Pyridinium-based nano material for targeting damaged mitochondria and preparation and application of pyridinium-based nano material

Abstract

The invention belongs to the technical field of biological medicines, and discloses a damaged mitochondria targeting pyridinium-based nano material as well as preparation and application thereof. The invention relates to a damaged mitochondria targeting pyridinium-based nano material, which comprises more than one of the following formulas I-II, p1 is poly (4-vinylpyridine) or polyethylene acrylate pyridine in which a pyridine group participates in forming a pyridinium group; p2 is NH2 or a secondary amine group which participates in forming a polyamide-amine dendrimer, a polypropyleneimine dendrimer, a polylysine dendrimer, linear polyethyleneimine, branched polyethyleneimine and linear polylysine which contain an N group in a Z structure. The invention also discloses a preparation method of the pyridinium-based nano material. The pyridinium-based nano material is stable in structure, has good damaged mitochondrial targeting ability, mitochondrial autophagy promoting ability and biocompatibility, and is used for preparing drugs for treating mitochondrial dysfunction related diseases, including drugs for treating atherosclerosis and non-alcoholic fatty liver diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a pyridinium-based nanomaterial targeting damaged mitochondria and preparation and application thereof. BACKGROUND

[0002] Mitochondria, as the key organelle for energy supply, redox regulation and metabolic homeostasis in cells, plays a core role in the occurrence and development of various chronic diseases. Mitochondrial dysfunction is closely related to cellular energy metabolism disorder, oxidative stress increase, inflammation activation and programmed cell death, and thus participates in the pathogenesis of various chronic diseases including atherosclerosis (AS) and non-alcoholic fatty liver disease (NAFLD).

[0003] In the pathogenesis of AS and NAFLD, mitochondrial dysfunction is at the core of pathological evolution. In AS, monocytes migrate to the vascular intima and differentiate into macrophages, which phagocytose a large amount of oxidized low-density lipoprotein (ox-LDL) to form foam cells. The continuous accumulation of foam cells leads to excessive intracellular lipid load, directly inducing cellular metabolic disorder and mitochondrial damage, further activating oxidative stress response and pro-inflammatory signaling pathways, accelerating the formation of arterial plaques and increasing their instability. In NAFLD, due to increased lipid intake and synthesis and decreased oxidative utilization capacity of hepatocytes, a large amount of lipids are abnormally accumulated in cells. The lipids that are not removed in time trigger lipid peroxidation and mitochondrial stress, and further induce a series of pathological changes such as hepatocyte steatosis, inflammatory cell infiltration and apoptosis. Studies have shown that in these two types of diseases, mitochondrial dysfunction is not only a direct cause of energy metabolism imbalance, but also an important driving factor for excessive ROS production, persistent inflammation activation and cell death. Therefore, intervention on the mechanism of mitochondrial dysfunction, especially restoration of mitochondrial function and maintenance of its homeostasis, has become a key entry point for the prevention and treatment of diseases such as AS and NAFLD. Developing drugs or delivery systems that can target damaged mitochondria and enhance the ability to restore mitochondrial function has important theoretical value and clinical application prospect.

[0004] Mitophagy is a process in which cells selectively recognize and eliminate damaged mitochondria under stress conditions, and is an important quality control mechanism for maintaining the number and function of mitochondria. In diseases such as AS and NAFLD, the chronic stress environment in which cells exist inhibits the expression or activation of key proteins related to mitochondrial autophagy (such as PINK1 and Parkin), resulting in a decrease in the level of mitochondrial autophagy, and the accumulation of dysfunctional mitochondria. The accumulation of damaged mitochondria not only exacerbates ROS generation and oxidative stress, but also can induce inflammatory response and programmed cell death. Therefore, how to activate mitochondrial autophagy and enhance the ability of cells to recognize, wrap and degrade damaged mitochondria is a key point for restoring mitochondrial function, improving energy supply and relieving oxidative damage and inflammatory response. Developing materials or drugs that can effectively promote mitochondrial autophagy has the potential to become a core strategy for treating mitochondrial dysfunction-related diseases.

[0005] At present, a variety of natural compounds and synthetic drugs have been found to have the ability to induce mitochondrial autophagy, such as resveratrol and rapamycin. However, their application in the treatment of mitochondrial dysfunction-related diseases such as AS and NAFLD still faces many challenges. First, low bioavailability. Most natural compounds and small molecule drugs are rapidly metabolized in vivo and have a short half-life, making it difficult to maintain an effective drug concentration, which seriously restricts the sustainability and stability of their efficacy. Second, non-specific activation of autophagy. These inducers mainly regulate the upstream autophagy pathway, which not only enhances the level of mitochondrial autophagy, but also enhances the autophagy of cells in general, and do not specifically act on mitochondria. Excessive or non-specific activation of autophagy can lead to the loss of healthy mitochondria.

[0006] With the continuous development of nanotechnology, the bioavailability of small molecule drugs has been significantly improved, and strategies for mitochondrial-targeted therapy have also made great progress. Among them, delocalized lipophilic cations (DLCs) have attracted attention due to their excellent mitochondrial targeting ability, such as triphenylphosphine (TPP), rhodamine 123 and decylammonium chloride (DQA). Although the above mitochondrial autophagy inducers have shown great potential in the treatment of mitochondrial dysfunction-related diseases, they still face many challenges. First, low stability and bioavailability. Many natural compounds or small molecule drugs are rapidly metabolized in vivo and have a short half-life, affecting their sustained efficacy. Second, non-specific activation of autophagy. These inducers mainly regulate the upstream autophagy pathway, which not only enhances the level of mitochondrial autophagy, but also enhances the autophagy of cells in general, and do not specifically act on mitochondria. Excessive or non-specific activation of autophagy can lead to the loss of healthy mitochondria, thereby exacerbating the disease. SUMMARY

[0007] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present application is to provide a pyridinium-based nanomaterial targeting damaged mitochondria and a preparation method thereof. The nanomaterial of the present application takes a polymer as a skeleton, introduces a functional side chain modified pyridinium group such as a fluoralkyl group on the surface thereof, and spontaneously assembles to form nanoparticles with uniform particle size and stable structure in a water environment. The nanomaterial of the present application has excellent damaged mitochondria targeting ability, can not only be specifically located in functionally damaged mitochondria, but also effectively activate the mitochondrial autophagy pathway, realize selective removal of diseased mitochondria, and further restore the mitochondrial membrane potential, respiratory chain function and cell energy metabolism. The nanomaterial of the present application shows significant effects in AS and NAFLD models, including improving lipid accumulation in foam cells and fatty liver cells, inhibiting oxidative stress and inflammatory response, promoting recovery of cell metabolic homeostasis, etc.

[0008] Another purpose of the present application is to provide the application of the above-mentioned pyridinium-based nanomaterial targeting damaged mitochondria. The nanomaterial of the present application is used for preparing a drug for treating diseases related to mitochondrial dysfunction, specifically including treating diseases such as atherosclerosis and non-alcoholic fatty liver. The nanomaterial of the present application can be used as a nano-injection. The nanomaterial is used for a damaged mitochondria fluorescence imaging agent.

[0009] The purpose of the present application is achieved by the following technical solutions:

[0010] A pyridinium-based nanomaterial targeting damaged mitochondria comprises one or more of the following formulae I to II:

[0011]

[0012] P1 is a poly-4-vinylpyridine or polyvinyl acrylate pyridine in which a pyridine group participates in forming a pyridinium group. In formula I, the pyridine group corresponding to the pyridinium group is from P1, i.e. from the pyridine group in poly-4-vinylpyridine or polyvinyl acrylate pyridine. The grafting rate of the pyridinium modification group, i.e. the percentage of the number of moles of the pyridinium modification group in the total number of moles of the pyridine group in P1, is 0.1-100%, preferably 10-90%.

[0013] Formula I includes formula I-1 and formula I-2:

[0014]

[0015] In Formula I, each X is selected from H or F, each Y is selected from H, F, -OH, -COOH, -SO3H, a is an integer from 1 to 12 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), b is an integer from 0 to 11 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11), and a+b is an integer from 1 to 12 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0016] In formula II, Z represents -NH-, -N=C-, -NHCH2CH(OH)-, -NHCH2CH(OH)CH2O-, Group; P2 is a polyamide-amine dendritic polymer, polypropylene imine dendritic polymer, polylysine dendritic polymer, linear polyethyleneimine, branched polyethyleneimine, or linear polylysine (specifically, P2 is a polymer formed by the removal of hydrogen from the terminal group NH2 or NH2 or NH); N in the Z structure comes from the terminal group NH2 in the polyamide-amine dendritic polymer, polypropylene imine dendritic polymer, or polylysine dendritic polymer, or from the terminal group NH2 or NH in the linear polyethyleneimine, branched polyethyleneimine, or linear polylysine;

[0017] X is selected from H or F, Y is selected from H, F, -OH, -COOH, -SO3H, a is an integer from 1 to 12 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), b is an integer from 0 to 11 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11), and a+b is an integer from 1 to 12 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12); c is the number of functionalized pyridinium-modified groups covalently linked to the polymer surface or the grafting rate of pyridinium-modified groups.

[0018] In Formula II, the polyamide-amine resin is PAMAM, and the terminal group of PAMAM is NH2; when the structure is Formula II-1: In the formula, m represents the number of dendritic polymer core M connected to -(CH2CH2-CONH-CH2CH2N), n represents the number of repeating branch units (-CH2CH2-CONH-CH2CH2N), the CH2 of the next branch unit is connected to the N of the previous branch unit, the N of each branch unit is connected to the CH2 of the next two branch units, and the terminal group of the last branch unit is NH2; at this time, c in formula II is an integer from 1 to 2048, M is a group formed by losing hydrogen from the amino group of ammonia, ethylenediamine, butanediamine, pentanediamine, hexanediamine, octanediamine, decanediamine or 1,12-dodecanediamine; n is an integer from 1 to 10 (preferably 2, 3, 4, 5, 6, 7, 8); m is an integer from 2 to 4.

[0019] Polypropylene imine dendrimer, structure of formula II-2: In the formula, m represents the number of the dendrimer core M connected with -(CH2CH2CH2N), and n represents the number of the repeating branch unit (-CH2CH2CH2N), the CH2 of the latter branch unit is connected with the N of the former branch unit, the N of each branch unit is connected with the CH2 of the two latter branch units, and the end group of the last branch unit is NH2; at this time, c in formula II is an integer from 1 to 64, M is a group formed by losing hydrogen from the amine group in ammonia, butanediamine, ethylenediamine or 1,12-dodecanediamine, n is an integer from 1 to 5, and m is an integer from 2 to 4.

[0020] Polylysine dendrimer, structure of formula II-3: In the formula, m represents the number of the dendrimer core M connected with -(COCH(CH2CH2CH2CH2NH)NH), and n represents the number of the repeating branch unit (COCH(CH2CH2CH2CH2NH)NH), the C=O of the latter branch unit is connected with the NH of the former branch unit, one or two NH of each branch unit is connected with the CH2 of the one or two latter branch units, and the end group of the last branch unit is NH2; at this time, c in formula II is the grafting rate of the pyridinium modification group, 0.1% to 100%, preferably 10% to 90%; M is a group formed by losing hydrogen from the amine group in ammonia, ethylenediamine, butanediamine, pentanediamine, hexanediamine, octanediamine, sunflower diamine or 1,12-dodecanediamine; n is an integer from 1 to 10; and m is an integer from 2 to 4.

[0021] Linear polyethylene imine, structure of formula II-4: -(CH2-CH2-NH) x - or x is an integer greater than or equal to 0; the linear polyethylene imine can also be ethylenediamine-terminated polyethylene imine. The molecular weight of the linear polyethylene imine is 1800 to 25000. At this time, c in formula II is the grafting rate of the pyridinium modification group, 0.1% to 100%, preferably 10% to 90%.

[0022] Branched polyethylene imine, structure of formula II-5:

[0023] n is an integer greater than or equal to 0, and x and y are integers greater than or equal to 0; the molecular weight of the branched polyethylene imine is 1800 to 25000. At this time, c in formula II is the grafting rate of the pyridinium modification group, 0.1% to 100%, preferably 10% to 90%.

[0024] Linear polylysine, structure of formula II-6: x is an integer of 0 or more; the linear polylysine has a molecular weight of 5000-100000. In this case, c in formula II is the grafting rate of the pyridinium-modified group, 0.1%-100%, preferably 10-90%.

[0025] The preparation method of the pyridinium-based nanomaterial targeting damaged mitochondria comprises the following steps: preparing one or more polymers in formula I-II into nanoparticles to obtain the pyridinium-based nanomaterial targeting damaged mitochondria.

[0026] The preparation method specifically comprises the following steps:

[0027] 1) reacting poly-4-vinylpyridine or polyvinyl acrylate pyridine with a halogen compound in an organic solvent as a reaction medium to obtain the pyridinium-based nanomaterial targeting damaged mitochondria in formula I; or reacting the polymer with a pyridinium group compound in an organic solvent as a reaction medium to obtain the pyridinium-based nanomaterial targeting damaged mitochondria in formula II.

[0028] The halogen compound is R' is halogen (-Cl, -Br, -I). The poly-4-vinylpyridine has a molecular weight of 5000-160000 Da.

[0029] The polyvinyl acrylate pyridine has a molecular weight of 6000-160000 Da.

[0030] The polymer is polyamide-amine dendrimer, polypropylene imine dendrimer, polylysine dendrimer, linear polyethylene imine, branched polyethylene imine, or linear polylysine.

[0031] The pyridinium group compound is wherein Q is halogen (-Cl, -Br, -I), -COOH, (R is -CF3, -CF2CF3, -CF2CF2CF3, -CF2CF2CF2CF3, or -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3).

[0032] In the preparation of formula I, the organic solvent is one or more of DMSO, methanol, acetonitrile, and DMF.

[0033] In the preparation of formula I, the reaction conditions are 40-60℃ for 5-9 days. The molar ratio of the pyridine group in the poly-4-vinylpyridine or polyvinyl acrylate pyridine to the halogen compound is 1:(0.1-2).

[0034] In the preparation of formula II, the organic solvent is one or more of methanol, DMSO, acetonitrile, and DMF.

[0035] The reaction condition is 8-12h at room temperature-60℃.

[0036] The amount of the polymer and the pyridinium group compound satisfies: 0.1 times to 2 times of the number of moles of -NH2 on the surface of the polymer per one mole of the polymer needs to be added with the pyridinium group compound.

[0037] When the polymer is linear polyethylene imine or branched polyethylene imine, the amount of the polymer and the pyridinium group compound satisfies: 0.1 times to 2 times of the number of moles of -NH2 and / or -NH on the surface of the polymer per one mole of the polymer needs to be added with the pyridinium group compound.

[0038] The pyridinium group compound is obtained by reacting a pyridine compound with a halogen-containing compound.

[0039] The pyridine compound is: The halogen-containing compound is R" is -Br, -I. The reaction is carried out in an organic solvent. The organic solvent is one or more of DMSO, methanol, acetonitrile, DMF. The reaction condition is 5-9 days at 40-60℃. The molar ratio of the pyridine compound to the halogen-containing compound is 1:(0.5-1.5).

[0040] In the preparation of formula I, after the reaction is completed, water dialysis and freeze-drying are adopted.

[0041] In the preparation of formula II, after the reaction is completed, water dialysis and freeze-drying are adopted.

[0042] The pyridinium group nanomaterial targeting damaged mitochondria is used for preparing a nanoinjection.

[0043] The pyridinium group nanomaterial targeting damaged mitochondria is used for preparing a damaged mitochondria fluorescence imaging agent.

[0044] The pyridinium group nanomaterial targeting damaged mitochondria is used for preparing a drug for treating diseases related to mitochondrial dysfunction, specifically including treating atherosclerosis, non-alcoholic fatty liver disease, etc.

[0045] The pyridinium group nanomaterial targeting damaged mitochondria of the application can effectively activate mitochondrial autophagy, promote the removal of dysfunctional mitochondria, restore the mitochondrial membrane potential difference and respiratory chain function, improve the efficiency of adenosine triphosphate (ATP) synthesis, reduce the level of reactive oxygen species (ROS) in cells, promote the expression of anti-inflammatory macrophage (M2 type) phenotype, and inhibit pro-inflammatory macrophage (M1 type).

[0046] The nanomaterial of the present application spontaneously assembles in a water environment to form nanoparticles with uniform particle size and stable structure. The nanoparticles have the ability to target mitochondria and can specifically bind to diseased mitochondria in foam cells, fatty liver cells and other mitochondria-damaged cells rich in lipid droplets. The nanomaterial of the present application has a high affinity for damaged mitochondria with lost membrane potential, thereby enhancing the ability to target mitochondria. Further, the material can regulate the interaction strength of the pyridinium group with the mitochondrial membrane protein by adjusting the density of the pyridinium group, thereby achieving precise regulation of the degree of activation of the mitochondrial autophagy pathway. This regulation mechanism can promote the removal of damaged mitochondria while avoiding excessive autophagy leading to the loss of normal mitochondrial function and cell energy depletion.

[0047] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0048] (1) The nanomaterial of the present application effectively improves its lipophilicity and the ability to enrich under the driving of the mitochondrial membrane potential, enabling it to efficiently target functionally damaged mitochondria and achieve specific action. This feature is significantly different from the non-specific autophagy activation induced by traditional small molecule inducers, avoiding damage to normal mitochondria and reducing potential toxic side effects.

[0049] (2) The nanomaterial of the present application can effectively induce mitochondrial autophagy activation, enhancing the recognition, wrapping and removal ability of damaged mitochondria, breaking the limitations of suppressed autophagy in chronic pathological environments, and effectively restoring the metabolic homeostasis of cells. Related experiments have confirmed that the material can alleviate the oxidative stress and inflammatory response caused by excessive lipid load in foam cells and fatty liver cells, thereby reversing the pathological progression at the mechanism level.

[0050] (3) Compared with the low bioavailability and poor drug efficacy persistence of existing inducers, the nanomaterial of the present application exhibits good stability and long-acting effect in vivo, effectively maintaining the effective concentration in the lesion tissue, enhancing the practicality and controllability.

[0051] In summary, the nanomaterial of the present application has stable structure, good damaged mitochondria targeting ability, mitochondrial autophagy promoting ability and biocompatibility. The nanomaterial of the present application can effectively activate mitochondrial autophagy and promote the removal of dysfunctional mitochondria, thereby achieving the intervention effect on mitochondrial dysfunction-related diseases. The material has good application prospects in the preparation of drugs for treating atherosclerosis, non-alcoholic fatty liver and other diseases, and has potential value as a mitochondrial function regulating therapeutic agent. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 One-dimensional nuclear magnetic hydrogen spectrum of PF prepared in Example 1 in d6-DMSO;

[0053] Figure 2 One-dimensional nuclear magnetic hydrogen spectrum of PC prepared in Example 1 in d6-DMSO;

[0054] Figure 3 TEM images of nanoparticles of PF and PC prepared in Example 1 in 20% FBS;

[0055] Figure 4 Sample images of PF and PC prepared in Example 1 incubated in solutions containing 10%-50% (volume fraction, v / v) FBS for 2 hours, respectively;

[0056] Figure 5 Qualitative analysis of protein adsorption behavior on the surface of PF and PC by SDS-PAGE

[0057] Figure 6 BrdU cell proliferation results of PF and PC on Raw 264.7 cells at different doses;

[0058] Figure 7 CCK8 toxicity results of PF and PC on NIH 3T3 cells at different doses;

[0059] Figure 8 Mitochondrial fluorescence colocalization images and material and mitochondrial colocalization coefficient analysis of PF prepared in Example 1 and stably transfected mitoGFP 143B cells co-incubated for different times;

[0060] Figure 9 LC3 and p62 protein immunofluorescence staining images of foam cells treated with PF prepared in Example 1 for 6 hours;

[0061] Figure 10 TOM20 protein immunofluorescence staining images of foam cells treated with PF prepared in Example 1 for 6 hours;

[0062] Figure 11 Western blot detection results of LC3, p62 and TOM20 proteins of foam cells treated with PF prepared in Example 1 for 6 hours;

[0063] Figure 12 Mitochondrial membrane potential confocal images of foam cells treated with PF prepared in Example 1 for 6 hours;

[0064] Figure 13 Intracellular ATP level detection results of foam cells treated with PF prepared in Example 1 for 6 hours;

[0065] Figure 14Transmission electron microscope (TEM) images of mitochondria in RAW264.7 normal cells (positive control group), foam cells (negative control group) and foam cells treated with PF (PF); red arrow indicates lipid droplet, green arrow indicates autophagosome wrapping mitochondria, blue arrow indicates autophagosome wrapping lipid droplet; scale bar: 2 μm;

[0066] Figure 15 Confocal images of ROS in Raw 264.7 cells treated with PF prepared in Example 1 for 6 hours; ROS level was detected by DCFH-DA fluorescent probe;

[0067] Figure 16 Confocal images of mitochondrial ROS in foam cells treated with PF prepared in Example 1 for 6 hours; mitochondrial ROS level was detected by MitoSOX GREEN fluorescent probe;

[0068] Figure 17 Confocal images of lipid droplets in foam cells treated with PF prepared in Example 1 for 6 hours;

[0069] Figure 18 Representative images of oil red O staining of aortic tissue of AS mice after different treatments;

[0070] Figure 19 Representative images of H&E staining of aortic root tissue sections of AS mice after different treatments;

[0071] Figure 20 Biological imaging images of PF fluorescent signal in aortic tissue of healthy mice and AS model mice;

[0072] Figure 21 Biological imaging images of PF fluorescent signal in main organs of AS model mice;

[0073] Figure 22 Collagen immunohistochemical staining images of aortic root sections of AS model mice after PF treatment;

[0074] Figure 23 iNOS immunofluorescence staining images of aortic root sections of AS model mice after PF treatment;

[0075] Figure 24 LC3B immunohistochemical staining images of aortic root sections of AS model mice after PF treatment;

[0076] Figure 25 γ-H2AX immunofluorescence staining images of aortic root sections of AS model mice after PF treatment;

[0077] Figure 26 H&E staining histological images of major organs of AS model mice after PF treatment;

[0078] Figure 27 H&E staining histological images of liver of AS model mice after PF treatment;

[0079] Figure 28 Mitochondrial membrane potential confocal images of fatty liver cells after 6 hours of PF14-88 treatment in Example 2; the CCCP treatment group was used as a negative control, and green fluorescence was rhodamine 123;

[0080] Figure 29 Intracellular ATP level detection results of fatty liver cells after 6 hours of PF14-PF88 treatment prepared in Example 2;

[0081] Figure 30 Confocal images of intracellular ROS of HepG2 cells after 6 hours of PF14-PF88 treatment prepared in Example 2; ROS levels were detected by DCFH-DA fluorescent probe;

[0082] Figure 31 Images of intracellular red lipid droplets of HepG2 cells after 6 hours of PF14-PF88 treatment prepared in Example 2;

[0083] Figure 32 Effect of PF14-PF88 prepared in Example 2 on NAFLD mice; photographs of the body size and liver of the mice after intravenous injection;

[0084] Figure 33 One-dimensional nuclear magnetic hydrogen spectrum of C3C50% in d6-DMSO prepared in Example 3;

[0085] Figure 34 Cell toxicity experiment results of pyridinium nanoparticles with different end groups prepared in Example 3 on 143B cells;

[0086] Figure 35 Effect of poly-pyridinium high molecular materials with different end groups prepared in Example 3 on the autophagy level of cells; the cells used were 143B cells stably expressing GFP-mCHerry-LC3 protein, which were used to detect the autophagy level of cells;

[0087] Figure 36 Intracellular ATP level detection results of foam cells after 6 hours of C3C50%, C3H50% and C3M50% treatment;

[0088] Figure 37Confocal images of intracellular ROS of Raw 264.7 cells treated with C3C50%, C3H50% and C3M50% prepared in Example 3 for 6 hours; ROS level was detected by DCFH-DA fluorescent probe;

[0089] Figure 38 Confocal images of intracellular lipid droplets of foam cells treated with C3C50%, C3H50% and C3M50% prepared in Example 3 for 6 hours;

[0090] Figure 39 Results of cytotoxicity experiment of G5F9, G5F11 and G5F13 pyridinium nanoparticles prepared in Example 4 on 143B cells;

[0091] Figure 40 Effects of G5F9, G5F11 and G5F13 pyridinium nanoparticles prepared in Example 4 on mitochondrial autophagy level; the cells used were 143B cells stably expressing mito-keima protein, which were used to detect the mitochondrial autophagy level of cells;

[0092] Figure 41 Results of intracellular ATP level detection of foam cells treated with G5F9, G5F11 and G5F13 for 6 hours;

[0093] Figure 42 Confocal images of intracellular ROS of Raw 264.7 cells treated with G5F9, G5F11 and G5F13 prepared in Example 4 for 6 hours; ROS level was detected by DCFH-DA fluorescent probe;

[0094] Figure 43 Confocal images of intracellular lipid droplets of foam cells treated with G5F9, G5F11 and G5F13 for 6 hours;

[0095] Figure 44 Results of cytotoxicity experiment of P4F9, P4F11 and P4F13 pyridinium nanoparticles prepared in Example 5 on 143B cells;

[0096] Figure 45 Effects of P4F9, P4F11 and P4F13 pyridinium nanoparticles prepared in Example 5 on mitochondrial autophagy level; the cells used were 143B cells stably expressing mito-keima protein, which were used to detect the mitochondrial autophagy level of cells;

[0097] Figure 46 Results of intracellular ATP level detection of foam cells treated with P4F9, P4F11 and P4F13 for 6 hours;

[0098] Figure 47Confocal images of intracellular ROS in Raw 264.7 cells treated with P4F9, P4F11 and P4F13 for 6 hours; ROS level was detected by DCFH-DA fluorescent probe;

[0099] Figure 48 Confocal images of intracellular lipid droplets in foam cells treated with P4F9, P4F11 and P4F13 prepared in Example 5 for 6 hours;

[0100] Figure 49 Results of cytotoxicity experiment of Plys4F9, Plys4F11 and Plys4F13 pyridinium nanoparticles prepared in Example 6 on 143B cells;

[0101] Figure 50 Effects of Plys4F9, Plys4F11 and Plys4F13 pyridinium nanoparticles prepared in Example 6 on mitochondrial autophagy level;

[0102] Figure 51 Results of intracellular ATP level detection of foam cells treated with Plys4F9, Plys4F11 and Plys4F13 prepared in Example 6 for 6 hours;

[0103] Figure 52 Confocal images of intracellular ROS in Raw 264.7 cells treated with Plys4F9, Plys4F11 and Plys4F13 for 6 hours;

[0104] Figure 53 Confocal images of intracellular lipid droplets in foam cells treated with Plys4F9, Plys4F11 and Plys4F13 for 6 hours;

[0105] Figure 54 Results of cytotoxicity experiment of bPEI-F9, bPEI-F11 and bPEI-F13 pyridinium nanoparticles prepared in Example 7 on 143B cells;

[0106] Figure 55 Effects of bPEI-F9, bPEI-F11 and bPEI-F13 pyridinium nanoparticles prepared in Example 7 on mitochondrial autophagy level;

[0107] Figure 56 Results of intracellular ATP level detection of foam cells treated with bPEI-F9, bPEI-F11 and bPEI-F13 prepared in Example 7 for 6 hours;

[0108] Figure 57Confocal images of intracellular ROS of Raw 264.7 cells treated with bPEI-F9, bPEI-F11 and bPEI-F13 prepared in Example 7 for 6 hours;

[0109] Figure 58 Confocal images of intracellular lipid droplets of foam cells treated with bPEI-F9, bPEI-F11 and bPEI-F13 prepared in Example 7 for 6 hours;

[0110] Figure 59 Cytotoxicity results of lPEI-F9, lPEI-F11 and lPEI-F13 pyridinium nanoparticles prepared in Example 8 on 143B cells;

[0111] Figure 60 Effects of lPEI-F9, lPEI-F11 and lPEI-F13 pyridinium nanoparticles prepared in Example 8 on mitochondrial autophagy level;

[0112] Figure 61 Intracellular ATP level detection results of foam cells treated with lPEI-F9, lPEI-F11 and lPEI-F13 for 6 hours;

[0113] Figure 62 Confocal images of intracellular ROS of Raw 264.7 cells treated with lPEI-F9, lPEI-F11 and lPEI-F13 prepared in Example 8 for 6 hours;

[0114] Figure 63 Confocal images of intracellular lipid droplets of foam cells treated with lPEI-F9, lPEI-F11 and lPEI-F13 prepared in Example 8 for 6 hours;

[0115] Figure 64 Cytotoxicity results of lPLyF9, lPLyF11 and lPLyF13 pyridinium nanoparticles prepared in Example 9 on 143B cells;

[0116] Figure 65 Effects of lPLyF9, lPLyF11 and lPLyF13 pyridinium nanoparticles prepared in Example 9 on mitochondrial autophagy level;

[0117] Figure 66 Intracellular ATP level detection results of foam cells treated with lPLyF9, lPLyF11 and lPLyF13 prepared in Example 9 for 6 hours;

[0118] Figure 67The confocal image of the intracellular ROS of Raw 264.7 cells treated with lPLyF9, lPLyF11 and lPLyF13 for 6 hours;

[0119] Figure 68 The confocal image of the intracellular lipid droplets of foam cells treated with lPLyF9, lPLyF11 and lPLyF13 prepared in Example 9 for 6 hours. DETAILED DESCRIPTION

[0120] The application will be further described in conjunction with specific embodiments, but the embodiments of the application are not limited thereto.

[0121] Preparation of nanomaterials PF and PC in Example 1

[0122] Poly-4-vinylpyridine (P4VP) with a molecular weight of 60000 Da, 1H, 1H, 2H, 2H-perfluoroiodohexane and iodohexane were respectively dissolved in DMSO to obtain solutions of the three substances; the 1H, 1H, 2H, 2H-perfluoroiodohexane solution and the iodohexane solution were respectively added dropwise into the poly-4-vinylpyridine solution, and after uniform dropwise addition, the reaction was carried out in a 50℃ oil bath for 7 days, followed by pure water dialysis and freeze-drying to obtain fluorine-containing pyridinium polymer (i.e. nanomaterial PF) and carbon chain-containing pyridinium polymer (i.e. nanomaterial PC) with surface modification. The molar ratio of the pyridine groups in poly-4-vinylpyridine (P4VP) to 1H, 1H, 2H, 2H-perfluoroiodohexane was 1:0.55, and the molar ratio of the pyridine groups to iodohexane was 1:0.55.

[0123] When 1H, 1H, 2H, 2H-perfluoroiodohexane was used, a in the structure of formula I of the pyridinium-based nanomaterial targeting damaged mitochondria was 1, b was 4, X was F, and Y was F, and at this time the nanomaterial was denoted as nanomaterial PF; when iodohexane was used, a in the structure of formula I was 1, b was 4, X was H, and Y was H, and at this time the nanomaterial was denoted as PC. The obtained polymers were structurally characterized by nuclear magnetic resonance hydrogen spectrum (1H NMR) analysis, and the hydrogen spectrum of the nanomaterials PF and PC is shown in 1 and Figure 1 2 The degree of quaternization of the nanomaterials PF and PC was calculated using the hydrogen spectrum, i.e. the pyridinium modification efficiency. The degree of quaternization of the nanomaterial PF was 52%, and the degree of quaternization of the nanomaterial PC was 53%.

[0124] Performance test of nanomaterials PF and PC:

[0125] Serum stability in Example 1-1

[0126] ​(1) The PF and PC prepared in Example 1 were dissolved in 20% FBS aqueous solution, and incubated at room temperature for 2 hours. The TEM images of PF, PC and PF and PC in 20% FBS are shown in Figure 3 . Figure 3 PF and PC correspond to nanoparticles before adding 20% FBS aqueous solution, and PF+FBS and PC+FBS correspond to after adding 20% FBS aqueous solution.

[0127] It can be seen that when PF is exposed to 20% fetal bovine serum (FBS) condition, the nanoparticle size only appears slight increase, still maintains uniform distribution, and no obvious aggregation phenomenon is observed. In comparison, the non-fluorine-containing polymer PC significantly aggregates under the same condition, forming micron-sized aggregates, and the particle heterogeneity is obviously increased. Figure 3

[0128] (2) The PF and PC (0.1 mg) prepared in Example 1 were respectively dissolved in 400 μL of 10%-50% (volume ratio) FBS-containing aqueous solution, and incubated at room temperature for 2 hours. After incubation, the polymer nanoparticles were collected by centrifugation at 22,000xg for 40 minutes (4°C). The obtained precipitate was washed with deionized water for three times to remove unbound serum proteins. The final washed precipitate and supernatant were respectively used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

[0129] In the SDS-PAGE analysis, the protein complex was first dissolved in 10 μL of 1x protein loading buffer containing 2-mercaptoethanol, and heated and denatured at 100°C for 10 minutes. The sample was loaded into a 12.5% polyacrylamide gel, and first electrophoresed at 80V for 30 minutes (concentration gel stage), and then continued to electrophorese at 120V for 1 hour (separation gel stage). The test results are shown in Figure 4 and 5 .

[0130] Figure 4 PF (i.e. PF52) and PC (i.e. PC53) respectively in 10%-50% (volume fraction) FBS-containing solution for 2 hours. Figure 5 SDS-PAGE qualitative analysis of the protein adsorption behavior on the surface of PF and PC.

[0131] As shown in Figure 4 ​As shown, PC exhibited poor colloidal stability in the presence of 10% FBS, and its stability decreased gradually with the increase of FBS content, which was manifested by the significant increase of solution turbidity, suggesting that PC was prone to form precipitates or aggregates in physiological environment. This instability might be attributed to two aspects: one was the electrostatic adsorption between the pyridinium cations on the surface of the material and the negatively charged protein components in the serum; the other was the enhanced hydrophobic association between molecules due to the hydrophobicity of the alkyl chain, which induced particle aggregation. The above effects weakened the colloidal stability of PC material. In the same system, even if the FBS volume fraction increased to 50%, the PF solution remained transparent, and the particles were uniform, showing good dispersibility and colloidal stability. Figure 5 As shown, it further verified the excellent performance of PC and PF in resisting non-specific serum protein adsorption and the good physical stability and anti-protein contamination ability of PF in high serum environment.

[0132] Example 1-2 cytotoxicity evaluation

[0133] (1) Different doses of PF and PC nanoparticles were added to mouse macrophage RAW264.7 cells, and EdU incorporation experiment was used to detect DNA synthesis activity. EdU (5-ethynyl-2'-deoxyuridine) can replace thymidine and be incorporated into newly synthesized DNA during DNA synthesis. On the other hand, the ethynyl group on EdU can covalently react with a fluorescently labeled small molecule azide probe (such as Azide Alexa Fluor 488) through the catalysis of monovalent copper ions to form a stable triazole ring. Through this reaction, newly synthesized DNA will be labeled with the corresponding fluorescent probe, so that the proliferating cells can be detected using the fluorescence detection equipment of Alexa Fluor 488. The test results are shown in Figure 6 . Figure 6 The BrdU cell proliferation results of PF and PC at different doses on Raw 264.7 cells are shown in Figure 6 It can be seen that the cell viability of the PC treatment group decreased significantly when the concentration reached 50 μg / mL, and the cell viability of the PF treatment group remained at a high level even when the concentration was as high as 150 μg / mL, and no significant toxic reaction was observed.

[0134] (2) Different doses of PF and PC nanoparticles prepared in Example 1 were added to fibroblast model NIH3T3, and CCK8 was used to detect cell activity. The test results are shown in Figure 7 .

[0135] As shown in Figure 7As shown, the results show that the PC treatment group significantly decreased in cell viability at a concentration of 32 μg / mL, and the PF treatment group maintained a high level of cell viability even at a concentration of 120 μg / mL, without significant toxic reactions.

[0136] Example 1-3 Mitochondrial targeting ability

[0137] Stable 143B cells expressing mitochondria-targeted green fluorescent protein (mitoGFP) were seeded in confocal dishes and cultured overnight in a constant temperature incubator. Then, 32 μg / mL of PF prepared in Example 1 was added to treat the cells, and different time points were set as 1 hour and 3 hours, respectively. After treatment, the cells were washed with PBS to remove free polymers. The distribution of PF in the cells and the mitochondrial localization signal were co-localized by confocal laser scanning microscopy. The test results are shown in Figure 8 Figure 8 Mitochondrial fluorescence co-localization diagram of PF and stable mitoGFP 143B cells incubated for different times, and material and mitochondrial co-localization coefficient analysis.

[0138] As can be seen from Figure 8 , after PF was added to the culture solution of 143B cells for 1 hour, the PF red fluorescence signal (PF itself has inherent red fluorescence signal, so fluorescence imaging and targeting localization analysis can be directly performed without additional fluorescence labeling) was highly overlapped with the mitoGFP green fluorescence, and the Pearson correlation coefficient calculated was as high as 0.97, showing a high co-localization consistency.

[0139] Further extending the incubation time to 3 hours, the fluorescence co-localization relationship remained highly consistent, indicating that PF not only can quickly target mitochondria, but also can stably enrich in the mitochondrial region and is not prone to transfer or diffusion. This result verifies that PF material has good mitochondrial targeting property.

[0140] Example 1-4 Effect of nanomaterial PF on the level of mitochondrial autophagy

[0141] (1) Mouse-derived monocyte / macrophage leukemia cell line RAW264.7 (ATCC) was cultured in modified Dulbecco's medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS), 100 μg / mL penicillin and 100 μg / mL streptomycin, and maintained in a conventional culture condition at 37°C in a 5% CO2 constant temperature incubator.

[0142] ​AS model cells: To induce inflammatory state, RAW264.7 cells were stimulated with 1 μg / mL lipopolysaccharide (LPS) for 12 hours. Subsequently, the cells were incubated in the medium containing 1 μg / mL LPS and 40 μg / mL oxidized low-density lipoprotein (oxLDL) for 12 hours to induce the formation of foam cells.

[0143] RAW264.7 macrophages were induced to differentiate into foam cells according to the above method. Then, PF prepared in Example 1 (32 μg / mL) was added for 6 hours of treatment. After the end of treatment, the cells were fixed, permeabilized and blocked, and then incubated overnight at 4°C, and the primary antibodies LC3B (Abeam, ab192890), TOM20 (Abeam, ab186734) and p62 (Abeam, ab280086) were added, respectively. The next day, under the condition of avoiding light, the fluorescently labeled secondary antibodies (Abeam, ab150079, ab150077) were added and incubated at room temperature for 1 hour. After the nuclei were stained with Hoechest 33342, the imaging was performed using a confocal laser scanning microscope (CLSM) to observe the localization and co-localization of the mitochondrial autophagy-related proteins. The test results are shown in Figure 9 and 10 The blank cells are normal Raw 264.7 cells without induction, serving as a positive control; and the foam cells are a negative control. Figure 9 LC3 and p62 protein immunofluorescence staining images of foam cells treated with PF prepared in Example 1 for 6 hours; Figure 10 TOM20 protein immunofluorescence staining images of foam cells treated with PF prepared in Example 1 for 6 hours.

[0144] The present application employs three classical autophagy and mitochondrial markers for combined detection, including microtubule-associated protein light chain 3 (LC3), autophagy adaptor protein p62 and mitochondrial outer membrane transporter TOM20. Among them, LC3 is converted from cytoplasmic form LC3-I to membrane-bound form LC3-II during autophagy, and the level of LC3-II is often used as an indicator of autophagy activity; p62 (also known as SQSTM1) can recognize ubiquitinated proteins and mediate their binding to LC3, and is involved in selective autophagy, and its expression level is negatively correlated with autophagy degradation activity; TOM20 is a structural protein of the mitochondrial outer membrane, which can be used to monitor the integrity and relative abundance of mitochondria.

[0145] From Figure 9 and 10As can be seen, compared with uninduced RAW264.7 control cells, the expression level of LC3-II in foam cells was significantly decreased, while the expression levels of p62 and TOM20 were significantly increased, indicating that mitophagy is significantly inhibited under atherosclerotic conditions. Furthermore, after treating foam cells with the PF material of this invention, the results showed the opposite trend: LC3-II levels recovered and increased, while p62 and TOM20 expression was downregulated, indicating that PF can significantly promote the activation and functional recovery of mitophagy.

[0146] (2) RAW264.7 macrophages were induced to differentiate into foam cells using the method described above. Subsequently, PF (32 μg / mL) prepared in Example 1 was added and the cells were treated for 6 hours. Total protein was extracted by lysis and analyzed by Western blotting. Protein samples were separated by SDS-PAGE electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skim milk powder at room temperature for 1 hour, followed by the addition of primary antibodies: LC3, TOM20, p62 (Abcam, ab109012) and internal control β-actin (Beyotime, AF5003), and incubated overnight at 4°C. After washing, secondary antibody (Beyotime, A0208) was added and incubated at room temperature for 1 hour. Protein bands were developed using the BeyoECLPlus chemiluminescence kit, and grayscale analysis and quantification were performed using software. Blank cells were uninduced normal RAW 264.7 cells, serving as a positive control; foam cells were used as a negative control. The test results are as follows: Figure 11 As shown.

[0147] from Figure 11 As can be seen, compared with uninduced RAW264.7 control cells, the expression level of LC3-II in foam cells was significantly decreased, while the expression levels of p62 and TOM20 were significantly increased, indicating that mitophagy is significantly inhibited under atherosclerotic conditions. Furthermore, after treating foam cells with the PF material of this invention, the results showed the opposite trend: LC3-II levels recovered and increased, while p62 and TOM20 expression was downregulated, indicating that PF can significantly promote the activation and functional recovery of mitophagy.

[0148] Examples 1-5: Effects of the nanomaterial PF on dysfunctional mitochondria

[0149] AS model cells: To induce an inflammatory state, RAW264.7 cells were stimulated with 1 μg / mL lipopolysaccharide (LPS) for 12 hours. Subsequently, they were incubated for another 12 hours in a medium containing 1 μg / mL LPS and 40 μg / mL oxidized low-density lipoprotein (oxLDL) to induce foam cell formation.

[0150] RAW264.7 macrophages were differentiated into foam cells and then treated with PF (32 μg / mL) prepared in Example 1 for 6 hours.

[0151] (1) The recovery of mitochondrial membrane potential of foam cells treated with PF was detected. Mitochondrial function is mainly reflected in its core role in cell energy supply, which is manifested as the ability to maintain mitochondrial membrane potential (ΔΨm). The recovery of mitochondrial function of foam cells treated with PF prepared in Example 1 for 6 hours is shown in FIG. 1A. As shown in FIG. 1A, compared with untreated RAW264.7 cells, ΔΨm in foam cells was significantly lost, suggesting that mitochondrial energy metabolism function was impaired. After treatment with PF, the mitochondrial membrane potential in cells also recovered to near normal levels, indicating that PF had good effects on stabilizing membrane potential. Figure 12

[0152] Figure 12 The recovery of mitochondrial function of foam cells treated with PF, i.e., the confocal image of mitochondrial membrane potential of foam cells treated with PF prepared in Example 1 for 6 hours, is shown in FIG. 1B. The group treated with CCCP (apoptosis inducer) was used as a negative control. As shown in FIG. 1B, compared with untreated RAW264.7 cells, ΔΨm in foam cells was significantly lost, suggesting that mitochondrial energy metabolism function was impaired. After treatment with PF, the mitochondrial membrane potential in cells also recovered to near normal levels, indicating that PF had good effects on stabilizing membrane potential. Figure 12

[0153] (2) The ATP level of foam cells treated with PF was detected. Mitochondrial function is mainly reflected in its core role in cell energy supply, which is manifested as the efficiency of ATP synthesis. The regulation of mitochondrial energy metabolism function by PF was tested in a foam cell model, and the results of ATP level detection are shown in FIG. 2A. As shown in FIG. 2A, compared with untreated RAW264.7 cells, the ATP level in foam cells was significantly decreased, suggesting that mitochondrial energy metabolism function was impaired. After treatment with PF, the ATP content in cells significantly increased, indicating that PF had good effects on improving mitochondrial energy output. Figure 13 Figure 13

[0154] (3) To more intuitively observe the repair effect of PF on mitochondrial structure, the cells were collected, embedded, and sectioned, and then transmission electron microscopy (TEM) was used to characterize the ultrastructure of mitochondria in foam cells. The results are shown in FIG. 3. As shown in FIG. 3, typical mitochondrial damage morphology was observed in untreated foam cells, including volume shrinkage, disappearance of ridge structure, and membrane structure blurring. After treatment with PF, the volume of mitochondria recovered, the ridge structure was visible again, and the membrane integrity was enhanced, showing that it had significant structural repair ability for pathological mitochondria. Figure 14 Figure 14

[0155] Anti-inflammatory effects of Examples 1-6 on disease model cells

[0156] ​​​​​​RAW264.7 cells were stimulated with 1 pg / mL lipopolysaccharide (LPS) for 12 hours to induce inflammatory phenotype, then PF52 (32 pg / mL) was added to treat for 6 hours.

[0157] (1) The active oxygen detection kit was used to detect the intracellular ROS level. The results were observed by confocal laser scanning microscope (CLSM) and quantitatively analyzed by flow cytometry. The test results are shown in Figure 15

[0158] As shown in Figure 15 ; after PF treatment, the intracellular ROS level of Raw 264.7 cells decreased significantly. This change was detected by 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe staining, and the fluorescence intensity decreased significantly, which confirmed that PF had the ability to scavenge ROS and relieve oxidative stress.

[0159] (2) MitoSOX GREEN probe was used to detect the intracellular mitochondrial ROS level. The results were observed by confocal laser scanning microscope (CLSM).

[0160] As shown in Figure 16 ; after PF treatment, the intracellular mitochondrial ROS level of Raw 264.7 cells decreased significantly. This change was detected by MitoSOX GREEN fluorescent probe staining, and the fluorescence intensity decreased significantly, which confirmed that PF had the ability to scavenge mitochondrial ROS and relieve mitochondrial oxidative stress.

[0161] Example 1-7 Lipid droplet metabolic capacity

[0162] AS model cells: To induce inflammatory state, RAW264.7 cells were stimulated with 1 pg / mL lipopolysaccharide (LPS) for 12 hours. Then, continue to incubate in the medium containing 1 pg / mL LPS and 40 pg / mL oxidized low density lipoprotein (oxLDL) for 12 hours to induce the formation of foam cells.

[0163] Different degrees of quaternary amine PF (32 pg / mL) were added to the foam cells to treat for 6 hours, and BODIPY493 / 503-containing lipid droplet green fluorescent staining kit was used for lipid droplet staining. The stained cells were observed under confocal laser scanning microscope (CLSM).

[0164] Experimental results: as shown in Figure 17 Figure 17 ​​The confocal images of the lipid droplets in the foam cells after 6 hours of PF treatment. In the untreated foam cells, a large number of fluorescent signals were observed, indicating that the lipid droplets were significantly increased, which was consistent with the typical characteristics of lipid metabolism disorder after the formation of foam cells. In the PF-treated group, the BODIPY fluorescence signal in the cells was significantly weakened, and the number of lipid droplets was significantly reduced, indicating that the material could effectively reduce the lipid accumulation level in the foam cells. The above results suggest that the PF pyridinium polymer can target mitochondria, restore fatty acid metabolism, and improve the lipid processing capacity in cells to some extent, thereby reducing pathological lipid droplet accumulation and exhibiting good potential for lipid metabolism regulation.

[0165] Inhibition of AS by the nanomaterials of Examples 1-8

[0166] ApoE gene knockout mice (ApoE- / -) were selected to establish an atherosclerosis model. ApoE- / - mice can spontaneously form atherosclerotic plaques under the condition of high-fat high-cholesterol (HFHC) diet intake.

[0167] C57BL / 6 ApoE- / - mice were continuously fed with HFHC feed for 9 weeks to induce the formation of atherosclerosis. After the modeling was completed, the mice were randomly divided into three groups, and each group was injected with different treatment solutions through the tail vein twice a week for 8 weeks. The three groups of treatment schemes were: phosphate buffer solution (PBS, as a control group), low-dose PF (PF-L, 0.67 mg / kg), and high-dose PF (PF-H, 1.33 mg / kg). After the treatment period, the aortic tissues of the mice were collected, and the lipid deposition in the lesion area was detected by Oil Red O staining. The test results are shown in Figure 18 .

[0168] As shown in Figure 18 , the aortic plaque area of the PBS treatment group was significantly enlarged, and a large amount of lipid accumulation was observed; in contrast, the lipid plaque area in the aortas of the PF-L and PF-H treatment groups was significantly reduced, from 25.1% in the control group to 7.4% and 3.1%, respectively, showing that PF has a good plaque inhibition effect and presents an obvious dose-dependent relationship. In this test, PF is PF52.

[0169] The above observation results were further verified by Hematoxylin and Eosin (H&E) tissue staining. As shown in Figure 19As shown, the aortic wall structure of PBS group was disordered, and there was obvious plaque accumulation. After treatment with PF-L and PF-H, the plaque burden of the lesion area was significantly reduced, and the aortic wall tended to be complete and the structure recovered well. Based on the above results, it was shown that PF nanoparticles could effectively inhibit the formation and progression of lipid plaques in ApoE- / - atherosclerotic animal models, and had good in vivo treatment potential.

[0170] Example 1-9 in vivo tracking

[0171] To further clarify the targeting delivery ability and distribution characteristics of the fluorine-containing pyridinium polymer PF nanoparticles in vivo, the distribution of PF in the atherosclerotic model mice was evaluated by fluorescence imaging. Six hours after intravenous injection of PF, in vivo imaging of the drug was performed by fluorescence platform. The test results are shown in Figure 20 .

[0172] As can be seen from Figure 20 , six hours after intravenous injection of PF, red fluorescence signals from PF can be clearly observed in the aortic tissue peeled off from ApoE – / – model mice, and the fluorescence signal was significantly enhanced in the lesion area of the artery, indicating that PF had good targeted enrichment ability and could selectively accumulate in the lesion site.

[0173] In contrast, almost no obvious fluorescence signal was observed in the aortic tissue of healthy control mice, indicating that PF had less distribution in normal vascular tissue and had high disease tissue selectivity. This distribution characteristic may be closely related to the enhanced vascular permeability and enhanced permeability and retention (EPR) effect widely existing in the chronic inflammatory area. In the inflammatory environment, nanoparticles can leak into the lesion area through the vascular wall and stay in the local tissue for a long time, which is called the selective retention effect at the inflammatory site (ELVIS effect). PF nanoparticles achieve passive targeted accumulation in the AS lesion area through this mechanism.

[0174] In addition to the aortic tissue, the distribution of PF in the main organs in vivo was also analyzed. As shown in Figure 21 . Figure 21 The fluorescence signal of PF prepared in Example 1 in the main organs of the AS model mice was bioimaged.

[0175] PF nanoparticles also detected strong fluorescence signals in the liver and kidney of the model mice, indicating that the material had a certain degree of distribution in the metabolic organs. This result is consistent with the pharmacokinetic characteristics of most nanomaterials being cleared by the liver and kidney system in vivo.

[0176] Example 1-10 mechanism of action of nanomaterials in AS

[0177] Eight-week-old male C57BL / 6 mice with ApoE- / - knockout were fed a high-cholesterol diet for nine weeks to induce an atherosclerosis model. After modeling, the mice were administered different treatment groups via tail vein injection: a PBS control group, a low-dose PF52 group (0.67 mg / kg), and a high-dose PF52 group (1.33 mg / kg), twice a week for eight weeks. Immunofluorescence and immunohistochemistry were used to detect the levels of collagen, iNOS, LC3, and γH2AX proteins in the mouse arterial plaque tissue.

[0178] Collagen is a major component of the fibrous cap of atherosclerotic plaques. The collagen content in arterial plaque tissue from ApoE– / – mice was detected using Masson's trichrome staining method. Figure 22 As shown in the figure, the collagen content in the plaques of the PBS-treated group was significantly lower than that of the healthy control group, at only 25.4%, indicating that the plaque structure was fragile and its stability was reduced. After PF treatment, the collagen content in the plaques of the low-dose group and the high-dose group increased to 46.4% and 40.9%, respectively, indicating that PF can effectively promote collagen deposition, enhance the strength of the fibrous cap, and thus improve the overall stability of the plaques.

[0179] Based on morphological verification, this study further explores the mechanism of PF's anti-atherosclerotic action in vivo, focusing on its regulatory effects on local inflammation levels, macrophage polarization, and autophagy and senescence. Figure 23 As shown, iNOS immunohistochemical staining analysis of the plaque area revealed that PF treatment significantly reduced the proportion of iNOS-positive cells, suggesting that it effectively inhibited the activation of pro-inflammatory M1 macrophages and promoted the transformation of macrophages to the anti-inflammatory M2 phenotype, thereby alleviating local inflammatory response and improving the lesion microenvironment.

[0180] In addition, to assess the ability of PF to activate autophagy in plaque tissue, the expression level of the autophagy-associated marker protein LC3 was examined, such as... Figure 24 As shown. Figure 24 The results showed that the LC3 signal intensity in the plaque area of ​​the PF treatment group was significantly enhanced, with a significant upregulation compared to the control group. This indicates that PF can effectively activate the local autophagy pathway, which may help clear damaged organelles, reduce inflammation and metabolic stress, and further improve plaque stability.

[0181] The detection of aging-related indicators further revealed the mechanism of action of PF in anti-aging. For example... Figure 25 As shown, γH2AX staining was used to assess the level of cellular senescence. The results showed that PF treatment significantly reduced the number of γH2AX-positive cells in the plaque area, suggesting that PF has a good anti-cellular senescence effect and may delay the senescence process of local tissues by alleviating DNA damage-related stress response.

[0182] Safety evaluation of the biological safety of Examples 1-11

[0183] 8-week-old male C57BL / 6 mice with ApoE - / - were fed a high-cholesterol diet for 9 weeks to induce the establishment of an atherosclerosis model. After modeling was completed, the mice were injected with different treatment groups via the tail vein: a PBS control group, a PF52 low-dose group (0.67 mg / kg), and a high-dose group (1.33 mg / kg), twice a week for 8 weeks of continuous treatment. The mice in different groups were subjected to histological analysis of the major organs of the whole body, covering key organs such as the heart, spleen, lung, liver, and kidney. After routine paraffin embedding and hematoxylin-eosin (H&E) staining, the tissue sections were observed.

[0184] Figure 26 PF52, i.e., the PF prepared in Example 1, after acting on the major organs of AS model mice. As shown in FIG. 8, the PF treatment group had complete organ structure, no bleeding, edema, necrosis, or obvious abnormal pathological changes such as cell apoptosis, and the morphology was basically consistent with that of the healthy control group, indicating that the PF material did not cause systemic organ toxicity under the condition of long-term intravenous injection, and exhibited good in vivo biocompatibility and safety. Figure 26

[0185] In addition, typical lipid droplet accumulation was observed in the liver tissue of ApoE– / –model mice, showing extensive white vacuole areas, indicating significant liver steatosis, consistent with the pathological manifestations of atherosclerosis combined with NAFLD. Figure 27 PF52, i.e., the PF prepared in Example 1, after acting on the major organs of AS model mice. As shown in FIG. 8, the PF treatment group had complete organ structure, no bleeding, edema, necrosis, or obvious abnormal pathological changes such as cell apoptosis, and the morphology was basically consistent with that of the healthy control group, indicating that the PF material did not cause systemic organ toxicity under the condition of long-term intravenous injection, and exhibited good in vivo biocompatibility and safety.

[0186] In summary, the PF nanoparticles described in the present application exhibit excellent systemic safety in vivo without obvious organ toxicity when administered intravenously, and also exhibit an inhibitory effect on liver lipid accumulation during atherosclerosis treatment.

[0187] Example 2 Preparation of PF nanomaterials with different degrees of quaternary amination

[0188] ​P4VP with a molecular weight of 60000 Da and 1H, 1H, 2H, 2H- perfluoroiodohexane were dissolved in DMSO to obtain a P4VP solution and a 1H, 1H, 2H, 2H- perfluoroiodohexane solution, respectively; the 1H, 1H, 2H, 2H- perfluoroiodohexane solution was gradually added to the P4VP solution (the molar ratio of pyridine groups in P4VP to 1H, 1H, 2H, 2H- perfluoroiodohexane was 1:0.25, 1:0.45, 1:0.75 or 1:1), and after the addition was completed, the reaction solution was reacted in a 50°C oil bath for 7 days, and after water dialysis and freeze-drying, nanomaterials PF14, PF40, PF60 and PF88 were obtained.

[0189] Through characterization and calculation, when the molar ratio of pyridine groups to 1H, 1H, 2H, 2H- perfluoroiodohexane was 1:0.25, 1:0.45, 1:0.75 and 1:1, the pyridinium modification efficiency (i.e. the degree of quaternization) in the nanomaterials was 14%, 40%, 60% and 88%, respectively, and were named PF14, PF40, PF60 and PF88, respectively.

[0190] Performance test of PF nanomaterials with different degrees of quaternization:

[0191] Example 2-1 Effect on dysfunctional mitochondria:

[0192] NAFLD model cells: To induce NAFLD model cells, HepG2 cells were stimulated with 400 μM palmitic acid for 24 hours. Then PF14, PF40, PF60 and PF88 (32 μg / mL) were added for 6 hours of treatment.

[0193] (1) The recovery of mitochondrial membrane potential of NAFLD model cells treated with PF prepared in Example 2 was detected. Mitochondrial function is mainly reflected in its core role in energy supply to cells, which is manifested as the ability to maintain mitochondrial membrane potential (ΔΨm).

[0194] The recovery of mitochondrial function of foam cells treated with PF14-88 is shown in Figure 28 . Figure 28 The confocal images of mitochondrial membrane potential (mitochondrial membrane potential) of NAFLD model cells treated with PF prepared in Example 2 for 6 hours; the CCCP (apoptosis inducer) treatment group was used as a negative control. As can be seen from Figure 28 , compared with uninduced HepG2 cells, ΔΨm in NAFLD model cells was significantly lost, indicating that mitochondrial energy metabolism function was impaired. After treatment with PF, the mitochondrial membrane potential in cells also recovered to near normal levels, indicating that PF had good effect in stabilizing membrane potential.

[0195] (2) Detection of ATP levels in foam cells prepared in Example 2. Mitochondrial function is mainly reflected in its core role in cell energy supply, which is manifested as the efficiency of ATP synthesis. As shown in FIG. 8, the ATP level in the fatty liver cells was significantly reduced compared to the uninduced HepG2 cells, suggesting that the mitochondrial energy metabolism function was impaired. After PF treatment, the intracellular ATP content increased significantly, indicating that PF had a good effect on improving mitochondrial energy output. This further confirmed its broad-spectrum effect on regulating mitochondrial function under different pathological backgrounds. Figure 29

[0196] Example 2-2 Anti-inflammatory effect of the nanomaterial on disease model cells:

[0197] NAFLD model cells were selected and treated with PF14, PF40, PF60 and PF88 (32 μg / mL) for 6 hours. After treatment, the intracellular ROS level was detected using an active oxygen detection kit. The results were observed by confocal laser scanning microscopy (CLSM).

[0198] As shown in FIG. 9, the ROS level in the NAFLD model cells was significantly reduced after PF treatment. This change was detected by 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe staining, and the fluorescence intensity was significantly reduced, confirming that PF had the ability to scavenge ROS and relieve oxidative stress. Figure 30

[0199] Example 2-3 Lipid droplet metabolism:

[0200] NAFLD model cells were selected and treated with PF14, PF40, PF60 and PF88 (32 μg / mL) for 6 hours. The intracellular neutral lipids were visualized and labeled by oil red O.

[0201] As shown in FIG. 10, a large number of lipid droplets accumulated in the palmitic acid-treated HepG2 cells, which was a typical pathological feature. However, after adding PF polymers for treatment, the number of red lipid droplets in the cells decreased significantly, indicating that PF could significantly reduce the intracellular lipid accumulation level in the NAFLD model. Figure 31 This result further confirmed that the nanomaterial of the present application had good lipid droplet scavenging ability in different types of lipid metabolism disorder models.

[0202] Example 2-4 Inhibitory effect of the nanomaterial in NAFLD:

[0203]

[0204] ​​​C57BL / 6 mice were selected to continuously ingest HFHC feed for 12 weeks to induce the occurrence and progression of NAFLD. After modeling was completed, the mice were randomly divided into groups (n=5) and divided into 6 groups, twice a week, for 8 weeks: normal diet, no treatment group; tail vein injection of equal volume of PBS group; PF14, PF40, PF60 and PF88 (1.2 mg / kg) intravenous injection of four groups. After the treatment period, the body weight and liver weight of the mice in each group were measured, and histological observation was performed.

[0205] As shown in Figure 32 , the HFHC diet combined with PBS treatment group (positive control group) mice significantly increased in size, with significantly increased average body weight and liver weight, and macroscopically visible liver volume expansion, color fading, suggesting severe intraliver lipid deposition and significant fatty liver lesions. However, in the PF14-88 series of materials intravenous treatment, the body size and liver weight of the model mice were significantly decreased, and the degree of liver lipid accumulation was significantly alleviated, suggesting that PF materials have good inhibitory effect on NAFLD, and the efficacy is enhanced with the increase of quaternization degree.

[0206] Example 3 Preparation of nanomaterials with different end groups

[0207] P4VP with a molecular weight of 60000 Da, iodopropane, iodopropanol and iodopropanoic acid were dissolved in DMSO to obtain P4VP solution, iodopropane solution, iodopropanol solution and iodopropanoic acid solution; iodopropane solution, iodopropanol solution and iodopropanoic acid solution were added dropwise to the P4VP solution, respectively, and after the dropwise addition was completed, the reaction solution was reacted in a 50°C oil bath for 7 days, and after water dialysis and freeze-drying, iodopropane pyridinium-containing nanomaterials, iodopropanol pyridinium-containing nanomaterials and iodopropanoic acid pyridinium-containing nanomaterials were obtained, respectively. The molar ratio of pyridine groups in P4VP to iodopropane was 1:0.55, the molar ratio of pyridine groups in P4VP to iodopropanol was 1:0.55, and the molar ratio of pyridine groups in P4VP to iodopropanoic acid was 1:0.55.

[0208] When iodopropane, iodopropanol and iodopropanoic acid are used respectively, the pyridinium-based nanomaterials targeting damaged mitochondria have the structure of formula I, wherein a is 1 and b is 1; when iodopropane is used, X is H and Y is H, and at this time the iodopropane pyridinium-containing nanomaterials are denoted as nanomaterial C3M50%; when iodopropanol is used, formula I has a structure wherein a is 1, b is 1, X is H and Y is OH, and at this time the iodopropanol pyridinium-containing nanomaterials are denoted as C3H50%; when iodopropanoic acid is used, formula I has a structure wherein a is 1, b is 1, X is H and Y is COOH, and at this time the iodopropanoic acid pyridinium-containing nanomaterials are denoted as C3C50%.

[0209] By characterization and calculation, the degree of quaternization of the propane pyridinium nanomaterial is 50%, the degree of quaternization of the propyl alcohol pyridinium nanomaterial is 50%, and the degree of quaternization of the propionic acid pyridinium nanomaterial is 50%. The hydrogen spectrum of the C3C50% pyridinium nanomaterial is shown in Figure 33 .

[0210] Performance test of nanomaterials with different end groups:

[0211] Example 3-1 cytotoxicity evaluation:

[0212] Different doses of different end group pyridinium nanoparticles were added to 143B cells, and CCK8 was used to detect cell activity. As shown in Figure 34 , the results showed that the cell viability of the C3H50% treatment group decreased to about 65% when the concentration reached 40 μg / mL, the cell viability of the C3M50% treatment group decreased to about 55% when the concentration reached 40 μg / mL, and the cell viability of the C3C50% treatment group remained at a high level even when the concentration was as high as 40 μg / mL, and no significant toxic reaction was observed.

[0213] Example 3-2 induction of autophagy ability:

[0214] 143B cells stably expressing fusion green fluorescent protein (GFP) and red fluorescent mCherry LC3 protein were seeded in confocal culture dishes and cultured in a constant temperature incubator overnight. Subsequently, 32 μg / mL of C3C50%, C3H50% and C3M50% were added to treat the cells, and different time points were set as 1 hour and 3 hour treatment groups, respectively. After treatment, PBS was used for washing to remove free polymer. The distribution of PF in cells and the localization signal of mitochondria were analyzed by confocal laser scanning microscope.

[0215] The present application adopts two kinds of classical autophagy and mitochondrial markers for combined detection, including microtubule associated protein light chain 3 (LC3), autophagy adaptor p62, among which LC3 is converted from cytoplasmic form LC3-I to membrane-bound form LC3-II in the autophagy process, and the level of LC3-II is often used as an indicator of autophagy activity; p62 (also known as SQSTM1) can recognize ubiquitinated proteins and mediate their binding with LC3, participate in selective autophagy, and its expression level is negatively correlated with autophagy degradation activity; the content of LC3 in autophagy can mark the level of autophagy, and according to the pH of the autophagy process, the combined expression of two proteins mcherry and GFP is selected. Autophagosomes are mainly divided into autophagosomes and autolysosomes, and the whole process is divided into five steps, namely autophagy initiation, nucleation, expansion, autophagosome formation, autophagosome and lysosome fusion into autolysosome and degradation. When the cell starts to initiate autophagy, the uniformly distributed LC3 protein in the cytoplasm will be recruited and gathered on the autophagosome membrane. In the autophagosome, the pH value is neutral, so mcherry and GFP emit red and green fluorescence respectively, and the cell shows yellow fluorescent spots, when the autophagosome and lysosome fuse, the pH value decreases, and the acidic autolysosome bubble environment can quench the green fluorescence of GFP.

[0216] The results are shown in Figure 35 As shown in the table, the control cells without treatment have few autophagosome (yellow) and autolysosome (red) fluorescent spots, indicating a low level of autophagy; the number of autophagy fluorescent spots in the 143B cells treated with C3M50% and C3H50% increases, indicating that the level of autophagy increases. The autophagy level of the cells treated with C3C50% is similar to that of the control cells, indicating that the autophagy level is not improved.

[0217] Example 3-3 Influence on dysfunctional mitochondria:

[0218] Using AS model cells, RAW264.7 macrophages were differentiated into foam cells, and then C3C50%, C3H50% and C3M50% (12 μg / mL) were added for 6 hours. The ATP levels of foam cells treated with different end groups of pyridinium nanoparticles were detected. The results are shown in Figure 36 As shown in the table, compared with untreated RAW264.7 cells, the ATP level in foam cells decreased significantly, indicating that the energy metabolism function of mitochondria was damaged. After treatment with the three materials, the ATP content in the cells increased significantly, indicating that PF has a good effect on improving mitochondrial energy output.

[0219] Example 3-4 Anti-inflammatory effect on disease model cells:

[0220] RAW264.7 cells were stimulated with 1 pg / mL lipopolysaccharide (LPS) for 12 hours to induce inflammatory phenotype, followed by the addition of C3C50%, C3H50% and C3M50% (12 pg / mL) for 6 hours. After treatment, the reactive oxygen species detection kit was used to detect the intracellular ROS level. The results were observed by confocal laser scanning microscope (CLSM). The effect of pyridinium nanoparticles with different end groups on the ROS level of M1 type macrophages in the inflammatory model cells was investigated.

[0221] The experimental results are shown in Figure 37 ; after C3C50%, C3H50% and C3M50% treatment, the ROS level in Raw 264.7 cells decreased significantly. This change was detected by 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe staining, and the fluorescence intensity decreased significantly, confirming that PF has the ability to scavenge ROS and relieve oxidative stress.

[0222] Example 3-4 Lipid droplet metabolic capacity:

[0223] AS model cells were selected, and C3C50%, C3H50% and C3M50% (12 pg / mL) were added to the foam cells for 6 hours, and the lipid droplet green fluorescent staining kit containing BODIPY 493 / 503 was used for lipid droplet staining. The cells after staining were observed under confocal laser scanning microscope (CLSM).

[0224] The experimental results are shown in Figure 38 , Figure 38 The metabolic effect of C3C50%, C3H50% and C3M50% on lipid droplets in foam cells. That is, the confocal images of the lipid droplets in the foam cells after 6 hours of treatment with C3C50%, C3H50% and C3M50%. In untreated foam cells, a large number of fluorescence signals were observed, indicating that the lipid droplet content was significantly increased, which is consistent with the typical lipid metabolism disorder characteristics after the formation of foam cells. In the C3C50%, C3H50% and C3M50% treatment groups, the BODIPY fluorescence signal in the cells was significantly weakened, and the number of lipid droplets was significantly reduced, indicating that the material can effectively reduce the lipid accumulation level in foam cells.

[0225] Example 4: Pyridinium modified G5 nanomaterials with different fluorine chain lengths

[0226] 4-pyridinecarboxaldehyde was dissolved in methanol to obtain a 4-pyridinecarboxaldehyde solution, 1H, 1H, 2H, 2H-perfluorohexane was dissolved in methanol to obtain a 1H, 1H, 2H, 2H-perfluorohexane solution, 1H, 1H, 2H, 2H-perfluorohexane was dissolved in methanol to obtain a 1H, 1H, 2H, 2H-perfluorohexane solution, and 1H, 1H, 2H, 2H-perfluorooctane was dissolved in methanol to obtain a 1H, 1H, 2H, 2H-perfluorooctane solution; the 4-pyridinecarboxaldehyde solution was mixed with the 1H, 1H, 2H, 2H-perfluorohexane solution, 1H, 1H, 2H, 2H-perfluorohexane solution, and 1H, 1H, 2H, 2H-perfluorooctane solution in a molar ratio of 1:1, and the reaction solution was reacted in a 50°C oil bath for 7 days; G5 dendrimer (fifth generation polyamidoamine dendrimer) was added to the reaction solution, and the molar ratio of G5 to pyridinium group was 1:64; 15-100 μL of acetic acid was added to the reaction solution overnight, and then NaBH4 was added; after dialysis and lyophilization, 1H, 1H, 2H, 2H-perfluorohexane-modified pyridinium grafted G5, 1H, 1H, 2H, 2H-perfluorohexane-modified pyridinium grafted G5, and 1H, 1H, 2H, 2H-perfluorooctane-modified pyridinium grafted G5 were obtained.

[0227] The fifth generation polyamidoamine dendrimer in this example was obtained by reacting ethylenediamine with methyl acrylate; in the structural formula II-1 of the polyamidoamine dendrimer, M is ethylenediamine, and the number of surface NH2 is 64. In the structure II of the nanomaterial, Z is -NH-CH2-. The 1H, 1H, 2H, 2H-perfluorohexane-modified pyridinium grafted G5 is named G5F9, and in the structure II of the corresponding nanomaterial, a is 2, b is 4, X is F, and Y is F. The 1H, 1H, 2H, 2H-perfluorohexane-modified pyridinium grafted G5 is named G5F11, and in the structure II of the corresponding nanomaterial, a is 2, b is 5, X is F, and Y is F. The 1H, 1H, 2H, 2H-perfluorooctane-modified pyridinium grafted G5 is named G5F13, and in the structure II of the corresponding nanomaterial, a is 2, b is 6, X is F, and Y is F.

[0228] Through characterization and calculation, the pyridinium modification efficiency of the pyridinium-modified G5 with different fluorine chain lengths was 62±2 pyridinium groups modified on the G5 with different fluorine chain lengths.

[0229] Performance test of the pyridinium-modified G5 nanomaterial with different fluorine chain lengths:

[0230] Example 4-1 cytotoxicity evaluation: test results are as follows Figure 39The results show that the cell viability of the G5F9 treatment group decreases to about 50% when the concentration reaches 20 μg / mL, the cell viability of the G5F11 treatment group decreases to about 55% when the concentration reaches 32 μg / mL, and the cell viability of the G5F13 treatment group remains at a high level even when the concentration is as high as 32 μg / mL, and no significant toxic reaction is observed.

[0231] Example 4-2 Inducing ability of mitochondrial autophagy:

[0232] The 143B cells stably expressing mito protein fused with keima protein (mito keima 143B) were inoculated in confocal culture dishes and cultured in a constant temperature incubator overnight. Subsequently, the cells were treated with 12 μg / mL of G5F9, G5F11 and G5F13, respectively, and incubated for 6 h, followed by washing with PBS to remove free polymers. The fluorescence in the cells was observed by confocal laser scanning microscopy.

[0233] When the mitochondria are in the normal cytoplasmic environment, Keima is excited at 440 nm at neutral pH; when the mitochondria are transported to the acidic lysosome by autophagy, the excitation peak is shifted to 586 nm, so that the entry of mitochondria into the lysosome can be quantitatively detected by dual-wavelength ratio imaging or flow cytometry, thereby accurately reporting the occurrence and degree of mitochondrial autophagy in the cell.

[0234] As shown in Figure 40 , the control cells without treatment have few red fluorescent spots, indicating a low level of mitochondrial autophagy; the red fluorescent spots increase in the mito keima 143B cells treated with G5F9, G5F11 and G5F13, indicating an increased level of mitochondrial autophagy.

[0235] Example 4-3 Effect on dysfunctional mitochondria:

[0236] The AS model cells were selected, and RAW264.7 macrophages were differentiated into foam cells, followed by treatment with G5F9, G5F11 and G5F13 (12 μg / mL) for 6 h. The ATP levels of the foam cells treated with the pyridinium nanoparticles with different end groups were detected. The experimental results are shown in Figure 41 , compared with the untreated RAW264.7 cells, the ATP level in the foam cells decreased significantly, indicating that the mitochondrial energy metabolism function was damaged. After treatment with the three materials, the ATP content in the cells increased significantly, indicating that the nanomaterials had a good effect on improving the energy output of mitochondria.

[0237] Example 4-4 Anti-inflammatory effect on disease model cells:

[0238] RAW264.7 cells were stimulated with 1 pg / mL lipopolysaccharide (LPS) for 12 hours to induce inflammatory phenotype, followed by the addition of C3C50%, C3H50% and C3M50% (32 pg / mL) for 6 hours. After treatment, reactive oxygen species detection kit was used to detect the intracellular ROS level. The results were observed by confocal laser scanning microscope (CLSM). The experimental results are shown in Figure 42 After G5F9, G5F11 and G5F13 treatment, the intracellular ROS level of Raw264.7 cells decreased significantly. This change was detected by 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe staining, and the fluorescence intensity decreased significantly, confirming that the nanomaterials had the ability to scavenge ROS and relieve oxidative stress.

[0239] Example 4-5 Lipid droplet metabolic capacity:

[0240] AS model cells were selected, and G5F9, G5F11 and G5F13 (12 pg / mL) were added to the foam cells for 6 hours, and BODIPY 493 / 503-containing lipid droplet green fluorescent staining kit was used for lipid droplet staining. The stained cells were observed under confocal laser scanning microscope (CLSM).

[0241] The experimental results are shown in Figure 43 Figure 43 The metabolic effect of C3C50%, C3H50% and C3M50% on lipid droplets in foam cells. That is, the confocal images of the intracellular lipid droplets after the foam cells were treated with C3C50%, C3H50% and C3M50% for 6 hours. A large number of fluorescence signal can be seen in the untreated foam cells, indicating that the lipid droplet content is significantly increased, which is consistent with the typical lipid metabolism disorder characteristics after the formation of foam cells. In the C3C50%, C3H50% and C3M50% treatment groups, the BODIPY fluorescence signal in the cells was significantly weakened, and the number of lipid droplets was significantly reduced, indicating that the material can effectively reduce the lipid accumulation level in foam cells.

[0242] Example 5: Fourth generation of pyridinium-modified polypropylene imine dendrimers (P4) with different fluorine chain lengths

[0243] ​4-pyridinecarboxaldehyde was dissolved in methanol to obtain a 4-pyridinecarboxaldehyde solution, 1H, 1H, 2H, 2H-perfluorohexane was dissolved in methanol to obtain a 1H, 1H, 2H, 2H-perfluorohexane solution, 1H, 1H, 2H, 2H-perfluoroheptane was dissolved in methanol to obtain a 1H, 1H, 2H, 2H-perfluoroheptane solution, and 1H, 1H, 2H, 2H-perfluorooctane was dissolved in methanol to obtain a 1H, 1H, 2H, 2H-perfluorooctane solution; the 4-pyridinecarboxaldehyde solution was mixed with the 1H, 1H, 2H, 2H-perfluorohexane solution, the 1H, 1H, 2H, 2H-perfluoroheptane solution, and the 1H, 1H, 2H, 2H-perfluorooctane solution in a molar ratio of 1:1, and the reaction solution was reacted in a 50°C oil bath for 7 days, and P4 dendrimer was added to the reaction solution, wherein the molar ratio of P4 to pyridinium groups was 1:64. Meanwhile, 15-100 μL of acetic acid was added for overnight reaction, and then NaBH4 was added, and after dialysis with pure water and lyophilization, pyridinium-modified P4 polymers with different fluorine chain lengths on the surface were obtained.

[0244] The fourth generation polypropylenimine dendrimer in this example was a dendrimer synthesized with butanediamine as the core and propylenediamine as the monomer, the structure of the fourth generation polypropylenimine dendrimer is formula II-2, wherein M is butanediamine and the number of surface NH2 is 32. In the structure of formula II of the nanomaterial, Z is -NH-CH2-. The 1H, 1H, 2H, 2H-perfluorohexane-modified pyridinium grafted P4 is named P4F9, and in the structure of formula II of the corresponding nanomaterial: a is 2, b is 4, X is F, and Y is F; the 1H, 1H, 2H, 2H-perfluoroheptane-modified pyridinium grafted P4 is named P4F11, and in the structure of formula II of the corresponding nanomaterial: a is 2, b is 5, X is F, and Y is F; the 1H, 1H, 2H, 2H-perfluorooctane-modified pyridinium grafted P4 is named P4F13, and in the structure of formula II of the corresponding nanomaterial: a is 2, b is 6, X is F, and Y is F.

[0245] Through characterization and calculation, the pyridinium modification efficiency of P4 modified with different fluorine chain lengths of pyridinium is 30±2.

[0246] Example 5-1 cytotoxicity evaluation: the results are shown in Figure 44 The results show that the cell viability of the P4F9 treatment group decreases to about 60% when the concentration reaches 32 μg / mL, the cell viability of the P4F11 treatment group decreases to about 60% when the concentration reaches 32 μg / mL, and the cell viability of the P4F13 treatment group decreases to about 60% even when the concentration is as high as 32 μg / mL.

[0247] Example 5-2 induction of mitochondrial autophagy: the experimental results are shown in Figure 45As shown, the red fluorescence points of the untreated control cells were few, indicating a low level of mitophagy; the red fluorescence points of the mitokeima 143B cells treated with P4F9, P4F11 and P4F13 increased, indicating an increased level of mitophagy.

[0248] Example 5-3: Effect on dysfunctional mitochondria: Results as shown in Figure 46 As shown, compared with untreated RAW264.7 cells, the ATP level in the foam cells decreased significantly, indicating that the mitochondrial energy metabolism function was impaired. After treatment with P4F9, P4F11 and P4F13, the intracellular ATP content increased significantly, indicating that PF had a good effect on improving mitochondrial energy output.

[0249] Example 5-4: Anti-inflammatory effect on disease model cells: Experimental results as shown in Figure 47 As shown, after treatment with P4F9, P4F11 and P4F13, the intracellular ROS level of Raw264.7 cells decreased significantly. This change was detected by 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe staining, and the fluorescence intensity decreased significantly, confirming that PF had the ability to scavenge ROS and relieve oxidative stress.

[0250] Example 5-5: Lipid droplet metabolism: Experimental results as shown in Figure 48 As shown, Figure 48 The effect of P4F9, P4F11 and P4F13 on lipid droplets in foam cells. After treatment of foam cells with P4F9, P4F11 and P4F13 for 6 hours, the confocal images of the intracellular lipid droplets were obtained. In the untreated foam cells, a large number of fluorescence signals were observed, indicating a significant increase in lipid droplets, which is consistent with the typical lipid metabolism disorder characteristics after the formation of foam cells. In the P4F9, P4F11 and P4F13 treatment groups, the BODIPY fluorescence signal in the cells was significantly weakened, and the number of lipid droplets was significantly reduced, indicating that the material could effectively reduce the lipid accumulation level in foam cells.

[0251] Example 6: Pyridinium-modified polylysine dendrimers (Plys4) with different fluorine chain lengths

[0252] 4-Pyridinecarboxaldehyde was dissolved in methanol (4-pyridinecarboxaldehyde concentration was 20 mg / mL) and added in a molar ratio of 1:1. The reaction solution was reacted in an oil bath at 50 °C for 7 days. Plys4 dendritic polymer (4th generation polylysine dendritic polymer) was then added and mixed with the reaction solution. The molar ratio of Plys4 to pyridinium was 1:64. Simultaneously, 15-100 μL of acetic acid was added and the reaction was allowed to proceed overnight. Then, NaBH4 was added, and after dialyzing with pure water and lyophilization, pyridinium-modified Plys4 polymers with different fluorine chain lengths were obtained, namely, pyridinium-grafted Plys4 modified with 1H,1H,2H,2H-perfluorohexane, pyridinium-grafted Plys4 modified with 1H,1H,2H,2H-perfluoroiodoheptane, and pyridinium-grafted Plys4 modified with 1H,1H,2H,2H-perfluorooctane.

[0253] In this embodiment, the fourth-generation polylysine dendritic polymer has the structural formula II-3, where M is ethylenediamine, n is 4, m is 4, and Z is -NH-CH2-. The pyridinium-grafted Plys4 modified with 1H,1H,2H,2H-perfluorohexane is named Plys4F9. In the corresponding nanomaterial's Formula II structure, a is 2, b is 4, c is 50%, X is F, and Y is F. The pyridinium-grafted Plys4 modified with 1H,1H,2H,2H-perfluoroiodoheptane is named Plys4F11. In the corresponding nanomaterial's Formula II structure, a is 2, b is 5, c is 50%, X is F, and Y is F. The pyridinium-grafted Plys4 modified with 1H,1H,2H,2H-perfluorooctane is named Plys4F13. In the corresponding nanomaterial's Formula II structure, a is 2, b is 6, c is 50%, X is F, and Y is F.

[0254] By nuclear magnetic resonance hydrogen spectrum ( 1 The obtained polymer was characterized by ¹H NMR analysis. The degree of pyridinium modification under different reaction conditions was determined, and the pyridinium modification efficiency was calculated. The number of pyridinium chains of different lengths modified on Plys4 was 16 ± 2.

[0255] Example 6-1 Cytotoxicity Assessment: Experimental Results: as follows Figure 49 As shown, the results indicate that when the concentration of Plys4F9 reached 15 μg / mL, the cell viability decreased to around 80%, when the concentration of PlysF11 reached 15 μg / mL, the cell viability decreased to around 50%, and when the concentration of P4F13 reached as high as 15 μg / mL, the cell viability decreased to around 50%.

[0256] Example 6-2 Induction of Mitochondrial Autophagy: Experimental Results: as followsFigure 50 As shown, the red fluorescence spots of the untreated control cells were few, indicating a low level of mitophagy; the red fluorescence spots increased in Plys4F9, Plys4F11 and Plys4F13 treated mitokeima 143B cells, indicating an increased level of mitophagy

[0257] Example 6-3 Effect on dysfunctional mitochondria: RAW264.7 macrophages were differentiated into foam cells and then treated with Plys4F9, Plys4F11 and Plys4F13 (8 pg / mL) for 6 hours. The ATP levels of foam cells were detected with different end groups of pyridinium nanoparticles. As shown in Figure 51 As shown, the ATP levels of foam cells were significantly decreased compared with untreated RAW264.7 cells, indicating that the mitochondrial energy metabolism function was impaired. After treatment with the three materials, the ATP content in the cells increased significantly, indicating that the nanomaterials had a good effect on improving mitochondrial energy output.

[0258] Example 6-4 Anti-inflammatory effect on disease model cells: Plys4F9, Plys4F11 and Plys4F13 (8 pg / mL) were used for treatment for 6 hours. The experimental results are shown in Figure 52 As shown, the ROS levels in Raw 264.7 cells were significantly reduced after treatment with Plys4F9, Plys4F11 and Plys4F13. This change was detected by 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe staining, and the fluorescence intensity was significantly reduced, confirming that PF has the ability to scavenge ROS and relieve oxidative stress.

[0259] Example 6-5 Lipid droplet metabolism: The experimental results are shown in Figure 53 As shown, Figure 53 Plys4F9, Plys4F11 and Plys4F13 on lipid droplets in foam cells. After treatment of foam cells with Plys4F9, Plys4F11 and Plys4F13 for 6 hours, the confocal images of the lipid droplets in the cells were obtained. A large number of fluorescence signals were observed in the untreated foam cells, indicating a significant increase in lipid droplets, which is consistent with the typical lipid metabolism disorder characteristics after the formation of foam cells. In the Plys4F9, Plys4F11 and Plys4F13 treatment groups, the BODIPY fluorescence signal in the cells was significantly weakened, and the number of lipid droplets was significantly reduced, indicating that the material can effectively reduce the level of lipid accumulation in foam cells.

[0260] Example 7: Pyridinium-modified branched polyethyleneimine (bPEI) with different fluorine chain lengths

[0261] 4-Pyridinecarboxaldehyde was dissolved in methanol (4-pyridinecarboxaldehyde concentration 20 mg / mL) along with 1H,1H,2H,2H-perfluoroiodoheptane and 1H,1H,2H,2H-perfluoroiodooctane at a molar ratio of 1:1. The reaction solution was then reacted in an oil bath at 50°C for 7 days. Then, bPEI polymer (1 mol of bPEI contains 426 mol of amine groups (primary and secondary amines) that can react) with a molecular weight of 25 kDa was added and mixed with the reaction solution, where the bPEI:pyridinium molar ratio was 1:256. Simultaneously, 15-100 μL of acetic acid was added and the reaction proceeded overnight. NaBH4 was then added, and the mixture was dialyzed against pure water and lyophilized to obtain bPEI polymers with different fluorine chain lengths.

[0262] In this embodiment, the pyridinium-grafted bPEI modified with 1H,1H,2H,2H-perfluorohexane is named bPEI-F9, and in the corresponding nanomaterial's Formula II structure: a is 2, b is 4, c is 50%, X is F, and Y is F; the pyridinium-grafted bPEI modified with 1H,1H,2H,2H-perfluoroiodoheptane is named bPEI-F11, and in the corresponding nanomaterial's Formula II structure: a is 2, b is 5, c is 50%, X is F, and Y is F; the pyridinium-grafted bPEI modified with 1H,1H,2H,2H-perfluorooctane is named bPEI-F13, and in the corresponding nanomaterial's Formula II structure: a is 2, b is 6, c is 50%, X is F, and Y is F.

[0263] Approximately 2 mg of the product was dissolved in 400 μL of deuterium water, and the samples were analyzed by 1H NMR spectroscopy. 1 The obtained polymer was characterized by H NMR analysis, the degree of pyridinium modification under different reaction conditions was determined, and the pyridinium modification efficiency was calculated.

[0264] Experimental results: The pyridinium content of different fluorine chain lengths modified on bPEI was 50% ± 2%.

[0265] Example 7-1 Cytotoxicity Assessment: Experimental Results: as follows Figure 54 As shown in the figure. The results showed that when the concentration of bPEI-F9 reached 32 μg / mL, the cell viability decreased to around 50%; when the concentration of bPEI-F11 reached 32 μg / mL, the cell viability decreased to around 85%; and even at a concentration as high as 32 μg / mL, the cell viability of bPEI-F13 decreased to around 70%.

[0266] Example 7-2 Induction of Mitochondrial Autophagy: Experimental Results: as follows Figure 55As shown, the red fluorescence points of the untreated control cells were few, indicating a low level of mitophagy; the red fluorescence points of the mitokeima 143B cells treated with bPEI-F9, bPEI-F11 and bPEI-F13 increased, indicating an increased level of mitophagy.

[0267] Example 7-3: Effect on dysfunctional mitochondria: As shown in Figure 56 As shown, compared with untreated RAW264.7 cells, the ATP level in the foam cells decreased significantly, indicating that the energy metabolism function of mitochondria was impaired. After treatment with the three materials, the intracellular ATP content increased significantly, indicating that PF had a good effect on improving mitochondrial energy output.

[0268] Example 7-4: Anti-inflammatory effect on disease model cells: As shown in Figure 57 As shown, after treatment with bPEI-F9, bPEI-F11 and bPEI-F13, the ROS level in Raw264.7 cells decreased significantly. This change was detected by 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe staining, and the fluorescence intensity decreased significantly, confirming that PF had the ability to scavenge ROS and relieve oxidative stress.

[0269] Example 7-5: Lipid droplet metabolism: As shown in Figure 58 Figure 58 As shown, the effect of bPEI-F9, bPEI-F11 and bPEI-F13 on lipid droplets in foam cells. After treatment of foam cells with bPEI-F9, bPEI-F11 and bPEI-F13 for 6 hours, the confocal images of the intracellular lipid droplets were obtained. In the untreated foam cells, a large number of fluorescence signals were observed, indicating a significant increase in lipid droplets, which is consistent with the typical lipid metabolism disorder characteristics after the formation of foam cells. In the bPEI-F9, bPEI-F11 and bPEI-F13 treatment groups, the BODIPY fluorescence signal in the cells was significantly weakened, and the number of lipid droplets was significantly reduced, indicating that the material could effectively reduce the lipid accumulation level in foam cells.

[0270] Example 8: Linear polyethyleneimine (lPEI) modified with pyridinium of different fluorine chain lengths

[0271] ​4-Pyridinecarboxaldehyde was dissolved in methanol along with 1H,1H,2H,2H-perfluoroiodohexane, 1H,1H,2H,2H-perfluoroiodoheptane, and 1H,1H,2H,2H-perfluoroiodooctane, respectively, at a molar ratio of 1:1. The reaction solution was then reacted in an oil bath at 50°C for 7 days. A 5000 Da lPEI dendritic polymer was then added and mixed with the reaction mixture, with a lPEI:pyridinium molar ratio of 1:64. Simultaneously, 15-100 μL of acetic acid was added, and the reaction proceeded overnight. NaBH4 was then added, and the mixture was dialyzed against pure water and lyophilized to obtain pyridinium-modified lPEI polymers with different fluorine chain lengths.

[0272] In this embodiment, the pyridinium-grafted 1H,1H,2H,2H-perfluorohexane-modified pyridinium is named 1PEI-F9, where: a is 2, b is 4, c is 50%, X is F, and Y is F; the pyridinium-grafted 1H,1H,2H,2H-perfluoroheptanyl is named bPEI-F11, where: a is 2, b is 5, c is 50%, X is F, and Y is F; the pyridinium-grafted 1H,1H,2H,2H-perfluorooctane-modified pyridinium is named bPEI-F13, where: a is 2, b is 6, c is 50%, X is F, and Y is F.

[0273] By nuclear magnetic resonance hydrogen spectrum ( 1 The obtained polymer was characterized by H NMR analysis, the degree of pyridinium modification under different reaction conditions was determined, and the pyridinium modification efficiency was calculated.

[0274] Experimental results: The pyridinium content of different fluorine chain lengths modified on lPEI was 50% ± 2%.

[0275] Example 8-1 Cytotoxicity assessment: such as Figure 59 As shown, the results indicate that when the concentration of lPEI-F9 reached 15 μg / mL, the cell viability decreased to around 60%; when the concentration of lPEI-F11 reached 15 μg / mL, the cell viability decreased to around 80%; and even when the concentration of lPEI-F13 reached as high as 15 μg / mL, the cell viability decreased to around 80%.

[0276] Example 8-2 Inducing Mitochondrial Autophagy: such as Figure 60 As shown, the control cells without treatment had very few red fluorescent spots, indicating a low level of mitophagy; in mitokeima 143B cells treated with lPEI-F9, lPEI-F11 and lPEI-F13, the number of red fluorescent spots increased, indicating an increased level of mitophagy.

[0277] Example 8-3 Effects on Dysfunctional Mitochondria: such as Figure 61As shown, compared with untreated RAW264.7 cells, the ATP level in foam cells was significantly reduced, suggesting that the mitochondrial energy metabolism function was impaired. After treatment with lPEI-F9, lPEI-F11 and lPEI-F13, the intracellular ATP content increased significantly, indicating that PF has a good effect on improving mitochondrial energy output.

[0278] Example 8-4 Anti-inflammatory effect on disease model cells: As shown, after treatment with lPEI-F9, lPEI-F11 and lPEI-F13, the intracellular ROS level of Raw 264.7 cells decreased significantly. This change was detected by 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe staining, and the fluorescence intensity decreased significantly, confirming that the nanomaterials have the ability to scavenge ROS and relieve oxidative stress. Figure 62

[0279] Example 8-5 Lipid droplet metabolic capacity: As shown, Figure 63 Figure 63 is the metabolic effect of lPEI-F9, lPEI-F11 and lPEI-F13 on lipid droplets in foam cells. After treatment of foam cells with lPEI-F9, lPEI-F11 and lPEI-F13 for 6 hours, the confocal images of the intracellular lipid droplets were obtained. In untreated foam cells, a large number of fluorescence signals were observed, indicating that the lipid droplet content was significantly increased, which is consistent with the typical lipid metabolism disorder characteristics after the formation of foam cells. In the lPEI-F9, lPEI-F11 and lPEI-F13 treatment groups, the BODIPY fluorescence signal in the cells was significantly weakened, and the number of lipid droplets was significantly reduced, indicating that the material can effectively reduce the lipid accumulation level in foam cells.

[0280] Example 9: Linear polylysine (lPLys) of different fluorine chain length pyridinium

[0281] 4-pyridine formaldehyde was dissolved in methanol with 1H,1H,2H,2H- perfluoroiodohexane, 1H,1H,2H,2H-perfluoroiodoheptane and 1H,1H,2H,2H- perfluoroiodooctane respectively, and the reaction solution was reacted in a 50°C oil bath for 7 days. Linear lPLys polymer (the number of NH2 of 1 mol of polylysine is 34 (including end groups), and the reactivity of polylysine is low) was added to the reaction solution, and the molar ratio of lPLy to pyridinium was 1:64. At the same time, 15-100 μL of acetic acid was added for overnight reaction, and then NaBH4 was added. After dialysis with pure water and lyophilization, lPLy polymer modified with pyridinium of different fluorine chain length on the surface was obtained.

[0282] ​​In this embodiment, the pyridinium-grafted lPLy modified with 1H,1H,2H,2H-perfluorohexane is named lPLy-F9, wherein: a is 2, b is 4, c is 50%, X is F, and Y is F; the pyridinium-grafted lPLy modified with 1H,1H,2H,2H-perfluoroheptanyl is named lPLy-F11, wherein: a is 2, b is 5, c is 50%, X is F, and Y is F; the pyridinium-grafted lPLy modified with 1H,1H,2H,2H-perfluorooctane is named lPLy-F13, wherein: a is 2, b is 6, c is 50%, X is F, and Y is F.

[0283] Approximately 2 mg of the product was dissolved in 400 μL of deuterium water, and the samples were analyzed by 1H NMR spectroscopy. 1 The obtained polymer was characterized by HNMR analysis, the degree of pyridinium modification under different reaction conditions was determined, and the pyridinium modification efficiency was calculated.

[0284] Experimental results: The pyridinium content of different fluorine chain lengths modified on lPLy was 50% ± 2%.

[0285] Example 9-1 Cytotoxicity assessment: as follows Figure 64 As shown, the results indicate that when the concentration of lPLyF9 reached 15 μg / mL, the cell viability of the lPLyF11 treatment group decreased to around 70% when the concentration reached 15 μg / mL, and the cell viability of the lPLyF13 treatment group decreased to around 85% even at a concentration as high as 15 μg / mL.

[0286] Example 9-2 Inducing Mitochondrial Autophagy: such as Figure 65 As shown, the control cells without treatment had very few red fluorescent spots, indicating a low level of mitophagy; in mitokeima 143B cells treated with lPLyF9, lPLyF11 and lPLyF13, the number of red fluorescent spots increased, indicating an increased level of mitophagy.

[0287] Examples 9-3: Effects on dysfunctional mitochondria: Figure 66 As shown, compared with untreated RAW264.7 cells, the ATP level in foam cells was significantly decreased, indicating impaired mitochondrial energy metabolism. After treatment with lPLyF9, lPLyF11, and lPLyF13 (12 μg / mL), the intracellular ATP content significantly increased, indicating that PF has a good effect on improving mitochondrial energy output.

[0288] Example 9-4 Anti-inflammatory effect on disease model cells: such as Figure 67The ROS level in Raw 264.7 cells was significantly reduced after treatment with lPLyF9, lPLyF11 and lPLyF13. This change was detected by 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe staining, and the fluorescence intensity was significantly reduced, confirming that lPLyF9, lPLyF11 and lPLyF13 have the ability to scavenge ROS and alleviate oxidative stress.

[0289] Example 9-5 Lipid droplet metabolic capacity: As shown in Figure 68 Figure 68 The metabolic effect of lPLyF9, lPLyF11 and lPLyF13 on lipid droplets in foam cells. That is, the confocal images of the intracellular lipid droplets of foam cells after 6 hours of treatment with lPLyF9, lPLyF11 and lPLyF13. A large number of fluorescence signals were observed in untreated foam cells, indicating a significant increase in lipid droplet content, consistent with the typical lipid metabolism disorder characteristic of foam cell formation. In the lPLyF9, lPLyF11 and lPLyF13 treatment groups, the BODIPY fluorescence signal in the cells was significantly reduced, and the number of lipid droplets was significantly reduced, indicating that the material can effectively reduce the lipid accumulation level in foam cells.​

Claims

1. A pyridinium-based nanomaterial targeting damaged mitochondria, characterized by: one or more of the following formula I- formula II: P1 is a pyridine group involved in the formation of a pyridinium group of poly-4-vinylpyridine or polyvinyl acrylate pyridine, the pyridine group corresponding to the pyridinium group in formula I is from P1, that is, from the pyridine group in poly-4-vinylpyridine or polyvinyl acrylate pyridine; the grafting rate of the pyridinium modification group, that is, the percentage of the number of moles of the pyridinium modification group to the total number of moles of the pyridine group in P1, is 0.1-100%; X in formula I is selected from H or F, Y is selected from H, F, -OH, -COOH, -SO3H, a is an integer of 1-12, b is an integer of 0-11, and a+b is an integer of 1-12; In formula II, Z represents -NH-, -N=C-, -NHCH2CH(OH)-, -NHCH2CH(OH)CH2O-, Group; P2 is a terminal NH2 group or a terminal NH2 group or a secondary amine group participating in the formation of a Z structure containing an N group, such as polyamide-amine dendritic polymer, polypropylene imine dendritic polymer, polylysine dendritic polymer, linear polyethyleneimine, branched polyethyleneimine, or linear polylysine; N in the Z structure comes from the terminal NH2 group in polyamide-amine dendritic polymer, polypropylene imine dendritic polymer, or polylysine dendritic polymer, or from the NH2 group or secondary amine group in linear polyethyleneimine, branched polyethyleneimine, or linear polylysine; X in formula II is selected from H or F, Y is selected from H, F, -OH, -COOH, -SO3H, a is an integer of 1-12, b is an integer of 0-11, and a+b is an integer of 1-12; c is the number of functionalized pyridinium modification groups covalently connected to the surface of the polymer or the grafting rate of the pyridinium modification group.

2. The pyridinium-based nanomaterial targeted to damaged mitochondria of claim 1, wherein: The formula I includes formula I-1, formula I-2, The grafting rate in formula I is 10-100%; The polyamide-amine dendrimer is a first to tenth generation polyamide-amine dendrimer; the polypropylene imine dendrimer is a first to tenth generation polypropylene imine dendrimer; and the polylysine dendrimer is a first to tenth generation polylysine dendrimer.

3. The pyridinium-based nanomaterial targeting damaged mitochondria according to claim 1, characterized in that: In formula II, the structure of the polyamide-amine dendrimer is formula II-1: In the formula, m represents the number of the connection of the dendrimer core M and -(CH2CH2-CONH-CH2CH2N), n represents the number of the repeat of the branch unit (-CH2CH2-CONH-CH2CH2N), the CH2 of the latter branch unit is connected with the N of the former branch unit, the N of each branch unit is connected with the CH2 of the two latter branch units, and the end group of the last branch unit is NH2; at this time, c in formula II is an integer from 1 to 2048, M is a group formed by losing hydrogen from the amine group in ammonia, ethylenediamine, butanediamine, pentanediamine, hexanediamine, octanediamine, sunflower diamine or 1,12-dodecanediamine; n is an integer from 1 to 10; and m is an integer from 2 to 4. polypropylenimine dendrimers of formula II-2: wherein m represents the number of dendrimer core M and -(CH2CH2CH2N) linkages, n represents the number of repeat branching units (-CH2CH2CH2N), the CH2 of the last branching unit is linked to the N of the previous branching unit, and the N of each branching unit is linked to the CH2 of the next two branching units, and the end group of the last branching unit is NH2; in this case, c in formula II is an integer from 1 to 64, M is a group formed by the loss of hydrogen from the amine group of ammonia, butylenediamine, ethylenediamine, or 1,12-dodecanediamine; n is an integer from 1 to 5, and m is an integer from 2 to 4; The polylysine dendrimer has a structure of formula II-3: In the formula, m represents the number of connections of the dendrimer core M and -(COCH(CH2CH2CH2CH2NH)NH), n represents the number of repetitions of the branch unit (COCH(CH2CH2CH2CH2NH)NH), the C=O of the latter branch unit is connected with the NH of the former branch unit, one or two NH of each branch unit is connected with the CH2 of the next one or two branch units, and the end group of the last branch unit is NH2; at this time, c in formula II is the grafting rate of the pyridinium modification group, 0.1%-100%; M is a group formed by losing hydrogen from the amine group in ammonia, ethylenediamine, butanediamine, pentanediamine, hexanediamine, octanediamine, eicosanediamine or 1,12-dodecanediamine; n is an integer from 1 to 10; and m is an integer from 2 to 4. when the linear polyethyleneimine is of formula II-4: -(CH2-CH2-NH) x - or x is an integer of 0 or more; the molecular weight of the linear polyethyleneimine is 1800-25000; at this time c in formula II is the grafting rate of the pyridinium modification group, 0.1%-100%; a branched polyethyleneimine having a structure of Formula II-5: n is an integer of 0 or more, x and y are integers of 0 or more; the branched polyethyleneimine has a molecular weight of 1800-25000; and in this case, c in Formula II is the grafting rate of the pyridinium-modified group, 0.1%-100%. Linear polylysine, when the structure is Formula II-6: x is an integer of 0 or more; the molecular weight of the linear polylysine is 5,000-100,000; and c in Formula II is the grafting rate of the pyridinium-modified group, 0.1%-100%.

4. The method of claim 1-3, wherein the preparation of the pyridinium-based nanomaterial targeting damaged mitochondria is characterized by: It comprises the following steps: preparing one or more of the polymers in formula I- formula II into nanoparticles to obtain the pyridinium-based nanomaterial targeting damaged mitochondria.

5. The method of claim 4, wherein the preparation of the pyridinium-based nanomaterial targeting damaged mitochondria is characterized by: Specifically, it comprises the following steps: 1) Using an organic solvent as a reaction medium, poly-4-vinylpyridine or polyvinyl acrylate pyridine is reacted with a halogen compound to obtain the pyridinium-based nanomaterial targeting damaged mitochondria in formula I; or using an organic solvent as a reaction medium, the polymer is reacted with a pyridinium group compound to obtain the pyridinium-based nanomaterial targeting damaged mitochondria in formula II; The halogen compound is R' is -CI, -Br, -I; the molecular weight of the poly-4-vinylpyridine is 5,000-160,000 Da; The molecular weight of the polyvinyl acrylate pyridine is 6000-160000 Da; The polymer is a polyamide-amine dendrimer, a polypropylene imine dendrimer, a polylysine dendrimer, a linear polyethylene imine, a branched polyethylene imine, or a linear polylysine; The pyridinium group compound is wherein Q is -CI, -Br, -I, -COOH, R is -CF3, -CF2CF3, -CF2CF2CF3, -CF2CF2CF2CF3, or -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3.

6. The method of claim 5, wherein the preparation of the pyridinium-based nanomaterial targeting damaged mitochondria is characterized by: In the preparation of formula I, the organic solvent is one or more of DMSO, methanol, acetonitrile, and DMF; In the preparation of formula I, the reaction conditions are 40-60℃ for 5-9 days; and the molar ratio of the pyridine group in the poly-4-vinylpyridine or polyvinyl acrylate pyridine to the halogen compound is 1:(0.1-2); In the preparation of formula II, the organic solvent is one or more of methanol, DMSO, acetonitrile, and DMF; The reaction conditions are room temperature-60℃ for 8-12h; The amount of the polymer and the pyridinium group compound satisfies the following relationship: for each mole of the polymer, 0.1 times to 2 times the number of moles of the polymer surface -NH2 requires the pyridinium group compound; The pyridinium group compound is obtained by reacting a pyridine compound with a halogen-containing compound; The pyridine compound is: The halogen-containing compound is R" is -Br, -I; the reaction is carried out in an organic solvent; the organic solvent is one or more of DMSO, methanol, acetonitrile, DMF; the reaction conditions are 40-60°C for 5-9 days; the molar ratio of the pyridine compound to the halogen-containing compound is 1:(0.5-1.5); In the preparation of formula I, after the reaction is completed, water dialysis and freeze-drying are adopted; In the preparation of formula II, after the reaction is completed, water dialysis and freeze-drying are adopted.

7. Use of a pyridinium-based nanomaterial targeting damaged mitochondria according to any one of claims 1 to 3, characterized in that: The pyridinium-based nanomaterial targeting damaged mitochondria is used for preparing a nano injection.

8. Use of a pyridinium-based nanomaterial targeting damaged mitochondria according to any one of claims 1 to 3, characterized in that: The pyridinium-based nanomaterial targeting damaged mitochondria is used for preparing a damaged mitochondria fluorescence imaging agent.

9. Use of a pyridinium-based nanomaterial targeting damaged mitochondria according to any one of claims 1 to 3, characterized in that: The pyridinium-based nanomaterial targeting damaged mitochondria is used for preparing a drug for treating diseases related to mitochondrial dysfunction, specifically including a drug for treating atherosclerosis and non-alcoholic fatty liver.

10. Use of a pyridinium-based nanomaterial targeting damaged mitochondria according to any one of claims 1 to 3, characterized in that: The pyridinium-based nanomaterial targeting damaged mitochondria is used for preparing a preparation capable of effectively activating mitochondrial autophagy, promoting the clearance of dysfunctional mitochondria, restoring the mitochondrial membrane potential difference and the respiratory chain function, improving the adenosine triphosphate synthesis efficiency, reducing the intracellular active oxygen level, promoting the expression of anti-inflammatory macrophage phenotypes, and inhibiting the pro-inflammatory macrophage.

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