CN107254016B - The preparation method of a kind of difunctional compolymer/nano grain inhibiting agent and in the inhibition of amyloid beta protein aggregation and the application of removing toxic substances - Google Patents
The preparation method of a kind of difunctional compolymer/nano grain inhibiting agent and in the inhibition of amyloid beta protein aggregation and the application of removing toxic substances Download PDFInfo
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Abstract
The present invention one is related to kind of the preparation method of difunctional compolymer/nano grain inhibiting agent and in the inhibition of amyloid beta protein aggregation and the application of removing toxic substances；Iminodiacetic acid (IDA) is synthesized by free radicals copolymerization reaction with metal chelation abilities as monomer with monomers such as n-isopropyl acrylamide (NIPAm), N-tert-butyl acrylamides (TBAm) to the connection product GMA-IDA of glycidyl methacrylate (GMA) and is inhibited the dual-functional nanometer grain inhibiting agent of albumen ability of aggregation.The average grain diameter of the difunctional compolymer/nano particle of this method synthesis is 80-120nm.The metal ion-chelant capacity of the nano particle of this method synthesis is greater than 700 μm of ol/g, can chelate the metal ion in environment, while inhibiting the fibrosis accumulation process of A β 42, is a kind of new drug raw material of potential treatment alzheimer's disease.
Description
Technical field
The compolymer/nano particle suppression with metal-chelating and the dual function for inhibiting fibrosis aggregation that the present invention relates to a kind of
The preparation method of preparation and the application in terms of the inhibition and removing toxic substances of the aggregation of metal inducement amyloid beta protein.
Background technique
Alzheimer's disease is most commonly seen senile dementia, and illness shows as cognition dysfunction, memory capability
The symptoms such as decline, emotional instability.A large number of studies show that along with amyloid beta (amyloid β-in AD pathogenic process
Protein, A β) accumulation process, fibrous patch is generated in patient's intracerebral neuronal cell.Therefore, it is considered that A β is poly-
There are close ties between integrated fibers and AD morbidity.
A β is successively to shear to obtain through β-and gamma-secretase by cross-film amyloid precusor protein (APP).But due to γ-points
The restriction enzyme site for secreting enzyme is different, therefore can produce out the A beta polypeptides with different C ' end amino acids, such as A β 43, A β
42,Aβ40,Aβ38,Aβ37.Wherein, content highest of the A β 40 in human brain, accounts for about the 50%-70% of A beta polypeptides sum, and its
The A β content of its type is all very low.And found in pathological research, A β 42 is the chief component of amyloid plaques, is had
The stronger ability for being gathered into fiber, and it was found that it secretes more in familial AD patient.Therefore, A β 40 and A β 42 is existing
Albumen is predominantly targeting in research AD treatment method.
Zinc ion is in close relations with nervous system, and brain is one of highest organ of internal zinc content.In studies have shown that
Mainly in conjunction with metalloprotein, Free Zinc contains in big intracerebral average content for Zn in serum about 85% zinc in pivot nervous system
10 times of amount.Appropriate concentration zinc ion plays a key effect to the internal eubolism of maintenance.It has been reported that at present and shows AD patient's brain
The zinc ion level of interior hippocampus and amygdaloid nucleus is about higher by 30% than normal person, this may be due to heavy metal ion such as zinc ions
It is related to the inducing action of protein aggregation.Zinc ion with A β quickly combined formed be unable to fibrosis zinc-A beta-aggregation body or
Zinc ion is bonded directly to fiber ends, to inhibit A beta.Generally believe that the aggregation of this zinc ion induction is made at present
There is stronger cytotoxicity with aggregation is formed by.
To overcome metal inducement A beta-aggregation, the method for a kind of " metal-chelating treatment " is proposed in recent years, this method is intended to
Metal-chelator is added, to eliminate the A beta-aggregation of metal ion induction.Some metal-chelators such as ethylenediamine tetra-acetic acid two
Sodium (EDTA), enteroquinol (CQ), His-Ala mutant of CQ structural derivative PBT-2, A β 42 etc. can be with
Effectively inhibit the aggregation of the aβ protein of metal ion induction.However these traditional small molecule metal-chelators are with non-specific
Property, toxic side effect and lower blood-brain barrier penetrability.
Iminodiacetic acid (IDA) is a kind of chemical raw material, is normally used for the manufacture of the pesticides such as glyphosate.It is raw with soda acid
At salt, also and various metals form chelate.Using the metal-chelating performance of IDA, have been reported in chromatographic isolation field by it
Applied to the raw material of ion exchange resin, and do not had been reported that in biomedicine field.
In addition, current " metal-chelating treatment " method is primarily directed to the ion domain of the existence in high metal concentration to gold
The albumen aggregation for belonging to induction is inhibited, so that the metal inducement of aβ protein be delayed to assemble, but forms amyloid fiber to A β
And its caused toxicity inhibition and detoxifying effect it is unsatisfactory.Aiming at the problem that A β forms amyloid fiber, Hong-chen
Liu et al. has synthesized a series of compolymer/nano particles with negative electrical charge by including acrylic monomers copolymerization and being formed, and obtains
Nano grain surface negative electrical charge assembles the inhibiting mechanism for generating inhibiting effect to A beta-amyloyd, and has obtained having and inhibited reconciliation
Nano particle inhibitor (the Negatively charged hydrophobic nanoparticles inhibit of toxic action
amyloid β-protein fibrillation:The presence of an optimal charge density[J]
.Reactive and Functional Polymers,2016,103:108-116.).To further expand compolymer/nano particle
The application in terms of A beta-aggregation in the presence of metal, the present invention utilize iminodiacetic acid (IDA), synthesize GMA-IDA monomer
(Synthesis of Magnetic Chelator for High-Capacity Immobilized Metal Affinity
Adsorption of Protein by Cerium Initiated Graft Polymerization,Langmuir,2005,
21,6987-6994) compolymer/nano particle, is modified using it, with metal-chelating and the dual function for inhibiting fibrosis to assemble
Energy.Nano particle inhibitor preparation process is simple, good biocompatibility, is highly suitable for as amyloid beta protein aggregation
The application of drug and health care product.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, and further exploitation has metal chelating groups and negative electrical charge
The dual-functional nanometer grain inhibiting agent of surface modification.The nano particle has metal-chelating effect simultaneously and inhibits albumen aggregation
Effect, can be effectively suppressed under metal ion existence condition aβ protein aggregation.The present invention is provided simultaneously as amyloid
The drug of albumen aggregation and the purposes of health care product.
Technical solution of the present invention is summarized as follows:
The present invention is by the connection product GMA-IDA of iminodiacetic acid (IDA) and glycidyl methacrylate (GMA)
Pass through as monomer and the monomers such as n-isopropyl acrylamide (NIPAm), N-tert-butyl acrylamide (TBAm) free-radical polymerized anti-
It should synthesize with metal chelation abilities and inhibit the dual-functional nanometer grain inhibiting agent of albumen ability of aggregation.This method synthesis is received
The average grain diameter of rice grain is 80-120nm.The metal ion-chelant capacity of the nano particle of this method synthesis reaches 700-800 μ
Mol/g can chelate the metal ion in environment, while inhibit the fibrosis accumulation process of A β 42.
The structural formula of above-mentioned GMA-IDA is as follows:
The structural formula of above-mentioned dual-functional nanometer particle is as follows:
Nano particle average grain diameter 80-120nm, nanoparticle metal ion chelating capacity reach 700-800 μm of ol/g,
The ratio of x, y, z is 38-53:35-50:5-20 in middle chemical formula.
The preparation method of difunctional compolymer/nano particle of the invention, its step are as follows:
1) the connection product GMA-IDA of synthesizing methyl glycidyl acrylate and iminodiacetic acid；
2) by polymerized monomer N ,-N '-methylene bisacrylamide, N-tert-butyl acrylamide, n-isopropyl acrylamide according to
The ratio and GMA-IDA of 1-5:38-53:35-50 mixes, wherein the molar ratio that control GMA-IDA monomer accounts for total monomer is 5-
20%.
3) to above-mentioned steps 2) in mixed liquor in surfactant, such as sodium hexadecyl sulfate is added, and make surface
The final concentration of 0.2-0.4mg/mL of activating agent；
4) by above-mentioned steps 3) mixed liquor ultrasonic treatment be allowed to be completely dissolved, and it is mixed to be discharged to be passed through nitrogen 10-60min
Close the air in liquid；
5) to above-mentioned steps 4) in mixed liquor in be added initiator sodium peroxydisulfate originate copolyreaction.Under nitrogen protection
Difunctional compolymer/nano particle is obtained after reacting 10-15h in water bath with thermostatic control shaking table.
The partial size for the difunctional compolymer/nano particle that the present invention obtains is 80-120nm, surface δ current potential in water about-
40mV, negative charged surface results in the intermolecular electrostatic repulsion of nano particle Yu 42 albumen of A β, to more effectively press down
The fibrosis of A β processed is assembled；In addition, a large amount of metal chelating groups of nanometer dielectric surface, metal ion-chelant capacity > 700 μ
Mol/g can effectively adsorb the metal ion in environment, hinder the amyloid aggregation of metal ion induction；Inhibit A β simultaneously
42 fibrosis accumulation process is a kind of new drug raw material of potential treatment alzheimer's disease.
Difunctional compolymer/nano particle of the present invention is used for the inhibition of metal ion induction amyloid aggregation
Method has the advantages that
First, compared with common metal chelating agent, difunctional compolymer/nano particle of the invention has better biofacies
Capacitive；
Second, compared with previous single function nano particle inhibitor, difunctional compolymer/nano particle of the invention has
Good metal chelation abilities can be used for " the metal-chelating treatment " of amyloid aggregation.
Third, the A β 42 that difunctional compolymer/nano grain inhibiting agent of the invention can inhibit metal ion to induce simultaneously are poly-
Collection and the aggregation of 42 fibrosis of A β.
4th, difunctional compolymer/nano grain inhibiting agent of the present invention can reduce caused by 42 accumulation process of A β
Cytotoxic effect.
Detailed description of the invention
Fig. 1: in embodiment 4 with zinc ion and zinc ion and difunctional compolymer/nano grain inhibiting agent mixture co-incubation
42 aggregation of A β ThT fluorescence intensity.
Fig. 2: in embodiment 5 with zinc ion and zinc ion and difunctional compolymer/nano grain inhibiting agent mixture co-incubation
42 aggregation of A β transmission electron microscope picture.
Fig. 3: in embodiment 6 with zinc ion and zinc ion and difunctional compolymer/nano grain inhibiting agent mixture co-incubation
42 aggregation of A β circular dichroism spectrogram.
Fig. 4: in embodiment 7 with zinc ion and zinc ion and difunctional compolymer/nano grain inhibiting agent mixture co-incubation
A β 42 to the cell survival rate influence diagram of SH-SY5Y.
Specific embodiment
Following example will be further described method provided by the invention:
Embodiment 1: the synthesis of difunctional compolymer/nano grain inhibiting agent.
Copolymer nano particle is prepared by free radicals copolymerization reaction.(1) by N ,-N '-methylene bisacrylamide, tert-butyl
Acrylamide, n-isopropyl acrylamide and GMA-IDA 1%, 53%, 41%, 5% mixing in molar ratio respectively.In mixture,
Surfactant sodium hexadecyl sulfate 15mg is added, mixed liquor final volume is 50mL.Obtained solution is blown about in nitrogen
30min, to empty air.60mg/mL sodium peroxydisulfate initiator is added, is reacted under nitrogen protection in 25 DEG C of 160rpm shaking tables
12h.Resulting polymers dialyse 14 days (changing water at least twice daily) with MilliQ water to remove unreacted monomer after reaction.It receives
It is stand-by that rice grain freeze-drying is stored in 4 DEG C of refrigerators.The hydraulics partial size of nano particle made from the condition is 118 ± 6nm, surface
Zeta potential is -37 ± 5mV.
Embodiment 2: the synthesis of difunctional compolymer/nano grain inhibiting agent.
Copolymer nano particle is prepared by free radicals copolymerization reaction.(1) by N ,-N '-methylene bisacrylamide, tert-butyl
Acrylamide, n-isopropyl acrylamide and GMA-IDA 2%, 40%, 48%, 10% mixing in molar ratio respectively.Mixture
In, surfactant sodium hexadecyl sulfate 10mg is added, mixed liquor final volume is 50mL.Obtained solution is blown in nitrogen
About 60min, to empty air.60mg/mL sodium peroxydisulfate initiator is added, is reacted under nitrogen protection in 25 DEG C of 160rpm shaking tables
15h.Resulting polymers dialyse 14 days (changing water at least twice daily) with MilliQ water to remove unreacted monomer after reaction.It receives
It is stand-by that rice grain freeze-drying is stored in 4 DEG C of refrigerators.The hydraulics partial size of nano particle made from the condition is 88 ± 6nm, surface
Zeta potential is -41 ± 8mV.
Embodiment 3: the synthesis of difunctional compolymer/nano grain inhibiting agent.
Copolymer nano particle is prepared by free radicals copolymerization reaction.(1) by N ,-N '-methylene bisacrylamide, tert-butyl
Acrylamide, n-isopropyl acrylamide and GMA-IDA 5%, 38%, 37%, 20% mixing in molar ratio respectively.Mixture
In, surfactant sodium hexadecyl sulfate 20mg is added, mixed liquor final volume is 50mL.Obtained solution is blown in nitrogen
About 10min, to empty air.60mg/mL sodium peroxydisulfate initiator is added, is reacted under nitrogen protection in 25 DEG C of 160rpm shaking tables
10h.Resulting polymers dialyse 14 days (changing water at least twice daily) with MilliQ water to remove unreacted monomer after reaction.It receives
It is stand-by that rice grain freeze-drying is stored in 4 DEG C of refrigerators.The hydraulics partial size of nano particle made from the condition is 80.0 ± 5nm, surface
Zeta potential is -49 ± 7mV.
Embodiment 4: the A β with zinc ion and zinc ion and difunctional compolymer/nano grain inhibiting agent mixture co-incubation
The ThT fluorescence intensity of 42 aggregations.
Firstly, ultrasonic disperse 20min is poly- to remove A β 42 by 42 solid powder of A β with the dissolution of 1mg/mL concentration with HFIP
Collective, then standing 2h dissolves A β 42 sufficiently.Then by the HFIP solution of A β 42 in 4 DEG C, refrigerated centrifuge under 12000g
20min takes 75% supernatant to be freeze-dried.42 solid powder of A β after freeze-drying is stored for use in -20 DEG C of low temperature refrigerators.
Before use, 42 solid powder of A β is redissolved in 20mM NaOH, then in 4 DEG C, refrigerated centrifuge 20min under 12000g,
75% supernatant is taken to be freeze-dried.Not metal ion Hepes buffer solution A (20mM, pH 7.4) is then added, or contains metal
IONS OF H epes buffer solution B (7.4,25 μM of ZnCl of 20mM, pH), or joined the single function nano particle of 50 μ g/mL and (pass through
Document Negatively charged hydrophobic nanoparticles inhibit amyloid β-protein
fibrillation:The presence of an optimal charge density[J].Reactive and
Functional Polymers, method synthesis in 2016,103:108-116.) Hepes buffer solution B in, or joined 50 μ
In the Hepes buffer solution B of the difunctional compolymer/nano particle prepared by the present invention of g/mL, final A β concentration is 25 μM.At 37 DEG C,
48h is cultivated in 160rpm shaking table.200 μ L samples are added in 40 μM of ThT solution of 2mL, Fluorescence spectrophotometer is used
(Perking Elmer LS-55, MA, USA) is measured 42 albumen of A β aggregation situation, and measurement carries out under the conditions of 25 DEG C,
Wherein exciting light is 440nm, and transmitting light is 480nm, slit width 5nm.Experimental result takes the average value measured three times.
By Fig. 1, it can be seen that, the ThT fluorescence intensity that the A β 42 containing zinc ion assembles is substantially reduced, and zinc ion is added
It is significantly risen with the A β 42 of the sample of the difunctional compolymer/nano particle ThT fluorescence assembled, but and zinc ion and single function is added
The ThT fluorescence that the A β 42 of the sample of nano particle assembles rises unobvious.This illustrates that the zinc ion induction aggregation formation of A β 42 does not have
There is the aggregation of β lamellar structure；42 aggregation of A β that single function nano particle can not inhibit zinc ion to induce is formed；And it is difunctional
Compolymer/nano particle can inhibit 42 aggregation of A β of zinc ion induction to be formed with chelated zinc ions.
Embodiment 5: the A β with zinc ion and zinc ion and difunctional compolymer/nano grain inhibiting agent mixture co-incubation
The varying topography of 42 aggregations.
It is handled according to method same as Example 4 and cultivates 42 sample of A β.After having cultivated 48h, the A β of 100 μ L is taken to train
5-10 μ L solution is added dropwise on the carbon film copper mesh of 400 mesh by nutrient solution, ultrasonic 10min, spontaneously dries.It will to the solution on copper mesh
When dry not dry, surplus liquid is sopped up after 2% Salkowski's solution (pH 6.5) negative staining 30s is added dropwise, is spontaneously dried.Sample obtained
It is observed using JEM-100CXII transmission electron microscope (acceleration voltage 100kV).Choose the image that scale after amplifying is 100nm
It is observed, as a result as shown in Figure 2.
By Fig. 2 it can be seen that, the A beta-aggregation of zinc ion is added into random metal-protein masses, joined zinc
42 the morphology of the aggregate of A β of ion and single function nano particle substantially be added zinc ion A beta-aggregation at random metal-egg
White aggregation is consistent, and the A β 42 that this instruction sheet function nano particle can not inhibit metal ion to induce assembles.And joined zinc from
42 aggregation of A β of sub and difunctional compolymer/nano particle forms short and thick fibrilla aggregates.This illustrates IDA not only
The A β 42 for preventing metal ion to induce assembles, and the fibrosis of A β is inhibited to assemble to a certain extent.
Embodiment 6: the A β with zinc ion and zinc ion and difunctional compolymer/nano grain inhibiting agent mixture co-incubation
The secondary structure of 42 aggregations changes
42 albumen of A β is handled and cultivated according to method identical with example 4.Taking 300 μ L culture solutions that light path is added is 0.1mm
Silica dish in, observed at extreme ultraviolet wavelength (190-260nm).Wherein sweep bandwidth is 1nm, scanning speed 100nm/
min.Continuous scanning takes average as measurement result three times, and measurement result is as shown in Figure 3.
From figure 3, it can be seen that after cultivating 48h, sample containing only A β 42 200nm or so there is positive maximum and
219nm or so goes out negative minimum.The curve shows to form β-lamellar structure in sample.And with 25 μM of zinc ion co-incubations
There is negative minimum in 200nm or so and has a very weak negative value in 220nm or so in sample, this shows that sample is induced in zinc ion
Under form irregular secondary structure.In the sample that single function nano particle is added, map and the result one that zinc ion is added
It causes, this shows the formation for the irregular secondary structure of A β 42 that single function nano particle can not inhibit metal ion to induce.It is being added
In the sample of difunctional compolymer/nano particle, curve has minimum value at 215-218nm, this may be α-helix to β-piece
The second structure characteristic of layer conversion.This shows that difunctional compolymer/nano particle can prevent 42 albumen of A β from forming zinc ion induction
Irregular secondary structure, and disturb the formation of β-lamellar structure.
Embodiment 7: the A β 42 of zinc ion and zinc ion and difunctional compolymer/nano grain inhibiting agent mixture co-incubation
Influence to the cell survival rate of SH-SY5Y.
Using SH-SY5Y cell strain (DMEM/F12 culture medium, wherein 20%FBS, 2mmol/L glutamine, 100U/
ML is dual anti-).Cell is cultivated in the constant incubator containing 5%CO2 in 37 DEG C.
Cell activity is analyzed using MTT detection.Steps are as follows for specific experiment: 96 orifice plates are added in cell culture fluid
In, wherein 90 μ L cell liquid are added in every hole, the cell density in every hole is 5000.After cultivating for 24 hours in the incubator, 10 μ L are added
250 μM of zinc ion mother liquor, or the single function nanoparticle containing 250 μM of zinc ions and 500 μ g/mL (pass through document
Negatively charged hydrophobic nanoparticles inhibit amyloid β-protein
fibrillation:The presence of an optimal charge density[J].Reactive and
Functional Polymers, method synthesis in 2016,103:108-116.) mother liquor, or contain 250 μM of zinc ions and 500 μ
The difunctional compolymer/nano particle mother liquor prepared by the present invention of g/mL.Final zinc ion concentration is 25 μM, and nanoparticle concentration is
50μg/mL.Continue to remove culture medium after cultivating 48h, is cleaned using Hepes buffer (pH 7.4), be then added in every hole
100 μ L serum free mediums and 10 μ L MTT solution (6mg/mL), continuation cultivate 4h in 37 DEG C of incubators.
Before detection, it is centrifuged off culture medium using plate first, 100 μ L DMSO dissolution is then added and is attached to orifice plate
The cell at bottom.96 orifice plates are protected from light centrifugation 10min, after purple crystals all dissolution, measure it 570nm's using microplate reader
Absorbance.Cell activity is calculated by absorbance.Wherein not celliferous sample is blank group (0%), without A β 42 and is inhibited
Agent, only sample containing cell is control group (100%).Each sample is arranged 6 multiple holes and measures, and is averaged to measurement result.
As a result as shown in Figure 4.
When being free of zinc ion, the addition of A β 42 will lead to cell activity and be reduced to 64.7%, and the A β 42 containing zinc ion
The cell activity of sample is reduced to 47.6%, hence it is evident that lower than the cell activity of 42 sample of A β without zinc ion.This show zinc from
The albumen aggregation of son induction has higher cytotoxicity.The sample cell with higher of difunctional compolymer/nano particle is added
Active (86.3%) is not only much higher than the sample of single function nano particle, and cell activity is apparently higher than the sample that A β 42 is only added
Product.This shows that difunctional compolymer/nano particle has not only chelated zinc ion, eliminates the toxicity of metal inducement protein aggregation, together
When the fibrosis of A β 42 is assembled caused by cytotoxicity also there is good detoxication.
The invention proposes radical copolymerization method is utilized, metal-chelating function monomer, hydrophobic function monomer are utilized
Etc. having synthesized the difunctional compolymer/nano particle with metal chelating groups, and proposes difunctional compolymer/nano particle and inhibiting
The application of metal inducement beta-amyloid aggregation and its relevant cytotoxic, and in Alzheimer disease drug and health care product
Develop the application of aspect.It is described by scene preferred embodiment, related technical personnel can obviously not depart from this
Method described herein is modified in summary of the invention, spirit and scope or appropriate changes and combinations, to realize skill of the present invention
Art.In particular, it should be pointed out that all similar replacements and change are apparent to those skilled in the art, he
Be considered as being included in spirit of that invention, range and content.
Claims (4)
1. a kind of difunctional compolymer/nano grain inhibiting agent；It is characterized in that it contains following structural unit:
Synthesis difunctional compolymer/nano particle average grain diameter be 80-120nm, this method synthesis nano particle metal from
Sub- chelating capacity is greater than 700 μm of ol/g, wherein the ratio of each structural unit is x:y:z=35-50:38-53:5-20.
2. the preparation method of bifunctional copolymer nano particle inhibitor described in claim 1, it is characterized in that preparation step is such as
Under:
1) the connection product GMA-IDA of synthesizing methyl glycidyl acrylate and iminodiacetic acid；
2) by polymerized monomer N ,-N '-methylene-bisacrylamide, N-tert-butyl acrylamide, n-isopropyl acrylamide is according to 1-
The ratio and GMA-IDA of 5:38-53:35-50 mixes, wherein the molar ratio that control GMA-IDA monomer accounts for total monomer is 5-20%；
3) to above-mentioned steps 2) in mixed liquor in surfactant, sodium hexadecyl sulfate is added, and keep surfactant whole
Concentration is 0.2-0.4mg/mL；
4) by above-mentioned steps 3) mixed liquor ultrasonic treatment be allowed to be completely dissolved, and be passed through nitrogen 10-60min so that mixed liquor is discharged
In air；
5) to above-mentioned steps 4) in mixed liquor in be added initiator originate copolyreaction；It is shaken under nitrogen protection in water bath with thermostatic control
Difunctional compolymer/nano particle is obtained after reacting 10-15h in bed.
3. difunctional compolymer/nano particle described in claim 1 is inhibiting metal inducement beta-amyloid aggregation and its correlation
The application of cytotoxicity.
4. difunctional compolymer/nano particle described in claim 1 is in terms of for Alzheimer disease drug and health care product exploitation
Application.
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