CN113214822A - AIE nanofiber probe for targeted staining of cell membrane and preparation method thereof - Google Patents

AIE nanofiber probe for targeted staining of cell membrane and preparation method thereof Download PDF

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CN113214822A
CN113214822A CN202110390197.4A CN202110390197A CN113214822A CN 113214822 A CN113214822 A CN 113214822A CN 202110390197 A CN202110390197 A CN 202110390197A CN 113214822 A CN113214822 A CN 113214822A
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nanofiber
aie
probe
dspe
peg
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CN113214822B (en
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王琳
郭锟忠
高蒙
陈军建
卞钲琪
薛永业
周海艳
石志锋
张敏杰
胡杨
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South China University of Technology SCUT
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Abstract

The invention discloses an AIE nanofiber probe for targeted staining of cell membranes and a preparation method thereof. The method comprises the following steps: using amphiphilic molecules DSPE-PEG2000-Mal and DSPE-PEG2000Wrapping fluorescent molecule DTPM with aggregation-induced emission characteristics to prepare nano fibers; and then, performing addition reaction on maleic anhydride (-Mal) on the surface of the nanofiber and a sulfhydryl (-SH) reaction group on cysteine (Cys) in a polypeptide sequence, introducing a flexible chain bioactive polypeptide chain with a targeting function on the nanofiber, and finally preparing the AIE nanofiber with positive charges and a hydrophilic and hydrophobic structure surface, wherein the targeting is realized through the charges of the polypeptide and the hydrophilic and hydrophobic structure. The method realizes targeting of AIE nanofiber to cell membrane staining, and has high resolution, no elution, and high staining speedAnd the performance is stable, and the like, and is expected to be a novel cell membrane fluorescent probe.

Description

AIE nanofiber probe for targeted staining of cell membrane and preparation method thereof
Technical Field
The invention relates to a fluorescent probe capable of staining cell membranes in a targeted manner, in particular to an AIE nanofiber probe capable of staining cell membranes in a targeted manner and a preparation method thereof.
Background
The cell membrane is a channel for information and substance exchange between cells and the surrounding environment, and the shape and the form of the cell membrane are continuously and dynamically changed, so that the cell membrane is an important structure for completing normal physiological activities of cells such as endocytosis, exocytosis, signal transmission and the like. The morphology of the cell membrane is a direct indicator of the state of the cell, for example, cell membrane rupture or partial phagocytosis is important evidence of apoptosis. In addition, the cell membrane serves as a first protective barrier for the cell, and once damaged by external harmful substances (such as heavy metal ions), changes in form and structure occur. Therefore, the real-time tracking and observation of the cell membrane has important values on cell biology, pharmacology, toxicology and the like. There are reports of cell membrane observation using bright field microscope, but these results have limited help for cell membrane-related observation studies due to low resolution, insignificant color discrimination, and the like. Fluorescence microscopy is a powerful, reliable method that provides intuitive, vivid information. However, this technique is highly dependent on the performance of the fluorescent probe.
Most of the traditional fluorescent probes used at present have aggregation-induced quenching (ACQ) effect, and the problems of low signal quantity, poor sensitivity, easy photobleaching and the like of biological detection exist in the using process. The problem is solved well by the discovery of aggregation-induced emission (AIE) materials, stronger fluorescence can be excited in an aggregation state, and the formed AIE aggregates have excellent light stability and photobleaching resistance and can realize long-term stable fluorescence tracking and monitoring. Nevertheless, since AIE molecules tend to have large molecular weights and strong structural hydrophobicity, their hydrophilicity and functional modification are difficult, and the modification may adversely affect their photophysical properties. Therefore, how to realize modification and functionalization of an AIE molecule in a simple manner is a problem that many researchers are studying, and packaging a nanostructure is a good way to solve the problem at present, but a problem still exists how to modify the nanostructure to realize a specific function, and a related fluorescent probe is in urgent need of development.
In recent years, many researchers are dedicated to the research of targeting AIE nano fluorescent probes, but the probes often have the problems of difficult targeting, complex preparation mode, poor light stability, difficulty in realizing long-term marking and tracking, toxicity to normal cells and tissues and the like, and the problems need to be solved urgently. For example, patent CN 110231316A discloses an AIE-Gd @ SiO2The nano composite fluorescent probe is prepared by mixing AIE fluorescent material and Gd @ SiO2The compound is combined to prepare the nano probe for targeted labeling of stem cells, but the nano probe still has the problems of certain cytotoxicity, long time for staining and labeling and the like.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the AIE fluorescent probe which has the functions of targeting cell membranes, simple preparation method, excellent light stability, long-term fluorescent labeling and good biocompatibility.
The invention aims to prepare an AIE nanofiber fluorescent probe capable of staining cell membranes in a targeted mode, and long-acting stable staining and observation of the cell membranes are achieved.
Another object of the present invention is to provide a method for preparing the above AIE nanofibers capable of targeting staining cell membranes.
The purpose of the invention is realized by at least one of the following technical solutions.
The preparation method of the AIE nanofiber probe for targeted staining of cell membranes, provided by the invention, comprises the following steps:
(1) mixing DTPM solution in Tetrahydrofuran (THF), DSPE-PEG2000-Mal in tetrahydrofuran solution, DSPE-PEG2000The tetrahydrofuran solution is evenly mixed to obtain a mixed solution, and the tetrahydrofuran is removed by rotary evaporation (the mixed solution is placed in a rotary evaporation bottle for rotary drying), so that a mixture is obtained;
(2) adding the mixture obtained in the step (1) into ultrapure water, carrying out ultrasonic oscillation treatment to obtain a dispersion liquid, and filtering to obtain nano fibers;
(3) re-suspending the nanofibers obtained in the step (2) in water to obtain a nanofiber dispersion liquid; adding bioactive polypeptide into the nanofiber dispersion liquid to obtain a mixed liquid, stirring for reaction, filtering and taking filter residue to obtain the AIE nanofiber probe for the target dyed cell membrane.
Further, the structural formula of the DTPM in the step (1) is shown as
Figure BDA0003016463420000031
The DTPM is English abbreviation of 2- {3- [5- (4-Diphenylamino-phenyl) -thiophen-2-yl ] -1-phenyl-allylidene } -Malononitrile.
Further, said DSPE-PEG of step (1)2000-Mal has the formula shown below:
Figure BDA0003016463420000032
further, said DSPE-PEG of step (1)2000The structural formula of (A) is as follows:
Figure BDA0003016463420000033
further, in the mixture in the step (1), the concentration of DTPM is 1-15 mg/mL;
further, in the mixture of step (1), DSPE-PEG2000-Mal concentration of 1-20 mg/mL;
further, in the mixture of step (1), DSPE-PEG2000The concentration of (b) is 1-25 mg/mL.
Further, the mass-to-volume ratio of the mixture in the step (2) to water is 0.1-1 mg/mL.
Preferably, the mass-to-volume ratio of the mixture in the step (2) to the water is 0.5-1 mg/mL.
Further preferably, the mass-to-volume ratio of the mixture of step (2) to water is 1 mg/mL.
Further, the time of the ultrasonic oscillation treatment in the step (2) is 1-3 min; the molecular weight of the nanofiber is 10000-5000000;
further, the length distribution of the nano-fibers in the step (2) is 0.5-10 μm, and the width distribution of the nano-fibers is 10-100 nm.
Preferably, the time of the ultrasonic oscillation treatment in the step (2) is 1 min.
Preferably, the filtering of step (2) comprises: firstly filtering the dispersion liquid with a filter head (the filtering molecular weight is 0-5000000), taking the filtrate, then passing the filtrate through an ultrafiltration tube (the filtering molecular weight is 0-10000), and taking the filter residue, namely the nano-fiber.
Further, in the nanofiber dispersion liquid in the step (3), the concentration of the nanofibers is 200-800. mu.g/mL.
Further, the bioactive polypeptide in the step (3) is a flexible chain bioactive polypeptide with a sulfhydryl group connected to an amino terminal; the sequence of the flexible chain bioactive polypeptide is (Lys)a-(Arg)b-(Trp)c-(Lys)d-(Arg)e-(Trp)f-(Lys)g-(Arg)hThe value range of a is 0-2; b ofThe value range is 0-2, the value range of c is 0-3, the value range of d is 0-2, the value range of e is 0-2, the value range of f is 0-3, the value range of g is 0-2, and the value range of h is 0-2;
further, in the mixed solution in the step (3), the concentration of the bioactive polypeptide is 1-30mg/mL,
further, the stirring reaction time of the step (3) is 0.5-48 h. The molecular formula of the flexible chain bioactive polypeptide can be shown in figure 4.
The flexible chain bioactive polypeptide with the amino terminal connected with a sulfhydryl group (-SH) is synthesized by a solid phase synthesis method and consists of arginine (Arg) with positive charge, lysine (Lys) and tryptophan (Trp) with hydrophobic group, and the sequence of the flexible chain bioactive polypeptide is (Lys)a-(Arg)b-(Trp)c-(Lys)d-(Arg)e-(Trp)f-(Lys)g-(Arg)h. Meanwhile, the amino end of the compound is connected with cysteine (Cys) through polyethylene glycol, so that a sulfhydryl (-SH) reaction group is introduced. The reaction is realized by the addition reaction of maleic anhydride groups (-Mal) and mercapto groups (-SH) on the surface of the nanofiber. The number n of polyethylene glycol repeating units connected by the polypeptide ranges from 4 to 24. The number m of cysteines ranged from 1.
Preferably, the filtering of step (3) comprises: and (3) carrying out centrifugal filtration on the mixed solution after the stirring reaction by adopting an ultrafiltration tube (the filtration molecular weight is 0-10000), wherein the rotating speed of the centrifugal filtration is 3000-8000rpm, and the time of the centrifugal filtration is 10-30min, and taking filter residue (namely the AIE nano fiber of the targeted staining cell membrane). The molecular weight of the AIE nanofiber targeting the stained cell membrane is 10000-1000000.
The AIE nanofiber probe for targeted staining of cell membranes, which is prepared by the preparation method, has the length of 0.5-10 mu m and the width of 10-100 nm.
The AIE nano-fiber probe for targeted staining of cell membranes provided by the invention is prepared by using AIE molecules DTPM with aggregation-induced emission characteristics and DSPE-PEG2000-Mal and DSPE-PEG2000Coated and grafted with targeting flexible chain bioactive polypeptide,a schematic diagram of which can be seen with reference to fig. 1.
The targeting of the AIE nanofiber for targeting and staining cell membranes is realized by the charge quantity and the hydrophilic and hydrophobic structures of surface polypeptides.
The preparation method provided by the invention utilizes amphiphilic molecule DSPE-PEG2000-Mal and DSPE-PEG2000Wrapping fluorescent molecule DTPM with aggregation-induced emission characteristics to prepare nano fibers; and then, performing addition reaction on maleic anhydride (-Mal) on the surface of the nanofiber and a sulfhydryl (-SH) reaction group on cysteine (Cys) in a polypeptide sequence, introducing a flexible chain bioactive polypeptide chain with a targeting function on the nanofiber, and finally preparing the AIE nanofiber with positive charges and a hydrophilic and hydrophobic structure surface, wherein the targeting is realized through the charges of the polypeptide and the hydrophilic and hydrophobic structure. The method realizes the targeting of AIE nano fiber to cell membrane dyeing, has the advantages of high resolution, no elution, high dyeing speed, stable performance and the like, and is expected to be used as a novel cell membrane fluorescent probe.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the AIE nanofiber probe for targeting dyeing of cell membranes, which is prepared by the invention, can target dyeing of cell membranes and has excellent photostability and photobleaching resistance;
(2) the AIE nanofiber probe for targeted staining of cell membranes, which is prepared by the invention, can be stably gathered on the cell membranes of cells for a long time, and bright fluorescence can be observed after the AIE nanofiber probe is co-cultured with the cells for 24 hours;
(3) the AIE nanofiber probe for targeted staining of cell membranes, which is prepared by the invention, has the function of rapidly and stably staining the cell membranes, is low in fluorescence background, does not need to be additionally eluted, and is simple and efficient in staining process.
(4) The AIE nanofiber probe for targeted staining of cell membranes prepared by the invention has excellent biocompatibility, and the influence on cells in a certain concentration range (0-80 mu g/mL) can be ignored.
Drawings
FIG. 1 is a schematic structural view of the AIE nanofibers targeting a stained cell membrane prepared in the example;
FIG. 2 is a structural formula of a DTPM molecule used in examples;
FIGS. 3a and 3b are respectively DSPE-PEG used in the examples2000-Mal and DSPE-PEG2000The structural formula (1);
FIG. 4 is a structural formula of a biologically active polypeptide used in the examples;
FIG. 5 is a Transmission Electron Microscope (TEM) picture of the AIE nanofibers targeting the stained cell membrane prepared in example 1;
FIG. 6 is the photostability of the AIE nanofibers targeting a stained cell membrane prepared in example 1 after 0, 5, 10min of continuous light exposure;
FIG. 7 is the fluorescence results of the AIE nanofibers targeting the stained cell membrane prepared in example 1 after incubation with HeLa cells for 5 min;
FIG. 8 is a graph showing the results of fluorescence after incubation of the AIE nanofibers targeting the stained cell membrane prepared in example 1 with HeLa cells for 24 hours;
FIG. 9 is a graph representing the biocompatibility of the AIE nanofibers targeting a stained cell membrane prepared in example 1;
FIG. 10 is a TEM picture of the AIE nanofibers targeting the stained cell membrane prepared in example 2;
FIG. 11 is a graph showing the results of fluorescence after incubation of the AIE nanofibers targeting a stained cell membrane prepared in example 2 with HeLa cells for 5 min;
FIG. 12 is a TEM picture of the AIE nanofibers targeting the stained cell membrane prepared in example 3;
FIG. 13 is a graph showing the results of fluorescence after incubation of the AIE nanofibers targeting the stained cell membrane prepared in example 3 with HeLa cells for 5 min;
FIG. 14 is a TEM picture of the nanoprobe prepared in comparative example 1;
FIG. 15 is a TEM picture of AIE nanofibers prepared in comparative example 2;
FIG. 16 is a graph showing the results of fluorescence obtained after incubating the AIE nanofibers prepared in comparative example 2 with HeLa cells for 5 min;
FIG. 17 is a TEM picture of AIE nanofibers prepared in comparative example 3;
FIG. 18 is a graph showing the results of fluorescence of AIE nanofibers prepared in comparative example 3 after incubation with HeLa cells for 5 min.
Detailed Description
The following examples are presented to further illustrate the practice of the invention, but the practice and protection of the invention is not limited thereto. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.
The structural formula of DTPM molecules used in the following examples is shown in FIG. 2, and DSPE-PEG used therein2000The structural formula of-Mal can be shown in FIG. 3a, and the DSPE-PEG used2000The structural formula of the polypeptide can be shown in FIG. 3b, and the structural formula of the biologically active polypeptide can be shown in FIG. 4.
Example 1
A preparation method of an AIE nanofiber probe for targeted staining of cell membranes comprises the following steps:
(1) mixing DTPM molecule and DSPE-PEG2000-Mal and DSPE-PEG2000Mixing the solution of (A) with the solution of (B) to obtain a mixture, and adding DTPM and DSPE-PEG to the mixture2000-Mal、DSPE-PEG2000The concentrations are 1mg/mL, 1mg/mL and 1mg/mL respectively;
(2) placing the mixed solution obtained in the step (1) into a rotary evaporation bottle for spin-drying (removing THF), adding ultrapure water, wherein the mass-volume ratio of the mixture to the ultrapure water is 1mg/mL, and performing ultrasonic oscillation for 1min to obtain a dispersion liquid;
(3) filtering the nanofiber by using a filter head filtering technology with the filter head specification of 450nm, and taking a filtrate (marked as a first filtrate); then, filtering the first filtrate by using an ultrafiltration tube centrifugation (filtration molecular weight is 0-10000) filtering technology, wherein the centrifugation speed is 8000rpm, and the centrifugation time is 30 min; the obtained nano-fiber has the length distribution of 2.53 +/-1.76 mu m and the width range of 22 +/-12 nm; resuspending the nanofibers in water to obtain a nanofiber dispersion (2 mg/mL);
(4) adding bioactive polypeptide into the prepared nanofiber dispersion liquid to obtain a mixed liquid, wherein the concentration of the bioactive polypeptide in the mixed liquid is 5 mg/mL; the bioactive polypeptide is a flexible chain bioactive polypeptide with an amino end connected with a sulfhydryl group; the sequence of the flexible chain bioactive polypeptide (marked as polypeptide 1) is shown in SEQ ID NO.1 and is Lys-Arg-Trp-Trp-Lys-Trp-Trp-Arg-Arg, the amino terminal of the flexible chain bioactive polypeptide is connected with cysteine (Cys) through polyethylene glycol so as to be connected with sulfydryl (-SH), the number of the repeating units of the polyethylene glycol at the amino terminal is 4, and the number of the cysteine is 1;
(5) and (3) stirring the mixed solution obtained in the step (4) for reaction (the time is 24h), then filtering the mixed solution by adopting an ultrafiltration tube (the filtering molecular weight is 0-10000) centrifugal filtering technology, wherein the centrifugation speed is 8000rpm, the centrifugation time is 30min, and taking filter residues to obtain the AIE nanofiber probe for the target dyed cell membrane, wherein the nanofiber length distribution of the AIE nanofiber probe for the target dyed cell membrane is 2.53 +/-1.76 mu m, and the width range is 22 +/-12 nm.
The appearance of the AIE nanofiber probe targeting to stain the cell membrane was observed by using a transmission electron microscope, and the result is shown in fig. 5, and it can be known from fig. 5 that the material prepared in this example is a fibrous nanostructure; the ultraviolet spectrophotometer is used for measuring the light stability of the nano-fiber, and the result is shown in fig. 6, and fig. 6 shows that the nano-fiber has excellent light stability under the continuous illumination for 10 min; the prepared AIE nanofiber probe for targeting staining of cell membranes is used for carrying out fluorescence labeling on HeLa cells, the concentration of AIE nanofibers is 10 mug/mL, the labeling time is 5min, and then observation is carried out by using a laser confocal microscope. As shown in fig. 7, it can be seen that the nanofibers prepared in this example have rapid and excellent cell staining effect, fluorescent-labeled cell membranes can be observed under a confocal microscope after 5min of reaction, and background fluorescence is weak without elution, which does not affect the fluorescent observation of the cell membranes; the AIE nano-fiber and the HeLa cell are continuously co-cultured, and the fluorescence imaging condition is observed after 24 hours, and the result is shown in figure 8, and the cell membrane still shows bright fluorescence effect; the AIE nano-fiber with the concentration range of 80 mu g/mL and mouse fibroblast NIH3T3 are co-cultured, and the CCK-8 dye is used for representing the cell activity after 24 hours, so that the result is shown in figure 9, the cell activity is good, the normal cells are not obviously influenced in activity at high concentration, and the AIE nano-fiber is expected to be used as a novel cell membrane staining fluorescent probe for relevant application.
Example 2
A preparation method of an AIE nanofiber probe for targeted staining of cell membranes comprises the following steps:
(1) mixing DTPM molecule and DSPE-PEG2000-Mal and DSPE-PEG2000Mixing the solution of (A) with the solution of (B) to obtain a mixture, and adding DTPM and DSPE-PEG to the mixture2000-Mal、DSPE-PEG2000The concentrations are respectively 15mg/mL, 20mg/mL and 25 mg/mL;
(2) placing the mixed solution obtained in the step (1) in a rotary evaporation bottle for spin-drying (removing THF) to obtain a mixture, adding ultrapure water, and performing ultrasonic oscillation for 3min to obtain a dispersion liquid, wherein the mass-volume ratio of the mixture to the ultrapure water is 0.5 mg/mL;
(3) filtering the nano-fiber by using a filter head filtering technology with the filter head specification of 800nm, and taking a filtrate (marked as a first filtrate); then, filtering the primary filtrate by using an ultrafiltration tube (the filtration molecular weight is 0-5000) centrifugal filtration technology, wherein the centrifugal speed is 3000rpm, the centrifugal time is 20min, and taking filter residues to obtain nano fibers; the obtained nano-fiber has the length distribution of 5.2 +/-4.32 mu m and the width range of 55 +/-43 nm; resuspending the nanofibers in water to obtain a nanofiber dispersion (1 mg/mL);
(4) adding bioactive polypeptide into the prepared nanofiber dispersion liquid to obtain a mixed liquid, wherein the concentration of the polypeptide in the mixed liquid is 20 mg/mL; wherein the bioactive polypeptide is a flexible chain bioactive polypeptide with an amino end connected with a sulfhydryl group; the sequence of the flexible chain bioactive polypeptide (marked as polypeptide 2) is shown in SEQ ID NO.2, and is Lys-Lys-Trp-Trp-Trp-Arg-Arg-Lys-Lys-Arg-Arg, the amino end of the flexible chain bioactive polypeptide is connected with cysteine (Cys) through polyethylene glycol so as to be connected with sulfydryl (-SH), the number of the repeating units of the polyethylene glycol is 10, and the number of the cysteine is 1;
(5) and (3) stirring the mixed solution obtained in the step (4) for reaction (the time is 24h), then filtering the mixed solution by adopting an ultrafiltration tube (the filtering molecular weight is 0-5000) centrifugal filtration technology, wherein the centrifugation speed is 3000rpm, the centrifugation time is 20min, and taking filter residues to obtain the AIE nanofiber probe for the targeted stained cell membrane, wherein the length distribution of the AIE nanofiber probe for the targeted stained cell membrane is 5.2 +/-4.32 mu m, and the width range is 55 +/-43 nm.
The morphology of the AIE nanofiber probe targeting the stained cell membrane was observed by a transmission electron microscope, and as a result, as shown in fig. 10, it can be seen from fig. 10 that the material prepared in this example is a fibrous nanostructure. The light stability of the composition is excellent when measured by an ultraviolet spectrophotometer. The prepared AIE nanofiber probe for targeting staining of cell membranes is used for carrying out fluorescence labeling on HeLa cells, the concentration of AIE nanofibers is 10 mug/mL, the labeling time is 5min, and then observation is carried out by using a laser confocal microscope. As shown in fig. 11, it can be seen that the nanofiber prepared in this example can effectively target cell membranes, and a significant fluorescence signal can be observed at the cell membrane position under the confocal condition, but due to the large size of the nanofiber prepared in this example, a large amount of DTPM molecules are aggregated, resulting in high background fluorescence, which seriously affects the fluorescence observation of the cell membranes.
Example 3
A preparation method of an AIE nanofiber probe for targeted staining of cell membranes comprises the following steps:
(1) mixing DTPM molecule and DSPE-PEG2000-Mal and DSPE-PEG2000Mixing the solution of (A) with the solution of (B) to obtain a mixture, and adding DTPM and DSPE-PEG to the mixture2000-Mal、DSPE-PEG2000The concentrations are respectively 10mg/mL, 18mg/mL and 22 mg/mL;
(2) placing the mixed solution obtained in the step (1) into a rotary evaporation bottle for spin-drying (removing THF), adding ultrapure water, wherein the mass-volume ratio of the mixture to the ultrapure water is 1mg/mL, and performing ultrasonic oscillation for 3min to obtain a dispersion solution;
(3) filtering the nanofiber by using a filter head filtering technology with the filter head specification of 450nm, and taking a filtrate (marked as a first filtrate); then, an ultrafiltration tube (the filtration molecular weight is 0-10000) centrifugal filtration technology is utilized, the parameter is set to be 8000rpm/30min, the primary filtrate is filtered, and filter residue is obtained, so that the nano-fiber is obtained; the obtained nano-fiber has the length distribution of 2.65 +/-1.46 mu m and the width range of 25 +/-14 nm; resuspending the nanofibers in water to give a nanofiber dispersion (0.5 mg/mL);
(4) adding bioactive polypeptide into the prepared nanofiber dispersion liquid to obtain a mixed liquid, wherein the concentration of the bioactive polypeptide in the mixed liquid is 30 mg/mL;
wherein the bioactive polypeptide is a flexible chain bioactive polypeptide with an amino end connected with a sulfhydryl group; the sequence of the flexible chain bioactive polypeptide (marked as polypeptide 3) is shown in SEQ ID NO.3 and is Arg-Arg-Trp-Lys-Lys-Trp-Trp-Trp-Lys-Lys; the amino end of the flexible chain bioactive polypeptide is connected with cysteine (Cys) through polyethylene glycol so as to be further connected with sulfydryl (-SH), the number of repeating units of the polyethylene glycol is 24, and the number of the cysteine is 1;
(5) stirring the mixed solution obtained in the step (4) for reaction (the time is 48h), and then filtering the mixed solution by adopting an ultrafiltration tube (the filtering molecular weight is 0-10000) centrifugal filtering technology, wherein the experimental parameters of the filtering are 8000rpm/30 min; and taking filter residues to obtain the AIE nanofiber probe for the targeted staining of the cell membrane, wherein the length distribution of the AIE nanofiber probe for the targeted staining of the cell membrane is 2.45 +/-1.42 mu m, and the width range of the AIE nanofiber probe for the targeted staining of the cell membrane is 22 +/-12 nm.
The morphology of the AIE nanofiber probe targeting the stained cell membrane was observed by a transmission electron microscope, and as a result, as shown in fig. 12, it can be seen from fig. 12 that the material prepared in this example is a fibrous nanostructure. The light stability of the composition is excellent when measured by an ultraviolet spectrophotometer. The prepared AIE nano-fiber is used for carrying out fluorescence labeling on the HeLa cell, the concentration of the AIE nano-fiber is 20 mug/mL, and after reaction for 5min, observation is carried out by using a laser confocal microscope. As shown in fig. 13, it can be seen that the nanofibers prepared in this example can achieve targeted staining of cell membrane, but the fluorescence observed in confocal after incubating with cells for 5min is weak, and only a small amount of nanofibers stain cell membrane, because the modified bioactive polypeptide is not the optimal targeting cell membrane sequence.
Comparative example 1
A preparation method of a nanoprobe comprises the following steps:
(1) mixing DTPM molecule and DSPE-PEG2000-Mal is dissolved in THF to obtain a mixture in which DTPM molecules and DSPE-PEG are present2000-Mal concentrations of 7mg/mL and 20mg/mL respectively, placing in a rotary evaporation bottle for spin-drying (removing THF) to obtain a mixture, adding ultrapure water, wherein the mass-to-volume ratio of the mixture to the ultrapure water is 0.8mg/mL, and performing ultrasonic oscillation for 2min to obtain a dispersion;
(2) filtering the nano-fiber by using a filter head filtering technology with the filter head specification of 220nm, and taking a filtrate (marked as a first filtrate); and then, filtering the primary filtrate by using an ultrafiltration tube (the filtration molecular weight is 0-10000) centrifugal filtration technology with the parameter set to be 5000rpm/10min, and taking filter residues to obtain the nano fibers.
The morphology of the nanofibers prepared in this example was characterized by a scanning electron microscope, and the results are shown in fig. 14, which indicates that no polymer structure was formed during the experimental operation, which may be due to the absence of DSPE-PEG2000 added to the raw material, and that no further experiments could be performed (the DTPM molecules of the dispersion could not pass through the DSPE-PEG molecules)2000Maleic anhydride groups of Mal grafted biologically active polypeptides and therefore do not meet the requirements of cell membrane targeted staining experiments).
Comparative example 2
A nanofiber for binding polypeptide is prepared by the following steps:
(1) mixing DTPM molecule and DSPE-PEG2000-Mal and DSPE-PEG2000Mixing the solution of (A) with the solution of (B) to obtain a mixture, and adding DTPM and DSPE-PEG to the mixture2000-Mal、DSPE-PEG2000The concentrations are respectively 5mg/mL, 15mg/mL and 20 mg/mL;
(2) placing the mixed solution obtained in the step (1) into a rotary evaporation bottle for spin-drying (removing THF), wherein the mass-volume ratio of the mixture to ultrapure water is 0.5mg/mL, and performing ultrasonic oscillation for 2.5min to obtain a dispersion liquid;
(3) filtering the nano-fiber by using a filter head filtering technology with the filter head specification of 220nm, and taking a filtrate (marked as a first filtrate); then, an ultrafiltration tube (the filtration molecular weight is 0-5000) centrifugal filtration technology is utilized, the parameter is set to be 6000rpm/15min, and the primary filtrate is filtered to obtain nano fibers; the obtained nano-fiber has the length distribution of 1.54 +/-0.86 mu m and the width range of 18 +/-5 nm; resuspending the nanofibers in water to obtain a nanofiber dispersion (1 mg/mL);
(4) adding bioactive polypeptide into the prepared nanofiber dispersion liquid to obtain a mixed liquid, wherein the concentration of the bioactive polypeptide in the mixed liquid is 30 mg/mL; wherein the bioactive polypeptide is a flexible chain bioactive polypeptide with an amino end connected with a sulfhydryl group; the sequence of the flexible chain bioactive polypeptide (marked as polypeptide 1) is shown in SEQ ID NO.1 and is Lys-Arg-Trp-Trp-Lys-Trp-Trp-Arg-Arg, the amino terminal of the flexible chain bioactive polypeptide is connected with polyethylene glycol, the number of repeating units of the polyethylene glycol is 20, but the polyethylene glycol is not connected with cysteine (Cys), the number of the cysteine is 0, and the polyethylene glycol is not connected with sulfydryl (-SH);
(5) stirring the mixed solution obtained in the step (4) for reaction (the time is 0.5h), and then filtering the mixed solution by adopting an ultrafiltration tube (the filtering molecular weight is 0-5000) centrifugal filtration technology, wherein the experimental parameters of filtration are 6000rpm/10 min; and (3) taking filter residues to obtain the AIE nanofiber probe for the targeted staining of the cell membrane, wherein the length distribution of the AIE nanofiber probe for the targeted staining of the cell membrane is 1.46 +/-1.33 mu m, and the width range of the AIE nanofiber probe for the targeted staining of the cell membrane is 18 +/-4 nm.
The morphology of the AIE nanofiber probe targeting the stained cell membrane was observed by a transmission electron microscope, and as a result, as shown in fig. 15, it was found from fig. 15 that the material prepared in this comparative example was a fibrous nanostructure. The light stability of the composition is excellent when measured by an ultraviolet spectrophotometer. The prepared AIE nano-fiber is used for carrying out fluorescence labeling on the HeLa cell, the concentration of the AIE nano-fiber is 10 mug/mL, and after reaction for 5min, observation is carried out by using a laser confocal microscope. As a result, as shown in fig. 16, it can be found that the nanofibers prepared in this comparative example did not achieve targeted staining of cell membranes, and fluorescence could not be observed in confocal, because there was no group on the polypeptide chain that reacted with the nanofibers to graft, and thus the polypeptide was not grafted to the nanofibers, and thus there was no targeting to cell membranes.
Comparative example 3
A preparation method of a fiber probe comprises the following steps:
(1) mixing DTPM molecule and DSPE-PEG2000-Mal and DSPE-PEG2000Mixing the solution of (A) with the solution of (B) to obtain a mixture, and adding DTPM and DSPE-PEG to the mixture2000-Mal、DSPE-PEG2000The concentrations are 1mg/mL, 1mg/mL and 1mg/mL respectively;
(2) placing the mixed solution obtained in the step (1) into a rotary evaporation bottle for spin-drying (removing THF), wherein the mass-volume ratio of the mixture to ultrapure water is 0.6mg/mL, and performing ultrasonic oscillation (for 3min) to obtain a dispersion liquid;
(3) filtering the nanofiber by using a filter head filtering technology with the filter head specification of 450nm, and taking a filtrate (marked as a first filtrate); then, an ultrafiltration tube (the filtration molecular weight is 0-10000) centrifugal filtration technology is utilized, the parameter is set to be 8000rpm/15min, the primary filtrate is filtered, and filter residue is obtained, so that the nano-fiber is obtained; the obtained nano-fiber has the length distribution of 2.71 +/-1.52 mu m and the width range of 27 +/-15 nm; resuspending the nanofibers in water to obtain a nanofiber dispersion (2 mg/mL);
(4) this example did not graft biologically active polypeptide.
The morphology of the AIE nanofiber probe targeting the stained cell membrane was observed by a transmission electron microscope, and as a result, as shown in fig. 17, it can be seen from fig. 17 that the material prepared in this comparative example has a fibrous nanostructure. The light stability of the composition is excellent when measured by an ultraviolet spectrophotometer. The prepared AIE nano-fiber is used for carrying out fluorescence labeling on the HeLa cell, the concentration of the AIE nano-fiber is 10 mug/mL, and after reaction for 5min, observation is carried out by using a laser confocal microscope. As a result, as shown in fig. 18, it was found that the nanofibers prepared in this comparative example did not achieve targeted staining of cell membranes, and fluorescence could not be observed in confocal, because the AIE nanofibers lacked surface targeting polypeptides and thus failed to emit luminescence by aggregation at the cell membrane site.
The above examples are only preferred embodiments of the present invention, which are intended to be illustrative and not limiting, and those skilled in the art should understand that they can make various changes, substitutions and alterations without departing from the spirit and scope of the invention.
Sequence listing
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Claims (10)

1. A preparation method of an AIE nanofiber probe for targeted staining of cell membranes is characterized by comprising the following steps:
(1) mixing DTPM solution in tetrahydrofuran, DSPE-PEG2000-Mal in tetrahydrofuran solution, DSPE-PEG2000The tetrahydrofuran solution is evenly mixed to obtain a mixed solution, and the mixed solution is dried in a spinning mode to obtain a mixture;
(2) adding the mixture obtained in the step (1) into water, carrying out ultrasonic oscillation treatment to obtain a dispersion liquid, and filtering to obtain nano fibers;
(3) re-suspending the nanofibers obtained in the step (2) in water to obtain a nanofiber dispersion liquid; adding bioactive polypeptide into the nanofiber dispersion liquid to obtain a mixed liquid, stirring for reaction, filtering and taking filter residue to obtain the AIE nanofiber probe for the target dyed cell membrane.
2. The method for preparing the AIE nanofiber probe for targeted staining of cell membranes according to claim 1, wherein the DTPM of step (1) has a structural formula of
Figure FDA0003016463410000011
3. The method for preparing AIE nanofiber probe for targeted staining of cell membrane according to claim 1, wherein the DSPE-PEG of step (1)2000-Mal has the formula shown below:
Figure FDA0003016463410000012
4. the method for preparing AIE nanofiber probe for targeted staining of cell membrane according to claim 1, wherein the DSPE-PEG of step (1)2000The structural formula of (A) is as follows:
Figure FDA0003016463410000021
5. the method for preparing AIE nano fiber for targeted staining of cell membranes according to claim 1, wherein the concentration of DTPM is 1-15mg/mL and DSPE-PEG is added to the mixture of step (1)2000-Mal concentration of 1-20mg/mL, DSPE-PEG2000The concentration of (b) is 1-25 mg/mL.
6. The method for preparing the AIE nanofiber probe for targeted staining of cell membranes according to claim 1, wherein the mass-to-volume ratio of the mixture of the step (2) to water is 0.5-1 mg/mL.
7. The method for preparing the AIE nanofiber probe for targeted staining of cell membranes according to claim 1, wherein the time of the ultrasonic concussion treatment in the step (2) is 1-3 min; the molecular weight of the nanofiber is 10000-5000000, the length distribution of the nanofiber is 0.5-10 mu m, and the width distribution of the nanofiber is 10-100 nm.
8. The method for preparing an AIE nanofiber probe for targeted staining of cell membranes as claimed in claim 1, wherein the concentration of the nanofibers in the nanofiber dispersion liquid in step (3) is 200-800 μ g/mL.
9. The AIE sodium of claim 1 for targeted staining of cell membranesThe preparation method of the rice fiber probe is characterized in that the bioactive polypeptide in the step (3) is a flexible chain bioactive polypeptide with an amino end connected with a sulfhydryl group; the sequence of the flexible chain bioactive polypeptide is (Lys)a-(Arg)b-(Trp)c-(Lys)d-(Arg)e-(Trp)f-(Lys)g-(Arg)hThe value range of a is 0-2; the value range of b is 0-2, the value range of c is 0-3, the value range of d is 0-2, the value range of e is 0-2, the value range of f is 0-3, the value range of g is 0-2, and the value range of h is 0-2; in the mixed solution, the concentration of the bioactive polypeptide is 1-30mg/mL, and the stirring reaction time is 0.5-48 h.
10. An AIE nanofiber probe targeting a stained cell membrane prepared by the preparation method of any one of claims 1 to 9, characterized in that the length is 0.5 to 10 μm and the width is 10 to 100 nm.
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