Materials and Characterization

1-Bromooctadecane (Aladdin, 97%), potassium tert-butanol (Aladdin, 95%), phosphoryl chloride (Xiya Reagent Co., Ltd., 99%), 2,2,2-trifluoroethyl methacrylate (TFEMA) (Aladdin, 98%), 6-chloro-2-hexanone (Aladdin, 97%), tetrabutylammoniumhydroxide solution (TBAH) (Aladdin, ~25% in methanol, ~0.8 mol/L), phenothiazine (Aladdin, 98%), sodium iodide, 1,2-dichloroethane (Aladdin, 99.8%), 4-hydroxyphenylacetonitrile (Aladdin, 98%), 2,2-azobisisobutyronitrile (AIBN) (Aladdin, 99%), Novozym 435, potassium carbonate, triethylamine (TEA) and sodium bicarbonate were purchased directly for use. Poly(ethylene glycol) monomethacrylate (PEGMA) (Aladdin, M_n=475, AR) was purified through an aluminum oxide column before use. The solvents such as n-hexane, methylene chloride, etc. used directly in the experiment were analytical reagent. 6-Chloro-2-hexanol (CHOL),10-octadecyl-10H-phenothiazine-3-carbaldehyde (OPC) and chain transfer agent (CTA) of 4-cyano-4-(ethylthiocarbonothioylthio) pentanoic acid were self-made in the laboratory according to the reported references.^[37−40]

The chemical structure of the target products was determined by hydrogen spectroscopy of nuclear magnetic resonance (¹H-NMR) and Fourier transform infrared (FTIR) spectroscopy. The ¹H-NMR spectroscopy was measured on a JEOL JNM-ECA 400 (400 MHz) spectrometer with chloroform (CDCl₃) as the solvent and tetramethylsilane (TMS) as the reference. Infrared spectra were acquired on a PerkinElmer TWO spectrometer. The molecular weight (M_n, g/mol) and the polydispersity index (PDI) were obtained from a gel permeation chromatograph (GPC) model of a Waters 1515 equipment using tetrahydrofuran (THF) as mobile phase at 1.0 mL/min flow rate. Transmission electron microscopy (TEM) images of copolymer nanoparticles were obtained on a transmission electron microscope model of JEM-1200EX at an operating voltage of 100 kV. The preparation process of the test sample was as follows: 10 mg of the target copolymers were added into 4 mL of deionized water and dissolved completely, and then added a drop of mixed solution onto the carbon-coated copper grids and let it stand until dry. Ultraviolet-visible spectra (UV-Vis) were recorded on a Perkin-Elmer LAMBDA 35 spectrophotometer. The fluorescence curves were acquired on a Hitachi F-2700 spectrophotometer. The specific rotation of the (R)-PVHMA monomer and PEG-PVH was measured on an SGW-3 autopolarizer in acetone (concentration=10 mg/mL) at 25 °C.

Synthesis of 2-(4-((5-Hydroxyhexyl)oxy)phenyl)- acetonitrile (HPA)

4-Hydroxyphenylacetonitrile (1.13 g, 8.5 mmol), CHOL (1.50 g, 11.0 mmol), potassium carbonate (2.93 g, 21.25 mmol), sodium iodide (140 mg), DMF (35.0 mL) and a magnetic stir bar were added into a Schlenk tube, and then this tube was sealed, frozen in liquid nitrogen and deoxidized for five times of vacuum-nitrogen cycles. The Schlenk tube was then heated and stirred at 100 °C for 24 h in an oil bath. Afterwards, the pH was neutralized to 4−5 with 3 mol/L of hydrochloric acid. An appropriate amount of distilled water was put into the Schlenk tube, and then the organic phase was extracted for three times using ethyl acetate and dried with anhydrous MgSO₄. The reaction mixture was further filtered to remove MgSO₄, and the crude product was acquired through removing the solvent on a rotary evaporator. The product was further purified on a silica column using petroleum ether/ethyl acetate (V/V=1/1) as mobile phase, and the pure product HPA was obtained after completely drying under vacuum (1.61 g, 81%). ¹H-NMR (400 MHz, CDCl₃, δ, ppm): 1.23−1.24 (d, J=4.0 Hz, 3H; CH₃), 1.53−1.56 (m, 4H; ―CH₂―), 1.60−1.64 (m, 1H; ―OH), 1.81−1.85 (m, 2H; ―CH₂―), 3.70 (s, 2H; ―CH₂CN), 3.84−3.88 (m, 1H; ―CH), 3.97−4.00 (t, J=6.0 Hz, 2H; ―CH₂Cl), 6.89−6.91 (d, J=6.0 Hz, 2H; ArH), 7.23−7.25 (d, J=8.0 Hz, 2H; ArH).

Synthesis of (R)-6-(4-(Cyanomethyl)phenoxy)hexan-2-yl Methacrylate ((R)-PHMA)

Novozym435 (294 mg) and a magnetic stir bar were added into a Schlenk tube, and then this tube was sealed and deoxidized for five times by vacuum-nitrogen cycling. HPA (1.63 g, 7.0 mmol), TFEMA (0.71 g, 4.2 mmol) and TEA (0.35 g, 3.5 mmol) were completely dissolved in toluene (20.0 mL), followed by injection of the mixture into the Schlenk tube, which was put into an oil bath at 55 °C and stirred for 24 h. Subsequently, Novozym435 catalytic agent was eliminated through centrifugation, and then the solvent of the crude product was eliminated through a rotary evaporator. Finally, the product was purified through a silica column taking petroleum ether/ethyl acetate (V/V=2/1) as eluent, and the product (R)-PHMA was obtained by drying under vacuum (0.66 g, 63%). ¹H-NMR (400 MHz, CDCl₃, δ, ppm): 1.28−1.30 (d, J=8.0 Hz, 3H; ―CH₃), 1.50−1.70 (m, 4H; ―CH₂―), 1.77−1.84 (m, 2H; ―CH₂―), 1.96 (s, 3H; ―CH₃), 3.70 (s, 2H; ―CH₂CN), 3.95−3.98 (t, J=6.0 Hz, 2H; ―CH₂Cl), 5.00−5.04 (m, 1H; ―CH), 5.56 (s, 1H; ―CH), 6.10 (s, 1H; ―CH), 6.89−6.90 (d, J=4.0 Hz, 2H; ArH), 7.23−7.25 (d, J=8.0 Hz, 2H; ArH).

Synthesis of (R)-6-(4-(1-Cyano-2-(10-octadecane-10H-phenothiazin-3-yl)vinyl)phenoxy)hexan-2-yl methacrylate ((R)-PVHMA)

OPC (1.16 g, 2.42 mmol), (R)-PHMA (0.66 g, 2.2 mmol), TBAH (5−6 drops), ethanol (20.0 mL) and a magnetic stir bar were put into a Schlenk tube, and then this tube was stirred magnetically for 0.5 h at 30 °C to make the mixture completely dissolve. The Schlenk tube was then heated and stirred for 5 h at 78 °C. At the end of the reaction, the product was obtained by removing the solvent on a rotary evaporator, which was further purified through a silica column taking dichloromethane/n-hexane (V/V=2:1) as eluent, and the pure product (R)-PVHMA was dried under vacuum (1.00 g, 60%). ¹H-NMR (400 MHz, CDCl₃, δ, ppm): 0.88−0.92 (t, J=8.0 Hz, 3H, ―CH₃), 1.20−1.34 (m, 29H, ―C₁₃H₂₆―, ―CH₃), 1.42−1.48 (m, 2H, ―CH₂―), 1.52−1.67 (m, 6H, ―CH₂―), 1.80−1.87 (m, 4H, ―CH₂―), 1.97 (s, 3H, ―CH₃), 3.90 (s, 2H, ―CH₂―), 4.00−4.03 (t, J=6.0 Hz, 2H, ―CH₂Cl), 5.00−5.04 (m, 1H, ―CH), 5.56 (s, 1H, ―CH), 6.11 (s, 1H, ―CH), 6.88−6.89 (d, J=4.0 Hz, 2H, ArH), 6.93−6.95 (d, J=8.0 Hz, 3H, ArH), 7.14 (m, 2H, ArH), 7.32−7.34 (d, J=4.0 Hz, 1H, ArH), 7.55 (s, 1H, Ar-CH), 7.56−7.58 (d, J=8.0 Hz, 2H, ArH), 7.80−7.82 (d, J=8.0 Hz, 1H, ArH). ¹³C-NMR (400 MHz, CDCl₃, Fig. S1 in the electronic supplementary information, ESI): 14.13, 17.93, 19.97, 21.31, 22.01, 22.70, 26.94, 29.67, 31.89, 35.75, 67.94, 70.86, 115.00, 124.73, 127.37, 137.11. The molecular weight by TOFMS=762.5 (Fig. S2 in ESI).

Synthesis of PEG-PVH Amphiphilic Copolymers

(R)-PVHMA (104.3 mg, 0.137 mmol), AIBN (1.0 mg, 0.006 mmol), PEGMA (237 mg, 0.499 mmol), CTA (3.1 mg, 0.012 mmol), toluene (2.0 mL) and a magnetic stir bar were placed into a Schlenk tube, subsequently, this tube was sealed, frozen in liquid N₂ and then deoxygenated by vacuum-nitrogen cycles for five times. The Schlenk tube was then heated and stirred at 70 °C for 36 h. After the reaction was completed, the mixture was continually dialyzed with acetone for three times for 12 h each. The crude product was obtained by removing the organic solvent on a rotary evaporator, and then purified by multiple cycles of tetrahydrofuran dissolution and petroleum ether precipitation, and the resulted PEG-PVH1 copolymers were dried under vacuum with the yield of 0.26 g. Another PEG-PVH2 copolymers were fabricated according to the same method as described above, where the molar ratio of (R)-PHMA was adjusted to 29.6%, yield: 0.27 g.

Characterization and Features of PEG-PVH FONs

Scheme 1 shows the synthetic route to the fluorescence monomer (R)-PVHMA and this process can be divided into three parts. Firstly, the nucleophilic substitution reaction of phenothiazine (PHT) and 1-bromooctadecane was used to obtain the intermediate product 10-octadecanephenothiazine (OPHT), which was followed by a Vilsmeier-Haack reaction with DMF catalyzed by phosphoryl chloride to obtain the OPC. Secondly, HPA was obtained by the nucleophilic substitution reaction of 4-hydroxyphenylacetonitrile and CHOL, and it would continually perform the transesterification reaction with TEFMA catalyzed by Novozym435 to obtain chiral molecule (R)-PHMA with R conformation through the efficient chiral resolution of racemic secondary alcohol. Thirdly, the aldehyde group in the OPC structure will undergo a Knoevenagel condensation reaction with the active methylene of (R)-PHMA and obtain the chiral fluorescence monomer (R)-PVHMA. Subsequently, the RAFT polymerization of (R)-PVHMA with PEGMA was used to prepare the corresponding amphiphilic PEG-PVH copolymers with chirality property, which tended to self-assemble into nanoparticles due to their amphiphilicity and could be easily absorbed by cells, as exhibited in Scheme 2.

1Full-screen View

Synthetic route to the fluorescence (R)-PVHMA monomer.

2Full-screen View

Fabrication diagram of the fluorescence PEG-PVH copolymers by RAFT polymerization and their biological imaging application.

The GPC data for the target PEG-PVH1 copolymers was exhibited in Fig. 1(A), the M_n value was about 2.2×10⁴, and their PDI was about 1.30. The structures of the individual target substances OPC, HPA, (R)-PHMA, (R)-PVHMA and PEG-PVH were investigated by ¹H-NMR as exhibited in Fig. 1(B). A simple substitution reaction of phenothiazine and 1-bromooctadecane was used to construct the intermediate product OPHT, which was formylated by DMF to produce OPC through the catalysis of phosphoryl chloride, and the peak assigned to the characteristic proton of the introduced formyl group appeared at 9.80 ppm. At the same time, the triplet peaks at 3.90 ppm originated from the methylene ―CH₂― adjacent to the N atom, indicating that the introduced octadecyl chain was not affected by the formylation reaction. Hydrocarbylation reaction of 4-hydroxyphenylacetonitrile and CHOL formed the corresponding aromatic ether HPA. In the ¹H-NMR spectrum of HPA, the peaks assigned to the proton of ―CH₂＝CN appeared at 3.70 ppm, and the characteristic proton peak of the methylene ―CH₂― linking to the O atom appeared at 3.98 ppm. More importantly, the characteristic peak of the H atom connecting to the chiral C atom appeared at 3.85 ppm. Due to the high enantioselectivity of Novozym435 to racemic secondary alcohol HPA, R configuration HPA had an absolute advantage in the transesterification reaction with TEFMA, and thus the corresponding R configuration chiral intermediate (R)-PHMA was obtained. As compared with HPA, the ¹H-NMR spectrum of (R)-PHMA not only retained the typical peaks at 3.70 and 3.98 ppm, but also presented characteristic single peaks at 5.56 and 6.10 ppm deriving from ―C＝CH₂. Otherwise, the resonant absorption peak of the H atom attached to the chiral C atom transferred to the lower field and appeared at 5.02 ppm from 3.85 ppm due to the successful introduction of the electron-withdrawing methacrylate group, which would result in a decrease of the electrons cloud density and an increase of chemical shift. The chiral fluorescent monomer (R)-PVHMA was constructed by the Knoevenagel condensation reaction of aldehyde group and active methylene group, which connected OPC and (R)-PHMA through a C＝C bond. For the ¹H-NMR spectrum of (R)-PVHMA, the peak at 3.70 ppm assigned to the ―CH₂―CN disappeared completely, while the characteristic proton peaks at 3.98, 5.02, 5.56 and 6.10 ppm were unchanged, in addition, the peak of ―CH₂― linking to the N atom still appeared at 3.90 ppm, and these results implied the successful Knoevenagel condensation reaction between OPC and (R)-PHMA. Basing on RAFT polymerization of (R)-PVHMA and PEGMA, the ―C＝CH₂ double bonds in the structure were broken and the characteristic proton peaks at 5.56 and 6.10 ppm assigned to ―C＝CH₂ completely disappeared. At the same time, the electron density of the H atom bonding to the chiral C atom increased slightly, and its characteristic peak shifted slightly towards the high field, appearing at 4.75 ppm. The peaks of methylene ―CH₂― linking to the ester group in the PEGMA were presented at 4.10 ppm, and the typical peaks of aromatic rings in (R)-PVHMA at 6.90−7.80 ppm. The disappearance of the peaks at 5.56 and 6.10 ppm implied that (R)-PVHMA and PEGMA polymerized successfully into the PEG-PVH copolymers through RAFT polymerization. A rough estimation demonstrated that the molar ratio of (R)-PVHMA in the PEG-PVH1 and PEG-PVH2 copolymers were about 18.1% and 25.7% according to the peaks area ratio of 4.10 ppm and 6.90−7.80 ppm, respectively.

1Full-screen View

(A) GPC curve of PEG-PVH1; (B) ¹H-NMR spectra (CDCl₃) of OPC, HPA, (R)-PHMA, fluorescent (R)-PVHMA dye and fluorescent PEG-PVH copolymers.

The (R)-PVHMA and PEGMA fragments were respectively hydrophobic and hydrophilic, so the obtained copolymers PEG-PVH were amphiphilic, making them easily disperse in aqueous solution and self-assemble into nanoparticles. PEGMA fragments were affinity with water and stretched into water, while hydrophobic (R)-PVHMA fragments were estranged from water and aggregated together which induced fluorescence emission. The stretching PEGMA fragments were considered as a shell and the aggregating (R)-PVHMA fragment as a core, so the copolymers were dispersed into aqueous solution as a fluorescent organic nanoparticle (FONs) with a core-shell structure. The TEM image confirmed this result, as shown in Fig. 2(a), where PEG-PVH1 FONs were spherical particles with about 100 nm in diameter, which also indirectly confirmed the successful combination of the two monomers (R)-PVHMA and PEGMA into the copolymers PEG-PVH.

2Full-screen View

Characterization of (R)-PVHMA monomer and PEG-PVH FONs: (a) TEM image of PEG-PVH dispersed in water, scale bar=1000 nm, (b) FTIR spectra of OPHT, OPC, HPA, (R)-PHMA and (R)-PVHMA dye monomer and fluorescent copolymers PEG-PVH; (c) fluorescence emission curves of PEG-PVH1 copolymers in the H₂O and THF solution with 405 nm excitation wavelength, and the inset shows the fluorescence intensity of PEG-PVH1 in water solution with different concentrations, and the concentration is about 0.50 mg/mL; (d) UV-Vis spectrum of PEG-PVH1 dispersed in aqueous solution, and the concentration is about 40 μg/mL.

In order to further research the structure of the individual target products OPHT, OPC, HPA, (R)-PHMA, (R)-PVHMA and PEG-PVH, their FTIR spectra were analyzed and the results were shown in Fig. 2(b). The introduction of octadecyl group into phenothiazine was performed through a simple nucleophilic substitution reaction, and the FTIR spectrum reflected the successful preparation of OPHT because the typical stretching bands of alkanes obviously were presented at 2850 and 2910 cm⁻¹. In the FTIR spectrum of OPC, a vibration band of the introduced C＝O group evidently appeared at 1690 cm⁻¹, and at the same time, the octadecyl structure remained unchanged. In the curve of HPA, the absorption bands at 2230, 3450 and 2960 cm⁻¹ were assigned to the vibration of cyano C≡N, hydroxy O―H and alkyl chains, respectively, which implied that the hydrocarbylation reaction of 4-hydroxyphenylacetonitrile and CHOL was successful. As compared with HPA, the band at 3450 cm⁻¹ completely disappeared in the curve of (R)-PHMA, while a typical vibration peak of C＝O group appeared at 1720 cm⁻¹, which indicated that the alcohol hydroxyl groups in the HPA structure were completely depleted and the transesterification reaction between HPA and TFEMA was successful. Moreover, the high enantioselectivity of Novozym435 to the racemic HPA was utilized to realize the construction of intermediate (R)-PHMA with R configuration. Compared with (R)-PHMA, the FTIR spectrum of (R)-PVHMA also contained absorption bands at 1720, 2230 and 2960 cm⁻¹, indicating that (R)-PVHMA structure contained (R)-PHMA fragments. The difference mainly concentrated on the range of 1580−1500 cm⁻¹ and 800−750 cm⁻¹, which reflected the stretching vibration and out-of-plane deformation vibration of the conjugated aromatic ring skeleton, respectively. The stretching vibrations of C＝O, ―CH₂― and C―O brought about three distinct absorption bands of 1720, 2870 and 1100 cm⁻¹ respectively, in the spectrum of the PEG-PVH1 copolymers. The structure of the obtained copolymers included typical characteristics of (R)-PVHMA and PEGMA, implying that (R)-PVHMA and PEGMA were copolymerized successfully into the PEG-PVH1 copolymers.

The fluorescence curves of PEG-PVH1 dispersed in THF/H₂O solvents with various volume ratios are exhibited in Fig. 2(c). In THF good solvent, the copolymers PEG-PVH1 could emit fluorescence. The reason was that (R)-PVHMA had a relatively strong electron-donating ability and formed a D-A structure with a strong electron-withdrawing phenylacetonitrile group, so the intramolecular charge transfer (ICT) effect would emit fluorescence.^[41−43] The fluorescence intensity was slightly reduced when the volume of water was about 10% in the mixed solution, which is caused by a twisted intramolecular charge transfer (TICT) with solvent polarity.^[44] With the volume fraction of H₂O in the mixed solvent continually increasing, the fluorescence intensity gradually increased. In water, the fluorescence intensity showed a very remarkable improvement and the maximum fluorescence wavelength of PEG-PVH1 FONs was about 580 nm with a shoulder peak at 630 nm. The reason was that the copolymers PEG-PVH1 gradually aggregated along with the increase of H₂O fraction in the solution, which hindered the intramolecular motion and activated the restriction of intramolecular motion (RIM), indicating the obvious aggregation-induced emission enhancement (AEE) phenomenon. Amphiphilic copolymers PEG-PVH exhibited self-assemble properties in water, forming corresponding PEG-PVH FONs, whose UV-Vis spectrum was exhibited in Fig. 2(d), where there were three peaks at 240, 330 and 410 nm, respectively. The π→π^* electron transition of polycyclic aromatic rings brought the absorption peak at 240 nm, at the same time, due to the presence of hetero atoms O and N in the molecular structure of the copolymers, the n→π^* electron transition of their lone electrons resulted in an absorption peak at 330 nm. As mentioned earlier, (R)-PVHMA had a relatively strong electron-donating capacity and there was an intramolecular charge transfer between electron-withdrawing groups such as cyano, carbonyl groups and phenothiazine, which leaded to an absorption peak at 410 nm.^[23]

Moreover, as shown in Fig. 3, the quantum calculations of (R)-PVHMA dye and its OPC substrate were achieved from B3LYP/6-31+G(d) method of the Gaussian 09 to investigate the D-A structure on the fluorescence property. For the OPC substrate, most of the electrons mainly concentrate on the aromatic rings in HOMO situation, and they would transfer to ―CHO group in LUMO situation, subsequently, these electrons continually transferred to ―CN group in LUMO situation of (R)-PVHMA dye. As compared with OPC, the more dispersed electrons in LUMO situation of (R)-PVHMA would reduce the energy level difference of HOMO and LUMO situations, which was consistent with the fluorescence emission curves as demonstrated in Fig. S3 (in ESI): The emission wavelength of (R)-PVHMA transferred from 530 nm to 600 nm of OPC with stronger intensity, and the emission wavelength red-shift of (R)-PVHMA dye was very profitable for application in bioimaging area. Otherwise, the PEG-PVH copolymers presented also excellent fluorescence emission in other solvents such as ACN, DCM, DMF and DMSO, as shown in Fig. S4 (in ESI).

3Full-screen View

The quantum calculations of LUMO and HOMO orbits of OPC and (R)-PVHMA dyes.

The kinetics analysis of RAFT polymerization was further studied in detail. The ¹H-NMR spectra of the samples with various reaction time are exhibited in Fig. S5 (in ESI). The characteristic peaks at 5.61 and 6.17 ppm were attributed to the ―C＝CH₂ of PEGMA, and the peaks at 5.56 and 6.13 ppm were attributed to the ―C＝CH₂ of (R)-PVHMA. Otherwise, the typical peak at 6.80 ppm assigned to trimethylbenzene was selected as interior label, and the conversion rate of PEGMA and (R)-PVHMA was calculated by monitoring the intensity change of these peaks mentioned above. From the Fig. S5 (in ESI), it can be observed that the intensity of the above four peaks gradually reduced as the polymerization proceeded, which meant that (R)-PVHMA and PEGMA have been gradually used up in polymerization reactions. For 24 h, their conversion rate was about 90% with a high level. The kinetic study of ln([M₀]/[M]) versus time presented a quasilinear first-order procedure as shown in Fig. 4(a). During the polymerization, the conversion rates of (R)-PVHMA and PEGMA remained essentially the same, and the resulted PEG-PVH copolymers should be random copolymers. As shown in Fig. 4(b), during RAFT polymerization, the molecular weights nearly rose linearly with narrow PDI as monomers conversion, indicating a controllable RAFT procedure.

4Full-screen View

Kinetics study of PEGMA and PEG-PVH RAFT polymerization: (a) conversion rates and the kinetic analysis of PEG-PVH and PEGMA monomers versus reaction time, (b) the molecular weights and PDI versus monomer conversion.

Subsequently, the optical activity of (R)-PVHMA fluorescent monomer and its PEG-PVH2 copolymers was investigated. In the transesterification procedure of HPA and TFEMA, the catalyst Novozym435 played a crucial role. Its high enantioselectivity ultimately led to the preferential consumption of the (R)-HPA enantiomer, which successfully achieved the transformation from racemate (±) HPA to levorotatory (R)-PVHMA. The specific rotation ${ \left[\alpha \right]}_{D}^{25 ^\circ {\rm{C}}}$ of the resulted (R)-PVHMA monomer was about −6.39°, and that of PEG-PVH2 copolymers was about −2.96°.

Cytotoxicity and Bioimaging Evaluation of PEG-PVH FONs

L929 cells were used as the reference to evaluate the cytotoxicity of the copolymers PEG-PVH. In the CCK-8 assay, L929 cells were subjected to special treatment: incubation with various concentrations of PEG-PVH1 FONs solution (0, 10, 20, 40, 80, 120 μg/mL) for 8 and 24 h.^[45−48] As exhibited in Fig. 5(a), the cell viability of each experimental group was above 90%, indicating that the prepared PEG-PVH1 FONs have low cytotoxicity and excellent biocompatibility, conferring the potential biomedical applications of the prepared PEG-PVH1 FONs.

5Full-screen View

Biocompatibility estimation and cells imaging of PEG-PVH1 FONs: (a) cell viability of PEG-PVH1 FONs with L929 cells; (b−d) CLSM images of L929 cells incubating with 20 μg/mL of PEG-PVH1 for 3 h at 37 °C, and then fixed with 4% paraformaldehyde for 10 min, (b) bright field, (c) excited by a 405 nm laser, (d) combined image of (b) and (c). Scale bars=100 μm.

Accordingly, the effect of the prepared PEG-PVH1 FONs on cell imaging was also evaluated with L929 cells as the representative, and they were incubated with PEG-PVH1 FONs solution at a concentration of 20 μg/mL.^[49,50] Then, their confocal laser scanning microscope (CLSM) images of experimental cells under 405 nm laser excitation were acquired. As shown in Figs. 5(b)−5(d), the experimental cells showed bright yellow fluorescence under excited light, implying that PEG-PVH1 FONs could readily enter into the cells through endocytosis. On closer inspection, very bright fluorescence could be observed in the periphery of each cell, while the interior was fainter. In the consideration of the sizes of cells, cells cores and PEG-PVH1 FONs, PEG-PVH1 FONs should mainly disperse in the cytoplasmic region after entering the cells. The maximum fluorescence emission wavelength tetraphenylethylene skeleton was generally around 470−510 nm, so the imaging performance of PEG-PVH1 FON was better than them owing to red-shift of the emission wavelength. This was because certain molecules or tissues in the organism would fluoresce on their own, which would interfere with the signal during the imaging process, reduce the sensitivity, and affect the imaging effect. The longer fluorescence wavelength of PEG-PVH1 FONs could reduce the background fluorescence of the organism itself to a certain extent, which also meant that the application prospect of PEG-PVH1 FONs in the field of biomedicine was more prospective.