CN110092858B - Preparation method of carboxyl and polypeptide modified AIE polymer nanoparticles - Google Patents

Preparation method of carboxyl and polypeptide modified AIE polymer nanoparticles Download PDF

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CN110092858B
CN110092858B CN201910400547.3A CN201910400547A CN110092858B CN 110092858 B CN110092858 B CN 110092858B CN 201910400547 A CN201910400547 A CN 201910400547A CN 110092858 B CN110092858 B CN 110092858B
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carboxyl
emulsion
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曹志海
梁小琴
齐琪
余棒
方星宇
裘乘锦
胡亚新
秘一芳
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Zhejiang University of Technology ZJUT
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Abstract

A preparation method of AIE polymer nanoparticles modified by carboxyl and polypeptide comprises the following steps: 1) preparing an emulsifier aqueous solution; 2) dissolving AIE molecules and an austenite curing effect inhibitor in a mixed solution of a carboxyl functional monomer and a hydrophobic monomer to obtain an oil phase solution; 3) adding the emulsifier aqueous solution into the oil phase solution, stirring and pre-emulsifying to obtain a coarse emulsion, and performing ultrasonic treatment to obtain a monomer fine emulsion; introducing nitrogen to remove oxygen, and adding a water-soluble initiator to react; 4) dissolving a carbodiimide condensing agent in an acidic pH buffer solution, and adding the carbodiimide condensing agent into the emulsion prepared in the step 3) for activation reaction; 5) dissolving omega-amino maleimide in an alkaline pH buffer solution to obtain an omega-amino maleimide solution; 6) adding the omega-amino maleimide solution into the emulsion prepared in the step 4) for reaction; 7) adding a polypeptide aqueous solution with a terminal containing a cysteine sequence unit into the emulsion prepared in the step 6) to react to prepare the AIE polymer nano particle modified by carboxyl and polypeptide.

Description

Preparation method of carboxyl and polypeptide modified AIE polymer nanoparticles
(I) technical field
The invention relates to a preparation method of surface-modified aggregation-induced emission (AIE) polymer nanoparticles.
(II) background of the invention
The fluorescent nanoparticles are important functional nano materials, have the characteristics of good water dispersibility, high light stability, low cytotoxicity, surface modification and the like, and have important application values in a plurality of high-end fields such as cell imaging, disease diagnosis, biosensing and the like. However, the nanoparticles using the conventional aggregation-induced quenching (ACQ) fluorescent molecules as functional components often have the defects of low fluorescence quantum yield, narrow fluorescence intensity control interval and the like, and the application of the nanoparticles is limited to a certain extent. In 2001, the group of subjects in the Tang-loyal academy first reported a new class of fluorescent molecules with AIE effect, which in the aggregated state, would emit intense fluorescence due to restricted intramolecular rotation [ Chem Soc Rev 2011,40, 5361-. The AIE molecules are well suited for the preparation of high fluorescence intensity and high phiFThe polymer nanoparticles of (1). Because, on the one hand, the higher the concentration of AIE molecules, the greater the degree of molecular aggregation and the brighter the fluorescence(ii) a On the other hand, the molecular confinement degree inside the particles taking the polymer as the matrix is high, which is also beneficial to enhancing the fluorescence of the AIE molecules.
Currently, various methods for preparing AIE polymer nanoparticles have been developed, such as researchers developed a series of self-assembly combined with RAFT polymerization, Schiff base condensation reactions, Mannich reactions, click reactions, and dynamic covalent bond interactions to prepare different AIE polymer nanoparticles [ Colloids Surf, B2017, 150, 114-; j Colloid Interface Sci 2018,519, 137-144; polym Chem 2017,8, 4746-4751; dye Pigm 2018,151, 123-. However, the self-assembly method generally requires the block copolymer to be synthesized in advance, and the assembly process is often controlled to be performed at a low concentration, so that it has a problem of low synthesis efficiency.
The miniemulsion polymerization system is a heterogeneous polymerization system with monomer droplets as a dispersed phase and water as a continuous phase, and polymer particles are nucleated by the monomer droplets during polymerization to form [ Prog Polym Sci 2002,27, 1283-1346. Adv Polym Sci 2005,175, 129-. The monomer droplets are both storage sites for the monomer and polymerization sites. Various polymeric nanoparticles [ Angew Chem Int Ed 2009,48, 4488-. The inventors first proposed the preparation of AIE polymer nanoparticles by miniemulsion polymerization [ Polym Chem 2015,6, 6378-6385 ]. So far, the surface of AIE polymer nanoparticles prepared by miniemulsion polymerization is not modified by specific polypeptide, so that the application value of the AIE polymer nanoparticles in the field of cell selective imaging is limited. Therefore, on the basis of the existing research, the invention provides that styrene, acrylic ester and/or methacrylic ester are taken as monomers, carboxyl functional monomers are added, AIE molecules are taken as fluorescent components, and the AIE polymer nano particles with surface carboxyl modified are prepared by one-step emulsion polymerization; then, the carboxyl reaction of the omega-amino maleimide and the surface of the AIE polymer nanoparticle activated by the carbodiimide condensing agent is utilized to realize the modification of the maleimide group on the surface of the nanoparticle; and finally, realizing the polypeptide modification on the surface of the AIE polymer nanoparticle by using a Michelal addition reaction between sulfydryl on a cysteine sequence unit in a polypeptide molecule and a maleimide group. The prepared AIE polymer nano particles modified by carboxyl and polypeptide have the advantages of high fluorescence brightness, good light stability, good storage stability, strong cell imaging capability and the like.
Disclosure of the invention
The invention aims to provide a novel method for preparing carboxyl and polypeptide modified AIE polymer nanoparticles based on miniemulsion polymerization technology, and the prepared carboxyl and polypeptide modified AIE polymer nanoparticles have the characteristics of high fluorescence intensity, good light stability, good storage stability, strong cell imaging capability and the like.
The technical scheme adopted by the invention is as follows:
a method for preparing carboxyl-and polypeptide-modified AIE polymer nanoparticles, the method comprising the steps of:
(1) dissolving an emulsifier in deionized water to obtain an emulsifier aqueous solution, wherein the mass consumption of the emulsifier is 0.1-10% of the mass consumption of the deionized water;
the emulsifier is selected from at least one of the following: anionic, amphoteric and nonionic emulsifiers;
(2) dissolving AIE molecules and an austenite curing effect inhibitor in a mixed solution of a carboxyl functional monomer and a hydrophobic monomer to obtain an oil phase solution, wherein the mass consumption of the AIE molecules is 0.1-30% of the total mass of the monomers; the mass usage of the austenite curing effect inhibitor is 1-12% of the total mass of the monomers; the mass usage of the carboxyl functional monomer is 0.1-20% of the total mass usage of the monomer; the total mass of the monomers refers to the total mass of the carboxyl functional monomers and the hydrophobic monomers;
the carboxyl functional monomer is at least one of acrylic acid and methacrylic acid shown in a formula (I) and itaconic acid monomer shown in a formula (II);
Figure GDA0003024875590000031
in the formula (I), R1Is H or CH3，
Figure GDA0003024875590000032
The AIE molecule is selected from at least one of the following AIE-1 to AIE-42 molecules:
Figure GDA0003024875590000041
1. AIE molecules with a typical infrastructure
Figure GDA0003024875590000042
Figure GDA0003024875590000051
2. Cyano group modified AIE compounds
Figure GDA0003024875590000052
Figure GDA0003024875590000061
TPE modified cyanine dye derivatives
Figure GDA0003024875590000071
Figure GDA0003024875590000081
Figure GDA0003024875590000091
TPE modified BODIPY derivatives
Figure GDA0003024875590000092
Figure GDA0003024875590000101
Figure GDA0003024875590000111
TPE, TPA or anthrone modified DDP derivatives
Figure GDA0003024875590000112
Figure GDA0003024875590000121
6. dicyanomethylene-4H-pyran derivatives
Figure GDA0003024875590000122
Figure GDA0003024875590000131
7. AIE compound composed of benzobis (thiadiazole) and TPA
Figure GDA0003024875590000132
Figure GDA0003024875590000141
Figure GDA0003024875590000151
TPE-modified benzothiadiazole derivatives
The inhibitor of the austenite ripening effect is selected from at least one of the following: aliphatic hydrocarbon of C14-C22, aliphatic alcohol of C14-C22;
the hydrophobic monomer is selected from at least one of the following: styrene, acrylates and methacrylates represented by formula (III);
Figure GDA0003024875590000161
in the formula (III), R7Is H or methyl; r8Is C1-C5 alkyl;
(3) adding the emulsifier aqueous solution prepared in the step (1) into the oil phase solution prepared in the step (2) to ensure that the total mass of monomers in the oil phase solution is 1-50% of the mass of water in the emulsifier aqueous solution, and stirring for pre-emulsification to obtain a crude emulsion; placing the container filled with the coarse emulsion in an ice-water bath, and carrying out ultrasonic treatment for 0.5-60 min under the power of 25-950W to prepare a monomer fine emulsion; introducing nitrogen to remove oxygen, adding a water-soluble initiator, reacting for 1-48 h at the temperature of 25-90 ℃ under the protection of nitrogen to prepare AIE polymer nanoparticle emulsion with carboxyl modified on the surface, and dialyzing to obtain purified AIE polymer nanoparticle emulsion with carboxyl modified;
the water-soluble initiator is selected from at least one of the following: persulfate, water-soluble azo, oxidant and reductant form a redox system; the electrical properties of the water-soluble initiator and the emulsifier need to be matched; the mass usage of the water-soluble initiator is 0.05-10% of the total mass of the monomers;
(4) dissolving a carbodiimide condensing agent in an acidic pH buffer solution with the pH value of 5.0-7.0 to prepare a carbodiimide condensing agent buffer solution with the mass fraction of 0.005-10%, and adding the carbodiimide condensing agent buffer solution into the carboxyl modified AIE polymer nanoparticle emulsion prepared in the step (3), wherein the mass consumption of the carbodiimide condensing agent is 50-600% of the consumption of the carboxyl functional monomer, and activating for 1 min-6 h to prepare the activated carboxyl modified AIE polymer nanoparticle emulsion; the acidic pH buffer is selected from one of: disodium hydrogen phosphate-citric acid buffer solution, citric acid-sodium citrate buffer solution, acetic acid-sodium acetate buffer solution, 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution and potassium dihydrogen phosphate-sodium hydroxide buffer solution; the carbodiimide condensing agent is one selected from N, N '-dicyclohexylcarbodiimide, N' -diisopropylcarbodiimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride;
(5) dissolving omega-amino maleimide in an alkaline pH buffer solution with the pH value of 7.0-9.0 to obtain an omega-amino maleimide solution, wherein the mass fraction of the omega-amino maleimide is controlled within the range of 0.01-10%; the alkaline pH buffer is selected from one of the following: tris-hydroxymethyl aminomethane-hydrochloric acid buffer solution, boric acid-borax buffer solution, Phosphate Buffer Salt (PBS) solution; the omega-amino maleimide is selected from one of the compounds shown in the formula (IV):
Figure GDA0003024875590000171
in the formula (IV), R9Is C1-C6 alkyl;
(6) adding the omega-amino maleimide solution prepared in the step (5) into the activated carboxyl modified AIE polymer nanoparticle emulsion prepared in the step (4), enabling the mass usage of omega-amino maleimide to be 10% -1000% of the mass usage of carboxyl functional monomers, adjusting the pH value of the emulsion to 7.0-9.0 by using a pH regulator, reacting at room temperature for 1-72 h to prepare a maleimide modified AIE polymer nanoparticle emulsion, and dialyzing to obtain a purified maleimide modified AIE polymer nanoparticle emulsion; the pH regulator is selected from at least one of the following: sodium citrate, borax, sodium hydroxide, carbonate and hydrate thereof, bicarbonate and ammonia water;
(7) dissolving polypeptide with a tail end containing a cysteine sequence unit into deionized water to prepare a polypeptide aqueous solution, wherein the mass fraction of the polypeptide is controlled within the range of 0.01-10%; adding a polypeptide aqueous solution into the AIE polymer nanoparticle emulsion modified by maleimide prepared in the step (6), enabling the mass usage of the polypeptide to be 5% -100% of the mass usage of the carboxyl functional monomer, reacting at room temperature for 1-72 h to prepare AIE polymer nanoparticles modified by carboxyl and polypeptide, and dialyzing to obtain purified AIE polymer nanoparticle emulsion modified by carboxyl and polypeptide;
the polypeptide containing a cysteine sequence unit at the tail end is selected from at least one of the polypeptides in the table 1;
TABLE 1 Polypeptides with terminal cysteine sequence units
Figure GDA0003024875590000181
In the miniemulsion polymerization system of the present invention, the selection of the emulsifier and initiator has an effect on the particle size of the nanoparticles, the stability of the system and the cell uptake capacity. Considering that electrostatic interaction between the initiator and the emulsifier with opposite charges can cause system instability, the electrical properties of the initiator and the emulsifier need to be matched, for example, when a water-soluble anionic initiator is selected, the emulsifier is selected from an anionic emulsifier, a nonionic emulsifier or a composite emulsifier formed by the anionic emulsifier and the nonionic emulsifier; when the water-soluble cationic initiator is selected, a nonionic emulsifier is selected.
In step (1) of the present invention, the emulsifier is selected from at least one of the following: anionic emulsifiers, amphoteric emulsifiers and nonionic emulsifiers. The anionic emulsifier can be selected from at least one of the following: alkyl sulfonate emulsifier R10-SO3M, alkyl sulfate emulsifier R11-OSO3M and alkyl benzene sulfonate emulsifier R12-C6H4-SO3M, wherein R10And R11Is a fatty chain of C10-C20, R12Is a fatty chain of C10-C18, M is Na+Or K+. The amphoteric emulsifier can be selected from at least one of the following: dodecyl amino propionic acid, octadecyl dihydroxyEthylamine oxide, Carboxylic betaine R13N+(CH3)2CH2COO-, sulfobetaine R14N+(CH3)2CH2CH2SO3-or R15N+(CH3)2CH2CH2CH2SO3-, wherein R13、R14And R15Is a fatty chain of C12-C18. The nonionic emulsifier can be selected from at least one of the following: OP-series emulsifier, O-series emulsifier, MOA-series emulsifier, Tween-series emulsifier and SG-series emulsifier. Wherein the OP-series emulsifier can be at least one of OP-9, OP-10 and OP-15. The O-series emulsifier may be at least one of O-10, O-20, O-30 and O-50. The MOA series emulsifier may be at least one of MOA-7, MOA-9, MOA-15 and MOA-23. The Tween series emulsifier can be at least one of Tween-20, Tween-40, Tween-60, Tween-80 and Tween-85. The SG-series emulsifier may be SG-40 and/or SG-100. In view of the stability of the miniemulsion system and the biocompatibility of the AIE polymer nanoparticles, the emulsifier is preferably an anionic emulsifier, a nonionic emulsifier, or a composite emulsifier consisting of an anionic emulsifier and a nonionic emulsifier, the nonionic emulsifier is more preferably at least one of O-series emulsifiers, MOA-series emulsifiers, and tween-series emulsifiers, and the anionic emulsifier is more preferably sodium lauryl sulfate or sodium dodecyl benzene sulfonate.
In step (2) of the present invention, considering that the AIE polymer nanoparticles prepared by the present invention are mainly applied to the field of cell imaging, it is preferable that AIE molecules with an emission wavelength of more than 500nm be used as a fluorescent component, and the mass usage amount of the AIE molecules is preferably 0.1% to 10% of the total mass usage amount of the monomers.
In step (2) of the present invention, the austenite ripening effect inhibitor is preferably a C16-C22 alkane, in view of stability of the fine emulsion droplets.
The inventors have found that, in the preparation of carboxyl modified AIE polymer nanoparticles by miniemulsion polymerization, the amount of carboxyl functional monomer has an influence on the colloidal stability of the system, the size and distribution of the particles, and the modification degree of the polypeptide. Generally speaking, with the increase of the dosage of the carboxyl functional monomer, the carboxyl modification density on the surface of the AIE polymer nanoparticle is increased, which is beneficial to the subsequent modification of maleimide group and polypeptide. Within a certain range, the increase of the dosage of the carboxyl functional monomer has little influence on the colloidal stability of the system, the particle size of the nano particles and the distribution thereof, but the excessively high dosage of the carboxyl monomer can lead to the deterioration of the colloidal stability of the system, the enlargement of the particle size and the widening of the size distribution. Therefore, the proper amount of carboxyl functional monomer should be determined according to the requirements of the colloidal stability of the system, the particle size of the AIE polymer nanoparticles and the subsequent degree of carboxyl and polypeptide modification.
In step (2), in consideration of providing sufficient reaction sites for subsequent polypeptide modification and ensuring that the particles have good colloidal stability, the dosage of the carboxyl functional monomer is controlled to be 0.1-20%, preferably 0.5-15% of the mass dosage of the monomer.
In the step (3), in order to prevent the miniemulsion from being overheated in the ultrasonic process, the container filled with the macroemulsion is placed in an ice-water bath for ultrasonic treatment, the ultrasonic power is preferably 50W-600W, and the ultrasonic time is preferably 5 min-30 min.
In step (3) of the present invention, the water-soluble initiator is selected from at least one of the following: persulfate, water-soluble azo, oxidant and reductant constitute redox system. The persulfate type initiator may be selected from at least one of: sodium persulfate, potassium persulfate and ammonium persulfate. The water-soluble azo initiator may be selected from at least one of: azodiisobutyl amidine hydrochloride, azodiisobutyl imidazoline hydrochloride, azodicyano valeric acid and azodiisopropyl imidazole. In the redox system, the reducing agent can be sodium bisulfite, sodium thiosulfate or sodium sulfite, and the oxidizing agent can be persulfate or hydrogen peroxide. The water-soluble initiator is preferably ammonium persulfate, potassium persulfate, azobisisobutyramidine hydrochloride in view of colloidal stability of the polymerization reaction system.
In step (3) of the present invention, the dialysis is preferably performed as follows: utilizing the molecular weight cut-off of 5000 g/mol–1Dialyzing the dialysis bag for three days, and changing waterThe frequency is changed every 2h for the first day, every 3h for the second day and every 6h for the third day.
In the step (4), the reaction of activating the carboxyl AIE polymer nanoparticles by the carbodiimide condensing agent is carried out in a weakly acidic buffer solution, so that deprotonation of carboxyl can be avoided, and the activation efficiency is improved. Preferably, a buffer solution with the pH value within the range of 6.0-6.5 is used as a reaction medium. Comprehensively considering the activation efficiency and degree of carboxyl on the surface of the AIE polymer nanoparticles, the activation reaction time is preferably 5 min-3 h.
In the step (5), the reaction of the activated carboxyl modified AIE polymer nanoparticles and omega-amino maleimide is carried out in a weakly alkaline reaction medium, so that the protonation of amino can be avoided, and the efficiency of amide condensation reaction is improved; in view of the efficiency of the reaction of omega-amino maleimide with the carboxyl groups on the surface of the nanoparticles, omega-amino maleimide is preferably N- (2-aminoethyl) maleimide.
In the step (6), the amide condensation reaction of the activated carboxyl AIE polymer nanoparticles and omega-amino maleimide is carried out in a weak alkaline medium, so that the protonation of amino groups can be avoided, and the efficiency of the amide condensation reaction is improved. The pH value of the reaction medium is preferably 7.0-8.5. The reaction time is preferably 1 to 36 hours, taking the efficiency and degree of the reaction between the carboxyl group and the omega-amino maleimide into comprehensive consideration.
In step (6) of the present invention, the dialysis is preferably performed as follows: the molecular weight cut-off of the prepared maleimide modified AIE polymer nano particle emulsion is 5000 g.mol–1The dialysis bag is dialyzed for three days, the water changing frequency is changed every 2h for the first day, every 3h for the second day, and every 6h for the third day.
In the step (7) of the invention, the polypeptide is connected to the surface of the AIE polymer nanoparticle through a Michelal addition reaction between a sulfydryl of a cysteine sequence unit and a double bond of maleimide. In order to ensure that the polypeptide and the maleimide group on the surface of the nanoparticle are fully reacted, the reaction time is preferably 3-48 h.
In step (7) of the present invention, the dialysis is preferably performed as follows: modifying the carboxyl group and polypeptideThe molecular weight cut-off of the AIE polymer nanoparticle emulsion is 5000 g.mol–1The dialysis bag is dialyzed for three days, the water changing frequency is changed every 2h for the first day, every 3h for the second day, and every 6h for the third day.
When the AIE polymer nano particle modified by carboxyl and polypeptide is applied to cell imaging, the AIE polymer nano particle has higher fluorescence intensity, can be efficiently absorbed by cells, and even can be selectively and efficiently absorbed, so that a better imaging effect can be obtained. Depending on the type of cell being imaged, different polypeptides may be selected to modify the AIE polymer nanoparticles. For example, the RGD peptide contains an arginine-glycine-aspartic acid sequence and is a specific ligand of integrin α v β 3 overexpressed in tumor cells, and thus, the AIE polymer nanoparticles modified with the RGD peptide can be used for detection of tumor cells. The cell-penetrating peptide HIV-1TAT can promote the uptake rate of the cell to the nano-particle and can carry out fluorescence tracing on the physiological activity of the cell, so that the AIE polymer nano-particle modified by the HIV-1TAT can be used for researching the physiological processes of apoptosis and the like.
Compared with the prior art, the invention has the following beneficial effects: provides a novel method for efficiently preparing AIE polymer nano particles modified by carboxyl and polypeptide by combining a miniemulsion polymerization technology and a click reaction. Through the miniemulsion copolymerization reaction with the participation of carboxyl functional monomers, the carboxyl modification of the surface of the nanoparticle is realized while the AIE molecule is embedded in the polymer matrix. Maleimide groups are attached to the particle surface by means of an amide condensation reaction of omega-amino maleimide and AIE polymer nanoparticles whose surface carboxyl groups are activated by a carbodiimide condensing agent. And finally, efficiently modifying the polypeptide on the surface of the AIE polymer nanoparticle through one-step Michelal addition reaction with the polypeptide containing sulfhydryl groups to prepare the carboxyl and polypeptide modified AIE polymer nanoparticle with good biocompatibility and cell imaging effect. The method has the advantages that: (1) the particle characteristics of the AIE polymer nanoparticles can be effectively regulated and controlled by adjusting the synthesis parameters of the miniemulsion; (2) the emission wavelength and the emission intensity of the AIE polymer nano-particles can be flexibly regulated and controlled in a wider range through the type and the loading capacity of AIE molecules; (3) the carboxyl modification amount on the surface of the AIE polymer nano-particle and the modification capability of the polypeptide can be conveniently adjusted by the using amount of the carboxyl functional monomer; (4) the Michelal addition reaction of maleimide and sulfydryl has the characteristic of click reaction, and can efficiently connect polypeptides with different functions to the surface of the AIE polymer nanoparticle. The prepared AIE polymer nano-particle modified by carboxyl and polypeptide has large emission wavelength and intensity regulation and control interval and strong cell uptake capacity, and has important potential application value in the field of cell imaging.
(V) description of the drawings
FIG. 1 is a transmission electron microscope image of carboxyl and polypeptide modified AIE polymer nanoparticles prepared in example 1.
FIG. 2 shows fluorescence emission spectra of carboxyl-and polypeptide-modified AIE polymer nanoparticle emulsion prepared in example 1 and photographs of the emulsion excited by ultraviolet light.
(VI) detailed description of the preferred embodiment
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
the polypeptides used in the examples of the present invention are from Jier Biochemical Co., Ltd, Shanghai.
Example 1:
0.2g O-50 emulsifier was weighed and dissolved in 12.5g deionized water to obtain an aqueous solution.
Weighing 0.01g of AIE-6 molecule, and dissolving in a mixed solution of 0.06g of n-hexadecane, 0.1g of acrylic acid, 0.45g of styrene and 0.45g of methyl methacrylate to obtain an oil phase solution; adding the water phase solution into the oil phase solution, and stirring and pre-emulsifying to obtain a coarse emulsion; placing the container filled with the coarse emulsion in an ice-water bath, and performing ultrasonic treatment for 9min by using ultrasonic waves with the power of 400W to prepare stable monomer fine emulsion; introducing nitrogen to remove oxygen, adding 0.02g of potassium persulfate, heating to 70 ℃, and reacting for 6 hours under the protection of nitrogen to prepare the carboxyl modified AIE polymer nanoparticle emulsion. Using a molecular weight cut-off of 5000 g.mol–1Dialyzing for three days, changing water at intervals of 2h for the first day, 3h for the second day, and 6h for the third day to obtain purified carboxyl modified AIE polymer nanoparticle emulsion, and measuringThe solid content of the obtained emulsion is 2.2 wt%;
0.19g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride was weighed and dissolved in 200g of 0.1M MES buffer (pH 6), and added to the purified carboxyl-modified AIE polymer nanoparticle emulsion and activated for 60min to prepare an activated carboxyl-modified AIE polymer nanoparticle emulsion.
0.16g of N- (2-aminoethyl) maleimide was weighed out and dissolved in 100g of 1M Tris-HCl buffer (pH 7.5) and added to the activated carboxy modified AIE polymer nanoparticle emulsion with 0.1M NaHCO3Adjusting the pH value of the system to 7.5, and reacting at room temperature for 12 hours to prepare a maleimide modified AIE polymer nanoparticle emulsion; the molecular weight cut-off of the prepared maleimide modified AIE polymer nano particle emulsion is 5000 g.mol–1The dialysis bag is dialyzed for three days, the water changing frequency is changed every 2h for the first day, every 3h for the second day and every 6h for the third day, and the purified maleimide modified AIE polymer nano particle emulsion is obtained.
0.015g of HIV-1TAT peptide of which the tail end contains a cysteine sequence unit is dissolved in 15g of deionized water, polypeptide aqueous solution is uniformly mixed with purified maleimide modified AIE polymer nanoparticle emulsion, and the mixture reacts for 12 hours at room temperature to prepare carboxyl and polypeptide modified AIE polymer nanoparticles; the molecular weight cut-off of the prepared carboxyl and polypeptide modified AIE polymer nano particle emulsion is 5000 g.mol–1The dialysis bag is dialyzed for three days, the water changing frequency is changed every 2 hours for the first day, every 3 hours for the second day and every 6 hours for the third day, and the purified carboxyl and polypeptide modified AIE polymer nano particle emulsion is obtained.
The morphology of the carboxyl and polypeptide modified AIE polymer nanoparticles is observed by a transmission electron microscope, and the result shows that the carboxyl and polypeptide modified AIE polymer nanoparticles are spherical particles with the number average particle diameter of about 89 nm. The fluorescence spectrum of the AIE polymer nanoparticle emulsion modified by carboxyl and polypeptide shows that the maximum fluorescence emission wavelength of the AIE polymer nanoparticle emulsion is 599nm, and the AIE polymer nanoparticle emulsion emits obvious orange fluorescence under the excitation of ultraviolet light. The experimental results of the double-cinchoninic acid (BCA) method polypeptide assay show that HIV-1TAT peptide is successfully modified on the surface of AIE polymer nanoparticles.
Comparative example 1:
the same formulation and preparation conditions as in example 1 were used to prepare carboxyl-modified AIE polymer nanoparticle emulsions. Using a molecular weight cut-off of 5000 g.mol–1The dialysis bag was dialyzed for three days with water exchange frequency of once every 2h for the first day, once every 3h for the second day, and once every 6h for the third day to obtain a purified carboxyl-modified AIE polymer nanoparticle emulsion having a solid content of 2.2 wt%.
0.19g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride was weighed and dissolved in 200g of 0.1M MES buffer (pH 6), and added to the purified carboxyl-modified AIE polymer nanoparticle emulsion and activated for 60min to prepare an activated carboxyl-modified AIE polymer nanoparticle emulsion.
Weighing 0.16g of N- (2-aminoethyl) maleimide, dissolving the N- (2-aminoethyl) maleimide in 100g of 1M tris-hydroxymethyl aminomethane-hydrochloric acid buffer solution (pH 7.5), adding the solution into the activated carboxyl modified AIE polymer nanoparticle emulsion, adjusting the pH value of the system to 10 by using NaOH, and reacting at room temperature for 12 hours to obtain a product; the obtained product is treated by the method of molecular weight cut-off 5000g & mol–1The dialysis bag is dialyzed for three days, the water changing frequency is changed every 2 hours for the first day, every 3 hours for the second day and every 6 hours for the third day, and a purified product is obtained. The maleimide group undergoes hydrolysis side reaction due to the over-high pH of the reaction system, and the product is obtained1No characteristic peak of maleimide group was observed in H NMR spectrum, and maleimide modification of the particle surface failed.
Comparative example 2:
the same formulation and preparation conditions as in example 1 were used to prepare carboxyl-modified AIE polymer nanoparticle emulsions. Using a molecular weight cut-off of 5000 g.mol–1The dialysis bag was dialyzed for three days with water exchange frequency of once every 2h for the first day, once every 3h for the second day, and once every 6h for the third day to obtain a purified carboxyl-modified AIE polymer nanoparticle emulsion having a solid content of 2.2 wt%.
0.19g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride was weighed and dissolved in 200g of 0.1M MES buffer (pH 6), and added to the purified carboxyl-modified AIE polymer nanoparticle emulsion and activated for 60min to prepare an activated carboxyl-modified AIE polymer nanoparticle emulsion.
Weighing 0.16g of N- (2-aminoethyl) maleimide, dissolving the N- (2-aminoethyl) maleimide in 100g of 1M tris-hydroxymethyl aminomethane-hydrochloric acid buffer solution (pH 7.5), adding the solution into the activated carboxyl modified AIE polymer nanoparticle emulsion, adjusting the pH value of the system to 2 by using HCl, and reacting at room temperature for 12 hours to obtain a product; the obtained product is treated by the method of molecular weight cut-off 5000g & mol–1The dialysis bag is dialyzed for three days, the water changing frequency is changed every 2 hours for the first day, every 3 hours for the second day and every 6 hours for the third day, and a purified product is obtained. Purification of the product due to protonation of the amino group under acidic reaction conditions, which significantly reduces its ability to participate in the amide condensation reaction1The characteristic peak of maleimide group is not observed in HNMR spectrogram, and the maleimide modification on the particle surface fails.
Example 2:
0.5g of Tween 20 was weighed out and dissolved in 15g of deionized water to obtain an aqueous solution.
Weighing 0.0075g of AIE-12 molecule, and dissolving in a mixed solution of 0.08g of n-hexadecane, 0.15g of methacrylic acid and 1.35g of methyl methacrylate to obtain an oil phase solution; adding the water phase solution into the oil phase solution, and stirring and pre-emulsifying to obtain a coarse emulsion; placing the container filled with the coarse emulsion in an ice-water bath, and performing ultrasonic treatment for 20min by using ultrasonic waves with the power of 200W to prepare stable monomer fine emulsion; introducing nitrogen to remove oxygen, adding 0.015g of ammonium persulfate, heating to 70 ℃, and reacting for 10 hours under the protection of nitrogen to prepare the carboxyl modified AIE polymer nanoparticle emulsion. Using a molecular weight cut-off of 5000 g.mol–1Dialyzing the dialysis bag for three days, wherein the water changing frequency is changed every 2h for the first day, every 3h for the second day and every 6h for the third day to prepare the purified carboxyl modified AIE polymer nano particle emulsion, and the solid content of the emulsion is measured to be 2.9 wt%;
0.25g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride was weighed and dissolved in 200g of 0.1M MES buffer (pH 6), and added to the purified carboxyl-modified AIE polymer nanoparticle emulsion and activated for 60min to prepare an activated carboxyl-modified AIE polymer nanoparticle emulsion.
Weighing 0.16g of N- (2-aminoethyl) maleimide, dissolving the N- (2-aminoethyl) maleimide in 100g of 0.1M tris-hydroxymethyl aminomethane-hydrochloric acid buffer solution (pH 7.5), adding the solution into the activated carboxyl modified AIE polymer nanoparticle emulsion, adjusting the pH value of the system to 7.5 by using 0.1M NaOH, and reacting at room temperature for 12 hours to obtain the maleimide modified AIE polymer nanoparticle emulsion; the molecular weight cut-off of the prepared maleimide modified AIE polymer nano particle emulsion is 5000 g.mol–1The dialysis bag is dialyzed for three days, the water changing frequency is changed every 2h for the first day, every 3h for the second day and every 6h for the third day, and the purified maleimide modified AIE polymer nano particle emulsion is obtained.
0.015g of RGD peptide with a cysteine sequence unit at the end is dissolved in 15g of deionized water, a polypeptide aqueous solution is uniformly mixed with the purified maleimide modified AIE polymer nano particle emulsion, and the mixture reacts for 12 hours at room temperature to prepare carboxyl and polypeptide modified AIE polymer nano particles; the molecular weight cut-off of the prepared carboxyl and polypeptide modified AIE polymer nano particle emulsion is 5000 g.mol–1The dialysis bag is dialyzed for three days, the water changing frequency is changed every 2 hours for the first day, every 3 hours for the second day and every 6 hours for the third day, and the purified carboxyl and polypeptide modified AIE polymer nano particle emulsion is obtained.
The morphology of the carboxyl and polypeptide modified AIE polymer nanoparticles is observed by a transmission electron microscope, and the result shows that the carboxyl and polypeptide modified AIE polymer nanoparticles are spherical particles with the number average particle diameter of about 95 nm. The fluorescence spectrum of the AIE polymer nanoparticle emulsion modified by carboxyl and polypeptide shows that the maximum fluorescence emission wavelength is 640nm, and the emulsion emits obvious red under the excitation of ultraviolet light. The BCA method polypeptide determination experiment result shows that RGD peptide is successfully modified to the surface of AIE polymer nanoparticles.
Example 3:
0.3g of MOA-9 was weighed out and dissolved in 15g of deionized water to obtain an aqueous solution.
Weighing 0.003g of AIE-15 molecule, and dissolving in a mixed solution of 0.08g of n-hexadecane, 0.05g of methacrylic acid, 0.45g of methyl methacrylate and 0.5g of styrene to obtain an oil phase solution; adding the water phase solution into the oil phase solution, and stirring and pre-emulsifying to obtain a coarse emulsion; placing the container filled with the coarse emulsion in an ice-water bath, and performing ultrasonic treatment for 5min by using ultrasonic waves with the power of 600W to prepare stable monomer fine emulsion; introducing nitrogen to remove oxygen, adding 0.05g of potassium persulfate, heating to 72 ℃, and reacting for 6 hours under the protection of nitrogen to prepare the carboxyl modified AIE polymer nanoparticle emulsion. Using a molecular weight cut-off of 5000 g.mol–1Dialyzing the dialysis bag for three days, wherein the water changing frequency is changed every 2 hours on the first day, every 3 hours on the second day and every 6 hours on the third day to prepare the purified carboxyl modified AIE polymer nano particle emulsion, and the solid content of the emulsion is measured to be 2.1 wt%;
0.2g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride was weighed and dissolved in 200g of 0.1M citric acid-sodium citrate buffer (pH 6.2), and the purified carboxyl-modified AIE polymer nanoparticle emulsion was added and activated for 60min to prepare an activated carboxyl-modified AIE polymer nanoparticle emulsion.
0.16g of N- (2-aminoethyl) maleimide was weighed out and dissolved in 120g of 0.2M boric acid-borax buffer (pH 7.5) and added to the carboxyl-modified AIE polymer nanoparticle emulsion using 0.1M Na2CO3Adjusting the pH value of the system to 7.6 by the solution, and reacting at room temperature for 8 hours to prepare a maleimide modified AIE polymer nanoparticle emulsion; the molecular weight cut-off of the prepared maleimide modified AIE polymer nano particle emulsion is 5000 g.mol–1The dialysis bag is dialyzed for three days, the water changing frequency is changed every 2h for the first day, every 3h for the second day and every 6h for the third day, and the purified maleimide modified AIE polymer nano particle emulsion is obtained.
0.04g of RGD peptide with a cysteine sequence unit at the tail end is dissolved in 10g of deionized water, and the polypeptide aqueous solution and the purified maleimide modified AIE polymer sodium are mixedUniformly mixing the rice grain emulsion, and reacting at room temperature for 10 hours to prepare carboxyl and polypeptide modified AIE polymer nanoparticles; the molecular weight cut-off of the prepared carboxyl and polypeptide modified AIE polymer nano particle emulsion is 5000 g.mol–1The dialysis bag is dialyzed for three days, the water changing frequency is changed every 2 hours for the first day, every 3 hours for the second day and every 6 hours for the third day, and the purified carboxyl and polypeptide modified AIE polymer nano particle emulsion is obtained.
The morphology of the carboxyl and polypeptide modified AIE polymer nanoparticles is observed by a transmission electron microscope, and the result shows that the carboxyl and polypeptide modified AIE polymer nanoparticles are spherical particles with the number average particle diameter of about 74 nm. The fluorescence spectrum of the AIE polymer nanoparticle emulsion modified by carboxyl and polypeptide shows that the maximum fluorescence emission wavelength is 645nm, and the emulsion emits obvious red fluorescence under the excitation of ultraviolet light. The BCA method polypeptide determination experiment result shows that RGD peptide is successfully modified to the surface of AIE polymer nanoparticles.
Example 4:
0.2g of sodium lauryl sulfate was weighed and dissolved in 50g of deionized water to obtain an aqueous phase solution.
Weighing 0.006g of AIE-21 molecule, and dissolving in a mixed solution of 0.3g of n-hexadecane, 0.54g of itaconic acid, 1g of butyl acrylate and 4.46g of methyl methacrylate to obtain an oil phase solution; adding the water phase solution into the oil phase solution, and stirring and pre-emulsifying to obtain a coarse emulsion; placing the container filled with the coarse emulsion in an ice-water bath, and performing ultrasonic treatment for 10min by using ultrasonic waves with the power of 500W to prepare stable monomer fine emulsion; introducing nitrogen to remove oxygen, adding 0.05g of potassium persulfate, heating to 70 ℃, and reacting for 5 hours under the protection of nitrogen to prepare the carboxyl modified AIE polymer nanoparticle emulsion. Using a molecular weight cut-off of 5000 g.mol–1Dialyzing the dialysis bag for three days, wherein the water changing frequency is changed every 2h for the first day, every 3h for the second day and every 6h for the third day to prepare the purified carboxyl modified AIE polymer nano particle emulsion, and the solid content of the emulsion is measured to be 3.7 wt%;
2.5g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride was weighed and dissolved in 500g of 0.2M acetic acid-sodium acetate buffer (pH 6.2), and the purified carboxyl-modified AIE polymer nanoparticle emulsion was added and activated for 2 hours to prepare an activated carboxyl-modified AIE polymer nanoparticle emulsion.
Weighing 1.35g N- (2-aminoethyl) maleimide, dissolving in 135g of 0.2M PBS buffer (pH 7.5), adding into carboxyl modified AIE polymer nanoparticle emulsion, adjusting the pH value of the system to 7.5 with 0.1M NaOH solution, and reacting at room temperature for 36 hours to obtain maleimide modified AIE polymer nanoparticle emulsion; the molecular weight cut-off of the prepared maleimide modified AIE polymer nano particle emulsion is 5000 g.mol–1The dialysis bag is dialyzed for three days, the water changing frequency is changed every 2h for the first day, every 3h for the second day and every 6h for the third day, and the purified maleimide modified AIE polymer nano particle emulsion is obtained.
0.16g of T7 peptide with the end containing a cysteine sequence unit is dissolved in 20g of deionized water, polypeptide aqueous solution is evenly mixed with purified maleimide modified AIE polymer nano particle emulsion, and the mixture reacts for 24 hours at room temperature to prepare carboxyl and polypeptide modified AIE polymer nano particles; the molecular weight cut-off of the prepared carboxyl and polypeptide modified AIE polymer nano particle emulsion is 5000 g.mol–1The dialysis bag is dialyzed for three days, the water changing frequency is changed every 2 hours for the first day, every 3 hours for the second day and every 6 hours for the third day, and the purified carboxyl and polypeptide modified AIE polymer nano particle emulsion is obtained.
The morphology of the carboxyl and polypeptide modified AIE polymer nanoparticles is observed by a transmission electron microscope, and the result shows that the carboxyl and polypeptide modified AIE polymer nanoparticles are spherical particles with the number average particle diameter of about 82 nm. The fluorescence spectrum of the AIE polymer nanoparticle emulsion modified by carboxyl and polypeptide shows that the maximum fluorescence emission wavelength of the AIE polymer nanoparticle emulsion is 593nm, and the AIE polymer nanoparticle emulsion emits obvious orange fluorescence under the excitation of ultraviolet light. The BCA method polypeptide determination experiment result shows that the T7 peptide is successfully modified on the surface of the AIE polymer nanoparticle.
Example 5:
0.25g of sodium lauryl sulfate and 0.5g of Tween 80 were weighed out and dissolved in 50g of deionized water, respectively, to obtain aqueous solutions.
Weighing 0.12g of AIE-28 molecules, and dissolving in a mixed solution of 0.675g of n-hexadecane, 0.8g of itaconic acid, 1.25g of methyl acrylate and 7.95g of methyl methacrylate to obtain an oil phase solution; adding the water phase solution into the oil phase solution, and stirring and pre-emulsifying to obtain a coarse emulsion; placing the container filled with the coarse emulsion in an ice-water bath, and performing ultrasonic treatment for 10min by using ultrasonic waves with the power of 400W to prepare stable monomer fine emulsion; introducing nitrogen to remove oxygen, adding 0.225g of azobisisobutyramidine hydrochloride, raising the temperature to 70 ℃, and reacting for 4 hours under the protection of nitrogen to prepare the carboxyl modified AIE polymer nanoparticle emulsion. Using a molecular weight cut-off of 5000 g.mol–1Dialyzing the dialysis bag for three days, wherein the water changing frequency is changed every 2h for the first day, every 3h for the second day and every 6h for the third day to prepare the purified carboxyl modified AIE polymer nano particle emulsion, and the solid content of the emulsion is measured to be 4.5 wt%;
2.7g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride was weighed and dissolved in 100g of 0.5M potassium dihydrogen phosphate-sodium hydroxide buffer (pH 6.4), and the purified carboxyl-modified AIE polymer nanoparticle emulsion was added and activated for 3 hours to prepare an activated carboxyl-modified AIE polymer nanoparticle emulsion.
Weighing 1.8g N- (2-aminoethyl) maleimide, dissolving in 100g of 0.1M PBS buffer (pH 7.2), adding into carboxyl modified AIE polymer nanoparticle emulsion, adjusting the pH value of the system to 7.5 with 0.1M NaOH solution, and reacting at room temperature for 36 hours to obtain maleimide modified AIE polymer nanoparticle emulsion; the molecular weight cut-off of the prepared maleimide modified AIE polymer nano particle emulsion is 5000 g.mol–1The dialysis bag is dialyzed for three days, the water changing frequency is changed every 2h for the first day, every 3h for the second day and every 6h for the third day, and the purified maleimide modified AIE polymer nano particle emulsion is obtained.
0.225g RGD peptide with a cysteine sequence unit at the end is dissolved in 100g deionized water, the polypeptide water solution is evenly mixed with the purified maleimide modified AIE polymer nano particle emulsion, and the mixture is reacted for 30 hours at room temperature to prepare carboxyl and carboxylPolypeptide-modified AIE polymer nanoparticles; the molecular weight cut-off of the prepared carboxyl and polypeptide modified AIE polymer nano particle emulsion is 5000 g.mol–1The dialysis bag is dialyzed for three days, the water changing frequency is changed every 2 hours for the first day, every 3 hours for the second day and every 6 hours for the third day, and the purified carboxyl and polypeptide modified AIE polymer nano particle emulsion is obtained.
The morphology of the carboxyl and polypeptide modified AIE polymer nanoparticles is observed by a transmission electron microscope, and the result shows that the carboxyl and polypeptide modified AIE polymer nanoparticles are spherical particles with the number average particle diameter of about 75 nm. The fluorescence spectrum of the AIE polymer nanoparticle emulsion modified by carboxyl and polypeptide shows that the maximum fluorescence emission wavelength of the AIE polymer nanoparticle emulsion is 600nm, and the AIE polymer nanoparticle emulsion emits obvious orange fluorescence under the excitation of ultraviolet light. The BCA method polypeptide determination experiment result shows that RGD peptide is successfully modified to the surface of AIE polymer nanoparticles.
Example 6:
0.2g O-50 g was weighed and dissolved in 12.5g deionized water to obtain an aqueous solution.
Weighing 0.012g of AIE-33 molecule, and dissolving in a mixed solution of 0.06g of n-hexadecane, 0.15g of methacrylic acid and 0.85g of methyl methacrylate to obtain an oil phase solution; adding the water phase solution into the oil phase solution, and stirring and pre-emulsifying to obtain a coarse emulsion; placing the container filled with the coarse emulsion in an ice-water bath, and performing ultrasonic treatment for 9min by using ultrasonic waves with the power of 400W to prepare stable monomer fine emulsion; introducing nitrogen to remove oxygen, adding 0.02g of azobisisobutyramidine hydrochloride, heating to 65 ℃, and reacting for 6 hours under the protection of nitrogen to obtain the carboxyl modified AIE polymer nanoparticle emulsion. Using a molecular weight cut-off of 5000 g.mol–1Dialyzing the dialysis bag for three days, wherein the water changing frequency is changed every 2 hours on the first day, every 3 hours on the second day and every 6 hours on the third day to prepare the purified carboxyl modified AIE polymer nano particle emulsion, and the solid content of the emulsion is measured to be 2.3 wt%;
0.19g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride was weighed and dissolved in 200g of 0.1M MES buffer (pH 6.0), and the purified carboxyl-modified AIE polymer nanoparticle emulsion was added and activated for 60min to prepare an activated carboxyl-modified AIE polymer nanoparticle emulsion.
Weighing 0.15g N- (2-aminoethyl) maleimide, dissolving in 100g of 0.1M PBS buffer (pH 7.2), adding into carboxyl modified AIE polymer nanoparticle emulsion, adjusting the pH value of the system to 7.5 with 0.1M NaOH solution, and reacting at room temperature for 12 hours to obtain maleimide modified AIE polymer nanoparticle emulsion; the molecular weight cut-off of the prepared maleimide modified AIE polymer nano particle emulsion is 5000 g.mol–1The dialysis bag is dialyzed for three days, the water changing frequency is changed every 2h for the first day, every 3h for the second day and every 6h for the third day, and the purified maleimide modified AIE polymer nano particle emulsion is obtained.
Dissolving 0.03g of HIV-1TAT peptide of which the tail end contains a cysteine sequence unit in 10g of deionized water, uniformly mixing a polypeptide aqueous solution with the purified maleimide modified AIE polymer nanoparticle emulsion, and reacting at room temperature for 12 hours to prepare carboxyl and polypeptide modified AIE polymer nanoparticles; the molecular weight cut-off of the prepared carboxyl and polypeptide modified AIE polymer nano particle emulsion is 5000 g.mol–1The dialysis bag is dialyzed for three days, the water changing frequency is changed every 2 hours for the first day, every 3 hours for the second day and every 6 hours for the third day, and the purified carboxyl and polypeptide modified AIE polymer nano particle emulsion is obtained.
The morphology of the carboxyl and polypeptide modified AIE polymer nanoparticles is observed by a transmission electron microscope, and the result shows that the carboxyl and polypeptide modified AIE polymer nanoparticles are spherical particles with the number average particle diameter of about 84 nm. The fluorescence spectrum of the AIE polymer nanoparticle emulsion modified by carboxyl and polypeptide shows that the maximum fluorescence emission wavelength of the AIE polymer nanoparticle emulsion is 603nm, and the AIE polymer nanoparticle emulsion emits obvious orange fluorescence under the excitation of ultraviolet light. The BCA method polypeptide determination experiment result shows that HIV-1TAT peptide is successfully modified on the surface of AIE polymer nanoparticles.
Example 7:
0.06g of sodium dodecylbenzenesulfonate was weighed out and dissolved in 15g of deionized water to obtain an aqueous solution.
0.005g of AIE-37 molecule was weighed out and dissolved in 0.06g of n-hexadecane, 0.05gMixing acrylic acid and 0.95g of styrene to obtain an oil phase solution; adding the water phase solution into the oil phase solution, and stirring and pre-emulsifying to obtain a coarse emulsion; placing the container filled with the coarse emulsion in an ice-water bath, and performing ultrasonic treatment for 9min by using ultrasonic waves with the power of 400W to prepare stable monomer fine emulsion; introducing nitrogen to remove oxygen, adding 0.075g of potassium persulfate, heating to 65 ℃, and reacting for 24 hours under the protection of nitrogen to prepare the carboxyl modified AIE polymer nanoparticle emulsion. Using a molecular weight cut-off of 5000 g.mol–1Dialyzing the dialysis bag for three days, wherein the water changing frequency is changed every 2 hours on the first day, every 3 hours on the second day and every 6 hours on the third day to prepare the purified carboxyl modified AIE polymer nano particle emulsion, and the solid content of the emulsion is measured to be 2.5 wt%;
0.15g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride was weighed and dissolved in 200g of 0.1M MES buffer (pH 6.2), and the purified carboxyl-modified AIE polymer nanoparticle emulsion was added and activated for 60min to prepare an activated carboxyl-modified AIE polymer nanoparticle emulsion.
Weighing 0.15g N- (2-aminoethyl) maleimide, dissolving in 100g of 0.1M PBS buffer (pH 7.2), adding into carboxyl modified AIE polymer nanoparticle emulsion, adjusting the pH value of the system to 7.5 with 0.1M NaOH solution, and reacting at room temperature for 12 hours to obtain maleimide modified AIE polymer nanoparticle emulsion; the molecular weight cut-off of the prepared maleimide modified AIE polymer nano particle emulsion is 5000 g.mol–1The dialysis bag is dialyzed for three days, the water changing frequency is changed every 2h for the first day, every 3h for the second day and every 6h for the third day, and the purified maleimide modified AIE polymer nano particle emulsion is obtained.
Dissolving 0.03g of HIV-1TAT peptide of which the tail end contains a cysteine sequence unit in 15g of deionized water, uniformly mixing a polypeptide aqueous solution with the purified maleimide modified AIE polymer nanoparticle emulsion, and reacting at room temperature for 12 hours to prepare carboxyl and polypeptide modified AIE polymer nanoparticles; the molecular weight cut-off of the prepared carboxyl and polypeptide modified AIE polymer nano particle emulsion is 5000 g.mol–1Dialyzing the dialysis bag for three days,the water changing frequency is changed every 2h for the first day, every 3h for the second day and every 6h for the third day, and the purified carboxyl and polypeptide modified AIE polymer nano particle emulsion is obtained.
The morphology of the carboxyl and polypeptide modified AIE polymer nanoparticles is observed by a transmission electron microscope, and the result shows that the carboxyl and polypeptide modified AIE polymer nanoparticles are spherical particles with the number average particle diameter of about 76 nm. The fluorescence spectrum of the AIE polymer nanoparticle emulsion modified by carboxyl and polypeptide shows that the maximum fluorescence emission wavelength of the AIE polymer nanoparticle emulsion is 598nm, and the AIE polymer nanoparticle emulsion emits obvious orange fluorescence under the excitation of ultraviolet light. The BCA method polypeptide determination experiment result shows that RGD peptide is successfully modified to the surface of AIE polymer nanoparticles.
The above-described embodiments of the invention are intended to be illustrative of the invention and are not to be construed as limiting the invention, and any variations that fall within the meaning and scope of the invention equivalent to the claims are intended to be embraced therein.

Claims (10)

1. A preparation method of AIE polymer nano particles modified by carboxyl and polypeptide is characterized in that: the method comprises the following steps:
(1) dissolving an emulsifier in deionized water to obtain an emulsifier aqueous solution, wherein the mass consumption of the emulsifier is 0.1-10% of the mass consumption of the deionized water;
the emulsifier is selected from at least one of the following: anionic, amphoteric and nonionic emulsifiers;
(2) dissolving AIE molecules and an austenite curing effect inhibitor in a mixed solution of a carboxyl functional monomer and a hydrophobic monomer to obtain an oil phase solution, wherein the mass consumption of the AIE molecules is 0.1-30% of the total mass of the monomers; the mass usage of the austenite curing effect inhibitor is 1-12% of the total mass of the monomers; the mass usage of the carboxyl functional monomer is 0.1-20% of the total mass usage of the monomer; the total mass of the monomers refers to the total mass of the carboxyl functional monomers and the hydrophobic monomers;
the carboxyl functional monomer is at least one of acrylic acid and methacrylic acid shown in a formula (I) and itaconic acid monomer shown in a formula (II);
Figure FDA0003097184560000011
in the formula (I), R1Is H or CH3，
Figure FDA0003097184560000012
The AIE molecule is selected from at least one of the following AIE-1 to AIE-42 molecules:
Figure FDA0003097184560000021
AIE-7 molecule, wherein R2＝H，R3＝N(C2H5)2
Figure FDA0003097184560000022
Figure FDA0003097184560000031
An AIE-13 molecule, wherein
R4＝OC6H13，
R5＝OC12H25，
R6＝H
Figure FDA0003097184560000041
Figure FDA0003097184560000051
Figure FDA0003097184560000061
Figure FDA0003097184560000071
Figure FDA0003097184560000081
Figure FDA0003097184560000091
Figure FDA0003097184560000101
Figure FDA0003097184560000111
Figure FDA0003097184560000121
The inhibitor of the austenite ripening effect is selected from at least one of the following: aliphatic hydrocarbon of C14-C22, aliphatic alcohol of C14-C22;
the hydrophobic monomer is selected from at least one of the following: styrene, acrylates and methacrylates represented by formula (III);
Figure FDA0003097184560000122
in the formula (III), R7Is H or methyl; r8Is C1-C5 alkyl;
(3) adding the emulsifier aqueous solution prepared in the step (1) into the oil phase solution prepared in the step (2) to ensure that the total mass of monomers in the oil phase solution is 1-50% of the mass of water in the emulsifier aqueous solution, and stirring for pre-emulsification to obtain a crude emulsion; placing the container filled with the coarse emulsion in an ice-water bath, and carrying out ultrasonic treatment for 0.5-60 min under the power of 25-950W to prepare a monomer fine emulsion; introducing nitrogen to remove oxygen, adding a water-soluble initiator, reacting for 1-48 h at the temperature of 25-90 ℃ under the protection of nitrogen to prepare AIE polymer nanoparticle emulsion with carboxyl modified on the surface, and dialyzing to obtain purified AIE polymer nanoparticle emulsion with carboxyl modified;
the water-soluble initiator is selected from at least one of the following: persulfate, water-soluble azo, oxidant and reductant form a redox system; the electrical properties of the water-soluble initiator and the emulsifier need to be matched, and when the water-soluble anionic initiator is selected, the emulsifier is selected from an anionic emulsifier, a nonionic emulsifier or a composite emulsifier consisting of the anionic emulsifier and the nonionic emulsifier; when the water-soluble cationic initiator is selected, a nonionic emulsifier is selected; the mass usage of the water-soluble initiator is 0.05-10% of the total mass of the monomers;
(4) dissolving a carbodiimide condensing agent in an acidic pH buffer solution with the pH value of 6.0-6.5 to prepare a carbodiimide condensing agent buffer solution with the mass fraction of 0.005-10%, and adding the carbodiimide condensing agent buffer solution into the carboxyl modified AIE polymer nanoparticle emulsion prepared in the step (3), wherein the mass consumption of the carbodiimide condensing agent is 50-600% of the consumption of the carboxyl functional monomer, and activating for 1 min-6 h to prepare the activated carboxyl modified AIE polymer nanoparticle emulsion; the acidic pH buffer is selected from one of: disodium hydrogen phosphate-citric acid buffer solution, citric acid-sodium citrate buffer solution, acetic acid-sodium acetate buffer solution, 2- (N-morpholine) ethanesulfonic acid buffer solution and potassium dihydrogen phosphate-sodium hydroxide buffer solution; the carbodiimide condensing agent is one selected from N, N '-dicyclohexylcarbodiimide, N' -diisopropylcarbodiimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride;
(5) dissolving omega-amino maleimide in an alkaline pH buffer solution with the pH value of 7.0-9.0 to obtain an omega-amino maleimide solution, wherein the mass fraction of the omega-amino maleimide is controlled within the range of 0.01-10%; the alkaline pH buffer is selected from one of the following: trihydroxymethyl aminomethane-hydrochloric acid buffer solution, boric acid-borax buffer solution and phosphate buffer salt solution; the omega-amino maleimide is selected from one of the compounds shown in the formula (IV):
Figure FDA0003097184560000141
in the formula (IV), R9Is C1-C6 alkyl;
(6) adding the omega-amino maleimide solution prepared in the step (5) into the activated carboxyl modified AIE polymer nanoparticle emulsion prepared in the step (4), enabling the mass usage of omega-amino maleimide to be 10% -1000% of the mass usage of carboxyl functional monomers, adjusting the pH value of the emulsion to 7.5-8.5 by using a pH regulator, reacting at room temperature for 1-72 h to prepare a maleimide modified AIE polymer nanoparticle emulsion, and dialyzing to obtain a purified maleimide modified AIE polymer nanoparticle emulsion; the pH regulator is selected from at least one of the following: sodium citrate, borax, sodium hydroxide, carbonate and hydrate thereof, bicarbonate and ammonia water;
(7) dissolving polypeptide with a tail end containing a cysteine sequence unit into deionized water to prepare a polypeptide aqueous solution, wherein the mass fraction of the polypeptide is controlled within the range of 0.01-10%; adding a polypeptide aqueous solution into the AIE polymer nanoparticle emulsion modified by maleimide prepared in the step (6), enabling the mass usage of the polypeptide to be 5% -100% of the mass usage of the carboxyl functional monomer, reacting at room temperature for 1-72 h to prepare AIE polymer nanoparticles modified by carboxyl and polypeptide, and dialyzing to obtain purified AIE polymer nanoparticle emulsion modified by carboxyl and polypeptide;
the polypeptide containing a cysteine sequence unit at the tail end is selected from at least one of the following polypeptides: an HIV-1Tat (47-57) peptide with the sequence of YGRKKRRQRRRC, an RGD peptide with the sequence of RGDC, an NGR peptide with the sequence of NGRC, an REDV peptide with the sequence of REDVC, a T7 peptide with the sequence of HAIYPRHC, and an AG73 peptide with the sequence of RKRLQVQLSIRTC.
2. The method of claim 1, wherein: in the step (1), the anionic emulsifier is selected from at least one of the following: alkyl sulfonate emulsifier R10-SO3M, alkyl sulfate emulsifier R11-OSO3M and alkyl benzene sulfonate emulsifier R12-C6H4-SO3M, wherein R10And R11Is a fatty chain of C10-C20, R12Is a fatty chain of C10-C18, M is Na+Or K+；
The amphoteric emulsifier is selected from at least one of the following: dodecyl amino propionic acid, octadecyl dihydroxyethyl amine oxide, and carboxyl betaine R13N+(CH3)2CH2COO–Sulfobetaine R14N+(CH3)2CH2CH2SO3 –Or R15N+(CH3)2CH2CH2CH2SO3 –Wherein R is13、R14And R15Is a C12-C18 fatty chain;
the nonionic emulsifier is selected from at least one of the following: OP-series emulsifier, O-series emulsifier, MOA-series emulsifier, Tween-series emulsifier and SG-series emulsifier.
3. The method of claim 1 or 2, wherein: in the step (2), the AIE molecules are AIE molecules with the emission wavelength of more than 500nm, and the mass dosage of the AIE molecules is 0.1-10% of the total mass dosage of the monomers.
4. The method of claim 1 or 2, wherein: in the step (2), the dosage of the carboxyl functional monomer is controlled to be 0.5-15% of the mass dosage of the monomer.
5. The method of claim 1 or 2, wherein: in the step (3), the water-soluble initiator is selected from at least one of the following: persulfate, water-soluble azo, oxidant and reductant form a redox system; the persulfate initiator is selected from at least one of the following: sodium persulfate, potassium persulfate, ammonium persulfate; the water-soluble azo initiator is selected from at least one of the following: azobisisobutyramidine hydrochloride, azobisisobutyrimidazoline hydrochloride, azobiscyanovaleric acid, azobisisopropylimidazole; in the redox system, the reducing agent is sodium bisulfite, sodium thiosulfate or sodium sulfite, and the oxidizing agent is persulfate or hydrogen peroxide.
6. The method of claim 1 or 2, wherein: in the step (4), the pH value of the acidic pH buffer solution is within 6.0-6.5, and the activation reaction time is 5 min-3 h.
7. The method of claim 1 or 2, wherein: in the step (5), the omega-amino maleimide is N- (2-aminoethyl) maleimide.
8. The method of claim 1 or 2, wherein: in the step (6), the pH value of the emulsion is adjusted to 7.5-8.5 by using a pH regulator, and the emulsion is reacted for 1-36 h at room temperature.
9. The method of claim 1 or 2, wherein: in the step (7), the reaction time is 3-48 h.
10. The method of claim 3, wherein: the emulsifier is anionic emulsifier and/or nonionic emulsifier, wherein the nonionic emulsifier is at least one selected from O series emulsifier, MOA series emulsifier and Tween series emulsifier, and the anionic emulsifier is sodium dodecyl sulfate or sodium dodecyl benzene sulfonate; the austenite curing effect inhibitor is C16-C22 alkane; the water-soluble initiator is ammonium persulfate, potassium persulfate and azodiisobutyl amidine hydrochloride; the omega-amino maleimide is N- (2-aminoethyl) maleimide; the pH value of the acidic pH buffer solution is within the range of 6.0-6.5; in the step (6), the pH value of the emulsion is adjusted to 7.5-8.5 by using a pH regulator.
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