阅读说明：本技术 一种制备PPEs高荧光纳米粒子方法 (Method for preparing PPEs high-fluorescence nanoparticles ) 是由 权莉 宋钦涌 童捷 赵应时 魏言春 王毅庆 于 2020-12-11 设计创作，主要内容包括：本发明公开了聚苯醚(PPEs)荧光染料改性技术,特别涉及一种制备PPEs高荧光纳米粒子方法,其步骤包含：PPEs-OH的合成：在施楞克烧瓶中,将2-(2,5-二碘-4-甲基)苯乙醇、1,4-二乙炔基-2,5-二辛氧基苯和哌啶按照物质的量1～3:1～3:1～3.3混合溶解到四氢呋喃中,加入0.00001～1当量的双三苯基磷二氯化钯((PPh-3)-2PdCl-2)和0.00001～1当量的碘化亚铜(CuI),在室温下搅拌20-28h；本发明首先合成PPEs-OH,用羟基引发己内酯开环聚合得到PCL修饰的PPEs,然后与嵌段共聚物PEG-PCL自组装成纳米粒子,PCL的修饰有效阻滞PPEs聚集改善PPEs荧光性能,同时提高PPEs的光稳定性。本发明改善了PPEs的亲水性和生物相容性,使PPEs具有更广泛的应用。(The invention discloses a modification technology of polyphenylene oxide (PPEs) fluorescent dye, and particularly relates to a method for preparing PPEs high-fluorescence nanoparticles, which comprises the following steps: and (3) synthesis of PPEs-OH: in a Schlenk flask, 2- (2, 5-diiodo-4-methyl) phenethyl alcohol and 1, 4-diacetylene are addedMixing and dissolving 2, 5-dioctyloxybenzene and piperidine in an amount of 1-3: 1-3.3 in tetrahydrofuran, and adding 0.00001-1 equivalent of bis (triphenylphosphine) palladium dichloride ((PPh) 3 ) 2 PdCl 2 ) And 0.00001-1 equivalent of cuprous iodide (CuI), and stirring at room temperature for 20-28 h; according to the invention, PPEs-OH is synthesized firstly, caprolactone ring-opening polymerization is initiated by hydroxyl to obtain PPEs modified by PCL, and then the PPEs modified by PCL and a segmented copolymer PEG-PCL are self-assembled into nanoparticles, wherein the modification of PCL effectively retards PPEs from aggregating to improve the fluorescence property of PPEs, and simultaneously improves the light stability of PPEs. The invention improves the hydrophilicity and biocompatibility of the PPEs and enables the PPEs to have wider application.)

1. A method for preparing PPEs high-fluorescence nanoparticles is characterized by comprising the following steps:

s1, synthesizing PPEs-OH

In a Schlenk flask, 2- (2, 5-diiodo-4-methyl)) Phenethyl alcohol, 1, 4-diacetylene-2, 5-dioctyloxybenzene and piperidine are mixed and dissolved in tetrahydrofuran according to the amount of 1-3: 1-3.3, and bis (triphenylphosphine) palladium dichloride with 0.00001-1 equivalent of bis (triphenylphosphine) is added₃)₂PdCl₂) And 0.00001-1 equivalent of cuprous iodide (CuI), and stirring at room temperature for 20-28 h; extracting the obtained polymer with dichloromethane for at least 2 times, removing the solvent by vacuum evaporation to obtain a solid, and washing with 5-20% ammonia and 5-20% hydrochloric acid in sequence; removing the solvent by vacuum filtration; obtaining a product, dissolving the product by using dichloromethane, settling the product by using methanol, filtering the product, and repeating the process for at least 2 times to obtain a dark green solid;

s2, modifying the PPEs-OH prepared by S1 to obtain PCL-PPEs

Drying the PPEs-OH sample prepared in the step S1 at 50-90 ℃ under the vacuum of an oil pump, adding epsilon-caprolactone, heating the mixture to 70-140 ℃ until a polymer is dissolved, adding 0.01-1 equivalent of stannous compound serving as a catalyst after the mixture becomes solid, stopping reaction, dissolving the product in chloroform, settling with methanol, filtering, and drying under the vacuum of the oil pump to obtain the PPEs modified by the PCL;

s3, assembling the PPEs-PCL and the PEG-PCL together

Mixing 0.1-100mg of the PCL modified PPEs (PPEs-PCL) with 0-1000mg of PCL_2K-PEG_5KDissolving in 1-100ml of tetrahydrofuran, slowly dripping the tetrahydrofuran solution into 10-1000ml of water to obtain a solution, removing the tetrahydrofuran, and freeze-drying at-20 ℃ to-50 ℃ to obtain yellow powdery PPEs fluorescent nanoparticles.

2. The method as claimed in claim 1, wherein the molecular weight of PPEs-OH is 500-10000g/mol, the concentration is 0.01mM-100mM, the molecular weight of PCL is 0-150000g/mol, and the concentration is 0.1mM-10000 mM.

3. The method for preparing PPEs high-fluorescence nanoparticles according to claim 1, wherein the molecular weight of epsilon-caprolactone (PCL) in S2 is 0-150000 g/mol.

4. The method for preparing the PPEs high-fluorescence nanoparticles as claimed in claim 1, wherein the catalyst is stannous octoate or tin (II) 2-ethyl hexanoic acid.

5. The method for preparing PPEs high-fluorescence nanoparticles according to claim 4, wherein the amount of the stannous octoate or tin (II) 2-ethyl hexanoate substance is in the range of 0.1mM-10000 mM.

6. The method for preparing PPEs high-fluorescence nanoparticles according to claim 1, wherein the step of removing tetrahydrofuran in S3 is dialysis with a dialysis bag.

Technical Field

The invention relates to a modification technology of polyphenylene oxide (PPEs) fluorescent dye, in particular to a method for preparing PPEs high-fluorescence nanoparticles.

Background

Conjugated polymers such as Polythiophene (PT), poly-p-phenylene vinylene (PPV) and polyphenylene oxides (PPEs) are a class of organic semiconductor materials characterized by large, non-localized hydrogen bonds present in the polymer backbone. They exhibit excellent photophysical properties, such as high fluorescence and photostability, which are key parameters for various biological applications, such as biosensing and cell imaging. The optical properties of conjugated polymers are sensitively dependent on the chemical composition of the polymer backbone and the physical filling of the side chains. Although the theory of energy migration within and between chains is complex and controversial, it is widely accepted that in the solid state, conjugated polymers tend to form H aggregates due to strong dual stacking between chains, leading to a significant decrease in fluorescence quantum yield.

Fluorescent probes are widely studied in biomedical research, such as cellular imaging, biosensing, and even clinical applications for disease diagnosis. Ideally, the probe should have high fluorescence quantum yield and photostability with minimal non-specific interaction with cells or other biological materials. Polyphenylene Ethers (PPEs), one of the important components of conjugated polymers, exhibit extremely high fluorescence Quantum Yields (QYs) in organic solvents, almost reaching 100%. Since the pioneer work report of Bunz in 2003, a number of PPE-based probes have been developed for a wide variety of biosensing applications. In order to develop a water-soluble conjugated polymer probe, carboxylate, sulfate, quaternary ammonium salt or polyethylene glycol plasma side chains are usually added on a polymer main chain, however, a hydrophobic main chain of PPEs still easily aggregates due to strong latent polymerization to form an excimer state, which greatly reduces fluorescence by 10-100 times, and QYs of the probes is usually reduced to 1-10%, which brings great trouble to the accuracy and sensitivity of the PPEs fluorescent probe in application.

Disclosure of Invention

The invention aims to provide a simple and universal method for preparing PPEs high-fluorescence nanoparticles, which comprises the steps of firstly synthesizing PPEs-OH, initiating caprolactone ring-opening polymerization by hydroxyl to obtain PPEs modified by PCL, and then self-assembling the PPEs modified by the PCL and a segmented copolymer PEG-PCL to form the nanoparticles.

A method for preparing PPEs high-fluorescence nanoparticles comprises the following steps:

s1, synthesizing PPEs-OH

In a Schlenk flask, 2- (2, 5-diiodo-4-methyl) phenethyl alcohol, 1, 4-diacetylene-2, 5-dioctyloxybenzene and piperidine are mixed and dissolved into tetrahydrofuran according to the amount of 1-3: 1-3.3, and then 0.00001-1 equivalent of bis (triphenylphosphine) palladium dichloride ((PPh)₃)₂PdCl₂) And 0.00001-1 equivalent of cuprous iodide (CuI), and stirring at room temperature for 20-28 h; extracting the obtained polymer with dichloromethane for at least 2 times, removing the solvent by vacuum evaporation to obtain a solid, and washing with 5-20% ammonia and 5-20% hydrochloric acid in sequence; removing the solvent by vacuum filtration; obtaining a product, dissolving the product by using dichloromethane, settling the product by using methanol, filtering the product, and repeating the process for at least 2 times to obtain a dark green solid;

s2, modifying the PPEs-OH prepared by S1 to obtain PCL-PPEs

Drying the PPEs-OH sample prepared in the step S1 at 50-90 ℃ under the vacuum of an oil pump, adding epsilon-caprolactone, heating the mixture to 70-140 ℃ until a polymer is dissolved, adding 0.01-1 equivalent of stannous compound serving as a catalyst after the mixture becomes solid, stopping reaction, dissolving the product in chloroform, settling with methanol, filtering, and drying under the vacuum of the oil pump to obtain the PPEs modified by the PCL;

s3, assembling the PPEs-PCL and the PEG-PCL together

Mixing 0.1-100mg of the PCL modified PPEs (PPEs-PCL) with 0-1000mg of PCL_2K-PEG_5KDissolving in 1-100ml of tetrahydrofuran, slowly dripping the tetrahydrofuran solution into 10-1000ml of water to obtain a solution, removing the tetrahydrofuran, and freeze-drying at-20 ℃ to-50 ℃ to obtain yellow powdery PPEs fluorescent nanoparticles.

Further, the molecular weight of the PPEs-OH is in the range of 500-10000g/mol, the concentration is in the range of 0.01mM-100mM, the molecular weight of the PCL is in the range of 0-150000g/mol, and the concentration is in the range of 0.1mM-10000 mM.

Further, the molecular weight of epsilon-caprolactone (PCL) in S2 is 0-150000 g/mol.

Further, the catalyst is stannous octoate or tin (II) 2-ethyl hexanoic acid.

Preferably, the amount of stannous octoate or tin (II) 2-ethylhexanoate species ranges from 0.1mM to 10000 mM. Further, the method for removing tetrahydrofuran in S3 is dialysis with a dialysis bag.

The synthesis route of PCL-PPEs is as follows:

compared with the prior art, the invention has the beneficial effects that:

firstly, epsilon-caprolactone (PCL) is used for modification, and the epsilon-caprolactone (PCL) can effectively retard the accumulation of PPEs and protect fluorescence;

modifying with epsilon-caprolactone (PCL) to be equivalent to adding a protective shell to each PPEs, so that the light stability of the PPEs is improved while the fluorescence is protected;

and thirdly, the polyphenylene oxide (PPEs) is prepared into nano particles, so that the water solubility and the biocompatibility of the polyphenylene oxide (PPEs) are increased, and the application of the polyphenylene oxide (PPEs) in the fields of organic electronics and sensing is expanded.

Drawings

FIG. 1 is a fluorescence diagram of PPEs modified with different PCL chain lengths, as shown in the case that the molecular weight of PPEs is less than or equal to 10000da, the fluorescence intensity of PPEs increases with the increase of PCL molecular weight;

FIG. 2 is an electron microscope image and a dynamic light scattering image of PPEs with different PCL chain length modifications and PEG-PCL co-assembled nanoparticles, and the images are specifically illustrated as follows: before and after PCL modification of PPEs, the particle size of the nanoparticle co-assembled with the PEG-PCL is slowly increased along with the increase of the molecular weight of the PCL, for example, the particle size of the nanoparticle co-assembled with the PCL0-PPEs and the PEG-PCL is 20nm, and the PCL is₂₂₈₀The particle diameter of the nanoparticle with PPEs and PEG-PCL assembled together is 23nm, and PCL₆₈₄₀-the particle size of the nanoparticles co-assembled with PPEs and PEG-PCL is 25 nm;

FIG. 3 shows the light stability before and after PCL modification of PPEs, and the stability is specifically shown as follows: after PCL modification, the light stability of PPEs is greatly improved, and after 1h of illumination, the fluorescence intensity is reduced by 4%.

Detailed Description

Chemical Agents stannous octoate, ε -caprolactone and other Agents involved the highest purity, PCL, was obtained from Sigma Aldrich_2K-PEG_5KPurchased from (Nanjing Juyou).

Using an instrument: dynamic light scattering nanometer particle size potentiometer, melvin instrument; transmission electron microscope JEOL JEM-2010 (HR); an ultraviolet-visible spectrophotometer (UV-3600, Shimadzu, Japan); horiba PTI QuantaMaster 400 steady state fluorescence system; bruker1H proton NMR 400DRX spectrometer.

The fluorescence detection method related to the following examples is specifically as follows: testing the absorbance and emission spectra of the PPEs nanoparticles in a diluted chloroform solution; relative fluorescence quantum yields of PPEs nanoparticles were measured by absorption and emission spectroscopy with quinine sulfate as a reference.

Example 1

S1. Synthesis of PPEs-OH

Into a Schlenk flask were added 1.00mmol of 2- (2, 5-diiodo-4-methyl) phenethyl alcohol, 1.00mmol of 1, 4-diacetylene-2, 5-dioctyloxybenzene and 1.60mL of piperidine, the three substances were dissolved with 1.60mL of Tetrahydrofuran (THF), and 0.1. mu. mol of bis (triphenylphosphine) palladium dichloride ((PPh)₃)₂PdCl₂) 1.80 mu mol of cuprous iodide (CuI) are mixed and stirred for 24 hours at room temperature;

extracting the obtained polymer with 1ml dichloromethane for 3 times, vacuum evaporating to remove solvent to obtain solid, and washing with 10% ammonia water and 10% hydrochloric acid; vacuum filtering to remove solvent to obtain product, extracting with 1ml dichloromethane for 3 times, precipitating with methanol, and filtering to obtain dark green solid P0;

s2, synthesis of PPEs-PCL: taking 10mg of sample PPEs-OH prepared by S1, drying at 70 ℃ under vacuum of an oil pump, adding 0.2ml of epsilon-caprolactone, heating the mixture to 110 ℃ until the polymer is dissolved, then adding stannous octoate (0.1mg, 0.3mol), stopping the reaction after the mixture becomes solid, dissolving the product in 2ml of chloroform, settling with methanol, filtering, and drying under vacuum of the oil pump to obtain a product P20;

s3, assembling PPEs-PCL: 1.00mg of PPEs-PCL prepared in S2 was dissolved in 1ml of tetrahydrofuran, and the mixture was dropped into 10ml of deionized water under the vortex action for 1 hour, and then the tetrahydrofuran was removed by dialysis with a dialysis bag and lyophilized at-50 ℃ to obtain yellow powder.

The fluorescence quantum yield of the target prepared in example 1 was 0.05 according to the fluorescence detection method described above.

Example 2

S1. Synthesis of PPEs-OH

2.00mmol of 2- (2, 5-diiodo-4-methyl) phenethyl alcohol, 2.00mmol of 1, 4-diacetylene-2, 5-dioctyloxybenzene and 3.20mL of piperidine were added to a Schlenk flask, the three substances were dissolved with 3.20mL of Tetrahydrofuran (THF), and 0.2. mu. mol of bis-triphenylene was addedPhosphorus palladium dichloride ((PPh)₃)₂PdCl₂) 3.60. mu. mol of cuprous iodide (CuI) and stirring at room temperature for 24 hours;

extracting the obtained polymer with 2ml dichloromethane each time for 3 times, removing the solvent by vacuum evaporation to obtain a solid, and washing with 10% ammonia water and 10% hydrochloric acid in sequence; vacuum-filtering to remove solvent to obtain product, extracting with 2ml dichloromethane for 3 times, precipitating with methanol, and filtering to obtain dark green solid P0;

s2, synthesis of PPEs-PCL: taking 100mg of sample PPEs-OH prepared by S1, drying at 70 ℃ under vacuum of an oil pump, adding 2ml of epsilon-caprolactone, heating the mixture to 110 ℃ until the polymer is dissolved, then adding tin (II) 2-ethylhexanoic acid (1mg,3mol), stopping the reaction after the mixture becomes solid, dissolving the product in 5ml of chloroform, settling with methanol, filtering, and drying under vacuum of the oil pump to obtain a product P20;

s3.PPEs-PCL and PCL_2K-PEG_5KAnd (3) assembling: 5mgPPEs-PCL and 55mgPCL prepared from S2_2K-PEG_5K(commercially available) was dissolved in 4ml of tetrahydrofuran, and the mixture was dropped into 15ml of deionized water under vortex action for 1 hour, then dialyzed with a dialysis bag to remove tetrahydrofuran, and lyophilized at-50 ℃ to give a yellow powder.

The fluorescence quantum yield of the target prepared in example 2 was 0.37 according to the fluorescence detection method described above.

Example 3

And A, synthesizing PPEs-PCL: 100mg of sample P20 prepared in S2 of example 2 was dried under oil pump vacuum at 70 ℃ and 4ml of epsilon-caprolactone was added, the mixture was heated to 110 ℃ until the polymer dissolved, stannous octoate (1mg,3mol) was added, the reaction was stopped after the mixture solidified, the product was dissolved in chloroform, settled with methanol, filtered and dried under oil pump vacuum to give product P60.

PPEs-PCL and PCL_2K-PEG_5KAnd (3) assembling: mixing the 1.00mgPPEs-PCL and 11mgPCL prepared in the step A_2K-PEG_5K(commercially available) dissolving in 1ml, dropping the mixture into 15ml deionized water under vortex action for 1h, and dialyzingThe bag was dialyzed to remove tetrahydrofuran and lyophilized at-50 ℃ to give a yellow powder.

The fluorescence quantum yield of the target prepared in example 3 was 0.78 according to the fluorescence detection method described above.

Example 4

The technical scheme is a further experiment based on the scheme of the embodiment 3, the implementation mode is the same as that of the embodiment 3, and the only difference is that 100mg of P60 is reacted with 2ml of epsilon-caprolactone to obtain P100; the rest of the embodiments are the same as embodiment 3, and are not described herein.

The fluorescence quantum yield of the target prepared in example 4 was 0.87 according to the fluorescence detection method described above.

Example 5

The technical scheme is a further experiment based on the scheme of the embodiment 4, the implementation mode is the same as that of the embodiment 3, and the only difference is that 100mg of P100 is reacted with 5ml of epsilon-caprolactone to obtain P200; the rest of the embodiments are the same as embodiment 3, and are not described herein.

The fluorescence quantum yield of the target prepared in example 5 was 0.96 according to the fluorescence detection method described above.

Example 6

The technical scheme is a further experiment based on the scheme of the embodiment 5, the implementation mode is the same as that of the embodiment 3, and the only difference is that 100mg of P200 is reacted with 10ml of caprolactone to obtain P400; the rest of the embodiments are the same as embodiment 3, and are not described herein.

The fluorescence quantum yield of the target prepared in example 6 was 0.68 according to the fluorescence detection method described above.

The invention adopts the technical scheme that firstly PCL is used for modifying PPEs and then is assembled with PEG-PCL to form nano particles, the length of the PCL has certain influence on the fluorescence performance of the PPEs, namely, under the condition that the molecular weight of the PPEs is less than or equal to 10000da, the molecular weight of the PCL and the fluorescence performance of the PPEs form a parabolic relation: that is, as the molecular weight of PCL increases, the fluorescence quantum yield of PPEs increases, and when the molecular weight of PCL increases to a certain value (the specific value depends on the molecular weight of PPEs), the fluorescence quantum yield of PPEs reaches an optimal value, and then the fluorescence quantum yield of PPEs decreases as the molecular weight of PCL increases.

Comparative example

The prior preparation process of the polyphenyl ether fluorescent material comprises the following specific steps: (Macromolecules, Vol.40, No.6,2007) 1, 4-diiodo-2-methylbenzene (482mg,1.40mmol) was mixed with piperidine (1.5mL), THF (1.5mL), (PPh)₃)₂PdCl₂(2mg,2mol,0.2 mol%) and CuI (1mg,5mol,0.4 mol%) were mixed in a Schlenk flask. Protected with acetylene gas (34mL,1.40mmol) and the mixture stirred at room temperature for 24h, then extracted with dichloromethane (50 mL). The organic layer was washed with ammonium hydroxide (10%, 250 mL) and hydrochloric acid (10%, 50 mL). The organic layer was over MgSO₄Dried above and concentrated under reduced pressure. The remaining mixture (10ml) was precipitated with acidified methanol. The polymer was collected by filtration through a sintered funnel, redissolved in dichloromethane and purified by reprecipitation with methanol. A yellow-green polymer was obtained.

According to the fluorescence detection method described above, the fluorescence quantum yield of the target prepared in the comparative example was 0.38.

The quantum yield of the product of the existing method for preparing the polyphenyl ether fluorescent material is lower due to the accumulation effect of PPEs molecules, and the fluorescence performance of the product obtained by the scheme can be well protected and improved.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.