阅读说明：本技术 经呼吸道递送氧化铁纳米颗粒的生物安全性评价方法 (Biosafety evaluation method for delivering iron oxide nanoparticles through respiratory tract ) 是由 孙夕林 李迎波 郑利敏 王凯 吴丽娜 杨洁 徐佐宇 杨丽丽 方芳 于 2021-09-13 设计创作，主要内容包括：本发明提出了一种经呼吸道递送氧化铁纳米颗粒的生物安全性评价方法,包括对纳米颗粒进行细菌内毒素检测；使用不同浓度纳米颗粒处理肺上皮细胞和巨噬细胞,测定细胞存活率；健康小鼠体内经呼吸道递送纳米材料后不同时间点采血进行血生化分析、肺泡灌洗液分析、组织标本切片染色以及各重要器官进行RT-PCR炎症细胞因子表达水平检测。本发明为经呼吸道递送氧化铁纳米颗粒的安全性检测提供了方法,也为其他纳米颗粒经呼吸道递送的安全性评价提供了参考。(The invention provides a biosafety evaluation method for delivering iron oxide nanoparticles through a respiratory tract, which comprises the steps of detecting bacterial endotoxin of the nanoparticles; treating lung epithelial cells and macrophages by using nanoparticles with different concentrations, and determining the cell survival rate; blood is collected at different time points after nanometer materials are delivered through respiratory tracts in healthy mice for blood biochemical analysis, alveolar lavage fluid analysis, tissue specimen section staining and RT-PCR inflammatory cytokine expression level detection of various important organs. The invention provides a method for detecting the safety of the iron oxide nanoparticles delivered through the respiratory tract, and also provides a reference for evaluating the safety of other nanoparticles delivered through the respiratory tract.)

1. A method for assessing the biological safety of iron oxide nanoparticles delivered via the respiratory tract route, characterized in that it comprises the following steps:

(1) and (3) internal toxicity detection: performing endotoxin detection on SPIONs by using a limulus reagent by a gel method;

(2) cell activity assay: after different concentrations of SPIONs are respectively incubated with macrophages and human lung epithelial cells for 24-48 hours, the cell survival rate is determined by using an MTT method;

(3) blood analysis: after SPIONs are delivered through a respiratory tract in a healthy BALB/c mouse body, venous blood sampling is carried out at different time points, and blood biochemical analysis is carried out on liver and kidney functions and blood conventional indexes;

(4) alveolar lavage fluid analysis: after SPIONs are delivered through a respiratory tract in a healthy BALB/c mouse, alveolar lavage is carried out at different time points, and tissue inflammatory factor detection and cell smear analysis are carried out on alveolar lavage fluid;

(5) inflammatory cytokine detection: after SPIONs are delivered through a respiratory tract in a healthy BALB/c mouse, all main organs are taken at different time points, tissue mRNA is extracted, and RT-PCR detection of inflammatory cytokine mRNA is carried out;

(6) and (3) biodistribution of the nanoparticles: after SPIONs are delivered through a respiratory tract in a healthy BALB/c mouse, taking each main organ at different time points, and detecting the content of iron ions in each tissue by using ICP-MS after treatment;

(7) nanoparticle intrapulmonary distribution: after SPIONs are delivered through a respiratory tract in a healthy BALB/c mouse, lung tissues are taken at different time points, iron staining is carried out after slicing, and the distribution condition of nanoparticles at alveolus and bronchial positions is observed;

(8) analyzing the tissue pathological damage: after SPIONs are delivered through a respiratory tract in a healthy BALB/c mouse, each main organ is taken at different time points to be pathologically sliced and observed by an optical microscope;

(9) and (3) pulmonary fibrosis analysis: after SPIONs are delivered through the respiratory tract in a healthy BALB/c mouse, lung tissues are taken at different time points, and are sliced and then subjected to specific staining of masson, so that the pulmonary fibrosis condition is observed.

2. The biosafety assessment method for the delivery of iron oxide nanoparticles via the respiratory route according to claim 1, characterized in that: in the step (2), the concentration of SPIONs co-incubated with macrophages and human lung epithelial cells is 0-1000 mug/mL.

3. The biosafety assessment method for the delivery of iron oxide nanoparticles via the respiratory route according to claim 1, characterized in that: in steps (3) - (9), the concentration of SPIONs delivered via the respiratory tract route is 1mg Fe/mL, and the volume is 0-100 μ L.

Technical Field

The invention relates to a biological safety evaluation method for delivering nanoparticles through a respiratory tract, in particular to a biological safety evaluation method for delivering superparamagnetic iron oxide nanoparticles through a respiratory tract.

Background

In recent years, with the increasing incidence of respiratory diseases such as lung cancer, diagnosis and treatment of lung diseases have been a great challenge. The traditional treatment modes such as intravenous administration or oral administration have the defects of first-pass elimination of liver, low concentration of medicine at the lesion part and the like. Due to the unique physiological structure of the lung, drug delivery via the respiratory tract shows great potential for clinical use. Compared with other organs of the human body, the lung is directly communicated with the outside, and convenience is provided for noninvasive drug delivery; and the organ has extremely large surface area, extremely thin air-blood barrier, high blood flow and less enzymatic activity, and administration through the respiratory tract also provides a novel non-invasive method for systemic delivery of drugs. Respiratory tract delivery can achieve high concentration accumulation of lung disease with low dose of drug, avoids first pass effect, and has low concentration outside target organs and high biological safety.

With the development of nanotechnology in recent years, more and more nanoparticles are used in the biomedical field, especially for the diagnosis and treatment of diseases. The superparamagnetic iron oxide nanoparticles (SPIONs) are widely applied to Magnetic Resonance Imaging (MRI), Magnetic Particle Imaging (MPI) and the like due to good biocompatibility and excellent surface chemical properties, and can also be used as delivery carriers of chemotherapeutic drugs, photothermal materials, photosensitizers, nucleic acids and other drugs for treating diseases, especially in the aspects of targeted therapy, chemokinetic therapy, photodynamic therapy, photothermal therapy, magnetocaloric therapy, immunotherapy and the like. However, there are very limited research reports in the diagnosis and treatment of pulmonary diseases, especially the delivery of SPIONs via the respiratory route.

Although nanoparticles have been widely used in research fields such as diagnosis and treatment of diseases, few nanoparticles for clinical use are approved for the market. The reason for this is that the biological safety of nanoparticles is seriously insufficient for clinical studies, especially nanoparticles delivered via the respiratory tract. Biological safety evaluation of nanoparticles prepared by previous research is carried out by evaluating biological safety problems after intravenous route administration, and the biological safety evaluation after respiratory tract delivery is not carried out, so that the influence and safety problems on organisms after respiratory tract delivery cannot be truly reflected.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a biosafety evaluation method for delivering SPIONs through a respiratory tract, and the biosafety of nanoparticles delivered through the respiratory tract is systematically evaluated, so that the conversion of the nanoparticles delivered through the respiratory tract in clinical application is facilitated.

In order to achieve the above purpose, the invention is realized by the following technical scheme:

a method for assessing the biological safety of iron oxide nanoparticles delivered via the respiratory tract comprising the steps of:

(1) and (3) internal toxicity detection: performing endotoxin detection on SPIONs by using a limulus reagent by a gel method; endotoxin is a phospholipid polysaccharide-protein compound produced by gram-negative bacteria, can cause fever, endotoxin shock, intravascular coagulation and the like, and the detection of SPIONs can evaluate whether endotoxin pollution exists or not;

(2) cell activity assay: after different concentrations of SPIONs are respectively incubated with macrophages and human lung epithelial cells for 24-48 hours, the cell survival rate is determined by using an MTT method; the biological safety of the nanoparticles is firstly reflected in the influence on the cell proliferation capacity, and the biological safety of the SPIONs can be preliminarily determined by in vitro co-incubation with the cells and observation of the activity of the cells; because the invention aims to establish the safety evaluation of the nanoparticles delivered by the respiratory tract, human lung epithelial cells and macrophages are selected and used, and the influence of the SPIONs on the activities of the two cells is observed;

the biological safety of the nanoparticles to be used for respiratory tract delivery can be preliminarily and rapidly evaluated under in-vitro conditions through the steps (1) and (2), the concentration and the dosage of the SPIONs delivered through the respiratory tract can be guided, and the nanoparticles with high safety can be screened;

(3) blood analysis: after SPIONs are delivered through a respiratory tract in a healthy BALB/c mouse body, venous blood sampling is carried out at different time points, and blood biochemical analysis is carried out on liver and kidney functions and blood conventional indexes; the nanoparticles enter into a living body and can be discharged out of the body through organ metabolism of liver and kidney, and in the biological process, the nanoparticles can be accumulated in organs to damage liver and kidney organs, influence the normal physiological functions of the organs, and are expressed as the abnormality of blood biochemical indexes, and whether the SPIONs have toxic effects on the organs or not can be evaluated through blood biochemical analysis;

(4) alveolar lavage fluid analysis: after SPIONs are delivered through a respiratory tract in a healthy BALB/c mouse, alveolar lavage is carried out at different time points, and tissue inflammatory factor detection and cell smear analysis are carried out on alveolar lavage fluid;

(5) inflammatory cytokine detection: after SPIONs are delivered through a respiratory tract in a healthy BALB/c mouse, all main organs are taken at different time points, tissue messenger ribonucleic acid (mRNA) is extracted, and RT-PCR detection of inflammatory cytokine mRNA is carried out;

TNF-alpha, IL-6, IL-8, IL-1 beta, IL-13 inflammatory cytokines are highly expressed in the alveoli of a variety of pulmonary inflammatory diseases; compared with normal tissues, the inflammatory tissues show the mRNA transcription increase of the cytokines which are obviously up-regulated, and all the indexes are inflammation and pathological markers of damaged tissues; whether the SPIONs induce the lung tissues to generate inflammatory responses can be evaluated by evaluating the expression conditions of the alveolar lavage fluid and inflammatory factors of various tissues after the SPIONs are delivered through the respiratory tract and performing smear observation on the alveolar lavage fluid to observe cell types;

the steps (3), (4) and (5) can evaluate whether the SPIONs delivered by the respiratory tract have acute, subacute and chronic toxic and side effects on main organs of the organism or not, and identify the biological safety of the SPIONs;

(6) and (3) biodistribution of the nanoparticles: after SPIONs are delivered through a respiratory tract in a healthy BALB/c mouse, taking each main organ at different time points, and detecting the content of iron ions in each tissue by using ICP-MS after treatment; the distribution of the nanoparticles in organisms, namely the metabolic dynamics, is closely related to the intensity and the persistence of toxic and side effects of the SPIONs in the organisms and the metabolic process in the organisms, and the in-vivo distribution state of the SPIONs can be identified and the safety of the SPIONs can be evaluated by researching the biological distribution of the SPIONs;

(7) nanoparticle intrapulmonary distribution: after SPIONs are delivered through a respiratory tract in a healthy BALB/c mouse, lung tissues are taken at different time points, iron staining is carried out after slicing, and the distribution condition of the nanoparticles in alveoli and bronchial parts at all levels is observed; by specifically studying the distribution in the lungs at different time points after the SPIONs are delivered through the respiratory tract, the distribution state and the residence time of the SPIONs in the bronchi including alveoli and all levels can be effectively evaluated, and the implementation of the SPIONs safety delivery scheme is guided;

the metabolic mode and the route of the SPIONs can be evaluated according to the distribution characteristics in vivo after the SPIONs are delivered through the respiratory tract route, the biological applications of imaging, treatment and the like of the SPIONs are guided, and the safe and effective delivery scheme of the SPIONs is guided;

(8) analyzing the tissue pathological damage: after SPIONs are delivered through a respiratory tract in a healthy BALB/c mouse, each main organ is taken at different time points to be pathologically sliced and observed by an optical microscope; histopathology is a common method for analyzing pathological changes of tissue structures in clinic, and the in-vivo biological safety of the tissues and organs can be judged intuitively by analyzing pathological sections of the tissues and organs after SPIONs are delivered through a respiratory tract;

(9) and (3) pulmonary fibrosis analysis: after SPIONs are delivered through a respiratory tract in a healthy BALB/c mouse, lung tissues are taken at different time points, and are sliced, and then Masson (Masson) specific staining is carried out to observe the pulmonary fibrosis condition; previous researches prove that after being inhaled by the lung, part of nanoparticles can cause acute and chronic inflammatory reactions of lung tissues and gradually develop into fibrosis of the lung tissues; therefore, the evaluation of the presence or absence of fibrosis in lung tissue after the SPIONs are delivered through the respiratory tract is also one of the indexes for judging the biological safety of the lung tissue;

and (8) accurately and effectively evaluating whether the SPIONs delivered through the respiratory tract generate tissue damage and pathological states to the organism or not by evaluating the histological morphology of the tissue sections of each main organ, and visually evaluating the biological safety of the organism.

Further, in the step (2), the concentration of the SPIONs co-incubated with the macrophages and the human lung epithelial cells is 0-1000 mu g/mL.

Further, in steps (3) - (9), the concentration of SPIONs delivered via the respiratory pathway is 1mg Fe/mL and the volume is 0-100. mu.L.

The invention has the following advantages:

1. the method for detecting the biological performances of the SPIONs such as bacterial endotoxin, cell survival rate and the like is researched, the biological safety of the SPIONs at the in vitro and cell level is explored, and the method has the advantages of high sensitivity, low cost, quick and reliable result. 2. Through biodistribution, histopathological injury analysis and inflammatory cytokine detection after SPIONs are delivered through the respiratory tract, the monitoring time is long, evaluation indexes are comprehensive, and systemic in-vivo evaluation of the tissue level biosafety of nanoparticles delivered through the respiratory tract is facilitated. 3. The evaluation methods used by the invention have the advantages of simple method, high repeatability and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a technical scheme of the method of use of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.

As shown in fig. 1, a biosafety assessment method for delivering iron oxide nanoparticles via the respiratory tract, comprising the steps of:

(1) the endotoxin detection of SPIONs was performed by gel method. Preparing endotoxin standard solution, respectively carrying out limulus reagent sensitivity composite test, interference pre-test and interference formal test, and finally carrying out endotoxin detection of SPIONs.

(2) Culturing human lung epithelial cell BEAS-2B cell and macrophage RAW264.7 in cell culture medium, inoculating log-phase grown cells into 96-well plate, and culturing at 1 × 10⁵cells/mL (200. mu.L per well) were plated, incubated overnight, then added with different concentrations (0-1000. mu.g Fe/mL) of SPIONs and cells incubated for 24 hours or 48 hours, PBS washed three times after removal of SPIONs, and FBS-free MTT (3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2-H-tetrazolium bromide) solution (1mg/mL) was added to each well. After 4 hours incubation, the supernatant was discarded and washed three times with PBS. The Formazan formed by the reaction was extracted with 150. mu.L DMSO and the absorbance value (OD) was measured at a wavelength of 560nm using a multifunctional microplate detector. Cell viability was calculated by the following formula:

cell survival rate was ═ 100% x [ (experimental OD-blank OD)/(control OD-blank OD) ].

Whether SPIONs are cytotoxic and the effect of SPIONs on cell proliferation are monitored by determining the application of different concentrations of SPIONs to the cell viability of lung epithelial cells and macrophages.

(3) Healthy BALB/c mice were anesthetized and delivered 0-100 μ g SPIONs via the respiratory tract, blood was drawn from the retroorbital sinus of the mice on days 1, 7, and 30, centrifuged at room temperature and serum was isolated. Analyzing blood sample for leukocyte count, erythrocyte count, hematocrit, platelet count, hemoglobin, blood general index of leukocyte classification, and liver and kidney function index of AST, ALT, LDH, total protein, total bilirubin, albumin, globulin, creatinine and urea nitrogen. After different concentrations of SPIONs are delivered through a respiratory tract way, the change trend of each index is detected and compared, and whether the SPIONs have influence on the physiological indexes is determined.

(4) After 0-100 mu g of SPIONs are delivered through a respiratory tract after a healthy BALB/c mouse is anesthetized, the mouse is sacrificed after the mouse is anesthetized on days 1, 7 and 30, the trachea is exposed after the body surface is disinfected, a syringe with a needle head is inserted after the trachea is cut and fixed by a thin line, 1ml of sterile physiological saline is extracted and slowly injected into the alveolus for lavage, the lavage liquid of the alveolus is collected after 2-4 times of slow suction, and the lavage liquid is 5ml in total. Centrifuging the collected alveolar lavage fluid at 4 ℃, taking the supernatant for detecting cytokines, taking cell precipitates for cell counting, smearing and HE staining, and then classifying cells.

(5) After 0-100 μ g of SPIONs are delivered through respiratory tract after a healthy BALB/c mouse is anesthetized, the mouse is sacrificed after the mouse is anesthetized at days 1, 7 and 30, lung tissues and various important organs (heart, liver, spleen, brain, kidney, intestine and skeletal muscle) are taken, and mRNA of the tissues is extracted for RT-PCR detection. GAPDH or beta-actin (Actb) cDNA is used as a housekeeping gene, and TNF-alpha, IL-6, IL-8, IL-1 beta and IL-13 inflammatory cytokines are used as target genes, and the mRNA expression level of the inflammatory cytokines is analyzed.

(6) After 0-100 mu g of SPIONs are delivered through a respiratory tract after a healthy BALB/c mouse is anesthetized, the mouse is killed after the mouse is anesthetized at days 1, 7 and 30, lung tissues and various important organs (heart, liver, spleen, brain, kidney, intestine and skeletal muscle) are taken, the tissues are dissolved by wet digestion and are subjected to acid removal treatment, and the content of iron ions in various tissues is detected by ICP-MS. Metabolic pathways of SPIONs are determined by short, medium and long term observations of the distribution of SPIONs delivered via respiratory pathways in organisms.

(7) After 0-100 μ g of SPIONs were delivered via the respiratory tract after anaesthetizing healthy BALB/c mice, the mice were sacrificed after anaesthetizing the mice on days 1, 7, and 30, lung tissues were taken for histopathological sectioning, and the sectioned lung tissues were stained with prussian blue iron. The distribution of SPIONs in the lungs at various times following respiratory tract delivery of SPIONs was observed.

(8) After 0-100 μ g of SPIONs were delivered via the respiratory tract after anaesthesia in healthy BALB/c mice, the mice were sacrificed after anaesthesia on days 1, 7, and 30, and lung tissues and various vital organs (heart, liver, spleen, brain, kidney, intestine, and skeletal muscle) were taken for HE staining of histopathological sections. And observing whether pathological damage exists in each organ of the mouse at different time after SPIONs are delivered through the respiratory tract.

(9) Healthy BALB/c mice were anesthetized and then delivered with 0-100 μ g SPIONs via the respiratory tract, mice were sacrificed after anesthetizing the mice on days 1, 7, and 30, lung tissue was taken for pathological sectioning, and Masson's (Masson) staining of sectioned lung tissue was performed. Mice were observed for lung tissue damage at various times following delivery via the respiratory route.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.