CN109576340A - It is distributed and Evaluation of Biocompatibility method in fluorescent nano particles body in vinegar - Google Patents
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Abstract
The invention discloses the method for distribution and biological safety in fluorescent nano particles body in a kind of evaluation vinegar, fluorescent nano particles handle rats renal cells in the vinegar including using various concentration, measure cell survival rate；It selects the concentration for the vinegar fluorescent nano particles for making NRK cell survival rate reach 50%~90% as potential danger concentration, detects Apoptosis situation；The experiment of NRK cell imaging is carried out using the safe concentration that cell survival rate reaches 90% or more, observes distribution situation of the fluorescent nano particles in cell in vinegar；By the way that fluorescent nano particles in BALB/c mouse stomach-filling vinegar, after different time is handled, fluorescent nano particles are in the intracorporal distribution of mouse in observation vinegar；Finally by hemolytic experiment measurement vinegar fluorescent nano particles to the extent of the destruction of mouse erythrocyte.The present invention provides reliable scientific basis for the safety of fluorescent nano particles in vinegar, also provides reference for the safety evaluatio of other nano materials.
Description
Technical field
The present invention relates to the evaluation methods being distributed in fluorescent nano particles body in a kind of vinegar with biological safety.
Background technique
In process, organic molecule and oligomer in raw material are heated to occur series reaction to food, this and chemistry
The process for synthesizing fluorescent nano particles by hydrothermal/solvent thermal method in synthesis is similar, and vinegar is mainly by sorghum, yeast, wheat bran, paddy
The raw materials such as health are brewed through techniques such as gelatinization, fermentation, smoked unstrained spirits, exist inevitably add to raw material in process of production
The process of heat, in the process, protein and polysaccharide in cereal materials are degraded to short peptide chain, amino acid and oligosaccharides, these
It mutually reacts between substance, forms the fluorescent nano particles in vinegar, this fluorescent nano particles are the classes by dispersing
Spherical carbon particle composition, size is minimum, 10 nm hereinafter, its essential element contained is carbon (C), hydrogen (H), oxygen (O), nitrogen (N),
General chemical structure has hydroxyl (- OH), carboxyl (- COOH), and carbonyl (- C=O) and amino (- NH2) etc., it also has good water
Dissolubility and its aqueous solution can issue bright blue-fluorescence under 365 nm ultraviolet lights, therefore referred to as fluorescent nano particles.
Food nanoparticle safety problem is always focus concerned by people, vinegar as flavouring essential in daily life,
It is particularly significant to the research of its safety, currently, people are usually negative to the view of the food containing novel fluorescence nanoparticle
Face, however any one place in the world, all there are no the mandatory label about fluorescent nano particles in food, consumption
Person is difficult to understand the relevant information of fluorescent nano particles, does not also know in commercialized product with the presence or absence of fluorescence nano grain
Son, it is therefore, particularly significant to the internal distribution situation and bio-safety Journal of Sex Research of fluorescent nano particles in vinegar.Currently, report
The evaluation method for the safety for nano material led includes building cell model and animal model, observes nano material thin
The influence of distribution and cell proliferation ability and apoptosis situation in born of the same parents, finding nano material influences Cell apoptosis and proliferation
Reason, biological safety of the comprehensive analysis nano material in cellular level.
Summary of the invention
The object of the present invention is to provide the sides of distribution and biological safety in fluorescent nano particles body in a kind of evaluation vinegar
Method.
Present invention technical solution used for the above purpose is: in a kind of evaluation vinegar in fluorescent nano particles body
The method of distribution and biological safety, comprising the following steps:
A, rat renal tubular epithelial cells inoculation is cultivated in cell culture medium, is grown to pair to rat renal tubular epithelial cells
It is digested when the number phase, postdigestive rat renal tubular epithelial cells suspension carries out 24 h of adhere-wall culture, obtains adherent growth
Rat renal tubular epithelial cells；
B, in cell culture medium by fluorescent nano particles dispersion in vinegar, whirlpool concussion obtains fluorescent nano particles solution；
C, the fluorescent nano particles solution gradient in step B is diluted to several concentration is 1.25 mg/mL, 2.5 mg/mL, 5
Mg/mL, 10 mg/mL and 20 mg/mL, are added separately in the rat renal tubular epithelial cells for the adherent growth that step A is obtained,
Cultivate 24 h；
D, the rat renal tubular epithelial cells after culture are continued to cultivate in 100 μ L tetrazole indigo plant solution, it is molten to tetrazole indigo plant
200 μ L dimethyl sulfoxides are added in rat renal tubular epithelial cells after liquid culture, measure light absorption value under 492 nm wavelength, make
For test results, control group is set, the result of control group is by rat renal tubular epithelial cells successively through cell culture medium, four
After nitrogen azoles indigo plant solution and dimethyl sulphoxide solution processing, light absorption value is measured under 492 nm wavelength, is measured as test results
Cell survival rate is calculated by test results and control group result in absorbance value；
E, the vinegar fluorescent nano particles solution of the potential danger concentration obtained in step D, concentration gradient be 0.2 mg/mL and
0.4 mg/mL；Postdigestive 24 h of logarithmic phase rat renal tubular epithelial cells in processing step A is formed single after pancreatin digests
Cell suspension is centrifuged after being rinsed with phosphate buffer solution, and fluidic cell buffer is added, measures the Survival of cell, if
Set control group, the result of control group be by rat renal tubular epithelial cells successively through cell culture medium, phosphate buffer solution and
The cells survival situation measured after the processing of fluidic cell buffer solution, is calculated carefully by test results and control group result
Born of the same parents' apoptosis rate；
F, young rat is divided into two groups, fluorescent nano particles in vinegar are dispersed in ultrapure water by control group and experimental group, whirlpool shake
It swings, obtains fluorescent nano particles solution, the reconditioning of Mouse oral is 2 g kg-1Weight, experimental mice take orally fluorescence and receive
Rice corpuscles solution, control group mice take orally NaCl aqueous solution, and control group and experimental group is taken to take orally the mouse master of different time respectively
Want accumulation of the organ observation vinegar fluorescent nano particles in mouse organs；
G, fluorescent nano particles in vinegar are dispersed in phosphate buffer solution, it is molten to obtain fluorescent nano particles for whirlpool concussion
Simultaneously gradient dilution carries out intragastric administration on mice test at several concentration to liquid；Test is using 4-5 week old male mice, fluorescence nano
The concentration of particle is 2 g kg-1 × mouse weight (kg)/400 μ L；Mouse major organs include stomach, intestines, kidney, liver, lung and
Brain.
H, control group mice blood is taken, by centrifugal blood except serum deprivation obtains erythrocyte, the parameter of centrifugation is set as 4
DEG C, 3500 rpm, 15 min；It is spare after being rinsed with phosphate buffer solution, it is added obtained in step G in cell culture fluid
Fluorescent nano particles solution, concentration gradient be respectively 0.5 mg/mL, 1 mg/mL, 2.5 mg/mL, 5 mg/mL, 10 mg/mL,
15 mg/mL and 20 mg/mL；120 min of incubated at room temperature, negative control group are that isometric phosphoric acid is only added into cell culture fluid
Salt buffer solution is remained unchanged with cellular morphology, and positive controls are that isometric ultrapure water is only added to make cell rupture, 120 min
Centrifuging and taking supernatant surveys absorbance at 541 nm after incubation；
I, according to the bio distribution and biological safety of fluorescent nano particles in the above test overall merit vinegar.
It is distributed in fluorescent nano particles body in a kind of evaluation vinegar of the present invention and the method for biological safety, raw material is inexpensively easy
Purchase；Fluorescent nano particles preparation method is simple, and test repeatability is good；By test cell line with preliminary animal experiment to glimmering in vinegar
Light nanoparticle carries out internal distribution situation and bio-safety Journal of Sex Research, and the safety of fluorescent nano particles in vinegar is evaluated with this
Property, and the evaluation method of fluorescent nano particles safety in vinegar is established, it is provided for fluorescent nano particles safety evaluation in vinegar
Method.
Detailed description of the invention
Fig. 1 is that vinegar fluorescent nano particles MTT tests cell survival rate figure in the embodiment of the present invention 1.
Fig. 2 is vinegar fluorescent nano particles cell apoptosis assay result figure in the embodiment of the present invention 2.
Fig. 3 be in the embodiment of the present invention 4 vinegar fluorescent nano particles in rat kidney tissue situation map.
Fig. 4 is vinegar fluorescent nano particles hemolytic experiment result figure in the embodiment of the present invention 5.
Fig. 5 be in the embodiment of the present invention 5 vinegar fluorescent nano particles to erythrocyte morphology influence result figure.
Specific embodiment
The method for evaluating distribution and biological safety in fluorescent nano particles body in vinegar, specific steps are as follows: A, by rat
Renal cells NRK is seeded in culture in cell culture medium (DMEM), grows to pair to rat renal tubular epithelial cells NRK
It is digested when the number phase, postdigestive rat renal tubular epithelial cells NRK suspension carries out 24 h of adhere-wall culture, obtains adherent growth
NRK cell；B, fluorescent nano particles in vinegar are dispersed in DMEM, whirlpool concussion obtains fluorescent nano particles solution；C,
Fluorescent nano particles solution gradient in step B is diluted to several concentration, is added separately to the adherent growth that step A is obtained
In NRK cell, 24 h are cultivated, the concentration gradient of fluorescent nano particles is 20 mg/mL of 0-；D, the NRK cell after culture is existed
Continue to cultivate in blue (MTT) solution of 100 μ L tetrazoles, 200 μ L dimethyl are added into the NRK cell after MTT hydroponics
Sulfoxide (DMSO) measures light absorption value under 492 nm wavelength, as test results；Control group is set, and the result of control group is will
The absorbance value that NRK cell successively measures after DMEM, MTT solution and DMSO solution processing；By test results and experiment pair
Formula is substituted into according to group result, cell survival rate is calculated: cell survival rate=ODexp/ODcon× 100%, wherein ODexpIt represents real
Test a group absorbance value, ODconExperimental comparison group absorbance value is represented, according to cell survival rate in experimental group in 50%~90% institute
Use the concentration of vinegar fluorescent nano particles solution as the potential danger concentration of vinegar fluorescent nano particles；E, it is obtained in step D
To potential danger concentration vinegar fluorescent nano particles solution treatment steps A in postdigestive 24 h of logarithmic phase NRK cell, warp
Single cell suspension is formed after pancreatin digestion, is centrifuged afterwards twice with phosphate buffer solution (PBS) flushing, fluidic cell buffering is added
Liquid, measures the Survival of cell, is arranged control group, the result of control group be by NRK cell successively through DMEM, PBS solution and
The cells survival situation measured after the processing of fluidic cell buffer solution；Test results and experimental comparison group result are substituted into formula
Apoptosis rate, apoptosis rate=apoptotic cell number/total cell number × 100% is calculated；F, Dalian medical treatment is followed
Institutional Animal nursing and animal using the committee approval protocol in all zoopery programs, by BALB/c young rat mouse (18~
22 g) are divided into two groups, control group: male mice 5, experimental group: male mice 5, chow diet and drinking-water are provided, by vinegar
Middle fluorescent nano particles are dispersed in ultrapure water, and whirlpool concussion obtains fluorescent nano particles solution, the reconditioning of Mouse oral
For 2 g kg-1Weight, experimental mice take orally 400 μ L of fluorescent nano particles solution, control group mice, and male mice 5, mouth
400 μ L, 0.9% NaCl aqueous solution is taken, control group and experimental group is taken to take orally 0 h, 2 h, 6 h, the main device of the mouse of 24 h respectively
Official observes accumulation of the vinegar fluorescent nano particles in mouse organs；G, fluorescent nano particles in vinegar are dispersed in PBS
In, whirlpool concussion obtains fluorescent nano particles solution and gradient dilution into several concentration；H, control group mice blood in step G is taken
Liquid, by centrifugal blood except serum deprivation obtain erythrocyte, flushed three times with PBS solution it is rear spare, in cell culture fluid (MEM)
Fluorescent nano particles solution obtained in step H, 120 min of incubated at room temperature is added, negative control group is only to cell culture fluid
The middle isometric PBS of addition is remained unchanged with cellular morphology, and positive controls are that isometric ultrapure water is only added to make cell rupture,
Centrifuging and taking supernatant surveys absorbance at 541 nm after 120 min are incubated for；I, according to glimmering in the above test overall merit vinegar
The bio distribution and biological safety of light nanoparticle.
Embodiment 1, fluorescent nano particles MTT cell survival rate experiment in vinegar
Cell culture: rat renal tubular epithelial cells NRK is containing 10% fetal calf serum, 1% penicillin and streptomysin
It is cultivated in DMEM complete medium, CO2 and 37 DEG C of constant incubator that culture environment is 5% changes liquid, logarithmic growth phase daily
Cell tested；The preparation of vinegar fluorescent nano particles solution: accurately weighing 20 mg fluorescent nano particles, and 1 mL is added
After DMEM mixing, vortex vibrates 1 min, and it is spare to be diluted to 1.25,2.5,5,10,20 mg/mL with culture medium, takes logarithmic growth
The NRK cell of phase is added 0.25% pancreatin and digests, 1 × 105/mL of cell suspension, in 96 orifice plates of every 100 μ L of hole addition,
It is cultivated 24 hours in incubator, fluorescent nano particles DMEM is diluted into 1.25,2.5,5,10, the 20 every hole mg/mL of various concentration
100 μ L are added, sequentially add, while the DMEM culture cell without fluorescent nano particles is set as a control group, every group of setting
6 multiple holes inhale after culture 24 hours and abandon the supernatant containing fluorescent nano particles, cleaned 2 times with PBS buffer solution, are added 0.05
The MTT solution of mg/mL continues culture 4 hours, discards supernatant, and 150 μ L DMSO are added, after sufficiently dissolving purple crystal thing,
Light absorption value is measured in 492 nm of microplate reader；The result of control group is by NRK cell successively through DMEM, MTT solution and DMSO solution
The absorbance value measured after processing；Experimental result is analyzed using origin2015 software, by test results and experiment
Control group result substitutes into formula and cell survival rate is calculated: cell survival rate=ODexp/ODcon × 100%, wherein ODexp
Represent experimental group absorbance value, ODcon represents experimental comparison group absorbance value, experimental result: NRK cell is by various concentration
After fluorescent nano particles processing, cell activity is reduced with the raising of fluorescent nano particles concentration, if Fig. 1 is in low dosage water
Under flat (1.25 mg/mL, 2.5 mg/mL, 5 mg/mL), fluorescent nano particles compare almost the survival rate of cell with control group
It does not influence, cell survival rate is 90% or more；But under middle high dose under (10 mg/mL, 20 mg/mL), cytotoxicity
It obviously increases, cell survival rate is below 80%.
Embodiment 2, fluorescent nano particles cell apoptosis assay in vinegar
PI decoration method detects Apoptosis: NRK cell is handled with the fluorescent nano particles that concentration is respectively 0.2,0.4 mg/mL,
Trypsin digestion cell is used after 24 h, 70% ethyl alcohol is fixed, and is then added and contains 5 μ g/mL propidium iodides (PI) and 1 mg/mL
Cell is resuspended in the PBS of RNase, and Annexin V/PI fluorescence colour detects Apoptosis: adjustment NRK cell density is 1 ×
105/mL is inoculated in 6 orifice plates, 37 DEG C, cultivates under the conditions of 5%CO2 and is separately added into 0.2,0.4 mg/mL after 24 h and receives containing fluorescence
The culture medium of rice corpuscles removes culture solution if Normal group after 24 h culture, PBS is washed 2 times, and each group is added 195 μ L and combines
5 μ L Annexin V dye liquors are then added in liquid (binding buffer), 190 μ L combination liquid are added after sucking liquid, then
5 μ L PI dye liquors are added, show emblem microscopic observation Apoptosis situation being inverted fluorescence, Annexin V is to phosphatidylserine height
The compatibility of degree can detecte the early apoptosis of cell, and PI is only capable of the cell being damaged through after birth, is embedded in core DNA, send out Chinese red
Fluorescence, experimental result: thin with the fluorescent nano particles processing NRK of flow cytometer Annexin V/PI method detection various concentration
The apoptosis percentage of 24 h of born of the same parents, as seen from Figure 2, as fluorescent nano particles concentration increases, the cell number of apoptosis constantly increases
Add, apoptosis percentage is respectively 8.25%, 14.6%, 14.2%.
Embodiment 3, fluorescent nano particles rat kidney tissue experiment in vinegar
It follows Dalian medical institutions animal care and animal and ratifies all zoopery programs in protocol using the committee, it will
BALB/c mouse, age of mouse were divided into two groups at 4-5 weeks, weight 18-22g, and experimental group totally 5 male mices take orally from vinegar
The fluorescent nano particles of extraction, the reconditioning of Mouse oral are 2 g kg-1 weight, and 5 male mices of control group take orally
400 μ L, 0.9% NaCl aqueous solution takes 0 h of control group and experimental group stomach-filling, 2 h, 6 h, the main device of the mouse of 24 h respectively
Official, stomach, intestines, kidney, liver, lung and brain observe fluorescent nano particles in mouse organs by toy fluorescent vital imager
Accumulation, experimental result: after the bio distribution situation of fluorescent nano particles passes through stomach-filling mouse in vinegar, to its major organs
Fluorescence analysis is carried out including stomach, intestines, kidney, liver, lung and brain, in the intracorporal distribution of mouse, such as Fig. 3 after taking orally with determining its
The accumulation of shown fluorescent nano particles occurs mainly in alimentary canal position, such as stomach, intestines early period, with the extension of time, mouse
Kidney and other organs, liver, lung and brain start the fluorescence for occurring stronger than control group, these results indicate that intracorporal
Fluorescent nano particles can pass through blood-brain barrier as blood transportation is into each organ, accumulate in mouse brain, fluorescence nano
The discharge regime of particle may be by excrement and be excreted in vitro.
Embodiment 4, fluorescent nano particles hemolytic experiment in vinegar
Control group (non-stomach-filling fluorescent nano particles) mouse blood is taken, (4 DEG C, 3500 rpm, 15 min) of centrifugal blood are removed
Serum obtains erythrocyte, and rear spare, the addition gradient concentration 0.5 in cell culture fluid (MEM) is flushed three times with PBS solution,
The fluorescent nano particles solution of 1,2.5,5,10,15,20 mg/mL cultivates 120 min in insulating box；Negative control group be only to
Isometric PBS is added in cell culture fluid to remain unchanged with cellular morphology, positive controls are that isometric ultrapure water is only added to make
Cell rupture, centrifuging and taking supernatant surveys absorbance at 541 nm after 120 min are incubated for, using origin2015 software to reality
It tests result to be analyzed, experimental result: as shown in figure 4, after the fluorescent nano particles culture cell of gradient concentration is added, on cell
The no conspicuousness variation compared with negative control of the absorbance of clear liquid, i.e., fluorescent nano particles are to erythrocyte without significant molten
Blood effect, Fig. 5 are negative control (PBS), positive control (Water), low concentration fluorescent nano particles (0.5 mg/mL), in it is dense
The aspect graph of erythrocyte after degree fluorescent nano particles (5 mg/mL) and high concentration fluorescent nano particles (20 mg/mL) processing,
Erythrocyte form does not change compared with negative control group under the processing of low dosage fluorescent nano particles；Middle dosage fluorescence nano
There is the phenomenon identical as positive controls in erythrocyte part after particle processing, i.e. slight change occurs for cellular morphology；High agent
Treated erythrocyte the occurs apparent cellular morphology variation of amount fluorescent nano particles.
Claims (8)
1. in a kind of vinegar in fluorescent nano particles body distribution and biological safety evaluation method, which is characterized in that including with
Lower step:
A, rat renal tubular epithelial cells inoculation is cultivated in cell culture medium, is grown to pair to rat renal tubular epithelial cells
It is digested when the number phase, postdigestive rat renal tubular epithelial cells suspension carries out 24 h of adhere-wall culture, obtains adherent growth
Rat renal tubular epithelial cells；
B, in cell culture medium by fluorescent nano particles dispersion in vinegar, whirlpool concussion obtains fluorescent nano particles solution；
C, the fluorescent nano particles solution gradient in step B is diluted to several concentration, be added separately to step A obtain it is adherent
In the rat renal tubular epithelial cells of growth, 24 h are cultivated；
D, the rat renal tubular epithelial cells after culture are continued to cultivate in tetrazole indigo plant solution, to tetrazole indigo plant hydroponics
Dimethyl sulfoxide is added in rat renal tubular epithelial cells afterwards, measures light absorption value under wavelength, as test results, setting
Control group, the result of control group are by rat renal tubular epithelial cells successively through cell culture medium, tetrazole indigo plant solution and diformazan
The absorbance value measured after the processing of base sulfoxide solution, is calculated cell survival rate by test results and control group result；
E, postdigestive right in the vinegar fluorescent nano particles solution treatment steps A of the potential danger concentration obtained in step D
Number 24 h of phase rat renal tubular epithelial cells, forms single cell suspension after pancreatin digests, after being rinsed with phosphate buffer solution
Centrifugation is added fluidic cell buffer, measures the Survival of cell；Control group is set, and the result of control group is by Rat renal
The cell that Tubular epithelial cell successively measures after the processing of cell culture medium, phosphate buffer solution and fluidic cell buffer solution
Apoptosis rate is calculated by test results and control group result in Survival；
F, young rat is divided into two groups, fluorescent nano particles in vinegar are dispersed in ultrapure water by control group and experimental group, whirlpool shake
It swings, obtains fluorescent nano particles solution, exposure, the experimental mice under the conditions of a certain concentration of Mouse oral take orally fluorescence nano
Particle solution, control group mice take orally NaCl aqueous solution, and the mouse for taking control group and experimental group to take orally different time respectively is main
Organ observes accumulation of the vinegar fluorescent nano particles in mouse organs；
G, fluorescent nano particles in vinegar are dispersed in phosphate buffer solution, it is molten to obtain fluorescent nano particles for whirlpool concussion
Liquid and gradient dilution are at several concentration；
H, control group mice blood is taken, by centrifugal blood except serum deprivation obtains erythrocyte, after being rinsed with phosphate buffer solution
It is spare, fluorescent nano particles solution obtained in step G, 120 min of incubated at room temperature, negative control are added in cell culture fluid
Group is remained unchanged for isometric phosphate buffer solution is only added into cell culture fluid with cellular morphology, and positive controls are only
Isometric ultrapure water, which is added, makes cell rupture, and centrifuging and taking supernatant surveys absorbance at 541 nm after 120 min incubation；
I, according to the bio distribution and biological safety of fluorescent nano particles in the above test overall merit vinegar.
2. the side with biological safety is distributed in a kind of evaluation vinegar according to claim 1 in fluorescent nano particles body
Method, it is characterised in that: the concentration gradient of fluorescent nano particles is 20 mg/mL of 0- in the step C.
3. the side with biological safety is distributed in a kind of evaluation vinegar according to claim 1 in fluorescent nano particles body
Method, it is characterised in that: the concentration gradient of fluorescent nano particles is 20 mg/mL of 0- in the step D, by the Rat renal after culture
Tubular epithelial cell continues to cultivate in 2-100 μ L tetrazole indigo plant solution, the rat renal tubule to after tetrazole indigo plant hydroponics
200 μ L dimethyl sulfoxides are added in epithelial cell, light absorption value are measured under 492 nm wavelength, as test results.
4. the side with biological safety is distributed in a kind of evaluation vinegar according to claim 1 in fluorescent nano particles body
Method, it is characterised in that: the concentration gradient of fluorescent nano particles is 0.01-2 mg/mL in the step E.
5. the side with biological safety is distributed in a kind of evaluation vinegar according to claim 1 in fluorescent nano particles body
Method, it is characterised in that: using 4-5 week old male mice, the exposure concentrations of fluorescent nano particles are for test in the step F
0.1-2 g kg-1× mouse weight (kg).
6. the side with biological safety is distributed in a kind of evaluation vinegar according to claim 1 in fluorescent nano particles body
Method, it is characterised in that: mouse major organs include stomach, intestines, kidney, liver, lung and brain in the step F.
7. the side with biological safety is distributed in a kind of evaluation vinegar according to claim 1 in fluorescent nano particles body
Method, it is characterised in that: in the step G concentration gradient of fluorescent nano particles be 0.5 mg/mL, 1 mg/mL, 2.5 mg/mL,
5 mg/mL, 10 mg/mL, 15 mg/mL and 20 mg/mL.
8. the side with biological safety is distributed in a kind of evaluation vinegar according to claim 1 in fluorescent nano particles body
Method, it is characterised in that: the parameter being centrifuged in the step H is set as 4 DEG C, 3500 rpm, 15 min.
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