CN107412782A - A kind of polypeptide polymer nano material and its preparation method and application - Google Patents
A kind of polypeptide polymer nano material and its preparation method and application Download PDFInfo
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- 0 CCC(C)(C(C(*C(CC*(C)*)=O)C(C1)OC2)OC(CO)C1C2(C)OC(C(C1O)N=C)OC(CO)C1OCC(*C(C*)C(**)C1*)C1NC(C)=O)OC(C)(CC1C(CO)OC2)COC1C2N(*C)C(CCS*)=[O+] Chemical compound CCC(C)(C(C(*C(CC*(C)*)=O)C(C1)OC2)OC(CO)C1C2(C)OC(C(C1O)N=C)OC(CO)C1OCC(*C(C*)C(**)C1*)C1NC(C)=O)OC(C)(CC1C(CO)OC2)COC1C2N(*C)C(CCS*)=[O+] 0.000 description 2
- YNYNBKWDUWFHJJ-UHFFFAOYSA-N CC(C)C(NCC(C)=O)=O Chemical compound CC(C)C(NCC(C)=O)=O YNYNBKWDUWFHJJ-UHFFFAOYSA-N 0.000 description 1
- UTJLYWDCEOAPOK-UHFFFAOYSA-N CCC(C(N(C)C(CC(O)=O)C(NCCc1ccccc1)=O)=O)N Chemical compound CCC(C(N(C)C(CC(O)=O)C(NCCc1ccccc1)=O)=O)N UTJLYWDCEOAPOK-UHFFFAOYSA-N 0.000 description 1
- XLSZMDLNRCVEIJ-UHFFFAOYSA-N Cc1c[nH]cn1 Chemical compound Cc1c[nH]cn1 XLSZMDLNRCVEIJ-UHFFFAOYSA-N 0.000 description 1
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- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
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- A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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Abstract
The present invention provides a kind of polypeptide polymer nano material and its preparation method and application, the peptide sequence of identification amyloid beta and the peptide sequence of active cell autophagy that the polypeptide polymer nano material includes chitosan and is connected on chitosan.The polypeptide polymer that the present invention is prepared by synthesis in solid state and Michael additions has good bio-compatibility and anti-A β neurotoxicities, polypeptide polymer nanosphere therefrom can assemble so as to effectively prevent its aggregation altogether with A β, reduce A β neurotoxicity, assembly being capable of active cell autophagy after cell is entered altogether simultaneously, pass through cell autophagy degraded A β, so as to realize synergistic treatment Alzheimer's disease, the efficiency of the treatment Alzheimer's disease of polypeptide nano material is greatly improved, is with a wide range of applications.
Description
Technical field
The invention belongs to polymeric material field, is related to polypeptide polymer nano material and its preparation method and application, especially
It is related to a kind of multi-functional polypeptide polymer nano material and preparation method thereof and the application in Alzheimer's disease.
Background technology
Alzheimer's disease is considered as most common neurodegenerative diseases, and 2015, the whole world had nearly 50,000,000
People suffers from senile dementia.Up to the present, for Alzheimer's disease also without effective treatment method, for Alzheimer's disease
Research is significant.Exist it is well known that an important pathological hallmark of Alzheimer's disease is A β (amyloid-beta)
Extracellular deposition and intracellular NFT.In the past few decades, the research on Alzheimer's disease
A main policies be inhibitor for A β designs, it is therefore an objective to it is prevented extracellular heavy by A β self assembling process
Accumulate to reach therapeutic purposes.Inhibitor has devised a lot, but its effect and bad.In recent years, with cell autophagy
It was found that and its influence to cancer and neurogenic disease, it has been found that, many neurogenic disease patients, as Alzheimer's disease suffer from
Person, their nerve cell autophagy function are chaotic.From this point of view, autophagy is also one for the treatment of Alzheimer's disease potential
Target spot.Except using autophagy as therapy target come in addition to studying Alzheimer's disease, enzyme when also someone forms A β is as controlling
Treat target spot, it is intended to A β generation is reduced from upstream.From the point of view of the current research situation to Alzheimer's disease, although obtaining
Certain achievement, but therapeutic effect is all bad.Therefore, open up a kind of new strategy and be used for the treatment of Alzheimer very
It is necessary.
In the art, need badly searching how the strategy that two kinds of strategies are combined together, such as design a kind of material, can
Prevent A β aggregation, while and can regulation cell autophagy and degraded A β by autophagy, by both strategies come synergistic treatment Ah
Er Cihaimo diseases, the treatment that this will be to Alzheimer's disease are significant.
The content of the invention
In view of the shortcomings of the prior art, it is an object of the invention to provide a kind of polypeptide polymer nano material and its preparation
Methods and applications, especially it is to provide a kind of multi-functional polypeptide polymer nano material and preparation method thereof and in Alzheimer
Application in disease.
To reach this goal of the invention, the present invention uses following technical scheme：
On the one hand, the present invention provides a kind of polypeptide polymer nano material, and the polypeptide polymer nano material includes shell
Glycan and the peptide sequence of identification amyloid-beta (A β) and the polypeptide of active cell autophagy being connected on chitosan
Sequence.
In the present invention, the polypeptide polymer nano material can assemble so as to prevent its aggregation altogether with A β, reduce A β
Neurotoxicity, while altogether assembly enter cell after can active cell autophagy, degrade A β, so as to realize synergistic treatment
Alzheimer's disease.
Preferably, the polypeptide polymer nano material has the structure as shown in following formula I：
Wherein, R1For the peptide sequence of identification amyloid-beta (A β), R2For the peptide sequence of active cell autophagy, R3
For hydrophilic polyglycol chain, R1And R3Between by acid amides key connection, m, n are the integer more than or equal to 0, and m/n is 1:
3~3:1.
In the present invention, represented by Formulas I be connected with chitosan chain identification amyloid-beta peptide sequence
With the peptide sequence of active cell autophagy, can also there are the fragment for not connecting these peptide sequences, molecule on chitosan chain
In formulaRepresent that chitosan polymer chain stretches out.
M, n are the integer more than or equal to 0 in the present invention, for example, m, n can independently be 1,2,3,4,5,6,7,8,
9th, 10,15,20 etc., and need to meet that m/n is 1:3~3:1, such as 1:3、1:2.8、1:2.5、1:2.3、1:2、1:1.8、1:
1.6、1:1.4、1:1.2、1:1、1.3:1、1.5:1、1.8:1、2:1、2.3:1、2.5:1、2.8:1 or 3:1.
Preferably, in the polypeptide polymer nano material donor of main polymer chain compound group for Formula II institute
Show the acrylated chitosan of structure：
Wherein, m, n are the integer more than or equal to 0, and m/n is 1:3~3:1.
Preferably, in the polypeptide polymer nano material donor of main polymer chain compound group weight average molecular weight
For 3000-7000, such as 3000,3500,4000,4500,5000,5500,6000,6500 or 7000.
In the present invention, the acrylated chitosan using structure shown in the Formula II and the R with sulfydryl1Donor enters
Row reaction so that double bond and sulfydryl react in acrylated chitosan, so as to by R1Group is connected on chitosan.
Preferably, R1For：
OrWhereinRepresent the connection site of group, " * * "
The group of position and the site with the carbon-carbon double bond reaction in chitosan side base, R1With R3Between group connection site.I.e. in this hair
In bright, R1Donor constructions be：
(being represented by CFFVLKG) or(being represented by CDFFPLG).
Preferably, R2For：
,
WhereinRepresent the connection site of group.I.e. in the present invention, R2Donor be：
(being represented by CTNVFNATFHIWHSGQFGT).
Preferably, R3Donor be carboxy blocking polyethylene glycol, its weight average molecular weight is 300-50000, for example, 300,
400、500、600、700、800、900、1000、1200、1400、1600、1800、2000、4000、6000、8000、10000、
30000 or 50000, preferably 300-2000.
I.e. in the present invention, the R3It can be expressed asWith carbonyl end and R2In amino formed acid amides
Key and link together.
On the other hand, it is described the invention provides a kind of preparation method of polypeptide polymer nano material as described above
Method comprises the following steps：
(1) using resin as carrier, using the polyethylene glycol of carboxy blocking and amino acid as raw material, solid phase synthesis process is utilized
Polypeptide compounds HS-R is prepared1-R3And R2-SH；
(2) the polypeptide compounds HS-R obtained using acrylated chitosan with step (1)1-R3And R2- SH is carried out
Michael addition reactions obtain the polypeptide polymer nano material；
Wherein polypeptide compounds HS-R1-R3And R2- SH mol ratio is 1:3~3:1.
Synthesis in solid state and Michael addition reactions are technologies well known to those skilled in the art in the present invention, can be with
Its preparation condition is selected according to prior art, to complete the purpose for preparing the polypeptide polymer nano material, here, right
No longer make and being particularly limited in synthesis in solid state and Michael addition reactions.
In the present invention, the acrylated chitosan can utilize the known technology of this area to synthesize to obtain, such as
1mmol chitosans can be dissolved in 2mL ionized waters and be heated to 50-60 DEG C (such as 50 DEG C, 53 DEG C, 55 DEG C or 58 DEG C), stirred
Lower holding 30-60min (such as 30min, 35min, 40min, 45min, 55min or 60min) is mixed, is cooled to room temperature, is added
5-10mmol (such as 5mmol, 5.5mmol, 7mmol, 7.5mmol, 8mmol, 8.5mmol, 9mmol, 9.5mmol or 10mmol)
Triethylamine, be slowly added under ice bath 3-5mmol (such as 3mmol, 3.5mmol, 3.8mmol, 4mmol, 4.3mmol, 4.5mmol,
4.8mmol or 5mmol) acryloyl chloride, reaction 12-24 hours (such as 12 hours, 14 hours, 16 hours, 18 hours, 20 hours,
22 hours or 24 hours), dialysis, lyophilized obtain acrylated chitosan.
On the other hand, the invention provides the self-assembling method of polypeptide polymer nano material as described above, the side
Method comprises the following steps：Polypeptide polymer nano material is dissolved in organic solvent, obtains polypeptide polymer nanomaterial solution,
Obtained polypeptide polymer nanomaterial solution is added in cushioning liquid, self assembly obtains nanosphere.
Preferably, the organic solvent is that (hexafluoro is different by DMSO (dimethyl sulfoxide (DMSO)), DMF (dimethylformamide) or HFIP
Propyl alcohol) in any one or at least two combination, preferably DMSO.
Preferably, the concentration of the polypeptide polymer nanomaterial solution is 10-200 μ g mL-1, such as 10 μ g mL-1、
20μg mL-1、30μg mL-1、40μg mL-1、50μg mL-1、60μg mL-1、70μg mL-1、80μg mL-1、90μg mL-1、
100μg mL-1Or 200 μ g mL-1。
Preferably, the cushioning liquid is PBS cushioning liquid.
Preferably, the pH value of the cushioning liquid is 6-8, such as 6,6.4,6.8,7,7.2,7.4,7.6,7.8 or 8.
Preferably, the self assembly is carried out under ultrasound, and the ultrasonic time is 1-10min, such as 1min, 2min,
3min, 4min, 5min, 6min, 7min, 8min, 9min or 10min.
Preferably, protected after the ultrasound at 20-40 DEG C (such as 22 DEG C, 25 DEG C, 28 DEG C, 30 DEG C, 32 DEG C, 35 DEG C or 38 DEG C)
Hold 0.5-12h (such as 0.8h, 1h, 3h, 5h, 8h, 10h, 11h or 12h).
The polypeptide polymer of the present invention has good bio-compatibility and anti-A β neurotoxicities.What the present invention was prepared
Polypeptide polymer nanosphere can assemble so as to prevent its aggregation altogether with A β, reduce A β neurotoxicity, while assembly exists altogether
Into after cell can active cell autophagy, degraded A β by cell autophagy, so as to realize synergistic treatment Alzheimer's disease.
On the other hand, controlled the invention provides polypeptide polymer nano material as described above preparing Alzheimer's disease
Treat the application in medicine.
Polypeptide polymer nano material provided by the present invention, good recognition capability and polymerization inhibiting capacity based on it to A β,
And effectively active cell autophagy and the ability for the A β of being degraded by autophagy, greatly improve the treatment alzheimer ' of polypeptide nano material
The efficiency of silent disease, is with a wide range of applications.
Relative to prior art, the invention has the advantages that：
The polypeptide polymer of the present invention has good bio-compatibility and anti-A β neurotoxicities.What the present invention was prepared
Polypeptide polymer nanosphere can assemble so as to effectively prevent its aggregation altogether with A β, reduce A β neurotoxicity, while assembling altogether
Body enter cell after can active cell autophagy, by cell autophagy degrade A β, so as to realize synergistic treatment alzheimer '
Silent disease, the efficiency of the treatment Alzheimer's disease of polypeptide nano material is greatly improved, is with a wide range of applications.
Brief description of the drawings
Fig. 1 is the nucleus magnetic hydrogen spectrum figure of the polypeptide polymer nano material of embodiment 1；
Fig. 2A is the cytotoxicity experiment result figure of the polypeptide polymer nano material of embodiment 2；
Fig. 2 B are the anti-A β neurotoxicities cell experiment result figure of the polypeptide polymer nano material of embodiment 2；
Fig. 3 is that the polypeptide polymer nano material of embodiment 3 is incubated altogether with A β and independent A β are in excitation wavelength 440nm, hair
The fluorescence intensity curves figure determined under the long 480nm of ejected wave；
Fig. 4 A are polypeptide polymer nano material M1-1Transmission electron microscope picture, scale 100nm；
Fig. 4 B are polypeptide polymer nano material M1-1The transmission electron microscope picture being incubated altogether with A β, scale 100nm；
Fig. 5 A are polypeptide polymer nano material M1-0Transmission electron microscope picture, scale 100nm；
Fig. 5 B are polypeptide polymer nano material M1-0The transmission electron microscope picture being incubated altogether with A β, scale 100nm；
Fig. 6 A are polypeptide polymer nano material M0-1Transmission electron microscope picture, scale 100nm；；
Fig. 6 B are polypeptide polymer nano material M0-1The transmission electron microscope picture being incubated altogether with A β, scale 100nm；
Fig. 7 is the Laser Scanning Confocal Microscope photo that the polypeptide polymer nano material determined in embodiment 6 is used to capture A β；
When Fig. 8 is that the polypeptide polymer nano material determined in embodiment 7 is used for mouse Nerve knurl mother cell (N2a), a word used for translation
The cell of pyridine orange (Acridine Orange, AO) dyeing is copolymerized burnt picture；
When Fig. 9 is that the polypeptide polymer nano material determined in embodiment 8 is used for N2a cells, the biological electricity of cell section
Mirror picture；Wherein A figures are the electron microscope of undressed cell, and B figures are M1-0The electron microscope of cell after processing, C2 figures are M1-1
The electron microscope of cell after processing, C1 figures are the enlarged drawing in region in dotted line frame in C2 figures；D2 figures are M0-1Cell after processing
Electron microscope, D1 figures are the enlarged drawing in region in dotted line frame in D2 figures；
Figure 10 is that the polypeptide polymer nano material that determines and A β and the copolymerization that N2a cells are incubated altogether are burnt aobvious in embodiment 9
Micro mirror photo；
Figure 11 is the polypeptide polymer nano material of embodiment 10 by being injected intravenously to latent after APP/PS1 transgenic mices
Lie prostrate the result figure of time；Wherein, WT represents normal mouse group, and AD groups represent the senile dementia group of injection PBS, M1-1Generation
The treatment group that table injection polypeptide polymer nano material is treated；
Figure 12 be the polypeptide polymer nano material of embodiment 10 by be injected intravenously to APP/PS1 transgenic mices after, take
The immunostaining picture that mouse head tissue is obtained by immunostaining, wherein A figures are wild normal mouse group (WT groups), and B schemes
To inject the Elderly dementia patients group (AD groups) of PBS, C figures are experimental group (M1-1Group)；
After Figure 13 is the polypeptide polymer nano material of embodiment 10 by being injected intravenously to APP/PS1 transgenic mouses, take
The Nissl's staining picture that mouse head tissue is obtained by Nissl's staining, wherein A figures are wild normal mouse group (WT groups), and B schemes
To inject the Elderly dementia patients group (AD groups) of PBS, C figures are experimental group (M1-1Group).
Embodiment
Technical scheme is further illustrated below by embodiment.Those skilled in the art should be bright
, the embodiment be only to aid in understand the present invention, be not construed as to the present invention concrete restriction.
Embodiment 1
In the present embodiment, the structure of polypeptide polymer nano material is as follows：
I.e. in Formulas I provided by the present invention, R1Donor be CFFVLKG, R2Donor be
CTNVFNATFHIWHSGQFGT, R3Donor be carboxy blocking polyethylene glycol, its molecular weight is 368g mol-1, m/n=1/1
For M1-1。
The synthetic method of the polypeptide polymer is as follows：
Using resin as carrier, using the polyethylene glycol of carboxy blocking and amino acid as raw material, according to the order of connection of polypeptide,
Using solid phase synthesis process, the polyethylene glycol of carboxy blocking is coupled in sequence with amino acid, will most be produced through trifluoroacetic acid afterwards
Thing is cleaved from carrier, and purifying obtains polypeptide compounds HS-R1-R3And R2-SH；
By HS-R1-R3And R2- SH gathers according to 1/0,1/3,1/1,3/1,0/1 ratio and the shell after the acrylated of excess
Sugar carries out Michale addition reactions, i.e., being reacted by the sulfydryl on amino acid C (i.e. cysteine) and carbon-carbon double bond will
Polypeptide compounds and acrylated chitosan connect, finally dialyse, be lyophilized and obtain.
Embodiment 2
Difference from Example 1 is only that, by HS-R1-R3And R2- SH according to 1/3 ratio with excess it is acrylated
Chitosan afterwards carries out Michale addition reactions, obtains polypeptide polymer, due to m/n=1/3, is designated as M1-3。
Embodiment 3
Difference from Example 1 is only that, by HS-R1-R3And R2- SH according to 3/1 ratio with excess it is acrylated
Chitosan afterwards carries out Michale addition reactions, obtains polypeptide polymer, due to m/n=3/1, is designated as M3-1。
Comparative example 1
Difference from Example 1 is only that, by HS-R1-R3And R2- SH according to 1/0 ratio with excess it is acrylated
Chitosan afterwards carries out Michale addition reactions, obtains polypeptide polymer, due to m/n=1/0, is designated as M1-0。
Comparative example 2
Difference from Example 1 is only that, by HS-R1-R3And R2- SH according to 0/1 ratio with excess it is acrylated
Chitosan afterwards carries out Michale addition reactions, obtains polypeptide polymer, due to m/n=0/1, is designated as M0-1。
Nuclear-magnetism sign is carried out to the polypeptide polymer nano material that embodiment 1-3 and comparative example 1-2 is synthesized, as a result as schemed
Shown in 1, it can be seen that in acrylated chitosan collection of illustrative plates, the double bond characteristic peak at the nuclear-magnetism peak at ppm=5.8-6.3,
And in the spectrogram of product, at this, characteristic peak disappears, and shows that reaction is complete, meanwhile, it is inferred to what is occurred by integral area
Nuclear-magnetism peak meets the characteristic peak of the molecular weight of material molecule, and is calculated by integral area and to infer that material molecule is according to feeding intake
Ratio reacted.
Embodiment 4
The polypeptide polymer nano material that embodiment 1-3 and comparative example 1-2 is prepared is investigated in this embodiment
Bio-compatibility and anti-A β toxicity, method is as follows：
The experiment of its bio-compatibility is tested first：N2a cell suspending liquids are got out, 5000 are added carefully per hole to 96 orifice plates
Born of the same parents, per the μ L of hole 200.12h is cultivated, takes out culture medium, experimental group adds (10,20,50,100, the 200 μ g of various concentrations per hole
mL-1) polypeptide polymer nano material (M1-0,M3-1,M1-1,M1-3,M0-1) the culture medium without serum, blank group add volume
For the 200 μ L culture medium without serum, that not celliferous group is equally handled, and cultivates 12h.Then take out culture medium,
With PBS rinses 3 times, CCK-8 solution (V is addedCCK-8：VCulture medium=1：9) and then by 450nm excite, 690nm transmitting tests.Survey
As shown in Figure 2 A, from result, the biocompatibility of polypeptide polymer nano material sample is fine for test result.
Secondly, its anti-A β neurotoxicities experiment is investigated：N2a cell suspending liquids are got out, 5000 are added per hole to 96 orifice plates
Individual cell, per the μ L of hole 200.12h is cultivated, takes out culture medium, experimental group is (with A β/M1-1Represent) per hole, addition polypeptide polymer is received
Rice material concentration is 20 μ g mL-1, 20 μM of aβ protein concentration the culture medium without serum, control group (being represented with A β) is per hole
The culture medium without serum of 20 μM of aβ protein concentration is added, blank group adds the culture medium without serum that volume is 200 μ L,
That not celliferous group is equally handled, and cultivates 12h.Culture medium is then taken out, with PBS rinses 3 times, adds CCK-8 solution
(VCCK-8：VCulture medium=1：9) and then by 450nm excite, 690nm transmitting tests.Test result as shown in Figure 2 B, can by result
Know, polypeptide polymer nano material samples can effectively reduce A β toxicity, wherein M3-1、M1-1、M1-3Effect compares M1-0With
M0-1It is good, illustrate that identifying that both the peptide sequence of amyloid-beta (A β) and the peptide sequence of active cell autophagy cooperate with makees
With reduction A β toxicity；And wherein M1-1Effect it is best.
Embodiment 5
The polypeptide polymer nano material that embodiment 1-3 and comparative example 1-2 are prepared in the present embodiment and A β
Tht fluorescence experiments are carried out, investigate the ability that the material prevents A β aggregation.
First, the ability that polypeptide polymer nano material prevents A beta peptide aggregations is tested, embodiment 1 is prepared multi-functional
Polypeptide polymer nano material be dissolved in DMSO (5mg mL-1), A β are made into 200 μM of (DMSO：H2O=1:4), 200 μM of Tht,
Each hole sequentially adds 2 times of μ L of PBS solution 100,62 μ L H in 96 orifice plates2O, 8 μ L polypeptide polymers nano materials, 20 μ LA β,
10 μ L Tht, then carried out according to time point 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 36h, 48h, 72h, 96h
Test；For single A β groups except being not added with multifunctional polymer nano material, other processing modes are consistent.Last test result is as schemed
Shown in 3.
From figure 3, it can be seen that stronger to the single A β group fluorescence of multi-functional polypeptide polymer nano material, and add
Cross that group of polypeptide polymer nano material is weaker, show that material can effectively prevent the aggregation of A β groups, and wherein M1-0,
M3-1,M1-1,M1-3Compare M0-1Effect it is good, i.e., higher with R1 content, Tht fluorescence intensities are lower, it was demonstrated that R1Partial is more
Peptide can prevent A beta peptide aggregations.
Polypeptide polymer nano material M prepared by embodiment 11-1And polypeptide polymer nano material M1-1It is incubated altogether with A β
Transmission electron microscope picture respectively as shown in Figure 4 A and 4 B shown in FIG., as seen from the figure, M1-1It can form nano particle (Fig. 4 A), and M1-1It is total to A β
With obvious fiber (Fig. 4 B) will not be formed after being incubated, A betas can be effectively prevented, also can prove that of the present invention
Polypeptide polymer nano material prevents the ability of A beta peptide aggregations.M1-0And M1-1The transmission electron microscope picture being incubated altogether with A β such as Fig. 5 A and figure
Shown in 5B, as seen from the figure, M1-0It can form nano particle (Fig. 5 A), and M1-0Obvious fibre will not be formed after being incubated jointly with A β
Tie up (Fig. 5 B), can effectively prevent A betas.M0-1And M0-1The transmission electron microscope picture being incubated altogether with A β such as Fig. 6 A and Fig. 6 B
It is shown, as seen from the figure, M0-1It can form nano particle (Fig. 6 A), and M0-1A β can also form obvious fibre after being incubated jointly with A β
Tie up (Fig. 6 B), it is impossible to prevent A betas.Result above shows, the R in polypeptide polymer nano material1Polypeptide portion be can
To prevent A betas.
Embodiment 6
The polypeptide polymer nano material that embodiment 1 and comparative example 1-2 are prepared and A β are entered in the present embodiment
Row common location is tested, and investigates capture ability of the material to A β, and method is as follows：
The polypeptide polymer nano material of red fluorescence mark and the A β of green fluorescent label are incubated altogether, and 2h is total to using fluorescence
Focusing microscope is observed, as a result as shown in fig. 7, M1-1And M1-0Can be with the good common locations of A β, and M0-1Then can not be fixed altogether with A β
Position, to M3-1And M1-3Same experiment is carried out, can equally prove M3-1And M1-3Can be with the good common locations of A β, result above
Show, the R in polypeptide polymer nano material of the invention1Polypeptide portion can capture A β, carry out common assembling with A β.
Embodiment 7
The polypeptide polymer nano material that embodiment 1 and comparative example 1-2 are prepared carries out the burnt test of cell copolymerization,
Its influence to cell autophagy is investigated, method is as follows：
Prepare cell suspending liquid, be each copolymerized burnt culture dish 1mL, overnight incubation.Culture medium is taken out, is added multi-functional more
The μ g ML-1 of peptide polymer nano material concentration 20 culture medium 1mL, 10 μM of acridine orange dye dyeing 2h；With PBS 3 times,
Co-focusing imaging experiment is carried out, using 488nm laser channelings, collects red spectral band 400-600nm.
As a result as shown in Fig. 8 (wherein control represents control group), cell and M1-1And M0-1Material is incubated altogether, single photon
Confocal experiments, when 488nm is excited, when gathering 400-600nm scopes, cytoplasmic matrix major part limegreen is acid
Lysosome, autophagosome take on a red color；And individually groups of cells and use M1-0The groups of cells of processing does not almost have red fluorescence, only green
Color fluorescence, it was demonstrated that polypeptide polymer nano material M1-1And M0-1Can active cell autophagy, and M1-0Then can not.To M3-1And M1-3
Same experiment is carried out, can equally prove M3-1And M1-3Can active cell autophagy, therefore, finally illustrate of the present invention
Polypeptide polymer nano material B being capable of Induces Autophagy.
Embodiment 8
The polypeptide polymer being prepared in this embodiment by biological electron microscope investigation embodiment 1 and comparative example 1 and 2
Nano material influences the situation of cell autophagy, and method is as follows：
Cell culture 24h, culture medium is then taken out, PBS 3 times, then adds 20 μ g ML-1Multi-functional polypeptide
Polymer nano material not serum-containing media 10mL, cultivate 4h.Take out cell, 20% glutaraldehyde solution (glutaraldehyde：PBS
Buffer solution=1:4) 2-3h is solidified, then, each 10min, then 2h is fixed with 1% osmic acid, then it is clear with PBS. with PBS three times
Wash 3 times, then with 50%, 70%, 80%, 90%, 100% ethanol dehydration, each concentration is dehydrated 1 time, every time 10 minutes.So
Ethanol/acetone (1 is used afterwards:1) and 100% acetone is dehydrated each dehydration twice.Acetone/embedding medium (1:1、1：2) infiltration is handled, room
Each 1h under temperature, acetone/embedding medium (1：3) stay overnight for 4 DEG C, then 4 DEG C of pure embedding medium is overnight.Optimal pure embedding medium 37,45,60 DEG C of each guarantors
Hold 24 hours, then cut into slices, dye, electron microscopic observation.
As a result as shown in figure 9, wherein A figures are the electron microscope of undressed cell, B figures are M1-0Cell after processing
Electron microscope, C2 figures are M1-1The electron microscope of cell after processing, C1 figures are the enlarged drawing in region in dotted line frame in C2 figures；D2 figures are
M0-1The electron microscope of cell after processing, D1 figures are the enlarged drawing in region in dotted line frame in D2 figures；It can be seen from the results that cell
Through polypeptide polymer nano material M1-1And M0-1After processing, cell interior has the autophagy of substantial amounts of Giant Vesicles and duplicature small
Body, this is the characteristic feature of cell autophagy；Without passing through material process and using M1-0The cell of processing (does not observe then obvious
Vesica and autophagosome, it was demonstrated that polypeptide polymer nano material M1-1And M0-1Can active cell autophagy, and M1-0Then can not.Most
Explanation polypeptide polymer nano material R eventually2Polypeptide portion being capable of Induces Autophagy.
So far, two polypeptide portions (identify β-shallow lakes in our the results show polypeptide polymer nano material
The peptide sequence of powder sample albumen (A β) and the peptide sequence of active cell autophagy) each serve as capture A β and activation autophagy degraded
Effect, the effect of competence exertion synergistic treatment is used in combination, A β toxicity is preferably minimized.
Embodiment 9
Investigating the polypeptide polymer nano material that embodiment 1 is prepared in the present embodiment sends out the effect of A β and cell
It is raw to change, the A β for being attached on cell surface can be brought into cell interior, method is as follows：
N2a cells and A β, A β+M1-0、Aβ+M1-1、Aβ+M0-1Blend be incubated 2h altogether, pass through laser confocal microscope
To observe, shown in Figure 10, A β (green fluorescence) are attached on cell surface in itself；And A β+M1-0、Aβ+M1-1Then in endochylema common location,
It may infer that M1-0And M1-1A β are captured and carried it and enter born of the same parents, and M0-1Do not have then.The results show, polypeptide polymer nanometer
The R of material1Polypeptide portion there is capturing function, and can Table A β and cell interaction, carry it and enter born of the same parents.
Embodiment 10
The polypeptide polymer nano material M that embodiment 1 is prepared is investigated in the present embodiment1-1To transgenic mice
Treatment, method are as follows：
The μ L cyclosporins (Cyclosporin) of 6 monthly age mouse mainline 200, to open blood-brain barrier, concentration is 10 μ
M；After 0.5 hour, 20 μ g mL are injected intravenously-1The μ L of multifunctional polypeptides polymer nano material solution 200, be administered every other day, treat
One month.Then water maze laboratory, immunostaining and Nissl's staining are carried out.
Water maze laboratory result is as shown in figure 11, wherein normal mouse group (being represented with WT), injects the old age of PBS
Dull-witted group (being represented with AD), the treatment group that injection polypeptide polymer nano material is treated is (with M1-1Represent), by pair of three
Than understanding, (M after being treated with the polypeptide polymer nano material1-1Group), the latent time of mouse (finds target in labyrinth
The time of thing) more untreated mouse group (AD groups) time is short, illustrate mouse learning ability and memory capability all than not treating
It is good, show the present invention polypeptide polymer nano material can effectively treat Alzheimer's disease.
Immunostaining experiment is as shown in figure 12, and wherein A figures are normal mouse group (being represented with WT), and B figures are injection PBS bufferings
The senile dementia group (being represented with AD) of liquid, C figures are to inject the treatment group treated of polypeptide polymer nano material (with M1-1Table
Show), schemed from A, B schemes, the contrast of three figures of C figures, the brain the inside of mouse after being treated with polypeptide polymer nano material
A β plaque block significantly reduce, patch can effectively be reduced and formed and treat A Erci by showing the polypeptide polymer nano material of the present invention
The silent disease in sea.
Nissl's staining experiment is as shown in figure 13, and A figures are normal mouse group (being represented with WT), and B figures are injection PBS
Senile dementia group (represents) with AD, and C figures are to inject treatment group that polypeptide polymer nano material is treated (with M1-1Represent),
Schemed from A, B schemes, the contrast of three figures of C figures, (M after being treated with polypeptide polymer nano material1-1Group), the brain of mouse
Nerve cell quantity increase, show the present invention polypeptide polymer nano material can effectively make up neurological deficits and treat Ah
Er Cihaimo diseases.
Applicant states that the present invention illustrates the polypeptide polymer nano material and its system of the present invention by above-described embodiment
Preparation Method and application, but the invention is not limited in above-described embodiment, that is, do not mean that the present invention has to rely on above-described embodiment
It could implement.Person of ordinary skill in the field is it will be clearly understood that any improvement in the present invention, to raw material selected by the present invention
Equivalence replacement and the addition of auxiliary element, the selection of concrete mode etc., all fall within protection scope of the present invention and open scope
Within.
Claims (10)
- A kind of 1. polypeptide polymer nano material, it is characterised in that the polypeptide polymer nano material include chitosan and The peptide sequence of identification amyloid-beta and the peptide sequence of active cell autophagy being connected on chitosan.
- 2. polypeptide polymer nano material according to claim 1, it is characterised in that the polypeptide polymer nano material With the structure as shown in following formula I：Wherein, R1For the peptide sequence of identification amyloid-beta (A β), R2For the peptide sequence of active cell autophagy, R3For parent Water-based polyglycol chain, R1And R3Between by acid amides key connection, m, n are the integer more than or equal to 0, and m/n is 1:3~ 3:1。
- 3. polypeptide polymer nano material according to claim 1 or 2, it is characterised in that the polypeptide polymer nanometer The donor of main polymer chain compound group is the acrylated chitosan with structure shown in Formula II in material：Wherein, m, n are the integer more than or equal to 0, and m/n is 1:3~3:1.
- 4. the polypeptide polymer nano material according to any one of claim 1-3, it is characterised in that the polypeptide polymerization The weight average molecular weight of the donor of main polymer chain compound group is 3000-7000 in thing nano material.
- 5. the polypeptide polymer nano material according to any one of claim 1-4, it is characterised in that R1For：WhereinRepresent the connection site of group, " * * " positions The group put and the site with the carbon-carbon double bond reaction in chitosan side base, R1With R3Between group connection site；Preferably, R2For：,WhereinRepresent the connection site of group；Preferably, R3Donor be carboxy blocking polyethylene glycol, its weight average molecular weight is 300-50000, preferably 300-2000.
- 6. the preparation method of the polypeptide polymer nano material according to any one of claim 1-5, it is characterised in that institute The method of stating comprises the following steps：(1) using resin as carrier, using the polyethylene glycol of carboxy blocking and amino acid as raw material, prepared using solid phase synthesis process Obtain polypeptide compounds HS-R1-R3And R2-SH；(2) the polypeptide compounds HS-R obtained using acrylated chitosan with step (1)1-R3And R2- SH is carried out Michael addition reactions obtain the polypeptide polymer nano material；Wherein polypeptide compounds HS-R1-R3And R2- SH mol ratio is 1:3~3:1.
- 7. the self-assembling method of the polypeptide polymer nano material according to any one of claim 1-5, it is characterised in that It the described method comprises the following steps：Polypeptide polymer nano material is dissolved in organic solvent, obtains polypeptide polymer nanometer material Expect solution, obtained polypeptide polymer nanomaterial solution is added in cushioning liquid, self assembly obtains nanosphere.
- 8. self-assembling method according to claim 7, it is characterised in that the organic solvent is dimethyl sulfoxide (DMSO), diformazan In base formamide or hexafluoroisopropanol any one or at least two combination, preferred dimethyl sulfoxide (DMSO).
- 9. the self-assembling method according to claim 7 or 8, it is characterised in that the polypeptide polymer nanomaterial solution Concentration be 10-200 μ g mL-1；Preferably, the cushioning liquid is PBS cushioning liquid；Preferably, the pH value of the cushioning liquid is 6-8；Preferably, the self assembly is carried out under ultrasound, and the ultrasonic time is 1-10min；Preferably, 0.5-12h is kept at 20-40 DEG C after the ultrasound.
- 10. the polypeptide polymer nano material according to any one of claim 1-5 is preparing Alzheimer's disease treatment Application in medicine.
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