CN110479205A - A kind of drug screening magnetic Nano material and preparation method and application based on cell membrane - Google Patents
A kind of drug screening magnetic Nano material and preparation method and application based on cell membrane Download PDFInfo
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Abstract
High expressed receptor cell membrane puppet is mounted in the magnetic nano-particle surface containing aldehyde radical, obtains the drug screening magnetic Nano material based on cell membrane by a kind of drug screening magnetic Nano material and preparation method and application based on cell membrane.The abundant surface aldehydes of nano particle make nano particle covalent coupling stablize adherent cell film and reacting with the amino of exposure on cell membrane phospholipid surface in the material, and cell membrane will not be destroyed in whole process.Drug screening magnetic Nano material based on cell membrane has preferable adsorptivity, compared with strong selectivity and affinity, in the screening that can be applied to complicated natural products.
Description
Technical field
The invention belongs to biomimetic material technology field, be related to a kind of drug screening magnetic Nano material based on cell membrane and
Preparation method and application.
Background technique
Magnetic nano-particle has the unique physicochemical characteristics such as efficient superparamagnetism energy and bigger serface, in life
Application is more and more common in object field.In recent years magnetic Nano technology achieves major progress during natural products is found.
Most importantly the suitable coating of nanotechnology, suitable coating are magnetic nano-particles in entire natural products discovery procedure
Prerequisite, it not only increases the stability of magnetic nano-particle, and specific target spot can pass through and increase specific target
Into reduction simultaneously and complex sample, the combination of non-specific compound, enables magnetic nano-particle effectively to catch theirs
Target.Therefore, the method for selecting efficient functional magnetic nano particle can aid in efficient to the generation of its target special
Property screening.
In the recent period, cell membrane is used to cause more concerns as the Bionic Design strategy of functional coating, by from nature
Mentality of designing is obtained in boundary, the basic biological form of cell membrane such as can be integrated into synthesis to its shape and flexibility and receive
In rice system.Especially it can simulate the overall surface structure of source cell, and nano particle is made to have membrane molecule not
Same inherent characteristic.Currently, cell membrane camouflage nanoparticle technology has been used for screening bioactive compound from complex matrices,
This drug discovery method is compared with the traditional method the discovery efficiency for substantially increasing reactive compound in natural products.However,
It is worth noting that since cell membrane is easy to fall off from carrier during recycling, currently used cell membrane stealth side
The stability of method is lower.Improve the stability of cell membrane stealth nano particle, develops more stable and effective cell membrane stealth work
Tool is of great significance.
Chinese medicine is the rarity of Chinese traditional culture, and the Chinese material medicine resource in China is abundant.Nobel's physiology in 2015
Or medicine winner slaughter cry of a deer once said " traditional Chinese medicine is a great treasure-house, should make great efforts to excavate, be improved ".Therefore,
Chinese medicine can be used as the natural origin of drug screening and discovery.However, due to Chinese medicinal composition complicated component and trace constituent is more,
Screening active constituent is difficult directly from Chinese medicine and takes time and effort.There is theory studies have shown that in this complex system of Chinese medicine
In, not all ingredient is all generating pharmacological action, wherein only a small amount of active constituent has purpose activity, remaining
Fractions can not generate the pharmacological action of expectation, may contain serious toxic side effect what is more, aggravate the state of an illness.Therefore,
It is imperative to guarantee that tcm product is safe and effective to illustrate Chinese medicine mechanism of action, find and confirm active constituent in Chinese medicine.Base
In this, inventing one kind there is high stability cellular membrane biomimetic magnetic Nano material to screen reactive compound from Chinese medicine is very
Significant.
Summary of the invention
The drug screening magnetic Nano material and preparation method and answer that the object of the present invention is to provide a kind of based on cell membrane
With.
The present invention to achieve the above object, adopts the following technical scheme that:
A kind of preparation method of the drug screening magnetic Nano material based on cell membrane, by Fe3O4@SiO2@NH2Nanometer
Grain is handled through glutaraldehyde solution, obtains Fe3O4- CHO nano particle；
Cultivate high express alpha1A293 cell of HEK of-AR, and cell is collected in logarithmic phase, cell is resuspended in Tris-HCl
In, by ultrasound, centrifugation obtains membrane suspensions；
Utilize 2~6mg of 2mL mL-1Membrane suspensions and 8~12mg Fe3O4Covalently knot occurs for-CHO nano particle
It closes, the drug screening magnetic Nano material based on cell membrane is made.
A further improvement of the present invention lies in that Fe3O4@SiO2@NH2Nano particle is obtained by following procedure: by Fe3O4@
SiO2Nano particle is dispersed in dry toluene, and APTES is added after ultrasonic treatment, then in 110~130 DEG C, N2The lower stirring of protection
22~26 hours, obtain Fe3O4@SiO2@NH2Nano particle.
A further improvement of the present invention lies in that Fe3O4@SiO2Nano particle is obtained by following procedure: by 0.1g Fe3O4
Magnetic nanoparticle is dispersed in the mixed solution of ethyl alcohol and ultrapure water, is then added with stirring ammonia spirit, is mixed
Object is closed, TEOS is then added and is separated with external magnetic field after uniform stirring 7~9 hours, obtains Fe3O4@SiO2Nano particle.
A further improvement of the present invention lies in that by FeCl under ultrasound condition3·H2O and trisodium citrate are dissolved in second two
In alcohol, after the lower stirring of NaAc heating being then added 30 minutes, then carries out hydro-thermal reaction 9~11 hours at 190~210 DEG C, obtain
To Fe3O4Magnetic nano-particle.
A further improvement of the present invention lies in that by high express alpha1A293 cell of HEK of-AR is cultivated in DMEM culture medium,
High express alpha1A293 cell of-AR HEK contains 5%CO at 37 DEG C2Humid atmosphere in grow, collect the height that grows from exponential phase
Express alpha1AThen the cell of harvest is resuspended in Tris-HCl and is passed through ultrasonic disruption, filters by 293 cell of-AR HEK,
Film sediment is obtained, film sediment is suspended in PBS solution, membrane suspensions are obtained.
A further improvement of the present invention lies in that containing 10% fetal calf serum of volumetric concentration, 100UmL in DMEM culture medium-1
Streptomysin, 300mgL-1Geneticin and 100UmL-1Penicillin；The pH=7.4 of Tris-HCl.
A further improvement of the present invention lies in that utilizing membrane suspensions and Fe3O4Covalently knot occurs for-CHO nano particle
It closes, the detailed process of the drug screening magnetic Nano material based on cell membrane is made are as follows: by membrane suspensions and Fe3O4-CHO
Nano particle ultrasonic mixing at vacuum, 2~6 DEG C is uniform, obtains the drug screening magnetic Nano material based on cell membrane；Its
In, the concentration of membrane suspensions is 2~6mg mL-1, volume 2mL, Fe3O4The quality of-CHO nano particle is 8~12mg.
A kind of drug screening magnetic Nano material based on cell membrane being prepared according to the above method.
A kind of drug screening magnetic Nano material based on cell membrane as described above is in the screening of complicated natural products
Application.
A further improvement of the present invention lies in that complicated natural products is natural extracts, Chinese medical extract or contains more
The mixture of kind chemical component.
Compared with prior art, the invention has the following advantages:
Bioabsorbable carrier material of the present invention is Fe3O4@SiO2@NH2Magnetic nano-particle, magnetic nano-particle tool
Have higher specific surface area, superparamagnetism, while there is magnetic responsiveness and surface-functional, can under externally-applied magnetic field quickly,
Target molecule is efficiently separated from medium.Therefore, competent cell film is combined with magnetic nano-particle, preparation keeps cell
The stereochemical structure and bioactivity of film, while having the characteristics that the magnetic cell film solid phase extraction material of quick separating, pass through magnetism
Cell membrane Solid Phase Extraction new method, may be implemented the fast enriching of micro constitutent in complex sample, have to analysis efficiency is improved
Important meaning.
The present invention has high stability using the cell membrane magnetic Nano material of aldehyde radical method of modifying preparation.It is common at present
Method in, the binding force between nano particle and cell membrane is hydrophobic interaction, but since hydrophobic interaction is too weak
And causing the stability in actual cycle application very poor, cell membrane may fall off from carrier surface in screening process.And
Aldehyde radical can be reacted with the amino of cell surface, form stable amine key without being broken.The present invention utilizes Fe3O4- CHO nanometers
The abundant surface aldehydes of particle make nano particle covalent coupling and reacting with the amino of exposure on cell membrane phospholipid surface
Stablize adherent cell film, and cell membrane will not be destroyed in whole process.Aldehyde group modified is to cell membrane stealth nanotechnology
The suitable and effective improvement of stability.
Biomaterial of the present invention is cell membrane, modern pharmacology research show on drug and cell membrane by
Body specific binding is the key that it plays a role in vivo.Current verified cell-membrane receptor is main in conjunction with drug
Target plays an important role in the bioelectric interface of cell.At least the drug of 50-60% be proved with its specific cell membrane by
Body interacts to generate healing effect.Therefore, cell membrane pretends nano particle strategy compared with traditional drug discovery method
Have the advantages that specific high and rapidly and efficiently.
Drug screening magnetic Nano material prepared by the present invention based on cell membrane has preferable adsorptivity, relatively strong selection
Property and affinity, in the screening that can be applied to complicated natural products.
Detailed description of the invention
Fig. 1 is α1A/ MNGs and α1A/Fe3O4The rate of recovery of-the OHs to Tamsulosin.
Fig. 2 is α1ADLS, FT-IR, XRD and the VSM characterization result of/MNGs, wherein A is DLS as a result, a is high in figure A
α1AThe particle size and zeta current potential that-AR expresses HEK293 Cell membrane vesicles are as a result, b is Fe3O4The partial size of-CHO nano particle
Size and zeta current potential are as a result, c is α1AThe particle size and zeta current potential result of/MNGs；B is FT-IR spectral results, is schemed in B,
A is Fe3O4, b Fe3O4-SiO2, c Fe3O4-CHO；C is XRD, is schemed in C, a Fe3O4, b Fe3O4- CHO, c α1A/
The XRD spectrum of MNGs；Figure D is VSM curve, is schemed in D, a Fe3O4, b Fe3O4- CHO, c α1A/MNGs。
Fig. 3 is MNGs and α1AThe TEM image and MNGs and α of/MNGs1AThe confocal images of/MNGs.Wherein,
A is the TEM image of MNGs, b α1AThe TEM image of/MNGs；C is Laser Scanning Confocal Microscope figure of the MNGs under 488 excitation wavelengths
Picture, d are confocal images of the MNGs under 549nm excitation wavelength, e α1A/ MNGs being total under 488nm excitation wavelength
Focusing microscope image, f α1AConfocal images of/the MNGs under 549nm excitation wavelength, green fluorescence (c, e) generation
Table Fe3O4- CHO nano particle, red fluorescence (d, f) represent cell membrane.
Fig. 4 is α1AThe absorption property result of/MNGs.Wherein, A is Static Adsorption as a result, B is that Freundlich thermoisopleth is quasi-
Close result；C is α1AExtraction results of/the MNGs and MNGs to five kinds of different drugs.
Fig. 5 is for five kinds of different solutions to positive drug in α1AThe elution efficiency of/MNGs and MNGs, wherein a is water, and b is
PBS, c are water-methanol (2:8, v/v), and d is 0.1% acetic acid solution, and e is 1% acetic acid solution.
Fig. 6 is α1AThe chromatogram of radix aconiti agrestis extract before and after/MNGs Screening Treatment.Wherein, (a) is to scheme in (b) at peak 1
Enlarged drawing (b) is chromatogram, is (c) enlarged drawing at peak 2 in figure (b).Scheme in (b), a is that initial radix aconiti agrestis extracts solution, and b is
Solution after sample-adding, c are solution after elution, and d is the TOFMS and chemical structure at solution and the peak 1 and 2 in eluent after elution.
Specific embodiment
The present invention is described in detail with reference to the accompanying drawing.
High expressed receptor cell membrane puppet is mounted in the magnetic nano particle sublist containing aldehyde radical using covalent bond approach by the present invention
Face obtains the drug screening magnetic Nano material based on cell membrane.In the material the abundant surface aldehydes of nano particle by with
The amino of exposure reacts and nano particle covalent coupling is made to stablize adherent cell film, and entire mistake on cell membrane phospholipid surface
Cell membrane will not be destroyed in journey.
The cell membrane drug screening magnetic Nano material can be applied in the screening of complicated natural products.
Complicated natural products includes natural extracts, Chinese medical extract or the mixture containing a variety of chemical components.Especially
It is applied in Chinese medical extract in the screening of active constituent.
Magnetic nano-particle is prepared using hydro-thermal method, Fe will be obtained after the siliconized processing of magnetic nano-particle3O4@SiO2Grain
Son handles obtained magnetic nano-particle using silylating reagent APTES, obtains Fe3O4@SiO2@NH2Nano particle；Finally
Handling through glutaraldehyde solution can be obtained Fe3O4- CHO nano particle.
On the other hand, high express alpha is cultivated1A293 cell of HEK of-AR, and cell is collected in logarithmic phase to prepare cell membrane
Material obtains membrane suspensions after a series of operation by centrifugation ultrasounds.Utilize membrane suspensions and Fe3O4- CHO receives
Covalent bond occurs for rice grain, finally successfully prepares bionical high stability cell membrane magnetic Nano material α1A/MNGs。
Embodiment 1
One, materials and methods
1.α1AThe preparation of/MNGs
1.1 Fe3O4The synthesis of-CHO nano particle
Pass through solvent structure Fe3O4Magnetic nano-particle.Concrete operations are in brief are as follows: will under ultrasound condition
FeCl3·H2O (3.60g) and trisodium citrate (0.72g) are dissolved in ethylene glycol (100mL), and NaAc (4.80g) then is added
After 50 DEG C are vigorously stirred 30 minutes, then mixture is sealed in the stainless steel autoclave of the Teflon lining of 100mL capacity
In.By stainless steel autoclave at 200 DEG C after 10 hours, products therefrom is washed six times altogether with first alcohol and water, can be obtained
Neutral products, as Fe3O4Magnetic nano-particle.
Synthesize Fe3O4@SiO2The datail description of nano particle, preparation is as follows: by 0.1g Fe3O4Magnetic nanoparticle is uniform
It is dispersed in the mixed solution of the ethyl alcohol after ultrasonic disperse (80mL) and ultrapure water (12mL), adds under then being stirred at 40 DEG C
Enter ammonia spirit (4mL, volumetric concentration 25%), obtain mixture, then TEOS (tetraethyl orthosilicate, 0.8mL) is added and is mixed
In object, after uniform stirring 8 hours, Fe is separated with external magnetic field3O4@SiO2Nano particle is finally washed altogether with methanol and ultrapure water
It washs six times, neutral products, as Fe can be obtained3O4@SiO2Nano particle.
Fe is prepared finally by APTES (3-aminopropyltriethoxysilane)3O4@SiO2@NH2Nano particle.Firstly,
By 0.4g Fe3O4@SiO2Nano particle is dispersed in 50mL dry toluene, and 4mL APTES is added in ultrasonic treatment after 30 minutes, will
N of the reaction mixture at 120 DEG C2Then gained nano particle is used dry toluene by the lower stirring of protection 24 hours respectively, methanol and
Water washing three times, obtains Fe3O4@SiO2@NH2Nano particle.
By 100mg Fe3O4@SiO2@NH2Nano particle immerses and in 100mL (2%v/v) glutaraldehyde solution, and in room
Temperature lower stirring 0.5-2 hours.After being finally washed with water three times, Fe can be synthesized3O4- CHO nano particle.
1.2 α1AThe synthesis of/MNGs
By high α1A293 cell line of HEK of-AR expression is containing 10% fetal calf serum of volumetric concentration, 100UmL-1Strepto-
Element, 300mgL-1Geneticin and 100UmL-1It is cultivated in the DMEM culture medium of penicillin.High α 1A-AR expresses HEK 293
Cell contains 5%CO at 37 DEG C2Humid atmosphere in grow.It is thin to collect the high α 1A-AR expression HEK 293 grown from exponential phase
Born of the same parents, and washed three times with physiological saline (pH=7.4) to prepare to prepare α1A/MNGs。
The separation of cell membrane is carried out according to the method for report.After being washed repeatedly three times with PBS solution, by the cell weight of harvest
It is suspended from 50mmolL-1In Tris-HCl (pH=7.4) and by ultrasonic disruption, three times, 30 minutes every time (42kHz,
100W).Filtering, obtains thick film sediment, then washs thick film sediment three times with 10mLPBS solution, in 12,000 × g, 4
It is centrifuged 20 minutes at DEG C, filters, obtain film sediment.It takes film sediment to be suspended in it in PBS solution, obtains 2~6mg mL-1
Membrane suspensions, then by 8~12mg Fe3O4- CHO nano particle is well dispersed in the membrane suspensions of 2mL, then
Ultrasound uniformly mixing under vacuum, can prepare α at 4 DEG C1A/MNGs。
The preparation process of MNG:MNG is prepared as it appears from the above, by cell membrane suspension same amount of normal saline unlike unique
It is substituted.
2.α1AThe absorption property of/MNGs is investigated
In order to investigate α1AThe adsorption property of/MNGs carries out adsorption experiment as follows.All experiments are divided into triplicate below
It carries out.
By using 2mL centrifuge tube by 5.0mg α1A/ MNGs, MNGs are added to 60 to 3500mg L-1The Tan Suo of (1mL)
Adsorption isotherm experiment is carried out in the graded series concentration of Rosin.It seals all mixtures and is vibrated 2 hours at 37 DEG C.It is additional
Magnetic field separation analyzes supernatant by HPLC to determine the concentration of Tamsulosin.
Adsorption capacity (Q) can be calculated according to the following formula:
Wherein, Q (mg g-1) it is Tamsulosin to α1AThe adsorbance of/MNGs or MNGs, Co(mol L-1) it is that absorption is preceding smooth
The initial concentration of Suo Luoxin, CeIt is the Tamsulosin concentration of supernatant after adsorbing, V (mL) is the body of initial Tamsulosin solution
Product, m (g) is α1AThe quality of/MNGs or MNGs.
3.α1AThe practical application of/MNGs
Radix aconiti agrestis is purchased from Xi'an medical market (Xi'an, China).In order to extract the more multicomponent in radix aconiti agrestis as far as possible, extract
Process is by 10g radix aconiti agrestis grind into powder and reflux 2 hours in 100mL ethyl alcohol (60%, v/v).After extracting three times, Bu Shi is used
Funnel filtering total extract is simultaneously condensed into dark brown color substance, can be used as sample dissolution.
By 50mg α1A/ MNGs is mixed with radix aconiti agrestis total extract solution.Then mixed liquor is ultrasonically treated and is vibrated at 37 DEG C
20 minutes, then additional magnet separating alpha1A/ MNGs uses 1% acetic acid of 0.1% acetic acid solution of 5mL and 5mL under ultrasonic treatment respectively
Solution elution and elution α1AEach 20 minutes of compound on/MNGs.Externally-applied magnetic field separates and collects each section liquid and is evaporated.With
0.5mL methanol re-dissolves and is used for further HPLC analysis.
Two, experimental result
1.α1AThe preparation of/MNGs
As reactive compound screening implement, α1A/ MNGs should be more effective and be stablized.The cell membrane camouflage magnetic reported before
Property nanoparticle using the defect that for several times will appear stability difference afterwards.Therefore, α is improved1AThe stability of/MNGs is this research
It is crucial.In order to study their stability, it is prepared for three crowdes of α1A/ MNGs and high α1A- AR expresses the camouflage of HEK293 cell membrane
Fe3O4- OH nano particle (α1A/Fe3O4- OHs) and be added separately in Tamsulosin reference substance solution entirely screened
Journey.During this investigation it turned out, being chosen as α according to document Tamsulosin1APositive drug.It will be seen from figure 1 that with absorption-solution
The increase of sorption cycle number, α1A/Fe3O4- OHs is decreased obviously the rate of recovery of Tamsulosin.It is attached by 5 absorption-desorptions
After circulation, α1A/Fe3O4The rate of recovery of-OHs is only 65.4%, and compared with the rate of recovery after use for the first time, loss late is close
25%.However, the α after 5 attached circulations of absorption-desorption1AThe adsorption capacity of/MNGs only damages 3.4%, under the rate of recovery is not apparent
Drop, α1A/ MNGs shows stable performance.α1A/Fe3O4The rate of recovery loss of-OHs may be due to cell membrane and nano particle
Between weak hydrophobic interaction binding force it is weaker, lead to falling off to final in cycling elution and elution process cell membrane
Cause adsorption capacity constantly to reduce.However for α1AFor/MNGs, Fe3O4The free aldehyde of-CHO nano grain surface can be with
It is reacted with the amino rich in cell membrane phospholipid, covalent bond more stronger than hydrophobic interaction is formed, so that α1A/ MNGs has good
Stability.Therefore it is concluded that α1A/ MNGs is to have good stability for subsequent practical application.
2.α1AThe characterization of/MNGs
In order to study α1AThe physicochemical property of/MNGs has carried out DLS, FT-IR, XRD and VSM characterization research to it.Characterization knot
Fruit is as follows, as shown in the result of the DLS of A in Fig. 2, α1AThe size of/MNGs is slightly larger than Fe3O4The size of-CHO nano particle
And α1AThe zeta current potential and high α on the surface /MNGs1AThe zeta current potential that-AR expresses HEK293 Cell membrane vesicles is similar, thus may be used
To think that cell membrane is successfully wrapped in Fe3O4The surface of-CHO, in other words, α1A/ MNGs is successfully prepared.This
Outside, as in Fig. 2 B show Fe3O4,Fe3O4-SiO2And Fe3O4The FT-IR characterization result of-CHO, 578cm-1The absorption peak at place
It is attributed to the stretching vibration of Fe-O, illustrates Fe3O4Magnetic core is successfully prepared.802,948,1095cm-1The absorption peak at place illustrates SiO2
The presence of shell, this is attributed to the stretching vibration of Si-O, Si-O-H and Si-O-Si respectively.2830cm-1and 1720cm-1Place
Peak is attributed to the stretching vibration of C-H and C=O respectively, it was demonstrated that the successful synthesis of aldehyde radical.Therefore it can be concluded that Fe3O4- CHO nanometers
The conclusion that particle is successfully prepared.
As C is the Fe of preparation in Fig. 23O4, Fe3O4- CHO's and α1AThe XRD of/MNGs is investigated as a result, XRD can study object
The crystal structure of matter.Obviously six characteristic peaks of three kinds of materials are mainly observed within the scope of 20-80 ° of 2 θ.Their XRD spectrum
With Fe3O4Standard pattern XRD spectrum it is consistent, be six diffraction maximums: (220), (311), (400), (422), (511) and
(440), this standard diagram is obtained in JCPDS-International diffraction data center (No. JCPDS: 19-629) file
's.It was therefore concluded that the modification of Si-OH and cell membrane, which wrap up these surface modifications, will not change Fe3O4Magnetic core knot
Structure.Magnetic characteristic for preparation nano-bionic material screening and isolating target compound in application be vital.
Therefore, Fe3O4,, Fe3O4- CHO's and α1AThe hysteresis loop of/MNGs is as shown in D in Fig. 2.Hysteresis loop to three kinds of materials almost
There is no coercivity and remanent magnetism, shows Fe3O4,, Fe3O4- CHO's and α1A/ MNGs has satisfactory superparamagnetism, they
Saturation magnetization is respectively 52,28,15emug-1.These three materials can be controlled by magnetic field, even if saturation magnetization
Due to SiO2The formation of shell and cell putamina wrapping layer and reduce, also can in seconds by external magnetic field completely rapidly
It is separated from complex sample.The results show that once applying or removing external magnets, α during practical application1A/ MNGs energy
Enough rapid aggregations form stable and uniform suspension.
Fe is wrapped in order to verify3O4The presence of cell membrane on-CHO nano particle, has carried out TEM and copolymerization is burnt micro-
Mirror visualization is investigated.The TEM as shown in a and b in Fig. 3 is as a result, and Fe3O4- CHO nuclear phase ratio, α1A/ MNGs shows cell membrane
The typical nucleocapsid structure of Encapsulation nanoparticle.Cell membrane coat is in Fe3O4Ring is formed around-CHO, shows to be successfully prepared α1A/
MNGs.Confocal microscopy is additionally carried out, (FITC, excitation/emission are with Green fluorescent dye during the preparation process
488nm/520nm) construct Fe3O4- CHO nano particle, and with Erythrocyte membrane lipid bilayer red fluorescence dyestuff (DiI, excitation/hair
It penetrates and marks cell membrane for 549nm/565nm).As a result as shown in c, d, e and f in Fig. 3, green fluorescence comes from Fe3O4- CHO core,
Red fluorescent comes from cell membrane, and the two shows good common location.Therefore it may be concluded that cell membrane successfully wraps
Wrap up in Fe3O4On the surface of-CHO nano particle.
3.α1AThe absorption property of/MNGs is investigated
α1AThe absorption property of/MNGs is influence factor important in practical applications, therefore is ground by static and selectivity
Study carefully and is investigated.As a result as shown in A in Fig. 4, as Tamsulosin concentration is from 60mg mL-1Increase to 2000mg mL-1, α1A/ MNGs increases sharply to the adsorption capacity of Tamsulosin when starting, and then slows down, then adsorption capacity becomes stable
And reach saturation.Final saturated extent of adsorption is 43.35mg g-1.In addition, α1A/ MNGs is clearly remote to the adsorbance of Tamsulosin
Higher than MNGs to the adsorbance of Tamsulosin.Then respectively by Freundlich, Langmuir, Scatchard and Dubinin-
Radushkevich isotherms is further processed the data obtained, to assess α1ABinding characteristic (B and the table in Fig. 4 of/MNGs
1).It can clearly observe, Freundlich thermoisopleth (FI) model can describe Static Adsorption well, and have most
High related coefficient is (for α1A/ MNGs, r 0.9917, for MNGs, r 0.9746).FI is based on equation (1) and equation
(2) widely used model.
lgQe=lgKF+mlgCe (2)
Wherein, Qe(mg g-1) and Ce(mg L-1) concentration and liquid phase of the Tamsulosin of solid phase when respectively representing adsorption equilibrium
In Tamsulosin concentration.KFIt is constant with m.KFTotal binding number of loci, i.e. adsorption capacity are represented, m represents heterogencity
Index.The results are shown in Table 1, α1AThe K of/MNGsFFor 8.61Lmg-1, m 0.9079.
In order to study α1AThe selectivity of/MNGs, 5 kinds of different drugs such as Valsartans of experimental selection, captopril, Xi Luoduo
Pungent, oxymetazoline and Tamsulosin carry out α1A/ MNGs is selectively investigated.Wherein, silodosin, oxymetazoline and Tamsulosin are made
To act on α1AThe positive drug of-AR.As a result as shown in C in Fig. 4, hence it is evident that observe silodosin, oxymetazoline and Tan Suoluo
It is pungent by α1AThe complete slective extraction of/MNGs, their rate of recovery are apparently higher than MNGs to their rate of recovery.At the same time, α1A/
MNGs and MNGs does not extract the two negative drugs of Valsartan and captopril.These are the result shows that α1A/ MNGs pairs with
α1AThe drug that-AR can interact has strong selectivity and affinity.
When elution, the selection of eluent is most important in application process.This experimental study water, phosphate buffer
(PBS), five kinds of water-methanol (2:8, v/v), 0.1% glacial acetic acid and 1% glacial acetic acid solvents are to MNGs and α1AThe sun of/MNGs absorption
The rate of recovery of property medicine Tamsulosin.From figure 5 it can be seen that 0.1% glacial acetic acid only has good eluting power to MNGs, and
1% glacial acetic acid is to MNGs and α1A/ MNGs has preferable eluting rate.Therefore, 0.1% acetic acid solution and 1% acetic acid solution are selected
Respectively as leacheate and eluent.
4.α1AThe practical application of/MNGs
The magnetic bio material of this experiment invention is intended to provide quickly, simply, targeting and effective Sample Pretreatment Technique Used,
The technology can be used for from compound quickly finding and screen target compound.In order to determine the applicability of proposed material, make
Use α1AThe extract of/MNGs centering medicinal herbs crow is extracted, as the result is shown in Fig. 5.(a), (b) can with (c) from Fig. 6
It arrives, compared with Chinese medicine radix aconiti agrestis extract solution chromatographic peak, a large amount of components are leached in screening process, final two radix aconiti agrestis
Main component is by α1A/ MNGs is screened.It in order to identify two kinds of screen fractions, is further analyzed by TOFMS, as the result is shown two
A peak is accredited as benzoyl time aconine and bulleyaconitine A respectively.It is inferred above in order to verify, use α1AAt/MNGs screening
Managed the reference substance solution of processing benzoyl time aconine and bulleyaconitine A, acquired results and front it is consistent.Prove experiment
The component filtered out is determined as benzoyl time aconine and bulleyaconitine A.
Embodiment 2
By FeCl under ultrasound condition3·H2O and trisodium citrate are dissolved in ethylene glycol, are then added under NaAc heating
After stirring 30 minutes, then carries out hydro-thermal reaction 11 hours at 190 DEG C, obtain Fe3O4Magnetic nano-particle.
By Fe3O4Magnetic nanoparticle is dispersed in the mixed solution of ethyl alcohol and ultrapure water, is then added with stirring ammonia
Aqueous solution obtains mixture, and TEOS is then added and is separated with external magnetic field after uniform stirring 7 hours, obtains Fe3O4@SiO2It receives
Rice grain.
By Fe3O4@SiO2Nano particle is dispersed in dry toluene, and APTES is added after ultrasonic treatment, then 110 DEG C,
N2Protection lower stirring 26 hours, obtain Fe3O4@SiO2@NH2Nano particle.
By Fe3O4@SiO2@NH2Nano particle is handled through the glutaraldehyde solution that volumetric concentration is 1%, obtains Fe3O4- CHO receives
Rice grain；
By high express alpha1A293 cell of HEK of-AR is cultivated in DMEM culture medium, high express alpha1A293 cell of-AR HEK
At 37 DEG C, contain 5%CO2Humid atmosphere in grow, collect the high express alpha that grows from exponential phase1A293 cell of-AR HEK,
Then the cell of harvest is resuspended in Tris-HCl to and is passed through ultrasonic disruption, filtering obtains film sediment, by film sediment
It is suspended in PBS solution, obtains membrane suspensions.Wherein, 10% fetal calf serum of volumetric concentration is contained in DMEM culture medium,
100U·mL-1Streptomysin, 300mgL-1Geneticin and 100UmL-1Penicillin；The pH=7.4 of Tris-HCl.
By membrane suspensions and Fe3O4- CHO nano particle ultrasonic mixing at vacuum, 2 DEG C is uniform, obtains based on cell
The drug screening magnetic Nano material of film；Wherein, the concentration of membrane suspensions is 2mgmL-1, volume 2mL, Fe3O4-
The quality of CHO nano particle is 8mg.
Embodiment 3
By FeCl under ultrasound condition3·H2O and trisodium citrate are dissolved in ethylene glycol, are then added under NaAc heating
After stirring 30 minutes, then carries out hydro-thermal reaction 9 hours at 210 DEG C, obtain Fe3O4Magnetic nano-particle.
By Fe3O4Magnetic nanoparticle is dispersed in the mixed solution of ethyl alcohol and ultrapure water, is then added with stirring ammonia
Aqueous solution obtains mixture, and TEOS is then added and is separated with external magnetic field after uniform stirring 9 hours, obtains Fe3O4@SiO2It receives
Rice grain.
By Fe3O4@SiO2Nano particle is dispersed in dry toluene, and APTES is added after ultrasonic treatment, then 130 DEG C,
N2Protection lower stirring 22 hours, obtain Fe3O4@SiO2@NH2Nano particle.
By Fe3O4@SiO2@NH2Nano particle is handled through the glutaraldehyde solution that volumetric concentration is 3%, obtains Fe3O4- CHO receives
Rice grain；
By high express alpha1A293 cell of HEK of-AR is cultivated in DMEM culture medium, high express alpha1A293 cell of-ARHEK
At 37 DEG C, contain 5%CO2Humid atmosphere in grow, collect the high express alpha that grows from exponential phase1A293 cell of-AR HEK,
Then the cell of harvest is resuspended in Tris-HCl to and is passed through ultrasonic disruption, filtering obtains film sediment, by film sediment
It is suspended in PBS solution, obtains membrane suspensions.Wherein, 10% fetal calf serum of volumetric concentration is contained in DMEM culture medium,
100U·mL-1Streptomysin, 300mgL-1Geneticin and 100UmL-1Penicillin；The pH=7.4 of Tris-HCl.
By membrane suspensions and Fe3O4- CHO nano particle ultrasonic mixing at vacuum, 6 DEG C is uniform, obtains based on cell
The drug screening magnetic Nano material of film；Wherein, the concentration of membrane suspensions is 6mgmL-1, volume 2mL, Fe3O4-
The quality of CHO nano particle is 12mg.
The present invention provides a kind of cell membrane camouflage strategy based on functionalized nano particle of stability with enhancing.And
This strategy is used to target and screen the bioactive compound from Chinese medicine for the first time.Pass through aldehyde group modified preparation α1A/MNGs。
After five adsorption-desorption cycle uses, α1AThe rate of recovery of/MNGs is stablized, and the present invention takes in terms of improving quality and stability
Obtained remarkable progress.The present invention provides unique function for the magnetic nano-particle that cell membrane pretends, in screening bioactivity
There is advanced stability, and modified can be further improved of effective aldehyde radical finds day from complex matrices in terms of compound
The stability of right product.The present invention can widen the approach and method of cell membrane Yu nanoparticle Platform integration.
Claims (10)
1. a kind of preparation method of the drug screening magnetic Nano material based on cell membrane, which is characterized in that
By Fe3O4@SiO2@NH2Nano particle is handled through glutaraldehyde solution, obtains Fe3O4- CHO nano particle；
Cultivate high express alpha1A293 cell of HEK of-AR, and cell is collected in logarithmic phase, cell is resuspended in Tris-HCl, is led to
Ultrasound is crossed, is centrifuged, obtains membrane suspensions；
Utilize membrane suspensions and Fe3O4Covalent bond occurs for-CHO nano particle, and the drug screening magnetic based on cell membrane is made
Property nano material.
2. the preparation method of the drug screening magnetic Nano material according to claim 1 based on cell membrane, feature exist
In Fe3O4@SiO2@NH2Nano particle is obtained by following procedure: by Fe3O4@SiO2Nano particle is dispersed in dry toluene,
APTES is added after ultrasonic treatment, then in 110~130 DEG C, N2Protection lower stirring 22~26 hours, obtain Fe3O4@SiO2@NH2
Nano particle.
3. the preparation method of the drug screening magnetic Nano material according to claim 2 based on cell membrane, feature exist
In Fe3O4@SiO2Nano particle is obtained by following procedure: by Fe3O4Magnetic nanoparticle is dispersed in ethyl alcohol and ultrapure water
Mixed solution in, be then added with stirring ammonia spirit, obtain mixture, be then added TEOS, uniform stirring 7~9 hours
Afterwards, it is separated with external magnetic field, obtains Fe3O4@SiO2Nano particle.
4. the preparation method of the drug screening magnetic Nano material according to claim 3 based on cell membrane, feature exist
In by FeCl under ultrasound condition3·H2O and trisodium citrate are dissolved in ethylene glycol, and the lower stirring 30 of NaAc heating is then added
After minute, then carries out hydro-thermal reaction 9~11 hours at 190~210 DEG C, obtain Fe3O4Magnetic nano-particle.
5. the preparation method of the drug screening magnetic Nano material according to claim 1 based on cell membrane, feature exist
In by high express alpha1A293 cell of HEK of-AR is cultivated in DMEM culture medium, high express alpha1A293 cell of-AR HEK is 37
DEG C, contain 5%CO2Humid atmosphere in grow, collect the high express alpha that grows from exponential phase1A293 cell of-AR HEK, then
The cell of harvest is resuspended in Tris-HCl to and is passed through ultrasonic disruption, filtering obtains film sediment, film sediment is suspended
In PBS solution, membrane suspensions are obtained.
6. the preparation method of the drug screening magnetic Nano material according to claim 5 based on cell membrane, feature exist
In, in DMEM culture medium contain 10% fetal calf serum of volumetric concentration, 100UmL-1Streptomysin, 300mgL-1Geneticin and
100U·mL-1Penicillin；The pH=7.4 of Tris-HCl.
7. the preparation method of the drug screening magnetic Nano material according to claim 1 based on cell membrane, feature exist
In utilizing membrane suspensions and Fe3O4Covalent bond occurs for-CHO nano particle, and the drug screening magnetic based on cell membrane is made
The detailed process of property nano material are as follows: by membrane suspensions and Fe3O4- CHO nano particle ultrasound at vacuum, 2~6 DEG C is mixed
It closes uniformly, obtains the drug screening magnetic Nano material based on cell membrane；Wherein, the concentration of membrane suspensions is 2~6mg
mL-1, volume 2mL, Fe3O4The quality of-CHO nano particle is 8~12mg.
8. a kind of drug screening based on cell membrane that any one of -7 the methods according to claim 1 are prepared is magnetic
Nano material.
9. a kind of sieve of the drug screening magnetic Nano material based on cell membrane in complicated natural products as claimed in claim 8
The application chosen.
10. application according to claim 9, complicated natural products is natural extracts, Chinese medical extract or contains more
The mixture of kind chemical component.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113189217A (en) * | 2021-04-12 | 2021-07-30 | 西安交通大学 | Magnetic nano material with cell membrane reversely coated and preparation method and application thereof |
| CN115121225A (en) * | 2022-06-27 | 2022-09-30 | 中国药科大学 | Magnetic liposome for enriching THC, preparation method and application thereof |
| CN115389659A (en) * | 2022-08-23 | 2022-11-25 | 浙江中医药大学 | Cell membrane bonded magnetic carbon sphere composite material and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337247A (en) * | 2011-09-13 | 2012-02-01 | 西安交通大学 | Recombinant HEK293 cell with high FGFR4 expression and application thereof |
| US20130243685A1 (en) * | 2012-03-15 | 2013-09-19 | National Tsing Hua University | Oil Extractor and The Preparation Method Thereof |
| CN107064333A (en) * | 2016-10-19 | 2017-08-18 | 中国人民解放军第二军医大学 | A kind of membrane flexibility post of utilization APTES modifications and its preparation method and application |
-
2019
- 2019-08-15 CN CN201910754782.0A patent/CN110479205A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337247A (en) * | 2011-09-13 | 2012-02-01 | 西安交通大学 | Recombinant HEK293 cell with high FGFR4 expression and application thereof |
| US20130243685A1 (en) * | 2012-03-15 | 2013-09-19 | National Tsing Hua University | Oil Extractor and The Preparation Method Thereof |
| CN107064333A (en) * | 2016-10-19 | 2017-08-18 | 中国人民解放军第二军医大学 | A kind of membrane flexibility post of utilization APTES modifications and its preparation method and application |
Non-Patent Citations (1)
| Title |
|---|
| YUSI BU ETAL.: "Cell membrane camouflaged magnetic nanoparticles as a biomimetic drug discovery platform", 《CHEMICAL COMMUNICATION》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113189217A (en) * | 2021-04-12 | 2021-07-30 | 西安交通大学 | Magnetic nano material with cell membrane reversely coated and preparation method and application thereof |
| CN115121225A (en) * | 2022-06-27 | 2022-09-30 | 中国药科大学 | Magnetic liposome for enriching THC, preparation method and application thereof |
| CN115121225B (en) * | 2022-06-27 | 2024-04-19 | 中国药科大学 | Magnetic liposome for enriching THC, preparation method and application thereof |
| CN115389659A (en) * | 2022-08-23 | 2022-11-25 | 浙江中医药大学 | Cell membrane bonded magnetic carbon sphere composite material and preparation method and application thereof |
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