Histone-derived peptide nanoparticles targeting the degradation of cytoplasmic cGAS, their preparation method and applications

Abstract

This invention discloses a histone-derived peptide nanoparticle targeting the degradation of cytoplasmic cGAS, its preparation method, and its applications. The invention is characterized by: dissolving a peptide complex in phosphate buffer, sonicating, allowing it to self-assemble at room temperature to form nanoparticles, and purifying by centrifugation to obtain histone-derived peptide nanoparticles A4@CMA NPs targeting the degradation of cytoplasmic cGAS. These nanoparticles (A4@CMA NPs) can target and bind to cytoplasmic cGAS, mediating its degradation, blocking the activation of the cGAS-STING pathway, effectively reducing periodontal inflammation and alveolar bone resorption, and providing a new strategy for the treatment of periodontitis.

Description

TECHNICAL FIELD

[0001] The present application relates to a histone-derived peptide nanoparticle targeting cytosolic cGAS and its preparation method and application, and belongs to the technical field of biological medicine. BACKGROUND

[0002] Periodontitis is a global high-incidence chronic inflammatory oral disease, and the incidence of periodontal disease in Chinese adults is as high as 86%. Its core pathological feature is the progressive destruction of periodontal support tissue (including gingiva, periodontal membrane, alveolar bone and cementum), which is the primary cause of tooth loss in adults. The pathogenesis of periodontitis is complex, in which pathogenic bacteria (such as Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans) and their metabolites can induce abnormal leakage of host cell DNA (including nuclear DNA and mitochondrial DNA) into the cytoplasm, activate the cytosolic key DNA sensor cGAS (cytosolic DNA sensor), and then activate the downstream stimulator of interferon genes (STING) signaling pathway, forming a vicious cycle of "DNA-cGAS-STING-inflammation factor-tissue destruction". The activation of this pathway can promote the release of type I interferon (IFN-β) and pro-inflammatory factors (IL-1β, TNF-α, iNOS), exacerbating the inflammatory response of periodontal tissue, while inhibiting the final alveolar bone absorption.

[0003] Recent studies have found that the activation of cGAS has strict spatial specificity: cGAS in the cytoplasm can be activated by abnormal DNA and trigger inflammation, while cGAS in the nucleus is inhibited by histone H2A in the nucleosome. The "acidic patch" domain of histone H2A can form a stable complex with site B of cGAS through hydrogen bonds and salt bridges, and its binding ability is significantly stronger than that of DNA, thereby competitively inhibiting the binding of cGAS and DNA and the dimerization activation. This finding suggests that histone H2A is a natural inhibitor of intracellular cGAS, and a derivative peptide targeting cGAS based on its structure is expected to become a new strategy for blocking the cGAS-STING pathway and treating periodontitis.

[0004] However, natural polypeptides have significant defects as therapeutic agents: ① poor stability, easily degraded by intracellular proteases (such as trypsin and elastase), short half-life; ② weak transmembrane ability, difficult to penetrate the cell membrane and enter the cytoplasm to play a role; ③ insufficient targeting, unable to precisely enrich cytosolic cGAS, and easy to have non-specific effects on normal tissues; ④ lack of degradation activity, only able to inhibit cGAS through competitive binding, unable to eliminate activated cytosolic cGAS, and limited therapeutic effect.

[0005] The existing intervention means for cGAS have obvious limitations: ① Small molecule inhibitors can inhibit cGAS activity, but have poor targeting, are easy to cross-react with other DNA binding proteins, and have poor biocompatibility; ② Traditional polypeptide delivery systems (such as liposomes and polymer nanoparticles) can improve the stability of polypeptides, but lack microenvironment responsiveness, cannot achieve precise drug release at the lesion site, and are difficult to mediate cGAS degradation; ③ Gene silencing technology (such as siRNA targeting cGAS) is easy to cause off-target effects, and the delivery efficiency is significantly affected by the complex microenvironment of the oral cavity (saliva flushing, pH fluctuation). Therefore, developing a new nano-therapy system with targeting, degradation activity, biocompatibility and stability is of great significance for the effective treatment of periodontitis. SUMMARY

[0006] In view of the above technical problems, the first object of the present application is to provide a preparation method of histone-derived peptide nanoparticles for targeted degradation of cytosolic cGAS, the second object is to provide histone-derived peptide nanoparticles for targeted degradation of cytosolic cGAS prepared by the method, and the third object is to provide applications thereof. The nanoparticles (A4@CMANPs) can target and bind to cytosolic cGAS and mediate its degradation, block the activation of the cGAS-STING pathway, effectively reduce periodontal tissue inflammation and alveolar bone resorption, and provide a new strategy for the treatment of periodontitis.

[0007] To achieve the above-mentioned first object, the technical solution of the present application is: a preparation method of histone-derived peptide nanoparticles for targeted degradation of cytosolic cGAS, characterized by: dissolving a polypeptide complex in a phosphate buffer, ultrasonic treatment, room temperature standing self-assembly to form nanoparticles, centrifugal purification to obtain histone-derived peptide nanoparticles A4@CMANPs for targeted degradation of cytosolic cGAS, the polypeptide complex is:

[0008] ;

[0009] including:

[0010] hydrophobic core: alkyl chain C16;

[0011] intermediate responsive connection unit: cathepsin B responsive peptide GFLG and endosome escape peptide GSVSHHHHHHGGHHHH in series;

[0012] hydrophilic surface layer: modified cGAS binding peptide NDEALNKLLG and CMA targeting motif KFERQKILDQRFFE are connected by lysine to form a dendritic structure. That is, the cGAS binding peptide NDEALNKLLG is connected to the carboxy terminus of lysine, and the carboxy terminus of KFERQKILDQRFFE is connected to the amino terminus of the side chain of lysine.

[0013] The polypeptide complex is synthesized by solid-phase polypeptide synthesis method (synthesis method is prior art), verified by HPLC / MS, purity is ≥95-98%, and molecular weight error is <2ppm.

[0014] In the above scheme: the concentration of the polypeptide complex is 0.5-2mg / mL.

[0015] In the above scheme: ultrasonic treatment for 2-3min, and standing at room temperature for 30-60min.

[0016] In the above scheme: the centrifugal parameter is 13000-15000g centrifugation for 30-40min.

[0017] A histone-derived peptide nanoparticle for targeted degradation of cytosolic cGAS is prepared by a preparation method of the histone-derived peptide nanoparticle for targeted degradation of cytosolic cGAS. The nanoparticle is a uniform spherical structure, the average particle size is 20-40nm, the zeta potential is-25mV-15mV, and the nanoparticle has cathepsin B responsiveness and lysosome escape ability.

[0018] The histone-derived peptide nanoparticle for targeted degradation of cytosolic cGAS is applied to preparation of a medicine for treating a disease related to abnormal activation of a cGAS-STING pathway. The disease is periodontitis. The nanoparticle inhibits STING phosphorylation and release of downstream inflammatory factors (IFN-β and iNOS) by targeted degradation of cytosolic cGAS, and reduces periodontal tissue inflammation and alveolar bone absorption. The nanoparticle exhibits excellent cGAS targeting ability and degradation activity, and effectively regulates cGAS-STING pathway homeostasis.

[0019] Design and screening of cGAS-binding peptide NDEALNKLLG: ① Construct a continuous peptide library of histone H2A, and screen a concentrated region of a peptide segment with excellent cGAS activity by molecular docking; ② Construct an overlapping peptide library for the "acidic patch" domain of H2A, and screen out a histone-derived peptide H8 that inhibits cGAS activation by WB and RT-qPCR, and the amino acid sequence of the histone-derived peptide H8 is NDEELNKLLG; ③ Construct an alanine point mutation peptide library based on the peptide H8, and screen out a histone-derived peptide A4 with the most optimal activity by WB, and the amino acid sequence of the histone-derived peptide A4 is NDEALNKLLG.

[0020] The application is an integrated treatment system capable of simultaneously realizing inflammation microenvironment-responsive drug release, cytoplasmic precise targeting and cGAS degradation. The nanoparticles take histone-derived peptide A4 as a cGAS binding unit, specifically bind to cGAS by simulating the inhibition mechanism of histone H2A, mediate the chaperone-mediated autophagy pathway through the CMA targeting motif to realize the degradation of cGAS, realize intelligent drug release in the inflammation microenvironment through the cathepsin B response unit, and promote the nanoparticles to escape from the lysosome to the cytoplasm through the endosome escape unit. The nanoparticles (A4@CMANPs) can effectively target and degrade cytoplasmic cGAS, inhibit the activation of the STING pathway, reduce the inflammatory response, and thus promote alveolar bone regeneration.

[0021] Compared with the prior art, the histone-derived peptide nanoparticles (A4@CMANPs) for targeted degradation of cytoplasmic cGAS provided by the application have the following remarkable advantages and beneficial effects:

[0022] (1) The preparation process is simple and reliable, and the material performance is excellent: the application adopts solid-phase polypeptide synthesis and self-assembly technology, which are both conventional nanometer preparation methods, simple to operate, mild in conditions and good in reproducibility. The obtained nanoparticles have uniform particle size distribution (about 20-40 nm), good stability, are suitable for large-scale preparation and storage, and provide feasibility for clinical application and transformation.

[0023] (2) Precise targeting and degradation of cGAS are realized: through the specific binding of histone-derived peptide A4 to cGAS and the mediation of the CMA targeting motif, the nanoparticles of the application can actively accumulate in cytoplasmic cGAS and realize its degradation through the chaperone-mediated autophagy pathway, overcoming the limitation of traditional polypeptides that can only inhibit but cannot remove, and laying a foundation for realizing radical treatment of periodontitis.

[0024] (3) It has the pathological microenvironment-responsive drug release characteristic: by using the cathepsin B response unit, the nanoparticles of the application can be specifically cut in the inflammation microenvironment specific to periodontitis, realizing the on-demand release of functional peptides. This intelligent drug release mode effectively improves the bioavailability of the drug and reduces its potential side effects on normal tissues.

[0025] (4) Block the key pathological links of periodontitis through multiple synergistic effects: the nanoparticles of the application not only effectively inhibit the activation of the STING pathway by degrading cGAS, but more importantly, the functional peptides delivered by the nanoparticles can directly block the release of inflammatory factors, reduce the inflammation and osteoclast activity of periodontal tissue, and thus reverse the alveolar bone resorption from the root. The process cooperatively restores the homeostasis of periodontal tissue and ultimately promotes alveolar bone regeneration. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1Design and screening of histone-derived peptides. A, Schematic diagram of constructing H2A continuous peptide library. B, Molecular docking screening of peptides with excellent cGAS activity binding to acidic patch domain. C, Schematic diagram of constructing overlapping peptide library against H2A acidic patch domain. D, WB screening results of overlapping peptide library. E, RT-qPCR screening results of overlapping peptide library. F, Schematic diagram of constructing alanine point mutation peptide library based on peptide H8. G, WB screening of the most active histone-derived peptide A4.

[0027] Figure 2 Synthesis and characterization of A4@CMANPs (as shown in

[0028] Figure 3 In vitro functional verification of A4@CMANPs. A, In vitro mode diagram. B, CCK8 detection of cell proliferation. C, Calcein / PI staining to observe cell live and dead state. D, WB detection of cGAS and STING phosphorylated protein expression and quantitative analysis. E, RT-qPCR detection of interferon factor IFNβ expression. F, WB concentration-dependent protein expression. G, RT-qPCR detection of concentration-dependent interferon factor expression.

[0029] Figure 4 In vivo treatment effect of A4@CMANPs. A, Schematic diagram of periodontal local treatment. B, Hemolysis experiment detection. C, H&E staining to observe heart, liver, spleen, lung and kidney to evaluate biocompatibility. D, Micro-CT detection of alveolar bone resorption. E, Quantitative analysis of CEJ to ABC distance. F, H&E staining to observe periodontal tissue inflammation. G, IHC detection of inflammatory factor iNOS expression. H, TRAP staining to detect osteoclast activity.

[0030] Figure 5 Flow chart of screening of peptide A4 of the present application, A4@CMANPs assembly and schematic diagram of action principle. DETAILED DESCRIPTION

[0031] The present application will be further described below by way of examples in conjunction with the accompanying drawings:

[0032] Example 1

[0033] Design and screening of histone-derived peptides (as shown in Figure 1 ​

[0034] Experimental materials: THP1 cells, histone H2A antibody, cGAS antibody, STING antibody, pSTING antibody, HtDNA, Lipofectamine 3000 transfection reagent, etc.

[0035] Experimental content: (1) Continuous peptide library construction and screening: Taking histone H2A as a template, 13 continuous 10-peptide libraries (P1-P13) were constructed. The binding conformation and binding energy of polypeptides and cGAS were evaluated by molecular docking. The results showed that the peptide segments with excellent binding activity were concentrated in the "acidic patch" region of H2A (residues 61-98) (A-B of FIG. 1). Figure 1 (2) Overlapping peptide library construction and screening: For the "acidic patch" domain of H2A (residues 61-98), 8 overlapping 10-peptide libraries (H1-H8) were constructed. After HtDNA (deoxyribonucleic acid sodium salt from herring testis) (2 μg / mL) stimulated THP1 cells for 6 h, the polypeptides (2 μg / mL) of the overlapping peptide library were added for 24 h. Subsequently, the WB (Western blot) detection results showed that the peptide segment H8 had the best inhibitory effect on the phosphorylation level of STING protein, and the RT-qPCR (reverse transcription-real-time fluorescent quantitative PCR) results showed that the peptide segment H8 had the most significant inhibitory effect on the expression of IFN-β gene. Therefore, the peptide H8 (sequence NDEELNKLLG) with the best inhibitory effect on cGAS activation was screened (C-E of FIG. 1). Figure 1 (3) Point mutation peptide library screening: The alanine point mutation peptide library (A1-A10) of peptide H8 was constructed. After HtDNA (2 μg / mL) stimulated THP1 cells for 6 h, the point mutation polypeptides (2 μg / mL) were added. The WB detection results showed that the peptide A4 (sequence NDEALNKLLG) had the best activity (F-G of FIG. 1). Figure 1

[0036] Example 2: Preparation and characterization of histone-derived peptide nanoparticle A4@CMANPs Figure 2

[0037] Experimental materials: alkyl chain C16, cathepsin B responsive peptide (GFLG), endosome escape peptide (GSVSHHHHHHGGHHHH), peptide A4 (NDEALNKLLG), CMA targeting motif (KFERQKILDQRFFE), HPLC instrument, mass spectrometer, SEM, TEM, AFM, Malvern particle size instrument, etc.

[0038] Experimental content: A polypeptide complex containing a hydrophobic core, an intermediate connecting unit and a hydrophilic surface layer was synthesized by solid-phase polypeptide synthesis method (interacted with Shanghai Huacheng Peptide Biological Synthesis).

[0039] Hydrophobic core: alkyl chain C16;

[0040] ​​Intermediate responsive linker unit: formed by the cathepsin B responsive peptide GFLG and the endosome escape peptide GSVSHHHHHHGGHHHH in tandem;

[0041] Hydrophilic surface layer: Modified cGAS-binding peptide NDEALNKLLG and CMA-targeting motif KFERQKILDQRFFE form a dendritic structure linked by lysine residues. Specifically, cGAS-binding peptide NDEALNKLLG is attached to the carboxyl terminus of lysine residues, and the carboxyl terminus of KFERQKILDQRFFE is attached to the amino terminus of the lysine side chain.

[0042] HPLC / MS was used to verify the purity (≥95%) and molecular weight (error from theoretical value <2ppm) of the synthesized peptide complex. Figure 2 The peptide complex was then dissolved in 10 mM phosphate buffer (PB) to prepare a concentration of 1 mg / mL. The mixture was sonicated for 3 min, allowed to stand at room temperature for 30 min, and purified by centrifugation at 15000 g for 30 min to obtain histone-derived peptide nanoparticles that target the degradation of cytoplasmic cGAS and self-assembled to form nanoparticles A4@CMANPs.

[0043] The critical micelle concentration (CMC) of the material was determined using the Nile Red method. Nile Red exhibits weak fluorescence in an aqueous environment, but once it enters the hydrophobic pocket of the micelles, it shows a significant blue shift and a sharp increase in fluorescence intensity. The results showed that the fluorescence intensity of Nile Red gradually increased with increasing material concentration. When the concentration reached the critical value, the fluorescence intensity of Nile Red increased significantly and showed a marked blue shift, confirming the formation of the hydrophobic pocket and the generation of micelles. The maximum intensity was corrected based on the method described in the literature, and the intersection of the regression curves for A4@CMANPs was determined to be 1.29, corresponding to a CMC of approximately 19.5 µM. Figure 2 The nanoparticles were observed to be uniformly spherical in shape using SEM / TEM / AFM. Figure 2 The particle size is 20-40 nm, and the zeta potential is -25 mV to 15 mV. Figure 2 (FG).

[0044] Fluorescence spectroscopy results showed that no significant differences were observed in the fluorescence spectra of the group without cathepsin B at 12 h, while in the group with cathepsin B, the fluorescence intensity gradually increased with time, reaching a peak at 6 h. This indicates that A4@CMANPs nanoparticles can respond well to cathepsin B in vitro and undergo structural deconstruction within 6 h, thus achieving an effective response within 6 h under the action of cathepsin B. Figure 2 H).

[0045] The lysosomal escape of A4@CMANP nanoparticles was further detected using a lysosomal red fluorescent probe. After treating THP1 cells with 5-FAM fluorescently labeled A4@CMANPs for different time periods, the observation results and fluorescence colocalization statistics showed that 5-FAM-A4@CMANPs (green fluorescence) exhibited strong colocalization with lysosomes (red fluorescence) 1 h after cell addition. This colocalization gradually decreased after 2 h, and after 4 h, there was almost no obvious colocalization area, indicating that the A4@CMANPs nanoparticles had essentially escaped the lysosomes by this time. These results indicate that A4@CMANPs nanoparticles possess good lysosomal escape characteristics after 4 h, which can effectively improve the bioavailability of A4@CMANPs nanoparticles in vivo. Figure 2 (I).

[0046] Example 3: In vitro functional validation of histone-derived peptide nanoparticles A4@CMANPs ( Figure 3 )

[0047] Experimental materials: THP1 cells, control group nanoparticles H8@CMANPs, CMANPs, A4NPs, CCK8 kit, Calcein / PI staining kit, cytoplasmic / nuclear protein extraction kit, etc.

[0048] Wherein: H8@CMANPs refers to peptide A4 replaced with the control peptide H8 (NDEELNKLLG), prepared according to the method of Example 2. CMANPs and A4NPs refer to the hydrophilic surface layer peptides of nanoparticles containing only CMA or A4, with other conditions unchanged, prepared according to the method of Example 2. Dissolved in 10mM phosphate buffer (PB) to a concentration of 1 mg / mL, sonicated for 3 min, incubated at room temperature for 30 min, and purified by centrifugation at 15000g for 30 min.

[0049] Experimental content: First, the CCK8 assay was used to detect the effect of different concentrations of nanoparticles (1-20 μg / mL) on the proliferation of THP1 cells, and Calcein / PI staining was used to observe the cell viability. The results showed that the nanoparticles had good biocompatibility within a concentration of 10 μg / mL. Figure 3 The BC of the cells was then used to stimulate THP1 cells with HtDNA (2 μg / mL), followed by treatment with A4@CMANPs nanoparticles (1 μg / mL). Cytoplasmic proteins were extracted, and Western blotting was used to detect cGAS protein expression levels and downstream STING phosphorylation levels. RT-qPCR was used to detect the expression levels of type I interferon IFN-β and other genes. The results showed that A4@CMANPs significantly degraded cytoplasmic cGAS, inhibited STING phosphorylation, and reduced downstream interferon factor expression. Figure 3The inhibitory effect of A4@CMANPs is noteworthy as it exhibits a significant concentration-dependent effect. Figure 3 (FG).

[0050] Example 4: In vivo therapeutic efficacy verification of histone-derived peptide nanoparticles A4@CMANPs ( Figure 4 )

[0051] Experimental materials: C57BL / 6 mice, 5-0 silk thread, A4@CMANPs nanoparticles, Micro-CT, H&E staining kit, IHC kit, TRAP staining kit, etc.

[0052] Experimental procedure: A periodontitis mouse model induced by suture ligation of the cervical region of the second molar in C57BL / 6 mice was established. Simultaneously, nanoparticles (5 mg / kg / day) were injected locally into the periodontal region for 7 consecutive days. Figure 4 (A). First, biocompatibility testing was performed: a hemolysis experiment was conducted to detect the effect of the nanoparticles on erythrocytes; heart, liver, spleen, lung, and kidney tissues from mice were collected, and H&E staining was used to observe organ morphology. The results showed that the histone-derived peptide nanoparticles A4@CMANPs did not exhibit significant hemolysis and did not cause damage to the major organs of mice. Figure 4 The treatment effect was then evaluated: Micro-CT was used to detect alveolar bone resorption (CEJ-ABC distance); H&E staining was used to observe periodontal tissue inflammatory infiltration; IHC was used to detect iNOS protein expression; and TRAP staining was used to detect osteoclast activity. The results showed that A4@CMANPs could significantly reduce alveolar bone resorption, alleviate periodontal tissue inflammatory infiltration, reduce inflammatory factor expression, and inhibit osteoclast activity, with excellent treatment effect. Figure 4 (EH).

[0053] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, such as sonication for 2-3 min, standing at room temperature for 30-60 min, centrifugation at 13000-15000g for 30-40 min, and selection of peptide complex concentration between 0.5-2 mg / mL. The scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method of preparing a histone-derived peptide nanoparticle that targets degradation of cytosolic cGAS, characterized by: The polypeptide complex is dissolved in a phosphate buffer, ultrasonically treated, and left to stand at room temperature to self-assemble into nanoparticles, which are purified by centrifugation to obtain the histone-derived peptide nanoparticles A4@CMA NPs that target cytosolic cGAS degradation, wherein the polypeptide complex is: comprising: Hydrophobic core: alkyl chain C16; Intermediate responsive connection unit: cathepsin B responsive peptide GFLG connected in series with endosome escape peptide GSVSHHHHHHGGHHHH; Hydrophilic surface layer: modified cGAS binding peptide NDEALNKLLG and CMA targeting motif KFERQKILDQRFFE are connected by lysine to form a dendritic structure, wherein the cGAS binding peptide NDEALNKLLG is connected at the carboxy terminus of lysine, and the carboxy terminus of KFERQKILDQRFFE is connected at the amino terminus of the side chain of lysine.

2. The method of claim 1, wherein the histone-derived peptide nanoparticles targeting cytosolic cGAS for degradation are prepared by: The concentration of the polypeptide complex is 0.5-2 mg / mL.

3. The method of claim 2, wherein the histone-derived peptide nanoparticles targeting cytosolic cGAS for degradation are prepared by: Ultrasonic treatment for 2-3 min, and left to stand at room temperature for 30-60 min.

4. The method of claim 3, wherein the histone-derived peptide nanoparticles targeting cytosolic cGAS for degradation are prepared by: The centrifugation parameters are 13000-15000g for 30-40 min.

5. A preparation method of the histone-derived peptide nanoparticles that target cytosolic cGAS degradation according to any one of claims 1-4.

6. The histone-derived peptide nanoparticle targeting degradation of cytosolic cGAS of claim 5, wherein: The histone-derived peptide nanoparticles that target cytosolic cGAS degradation are uniform spherical structures with an average particle size of 20-40 nm and a zeta potential of -25 mV to -15 mV.

7. Use of the histone-derived peptide nanoparticles that target cytosolic cGAS degradation according to claim 5 in the preparation of a medicament for treating periodontitis.

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