CN106279464A - Modification of chitosan transgene carrier that a kind of pH is sensitive and preparation method and application - Google Patents
Modification of chitosan transgene carrier that a kind of pH is sensitive and preparation method and application Download PDFInfo
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- CN106279464A CN106279464A CN201510270045.5A CN201510270045A CN106279464A CN 106279464 A CN106279464 A CN 106279464A CN 201510270045 A CN201510270045 A CN 201510270045A CN 106279464 A CN106279464 A CN 106279464A
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Abstract
The present invention discloses modification of chitosan transgene carrier sensitive for a kind of pH and preparation method and application, gamatine and dimethyl maleic anhydride are grafted on chitosan macromolecular main chain respectively, form the biomacromolecule simultaneous with positive and negative charge, and prepared material is used for gene transfection.It is an advantage of the current invention that its preparation method is simple, reaction condition is gentle, and resulting vehicle can be effectively compressed DNA under conditions of neutral ph and form the nano-complex that surface is electronegative, the beneficially absorption of complex anti-serum protein.And under condition of acidic pH, there is charge reversal, the surface charge of nano-complex is changed to positive by negative, the beneficially cell endocytic of complex, and then improves transfection efficiency.
Description
Technical field
The present invention relates to a kind of transgene carrier and preparation method, more particularly, it relates to the changing of a kind of pH of having sensitivity
Property chitosan transgene carrier and preparation method thereof.
Background technology
Gene therapy, refers to the normal gene of the mankind or the base with certain therapeutical effect by certain external source mode
Because fragment imports the normal cell of the mankind, so as to correcting the genetic flaw of produced disease or playing the one of therapeutical effect
Emerging technology.So developing and selecting suitable genophore, it is possible to efficiently genetic fragment is delivered to lesions position,
And enable the high efficient expression of gene, become the key link of gene therapy technology.In the development of recent decades, mainly
Occur in that two kinds of genophore: viral vector and non-virus carrier.Viral vector is due to the cell infection of its uniqueness
Mechanism and the ability of self replication, it is possible to obtain the highest cell transfecting efficiency, obtain good therapeutic effect.But by
The shortcomings such as the carrying capacity in viral vector is low, guidance quality is poor, potential carcinogenecity and immunogenicity, limit it in clinic
Application in treatment.Non-virus carrier has hypotoxicity, low carcinogenecity, reduced immunogenicity, carries genes of interest ability height
With advantages such as easy preparations, achieve huge progress in recent years.Non-virus carrier mainly includes naked DNA, liposome,
Cation superpolymer etc..Wherein cation superpolymer is due to wide material sources, prepares simple and diversified, is the most extensively ground
The person of studying carefully favors, and has evolved into a class maximum in non-viral gene vector.But cation superpolymer is as genophore
During application, still presenting some problems, such as poor biocompatibility, toxic, biological degradability is low and transfection effect
Rate is low.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, it is provided that a kind of good biocompatibility, toxicity are little, transfection effect
The cation superpolymer that rate is high uses as non-viral type transgene carrier.
It is an object of the invention to overcome the deficiencies in the prior art, it is provided that a kind of method preparing above-mentioned cation superpolymer.
The present invention also aims to provide the application of a kind of above-mentioned cation superpolymer, as the application of transgene carrier.
The above-mentioned purpose of the present invention is achieved by following technical proposals:
Modification of chitosan transgene carrier, as cation superpolymer, uses as transgene carrier.
Modification of chitosan transgene carrier, is grafted on chitosan macromole master respectively by gamatine and dimethyl maleic anhydride
Forming the biomacromolecule simultaneous with positive and negative charge on chain, molecular structure is shown below:
With chitosan molecule as main chain, the amino of chitosan is grafted dimethyl maleic anhydride, at the methylol of chitosan
Upper grafting gamatine.
The number-average molecular weight of above-mentioned modification of chitosan is 12 20 ten thousand.
In above-mentioned modification of chitosan, the substitution value of dimethyl maleic anhydride is 90 95%, and the substitution value of gamatine is
9% 14%.
A kind of method preparing modification of chitosan transgene carrier, is first grafted on the ammonia of chitosan by dimethyl maleic anhydride
On base, generate the chitosan that dimethyl maleic anhydride is modified；Secondly with paratoluensulfonyl chloride, dimethyl maleic anhydride is modified
The hydroxymethyl position of chitosan be modified, generate the p-toluenesulfonyl chitosan that dimethyl maleic anhydride is modified；?
After by dimethyl maleic anhydride modify p-toluenesulfonyl chitosan react with gamatine, generation dimethyl maleic anhydride
The chitosan jointly modified with gamatine, concrete preparation process is as follows:
Step 1, is grafted on dimethyl maleic anhydride on the amino of chitosan, obtains the shell that dimethyl maleic anhydride is modified
Polysaccharide；
Dimethyl maleic anhydride is dissolved in the mixed solvent of DMF and water, is added thereto to chitosan,
After mix homogeneously, in an inert atmosphere, reaction temperature 100 150 DEG C, 35 hours response time.Reaction will after terminating
Product is cooled to room temperature, precipitates with ethanol and washs three times, and the most vacuum dried dimethyl maleic anhydride that i.e. can get is repaiied
The chitosan of decorations；
In step 1, sufficiently high to the substitution value of amino of chitosan for ensureing dimethyl maleic anhydride, chitosan sugar ring is tied
Structure unit is 1:(1 4 with the mol ratio of dimethyl maleic anhydride), preferably 1:(2 3)；N, N-dimethyl formyl
Amine is with the mixed solvent of water, and the volume ratio of water and DMF is 1:(20 25), preferably 1:(22
24)；For guaranteeing that reaction is smoothed out, reaction need to be carried out under anaerobic, by be passed through noble gas (as nitrogen,
Helium, argon) realize.
In step 1, carry out continuously stirred in course of reaction, to realize the uniform mixing of system, such as 150 300
Turn/min.
Step 2, the hydroxymethyl position of the chitosan that dimethyl maleic anhydride is modified is modified by paratoluensulfonyl chloride,
The p-toluenesulfonyl chitosan modified to dimethyl maleic anhydride；
The chitosan that dimethyl maleic anhydride step 1 prepared is modified is dissolved in the sodium hydrate aqueous solution of 1M, places
Stir in ice-water bath, then paratoluensulfonyl chloride is dissolved in acetonitrile, be added drop-wise to dimethyl maleic anhydride modification shell and gather
In the sodium hydrate aqueous solution of sugar, ice-water bath stirring reaction forwards room temperature 20 25 degrees Celsius to after at least 2 hours and continues reaction
At least 3 hours.
In step 2, chitosan sugar ring structure unit is 1:(6 20 with the mol ratio of paratoluensulfonyl chloride), preferably
1:(8 15)；Course of reaction is carried out continuously stirred, to realize the uniform mixing of system, such as 150 300 turns
/min；Rate of addition is 1 5ml/min；Reaction carries out the ice-water bath reaction of 24 hours at the beginning, is then warming up to 20
25 degrees Celsius are continued reaction 45 hours.After completion of the reaction, precipitate in order to avoid the degeneration ethanol of product and wash three times,
Then with phosphate buffer (pH=7.4) washing until neutral, the most vacuum dried i.e. can get dimethyl maleic acid
The p-toluenesulfonyl chitosan that acid anhydride is modified.
Step 3, reacts the p-toluenesulfonyl chitosan that dimethyl maleic anhydride is modified with gamatine, and generates dimethyl
The chitosan that maleic anhydride and gamatine are modified jointly；
The p-toluenesulfonyl chitosan that the dimethyl maleic anhydride step 2 prepared is modified be dissolved in gamatine mix molten
In agent, after stirring in an inert atmosphere, 80 90 DEG C of stirrings are reacted at least 24 hours；Described mixed solvent is by 0.2M
Sodium hydrate aqueous solution and the mixed solvent of triethanolamine composition, 0.2M sodium hydrate aqueous solution and the volume ratio of triethanolamine
For (1 2): 1, preferably (1.5 2): 1.
In step 3, chitosan sugar ring structure unit is 1:(6 20 with the mol ratio of gamatine), preferably 1:
(8—15)；In step 3, carry out continuously stirred in course of reaction, to realize the uniform mixing of system, such as 150
300 turns/min.
In step 3, in order to avoid the degeneration of product, final reactant liquor is placed in the dialysis that molecular cut off is 3500
Dialysing 5 days with phosphate buffer (pH=7.4) in Dai, lyophilizing i.e. obtains dimethyl maleic anhydride and jointly repaiies with gamatine
The chitosan of decorations.
In step 3, for guaranteeing that reaction is smoothed out, reaction need to be carried out under anaerobic, by being passed through noble gas
(such as nitrogen, helium, argon) realizes.
In step 3, preferably 80 90 DEG C of stirring reactions 24 48 hours.
In the inventive solutions, chitosan sugar ring structure unit refers to as in above-mentioned molecular formula, on chitosan main chain
The circulus of hexa-atomic sugar, the sugared ring being i.e. made up of five carbon and an oxygen.
Utilize 500MHz HIGH RESOLUTION SUPERCONDUCTING nuclear magnetic resonance analyser Varian UNITY plus-500 to raw material, intermediate product and
Whole sample detects, and obtains hydrogen spectrum, and result is as shown in accompanying drawing 1,3,4.By accompanying drawing 4 it can be seen that chemical shift
Peak value correspondence chemical group-C (CH at 1.703)=C (CH3)-in hydrogen atom, and by chitosan shown in accompanying drawing 3 at this
Peak does not occur at chemical shift, illustrates that dimethyl maleic anhydride is modified successfully.From accompanying drawing 1 it can be seen that chemical potential
Move the peak value correspondence chemical group-C (CH at 1.723)=C (CH3)-in hydrogen atom, illustrate that dimethyl maleic anhydride is modified
Success.Peak value at chemical shift 3.04,2.83,1.42 3 corresponding gamatine respectively is in the hydrogen of three kinds of chemical environments
Atom, illustrates that gamatine is modified successfully, illustrates further the shell that dimethyl maleic anhydride is modified jointly with gamatine
Polysaccharide is successfully prepared.
The chitosan that dimethyl maleic anhydride prepared by the present invention and gamatine are modified jointly can be at the bar of pH=7.4
It is effectively compressed DNA under part and forms the nano-complex that surface is electronegative, and under conditions of pH=6.5, charge reversal
Occurring, the surface charge of nano-complex is changed to positive by negative.Dimethyl maleic anhydride and gamatine are modified jointly
The complex that chitosan is formed with DNA is respectively placed in the buffer of pH=7.4 and pH=6.5, with Malvern company
The current potential of Zetasizer Nano ZS90 Instrument measuring different time points, result is as shown in Figure 2.Can from accompanying drawing 2
Going out, under conditions of pH=7.4, the chitosan that dimethyl maleic anhydride and gamatine are modified jointly is formed with DNA
The change over time of composite surface current potential is held essentially constant, and maintains about-12mv all the time.And pH=6.5's
Under the conditions of, the surface potential of formed complex was changed to positive by negative rapidly within half an hour, finally maintained 12mv left
Right.It can be seen that the complex that the chitosan jointly modified of dimethyl maleic anhydride and gamatine and DNA are formed has
PH sensitivity, along with the reduction of pH value, charge reversal phenomenon occurs.
It is an advantage of the current invention that preparation method is simple, reaction condition is gentle, and choosing biomacromolecule chitosan is main chain,
There is good biocompatibility, the advantages such as cytotoxicity is little, degradable.Prepared carrier can compress DNA shape effectively
Become nano-complex, reach to increase substantially the purpose of transfection efficiency by regulation pH value.
Accompanying drawing explanation
Fig. 1 is the proton nmr spectra of the modification of chitosan CS-DM-Agm10 of the present invention.
Fig. 2 is that the current potential of modification of chitosan CS-DM-Agm10 Yu the DNA formation complex of the present invention changes over figure, 1
The complex current potential versus time curve under conditions of pH=7.4 formed for CS-DM-Agm10 and DNA, 2
The complex current potential versus time curve under conditions of pH=6.5 formed for CS-DM-Agm10 and DNA.
Fig. 3 is the proton nmr spectra of the chitosan that the present invention uses.
Fig. 4 is to use dimethyl maleic anhydride to modify the proton nmr spectra of chitosan in the present invention.
Detailed description of the invention
Technical scheme is further illustrated below in conjunction with specific embodiment.
Embodiment 1:
Dimethyl maleic anhydride (5.86g, 46.5mmol, sigma) is dissolved in DMF (48mL) and water
(2mL), in mixed solution, it is added thereto to chitosan (2.5g, 15.5mmol sugar ring structure unit, sigma),
After mix homogeneously, in nitrogen atmosphere, 130 DEG C of stirrings are reacted 5 hours.Product is cooled to room temperature after terminating by reaction, uses
Ethanol precipitates and washs three times, the most vacuum dried chitosan that i.e. can get dimethyl maleic anhydride modification, named
CS-DMA,1H-NMR assay products structure: the substitution value of dimethyl maleic anhydride is about 95%.
The chitosan (the sugared ring structure unit of 1.2g, 5mmol) modified by dimethyl maleic anhydride obtained above is dissolved in 1M
Sodium hydroxide solution (100mL) in, be placed in ice-water bath and stir.Then by paratoluensulfonyl chloride (7.62g,
40mmol, sigma) it is dissolved in acetonitrile (40mL), it is added dropwise to dimethyl maleic anhydride and modifies the hydrogen-oxygen of chitosan
Changing in sodium solution, ice-water bath stirring reaction forwards room temperature to after 2 hours and continues reaction 3 hours.After completion of the reaction, ethanol is used
Precipitate and wash three times, then with phosphate buffer (pH=7.4) washing until neutrality, the most vacuum dried
Obtain the p-toluenesulfonyl chitosan that dimethyl maleic anhydride is modified, named CS-DMA-Ts8.
P-toluenesulfonyl chitosan and gamatine that all dimethyl maleic anhydrides obtained above are modified (5.7g, 25
Mmol, sigma) it is dissolved in the sodium hydroxide (60mL) of 0.2M and the mixed solution of triethanolamine (40mL), stir
After mixing uniformly, in nitrogen atmosphere, 85 DEG C of stirrings are reacted 48 hours, and it is 3500 that final reactant liquor is placed in molecular cut off
Bag filter in dialyse 5 days with phosphate buffer (pH=7.4), lyophilizing i.e. obtains dimethyl maleic anhydride and gamatine
The common chitosan modified, named CS-DM-Agm8,1H-NMR assay products structure: the substitution value of gamatine
It is about 9%.
The end product 2mg obtained is dissolved in 1mL ultra-pure water, with 0.22 μm filter bacteriological filtration.By above-mentioned solution and body such as grade
Long-pending pGL3 plasmid solution is hybridly prepared into CS-DM-Agm8/DNA complex, and composite quality ratio is 12/1, stands
After 30 minutes, complex solution is joined the human cervical carcinoma cell (HeLa) cultivated to exponential phase of growth containing serum
In training base (pH=7.4 or 6.5), after transfecting 4 hours, change liquid, continue to cultivate 44 hours.Cell lysis, measures it
In uciferase activity be: 7.94 × 104RLU/mg protein (pH=7.4), 2.22 × 107RLU/mg protein
(pH=6.5).
Embodiment 2:
Dimethyl maleic anhydride (5.86g, 46.5mmol, sigma) is dissolved in DMF (48mL) and water
(2mL), in mixed solution, it is added thereto to chitosan (2.5g, 15.5mmol sugar ring structure unit, sigma),
After mix homogeneously, in nitrogen atmosphere, 130 DEG C of stirrings are reacted 5 hours.Product is cooled to room temperature after terminating by reaction, uses
Ethanol precipitates and washs three times, the most vacuum dried chitosan that i.e. can get dimethyl maleic anhydride modification, named
CS-DMA,1H-NMR assay products structure: the substitution value of dimethyl maleic anhydride is about 95%.
The chitosan (the sugared ring structure unit of 1.2g, 5mmol) modified by dimethyl maleic anhydride obtained above is dissolved in 1M
Sodium hydroxide solution (100mL) in, be placed in ice-water bath and stir.Then by paratoluensulfonyl chloride (9.53g,
50mmol, sigma) it is dissolved in acetonitrile (40mL), it is added dropwise to dimethyl maleic anhydride and modifies the hydrogen-oxygen of chitosan
Changing in sodium solution, ice-water bath stirring reaction forwards room temperature to after 2 hours and continues reaction 3 hours.After completion of the reaction, ethanol is used
Precipitate and wash three times, then with phosphate buffer (pH=7.4) washing until neutrality, the most vacuum dried
Obtain the p-toluenesulfonyl chitosan that dimethyl maleic anhydride is modified, named CS-DMA-Ts10.
P-toluenesulfonyl chitosan and gamatine that all dimethyl maleic anhydrides obtained above are modified (5.7g, 25
Mmol, sigma) it is dissolved in the sodium hydroxide (60mL) of 0.2M and the mixed solution of triethanolamine (40mL), stir
After mixing uniformly, in nitrogen atmosphere, 85 DEG C of stirrings are reacted 48 hours, and it is 3500 that final reactant liquor is placed in molecular cut off
Bag filter in dialyse 5 days with phosphate buffer (pH=7.4), lyophilizing i.e. obtains dimethyl maleic anhydride and gamatine
The common chitosan modified, named CS-DM-Agm10,1H-NMR assay products structure: the substitution value of gamatine
It is about 13%.
The end product 2mg obtained is dissolved in 1mL ultra-pure water, with 0.22 μm filter bacteriological filtration.By above-mentioned solution and body such as grade
Long-pending pGL3 plasmid solution is hybridly prepared into CS-DM-Agm10/DNA complex, and composite quality ratio is 10/1, stands
After 30 minutes, complex solution is joined the human cervical carcinoma cell (HeLa) cultivated to exponential phase of growth containing serum
In training base (pH=7.4 or 6.5), after transfecting 4 hours, change liquid, continue to cultivate 44 hours.Cell lysis, measures it
In uciferase activity be: 1.38 × 105RLU/mg protein (pH=7.4), 4.04 × 107RLU/mg protein
(pH=6.5).
Embodiment 3:
Dimethyl maleic anhydride (5.86g, 46.5mmol, sigma) is dissolved in DMF (48mL) and water
(2mL), in mixed solution, it is added thereto to chitosan (2.5g, 15.5mmol sugar ring structure unit, sigma),
After mix homogeneously, in nitrogen atmosphere, 130 DEG C of stirrings are reacted 5 hours.Product is cooled to room temperature after terminating by reaction, uses
Ethanol precipitates and washs three times, the most vacuum dried chitosan that i.e. can get dimethyl maleic anhydride modification, named
CS-DMA,1H-NMR assay products structure: the substitution value of dimethyl maleic anhydride is about 95%.
The chitosan (the sugared ring structure unit of 1.2g, 5mmol) modified by dimethyl maleic anhydride obtained above is dissolved in 1M
Sodium hydroxide solution (100mL) in, be placed in ice-water bath and stir.Then by paratoluensulfonyl chloride (14.29
G, 75mmol, sigma) it is dissolved in acetonitrile (40mL), it is added dropwise to dimethyl maleic anhydride and modifies chitosan
In sodium hydroxide solution, ice-water bath stirring reaction forwards room temperature to after 2 hours and continues reaction 3 hours.After completion of the reaction, use
Ethanol precipitates and washs three times, then with phosphate buffer (pH=7.4) washing until neutrality, the most vacuum dried
I.e. can get the p-toluenesulfonyl chitosan that dimethyl maleic anhydride is modified, named CS-DMA-Ts15.
P-toluenesulfonyl chitosan and gamatine that all dimethyl maleic anhydrides obtained above are modified (5.7g, 25
Mmol, sigma) it is dissolved in the sodium hydroxide (60mL) of 0.2M and the mixed solution of triethanolamine (40mL), stir
After mixing uniformly, in nitrogen atmosphere, 85 DEG C of stirrings are reacted 48 hours, and it is 3500 that final reactant liquor is placed in molecular cut off
Bag filter in dialyse 5 days with phosphate buffer (pH=7.4), lyophilizing i.e. obtains dimethyl maleic anhydride and gamatine
The common chitosan modified, named CS-DM-Agm15,1H-NMR assay products structure: the substitution value of gamatine
It is about 14%.
The end product 2mg obtained is dissolved in 1mL ultra-pure water, with 0.22 μm filter bacteriological filtration.By above-mentioned solution and body such as grade
Long-pending pGL3 plasmid solution is hybridly prepared into CS-DM-Agm15/DNA complex, and composite quality ratio is 10/1, stands
After 30 minutes, complex solution is joined the human cervical carcinoma cell (HeLa) cultivated to exponential phase of growth containing serum
In training base (pH=7.4 or 6.5), after transfecting 4 hours, change liquid, continue to cultivate 44 hours.Cell lysis, measures it
In uciferase activity be: 8.30 × 104RLU/mg protein (pH=7.4), 2.29 × 107RLU/mg protein
(pH=6.5).
The experimental detail recorded according to technical scheme and embodiment, being adjusted under the process conditions all may system
The modification of chitosan of the standby present invention, and show the character essentially identical with embodiment.
Above the present invention is done exemplary description, it should explanation, in the case of without departing from the core of the present invention,
Any simple deformation, amendment or other those skilled in the art can not spend the equivalent of creative work all to fall
Enter protection scope of the present invention.
Claims (10)
1. a modification of chitosan transgene carrier sensitive for pH, it is characterised in that by gamatine and dimethyl Malaysia
Anhydride is grafted on respectively on chitosan macromolecular main chain and forms the biomacromolecule simultaneous with positive and negative charge, divides with chitosan
Son is main chain, is grafted dimethyl maleic anhydride on the amino of chitosan, is grafted gamatine on the methylol of chitosan.
The modification of chitosan transgene carrier that a kind of pH the most according to claim 1 is sensitive, it is characterised in that modified
The number-average molecular weight of chitosan is 12 20 ten thousand, and the substitution value of dimethyl maleic anhydride is 90 95%, taking of gamatine
Dai Du is 9% 14%.
3. the preparation method of a modification of chitosan transgene carrier sensitive for pH, it is characterised in that first by dimethyl
Maleic anhydride is grafted on the amino of chitosan, generates the chitosan that dimethyl maleic anhydride is modified；Secondly with to toluene sulphur
The hydroxymethyl position of the chitosan that dimethyl maleic anhydride is modified is modified by acyl chlorides, generates dimethyl maleic anhydride and modifies
P-toluenesulfonyl chitosan；The p-toluenesulfonyl chitosan finally dimethyl maleic anhydride modified and gamatine
Reaction, generates the chitosan that dimethyl maleic anhydride is modified jointly with gamatine, and concrete preparation process is as follows:
Step 1, is grafted on dimethyl maleic anhydride on the amino of chitosan, and the shell obtaining dimethyl maleic anhydride modification gathers
Sugar；Dimethyl maleic anhydride is dissolved in the mixed solvent of DMF and water, is added thereto to chitosan,
After mix homogeneously, in an inert atmosphere, reaction temperature 100 150 DEG C, 35 hours response time；In step 1,
Chitosan sugar ring structure unit is 1:(1 4 with the mol ratio of dimethyl maleic anhydride)；N,N-dimethylformamide and water
Mixed solvent in, the volume ratio of water and DMF is 1:(20 25)；
Step 2, the hydroxymethyl position of the chitosan that dimethyl maleic anhydride is modified is modified by paratoluensulfonyl chloride,
The p-toluenesulfonyl chitosan modified to dimethyl maleic anhydride；Dimethyl maleic anhydride step 1 prepared is modified
Chitosan is dissolved in the sodium hydrate aqueous solution of 1M, is placed in ice-water bath and stirs, then by paratoluensulfonyl chloride
Being dissolved in acetonitrile, be added drop-wise in the sodium hydrate aqueous solution that dimethyl maleic anhydride modifies chitosan, ice-water bath stirring reaction is extremely
Forward room temperature 20 25 degrees Celsius after few 2 hours to and continue reaction at least 3 hours；In step 2, chitosan sugar ring structure
Unit is 1:(6 20 with the mol ratio of paratoluensulfonyl chloride)；
Step 3, reacts the p-toluenesulfonyl chitosan that dimethyl maleic anhydride is modified with gamatine, and generates dimethyl
The chitosan that maleic anhydride and gamatine are modified jointly；Dimethyl maleic anhydride prepared by step 2 modify to toluene
Sulfonyl chitosan and gamatine are dissolved in mixed solvent, and after stirring in an inert atmosphere, 80 90 DEG C of stirrings are anti-
Should at least 24 hours；The mixed solvent that described mixed solvent is made up of 0.2M sodium hydrate aqueous solution and triethanolamine, 0.2M
The volume ratio of sodium hydrate aqueous solution and triethanolamine is (1 2): 1；Chitosan sugar ring structure unit and gamatine
Mol ratio is 1:(6 20).
The preparation method of the modification of chitosan transgene carrier that a kind of pH the most according to claim 3 is sensitive, it is characterised in that
In step 1, chitosan sugar ring structure unit is 1:(2 3 with the mol ratio of dimethyl maleic anhydride)；N, N-diformazan
Base Methanamide is with the mixed solvent of water, and the volume ratio of water and DMF is 1:(22 24).
The preparation method of the modification of chitosan transgene carrier that a kind of pH the most according to claim 3 is sensitive, its feature exists
In, in step 1, for guaranteeing that reaction is smoothed out, reaction need to be carried out under anaerobic, by being passed through noble gas
Realizing, noble gas is nitrogen, helium or argon；In step 1, carry out continuously stirred in course of reaction,
To realize the uniform mixing of system, such as 150 300 turns/min.
The preparation method of the modification of chitosan transgene carrier that a kind of pH the most according to claim 3 is sensitive, its feature exists
In, in step 2, chitosan sugar ring structure unit is 1:(8 15 with the mol ratio of paratoluensulfonyl chloride).
The preparation method of the modification of chitosan transgene carrier that a kind of pH the most according to claim 3 is sensitive, its feature exists
In, in step 2, carry out continuously stirred in course of reaction, to realize the uniform mixing of system, such as 150 300
Turn/min；Rate of addition is 1 5ml/min；Reaction carries out the ice-water bath reaction of 24 hours at the beginning, is then warming up to
20 25 degrees Celsius are continued reaction 45 hours.
The preparation method of the modification of chitosan transgene carrier that a kind of pH the most according to claim 3 is sensitive, its feature exists
In, in step 3, the volume ratio of 0.2M sodium hydrate aqueous solution and triethanolamine is (1.5 2): 1；Chitosan sugar
Ring structure unit is 1:(8 15 with the mol ratio of gamatine)；Preferably little 80 90 DEG C of stirring reactions 24 48
Time.
The preparation method of the modification of chitosan transgene carrier that a kind of pH the most according to claim 3 is sensitive, its feature exists
In, in step 3, carry out continuously stirred in course of reaction, to realize the uniform mixing of system, such as 150 300
Turn/min；For guaranteeing that reaction is smoothed out, reaction need to be carried out under anaerobic, realizes by being passed through noble gas,
Described noble gas is nitrogen, helium or argon.
The modification of chitosan transgene carrier that a kind of pH the most as described in claim 1 or 2 is sensitive, gathers as cation height
Thing, the application in transgenic field.
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| CN114213570A (en) * | 2020-12-18 | 2022-03-22 | 四川大学华西医院 | Acid-sensitive antibacterial coating and preparation method and application thereof |
| CN114213570B (en) * | 2020-12-18 | 2022-12-02 | 四川大学华西医院 | Acid-sensitive antibacterial coating and preparation method and application thereof |
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