CN109897187A - A kind of grafting polyethylene imine rice bran polysaccharide and preparation method thereof and genophore - Google Patents
A kind of grafting polyethylene imine rice bran polysaccharide and preparation method thereof and genophore Download PDFInfo
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- CN109897187A CN109897187A CN201910170445.7A CN201910170445A CN109897187A CN 109897187 A CN109897187 A CN 109897187A CN 201910170445 A CN201910170445 A CN 201910170445A CN 109897187 A CN109897187 A CN 109897187A
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Abstract
The present invention discloses a kind of grafting polyethylene imine rice bran polysaccharide and preparation method thereof and genophore, is related to genophore technical field.The preparation method of the grafting polyethylene imine rice bran polysaccharide includes: after potassium metaperiodate formation reaction solution is added into the aqueous solution of rice bran polysaccharide, to dialyse to reaction solution, obtain dialyzate；Polyethyleneimine is dissolved in buffer, polyethylenimine solution is formed；Dialyzate is added in polyethylenimine solution to be stirred to react to form reaction solution, and controls reaction solution in alkalinity；Sodium borohydride is added in reaction solution, just sample liquid is formed；It dialyses to first sample liquid, obtains grafting polyethylene imine rice bran polysaccharide after taking permeate alcohol precipitation, centrifugation, freeze-drying.The present invention also proposes a kind of genophore, including grafting polyethylene imine rice bran polysaccharide.Grafting polyethylene imine rice bran polysaccharide produced by the present invention has lower cytotoxicity and good bio-compatibility, can be used as a kind of good genophore application.
Description
Technical field
The present invention relates to genophore technical field, in particular to a kind of grafting polyethylene imine rice bran polysaccharide and its preparation
Method and genophore.
Background technique
Exposed foreign gene DNA is difficult to enter cell and is easy by body or cell degradation, thus beyond expression of words, so
It should find and assist target gene to enter target cell in the carrier that pathological tissues have selectivity, high transfection efficiency and hypotoxicity, be used for
It is genophore that transhipment target gene, which enters the carrier of target cell, and selecting suitable genophore is the successful pass of gene therapy
Key.Genophore mainly has two class of viral vectors and non-virus carrier.
Polyethyleneimine is one of current most representative high efficiency gene carrier, but its that there are bio-compatibilities is poor, carefully
The higher disadvantage of cellular toxicity, it is still necessary to improve.
Summary of the invention
The main object of the present invention is to propose that a kind of grafting polyethylene imine rice bran polysaccharide and preparation method thereof and gene carry
Body, it is intended to a kind of grafting polyethylene imine rice bran polysaccharide is prepared, there is lower cytotoxicity and good bio-compatibility, it can
Using as a kind of good genophore application.
To achieve the above object, the present invention proposes that a kind of grafting polyethylene imine rice bran polysaccharide, the polyethyleneimine connect
Branch rice bran polysaccharide has following structure structure shown in formula (1).
Structural formula (1):
To achieve the above object, the invention also provides a kind of preparation method of grafting polyethylene imine rice bran polysaccharide, institutes
State the preparation method of grafting polyethylene imine rice bran polysaccharide the following steps are included:
Potassium metaperiodate is added into the aqueous solution of rice bran polysaccharide, is protected from light after stirring forms reaction solution, to the reaction solution into
Row dialysis, obtains dialyzate；
Polyethyleneimine is dissolved in buffer, polyethylenimine solution is formed；
The dialyzate is added to and is stirred to react to form reaction solution in the polyethylenimine solution, and stirred
Reaction solution is controlled in journey in alkalinity；
Sodium borohydride is added in the reaction solution, is stirred to react to form just sample liquid；
It dialyses to the just sample liquid, obtains grafting polyethylene imine rice bran after taking permeate alcohol precipitation, centrifugation, freeze-drying
Polysaccharide.
Preferably, potassium metaperiodate is added in the aqueous solution to rice bran polysaccharide, is protected from light after stirring forms reaction solution, to institute
It states in the step of reaction solution dialyses, obtains dialyzate,
The mixing time that is protected from light is 24~48h；And/or
The time of the dialysis is 36~48h.
Preferably, described that polyethyleneimine is dissolved in buffer, formed polyethylenimine solution the step of in,
The buffer is pH9.0 phosphate buffer.
Preferably, the dialyzate is added to and is stirred to react to form reaction solution in the polyethylenimine solution, and
Reaction solution is controlled in whipping process in the step of alkalinity includes:
The dialyzate is added drop-wise in the polyethylenimine solution and is stirred to react 30~36h formation reaction solution, and
Sodium hydroxide solution is added dropwise during dropwise addition, in Xiang Suoshu reaction solution with control the pH value of the reaction solution 8.8~
In 9.2 ranges；
Wherein, described that the dialyzate is added drop-wise to the time in the polyethylenimine solution no more than 1h.
Preferably, the mass ratio of rice bran polysaccharide, potassium metaperiodate, polyethyleneimine and sodium borohydride is 1:(1.5~2.0):
(2.5~4.0): (1.5~4).
Preferably, described that sodium borohydride is added in the reaction solution, it is stirred to react the step of forming just sample liquid, packet
It includes:
Sodium borohydride is added in the reaction solution, 40~48h is stirred to react and forms the first mixed liquor；
Sodium borohydride is added in first mixed liquor, 22~formation for 24 hours just sample liquid is stirred to react.
Preferably, the additional amount that sodium borohydride is added to the sodium borohydride in the step in the reaction solution with
The mass ratio of rice bran polysaccharide additional amount is (1~1.5): 1；And/or
The additional amount and rice bran that sodium borohydride is added to the sodium borohydride in the step in first mixed liquor
The mass ratio of polysaccharide additional amount is (0.5~2.5): 1.
Preferably, described to dialyse to the just sample liquid, polyethylene is obtained after taking permeate alcohol precipitation, centrifugation, freeze-drying
Imines was grafted in the step of rice bran polysaccharide,
When the dialysis, the molecular cut off of the bag filter used is 3000~3200；And/or
The dialysis time is 24~48h；And/or
The alcohol precipitation time is 48~50h.
In addition, the genophore includes above-mentioned grafting polyethylene imine rice the invention also provides a kind of genophore
Chaff polysaccharide.
In technical solution of the present invention, by the way that a kind of poly- second is made so that polyethyleneimine is cation-modified to rice bran polysaccharide progress
Alkene imines is grafted rice bran polysaccharide, can agglomerate DNA and enter intracellular rear release by the endocytosis of cell membrane, therefore, can
For use as genophore.It is from a wealth of sources since rice bran polysaccharide is abundant in china natural resources, and there is good biocompatibility, can
Biological degradability, therefore using rice bran polysaccharide as substrate grafting polyethylene imine, cytotoxicity can be reduced, bio-compatible is improved
Property, and then improve using grafting polyethylene imine rice bran polysaccharide as the quality of the genophore of raw material.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
Other relevant attached drawings are obtained according to these attached drawings.
Fig. 1 is the process signal of an embodiment of the preparation method of grafting polyethylene imine rice bran polysaccharide provided by the invention
Figure；
Fig. 2 is the gel electrophoresis for the product that grafting polyethylene imine rice bran polysaccharide made from Examples 1 to 6 agglomerates after DNA
Figure；
Fig. 3 is the fluorescence microscope of the detection of grafting polyethylene imine rice bran polysaccharide biocompatibility made from Examples 1 to 6
Photo.
The embodiments will be further described with reference to the accompanying drawings for the realization, the function and the advantages of the object of the present invention.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Polyethyleneimine is one of current most representative high efficiency gene carrier, but its that there are bio-compatibilities is poor, carefully
The higher disadvantage of cellular toxicity, it is still necessary to improve.
In consideration of it, the present invention proposes that a kind of genophore, the genophore include grafting polyethylene imine rice bran polysaccharide.
Meanwhile the invention also provides above-mentioned grafting polyethylene imine rice bran polysaccharide, the grafting polyethylene imine rice
Chaff polysaccharide has the structure of following structural formula (1):
Wherein, x, y, n are integer, it should be understood that above-mentioned grafting polyethylene imine rice bran polysaccharide is with above structure
The polymer of formula structure, all compounds with structure above structure are within the scope of the invention.
China's rice yield is extremely abundant, and there are a kind of rice bran polysaccharides in the caryopsis cortex of paddy, has good life
Object compatibility and biodegradable, and DNA can be effectively compressed and transfected to intracellular, but since rice bran polysaccharide is free of
There is cation group, rice bran polysaccharide agglomerates the ineffective of DNA；Contain a large amount of amino in the structure of polyethyleneimine, that is, carries
A large amount of positive charges can reinforce the combination of polyethyleneimine and negatively charged DNA, both make to be formed stable nano combined
Object, and can be entered into the cell by the endocytosis of cell membrane, while passing through proton sponge effect released dna in the cell,
Achieve the purpose that transport DNA, but polyethyleneimine bio-compatibility difference and cytotoxicity with higher.In the knot of rice bran polysaccharide
Grafting polyethylene imine in structure, can not only make product rich in positive charge, agglomerate DNA, transporter gene so as to stablize, improve
Transfection efficiency, and there is good biocompatibility and lower cytotoxicity, it is a kind of material of good genophore,
That is, can be used grafting polyethylene imine rice bran polysaccharide as a kind of genophore, it can also be with polyethyleneimine
Grafting rice bran polysaccharide is core, adds other supplementary materials (such as polyethylene glycol, mucopolysaccharide, polyglutamic acid etc.) building cladding shell,
Genophore is made.Said gene carrier has that raw material sources are extensive, cheap, transfection efficiency is high, good biocompatibility, thin
The low advantage of cellular toxicity.
In addition, the invention also provides a kind of preparation methods of grafting polyethylene imine rice bran polysaccharide, as shown in connection with fig. 1
The flow diagram of one embodiment of the preparation method of grafting polyethylene imine rice bran polysaccharide, the grafting polyethylene imine rice bran
The preparation method of polysaccharide the following steps are included:
Step S10, potassium metaperiodate is added into the aqueous solution of rice bran polysaccharide, is protected from light after stirring forms reaction solution, to described
Reaction solution is dialysed, and dialyzate is obtained.
Step S20, polyethyleneimine is dissolved in buffer, forms polyethylenimine solution.
Step S30, the dialyzate is added to and is stirred to react to form reaction solution in the polyethylenimine solution, and
Reaction solution is controlled in whipping process in alkalinity.
Step S40, sodium borohydride is added in the reaction solution, is stirred to react to form just sample liquid.
Wherein, the mass ratio of rice bran polysaccharide, potassium metaperiodate, polyethyleneimine and sodium borohydride is 1:(1.5~2.0):
(2.5~4.0): (1.5~4).
In step S10, the mixing time that is protected from light is 24~48h, and the time of the dialysis is 36~48h；Step S20
In, the buffer is pH9.0 phosphate buffer.
In above-mentioned steps, potassium metaperiodate has strong oxidizing property when being dissolved in water, can aoxidize rice bran polysaccharide, obtains containing aldehyde radical
Rice bran polysaccharide, buffer can provide stable acid or alkali environment for reaction system, smoothly be grafted to oxygen convenient for polyethyleneimine
Change in processed rice bran polysaccharide structure, sodium borohydride is reducing agent, poly- to be formed to carry out reduction treatment to graft product
Aziridine is grafted rice bran polysaccharide.
To guarantee reaction forward, sufficiently carrying out, potassium metaperiodate and sodium borohydride should be more than that participation oxidation or reduction are anti-
The theoretical weight answered, therefore in the present embodiment, rice bran polysaccharide, potassium metaperiodate, the mass ratio of polyethyleneimine and sodium borohydride are excellent
It is selected as 1:(1.5~2.0): (2.5~4.0): (1.5~4).Excessive potassium metaperiodate and sodium borohydride are cleared up after the completion of reaction
Fall, in step slo, dialyse to the reaction solution, is to dispose potassium metaperiodate.
Meanwhile in step S20 and S30, the appropriate pH of above-mentioned graft reaction is 9.0, therefore in the present embodiment, dissolve poly- second
The solvent of alkene imines is preferably the phosphate buffer of pH9.0, and in process of grafting, and control reaction solution is in alkalinity,
In the specific implementation, step S30 may include: and the dialyzate be added drop-wise in the polyethylenimine solution to be stirred to react 30
~36h forms reaction solution, and during dropwise addition, and it is described anti-to control that sodium hydroxide solution is added dropwise in Xiang Suoshu reaction solution
Answer the pH value of solution in 8.8~9.2 ranges；
Wherein, described that the dialyzate is added drop-wise to the time in the polyethylenimine solution no more than 1h.
This is because polyethyleneimine can be wrapped in rapidly rice bran polysaccharide structure after polyethyleneimine is contacted with dialyzate
Surface, if the amount of being added at one time is more, the part rice bran polysaccharide that will lead to the inside can not participate in reacting, and then make synthetic yield
It reduces, and dialyzate single additional amount crosses conference and the pH of polyethylenimine solution is caused to reduce, therefore, in the present embodiment, choosing
With the mode of dropwise addition to promote reaction to go on smoothly, yield is improved.Meanwhile to improve production efficiency, rate of addition should not be excessively slow,
Therefore, the time that the dialyzate is added drop-wise in the polyethylenimine solution is preferably no more than 1h.
Further, in step S40, to carry out reduction reaction sufficiently, in the specific implementation, the addition step of reducing agent
May include:
Step S410, sodium borohydride is added in the reaction solution, is stirred to react 40~48h and forms the first mixing
Liquid；
Step S420, sodium borohydride is added in first mixed liquor, is stirred to react 22~formation for 24 hours just sample liquid.
Sodium borohydride is divided into and is added twice to ensure that it is abundant that reduction reaction carries out.
Wherein, the mass ratio of sodium borohydride additional amount used in step S410 and rice bran polysaccharide additional amount be preferably (1~
1.5): 1；The mass ratio of sodium borohydride additional amount used in step S420 and rice bran polysaccharide additional amount be preferably (0.5~
2.5): 1.
Step S50, it dialyses to the just sample liquid, obtains polyethyleneimine after taking permeate alcohol precipitation, centrifugation, freeze-drying
It is grafted rice bran polysaccharide.
Wherein, when the dialysis, the molecular cut off of the bag filter used is 3000~3200；Dialysis time be 24~
48h；The alcohol precipitation time is 48~50h.
Due to also containing the impurity components such as excessive sodium borohydride in first sample liquid in addition to grafting polyethylene imine rice bran polysaccharide,
It therefore, it is necessary to be purified, in the present embodiment, preferably dialyses, when dialysis, the molecular cut off of the bag filter used is preferably
3000~3200；Dialysis time is preferably 24~48h.It after dialysis, is handled by alcohol precipitation, the poly- second insoluble in Organic Alcohol can be made
Alkene imines is grafted rice bran polysaccharide precipitation and separation, and in the specific implementation, the Organic Alcohol of selection can be dehydrated alcohol, and the alcohol precipitation time is excellent
It is selected as 48~50h.And in order to further separate thoroughly, alcohol precipitation can be precipitated and be centrifuged, when centrifugation, revolving speed can be 8000
~10000r/min, centrifugation time are 5~10min, and centrifugation number can be primary or multiple, and in order to further will
It is attached to the impurity washes clean of precipitation surface, precipitating can be washed with dehydrated alcohol between adjacent centrifugation twice.
Technical solution of the present invention is described in further detail below in conjunction with specific embodiments and the drawings, it should be understood that
Following embodiment is only used to explain the present invention, is not intended to limit the present invention.
Embodiment 1
1g rice bran polysaccharide is added in the round-bottomed flask equipped with 10mL deionized water, 1.5g periodic acid is added while stirring
Potassium is protected from light stirring and reaction solution is fitted into bag filter the 48h that dialyses afterwards for 24 hours, obtains dialyzate.30g polyethyleneimine is dissolved in 100mL
PH9.0 phosphate buffer in, formed polyethylenimine solution.It is molten that the above-mentioned polyethyleneimine of 10mL is added dropwise into dialyzate
Liquid, and in being added dropwise in 1h, 34h is then stirred at room temperature, forms reaction solution.It is molten that 1g sodium borohydride is added to above-mentioned reaction
In liquid, stirring 40h forms the first mixed liquor.2.5g sodium borohydride is added in above-mentioned first mixed liquor again, continues to stir
23h forms just sample liquid.First sample liquid is placed in the bag filter that molecular cut off is 3000 48h that dialyses, takes permeate.To transmission
The dehydrated alcohol of 3 times of permeate volumes is added in liquid, after alcohol precipitation 48h, is centrifuged, is freeze-dried to obtain grafting polyethylene imine rice bran
Polysaccharide.
Embodiment 2
1g rice bran polysaccharide is added in the round-bottomed flask equipped with 10mL deionized water, 1.6g periodic acid is added while stirring
Potassium is protected from light after stirring 30h reaction solution being fitted into bag filter the 40h that dialyses, obtains dialyzate.25g polyethyleneimine is dissolved in 100mL
PH9.0 phosphate buffer in, formed polyethylenimine solution.It is molten that the above-mentioned polyethyleneimine of 10mL is added dropwise into dialyzate
Liquid, and in being added dropwise in 1h, 31h is then stirred at room temperature, forms reaction solution.1.1g sodium borohydride is added to above-mentioned reaction
In solution, stirring 42h forms the first mixed liquor.2g sodium borohydride is added in above-mentioned first mixed liquor again, continues to stir
For 24 hours, just sample liquid is formed.First sample liquid is placed in the bag filter that molecular cut off is 3200 36h that dialyses, takes permeate.To transmission
The dehydrated alcohol of 3 times of permeate volumes is added in liquid, after alcohol precipitation 50h, is centrifuged, is freeze-dried to obtain grafting polyethylene imine rice bran
Polysaccharide.
Embodiment 3
1g rice bran polysaccharide is added in the round-bottomed flask equipped with 10mL deionized water, 1.7g periodic acid is added while stirring
Potassium is protected from light after stirring 35h reaction solution being fitted into bag filter the 37h that dialyses, obtains dialyzate.26g polyethyleneimine is dissolved in 100mL
PH9.0 phosphate buffer in, formed polyethylenimine solution.It is molten that the above-mentioned polyethyleneimine of 10mL is added dropwise into dialyzate
Liquid, and in being added dropwise in 1h, 32h is then stirred at room temperature, forms reaction solution.1.2g sodium borohydride is added to above-mentioned reaction
In solution, stirring 48h forms the first mixed liquor.1g sodium borohydride is added in above-mentioned first mixed liquor again, continues to stir
22h forms just sample liquid.First sample liquid is placed in the bag filter that molecular cut off is 3100 and is dialysed for 24 hours, permeate is taken.To transmission
The dehydrated alcohol of 3 times of permeate volumes is added in liquid, after alcohol precipitation 49h, is centrifuged, is freeze-dried to obtain grafting polyethylene imine rice bran
Polysaccharide.
Embodiment 4
1g rice bran polysaccharide is added in the round-bottomed flask equipped with 10mL deionized water, 1.8g periodic acid is added while stirring
Potassium is protected from light after stirring 40h reaction solution being fitted into bag filter the 45h that dialyses, obtains dialyzate.40g polyethyleneimine is dissolved in 100mL
PH9.0 phosphate buffer in, formed polyethylenimine solution.It is molten that the above-mentioned polyethyleneimine of 10mL is added dropwise into dialyzate
Liquid, and in being added dropwise in 1h, 36h is then stirred at room temperature, forms reaction solution.1.3g sodium borohydride is added to above-mentioned reaction
In solution, stirring 46h forms the first mixed liquor.1.5g sodium borohydride is added in above-mentioned first mixed liquor again, continues to stir
22.5h is mixed, just sample liquid is formed.First sample liquid is placed in the bag filter that molecular cut off is 3000 30h that dialyses, takes permeate.To
The dehydrated alcohol of 3 times of permeate volumes is added in permeate, after alcohol precipitation 48h, is centrifuged, is freeze-dried to obtain grafting polyethylene imine
Rice bran polysaccharide.
Embodiment 5
1g rice bran polysaccharide is added in the round-bottomed flask equipped with 10mL deionized water, 1.9g periodic acid is added while stirring
Potassium is protected from light after stirring 45h reaction solution being fitted into bag filter the 36h that dialyses, obtains dialyzate.28g polyethyleneimine is dissolved in 100mL
PH9.0 phosphate buffer in, formed polyethylenimine solution.It is molten that the above-mentioned polyethyleneimine of 10mL is added dropwise into dialyzate
Liquid, and in being added dropwise in 1h, 35h is then stirred at room temperature, forms reaction solution.1.4g sodium borohydride is added to above-mentioned reaction
In solution, stirring 45h forms the first mixed liquor.0.8g sodium borohydride is added in above-mentioned first mixed liquor again, continues to stir
23.5h is mixed, just sample liquid is formed.First sample liquid is placed in the bag filter that molecular cut off is 3000 40h that dialyses, takes permeate.To
The dehydrated alcohol of 3 times of permeate volumes is added in permeate, after alcohol precipitation 50h, is centrifuged, is freeze-dried to obtain grafting polyethylene imine
Rice bran polysaccharide.
Embodiment 6
1g rice bran polysaccharide is added in the round-bottomed flask equipped with 10mL deionized water, 2.0g periodic acid is added while stirring
Potassium is protected from light after stirring 48h reaction solution being fitted into bag filter the 42h that dialyses, obtains dialyzate.35g polyethyleneimine is dissolved in 100mL
PH9.0 phosphate buffer in, formed polyethylenimine solution.It is molten that the above-mentioned polyethyleneimine of 10mL is added dropwise into dialyzate
Liquid, and in being added dropwise in 1h, 33h is then stirred at room temperature, forms reaction solution.1.5g sodium borohydride is added to above-mentioned reaction
In solution, stirring 44h forms the first mixed liquor.0.5g sodium borohydride is added in above-mentioned first mixed liquor again, continues to stir
It mixes for 24 hours, forms just sample liquid.First sample liquid is placed in the bag filter that molecular cut off is 3200 45h that dialyses, takes permeate.To saturating
It crosses in liquid and the dehydrated alcohol of 3 times of permeate volumes is added, after alcohol precipitation 49h, be centrifuged, be freeze-dried to obtain grafting polyethylene imine rice
Chaff polysaccharide.
Grafting polyethylene imine rice bran polysaccharide made from Examples 1 to 6 is detected, detection project includes: DNA cohesion
And releasing effect detection；Biocompatibility detection；And cytotoxicity detection.Specific detection process and result are as follows:
(1) DNA flocculating result detects
Each 0.005g of grafting polyethylene imine rice bran polysaccharide made from Example 1~6, is dissolved in 1mL water, as sample
Solution.
By sample solution and Plasmid DNA, by different mass ratio mixing, (sample solution and the mass ratio of Plasmid DNA are respectively
0.5:1,1:1,1.5:1,2:1,2.5:1 and 3:1), sample-loading buffer is added in hybrid reaction 30min after reaction, forms point
Sample solution.Spotting solution is taken to carry out agarose gel electrophoresis, wherein gel is 1% Ago-Gel, and the voltage of electrophoresis apparatus is
90V, electric current 80mA.Naked DNA control is set simultaneously.It observes and takes pictures in gel imager after the completion of electrophoresis, gel electricity
Swimming figure corresponds to naked DNA control labeled as 0 well, is corresponding in turn to sample labeled as 1,2,3,4,5,6 as shown in Fig. 2, in figure
Solution and the mass ratio of Plasmid DNA are respectively the spotting solution of 0.5:1,1:1,1.5:1,2:1,2.5:1 and 3:1.
As can be seen from Figure 2, in the corresponding each group spotting solution of Examples 1 to 6, with the concentration of sample solution in spotting solution
Increase, retention effect is more obvious in gel pore, this illustrates that grafting polyethylene imine rice bran polysaccharide is positively charged, when its concentration of increase
When, then it is trapped in gel pore in conjunction with electronegative DNA and by DNA.Grafting polyethylene imine rice bran can be shown from Fig. 2
Polysaccharide is smoothly agglomerated with DNA.
(2) biocompatibility detects
The HeLA cell frozen is taken out from -80 DEG C of refrigerators, and after cell recovery is grown well, Examples 1 to 6 is made respectively
Grafting polyethylene imine rice bran polysaccharide be put into after ultraviolet-sterilization is handled culture for 24 hours；Then cell is contaminated with DAPI
Color the case where growth on the surface of the material using fluorescence microscope HeLA cell, and records fluorescent microscopy images such as Fig. 3 institute
Show.
As can be seen from Figure 3, grafting polyethylene imine rice bran polysaccharide surface made from each embodiment is attached to a certain number of
HeLA cell, and do not present and substantially reduce phenomenon, illustrate that grafting polyethylene imine rice bran polysaccharide has good cell compatibility
And hypotoxicity.
(3) cytotoxicity detects
It is more that the grafting polyethylene imine rice bran prepared in Examples 1 to 6 under same concentration is measured using thiazolyl blue (MTT) method
Influence of the sugar to Hela cell survival rate.Plating density of the Hela cell in 96 orifice plates is 1x104/ hole is placed in 37 DEG C, 5%
CO2Under the conditions of cultivate for 24 hours, when cell growth reach 70-80% convergence degree when, respectively into each hole be added concentration be 0.5mg/mL
10 μ L of grafting polyethylene imine rice bran polysaccharide solution, under the same terms stimulation cell for 24 hours after, every hole is added after siphoning away culture solution
The MTT solution of 100 μ L 5mg/mL continues under the same terms after placing 4h, sucks cell culture fluid, 100 μ L bis- are added in each hole
Methyl sulfoxide dissolves the crystal that cell generates, with the absorbance value A1 at microplate reader measurement 570nm.Not to be loaded the cell of sample wells
Survival rate is 100%, absorbance value A0.Cell survival rate is calculated by formula 1, and the results are shown in Table 1.
Formula 1: cell survival rate (%)=A1/A0 × 100
The cytotoxicity of grafting polyethylene imine rice bran polysaccharide obtained in 1 Examples 1 to 6 of table
| Hela cell survival rate (%) | |
| Embodiment 1 | 95.6 |
| Embodiment 2 | 97.7 |
| Embodiment 3 | 96.2 |
| Embodiment 4 | 96.8 |
| Embodiment 5 | 98.0 |
| Embodiment 6 | 97.8 |
As can be seen from Table 1, when the grafting polyethylene imine rice bran polysaccharide concentration of each embodiment preparation is 0.05mg/mL,
The cell survival rate of Hela cell is 95% or more, illustrates the grafting polyethylene imine rice bran polysaccharide prepared by the method
Cytotoxicity is very low, is especially advantageous for grafting polyethylene imine rice bran polysaccharide passenger gene and enters cell.
The above is only a preferred embodiment of the present invention, is not intended to limit the scope of the invention, for this field
For technical staff, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any
Modification, equivalent replacement, improvement etc. should all be included within the scope of the present invention.
Claims (10)
1. a kind of grafting polyethylene imine rice bran polysaccharide, which is characterized in that the grafting polyethylene imine rice bran polysaccharide has knot
Structure shown in structure formula (1).
Structural formula (1):
2. a kind of preparation method of grafting polyethylene imine rice bran polysaccharide as described in claim 1, which is characterized in that including with
Lower step:
Potassium metaperiodate is added into the aqueous solution of rice bran polysaccharide, is protected from light after stirring forms reaction solution, the reaction solution is carried out saturating
Analysis, obtains dialyzate；
Polyethyleneimine is dissolved in buffer, polyethylenimine solution is formed；
The dialyzate is added to and is stirred to react to form reaction solution in the polyethylenimine solution, and in whipping process
Reaction solution is controlled in alkalinity；
Sodium borohydride is added in the reaction solution, is stirred to react to form just sample liquid；
Dialyse to the just sample liquid, take after permeate alcohol precipitation, centrifugation, freeze-drying grafting polyethylene imine rice bran is more
Sugar.
3. the preparation method of grafting polyethylene imine rice bran polysaccharide as claimed in claim 2, which is characterized in that described to rice bran
Potassium metaperiodate is added in the aqueous solution of polysaccharide, is protected from light after stirring forms reaction solution, dialyses to the reaction solution, obtain dialyzate
The step of in,
The mixing time that is protected from light is 24~48h；And/or
The time of the dialysis is 36~48h.
4. the preparation method of grafting polyethylene imine rice bran polysaccharide as claimed in claim 2, which is characterized in that described by poly- second
In the step of alkene imines is dissolved in buffer, forms polyethylenimine solution,
The buffer is pH9.0 phosphate buffer.
5. the preparation method of grafting polyethylene imine rice bran polysaccharide as claimed in claim 2, which is characterized in that by the dialysis
Liquid is added in the polyethylenimine solution and is stirred to react to form reaction solution, and controls reaction solution in whipping process and be in
Alkalinity step include:
The dialyzate is added drop-wise in the polyethylenimine solution and is stirred to react 30~36h formation reaction solution, and is being dripped
Sodium hydroxide solution is added dropwise during adding, in Xiang Suoshu reaction solution to control the pH value of the reaction solution in 8.8~9.2 models
In enclosing；
Wherein, described that the dialyzate is added drop-wise to the time in the polyethylenimine solution no more than 1h.
6. the preparation method of grafting polyethylene imine rice bran polysaccharide as claimed in claim 2, which is characterized in that rice bran polysaccharide,
The mass ratio of potassium metaperiodate, polyethyleneimine and sodium borohydride is 1:(1.5~2.0): (2.5~4.0): (1.5~4).
7. the preparation method of grafting polyethylene imine rice bran polysaccharide as claimed in claim 6, which is characterized in that described by boron hydrogen
Change sodium to be added in the reaction solution, be stirred to react the step of forming just sample liquid, comprising:
Sodium borohydride is added in the reaction solution, 40~48h is stirred to react and forms the first mixed liquor；
Sodium borohydride is added in first mixed liquor, 22~formation for 24 hours just sample liquid is stirred to react.
8. the preparation method of grafting polyethylene imine rice bran polysaccharide as claimed in claim 7, which is characterized in that described by boron hydrogen
The mass ratio for changing the additional amount and rice bran polysaccharide additional amount of the sodium borohydride in the step that is added in the reaction solution of sodium is
(1~1.5): 1；And/or
The additional amount and rice bran polysaccharide that sodium borohydride is added to the sodium borohydride in the step in first mixed liquor
The mass ratio of additional amount is (0.5~2.5): 1.
9. the preparation method of grafting polyethylene imine rice bran polysaccharide as claimed in claim 2, which is characterized in that described to described
First sample liquid is dialysed, take after permeate alcohol precipitation, centrifugation, freeze-drying grafting polyethylene imine rice bran polysaccharide the step of in,
When the dialysis, the molecular cut off of the bag filter used is 3000~3200；And/or
The dialysis time is 24~48h；And/or
The alcohol precipitation time is 48~50h.
10. a kind of genophore, which is characterized in that including grafting polyethylene imine rice bran polysaccharide as described in claim 1.
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