Fluorescent nanoparticles based on fluorinated zwitterionic random copolymers, methods of making and uses thereof

Abstract

The present application relates to fluorescent nanoparticles based on fluorinated zwitterionic random copolymer and its preparation method and application, the fluorescent nanoparticles include the single chain nanoparticles formed by poly trifluoroethyl methacrylate-co-methacryloyloxyethyl sulfobetaine, the molecular chain end of the poly trifluoroethyl methacrylate-co-methacryloyloxyethyl sulfobetaine is connected with ACQ type group selected from anthryl, perylene imide group and pyrene base or AIE type group selected from tetraphenyl ethylene base, carbazole base and cyanine-containing triphenyl ethylene group.The fluorescent nanoparticles based on the poly trifluoroethyl methacrylate-co-methacryloyloxyethyl sulfobetaine of the present application that terminal fluorescent group is terminated, has excellent controllable fluorescent luminescence performance.It is considered that the single chain nanoparticles have isolation effect, avoid the fluorescence quenching effect of ACQ type group, and can provide limited microenvironment for AIE type group, ensure the fluorescent luminescence performance of nanoparticles.

Description

Technical Field

[0001] This invention belongs to the field of functional polymer nanomaterials, specifically relating to fluorescent nanoparticles based on fluorinated zwitterionic random copolymers, their preparation methods, and applications. Background Technology

[0002] Fluorescence is an important stimulated light behavior. The development of fluorescent materials and the use of fluorescence properties to achieve fluorescent bioimaging or to develop highly sensitive fluorescent sensors are among the current research hotspots (see SW Thomas, GD Joly, TMSwager, Chem. Rev., 2007, 107, 1339-1386; W. Li, G. S. Kaminski Schierle, B. Lei, Y. Liu, C. C. Kaminski, Chem. Rev., 2022, 122, 12495-12543). Pyrene compounds are a representative class of classic fluorescent molecules. Due to the π-π stacking interaction of their coplanar aromatic structures, the luminescence intensity is reduced in concentrated solutions or in aggregated states, or even disappears completely. This exhibits a typical "aggregation-caused quenching (ACQ)" effect (see SAJenekhe, JAOsaheni, Science, 1994, 265, 765-768; MMIslam, Z.Hu, Q.Wang, C.Redshaw, X.Feng, Mater. Chem. Front., 2019, 3, 762-781). In 2001, the team led by Benzhong Tang at the Hong Kong University of Science and Technology discovered the anomalous luminescence phenomenon of non-coplanar organic molecules such as polysubstituted thiophenes in a condensed state, pioneering the theory of "aggregation-induced emission (AIE)." This discovery quickly attracted widespread research interest and opened up a new field for organic luminescent materials (see J. Luo, Z. Xie, JWY Lam, L. Cheng, H. Chen, C. Qiu, HSKwok, X. Zhan, Y. Liu, D. Zhu, BZ Tang, Chem. Commun., 2001, 1740-1741; D. Wang, BZ Tang, Acc. Chem. Res., 2019, 52, 2559-2570). Currently, a series of AIE luminescent materials containing aromatic ring structures have been designed and developed. These materials exhibit weak or no luminescence in dilute solutions with good solvents, but strong luminescence in concentrated solutions or in a condensed state of bulk aggregation.This type of material, which exhibits strong fluorescence in an aggregated state and whose luminescence properties are tunable, has broad application prospects in many technological fields, including bioimaging, random laser materials, solar energy capture and conversion, photochromic glass, and can also be used for circularly polarized light in 3D stereoscopic image display (see J.Mei, NLCLeung, RTKKwok, JWYLam, BZTang, Chem. Rev., 2015, 115, 11718-11940; X.Li, Y.Sun, J.Chen, Z.Wu, P.Cheng, Q.Li, J.Fang, D.Chen, Polym. Chem., 2019, 10, 1575-1584; B.Kulkarni, S.Qutub, V.Ladelta, NMKhashab, N.Hadjichristidis, Biomacromolecules, 2021, 22, 5243-5255).

[0003] To prevent the ACQ effect caused by π-π stacking of pyrene nuclei, Tang Benzhong's team recently introduced large-volume aromatic ring units with twisted structures, such as carbazole groups or tetraphenylethylene (TPE), into the pyrene molecule. The resulting pyrene derivatives exhibit strong AIE luminescence properties and can be used as functional fluorescent pigments for the production of fluorescent anti-counterfeiting inks (see X.Wang, L.Wang, X.Mao, Q.Wang, Z.Mu, L.An, W.Zhang, X.Feng, C.Redshaw, C.Cao, A.Qin, BZTang, J.Mater.Chem.C, 2021, 9, 12828-12838). Given the long fluorescence lifetime and pure blue fluorescence of modified pyrene derivatives, Wu et al. covalently introduced them into the surface of electron beam irradiated nonwoven fabrics to modulate their fluorescence properties. The resulting fluorescent fabrics not only exhibited significant luminescence properties but also excellent wash-resistant fluorescence stability. Furthermore, the fluorescence intensity and quantum yield of these fluorescent fabrics were tunable (see M. Zhang, J. Chen, M. Wang, M. Yuan, R. Li, X. Feng, Y. He, X. Mao, Y. Li, Z. Xiong, Z. Xing, J. Hu, G. Wu, ACS Appl. Mater. Interfaces, 2021, 13, 9036-9042).

[0004] Given the high sensitivity of the AIE-inducing group (AIEgen) to minute changes in solution viscosity, it has been reported that introducing AIEgen into chain transfer agents (CTAs) as AIE-type CTA reagents, followed by reversible addition-fragmentation chain transfer (RAFT) polymerization or atom transfer radical polymerization (ATRP) using initiators incorporating AIEgen, allows for in-situ monitoring of the polymerization process and monomer conversion. This provides direct verification of the "active" / controllable kinetic behavior of the polymerization reaction (see X. Wang, X. Qiao, X. Yin, Z. Cui, P. Fu, M. Liu, G. Wang, X. Pan, X. Pang, Chem. Asian J., 2020, 15, 1014-1017; C. Ma, T. Han, M. Kang, E. Liarou, AM. Wemyss, S. Efstathiou, B. Z. Tang, D. Haddleton, ACS Macro Lett., 2020, 9, 769-775. Yuan Jinying et al. from Tsinghua University combined the AIE effect with polymerization-induced self-assembly (PISA) to successfully construct AIE luminescent polymer nanoassemblies with controllable assembly morphology and size to a certain extent. This provides an effective technical path and evaluation platform for studying the photophysical behavior of fluorescent nanomaterials related to nanostructures (see M.Huo, Q.Ye, H.Che, X.Wang, Y.Wei, J.Yuan, Macromolecules, 2017, 50, 1126-1133). In stark contrast to the excellent luminescent properties of AIE-type groups, ACQ-type chromophores are difficult to apply in practical luminescent states such as solid films due to fluorescence quenching effects (see MMIslam, Z.Hu, Q.Wang, C.Redshaw, X.Feng, Mater. Chem. Front., 2019, 3, 762-781).

[0005] On the other hand, single-chain nanoparticles (SCNPs) are small-sized polymeric soft nanomaterials, typically prepared from precursor polymers with reactive side groups through intrachain crosslinking. They have very attractive application prospects in fields such as sensing, catalysis, drug delivery, and biomedicine (see R. Chen, E.B. Berda, ACS Macro Lett., 2020, 9, 1836-1843; J. Chen, E.S. Garcia, SC. ​​Zimmerman, Acc. Chem. Res., 2020, 53, 1244-1256; L. Deng, L. Albertazzi, ARA Palmans, Biomacromolecules, 2022, 23, 326-338).

[0006] The combination of AIEgens and SCNPs is a novel method for constructing new fluorescent SCNP probes. These polymer nanoparticle probes have smaller size (5–20 nm), higher fluorescence intensity, and tunable fluorescence properties, and exhibit more stable luminescence performance than large-size (100 nm–1 μm) luminescent materials (refer to JF Hoffmann, AHRoos, FJSchmitt, D. Hinderberger, WHBinder, Angew. Chem. Int. Ed., 2021, 60, 7820-7827). Barner-Kowollik et al. prepared fluorescent single-chain nanoparticles with efficient intra-chain folding by reacting polystyrene with a newly designed and synthesized optically active bifunctional crosslinking agent via reversible photocontrolled crosslinking. They then controlled the unfolding of the nanoparticles by oxidative reversible bond-breaking reactions, thereby eliminating their fluorescence properties and achieving "visualized" self-reported monitoring of the polymer chain folding process and reversible bond-breaking and unfolding (refer to T.S.Fischer, S.Spann, Q.An, B.Luy, M.Tsotsalas, J.P.Blinco, H.M.Mutlu, C.Barner-Kowollik, Chem. Sci., 2018, 9, 4696-4702).

[0007] However, to date, there is no effective modification strategy that can simultaneously excite the fluorescence properties of both ACQ and AIE type groups. Summary of the Invention

[0008] In view of this, the purpose of this invention is to address the problems existing in the prior art by providing fluorescent nanoparticles based on fluorinated zwitterionic random copolymers, their preparation methods, and applications.

[0009] The objective of this invention is achieved through the following technical solutions.

[0010] In a first aspect, the present invention provides fluorescent nanoparticles based on fluorinated zwitterionic random copolymers, wherein the fluorescent nanoparticles comprise single-chain nanoparticles formed from poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine, wherein the molecular chain end of the poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine is connected to an ACQ-type group selected from anthracene, perylene imide, and pyrene groups, or an AIE-type group selected from tetraphenylethylene, carbazole, and cyanotriphenylethylene groups.

[0011] In this invention, fluorescent nanoparticles prepared by assembling a solution of a fluorinated zwitterionic amphiphilic random copolymer (poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine) terminated with potential fluorescent functional groups (ACQ-type groups or AIE-type groups) exhibit excellent tunable fluorescence luminescence properties. It is believed that the single-chain nanoparticles provide an isolating effect, avoiding the fluorescence quenching effect of the ACQ-type groups, and can provide a confined microenvironment for the AIE-type groups, thereby ensuring the fluorescence luminescence properties of the nanoparticles.

[0012] In the fluorescent nanoparticles provided by the present invention, the molar ratio of poly(trifluoroethyl methacrylate) repeating units to methacryloyloxyethyl sulfonate repeating units in the poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine is 10:90 to 90:10, preferably 10:90 to 50:50, more preferably 10:90 to 40:60, and even more preferably 10:90 to 30:70.

[0013] The fluorescent nanoparticles provided according to the present invention, wherein, as measured by 1H NMR spectroscopy, the molecular weight of the poly(trifluoroethyl methacrylate-co-methacryloyloxyethyl sulfonate betaine) is 10–80 kDa, preferably 15–50 kDa.

[0014] According to the fluorescent nanoparticles provided by the present invention, the average particle size of the fluorescent nanoparticles, as measured by dynamic light scattering, is less than 20 nm, preferably 5 to 20 nm, more preferably 7.5 to 15 nm, and / or the particle size distribution is less than 0.10, preferably 0.01 to 0.10, more preferably 0.01 to 0.08, and even more preferably 0.01 to 0.05.

[0015] According to the fluorescent nanoparticles provided by the present invention, the fluorescent nanoparticles may exist in the form of a dispersion or a solid, preferably in the form of a dispersion, such as an aqueous dispersion.

[0016] In some embodiments, the concentration of poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine in the aqueous dispersion is 0.05–5.0 mg / mL, preferably 0.1–4.5 mg / mL.

[0017] In a second aspect, the present invention provides a method for preparing fluorescent nanoparticles based on fluorinated zwitterionic random copolymers, wherein the preparation method includes the following steps:

[0018] S100, providing α-bromophenylacetic acid ester initiators containing ACQ-type or AIE-type groups;

[0019] S200 provides a polymerization reaction solution containing trifluoroethyl methacrylate, methacryloxyethyl sulfonate betaine, α-bromophenylacetic acid ester initiator, metal salt catalyst, ligand, reducing agent and polymerization solvent, and performs ATRP polymerization reaction in the polymerization reaction solution at a temperature of 60-90°C and under inert gas protection to obtain the reaction product.

[0020] S300. The reaction product is purified to obtain a random copolymer of poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl betaine with ACQ-type or AIE-type groups attached to the ends of the molecular chains.

[0021] S400: Poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine is dissolved and dispersed in water, then sonicated and allowed to stand to obtain fluorescent nanoparticles.

[0022] In this invention, α-bromophenylacetic acid esters containing ACQ-type or AIE-type groups are used as ATRP initiators. These ATRP initiators introduce different fluorescent structural units through the ester group, but all possess the same degree of substitution, radical stabilizing groups, and transferable bromine atoms. These ATRP initiators exhibit almost identical initiation efficiencies. Therefore, the copolymerization reaction between trifluoroethyl methacrylate (TFMA) and methacryloyloxyethyl sulfonate betaine (SBMA) can be carried out efficiently, and the resulting fluorinated zwitterionic random copolymer has a controllable target composition and molecular weight.

[0023] According to the preparation method provided by the present invention, the α-bromophenylacetic acid ester initiator is 2-bromo-2-phenylacetic acid pyrene ester or 2-bromo-2-phenylacetic acid-4-tetraphenyl ester.

[0024] In some implementations, step S100 includes:

[0025] S110. Prepare an esterification reaction solution containing α-bromophenylacetic acid, an aromatic compound containing phenolic hydroxyl groups selected from 1-hydroxypyrene and 4-(1,2,2-triphenylvinyl)phenol, an esterification catalytic system, and an esterification solvent. The esterification reaction solution is subjected to an esterification reaction at 15–30 °C and under an inert atmosphere to obtain the esterification product.

[0026] S120. The esterification product is filtered, dried for the first time, purified by silica gel column, and dried for the second time to obtain α-bromophenylacetic acid ester initiator.

[0027] Preferably, in step S110, the molar ratio of α-bromophenylacetic acid to the aromatic compound containing phenolic hydroxyl groups is 0.5 to 2:1, more preferably 1 to 1.5:1.

[0028] Preferably, in step S110, the esterification catalytic system is selected from N,N-cyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP).

[0029] Preferably, the molar ratio of the esterification catalysis system to the aromatic compound containing phenolic hydroxyl groups is 0.1 to 0.3:1, and more preferably 0.1 to 0.2:1.

[0030] Preferably, the esterification solvent is selected from anhydrous dichloromethane or anhydrous tetrahydrofuran.

[0031] Preferably, the concentration of α-bromophenylacetic acid in the esterification reaction solution is 10–100 mg / mL, and more preferably 20–50 mg / mL.

[0032] Preferably, in step S110, the esterification reaction solution is prepared by dropwise addition of an esterification catalytic system solution to a solution containing α-bromophenylacetic acid and an aromatic compound containing a phenolic hydroxyl group. This operation is preferably carried out at a temperature of 0–5°C.

[0033] Preferably, the esterification reaction in step S110 takes 12 to 24 hours.

[0034] Preferably, in step S120, the first drying is carried out by rotary evaporation.

[0035] Preferably, in step S120, the silica gel column purification uses a petroleum ether / ethyl acetate mixed solvent with a volume ratio of 20 to 50:1 as the mobile phase.

[0036] Preferably, in step S120, the second drying includes rotary evaporation and vacuum drying at 40–50°C.

[0037] According to the preparation method provided by the present invention, in step S200, based on a total monomer content of 100 mol of trifluoroethyl methacrylate and methacryloyloxyethyl sulfonate betaine, the molar ratio of total monomer, metal salt catalyst, ligand and reducing agent is 100:0.1~1:0.1~2:1~6:0.1~4, preferably 100:0.5~1:0.5~2:2~6:0.5~2, more preferably 100:0.5~1:1~2:3~6:1~2, for example, 100:1:2:6:2.

[0038] According to the preparation method provided by the present invention, in step S200, the molar ratio of trifluoroethyl methacrylate and methacryloxyethyl sulfonate betaine is 10:90 to 90:10, preferably 10:90 to 50:50, more preferably 10:90 to 40:60, and even more preferably 10:90 to 30:70.

[0039] According to the preparation method provided by the present invention, the metal salt catalyst is selected from FeCl3, FeBr3 and CuBr2.

[0040] According to the preparation method provided by the present invention, the ligand is selected from triethylamine (Et3N), triphenylphosphine (PPh3) and tris(3,6-dioxaheptyl)amine (TDA).

[0041] According to the preparation method provided by the present invention, the reducing agent is selected from azobisisobutyronitrile (AIBN), ascorbic acid (AsA), and sodium ascorbate (AsA-Na).

[0042] According to the preparation method provided by the present invention, the polymerization solvent is trifluoroethanol (TFE). TFE is an ideal solvent for the homopolymerization of TFMA and SBMA, as well as for the random copolymerization of the two.

[0043] According to the preparation method provided by the present invention, the total molar concentration of trifluoroethyl methacrylate and methacryloxyethyl sulfonate betaine in the polymerization reaction solution is 0.5 to 2.0 mmol / mL.

[0044] According to the preparation method provided by the present invention, the reaction conditions for the ATRP polymerization reaction include: a temperature of 75-85°C, preferably 80°C; stirring at a speed of 800-3000 rpm; and a time of 1-24 hours, preferably 6-24 hours, for example, 8 hours.

[0045] According to the preparation method provided by the present invention, the method for controllably synthesizing fluorinated zwitterionic amphiphilic random copolymers via RAFT polymerization and preparing single-chain nanoparticles (SCNPs) in aqueous solution via an intrachain self-folding strategy can be referred to Chinese Invention Patent Application 202110493579.X, "Fluorinated polyzwitterionic copolymer nanoparticles with stabilizing protease activity, preparation method and application"; characterization techniques such as dynamic light scattering (DLS) and high-resolution transmission electron microscopy (TEM) can be found in Y. Zeng, T. Xu, X. Hou, J. Liu, C. Liu, Z. Chang, J. Fang, D. Chen, ACS Appl. Polym. Mater., 2023, 5, 3777-3791, which are incorporated herein by reference to the extent consistent with this application.

[0046] According to the preparation method provided by the present invention, the purification in step S300 includes:

[0047] S310. The reaction product is diluted with a polymerization solvent and dialyzed in water and methanol in sequence.

[0048] S320. Vacuum dry the product obtained from dialysis.

[0049] Preferably, in step S310, the reaction product is diluted 2 to 4 times using a polymerization solvent.

[0050] Preferably, the dialyzing conditions in water and methanol in step S310 include: a molecular weight cutoff of 0.5 to 2 kDa and a time of 12 to 48 hours, for example, 24 hours.

[0051] Preferably, in step S320, the vacuum drying conditions include a temperature of 30–40°C.

[0052] According to the preparation method provided by the present invention, in step S400, the weight ratio of water to poly(trifluoroethyl methacrylate-co-methacryloyloxyethyl sulfonate betaine) is 1000:0.1 to 5, for example, 1000:1.

[0053] According to the preparation method provided by the present invention, the ultrasonic conditions in step S400 include: power 200-300W and time 15-25 minutes.

[0054] According to the preparation method provided by the present invention, the standing time in step S400 is 5 to 20 minutes, for example 10 minutes.

[0055] According to the preparation method provided by the present invention, the inert gas is argon or nitrogen.

[0056] Thirdly, the present invention also provides the application of the fluorescent nanoparticles of the first aspect in cell imaging for non-diagnostic and therapeutic purposes.

[0057] According to the application provided by the present invention, the cells may be HeLa cells.

[0058] According to the application provided by the present invention, the application includes: adding a fluorescent nanoparticle dispersion to a cell culture medium for culturing.

[0059] Preferably, cell imaging is performed on a well plate, and the concentration of HeLa cells in the HeLa cell culture medium used for cell imaging is 2.0 × 10⁻⁶. 3 ~10.0×10 3 The number of holes per hole is preferably 5.0 × 10⁻⁶. 3 One hole / hole.

[0060] Preferably, the concentration of the fluorescent nanoparticle solution used for cell imaging is 0.05–0.5 mg / mL, and more preferably 0.1 mg / mL.

[0061] Preferably, the volume ratio of the fluorescent nanoparticle solution system to the cell culture medium is 1-5:1-50, and more preferably 1:10.

[0062] In this invention, the term "Pyr-P(TFMA)" is used. m -r-SBMA n ")" and "TPE-P(TFMA" m -r-SBMA n ")" respectively represent random copolymers of poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine with pyrene Pyr and tetraphenylethylene TPE attached to the ends of the molecular chains. The subscripts m and n represent the degree of polymerization of the repeating units of TFMA and SBMA, respectively.

[0063] The terms "single-chain nanoparticles" and "SCNPs" have the same meaning and can be used interchangeably; the terms "ATRP," "ATRP polymerization," and "atom transfer radical polymerization" have the same meaning and can be used interchangeably; the terms "poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine" and "poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine random copolymer" have the same meaning and can be used interchangeably; the terms "ACQ" and "aggregation-induced fluorescence quenching" have the same meaning and can be used interchangeably; the terms "AIE" and "aggregation-induced emission" have the same meaning and can be used interchangeably.

[0064] This invention has the following advantages:

[0065] (1) In this invention, by introducing a single fluorescent group at the end of the chain of a fluorinated zwitterionic random copolymer, the fluorescence quenching effect of ACQ type groups is eliminated by utilizing the isolation and confinement effect of SCNPs assembled by single chain folding, and a confined microenvironment is provided for AIE type groups, thereby realizing the preparation of a series of novel quasi-nanoscale fluorescent probe materials.

[0066] (2) In the prior art, AIE-type groups are usually attached to the hydrophobic segments of the amphiphilic block copolymer. Under the hydrophobic effect, the block copolymer spontaneously forms submicron (100nm~1μm) block copolymer assemblies such as micelles and vesicles. In this process, the AIE-type groups gather in the hydrophobic region, and the molecular motion is hindered, which “lights up” the assembly and causes it to exhibit fluorescence luminescence.

[0067] In contrast, this invention introduces AIE-type or ACQ-type groups into the polymer chain ends using an ATRP initiator, and utilizes ATRP controlled free radical polymerization to prepare a fluorinated zwitterionic random copolymer (poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine) with potentially fluorescent functional groups attached. By covalently bonding the potentially fluorescent functional groups (AIE-type or ACQ-type groups) to the polymer chain in a "one-to-one" manner, polymer single-chain fluorescent nanoparticles (FSCNPs) are prepared, which have a series of advantages such as precise small nanoscale size (e.g., about 15 nm), high fluorescence intensity, and tunable fluorescence luminescence performance.

[0068] (3) In the preparation of most existing luminescent materials, compounds with planar conjugated aromatic structures exhibit strong π-π stacking interactions, leading to a sharp decrease or even complete disappearance of fluorescence intensity in concentrated solutions or aggregated states. This is the so-called "aggregation-induced fluorescence quenching (ACQ)" effect. This invention introduces ACQ-type groups into an ATRP initiator, synthesizing a fluorinated zwitterionic random copolymer (poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine) with ACQ-type chromophores at its ends via controlled ATRP polymerization. Single-chain fluorescent nanoparticles are then prepared through solution assembly. The small, confined space of approximately 15 nm in the single-chain nanoparticles (SCNPs) effectively prevents fluorescence quenching of the ACQ-type groups, a method completely different from existing chemical modification schemes involving ACQ-type groups.

[0069] (4) In most existing AIE luminescent materials preparation, AIE-type groups are mainly introduced into a block of polymer backbone, side group or copolymer. The synthesis process is complicated, and the size of the fluorescent nanoparticles obtained is generally in the hundreds of nanometers (100nm~1μm).

[0070] This invention introduces AIE-type groups into an ATRP initiator, and synthesizes a fluorinated zwitterionic random copolymer (poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine) with AIE-type groups at the ends through ATRP controlled polymerization. Fluorescent nanoparticles with ultrafine polymer single chains of about 15 nm are prepared by solution assembly. The fluorescence emission of AIE-type groups is induced by the small size confinement effect of the hydrophobic core of SCNPs, and the luminescence intensity of the fluorescent nanoparticles is linearly related to the solution concentration.

[0071] (5) Cell imaging technology is an imaging technique that uses advanced microscopes to study the life processes of cells and uses this as a basis for targeted treatment of diseases. Currently, imaging technologies such as optical microscopy, fluorescence imaging, and electron microscopy are available to visualize and monitor the structure and physiological state of cells. Among them, fluorescence imaging uses fluorescent dyes to label cells, which can obtain high-resolution three-dimensional morphological images of cell structures. Currently, fluorescent dyes mainly include two categories: organic compounds with large conjugated structures and coordination complexes of rare metals and organic ligands. Among them, the synthesis process of organic compounds with large conjugated structures is extremely complicated and the yield is extremely low; while although rare metal and organic ligand complexes have stable fluorescence properties, rare metals are expensive and have high biotoxicity.

[0072] In comparison, the fluorescent nanoparticles prepared by the present invention using a "one-to-one" covalent bonding scheme between fluorescent groups and SCNPs are not only applicable to various types of fluorescent groups, but also allow for efficient and convenient adjustment of the fluorescence luminescence performance of the fluorescent nanoparticles by selecting fluorescent groups with different structures. In addition, they have excellent biocompatibility and are non-toxic.

[0073] (6) The preparation method of this invention employs ATRP controlled free radical polymerization, allowing for the controllability of the degree of polymerization and fluorinated side group content of the synthesized fluorinated zwitterionic amphiphilic random copolymer. This enables the preparation of different fluorescent nanoparticles to meet specific needs, resulting in structurally stable and uniformly sized particles. Furthermore, the preparation method of this invention can be carried out under routine laboratory conditions without the need for special operating instruments (such as a glove box), facilitating the economical and efficient synthesis of fluorinated zwitterionic amphiphilic random copolymers terminated with fluorescent groups. The subsequent solution assembly for preparing fluorescent nanoprobes can be completed in pure water, making it environmentally friendly and conducive to the systematic development of research on this type of fluorescent nanoparticle material and the development of more efficient fluorescent nanoprobes for different biological systems. Attached Figure Description

[0074] Figure 1These are the 1H NMR spectra of the initiators, where (a) is the 1H NMR spectrum of phenyl 2-bromo-2-phenylacetic acid (PBPA); (b) is the 1H NMR spectrum of pyrene 2-bromo-2-phenylacetic acid (PyrBPA); and (c) is the 1H NMR spectrum of 4-tetraphenyl 2-bromo-2-phenylacetic acid (TPEBPA).

[0075] Figure 2 The reaction kinetics curves of ln([M]0 / [M]) versus polymerization time for two monomers, trifluoroethyl methacrylate (TFMA) and methacryloyloxyethyl sulfonate betaine (SBMA), under different initial feed ratios; wherein, in (a), the initial feed ratio of TFMA and SBMA is 60:40; and in (b), the initial feed ratio of TFMA and SBMA is 40:60.

[0076] Figure 3 These are the 1H NMR spectra of fluorinated zwitterionic random copolymers; where (a) is PBPA-P (TFMA-r-SBMA); (b) is Pyr-P (TFMA-r-SBMA); and (c) is TPE-P (TFMA-r-SBMA).

[0077] Figure 4 This is a schematic diagram of the preparation of fluorescent nanoparticles by self-folding assembly of fluorinated zwitterionic random copolymers according to the present invention;

[0078] Figure 5 These are DLS images of fluorescent nanoparticles assembled from solutions of fluorinated zwitterionic random copolymers; where (a) is Pyr-P(TFMA) 10 -r-SBMA 90 (b) is Pyr-P(TFMA) 20 -r-SBMA 80 (c) is Pyr-P(TFMA) 30 -r-SBMA 70 (d) is TPE-P (TFMA) 10 -r-SBMA 90 (e) is TPE-P (TFMA) 20 -r-SBMA 80 (f) is TPE-P (TFMA) 30 -r-SBMA 70 );

[0079] Figure 6 These are TEM images and particle size distribution diagrams of fluorescent nanoparticles assembled from fluorinated zwitterionic random copolymer solutions; where (a) is Pyr-P(TFMA) 10 -r-SBMA 90 (b) is Pyr-P(TFMA)20 -r-SBMA 80 (c) is Pyr-P(TFMA) 30 -r-SBMA 70 (d) is TPE-P (TFMA) 10 -r-SBMA 90 (e) is TPE-P (TFMA) 20 -r-SBMA 80 (f) is TPE-P (TFMA) 30 -r-SBMA 70 In the particle size distribution diagram, the horizontal axis represents the diameter (nm) and the vertical axis represents the number of particles.

[0080] Figure 7 This is a graph showing the fluorescence intensity of Pyr-P(TFMA-r-SBMA) based fluorescent nanoparticles as a function of solution concentration.

[0081] Figure 8 This is a graph showing the fluorescence intensity of TPE-P (TFMA-r-SBMA) based fluorescent nanoparticles as a function of solution concentration.

[0082] Figure 9 These are the results of cytotoxicity evaluation tests on fluorescent nanoparticles and fluorescence imaging of HeLa cells. Detailed Implementation

[0083] The present invention will be further described below with reference to the accompanying drawings and embodiments. The description below is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various alternatives, modifications, and changes. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0084] The chemical reagents used in this invention are as follows:

[0085] α-Bromophenylacetic acid (97%, Maclean's); Phenol (98%, Maclean's); 1-Hydroxypyrene (98%, Maclean's); 4-(1,2,2-Triphenylvinyl)phenol (99%, Maclean's); Trifluoroethyl methacrylate (98%, Maclean's); Methacryloxyethyl sulfonate betaine (>97%, Aladdin); 4-Dimethylaminopyridine (99%, Aladdin); N,N-Dicyclohexylcarbodiimide (99%, Aladdin); Ascorbic acid (99%, Aladdin); Sodium ascorbate (99%, Aladdin); Trifluoroethanol (99.5%, Maclean's); Copper bromide (99%, Maclean's); Bromine Ferric chloride (98%, Maclean), tris(2-(2-methoxyethoxy)ethyl)amine (TDA, 97%, Maclean), triphenylphosphine (99%, Aladdin), ferric chloride hexahydrate (99%, Aladdin), trypsin-EDTA solution (sterile filtration, Yuanye Biotechnology), tetrahydronaphthalene (97%, Aladdin), phosphate buffer (pH=7.2, Yuanye Biotechnology), tetrahydrofuran (AR, Sinopharm-Shanghai Testing), methanol (AR, Sinopharm-Shanghai Testing), ethanol (AR, Sinopharm-Shanghai Testing), dichloromethane (AR, Sinopharm-Shanghai Testing), ethyl acetate (AR, Sinopharm-Shanghai Testing), petroleum ether (AR, Sinopharm-Shanghai Testing).

[0086] In addition, reagents or instruments used in this application whose manufacturers are not specified can be obtained commercially or by self-production.

[0087] Thin-plate chromatography (TLC)

[0088] Column chromatography uses 200-300 mesh silica gel, and the reaction is observed using ultraviolet light or an iodine bath. Alternatively, the reaction can be monitored using thin-plate chromatography (TLC) with ultraviolet light.

[0089] 1 H-NMR and 13 C-NMR

[0090] The 400MHz nuclear magnetic resonance spectrometer, model AVANCE AVIII, from Bruker GmbH, Germany, was used for the examination. 1 H-NMR and 13 C NMR analysis was performed using CDCl3, D2O, DMSO-d6, or CF3COOD as solvents, with tetramethylsilane (TMS, δ=0) as an internal standard.

[0091] High-resolution mass spectrometry (HRMS) analysis

[0092] High-resolution mass spectrometry (HRMS) tests were performed using a Thermo Fisher Q Exactive high-resolution mass spectrometer. Electrospray ionization (ESI) or atmospheric chemical ionization (APCI) ion sources were used. The test range was 100-1500 Da with an accuracy of four decimal places. The samples were dissolved in methanol or dichloromethane to prepare a 1 μg / mL solution for testing.

[0093] GPC Analysis

[0094] Number average molecular weight (M) of oil-soluble homopolymer PTFMA n,GPC ) and molecular weight distribution (M w / M n The molecular weight was measured using a Viscotek TDAmax gel permeation chromatography (GPC) system from Malvern, UK. This system is equipped with a TDA305 differential refractive index detector, a TGard guard column, and two test columns (T4000 and T2500). The measurable molecular weight range is 5 × 10⁻⁶. 2 ~5×10 5 g / mol. During the test, the temperature was 35℃, tetrahydrofuran (THF) was used as the mobile phase, and the flow rate was 1.0 mL / min. The sample was injected through a Malvern autosampler, and the relative molecular weight was determined using polystyrene (PSt) as a standard.

[0095] Number-average molecular weight (Mn) of water-soluble polymer PSBMA n,GPC ) and molecular weight distribution (M w / M n Measurements were taken using an Agilent gel permeation chromatograph (PL-GPC50), equipped with a differential refractive index detector. It features one PL aquagel-OH Gard guard column (8 μm, 50 × 7.5 mm), and two test columns: PL aquagel-OH 30 (8 μm, 300 × 7.5 mm) and PL aquagel-OH 40 (8 μm, 300 × 7.5 mm). The measurable molecular weight range is 5 × 10⁻⁶. 2 ~5×10 5 g / mol. During the test, the temperature was 35℃, ultrapure water (0.1M NaNO3) was used as the mobile phase, and the flow rate was 1.0 mL / min. The relative molecular weight was determined using polyethylene glycol (PEG) as a standard.

[0096] Dynamic light scattering (DLS) test

[0097] Measurements were performed using a He-Ne laser (wavelength: 640 nm, measurement angle: 90°) on the Brookhaven BI-200SM instrument, and the data were analyzed using the CONTIN method.

[0098] High-resolution transmission electron microscopy (TEM) testing

[0099] TEM testing was performed using a JEOL JEM-1100 transmission electron microscope (TEM). The assembly structure morphology of the fluorinated zwitterionic amphiphilic random copolymer was observed at 200 kV after a dispersion of the polymer assembly solution was dropped onto a carbon-coated copper mesh and dried.

[0100] Ultraviolet-Vis spectrum (UV-vis)

[0101] UV-vis tests were performed using a Shimadzu UV-3600 UV-Vis spectrophotometer.

[0102] Fluorescence spectrum

[0103] Fluorescence spectra were measured using a Horiba Fluoromax-4 fluorescence spectrometer (USA), and the fluorescence quantum yield (Φ) of the fluorescent nanoparticles was calculated with reference to the literature (J. De-La-Cuesta, JAPomposo, ACS Omega, 2018, 3, 15193-15199). F,a ).

[0104] Laser confocal microscopy (CLSM)

[0105] Fluorescence imaging tests on live cells were performed using a Zeiss LSM-710 instrument from Germany.

[0106] Example 1

[0107] (1) Synthesis of phenyl 2-bromo-2-phenylacetic acid (PBPA) via esterification, a conventional ATRP initiator.

[0108] Under a nitrogen atmosphere and in an ice bath at 0°C, 8.1 g (37.5 mmol) of α-bromophenylacetic acid, 2.35 g (25.0 mmol) of phenol, 0.46 g (3.8 mmol) of 4-dimethylaminopyridine (DMAP), and 100 mL of dry dichloromethane solvent were placed in a 250 mL three-necked round-bottom flask. 7.74 g (37.5 mmol) of N,N-dicyclohexylcarbodiimide (DCC) was dissolved in 60 mL of dry dichloromethane and added dropwise to the flask using a constant-pressure dropping funnel. The mixture was stirred at room temperature for 24 hours, and the reaction was monitored by thin-plate chromatography (TLC).

[0109] After the reaction was completed, the insoluble matter was removed by filtration, and the filtrate was evaporated to dryness by rotary evaporation. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (volume ratio of 50:1). The mobile phase was collected and evaporated to dryness by rotary evaporation, and then dried in a vacuum oven at 40°C to constant weight to obtain the target product PBPA with a yield of 82.4%.

[0110] (2) Synthesis of 2-bromo-2-phenylacetic acid pyrene ester, an ATRP initiator containing a pyrene chromophore, via esterification reaction. (PyrBPA)

[0111] The ATRP initiator 2-bromo-2-phenylacetic acid pyrene ester (PyrBPA) containing a pyrene chromophore was prepared using essentially the same method as in (1), the only difference being that 25.0 mmol of 1-hydroxypyrene was used instead of phenol. The yield of the target product PyrBPA was 70.0%.

[0112] (3) Synthesize ATRP initiator 2-bromo-2-phenylacetic acid-4-tetraphenylene containing tetraphenylethylene group by esterification reaction. Ester (TPEBPA)

[0113] 2-Bromo-2-phenylacetic acid-4-tetraphenylethylene ester (TPEBPA), an ATRP initiator containing a tetraphenylethylene group, was prepared using essentially the same method as in (1), except that 25.0 mmol of 4-(1,2,2-triphenylvinyl)phenol was used instead of phenol. The yield of the target product TPEBPA was 75.2%.

[0114] Figure 1 The 1H NMR spectra of the three initiators are shown. Figure 1 (a) is the 1H NMR spectrum of PBPA in deuterated dimethyl sulfoxide solvent. The characteristic hydrogen proton c-shift located on the same carbon atom as bromine is at 6.27 ppm, while the remaining hydrogen protons on the aromatic ring are labeled. The proton peak intensity ratio is consistent with the target structure product. Additionally, high-resolution mass spectrometry analysis showed that [PBPA + Na]... + The molecular weight of [ ] is 312.9835, which is consistent with the theoretical value (C 14 H 11 BrO2Na + The results (312.9835) are consistent, further proving that the target product was successfully prepared.

[0115] Figure 1 (b) and (c) are the 1H NMR spectra of the prepared catalysts PyrBPA and TPEBPA in deuterated chloroform solvent, respectively. Figure 1 The chemical shifts of each hydrogen proton were marked in (b) and (c), and the proton peak intensity ratios matched the target structure product, thus the target initiator was obtained.

[0116] Example 2

[0117] This example illustrates the ATRP polymerization conditions for trifluoroethyl methacrylate (TFMA).

[0118] Experiment 2-2: According to [TFMA]0:[PBPA]0:[FeCl3] · The initial feed ratio of [6H2O]0:[TDA]0:[AsA]0 = 100:1:2:6:2 was used to feed 0.67g (4.0mmol) of monomer TFMA, 12.5mg (0.04mmol) of initiator PBPA, and 21.6mg (0.08mmol) of metal salt FeCl3. · 6H₂O, 77.6 mg (0.24 mmol) of the ligand tris(3,6-dioxaheptyl)amine (TDA), 15.8 mg (0.08 mmol) of the reducing agent ascorbic acid (AsA), and 2.0 mL of anhydrous solvent trifluoroethanol (TFE) were added to an ampoule equipped with a magnetic stir bar. After at least one oxygen removal and nitrogen purging operation, the ampoule was sealed. The ampoule was then transferred to an oil bath on a magnetic stirrer at 1800 rpm and heated at 80 °C for ATRP polymerization. After a given time of 8 hours, the sealed container was removed and opened. The polymerization system was diluted with 2 mL of TFE and dialyzed in methanol (dialysis membrane with a molecular weight cutoff of 1 kDa) for 48 hours to remove unreacted monomers, metal catalysts, and other impurities. The mixture was then dried in a vacuum oven at 35 °C to constant weight to obtain the target polymer.

[0119] The experiment was conducted using essentially the same method as Experiment 2-2, with the only difference being:

[0120] In Experiment 2-1, an equimolar amount of triphenylphosphine (PPh3) was used instead of TDA;

[0121] In experiments 2-3, equimolar amounts of sodium ascorbate (AsA-Naa) were used instead of ascorbic acid (AsA);

[0122] In experiments 2-4 and 2-5, equimolar amounts of FeBr3 and CuBr2 were used instead of FeCl3, respectively. · 6H2O;

[0123] In experiments 2-6, the polymerization temperature was adjusted to 70℃;

[0124] In Experiments 2-7, the polymerization temperature was adjusted to 60℃.

[0125] The polymerization conditions and results of experiments 2-1 to 2-7 are shown in Table 1.

[0126] Table 1. ATRP polymerization conditions and results of TFMA monomers

[0127]

[0128] As shown in Table 1, for the four catalytic systems FeCl3 / PPh3 (Experiment 2-1), FeCl3 / TDA (Experiment 2-2), FeBr3 / TDA (Experiment 2-4), and CuBr2 / TDA (Experiment 2-5), when FeCl3 / TDA was used as the catalytic system, the ideal polymer was successfully prepared. The monomer conversion rate of TFMA was 58.8%, and the GPC molecular weight (M) of the synthesized PTFMA was [missing information]. n,GPC The molecular weight is 43.5 kDa, and the molecular weight distribution (M) obtained by GPC is... w / M n The value is 1.34. Furthermore, through... 1 The NMR molecular weight M of PTFMA obtained by H NMR n,NMR It is 13.0 kDa, which is consistent with the theoretical molecular weight M calculated based on the conversion rate. n,th Relatively consistent (M) n,th =9.9kDa), where M n,GPC The result is significantly higher than expected, which may be due to the significant difference in solubility and solution behavior between the fluorinated side-group polymer PTFMA and the standard sample polystyrene (PSt) used in the GPC test.

[0129] Experiments 2-2, 2-3, 2-6, and 2-7 show that as the polymerization temperature increases to 80℃, the M of the synthesized polymer PTFMA increases. w / M n The value dropped to 1.34, indicating enhanced polymerization controllability. This may be due to the faster establishment of a reversible equilibrium between active free radicals and dormant species.

[0130] Example 3

[0131] This example is used to determine the ATRP polymerization feed ratio and reaction time.

[0132] The experiment was conducted using the method described in Experiment 2-2 of Example 2, according to the feed ratio and polymerization time in Table 2. The results are shown in Table 2. The feed ratio in Table 2 refers to the initial feed ratio [TFMA] 0: [PBPA] 0: [FeCl3]. . [6H2O]0:[TDA]0:[AsA]0, and in experiments 3-6 and 3-7, equimolar amounts of the monomer methacryloyloxyethyl sulfonate betaine (SBMA) were used instead of TFMA, and the amount of solvent TFE was increased to 4.0 mL.

[0133] Table 2. ATRP polymerization feed ratio and results

[0134]

[0135] Experiments 3-1, 3-2, and 3-3 show that the metal salt FeCl3 .Doubling the amount of 6H2O can prepare a more desirable polymer in a shorter reaction time, in which the monomer conversion rate of TFMA increased from 20.4% to 33.6% (Experiment 3-2) and 31.8% (Experiment 3-3), and the GPC molecular weight (M) of PTFMA also increased. n,GPC The molecular weight distribution values ​​(M) are 16.4 kDa and 12.0 kDa, respectively. w / M n The values ​​are 1.17 and 1.18 respectively. Therefore, a relatively high metal feed rate can ensure efficient polymerization.

[0136] Experiments 3-2 to 3-4 show that increasing the amount of reducing agent reduces the conversion rate, especially in experiment 3-4, where the reducing agent ratio [PBPA]0:[FeCl3] is significantly lower. . When the ratio of [6H2O]0:[AsA]0 is increased to 1:2:4, the monomer conversion rate of TFMA after 15 hours of polymerization is 32.0%, which is comparable to that in experiment 3-3. Therefore, the optimal reducing agent ratio [PBPA]0:[FeCl3] is selected. . The ratio of 6H2O]0:[AsA]0 is 1:2:2.

[0137] From experiments 3-2, 3-3, and 3-5, it can be seen that when [FeCl3] . When the feed ratio of [6H2O]0:[TDA]0 was increased to 2:6 (experiments 3-5), a higher monomer conversion rate (58.8%) was achieved, and the polymerized GPC molecular weight (M) was also higher. n,GPC The molecular weight is as high as 43.5 kDa, and the molecular weight distribution is controlled within 1.4. The reason may be that sufficient ligands can not only improve the solubility of metal salts and ensure that the metal salts and ligands in the system are completely complexed to form metal complexes and participate efficiently in the ATRP catalytic process, but also significantly improve the controllability of the polymerization process by rapidly establishing a reversible equilibrium between catalytically active free radicals and dormant species.

[0138] As can be seen from experiments 3-6 and 3-7, when SBMA monomer is used instead of TFMA, a relatively ideal polymerization result is also obtained under the condition of initial feed ratio of 100:1:2:6:2.

[0139] Example 4

[0140] This example demonstrates the ATRP random copolymerization of trifluoroethyl methacrylate (TFMA) and methacryloyloxyethyl sulfonate betaine (SBMA).

[0141] According to [TFMA]0:[SBMA]0:[PBPA]0:[FeCl3 ·The initial feed ratio of [6H2O]0:[TDA]0:[AsA]0 = 40:60:1:2:6:2 was used to add 0.27g (1.6mmol) TFMA, 0.68g (2.4mmol) SBMA, 12.5mg (0.04mmol) initiator PBPA, and 21.6mg (0.08mmol) metal salt FeCl3. · 6H₂O, 77.6 mg (0.24 mmol) of ligand TDA, 15.8 mg (0.08 mmol) of reducing agent AsA, and 4.0 mL of anhydrous solvent TFE were added to an ampoule equipped with a magnetic stirrer. After at least one deoxygenation and purging with inert nitrogen gas, the ampoule was sealed. The ampoule was then transferred to an oil bath on a magnetic stirrer at 1800 rpm and heated at 80 °C for ATRP polymerization. After the given time, the sealed container was removed and opened. The polymerization system was diluted with 4 mL of TFE and then dialyzed sequentially in large volumes of deionized water (dialysis membrane with a molecular weight cutoff of 1 kDa) for 24 hours and in methanol for 48 hours to remove unreacted monomers, metal catalysts, and other impurities. Finally, the ampoule was dried in a vacuum oven at 35 °C to constant weight to obtain the target polymer.

[0142] In addition, the ratio of the two monomers [TFMA]0:[SBMA]0 was adjusted to 60:40, and ATRP random copolymerization was carried out according to the above method. Figure 2 The reaction kinetic curves show the changes in the ln([M]0 / [M]) values ​​of the two monomers with polymerization time under different initial feed ratios.

[0143] Depend on Figure 2 It can be seen that changing the monomer feed ratio ([TFMA]0:[SBMA]0) has no significant effect on the polymerization behavior of ATRP random copolymerization; the monomer conversion rates of both TFMA and SBMA increase synchronously with polymerization time. It is believed that under different feed ratios, the synchronous consumption of TFMA and SBMA ensures that the hydrophilic sulfonate betaine SBMA and the hydrophobic trifluoroethyl monomer TFMA units can be randomly polymerized into the copolymer chain according to the feed ratio. Furthermore, the reaction kinetics of the monomer ln([M]0 / [M]) value changing with reaction time are approximately linear, confirming that the ATRP copolymerization of TFMA and SBMA exhibits first-order kinetic characteristics. This indicates that ATRP polymerization can achieve effective control over the composition and molecular weight of the random copolymer P(TFMA-r-SBMA), and that fluorinated zwitterionic random copolymer P(TFMA-r-SBMA) with the target structure can be prepared in a controlled manner through ATRP polymerization.

[0144] Example 5

[0145] This example illustrates the preparation of functionalized zwitterionic random copolymers via copolymerization of TFMA and SBMA using an ATRP initiator with introduced fluorescent groups.

[0146] According to Table 3, the initial feed ratio of monomers TFMA and SBMA was adjusted to 10–30:90–70. Functionalized fluorinated zwitterionic random copolymers were prepared using the initiators PyrBPA and TPEBPA prepared in Example 1, respectively, according to the method described in Example 4. The polymerization conditions were as follows: the polymerization system composition was [TFMA]0:[SBMA]0:[Initiator]0:[FeCl3]0. · [6H2O]0:[TDA]0:[AsA]0=m:n:1:2:6:2, where m and n are the theoretical degree of polymerization of TFMA and SBMA units, respectively, as shown in Table 3 (m / n). th One column; total monomer usage n Monomer = 4.0 mmol; the amount of anhydrous solvent TFE was 2.0 mL; the reaction temperature was 80 °C; and the reaction time was 24 hours.

[0147] Table 3. Preparation and properties of functionalized fluorinated zwitterionic random copolymers

[0148]

[0149] Note: m / n th The theoretical degree of polymerization of the repeating units of trifluoroethyl and sulfonate betaine; theoretical molecular weight M. n,th This represents the number-average molecular weight calculated based on the theoretical degree of polymerization and conversion rate; m / n exp The degree of polymerization obtained from the experiment is indicated; TFMA (mol%) represents the molar percentage content calculated based on the degree of polymerization using NMR; M n,th =(M TFMA ×m+M SBMA ×n)×Conv.%+M Initiator .

[0150] As shown in Table 3, this invention controllably prepares functionalized fluorinated zwitterionic random copolymers via ATRP polymerization. For example, when using the initiator PyrBPA, Pyr-P(TFMA) m -r-SBMA n Total aggregation degree (DP) total =m+n) was set to 100, and the theoretical molar ratio of TFMA was increased from 10% to 30% (experiments 5-1 to 5-3), through 1The molar ratio of TFMA measured by the integrated area ratio in the H-NMR spectrum increased from 9.1% to 28.4%, which is in high agreement with the theoretical molar ratio calculated from the conversion rate, and the theoretical molecular weight M based on the monomer conversion rate is also consistent. n,th With Pass 1 Molecular weight M measured by H NMR n,NMR There is also a high degree of consistency among them.

[0151] Figure 3 The 1H NMR spectrum of the fluorinated zwitterionic random copolymer is shown, wherein (a) is the NMR spectrum of the copolymer prepared according to Example 4 in the manner described in [TFMA]0:[SBMA]0:[PBPA]0:[FeCl3]. · The 1H NMR spectrum of the fluorinated zwitterionic random copolymer prepared at an initial feed ratio of 6H2O]0:[TDA]0:[AsA]0 = 40:60:1:2:6:2 is denoted as "PBPA-P(TFMA)". 40 -r-SBMA 60 (b) is the 1H NMR spectrum of the fluorinated zwitterionic random copolymer prepared in Experiment 5-2 of Example 5, denoted as "Pyr-P(TFMA)". 20 -r-SBMA 80 (c) is the 1H NMR spectrum of the fluorinated zwitterionic random copolymer prepared in experiments 5-8 of Example 5, denoted as "TPE-P(TFMA)". 20 -r-SBMA 80 )".

[0152] See Figure 3 (b) The signal peak between 7.20 and 8.00 ppm is the hydrogen proton peak of the pyrene group of the initiator, indicating that the pyrene group of the initiator PyrBPA has been successfully introduced into the chain end of the copolymer. Specifically, in the polymerization feed ratio [TFMA]0:[SBMA]0:[PyrBPA]0:[FeCl3]... · The copolymer Pyr-P(TFMA) was synthesized by reacting [6H2O]0:[TDA]0:[AsA]0 = 20:80:1:2:6:2 at 80°C for 24 hours under a nitrogen atmosphere. 20 -r-SBMA 80 The reaction conversion rate reached 71.0%, and the molecular weight (M) of the synthesized random copolymer was... n,NMR The value is 18.6 kDa.

[0153] See Figure 3 (c) The signal peak between 7.20 and 8.00 ppm is the hydrogen proton peak of the initiator tetraphenylene group, indicating that the tetraphenylene group of the initiator TPEBPA has been successfully introduced into the chain end of the copolymer.

[0154] therefore, Figure 3The correctness of the structure of the three fluorinated zwitterionic random copolymers P (TFMA-r-SBMA) was confirmed. The target functional group structure at the end of the polymer chain was well preserved. The preparation method of the present invention can efficiently prepare the target structure fluorinated zwitterionic random copolymer, ensuring the preparation of fluorescent nanoparticles through polymer composition structure design in the later stage.

[0155] Example 6

[0156] This embodiment illustrates the preparation of single-chain nanoparticles from fluorinated zwitterionic random copolymer P(TFMA-r-SBMA).

[0157] Specifically, 5 mg of fluorinated zwitterionic random copolymer P (TFMA-r-SBMA) was weighed, dissolved in 5 mL of deionized water, sonicated at room temperature (power 250 W) for 20 minutes, and then allowed to stand for 10 minutes to obtain fluorescent nanoparticles assembled from polymer solution.

[0158] 20 μL of sample solution was pipetted onto a carbon film copper grid and dried at room temperature for 24 hours before being characterized by transmission electron microscopy (TEM). Dynamic light scattering (DLS) was also performed on the freshly prepared polymer solution (1 mg / mL).

[0159] Figure 4 A schematic diagram is shown showing the preparation of single-chain nanoparticles by self-folding assembly of fluorinated zwitterionic random copolymers. Figure 5 The DLS characterization results of the solution assembly of the fluorinated zwitterionic random copolymer are shown. Figure 5 As shown, at a solution concentration of 1.0 mg / mL, two series of amphiphilic random copolymers Pyr-P(TFMA) m -r-SBMA n ) and TPE-P (TFMA m -r-SBMA n The average hydrodynamic particle size (D) h All particles are less than 15.0 nm, and the particle size distribution (PSD) value is less than 0.10, which are typical particle size characteristics of SCNPs.

[0160] Figure 6 TEM images and particle size distribution diagrams of the fluorinated zwitterionic random copolymer solution assembly are shown. Figure 6 As shown, the nanoparticles obtained by this invention are spherical with a diameter of approximately 15 nm, slightly larger than the particle size measured by DLS. This may be because the copolymer nanoparticles in the non-crosslinked system become flattened and larger in size on the carbon-coated TEM copper grid.

[0161] Furthermore, when the solution concentration was increased to 5.0 mg / mL, DLS and TEM characterization results showed that the properties and size of the solution-assembled single-chain nanoparticles remained largely unchanged, indicating that this type of nanoparticle has a wide range of preparation concentration windows.

[0162] Example 7

[0163] This example illustrates the photophysical properties of fluorescent nanoparticles prepared by assembling a solution of a fluorinated zwitterionic random copolymer.

[0164] 1 mg of fluorinated zwitterionic random copolymer P(TFMA-r-SBMA) was dissolved in 5 mL of deionized water. The solution was sonicated at room temperature (250 W) for 20 minutes and then allowed to stand for 10 minutes to obtain a fluorescent nanoparticle solution (0.2 mg / mL). Its photophysical properties were investigated using a UV-Vis spectrophotometer and a fluorescence spectrometer. Initial UV-Vis absorption tests of the solution indicated that the representative random copolymer P(TFMA-r-SBMA) incorporating Pyr and TPE groups... 20 -r-SBMA 80 Typical absorption peaks are observed at 340 nm and 300 nm, respectively.

[0165] Prepare a series of Pyr-P (TFMA) solutions of different concentrations m -r-SBMA n The fluorescent nanoparticle solution was tested. Figure 7 Pyr-P(TFMA) was displayed. m -r-SBMA n The fluorescence emission spectra of assembled FSCNPs, where (a) is Pyr-P(TFMA) 10 -r-SBMA 90 (a) The fluorescent nanoparticles Pyr-SCNP1 formed by Pyr-P(TFMA) were shown in Figure 1. 20 -r-SBMA 80 (c) is the fluorescent nanoparticle Pyr-SCNP2 formed by Pyr-P(TFMA) 30 -r-SBMA 70 The fluorescent nanoparticles Pyr-SCNP3 formed by the formation of the fluorescent nanoparticles are shown in (d), which illustrates the relationship between the fluorescence intensity at 396 nm and the fluorescence enhancement ratio (I / I0) and the solution concentration.

[0166] from Figure 7 As can be seen from (a)-(c), the intensity of all FL gradually increases with increasing solution concentration. From... Figure 7 (d) It can be seen that among the three random copolymers, Pyr-P(TFMA) has the largest molar ratio of TFMA. 30 -r-SBMA 70The assembled fluorescent nanoparticles not only exhibited the highest fluorescence intensity but also the largest I / I0 value. It is inferred that the single-chain nanoparticles prepared from fluorinated zwitterionic random copolymers possess a superhydrophilic zwitterionic stable shell and a fluoroalkyl core. Therefore, increasing the molar proportion of trifluoroethyl groups in the random copolymers incorporating fluorescent groups is more conducive to their intrachain self-folding assembly and greatly enhances the stability of the resulting FSCNPs, thus providing better isolation and effectively suppressing their ACQ effect, significantly promoting the PL performance of the fluorescent nanoparticles.

[0167] Prepare a series of TPE-P (TFMA) solutions of different concentrations m -r-SBMA n The fluorescent nanoparticle solution was tested. Figure 8 TPE-P (TFMA) was displayed. m -r-SBMA n The fluorescence emission spectra of assembled FSCNPs, where (a) is TPE-P (TFMA) 10 -r-SBMA 90 (a) Fluorescent nanoparticles TPE-SCNP1 formed by TPE-P (TFMA) 20 -r-SBMA 80 (c) is the fluorescent nanoparticle TPE-SCNP2 formed by TPE-P(TFMA). 30 -r-SBMA 70 The fluorescent nanoparticles TPE-SCNP3 formed by the formation of the fluorescent nanoparticles are shown in (d), which shows the relationship between the fluorescence intensity at 465 nm and the fluorescence enhancement ratio (I / I0) and the solution concentration.

[0168] from Figure 8 As can be seen from (a)-(c), when the solution concentration increases from 0.1 mg / mL to 4.0 mg / mL, the intensity of all FLs gradually increases, with the maximum emission peak around 465 nm. Based on the AIE characteristics of the TPE unit, TPE-P(TFMA) m -r-SBMA n The assembled FSCNPs, due to the fluorinated core providing a somewhat restricted microenvironment for TPE, exhibit AIE fluorescence emission, and the intensity gradually increases with the increase of the molar content of fluorinated side groups and the concentration of nanoparticles.

[0169] Example 8

[0170] This embodiment is used to demonstrate the in vitro cytotoxicity of fluorescent nanoparticles.

[0171] Weigh 5 mg of the fluorinated zwitterionic random copolymer P (TFMA-r-SBMA) prepared in experiments 5-1 to 5-6 of Example 5 and dissolve it in deionized water (5 mL). After sonicating at room temperature (power 250 W) for 20 minutes, let it stand for 10 minutes to obtain the fluorescent nanoparticle stock solution with a concentration of 1.0 mg / mL.

[0172] The cytotoxicity of fluorescent single-chain nanoparticles was evaluated using the MTT assay, with HeLa cells as the selected cell line. Specifically, HeLa cells were first seeded in 96-well plates at a cell density of 5 × 10⁶ cells / well. 3 Cells were cultured at 37°C, 5% CO2, and full humidity for 24 hours in DMEM medium (containing 10% fetal bovine whey, 100 units / mL penicillin, and 100 mg / mL streptomycin). HeLa cells were then co-cultured with PBS solutions containing different concentrations (0, 20, 50, 100, 200, 500, 1000 μg / mL) of fluorescent nanoparticles for 24 hours. 20 μL of PBS solution (5.0 mg / mL) was added to each well, and the cells were cultured for another 4 hours. After removing the medium and MTT, 150 μL of DMSO was added to each well to dissolve formazan crystals in the cells. The absorbance at 570 nm was measured using enzyme-linked immunosorbent assay (ELISA), and the cell viability at different concentrations was calculated.

[0173] The cell staining ability and fluorescence properties of FSCNPs were tested using confocal laser scanning fluorescence microscopy (CLSM).

[0174] Figure 9 The study demonstrates cytotoxicity evaluation tests of fluorescent nanoparticles and fluorescence imaging of HeLa cells.

[0175] Reference Figure 9 (a) In Pyr-SCNP1(Pyr-TFMA) 10 -r-SBMA 90 Experiment 5-1), Pyr-SCNP2 (Pyr-TFMA) 20 -r-SBMA 80 Experiment 5-2) and Pyr-SCNP3 (Pyr-TFMA) 30 -r-SBMA 70 After treating HeLa cells with FSCNPs solution (Experiment 5-3) for 4 hours, bright blue fluorescence was clearly observed in the HeLa cells. Figure 9 (a)(ii), (v), (viii)). By comparing with bright-field cell images ( Figure 9(a)(i), (iv), (vii)) compared, these FSCNPs specifically localized and stained lysosomes in cells. In the case of DP... total Pyr-P (TFMA) of 200 m -r-SBMA n The fluorescent nanoparticles Pyr-SCNP4 (Pyr-TFMA) assembled 20 -r-SBMA 180 Experiment 5-4), Pyr-SCPN5 (Pyr-TFMA) 40 -r-SBMA 160 Experiment 5-5) and Pyr-SCNP6 (Pyr-TFMA) 60 -r-SBMA 140 Similar cell staining behavior was observed in experiment 5-5.

[0176] Reference Figure 9 (b) shows the quantitative fluorescence intensity of Pyr-SCNPs. From Figure 9 (b) It can be seen that DP total The fluorescence intensity of Pyr-SCNPs with a value of 100 is slightly higher than that of DP. total The reason for using a system with a concentration of 200 is likely that, in FSCNP solutions of the same mass concentration, copolymers with shorter polymer chains have a much higher molar number of fluorescent groups. Therefore, assuming successful preparation of FSCNPs, DP is preferred for high-performance fluorescent FSCNPs. total A copolymer with a strength of 100.

[0177] from Figure 9 (c) It can be seen that even when the concentration of Pyr-SCNPs is increased to as high as 1000 μg / mL, the survival rate of all cells is above 85% after 24 hours of incubation in the presence of Pyr-SCNPs with different compositions. Furthermore, the copolymer nanoparticles Pyr-SCNP3 (Pyr-TFMA) with the highest proportion of fluorinated side groups were also observed. 30 -r-SBMA 70 Its biocompatibility is even slightly higher than that of Pyr-SCNP1 (Pyr-TFMA) with lower fluorine content. 10 -r-SBMA 90This not only demonstrates the excellent biocompatibility of Pyr-SCNPs but also corroborates the bioinertness of fluorinated chemicals. In the presence of TPE-SCNPs, cell viability is approximately 100%, indicating that the introduced TPE group has minimal impact on the nanoparticles. In fact, the type of fluorescent group used has almost no substantial impact on the biocompatibility of FSCNP nanoparticles because the fluorescent group is loaded and coated on the core of the FSCNPs, surrounded by fluorinated side chain segments and zwitterionic segments (see...). Figure 4 ).

Claims

1. Fluorescent nanoparticles based on fluorinated zwitterionic random copolymers, wherein, The fluorescent nanoparticles include single-chain nanoparticles formed from poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine, wherein the ends of the molecular chains of poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine are connected to ACQ-type groups selected from anthracene, perylene imide, and pyrene groups, or AIE-type groups selected from tetraphenylethylene, carbazole, and cyanotriphenylethylene groups. In the poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine, the molar ratio of poly(trifluoroethyl methacrylate) repeating units to methacryloyloxyethyl sulfonate repeating units is 10:90 to 90:10; as measured by 1H NMR spectroscopy, the number-average molecular weight of the poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine is 10 to 80 kDa; as measured by dynamic light scattering, the average particle size of the fluorescent nanoparticles is below 20 nm, and the particle size distribution is below 0.

10. The preparation method of the fluorescent nanoparticles includes the following steps: S100, providing α-bromophenylacetic acid ester initiators containing ACQ-type or AIE-type groups; S200 provides a polymerization reaction solution containing trifluoroethyl methacrylate, methacryloxyethyl sulfonate betaine, α-bromophenylacetic acid ester initiator, metal salt catalyst, ligand, reducing agent, and polymerization solvent, wherein the polymerization reaction solution is kept at 60~90°C. o The ATRP polymerization reaction was carried out at a temperature of C and under inert gas protection to obtain the reaction product. S300. The reaction product is purified to obtain a random copolymer of poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl betaine with ACQ-type or AIE-type groups attached to the ends of the molecular chains. S400: Poly(trifluoroethyl methacrylate-co-methacryloyloxyethyl sulfonate betaine) was dissolved and dispersed in water, and then ultrasonicated and allowed to stand to obtain fluorescent nanoparticles. The ligand is selected from triethylamine and tris(3,6-dioxaheptyl)amine.

2. The fluorescent nanoparticles according to claim 1, wherein, In poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine, the molar ratio of poly(trifluoroethyl methacrylate) repeating units to methacryloyloxyethyl sulfonate repeating units is 10:90 to 50:

50. And / or, as measured by 1H NMR spectroscopy, the number-average molecular weight of the poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine is 15-50 kDa. And / or, as measured by dynamic light scattering, the average particle size of the fluorescent nanoparticles is 5–20 nm, and / or the particle size distribution is 0.01–0.

10.

3. The fluorescent nanoparticles according to claim 1, wherein, In poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine, the molar ratio of poly(trifluoroethyl methacrylate) repeating units to methacryloyloxyethyl sulfonate repeating units is 10:90 to 40:

60. And / or, as measured by dynamic light scattering, the average particle size of the fluorescent nanoparticles is 7.5–15 nm; and / or, the particle size distribution is 0.01–0.

08.

4. The fluorescent nanoparticles according to claim 1, wherein, In poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine, the molar ratio of poly(trifluoroethyl methacrylate) repeating units to methacryloyloxyethyl sulfonate repeating units is 10:90 to 30:

70. And / or, as measured by dynamic light scattering, the particle size distribution of the fluorescent nanoparticles is 0.01 to 0.

05.

5. The fluorescent nanoparticles according to any one of claims 1 to 4, wherein, The fluorescent nanoparticles are in the form of a dispersion.

6. The fluorescent nanoparticles according to any one of claims 1 to 4, wherein, The fluorescent nanoparticles are in the form of an aqueous dispersion.

7. The fluorescent nanoparticles according to claim 6, wherein, The concentration of poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine in the aqueous dispersion is 0.05~5.0 mg / mL.

8. The fluorescent nanoparticles according to claim 6, wherein, The concentration of poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl sulfonate betaine in the aqueous dispersion is 0.1~4.5 mg / mL.

9. The method for preparing fluorescent nanoparticles according to any one of claims 1 to 8, wherein, The preparation method includes the following steps: S100, providing α-bromophenylacetic acid ester initiators containing ACQ-type or AIE-type groups; S200 provides a polymerization reaction solution containing trifluoroethyl methacrylate, methacryloxyethyl sulfonate betaine, α-bromophenylacetic acid ester initiator, metal salt catalyst, ligand, reducing agent, and polymerization solvent, wherein the polymerization reaction solution is kept at 60~90°C. o The ATRP polymerization reaction was carried out at a temperature of C and under inert gas protection to obtain the reaction product. S300. The reaction product is purified to obtain a random copolymer of poly(trifluoroethyl methacrylate)-co-methacryloyloxyethyl betaine with ACQ-type or AIE-type groups attached to the ends of the molecular chains. S400: Poly(trifluoroethyl methacrylate-co-methacryloyloxyethyl sulfonate betaine) was dissolved and dispersed in water, and then ultrasonicated and allowed to stand to obtain fluorescent nanoparticles. The ligand is selected from triethylamine and tris(3,6-dioxaheptyl)amine.

10. The preparation method according to claim 9, wherein, The α-bromophenylacetic acid ester initiator is 2-bromo-2-phenylacetic acid pyrene ester or 2-bromo-2-phenylacetic acid-4-tetraphenyl ester.

11. The preparation method according to claim 10, wherein, Step S100 includes: S110, Prepare an esterification reaction solution containing α-bromophenylacetic acid, an aromatic compound containing phenolic hydroxyl groups selected from 1-hydroxypyrene and 4-(1,2,2-triphenylvinyl)phenol, an esterification catalytic system, and an esterification solvent. The esterification reaction solution is heated at 15~30°C. o The esterification reaction was carried out under C and an inert atmosphere to obtain the esterified product; S120. The esterification product is filtered, dried for the first time, purified by silica gel column, and dried for the second time to obtain α-bromophenylacetic acid ester initiator.

12. The preparation method according to claim 11, wherein, In step S110, the molar ratio of α-bromophenylacetic acid to the aromatic compound containing a phenolic hydroxyl group is 0.5~2:1; And / or, the esterification catalytic system described in step S110 is selected from N,N-cyclohexylcarbodiimide and 4-dimethylaminopyridine; And / or, the molar ratio of the esterification catalysis system to the aromatic compound containing the phenolic hydroxyl group is 0.1~0.3:1; And / or, the esterification solvent is selected from anhydrous dichloromethane or anhydrous tetrahydrofuran; And / or, the concentration of α-bromophenylacetic acid in the esterification reaction solution is 10~100 mg / mL.

13. The preparation method according to claim 12, wherein, In step S110, the molar ratio of α-bromophenylacetic acid to the aromatic compound containing a phenolic hydroxyl group is 1~1.5:1; And / or, the molar ratio of the esterification catalysis system to the aromatic compound containing the phenolic hydroxyl group is 0.1~0.2:1; And / or, the concentration of α-bromophenylacetic acid in the esterification reaction solution is 20~50 mg / mL.

14. The preparation method according to any one of claims 9 to 13, wherein, In step S200, the molar ratio of trifluoroethyl methacrylate and methacryloyloxyethyl sulfonate betaine is 10:90 to 90:

10.

15. The preparation method according to claim 14, wherein, In step S200, the molar ratio of trifluoroethyl methacrylate and methacryloyloxyethyl sulfonate betaine is 10:90 to 50:

50.

16. The preparation method according to claim 14, wherein, In step S200, the molar ratio of trifluoroethyl methacrylate and methacryloyloxyethyl sulfonate betaine is 10:90 to 40:

60.

17. The preparation method according to claim 14, wherein, In step S200, the molar ratio of trifluoroethyl methacrylate and methacryloyloxyethyl sulfonate betaine is 10:90 to 30:

70.

18. The preparation method according to any one of claims 9 to 13, wherein, The metal salt catalyst is selected from FeCl3, FeBr3, and CuBr2; And / or, the reducing agent is selected from azobisisobutyronitrile, ascorbic acid, and sodium ascorbate; And / or, the polymerization solvent is trifluoroethanol; And / or, the total molar concentration of trifluoroethyl methacrylate and methacryloyloxyethyl sulfonate betaine in the polymerization reaction solution is 0.5~2.0 mmol / mL.

19. The preparation method according to any one of claims 9 to 13, wherein, The purification process in step S300 includes: S310. The reaction product is diluted with a polymerization solvent and dialyzed in water and methanol in sequence. S320. Vacuum dry the product obtained from dialysis.

20. The preparation method according to claim 19, wherein, In step S310, the reaction product is diluted 2 to 4 times with a polymerization solvent; And / or, the dialyzing conditions in water and methanol in step S310 include: molecular weight cutoff of 0.5~2 kDa, time of 12~48 hours.

21. The preparation method according to any one of claims 9 to 13, wherein, In step S400, the weight ratio of water to poly(trifluoroethyl methacrylate-co-methacryloyloxyethyl sulfonic acid betaine) is 1000:0.1~5. And / or, the conditions for ultrasound in step S400 include: power 200-300 W, time 15-25 minutes; And / or, the settling time in step S400 is 5 to 20 minutes.

22. The use of the fluorescent nanoparticles according to any one of claims 1 to 8 in cell imaging for non-diagnostic and non-therapeutic purposes.

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