CN107412782B - Polypeptide polymer nano material and preparation method and application thereof - Google Patents
Polypeptide polymer nano material and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a polypeptide polymer nano material, a preparation method and an application thereof, wherein the polypeptide polymer nano material comprises chitosan, a polypeptide sequence which is connected with the chitosan and is used for identifying beta-amyloid protein, and a polypeptide sequence for activating autophagy of cells. The polypeptide polymer prepared by solid phase synthesis and Michael addition has good biocompatibility and anti-Abeta neurotoxicity, the polypeptide polymer nanospheres obtained by the method can be assembled with Abeta together so as to effectively prevent aggregation of Abeta and reduce the neurotoxicity of Abeta, and meanwhile, the co-assembly can activate autophagy of cells after entering the cells, so that the Abeta is degraded by the autophagy of the cells, thereby realizing the cooperative treatment of Alzheimer's disease, greatly improving the efficiency of the polypeptide nanomaterial for treating Alzheimer's disease, and having wide application prospect.
Description
Technical Field
The invention belongs to the field of high molecular materials, relates to a polypeptide polymer nano material, a preparation method and application thereof, and particularly relates to a multifunctional polypeptide polymer nano material, a preparation method thereof and application thereof in Alzheimer's disease.
Background
Alzheimer's disease is considered the most common neurodegenerative disease, with nearly 5000 million people worldwide suffering from senile dementia in 2015. To date, there is no effective treatment for alzheimer's disease, and it is of great significance to the study of alzheimer's disease. It is well known that one of the important pathological hallmarks of alzheimer's disease is extracellular deposition of a β (β -amyloid) and intracellular neurofibrillary tangles. Over the past several decades, one of the main strategies for the study of alzheimer's disease has been to target inhibitors designed to the a β in order to prevent its extracellular deposition by the self-assembly process of a β for therapeutic purposes. Many inhibitors have been designed, but their effectiveness is not good. In recent years, with the discovery of autophagy and its effect on cancer and neurological diseases, it has been discovered that many patients with neurological diseases, such as alzheimer patients, have a disturbed autophagy function of their nerve cells. From this point of view, autophagy is also a potential target for the treatment of alzheimer's disease. In addition to studies of alzheimer's disease using autophagy as a therapeutic target, there are also attempts to reduce a β production from upstream using an enzyme in a β formation as a therapeutic target. Although certain achievements are achieved in the current research situation of alzheimer disease, the treatment effect is not good. Therefore, it is necessary to develop a new strategy for the treatment of alzheimer's disease.
In the art, there is a great need to find a strategy for combining two strategies together, such as designing a material capable of preventing aggregation of a β, and simultaneously regulating autophagy and degrading a β by autophagy, so that the two strategies can be used for synergistic treatment of alzheimer's disease, which will have great significance for the treatment of alzheimer's disease.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a polypeptide polymer nano material, a preparation method and application thereof, and particularly provides a multifunctional polypeptide polymer nano material, a preparation method and application thereof in Alzheimer's disease.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the present invention provides a polypeptide polymer nanomaterial comprising chitosan and a polypeptide sequence that recognizes beta-amyloid (a β) and a polypeptide sequence that activates autophagy linked to the chitosan.
In the invention, the polypeptide polymer nano material can be assembled with the A beta to prevent the A beta from aggregating, the neurotoxicity of the A beta is reduced, and meanwhile, the co-assembly can activate autophagy of cells after entering the cells to degrade the A beta, so that the synergistic treatment of the Alzheimer disease is realized.
Preferably, the polypeptide polymer nanomaterial has a structure shown as the following formula I:
wherein R is1To recognize the β -amyloid (A β) polypeptide sequence, R2Polypeptide sequence for activating autophagy of cells, R3Polyethylene glycol chain being hydrophilic, R1And R3The two groups are connected by amido bonds, m and n are integers which are more than or equal to 0, and m/n is 1: 3-3: 1.
In the present invention, formula I shows that the polypeptide sequence recognizing β -amyloid and the polypeptide sequence activating autophagy are linked to chitosan chain, and there will be fragments not linked to these polypeptide sequences on the chitosan chain, in the formulaIndicating that the chitosan polymer chains are extended outward.
In the present invention, m and n are each an integer of 0 or more, and for example, m and n may be independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, etc., and m/n is 1:3 to 3:1, for example, 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, the donor of the polymer backbone compound group in the polypeptide polymer nanomaterial is an acrylated chitosan having a structure represented by formula II:
wherein m and n are integers more than or equal to 0, and m/n is 1: 3-3: 1.
Preferably, the weight average molecular weight of the donor of the polymer backbone compound group in the polypeptide polymer nanomaterial is 3000-7000, such as 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500 or 7000.
In the invention, the acryloyl chitosan with the structure shown in the formula II and the R with sulfydryl are utilized1The donor reacts to react the double bond of the acrylated chitosan with the thiol group, thereby reacting R1The group is attached to chitosan.
Preferably, R1Comprises the following steps:
orWhereinDenotes the attachment site of a group, the site of the group at the ". about.", position, R, which reacts with a carbon-carbon double bond on the chitosan side group1And R3The site of attachment of the group. That is, in the present invention, R1The donor structure of (a) is:
Preferably, R2Comprises the following steps:
whereinIndicates the attachment site of the group. That is, in the present invention, R2The donors of (a) are:
Preferably, R3The donor of (a) is a carboxyl-terminated polyethylene glycol having a weight average molecular weight of 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.
That is, in the present invention, R is3Can be expressed asWith the carbonyl end and R2The amino groups in (b) are linked together by amide bond formation.
In another aspect, the present invention provides a method for preparing a polypeptide polymer nanomaterial as described above, the method comprising the steps of:
(1) resin is taken as a carrier, carboxyl-terminated polyethylene glycol and amino acid are taken as raw materials, and a solid-phase synthesis method is utilized to prepare the polypeptide compound HS-R1-R3And R2-SH;
(2) Utilizing acrylation chitosan and the polypeptide compound HS-R obtained in the step (1)1-R3And R2Carrying out Michael addition reaction on the-SH to obtain the polypeptide polymer nano material;
wherein the polypeptide compound HS-R1-R3And R2The molar ratio of-SH is 1:3 to 3: 1.
In the present invention, both the solid phase synthesis and the Michael addition reaction are well known techniques to those skilled in the art, and the preparation conditions thereof can be selected according to the prior art to accomplish the purpose of preparing the polypeptide polymer nanomaterial, and no particular limitation is imposed on the solid phase synthesis and the Michael addition reaction.
In the present invention, the acrylated chitosan can be synthesized using techniques known in the art, for example, 1mmol of chitosan can be dissolved in 2mL of deionized water and heated to 50-60 deg.C (e.g., 50 deg.C, 53 deg.C, 55 deg.C or 58 deg.C), stirred for 30-60min (e.g., 30min, 35min, 40min, 45min, 55min or 60min), cooled to room temperature, added with 5-10mmol (e.g., 5mmol, 5.5mmol, 7mmol, 7.5mmol, 8mmol, 8.5mmol, 9mmol, 9.5mmol or 10mmol) of triethylamine, slowly added with 3-5mmol (e.g., 3mmol, 3.5mmol, 3.8mmol, 4mmol, 4.3mmol, 4.5mmol, 4.8mmol or 5mmol) of acryloyl chloride under ice bath, reacted for 12-24 hours (e.g., 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours or 24 hours), dialyzed, and lyophilized to obtain acryloylated chitosan.
In another aspect, the present invention provides a method for self-assembly of a polypeptide polymer nanomaterial as described above, the method comprising the steps of: dissolving the polypeptide polymer nano material in an organic solvent to obtain a polypeptide polymer nano material solution, adding the obtained polypeptide polymer nano material solution into a buffer solution, and carrying out self-assembly to obtain the nanosphere.
Preferably, the organic solvent is any one of DMSO (dimethyl sulfoxide), DMF (dimethylformamide), or HFIP (hexafluoroisopropanol) or a combination of at least two thereof, preferably DMSO.
Preferably, the concentration of the polypeptide polymer nano material solution is 10-200 mug mL-1For example, 10. mu.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. mu.g mL-1。
Preferably, the buffer solution is a PBS buffer solution.
Preferably, the pH of the buffer solution 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 performed under ultrasound for a time of 1-10min, such as 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min or 10 min.
Preferably, the sonication is followed by a hold of from 0.5 to 12h (e.g. 0.8h, 1h, 3h, 5h, 8h, 10h, 11h or 12h) at from 20 to 40 ℃ (e.g. 22 ℃, 25 ℃, 28 ℃, 30 ℃, 32 ℃, 35 ℃ or 38 ℃).
The polypeptide polymer of the invention has good biocompatibility and anti-A beta neurotoxicity. The polypeptide polymer nanosphere prepared by the invention can be assembled with the A beta together to prevent aggregation of the A beta and reduce the neurotoxicity of the A beta, and meanwhile, the co-assembly can activate autophagy of cells after entering the cells and degrade the A beta through autophagy of the cells, so that the synergistic treatment of the Alzheimer disease is realized.
In another aspect, the invention provides the application of the polypeptide polymer nano material in the preparation of the medicine for treating alzheimer disease.
The polypeptide polymer nano material provided by the invention has good recognition capability and energy resistance capability on the A beta, and capability of effectively activating autophagy of cells and degrading the A beta through autophagy, greatly improves the efficiency of treating the Alzheimer disease by the polypeptide nano material, and has wide application prospect.
Compared with the prior art, the invention has the following beneficial effects:
the polypeptide polymer of the invention has good biocompatibility and anti-A beta neurotoxicity. The polypeptide polymer nanosphere prepared by the invention can be assembled with the Abeta so as to effectively prevent aggregation of the Abeta and reduce neurotoxicity of the Abeta, and meanwhile, the co-assembly can activate autophagy of cells after entering the cells, and the Abeta is degraded by the autophagy of the cells, so that the synergistic treatment of the Alzheimer disease is realized, the efficiency of the polypeptide nanomaterial for treating the Alzheimer disease is greatly improved, and the polypeptide polymer nanosphere has a wide application prospect.
Drawings
FIG. 1 is the nuclear magnetic hydrogen spectrum of the polypeptide polymer nanomaterial of example 1;
FIG. 2A is a graph showing the results of the cytotoxicity test of the polypeptide polymer nanomaterial of example 2;
FIG. 2B is a graph of the results of the anti-A β neurotoxicity cell assay of the polypeptide polymer nanomaterial of example 2;
FIG. 3 is a graph of fluorescence intensity of the polypeptide polymer nanomaterial of example 3 incubated with A β and A β alone measured at an excitation wavelength of 440nm and an emission wavelength of 480 nm;
FIG. 4A shows a polypeptide polymer nanomaterial M1-1The transmission electron microscope image of (2), the scale is 100 nm;
FIG. 4B shows a polypeptide polymer nanomaterial M1-1Transmission electron micrograph at 100nm of co-incubation with a β;
FIG. 5A shows a polypeptide polymer nanomaterial M1-0The transmission electron microscope image of (2), the scale is 100 nm;
FIG. 5B shows a polypeptide polymer nanomaterial M1-0Transmission electron micrograph at 100nm of co-incubation with a β;
FIG. 6A shows a polypeptide polymer nanomaterial M0-1The transmission electron microscope image of (2), the scale is 100 nm; (ii) a
FIG. 6B shows a polypeptide polymer nanomaterial M0-1Transmission electron micrograph at 100nm of co-incubation with a β;
FIG. 7 is a confocal microscope photograph of the polypeptide polymer nanomaterial determined in example 6 for capturing A β;
FIG. 8 is a confocal picture of Acridine Orange (AO) stained cells when the polypeptide polymer nanomaterial determined in example 7 was applied to mouse neuroma blast (N2 a);
FIG. 9 is a biomicroelectroscopic picture of a cell section when the polypeptide polymer nanomaterial determined in example 8 is applied to N2a cells; wherein, the A picture is the electron micrograph of the untreated cell, and the B picture is the M picture1-0The electron micrograph of the treated cells, C2 is M1-1An electron micrograph of the treated cells, panel C1, which is an enlargement of the area in the dashed box of panel C2; d2 at diagram M0-1The electron micrograph of the treated cells, D1 is the dotted line in D2An enlarged view of the area in the box;
fig. 10 is a confocal microscope photograph of co-incubation of polypeptide polymer nanomaterials and a β with N2a cells as determined in example 9;
FIG. 11 is a graph showing the results of the latency of the polypeptide polymer nanomaterial of example 10 after intravenous injection into APP/PS1 transgenic mice; wherein WT represents a normal mouse group, AD group represents a senile dementia group injected with PBS buffer, and M1-1Represents a treatment group for injecting polypeptide polymer nano-materials for treatment;
FIG. 12 is an immunostaining image obtained by immunostaining the brain pocket tissue of a mouse after the polypeptide polymer nanomaterial of example 10 was intravenously injected to APP/PS1 transgenic mice, wherein A is a wild normal mouse group (WT group), B is an Alzheimer's disease mouse group (AD group) injected with PBS buffer, and C is an experimental group (M)1-1Group);
FIG. 13 is a Nie staining image of the mouse brain pocket tissue obtained by Nie staining after the polypeptide polymer nanomaterial of example 10 was intravenously injected to APP/PS1 transgenic mice, wherein A is a wild normal mouse group (WT group), B is an Alzheimer's disease mouse group (AD group) injected with PBS buffer, and C is an experimental group (M)1-1Groups).
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
In this example, the structure of the polypeptide polymer nanomaterial is shown below:
that is, in the formula I provided by the invention, R1The donor is CFFVLKG, R2The donor of (A) is CTNVFNATFHIWHSGQFGT, R3The donor of (a) is a carboxyl-terminated polyethylene glycol having a molecular weight of 368g mol-1M/n is 1/1M1-1。
The synthesis method of the polypeptide polymer comprises the following steps:
resin is taken as a carrier, carboxyl-terminated polyethylene glycol and amino acid are taken as raw materials, the carboxyl-terminated polyethylene glycol and the amino acid are coupled in sequence according to the connection sequence of the polypeptide by using a solid-phase synthesis method, and finally, the product is cracked from the carrier by trifluoroacetic acid, and is purified to obtain the polypeptide compound HS-R1-R3And R2-SH;
Reacting HS-R1-R3And R2the-SH is prepared by carrying out Michale addition reaction on excess acrylated chitosan according to the proportion of 1/0, 1/3, 1/1, 3/1 and 0/1, namely, connecting the polypeptide compound and the acrylated chitosan by reacting sulfydryl on amino acid C (namely cysteine) with carbon-carbon double bonds, and finally dialyzing and freeze-drying.
Example 2
Differs from example 1 only in that HS-R1-R3And R2The Michale addition reaction of the-SH with excessive amount of the chitosan after the acylation according to the proportion of 1/3 to obtain the polypeptide polymer, wherein the M/n is 1/3 and is marked as M1-3。
Example 3
Differs from example 1 only in that HS-R1-R3And R2The Michale addition reaction of the-SH with excessive amount of the chitosan after the acylation according to the proportion of 3/1 to obtain the polypeptide polymer, wherein the M/n is 3/1 and is marked as M3-1。
Comparative example 1
Differs from example 1 only in that HS-R1-R3And R2The Michale addition reaction of the-SH with excessive amount of the chitosan after the acylation according to the proportion of 1/0 to obtain the polypeptide polymer, wherein the M/n is 1/0 and is marked as M1-0。
Comparative example 2
Differs from example 1 only in that HS-R1-R3And R2-SH in a ratio of 0/1 toThe excess of the acrylated chitosan was Michale-added to give a polypeptide polymer, which was designated as M because M/n is 0/10-1。
The nuclear magnetic characterization of the polypeptide polymer nanomaterials synthesized in examples 1 to 3 and comparative examples 1 to 2 revealed that, as shown in fig. 1, the double bond characteristic peak of the nuclear magnetic peak at the site of ppm 5.8 to 6.3 in the spectra of the acrylated chitosan was disappeared to show that the reaction was complete, and the nuclear magnetic peak appeared to satisfy the characteristic peak of the molecular weight of the material molecule by the integral area calculation, and the material molecule was estimated to react according to the ratio of the material to be charged by the integral area calculation.
Example 4
In this example, the polypeptide polymer nanomaterials prepared in examples 1-3 and comparative examples 1-2 were examined for their biocompatibility and anti-A β toxicity by the following methods:
the biocompatibility experiments were first tested: a suspension of N2a cells was prepared and 5000 cells per well of a 96-well plate were added at 200 μ L per well. After 12h incubation, the medium was removed and different concentrations (10, 20, 50, 100, 200. mu.gmL) were added to each well of the experimental group-1) Polypeptide polymer nanomaterial (M)1-0,M3-1,M1-1,M1-3,M0-1) The blank group was added with a volume of 200. mu.L of serum-free medium, and the cell-free group was treated similarly and cultured for 12 hours. Then the medium was taken out, rinsed 3 times with PBS, and added with CCK-8 solution (V)CCK-8:VCulture medium1: 9) then through 450nm excitation, 690nm emission test. The test results are shown in fig. 2A, and it can be seen from the results that the biocompatibility of the polypeptide polymer nanomaterial sample is good.
Next, examination of anti-A β neurotoxicity was carried out by preparing N2a cell suspension, adding 5000 cells per well of 96-well plate at 200. mu.L/well, culturing for 12 hours, removing the medium, and testing the group (at A β/M)1-1Expressed) per well, the concentration of the added polypeptide polymer nano material is 20 mu g mL-1A β protein concentration 20. mu.M serum-free medium, control group (indicated as A β)) Serum-free medium with a protein concentration of 20. mu.M A β was added to each well, a blank was added to a volume of 200. mu.L of serum-free medium, and the cell-free group was treated the same, cultured for 12 h. then the medium was taken out, rinsed 3 times with PBS, and CCK-8 solution (V) was addedCCK-8:VCulture medium1: 9) and then passes through a 450nm excitation and 690nm emission test, the test result is shown in fig. 2B, and the result shows that the polypeptide polymer nano material sample can effectively reduce the toxicity of A β, wherein M is3-1、M1-1、M1-3Ratio of effects M1-0And M0-1It is well demonstrated that the synergistic effect of the polypeptide sequence recognizing β -amyloid protein (A β) and the polypeptide sequence activating autophagy reduces A β toxicity, and wherein M1-1The effect of (2) is the best.
Example 5
In this example, the polypeptide polymer nanomaterials prepared in examples 1 to 3 and comparative examples 1 to 2 were subjected to a Tht fluorescence experiment with a β, and the ability of the materials to inhibit the aggregation of a β was examined.
First, to test the ability of the polypeptide polymer nanomaterial to prevent aggregation of A β, the multifunctional polypeptide polymer nanomaterial prepared in example 1 was dissolved in DMSO (5mg mL)-1) A β was prepared at 200. mu.M (DMSO: H)2O ═ 1:4), Tht 200 μ M, and 100 μ L of 2-fold PBS solution and 62 μ L H were added to each well of a 96-well plate in this order2O, 8 mu L of polypeptide polymer nano material, 20 mu LA β and 10 mu L Tht, then testing according to time points of 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 36h, 48h, 72h and 96h, wherein the treatment modes of the independent group A β are consistent except that the multifunctional polymer nano material is not added, and the final test result is shown in figure 3.
As can be seen in FIG. 3, the fluorescence was stronger in group A β alone and weaker in the group with the addition of the polypeptidic polymer nanomaterial, indicating that the material is effective in preventing aggregation in group A β, and where M is1-0,M3-1,M1-1,M1-3Ratio M0-1That is, as the content of R1 is higher, the Tht fluorescence intensity is lower, which proves that R is1A portion of the polypeptide may prevent aggregation of a β.
Example 1 preparation of polypeptide Polymer nanomaterial M1-1And polypeptide polymer nanomaterial M1-1Transmission electron micrographs of incubation with A β are shown in FIGS. 4A and 4B, respectively, from which it can be seen that M1-1Nanoparticles will be formed (FIG. 4A), and M1-1No significant fibers were formed after incubation with A β (FIG. 4B), which was effective in preventing A β fibrosis, and also demonstrated the ability of the polypeptide polymer nanomaterials of the invention to prevent A β aggregation1-0And M1-1Transmission Electron microscopy of Co-incubation with A β As shown in FIGS. 5A and 5B, it can be seen that M1-0Nanoparticles will be formed (FIG. 5A), and M1-0M was effective in preventing A β fibrosis without significant fiber formation after incubation with A β (FIG. 5B)0-1And M0-1Transmission Electron microscopy of Co-incubation with A β As shown in FIGS. 6A and 6B, it can be seen that M0-1Nanoparticles will be formed (FIG. 6A), and M0-1A β also formed significant fibers after incubation with A β (FIG. 6B), and did not prevent A β fibrosis1The polypeptide moiety of (a) is capable of preventing fibrosis of a β.
Example 6
In this example, a co-localization experiment was performed on the polypeptide polymer nanomaterials prepared in example 1 and comparative examples 1-2 and a β, and the capture ability of the materials on a β was examined by the following method:
the red fluorescence labeled polypeptide polymer nano material and the green fluorescence labeled A β were incubated together, and observed by fluorescence confocal microscope for 2h, the result is shown in FIG. 7, M1-1And M1-0Can be well co-located with A β, and M0-1Then it cannot co-locate with A β, for M3-1And M1-3The same experiment was carried out, and M was also confirmed3-1And M1-3Can be well co-positioned with A β, and the results show that R in the polypeptide polymer nano material of the invention1The polypeptide moiety of (a) may capture a β, co-assembling with a β.
Example 7
The polypeptide polymer nano-materials prepared in example 1 and comparative examples 1-2 were subjected to a cell confocal test to examine the influence on autophagy, and the method is as follows:
cell suspensions were prepared, 1mL per confocal dish, and incubated overnight. Taking out the culture medium, adding 1mL of culture medium with the concentration of the multifunctional polypeptide polymer nano material being 20 mu g ML-1, and dyeing for 2h by using 10 mu M acridine orange dye; and (3) washing by PBS (phosphate buffer solution) for 3 times, performing a confocal imaging experiment, and collecting red light wave band 400-600nm by adopting a 488nm laser channel.
The results are shown in FIG. 8 (wherein control represents control), where cells and M1-1And M0-1Materials are incubated together, and a single-photon confocal experiment is carried out, wherein when the materials are excited at 488nm and collected within the range of 400-600nm, most of cytoplasm matrix is orange green, and acidic lysosomes and autophagosomes are red; and the group of cells alone and with M1-0The treated cell group has almost no red fluorescence and only green fluorescence, and the polypeptide polymer nano material M is proved1-1And M0-1Capable of activating autophagy, M1-0It cannot. To M3-1And M1-3The same experiment was carried out, and M was also confirmed3-1And M1-3Can activate autophagy, and therefore, the polypeptide polymer nanomaterial B provided by the invention can be finally demonstrated to induce autophagy.
Example 8
In this example, the condition that the polypeptide polymer nanomaterials prepared in example 1 and comparative examples 1 and 2 affect autophagy is examined by a biological electron microscope, and the method is as follows:
the cells were cultured for 24h, then the medium was removed, washed 3 times with PBS, and then 20. mu.g ML was added-1The multifunctional polypeptide polymer nano material does not contain 10mL of serum culture medium and is cultured for 4 h. The cells were removed, solidified for 2-3h with 20% glutaraldehyde solution (glutaraldehyde: PBS buffer ═ 1:4), washed three times with PBS for 10min each, fixed for 2h with 1% osmic acid, washed 3 times with PBS, and then dehydrated with 50%, 70%, 80%, 90%, 100% ethanol for 1 time at each concentration for 10min each. Then using ethanol/acetone (1:1), and 100% acetone twice each. The acetone/embedding agent (1:1, 1: 2) is permeated for 1h at room temperature, the acetone/embedding agent (1: 3) is permeated at 4 ℃ overnight, and the pure embedding agent is permeated at 4 ℃ overnight. The optimal pure embedding medium is kept for 24 hours at 37 ℃, 45 ℃ and 60 ℃ respectively, and then is sliced, dyed and observed by an electron microscope.
The results are shown in FIG. 9, in which panel A is an electron micrograph of untreated cells and panel B is M1-0The electron micrograph of the treated cells, C2 is M1-1An electron micrograph of the treated cells, panel C1, which is an enlargement of the area in the dashed box of panel C2; d2 at diagram M0-1An electron micrograph of the treated cells, Panel D1, which is an enlargement of the area in the dashed box of Panel D2; as can be seen from the results, the polypeptide polymer nanomaterial M for cells1-1And M0-1After treatment, a large number of giant vesicles and bilayer membrane autophagosomes are arranged inside the cells, and are typical characteristics of autophagy; without material treatment and with M1-0Treated cells (no visible vesicles and autophagosomes were observed, demonstrating the polypeptide polymer nanomaterial M1-1And M0-1Capable of activating autophagy, M1-0It cannot. Finally, the polypeptide polymer nano material R is illustrated2The polypeptide portion of (a) is capable of inducing autophagy in a cell.
So far, our experimental results prove that two polypeptide parts (i.e. polypeptide sequence for recognizing beta-amyloid (A beta) and polypeptide sequence for activating autophagy) in the polypeptide polymer nanomaterial respectively play roles in capturing A beta and activating autophagy degradation, and the combined use can play a synergistic treatment effect to minimize the toxicity of A beta.
Example 9
In this example, the polypeptide polymer nanomaterial prepared in example 1 is examined to change the effects of A β and cells, and A β adhered to the cell surface can be brought into the cells by the following method:
n2a cells with A β, A β + M1-0、Aβ+M1-1、Aβ+M0-1The blend was incubated for 2h and observed by confocal laser microscopy, as shown in FIG. 10, A β (green fluorescence) itself adhered to the cell surface, and A β + M1-0、Aβ+M1-1Then at cytoplasmic co-localization, M can be inferred1-0And M1-1Capture and load A β into cells, and M0-1Then it is not. The experimental result proves that the R of the polypeptide polymer nano material1The polypeptide part of (a) has a capturing function and can be loaded into cells by the interaction with cells of table a β.
Example 10
In this example, the polypeptide polymer nanomaterial M prepared in example 1 was examined1-1For the treatment of transgenic mice, the procedure was as follows:
mice of 6 months of age were intravenously injected with 200 μ L of Cyclosporin (cyclosporine) to open the blood-brain barrier at a concentration of 10 μ M; after 0.5 hour, 20. mu.g mL of the solution was injected intravenously-1The multifunctional polypeptide polymer nano material solution is 200 mu L, and is administrated every other day for one month. Water maze experiments, immunostaining and nissl staining were then performed.
The results of the water maze experiment are shown in FIG. 11, in which the normal mice group (WT) was treated, the senile dementia group (AD) was treated by injecting PBS buffer, and the treatment group (M) was treated by injecting polypeptide polymer nanomaterial1-1Expressed), it was found from the comparison of the three that the polypeptide polymer nanomaterial was used after the treatment (M)1-1Group), the latency time of the mouse (i.e. the time for finding the target object in the maze) is shorter than that of the untreated mouse group (AD group), which indicates that the learning ability and the memory ability of the mouse are better than those of the untreated mouse, and indicates that the polypeptide polymer nano material can effectively treat the Alzheimer disease.
The immunostaining experiment is shown in FIG. 12, wherein panel A is normal mouse (WT), panel B is senile dementia (AD) injected with PBS buffer, and panel C is therapeutic group (M) injected with polypeptide polymer nano material1-1Shown in the figure A, the figure B and the figure C are compared, the A β plaque in the mouse brain is obviously reduced after the polypeptide polymer nano material is used for treating, and the polypeptide polymer nano material provided by the invention is capable of effectively reducing the plaque formation and treating the Alzheimer disease.
Nie's staining experiment is shown in FIG. 13, panel A is normal mouse (WT), panel B is senile dementia (AD) injected with PBS buffer, and panel C is therapeutic group (M) injected with polypeptide polymer nano material1-1Shown in (B), as can be seen from comparison of the three graphs in graphs A, B and C, after the treatment with the polypeptide polymer nanomaterial (M)1-1Group), the number of brain neurogenic cells of the mouse is increased, which shows that the polypeptide polymer nanomaterial of the invention can effectively compensate neurogenic deficiency and treat Alzheimer's disease.
The applicant states that the polypeptide polymer nanomaterial and the preparation method and application thereof are illustrated by the above examples, but the invention is not limited to the above examples, i.e., the invention is not limited to the above examples. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.
Claims (15)
1. A polypeptide polymer nanomaterial, wherein the polypeptide polymer nanomaterial comprises chitosan, a polypeptide sequence capable of recognizing beta-amyloid protein and a polypeptide sequence capable of activating autophagy of cells, wherein the polypeptide sequence is connected to the chitosan;
the polypeptide polymer nano material has a structure shown in the following formula I:
wherein R is1To recognize the β -amyloid polypeptide sequence, R2Polypeptide sequence for activating autophagy of cells, R3Polyethylene glycol chain being hydrophilic, R1And R3The two groups of the compounds are connected by amido bonds, m and n are integers more than or equal to 0, and m/n is 1: 3-3: 1;
R1comprises the following steps:
whereinRepresents the attachment site of the group, "+" position of the group and the site of the chitosan side group with carbon-carbon double bond reaction, "+" represents R1And R3(iii) a site of intersubgroup attachment;
R2comprises the following steps:
3. The polypeptide polymer nanomaterial as claimed in claim 2, wherein the weight average molecular weight of the donor of the polymer backbone compound group in the polypeptide polymer nanomaterial is 3000-7000.
4. The polypeptide polymer nanomaterial of claim 1, wherein R is3The donor of (a) is carboxyl-terminated polyethylene glycol with a weight-average molecular weight of 300-5000.
5. The polypeptide polymer nanomaterial according to claim 4, wherein the polypeptide polymer nanomaterial is characterized by beingIn that R is3The weight average molecular weight of the donor (1) was 300-2000.
6. The method for preparing polypeptide polymer nanomaterial according to any of claims 1-5, wherein the method comprises the following steps:
(1) resin is taken as a carrier, carboxyl-terminated polyethylene glycol and amino acid are taken as raw materials, and a solid-phase synthesis method is utilized to prepare the polypeptide compound HS-R1-R3And R2-SH;
(2) Utilizing acrylation chitosan and the polypeptide compound HS-R obtained in the step (1)1-R3And R2Carrying out Michael addition reaction on the-SH to obtain the polypeptide polymer nano material;
wherein the polypeptide compound HS-R1-R3And R2The molar ratio of-SH is 1:3 to 3: 1.
7. The method for self-assembly of polypeptide polymer nanomaterial according to any of claims 1-5, characterized in that it comprises the following steps: dissolving the polypeptide polymer nano material in an organic solvent to obtain a polypeptide polymer nano material solution, adding the obtained polypeptide polymer nano material solution into a buffer solution, and carrying out self-assembly to obtain the nanosphere.
8. The self-assembly method according to claim 7, wherein the organic solvent is any one of or a combination of at least two of dimethylsulfoxide, dimethylformamide or hexafluoroisopropanol.
9. The self-assembly method of claim 8, wherein the organic solvent is dimethyl sulfoxide.
10. The self-assembly method of claim 7, wherein the concentration of the polypeptide polymer nanomaterial solution is 10-200 μ g mL-1。
11. The self-assembly method of claim 7, wherein the buffer solution is a PBS buffer solution.
12. The self-assembly method of claim 7, wherein the buffer solution has a pH of 6-8.
13. The self-assembly method according to claim 7, wherein the self-assembly is performed under ultrasound for a time of 1-10 min.
14. The self-assembly method according to claim 7, wherein the temperature is maintained at 20-40 ℃ for 0.5-12h after the ultrasonication.
15. Use of the polypeptide polymer nanomaterial according to any one of claims 1 to 5 in the preparation of a medicament for the treatment of alzheimer's disease.
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