CN104316500B - A kind of long-time cell membrane imaging agents and preparation method thereof - Google Patents
A kind of long-time cell membrane imaging agents and preparation method thereof Download PDFInfo
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- CN104316500B CN104316500B CN201410485961.6A CN201410485961A CN104316500B CN 104316500 B CN104316500 B CN 104316500B CN 201410485961 A CN201410485961 A CN 201410485961A CN 104316500 B CN104316500 B CN 104316500B
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Abstract
The invention discloses a kind of long-time cell membrane imaging agents, it is characterised in that:It is made up of component A and component B;The component A is glycol-chitosan (glycol chitosan) macromolecule that side chain contains biotin and hydrophobic units;The component B is the avidin molecule for being grafted with fluorescein isothiocynate FITC.Cell membrane imaging agent good biocompatibility provided by the invention, imaging effect are excellent, and can be maintained at for a long time on cell membrane without that by endocytosis, can be used as new class cell membrane imaging agent.
Description
Technical field
The invention belongs to biological technical field, a kind of more particularly to long-time cell membrane fluorescence imaging reagent further relates to
The preparation method of the reagent.
Background technology
The cell membrane not only border in structure as cell, provides a metastable interior environment, simultaneously for cell
Vital effect is also played to the matter transportation between cell and external environment, energy conversion and information exchanging process.Carefully
After birth imaging technique is marked and traced to cell membrane using fluorescent dye, disclosed as a kind of strong research meanses
Many important biological processes including pinocytosis, exocytosis, cell division and apoptosis.
However, endocytosis phenomenon can occur quickly for some traditional lipophilic dyes, imaging time is short, is brought to research very big
Limitation.Such as presently commercially available fluorescent dye DiI, DiO and DiD, effective imaging time only have 10 minutes or so, and dyestuff can be by
It is progressive enter cytoplasm and by whole cell dyeing, so as to lose the effect of research cell membrane.In order to extend effective dyeing time,
Dyestuff endocytosis is reduced, existing company develops new dye molecule, such as CellMaskTM Orang, DeepRed series, main profit
Extend cell membrane dyeing time with the lipophilic group with negative electrical charge is with anchoring effect, but dyeing time is also only confined to 30 points
Clock was to 90 minutes.In order to preferably study the 26S Proteasome Structure and Function of cell membrane, a kind of long-time cell membrane imaging examination is developed
Agent is just particularly important.
The content of the invention
Goal of the invention:It is an object of the invention to provide a kind of cell membrane imaging agent of imaging time length.
Technical scheme:A kind of long-time cell membrane imaging agents, it is characterised in that:It is made up of component A and component B;It is described
Component A is the glycol-chitosan (glycol chitosan) that side chain contains biotin (biotin) and hydrophobic units;Described group
It is Avidin (avidin) molecule for being grafted with fluorescence molecule to divide B.Component A from glycol-chitosan as connection biotin and
The skeleton of hydrophobic units is due to that it has good dissolubility in the aqueous solution of any pH value, and has low immunogene
The features such as property, biocompatibility, biodegradability, bioactivity.Hydrophobic units in component A can pass through hydrophobic interaction
The hydrophobic tail region for the phospholipid bilayer being inserted into a manner of more sites on cell membrane, so as to by shitosan macromolecule and
Biotin molecule is anchored on cell membrane.Avidin in component B is introduced fluorescence molecule by being specifically bound with biotin
To polymer side chain, and then realize long-time cell membrane imaging.Preferably, the biotin accounts for the repetition of glycol-chitosan
The 5-30% of unit number;The hydrophobic units account for the 5-30% of the number of repeat unit of glycol-chitosan.
As another kind preferably, the molecular weight of the glycol-chitosan is more than 10000, preferably 10000-100000.
As it is another preferably, the biotin include (+) biotin-N- succinimides base ester (NHS-biotin,
CAS 35013-72-0);The biotin by with amino reactive grafting in shitosan macromolecule, for be grafted with it is glimmering
The avidin molecule specific binding (i.e. bitoin and avidin specific recognition) of light element molecule.
As another kind preferably, the hydrophobic units include cholesterol, phospholipid molecule, alkane and vitamin E, for thin
The grappling of after birth;The hydrophobic units are linked on the amino of glycol-chitosan by polyethylene glycol polymer (PEG), to increase
Adding water soluble;Wherein polyethylene glycol polymer (PEG) molecular weight is more than 500.
As another kind preferably, the fluorescence molecule includes fluorescein isothiocynate or Sulforhodamine B sulfonic acid chlorides, and it is logical
Cross with amino reactive grafting on avidin molecule surface.
Present invention also offers the preparation method of above-mentioned long-time cell membrane imaging agents, comprise the following steps:
(1) biotin, hydrophobic units and glycol-chitosan are dissolved in PBS respectively, mixed, room temperature reaction;
Product dialysis, freeze, produce component A;
(2) mixing fluorescein isothiocynate and avidin solution makes reaction;Product dialysis, freeze, produce component B.
Beneficial effect:Cell membrane imaging agent good biocompatibility provided by the invention, imaging effect are excellent, and can be long when
Between be maintained on cell membrane without by endocytosis, new class cell membrane imaging agent can be used as.
Specifically, the present invention has advantage following prominent relative to prior art:
(1) reagent good biocompatibility provided by the invention, imaging effect are excellent, and can be maintained at for a long time on cell membrane
Without by endocytosis, it utilizes cholesterol modification shitosan polymer side chain to carry out more site grapplings, forms a floor height molecular imaging
Substrate, the specific binding of biotin and avidin is recycled to introduce fluorescence molecule, so as to realize the high quality up to 18 hours
Cell membrane imaging.
(2) present invention is using cholesterol modification shitosan polymer side chain, because cholesterol can be carried out on cell membrane
Insertion, plays a part of more being the increase in the efficiency of grappling to macromolecule grappling, multiple cholesterol molecules.Therefore component A conducts
Imageable substrate quickly can surround cell membrane, and the multiple biotin molecules being grafted in shitosan macromolecule are exposed,
For being combined afterwards with the avidin molecule with fluorescence molecule, the imaging of cell membrane is realized.This " more site grapplings " simultaneously combines
The strategy of " dual cladding ", image quality and imaging time can be greatly enhanced;Experiment display, the imaging agents are contaminated cell
Color does not have obvious endocytosis phenomenon after 18 hours yet.
(3) present invention uses the high specific between biotin and avidin is firm to combine, when enabling fluorescence molecule long
Between be enclosed in cell membrane surface without by endocytosis.Further, since the joint efficiency of biotin and avidin is high, can greatly save
Dyeing time.Avidin also ensure that fluorescence molecule is not easy by endocytosis to a certain extent as large biological molecule.
(4) constituent chitosan, PEG, biotin and the Avidin of reagent of the present invention all possess good biofacies
Capacitive, and cholesterol with anchoring effect is even more the cell membrane proper constituent of zooblast, therefore the poison of the agents on cellular
Property is low.Component A is below 100 μ g/mL, and for component B below 200 μ g/mL, MTT experiment shows the imaging agents to cell viability
Almost without influence, so as to demonstrate the good biocompatibility of the reagent.
(5) positive charge that remaining amino carries in the shitosan macromolecule of reagent of the present invention, can with it is negatively charged thin
Electrostatic interaction, increase and the affinity of cell membrane occur for after birth, thus further extend guarantor of the dyestuff on cell membrane
Hold the time.
(6) reagent of the present invention can be as a kind of application platform of cell membrane dyeing for a long time, by replacing different fluorescence
Molecule can realize the fluorescence imaging of multiple color.
Brief description of the drawings
Fig. 1 is the molecular structure of long-time cell membrane fluorescence imaging reagent of the present invention.
Fig. 2 is influence evaluation test of the cell membrane fluorescence imaging reagent of the present invention to A549 lung carcinoma cell survival activities
(MTT is tested, and wherein chitosan and component A concentration are 100 μ g/mL, and component B concentration is 200 μ g/mL);
The cell membrane fluorescence imaging reagent of Fig. 3 (a), 3 (b), 3 (c) the respectively present invention are to A549 lung carcinoma cell cell membranes
Design sketch after dyeing 1 hour, 8 hours and 18 hours.
Embodiment
Embodiment 1
Long-time cell membrane fluorescence imaging reagent (component A:Chitosan-10%cholesterol-10%biotin with
And component B:Avidin-FITC design), synthesis and cell membrane fluorescent staining method are as follows:
The design of reagent:The molecular structure of the imaging agents is shown in Fig. 1, and the reagent is made up of two kinds of components:Component A with
Glycol-chitosan macromolecule (glycol chitosan) is skeleton, and side chain contains 10% polyethylene glycol 2000-cholesterol
(PEG2000-cholesterol) hydrophobic units and 10% biotin molecule (biotin);Component B is by being grafted with isothiocyanic acid
Fluorescein FITC Avidin (avidin) is formed.Described glycol-chitosan macromolecule, there is good biocompatibility
And water solubility.Described cholesterol hydrophobic side chain, it is connected to by PEG2000 of the molecular weight more than 500 in amino of chitosan,
That is NHS-PEG2000-cholesterol, increase are water-soluble.Described FITC fluorescence molecules, by being arrived with amino reaction forming
In avidin molecule.Cholesterol hydrophobic units can insert cell membrane by the more sites of hydrophobic interaction, by macromolecular scaffold
It is anchored on cell membrane;Then, fluorescence molecule is grafted on by high score by the strong bonded of biotin and avidin high-affinity
On son, as fluorescence imaging unit.
The synthesis of reagent, comprises the following steps:
(1) component A preparation:With n-hydroxysuccinimide-polyethylene glycol 2000-cholesterol (NHS-PEG2000-
Cholesterol), (+) biotin-N- succinimides base ester (NHS-Biotin) and molecular weight are more than 10000 second two
Alcohol shitosan macromolecule is raw material, and the percentage that hydrophobic synthetic unit accounts for shitosan number of repeat unit is 10%, biotin molecule
The percentage for accounting for shitosan number of repeat unit is 10% component A, i.e. chitosan-10%cholesterol-10%
biotin.Specially:It is (poly- that 16mg NHS-PEG2000-cholesterol, 15.2mg glycol chitosan are weighed respectively
It is right:More than 400;About 80000) and 2.5mg NHS-Biotin molecular weight, three is dissolved in respectively PBS (pH
7.4,50mM) after, being well mixed rapidly, reaction is stayed overnight at room temperature in shaking table;Reaction with molecular cut off is 10k's after terminating
Bag filter is dialysed 3 days and purified, and is finally freezed in freeze dryer and component A is made:Chitosan-10%cholesterol-
10%biotin;
(2) component B preparation:The sodium carbonate-bicarbonate buffering that 5mg avidin is dissolved in 2.5mL pH=9.5 is molten
Liquid;Take FITC and be dissolved in dimethylformamide (DMF), be configured to concentration 2mg/mL FITC solution;Into avidin solution
250 μ L FITC solution is added, after being well mixed, is reacted overnight in 4 DEG C of refrigerators;After reaction terminates, with molecular cut off 2k
Bag filter dialyse and remove unreacted FITC within three days and purified, be finally freeze-dried in freeze dryer and component B be made:
avidin-FITC。
Component A and component B freezen protectives in -20 DEG C.
Cell membrane dyes imaging experiment:Component A and component B are dissolved in the storage that 1mg/mL is made of PBS in cell culture respectively
Liquid storage, and be filtered to remove bacterium with 0.45 μm of aseptic filtration head and working solution is made.A549 lung carcinoma cells are in the burnt training of copolymerization
Support after being cultivated 12 hours on plate, first suck original culture medium, PBS twice, adds the fresh DMEM complete mediums of 2mL
(DMEM incomplete culture mediums+1% Pen .- Strep of+10% hyclone is dual anti-).First add the μ L of component A 200 and put back to
CO2gas incubator is incubated 30 minutes, is then suctioned out liquid simultaneously PBS 1 time in culture plate, is changed to DMEM complete mediums;
Add 400 μ L components B to be incubated in incubator 10 minutes, dyeing terminates rear PBS 3 times, rejoins DMEM and trains completely
Support base and cell is placed under laser scanning co-focusing microscope and observe and take pictures, and Follow-up observation cell membrane at regular intervals
Staining conditions.
Coloration result:Fig. 3 (a), 3 (b), 3 (c) are respectively cell membrane image after dyeing 1 hour, 8 hours and 18 hours;
Visible cell film is equably marked upper green fluorescence, obvious endocytosis is not occurred with the extension of dyeing time, is realized
Cell membrane long-time fluorescence imaging.
Embodiment 2
The implementation of the present embodiment is consistent with the method in embodiment 1, the glycol-chitosan macromolecule simply selected
Cholesterol hydrophobic units in molecular weight about 10000, NHS-PEG2000-cholesterol molecules make phospholipid molecule into successively
The hydrophobic side such as (1,2- myristoyl phosphatidyl-ethanolamine, DMPE), alkane (18 carbon atoms, C18) or vitamin E
Chain, synthesis obtain three kinds of cell membrane fluorescence imaging reagents.
Embodiment 3
In order to realize the cell membrane fluorescence imaging of other colors, the implementation and the method in embodiment 1 of the present embodiment
It is similar, the glycol-chitosan molecular weight polymeric simply selected about 100000, the fluorescence molecule FITC in embodiment 1 is replaced
As Sulforhodamine B sulfonic acid chloride fluorometric reagents, implementation is consistent with method in embodiment 1.
Embodiment 4
The implementation of the present embodiment is similar with the method in embodiment 1, simply the anchor unit in imaging agents component A
The percentage for accounting for shitosan number of repeat unit is changed to 30%, and the percentage that biotin units account for shitosan number of repeat unit is changed to
5%, obtained imaging agents composition is chitosan-30%cholesterol-5%biotin.
Embodiment 5
The implementation of the present embodiment is similar with the method in embodiment 1, simply the anchor unit in imaging agents component A
The percentage for accounting for shitosan number of repeat unit is changed to 5%, and the percentage that biotin units account for shitosan number of repeat unit is changed to
30%, obtained imaging agents composition is chitosan-5%cholesterol-30%biotin.
Claims (2)
- A kind of 1. long-time cell membrane imaging agents, it is characterised in that:It is made up of component A and component B;The component A is side chain Glycol-chitosan macromolecule containing biotin and hydrophobic units;The component B is the Avidin point for being grafted with fluorescence molecule Son, the biotin account for the 5-30% of component A glycol-chitosan number of repeat unit;The hydrophobic units account for component A second The 5-30% of glycol shitosan macromolecule number of repeat unit, the glycol-chitosan molecular weight are described in 10000-100000 Biotin includes (+) biotin-N- succinimide base esters;The biotin by with amino reactive grafting in chitosan high score On son, the hydrophobic units include cholesterol, phospholipid molecule, alkane and vitamin E;The hydrophobic units pass through polyethylene glycol height Molecule is linked on the amino of glycol-chitosan;The polyethylene glycol polymer molecular weight is more than 500, the fluorescence molecule Including fluorescein isothiocynate or Sulforhodamine B sulfonic acid chlorides, its by with amino reactive grafting on avidin molecule surface.
- A kind of 2. preparation method of long-time cell membrane imaging agents according to claim 1, it is characterised in that:Including with Lower step:(1) biotin, hydrophobic units and glycol-chitosan are dissolved in PBS respectively, mixed, room temperature reaction;Product Dialysis, freeze, produce component A;(2) mixing fluorescein isothiocynate and avidin solution makes reaction;Product dialysis, freeze, produce component B.
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