Natural linoleic acid nanoparticles for targeting T cells

Abstract

The invention discloses natural linoleic acid nanoparticles targeting T cells. A preparation method of the natural linoleic acid nanoparticles comprises the following steps: step 1, firstly, adding anti-CD3 protein molecules into a deionized water solution containing fetal calf serum (BSA) to form a stable system I; 2, dropwise adding the LA liquid into the liquid phase I under the ultrasonic condition, and forming a self-assembled nano core-shell structure (TTLANPs) which takes the LA molecule as a core and anti-CD3 protein / BSA as a shell and has a T cell targeting function through the interaction between hydrophobic / hydrophilic groups of the hydrophobic LA molecule and the protein molecule; according to the T-cell-targeting natural linoleic acid nano-particles, the regulation mechanism of linoleic acid on periodontitis is analyzed from the perspective of metabolic immunity, and the problems that periodontitis is prone to relapse, inflammation and tissue damage cannot be completely eliminated through traditional periodontal basic treatment, and previous research focuses on research and development of antibacterial drugs are solved. Or the immune response level of the organism is systematically regulated, but drug resistance is easily generated or toxic and side effects caused by systemic application exist.

Description

Technical Field

[0001] The invention relates to the field of medical technology, in particular to a natural linoleic acid nanoparticle targeting T cells. Background Art

[0002] Periodontal disease is a major oral health problem prevalent worldwide. It is caused by the host immune response triggered by dental plaque biofilm, which leads to continuous destruction of periodontal tissues, and T cell immunity in particular plays a key role. Existing treatment options are mainly mechanical periodontal treatment, supplemented by antibacterial or systemic immunomodulators, but there are still problems such as unstable treatment effects, high recurrence rates, and significant individual differences. Exploring more stable, efficient, safe and controllable treatment methods has become an urgent issue to be solved. The applicant previously discovered for the first time that the natural plant ingredient linoleic acid (LA) plays an anti-inflammatory function by regulating the differentiation of T cell subpopulations in the process of periodontitis pathogens crosstalking intestinal flora to aggravate intestinal inflammation. Therefore, this patent aims to construct a T-cell targeted LA nanodelivery system (TTLA NPs) using the antisolvent method, use a variety of methods to evaluate the therapeutic effects of TTLA NPs on periodontitis, and analyze the T cell immune regulation mechanism of TTLA NPs in the periodontal inflammation microenvironment from multiple dimensions;

[0003] The linoleic acid-AHR axis may mediate the progression of periodontal disease by regulating T cell immune responses;

[0004] Recently, the applicant discovered for the first time that the periodontal pathogen Porphyromonas gingivalis (Pg) crosstalks the disordered structure and metabolic abnormalities of the intestinal flora, inhibiting the production of the intestinal metabolite LA, thereby aggravating intestinal inflammation. During this process, the Th17 cells in the colonic lamina propria lymphocytes increased significantly, while the immunosuppressive Treg cells decreased significantly. At the same time, LA, as a ligand of the aromatic hydrocarbon receptor (AHR), can inhibit the secretion of IL-17 by Th17 cells induced in vitro by regulating the phosphorylation of Stat1 (Ser727) (the relevant results were published in Nature Communications as an independent first author). Linoleic acid (LA, C18: 2△9, 12) is a polyunsaturated fatty acid, one of the essential fatty acids for the human body, and is widely present in vegetable oils. Linoleic acid and its metabolites have a variety of biological activities, such as anti-inflammatory, immunomodulatory, cell proliferation and apoptosis regulation. The applicant's latest research shows that antibiotics eliminate intestinal flora, leading to aggravated periodontitis in mice, and the content of LA in their feces increases. This suggests that LA may play an important role in the pathological progression of periodontal disease. In previous studies, we have confirmed that LA is a strong ligand of AHR, which can downregulate pro-inflammatory Th17 cells in the inflammatory environment and upregulate immunosuppressive Treg cells.

[0005] In addition, the applicant has confirmed in previous experiments that the percentage of pro-inflammatory IL-17+Th17 cells in submandibular lymph node cells (SLNs) in the mouse periodontal disease model is significantly increased (the relevant results were published as an independent first author in the journal "Modern Stomatology"). Moreover, Porphyromonas gingivalis (Pg) can promote differentiation into Th17 cells by activating the TLR4 receptor on the surface of T cells (the relevant results were published as an independent first author in "Clinical & Translational Immunology"). Clinical data show that the concentration of IL-17 in the gingival crevicular fluid and serum of patients with periodontal disease is significantly increased. These results all prove that pro-inflammatory Th17 cells play a key role in the pathogenesis and pathological process of periodontal disease.

[0006] The role of AHR activation in Th17 cell differentiation based on different ligands is dual. AHR activation can promote the generation of Th17 cells and the release of IL-17, thereby exacerbating the inflammatory response. However, in periodontitis, moderate AHR activation can indirectly inhibit the inflammatory effect of Th17 cells by promoting the generation of Treg cells. However, the role of T cell immune regulatory signals mediated by the linoleic acid-AHR axis in the progression of periodontal disease and its specific mechanism are still unknown.

[0007] By constructing a nanosystem targeting T cells to amplify the immunoregulatory effect of linoleic acid by an order of magnitude, it may be possible to achieve precise nutritional intervention for periodontal disease.

[0008] The direct application of drugs generally has two major problems, namely poor water solubility and difficulty in entering cells. The nano-design of drugs provides an effective strategy to improve the bioavailability of drugs. Drug nano-design methods usually include nano-micelles, nanoparticles, nanocrystals and nano-emulsions. Among them, the preparation process of nano-micelles is complicated, they are easy to dissociate, the in vivo stability is not high, and they may cause immune reactions; nano-particles have good controlled release effects, can achieve targeted delivery, and increase biological half-life, but the preparation process is complicated, and the material selection and preparation process need to be optimized, and there may be toxicity and immune reactions; nano-crystal preparation equipment has high requirements, and physical stability (such as crystal morphology) needs to be strictly controlled; nano-emulsions prepare stable nano-scale droplets through oil / water emulsification, and the preparation method is simple and easy, with good stability.

[0009] Antisolvent-mediated drug self-assembly is a new method for constructing nanomedicines. Lipophilic drugs are dissolved in organic solvents and added to the aqueous phase system under ultrasound or high-speed stirring. Based on the lipophilic and hydrophobic properties of the drugs, the molecules self-assemble to form the desired nanoparticles. In addition, nanomedicines can be functionalized through surface modification to achieve accurate recognition and targeted delivery of specific targets, improve uptake efficiency, enhance therapeutic effects, avoid side effects caused by non-specific delivery, and provide new possibilities for personalized and precise treatment. Studies have shown that indocyaninegreen (ICG) functionalized with mannose self-assembles into BSA nanoparticles (MφL@BSA@Man-ICG NPs) based on non-covalent interactions, which are used for targeted delivery to macrophages. The self-assembled nanoparticles induced by in situ photodynamic therapy arm the macrophages with an instantaneous ROS burst to eliminate residual pathogens, providing a new strategy for infection treatment targeting macrophages. Studies have also shown that nanoparticle-based bispecific T cell binders (nanoBiTEs), one end of which is connected to a liposome modified with an anti-CD3 monoclonal antibody targeting T cells and the other end is a monoclonal antibody targeting cancer antigens, have successfully achieved an order of magnitude expansion of the efficacy of myeloma cells and prevented the development of antigen-free tumor escape. The above research and development cases all illustrate that the targeted nano drug delivery system is an efficient technology based on the targeted cell-based precision treatment of target tissue inflammation.

[0010] Based on the results of previous studies, this project uses the antisolvent method and intermolecular hydrophilic-hydrophobic interactions to design and construct self-assembled nanoparticles with LA as the core and anti-CD3 protein / BSA as the shell - T cell-targeted LA nanoparticles (TTLA NPs), and then evaluates the therapeutic effect and immunomodulatory effect of TTLA NPs on periodontitis, and analyzes the T cell immunomodulatory mechanism of TTLA NPs in the inflammatory microenvironment in multiple dimensions. This targeted nanosystem can accurately deliver linoleic acid to T cells, thereby enhancing its anti-inflammatory effect, regulating immune response, and inhibiting the progression of periodontal disease. This method not only improves the bioavailability and efficacy of linoleic acid, but also reduces potential side effects, showing good application prospects. At the same time, the combination of nanotechnology and natural active substances has opened up a new way for the prevention and treatment of periodontal disease, and is a new strategy for intervening in periodontal disease from the perspective of precision "medicine and food are of the same origin".

[0011] Therefore, we proposed a natural linoleic acid nanoparticle targeting T cells to solve the above problems. Summary of the invention

[0012] The purpose of the present invention is to provide a natural linoleic acid nanoparticle targeting T cells to solve the problems in the current market raised by the above background technology.

[0013] To achieve the above object, the present invention provides the following technical solution: a natural linoleic acid nanoparticle targeting T cells, comprising the following steps:

[0014] Step 1: first, add the anti-CD3 protein molecule into a deionized water solution containing fetal bovine serum (BSA) to form a stable system I;

[0015] Step 2: Then, under ultrasound, LA droplets are added to liquid phase I, and through the interaction between the hydrophobic LA molecules and the hydrophobic / hydrophilic groups of the protein molecules, a self-assembled nano core-shell structure (TTLA NPs) with T cell targeting function is formed with LA molecules as the core and anti-CD3 protein / BSA as the shell;

[0016] Step 3: Take an appropriate amount of TTLA NPs suspension and dilute it to an appropriate concentration with deionized water;

[0017] Step 4: Add a small amount of diluted suspension onto the copper mesh carrier and allow to dry naturally.

[0018] Preferably, the LA must be carried out in a liquid phase I system containing BSA.

[0019] Preferably, the carrier in step 4 may be other appropriate carriers.

[0020] Compared with the prior art, the present invention has the following beneficial effects: the natural linoleic acid nanoparticles targeting T cells:

[0021] 1. Analyze the regulatory mechanism of linoleic acid on periodontitis from the perspective of metabolic immunity

[0022] As periodontitis is prone to relapse, traditional periodontal basic treatment cannot completely eliminate inflammation and tissue damage. Previous research focuses on the development of antibacterial drugs or systemic regulation of the body's immune response level, which is prone to drug resistance or toxic side effects of systemic application. This project takes a unique approach and focuses on using linoleic acid, a natural ingredient obtained from plants, to analyze its immune metabolic regulation in the periodontal disease microenvironment, clarify its targets for mediating T cell immune responses, and achieve targeted regulation. It is an attempt to construct a "food therapy" for periodontitis from a safe and efficient perspective.

[0023] 2. Construct a targeted drug delivery system to enhance local T cell immune response

[0024] Previous studies have confirmed that linoleic acid (LA) has a good anti-inflammatory effect and metabolic immune regulation in inflammatory diseases. However, how to accurately regulate the immune response of T cells in the microenvironment to achieve efficient and safe treatment of periodontitis is an urgent problem to be solved. Therefore, this project uses the anti-solvent method to form a self-assembled nano core-shell structure with LA molecules as the core and anti-CD3 protein / BSA as the shell through the interaction between the hydrophobic LA molecules and the hydrophobic / hydrophilic groups of the anti-CD3 protein molecules to prepare T-cell targeted linoleic acid nanoparticles (TTLANPs). Technically, it is possible to achieve efficient immune regulation of T cells with a trace amount of LA, avoiding excessive accumulation of fatty acids and uncertainty in cellular uptake. It is a new breakthrough in the treatment and intervention of periodontitis with the new concept of "medicine and food are of the same origin". BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The overall view of the construction of the T cell targeted linoleic acid nanoparticles (TTLA NPs) system. (a) LA droplets are added to the BSA-water solution containing the targeting protein anti-CD3 to form a homogeneous and stable liquid phase system. (b) When LA droplets are added to pure water, the oil droplets aggregate and float on the surface and cannot form a stable dispersion system;

[0026] Figure 2 This is a schematic diagram of the preparation process of the present invention; DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] See also Figure 1-2 The present invention provides a technical solution: a natural linoleic acid nanoparticle targeting T cells, comprising the following steps:

[0029] (1) Construction of T cell-targeted linoleic acid nanoparticles (TTLA NPs)

[0030] Under ultrasound, linoleic acid liquid was added dropwise to the BSA aqueous solution containing anti-CD3 protein. Through the interaction between the hydrophobic oleic acid molecules and the hydrophobic / hydrophilic groups of the protein molecules, a self-assembled nano core-shell structure (TTLA NPs) with LA molecules as the core and anti-CD3 protein / BSA as the shell was formed.

[0031] (2) TEM characterization of morphology and size of TTLA NPs

[0032] Take an appropriate amount of TTLA NPs suspension and dilute it with deionized water to an appropriate concentration. Add a small amount of the diluted suspension on a copper mesh or other appropriate carrier and dry it naturally. Observe the dried sample using a transmission electron microscope (TEM). Take and analyze the morphology and size images of TTLA NPs. Use image analysis software to measure the average size and particle size distribution of the nanoparticles.

[0033] (3) HPLC detection of LA encapsulation efficiency and release characteristics

[0034] Encapsulation efficiency test: Prepare a standard solution of LA with known concentration for drawing a standard curve. Take an appropriate amount of TTLA NPs suspension and separate the nanoparticles from free LA by centrifugation. Take the supernatant and analyze the unencapsulated LA content by high-performance liquid chromatography (HPLC). Calculate the concentration of LA in the supernatant using the standard curve to determine the encapsulation efficiency.

[0035] Release characteristics detection: Place an appropriate amount of TTLA NPs suspension in a simulated body fluid (such as phosphate buffer) and place it in a constant temperature oscillator at 37°C. Samples are taken at different time points and centrifuged to separate the nanoparticles and released LA. HPLC is used to detect the LA concentration in the supernatant, and the release curve is drawn to analyze the release characteristics of LA.

[0036] (4) Absorbance and morphology changes to investigate the long-term dispersion stability of TTLA NPs

[0037] Absorbance determination: Take an appropriate amount of TTLNPs suspension and measure its initial absorbance using a UV-visible spectrophotometer. Store the suspension at room temperature or other set temperature. Take samples at set time intervals (such as 1 day, 7 days, 14 days, etc.), measure the absorbance, and compare the changes.

[0038] Observation of morphology changes: Samples were taken at different storage time points, and the morphology of TTLA NPs was observed using a transmission electron microscope (TEM). The morphological changes of the nanoparticles were recorded and analyzed to evaluate their dispersion stability.

[0039] (5) By using fluorescent dye and flow cytometry technology, the ratio of LA and anti-CD3 proteins was adjusted by detecting the effective targeting binding rate of TTLA NPs on CD4+ T cells, and the concentration gradient of surface-modified anti-CD3 was set to optimize the minimum amount that can meet effective targeting.

[0040] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A natural linoleic acid nanoparticle targeting T cells, characterized in that: The following steps are involved: Step 1: first, add the anti-CD3 protein molecule into a deionized water solution containing fetal bovine serum (BSA) to form a stable system I; Step 2: Then, under ultrasound, LA droplets are added to liquid phase I, and through the interaction between the hydrophobic LA molecules and the hydrophobic / hydrophilic groups of the protein molecules, a self-assembled nano core-shell structure (TTLA NPs) with T cell targeting function is formed with LA molecules as the core and anti-CD3 protein / BSA as the shell; Step 3: Take an appropriate amount of TTLA NPs suspension and dilute it to an appropriate concentration with deionized water; Step 4: Add a small amount of diluted suspension onto the copper mesh carrier and allow to dry naturally.

2. The natural linoleic acid nanoparticles targeting T cells according to claim 1, characterized in that: The LA must be carried out in a liquid phase I system containing BSA.

3. The natural linoleic acid nanoparticles targeting T cells according to claim 1, characterized in that: The carrier in step 4 may be other appropriate carriers.

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