CN109576206A - A method of excretion body is separated based on combination method - Google Patents
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Abstract
The present invention provides a kind of method based on combination method separation excretion body, method including using filtering with microporous membrane removes the large scale microvesicle in sample, the small size foreign protein in sample is removed using the method for ultrafiltration, the higher excretion body of purity is obtained and cooperation can be separated by the compound of superparamagnetic nanoparticle and ligand.Excretion body size prepared by the present invention meets the theoretical size of excretion body in 50-100nm, and membrane structure is complete.This method is easy to operate, at low cost, and time-consuming is few.
Description
Technical field
The present invention relates to field of biotechnology, more specifically to a kind of method based on combination method separation excretion body.
Background technique
Excretion body is membrane vesicle of a kind of diameter in 40-100nm, is discharged by most cells from multivesicular body (MVB)
It include various biomolecule, such as DNA, RNA, protein in extracellular space.In recent years, excretion body was in cell communication
Important function caused extensive concern.For example, being originated from the excretion body of mescenchymal stem cell to Ischemic/reperfusion
Damage have certain protective role { Lai, Ruenn Chai, et al. " Exosome secreted by MSC reduces
myocardial ischemia/reperfusion injury."Stem cell research 4.3(2010)}.Due to outer
Secrete body from cell and can in intercellular trafficking different kind organism molecule, they be also predicted to be a new generation have good life
The drug delivery carrier of object compatibility and high-efficiency transfection efficiency.
In order to realize more efficient drug delivery, high-purity, high yield, the excretion body of structural integrity are quickly and effectively obtained
As a current big problem urgently to be solved.Currently, the main source of excretion body is cell culture medium, including tumor cell culture
Base and all kinds of normal tissue cell culture mediums.It is well known, however, that many membranaceous vesicas are discharged into microenvironment by eukaryocyte
In, these include excretion body, microvesicle, apoptosis vesicle and some cell debris etc..It is used at present separated from culture medium it is pure
It is most of to use differential supercentrifugation in the method for changing allochthon, first by low-speed centrifugal (such as 500g, 2000g) to remove
Complete cell, bulk cell fragment and some large scale vesicas are removed, is then precipitated with high speed centrifugation (for example, 100,000g)
Excretion body.This method pollution risk is low, and can collect a large amount of excretion body { Ohno, Shin-ichiro, et al. "
Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to
breast cancer cells."Molecular Therapy 21.1(2013)}.However, the centrifugal force of high speed may be broken
The membrane structure of bad excretion body is lost hereditary information molecule entrained on excretion body.The destruction of phospholipid bilayer also can shadow
Ring drug loading and releasability of the excretion body as Delivery vehicles.
The body fluids such as blood of healthy animal, saliva, urine, sperm, breast milk etc. are another preferred excretion body sources, because
Its biological safety is higher and quantity is more.The ultracentrifugal method of differential is applied equally to the excretion in separation body fluid source
Body.However for example each micromolecular foreign protein of the complicated ingredient in body fluid, protein aggregate and outer can be caused under high speed centrifugation
The co-precipitation for secreting body reduces excretion body purity.Recently, a kind of method for having developed immunomagnetic isolation, by by magnetic
Then pearl and ligand coupling specifically bind the receptor in excretion body surface face and then realize the separation of excretion body under magnetic fields
{Qi,Hongzhao,et al."Blood exosomes endowed with magnetic and targeting
properties for cancer therapy."ACS nano 10.3(2016)}.Although this method improves excretion body
Purity, but the smaller ligand to dissociate in body fluid can form competitive relation with magnetic bead-ligand complex, affect magnetic bead-and match
The combination of nanocrystal composition and excretion body, to reduce the efficiency of Magneto separate.
Summary of the invention
The purpose of the present invention overcomes deficiency in the prior art, provides a kind of side based on combination method separation excretion body
Method is removed in sample specifically, the present invention removes the large scale vesica in sample using miillpore filter using the method for ultrafiltration
Small molecule foreign protein, obtain the higher excretion body of purity in simple and easy method, and can by with superparamagnetic nanoparticle
Separating effect is further promoted with the compound of ligand.
Technical purpose of the invention is achieved by following technical proposals
A method of based on combination method separate excretion body, using micro porous filtration and ultrafiltration centrifugation combine by the way of into
Row:
Processing is filtered to sample to be separated using miillpore filter, to remove large scale microvesicle, controls miillpore filter
Aperture is greater than 0.1 μm；
In micro porous filtration, sample to be separated is tumor cell culture base, normal tissue cell culture medium, various organisms
Liquid, such as blood (serum), saliva, urine, sperm, breast milk.
In micro porous filtration, the material of miillpore filter is fibre resin film or nylon membrane, and the aperture of miillpore filter is greater than
0.12 μm, preferably greater than 0.15 μm, such as 0.1 μm-10 μm, micro porous filtration number are 1-100 times, preferably 10-80 times.
The filtrate that micro porous filtration obtains is carried out ultrafiltration centrifugal treating to obtain to remove small size foreign protein containing excretion
The supernatant of body, the molecular cut off of super filter tube are 1kDa-400kDa, in 3-5 DEG C of centrifugation 10-20min, centrifugal speed for 80 ×
g-12000×g。
In ultrafiltration centrifugation, the size of small size foreign protein be 30nm once, such as 10-30nm.
In ultrafiltration centrifugation, the molecular cut off 3kDa-300kDa (number is equal) of super filter tube, ultra-filtration conditions are 4 DEG C, 12-
18min, centrifugal speed are 100 × g-10000 × g；Ultra-filtration filters number is 1-100 times, preferably 10-60 times.
Ultrafiltration centrifugal treating sample to be separated can also be first carried out, to remove small size foreign protein, then carries out miillpore filter mistake
Filter, to remove large scale microvesicle, obtains the supernatant containing excretion body.
After carrying out excretion body separation by the way of combining using micro porous filtration and ultrafiltration centrifugation, may be optionally added super
The compound of paramagnetic nano particle and ligand further promotes separating effect,
The compound of superparamagnetic nanoparticle and ligand is superparamagnetic nanoparticle-ligand complex (SPMN- ligand), choosing
Prepared by the mercapto ligand reaction for selecting superparamagnetic nanoparticle and activation, ligand is reacted with 2- imino group thiophane to be activated
Mercaptan ylidene ligands, bibliography Qi, Hongzhao, et al. " Blood exosomes endowed with magnetic
And targeting properties for cancer therapy. " ACS nano 10.3 (2016) or superparamagnetic are received
Rice grain-polyethylene glycol-ligand complex (SPMN-PEG- ligand) selects the superparamagnetic nanoparticle of Pegylation and swashs
Mercapto ligand reaction preparation living, the superparamagnetism Fe of aminofunctional3O4Nano particle and active ester polyethylene glycol Malaysia acyl
Imines is reacted, to realize the Pegylation of superparamagnetic nanoparticle；Ligand reacts to obtain with 2- imino group thiophane
The mercaptan ylidene ligands of activation, specifically:
By the superparamagnetism Fe of aminofunctional3O4Nanoparticles solution and active ester polyethylene glycol maleimide (NHS-
PEG-MAL it) is mixed in borate buffer solution and mixture is incubated for 1-3h under room temperature (20-25 DEG C), to realize that superparamagnetic is received
The Pegylation of rice grain；Ligand is mixed in borate buffer solution with 2- imino group thiophane, in room temperature (20-25
DEG C) under be incubated for 1-3h, with the mercaptan ylidene ligands activated, the borate buffer solution containing EDTA is used to dialyse 3-5h；Finally will
Pegylation superparamagnetic nanoparticle and the ligand of activation are dispersed in borate buffer solution and at room temperature (20-25 DEG C)
After lower incubation 1-3h, obtain SPMN-PEG- ligand complex, or in isolated Pegylation superparamagnetic nanoparticle and
After the ligand of activation, directly the two is dispersed in again in aqueous solution or borate buffer solution, obtains the solution of the two
After carry out mixing incubation reaction.
Ligand is using CD63 antibody, CD9 antibody, CD81 antibody, CD82 antibody, ALIX antibody, Tsg101 antibody or turns
One of ferritin.
In the Pegylation of superparamagnetic nanoparticle, the number-average molecular weight of active ester polyethylene glycol maleimide is
1000-100000, Magneto separate time are 10-5000min；It is preferred that the number-average molecular weight of active ester polyethylene glycol maleimide is
4000-10000, Magneto separate time are 60-200min.
Ligand is with 2- imino group thiophane with molar ratio for (1-10000): (1-10000), preferably (10-1000):
(10-1000), more preferred (10-100): (600-1000), directly according to the quality of ligand and 2- imino group thiophane
The molal quantity of the two is calculated with molecular mass.
The superparamagnetism Fe of aminofunctional3O4The molar ratio of nano particle and active ester polyethylene glycol maleimide is
(1-10000): (1-10000), preferably (10-1000): (10-1000), more preferred (10-100): (500-1000), root
Its molal quantity is calculated according to the quality and molecular mass of active ester polyethylene glycol maleimide, according to the superparamagnetic of aminofunctional
Property Fe3O4The concentration and volume of nano particle calculate the superparamagnetism Fe of aminofunctional3O4The number of nano particle, then divided by
NA。
Compared with prior art, the separated excretion body purity of the present invention is higher, no protein masses and other particles
Pollution, and it is smaller to the damage of the membrane structure of excretion body, biomolecule therein is completely protected, and be conducive to subsequent with excretion
Body is drug loading and the release of pharmaceutical carrier；It prepares that resulting excretion body purity is higher and membrane structure is complete, carries a large amount of
Hereditary information molecule, and a series of carrier for therapeutic agents such as can be used as curative drug, human cytokines, therapeutic genes
(application of the excretion body obtained in such a way that micro porous filtration and ultrafiltration centrifugation combine in preparation therapeutic carrier),
It plays an important role in the various diseases such as all kinds of cancers, Parkinson's disease, skin injury, liver class disease, obesity, diabetes.For
Excretion body separating effect is further increased, selection uses the compound of superparamagnetic nanoparticle and ligand, can be quickly and effectively
Excretion body is separated from serum, the time of excretion body preparation is shortened, since isolated excretion body carries superparamagnetism
Nano particle (i.e. SMNC-EXOs), can be as the pharmaceutical carrier of targeted therapy, i.e., according to isolated outer of the method for the present invention
It secretes body and is preparing the application in target therapeutic agent carrier.
Detailed description of the invention
Fig. 1 is the particle size that the isolated excretion body of mode is combined using micro porous filtration of the present invention and ultrafiltration centrifugation
Characterization result schematic diagram.
Fig. 2 is the transmitted electron that the isolated excretion body of mode is combined using micro porous filtration of the present invention and ultrafiltration centrifugation
Microscope photo (1).
Fig. 3 is the transmitted electron that the isolated excretion body of mode is combined using micro porous filtration of the present invention and ultrafiltration centrifugation
Microscope photo (2).
Fig. 4 is the characterization schematic diagram for carrying out specific proteins to separated excretion body using immunoblotting.
Fig. 5 be using micro porous filtration of the present invention and ultrafiltration centrifugation combine mode and with the use of superparamagnetic nanoparticle and
The compound of ligand carries out the transmission electron microscope photo of isolated SMNC-EXOs.
Fig. 6 is to measure the contrast test of (remnants) ligand concentration that dissociates in serum after the ultrafiltration of different time or dialysis
Curve graph.
Fig. 7 is the separating rate contrast test curve of SPMN- ligand and SPMN-PEG- ligand to the excretion body of ultrafiltration serum
Figure.
Fig. 8 be using micro porous filtration of the present invention and ultrafiltration centrifugation combine mode and with the use of superparamagnetic nanoparticle and
The compound of ligand carries out the particle diameter distribution phenogram of isolated SMNC-EXOs.
Specific embodiment
The embodiment of the present invention is further illustrated combined with specific embodiments below.
Key instrument and reagent used in embodiment: aminofunctional superparamagnetism Fe3O4Nano particle (Nanjing nanometer
Biotechnology Co., Ltd)；CD63 antibody, CD9 antibody, CD81 antibody, CD82 antibody, ALIX antibody, Tsg101 antibody and turn
Ferritin (Sigma-Aldrich company)；Miillpore filter (Merck Millipore)；Super filter tube (Merck Millipore)；
TG16-WS table model high speed centrifuge (Hunan Xiang Yi Laboratory Instruments Co., Ltd)；Dynamic light scattering particle size instrument (U.S.'s Brooker
Hai Wen Co., Ltd)；Transmission electron microscope (Jeol Ltd.).
Embodiment 1
1. being filtered processing to MCF-7 cell culture medium using miillpore filter
The filtering with microporous membrane for being 0.1 μm by the MCF-7 cell culture medium via hole diameter of 100mL, is repeated 1 times, takes filtrate.
2. the filtrate that step 1 is obtained carries out hyperfiltration treatment
1 gained filtrate of above-mentioned steps is taken to be added in super filter tube in (molecular cut off 3kDa), in 4 DEG C of centrifugation 15min, from
Heart speed is 500 × g, is repeated 1 times.
3. the supernatant that above-mentioned steps 2 are obtained is resuspended, excretion body is obtained.
Embodiment 2
1. pair RAW264.7 cell culture medium carries out hyperfiltration treatment
The RAW264.7 cell culture medium of 100mL is added in super filter tube in (molecular cut off 400kDa), 3 DEG C from
Heart 20min, centrifugal speed are 2000 × g, are repeated 20 times, and are resuspended, take supernatant.
2. being filtered processing to the supernatant that step 1 obtains using miillpore filter
The filtering with microporous membrane that the supernatant via hole diameter that step 1 is obtained is 0.05 μm, is repeated 10 times, takes filtrate.
3. collecting filtrate, excretion body is obtained.
Embodiment 3
1. pair serum carries out hyperfiltration treatment
The serum of 1mL is added in super filter tube in (molecular cut off 10kDa), in 5 DEG C of centrifugation 10min, centrifugal speed
It for 5000 × g, repeats 30 times, resuspension takes supernatant.
2. being filtered processing to the supernatant that step 1 obtains using miillpore filter
The filtering with microporous membrane that the supernatant via hole diameter that step 1 is obtained is 12 μm repeats 50 times, takes filtrate.
3. collecting filtrate, excretion body is obtained.
Embodiment 4
1. pair breast milk carries out hyperfiltration treatment
The breast milk of 1mL is added in super filter tube in (molecular cut off 1kDa), in 4 DEG C of centrifugation 20min, centrifugal speed is
100 × g is repeated 100 times, and resuspension takes supernatant.
2. being filtered processing to the supernatant that step 1 obtains using miillpore filter
The filtering with microporous membrane that the supernatant via hole diameter that step 1 is obtained is 10 μm, is repeated 20 times, takes filtrate.
3. collecting filtrate, excretion body is obtained.
Embodiment 5
1. pair urine carries out hyperfiltration treatment
The urine of 1mL is added in super filter tube in (molecular cut off 150kDa), in 5 DEG C of centrifugation 12min, centrifugal speed
It for 12000 × g, repeats 70 times, resuspension takes supernatant.
2. being filtered processing to the supernatant that step 1 obtains using miillpore filter
The filtering with microporous membrane that the supernatant via hole diameter that step 1 is obtained is 0.05 μm repeats 90 times, takes filtrate.
3. collecting filtrate, excretion body is obtained.
Embodiment 6
1. pair saliva carries out hyperfiltration treatment
The saliva of 1mL is added in super filter tube in (molecular cut off 18kDa), in 5 DEG C of centrifugation 10min, centrifugal speed
It for 80 × g, is repeated 10 times, resuspension takes supernatant.
2. being filtered processing to the supernatant that step 1 obtains using miillpore filter
The filtering with microporous membrane that the supernatant via hole diameter that step 1 is obtained is 5 μm repeats 40 times, takes filtrate.
3. collecting filtrate, excretion body is obtained.
Using transmission electron microscope to excretion body prepared in the above embodiments carry out morphology characterization, the partial size of excretion body and
Pattern is consistent substantially, and as a result as shown in Fig. 1-3, the hydrodynamic diameter of prepared excretion body is 40-100nm, should
Size meets the theoretical ruler of excretion body, and partial size is more uniform, and the pollution of no large scale vesica and small molecule foreign protein shows made
Standby excretion body purity is higher, the pollution of no macromolecular vesica and small molecule foreign protein, and excretion body membrane structure keep compared with
Completely.Prepared excretion body white spherical shape under transmission electron microscope, has phospholipid bilayer film structure
Nanoscaled vesicle, for diameter in 40-100nm, partial size is more uniform.Excretion body purity prepared by the present invention of this surface is higher, without big
The pollution of molecule vesica and small molecule foreign protein, and the membrane structure of excretion body be kept as it is more complete.As shown in figure 4, prepared
Excretion body surface face detect two species specificity PROTEIN C D63 (about 32KDa) and transferrins (about 60KDa), this shows prepared
Product be excretion body.
Carry out cooperation processing using the two ligand on the basis of the embodiment of the present invention, using dynamic light scattering particle size instrument to point
From the characterization that product carries out size.As a result as shown in figure 8, the hydrodynamic diameter of prepared excretion body shows always,
For 50-150nm, partial size is more uniform, and purity is higher, the pollution of no large scale vesica and small molecule foreign protein, and the size meets
The theoretical size of SMNC-EXOs.The characterization of size is carried out to products therefrom using transmission electron microscope.As a result as shown in figure 5,
The form of prepared SMNC-EXOs be stain surround sacculus rotundus, superparamagnetic nano particle with radial distribution outside
It secretes around body.This radial distribution increases the number for taking aim at the superparamagnetic nano particle being scheduled on excretion body, by original
6-8 increases 12-16, and this give the higher magnetic responsivenesses of SMNC-EXOs.
For characterization the present invention in ultrafiltration centrifugation and SPMN-PEG- ligand efficiency, tested as follows: (1) through it is different when
Between ultrafiltration or dialysis after, measure the concentration of free ligand in serum, i.e., the concentration of remaining ligand in serum, not with SPMN-PEG
The ligand of functionalization is carried out, interference can be generated to SPMN-PEG- ligand, the fewer residual volume the better when separating excretion body；It is super
After filter centrifugation, the concentration of ligandin in retentate is measured by BCA assay kit；After dialysis, ligandin in retentate
Concentration be measured also by BCA assay kit.Shown in result figure 6, after ultrafiltration 30 minutes, free ligand is dense in serum
Degree is down to~0.08mg/mL, concentration of 30 minutes well below dialysis.In order to reach identical concentration, the time for needs of dialysing
Be far longer than ultrafiltration, show ultrafiltration can it is significant accelerate serum in free ligand elimination, when this can shorten the separation of excretion body
Between；(2) by SPMN- ligand (prior art before seminar) and SPMN-PEG- ligand (present invention preparation) respectively with ultrafiltration blood
Clear mixing, is incubated for same time.After incubation, mixture is placed in different time sections in magnetic field.Then supernatant is abandoned, PBS is used
Three times by the sample washing of Magnetic Isolation.Finally, sample is resuspended in PBS respectively, and institute is measured by BCA assay kit
Obtain the protein concentration of solution.SPMN- ligand and SPMN-PEG- ligand are compared to the separation speed of the excretion body of ultrafiltration serum
Degree, protein concentration are used to indicate the quantity of excretion body.As a result as shown in fig. 7, SPMN-PEG- ligand separation after forty minutes it is outer
It secretes body protein matter concentration and reaches 3000 μ g/mL.However, similar in order to achieve the effect that, SPMN- ligand needs 100 minutes, shows
SPMN-PEG- ligand wants much higher to the Magneto separate efficiency of excretion body.
According to the present invention content carry out preparation parameter adjustment, can be achieved excretion body separation, after tested performance with it is upper
It is almost the same to state embodiment.Illustrative description is done to the present invention above, it should which explanation is not departing from core of the invention
In the case where the heart, it is any it is simple deformation, modification or other skilled in the art can not spend creative work etc.
Protection scope of the present invention is each fallen with replacement.
Claims (10)
1. a kind of method based on combination method separation excretion body, which is characterized in that combined using micro porous filtration and ultrafiltration centrifugation
Mode carry out: processing is filtered to sample to be separated using miillpore filter, to remove large scale microvesicle, controls miillpore filter
Aperture be greater than 0.1 μm；The filtrate that micro porous filtration obtains is carried out ultrafiltration centrifugal treating to obtain to remove small size foreign protein
To the supernatant containing excretion body, the molecular cut off of super filter tube is 1kDa-400kDa, in 3-5 DEG C of centrifugation 10-20min, centrifugation
Speed is 80 × g-12000 × g.
2. a kind of method based on combination method separation excretion body according to claim 1, which is characterized in that first carry out ultrafiltration
Centrifugal treating sample to be separated to remove small size foreign protein, then carries out filtering with microporous membrane, to remove large scale microvesicle, obtains
To the supernatant containing excretion body.
3. a kind of method based on combination method separation excretion body according to claim 1, which is characterized in that in micro porous filtration
In, sample to be separated is tumor cell culture base, normal tissue cell culture medium, various biological fluids, such as blood (serum), saliva
Liquid, urine, sperm, breast milk.
4. a kind of method based on combination method separation excretion body according to claim 1, which is characterized in that in micro porous filtration
In, the material of miillpore filter is fibre resin film or nylon membrane, and the aperture of miillpore filter is greater than 0.12 μm, preferably greater than 0.15 μ
M, such as 0.1 μm-10 μm, micro porous filtration number are 1-100 times, preferably 10-80 times.
5. a kind of method based on combination method separation excretion body according to claim 1, which is characterized in that be centrifuged in ultrafiltration
In, the size of small size foreign protein be 30nm once, such as 10-30nm.
6. a kind of method based on combination method separation excretion body according to claim 1, which is characterized in that be centrifuged in ultrafiltration
In, the molecular cut off 3kDa-300kDa of super filter tube (number is equal), ultra-filtration conditions are 4 DEG C, 12-18min, centrifugal speed is 100 ×
g-10000×g；Ultra-filtration filters number is 1-100 times, preferably 10-60 times.
7. a kind of method based on combination method separation excretion body according to claim 1, which is characterized in that selection is added super
The compound of paramagnetic nano particle and ligand further promotes separating effect, and the compound of superparamagnetic nanoparticle and ligand is super
Paramagnetic nano particle-ligand complex selects superparamagnetic nanoparticle and the mercapto ligand reaction of activation to prepare, ligand and 2-
Imino group thiophane reacts the mercaptan ylidene ligands activated or superparamagnetic nanoparticle-polyethylene glycol-with bluk recombination
Object, select Pegylation superparamagnetic nanoparticle and activation mercapto ligand reaction prepare, aminofunctional it is super suitable
Magnetic Fe3O4Nano particle is reacted with active ester polyethylene glycol maleimide, to realize the poly- second of superparamagnetic nanoparticle
It is diolation；Ligand reacts the mercaptan ylidene ligands activated with 2- imino group thiophane.
8. a kind of method based on combination method separation excretion body according to claim 7, which is characterized in that ligand uses
One of CD63 antibody, CD9 antibody, CD81 antibody, CD82 antibody, ALIX antibody, Tsg101 antibody or transferrins；?
In the Pegylation of superparamagnetic nanoparticle, the number-average molecular weight of active ester polyethylene glycol maleimide is 1000-
100000, the Magneto separate time is 10-5000min；It is preferred that the number-average molecular weight of active ester polyethylene glycol maleimide is 4000-
10000, the Magneto separate time is 60-200min；Ligand is with 2- imino group thiophane with molar ratio for (1-10000): (1-
10000), preferably (10-1000): (10-1000), more preferred (10-100): (600-1000)；Aminofunctional it is super suitable
Magnetic Fe3O4The molar ratio of nano particle and active ester polyethylene glycol maleimide is (1-10000): (1-10000), preferably
(10-1000): (10-1000), more preferred (10-100): (500-1000).
9. application of the excretion body that the method as described in claim 1 obtains in preparation therapeutic carrier.
10. the excretion body obtained the method for claim 7 is preparing the application in target therapeutic agent carrier.
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| WO2022111625A1 (en) * | 2020-11-30 | 2022-06-02 | 康码（上海）生物科技有限公司 | Microporous filter cloth and application thereof in nucleic acid extraction |
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