CN105061561B - The gold nanoparticle of polypeptide and polypeptide functionalization for inhibiting amyloid beta to assemble and preparation and application - Google Patents
The gold nanoparticle of polypeptide and polypeptide functionalization for inhibiting amyloid beta to assemble and preparation and application Download PDFInfo
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Abstract
本发明涉及一种用于抑制β淀粉样蛋白聚集的多肽和多肽功能化的金纳米粒子及制备和应用。首先从Aβ的经典抑制剂LPFFD和C末端序列VVIA出发，经过理性设计得到特定多肽VVIACLPFFD，这一多肽分子能够同时发挥LPFFD和VVIA的抑制性质，不会彼此冲突，能够在金纳米粒子表面形成特定的结构，放大其抑制聚集的效果。将其修饰在金纳米粒子上，通过多肽自身的抑制作用和与纳米粒子的协同作用，提高其与Aβ间的相互作用，有效抑制Aβ聚集。氨基酸序列为：Val‑Val‑Ile‑Ala‑Cys‑Leu‑Pro‑Phe‑Phe‑Asp。作为制备治疗阿尔茨海默氏症的药物的应用有广阔的前景。
The present invention relates to a polypeptide and a polypeptide-functionalized gold nanoparticle for inhibiting the aggregation of beta amyloid and its preparation and application. First, starting from the classical Aβ inhibitor LPFFD and C-terminal sequence VVIA, a specific polypeptide VVIACLPFFD was obtained through rational design. This polypeptide molecule can simultaneously exert the inhibitory properties of LPFFD and VVIA without conflicting with each other, and can form on the surface of gold nanoparticles A specific structure amplifies its aggregation-inhibiting effect. It is modified on gold nanoparticles to improve the interaction between Aβ and Aβ through the inhibitory effect of the polypeptide itself and the synergistic effect with the nanoparticles, and effectively inhibit the aggregation of Aβ. The amino acid sequence is: Val‑Val‑Ile‑Ala‑Cys‑Leu‑Pro‑Phe‑Phe‑Asp. The application as a medicine for the preparation of Alzheimer's disease has broad prospects.
Description
Technical field
The present invention relates to it is a kind of for inhibit amyloid beta assemble polypeptide and polypeptide functionalization gold nanoparticle and
Preparation and application.
Background technique
Alzheimer's disease (AD) is the main morbidity form of senile dementia, ends 2011, and the whole world is existing
35000000 AD patients, and death relevant to AD about 48.6 ten thousand.The number of the infected of AD has been only second to cancer
And heart disease, become one of the disease for most expending social expenditure.The pathological characteristics of AD are in nerve cell by Protein tau
The nerve fibre of formation twists together and extracellular amyloid beta (A β) deposition.A β is containing 39-43 amino acid residue
Polypeptide successively cuts shape through β and gamma secretase by amyloid precusor protein (amyloid- β precursor protein, APP)
At.Two kinds of main Types of A β are A β 40 and A β 42 respectively, and wherein 40 content of A β is compared with horn of plenty, but 42 toxicity of A β is stronger.It is many
Research has shown that the morbidity of the aggregation especially oligomerization and AD of A β is closely related.Therefore early stage A beta-aggregation to its into
Row inhibits to be feasible treatment means, can prevent or postpone the morbidity of AD.
Researcher has developed a variety of inhibitor for A β, including Polyphenols, polypeptide and its derivative, antibody and receives
Rice corpuscles etc..These inhibitor test in vitro and test cell line in there is certain effect, but their biocompatibility, body
There is problem in internal stability etc..Polypeptide has the characteristics that small molecular weight, non-immunogenicity and high specificity, and it is readily synthesized
And modification, physicochemical property are clear.Therefore ideal A beta polypeptides inhibitor can be obtained by design and rational.Due to the hydrophobic core of A β
Heart sequence (KLVFFAE) plays an important role to the aggregation of A β, therefore researcher is based on this polypeptide design and develops using LPFFD as generation
The a series of of table have an obvious peptide inhibitors for inhibiting A beta-aggregation effect, however these peptide inhibitors that there are stability is poor,
It is easily degraded and the disadvantages of autohemagglutination, does not obtain practical application so far.
In recent years with the development of nanotechnology, nanoparticle plays a significant role in terms of drug delivery.Especially Jenner
Rice corpuscles (GNP) has the characteristics that hypotoxicity, is easy to detect and be imaged, synthetic method is mature, can pass through blood-brain barrier, commonly uses
In drug delivery and mechanism study.Its surface is easy to carry out functional modification.If modifying peptide inhibitor in nanoparticle
(NP) the defects of surface researches and develops polypeptide functionalization GNP, can not only improve the functioning efficiency and dissolubility of polypeptide aglucon, moreover it is possible to draw
Enter nano effect, using the property of gold nanoparticle, its inhibiting effect to A β can be greatly enhanced, becomes the promising treatment of tool
The drug candidate of AD.
Summary of the invention
The purpose of the present invention is overcome the deficiencies in the prior art, design synthesizes the gold nano of a kind of polypeptide and polypeptide functionalization
Particle, and the gold nanoparticle for providing polypeptide functionalization inhibits the purposes of amyloid beta aggregation drug in preparation.
A kind of polypeptide for inhibiting amyloid beta to assemble, has SEQ ID No.1 amino acid sequence: Val-Val-
Ile-Ala-Cys-Leu-Pro-Phe-Phe-Asp.Through experiment exam, the polypeptide itself has certain inhibition beta-amyloyd egg
The ability of white aggregation.
Since the nanoparticle of polypeptide functionalization inhibits the effect of amyloid beta aggregation to be generally better than free polypeptide,
So the present invention is prepared for the gold nanoparticle of functionalization using aforementioned polypeptides amino acid sequence, structure representation is as follows:
The preparation method of gold nanoparticle of the invention is gone out from the classical inhibitor LPFFD and C-terminal sequence VVIA of A β
Hair, design obtains particular polypeptide VVIACLPFFD, and forms specific structure on gold nanoparticle surface.
Of the invention is described as follows: the gold nanoparticle of polypeptide functionalization of the present invention, nanoparticle used
Son is the gold nanoparticle (GNP) of diameter 15nm, and particular polypeptide VVIACLPFFD used has SEQ ID No.1 amino acid
Sequence: Val-Val-Ile-Ala-Cys-Leu-Pro-Phe-Phe-Asp.
The present invention from the classical inhibitor LPFFD of A β and C-terminal sequence VVIA, obtains spy by design and rational first
Determine polypeptide VVIACLPFFD, this peptide molecule can play the inhibition activity of LPFFD and VVIA simultaneously, it will not collide with one another,
And specific structure can be formed on gold nanoparticle surface, amplify its effect for inhibiting A beta-aggregation.It is modified and is in diameter
On the gold nanoparticle of 15nm, by the inhibiting effect of polypeptide itself and with the synergistic effect of nanoparticle, improve it between A β
Interaction, effectively inhibit A beta-aggregation.
Preferred preparation process:
1) by aqueous solution of chloraurate (concentration 1mmol/L) boiling reflux 10-30min；The sodium citrate that heat is added is water-soluble
After liquid (concentration 38.8mmol/L, aqueous solution of chloraurate: sodium citrate aqueous solution volume ratio=10:1), persistently it is stirred at reflux
30-60min；Reaction solution cooled and filtered removal precipitating, obtains GNP solution；
2) aqueous solution (concentration 1-3mmol/L) of polypeptide VVIACLPFFD is prepared；Add it to 1-5nmol/L's
In GNP solution (polypeptid solution: GNP aqueous solution volume ratio=1:10), the reaction 2-6h (polypeptide being added when reaction is stirred at room temperature
The excessive complete modification to guarantee the surface GNP).
3) solution made from first passes through 0.45 micron membrane filter of aperture and removes precipitating, and filtrate is then used deionized water dialysis
3-7 days (dialysis shut off molecular weight 3000-10000 dalton) removes wherein remaining soluble matter, obtains the Jenner of polypeptide functionalization
Rice corpuscles aqueous solution.
4) the gold nanoparticle aqueous solution of polypeptide functionalization is stored at 4 DEG C.
Key technology of the invention has at 3 points, is the structure of polypeptide sequence first, and polypeptide VVIACLPFFD is by the C-terminal of A β
Sequence VVIA is connected with the classical inhibitor LPFFD of A β with cysteine C, the hydrophobic part of VVIA and the live part of LPFFD
The both ends of peptide chain are exposed to, the two peptide fragments is enabled to play respective effect；Followed by when polypeptide VVIACLPFFD is crosslinked
When on gold nanoparticle, the position of crosslinking is on the side chain thiol of cysteine C, and at this moment the both ends of polypeptide are respectively in nanoparticle
Sub- surface extension so that VVIA and LPFFD with certain conformation exposure in the solution, can simultaneously in conjunction with A beta molecule, thus plus
Fast inhibiting effect simultaneously improves inhibitory effect；It is finally the gold nanoparticle of the later polypeptide functionalization of crosslinking in the solution at stabilization
Colloidal solution, stability greatly improves compared to gold nanoparticle, the gold nanoparticle of polypeptide functionalization be it is monodispersed, favorably
In in conjunction with smaller A beta-aggregation body and inhibiting its aggregation.
Advantages of the present invention:
Kinds of experiments means prove that the gold nanoparticle of polypeptide functionalization can effectively inhibit the aggregation of A β 42；Change A
The pattern of 42 aggregation of β, reduces the generation of 42 fiber of A β；And effectively inhibit A β's 42 at low concentration (0.1nmol/L)
Cytotoxicity.The gold nanoparticle of polypeptide functionalization can effectively inhibit A β's 42 as a kind of potential AD therapeutic agent
Oligomerization and fiber forming process, and generated cytotoxic effect in 42 accumulation process of A β is reduced, it is that A β 42 assembles
Ideal inhibitor.Gold nanoparticle holds out broad prospects as the application of the drug of preparation treatment Alzheimer's disease.
Detailed description of the invention
Fig. 1: the transmission electron microscope picture of the gold nanoparticle of polypeptide functionalization in embodiment 1
Fig. 2A: the thermogravimetric analysis figure of unmodified GNP in embodiment 2
Fig. 2 B: the thermogravimetric analysis figure of the gold nanoparticle of polypeptide functionalization in embodiment 2
Fig. 3: the gold nanoparticle Yu A β 42 of the polypeptide functionalization of 0.1nmol/L-3.5nmol/L concentration are total in embodiment 3
The ThT fluorogram of culture after culture for 24 hours
Fig. 4 A: the transmission electron microscope picture of culture after A β 42 is individually cultivated for 24 hours in embodiment 4
Fig. 4 B: culture after the gold nanoparticle Yu A β 42 of the polypeptide functionalization of 1nmol/L co-culture for 24 hours in embodiment 4
Transmission electron microscope picture
Fig. 5: culture after the gold nanoparticle Yu A β 42 of the polypeptide functionalization of 1nmol/L co-culture for 24 hours in embodiment 5
Grain size distribution.
Fig. 6 A: the gold nanoparticle Yu A β 42 of the polypeptide functionalization of 0.1nmol/L-3.5nmol/L concentration are total in embodiment 6
Cytotoxicity (MTT experiment) of the culture to SH-SY5Y cell after culture for 24 hours.
Fig. 6 B: the gold nanoparticle Yu A β 42 of the polypeptide functionalization of 0.1nmol/L-3.5nmol/L concentration are total in embodiment 6
Cytotoxicity (LDH release experiment) of the culture to SH-SY5Y cell after culture for 24 hours.
Specific embodiment
The present invention is described further combined with specific embodiments below.
The present invention from the classical inhibitor LPFFD of A β and C-terminal sequence VVIA, obtains spy by design and rational first
Determine polypeptide VVIACLPFFD amino acid sequence: Val-Val-Ile-Ala-Cys-Leu-Pro-Phe-Phe-Asp.
The structural formula of polypeptide is as follows:
This peptide molecule can play the inhibition activity of LPFFD and VVIA simultaneously, will not collide with one another, and can be in gold
Nanoparticle surface forms specific structure, amplifies its effect for inhibiting aggregation；It is modified in the gold nano that diameter is 15nm
On particle, by the inhibiting effect of polypeptide itself and with the synergistic effect of nanoparticle, improve its interaction between A β, have
Effect inhibits A beta-aggregation；Structural representation is as follows:
The preparation method of the gold nanoparticle of polypeptide functionalization of the invention, its step are as follows:
1) by aqueous solution of chloraurate (concentration 1mmol/L) boiling reflux 10-30min；The sodium citrate that heat is added is water-soluble
After liquid (concentration 38.8mmol/L, aqueous solution of chloraurate: sodium citrate aqueous solution volume ratio=10:1), persistently it is stirred at reflux
30-60min；Reaction solution cooled and filtered removal precipitating, obtains GNP solution；
2) aqueous solution (concentration 1-3mmol/L) of polypeptide VVIACLPFFD is prepared；Add it to 1-5nmol/L's
In GNP solution (polypeptid solution: GNP aqueous solution volume ratio=1:10), the reaction 2-6h (polypeptide being added when reaction is stirred at room temperature
The excessive complete modification to guarantee the surface GNP).
3) solution made from first passes through 0.45 micron membrane filter of aperture and removes precipitating, and filtrate is then used deionized water dialysis
3-7 days (dialysis shut off molecular weight 3000-10000 dalton) removes wherein remaining soluble matter, obtains the Jenner of polypeptide functionalization
Rice corpuscles aqueous solution.
4) the gold nanoparticle aqueous solution of polypeptide functionalization is stored at 4 DEG C.
It is proved using kinds of experiments means, the gold nanoparticle of polypeptide functionalization can effectively inhibit A β 42 to assemble, and energy
Reduce generated cytotoxicity in 42 accumulation process of A β.
Embodiment 1: the pattern of the gold nanoparticle of polypeptide functionalization
The aqueous solution of the gold nanoparticle of polypeptide functionalization is added dropwise on the carbon film copper mesh of 400 mesh, is spontaneously dried.Then
It is observed with transmission electron microscope (Tecnai G2 F20), detection voltage is 200kV, chooses the image that scale after amplifying is 50nm
It is observed, as a result as shown in Figure 1.
As shown in Figure 1, the pattern of the gold nanoparticle of polypeptide functionalization is relatively regular spherical shape, is analyzed by software, more
The diameter of the gold nanoparticle of peptide functionalization is 15.0 ± 1.2nm.
Embodiment 2: the mass change of the gold nanoparticle of unmodified GNP and polypeptide functionalization
The aqueous solution of unmodified GNP and the gold nanoparticle of polypeptide functionalization are concentrated and are lyophilized into brown nano powder
End is put into thermogravimetric analyzer and is analyzed.By nanometer powder by room temperature temperature programming (5 DEG C/min) to 900 DEG C, and with nitrogen
(gas velocity 20mL/min) is swept in air-blowing, and the polypeptide breakdown of GNP surface modification is made to volatilize, and records mass loss.As a result as shown in Figure 2.
Such as Fig. 2A it is found that unmodified GNP before heating after quality without significant change.By Fig. 2 B it is found that polypeptide functionalization
Gold nanoparticle had lost after heating 2.148% quality.The GNP relative molecular mass of diameter 15nm is 20,539,000
Dalton, the molecular mass of loss are about 441 kilodaltons.One VVIACLPFFD molecule of every modification, molecular weight increase
1123.38 dalton.So the average modification rate of the gold nanoparticle of polypeptide functionalization is about 400, i.e., repaired on average each GNP
About 400 VVIACLPFFD peptide molecules are adornd.
The gold nanoparticle and A β 42 of the polypeptide functionalization of embodiment 3:0.1nmol/L-3.5nmol/L concentration co-culture
The ThT fluorescence intensity change of culture after for 24 hours
A β 42 is dissolved in hexafluoroisopropanol solution first, obtains 42 solution of A β of 1mg/mL, ultrasonic 10min is in A β
Monodisperse status, freeze-drying obtain 42 dry powder of A β, save in -20 DEG C.
Weighing the ThT of 3.99mg, to be dissolved in the phosphate buffer of 500mL (PBS, phosphate buffer saline) molten
Liquid (wherein phosphate concn is 100mmol/L, NaCl concentration 10mmol/L), the ThT for obtaining final concentration of 25 μm of ol/L is molten
Liquid.
The A β 42 for weighing 5mg, is dissolved in the NaOH solution (concentration 20mmol/L) of 4.03mL, and ultrasonic 10min is allowed to fill
Divide dissolution, 4 DEG C, 16000g is centrifuged 20min, takes the supernatant of total volume 75%, and obtaining 42 solution of A β, (concentration is 275 μm of ol/
L).It is diluted with ThT solution, obtains 42 solution of A β of final concentration of 25 μm of ol/L.It is added dropwise by every 200 μ L of hole in 96 orifice plates
In, it is cultivated under the conditions of being placed in 37 DEG C.
The gold nanoparticle aqueous solution of polypeptide functionalization is diluted using ThT solution, obtaining concentration is 3.85nmol/L's
The solution of gold nanoparticles of polypeptide functionalization, and obtaining concentration by gradient dilution is respectively 2.2,1.1,0.55 and 0.11nmol/L
Polypeptide functionalization solution of gold nanoparticles.Taking concentration is 42 solution of A β of 275 μm of ol/L, uses the polypeptide of various concentration respectively
The solution of gold nanoparticles of functionalization dilutes, and obtains the gold nanoparticle of the polypeptide functionalization Han 3.5,2,1,0.5 and 0.1nmol/L
42 solution of A β (final concentration of 25 μm of ol/L of A β 42).Five kinds of solution are added dropwise in 96 orifice plates respectively by every 200 μ L of hole, are set
It is cultivated under the conditions of 37 DEG C.
96 orifice plates are placed in microplate reader, the constant temperature incubation at 37 DEG C.Every 10min vibratory orifice plate 10s.Incubation time reaches
After for 24 hours, the fluorescence intensity under 440nm excitation wavelength and 480nm launch wavelength, exciting light bandwidth is 9nm, and transmitting light belt is wide
For 20nm.Measurement result takes the average value of three parallel samples.With containing only the sample of A β be 100% control group, at 480nm
ThT fluorescence intensity (%) maps to the concentration of the gold nanoparticle of polypeptide functionalization, as a result as shown in Figure 3.
From the figure 3, it may be seen that the ThT fluorescence intensity that A β 42 after the gold nanoparticle of polypeptide functionalization is added is decreased obviously, explanation
The gold nanoparticle of polypeptide functionalization effectively enhances the effect for inhibiting A β 42 to assemble.ThT fluorescence intensity declines degree and polypeptide
The concentration of the gold nanoparticle of functionalization is proportional, illustrates that the gold nanoparticle concentration of polypeptide functionalization is higher, inhibits A β 42
Congregational rate is better.
Culture after the gold nanoparticle and A β 42 of the polypeptide functionalization of embodiment 4:1nmol/L concentration co-culture for 24 hours
The morphology of the aggregate variation
A β 42 is handled according to method same as Example 3, and prepares the gold nanoparticle of the functionalization of polypeptide containing 1nmol/L
42 solution of A β, final concentration of 25 μm of ol/L of A β 42 in solution.By above-mentioned solution in 37 DEG C, trained under the conditions of 150rpm
It supports.
After incubation time reaches for 24 hours, 42 culture solution of A β of 100 μ L is taken, 5-10 μ L solution is added dropwise 300 ultrasonic 10min
On purpose carbon film copper mesh, spontaneously dry.When the solution on copper mesh will be dry not dry, 1% Salkowski's solution (pH=6.5) is added dropwise
Surplus liquid is sopped up after dyeing 30s, is spontaneously dried.Then it is observed with transmission electron microscope (JEM100CXII), detection voltage is
100kV, scale is observed after choosing amplification for the image of 100nm, as a result as shown in Figure 4.
By Fig. 4 A it is found that A β 42 forms 10-20nm wide, the band-like shape dimension of hundreds of nanometers of length in independent culture afterwards for 24 hours；By
After Fig. 4 B is it is found that be added the gold nanoparticle of the polypeptide functionalization of 1nmol/L, the form of 42 fiber of A β is changed.It is added
The quantity of 42 fiber of A β significantly reduces in the sample of the gold nanoparticle of polypeptide functionalization, and fibre length shortens in irregular
Long rodlike disperse agglomerations.This explanation, the gold nanoparticle of polypeptide functionalization change the pattern of 42 aggregation of A β, it is suppressed that A β
The generation of 42 fibers.
Culture after the gold nanoparticle and A β 42 of the polypeptide functionalization of embodiment 5:1nmol/L concentration co-culture for 24 hours
Particle diameter distribution
A β 42 is handled according to method same as Example 3, and prepares Jenner's grain of rice of the polypeptide functionalization containing 1nmol/L
42 culture solution of A β of son, wherein 42 final concentration of 25 μm of ol/L of A β.Above-mentioned solution is placed in 37 DEG C, is trained under the conditions of 150rpm
It supports.After culture for 24 hours, takes culture solution to be diluted to the final concentration of 10 μ g/mL of A β using deionized water, pass through particle size analyzer
(Malvern Instruments), analyzes sample at 25 DEG C, as a result as shown in Figure 5.
From figure 5 it can be seen that the A β 42 as control is individually cultivated and partial size is occurred for the poly- of 200-1000nm or so
Collective.And in the sample that the gold nanoparticle of polypeptide functionalization and A β 42 are co-cultured, it can observe that partial size is 20-40nm simultaneously
The peak and partial size that characterize the gold nanoparticle of polypeptide functionalization are the peak that 200-1000nm or so characterizes 42 aggregation of A β.Polypeptide function
The gold nanoparticle of energyization reduces the quantity of 42 aggregation of A β generation, and the average grain diameter of aggregation is substantially reduced.The above results
Illustrate, the gold nanoparticle of polypeptide functionalization inhibits the generation of 42 aggregation of A β, changes the aggregation paths of A β.
The gold nanoparticle and A β 42 of the polypeptide functionalization of embodiment 6:0.1nmol/L-3.5nmol/L concentration co-culture
Cytotoxicity of the culture to SH-SY5Y cell after for 24 hours
Cell used in cytotoxicity experiment is people's neuroblastoma cell line (SH-SY5Y).Culture medium is DMEM/
F-12 culture medium, containing 20%FBS, 2mmol/L glutamine, 100U/mL is dual anti-.Culture environment is 5%CO2,37 DEG C of constant temperature trainings
It supports.
A β 42 is handled according to method same as Example 3, and is prepared containing various concentration (35,10,5 and 1nmol/L)
42 solution of A β of the gold nanoparticle of polypeptide functionalization, final concentration of 250 μm of ol/L of A β 42 in solution.
In MTT experiment, cell is pressed into every 90 μ L culture solution of hole, the density of 5000 cells is added in 96 orifice plates.Culture
After for 24 hours, the sample (10 μ L) that the gold nanoparticle of A β 42 or A β 42 after aging is added and polypeptide functionalization co-cultures finally makes
Final concentration of 3.5,1,0.5 and 0.1nmol/L, A β 42 of GNP or the gold nanoparticle of polypeptide functionalization in the every hole of medicine feeding hole
Final concentration of 25 μm of ol/L.Continue culture 48h to move back except culture medium, cell is cleaned and replaced using the PBS solution of pH=7.4
For serum free medium.Then 10 μ L MTT solution (concentration 6mg/mL) are added in every hole, continue to cultivate 4h.When detection,
96 orifice plates are centrifuged 10min with the speed of 1500rpm, centrifugation is moved back except solution in 96 orifice plates, and the DMSO that 100 μ L are added in every hole shakes
Bed concussion 10min.After purple crystals are completely dissolved, absorbance is measured at 570nm using microplate reader, calculates cell survival
Rate.Using not celliferous sample as blank group (0%)；Without A β and inhibitor, only sample containing cell is as a control group
(100%), the cell survival rate of dosing group is calculated.6 multiple holes are arranged in each nanoparticle concentration gradient in experiment.As a result as schemed
Shown in 6A.
In LDH release experiment, cell is pressed into every 90 μ L culture solution of hole, the density of 5000 cells is added in 96 orifice plates.
After culture for 24 hours, culture medium is changed to serum free medium, and the gold of the A β 42 after aging or A β 42 and polypeptide functionalization is added
The sample (10 μ L) that nanoparticle co-cultures, finally makes the end of GNP or the gold nanoparticle of polypeptide functionalization in the every hole of medicine feeding hole
Concentration is 3.5,1,0.5 and 0.1nmol/L, final concentration of 25 μm of ol/L of A β 42.Continue after cultivating 48h, carries out LDH release inspection
It surveys.Before detection, the Triton X-100 that 1% (V/V) is added in Positive control wells cultivates 1h, and cell is cracked completely, as
The positive controls (cell mortality 100%) of LDH release maximum value.Extracellular LDH is released through kit and is measured.Letter
For it, 96 orifice plates are centrifuged 5min at 400g, collect every hole supernatant (80 μ L) be added 40 μ L LDH reaction solutions, mix
It is measured after reacting 30min at room temperature afterwards.Microplate reader is used when measurement, in experiment wavelength 490nm, reference wavelength 630nm
Lower progress light absorption value detection.It is removed when detection using absorption value caused by GNP as background.To contain only the sample of cell as sky
White group, the cell sample that addition triton X-100 is cracked completely calculates the LDH of dosing group as positive controls (100%)
Release rate.6 multiple holes are arranged in each nanoparticle concentration gradient in experiment.As a result as shown in Figure 6B.
By Fig. 6 A it is found that when A 42 individualism of β, cell survival rate 48.46%.And the polypeptide function of various concentration is added
The gold nanoparticle (1,0.5 and 0.1nmol/L) of energyization afterwards SH-SY5Y cell survival rate be increased to 60.31%, 72.09%,
82.18%, illustrate the cytotoxicity that the gold nanoparticle of polypeptide functionalization can effectively inhibit 42 oligomer of A β to generate.By Fig. 6 B
It is found that cell LDH release rate is 31.67% when A 42 individualism of β.And the gold nano of the polypeptide functionalization of various concentration is added
SH-SY5Y cell LDH release rate is reduced to 21.54%, 14.52%, 12.38% to particle (1,0.5 and 0.1nmol/L) afterwards, says
The gold nanoparticle of bright polypeptide functionalization can effectively inhibit Apoptosis caused by 42 oligomer of A β.
The invention proposes on the basis of gold nanoparticle, by the method for chemical modification, make its surface with available
In the polypeptide aglucon (VVIACLPFFD) for inhibiting amyloid beta aggregation, the gold nanoparticle of polypeptide functionalization is prepared.It mentions
Gone out the gold nanoparticle of polypeptide functionalization preparation inhibit beta-amyloid aggregation in terms of purposes, and by its
Applied to the conformation change of A β 42, aggregation and cytotoxicity Inhibition test.It is described by scene preferred embodiment,
Related technical personnel can obviously not depart from the content of present invention, method described herein is being modified or fitted in spirit and scope
Work as changes and combinations, Lai Shixian the technology of the present invention.In particular, it should be pointed out that all similar substitutions and change are to this field
It is it will be apparent that they are considered as being included in spirit of that invention, range and content for technical staff.
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