Autofluorescent Polymers: 1H,1H,2H,2H-Perfluoro-1-decanol Grafted

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Autofluorescent polymers: 1H,1H,2H,2H-perfluoro-1decanol grafted poly(styrene-b-acrylic acid) block copolymers without conventional fluorophore Wenting Li, Chaoyue Che, Juanjuan Pang, Zhenhao Cao, Yapei Jiao, Jingjing Xu, Yufang Ren, and Xue Li Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00791 • Publication Date (Web): 17 Apr 2018 Downloaded from http://pubs.acs.org on April 18, 2018

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Autofluorescent polymers: 1H,1H,2H,2H-perfluoro1-decanol grafted poly(styrene-b-acrylic acid) block copolymers without conventional fluorophore Wenting Li, Chaoyue Che, Juanjuan Pang, Zhenhao Cao,Yapei Jiao, Jingjing Xu, Yufang Ren and Xue Li* Shandong Provincial Key Laboratory of Fluorine Chemistry and Chemical Materials, School of Chemistry and Chemical Engineering, University of Jinan, 336 West Road of Nan Xinzhuang, Jinan 250022, People’s Republic of China KEYWORDS: Block copolymers; 1H, 1H, 2H, 2H-perfluoro-1-decanol; poly(styrene-b-acrylic acid); fluorescence

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ABSTRACT

Recently, although several unconventional luminescent polymers have been synthesized, it still remains a significant challenge to prepare various new fluorescent polymers by functionalization of nonflurescent polymers. In the present study, a nonfluorescent 1H,1H,2H,2H-perfluoro-1decanol grafted to nonflurescent polystyrene-b-poly(acrylic acid) block copolymers through simply esterification reaction can exhibit strong blue emission. Based on control experiments and theoretical simulation, we have proposed that the luminescence stems from interchain n→π* interaction between the lone pair (n) of hydroxyl O atoms of carboxyl units and empty π* orbital of ester carbonyl unit. In addition, the fluorescent polymers are successfully employed for fluorescence imaging in living HeLa cell.


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INTRODUCTION Fluorescent polymers have been extensively investigated due to their applications in bacteria detecting, DNA probing, and signal amplification of diagnostics tests, as well as light-harvesting or antennae.1-7 In general, luminescent polymers are referred to the conjugated polymers, which are constructed by π-aromatic building blocks, functioning as emitting units.8 Recently, luminescent polymers without conventional aromatic groups, only containing auxochromophores or unconventional chromophores such as aliphatic tertiary amine, carbonyl, ester, and amide, have attracted extensive attention.9-15 For example, poly(amido amine)s (PAMAMs)16-21 can emit strong blue luminescence without any extra fluorescent reagent. This phenomenon was caused by the aggregations of carbonyl groups. Maleic anhydride polymers22-23 also exhibit strong fluorescence emission and its emission originated from anhydride cluster interacting. These reports demonstrate that without the need of high conjugated system, the clustering of certain functional group could generate chromophore which can emit luminescence under proper conditions. Although many unconventional fluorescent materials have been prepared, the luminescent mechanisms of these materials are still debatable. For example, oxidation of the nitrogen atom in the amino-containing polymers,24 aggregation of multiple carbonyl groups in the carbonylcontaining polymers,9,25 the interaction between carbonyl and phenyl groups26-27 have been proposed to be the mechanism of the emission. For example, π-π interactions of phenyl units and neighboring carbonyl units in the poly(N-isopropyl acrylamide) (PNIPAM)27 and a heterodox cluster by collection of many carbonyl groups26 have been reported. Recently, ‘‘non-conjugated polymer dots (NCPDs)’’ whose fluorescent behaviour are quite similar to that of the emissive carbon dots have been found.28 The functional groups such as C=O, C=N, N=O, –NH2 are


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proposed as sub-fluorophores. The photoluminescence (PL) of these sub-fluorophores can be enhanced by chemical cross linking or physical immobilization of polymer chains.28 Since luminescent polymers without conventional aromatic groups have great potential to be applied in fluorescent bioprobe, drug delivery and gene carrier due to their excellent flexibility, hydrophilicity, structural adjustability, biocompatibility and good biodegradability,3 several unconventional luminescent synthetic polymers have been generated.9,26-30 Currently, most of them are in-situ synthesized via RAFT polymerization,27 free-radical polymerization,9,26 and condensation polymerization,20-30 etc. For example, poly[(maleic anhydride)-alt-(vinyl acetate)] (PMV), a well-known nonconjugated alternating polymers was synthesized by free-radical polymerization.9 PNIPAM with strong blue emission was synthesized by RAFT polymerization27 and polyether amide (PEA) with an aggregation-induced emission feature was synthesized via condensation polymerization.29 Although autofluorescent polymers mentioned above have been synthesized, it still remains a significant challenge to develop a new type of autofluorescent polymers with different chain structures and novel properties for large-scale applications. During the preparation of super-hydrophobic polymeric nanoparticles of an 1H,1H,2H,2Hperfluoro-1-decanol grafted amphiphilic polystyrene-b-poly(acrylic acid) (PS-b-PAA-g-HFD) block copolymers, it is surprising to find that without using any fluorescent compound as part of the esterification reaction process, the obtained PS-b-PAA-g-HFD molecules and their micellar films without π-π conjugated structures exhibited strong blue emission under 350 nm UV light irradiation. However, the esterified amphiphilic PS-b-PAA-g-HFD molecules in solution are non-emissive or feebly luminescent. The remarkable and unconventional blue emission in solid states sparked our tremendous interest in the investigation of its emission mechanism and potential applications. In this paper, we think that the interchain n→π* interaction of the ester


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groups and neighboring carboxyl are responsible for the emission of esterified amphiphilic block copolymer molecules.

EXPERIMENTAL Materials Poly(styrene-b-acrylic acid) (PS-b-PAA, Mn,PS=15000, Mn,PAA=3600, Mw/Mn=1.2), carboxy terminated PS (PS-COOH, Mn=950), polyacrylic acid (PAA, Mn=2992) were all purchased from Polymer Source, Inc. Canada. 1H,1H,2H,2H-perfluoro-1-decanol (HFD, 97%), 1H,1H,2H,2Hperfluoro-1-dodecanol (PFD, 97%), 1H,1H-perfluoro-1-tetradecanol (PFTD, 97%), N,N'diisopropylcarbodiimide (DIPC, 99%), p-toluenesulfonic acid monohydrate (98.5%), and 4(dimethylamino)pyridine (DMAP, 99%) were obtained from Alfa Aesar chemical Co., Ltd. 1,4dioxane, dichloromethane (CH2Cl2), tetrahydrofuran (THF) and ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. n-decanol (DA, 98%) was purchased from J&K Scientific Ltd., China. All Reagents were used as received.

The esterification of PS-b-PAA PS-b-PAA-g-HFD was prepared by the esterification reaction between PS-b-PAA and HFD with the catalysis of 4-(N,N-dimethylamino)pyridinium-4-p-toluenesulfonate (DPTS), which was synthesized according to the previous work.31 The mixture of PS-b-PAA/1,4-dioxane solution (10.0 mL, 5.0 mg/mL) and CH2Cl2 (10.0 mL) was added to a round-bottom flask at room temperature under nitrogen atmosphere. The DIPC/CH2Cl2 (5.49×10-4 mol) and DPTS


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(5.49×10-4 mol) were introduced into the above mixture dropwise (~100 µL/min), and the solution was stirred for 40 min at room temperature. After that, the HFD (623.5 mg) was added into the reaction system and stirring for 48 hours. The product was dialyzed in THF to remove the unreacted HFD and other low molar mass components, and dried under vacuum at room temperature. The PAA-g-HFD and PS-b-HFD samples were also prepared with the similar procedures.

Cell Culture and Fluorescence Imaging HeLa cells were cultured in Dulbecco’s Modified Eagle media (DMEM) supplemented with 10% fetal bovine serum (FBS, Sijiqing), 1% penicillin/streptomycinsulphate (v/v=1/1, Hyclone) under a humidified atmosphere of 5% CO2 at 37 °C oven. HeLa cells were incubated in confocal plates (2 × 104 cells/mL). After 24 h, PS-b-PAA-g-HFD solution (5 µM) was added into the confocal plate and further incubated for 20 min at 37 °C. The imaging process was performed after the cells were washed for twice with PBS buffer (pH=7.4). Fluorescence images were acquired through a high-sensitivity laser confocal microscopy with a 40× objective lens and collected for the blue channel (425−475 nm) upon excitation at 405 nm and for the red channel (570−620 nm) upon excitation at 561 nm.

Characterization Fourier transform infrared (FTIR) spectra were recorded with a FTIR-8400S spectro photometer in the range of 4000-400 cm−1 with KBr plates. The 1H NMR and

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F spectra were

recorded on solutions in CDCl3 on a Bruker BioSpin (400 MHz) spectrometer. X-ray


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photoelectron spectroscopy (XPS) measurements were performed on a Thermo ESCALAB 250 with Al Ka excitation. PL spectra were measured using FLS920 instrument (Edinburgh Instruments, UK) with an excitation wavelength of 350 nm. The slit width used in the measurements is 0.8 nm. Fluorescence imaging experiments were performed with LSM780 confocal microscopy.

RESULTS AND DISCUSSION Preparation and characterization of PS-b-PAA-g-HFD copolymers As shown in Figure 1a, the PS-b-PAA-g-HFD was prepared by esterification reaction of PS-bPAA and HFD, catalyzed with DPTS, at room temperature in the mixed solvent of 1,4-dioxane and CH2Cl2. Figure 1b shows the FTIR spectrum of PS-b-PAA, HDF, and PS-b-PAA-g-HFD. The peak of C-F stretching vibration located at 1183 cm-1 can be found in the HFD grafted on PS-b-PAA block copolymers. More clearly, new peaks located at 1772 and 1029 cm-1, corresponding to the ester bond, also indicates that the HFD has been grafted on the PS-b-PAA chains by esterification reaction. The chemical composition of the PS-b-PAA-g-HFD product was further studied by 1H NMR and 19F NMR technique. As seen in Figure 1c and Figure S1, comparing to the 1H NMR spectra of the PS-b-PAA and HFD, the new signals at 3.99 and 2.36 ppm, which ascribe to H-C-O and H-C-C-F, confirm that HFD molecules are covalently anchored on the PAA block. The above results can also be proved by the

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F NMR spectrum of PS-b-PAA-g-HFD (Figure 1d). The

peaks at -81.01 and -113.7 ppm are ascribed to C-CF3 and -CF2-CH2, respectively. A series of peaks between -121.9 to -126.35 ppm are attributable to -CF2.32 Furthermore, the PS-b-PAA


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block copolymers used in this work is insoluble in THF, while the PS-b-PAA-g-HFD sample can be dissolved in THF very well, also indicating that the chemical structure of PAA block has been changed.

Figure 1. (a) Schematic illustration of the synthetic process of the PS-b-PAA-g-HFD copolymers. (b) The FTIR spectra of PS-b-PAA, HFD, and PS-b-PAA-g-HFD. (c) The 1H NMR spectra of PS-b-PAA (CDCl3/DMSO-d6), HFD and PS-b-PAA-g-HFD (THF/CDCl3). (d)

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F

NMR spectrum of PS-b-PAA-g-HFD (THF/CDCl3).


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XPS results (Figure 2) reveal that the PS-b-PAA-g-HFD product contains C, O, and F elements. The appearance of peaks at 284.1, 287.0 and 288.6 eV on C 1s spectrum are corresponding to the C-C, C-O or C=O and C-F components, respectively. The binding energy of F 1s core electrons at 689.3 eV corresponds to the C-F. In contrast to the HFD composite, the PS-b-PAA-g-HFD sample displays a binding energy shift toward high energy by 2.3 eV for F, which may be caused by the inter-chain interaction. The atomic percentage of C1s, O1s and F1s are 72.43, 24.59 and 2.98, respectively. Therefore, it can be concluded from FTIR, 1H NMR, 19F NMR and XPS results that HFD molecules are grafted on the PAA blocks. The grafting yield of HFD with PS-b-PAA was about 2.02%.


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Figure 2. XPS spectra of PS-b-PAA-g-HFD copolymers. (a) survey, (b) C1s, (c) O1s and (d) F1s, (e) F1s spectra of HFD.

Photophysical properties After confirming the chemical structures of the prepared PS-b-PAA-g-HFD copolymers, we further investigated its photophysical properties. According to our study, the PL spectra of HFD


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and PS-b-PAA illustrate that HFD and PS-b-PAA are non-emissive or feebly luminescent materials (Figure 3a). Excitedly, PS-b-PAA-g-HFD powders display a strong luminescence emission around 425 nm with the excitation of 350 nm UV light. This shining behavior is very similar to those fluorophore-containing polymers,17,18 and promotes us to make sense of the detailed mechanism. Generally, the esterification reaction between the carboxyl groups of nonfluorescent polymers and hydroxyl groups of nonfluorescent alcohol only produces nonfluorescent function polymers. Thus, we were quite surprised to observe strong PL from the PS-b-PAA-g-HFD because no other fluorophore-containing molecules were employed in the esterification reaction. As shown in Figure 3a, no emission was observed in PS-b-PAA-gHFD/THF solution when its concentration was 0.1 wt%, suggesting that no aggregation of fluorophores units was formed in the dilute solution. The fluorescent quantum yield of the PS-bPAA-g-HFD in solid was 4%, which was larger than that (1%) in solution (Table S1). At the same time, we investigated the emission behaviour of PS-b-PAA-g-HFD micelles. The micellar solution was prepared by adding water to the THF solution of PS-b-PAA-g-HFD copolymers to induce self-assembly, and then THF was removed by dialysis against ultrapure water to fix and stabilize the micelles. The TEM images in Figure 3b clearly confirmed the successful preparation polymeric porous micelles based on PS-b-PAA-g-HFD. The porous micellar solution was casted on a silicon substrate to form micellar thin film. The fluorescence microscope images (Figure 3c) show that PS-b-PAA-g-HFD emitted a strong blue and green fluorescence and displayed a weak red fluorescence taken under illumination of UV light, which is consistence with the PL spectra (Figure 4a).


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Figure 3. (a) PL spectra of PS-b-PAA, HFD, PS-b-PAA-g-HFD in solid and PS-b-PAA-g-HFD in THF (λex = 350 nm); (b) TEM image of the micelles formed by PS-b-PAA-g-HFD in water; (c) Fluorescence microscope images of PS-b-PAA-g-HFD micelles casted on a silicon substrate. The images were collected at the blue, green and red channels, respectively. The scale bar in (c) is 25 µm.

In order to understand the mechanism of the fluorescence, more control experiments have been conducted. The poly(styrene-b-acrylic acid-g-decyl alcohol) (PS-b-PAA-g-DA) was synthesized using the same reaction. Figure 4a shows the fluorescence emission spectrum of the PS-b-PAAg-DA in solid phase. Compared to PS-b-PAA-g-HFD (Figure 3a), the fluorescence emission intensity of PS-b-PAA-g-DA decreased, and the fluorescent quantum yield was reduced to 2%, indicating that fluorine atoms have an important influence on the emission behaviors. The


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maximum emission wavelength was also shifted from 425 nm to 411 nm (Figure 4a). The blue shift phenomenon may be due to the electrons delocalization along the fluorocarbon side chains.33 We thus speculated that the emission behaviour of PS-b-PAA-g-HFD and PS-b-PAA-gDA are related to the aggregation of ester groups. It has been reported that PL emission of the PMV polymer is associated with the clustering of the locked carbonyl groups.27 In the poly(amido amine) (PAMAM) system, the emission was attributed to the creation of a more rigid environment, which prevents the excitation to relax through nonradiative pathways.34-36

Figure 4. (a) PL spectrum of PS-b-PAA-g-DA (λex = 350 nm); (b) PL spectra of PS-b-HFD, and PAA-g-HFD (λex = 350 nm).

To further confirm this speculation, PS-b-HFD block copolymers and PAA-g-HFD graft polymers are used for comparison. The emission of PS-b-HFD (Figure 4b) is very weak after having been subjected to esterification between carboxyl-terminal PS and HFD. However, PAAg-HFD graft copolymers exhibit a much stronger blue emission than that of PS-b-PAA-g-HFD (Figure 4b). The measured fluorescent quantum yields of PS-b-HFD and PAA-g-HFD in solid state are 1% and 13%, respectively. To further compare with PAA-g-HFD, the fluorescence emission intensity of the mixture film of PS/PAA-g-HFD prepared by casting the mixed solution


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in THF on a substrate also degreased (Figure S2). These additional evidences essentially rule out the possible role played by phenyl groups in PS blocks in contributing to the PL. Based on this and previous observations, we conclude that the strong PL may come from the aggregation of sub-fluorophores clusters which are composed of the ester carbonyl groups and the carboxyl group in solid state. Aggregation of multiple carbonyl groups induced emission has been adopted to explain the mechanism of the emission of PMV.9 The interaction between the carbonyl groups, linked with fluorocarbon chains, and neighboring carboxyl groups would cause a strong fluorescence emission. Based on the above mechanism, the fluorescence emission intensity should be related to the length of fluorocarbon chains. In order to make sense of the presumption, we also investigated the effect of fluorocarbon chains length on its photophysical behaviour. 1H,1H,2H,2H-perfluoro-1-dodecanol (PFD) and 1H,1Hperfluoro-1-tetradecanol (PFTD) grafted PS-b-PAA were there by prepared. As shown in Figure 5, the longer fluorocarbon chain gives a relatively stronger fluorescence emission. These results indicate that the stronger interactions between the carbonyl groups linked to fluorocarbon chains and its neighboring carboxyl units were formed due to the strong electron-withdrawing ability of fluorocarbon chains which induce protonation of carbon atoms of ester group.


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Figure 5. The PL spectra of PS-b-PAA-g-HFD, PS-b-PAA-g-PFD and PS-b-PAA-g-PFTD.

Theoretical calculation In order to gain insights into the structures of PS-b-PAA-g-DA copolymers, density functional theory (DFT) was employed to calculate the molecular orbitals (MO) and optimize their conformations. To simplify the calculation, the PS-b-PAA-g-DA with five repeating units was employed as the model. It was found that the HOMO orbital is located in the carboxyl units and the LUMO orbital is located in the ester carbonyl unit which has strong interaction with the neighboring carboxyl units27 (Figure 6a). In such interactions, the hydroxyl O atoms of carboxyl units use the lone pair (n) to form n →π* interaction with the empty π* orbital of ester carbonyl unit, which mixes these orbitals and releases the excited energy.37-39 The fluorescence can be enhanced when the hydroxyl O atoms of the electron-pair donator contacted with the carbon of the acceptor carbonyl group along with electron-with drawing group which is consistent with the result of PS-b-PAA-g-HFD (Figure 5).40 Furthermore, no emission of PS-b-PAA-g-DA and PSb-PAA-g-HFD in solution (Figure 3a) suggested that the interactions between ester carbonyl unit


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and the neighboring carboxyl units are insufficient to generate adequate electron delocalization due to the large distance between ester carbonyl unit and the neighboring carboxyl units in solution. However, ester carbonyl unit and the neighboring carboxyl units tended to pack together in solid state due to the inter-chain interactions. In order to probe the effect of fluorine atoms on emission, we also calculated the distance between the ester carbonyl unit of carbon and neighboring carboxyl unit of oxygen. The distance between the ester carbonyl unit of carbon and neighboring carboxyl unit of oxygen decrease from 5.20 to 2.86 Å when the fluorine atoms instead of hydrogen atoms (Figure 6b and 6c). This result indicates that fluorine atoms on the pendent carbon chain further decreases the distance between the ester carbonyl unit of carbon and neighboring carboxyl unit of oxygen. As squeezing action generates a much more rigid and compact conformation, PS-b-PAA-g-HFD chain can be restricted the vibration and rotation and thus prevent dissipating the absorbed energy through non-radiative decay pathways, such as distortion, collision or rotations of these molecules9. Therefore, the intense emission of PS-b-PAA-g-HFD in solid states was observed.4143

For PS-b-HFD, it is difficulty for carbonyl units to form clusters due to the only one carbonyl

group in one PS-b-HFD molecule. Therefore, very weak emission of PS-b-HFD was detected in solid states.


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Figure 6. (a) Molecular orbitals calculate of PS-b-PAA-g-DA. Red is oxygen atom, cyan-blue is fluorine atom, gray is carbon atom. Wine red is positive charge, olive green is negative charge; (b) calculated distances between the ester carbonyl unit and neighboring carboxyl unit of PS-bPAA-g-HFD; (c) Calculated distances between the ester carbonyl unit and neighboring carboxyl unit of PS-b-PAA-g-DA.

Fluorescence Imaging in Living Cells Encouraged by the strong fluorescence emission, we further evaluated the fluorescence imaging of living cells by PS-b-PAA-g-HFD. Here, the Hela cells were chose as the model to test the imaging by incubating them in the solution of 5 µM PS-b-PAA-g-HFD, and the results are shown in Figure7. Firstly, after 20 min incubated with 5 µM PS-b-PAA-g-HFD, the morphology of the HeLa cells can be maintained, indicating that PS-b-PAA-g-HFD has low


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cytotoxicity to HeLa cells and the polymer may be potentially suitable for imaging in living cells. We can also found that the strong blue and green fluorescence emission appeared in the membrane of cells. In comparison, the cells can hardly be dyed to red, which is consistent with the PL spectrum. The above results illustrate that PS-b-PAA-g-HFD has good membrane permeability.

Figure 7. Fluorescence images of HeLa cells treated with 5 µM PS-b-PAA-g-HFD solution. The scale bar is 25 µm.

Conclusions In summary, the esterification reaction between the carboxyl groups and hydroxyl groups was employed to synthesize 1H,1H,2H,2H-perfluoro-1-decanol (HFD) grafted PS-b-PAA block copolymers (PS-b-PAA-g-HFD). To our best knowledge, this is the first discovery of fluorescent polymers just using non-fluorescent polymer functioned with HFD to restrict the intramolecular motion process. Moreover, through theoretical calculation of model of PS-b-PAA-g-DA and PSb-PAA-g-HFD, its emission can be ascribed to the n-π* interactions between the lone pair (n) of hydroxyl O atoms of carboxyl units and empty π* orbital of ester carbonyl unit. Importantly, PSb-PAA-g-HFD can be used as fluorescence imaging probe in the HeLa cells. Therefore, this is a versatile platform to prepare various new fluorescent polymers by functionalization of


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nonflurescent polymers through simply grafting technique, which opens a new way to design fluorescent materials and has great applications.


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AUTHOR INFORMATION Corresponding Author * E-mail : [email protected], [email protected]. (X. L.) ACKNOWLEDGMENT This work was funded by the National Natural Science Foundation of China (51173069, 51473068) and the Shandong Provincial Key Research and Development Plan, China (2017GGX20102). Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/xxxx.

The expanded 1H NMR spectra of PS-b-PAA, HFD and PS-b-PAA-g-HFD; PL spectra of the PS/PAA-g-HFD film; The fluorescent quantum yields of the samples


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For Table of Contents Use Only

A nonfluorescent 1H,1H,2H,2H-perfluoro-1-decanol grafted to nonflurescent polystyrene-bpoly(acrylic acid) block copolymers through simply esterification reaction can exhibit strong blue emission.


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Autofluorescent Polymers: 1H,1H,2H,2H-Perfluoro-1-decanol Grafted

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