CN113975405A

CN113975405A - Nanoparticle targeting TRPV1, and preparation method and application thereof

Info

Publication number: CN113975405A
Application number: CN202110847784.1A
Authority: CN
Inventors: 聂怡初; 邓文斌; 张祎迪; 麦扬; 刘赣; 赵景新; 徐健; 谢芫; 萧倩
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2021-07-27
Filing date: 2021-07-27
Publication date: 2022-01-28
Anticipated expiration: 2041-07-27
Also published as: CN113975405B

Abstract

The invention relates to a TRPV 1-targeted nanoparticle, a preparation method and application thereof, and relates to the field of pharmaceutical preparations. The preparation method comprises the following steps: preparing nanoparticles with photothermal conversion performance, selecting a block copolymer with surface carboxyl, dissolving the block copolymer and a photosensitizer respectively by using a solvent, mixing, adding into water, homogenizing, removing the solvent, centrifuging, and carrying out heavy suspension precipitation to obtain the nanoparticles; and (3) targeted modification, namely activating the surface carboxyl of the nanoparticle with the photothermal conversion performance, and then incubating the nanoparticle with the TRPV1mAb to covalently connect the TRPV1mAb to the carboxyl of the nanoparticle, so as to obtain the target product. The prepared nano-particles can be excited by near infrared light, selectively kill TRPV1 over-expressed cells in bronchial and lung tissues by utilizing the photothermal effect of the nano-particles, remarkably reduce the infiltration of airway inflammatory cells, inhibit the hyperproliferation of goblet cells and columnar epithelial cells, obviously improve the airflow limitation, and effectively treat allergic asthma.

Description

Nanoparticle targeting TRPV1, and preparation method and application thereof

Technical Field

The invention relates to the field of pharmaceutical preparations, in particular to a TRPV 1-targeted nanoparticle and a preparation method and application thereof.

Background

Asthma is a chronic airway inflammatory disease, the clinical manifestations are respiratory system related symptoms, such as asthma, dyspnea, chest distress and the like, and airflow limitation which is aggravated along with time change and the like, and the pathological features are airway inflammation, airway remodeling and airway stenosis.

Allergic asthma is the most common type of asthma. Various allergic factors (e.g., dust mites, cockroach residues, animal hair, molds, pollen) and non-allergic factors (e.g., infection, cigarette smoke, cold air, exercise, etc.) can trigger T helper type 2 (Th 2) immune responses, resulting in chronic airway inflammation. Th2 cell level is increased in the airway, cytokines including Interleukin (Interleukin)4(IL-4), IL-13, IL-5 and IL-13 are released, mast cell eosinophilic inflammation and Immunoglobulin E (IgE) production are promoted, inflammatory mediators such as histamine and cysteinyl leukotriene are released, and bronchospasm, edema and mucus secretion are increased. The clinical manifestations of the airway hyperreactivity and reversible trachea obstruction are the pathological characteristics of airway inflammation mainly caused by airway columnar epithelial eosinophil infiltration, goblet cell metaplasia, airway obstruction caused by mucus secretion increase, airway stenosis caused by airway smooth muscle excessive proliferation and the like.

The drugs for treating asthma are classified into control drugs, relief drugs and targeting drugs. Control drugs refer to drugs that require long-term daily use, including inhaled glucocorticoids (ICS), leukotriene modulators, long-acting β 2 receptor agonists, and the like. The relief drug is mainly used for relieving asthma symptoms by rapidly relieving bronchospasm, and comprises a beta 2 receptor agonist, an inhalant anticholinergic drug, short-acting theophylline, systemic hormone and the like which are quickly inhaled and orally taken. The targeted therapeutic drugs include anti-IgE monoclonal antibodies, anti-IL-5 receptor monoclonal antibodies, anti-IL-4 receptor monoclonal antibodies and the like.

At present, four monoclonal antibodies are approved by the U.S. food and drug administration for treating moderate-severe allergic asthma, namely omalizumab, meperilizumab, rosuvastatin and dolabrumab.

IgE has been shown to play a key role in the pathogenesis of allergic inflammation. Omalizumab binds specifically to IgE, inhibits the production of bronchitis and alleviates airway remodeling, and was approved by the FDA for the treatment of moderate to severe asthma in 2003. Omalizumab inhibits early and late allergic reactions in patients with allergic asthma, alleviates asthma symptoms, reduces the number of asthma exacerbations and the associated hospital and emergency visits to patients, and may allow patients to reduce or partially discontinue use of inhaled glucocorticoids. However, lung function was not significantly improved in asthmatic patients and was only effective in patients with a history of frequent asthma exacerbations who received high doses of glucocorticosteroid hormone therapy.

Eosinophils are key cells in allergic asthma, and IL-5 induces the differentiation, survival and activation of eosinophils. The mesmerizumab and the reluzumab target Interleukin 5(Interleukin-5, IL-5), and the interaction of IL-5 and an eosinophil surface IL-5 receptor is blocked by combining with IL-5. The evaluation of the efficacy of the anti-IL-5 monoclonal antibody in patients with moderate-severe asthma shows that although the monoclonal antibody can pathologically reduce the generation of eosinophil and promote apoptosis, the clinical outcome of most of the patients with asthma is not obviously improved. Research shows that the monoclonal antibody targeting IL-5 has a better treatment effect only on asthma patients with a specific phenotype, for example, the monoclonal antibody of MEIPELIA can effectively slow down the progress of asthma in part of eosinophilic asthma patients difficult to treat, and has a certain improvement effect on the life quality of the patients. Therefore, monoclonal antibodies targeting IL-5 require further intensive research into the pathology and genotyping of patients, on the basis of which specific asthma patients can achieve better therapeutic results.

In addition, the dolabrumab targeting Interleukin 4(Interleukin-4, IL-4) receptor alpha subunit also becomes the fourth FDA-approved targeting agent for the treatment of severe allergic asthma following omalizumab, meperilizumab and reluzumab. IL-4Ra binds to IL-4 and Interleukin 13(Interleukin-13, IL-13), preventing activation of macrophages and basophils. Dolbitumumab has better curative effect on patients with moderate and severe asthma accompanied with eosinophilia, and reduces biomarkers related to Th 2-driven inflammation. However, the incidence of injection site reactions, nasopharyngitis, nausea and headache in patients was higher than that in the placebo group.

The targeting preparation can relieve the condition of partial asthma patients and improve the life quality of the patients, but has high cost, is only effective for specific patient groups and is accompanied with certain side effects. Targeted formulations therefore require more clinical phenotype screening and cost-effective economic evaluation to assist physicians and patients in making rational choices.

For patients with severe and refractory Asthma for whom existing pharmacotherapeutic strategies are ineffective, the Global Initiative for Asthma prevention (GINA) recommends the use of bronchial thermoplasty for treatment.

Bronchial Thermoplasty (BT) is a non-pharmaceutical intervention that reduces the quality of the smooth muscles of adult asthmatics by controlled delivery of heat energy (above 65 ℃) through a bronchoscope to the Bronchial wall of airways 3-10mm in diameter, inhibiting the contraction function of the smooth muscles to relieve asthma symptoms. The research on RISA and AIR-2 followed in 5 years proves that the adverse reaction of respiratory tract and the acute attack frequency of asthma of a patient are reduced within 3-5 years after BT operation, so that the hospitalization rate and the emergency rate are both obviously reduced, and the dosage of the inhaled steroid hormone is reduced. The volume percentage of expired air (FEV 1%) in one second before and after lung CT and bronchodilator treatment of the patient has no obvious change, which indicates that the patient has no bronchiectasis, and verifies the long-term safety of BT treatment. PAS2 is an extension of the AIR-2 study, and compared with AIR-2, patients with higher ICS usage, obesity, cardiovascular disease, diabetes and the like combined with basic diseases are included, and similar results to AIR-2 are obtained. However, the above studies have strict screening criteria for patients, excluding the most clinically common patients with allergic asthma which are the most severe and have various basic diseases, and it is worth discussing whether the patients can obtain obvious treatment effects.

Although BT therapy has a good long-term outcome, there are still a number of problems to be solved. First, BT treatment requires 3 separate bronchoscopies to cover different areas of the lung, with adverse reactions such as exacerbation of asthma symptoms, increased respiratory symptoms (e.g. cough, chest distress, wheezing, etc.) and few severe adverse reactions such as atelectasis, pulmonary edema, etc. Furthermore, the bronchofiberscope is limited by the diameter of the tube and cannot penetrate into the tiny airway, so that the BT therapy is difficult to improve the airflow limitation condition of the tiny airway of the patient. Second, BT therapy is limited in the population to which it is suitable, with built-in defibrillators, pacemakers or other implanted devices, bronchoscopy drug allergies (such as lidocaine, atropine and diazepam) and BT therapy prohibited in patients who have previously received BT therapy; the therapeutic effects on asthma patients with a combination of various basic diseases are yet to be studied. Furthermore, BT therapy requires expensive equipment, requires high skill on the part of the operator, and is not universally applicable. For patients, the cost-benefit of BT therapy for treatment of severe and refractory asthma remains to be evaluated, is not in the medical insurance range, has a high self-cost price, and has low patient acceptance; multiple interventional procedures result in poor patient compliance. Therefore, a method which does not need an intervention operation, has low cost, high safety and simple operation and is used for replacing BT therapy to treat severe and refractory allergic asthma is needed to be searched.

Disclosure of Invention

Aiming at the problems, the invention provides a preparation method of a nanoparticle targeting TRPV1, and the prepared nanoparticle can convert light energy into heat energy and selectively kill TRPV1 overexpression cells.

In the process of investigation and research on asthma treatment technology, the inventor finds that although bronchial thermoplasty ablates airway smooth muscle through high temperature generated by radio frequency to relieve airway stenosis and airflow limitation symptoms of asthma patients, patients treated by bronchial thermoplasty have adverse reactions such as postoperative respiratory symptoms increase and asthma symptoms aggravation, and may be related to over-high radio frequency temperature and nonselective killing of tissues except airway smooth muscle. Meanwhile, the current Photothermal therapy (PTT) which is intensively applied to the research of tumor treatment can irradiate a photosensitizer with light of a specific wavelength, convert the absorbed light energy into heat energy to increase the temperature of cells or tissues in a local area, and induce apoptosis. Compared with the radio frequency ablation mode, the photothermal therapy requires lower temperature for obtaining similar treatment effect, and can reduce the toxicity to surrounding tissues by adjusting the position and the intensity of the laser light source, thereby having better safety and less adverse reaction. In addition, the near infrared light dye has strong tissue penetrability and potential for noninvasive treatment, so that the photothermal therapy is hoped to be introduced into the asthma treatment to replace the radio frequency-based bronchial thermoplasty.

Considering that the side effects caused by indiscriminate apoptosis of airway cells by heating are required to be reduced as much as possible in treating asthma by PTT, the targeting of PTT needs to be increased, namely, a suitable therapeutic target is searched. The inventor finds that in the process of treatment and research of asthma patients: TRPV1 is expressed in various cells as a nonselective cation channel and can be activated by various exogenous and endogenous physical and chemical stimuli, such as temperature higher than 43 deg.C, acidic condition, capsaicin, etc. Metabonomic analysis of the serum of patients with mild and severe asthma shows that the metabolic products such as oleoyl ethanolamide, sphingosine-1-phosphate, N-palmitoyl taurine and the like related to the activation of TRPV1 are obviously increased. Gene analysis of a fiber bronchoscopy sample of a severe asthma patient shows that the expression level of TRPV1 of the severe asthma patient is obviously increased in a patient with mild symptoms, and the severe asthma patient also has similar expression in epithelial tissues of a refractory (steroid hormone resistant) asthma patient. Both the above genomic and metabolomic analyses indicate that TRPV1 is overexpressed in the airways of patients with severe and refractory asthma. In addition, the expression of the TRPV1 gene is also significantly up-regulated in airway epithelium and smooth muscle primary cells of asthma patients, and loss of function after mutation of the TRPV1 gene is closely related to reduction of asthma susceptibility of patients, indicating that TRPV1 is a potential target for asthma treatment. Meanwhile, the inventor finds out in the research process that: (1) TRPV1 is closely related to airway inflammation. In the respiratory system, TRPV1 is widely distributed among sensory nerve fibers, particularly on unmyelinated C-type and myelinated a δ -type sensory nerve fibers, accounting for approximately 75% of afferent fibers in the pulmonary branches of the vagus nerve. TRPV1 activation in sensory neurons is associated with calcium influx and depolarization, and acts on respiratory effector cells such as cholinergic neurons, mucous gland cells, inflammatory cells, tracheal and vascular smooth muscles, etc. through tachykinin and calcitonin gene-related peptide (GCRP), resulting in neurogenic inflammation. In asthma, it is manifested as bronchoconstriction, edema of the tracheal mucosa and chemotaxis of inflammatory cells. Acute ablation of sensory neurons of TRPV1 in the vagus nerve ganglion can reduce allergic inflammation and bronchial hyperreactivity. In bronchoscopic radiofrequency ablation therapy, targeted removal of the parasympathetic nerve of the lungs of a patient can reduce airway resistance, mucus secretion and airway inflammation. (2) Studies in the OVA-induced guinea pig asthma model have shown that TRPV1 inhibitors may reduce airway hyperresponsiveness. The TRPV1 siRNA has the function of antagonizing TRPV1, and can relieve IL-13 induced respiratory inflammation, reduce airway hyperreactivity, airway remodeling change and epithelial fibrosis and relieve asthma symptoms of mice after treating OVA and IL-13 induced Balb/c acute asthma mice with TRPV1 siRNA. TRPV1 siRNA also had the same effect in OVA-induced chronic mouse allergy model. In addition to the OVA-induced asthma model, TRPV1 was also shown to be involved in the pathological process of AHR in asthma triggered by the chemical agent toluene 2, 4-diisocyanate (TDI). (3) Airway remodeling is an important pathological feature of asthma and also the pathological basis for irreversible airflow obstruction in asthma. As a major component of the airway wall, proliferation of Airway Smooth Muscle Cells (ASMCs) is one of the major causes of airway remodeling. In the rat asthma model, TRPV1 channel expression was significantly increased in rat airway smooth muscle and improved airway remodeling by affecting airway smooth muscle cell proliferation and apoptotic processes.

TRPV1 is also found widely in airway epithelium, smooth muscle cells, fibroblasts, and non-neural cells such as T cells. Inflammatory mediators such as tumor necrosis factor-alpha, prostaglandin E2, NGF and Interleukins (ILs) can reduce the TRPV1 activation threshold in the cells, improve the sensitivity of the TRPV1 of the organism and participate in the development of asthma.

Therefore, the inventors have selected TRPV1 as a target to increase the targeting of PTT by TRPV1mAb, and based on this, they have tried and tried through trial and error to provide a method for preparing nanoparticles targeting TRPV1, comprising:

preparing nanoparticles with photothermal conversion performance: selecting a block copolymer with surface carboxyl, respectively dissolving the block copolymer and a photosensitizer by using a solvent, mixing, adding into water, homogenizing, removing the solvent, centrifuging, and carrying out heavy suspension precipitation to obtain the photosensitizer;

targeted modification: and activating surface carboxyl of the nanoparticle with the photothermal conversion performance, and incubating the nanoparticle with the TRPV1mAb to covalently connect the TRPV1mAb to the carboxyl of the nanoparticle to obtain the compound.

The nanoparticles obtained by the preparation method can be excited by near infrared light, selectively kill TRPV1 over-expressed cells in bronchial and lung tissues by utilizing the photothermal effect of the nanoparticles, remarkably reduce the infiltration of airway inflammatory cells, inhibit the hyperproliferation of goblet cells and columnar epithelial cells, obviously improve the airflow limitation, and effectively treat allergic asthma.

In one embodiment, in the step of preparing the nanoparticle having the photothermal conversion property, the solvent dissolving the block copolymer is acetonitrile, and the solvent dissolving the photosensitizer is double distilled water. The solvent has better safety and can fully dissolve the material.

In one embodiment, the mass ratio of the block copolymer to the photosensitizer is 30 to 50: 1. By adopting the mass ratio, the copolymer is fully combined with the photosensitizer, and the encapsulation efficiency of the photosensitizer is improved.

In one embodiment, the rotation speed of the homogenization treatment is 20000-25000rpm, and the separation factor of the centrifugation is 20000-25000 g. By adopting the preparation conditions, the size of the dispersion can be reduced, the distribution uniformity of the dispersion can be improved, the photosensitizer can be uniformly mixed, and then the nano-particles with the size meeting the use requirement can be obtained.

In one embodiment, the activation is performed with 0.05-0.15mg/mL of carbodiimide salt. The active agent with the concentration can effectively activate carboxyl groups on the surface of the nano particles.

In one embodiment, the nanoparticle with photothermal conversion properties is incubated with TRPV1mAb for 2-6 hours. By adopting the incubation time, the nanoparticles can be in full contact reaction with the TRPV1mAb, the nanoparticles are in full contact with the TRPV1mAb, and the TRPV1mAb is firmly combined and is not easy to fall off.

In one embodiment, the photosensitizer is ICG. The inventor finds that the photosensitizer applied to the PTT in the research process needs to have the following characteristics: ICG is used as a typical photosensitizer for absorbing Near-infrared light (NIR), and has been used for angiography in operations such as coronary artery bypass surgery, peripheral tissue perfusion in solid organ transplantation and the like; in fluorescence probing of tumor tissue and lymph node boundaries and liver function evaluation in tumor resection, red fluorescence is generated after NIR excitation, and the red fluorescence can be used as an in vivo fluorescence diagnostic and photothermal therapeutic agent, and can generate a small amount of singlet oxygen, has a weak photodynamic effect, and has been approved by the U.S. Food and Drug Administration (FDA) as an optical imaging agent, so the present inventors selected ICG as a photosensitizer, while achieving excellent photothermal conversion efficiency, and enhancing the in vivo safety of the present invention.

In one embodiment, the block copolymer is 40k-5kDa PLGA-PEG-COOH. Polylactic acid-polyglycolic acid (PLGA) is one of biodegradable copolymers, and the polymer can be degraded in vivo through ester bond hydrolysis into anions (lactate and glycolate) and further decomposed into water and carbon dioxide to be removed from the body, so that the polylactic acid-polyglycolic acid copolymer has good biocompatibility; polyethylene glycol (PEG) is also an FDA approved polymer, increasing the hydrophilicity of PLGA and reducing the toxicity and immunogenicity of drug carriers; therefore, the block copolymer formed by PLGA and PEG can improve the stability of ICG, solve the defects that the ICG is easy to combine with plasma albumin, is aggregated in an aqueous medium, has activity easily influenced by conditions such as pH, temperature and the like, is quickly removed in vivo, lacks specificity and the like, and the modified ICG nano-particles have good photothermal effect and treatment effect.

The invention also provides application of the nanoparticle in preparing a medicament for preventing or treating TRPV1 overexpression diseases.

In one embodiment, the TRPV1 overexpression disease is allergic asthma.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a preparation method of nanoparticles of a targeted TRPV1, the nanoparticles obtained by the preparation method increase the in vitro photothermal stability of ICG, have good TRPV1 targeting property, can convert light energy into heat energy through the photothermal effect of the nanoparticles after being excited by near infrared light, selectively kill TRPV1 over-expression cells, further remarkably reduce the infiltration of airway inflammatory cells, inhibit the hyperproliferation of goblet cells and columnar epithelial cells, obviously improve the air flow limitation, and effectively treat allergic asthma.

Drawings

FIG. 1 is a schematic diagram of the synthesis route of PLGA-PEG-ICG-TRPV1mAb photothermal nanoparticles;

FIG. 2 is a transmission electron microscope morphology image, wherein a is PLGA-PEG, b is PLGA-PEG-ICG, c is PLGA-PEG-ICG-TRPV1mAb nanoparticles, scale: 200 nm;

FIG. 3 is a graph showing the average particle size of PLGA-PEG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb nanoparticles;

FIG. 4 is a Zeta potential diagram of PLGA-PEG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb nanoparticles;

FIG. 5 is a diagram of the ultraviolet spectra of ICG, PLGA-PEG-ICG-TRPV1 mAb;

FIG. 6 is a graph showing the evaluation of photothermal effect and photothermal stability in vitro of ICG, PLGA-PEG-ICG, and PLGA-PEG-ICG-TRPV1mAb nanoparticles, wherein the evaluation is stopped after 808nm laser irradiation for 10min (1W/cm2), and the temperature is raised and then returned to room temperature, wherein the evaluation is performed in a cycle of continuously exciting for 5 cycles, comparing the magnitude and speed of temperature rise;

FIG. 7 is a graph showing the comparison of the temperature rise of each group at the same ICG concentration in the in vivo photothermal effect evaluation of PLGA-PEG-ICG-TRPV1mAb nanoparticles;

FIG. 8 is a graph showing the temperature change of mice as the concentration and illumination time of PLGA-PEG-ICG-TRPV1mAb nanoparticles increase in the in vivo photothermal effect evaluation of PLGA-PEG-ICG-TRPV1mAb nanoparticles;

FIG. 9 is a thermal map of in vivo imaging of PLGA-PEG-ICG-TRPV1mAb nanoparticles at the highest temperature after exposure to light in vivo for evaluation of photothermal effects in vivo in mice of each dosing group;

FIG. 10 is a fluorescence microscope image of TRPV1-A549 and Vector-A549 cells in the identification of TRPV1-A549 cells;

FIG. 11 is a schematic of the detection of TRPV1 mRNA levels in cells by RT-qPCR in the identification of TRPV1-A549 cells;

FIG. 12 is a graph showing the detection of TRPV1 protein expression levels in cells by Western Blot in the identification of TRPV1-A549 cells;

FIG. 13 is a fluorescence microscopy image of different cells with different differential uptake of PLGA-PEG-ICG-TRPV1mAb in the evaluation of in vitro targeting of PLGA-PEG-ICG-TRPV1mAb nanoparticles, wherein DAPI is the nucleus, GFP is the green fluorescent protein carried by the cell, and PLGA-PEG-ICG-TRPV1mAb is red dot;

FIG. 14 is a focused fluorescence microscope image of the difference in uptake of three nanoparticles by the PLGA-PEG-ICG-TRPV1mAb nanoparticles in vitro targeting evaluation, in which ICG, PLGA-PEG-ICG, and PLGA-PEG-ICG-TRPV1mAb were incubated with TRPV1 mAb-pretreated TRPV1-A549 cells for 2h, versus TRPV1 mAb-pretreated TRPV1-A549 cells;

FIG. 15 is a focused fluorescence microscope image of the difference in uptake of three nanoparticles by TRPV1-A549 cells observed under a confocal fluorescence microscope after incubating three nanoparticles of 25 μ M ICG, PLGA-PEG-ICG and PLGA-PEG-TRPV 1mAb with TRPV1-A549 cells for 2 hours, respectively, wherein DAPI is the nucleus and GFP is the green fluorescent protein carried by the cells; PLGA-PEG-ICG-TRPV1mAb appeared red dotted;

FIG. 16 is a graph showing the results of measuring the fluorescence intensity of cells by flow analysis of 750nm excitation light after incubating three kinds of nanoparticles of 25. mu.M ICG, PLGA-PEG-ICG, and PLGA-PEG-TRPV 1mAb with TRPV1-A549 cells for 20 minutes, respectively, wherein the horizontal axis represents the number of cells and the vertical axis represents the fluorescence intensity;

FIG. 17 is a graph showing the results of flow-assay of fluorescence intensity of cells incubated with 25 μ M PLGA-PEG-TRPV 1mAb nanoparticles for 10min and 20min with TRPV1-A549 and Vector-A549, respectively;

FIG. 18 is a graph showing the results of different concentrations of ICG, PLGA-PEG-ICG, and PLGA-PEG-ICG-TRPV1mAb nanoparticles on killing of TRPV1-A549 cells;

FIG. 19 is a graph showing the results of different concentrations of ICG, PLGA-PEG-ICG, and PLGA-PEG-ICG-TRPV1mAb nanoparticles on Vector-A549 cell killing;

FIG. 20 is a graph showing the results of killing after incubating three nanoparticles of 25. mu.M ICG, PLGA-PEG-ICG, and PLGA-PEG-TRPV 1mAb with Vector-A549 cells and TRPV1-A549 cells, respectively, for 2 hours;

FIG. 21 is a graph showing the results of killing after incubation of PLGA-PEG-ICG-TRPV1mAb nanoparticles with Vector-A549 cells and TRPV1-A549 cells at concentration gradients of 6.25, 12.5, 25, 50 μ M for 2 hours, respectively;

FIG. 22 is a schematic diagram of experimental mouse grouping and dosing regimens;

FIG. 23 is a schematic representation of the modeling method for OVA-induced allergic asthma mouse model;

figure 24 is a flowchart of asthma mouse treatment;

FIG. 25 is a graph showing the results of immunohistochemical staining, such as H & E, Masson, PAS, etc., on paraffin sections of a normal saline group mouse and a model group mouse, respectively, in the establishment of an OVA-induced mouse allergic asthma model with high expression of TRPV1 gene;

FIG. 26 is a graph showing the results of detecting airway hyperresponsiveness of mice 24h after OVA final challenge by a Buxco noninvasive lung function (PFA) instrument in the establishment of an OVA-induced mouse allergic asthma model with high expression of TRPV1 gene;

FIG. 27 is a graph showing the results of immunohistochemical staining of TRPV1 (green) and α -SMA on frozen sections of a saline group mouse and a model group mouse, respectively, in the establishment of an OVA-induced mouse allergic asthma model in which the TRPV1 gene is highly expressed;

FIG. 28 is a graph of mouse survival;

FIG. 29 is a graph showing the results of H & E, Masson, and PAS staining of asthma mice administered with PLGA-PEG-ICG-TRPV1mAb nanoparticles at concentrations of 62.5, 125, and 250 μm via airway and with physiological saline at the same amount in 0 μm group, administered with light 24;

FIG. 30 is a graph showing the results of measuring airway responsiveness of mice 24 hours after light irradiation by a Buxco noninvasive lung function apparatus;

FIG. 31 is a graph showing the results of H & E, Masson, and PAS staining of asthmatic mice administered with 125 μm PLGA-PEG-ICG-TRPV1mAb nanoparticles and dexamethasone via airway, respectively, and control group of normal saline, administered with 24 light;

FIG. 32 is a graph showing the results of measuring airway responsiveness of mice 24 hours after different treatments by Buxco noninvasive pulmonary function;

FIG. 33 is a graph of asthma mice administered with 125 μm of the same concentration of ICG, PLGA-PEG-ICG, and PLGA-PEG-ICG-TRPV1mAb nanoparticles via the airways, and 0 μm of the same amount of saline. Dosing, lighting 24 parts of the materials, and taking the materials, and performing H & E, Masson and PAS dyeing to obtain a result graph;

FIG. 34 is a graph showing the results of measuring airway responsiveness of mice 24 hours after different treatments by Buxco noninvasive pulmonary function;

FIG. 35 is a graph showing the results of TRPV1 (green) and α -SMA immunofluorescence staining of frozen sections of various groups of mice.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Defining:

the photosensitizer of the invention: the material is irradiated by light, photon energy interacts with crystal lattices, vibration is intensified, and temperature is increased.

TRPV1 mAb: the monoclonal antibody of TRPV1 is a specific antibody aiming at TRPV1 epitope.

ICG: indocyanine green is a near-infrared fluorescent dye, has good tissue penetrability and high in-vivo safety, and is widely used for research of photothermal therapy.

PLGA: the polylactic acid-glycolic acid copolymer is formed by random polymerization of two monomers, namely lactic acid and glycolic acid, is a degradable functional polymer organic compound, and has good biocompatibility, no toxicity and good encapsulation and film forming performances.

And (3) reagent sources:

PLGA-PEG-COOH (40k-5kDa, Shanghai Bidi Biotech Co., Ltd.), carbodiimide salt (Thermo), ICG (Sigma), TRPV1mAb (Abstract), acetonitrile (Shanghai Chemicals Co., Ltd.), dimethyl sulfoxide (Shanghai Chemicals Co., Ltd.), disodium hydrogen phosphate (national drug group Chemicals Co., Ltd.), fetal bovine serum (Gibco), DMEM high-sugar medium (Gibco), trypsin (Thermo), PBS (Thermo), plasmid DNA Mass extraction kit (Tiangen), Lipofite (Hanbio Biotechnology), Ploylene (Solebao), puromycin (Solebao), RNA Rapid extraction kit (Yichen Biotech), One-Step-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech), Green qPCR Mierx (Trans Biotech), Gengel preparation kit (French Bioluminescence solution), Biochemical luminescence kit (ECVa), protein Synthesis kit (Advanta Biotechnology), BCA Biotechnology (Biotech), BCA Biotech), and BCA, Vanilloid R1/TRPV1 Antibody (Novus), GAPDH Monoclonal Antibody (Proteintech), Goat Anti-mouse IgG H & L (HRP) pre-adsorbed secondary Antibody (Abcam), Goat Anti-Rabbit IgG (H + L) (HRP Bioworld), BCA (Sigma), 20 × TBS (Solaibao), Tween 20(Sangon Biotech), running buffer (Biyunyyunyan), electrotransfer buffer (Biyunyan), methanol (national drug), Counting Kit-8(CCK-8, APEBiceIO), physiological saline (Shandong Duyu pharmaceutical Co., Ltd.), sodium pentobarbital (Sigma, American can), Valbumin Sigma), project Alum (Thermo), LPS (O55: B5E. coli, dexamethasone (Sigma), 4% Paracetal (Servicol), sucrose (Subtol), Chemicals Co., Ltd.), and reagent for chemical encapsulation of Chemicals, Inc. (Chemicals group Co., Ltd.), and reagent for chemical encapsulation of Zealand group (Chemicals Co., Ltd.), and reagent for chemical encapsulation of God HE staining kit (Servicebio), Masson staining kit (Servicebio), PAS staining kit (Servicebio), neutral gum (national drug group chemical agents limited), 10% Anti-goat serum (Boster), 20xDPBS (solibao), Vanilloid R1/TRPV1 Antibody (Novus), Alpha-smoothen active Antibody (Novus), Anti-mouse IgG (H + L), F (ab ')2fragment (cst), Anti-rabbitigg (H + L), F (ab')2fragment (cst), Anti-fluorescence quencher (laibao), slide glass (serviceo), injection aluminum solution (heat specimen), and injection aluminum solution (blood specimen (poison).

Material sources are as follows:

human non-small cell lung carcinoma cell a549 cell (commercially available), human embryonic kidney cell HEK 293T cell (commercially available), escherichia coli strain DH 5-alpha (commercially available), lentiviral packaging system (pSPAX2, pMD2G and shuttle plasmid, commercially available), 3-5 week old SPF-grade female Balb/c mice (central experimental animals in eastern school district of middle school university).

Equipment source:

SB20001 precision electronic balance (Shanghai Huyue science apparatus Co., Ltd.), T10 homogenizer (Germany IKA), model 752 ultraviolet spectrophotometer (Shanghai optical apparatus Co., Ltd.), L-90K ultra-high speed centrifuge (Beckman Co., USA), Mastersizer 3000 laser particle sizer (Marvin apparatus Co., Ltd., England), infrared imager (Fluke, USA), 808nm laser emitter (Shaanxi Kaiser electronics technology Co., Ltd.), ultrapure water system (Millipore), 4 ℃ refrigerator (Meiling), electronic balance (Saedolis), autoclave (Zealway Instrument Inc), carbon dioxide incubator (Thermo Fisher Scientific), ultra-clean bench (Suzhou purification), biosafety cabinet (Taishida), liquid nitrogen tank (Thermo Fisher Scientific), -20 ℃ refrigerator (Meiling), -80 ℃ refrigerator (Meiling), constant temperature water bath (park), Inverted fluorescence microscope (Nikon), confocal laser microscope (Olympus), flow cytometer (analytical, Beckman), nucleic acid micro-positioning instrument (Thermo), real-time quantitative PCR instrument (Roche), PCR instrument (Bio-Rad), all-band microplate reader (Bio-tek), chemiluminescence instrument (General Electric), ice maker (Panasonic), 8-channel manual range-adjustable pipettor (Thermo), Electric pipettor (Thermo), horizontal temperature-controlled shaker (beijing hardto), centrifuge (xiang instrument), 37 ℃ constant temperature incubator (fine macro), constant temperature water bath (Yi Heng), super clean bench (sujing), ultraviolet imager (sky energy), nucleic acid electrophoresis instrument (sky energy), mouse laryngoscope (shanghai jade scientific instrument limited), intratracheal micro-atomization drug delivery device (shanghai jade scientific instrument limited), tracheal platform (shanghai jade research instrument limited), and intubation, Ultrasonic atomizer 402AI (jiangsu yuejie medical equipment gmbh), Dehydrator (DIAPATH), embedding machine (wuhan junjie electronics gmbh), pathological section cutter (shanghai laika instruments gmbh), freezing table (wuhan junjie electronics gmbh), tissue slide machine (kedi instruments gmbh, jinjiang), oven (striki instruments gmbh, tianjin), freezing section cutter (Thermo), slicing knife (shanghai laika instruments gmbh), grouping pen (Servicebio), inverted fluorescence microscope (Nikon), laser confocal microscope (Olympus), and whole-body plethysmography detection system (Buxco).

Reagents and materials used in the present example are all commercially available sources unless otherwise specified; unless otherwise specified, all the experimental methods are routine in the art.

Example 1

PLGA-PEG-ICG-TRPV1mAb photothermal nanoparticles were synthesized.

1. And preparing PLGA-PEG-ICG nano particles.

Dissolving 10mg of PLGA-PEG-COOH in 1mL of acetonitrile under the condition of warm bath (30-40 ℃); ICG was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/mL. Adding 25 mu L of ICG solution into the 1mL of PLGA-PEG acetonitrile solution, shaking up to form a mixed solution. Then, the mixed solution was added dropwise to 5mL of pure water, and the IKA-T10 homogenizer was homogenized at 22000rpm for 30 s; the organic reagent was removed by evaporation with stirring overnight. After a high-speed centrifuge centrifuger is used for centrifuging at 22000g for 5min, pure water is used for resuspending and precipitating, and ICG-PLGA-PEG nanoparticles are obtained. The same method was used to prepare unloaded PLGA-PEG nanoparticles as control particles.

2. Preparation of PLGA-PEG-ICG-TRPV1mAb nanoparticles.

Adjusting the pH value of the ICG-PLGA-PEG nanoparticle solution to 6.0 by using a disodium hydrogen phosphate solution, adding carbodiimide salt (EDC) to activate carboxyl groups on the surfaces of the nanoparticles at a concentration of 0.1mg/mL, and reacting for 2 hours; and incubating with the TRPV1mAb for 4h, removing unconnected antibodies through a Sephadex-25 gel column to obtain TRPV1mAb modified ICG nanoparticles, concentrating the nanoparticle solution through an ultrafiltration centrifugal column, and storing at 4 ℃ for later use.

The invention adopts the experimental method to wrap ICG in PLGA-PEG high molecular material to form PLGA-PEG-ICG nano particles with self-assembled hydrophilic end and hydrophobic end, and the PLGA carboxyl end of the PLGA-PEG-ICG nano particles is connected with the amino end of TRPV1mAb to obtain the PLGA-PEG-ICG-TRPV1mAb nano particles, and the reaction process is shown in figure 1.

Example 2

PLGA-PEG-ICG-TRPV1mAb photothermal nanoparticles were characterized.

1. Particle size and Zeta potential measurements.

Under the condition of room temperature, 50 times of distilled water is used for dissolving and diluting ICG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb nanoparticle solutions, the solutions are added into a quartz dish, and the particle size distribution and the Zeta potential of a sample are respectively detected by a Malvern Mastersizer 3000 laser particle sizer.

2. The PLGA-PEG-ICG-TRPV1mAb nanoparticles were identified by transmission electron microscopy.

And (3) taking 10 mu L of each nanoparticle sample diluted by 50 times, dropwise adding the nanoparticle samples on the surface of the copper mesh, and observing the appearance of the nanoparticles under a 120kV transmission electron microscope.

3. And the ultraviolet absorption spectrum of PLGA-PEG-ICG-TRPV1mAb is verified.

At room temperature, 50 times of distilled water is used for dissolving and diluting the ICG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb nanoparticle solution, the solution is added into a quartz dish, and the 200-ion 1000nm full-wavelength scanning is carried out by an ultraviolet spectrophotometer.

4. And verifying the photothermal effect of the PLGA-PEG-ICG-TRPV1 mAb.

Taking 100 μ L of each 25 μ M ICG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb nanoparticle solutions in an EP tube, and irradiating with 808nm laser emitter (1W/cm) at room temperature²) Irradiating for 10min, taking the temperature rise and room temperature recovery as a cycle, and recording the temperature change by an infrared thermometer for 5 cycles in total.

5. And (6) analyzing the data.

The data obtained were analyzed using GraphPad Prism 8 software and the statistics were expressed as Mean ± standard error (Mean ± SEM), t-test for two-group comparisons and 2way ANOVA for multiple-group comparisons, with P <0.05 having statistical significance, # P <0.05, # P <0.01, # P < 0.001.

The structure of the nanoparticles was characterized by particle size, Zeta potential and transmission electron microscopy. TEM image of TEM is shown in FIG. 2, which shows that PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb are dispersed round particles. The average particle size and Zeta potential of PLGA-PEG, PLGA-PEG-ICG-TRPV1mAb nanoparticles were measured by Dynamic Light Scattering (DLS), and the average particle size was as shown in FIG. 3, showing that the average particle size of PLGA-PEG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb nanoparticles was 144.80 + -1.37 nm, 162.36 + -1.19 nm, 176.93 + -0.95 nm in this order, where n is 3; the Zeta potential is shown in figure 4, and the Zeta potential is-33.13 +/-0.15 mV, -28.7 +/-0.3 mV, -23.9 +/-0.62 mV, and n is 3. Research data show that after TRPV1mAb modification, PLGA-PEG-ICG has increased particle size and reduced electronegativity. The ultraviolet spectrum full-wavelength scanning is shown in FIG. 5, and the results show that the PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb nanoparticles have absorption peaks with the same shape at the characteristic absorption peak of ICG, which indicates that the PLGA-PEG-ICG-TRPV1mAb nanoparticles are successfully synthesized.

Example 3

And detecting the encapsulation efficiency of the ICG of the PLGA-PEG-ICG-TRPV1mAb nano-particles.

The wavelength lambda max of the maximum absorption peak of the ICG acetonitrile solution is 778nm measured by ultraviolet spectrophotometry. According to the absorbance of different concentrations (0.5, 1, 2,4 and 8pg/mL) of the free ICG at the lambda max of 778nm, a concentration-absorbance related standard curve y is established as 0.3474x +0.1709 (in the range of 0-8 pg/mL). Thus calculating the drug content of the ICG nano-particle after the dissolution of the acetonitrile. By the formula: the ICG encapsulation rate in the nanoparticles is that the ICG dosage is wrapped by the nanoparticles/the total amount of ICG added for preparing the nanoparticles is multiplied by 100 percent. The encapsulation efficiency of the ICG-PLGA-PEG-TRPV1 and ICG-PLGA-PEG nanoparticles was calculated to be 58.0% and 60.2%, respectively, and the results are shown in the following table.

TABLE 1 encapsulation efficiency of nanoparticles

Example 4

And evaluating the photothermal effect of the PLGA-PEG-ICG-TRPV1mAb nanoparticles.

In order to investigate whether the PLGA-PEG high polymer material wrapping and TRPV1 targeting modification influence the photothermal property of the ICG, the invention carries out in-vitro and in-vivo photothermal effect evaluation and photothermal stability evaluation on the ICG, the PLGA-PEG-ICG and the PLGA-PEG-ICG-TRPV1mAb nanoparticles.

1. And evaluating the in vitro photothermal effect of the PLGA-PEG-ICG-TRPV1mAb nanoparticles.

(1) The invention evaluates whether the photo-thermal effect of the ICG is changed by the modification of the PLGA-PEG and TRPV1mAb by exploring the temperature change of the nano-particle solution of the ICG, the PLGA-PEG-ICG and the PLGA-PEG-ICG-TRPV1mAb with the same concentration in the irradiation process of 808nm laser (1W/cm 2).

The results show that: the PLGA-PEG-ICG-TRPV1mAb nanoparticles have the largest and fastest heating amplitude, and the PLGA-PEG-ICG has larger heating amplitude than the ICG, which shows that the nanoparticles modified by PLGA-PEG and TRPV1mAb can enhance the photothermal effect of the ICG.

(2) The nano particles are irradiated by near infrared light for 10min and then return to room temperature for laser switching circulation. In the invention, ICG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb are placed in 5 continuous laser switch cycles to evaluate the photo-thermal stability.

The results show that: as shown in FIG. 6, 25 μm PLGA-PEG-ICG-TRPV1mAb nanoparticle solution was able to stably increase the temperature to above 40 ℃ in 5 cycles, and the highest temperature of PLGA-PEG-ICG and ICG with the same concentration was below 40 ℃, indicating that PLGA-PEG-ICG-TRPV1mAb nanoparticles have good in vitro photo-thermal stability.

2. Evaluation of photothermal Effect of PLGA-PEG-ICG-TRPV1mAb nanoparticles in vivo.

Next, the present invention investigated whether PLGA-PEG-ICG-TRPV1mAb nanoparticles could produce better photothermal effects in vivo. Asthmatic mice were divided into 4 groups, in which three groups were administered 125 μm of ICG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb nanoparticles, respectively, and the control group was administered an equivalent physiological saline group (0 μm group). The normal saline and all materials are administrated in an airway atomization mode, and before administration, an asthma mouse is anesthetized by 1% sodium pentobarbital. After 15min of administration, the mixture is treated by 808nm laser at 2W/cm²The intensity of the infrared thermometer is used for irradiating the lung and the bronchus of the mouse for 10min, the body surface temperature of the mouse is measured and recorded by a handheld infrared temperature measurer, and the temperature change of the mouse within 10min is compared. The in vivo photothermal effects of ICG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb were compared by the maximum magnitude of temperature increase in mice of each dosing group.

The results show that: as shown in fig. 7, the temperature increase amplitude of the administered group was significantly increased compared to the normal saline group, indicating that all three nanoparticles have good photothermal effect. Wherein, the temperature rise range of PLGA-PEG-ICG-TRPV1mAb reaches 12 ℃, ICG is 7 ℃, and the difference is more obvious, which shows that PLGA-PEG-ICG-TRPV1mAb has better in vivo photothermal effect.

In order to understand the effect of PLGA-PEG-ICG-TRPV1mAb nanoparticle concentration on photothermal effect in vivo, PLGA-PEG-ICG-TRPV1mAb nanoparticles with concentration of 62.5, 125, 250, 500 μ M respectively are irradiated by laser after being administered to mouse airway, and temperature change (Δ T) of the mouse within 10 minutes is compared with 0 μ M group airway nebulization physiological saline. The mice were divided into 5 groups of 6 mice each.

The results show that: as shown in fig. 8 and 9, the body temperature of the mice continuously increased with the increase of the light irradiation time and concentration. Compared with the normal saline group (0 μm), the body temperature rise of the mice of the 62.5 μm treatment group is not significantly different, and the body temperature of the mice of the 125, 250 and 500 μm treatment groups is significantly increased, which indicates that the 62.5 μm is most likely to be the lowest effective dose in the mouse experiment. There was no significant difference in the temperature rise of the mice compared to the 500 μ M treatment group at 250 μ M. Suggesting that 250 μ M may be the optimal effective dose for asthmatic mice

Example 5

PLGA-PEG-ICG-TRPV1mAb targeting study in vitro.

1. And (3) constructing a lentivirus plasmid.

Through sequencing comparison verification, pHBLV-h-TRPV1-3 XFLAG-ZsGreen-PURO slow virus plasmid and pHBLV-ZsGreen-PURO over-expression control slow virus plasmid are constructed. Amplified by Escherichia coli strain DH 5-alpha, extracted by plasmid mass extraction kit, and used for packaging and purifying lentivirus.

2. And (5) packaging lentivirus, and concentrating and purifying.

(1)293T cells were passaged in 100mm culture dishes and transfected at a density of 70-80%.

(2) Mixing 10 μ g pSPAX2, 5 μ g pMD2G, 10 μ g lentiviral plasmid, and 75 μ g LLIPOFITER TM, incubating at room temperature for 15min, slowly adding dropwise into 293T cells, and adding 5% CO at 37 deg.C₂Culturing in a cell culture box.

(3) Fresh complete medium containing 10% fetal bovine serum FBS was changed 16h after transfection and two virus supernatants were collected at 48h and 72h post transfection, respectively.

(4) Ultracentrifugation: centrifuging the virus supernatant in a 50mL centrifuge tube at 2000g and 4 ℃ for 10min to remove cell debris; the virus stock supernatant was collected and placed in an ultracentrifuge tube and centrifuged at 82700g for 120min at 4 ℃. Resuspend the viral pellet with complete medium and dispense the supercentrifugal resuspension into sterilized viral tubes.

(5) The dilution count assay monitors lentivirus titers.

3. Infection of A549 cells.

(1) A549 cells are subcultured in a 100mm culture dish, when the cell density is 40-50%, TRPV1-GFP overexpression lentivirus and control Vector lentivirus are respectively added into corresponding cells, Polybrene with the final concentration of 5 mu g/mL is added into the cells, and the cells are gently shaken.

(2) After the cells are infected for 24 hours, the solution is changed, fresh DMEM high-sugar medium containing 10% FBS and 1% double antibody is added, and the cells are placed into an incubator for continuous culture.

(3) After 24h of incubation, puromycin was added to a final concentration of 2. mu.g/mL for selection. When most of cells without virus infection die, the screening is stopped, the cells are changed into maintenance culture containing puromycin of 1 mu g/mL and the screening is continued, and A549 cells (TRPV1-A549) with high TRPV1 expression and control Vector A549 cells (Vector-A549) are obtained.

4. RNA extraction, reverse transcription and real-time quantitative PCR.

(1) And extracting total RNA in the cells.

After the cultured TRPV1-A549 cells and Vector-A549 cells are digested, according to the steps in the RNA rapid extraction kit, the total RNA in the cells is extracted from 1 hole of a 6-hole plate through the steps of cracking, column loading/RNA combination, column cleaning, RNA elution and the like.

RNA concentration was determined using a Nanodrop, dispensed into several enzyme-free EP tubes, and stored in a-80 ℃ freezer after recording the concentration at the tube wall.

(2) Reverse transcription.

Based on the measured RNA concentration, the volume required for reverse transcription of 1. mu.g of RNA was added to the enzyme-free EP tube and the corresponding reverse transcription reagent was added in a 20. mu.L system. After mixing, the mixture was placed in a PCR instrument for reverse transcription. The reaction parameters were set as follows: the reaction was stopped at 42 ℃ for 15min, 85 ℃ for 5s and 4 ℃. The cDNA was stored at-20 ℃.

(3) And (3) fluorescent quantitative PCR.

A qPCR system was prepared according to the following table and added to the eight-tube, with three secondary wells per sample.

TABLE 2 qPCR reaction System

After the mixture was centrifuged at low speed and mixed uniformly, the octaplexed tubes were placed in a qPCR instrument with the reaction parameters set as follows: preheating for 30s at 95 ℃; amplifying 45 cycles by a 3-step method (denaturation at 95 ℃ for 10s, renaturation at 60 ℃ for 60s and denaturation at 97 ℃ for 1 s); cooling at 37 deg.C for 30 s. The primer sequences are shown in the following table.

TABLE 3 primer sequences

5. Western Blot analysis of TRPV 1.

(1) Extracting total cell protein and measuring the concentration.

After digestion of TRPV1-A549 cells and Vector-A549 cells, a small amount of lysis buffer and protease inhibitor are added, repeated beating is carried out, ice is kept still for 20min, 14000r is centrifuged for 30min, and total protein in cells is extracted.

Protein concentration by BCA method: operating according to the instructions. Briefly, absorbance at 562nm per well was determined and intracellular protein concentration was calculated against a standard curve.

(2) Western Blot procedure.

Preparing glue: a Friedel one-step gel preparation kit is adopted to prepare the separation gel with the concentration of 8 percent and the concentrated gel. And adding the separation glue and the concentrated glue into the clamped glass plate respectively, inserting a comb, and standing at room temperature until the gel is solid.

Protein denaturation: the volume required was calculated from 30. mu.g of the sample, 5 XLoading Buffer was added and the mixture was left in a 100 ℃ water bath for 15min to denature the protein.

Electrophoresis: after the gel is solidified, the glass plate is placed in an electrophoresis tank, electrophoresis buffer solution is added, and the comb is taken down. 20 mu L of TRPV1-A549 protein solution, Vector-A549 protein solution and 4 mu L of protein Marker are respectively added into the sample adding hole. The electrophoresis apparatus is adjusted, the voltage is kept constant at 200V, and the electrophoresis is stopped when the band runs to the bottom of bromophenol blue.

Film transfer: the gel was carefully removed and soaked in the electrotransfer solution. The PVDF membrane is activated by soaking in methanol for 5 min. The electric transfer is carried out by adopting a classic sandwich method, the spongy cushion, the filter paper, the glue and the PVDF membrane are sequentially placed and then air bubbles are discharged, the plastic clamping plate is clamped tightly, and the plastic clamping plate is placed into an electric transfer groove filled with a transfer membrane buffer solution according to the correct direction. The electric rotary tank is arranged in an ice box, the constant current is 300mA, and the film is rotated for 1.5 h.

After the membrane conversion is finished, the power supply is cut off, the PVDF membrane is carefully taken out, placed in 5% skimmed milk powder sealing liquid and sealed for 1 hour at room temperature. The membrane was washed 3 times with TBST buffer for 10min each.

Membrane shearing is performed according to protein positions. Each membrane was placed in a 1:1000 dilution of the TRPV1 and GAPDH primary antibody and incubated overnight in a shaker at 4 ℃.

The membrane was washed 3 times with TBST buffer for 10min each. Each segment is associated with a segment with a 1: 5000 dilution of secondary antibody was incubated for 1h at room temperature. The membrane was washed 3 times with TBST buffer for 10min each. The membrane was immersed in a chemiluminescent solution (solution a: solution B: 1 configuration) and developed using a fully automated chemiluminescent image analyzer.

6. And (3) observing the uptake condition of the cells to the nanoparticles under a laser confocal microscope.

(1) Plate paving: TRPV1-A549 and Vector-A549 cells were digested with 0.25% trypsin, and the number of cells was 5X 10⁵One/well was inoculated in a confocal dish and cultured overnight.

(2) Administration: mu.M of the ICG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb nanoparticle solutions were added to TRPV1-A549 and Vector-A549, respectively, at 1mL per confocal dish.

(3) And (3) incubation: incubating for 2h, washing off the redundant medicines by PBS,

(4) dyeing the core: the Hoechst viable cell dye (10. mu.g/mL) was added to the cells, stained for 15min, and washed 2 times with PBS for 5min each.

(5) And (4) observation: red fluorescence in TRPV1-A549 and Vector-A549 cells was observed at 780nm of a confocal fluorescence microscope.

7. Flow cytometry was used to analyze the uptake of nanoparticles by cells.

(1) Plate paving: TRPV1-A549 and Vector-A549 cells were digested with 0.25% trypsin, and the number of cells was 4X 10⁵One/well was inoculated in 24-well plates and cultured overnight.

(2) Administration: mu.M of the ICG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb nanoparticle solutions were added to TRPV1-A549 and Vector-A549, 500. mu.L each, respectively.

(3) And (3) incubation: each well was incubated with time gradient in dark for 10min, 20min, 30min, respectively.

(4) Flow quantitative analysis: and washing away redundant medicines by PBS, digesting by pancreatin, washing for 1-2 times by PBS, and then carrying out flow quantitative analysis. The average fluorescence intensity at 780nm was compared.

8. CCK-8 experiment for detecting influence of nanoparticles on cell activity

(1) TRPV1-A549 and Vector-A549 cells were digested with 0.25% trypsin, and the number of cells was 5X 10³One/well was inoculated in 96-well plates and cultured overnight.

(2) ICG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb nanoparticle solutions with a mother liquor concentration of 1mM were diluted with serum-free DMEM high-sugar medium at equal ratios of concentrations of 0, 6.25, 12.5, 25, 50 μm. The diluted solution is added into TRPV1-A549 and Vector-A549 respectively, each well is 100 mu L, and the mixture is incubated for 2h in a dark place.

(3) After incubation, cells were washed 3 times with PBS to remove unabsorbed nanoparticles.

(4) 808nm laser (1W/cm) per well of cells²) Irradiating for 10 min. After illumination, incubation was continued for 24 h.

(5) After medium replacement, 10. mu.L of CCK-8 solution was added to each well, and the incubators were incubated for 1h in the dark.

(6) The absorbance at a wavelength of 450nm was measured using a microplate reader.

(7) The average absorbance of the experimental group (n-4) and the control group (n-4) were compared to calculate cytotoxicity. The calculation formula of the cell viability is as follows:

cell viability (%) - [ a (dosed) -a (blank) ]/[ a (not dosed) -a (blank) ] × 100%;

a (dosing): has OD values of cells, CCK-8 solution and drug solution wells;

a (no drug added): OD of wells with cells, CCK-8 solution and no drug solution;

a (blank): OD values without wells.

9. And (6) analyzing the data.

10. And (6) analyzing results.

(1) Verification of A549 cells (TRPV1-A549) with high expression of TRPV1 gene.

In order to verify whether the synthesized PLGA-PEG-ICG-TRPV1mAb has in vitro targeting, the invention constructs an A549 cell line (TRPV1-A549) with high expression of TRPV1 gene with green fluorescence and a control Vector-A549 cell line, and verifies the cell line from mRNA and protein levels by RT-qPCR and Western Blot.

TRPV1-A549 and Vector-A549 cells fluoresced green under a fluorescence microscope, and the results are shown in FIG. 10.

TRPV1 mRNA levels, n-3, P <0.001 in cells were detected by RT-qPCR, and the results are shown in figure 11.

The expression level of TRPV1 protein in cells was measured by Western Blot, and the results are shown in fig. 12.

(2) The PLGA-PEG-ICG-TRPV1mAb nanoparticles have TRPV1 receptor targeting.

After successfully constructing the A549 cell over-expressed by the TRPV1 and the PLGA-PEG-ICG nanoparticle connected with the TRPV1mAb, the invention verifies whether the PLGA-PEG-ICG-TRPV1mAb nanoparticle interacts with the TRPV1 ion channel receptor on the surface of the target cell.

The TRPV1mAb specifically binds to the cell surface TRPV1 receptor, binding of which occupies the site of action of other signaling molecules targeting the TRPV1 receptor. TRPV1-A549 cells were pre-treated with TRPV1mAb for 4 hours, and TRPV1-A549 cells, TRPV1-A549 cells and Vector-A549 cells pre-treated with TRPV1mAb were incubated with PLGA-PEG-ICG-TRPV1mAb nanoparticles for 2 hours, respectively. After the nanoparticles combined with the cells are eluted, the uptake of PLGA-PEG-ICG-TRPV1mAb nanoparticles by three cells is observed under a laser confocal microscope. After the PLGA-PEG-ICG-TRPV1mAb nano-particles are excited by 780nm laser, the shape of the nano-particles is in a small red dot shape under a confocal microscope. Both TRPV1-A549 cells and Vector-A549 cells carry GFP fluorescent protein tags.

25 mu M PLGA-PEG-TRPV 1mAb was incubated with TRPV1-A549, TRPV1-A549 cells pretreated with TRPV1mAb, and Vector-A549 for 2h, and the difference in the uptake of PLGA-PEG-ICG-TRPV1mAb by different cells was observed under a confocal fluorescence microscope, the results are shown in FIG. 13.

ICG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb and TRPV1-A549 cells pretreated by TRPV1mAb were incubated for 2h, and the difference of the uptake of three nanoparticles by TRPV1-A549 cells pretreated by TRPV1mAb was compared, the results are shown in FIG. 14.

Through laser confocal microscope observation, the PLGA-PEG-ICG-TRPV1mAb nanoparticles can not be combined with TRPV1-A549 cells pretreated by the TRPV1mAb, and the ingestion of ICG and PLGA-PEG-ICG is not influenced, which indicates that the PLGA-PEG-ICG-TRPV1mAb nanoparticles have the targeting property of TRPV1 receptors.

For Vector-A549, TRPV1 is expressed on the cell membrane of Vector-A549 as a cation channel protein in a small amount, compared with the intracellular large-amount uptake of ICG and PLGA-PEG-ICG nanoparticles, the PLGA-PEG-ICG-TRPV1mAb nanoparticles are distributed around the cell membrane in a small amount and do not enter cytoplasm, and the result shows that the PLGA-PEG-ICG-TRPV1mAb has cell membrane protein TRPV1 receptor targeting.

(3) TRPV1-A549 cells exhibited differences in uptake of ICG, PLGA-PEG-ICG, and PLGA-PEG-ICG-TRPV1mAb nanoparticles.

Through confocal laser microscopy, the TRPV1-A549 cells take up ICG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb nanoparticles within 2 hours. This almost non-selective uptake is presumably due to the long incubation time of the nanoparticles with the cells, where the nanoparticles enter the cells mainly by non-specific endocytosis rather than by specific binding to cell membrane surface receptors. Therefore, the invention shortens the incubation time of the cells and the nanoparticles, and analyzes the change of the average fluorescence intensity of the cells in different times by flow cytometry.

When the incubation time of the nanoparticles and the TRPV1-A549 cells is less than 30 minutes, compared with ICG and PLGA-PEG-ICG, the PLGA-PEG-ICG-TRPV1mAb nanoparticle treatment group has stronger fluorescence, which indicates that the TRPV1-A549 cells take more PLGA-PEG-ICG-TRPV1mAb nanoparticles than the ICG and PLGA-PEG-ICG treatment group, and have selective uptake characteristics; after the PLGA-PEG-ICG-TRPV1mAb nanoparticle solution is respectively incubated with TRPV1-A549 cells and Vector-A549 cells for 10min and 20min to compare fluorescence intensity, it is found that the PLGA-PEG-ICG-TRPV1mAb nanoparticles taken by the Vector-A549 cells are far less than that taken by the TRPV1-A549 cells within the time points of 10min and 20min, and the PLGA-PEG-ICG-TRPV1mAb nanoparticles have targeting characteristics.

The ingestion experiment provides in vitro data support for the interval time between the animal administration and the illumination in the in vivo experiment.

Respectively incubating three nanoparticles of 25 mu M ICG, PLGA-PEG-ICG and PLGA-PEG-TRPV 1mAb with TRPV1-A549 cells for 2 hours, and observing the uptake difference of the three nanoparticles by the TRPV1-A549 cells under a confocal fluorescence microscope, wherein the results are shown in FIG. 15;

after incubating three nanoparticles of 25. mu.M ICG, PLGA-PEG-ICG and PLGA-PEG-TRPV 1mAb with TRPV1-A549 cells for 20 minutes, the fluorescence intensity of the cells was measured by flow analysis of 750nm excitation light, and the results are shown in FIG. 16;

25 mu.M PLGA-PEG-TRPV 1mAb nanoparticles were incubated with TRPV1-A549 and Vector-A549 for 10min and 20min, respectively, and then the fluorescence intensity of the cells was measured by flow method, the results are shown in FIG. 17.

(4) The PLGA-PEG-ICG-TRPV1mAb nanoparticles can specifically kill TRPV 1-A549.

After the PLGA-PEG-ICG-TRPV1mAb nanoparticles are verified to have TRPV1 receptor targeting property and can be specifically absorbed by TRPV1-A549 highly expressed by TRPV1 protein, the invention verifies the targeting property of the nanoparticles in vitro.

The ICG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb nanoparticle solutions were diluted at equal ratios of 6.25, 12.5, 25, 50. mu.M, incubated for 2 hours in TRPV1-A549 cells and Vector-A549 cells, respectively, and then irradiated at 808nm with 2W/cm laser²The laser intensity of the three types of nanoparticles is irradiated for 10min, and the influence of the three types of nanoparticles on the activities of two types of cells is determined through a CCK-8 experiment. As a result, the nanoparticle solutions of ICG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb with different concentrations have different degrees of toxicity on TRPV1-A549 cells, and the toxicity is increased along with the increase of the concentration; the toxicity of ICG and PLGA-PEG-ICG to Vector cells increased with increasing concentration, but PLGA-PEG-ICG-TRPV1 was almost non-toxic to Vector-A549, and the concentration increased and brought about significant cytotoxicity.

Then 25 μm was selected to further compare the toxic effects of ICG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb on TRPV1-A549 cells and Vector-A549 cells. The administration treatment was as described previously. Compared with a Vector-A549 cell, the TRPV1-A549 cell highly expressed by the TRPV1 gene is more sensitive to three nanoparticles, and the killing effect of the three nanoparticles on the TRPV1-A549 cell is obviously stronger than that of the Vector-A549 cell. The killing effect of PLGA-PEG-ICG-TRPV1mAb on Vector-A549 cells is not significant, and the killing effect on TRPV1-A549 cells is significant at each concentration. Thus, it can be concluded that: the PLGA-PEG-ICG-TRPV1mAb has the function of selectively killing TRPV1 high expression cells, and the in vitro targeting property of the PLGA-PEG-ICG-TRPV1mAb nanoparticles is further verified.

The killing effect of ICG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb nanoparticles on cells at different concentrations in TRPV1-A549 cells (A) and Vector-A549 cells (B) is shown in FIG. 18 and FIG. 19;

three kinds of nanoparticles of 25 mu M ICG, PLGA-PEG-ICG and PLGA-PEG-TRPV 1mAb are respectively incubated with Vector-A549 cells and TRPV1-A549 cells for 2 hours, compared with the Vector-A549 cells, the killing effect of the three kinds of nanoparticles on the TRPV1-A549 cells is obviously improved, and the result is shown in figure 20;

PLGA-PEG-ICG-TRPV1mAb nanoparticles were incubated with Vector-A549 cells and TRPV1-A549 cells at concentration gradients of 6.25, 12.5, 25, 50 μ M for 2 hours, and PLGA-PEG-ICG-TRPV1mAb nanoparticles at different concentrations all had selective killing effects on TRPV1-A549, with the results shown in FIG. 21.

Therefore, through lentivirus infection, the invention successfully constructs a TRPV1-A549 cell line with high TRPV1 gene expression and an unloaded Vector-A549 cell line, and verifies that the PLGA-PEG-ICG-TRPV1mAb nanoparticle has in vitro targeting property through the differential uptake of the TRPV1 high-expression A549 cell, the TRPV1-A549 cell blocked by the TRPV1mAb and the A540 cell to the nanoparticle and the selective killing effect of the PLGA-PEG-ICG-TRPV1mAb nanoparticle to the TRPV 1-A549.

Example 6

The therapeutic effect of PLGA-PEG-ICG-TRPV1mAb on OVA-induced allergic asthma mice was studied.

1. And (4) experimental design.

Mice were divided into saline group, model group and administration group. The administration components are dexamethasone group, ICG group, PLGA-PEG-ICG group and PLGA-PEG-ICG-TRPV1mAb group, and different concentration gradients are designed to discuss the safety and effectiveness of nanoparticle photothermal therapy. Specific groups of mice are shown in figure 22. Each group comprises 15, 3-4 pathological sections and 4-6 noninvasive pulmonary function evaluations.

2. An OVA-induced allergic asthma mouse molding method.

The existing research shows that mice are mostly adopted for the research of allergic asthma as research objects, wherein mouse OVA (ovalbumin) induced mouse and HDM (dermatophagoides pteronyssinus) induced mouse allergic asthma models are the most common, TRPV1 gene in the OVA induced allergic asthma mouse model is highly expressed, and in order to verify the photothermal treatment effect of the synthesized PLGA-PEG-ICG-TRPV1mAb nanoparticles, the OVA induced mouse asthma model is selected.

The specific molding method is as follows:

dissolving OVA in injection aluminum solution to obtain OVA suspension with concentration of 1mg/ml, storing at 4 deg.C, and mixing for 30min before use. Endotoxin was prepared to 0.25mg/ml with physiological saline and stored at 4 ℃ in the dark. 6g of OVA is dissolved in 100ml of normal saline to prepare 6 percent OVA solution.

Balb/c mice were sensitized by intraperitoneal injection of 100. mu.L of 1mg/ml OVA suspension on

days

0, 7, 14, and 21. On day 7, mice were anesthetized with 1% sodium pentobarbital, the trachea was exposed under laryngoscope, the airway microatomizer was inserted into the trachea, and 50 μ L, 0.25mg/ml LPS was administered rapidly. Continuously atomizing 6% OVA solution by using an atomizer on 28-31 days to excite the mouse asthma, continuously atomizing for 4 days, and once a day for 30min each time. The mice in the normal saline group are all inhaled with the same amount of normal saline through intraperitoneal injection, airway microinjection and atomization. On day 32, the saline group and the model group mice were used, 3-5 mice in each group were used for lung tissue extraction and section preparation, 4-8 mice were used for noninvasive lung function measurement, and the modeling method is shown in fig. 23.

3. A method of administering drugs to asthmatic mice.

After the model is made, the mouse is anesthetized by 1% pentobarbital sodium and then fixed, the neck is stretched, the trachea is exposed under the laryngoscope, and the glottis of the epiglottis is visible to be opened and closed. The airway micro nebulizer was inserted into the trachea and 50 μ L of drug was administered rapidly. ICG group, PLGA-PEG-ICG group and PLGA-PEG-ICG-TRPV1mAb nanoparticle administration group, after administration, the mice were placed in supine position for 15min, and laser emitter with 808nm and 2W/cm²The laser intensity of (2) was irradiated to the mouse lung for 10 min. And treating 24h after illumination, wherein each group comprises 3-5 mice for lung tissue extraction and slice preparation, and 4-8 mice for noninvasive lung function determination. The dexamethasone mice are treated after being administrated for 24h in an air passage, the treatment mode is the same as that of the nanoparticle administration group, and a treatment flow chart of asthma mice is shown in figure 24.

4. Extracting lung tissue of mice.

Mice were sacrificed by spondylolysis, with the abdomen facing up fixed to a dissecting plate, the neck wiped with 75% alcohol, and the lungs collapsed by sternal scissors. The trachea and lungs were exposed and the tissue, blood vessels and nerves below the cricoid were isolated. And (5) placing binding wires below the cricoid cartilage and the left bronchus and ligating the left bronchus. A small oblique opening is cut below the tracheal cricoid cartilage, a flat-head stainless steel sampling needle with the diameter of 0.22mm is inserted and is ligated and fixed. The end of the injection needle is connected with a 1mL injector, and 4% precooled paraformaldehyde is injected until the lung of the mouse is full. The right lung was cut and soaked in 4% paraformaldehyde solution for 24h for making paraffin sections and frozen sections. The left lung was cut and stored in liquid nitrogen.

5. Preparation and tissue staining of a paraffin section of mouse lung tissue.

(1) And (5) preparing a paraffin section of the lung tissue of the mouse.

Material taking: the tissue was removed from the fixative and placed in a dehydration box after smoothing the tissue with a scalpel in a fume hood.

Dehydrating and wax dipping: the tissue is dehydrated by gradient alcohol in turn. The method comprises the following steps of 4 hours of 75% alcohol, 2 hours of 85% alcohol, 2 hours of 90% alcohol, 1 hour of 95% alcohol, 30 minutes of absolute ethanol I, 30 minutes of absolute ethanol II, 5-10 minutes of a mixed solution of ethanol and xylene 1:1, 5-10 minutes of xylene I, 5-10 minutes of xylene II, melting paraffin I1h at 65 ℃, melting paraffin II 1 hour at 65 ℃ and melting paraffin III 1 hour at 65 ℃.

Embedding: embedding the wax-soaked tissue in an embedding machine. And (3) putting the melted wax into an embedding frame, taking the tissue out of the dehydration box before solidification, and putting the tissue into the embedding frame according to the requirements of an embedding surface. Cooling and solidifying in a freezing table at-20 ℃, and taking out the wax block from the embedding frame and finishing after solidification.

Slicing: and putting the trimmed wax blocks into a freezing table at the temperature of-20 ℃ again for cooling. The re-cooled wax block was sectioned in a paraffin slicer to a slice thickness of 4 μm. Adding water into the sheet spreading machine, cooling to 40 deg.C, and placing into the slices to spread the tissue. The tissue was scooped up with a glass slide and baked in an oven at 60 ℃. Storing at normal temperature for later use.

(2) H & E staining.

Paraffin section dewaxing and hydration: sequentially placing the slices in xylene I and xylene II for 20min, respectively, anhydrous ethanol I, anhydrous ethanol II, and 75% ethanol for 5min, and adding dd H₂And (4) flushing.

Hematoxylin staining: the section is subjected to hematoxylin staining solution staining for 3-5 min, differentiation solution differentiation and bluing solution bluing, and after each step is finishedAll using dd H₂And (4) flushing.

Eosin staining: the slices are sequentially immersed in gradient alcohol of 85% and 95% for dehydration for 5min respectively, and then are dyed for 5min by adding eosin dye solution.

Dewatering and sealing: the slices are sequentially put into absolute ethyl alcohol I, absolute ethyl alcohol II, absolute ethyl alcohol III, dimethyl I and xylene II for 5min respectively until the slices are transparent, and are subjected to microscopic examination after being sealed by neutral gum.

Microscopic examination shows that the cell nucleus and the cell nucleus are blue, and the cytoplasm is red.

(3) Masson staining.

Paraffin section dewaxing and hydration: the same as the first H & E dyeing step.

Dyeing with potassium dichromate: immersing the slices in potassium dichromate overnight, dd H₂And (4) flushing.

And (3) hematoxylin staining: and mixing the solution A and the solution B in equal ratio to prepare the hematoxylin staining solution. Soaking the slices in hematoxylin for 3min, differentiating with differentiation solution, returning blue with returning blue solution, and returning blue with dd H₂And (4) flushing.

Ponceau acid fuchsin dyeing: soaking the slices in ponceau acid fuchsin dye solution for 6min, and dyeing with dd H₂And (4) flushing.

Phosphomolybdic acid staining: the slices were immersed in aqueous phosphomolybdic acid for 1 min.

And (3) aniline blue dyeing: the slices are dyed by phosphomolybdic acid, and then are directly placed in aniline blue dye solution for 3min without water washing.

Differentiation: sections were differentiated with 1% glacial acetic acid.

Dewatering and sealing: the final step of H & E staining.

Microscopic examination shows that the collagen fiber is blue; muscle fibers, cellulose and red blood cells appear red.

(4) And PAS dyeing.

Staining with Alisin blue staining solution: soaking the slices in Alisin blue staining solution for 10-15min, dd H₂And flushing twice for 1-2 min each time.

Schiff staining: immersing the slices in Schiff dye solution for dip-dyeing for 25-30min in dark, dd H₂And flushing for 5min by using O.

Slicing into hematoxylin staining solution to stain nuclei for 30s, dd H₂Washing for 1min by using O; 2-5 s, dd H differentiation with hydrochloric acid aqueous solution₂Washing for 1min by using O; ddH, back-blueing of Scott bluing solution₂And O washing for 3 min.

Dewatering and sealing: the final step of H & E staining.

Microscopic examination revealed that goblet cells were purplish red and the nuclei were pale blue.

6. Mouse lung tissue frozen sections and immunofluorescence staining.

(1) And (5) preparing a frozen section of the lung tissue of the mouse.

And (3) dehydrating: taking out the tissue from the fixed solution, placing the tissue in 15% sucrose solution for dehydration and bottom precipitation, transferring the tissue into 30% sucrose solution for dehydration and bottom precipitation, and performing the dehydration process at 4 ℃.

OCT embedding: the dehydrated tissue was removed and the filter paper blotted to dry the surface water. And (3) flattening the tissue by using a scalpel, putting the cut surface upwards into a sample holder, and dripping OCT embedding medium around the tissue. And (3) placing the sample on a quick-freezing table of a freezing microtome for quick-freezing embedding, and slicing after the OCT becomes white and hard.

Slicing: fixing the sample holder on a slicer, roughly cutting, flattening the tissue surface, and then starting slicing, wherein the slicing thickness is 8-10 mu m. The clean slide was placed flat on the cut tissue pieces, the frozen sections were completed and stored at-20 ℃ for future use.

(2) DAPI/TRPV 1/alpha-SMA immunofluorescent staining.

Pretreatment of frozen sections: the frozen sections were left at room temperature for 15min and air-dried. Sections were soaked in PBS (pH7.4) and washed on a decolorizing shaker for 10min to remove OCT.

And (3) circling: the section is slightly dried and then the tissue area to be dyed is encircled by a grouping pen.

Serum blocking: PBS was thrown off, 10% goat serum resistant was added dropwise, and the reaction mixture was sealed at room temperature for 1 hour.

Primary antibody incubation: the blocking solution was gently spun off, TRPV1 and α -SMA mixed with a primary antibody (TRPV1 polyclonal rabbit antibody: 1:40, α -SMA monoclonal mouse antibody: 1:1000) diluted with a primary antibody diluent were added dropwise to the sections, and the sections were placed flat in a wet box and incubated overnight at 4 ℃. A small amount of water was added to the wet box to prevent evaporation of the antibody. Recovering the primary antibody.

And (3) incubation of fluorescent secondary antibody: the slides were placed in PBS (pH7.4) and washed 3 times for 10min each time with shaking on a destaining shaker. After the section is dried, fluorescent secondary antibody (1:1000) is added into the ring to cover the tissue, and the tissue is incubated for 1h at room temperature in a dark place.

Removal of lung tissue autofluorescence: the slides were placed in PBS (pH7.4) and washed 3 times for 10min each time with shaking on a destaining shaker. Lung tissue Autofluorescence was removed using an Autofluorescence Quenching kit, the specific steps refer to the instructions.

DAPI counterstained nuclei: the slides were washed in PBS (pH7.4) for 10min with shaking on a destaining shaker. After the section is dried, DAPI dye liquor (10 mu g/mL) is dripped into the circle, and the section is incubated for 10min at room temperature in a dark place.

Sealing: the slices are slightly dried, sealed by an anti-fluorescence quenching sealing agent and stored in a wet box at 4 ℃.

And (5) microscopic examination and photographing: and (5) observing the section under a laser confocal microscope and collecting an image.

7. A method for non-invasively enhancing the expiratory intermittence determination of a mouse.

The Enhanced expiratory pause value (Penh) determined by the noninvasive plethysmography can accurately reflect the airway reactivity of the mouse. The degree of airflow restriction, when restricted, is measured by the prolongation of the expiratory phase, i.e. the expiratory Pause (Pause). The Penh value was obtained by normalizing the Peak Inspiratory Pressure (PIP) and Peak Expiratory Pressure (PEP) ratios. The calculation formula is as follows: penh PEP/PIP × Pause. The higher the Penh value, the more reactive the airway, and the more severely the airflow is restricted.

The specific determination method is as follows: and connecting the Buxco noninvasive lung function instrument and calibrating. Putting a Balb/c mouse in a natural state into a body-drawing box, and measuring a basic Penh value; the subsequent challenge with methacholine at concentrations ranging from low to high of 0, 3.125, 6.25, 12.5, 25 and 50mg/ml was followed by recording the Penh value at that time, 2 minutes per aerosol inhalation and 3 minutes

8. And (6) analyzing the data.

The data obtained were analyzed using GraphPad Prism 8 software and the statistics were expressed as Mean ± standard error (Mean ± SEM), t-test for two-group comparisons and 2way ANOVA for multiple-group comparisons, with P <0.05 having statistical significance, # P <0.05, # P <0.01, # P < 0.001. The survival curve of the mice was evaluated by the Kaplan-Meier method.

9. And (6) analyzing results.

(1) Validation of OVA-induced mouse allergic asthma model with high expression of TRPV 1.

H & E staining can show that the mouse alveolar gaps are reduced, alveolar structures are damaged, and diffuse bronchioles and perivascular inflammatory cells are infiltrated, wherein eosinophils are remarkably increased; PAS staining revealed bronchial goblet cell proliferation, and fluid embolism and inflammatory exudation in the lumen. The collagen proliferation near the bronchial smooth muscle is obvious by Masson staining, which indicates that the airway smooth muscle is hyperproliferated.

The enhanced expiratory pause (Penh) after the mice were inhaled with increasing concentrations of Methacholine (Mch) of 3.125-50 mg/ml by nebulization 24h after the last OVA challenge was determined by Buxco noninvasive pulmonary function assay. It can be seen that Enhanced breathing pause (Penh) of the model group is significantly increased, indicating that the airway hyperresponsiveness, expiratory airflow severely limited and asthma symptoms severe of the model group mice. Through the pathological verification of the tissue section and the in vivo verification of the Penh value, the invention successfully establishes an OVA-induced mouse allergic asthma model.

Next, the present invention examined whether the established OVA-induced mouse model of allergic asthma has the characteristic of increased expression level of TRPV1 protein. alpha-SMA is a smooth muscle agonist protein involved in smooth muscle contraction and is a marker of airway smooth muscle. In order to locate the expression position of the TRPV1 gene in mouse lung tissues, the invention carries out TRPV1 and alpha-SMA immunofluorescence double staining on asthma mouse lung frozen sections. Under confocal laser microscopy (20 ×), it can be seen that the protein expression levels of TRPV1 and α -SMA were significantly increased in the model group mice compared to the saline group, and that the expression of TRPV1 was mainly concentrated in airway columnar epithelial cells, but not in airway smooth muscle cells. Therefore, the invention constructs an OVA-induced mouse allergic asthma model with high expression of TRPV1 protein.

The results of immunohistochemical staining with H & E, Masson, PAS, and the like on paraffin sections of the saline group mice and the model group mice, respectively, showed that the model group mice had a large amount of infiltration of airway inflammatory cells and a large amount of proliferation of airway smooth muscles and goblet cells, and the results are shown in fig. 25.

Airway hyperresponsiveness of mice 24h after the final challenge with OVA was measured by a Buxco noninvasive lung function instrument. The isocratic increase of Methacholine (Methacholine, Mch) from 3.125mg/ml to 50mg/ml, with n being 4-6, is shown in fig. 26.

The TRPV1 (green) frozen sections and the alpha-SMA immunohistochemistry staining of the saline group mice and the model group mice respectively show that the TRPV1 and the alpha-SMA protein of the model group mice are remarkably increased as shown in the result of figure 27.

(2) And (3) evaluating the safety of the PLGA-PEG-ICG-TRPV1mAb nanoparticles.

If the photothermal nanoparticles are applied to clinical treatment of asthma, the in vivo safety of the nanoparticles needs to be ensured firstly. Therefore, the present invention first evaluated the safety of PLGA-PEG-ICG-TRPV1mAb nanoparticles in mice. Survival of asthmatic mice evaluated by photothermal effect in vivo within 24 hours was observed and survival curves were plotted. It can be found that when the concentration of PLGA-PEG-ICG-TRPV1mAb is 62.5 μm, the safety is best, and all mice survive; mice all died at 500 μm. The half lethal dose of PLGA-PEG-ICG-TRPV1mAb was 250 μ M. Drug concentration ranges are provided for the following evaluation of nanoparticle effectiveness: 62.5-250 μm

The survival of mice within 24 hours after the nanoparticles of PLGA-PEG-ICG-TRPV1mAb were evaluated by airway administration and laser challenge is shown in FIG. 28.

(3) Evaluation of effectiveness of PLGA-PEG-ICG-TRPV1mAb nanoparticles in treating asthma.

After obtaining the in vivo safe dose range of PLGA-PEG-ICG-TRPV1mAb nanoparticles, the effectiveness of the nanoparticles was evaluated. Mainly proceeds from three aspects: firstly, the optimal concentration of PLGA-PEG-ICG-TRPV1mAb nanoparticles for treating asthma is expected to be screened; secondly, it is hoped to know whether the PLGA-PEG-ICG-TRPV1mAb nanoparticles have better effect on the photothermal therapy of asthma than the traditional dexamethasone aerosol inhalation therapy; finally, it was investigated whether the therapeutic effect of PLGA-PEG-ICG-TRPV1mAb nanoparticles was related to the targeting of TRPV 1.

1) The optimal dose of PLGA-PEG-ICG-TRPV1mAb nanoparticles was explored.

In the safety evaluation, a safe dose range of 62.5-250 mu m is obtained, and in the combined body photothermal effect evaluation, the photothermal effect of the PLGA-PEG-ICG-TRPV1mAb nanoparticles with 62.5 mu m is not obviously different from that of a normal saline group, so that 62.5 mu m is supposed to be the lowest effective dose for treating the asthma of the mice. On the basis, the invention sets 125 and 250 μm administration groups in equal ratio. Dosing and light illumination for 10 minutes, and material drawing and airway reactivity evaluation were performed after 24 hours.

Compared with mice in an untreated group, the asthma symptoms of the mice in the groups treated by 62.5 μm, 125 μm and 250 μm are obviously relieved, wherein the curative effect of the group treated by 125 μm is the best. H & E staining is visible, the structure of the mouse circular trachea is obvious in the treatment group with 125 μm, inflammatory cell infiltration is obviously less than 62.5 μm, and the treatment group with 250 μm is obtained; PAS staining revealed that 125 μm treated mice had fewer airway goblet cells than the other two groups. The percentage of collagen fiber area in Masson staining is 125 μ M, 250 μ M and 62.5 μ M from small to large, which shows that 125 μ M treatment group also plays a certain role in reducing airway smooth muscle. In reducing airway responsiveness, the Penh value of mice in the 125 μ M treated group was 2 times that of normal mice in the nebulized saline group, while those in the 62.5 μ M and 250 μ M treated groups were 6 times, with a significant difference, when stimulated by nebulization of 50mg/ml methacholine. Indicating that the 125 μ M treated mice had the most significant improvement in airway limitation symptoms. In conclusion, the optimal dose of PLGA-PEG-ICG-TRPV1mAb nanoparticles for treating mouse allergic asthma is 125 μ M.

Asthma mice were administered via the airways with PLGA-PEG-ICG-TRPV1mAb nanoparticles at concentrations of 62.5, 125, 250 μm, and groups of 0 μm administered with equal amounts of saline, respectively. Dosing and illumination 24, post-harvest, H & E, Masson, PAS staining, results are shown in figure 29.

Airway responsiveness of mice 24 hours after light exposure was measured by a Buxco noninvasive lung function instrument and the results are shown in fig. 30.

2) Compared with dexamethasone aerosol inhalation administration mode, the method has the advantage of higher curative effect.

After defining the optimal therapeutic dose of PLGA-PEG-ICG-TRPV1mAb, the present inventors wanted to define whether photothermal therapy with nanoparticles of PLGA-PEG-ICG-TRPV1mAb had better therapeutic efficacy than the traditional asthma treatment regimen represented by dexamethasone inhalational administration. The prior art shows that the optimal concentration of the mouse dexamethasone in the airway nebulization drug delivery is 1 mg/kg. Asthma mice were treated with 1mg/kg dexamethasone by nebulization or with PLGA-PEG-ICG-TRPV1mAb nanoparticles at a concentration of 125 μm by photothermal therapy, and then compared for therapeutic effect by pathological section and Penh value. The PLGA-PEG-ICG-TRPV1mAb treated groups had reduced inflammatory cell infiltration, smooth muscle and goblet cell proliferation compared to the dexamethasone group; the Penh value is obviously reduced, and the PLGA-PEG-ICG-TRPV1mAb has better asthma treatment effect.

Asthma mice were administered 125 μm PLGA-PEG-ICG-TRPV1mAb nanoparticles, dexamethasone, and normal saline, respectively, via the airways. Dosing and illumination 24, post-harvest, H & E, Masson, PAS staining, results are shown in figure 31.

Airway responsiveness of mice 24 hours after different treatments was measured by a Buxco noninvasive pulmonary function instrument and the results are shown in fig. 32.

3) Relation between TRPV1 targeting property and curative effect in PLGA-PEG-ICG-TRPV1mAb nanoparticles.

The PLGA-PEG-ICG-TRPV1mAb nanoparticles are verified to have TRPV1 gene targeting by in vitro experiments, and in order to determine whether the better curative effect on the allergic asthma of the mice is related to the targeting of the TRPV1 gene, the invention discloses the curative effect of the PLGA-PEG-ICG-TRPV1mAb on the asthmatic mice by ICG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb under the optimal administration dose of 125 mu m and the same concentration.

The results show that ICG and PLGA-PEG-ICG also have certain therapeutic effect on asthma mice. Compared with other two groups, the PLGA-PEG-ICG-TRPV1mAb treatment group has obvious asthma relieving indications of reducing inflammatory cell infiltration, reducing goblet cell proliferation, improving mouse airway compliance and the like. However, in Masson staining of muscle fibers reflecting changes of airway smooth muscles, the relative blue staining area of the PLGA-PEG-ICG-TRPV1mAb treatment group to collagen is significantly higher than that of the ICG treatment group and the PLGA-PEG-ICG treatment group, which indicates that the PLGA-PEG-ICG-TRPV1mAb treatment group has inferior effect on reducing airway smooth muscles compared with the ICG treatment group and the PLGA-PEG-ICG treatment group.

The tissue location of TRPV1 protein high expression in an OVA-induced mouse asthma model is combined, namely the TRPV1 protein high expression is mainly distributed in airway columnar epithelium and has no obvious distribution on airway smooth muscle. It is speculated whether the PLGA-PEG-ICG-TRPV1mAb nanoparticles have better targeting in vivo, so that the nanoparticles have no significant killing effect on airway smooth muscle cells with low TRPV1 gene expression level. Therefore, the invention takes 125 μm ICG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb treatment groups of mouse lung tissue frozen sections respectively for DAPI/TRPV 1/alpha-SMA immunofluorescence staining.

The results showed that the expression level of TRPV1 in the columnar epithelium of mouse airways in the PLGA-PEG-ICG-TRPV1 mAb-treated group was significantly reduced compared to the saline-administered group (0 μ Μ), and significantly lower than in the ICG and PLGA-PEG-ICG treated groups; the expression level of alpha-SMA protein was significantly lower than that of the saline administration group (0 μm), but higher than that of the ICG and PLGA-PEG-ICG treatment group. It is demonstrated that PLGA-PEG-ICG-TRPV1mAb nanoparticles can improve proliferation of columnar epithelium in mice, but have a weaker effect on smooth muscle proliferation. Therefore, the PLGA-PEG-ICG-TRPV1mAb nanoparticles have better in vivo targeting.

The prior art shows that the inhibition of TRPV1 ion channel can reduce the content of Th2 type cytokines such as IL-4, IL-5 and IL-13 and epithelial cell derived cytokines such as IL-25 and IL-33 in mouse alveolar lavage fluid, and relieve goblet cell proliferation. The vagal neurons highly expressing TRPV1 play a key role in acute constriction of bronchi when airway hyperresponsiveness occurs, and ablation of neurons highly expressing TRPV1 in mouse vagal ganglia eliminates airway hyperresponsiveness. This is consistent with the significant reduction in inflammatory infiltration and goblet cell proliferation and reduction in airway hyperreactivity in mice in the PLGA-PEG-ICG-TRPV1mAb nanoparticle photothermal treatment group found in the above experiments. From this, it can be concluded that the nanoparticle photothermal therapy with PLGA-PEG-ICG-TRPV1mAb acts as a therapeutic treatment for asthma by acting on TRPV1 receptors in mouse bronchial and lung tissues.

Asthma mice were administered with 125 μm of ICG, PLGA-PEG-ICG and PLGA-PEG-ICG-TRPV1mAb nanoparticles at the same concentrations via the airways, respectively, and with 0 μm of the group given with the same amount of physiological saline. Dosing and illumination 24, post-harvest, H & E, Masson, PAS staining, results are shown in figure 33.

Airway responsiveness of mice 24 hours after different treatments was measured by a Buxco noninvasive pulmonary function instrument and the results are shown in fig. 34.

TRPV1 (green) and alpha-SMA immunofluorescent staining were performed on the frozen sections of the mice in each group, respectively, and as a result, as shown in FIG. 35, the alpha-SMA protein expression of the mice in the PLGA-PEG-ICG-TRPV1mAb group was significantly reduced compared to the 0 μm group, but was not significantly different from the other administration groups.

In conclusion, the OVA-induced allergic asthma mouse model with high expression of TRPV1 protein is successfully constructed, and is used for evaluating the in vivo safety and asthma treatment effect of PLGA-PEG-ICG-TRPV1mAb photothermal nanoparticles. Anesthetizing a mouse by 1% sodium pentobarbital, atomizing nanoparticles into a mouse lung by using an air passage micro-atomization administration syringe, and irradiating the mouse bronchus and the lung by 808nm laser (2W/cm) 15 minutes after administration²) The photothermal treatment was completed in 10 minutes.

Through a series of concentration gradient settings, the median lethal dose of PLGA-PEG-ICG-TRPV1mAb photothermal nanoparticles in mice was found to be 250. mu.M. H & E, Masson, PAS pathological section staining and noninvasive airway reactivity evaluation show that 125 muM is the optimal treatment concentration within the concentration range of 62.5 muM-250 muM. Compared with the treatment effect of 125 mu M PLGA-PEG-ICG-TRPV1mAb nanoparticle photothermal therapy and the traditional dexamethasone aerosol inhalation on the mouse allergic asthma, the result shows that the mouse asthma symptoms treated by the 125 mu M PLGA-PEG-ICG-TRPV1mAb nanoparticle photothermal therapy are more remarkably improved than that of the dexamethasone inhalation therapy, and the symptoms are shown in that the alveolar structure is normal and the infiltration of airway inflammatory cells is reduced; decreased columnar epithelium, goblet cells, and airway smooth muscle content; the symptoms of expiratory airflow limitation caused by acetylcholine stimulation are obviously relieved.

Meanwhile, the action mechanism of the PLGA-PEG-ICG-TRPV1mAb nano-particles is further explored. ICG and PLGA-PEG-ICG nanoparticles also have certain asthma treatment effects. In addition to weak inhibition of airway smooth muscle proliferation, the PLGA-PEG-ICG-TRPV1mAb nanoparticles have better therapeutic effects in reducing inflammatory cell infiltration, columnar epithelial cell reduction and expiratory airflow limitation improvement. The killing effect of the PLGA-PEG-ICG-TRPV1mAb nano-particles on the columnar epithelial cells of the airways highly expressing TRPV1 is more obvious, and the killing effect on the smooth muscle cells of the airways without highly expressing TRPV1 gene is weaker than that of the ICG and PLGA-PEG-ICG nano-particles, which indicates that the PLGA-PEG-ICG-TRPV1mAb nano-particles have TRPV1 in vivo targeting. The prior art has shown that the antagonism of TRPV1 receptor can improve inflammatory cell infiltration, goblet cell proliferation and airway hyperresponsiveness of asthmatic airways, and meanwhile, the therapeutic effect of PLGA-PEG-ICG-TRPV1mAb nanoparticles on asthmatic mice is combined with the established PLGA-PEG-ICG-TRPV1mAb nanoparticles, so that the conclusion that the PLGA-PEG-ICG-TRPV1mAb nanoparticles can treat asthma by antagonizing TRPV1 receptor can be inferred.

Through in-vivo experiments of mice, PLGA-PEG-ICG-TRPV1mAb photo-thermal nanoparticles are proved to have better asthma treatment effect on OVA-induced allergic asthma mice, but have a certain distance from clinical application. The main limiting factors are that the difficulty of laser penetrating through multiple layers of tissues to directly reach the bronchus from the outside of the body is high, the loss of light energy can be caused when the laser penetrates through one layer of tissue, and the laser can reach the bronchus from the outside of the body by enough power and generate energy capable of exciting the photosensitizer. Therefore, if the photothermal technique is applied to noninvasive treatment of asthma, both the safety of high-power laser irradiation on human body and the effectiveness of asthma treatment need to be considered.

Without making a breakthrough in laser research, PLGA-PEG-ICG-TRPV1mAb photothermal nanoparticles require 808nm laser irradiation via the airways with the aid of bronchofiberscope. Although non-interventional therapy cannot be achieved, compared with the bronchial thermoplasty, the photothermal therapy of the PLGA-PEG-ICG-TRPV1mAb nanoparticles can kill cells in a targeted manner at a milder temperature, reduce the adverse reaction of respiratory tract in the treatment process and improve the safety; the photothermal particles can act on the tiny airways through deposition, and can relieve stenosis by killing epithelial cells, airway smooth muscle cells and the like of the tiny airways after illumination, improve the airflow limitation of the tiny airways and have better asthma treatment effect.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A method for preparing nanoparticles targeting TRPV1, comprising:

2. The method according to claim 1, wherein in the step of preparing the nanoparticles having photothermal conversion properties, the solvent dissolving the block copolymer is acetonitrile, and the solvent dissolving the photosensitizer is dimethyl sulfoxide.

3. The method according to claim 2, wherein the mass ratio of the block copolymer to the photosensitizer is 30 to 50: 1.

4. The method as claimed in claim 1, wherein the rotation speed of the homogenization treatment is 20000-25000rpm, and the separation factor of the centrifugation is 20000-25000 g.

5. The production method according to claim 4, wherein in the targeted modification step, the activation is performed with 0.05 to 0.15mg/mL of a carbodiimide salt.

6. The preparation method according to claim 1, wherein the nanoparticle having photothermal conversion properties is incubated with TRPV1mAb for 2-6 hours.

7. The method of any one of claims 1-6, wherein the photosensitizer is ICG.

8. The method of claims 1-6, wherein the block copolymer is PLGA-PEG-COOH of 5k-40 kDa.

9. Use of the nanoparticles obtained by the preparation method according to any one of claims 1 to 8 for the preparation of a medicament for the prevention or treatment of a TRPV1 overexpression disorder.

10. The method as claimed in claim 9, wherein the TRPV1 overexpression disease is allergic asthma.