure 5 B) after irradiation by exogenous light. The single S-NO traditional NO donor is unstable and causes certain toxicity to normal liver cells (AML12), while the HA-NO-CE-LU nanoparticle with the same concentration of material has good stability, and the survival rate of AML12 is > 80% Figure 5 C), confirming the selective toxicity of nanoparticles to activated HSCs. Therefore, HA-NO-CE-LU can selectively promote the apoptosis of activated HSCs without significant toxicity to other normal cells in the liver, thereby promoting the treatment of liver fibrosis and reducing the side effects of the preparation.

[0075] Example 8

[0076] After treating HSC-T6 cells, they were stained with 1 μg / mL of 4,6-diamidino-2-phenylindole (DAPI) solution for 15 min. After three PBS washes, the cell uptake efficiency was observed by confocal laser scanning microscopy (CLSM).

[0077] The effect of HA surface modified nanoparticles on the internalization of hematopoietic stem cells was studied. As shown in Figure 6 More Cy5-COOH modified HA-PEI-NO-CE-LU was internalized by HSC-T6 cells than non-HA coupled PEI-NO-CE-LU under the same cell state. Under different cell states, activated HSC cells uptake more than the resting state, which is attributed to the high affinity between HA and CD44 receptors on HSC-T6 cells. They express less in the resting state, and HSCs are the main cells expressing CD44 in the liver. When liver fibrosis occurs, the proliferation of HSCs is accompanied by a significant increase in CD44 expression. It is confirmed that HA modification increases the targeting of HSC-T6.

[0078] Example 9

[0079] NO production in HSC-T6 cells was evaluated by 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) staining. After treating HSC-T6 cells according to the kit instructions, the cells were stained and washed, and the intracellular NO release effect was observed by confocal laser scanning microscopy (CLSM).

[0080] As shown in Figure 7As shown, in the model cell group, ROS stimulation promoted luminol-induced NO release, with NO molecules binding to DAF-AM and emitting green fluorescence, while the normal group showed no fluorescence. Furthermore, in the same HA-target modified material, the fluorescence intensity of NO was proportional to H2O2, indicating that nanoparticles achieve ROS-responsive phototriggered NO release on demand within cells.

[0081] Example 10

[0082] A three- and five-week-old fibrotic mouse model was established and compared with a normal group. 200 μL (3 mg / ml) of the nanomedicines P-NO-CE-LU and HA-NO-CE-LU were injected into the mice via the tail vein. The modeling cycle-dependent changes in the HA-NO-CE-LU luminescence signal in different groups were examined using an IVIS imaging system (exposure time = 5 min, f / stop = 1, pixel binning = 8, no filter). Simultaneously, control mice were injected with saline. The mice were euthanized 10-15 minutes after imaging, and the entire liver was isolated. H2O2, AST, and ALT levels in the liver tissue at different modeling cycles were detected using a detection kit. The isolated liver was also stained with NO fluorescence to observe and compare the fluorescence intensity of NO released from the nanomedicine in the liver tissue.

[0083] Because NO release in the in vitro model is positively correlated with luminescence intensity, further investigation revealed that in vivo NO release is also positively correlated with the intensity of ROS-triggered chemiluminescence. Figure 8 First, pathological examination of the ex vivo livers of mice in the modeling group and the normal group was performed. By observing HE, Sirius Red, and Masson staining, it was observed that the livers of the modeling group mice showed obvious inflammatory cell infiltration and fibrotic pathological features. Red and blue fibrous features were observed in Sirius Red and Masson staining, respectively, indicating successful modeling. Next, the fluorescence intensity of the nanoparticles was observed at different modeling stages using IVIS imaging. The fluorescence intensity increased with the length of the modeling period, reaching its maximum intensity in the fifth week. Figure 8 (B) Next, the concentration of H2O2 in isolated liver tissue was measured using a detection kit, such as... Figure 8 As shown in Figure C, hydrogen peroxide accumulation reached its maximum at week 5 and gradually increased with each modeling cycle. Further investigation was conducted using NO in vitro section staining to explore the release intensity of NO in liver tissue. Figure 8 Compared to untargeted P-NO-CE-LU, HA-modified nanoparticles showed relatively higher fluorescence intensity, with the most significant green fluorescence intensity at week 5. The final results indicate that observing the chemiluminescence intensity of nanoparticles in the liver can aid in the observation of NO-on-demand release therapy.

[0084] Example 11

[0085] For in vivo anti-fibrosis studies, male C57 mice were randomly divided into 7 groups (5 mice per group) after 5W of 40% CCL4 administration. The mice were then treated twice a week for 3W with the following solutions: Group 1, control group; Group 2, PBS by tail vein injection; Groups 3 and 4, treatment groups, fibrotic mice injected intravenously with P-NO-CE-LU, HA-NO-CE-LU, with a nanomaterial dose of 50 mg / kg. Body weight was measured once a week. One day after the last injection, all mice were sacrificed and their livers were collected. The liver tissue was dissected and stored in 10% formalin for subsequent histological analysis. In addition, liver tissue sections were stained with hematoxylin and eosin (H&E), MASSON and Sirius red, and images were taken using an optical microscope (Olympus).

[0086] Fibrotic mouse models were established by 40% carbon tetrachloride injection for 5 weeks, followed by analysis using hematoxylin and eosin (H&E) staining, Sirius red staining, and Masson's trichrome staining Figure 9 Based on the distribution of the nanomedicine in the mouse liver, the therapeutic dose was calculated and injected intravenously twice a week Figure 6 After three weeks of treatment, the mice were sacrificed to evaluate the anti-fibrosis indicators.

[0087] From the gross morphology of the liver, Figure 9 Group B) The liver of the HA-NO-CE-LU group was red and regular in shape, far superior to the damage of the PBS group and the partial repair state of the P-NO-CE-LU group; HE staining showed that the liver cells of this group were arranged close to the normal liver lobule structure, with no necrotic foci, few inflammatory cells, and efficient repair of cell damage and inhibition of inflammation; Masson and Sirius Red staining further confirmed that it could significantly inhibit collagen fiber deposition and reduce fibrosis load, with sparse and discrete distribution of blue and red collagen fibers, superior to the residual deposition of the P-NO-CE-LU group. Relying on the precise enrichment of the CD44 receptor on the surface of hepatic stellate cells (HSC) through hyaluronic acid (HA) targeting, in summary, HA-NO-CE-LU nanoparticles can precisely control the release of NO as needed at the fibrotic liver lesion site to achieve anti-liver fibrosis, significantly surpassing the non-targeted system in repair integrity and anti-fibrosis strength, and providing direct and strong evidence for precise treatment of liver fibrosis.

Claims

1. A reactive oxygen species-responsive, self-luminous, controlled-release nitric oxide nanoparticle, characterized in that, The nanoparticles are obtained by grafting NO donor with a polyamino polymer, followed by grafting with carboxyl-modified cerium dioxide luminol via an amidation reaction to obtain a copolymer, and then modifying the outer edge of the copolymer with hyaluronic acid.

2. The nanoparticles according to claim 1, characterized in that, The carboxyl-modified cerium dioxide luminol is obtained by coating cerium dioxide with a multi-carboxyl compound for modification, and then coupling it with luminol.

3. The nanoparticles according to claim 2, characterized in that, The polycarboxylic acid compound is citric acid or oleic acid, and the mass ratio of carboxyl-modified cerium dioxide to luminol is 1:1 to 1:

3.

4. The nanoparticles according to claim 1, characterized in that, The polyamino polymer is polyethyleneimine, aminoPEG or its derivatives, the NO donor is a nitrate ester or nitrite ester NO donor with a carboxyl group at the end, and the grafting molar ratio of the NO donor to the polyamino polymer is 1:1 to 1:

5.

5. The nanoparticles according to claim 1, characterized in that, The outer edge modification is achieved by electrostatic adsorption or chemical coupling, and the mass ratio of hyaluronic acid to copolymer is 2:1 to 1:

1.

6. A method for preparing the reactive oxygen species-responsive self-luminous nitric oxide controlled-release nanoparticles according to claim 1, characterized in that, Includes the following steps: (1) Take the polyamino polymer as the graft copolymer, dissolve the NO donor with activated carboxyl group in the solvent according to the molar ratio, and synthesize the polyamino polymer-NO copolymer with the prepared polyamino polymer. (2) The polyamino polymer-NO copolymer and carboxyl-modified cerium dioxide luminol were dissolved in solvents respectively, and the carboxyl groups were activated and then prepared by amidation to obtain polyamino polymer-NO-cerium dioxide luminol. (3) Dissolve the polyamino polymer-NO-cerium dioxide luminol and hyaluronic acid, stir and react at room temperature in the dark, and freeze dry to obtain reactive oxygen-responsive self-luminous nitric oxide controlled-release nanoparticles.

7. The preparation method according to claim 6, characterized in that, In step (1), the molecular weight of the polyamino polymer is 0.6-40 kDa, and in step (2), the molecular weight of the hyaluronic acid is 1-40 kDa.

8. The preparation method according to claim 6, characterized in that, In step (1) or (2), the solvent includes dichloromethane, trichloromethane or tetrahydrofuran; in step (3), the solvent is deionized water.

9. The use of the reactive oxygen species responsive self-luminescent nitric oxide controlled-release nanoparticles of claim 1 in the preparation of drugs for anti-fibrosis, inflammation, and tumor or immune diseases.

10. The application according to claim 9, characterized in that, The reactive oxygen species-responsive self-luminescent nitric oxide controlled-release nanoparticles serve as a prodrug, triggering the on-demand release of NO at the lesion site through endogenous ROS-responsive self-luminescence.

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