CN108424766B - Preparation and application of TPE-PDEAM with multi-responsive polymer quantum dots - Google Patents

Abstract

The invention discloses a TPE-PDEAEAM with multi-responsive polymer quantum dots, which is formed by atom transfer radical polymerization by using TPE-BMP as an initiator. Due to the diethylamino and acrylamide groups contained in the TPE-PDEAEAM polymer quantum dots, the TPE-PDEAEAM polymer quantum dots responded accordingly to the stimulation of temperature, pH and _CO . Fluorescent polymers exhibit unique aggregation-induced emission (AIE) behavior, can be easily digested by HeLa cells and track cells for up to nine passages, and thus can be used as long-term tracking fluorophores for cell imaging.

Description

Preparation and application of a polyresponsive polymer quantum dot TPE-PDEAEAM

The invention relates to the preparation of a polymer fluorescent quantum dot, in particular to the preparation of a TPE-PDEAEAM polymer quantum dot with multiple responsiveness. The polymer quantum dot has AIE fluorescence characteristics and can be used as a fluorescent agent for cell imaging .

Background technique

In recent years, fluorescence imaging, as a non-invasive technology in the field of life sciences, has attracted extensive attention in the field of scientific research due to its excellent characteristics such as high sensitivity, simple operation, and low cost. So far, various fluorescent molecules have been synthesized and used as bioimaging reagents. Among these numerous reagents, the development of long-term cell tracers for long-term tracking of cellular processes is of great significance for biological researchers to monitor biological processes such as the occurrence, development, invasion, and metastasis of cancer cells.

Quantum dots (QDs), as well-known fluorescent inorganic nanoparticles, are used as fluorescent cell tracers for various biotechnological applications due to their unique high light brightness and light stability. For example, CdSe/ZnS quantum dots have been used as cell tracers for long-term cellular imaging of living cells, with relatively high emission and excellent photostability. Unfortunately, the heavy metal components of QDs (such as Cd ²⁺ ) are highly cytotoxic, and these weaknesses limit their many applications in bioimaging. In addition, green fluorescent protein (GFP) and others in other families, a highly fluorescent molecule, have been widely used as long-term in vitro cell tracers. However, due to some shortcomings of GFP itself, such as sensitivity to proteolytic enzymes, changes in Stokes shift, poor light stability, the infection of fluorescent proteins can interfere with many normal cell functions, etc., hindering the development of more GFP probes. practical application.

Aggregated light-emitting polymer quantum dots (AIEs) have obvious advantages over inorganic semiconductor quantum dots (QDs) and some small molecules of organic dyes. For example, the half-peak width is relatively narrow, the emission peak is relatively symmetrical, the cytotoxicity is small, and the biocompatibility is good. It has good application prospects in the fields of optics and biology. At present, aggregated luminescent small molecules and polymers have been prepared by different synthetic methods, and AIE-type polymers have been gradually synthesized as cell probes. TPE (tetraphenylene) is a classic AIE (Aggregation Induced Emission) molecule, that is, it has no fluorescence in solution, but has strong fluorescence in solids, which is just the opposite of traditional aggregation quenching molecules, so in organic Optoelectronic materials and other fields have important applications and are attracting more and more attention. For example, Tang Benzhong et al. then reported a new type of fluorescence by attaching a large amount of tetraphenylene (TPE) to chitosan (CS) chains, and then using the polymer as a cell imaging agent for long-term tracking of living cells. probe. Zhang et al. synthesized an ATRP temperature-sensitive organic polymer nanoparticle from tetrastyryl poly(N-isopropylacrylamide) (TPE-PNIPAM) as a cell tracer for long-term cell tracking, Cell passaging up to seven passages can be performed in HeLa cells. However, there are relatively few reports on such AIE-based polymers. Therefore, designing and synthesizing AIE-based polymers as long-term tracers for biological applications is a very challenging task.

Stimuli-responsive polymers that respond to relatively small or internal changes in environmental conditions have much value or broader bioimaging applications. Thermoresponsive polymers exhibit soluble-insoluble phase transition at lower critical solution temperature (LCST). However, pH-sensitive polymers respond to changes in the surrounding medium by changing the size of the pH, and thus the size of the polymer. Temperature and pH/CO ₂ responsive homopolymers of poly[N-[2-(diethylamino)ethyl]acrylamide] (PDEAEAM) have been reported in the literature (Song, Z.; Wang, K.; Gao, C.; Wang, S.; Zhang, W. A New Thermo-,pH-, and CO ₂ -Responsive Homopolymer of Poly[N-[2-(diethylamino)ethyl]acrylamide]:Is the Diethylamino Group Underestimated Macromolecules . 2016 , 49 , 162-171.), whose pH/CO ₂ is adjustable. However, only one kind of stimulating fluorescent polymer materials can not meet the requirements of biomedical applications. AIE-type-based multistimuli-responsive polymers combine the advantages of aggregated luminescence (AIE) and stimuli-responsive polymers, exhibiting good water solubility, biocompatibility, and aggregated luminescence properties, and exhibit structural and luminescent properties upon external stimuli. The luminescence changes accordingly. These multistimulus-responsive polymers combined with AIE-type fluorophores can be used to overcome major problems with long-term cell tracking. Therefore, it is very necessary to develop fluorescent polymers with multiple responsiveness. Among them, heat-sensitive, pH- and carbon dioxide-responsive polymers are the most popular in the design and synthesis of novel irritant materials.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a preparation method of TPE-PDEAEAM polymer quantum dots with multiple responsiveness;

Another object of the present invention is to analyze the structure, AIE properties and application of the above-mentioned polyresponsive TPE-PDEAEAM polymer quantum dots as cell tracers for long-term cellular imaging of living cells.

1. Preparation of TPE-PDEAEAM polymer quantum dots

(1) Synthesis of 4-hydroxyphenylethylene

Using tetrahydrofuran (THF) as solvent, zinc powder, TiCl ₄ (Zn as reducing agent, TiCl ₄ as catalyst, mixed reducing catalyst), under the protection of argon, 4-hydroxybenzophenone and benzophenone with 1 : 1 ~ 1: 1.2 molar ratio refluxed for 24h; should be cooled to room temperature after the end, added K ₂ CO ₃ solution to quench the reaction, then filtered, extracted with ethyl acetate, separated and purified by column chromatography to obtain a pale yellow solid 4- Hydroxytetraethylene (TPE).

The add-on of zinc powder is 3～4 times of 4-hydroxybenzophenone, benzophenone total molar weight; The add-on of TiCl is 2 times of ₄ -hydroxybenzophenone, benzophenone total molar weight. ~3 times.

(2) Synthesis of 4-(12-hydroxydodecyl)tetraphenylene (TPE-OH)

4-Hydroxytetraethylene, 12-bromo-1-dodecanol and K ₂ CO ₃ were dissolved in anhydrous acetonitrile, refluxed for 20-24 h under argon protection; cooled to room temperature after the reaction was completed, the reaction solution was filtered and The organic layer was evaporated, and the product was separated and purified by column chromatography to obtain 4-(12-hydroxydodecyl)tetraphenylene as a pale yellow solid.

The function of 12-bromo-1-dodecanol is to extend the carbon chain and enhance the hydrophobicity. The molar ratio of 4-hydroxytetraethylene and 12-bromo-1-dodecanol is 1:1~1:1.2.

_The role _of K2CO3 is to provide alkalinity and water absorption. The molar ratio of 4-hydroxytetraethylene to K ₂ CO ₃ is 1:1~1:1.2.

(3) Synthesis of TPE-BPM

4-(12-Hydroxydodecyl)tetraphenylene (TPE-OH), triethylamine and 2-bromo-2-methylpropionyl bromide were added to anhydrous tetrahydrofuran (THF) and stirred at room temperature The reaction is carried out for 20-24 hours; after the reaction is completed, the reaction solution is filtered; the filtrate is concentrated to obtain a crude product, which is separated and purified by column chromatography to obtain a light yellow solid product, which is TPE-BMP.

The role of triethylamine is to provide alkalinity. The molar ratio of TPE-OH to triethylamine is 1:1~1:1.25.

The function of 2-bromo-2-methylpropionyl bromide is to synthesize initiator, and the molar ratio of TPE-OH to 2-bromo-2-methylpropionyl bromide is 1:1~1:1.25.

(4) Synthesis of TPE-PDEAEAM

N(2-(diethylamino)ethyl)acrylamide (DEAEAM) was dissolved in water-methanol mixed solvent, and tris(2-dimethylaminoethyl)amine (Me ₆ TREN) was added successively under argon protection. ), CuBr and TPE-BMP, stirred at room temperature for 20~24h, then dialyzed the reaction stock solution with ultrapure water for 70~72 hours, and then freeze-dried the dialysate for 70~72 hours to obtain light yellow solid powder, which is TPE -PDEAEAM.

In the water-methanol mixed solvent, the volume ratio of water and methanol is 1.5:1 to 2:1.

Tris(2-dimethylaminoethyl)amine (Me ₆ TREN) acts as a Cu ²⁺ ligand; its addition amount is 19%~20% of the mass of DEAEAM.

CuBr acts as a catalyst. Its addition amount is 0.5%~1% of the quality of DEAEAM.

The mass ratio of PDEAEAM to TPE-BMP is 60:1~65:1.

2. Structure and properties of TPE-PDEAEAM polymer quantum dots

1. H NMR

Figures 1 and 2 are the H NMR spectra of TPE-BPM and TPE-PDEAEAM, respectively. The chemical shift appears at about 6.9ppm, the signal peak of aromatic ring proton hydrogen, the signal peak of methyl hydrogen at 1.0ppm, the hydrogen peak of imino group above TPE-PDEAEAM at 2.6ppm and 3.2ppm, respectively, 1.5ppm Methylene signal peaks on TPE-PDEAEAM appeared. This indicates that the TPE molecules and PDEAEAM were successfully covalently bound together.

2. Infrared spectrum

Figure 3 is the infrared spectrum of TPE-PDEAEAM. We can clearly observe the characteristic peak of amide at 1658 cm ^-1 , and the vibration of NH bond at 1553 cm ^-1 . It is further demonstrated that the structure of the polymer quantum dots is consistent with our design.

3. GPC test

Table 1 shows the GPC data of TPE-PDEAEAM.

The structure of TPE-PDEAEAM was further proved by GPC test, and the number-average molecular weight ( _Mn ) and weight-average molecular weight (Mw) were 1.1×10 ⁴ and 2.1×10 ⁴ , respectively.

4. Morphology of polymer TPE-PDEAEAM polymer quantum dots self-assembled in aqueous solution

Figure 4 shows the Tyndall effect of TPE-PDEAEAM polymer quantum dots in tetrahydrofuran solution and aqueous solution. When a beam of light passes through the colloid, a bright "path" in the colloid can be observed from the vertical direction of the incident light. This phenomenon is called the Tyndall phenomenon. Through this phenomenon, we initially judge the TPE-PDEAEAM polymer quantum dots. Self-assembly in aqueous solution is better.

Figure 5 shows the hydrodynamic size of the TPE-PDEAEAM polymer quantum dots dissolved in an aqueous solution with a concentration of 2 mg/mL, showing that the hydrodynamic size of the quantum dots is maintained at about 250 nm. The particle size of the polymer quantum dots is around 250.

Figure 6 shows the morphologies (SEM) of the self-assembled TPE-PDEAEAM polymer quantum dots in aqueous solution. It is proved that the TPE-PDEAEAM polymer nanoparticles are spherical with relatively regular shape, and the size of the nanoparticles is relatively uniform, with a particle size of about 250 nm.

5. AIE characteristics of TPE-PDEAEAM polymer quantum dots

Both THF and H ₂ O can dissolve the TPE-PDEAEAM polymer segment well. The polymer quantum dots TPE-PDEAEAM can be completely dissolved in water to form a transparent solution in a certain temperature range. Its aqueous solution is still luminescent under UV light (365 nm), while it is almost invisible to the naked eye in pure THF solution. This phenomenon is very similar to the AIE (aggregated emission) properties of TPE small molecules.

We investigated the effect of concentration changes on the fluorescence of the polymer, and found that when the concentration of the polymer TPE-PDEAEAM aqueous solution increased from 0.001 mg·mL ^-1 to 1 mg·mL ^-1 , the polymer concentration increased with the concentration of the polymer. The increased fluorescence also increases with excitation and emission wavelengths of 337 nm and 475 nm, respectively. With the increase of the concentration, the TPE-PNIPAM molecules must be aggregated to some extent, and the fluorescence is significantly enhanced, showing its AIE properties.

The fluorescence changes of TPE-PDEAEAM in different ratios of water/tetrahydrofuran mixed solvent were observed, which further proved the AIE properties of the polymer: with the increase of H ₂ O volume fraction, the fluorescence intensity of TPE-PDEAEAM in a nonlinear manner weaken. When the volume fraction of H ₂ O exceeds 80%, the fluorescence intensity increases sharply, which is very similar to the aggregation-induced fluorescence enhancement reported in the literature. The main reason is that with the increase of the volume fraction of H ₂ O, the polymer chain expands, which makes TPE aggregate, and the aggregation of TPE molecules increases the fluorescence emission.

6. The effect of temperature on the fluorescence intensity of TPE-PDEAEAM polymer quantum dots

The temperature-sensitive properties of TPE-PDEAEAM polymer quantum dots can be easily determined by heating aqueous solutions of TPE-PDEAEAM polymer quantum dots at a temperature above the LCST (phase transition temperature), followed by cooling to a temperature below the LCST. The aqueous solution of TPE-PDEAEAM polymer quantum dots is transparent at room temperature. The solution became cloudy and opaque after heating above the LCST, and when cooled to room temperature, it became cloudy and further became transparent, confirming the reversible phase transition of TPE-PDEAEAM at LCST. Fig. 7 shows the light transmittance as a function of temperature of the aqueous solution of TPE-PDEAEAM polymer quantum dots with a concentration of 2 g/L. When the polymer concentration is 2 g/L, the LCST of TPE-PDEAEAM in LCST aqueous solution is 60 °C.

In addition, the effect of temperature on the fluorescence properties of TPE-PDEAEAM polymer quantum dots was investigated. Figure 8 shows the change of fluorescence in TPE-PDEAEAM polymer quantum dot solution from 25°C to 66°C: the fluorescence intensity of TPE-PDEAEAM polymer quantum dots decreases with the increase of temperature. This is because the polymer segment of PDEAEAM is a temperature-sensitive material.

7. The pH-sensitive properties of TPE-PDEAEAM polymer quantum dots

The effect of pH change on the fluorescence intensity of TPE-PDEAEAM polymer quantum dots was investigated. It is well known that the addition of hydrochloric acid or sodium hydroxide can easily destroy intermolecular hydrogen bonds, resulting in changes in the original aggregation state of TPE-PDEAEAM polymer quantum dots. Due to the change in aggregation state, a change in the fluorescence intensity of the AIEgen-bearing polymer will occur. Figure 9 shows the fluorescence spectra of TPE-PDEAEAM polymer quantum dots in different pH solutions (left) and the relative fluorescence intensity (I /I ₀ ) in aqueous solutions at different pH (right) (TPE-PDEAEAM] = 2.0g L ^-1 ), indicating that the fluorescence of the polymer quantum dots increases with the increase of pH. Therefore, the pH-sensitive fluorescent TPE-PDEAEAM may be used as a promising optical sensor for low or high pH determination.

8. CO ₂ Sensitivity of TPE-PDEAEAM Polymer Quantum Dots

Carbon dioxide-responsive polymers and carbon dioxide capture materials have received great attention in recent years due to the many advantages of carbon dioxide, such as good biocompatibility, excellent membrane permeability, abundant availability, non-toxicity, and good reversibility. Carbon dioxide can easily react with reactive groups such as amines or amidines to generate hydrophilic compounds and amidine-based polymers, constituting the largest class of carbon dioxide-responsive polymers. Therefore, the bearing amidine groups of TPE-PDEAEAM polymer quantum dots, which can react with carbon dioxide and water to form charged amidinium bicarbonates, will be carbon dioxide responsive. Figure 10 shows the fluorescence spectra of TPE-PDEAEAM polymer quantum dots in different _CO2 volume solutions, the inset is the linear relationship of relative fluorescence intensity (I/I0) and CO2 volume. As shown in Fig. 10, the fluorescence intensity of TPE-PDEAEAM polymer quantum dots dropped sharply, accompanied by a small blue shift, when the _CO volume increased from 0.0 to 0.4 mL. The inset shows that the ratio of blank fluorescence intensity to quenched fluorescence intensity (I ₀ /I) has a linear relationship with the added amount of CO ₂ from 0.0 to 0.4 mL, indicating that TPE-PDEAEAM polymer quantum dots can be used as a fluorescence sensor for CO ₂ . The reason is attributed to the interaction between carbon dioxide and tertiary amine groups in TPE-tetraPDEAEAM, which confirms that the tertiary amine groups can be protonated by carbon dioxide to form charged ammonium bicarbonate.

2. Toxicity assessment of TPE-PDEAEAM on Hela cells

For biomedical applications, evaluating the cytotoxicity of materials is critical.

(1) Cell plating: Take HeLa cells in logarithmic growth phase, digest with 0.25% trypsin (containing EDTA), and count them on a hemocytometer so that the cell density is 1×10 ⁴ /well. (The edge holes should be filled with sterile PBS). Use a pipette to inoculate 100 µL of cell fluid per well into a 96-well culture plate, incubate at 37°C, and culture in a saturated humidity incubator with 5% CO ₂ . Each group needs to set 3 duplicate holes.

(2) Cell administration: cultured in a CO ₂ incubator (temperature of 37 °C, 5% CO ₂ content) for 24 hours, and then added with concentrations of 50 μg/mL, 100 μg/mL, 200 μg/mL, 300 μg/mL, and 400 μg, respectively. /mL of TPE-PDEAEAM.

(3) Adding MTT: After the medicated Hela cells were cultured in the incubator for 24 hours, the original medium was poured out, and 100 mL of MTT solution was added to each well, and then the cells were cultured in the incubator for 4 hours and then stopped re-culturing. . The operation of the experiment should be protected from light as much as possible.

(4) Dissolving Hela cells: Aspirate the heavy medium from the 96-well culture plate, add 150 mL of DMSO to each well and incubate for 40 minutes to completely dissolve the precipitate before detection (do not touch the bottom of the 96-well culture plate during the operation to avoid affecting the detection). experimental results).

(5) Determination of the OD value of each well of the 96-well culture plate with a microplate reader: To measure the shaken Hela cells with a microplate reader, first we need to turn on the computer, connect the computer to the microplate reader, and then turn on the measurement The software was used to measure the 96-well plate, and the measurement results were processed with Excel.

(6) Survival rate of Hela cells: measure the OD value, and calculate the survival rate (%) of Hela cells containing TPE-PDEAEAM according to the following formula. Then take the concentration of TPE-PAA solution as the abscissa, and take the survival rate of Hela cells containing TPE-PDEAEAM as the ordinate.

Cell viability (%)=(OD test group-OD zero adjustment group)/(OD blank group-OD zero adjustment group)×100%

HeLa cytotoxicity test after 48 hours of treatment with different concentrations of TPE-PNIPAM, the results are shown in Figure 11. It was shown that more than 95% of the HeLa cells survived, incubated with different concentrations of TPE-PNIPAM (50~400μg mL ^-1 ) for 48h. It shows that TPE-PDEAEAM has less toxicity and is expected to be used for cell imaging or more biological applications.

3. TPE-PDEAEAM imaging application of Hela cells

Based on the characteristics of AIE fluorescence, TPE-PDEAEAM polymer quantum dots can be used as fluorescent agents for cell imaging. HeLa cells were chosen as organisms to demonstrate the ability of TPE-PDEAEAM polymer quantum dots for in vitro cell tracking applications. HeLa cells were seeded in 96-well culture plates at a density of 10,000 cells/well. After 24 hours in the incubator, cells were treated with different concentrations of TPE-PDEAEAM. Hela cells were washed with prepared PBS, then MTT solution (5 mg/mL, 10 μL) and Hela cell culture medium (90 μL) were added to the cells in each well. The 96-well plate was incubated in a 37°C (CO ₂ 5%) incubator for 4 hours. The MTT-containing medium was removed and dimethyl sulfoxide (DMSO, 100 μL) was added to dissolve the formazan crystals formed by living cells. Absorbance was measured at 492 nm using an RT-6100 microplate reader.

First, HeLa cells were pre-seeded on a 12-well culture plate cell sheet. The medium was 1640 solution containing 1% streptomycin and 10% fetal bovine serum. The 12-well plate was then placed in a humidified incubator with 5% CO2 and a temperature of 37°C for 24 hours. Hela cells were treated with 100 μg mL-1TPE-PAA, and after 24 hours, the cell sheet was removed and the plate was washed three times with phosphate buffered saline. Cell imaging pictures were taken under a confocal fluorescence microscope. Excitation at 332 nm and emission at 460~490 nm were performed using an Olympus FV1000 confocal microscope (Olympus Tokyo Japan).

To assess whether TPE-PDEAEAM polymer quantum dots can be used as probes to track cells for a long time, cells treated with TPE-PDEAEAM polymer quantum dots were passaged repeatedly every 48 hours, and the TPE-PDEAEAM polymer quantum dots of each generation were determined by confocal microscopy. intracellular fluorescence. After HeLa cells were incubated with 100 μgmL ^-1 of TPE-PDEAEAM polymer quantum dots for 24 hours (first generation), the signal intensity of TPE-PDEAEAM polymer quantum dots in HeLa cells was observed (Figure 12). The region with relatively weak intracellular fluorescence intensity may be the location of the nucleus, indicating that the polymer quantum dots successfully escaped the fully aggregated endosomal compartment and selectively stained the cytoplasmic region of HeLa cells. A strong signal was detected from the TPE-PDEAEAM polymer quantum dots in HeLa cells, even after 48 hours of incubation with a concentration of 100 μgmL ^-1 . The stained cells maintained strong fluorescence intensity for up to 20 days (ie, up to 9 passages). This suggests that fluorescent polymers can be used as fluorescent bioprobes for long-term cell tracking.

In summary, multistimuli-responsive polymers of tetrastyrene-grafted poly[N-[2-(diethylamino)-ethyl]acrylamide] (TPE-PDEAEAM polymer quantum dots) were synthesized by ATRP polymerization . Due to the diethylamino and acrylamide groups in the TPE-PDEAEAM polymer quantum dots, the TPE-PDEAEAM polymer quantum dots respond accordingly to the stimulation of temperature, pH and _CO . When the concentration of polymer quantum dots is 2.0 g L ^-1 , the LCST of TPE-PDEAEAM polymer quantum dots in aqueous solution is 60 °C, indicating that its thermal transition temperature may be changed by changing the solution concentration. At the same time, the fluorescence emission intensity of TPE-PDEAEAM polymer quantum dots decreased from 25 °C to 66 °C with the increase of temperature, indicating that the fluorescence of the polymer quantum dots has a certain thermal responsiveness. The fluorescence intensity of the TPE-PDEAEAM polymer quantum dots changes due to the salting-out effect caused by the pH adjuster. Furthermore, since the tertiary amine groups in the TPE-PDEAEAM polymer quantum dots can be protonated by _CO to form charged ammonium bicarbonate, the fluorescence intensity of the polymer decreases significantly with the increase in the volume of _CO added, and I The ratio of /I ₀ to the added volume of carbon dioxide varies from 0.4 to 1.2 ml, and the fluorescence intensity of the polymer quantum dots does not change significantly. More interestingly, fluorescent polymers exhibit unique aggregation-induced emission (AIE) behavior and can be used as long-term tracking fluorophores for cellular imaging.

Description of drawings

Figure 1 shows the H NMR spectrum of TPE-BPM.

Figure 2 shows the hydrogen NMR spectrum of TPE-PDEAEAM.

Figure 3 is the infrared spectrum of TPE-PDEAEAM.

Figure 4 shows the Tyndall effect of TPE-PDEAEAM polymer quantum dots in tetrahydrofuran solution and aqueous solution.

Figure 5 shows the hydrodynamic size of TPE-PDEAEAM polymer quantum dots dissolved in water at a concentration of 2 mg/mL.

Figure 6 shows the morphologies (SEM) of the self-assembled TPE-PDEAEAM polymer quantum dots in aqueous solution.

Figure 7 shows the light transmittance of the TPE-PDEAEAM polymer quantum dot aqueous solution with a concentration of 2 g/L as a function of temperature.

Fig. 8 Variation of fluorescence as TPE-PDEAEAM polymer quantum dot solution from 25°C to 66°C

Figure 9 shows the fluorescence spectra of TPE-PDEAEAM polymer quantum dots in different pH solutions (left) and the relative fluorescence intensity (I/I ₀ ) in aqueous solutions at different pH (right) (TPE-PDEAEAM] = 2.0g L ^-1 ).

Figure 10 shows the fluorescence spectra of TPE-PDEAEAM polymer quantum dots in different CO ₂ volume solutions. Inset: Linearity of relative fluorescence intensity (I/I ₀ ) versus CO ₂ volume.

Figure 11 shows the cytotoxicity test of TPE-PDEAEAM polymer quantum dots.

Figure 12 is a confocal microscope image of HeLa cells stained with TPE-PDEAEAM polymer quantum dots at different passages.

Detailed ways

The synthesis and application of the TPE-PDEAEAM polymer quantum dots of the present invention will be further described below through specific examples.

1. Synthesis of TPE-PDEAEAM polymer quantum dots

(1) Synthesis of monomeric N(2-(diethylamino)ethyl)acrylamide (DEAEAM): N[2-(diethylamino)ethyl]acrylamide (DEAEAM) was synthesized according to literature procedures: in Under magnetic stirring, N,N-diethylethylenediamine (75 mmol, 8.72 g) and CHCl ₃ (75 mL) were added to a 250 mL single-neck round-bottomed flask, which was then placed in an ice-water bath. Then, acryloyl chloride (90 mmol, 7.2 mL) dissolved in _CHCl3 (50 mL) was slowly added dropwise over 1 hour. After complete addition of acryloyl chloride, the reaction was continued at room temperature for 1 h. The reaction solution was washed with NaOH aqueous solution (100 mL, 1 mol/L) and water (100 mL), finally dried with anhydrous MgSO ₄ , the solvent of CHCl ₃ was removed by rotary evaporation, and the crude product was passed through ethyl acetate as eluent to obtain DEAEAM (10.3 g, 85% yield).

(2) Synthesis of hydroxyphenylethylene: Zn powder (20 g, 0.31 mol), 4-hydroxybenzophenone (9.5 g, 0.05 mol) and benzophenone (8.7 g, 0.05 mol) were dissolved in 200 mL of THF , TiCl ₄ (30.0 mL, 0.27 mol) was added under argon protection and refluxed for 24 h. After the reaction was completed, the reaction mixture was cooled to room temperature, 150 mL of 10% K ₂ CO ₃ solution was added and vigorously stirred, then the mixture was filtered and extracted with ethyl acetate to obtain an organic layer. Finally, the product was analyzed by column chromatography. Carry out separation and purification (eluent: V _{ethyl acetate} : V _{petroleum ether} =1:10), finally obtain 6.1 g of pale yellow solid 4-hydroxytetraethylene, and the yield is about 61%.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.13 – 6.97 (m, 15H), 6.89 (d, J = 8.6 Hz, 2H), 6.55 (d, J = 8.6 Hz, 2H), 4.60 (s, 1H) .

¹³ C NMR (151 MHz, CDCl ₃ ) δ 153.96 (s), 144.28 – 143.62 (m), 140.29 (d, J = 33.3 Hz), 136.37 (s), 132.71 (s), 131.52 – 131.16 (m), 127.63 (d, J = 15.1 Hz), 126.24 (s), 114.57 (s).

ESI-MS: M = 348.48.

(3) Synthesis of 4-(12-hydroxydodecyl)tetraethylene (TPE-OH): 4-hydroxytetraethylene (3.48g, 0.01mol), 12-bromo-1-dodecanol ( 0.28 mL, 0.012 mol) and K ₂ CO ₃ (1.66 g, 0.012 mol) were dissolved in 100 mL of anhydrous acetonitrile, and refluxed for 24 h under argon protection. After the reaction, after the reaction solution was cooled to room temperature, the reaction solution was filtered, the organic layer was evaporated, and finally the product was separated and purified by column chromatography (eluent: V _{ethyl acetate} : V _{petroleum ether} =1: 5), 2.6 g of pale yellow solid 4-(12-hydroxydodecyl)tetraphenylene was finally obtained, and the yield was about 61%.

¹ H NMR (600 MHz, CDCl ₃ ) δ 7.16-7.05 (m, 15H), 6.98 (d, J = 8.8 Hz, 2H), 6.67 (d, J = 8.8 Hz, 2H), 3.92-3.85 (m, 2H), 3.65 (t, J = 6.7 Hz, 2H), 3.45- 3.37 (m, 1H), 2.03 -1.82 (m, 2H), 1.81 -1.75 (m, 2H), 1.67 – 1.54 (m, 4H) , 1.47 (dd, J = 14.2, 9.0 Hz, 4H), 1.35 (s, 8H).

¹³ C NMR (151 MHz, CDCl ₃ ) δ 153.96 (s), 144.28 - 143.62 (m), 140.29 (d, J = 33.3 Hz), 136.37 (s), 132.71 (s), 131.52 -131.16 (m), 127.63 (d, J = 15.1 Hz), 126.24 (s), 114.57 (s).

(4) Synthesis of TPE-BPM: take TPE-OH (5.26g, 0.01mol), triethylamine (1.75mL, 0.0125mol) and 2-bromo-2-methylpropionyl bromide (1.5mL, 0.0125mol) , was added to a three-necked flask (250 mL) containing anhydrous THF (150 mL). The mixture was stirred at room temperature for 24 hours, and after the reaction was completed, the reaction solution was filtered; the filtrate was concentrated, and the crude product was separated and purified by column chromatography (eluent: V _{ethyl acetate} : V _{petroleum ether} =1:10 ), and finally 7 g of light yellow solid product TPE-BMP was obtained with a yield of about 65%.

¹ H NMR (600 MHz, H ₂ O) δ 8.89 – 7.06 (m, 15H), 7.06 – 6.92 (m, 2H), 6.85 – 6.49 (m, 2H), 4.39 – 4.16 (m, 2H), 3.93 – 3.70 (m, 2H), 2.14 – 1.91 (m, 7H), 1.93 – 0.80 (m, 24H).

The characterization data of TPE-BPM are shown in Figure 1.

(5) Synthesis of TPE-PDEAEAM: DEAEAM (3 g) was dissolved in a mixed solvent of 10 mL of water and 5 mL of methanol (V _water : V _methanol = 2:1) and stirred for 20 min, and then added sequentially under argon protection. Tris(2-dimethylaminoethyl)amine Me ₆ TREN (0.6 g), CuBr (0.0282 g, 0.1960 mmol), TPE-BMP (0.05 g, 0.05 mmol), after stirring at room temperature for 24 h, the reaction stock solution was Dialyzed with ultrapure water for 72 hours, and freeze-dried the dialysate for 72 hours to obtain 2.3 g of pale yellow solid powder, which is TPE-PDEAEAM. The characterization data of TPE-PDEAEAM are shown in Figure 2 and Figure 3.

¹ H NMR (600 MHz, H ₂ O): δ 7.01-6.25 (m), 3.20 (m), 2.51 (m), 1.90-1.30 (m), 0.93 (m).

2. TPE-PDEAEAM imaging application of Hela cells

First, HeLa cells were pre-seeded on a 12-well culture plate cell sheet. The medium was 1640 solution containing 1% streptomycin and 10% fetal bovine serum. The 12-well plate was then placed in a humidified incubator at 5% concentration _CO and 37 °C temperature for 24 h. Hela cells were treated with 100 μg mL-1TPE-PAA, and after 24 hours, the cell sheet was removed and the plate was washed three times with phosphate buffered saline. Cell imaging pictures were taken under a confocal fluorescence microscope. Excitation at 332 nm and emission at 460~490 nm were performed using an Olympus FV1000 confocal microscope (Olympus Tokyo Japan).

Figure 12 is a confocal microscope image of HeLa cells stained with TPE-PDEAEAM polymer quantum dots at different passages, and the scale bar is 25 microns. Figure 12 shows that after HeLa cells were incubated with 100 μgmL ^-1 of TPE-PDEAEAM polymer quantum dots for 24 hours (first generation), the signal intensity of TPE-PDEAEAM polymer quantum dots in HeLa cells was observed. The region with relatively weak intracellular fluorescence intensity may be the location of the nucleus, indicating that the polymer quantum dots successfully escaped the fully aggregated endosomal compartment and selectively stained the cytoplasmic region of HeLa cells. In addition, a strong signal was detected from the TPE-PDEAEAM polymer quantum dots in HeLa cells, even after 48 hours of incubation with a concentration of 100 μgmL ^-1 (passage 1), we could still observe a strong fluorescent signal. Cells treated with TPE-PDEAEAM polymer quantum dots were passaged repeatedly every 48 hours, and the intracellular fluorescence of TPE-PDEAEAM polymer quantum dots at each passage was determined by confocal microscopy. The results showed that the stained cells maintained strong fluorescence intensity for up to 20 days (ie, up to 9 passages). Therefore, TPE-PDEAEAM polymer quantum dots may be an ideal long-term fluorescent cell tracer.

Claims (9)

1. A preparation method of TPE-PDEAM with multi-responsive polymer quantum dots comprises the following process steps:

(1) synthesis of 4-hydroxyphenyl ethylene: tetrahydrofuran as solvent, zinc powder as reductant and TiCl₄Under the protection of argon, 4-hydroxybenzophenone and benzophenone are refluxed for 22-24 hours in a molar ratio of 1: 1-1: 1.2; after the reaction is finished, cooling to room temperature, and adding K₂CO₃Quenching the solution for reaction, then filtering, extracting with ethyl acetate, and separating and purifying the product by column chromatography to obtain a light yellow solid 4-hydroxy tetraethylene TPE;

(2) synthesis of 4- (12-hydroxydodecyl) tetraphenylethylene: 4-hydroxy-tetraethylene TPE, 12-bromo-1-dodecanol and K₂CO₃Dissolving in anhydrous acetonitrile, and refluxing for 20-24 h under the protection of argon; cooling to room temperature after the reaction is finished, filtering the reaction solution, evaporating an organic layer, and separating and purifying a product by column chromatography to obtain a light yellow solid 4- (12-hydroxy dodecyl) tetraphenylethylene TPE-OH;

(3) and (3) synthesis of TPE-BPM: adding 4- (12-hydroxydodecyl) Tetraphenylethylene (TPE) -OH, triethylamine and 2-bromo-2-methylpropanoyl bromide into anhydrous tetrahydrofuran, and stirring at room temperature for reacting for 20-24 hours; after the reaction is finished, filtering the reaction solution, concentrating the filtrate to obtain a crude product, and separating and purifying the crude product by column chromatography to obtain a faint yellow solid product, namely TPE-BPM;

(4) and (3) synthesis of TPE-PDEAM: dissolving N (2- (diethylamino) ethyl) acrylamide in a water-methanol mixed solvent, sequentially adding tris (2-dimethylaminoethyl) amine, CuBr and TPE-BPM under the protection of argon, stirring at room temperature for 20-24 h, dialyzing the reaction stock solution with ultrapure water for 70-72 h, and freeze-drying the dialyzate for 70-72 h to obtain light yellow solid powder, namely the target product TPE-PDEAM.

2. The method of claim 1, wherein the TPE-PDEAM is prepared from a polymer quantum dot with multiple responsivitiesThe method comprises the following steps: in the step (1), the adding amount of the zinc powder is 3-4 times of the total molar amount of the 4-hydroxybenzophenone and the benzophenone; TiCl (titanium dioxide)₄The adding amount of the (b) is 2-3 times of the total molar amount of the 4-hydroxybenzophenone and the benzophenone.

3. The method for preparing the TPE-PDEAM with the multi-responsive polymer quantum dots as claimed in claim 1, wherein: in the step (2), the molar ratio of the 4-hydroxy tetraethylene to the 12-bromo-1-dodecanol is 1: 1-1: 1.2.

4. The method for preparing the TPE-PDEAM with the multi-responsive polymer quantum dots as claimed in claim 1, wherein: in step (2), 4-hydroxytetraethylene and K₂CO₃The molar ratio of (a) to (b) is 1:1 to 1: 1.2.

5. The method for preparing the TPE-PDEAM with the multi-responsive polymer quantum dots as claimed in claim 1, wherein: in the step (3), the molar ratio of TPE-OH to triethylamine is 1: 1-1: 1.25.

6. The method for preparing the TPE-PDEAM with the multi-responsive polymer quantum dots as claimed in claim 1, wherein: in the step (3), the molar ratio of the TPE-OH to the 2-bromo-2-methylpropanoyl bromide is 1: 1-1: 1.25.

7. The method for preparing the TPE-PDEAM with the multi-responsive polymer quantum dots as claimed in claim 1, wherein: in the step (4), the volume ratio of the water to the methanol in the water-methanol mixed solvent is 1.5: 1-2: 1.

8. The method for preparing the TPE-PDEAM with the multi-responsive polymer quantum dots as claimed in claim 1, wherein: in the step (4), the addition amount of the tris (2-dimethylaminoethyl) amine is 19-20% of the mass of DEAEAM; the addition amount of CuBr is 0.5-1% of the mass of DEAEAM.

9. The method for preparing the TPE-PDEAM with the multi-responsive polymer quantum dots as claimed in claim 1, wherein: in the step (4), the mass ratio of DEAEAM to TPE-BMP is 60: 1-65: 1.

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