CN103182087A - Trimethyl chitosan-graft-polyethylene glycol/nucleic acid brain-targeting micellar and preparation method thereof - Google Patents
Trimethyl chitosan-graft-polyethylene glycol/nucleic acid brain-targeting micellar and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of medicine, and relates to an acetylcholine-receptor-mediated trimethyl chitosan-graft-polyethylene glycol/nucleic acid brain-targeting micellar and a preparation method thereof. According to the invention, trimethyl chitosan-graft-polyethylene glycol-brain-targeting functional peptide is obtained through the steps that: chitosan is sequentially subjected to quaternary ammonium modification such that trimethyl chitosan is obtained; the amino group of trimethyl chitosan is subjected to a reaction with an active group of two-end-activated polyethylene glycol, such that trimethyl chitosan-graft-polyethylene glycol is obtained; and another active group of polyethylene glycol is subjected to a reaction with brain-targeting functional peptide RVG. According to the invention, ion composite micellar is adopted as a carrier, and brain-targeting functional peptide RVG is adopted as a targeting molecule, such that nucleic acid medicine can actively target and position through blood-brain barrier and brain. The ion composite micellar provided by the invention is formed through static composition of positively charged trimethyl chitosan-graft-polyethylene glycol-brain-targeting functional peptide and negatively charged nucleic acid. With the invention, nucleic acid medicine defects such as easy in-vivo degradation, poor stability, and low transfection efficiency are solved.
Description
Technical field
The invention belongs to medical field of gene, the preparation method and the technology that relate to new and effective non-virus carrier are specifically related to a kind of N-trimethyl chitosan TMC-grafting-Polyethylene Glycol-brain target function peptide/nucleic acid brain targeting micelle by acetylcholinergic receptor mediation and preparation method thereof.
Background technology
The number of suffering from brain diseasess such as nervus retrogression in the world increases just year by year, but it is also few in number to treat the active drug of this type of disease.Nucleic acid drug is because having the specificity height, acts on the forward position that advantage such as rapid, safe, efficient has become field of medicaments research.
But nucleic acid drug exists the problem that is difficult to overcome in application: the one, there are a large amount of nucleases in the body, and the phosphodiester bond that it can hydrolytic nucleic acid makes medicine lose efficacy with regard to being degraded before arriving target spot; The 2nd, the permeability of nucleic acid drug cell membrane is relatively poor, is difficult to arrive target site by cell membrane.Solution to the problems described above mainly adopts virus or non-virus carrier to realize the administration of nucleic acid drug at present.Wherein, do the seroimmunity reaction that carrier then may cause the host with virus coat.By contrast, non-virus carrier has that immunogenicity is low, cost is low, many advantages such as repressor gene injection volume not, become the important research direction of present nucleic acid drug carrier.The non-virus carrier of research mainly comprises at present: liposome, nanoparticle and polycation etc.Examples of such carriers is carried the nucleic acid cation lipids that have the high density positive charge or cationic polymerization material and electronegative nucleic acid of adopting more and is formed the transmission systems that controllability is strong, toxicity is low such as liposome, nanoparticle, micelle by electrostatic interaction.
The existence of blood brain barrier (BBB) between the body circulation because central nervous system is unified has limited many potential medicines (especially nucleic acid drug) and has entered brain from blood and play a role.The method that promotes medicine to see through BBB mainly contains following four kinds at present.Wherein, the densification of artificially opening on the BBB connects, and make harmful substance and the medicine of meeting non-selectivity are gone into brain simultaneously; Medicine is carried out structure of modification may cause pharmacokinetic parameters reduction or change in curative effect of medication and the body; Change route of administration and mainly comprise carotid artery infusion and nasal-cavity administration, the former easily damages brain, and latter's bioavailability is low, is difficult to satisfy clinical requirement; Then not having an above-mentioned shortcoming by receptor-mediated, is the focus of present brain drug delivery system research.
2007, (Kumar P such as Priti Kumar, Wu HQ, McBride JL, et al. Transvascular delivery of small interfering RNA to the central nervous system. Nature, 2007,448:39-43) research report is a kind of contains 29 amino acid whose little peptide RVG and has the targeting of acetylcholinergic receptor, acetylcholinergic receptor mainly is distributed on the brain cell, comprise the brain capillary endothelial cells that is used as the BBB model, this carrier that this peptide is modified has the ability of the efficient BBB of seeing through and targeting brain cell simultaneously.Experiment showed, that this little peptide not only oneself can pass through BBB, and the siRNA carrier band of FITC labelling can be gone into brain by 9 poly arginines that connect at c-terminus.Behind the intravenous administration, siRNA effectively realizes the reticent effect of target gene, and detects less than FITC fluorescence in the spleen regulating liver-QI, and this shows that this little peptide has become by force brain and brain cell targeting.
The graft copolymer that contains PEG side chain and polycation main chain can be by compound this micelle of polyion composite micelle that is self-assembled into of static as nucleic acid drug brain targeted delivery system with the nucleic acid that has negative charge, have the following advantages: core/shell structure can the holdup time of prolong drug in blood, can protect medicine to avoid contacting with enzyme again; The micelle particle diameter is little, and penetration power is strong, and good stability can be realized initiatively targeting behind the connection brain target function peptide; Can pass through blood brain barrier; The preparation process gentleness can keep pharmaceutically active to greatest extent.
To sum up, the present invention has prepared the nucleic acid polyion composite micelle with brain target function peptide RVG modification, realizing nucleic acid drug by blood brain barrier and brain targeting location initiatively, and solve its clinical practice defective such as easily degraded, instability and transfection efficiency be low in vivo.
Summary of the invention
The objective of the invention is to prepare a kind of brain targeting micelle of the N-trimethyl chitosan TMC-grafting-Polyethylene Glycol-brain target function peptide/nucleic acid by the acetylcholinergic receptor mediation, realizing nucleic acid drug by blood brain barrier and brain targeting location initiatively, and solve its clinical practice defective such as easily degraded, instability and transfection efficiency be low in vivo.
The present invention with the siRNA(sequence at Alzheimer's disease design be 5 '-GUGCCUACCUGGACAAGAAdTdT-3 ') as the model drug of nucleic acid drug, the technical scheme of employing following (as Fig. 1).
With the quaternized N-trimethyl chitosan TMC (TMC) that makes of chitosan (Chitosan), insert Polyethylene Glycol at N-trimethyl chitosan TMC and make N-trimethyl chitosan TMC-grafting-Polyethylene Glycol, then, the other end at Polyethylene Glycol inserts brain target function peptide RVG, make the N-trimethyl chitosan TMC-grafting-Polyethylene Glycol-brain target function peptide (TMC-g-PEG-RVG) of acetylcholinergic receptor mediation, make it have function through blood brain barrier and brain targeting, at last, the nucleic acid of this material and bear electricity is formed initiatively targeting micelle of brain certainly by the static compound action.
The preparation of N-trimethyl chitosan TMC-grafting of the present invention-Polyethylene Glycol-brain target function peptide comprises the steps:
1) purification of chitosan:
Chitosan weight average molecular weight of the present invention is 5 – 500kD, and deacetylation is 80% – 95%.
Concrete purification step is as described below:
Chitosan is dissolved in 1% aqueous acetic acid, regulates filtrate pH value to 7.0 precipitation chitosan, sucking filtration postlyophilization with ammonia behind the sucking filtration.
2) preparation of N-trimethyl chitosan TMC:
The present invention selects for use iodomethane as methylating reagent, and concrete preparation method is as described below:
Chitosan behind the purification (Chitosan) is added in the N-Methyl pyrrolidone (NMP) stirring at room swelling 12 hours.Add sodium iodide (NaI), sodium hydroxide (NaOH) solution and iodomethane (CH
3I), finished the first step in water-bath 30-60 minute and methylate for 60 ℃.Adding sodium hydroxide solution and iodomethane continues reaction and finished for second step in 30-120 minute and methylate.Reactant liquor is added absolute ethyl alcohol and stirring 1 hour precipitation N-trimethyl chitosan TMC, precipitate centrifugal back with washing with alcohol 3 times, ether washs 3 final vacuum dryings.After the drying product is dissolved in 50 gL
-1Sodium-chloride water solution in exchange I ion be the Cl ion, dialysis is 72 hours in the distilled water, the dialysis solution lyophilization namely gets N-trimethyl chitosan TMC (TMC).
The first step methylates and the methylated response time in second step by changing, and can make the TMC of the quaternized degree of a series of differences.The quaternized degree of N-trimethyl chitosan TMC of the present invention's preparation is 10% – 99%, mole percent.
The preparation of 3) N-trimethyl chitosan TMC-grafting-Polyethylene Glycol:
Concrete preparation process is as follows:
N-trimethyl chitosan TMC (TMC) reacts with the two ends activated polyglycol in water, and reaction temperature is 50 ℃ of 4 –, and the response time is 3 – 480 minutes.Reaction is removed unreacted Polyethylene Glycol by ultrafiltration is centrifugal, and ultrafiltration is centrifugal again after with distilled water diluting to obtain concentrated solution, repeats 3 times, at last with the ultrafiltrate lyophilization, namely.
When the Polyethylene Glycol consumption was the TMC of 3 times of quality in the above-mentioned preparation method, the Polyethylene Glycol percent grafting of products therefrom was 34%; When being the TMC of 1.5 times of quality, the Polyethylene Glycol percent grafting of products therefrom is 14%; When being the TMC of 1 times of quality, the Polyethylene Glycol percent grafting of products therefrom is 5%.
The general formula of described Polyethylene Glycol is R
1-(CH
2CH
2O)
n-R
2, n=20 – 200 in the formula, R
1For generating the active group of Polyethylene Glycol amido link with the amino reaction, comprise butanimide group, aceticoceptor, aldehyde group, isocyanate group, isocyanide sulfate group, acrylic acid groups, nitrophenols group, R
2For can with sulfydryl reactive activity group, comprise maleimide base group, vinyl sulfone(Remzaol group, thiol group.
The preparation of 4) N-trimethyl chitosan TMC-grafting-Polyethylene Glycol-brain target function peptide (TMC-g-PEG-RVG):
The present invention selects for use brain target function peptide RVG as targeted molecular, gives the initiatively function of targeting of micelle brain.
The selected brain target function peptide RVG(sequence of the present invention is YTIWMPENPRPGTPCDIFTNSRGKRASNG), it is characterized in that, contain 29 aminoacid (YTIWMPENPRPGTPCDIFTNSRGKRASNG), targeting with acetylcholinergic receptor has sulfydryl (SH) in the molecular structure.
Concrete preparation process is as follows:
N-trimethyl chitosan TMC-grafting-Polyethylene Glycol reacts with brain target function peptide RVG in the phosphate buffer of pH7 – 8, and reaction temperature is 30 ℃ of 15 –, and the response time is 8 – 24 hours.Unreacted RVG is removed so that ultrafiltration is centrifugal in reaction back, obtain concentrated solution with distilled water diluting after ultrafiltration again, repeat 3 times, at last with the ultrafiltrate lyophilization, namely get TMC-
g-PEG-RVG.
Above-mentioned N-trimethyl chitosan TMC-grafting-Polyethylene Glycol-brain target function peptide can be self-assembled into initiatively targeting micelle of brain with the nucleic acid of bear electricity, and concrete preparation process is as follows:
N-trimethyl chitosan TMC-grafting-Polyethylene Glycol-brain target function peptide (TMC-g-PEG-RVG) is water-soluble, pass through the filter membrane degerming of 0.22 μ m then.Nucleic acid is water-soluble.Respectively get a certain amount of above two kinds of solution in mixed at room temperature, vortex 5 – 30 s make it mix homogeneously, and room temperature left standstill 15 – 60 minutes.
The present invention prepared TMC-PEG-RVG and siRNA's+/-charge ratio is the micelle of 1:4 – 64:1, and particle size distribution is 30 – 700nm, and Zeta potential is+1mV –+15mV.
The N-trimethyl chitosan TMC-grafting-Polyethylene Glycol-brain target function peptide by the acetylcholinergic receptor mediation of the present invention preparation/siRNA brain initiatively targeting micelle is 83% to the transfection efficiency of Neuro-2a cell that acetylcholinergic receptor is arranged with cell surface expression, transfection efficiency with naked siRNA 0.41% is compared, and is significantly increased.
The Laser Scanning Confocal Microscope imaging shows, initiatively the targeting micelle can be relatively large enters the Neuro-2a cell that surface expression has acetylcholinergic receptor for the N-trimethyl chitosan TMC-grafting-Polyethylene Glycol-brain target function peptide by the acetylcholinergic receptor mediation of the present invention preparation/siRNA brain, and this result conforms to the flow cytometry result.
Description of drawings
Fig. 1 is technical scheme figure of the present invention.
Fig. 2 is example 2 infrared spectrum characterizations figure as a result.
Fig. 3 is example 2 H-NMR characterization result figure.
Fig. 4 is example 5 H-NMR characterization result figure.
Fig. 5 is example 6 H-NMR characterization result figure.
Fig. 6 is example 7 electrophoresis result figure.
Fig. 7 is example 9 Laser Scanning Confocal Microscopes figure as a result.
The specific embodiment
The purification of example 1 chitosan
Chitosan 13.8g is added in the aqueous acetic acid of 1120mL 1%, magnetic agitation made it dissolving, sucking filtration in 40 minutes, dropping ammonia is regulated filtrate pH value to 7.0 separates out the chitosan precipitation, with 0.45 μ m filter membrane sucking filtration, the chitosan product is with distilled water washing 3 times, lyophilization on the filter membrane.
The preparation of example 2 N-N-trimethyl chitosan TMCs
Take by weighing chitosan (molecular weight 50kD, deacetylation 95%) 0.5 g of purification in 50 mL three-neck flasks, add N-Methyl pyrrolidone 20 mL, stirring at room swelling 12 hours.Add 1.2 g sodium iodide, 2.8 mL mass concentrations are 0.2 gmL
-1Sodium hydroxide solution and 3 mL iodomethane, 60 ℃ of stirring in water bath were finished the first step in 45 minutes and are methylated.Add again 2.8 mL sodium hydroxide solutions and 1.25 mL iodomethane reactions finished in 45 minutes second the step methylate.Reactant liquor is added 200 mL absolute ethyl alcohol and stirring 1 hour precipitation N-trimethyl chitosan TMC, precipitate centrifugal (5000 rpm, 15 minutes) back with washing with alcohol 3 times, ether washs 3 final vacuum dryings.After the drying product is dissolved in 20 mL, 50 gL
-1Sodium-chloride water solution in exchange I ion be the Cl ion, be 48 hours purification of dialysis in the bag filter distilled water of 12 –, 14 kD with molecular cut off, the dialysis solution lyophilization namely gets N-N-trimethyl chitosan TMC (TMC).
The chitosan raw material (Chitosan) of purification and synthetic N-N-trimethyl chitosan TMC (TMC) are respectively got 5mg, and carry out IR spectrum scanning, result such as Fig. 2 behind the KBr tabletting.As seen from Figure 2, synthetic N-N-trimethyl chitosan TMC (TMC) is compared with chitosan (Chitosan), is positioned at 3200 cm
-1Near
ν NHPeak and be positioned at 1590 cm
-1 β NHThe peak obviously weakens; And at 1475 cm
-1The place has newly occurred
δ CH3The peak, proved chitosan-NH
2By quaternized generation the-N
+(CH
3)
3
Get the synthetic N-N-trimethyl chitosan TMC (TMC) of 5mg, with D
2O is that solvent carries out
1H-NMR(300 MHz) detects result such as Fig. 3.Chemical shift 3.343 peaks are among the TMC-N among Fig. 3
+(CH
3)
3, and the amino quaternized degree that can be calculated in the chitosan by formula 1 is 32%.
%DQ=[[(CH
3)
3]/[H]×1/9]×100 （1）
%DQ is quaternized degree amino in the chitosan in the formula, [(CH
3)
3] be the peak area integrated value at 3.343 trimethyl group peak for chemical shift, [H] for chemical shift between 5.0-5.7 in the chitosan
1The peak area integrated value at H peak.
Sample 2
Take by weighing chitosan (molecular weight 100kD, deacetylation 80%) 0.5 g of purification in 50 mL three-neck flasks, add N-Methyl pyrrolidone 20 mL, stirring at room swelling 12 hours.Add 1.2 g sodium iodide, 2.8 mL mass concentrations are 0.2 gmL
-1Sodium hydroxide solution and 3 mL iodomethane, 60 ℃ of stirring in water bath were finished the first step in 60 minutes and are methylated.Add again 2.8 mL sodium hydroxide solutions and 1.25 mL iodomethane reactions finished in 120 minutes second the step methylate.Reactant liquor is added 200 mL absolute ethyl alcohol and stirring 1 hour precipitation N-trimethyl chitosan TMC, precipitate centrifugal (5000 rpm, 15 minutes) back with washing with alcohol 3 times, ether washs 3 final vacuum dryings.After the drying product is dissolved in 20 mL, 50 gL
-1Sodium chloride l aqueous solution in exchange I
-1Be Cl
-1, be 48 hours purification of dialysis in the bag filter distilled water of 12-14 kD with the molecular cut off, the dialysis solution lyophilization.Characterizing quaternized rate through nuclear-magnetism is 99%.
The preparation of example 3 N-trimethyl chitosan TMCs-grafting-Polyethylene Glycol
N-trimethyl chitosan TMC 10 mg of sample 1 and 15 mg maleimide-Polyethylene Glycol-butanimides (MAL-PEG-SCM) are dissolved in 2 mL distilled water in the example 2, room temperature magnetic agitation 6 hours, ultrafiltration is centrifugal removes unreacted PEG dressing agent, ultrafiltration is centrifugal again after with distilled water diluting to obtain concentrated solution, repeat 3 times, with the ultrafiltrate lyophilization namely at last.
The N-trimethyl chitosan TMC (TMC) for the treatment of excess syndrome example 2 samples 1, synthetic N-trimethyl chitosan TMC-grafting-Polyethylene Glycol (TMC-PEG-MAL) and each 5mg of maleimide-Polyethylene Glycol-butanimide (MAL-PEG-SCM) are with D
2O is that solvent carries out
1H-NMR(300 MHz) characterize, the result goes into Fig. 4.TMC-PEG-MAL compares with TMC among Fig. 4, and the peak of the chemical shift 3.713 that has more is in the Polyethylene Glycol-CH
2CH
2-group peak (seeing Fig. 4 MAL-PEG-SCM), the peak of chemical shift 6.881 is the peak of H correspondence in the maleimide among the MAL-PEG-SCM (MAL), simultaneously, the bimodal of H correspondence disappears in TMC-PEG-MAL in the butanimide (SCM) of chemical shift about 2.9 among the MAL-PEG-SCM, more than all proved Polyethylene Glycol grafting success.The percent grafting that can be calculated Polyethylene Glycol by formula 2 is 15.4%.
Percent grafting (%)=[PEG]/([H] * 248) * 100 (2)
In the formula [PEG] for chemical shift among the TMC-PEG-MAL be 3.713-CH
2CH
2The peak area integrated value at-group peak, [H] for chemical shift between 5.0-5.7 in the chitosan skeleton
1The peak area integrated value at H peak, 248 is the proton number in every polyglycol chain.
Sample 2
N-trimethyl chitosan TMC 10 mg of sample 1 and 30 mg maleimide-Polyethylene Glycol-butanimides (MAL-PEG-SCM) are dissolved in 2 mL distilled water in the example 2, room temperature magnetic agitation 6 hours, ultrafiltration is centrifugal removes unreacted PEG dressing agent, ultrafiltration is centrifugal again after with distilled water diluting to obtain concentrated solution, repeat 3 times, with the ultrafiltrate lyophilization namely at last.The percent grafting that characterizes Polyethylene Glycol through nuclear-magnetism is 34%.
The preparation of example 4 N-trimethyl chitosan TMCs-grafting-Polyethylene Glycol-brain target function peptide (TMC-PEG-RVG)
Sample 2 in the example 3
ThreeMethyl chitosan-grafting-Polyethylene Glycol 10 mg and 6 mg RVG are dissolved in the phosphate buffer of 2 mL pH7, room temperature magnetic agitation 24 hours, centrifugal (the 5000g of ultrafiltration, 1 hour) remove unreacted RVG, ultrafiltration is centrifugal again after with distilled water diluting to obtain concentrated solution, repeat 3 times, at last with the ultrafiltrate lyophilization, namely get TMC-PEG-RVG.
With product with D
2O is that solvent carries out
1H-NMR(300 MHz) characterizes result such as Fig. 5.TMC-PEG-RVG compares with TMC-PEG-MAL among Fig. 5, two groups of peaks of the chemical shift 0.6-2.0 that has more and 6.7-7.6 should be the peak of brain target function peptide RVG, simultaneously, chemical shift is that 6.881 peak (peak of MAL among the TMC-PEG-MAL) disappears, more than proved that all brain target function peptide RVG successfully inserts.
The preparation of the N-trimethyl chitosan TMC-grafting-Polyethylene Glycol-brain target function peptide/siRNA brain targeting micelle of example 5 acetylcholinergic receptors mediation
The TMC-PEG-RVG 1.4mg of example 4 is dissolved in the 161 μ l DEPC treated waters, passes through the filter membrane degerming of 0.22 μ m then.16.5 μ g siRNA are dissolved in the 480 μ l DEPC treated waters.Get 5 parts of above-mentioned siRNA solution 80 μ l, add respectively above-mentioned TMC-PEG- RVG solution 5,10,20,40,80 μ l with preparation TMC-PEG-RVG and siRNA+/-charge ratio is the micelle of 2:1,4:1,8:1,16:1 and 32:1, vortex 20s makes it to mix, and room temperature left standstill 30 minutes.
With each+/-the siRNA micelle that carries of charge ratio is that electrophoresis experiment, result such as Fig. 2 are carried out in contrast with siRNA.First band is the electrophoretic band of siRNA contrast among Fig. 2, all the other each bands from left to right are followed successively by+/-charge ratio be 32:1,16:1,8:1,4:1 and 2:1 carry siRNA micelle electrophoretic band.As can be seen, when+/-when charge ratio reached 32:1, the siRNA band disappeared on the electrophoretogram, illustrated that siRNA is compound fully with TMC-PEG-RVG at this moment, and Bao Zaiyu protects by micelle.
Each+/-charge ratio carry siRNA micelle Zeta potential measurement result such as table 1.
Table 1 year siRNA micelle particle size distribution and Zeta potential
The Flow cytometry experiment of the Pegylation N-N-trimethyl chitosan TMC siRNA brain targeting micelle transfection Neuro-2a cell of example 6 acetylcholinergic receptors mediation
With the Neuro-2a model cell in 12 orifice plates (10
4/ hole) cultivated 24 hours, culture medium is changed to serum-free medium.Wherein, blank group every hole, 3 holes adds the PBS of 66 μ l pH7.4; Every hole, 3 holes of matched group adds the fluorescently-labeled siRNA solution of 66 μ l FAM (be equivalent to every hole and contain 100pmol siRNA); Every hole, 3 holes of preparation group add 66 μ l examples 7+/-charge ratio be 32:1 carry FAM-siRNA micellar solution (be equivalent to every hole and contain 100pmol siRNA), cultivated 4 hours, wash 2 times with the PBS of pH7.4, add the complete culture solution cultivation.After 16 hours, after cell dissociation is centrifugal, every porocyte heavily is scattered among the PBS of 300 μ l and carries out Flow cytometry, it is 0.41% that testing result is calculated the transfection efficiency that can get the siRNA contrast, preparation group transfection efficiency is 83.42%, and the Pegylation N-N-trimethyl chitosan TMC siRNA brain targeting micelle of visible acetylcholinergic receptor mediation has improved the transfection efficiency to the Neuro-2a cell of surface expression acetylcholinergic receptor significantly.
The Laser Scanning Confocal Microscope imaging experiment of the Pegylation N-N-trimethyl chitosan TMC siRNA brain targeting micelle transfection Neuro-2a cell of example 7 acetylcholinergic receptors mediation
With the Neuro-2a model cell in 6 orifice plates (10
4/ hole) cultivated 24 hours, culture medium is changed to serum-free medium.Every hole add 66 μ l examples 7+/-charge ratio be 32:1 carry FAM-siRNA micellar solution (be equivalent to every hole and contain 100pmol siRNA), cultivated 4 hours, wash 3 times with the PBS of pH7.4, add the complete culture solution cultivation.After 4 hours, fixing with 37 ℃ of 4% paraformaldehydes, DAPI dyeing after 50% glycerol stationary, is sent the survey Laser Scanning Confocal Microscope, the results are shown in Figure 3.
Claims (12)
1. N-trimethyl chitosan TMC-grafting-Polyethylene Glycol-brain target function peptide/nucleic acid brain targeting micelle, it is characterized in that described micelle is formed through the self assembly of static compound action by the N-trimethyl chitosan TMC-grafting-Polyethylene Glycol-brain target function peptide of lotus positive electricity and the nucleic acid of bear electricity.
2. N-trimethyl chitosan TMC-grafting according to claim 1-Polyethylene Glycol-brain target function peptide/nucleic acid brain targeting micelle, it is characterized in that, described N-trimethyl chitosan TMC-grafting-Polyethylene Glycol-brain target function peptide is that chitosan obtains N-trimethyl chitosan TMC through quaternized modification successively, an active group reaction of the amido of N-trimethyl chitosan TMC and two ends activated polyethylene glycol obtains N-trimethyl chitosan TMC-grafting-Polyethylene Glycol, another active group of Polyethylene Glycol and brain target function reactive polypeptide RVG gained.
3. N-trimethyl chitosan TMC-grafting according to claim 1-Polyethylene Glycol-brain target function peptide/nucleic acid brain targeting micelle is characterized in that described chitosan weight average molecular weight is 10-150kD, and deacetylation is 80% – 95%.
4. N-trimethyl chitosan TMC-grafting according to claim 2-Polyethylene Glycol-brain target function peptide/nucleic acid brain targeting micelle is characterized in that quaternized rate is 10% – 99%, mole percent.
5. N-trimethyl chitosan TMC-grafting according to claim 2-Polyethylene Glycol-brain target function peptide/nucleic acid brain targeting micelle is characterized in that the general formula of described Polyethylene Glycol is R
1-(CH
2CH
2O)
n-R
2, n=20 – 200 in the formula, R
1For generating the active group of Polyethylene Glycol amido link with the amino reaction, comprise butanimide group, aceticoceptor, aldehyde group, isocyanate group, isocyanide sulfate group, acrylic acid groups, nitrophenols group, R
2For can with sulfydryl reactive activity group, comprise maleimide base group, vinyl sulfone(Remzaol group, thiol group.
6. N-trimethyl chitosan TMC-grafting according to claim 1-Polyethylene Glycol-brain target function peptide/nucleic acid brain targeting micelle, it is characterized in that, described brain target function peptide is RVG, contains 29 aminoacid, and its sequence is YTIWMPENPRPGTPCDIFTNSRGKRASNG.
7. the preparation method of N-trimethyl chitosan TMC-grafting as claimed in claim 1-Polyethylene Glycol-brain target function peptide/nucleic acid brain targeting micelle is characterized in that, comprises the steps:
The purification of chitosan;
(2) preparation of N-trimethyl chitosan TMC;
The preparation of (3) N-trimethyl chitosan TMC-grafting-Polyethylene Glycol;
The preparation of (4) N-trimethyl chitosan TMC-grafting-Polyethylene Glycol-brain target function peptide;
(5) N-trimethyl chitosan TMC-grafting-Polyethylene Glycol-brain target function peptide can be self-assembled into initiatively targeting micelle of brain with the nucleic acid of bear electricity.
8. preparation method according to claim 7 is characterized in that, reaction temperature is 50 ℃ of 4 – in the step (3), and the response time is 3 – 480 minutes, and the percent grafting of Polyethylene Glycol is 5% – 35%(mole percent).
9. according to the preparation method described in the claim 7, it is characterized in that, in the step (4): N-trimethyl chitosan TMC-grafting-Polyethylene Glycol reacts with brain target function peptide RVG in the phosphate buffer of pH6 – 9, and reaction temperature is 30 ℃ of 15 –, and the response time is 8 – 24 hours.
10. preparation method according to claim 7, it is characterized in that, in the step (5), N-trimethyl chitosan TMC-grafting-Polyethylene Glycol-brain target function peptide is dissolved in the DEPC treated water, pass through the filter membrane degerming of 0.22 μ m then, nucleic acid is dissolved in the DEPC treated water, and in mixed at room temperature, vortex 5-30 s makes it mix homogeneously with above two kinds of solution.
11., it is characterized in that the particle size distribution of prepared micelle is 10 – 700nm according to the described preparation method of claim 7, Zeta potential is+1mV –+15mV.
12. preparation method according to claim 7 is characterized in that, the N-trimethyl chitosan TMC-grafting of prepared micelle-Polyethylene Glycol-brain target function peptide and nucleic acid+/-charge ratio is 1:4-64:1.
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| CN105273207A (en) * | 2014-06-09 | 2016-01-27 | 中国科学院上海药物研究所 | Polycation inclusion complex, and preparation method and purpose thereof |
| CN106390137A (en) * | 2016-11-21 | 2017-02-15 | 湖北工业大学 | TMC/genipin/pDNA nanometer carrier and preparation method and application thereof |
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| CN106667962B (en) * | 2016-12-30 | 2019-12-13 | 深圳大学 | Charge conversion type nano-drug carrier and preparation method thereof |
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