CN104892815A - Luminescent nano micro-spheres with positive charge on surface and possessing aggregation induced fluorescence enhancement property and biological application thereof - Google Patents

Abstract

The invention relates to a fluorescent nano-microsphere with positive charges on the surface and the property of aggregation-induced fluorescence enhancement and its application in cell imaging, belonging to the technical field of polymer materials. The invention firstly synthesizes a positively charged nano-microsphere emulsion on the surface, and modifies the negatively-charged fluorescent molecules with AIE effect on the surface of the nano-microspheres through electrostatic force. The intramolecular rotation of fluorescent molecules is limited by the Coulomb force, and the absorbed energy is basically released through fluorescent radiation. Therefore, the fluorescence of fluorescent molecules modified on nano-microspheres is enhanced hundreds of times, showing excellent AIE properties. The fluorescent nanospheres we prepared have stable fluorescent properties, good biocompatibility, low toxicity, positive charges on the surface and easy entry into cells and biological detection. Therefore, the positively charged fluorescent nanospheres we prepared have broad application prospects in biological fields such as cell imaging.

Description

Fluorescent nanospheres with positively charged surfaces and aggregation-induced fluorescence enhancement properties and their biological applications

technical field

The invention belongs to the technical field of polymer materials, and in particular relates to a fluorescent nano-microsphere with a positive charge on the surface and an aggregation-induced fluorescence enhancement property and its application in cell imaging, biological detection and the like.

Background technique

Bioimaging is an emerging field that integrates multiple technologies, integrates multiple disciplines, has wide applications, and develops rapidly. In recent years, with the development of biochemistry and life sciences, people's research on organisms has gradually shifted from the macroscopic to the microscopic. Therefore, bioluminescent imaging, one of its branches, has become the focus of research and attention. Fluorescence technology has the advantages of fast, sensitive, real-time, non-radioactive, and good repeatability, and multiple photophysical parameters (such as emission wavelength, excitation wavelength, fluorescence intensity, and fluorescence lifetime) can be used for detection. Fluorescent probes are the core technology for bioluminescence imaging. Traditional fluorescent probes are mainly based on organic fluorescent dyes, but due to the disadvantages of organic fluorescent dyes such as poor light resistance, poor water solubility, poor biocompatibility, and aggregation-induced fluorescence quenching, it is difficult to apply in practice. With the increasing application of bioluminescent imaging, it is urgent to develop new fluorescent probes to overcome the shortcomings of traditional probes. Therefore, fluorescent nanoparticle probes have been proposed as an effective means to solve this problem.

At present, fluorescent nanoparticle probes mainly include: quantum dots (quantum dot, QD), upconversion rare earth nanoparticles (Upconversion Rare Earth Nanoparticles, UCRE-NPs) and polymer fluorescent nanospheres. Inorganic quantum dots are widely used in biomarkers and other fields due to their high fluorescence efficiency. However, studies have found that when inorganic quantum dots are used for bioimaging and enter the body, the surface is easily oxidized into heavy metal ions by oxidants in the body, so the biological toxicity is not suitable for biological imaging. Polymer fluorescent nano-microspheres start with polystyrene, polyacrylamides, and polymethacrylates as the particle body, and fluorescent nano-microspheres that are bonded or adsorbed on the surface of fluorescent substances. Because a single nanoparticle can bind multiple fluorescent molecules, the fluorescence intensity is enhanced. Compared with small molecule organic dyes, polymer fluorescent nanospheres usually have good dispersion in the aqueous phase, strong fluorescence emission intensity, and high luminous efficiency, while the probability of photobleaching is very low; and in a variety of The particle stability and biocompatibility under the biological environment are very high. Therefore, many polymer fluorescent nanoparticles can be directly used in biological applications such as bioimaging fluorescent probes and fluorescent biosensors without tedious modification and improvement. However, most of the fluorescent molecules currently used in polymer fluorescent nanospheres have aggregation-induced quenching properties. When preparing high-efficiency polymer fluorescent nanospheres, the fluorescence signal cannot be significantly improved when the amount of fluorescent dye added is small, but when the amount is large, the fluorescence signal will also attenuate or have no fluorescence emission due to the aggregation-induced quenching effect, which limits its application. .

In recent years, fluorescent molecules with aggregation-induced emission (AIE) have attracted people's attention. It is different from traditional fluorescent molecules. The fluorescence of AIE molecules will be significantly enhanced in the aggregated state or in poor solvents. When in a free state, the absorbed energy will be consumed due to the intensification of intramolecular rotation and the fluorescence will decay. However, AIE molecules are mostly small molecules of organic dyes, which have poor biocompatibility, short circulation time in the blood and cannot effectively enter cells, so they cannot be used for bioimaging. Therefore, designing an effective carrier that can realize the AIE effect of fluorescent molecules and has good biocompatibility, low biotoxicity, appropriate size, good cell penetration and no quenching effect of the in vivo environment is very important in the field of biological imaging. of.

Contents of the invention

The purpose of the present invention is to provide a kind of fluorescent nano-microspheres with aggregation-induced fluorescence enhancement properties, which are positively charged on the surface and can be used for cell imaging and biological detection.

The invention firstly synthesizes a positively charged nano-microsphere emulsion on the surface, and modifies the negatively-charged fluorescent molecules with AIE effect on the surface of the nano-microspheres through electrostatic force. The intramolecular rotation of fluorescent molecules is limited by the Coulomb force, and the absorbed energy is basically released through fluorescent radiation. Therefore, the fluorescence intensity of fluorescent molecules modified on nanospheres is enhanced by a hundred times, showing excellent AIE properties. The fluorescent nanospheres we prepared have stable fluorescent properties, good biocompatibility, low toxicity, positive charges on the surface and easy entry into cells for cell fluorescence imaging and biological detection. Therefore, the positively charged fluorescent nanospheres we prepared have broad application prospects in biological fields such as cell imaging.

The fluorescent nanospheres with aggregation-induced fluorescence enhancement properties with positive charges on the surface of the present invention are prepared by the following steps:

(1) Take 5-10mL of polymerized monomer 1 and disperse it in 150-200mL of deionized water, then add 0.04g-0.5g of polymerized monomer 2; at room temperature, under the protection of nitrogen, mechanically stir (300-600rpm), remove Oxygen in the reaction system; after heating up to 70-90°C, add 10 mL of an aqueous solution containing 0.3-0.5 mmol of initiator to the reaction system, and polymerize under nitrogen protection and stirring for 5-20 hours to obtain positively charged nanospheres on the surface solution; high-speed centrifugation (15000-20000rpm) washing to remove unreacted monomers, oligomers, initiators and other impurities, nano-microspheres re-dispersed ultrasonically in 100mL deionized water;

(2) Measure 10-20 mL of the nanosphere solution obtained in step (1), add 10-20 mL of deionized water, and then add 3-5 mL of AIE-type fluorescent molecules at 80-150 μg/mL. Under the condition of ℃ and nitrogen protection, magnetically stir the reaction for 4-10 hours; high-speed centrifugation (15000-20000rpm) removes uncomplexed AIE fluorescent molecules, and obtains positively charged fluorescent nanospheres on the surface of AIE molecules; the obtained fluorescent nanoparticles The microspheres are dispersed in deionized water, so as to obtain the fluorescent nanometer microspheres with positively charged surface and aggregation-induced fluorescence enhancement property of the present invention.

In the above method, polymerizable monomer 1 is styrene (St), fluorostyrene (F-St), methyl methacrylate (MMA), ethyl methacrylate, ethyl acrylate, vinyl chloride, tert-butyl acrylate esters, divinylbenzene or alpha-methylstyrene or glycidyl methacrylate (GMA);

In the above method, polymerizable monomer 2 is N,N,N-trimethylvinylbenzyl ammonium chloride (VBTAC), 2-(diisopropylamino)ethyl methacrylate (DPA) or 2-amino Ethyl methacrylate hydrochloride (AMA);

In the above method, the initiator is azobisisobutylamidine hydrochloride (V ₅₀ ), azobisisobutylimidazoline hydrochloride (AIBI), azoisobutylcyanoformamide (V ₃₀ );

In the above method, the fluorescent molecules of AIE type are 9,10-bis(styryl)anthracene sulfonate (DSA), 1,2-bis[4-(3-sulfonic acid propoxy)phenyl]-1 , 2-Diphenylethylene disodium salt (BSTPE) or 1,1,2,2-tetrakis[4-(3-sulfonic acid propoxy)phenyl]ethylene tetrasodium salt (TSTPE).

The present invention has the following advantages:

1. The particle size of the fluorescent nanospheres is at the nanometer level and the size is controllable (50-140nm);

2. The fluorescent nanospheres have good biocompatibility and can exist stably in phosphate buffer solution and biomacromolecule solution for more than 3 months (Figure 5);

3. The fluorescence of the fluorescent molecule is basically stable in the range of pH=3-7, and the fluorescence decreases linearly in the range of pH=7-10, but the degree of reduction has no effect on fluorescence imaging;

4. The surface of the fluorescent nanometer microsphere is positively charged, so it is easy to enter cells for cell fluorescence imaging and biological detection.

5. The fluorescence signal of the fluorescent nanospheres is enhanced in the presence of biomacromolecules such as BSA, and the biomacromolecules can amplify the fluorescence signal. And the concentration of BSA and the fluorescence intensity change linearly, so this material can be used to detect the concentration of BSA (Figure 7).

6. The cytotoxicity of the fluorescent nano-microsphere is low ( FIG. 8 ), which is favorable for its biological application.

Description of drawings

Fig. 1: the scanning electron micrograph (SEM) of nano microsphere prepared for embodiment 1;

Fig. 2: the dynamic light scattering particle diameter diagram of the nano-microspheres and fluorescent nano-microspheres prepared in Example 1;

Fig. 3: the dynamic light scattering Zeta figure of nano microsphere and fluorescent nano microsphere prepared for embodiment 1;

Fig. 4: Fluorescence spectrogram (the concentration of fluorescent molecules is the same, λex=420nm) of fluorescent nanospheres and fluorescent molecule aqueous solution prepared for embodiment 1;

Fig. 5: Fluorescent nanosphere aqueous solution, fluorescent nanosphere fetal bovine serum (FBS) phosphate buffer solution (pH=7.4), fluorescent nanosphere fetal bovine serum (FBS) phosphate after 3 months prepared for embodiment 1 Dynamic light scattering particle size diagram of buffer solution (pH=7.4);

Figure 6: (1) is the fluorescence spectrum of the fluorescent nanospheres prepared in Example 1 under different pH phosphate buffer solutions; (2) is the fluorescence intensity and pH of the fluorescent nanospheres prepared in Example 1 relation chart;

Figure 7: (1) is the fluorescence spectrum of the nano-microspheres prepared in Example 1 at different BSA concentrations; (2) is the BSA concentration and fluorescence intensity curve;

Figure 8: (1) is the cytotoxicity test graph of embodiment 1 fluorescent nano-microspheres to human gastric mucosal epithelial cells (GES-1); (2) is the cytotoxicity test diagram of embodiment 1 fluorescent nano-microspheres to human gastric cancer cells (SGC-7901 ) cytotoxicity test chart;

Figure 9: (a) Fluorescence confocal bright field photos of the nanospheres prepared in Example 1 in normal cells human gastric mucosal epithelial cells (GES-1); (b) the nanospheres prepared in Example 1 in normal cells Superimposed photos of fluorescent confocal bright field and dark field of human gastric mucosal epithelial cells (GES-1); Field photo; (d) fluorescence confocal bright field photo of the nano-microspheres prepared in Example 1 in normal cell human gastric cancer cells (SGC-7901); (e) the nano-microspheres prepared in Example 1 in human gastric cancer cells (SGC-7901) SGC-7901) fluorescent confocal bright-field and dark-field superimposed photos; (f) fluorescence confocal dark-field photos of the nanospheres prepared in Example 1 in human gastric cancer cells (SGC-7901);

Fig. 10: scanning electron micrograph (SEM) for the nano-microsphere prepared in embodiment 2;

Fig. 11: scanning electron micrograph (SEM) of the nano microsphere prepared for embodiment 3;

FIG. 12 : Scanning electron micrograph (SEM) of the nanospheres prepared in Example 4.

Detailed ways

Example 1:

(1) Take 5mL styrene (analytically pure, the polymerization inhibitor is removed by distillation under reduced pressure) and 0.06g N,N,N-trimethylvinylbenzyl ammonium chloride (VBTAC) and add it to a tank containing 185mL deionized water In a 500mL three-necked flask, stir mechanically (400rpm) at room temperature and under nitrogen protection for 30 minutes to remove oxygen in the reaction system, then heat up to 70°C, add 10mL containing 0.37mmol azobisisobutylamidine hydrochloride (V ₅₀ ) The aqueous solution of the initiator initiates the polymerization, and the polymerization is carried out for 10 h under nitrogen protection and a stirring speed of 400 rpm. The nano-microspheres obtained by polymerization were centrifuged 3 times at a speed of 18500rpm, washed 3 times with deionized water to remove unreacted monomers, oligomers, initiators, etc., and redispersed into 100mL deionized water to obtain The mass concentration is 2.92% of the positively charged nanometer microsphere solution on the surface.

(2) Take 10mL of nanosphere solution, add 10mL of deionized water, add 3mL, 100μg/mLDSA into a three-neck flask, put it into a stirrer, and heat it to 50°C under magnetic stirring for 4 hours. After the reaction is completed, fluorescent nanospheres Centrifuge 3 times at 18500rpm, wash 3 times with deionized water to remove uncomplexed fluorescent molecules, and redisperse them in 23mL deionized water to obtain a positively charged surface with aggregation-induced fluorescence enhancement with a mass concentration of 1.27%. properties of fluorescent nanosphere solutions.

From the scanning electron micrograph (SEM) of the nano-microsphere (Fig. 1), it can be seen that the size of the nano-microsphere is 100 nm, the size is uniform, and the monodispersity is good. The size of the nanospheres in the aqueous solution measured by dynamic light scattering (DLS) is 120.7nm, and the monodispersity index PDI=0.002 (Figure 2). Due to the existence of the hydrated particle size, the measured size is larger than the value of the SEM. The surface of the nanospheres was positively charged (42.4mv) as measured by dynamic light scattering (DLS) (Figure 3). The negatively charged AIE-type fluorescent molecule DSA is compounded to the surface of fluorescent nanospheres through the action of Coulomb force, and the hydrated particle size of the obtained fluorescent nanospheres is 131.7nm (Fig. The dispersion index PDI=0.013, the surface charge also decreased to 40.6mv (Figure 3). Although the fluorescent molecule DSA itself has good biocompatibility and overcomes the traditional aggregation fluorescence-induced quenching phenomenon, due to its water solubility, most of the absorbed energy is released through intramolecular rotation, and the fluorescence efficiency is very low, so it cannot Suitable for biological imaging. After the fluorescent molecule DSA is compounded on our synthesized nano-microspheres, due to the restriction of intramolecular rotation, most of the absorbed energy is released through radiation. It can be seen from the fluorescence spectrum that the fluorescence efficiency is significantly enhanced (Fig. 4). The fluorescent nanospheres we synthesized have good biocompatibility and biostability. We prepared 10% fetal bovine serum (FBS) phosphate buffer solution (pH=7.4) containing 500 μg/mL fluorescent nanospheres. After 3 months of storage at low temperature, there was no significant change in size as measured by dynamic light scattering (Fig. 5 ). And we tested the fluorescence stability of the fluorescent nanospheres in different biological environments. When the fluorescent nanospheres were in different pH phosphate buffer solutions, the fluorescence intensity did not change significantly ( FIG. 6 ). When in the biomacromolecule BSA solution, since these biomacromolecules are negatively charged, the adsorption to the surface of fluorescent nanospheres further binds the rotation of DSA molecules, so that the fluorescence is enhanced and changes linearly (Fig. 7), so the intracellular Some of the biomolecules can amplify the fluorescent signal of our synthesized fluorescent nanospheres. We conducted a cytotoxicity test of fluorescent nanospheres, and it can be seen that the survival rate of cells cultured at a concentration of 100 mg/L for 72 hours still reached more than 85%, and the cytotoxicity was low, which can be used for biological imaging (Figure 8). We conducted cell imaging experiments on it, and through confocal cell imaging, we saw that the fluorescent nanospheres we synthesized can well enter the cytoplasm of normal cells and cancer cells, and image the cells well (Figure 9).

Example 2:

(1) Take 5mL styrene (analytically pure, the polymerization inhibitor is removed by distillation under reduced pressure) and 0.5g N,N,N-trimethylvinylbenzyl ammonium chloride (VBTAC) and add it to a tank containing 185mL deionized water In a 500mL three-necked flask, stir mechanically (400rpm) at room temperature and under nitrogen protection for 30 minutes to remove oxygen in the reaction system, then heat up to 70°C, add 10mL containing 0.37mmol azobisisobutylamidine hydrochloride (V ₅₀ ) The aqueous solution of the initiator initiates the polymerization, and the polymerization is carried out for 10 h under nitrogen protection and a stirring speed of 400 rpm. The polymerized nanospheres were centrifuged 3 times at a speed of 18500rpm, washed 3 times with deionized water to remove unreacted, oligomers, initiators, etc., and redispersed into 100mL deionized water to obtain the mass concentration It is a 2.92% solution of nano-microspheres with positive charges on the surface.

(2) Take 10mL of nanosphere solution, add 10mL of deionized water, add 3mL, 100μg/mLDSA into a three-neck flask, put it into a stirrer, and heat it to 50°C under magnetic stirring for 4 hours. After the reaction is completed, fluorescent nanospheres Centrifuge 3 times at 18500rpm, wash 3 times with deionized water to remove uncomplexed fluorescent molecules, and redisperse them in 23mL deionized water to obtain a positively charged surface with aggregation-induced fluorescence enhancement with a mass concentration of 1.27%. properties of fluorescent nanosphere solutions.

We found that its properties were similar to those of the sample prepared in Example 1, but the size became smaller, about 50 nm (see Figure 10). This is because the polymerized monomer N,N,N-trimethylvinylbenzyl ammonium chloride (VBTAC) is an amphiphilic monomer that acts as a surfactant. When its amount increases, it is equivalent to an increase in the amount of surfactant, so the size of the nano-microspheres will become smaller.

Example 3:

(1) Take 5mL styrene (analytically pure, the polymerization inhibitor is removed by distillation under reduced pressure) and 0.05g N,N,N-trimethylvinylbenzyl ammonium chloride (VBTAC) and add it to a tank containing 185mL deionized water In a 500mL three-necked flask, stir mechanically (400rpm) at room temperature and under nitrogen protection for 30 minutes to remove oxygen in the reaction system, then heat up to 70°C, add 10mL containing 0.37mmol azobisisobutylamidine hydrochloride (V ₅₀ ) The aqueous solution of the initiator initiates the polymerization, and the polymerization is carried out for 10 h under nitrogen protection and a stirring speed of 400 rpm. The polymerized nanospheres were centrifuged 3 times at a speed of 18500rpm, washed 3 times with deionized water to remove unreacted, oligomers, initiators, etc., and redispersed into 100mL deionized water to obtain the mass concentration It is a 2.92% solution of nano-microspheres with positive charges on the surface.

(2) Take 10mL of nanosphere solution, add 10mL of deionized water, add 3mL, 100μg/mLDSA into a three-neck flask, put it into a stirrer, and heat it to 50°C under magnetic stirring for 4 hours. After the reaction is completed, fluorescent nanospheres Centrifuge 3 times at 18500rpm, wash 3 times with deionized water to remove uncomplexed fluorescent molecules, and redisperse them in 23mL deionized water to obtain a positively charged surface with aggregation-induced fluorescence enhancement with a mass concentration of 1.27%. properties of fluorescent nanosphere solutions.

We found that its properties were similar to those of the sample prepared in Example 1, but the size became larger, about 110 nm (see FIG. 11 ). This is because the polymerized monomer N,N,N-trimethylvinylbenzyl ammonium chloride (VBTAC) is an amphiphilic monomer that acts as a surfactant. When its amount decreases, it is equivalent to a decrease in the amount of surfactant, so the size of the nano-microspheres will become larger.

Example 4:

(1) Take 5mL styrene (analytically pure, the polymerization inhibitor is removed by distillation under reduced pressure) and 0.04g N,N,N-trimethylvinylbenzyl ammonium chloride (VBTAC) and add it to a tank containing 185mL deionized water In a 500mL three-necked flask, stir mechanically (400rpm) at room temperature and under nitrogen protection for 30 minutes to remove oxygen in the reaction system, then heat up to 70°C, add 10mL containing 0.37mmol azobisisobutylamidine hydrochloride (V ₅₀ ) The aqueous solution of the initiator initiates the polymerization, and the polymerization is carried out for 10 h under nitrogen protection and a stirring speed of 400 rpm. The polymerized nanospheres were centrifuged 3 times at a speed of 18500rpm, washed 3 times with deionized water to remove unreacted, oligomers, initiators, etc., and redispersed into 100mL deionized water to obtain the mass concentration It is a 2.92% solution of nano-microspheres with positive charges on the surface.

(2) Take 10mL of nanosphere solution, add 10mL of deionized water, add 3mL, 100μg/mLDSA into a three-neck flask, put it into a stirrer, and heat it to 50°C under magnetic stirring for 4 hours. After the reaction is completed, fluorescent nanospheres Centrifuge 3 times at 18500rpm, wash 3 times with deionized water to remove uncomplexed fluorescent molecules, and redisperse them in 23mL deionized water to obtain a positively charged surface with aggregation-induced fluorescence enhancement with a mass concentration of 1.27%. properties of fluorescent nanosphere solutions.

We found that its properties were similar to those of the sample prepared in Example 1, but the size became larger, about 140 nm (see Figure 12). This is because the polymerized monomer N,N,N-trimethylvinylbenzyl ammonium chloride (VBTAC) is an amphiphilic monomer that acts as a surfactant. When its amount decreases, it is equivalent to a decrease in the amount of surfactant, so the size of the nano-microspheres will become larger.

Claims (4)

1. A fluorescent nanosphere with a positive charge on the surface and an aggregation-induced fluorescence enhancement property, which is prepared by the following steps: 1) Take 5-10mL of polymerized monomer 1 and disperse it in 150-200mL of deionized water, then add 0.04g-0.5g of polymerized monomer 2; at room temperature, under the protection of nitrogen, mechanically stir to remove the oxygen in the reaction system; raise the temperature Add 10mL aqueous solution containing 0.3-0.5mmol initiator to the reaction system after reaching 70-90°C, and polymerize under nitrogen protection and stirring for 5-20 hours to obtain a positively charged nanosphere solution on the surface; after high-speed centrifugation and washing Re-disperse the obtained nano-microspheres ultrasonically in 100mL deionized water; 2) Measure 10-20 mL of the nanosphere solution obtained in step 1), add 10-20 mL of deionized water, and then add 3-5 mL of AIE-type fluorescent molecules at 80-150 μg/mL. Under the condition of nitrogen protection, magnetic stirring reaction for 4 ~ 10h; high-speed centrifugation to remove uncomplexed AIE fluorescent molecules, to obtain fluorescent nanospheres with positive charges on the surface of AIE molecular complexes; disperse the obtained fluorescent nanospheres in deionized water , so as to obtain fluorescent nanospheres with aggregation-induced fluorescence enhancement properties with positive charges on the surface; Polymerized monomer 1 is styrene, fluorostyrene, methyl methacrylate or glycidyl methacrylate; polymerized monomer 2 is N,N,N-trimethylvinylbenzyl ammonium chloride, 2- (Diisopropylamino)ethyl methacrylate or 2-aminoethyl methacrylate hydrochloride. 2. A kind of surface is positively charged as claimed in claim 1 and has the fluorescent nanosphere of aggregation-induced fluorescence enhancement property, it is characterized in that: initiator is azobisisobutyrimidine hydrochloride, azobisisobutyrimazole phenoline hydrochloride or azoisobutyrocyanoformamide. 3. a kind of fluorescent nanosphere with positive charge on the surface as claimed in claim 1 has aggregation-induced fluorescence enhancement property, it is characterized in that: the fluorescent molecule of AIE type is 9,10-two (styryl) anthracene sulfonic acid salt, 1,2-bis[4-(3-sulfonic acid propoxy)phenyl]-1,2-diphenylethylene disodium salt or 1,1,2,2-tetrakis[4-(3- Propoxy)phenyl]ethylene sulfonate tetrasodium salt. 4. The application of a kind of fluorescent nanospheres with positively charged surface and aggregation-induced fluorescence enhancement property according to any one of claims 1 to 3 in biological imaging.