Self-assembled nanomaterials and their use in ROS scavenging

Abstract

The application discloses a kind of self-assembly nanomaterial and its application in ROS clearance.The self-assembly nanomaterial of the application is denoted as TPP-FFYp-O, and its structural formula is shown as follows:TPP-FFYp-O of the application is specifically cut to generate TPP-FFY-O by alkaline phosphatase (ALP) overexpressed after entering inflammatory cell, TPP-FFY-O is enriched near mitochondrion by the targeting effect of TPP, and is self-assembled to form nanoparticle and stays in inflammatory cell by π-π stacking.Nanoparticle surface enrichment of a large number of TEMPO can convert superoxide radical anion (O2 ·‑ ) in mitochondrion into hydrogen peroxide (H2O2), and then decompose into oxygen and water, thereby significantly reducing the ROS level in inflammatory cell.This enzyme-controlled self-assembly nanoparticle improves the stability of TEMPO and the ability of ROS clearance, and has good application prospect.

Description

Technical Field

[0001] This invention relates to a self-assembled nanomaterial and its application in ROS removal. Background Technology

[0002] Reactive oxygen species (ROS) are chemical substances formed during incomplete oxidation-reduction processes, mainly including hydrogen peroxide (H2O2) and superoxide anion (O2). ·- Singlet oxygen () 1 O2) and hydroxyl radicals ( · (OH). In the human system, reactive oxygen species (ROS) influence various physiological activities and play a crucial role in diverse life processes. ROS are important secondary messengers in cell signaling pathways, mitotic responses, cell proliferation / migration / differentiation, and the body's resistance to pathogen invasion. However, high levels of ROS can easily cause lipid peroxidation, protein denaturation, DNA damage, and oxidative damage to other biomolecules, thus having detrimental effects on cells and tissues. ROS-induced oxidative stress refers to the imbalance between oxidation and antioxidation in the body and is an important indicator of aging and disease. It is well known that excessive ROS production in inflamed tissues can further aggravate local tissue damage, leading to chronic inflammatory diseases such as wound sepsis, inflammatory bowel disease, acute liver / kidney injury, and liver fibrosis. Therefore, controlling ROS levels is essential for maintaining normal cell function and viability and controlling disease progression.

[0003] ROS scavengers that can neutralize ROS can effectively regulate ROS levels to achieve ROS balance in the body. Currently, commonly used exogenous ROS scavengers are a class of agents containing nitrile oxide free radicals (NO). ·Low-molecular-weight nitroxides, which function similarly to endogenous superoxide dismutase (SOD), can catalyze the neutralization of ROS. However, these low-molecular-weight ROS scavengers (e.g., 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)) have some inherent limitations, including short half-life in vivo, cytotoxicity, insufficient or excessive cellular uptake, and off-target effects (e.g., interference with cellular pathways such as the electron transport chain). To address the above problems, existing studies report that TEMPO is loaded into high-molecular-weight nanoparticles to improve its stability. For example, Yoshitomi et al. reported poly(ethylene glycol)-b-poly(methyl styrene) (PEG-b-PMS) block copolymer micelles containing TEMPO as a pH-activated ROS scavenger for alleviating acute kidney injury. These preparations greatly reduce off-target effects, improve the pharmacokinetic properties of TEMPO, and partially release or expose TEMPO to cells / tissues by decomposing the nanoparticles under stimuli such as pH, thereby playing a role in scavenging ROS. However, nanoprobe has the shortcomings of complicated preparation and slow cellular uptake. Supramolecular self-assembly is a convenient loading system. After the small-molecule precursor probe enters the cell, it self-assembles in situ to form a nanostructure under specific stimuli, thereby enriching the signal molecule and prolonging the residence time to improve bioavailability. Therefore, there is great development prospect for using a supramolecular self-assembly system to load TEMPO. SUMMARY

[0004] The present application is directed to the problems existing in the prior art, and provides a self-assembled nanomaterial and its application in ROS scavenging. The self-assembled nanomaterial of the present application is modified with a mitochondrial targeting group (TPP) and an active oxygen (ROS) scavenging group (TEMPO) at both ends of a self-assembled tripeptide molecule. TPP-FFYp-O is specifically cleaved by alkaline phosphatase (ALP) overexpressed in inflammatory cells to generate TPP-FFY-O, which is enriched near mitochondria by the targeting action of TPP and self-assembles to form nanoparticles by π-π stacking and stays in inflammatory cells. Figure 1 The large amount of TEMPO enriched on the surface of the nanoparticles can convert superoxide anion (O2 ·- ) in mitochondria into hydrogen peroxide (H2O2), which is then decomposed into oxygen and water, thereby significantly reducing the ROS level in inflammatory cells.

[0005] The self-assembled nanomaterial of the present application is abbreviated as TPP-FFYp-O, and its structure is shown below:

[0006]

[0007] The application discloses a preparation method of a self-assembled nanomaterial TPP-FFYp-O.

[0008] Step 1: TPP-FF (250 mg, 0.37 mmol) was fully dispersed in DMF, then 1-ethyl-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC·HCl, 87.71 mg, 0.55 mmol) and N-hydroxysuccinimide (NHS, 63.33 mg, 0.55 mmol) were added, and stirring was carried out for 8-12 h to carry out an activation ester reaction; then Tyr(H2PO3)-OH (252.48 mg, 0.48 mmol) and N,N-diisopropylethylamine (DIEPA, 884 μL) were added, and stirring was carried out at room temperature for 24-48 h until the reaction solution was clear, to obtain compound TPP-FFYp. Each step of reaction was separated and purified by HPLC, purity was confirmed, and molecular structure was confirmed by H NMR, 1 H NMR, 13 C NMR and MS Figure 3 , Figure 4 , Figure 5 ).

[0009] Step 2: TPP-FFYp (40 mg, 0.045 mmol), 1-hydroxybenzotriazole (HOBT, 9.14 mg, 0.068 mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 25.64 mg, 0.068 mmol) were dissolved in DMF, and activation was carried out at room temperature for 30 min, then DIEPA (12 μL) and TEMPO (11.58 mg, 0.068 mmol) were added, and stirring was carried out at room temperature for 4 h to obtain target compound TPP-FFYp-O. In addition, due to the paramagnetism of TEMPO, the nuclear magnetic spectrum is not suitable, and therefore MS is used to confirm the molecular structure Figure 6 ).

[0010] The synthesis route is shown in the following:

[0011]

[0012] The application discloses a preparation method of a self-assembled nanomaterial TPP-FFYp-O.

[0013] The TPP-FFYp-O is modified with a mitochondrial targeting group (TPP) and a reactive oxygen species (ROS) scavenging group (TEMPO) at both ends of the self-assembled tripeptide molecule (Phe-Phe-Tyr, FFY). TPP-FFYp-O is specifically cleaved by the alkaline phosphatase (ALP) overexpressed in inflammatory cells to generate TPP-FFY-O, which is enriched near the mitochondria by the targeting effect of TPP and self-assembles into nanoparticles by π-π stacking to stay in inflammatory cells Figure 1 ). The large amount of TEMPO enriched on the surface of the nanoparticles can convert the superoxide anion (O2 ·- ) in the mitochondria into hydrogen peroxide (H2O2), which is subsequently decomposed into oxygen and water, thereby significantly reducing the ROS level in inflammatory cells. This enzyme-controlled self-assembled nanoparticle improves the stability of TEMPO and its ability to scavenge ROS, and has good application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Schematic diagram of the effect of the self-assembled nanomaterial TPP-FFYp-O of the application on reducing the ROS level in inflammatory cells.

[0015] Figure 2 Synthetic route map of the application.

[0016] Figure 3 HPLC spectrum of TPP-FFYp.

[0017] Figure 4 Carbon spectrum of TPP-FFYp.

[0018] Figure 5 Mass spectrum of TPP-FFYp.

[0019] Figure 6 Mass spectrum of TPP-FFYp-O.

[0020] Figure 7 Mass spectrum of TPP-O.

[0021] Figure 8 In (a) HPLC dynamic tracking after TPP-FFYp-O enzymatic cleavage; (b) TEM image after TPP-FFYp-O enzymatic cleavage.

[0022] Figure 9 Mass spectrum of TPP-FFY-O.

[0023] Figure 10 In (a) TPP-FFYp-O enzymatic kinetics determination; (b) ROS scavenging test.

[0024] Figure 11 Cytotoxicity test.

[0025] Figure 12 Confocal images of four groups of cells under (a) DCFH-DA staining; (b) live and dead cell fluorescence images of four groups of cells. DETAILED DESCRIPTION

[0026] Example 1: Preparation of TPP-FFYp-O

[0027] 1. TPP-FF (250 mg, 0.37 mmol) was fully dispersed in DMF, then 1-ethyl-(3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC-HCl, 87.71 mg, 0.55 mmol) and N-hydroxysuccinimide (NHS, 63.33 mg, 0.55 mmol) were added, and stirred for 8-12 h to carry out the activation ester reaction; then Tyr(H2PO3)-OH (252.48 mg, 0.48 mmol) and N,N-diisopropylethylamine (DIEPA, 884 μL) were added, and the reaction was stirred at room temperature for 24-48 h until the reaction solution was clear to obtain compound TPP-FFYp. Each step of the reaction was separated and purified by HPLC, and the purity was confirmed by HPLC, and the molecular structure was confirmed by H NMR, C NMR and MS. 1 H NMR, 13 C NMR and MS. Figure 3 Figure 4 Figure 5 ).

[0028] Hydrogen spectrum analysis results of TPP-FFYp: 1 H NMR (400 MHz, DMSO-D6) δ 8.29 (d, J = 7.7 Hz, 1H), 8.16-8.04 (m, 2H), 7.90-7.70 (m, 13H), 7.18-6.92 (m, 13H), 4.56-4.31 (m, 4H), 3.16-2.48 (m, 10H), 2.19 (t, J = 6.7 Hz, 2H), 1.51 (dt, J = 11.6, 8.1 Hz, 2H).

[0029] Carbon spectrum analysis results of TPP-FFYp: 13 ​​C NMR (101 MHz, DMSO-D6) δ 173.10 (2C), 171.68 (2C), 171.59 (1C), 138.53 (1C), 137.83 (1C), 135.47 (4C), 134.01 (6C), 130.65 (6C), 129.69 (4C), 128.33 (4C), 126.74 (1C), 126.54 (1C), 120.33 (2C), 119.20 (3C), 118.35 (2C), 54.23 (1C), 37.98 (2C), 36.44 (1C), 35.39 (1C), 20.14 (1C), 18.84 (1C), 17.32 (1C).

[0030] 2. TPP-FFYp (40 mg, 0.045 mmol), 1-hydroxybenzotriazole (HOBT, 9.14 mg, 0.068 mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 25.64 mg, 0.068 mmol) were dissolved in DMF and activated at room temperature for 30 min, then DIEPA (12 μL) and TEMPO (11.58 mg, 0.068 mmol) were added and stirred at room temperature for 4 h to obtain the target compound TPP-FFYp-O. In addition, due to the paramagnetism of TEMPO, it is not suitable for nuclear magnetic spectrum, so MS confirms the molecular structure ( Figure 6 ).

[0031] 3. Synthesis of compound TPP-O: TPP (50 mg, 0.12 mmol), HOBT (23.58 mg, 0.18 mmol), HBTU (66.32 mg, 0.18 mmol) were dissolved in DMF and activated at room temperature for 30 min, then DIEPA (30 μL) and TEMPO (29.93 mg, 0.18 mmol) were added and stirred at room temperature for 4 h to obtain the control compound TPP-O. Each step of the reaction was separated and purified by HPLC, the purity was confirmed, and the molecular structure was confirmed by MS ( Figure 7 ).

[0032] Example 2: In vitro data characterization

[0033] First, we explored the self-assembly ability of TPP-FFYp-O in vitro. In phosphate buffer solution (PB, 50 mM, pH = 7.4, 1% DMSO), we first incubated 100 μΜ TPP-FFYp-O and 0.04 U / μL ALP enzyme, and tracked the dynamic process of enzyme cutting by HPLC ( Figure 8a). We found that new absorption peaks were generated after 4h incubation. The absorption peaks of TPP-FFYp-O were almost disappeared after 8h incubation. The product after enzyme digestion was verified as TPP-FFY-O by mass spectrometry. Figure 9 ) by TEM. The TEM images clearly showed that TPP-FFYp-O self-assembled into nanoparticles with an average diameter of 30.9 ± 4.4 nm through π-π stacking after ALP digestion.

[0034] Subsequently, we tested the kinetic parameters of ALP catalyzed dephosphorylation reaction of TPP-FFYp-O. We first obtained the HPLC peak area calibration curve of TPP-FFYp-O at 254 nm. Different concentrations of excess TPP-FFYp-O were incubated with ALP in PB buffer at 37°C for 10 min. Then the initial velocity was plotted according to the initial concentration of TPP-FFYp-O and fitted with the Michaelis-Menten model, using Lineweaver-Burk analysis, we obtained the Michaelis constant kcat / k M = 1.475 x 10 5 M -1 s -1 Figure 10 a), indicating that ALP has a high affinity and fast catalytic rate for TPP-FFYp-O, and the probe can quickly self-assemble after entering the cell.

[0035] To verify the ability of nanoparticles to scavenge ROS, we used the total antioxidant capacity (T-AOC) detection kit (FRAP method) to test the effect of TPP-FFY-O on ROS scavenging. We used the kit to detect 25 μM TEMPO, TPP-O, and TPP-FFYp-O, respectively, and scored according to the ultraviolet absorption. Compared with un-assembled TPP-O, the ability of TPP-FFY-O to scavenge ROS after enzyme digestion was significantly different (p < 0.001, Figure 10 b).

[0036] Example 3: Cell experiments

[0037] Next, we further explored the feasibility of TPP-FFYp-O in clearing ROS in cells. Before that, we first investigated the cytotoxicity of the material. Immortalized mouse bone-derived macrophages (iBMDMs) were incubated with 10, 20, and 30 μM TPP-FFYp-O or TPP-O for 12 h, respectively. When the concentration of TPP-FFYp-O was 30 μM, more than 80% of iBMDMs were still alive Figure 11 ​), indicating that these probes have good biocompatibility. Phorbol-12-myristate-13-acetate (PMA) can be used to increase the endogenous ROS of macrophages. We divided the cells into four groups: the first group was not treated; the second group was incubated with PMA for 1 h; the third group was incubated with TPP-FFYp-O for 5 h and then added PMA for 1 h; the fourth group was incubated with TPP-O for 5 h and then added PMA for 1 h. The four groups of cells were respectively stained with commercial DCFH-DA probe for 30 min, and then cell imaging was performed using confocal microscopy. As shown in FIG. 1 Figure 12 a, the PMA group has obvious green fluorescence, indicating that PMA induces the production of endogenous ROS. In comparison, the green fluorescence of the PMA+TPP-O group decreases a little, and the green fluorescence of the PMA+TPP-FFYp-O group decreases significantly, indicating that TPP-FFYp-O can indeed self-assemble in cells to enrich TEMPO to further enhance the ROS scavenging capacity. Subsequently, we performed live and dead cell staining on the four groups of cells, and the PMA group was basically red fluorescence representing dead cells. After co-incubation with TPP-FFYp-O, the red fluorescence decreased significantly, further illustrating that TPP-FFYp-O enhances the ROS scavenging treatment strategy Figure 12 b, scale bar: 25 μΜ).

[0038] In summary, the present application provides an enzymatically self-assembled nanomaterial TPP-FFYp-O, which can successfully scavenge high levels of ROS in cells to achieve anti-inflammatory effects. In vitro experiments show that ALP has a good catalytic rate for TPP-FFYp-O. Due to the aggregation of TEMPO after self-assembly, TPP-FFY-O has stronger ROS scavenging capacity than TPP-O. The results of cell experiments further confirm that the TPP-FFYp-O probe has good ROS scavenging effect in cells. In the near future, our enzyme-controlled self-assembled nanomaterial TPP-FFYp-O will be suitable for clinical anti-inflammatory treatment of related inflammatory diseases.

Claims

1. A self-assembled nanomaterial, characterized in that The structure is shown as follows:

2. The preparation method of the self-assembled nanomaterial of claim 1, obtained by solid-phase synthesis, comprising the following steps: Step 1: TPP-FF is dispersed in DMF, then 1-ethyl-(3-dimethylaminopropyl)-carbodiimide hydrochloride and N-hydroxysuccinimide are added, and stirring is carried out for 8-12 h to carry out an esterification reaction; then Tyr(H2PO3)-OH and N,N-diisopropylethylamine are added, and stirring is carried out at room temperature for 24-48 h until the reaction solution is clear, to obtain compound TPP-FFYp; Step 2: TPP-FFYp, 1-hydroxybenzotriazole, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate are dissolved in DMF, and activation is carried out at room temperature for 30 min, then DIEPA and TEMPO are added, and stirring is carried out at room temperature for 4 h to obtain the target compound TPP-FFYp-O; the synthesis route is shown as follows:

3. The use of the self-assembled nanomaterial TPP-FFYp-O of claim 1 in the preparation of a ROS scavenger.

4. The use of the self-assembled nanomaterial TPP-FFYp-O of claim 1 in the preparation of an anti-inflammatory preparation.

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