CN111154482A - Biological nano composite material and synthetic method and application thereof - Google Patents
Biological nano composite material and synthetic method and application thereof Download PDFInfo
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- CN111154482A CN111154482A CN201811316786.2A CN201811316786A CN111154482A CN 111154482 A CN111154482 A CN 111154482A CN 201811316786 A CN201811316786 A CN 201811316786A CN 111154482 A CN111154482 A CN 111154482A
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- synuclein
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- iron ion
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Abstract
The invention discloses a multifunctional biological nano composite material and a synthesis method thereof, wherein the nano composite material is formed by compounding an iron ion two-photon fluorescent probe, a α -synuclein two-photon fluorescent probe, porous hollow copper sulfide nano particles, a DNA nano gate and α -synuclein aggregate treatment medicine.
Description
Technical Field
The invention belongs to the technical field of analysis and detection of nano materials, and relates to a biological nano composite material, a synthetic method and application thereof.
Background
The pathogenesis and pathogenesis of the Parkinson disease are not clear, the pathology is mainly, the midbrain substantia nigra dopamine neuron degenerates and dies, the striatum is remarkably reduced, the lewy body appears in the cytoplasm of the substantia nigra neuron, and the like, α -synuclein is a main component of the lewy body, is positioned at the presynaptic terminal of the neuron, is a protein consisting of 140 amino acids, and the mutation, aggregation and over-accumulation of the protein are closely related to a series of neurodegenerative diseases including the Parkinson disease.
Until now, researchers have established several methods for analyzing α -synuclein, such as transmission electron microscopy, polyacrylamide gel electrophoresis and western blotting, but these methods are time consuming and not suitable for high volume applications3+Can induce α -synuclein oligomer formation, and Fe in nerve melanin granules also observed in brain tissue substantia nigra of Parkinson disease patients3+The concentration is higher. However, Fe associated with Parkinson's disease has not been clearly reported3+And α -the interaction between synuclein.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a multifunctional biological nano composite material, which realizes the two-photon fluorescence lifetime detection and imaging of ferric ions and α -synuclein aggregates in vitro and in vivo, analyzes the interaction relation of ferric ions and α -synuclein related to Parkinson's disease for the first time, is used for imaging cells and rat brain slices, constructs a diagnosis and treatment integrated platform of chemotherapy and photothermal therapy, and has important significance for researching the treatment and diagnosis of Parkinson's disease.
The invention provides a synthesis method of an iron ion two-photon fluorescent probe, which comprises the following steps:
in an alkali aqueous solution, carrying out reflux reaction on tetrabutylammonium hydrogen sulfate (TBAHS), 4-diethylaminobenzaldehyde and 2-amino-4, 6-dimethylpyrimidine to obtain an iron ion two-photon fluorescent probe shown as a formula (a), wherein the reaction process is shown as a reaction formula (I):
in the present invention, the molar concentration ratio of tetrabutylammonium hydrogen sulfate, 4-diethylaminobenzaldehyde and 2-amino-4, 6-dimethylpyrimidine is (0.5-2): (20-25): (8-12); preferably, 1: 22: 10.
in the invention, the temperature of the reflux reaction is 100-150 ℃; preferably, it is 120 ℃.
In the invention, the time of the reflux reaction is 1-4 h; preferably, it is 2 h.
In the invention, the alkali is one or more of sodium hydroxide, potassium hydroxide, barium hydroxide and the like; preferably, sodium hydroxide.
In the invention, the iron ion two-photon fluorescent probe is in a D-pi-A-pi-D configuration (D represents an electron donor, A represents an electron acceptor) and is a ratio type fluorescent probe with a high two-photon absorption cross section.
Wherein, after the reaction is finished, the method also comprises the following steps of purifying the product: the apparatus was cooled to room temperature and rotary evaporated to give the crude product which was purified by column chromatography (ethyl acetate: petroleum ether: 1).
The invention adopts a bond energy transfer strategy to design and synthesize a novel two-photon fluorescent probe. Specifically, the iron ion two-photon fluorescent probe is a two-photon molecule based on a pyrimidine skeleton (D-pi-A-structure derivative), and two N atoms in positions 2 and 4 can be specifically combined with iron ions to generate fluorescence quenching. In order to obtain larger two-photon absorption cross-section values, the invention uses an olefinic group to link a pyrimidine nucleus (electron acceptor) and a phenyl group (electron donor) to form a D-pi-A-pi-D quadrupole molecule. The methyl and phenyl groups in the D-pi-A-pi-D molecule provide strong electron donating capability, and the long distance between the donor and the acceptor generates effective charge transfer, so that the two-photon cross section value of the probe molecule is large. The maximum two-photon value of the probe under 800nm light excitation was estimated to be 593GM-687 GM. The invention adopts a one-step method of 'reflux reaction' to synthesize the iron ion two-photon fluorescent probe, and has the advantages of mild reaction conditions, simple and convenient operation, low raw material cost and the like compared with the traditional iron ion fluorescent probe.
In one embodiment of the present invention, the method for synthesizing the iron ion two-photon fluorescent probe comprises the following steps: 1mmol of tetrabutylammonium hydrogen sulfate, 22mmol of 4-diethylaminobenzaldehyde and 10mmol of 2-amino-4, 6-dimethylpyrimidine were refluxed in 50mL of a 5M NaOH solution at 120 ℃ for 2 hours. The orange product was obtained by rotary evaporation and the pure material was obtained by column chromatography (ethyl acetate: petroleum ether ═ 1:1) as shown in scheme (I'):
the invention also provides the iron ion two-photon fluorescent probe prepared by the method, wherein the iron ion two-photon fluorescent probe is in a D-pi-A-pi-D configuration (D represents an electron donor, A represents an electron acceptor) and is a ratio type fluorescent probe with a high two-photon absorption cross section.
The invention also provides a synthesis method of the α -synuclein two-photon fluorescent probe, which comprises the following steps:
(1) in thatIn a first organic solvent, ruthenium trichloride, phenanthroline and lithium chloride are subjected to reflux reaction to obtain bis (1, 10-phenanthroline) ruthenium dichloride (Ru (phen)2Cl2)。
(2) Then in a mixed solution of methanol and water, bis (1, 10-phenanthroline) ruthenium dichloride (Ru (phen)2Cl2) And bipyridyl [3,2-A:2'.3' -C]And (3) carrying out reflux reaction on phenazine (dppz) to obtain α -synuclein two-photon fluorescent probe shown as a formula (b), wherein the reaction process is shown as a reaction formula (II):
in the step (1), the first organic solvent is one or more of N, N-dimethylformamide, dimethyl sulfoxide and the like; preferably, it is N, N-dimethylformamide.
In the step (1), the molar concentration ratio of the ruthenium trichloride to the phenanthroline and the lithium chloride is (10-20): (25-35): (0.5-1.5); preferably, 15: 30: 1.
in the step (1), the temperature of the reflux reaction is 150-180 ℃; preferably 170 deg.c.
In the step (1), the reflux reaction time is 6-12 h; preferably, it is 8 h.
The invention also comprises adding a second organic solvent into the reaction mixture after the reflux reaction in the step (1) and cooling overnight, wherein the second organic solvent is added for dissolving impurities, and the cooling overnight is used for reducing the solubility so as to separate out the product.
Wherein the organic solvent is one or more of acetone, ethanol, isopropanol and the like; preferably, acetone.
Wherein the cooling temperature is 0-10 ℃; preferably, it is 4 ℃.
In the step (2), the bis (1, 10-phenanthroline) ruthenium dichloride (Ru (phen)2Cl2) And bipyridyl [3,2-A:2'.3' -C]The molar concentration ratio of phenazine (dppz) is (1-2): (1-2); preferably, 1.2: 1.
in the step (2), the volume ratio of the methanol to the water is (1-2): (1-2); preferably, 1: 1.
in the step (2), the temperature of the reflux reaction is 80-110 ℃; preferably, it is 100 ℃.
In the step (2), the reflux reaction time is 3-5 h; preferably, it is 4 h.
The invention also comprises the step of cooling and then adding hexafluorophosphate to precipitate the product from the solution after the reflux reaction in the step (2).
Wherein the hexafluorophosphate is one or more of ammonium hexafluorophosphate, sodium hexafluorophosphate, potassium hexafluorophosphate and the like; preferably ammonium hexafluorophosphate.
The invention can also comprise the following steps after the whole reaction is finished, and the product is purified: the unit was cooled to room temperature, ammonium hexafluorophosphate was added to precipitate the product from solution and filtered to give the crude product. Purification by column chromatography (dichloromethane: acetonitrile: 4: 1) and recrystallization (ethanol: water: 90:10) gave red orange crystals.
The invention designs and synthesizes a novel two-photon fluorescent probe by the idea of dipyridyl phenazine derivatives, in particular, the α -synuclein two-photon fluorescent probe is a ruthenium (II) complex, can be specifically combined with fibrosis α -synuclein or detects the aggregation of intracellular protein, and through the improvement of a synthesis route, the α -synuclein two-photon fluorescent probe has red shift of fluorescence emission wavelength, obtains large Stokes shift (more than 200nm) and high light stability (more than 95 percent), and is beneficial to research on the aggregation of various proteins in cells.
In a specific embodiment of the invention, the synthesis method of the α -synuclein two-photon fluorescent probe comprises the following steps of refluxing 56mmol of ruthenium trichloride, 112mmol of phenanthroline and 3.7mmol of lithium chloride in 25mL of DMF for 8 hours at the temperature of 170 ℃, then adding acetone into the reaction mixture, cooling the mixture at the temperature of 4 ℃ overnight to obtain a product Ru (phen)2Cl2It was used without further purification. 12mmol Ru (phen) at 100 DEG C2Cl2And 10mmol of bipyridineAnd [3,2-A:2'.3' -C]Phenazine in the molar ratio of 1:1 in aqueous methanol for 4 hours. The unit was cooled to room temperature, ammonium hexafluorophosphate was added to precipitate the product from solution and filtered to give the crude product. Purification by column chromatography (dichloromethane: acetonitrile: 4: 1) and recrystallization (ethanol: water: 90:10) gave red orange crystals.
The invention also provides a synthesis method of the multifunctional biological nano composite material, which comprises the following steps:
in a buffer solution, firstly, the iron ion two-photon fluorescent probe prepared by the method is connected to the surface of a copper sulfide nanosphere through an amido bond, then the α -synuclein two-photon fluorescent probe prepared by the method is connected to the surface of the copper sulfide nanosphere through electrostatic adsorption, and finally, a medicine is loaded into the copper sulfide nanosphere and is sealed by a DNA (deoxyribonucleic acid) nano gate, so that the multifunctional biological nano composite material is synthesized.
In the invention, the medicine is one or more of rapamycin, cyclosporine, tacrolimus and the like; preferably, it is rapamycin.
In the invention, the buffer solution is preferably PBS buffer solution; the pH value of the buffer solution is 6.0-8.0; preferably, it is 7.4.
In the invention, the concentration ratio of the iron ion two-photon fluorescent probe to the copper sulfide nanosphere is (0.5-1): (9-11) (M: g/L); preferably, 1: 10.
in the invention, the concentration ratio of the α -synuclein two-photon fluorescent probe to the copper sulfide nanospheres is (0.5-1): (9-11) (M: g/L), and preferably is 1: 10.
The specific steps of "the iron ion two-photon fluorescent probe is linked to the surface of the copper sulfide nanosphere through an amide bond" in the present invention are that the copper sulfide nanosphere is dispersed in PBS buffer at pH 7.4 so that the concentration thereof is 1mg/mL, then 20 μ g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is added to 10mL of the above solution and stirred at room temperature for 1 hour, then 20 μ g N-hydroxysuccinimide is added and stirred at room temperature for 1 hour, then 100 μ L of the iron ion two-photon fluorescent probe at concentration of 10mol/L is added, stirred overnight, then the product is collected by centrifugation at 800rpm for 10min, centrifuged and redispersed with deionized water for 2 times, and finally redispersed in 10mL of PBS buffer at pH 7.4, then 100 μ L of the α -synuclein two-photon fluorescent probe at concentration of 10mol/L is added to the above solution and stirred at room temperature overnight, the product is collected at 800rpm for 10min, centrifuged and redispersed for 2 times, and finally redispersed in PBS buffer at pH 7.4.
The gene sequence of the DNA in the DNA nanomesh is shown in SEQ ID NO. 1-3:
sub-DNA-7:5'-HS-TTTTTTGAGCGACTCACTATr(A)GGAAGA GATG(SEQ ID NO.1)
wherein SEQ ID NO.1 is a DNA sequence in which RNA is inserted, the RNA inserted in the middle is represented by r (), and A in r (A) is adenine in RNA.
Partzyme A-7:TGGAAGCAACAAAATCACGGTCGAAATAGTGAGTCGCTC(SEQ ID NO.2)
Partzyme B-7:CAT CTCTTC TCCGAGCCTAGTCTT CCA(SEQ ID NO.3)
The copper sulfide nanoparticles of the present invention can be prepared by a method referred to (Deng X, Li K, Cai X, et al, AHollow-Structured CuS @ Cu2S @ Au Nanohybrid: synthetic Enhanced polymeric efficiency and Photoswitchable Targeting efficiency for Cancer therapeutics [ J ]. Advanced Materials,2017,29(36): 1701266), specifically comprising the steps of: adding hydrazine hydrate into a copper sulfate and sodium hydroxide solution by using polyvinylpyrrolidone as an end-capping agent to prepare cuprous oxide nanospheres, and then synthesizing hydrophilic porous hollow copper sulfide nanoparticles by an ion exchange process at room temperature by using the cuprous oxide nanospheres as a template; finally, the electric property of the film is changed into positive electricity by using the phthalic acid diethylene glycol diacrylate.
The invention adopts a stepwise assembly method to synthesize a multifunctional biological nano composite material, concretely, copper sulfide porous hollow nano particles with uniform size are synthesized by a one-pot method, and then iron ion two-photon fluorescent probes, α -synuclein two-photon fluorescent probes, drug rapamycin and DNA nanomeshes are gradually assembled on the surfaces of the copper sulfide nano particles through chemical bonds and electrostatic adsorption.
In one embodiment of the present invention, the method for synthesizing the multifunctional biological nanocomposite comprises the following steps:
in PBS buffer solution at pH 7.4, 5mL of copper sulfide nanosphere at 10 μ g/mL was added 20 μ g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and stirred for 2 hours, then 20 μ g N-hydroxysuccinimide was added and stirred for half an hour, after which 100 μ L of 50 μ g/mL iron ion two-photon fluorescence probe was added to the mixture overnight under stirring, the product was collected by centrifugation and purified by washing with deionized water for a number of times, then resuspended in PBS buffer solution at pH 7.4, finally, 100 μ L of 50 μ g/mL α -synuclein two-photon fluorescence probe was added to the solution and stirred at room temperature overnight, the resultant was collected by centrifugation and purified by washing with deionized water for a number of times, then resuspended in PBS buffer solution at pH 7.4.
The invention also provides the multifunctional biological nano composite material prepared by the method.
The invention also provides a method for detecting Fe in vitro by the two-photon fluorescence lifetime imaging of the multifunctional biological nano composite material3+And α -synuclein aggregates.
The invention also provides a method for analyzing Fe in cells by the multifunctional biological nano composite material through two-photon fluorescence lifetime imaging3+And α -synuclein aggregates.
The invention also provides application of the multifunctional biological nano composite material in detecting α -synuclein aggregates to treat Parkinson's disease, Alzheimer's disease, synucleinopathy and the like through two-photon fluorescence imaging.
The invention also provides application of the multifunctional biological nano composite material as a carrier in treatment and diagnosis of Parkinson's disease, Alzheimer's disease, synucleinopathy and the like.
The invention utilizes Fe3+Can be specifically combined with two N atoms at2 and 4 positions of a pyrimidine group in the iron ion two-photon fluorescent probe, and the iron ion two-photon fluorescent probe is combined with Fe with different concentrations3+Reacting to obtain a fluorescence lifetime attenuation curve and a linear range thereof, and further detecting Fe3+A change in concentration, wherein the fluorescence lifetime decay curve is a curve based on the decay of the mean lifetime of fluorescence imaging.
The invention also provides an in vitro Fe detection method through iron ion two-photon fluorescence lifetime imaging3+The method of (a), the method comprising the steps of:
(1) the iron ion two-photon fluorescent probe shown in the formula (a) and Fe3+Carrying out a complexation reaction;
(2) measuring the fluorescence lifetime by measuring the resulting fluorescence lifetime and Fe3+To determine Fe3+And (4) content.
Wherein, in the step (1), the temperature of the complexation reaction is room temperature; preferably, it is 25 ℃.
Wherein in the step (1), the time of the complexation reaction is 4-20 s; preferably 5 s.
Among them, in the step (1), the complexing reaction is preferably carried out under normal pressure.
Wherein the concentration of the iron ion two-photon fluorescent probe applicable to the method is 40-80 mu M; preferably 50. mu.M.
Wherein the method is suitable for detecting Fe3+The linear range of (A) is 0-10 [ mu ] M; preferably, it is 1 to 10. mu.M.
Wherein the method is suitable for detecting Fe3+The lowest detection limit of (2) was 0.66. mu.M.
Wherein, the Fe3+From a cell lysate; the cell comprises: one or more of SHSY-5Y cell, Hela cell, PC12 cell, macrophage, etc.; preferably, SHSY-5Y cells.
The pyrimidine group of the iron ion two-photon fluorescent probe synthesized by the invention has two N atoms at2 and 4 positions, and can perform specific coordination with metal cations. In addition, the ultraviolet-visible absorption spectrum of the iron ion two-photon fluorescent probe shows that the iron ion two-photon fluorescent probe has an absorption band near 470nm, which is probably caused by pi-pi electron transition centering on a ligand. Iron ions show more interference in the absorption spectrum, resulting in quenching of the probe fluorescence and a decrease in fluorescence lifetime, indicating high selectivity for iron ions. In addition, the fluorescence lifetime of the molecule is detected by adopting 800nm two-photon excitation, and compared with other methods, the method is not influenced by light intensity, concentration, photobleaching, cell autofluorescence and the like; carrying out quantitative accurate analysis; can realize in vivo detection; the tissue penetrability is strong, and the method can be used for in vivo imaging and the like.
In one embodiment of the invention, the iron ion two-photon fluorescence lifetime imaging detects Fe in vitro3+The method of (1), comprising:
(1) making a standard curve:
mixing a 50 mu M iron ion two-photon fluorescent probe of formula (a) with Fe with different concentrations3+(0,2,4,6,8,10,20,30,40 mu M) cell lysate reacts at normal temperature and normal pressure, fluorescence lifetime decay curves at different concentrations are recorded, and then groups of fluorescence lifetime and Fe are made3+The linear range of the concentration relation curve is 0-10 mu M.
(2) Determination of Fe in samples3+Content (wt.)
Mixing a 50 mu M iron ion two-photon fluorescent probe of formula (a) with Fe in a sample3+The reaction is carried out at normal temperature and pressure, the fluorescence lifetime is measured, and the reaction is based on Fe3+Calculating Fe in the sample according to the relation between the concentration and the fluorescence lifetime3+And (4) content.
The iron ion two-photon fluorescence lifetime attenuation curve obtained by the method and the linear range thereof can be used for in vitro detection of Fe3+The change in concentration.
The invention utilizes Fe3+Can be specifically combined with two N atoms at2 and 4 positions of a pyrimidine group in the iron ion two-photon fluorescent probe, and the iron ion two-photon fluorescent probe is combined with Fe with different concentrations3+Reacting to obtain a fluorescence lifetime attenuation curve and a linear range thereof, and further detecting Fe3+A change in concentration, wherein the fluorescence lifetime decay curve is a curve based on the decay of the mean lifetime of fluorescence imaging.
The invention also provides a method for detecting α -synuclein aggregates in vitro by α -synuclein two-photon fluorescence lifetime imaging, the method comprising the steps of:
(1) reacting α -synuclein fluorescent probe shown in formula (b) with α -synuclein aggregate;
(2) the fluorescence lifetime was measured, and the α -synuclein aggregate content was determined by measuring the relationship between the obtained fluorescence lifetime and α -synuclein aggregates.
Wherein, in the step (1), the reaction temperature is room temperature; preferably, it is 25 ℃.
Wherein in the step (1), the reaction time is 4-20 s; preferably 10 s.
Among them, in the step (1), the reaction is preferably carried out under normal pressure.
Wherein, the reaction between the α -synuclein fluorescent probe shown in the formula (b) and the α -synuclein aggregate means that the functional group of the α -synuclein fluorescent probe is embedded into the fiber trench.
Wherein the concentration of α -synuclein two-photon fluorescent probe suitable for the method is 40-80 mu M, preferably 50 mu M.
Wherein the linear range of α -synuclein aggregates suitable for detection by the method is 0-10 mu M, preferably 2-10 mu M.
Wherein the method is applicable to detecting α -synuclein aggregates with the lowest detection limit of 0.82 mu M.
The α -synuclein fluorescent probe synthesized in the invention is an optical switch molecule, particularly in an aqueous solution, the probe is in an unexcited state and hardly emits fluorescence, and under the existence of α -synuclein fibril framework, the probe interacts with a main groove to change the polarity of a microenvironment sensed by a ligand, which is beneficial to the increase of a luminous state and the fluorescence lifetime, under the existence of a monomer α -synuclein, the probe hardly emits fluorescence and has a constant fluorescence lifetime, because the interaction between the probe and the α -synuclein monomer is weak, the probe can be used for specifically detecting α -synuclein aggregates, in addition, the invention adopts 800nm two-photon excitation to detect the fluorescence lifetime of the molecule, and compared with other methods, the probe is not influenced by light intensity, concentration, photobleaching, cell autofluorescence and the like, can be quantitatively and accurately analyzed, can realize in vivo detection, has strong tissue penetrability, can be used for imaging living bodies and the like.
In one embodiment of the invention, the two-photon fluorescence lifetime imaging method for detecting α -synuclein aggregates in vitro comprises:
(1) making a standard curve:
the α -synuclein two-photon fluorescent probe with the molecular weight of 50 mu M in the formula (b) reacts with α -synuclein aggregates (0,2,4,6,8,10,12,14,16 mu M) with different concentrations at normal temperature and normal pressure, the fluorescence lifetime attenuation curves under different concentrations are recorded, and then the relation curves of the fluorescence lifetimes of all groups and the α -synuclein aggregate concentration are made, so that the linear range is 0-10 mu M.
(2) Determination of α -synuclein aggregate content in samples
Reacting α -synuclein two-photon fluorescent probe with the concentration of 50 mu M of the formula (a) with α -synuclein aggregates in a sample at normal temperature and normal pressure, measuring the fluorescence lifetime, and calculating the content of the α -synuclein aggregates in the sample according to the relationship between the concentration of the α -synuclein aggregates and the fluorescence lifetime.
The two-photon fluorescence lifetime attenuation curve obtained by the method and the linear range thereof can be used for detecting α -synuclein aggregate concentration change in vitro.
According to the invention, by utilizing the characteristic that α -synuclein aggregates can be specifically combined with dppz ligands in α -synuclein two-photon fluorescent probes, α -synuclein two-photon fluorescent probes are reacted with α -synuclein aggregates with different concentrations, so that a fluorescence lifetime attenuation curve and a linear range thereof are obtained, and further, the change of the concentration of α -synuclein aggregates can be detected, wherein the fluorescence lifetime attenuation curve is a curve made according to the attenuation of the average lifetime of fluorescence imaging.
The invention also provides a method for detecting Fe in cells by fluorescence lifetime imaging3+And α -synuclein aggregates, the method comprises the following steps:
(1) co-culturing the multifunctional biological nano composite material and cells to enable the probe to enter the cells;
(2) measuring fluorescence lifetime, and judging Fe according to lifetime change conditions of different regions in single cells by fluorescence lifetime imaging3+And α -change and distribution of synuclein aggregates, to determine Fe3+And α -synuclein aggregate content.
Wherein, in the step (1), the cell may be a neuroma blast; preferably, the cells are cells cultured for 24 h.
Wherein in the step (1), the temperature of the co-culture is 25-40 ℃; preferably, it is 37 ℃.
Wherein in the step (1), the co-culture time is 60-120 min; preferably, it is 90 min.
Wherein, in the step (1), the co-culture condition is 95% O2,5%CO2。
Wherein, in the step (1), the concentration of the multifunctional biological nano composite material is 30-80 μ M; preferably 50. mu.M.
Wherein, in the step (2), the Fe3+And α -the concentration of synuclein aggregates is 5-25. mu.M, preferably 10. mu.M.
The fluorescence lifetime of the molecule is detected by adopting 800nm two-photon excitation, and compared with other methods, the method is not influenced by light intensity, concentration, photobleaching, cell autofluorescence and the like; carrying out quantitative accurate analysis; can realize in vivo detection; the tissue penetrability is strong, and the method can be used for in vivo imaging and the like.
In one embodiment of the invention, the intracellular Fe is detected intracellularly by fluorescence lifetime imaging3+And α -a method of synuclein aggregation, comprising:
(1) making a standard curve:
① addition of different concentrations of Fe to cells3+(0,10,20,30,40,50 μ M) or MG-132(0,20,40,60,80, 100 μ M), co-cultured for 24 h;
② adding 50 μ M multifunctional biological nanocomposite into a culture dish cultured for 24h, co-culturing in an incubator for 30min under the culture condition of 95% O2,5%CO2,37℃;
③ the samples were washed 3 times with PBS, 0.5ml PBS was added to the samples and fluorescence imaging was performed on a Leica confocal set (TCS SP 8);
④ lifetime fitting is carried out by SyPhotome-64 according to each group of imaging, and Fe is judged according to the lifetime change condition of different regions in a single cell3+And α -Change and distribution of synuclein aggregates.
(2) Determination of Fe in samples3+And α -synuclein aggregate content
The operation method is the same as the step (1), the sample is imaged on a Leica confocal lens (TCS SP 8); and obtaining Fe in the sample according to the life fitting curve or the imaging depth3+And α -synuclein aggregate content.
The invention also provides a method for detecting the effect of the mouse brain slice α -synuclein aggregate on treating the Parkinson disease by two-photon fluorescence imaging, which comprises the following steps:
(1) inducing with 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine to obtain a Parkinson disease model mouse;
(2) injecting the multifunctional biological nano composite material into striatum of right side brain of a mouse, irradiating by using near-infrared laser to release the medicine, and slicing;
(3) two-photon fluorescence lifetime imaging detection is carried out by taking two photons as exciting light, and the treatment effect is analyzed by measuring the relation between the fluorescence lifetime and α -synuclein aggregates.
Wherein, in the step (1), the mice are 6 male C57BL mice with the age of 8 weeks, and the average body weight is 24 +/-2 g.
Wherein, in the step (1), the 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine is injected intraperitoneally to obtain the Parkinson's disease model.
Wherein, in the step (2), the concentration of the multifunctional biological nano composite material is 40-60 mu M; preferably 50. mu.M.
Wherein, in the step (2), the wavelength of the near-infrared laser is 808 nm.
Wherein in the step (2), the laser irradiation time is 5-20 min; preferably, it is 10 min.
Wherein, in the step (2), the wavelength of the two-photon as the exciting light is 690-900 nm; preferably 800 nm.
The method adopts 800nm two-photon excitation to detect the fluorescence life of the molecule, and compared with other methods, the method is not influenced by light intensity, concentration, photobleaching, cell autofluorescence and the like; carrying out quantitative accurate analysis; can realize in vivo detection; the tissue penetrability is strong, and the method can be used for in vivo imaging and the like.
In one embodiment of the present invention, the method for detecting the therapeutic effect of the mouse brain slice α -synuclein aggregate by two-photon fluorescence imaging comprises:
(1) preparation of Parkinson disease model mouse
Male C57BL mice, 8 weeks old, were injected intraperitoneally with 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine (30mg/kg) once a day for 7 consecutive days, and behavioral performance of the 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine pair was studied by comparing 5 behavioral tests, thereby obtaining parkinson-disease model mice.
(2) Multifunctional biological nano composite material for injecting into rat brain
After the mouse Parkinson disease model is successfully constructed, the right striatum of the mouse brain is injected by a 50 mu M multifunctional biological nano composite material and is irradiated by laser with the wavelength of 808nm for 10 minutes.
(3) Analysis of α -synuclein aggregate treatment effects in Parkinson's disease mouse brain
The operation method is the same as the step (1), and Fe in the rat brain slice sample is subjected to3+And α -fluorescent lifetime imaging of synuclein aggregates (TCS SP8) and according to standard curves or imaging depthObtaining Fe in the rat brain slice sample3+And α -synuclein aggregate content.
In the invention, the multifunctional biological nano composite material is injected into a Parkinson model mouse brain, drug treatment is released through laser irradiation, and then the obtained mouse brain slice is subjected to two-photon fluorescence lifetime imaging research on Leica TCS SP 8. Comparing the color corresponding to the two-photon fluorescence lifetime spectrum with the color of the slice to judge the Fe in the brains of the mice suffering from the Parkinson's disease and treated mice3+And α -difference of synuclein aggregate content, and can be used for researching Fe in Parkinson's disease3+And α -synuclein, and treating α -synuclein toxic aggregation state.
The invention has the beneficial effects that the multifunctional biological nano composite material is synthesized by compounding the iron ion two-photon fluorescent probe, the α -synuclein aggregate two-photon fluorescent probe, the porous hollow copper sulfide nano particle, the DNA nanomesh and the α -synuclein aggregate treatment drug, the two synthesized two-photon fluorescent probes have the advantages of short synthetic steps, simple operation, high selectivity, high fluorescence quantum yield (0.36-0.42), high two-photon absorption cross section (593GM-687GM) and high sensitivity (the slope of a calibration curve is 7.7), the copper sulfide nano particle is synthesized by a one-pot method, the size distribution is uniform (97 nm-102 nm), and the specific surface area is large (8.7 m)2g-1-9.1m2g-1) The multifunctional biological nano composite material synthesized by the invention has good drug-loading capacity, can detect and image the two-photon fluorescence lifetime of iron ions and α -synuclein aggregates in vitro and in vivo, analyzes the interaction relation between iron ions related to the Parkinson disease and α -synuclein for the first time, is used for imaging cells and rat brain slices, constructs a diagnosis and treatment integrated platform of chemotherapy and photothermal therapy, and has important significance for researching the treatment and diagnosis of the Parkinson disease.
Drawings
FIG. 1: a multifunctional nano material schematic diagram.
FIG. 2: (a)50 mu M iron ion fluorescent probe at different concentrations of Fe3+Double light of (0-40 mu M) front and backSub-fluorescence lifetime curves (inset: probe fluorescence lifetime and Fe)3+Linear relationship of concentration), (b) two-photon fluorescence lifetime curves of 50. mu.M α -synuclein probe before and after different concentrations of α -synuclein aggregates (0-16. mu.M) (inset: linear relationship of probe fluorescence lifetime and α -synuclein aggregate concentration).
FIG. 3: adding Fe with different concentrations into neuroma blast cells cultured by 50 mu M multifunctional nanoparticles3+Or MG-132.
FIG. 4: the therapeutic effect graph of the Parkinson disease model mouse (A: nano material, B: nano material + illumination, C: nano material + drug, D: nano material + drug + illumination).
FIG. 5: a selective experimental graph of 50 mu M two fluorescent probes on common metal ions and proteins contained in organisms; wherein a is a metal ion and b is a protein.
FIG. 6: SHSY-5Y cytotoxicity test results.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
Example 1: synthesis of iron ion two-photon fluorescent probe
1mmol tetrabutylammonium hydrogen sulfate, 22mmol 4-diethylaminobenzaldehyde and 10mmol 2-amino-4, 6-dimethylpyrimidine were refluxed in 50mL 5M NaOH solution for 2 hours. An orange product was obtained by rotary evaporation and the pure material was obtained by column chromatography (ethyl acetate: petroleum ether ═ 1: 1).
Example 2 Synthesis of α -synuclein two-photon fluorescent Probe
56mmol of ruthenium trichloride, 112mmol of phenanthroline and 3.7mmol of lithium chloride are refluxed in 25mL of DMF for 8 hours. Acetone was then added to the reaction mixture, which was cooled at 4 ℃ overnight to give the product Ru (phen)2Cl2It was used without further purification. 12mmol Ru (phen)2Cl2And 10mmol of bipyridyl [3,2-A:2'.3' -C]Phenazine in the molar ratio of 1:1 in aqueous methanol for 4 hours. The unit was cooled to room temperature, ammonium hexafluorophosphate was added to precipitate the product from solution and filtered to give the crude product. Purification by column chromatography (dichloromethane: acetonitrile: 4: 1) and recrystallization (ethanol: water: 90:10) gave red orange crystals.
Example 3: synthesis of multifunctional biological nano composite material
In PBS buffer solution with pH 7.4, 5mL of copper sulfide nanosphere with 10 μ g/mL was added 20 μ g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and stirred for 2 hours, then 20 μ g N-hydroxysuccinimide was added and stirred for half an hour, after which 100 μ L of 50 μ g/mL of the iron ion two-photon fluorescence probe prepared in example 1 of the present invention was added to the mixture overnight with stirring, the product was collected by centrifugation and purified by washing with deionized water for a number of times, and then resuspended in 5mL of PBS buffer solution with pH 7.4, finally, 100 μ L of 50 μ g/mL of the α -synuclein two-photon fluorescence probe prepared in example 2 of the present invention was added to the solution and stirred at room temperature overnight, the resulting product was collected by centrifugation and purified by washing with deionized water for a number of times, then resuspended in 5mL of PBS buffer solution with pH 7.4, and finally, the drug rapamycin was loaded into copper sulfide nanosphere and capped with DNA nanopore, thereby constructing a composite nanomaterial.
Example 4: fe ion two-photon fluorescent probe for in-vitro detection of Fe3+
50 μ M of the iron ion two-photon fluorescent probe prepared in example 1 of the present invention represented by the formula (a) was mixed with a probe containing Fe at various concentrations3+(0,2,4,6,8,10,20,30,40 mu M) of cell lysate reacts at normal temperature and normal pressure, fluorescence lifetime decay curves at different concentrations are recorded, and then groups of fluorescence lifetimes and Fe are made3+The linear range of the concentration relation curve is 0-10 mu M. The fluorescence lifetime decay curve and its linear relationship were obtained (FIG. 2). FIG. 2 shows that the Fe ion two-photon fluorescent probe prepared in example 1 of the present invention has different Fe concentrations3+As can be seen from FIG. 2, the fluorescence lifetime curves under the conditions of (0,2,4,6,8,10,20,30, 40. mu.M)The change range of the lifetime is 1.46 ns-1.375 ns, which indicates that the probe can be well used for fluorescence lifetime imaging analysis.
Example 5 α in vitro detection of the two-photon fluorescent Probe for synuclein α -synuclein
50 mu M of the α -synuclein two-photon fluorescent probe prepared in the embodiment 2 of the invention shown in the formula (b) reacts with cell lysate containing α -synuclein aggregates (0,2,4,6,8,10,12,14,16 mu M) with different concentrations at normal temperature and normal pressure, fluorescence lifetime attenuation curves under different concentrations are recorded, then a relation curve of each group of fluorescence lifetime and α -synuclein aggregate concentration is made, a linear range of 0-10 mu M is obtained, a fluorescence lifetime attenuation curve and a linear relation thereof (figure 2) are obtained, and figure 2 shows that the α -synuclein two-photon fluorescent probe prepared in the embodiment 2 of the invention reacts with different concentrations of Fe3+As can be seen from FIG. 2, the fluorescence lifetime curve under the condition of (0,2,4,6,8,10,12,14, 16. mu.M) has a fluorescence lifetime variation range of 136.4 ns-198.3 ns, indicating that the probe can be well used for fluorescence lifetime imaging analysis.
Example 6: multifunctional biological nano composite material for two-photon fluorescence lifetime imaging of neuroma blast (SHSY-5Y)
The above-mentioned materials containing different concentrations of Fe3+Or performing fluorescence lifetime imaging experiment on the neuroma blast of MG-132, wherein the experiment is divided into five groups including a control group and experiment groups B-F, and the control group does not contain Fe3+Adding Fe with different concentrations into the experimental groups B to F respectively3+(10, 20,30,40, 50. mu.M) or MG-132(10, 20,30,40, 50. mu.M). Adding 50 μ M multifunctional nanometer material into culture dish for 24h, co-culturing in incubator for 30min under 95% O2,5%CO2At 37 ℃. The samples were washed 3 times with PBS and fluorescence lifetime imaged on a Leica confocal set (TCS SP8) and finally lifetime fitted with SyPhotome-64 based on each set of imaging. The experimental results are shown in FIG. 3, in the first row, after the cells were co-incubated with different concentrations of iron ions (0,10,20,30,40,50 μ M) for 24 hours, 50 μ M of the composite nanomaterial was added, and the lifetime of the iron ion two-photon fluorescence probe was observed from the fluorescence lifetime imaging graphSecond row, after cells were incubated with different concentrations of MG-132(0, 10,20,30,40, 50. mu.M) for 24 hours, 50. mu.M of the composite nanomaterial was added, and the lifetime of α -synuclein two-photon fluorescent probe was gradually increased and then gradually decreased as observed from the fluorescence lifetime imaging graph, which indicated that α -synuclein aggregation also promoted the increase in the iron ion concentration in return, as can be seen from FIG. 3, the multifunctional bio-nanocomposite material could be well used for fluorescence lifetime imaging of cells.
By measuring fluorescence lifetime imaging and by the fluorescence lifetime change conditions of different regions in the single cell, the method can be used for detecting Fe in different regions in the single cell3+And α -study of changes in synuclein aggregates.
Example 7: multifunctional biological nano composite material for two-photon fluorescence imaging of parkinsonism mouse brain slice
Comparing the color corresponding to the two-photon spectrum with the color of the slice to judge the Fe in the parkinsonism mouse brain and the drug-treated mouse brain3+And α -synuclein aggregate content, and further analyzing the treatment effect of α -synuclein aggregate in the brain of the Parkinson disease mouse.
Injecting 50 mu M of the multifunctional biological nano composite material into a Parkinson model mouse brain, releasing a medicament for treatment for 10min through laser irradiation at 808nm, and then carrying out two-photon imaging detection on a Leica TCS SP8 by using two-photon at 800nm as exciting light on the obtained mouse brain slice.
The experimental result is shown in fig. 4, a fluorescence lifetime imaging graph of the first behavior treatment effect is shown, and it can be known from the graph that independent nano materials (column A) and nano materials +808nm laser irradiation (column B) have almost no influence on the concentration of iron ions and α -synuclein aggregates in a mouse brain, when the nano materials (column C) load drug rapamycin and enter the mouse brain, the fluorescence lifetime of an iron ion two-photon fluorescence probe is slightly increased, and the fluorescence lifetime of α -synuclein two-photon fluorescence is slightly reduced, so that the effect on treatment is proved to be certain, on the basis, a DNA nano gate is opened by 808nm laser irradiation (column D), a large amount of drug rapamycin is released to treat fibrous α -synuclein, and the concentration of iron ions is greatly reduced, so that the nano composite material is proved to have a good treatment effect on a Parkinson model mouse.
The tyrosine hydroxylase activity of fig. 4 (second row), nanomaterial alone (column a) and nanomaterial +808nm laser irradiation (column B) had little effect on tyrosine hydroxylase activity; when the nano material is loaded with rapamycin, the nano material has a certain promotion effect on the activity of tyrosine hydroxylase; on the basis, the DNA nanomesh is opened by laser irradiation (column D) at 808nm, and a large amount of rapamycin is released for treatment, so that the activity of tyrosine hydroxylase is greatly improved.
α -synuclein activity (third row) in FIG. 4, the effect of individual nano material (column A) and laser irradiation of nano material +808nm (column B) on α -synuclein activity is not great, when the nano material is loaded with drug rapamycin, the nano material has a certain promotion effect on α -synuclein activity, and on the basis, the DNA nanomesh is opened by laser irradiation of 808nm (column D) to release a large amount of drug rapamycin for treatment, so that the α -synuclein activity is greatly improved, and the treatment effect on fibrous α -synuclein is proved.
HE staining (fourth row) and Tunel staining (fifth row) of fig. 4, with more apoptosis in both nanomaterial alone (column a) and nanomaterial +808nm laser irradiation (column B); when the nano material is loaded with rapamycin, the apoptosis is reduced to some extent; on the basis, the DNA nano gate is opened by using 808nm laser irradiation (column D), and a large amount of drug rapamycin is released for treatment, so that the cell activity is greatly improved, and the results all prove that the nano composite material has a good treatment effect on the Parkinson model mouse.
Example 8: selectivity test
The iron ion and α -synuclein two-photon fluorescent probes prepared in examples 1 and 2 were reacted with common metal ions, proteins, etc. contained in organisms, and a selective experiment was performed.
50 μ M of the invention was carried outIron ion two-photon fluorescent probe prepared in example 1 and metal ion (1.0mM K)+,Na+,Ca2+,Mg2+;25M for Fe3+,Pb2+,Fe2+,Cu2+,Al3+and Cu+) (FIG. 5) after the reaction, the corresponding fluorescence lifetime values were obtained and plotted according to each set of curves (L)0-L)/L0FIG. 50. mu.M α -synuclein probe reacted with protein (50. mu.M Tau, GFAP, Sph1, TH, ERK2, MAP-1B, Ub, GRK5, VMAT2, α -Tub) to obtain the corresponding fluorescence lifetime values, which were plotted according to each set of curves (L)0-L)/L0Figure (a).
The iron ion two-photon fluorescent probe with the concentration of 50 mu M prepared in the embodiment 1 of the invention measures the fluorescence lifetime value in the range of pH 4.0-9.0, and draws a relation graph between different pH values and fluorescence lifetimes.
The iron ion two-photon fluorescent probe with the thickness of 50 mu M prepared in the embodiment 1 of the invention is measured to obtain the fluorescence lifetime value in the temperature range of 0-50OC, and a relation graph of different temperatures and fluorescence lifetimes is drawn.
The experimental results are as follows: the two fluorescent probes have good selectivity, pH and temperature stability, and can be well used for in-vivo imaging experiments.
Example 9: cytotoxicity assays
The cytotoxicity test of the multifunctional bio-nanocomposite prepared in example 3 of the present invention was performed using the MTT test. The SHSY-5Y cells in the 96-well plate are divided into 8 groups, namely a negative group, a positive group, a blank control group and an experimental group (total 8 groups), wherein the experimental group is the multifunctional biological nanocomposite material prepared in the embodiment 3 of the invention, and the concentrations are respectively 30, 60, 90 and 120 mu g/mL. After the 8 groups are co-cultured for 24h and 48h respectively, 20 mu L of MTT reagent is added, after reaction for 4h, the supernatant in 96 holes is removed, 80 mu L of DMSO is added for color reaction, and finally, ultraviolet measurement is carried out.
Wherein, the negative control group is added with triton, and the cell is completely lethal.
Wherein the multifunctional biological nanocomposite material prepared in the embodiment 3 of the invention is not added to the positive control group.
Wherein, the blank control is the multifunctional biological nano composite material prepared by the embodiment 3 without adding cells.
The experimental result is shown in fig. 6, and it can be seen from fig. 6 that the cell activity is greater than 90% under the conditions that the concentrations of the multifunctional biological nanocomposite material are 30, 60, 90, and 120 μ g/mL, respectively, indicating that the multifunctional biological nanocomposite material prepared in example 3 of the present invention has very low cytotoxicity, and can be well used for in vivo imaging experiments.
Example 10: t test method
the invention utilizes a T test method to prove the detection accuracy of the fluorescent probe prepared by the invention, for the iron ion two-photon fluorescent probe, the commercial probe rhodamine hydrazide is selected as a comparison method, when the iron ion concentration is respectively 20 mu M and 40 mu M, the fluorescence intensity of the two probes is respectively measured, three groups of parallel experiments are carried out to calculate the variance value, and finally the T value is calculated to be compared with a standard value, for the α -synuclein two-photon fluorescent probe, the commercial probe thioflavin T is selected as a comparison method, when the α -synuclein fibril concentration is respectively 2 mu M and 6 mu M, the fluorescence intensity of the two probes is respectively measured, three groups of parallel experiments are carried out to calculate the variance value, and finally the T value is calculated to be compared with the standard value.
The experimental results are shown in table 1, and it can be seen from table 1 that, according to the statistical calculation of the test (α ═ 0.05), the concentration of the synthesized iron ion two-photon fluorescent probe of the present invention is consistent with the concentration detected by the commercial probe rhodamine hydrazide, specifically, when the concentration of the iron ion of the present invention is 20 μ M, T ═ 0.32, when the concentration of the iron ion is 40 μ M, T ═ 0.47, which is much smaller than the standard value of 2.78, indicating that the measurement result of the present method is consistent with the measurement result of rhodamine hydrazide, furthermore, the present invention compares the synthesized α -synuclein two-photon fluorescent probe with the commercial measurement result of thioflavin T, specifically, when the concentration of the α -synuclein fibril of the present invention is 6 μ M, T ═ 1.09, and when the concentration of the α -synuclein fibril is 10 μ M, T ═ 1.08, which is much smaller than the standard value of 2.78, indicating that the measurement result of the present method is consistent with the measurement result of thioflavin T.
TABLE 1 test results of t test method
T test method
α=0.05,f=4
The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected.
SEQUENCE LISTING
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Claims (12)
1. A method for synthesizing a multifunctional biological nano composite material is characterized by comprising the following steps:
(1) synthetic iron ion two-photon fluorescent probe
In an alkali aqueous solution, carrying out reflux reaction on tetrabutylammonium hydrogen sulfate (TBAHS), 4-diethylaminobenzaldehyde and 2-amino-4, 6-dimethylpyrimidine to obtain an iron ion two-photon fluorescent probe shown as a formula (a), wherein the reaction process is shown as a reaction formula (I):
(2) synthesis of α -synuclein two-photon fluorescent probe
(2.1) in an organic solvent, carrying out reflux reaction on ruthenium trichloride, phenanthroline and lithium chloride to obtain bis (1, 10-phenanthroline) ruthenium dichloride Ru (phen)2Cl2;
(2.2) then in a mixed solution of methanol and water, bis (1, 10-phenanthroline) ruthenium dichloride Ru (phen)2Cl2And bipyridyl [3,2-A:2'.3' -C]And (3) carrying out reflux reaction on phenazine dppz to obtain α -synuclein two-photon fluorescent probe shown as a formula (b), wherein the reaction process is shown as a reaction formula (II):
(3) synthesis of multifunctional biological nano composite material
In a buffer solution, firstly, connecting the iron ion two-photon fluorescent probe synthesized in the step (1) to the surface of a copper sulfide nanosphere through an amido bond, then connecting the α -synuclein two-photon fluorescent probe synthesized in the step (2) to the surface of the copper sulfide nanosphere through electrostatic adsorption, and finally loading a drug into the copper sulfide nanosphere and sealing the hole with a DNA nano gate, thereby synthesizing the multifunctional biological nanocomposite.
2. The method according to claim 1, wherein in the step (1), the alkali is one or more of sodium hydroxide, potassium hydroxide and barium hydroxide; and/or the molar concentration ratio of tetrabutylammonium hydrogen sulfate, 4-diethylaminobenzaldehyde and 2-amino-4, 6-dimethylpyrimidine is (0.5-2): (20-25): (8-12).
3. The method of claim 1, wherein in step (1), the temperature of the reflux reaction is from 100 ℃ to 150 ℃.
4. The method according to claim 1, wherein in the step (2.1), the molar concentration ratio of the ruthenium trichloride to the phenanthroline to the lithium chloride is (10-20): (25-35): (0.5-1.5); and/or the temperature of the reflux reaction is 150-180 ℃.
5. The method according to claim 1, wherein in step (2.2), the bis (1, 10-phenanthroline) dichloride ruthenium Ru (phen)2Cl2And bipyridyl [3,2-A:2'.3' -C]The molar concentration ratio of phenazine dppz is (1-2): (1-2); and/or the temperature of the reflux reaction is 80-110 ℃.
6. The method according to claim 1, wherein in step (2.1), the organic solvent is one or both of N, N-dimethylformamide and dimethyl sulfoxide.
7. The method of claim 1, wherein in step (3), the concentration ratio of the iron ion two-photon fluorescent probe to the copper sulfide nanospheres is (0.5-1): 9-11) (M: g/L), and/or the concentration ratio of the α -synuclein two-photon fluorescent probe to the copper sulfide nanospheres is (0.5-1): 9-11 (M: g/L).
8. The method of claim 1, wherein in step (3), the buffer solution is a PBS buffer solution; and/or the pH of the buffer solution is 6.0-8.0; and/or the gene sequence of the DNA is selected from any one or more of SEQ ID NO. 1-3; and/or the medicament is one or more of rapamycin, cyclosporine and tacrolimus.
9. A multifunctional biological nanocomposite material prepared by the method of any one of claims 1 to 8.
10. The multifunctional biological nanocomposite material of claim 9 for detecting Fe by two-photon fluorescence lifetime imaging in vitro or in cells3+And α -synuclein aggregates.
11. The multifunctional biological nanocomposite material of claim 9, for use in detecting α -synuclein aggregates by two-photon fluorescence imaging for treating parkinson's disease.
12. The multifunctional biological nanocomposite material according to claim 9 for use in the treatment and diagnosis of parkinson's disease, alzheimer's disease, synucleinopathies.
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112870377A (en) * | 2021-01-29 | 2021-06-01 | 中南大学湘雅医院 | Composite nanoparticle for tumor photothermal and photodynamic cooperative treatment and preparation method thereof |
CN112870377B (en) * | 2021-01-29 | 2022-09-16 | 中南大学湘雅医院 | Composite nanoparticle for tumor photothermal and photodynamic cooperative treatment and preparation method thereof |
CN115266663A (en) * | 2022-06-30 | 2022-11-01 | 四川省医学科学院·四川省人民医院 | Biological probe for non-invasive diagnosis of Parkinson's disease triggered by intestinal microenvironment as well as preparation method and application of biological probe |
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