AIE-active polysiloxane-based fluorescent probe for identifying cancer cells by locating lipid drops

Analytica Chimica Acta 1091 (2019) 88e94

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AIE-active polysiloxane-based fluorescent probe for identifying cancer cells by locating lipid drops Tingxin Yang, Yujing Zuo, Yu Zhang, Zhiming Gou, Xiaoni Wang, Weiying Lin* Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Materials Science and Engineering, University of Jinan, Shandong, 250022, PR China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


A novel polysiloxane-based fluorescent probe TR-1 based on its D-p-A structure, which exhibited solvatochromism property.
The first polysiloxane-based fluorescent probe TR-1 with AIE characteristic can be used in potential cancer diagnosis.
The excellent photostability of TR-1 enable stable fluorescence to exhibit in cancer cells during effective time.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 June 2019 Received in revised form 5 September 2019 Accepted 8 September 2019 Available online 11 September 2019

Comparing with normal cells, Lipid droplets (LDs) of cancer cells show lower polarity and less quantity, which can be utilized as a marker for cancer diagnosis. However, the investigation of LDs in living cancer cells is restricted by the lack of effective molecular tools. Herein, we first reported a novel polysiloxanebased polymer fluorescent polar probe TR-1 with AIE properties, which realized the possibilities for locating LDs. It can aggregate in the LDs of cancer cells and show a stronger fluorescent signal to conduct cancer diagnosis. Moreover, the excellent photostability of TR-1 enable stable fluorescence to exhibit in cancer cells during effective time. © 2019 Elsevier B.V. All rights reserved.

Keywords: LDs Polymer fluorescent probe AIE Cancer cells

1. Introduction Polarity is an important parameter, which can greatly control the reaction processes [1] and its abnormal changes in biological micro-environment are tightly relevant with disorders and sickness [2e4]. Thus it has received wide attentions [5,6] and has been thoroughly researched in chemistry and chemical technology [7].

* Corresponding author. E-mail address: [email protected] (W. Lin). https://doi.org/10.1016/j.aca.2019.09.020 0003-2670/© 2019 Elsevier B.V. All rights reserved.

Furthermore, the polarity of cells could reflect cellular regulating mechanisms which maintain the functions of subcellular organelles [8]. According numerous literatures reported, the physiological activity of Lipid droplets (LDs) is related to stabilizing the polarity of microenvironments homeostasis [9]. LDs, well-connected organelles, can regulate the storage and metabolism of neutral lipids [10,11]. It can prevent cells from lipotoxicity induced by the buildup of excessive lipids, which is relevant to obesity and virus infections [12e14]. Due to the growth of cancer cells needs plenty of fatty acids and phospholipids, LDs has seen as the marker of cancer diagnosis [15]. Increasing evidence

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indicates that the change of LDs polarity and quantity are associated with cancer diagnosis [16]. The LDs' number in cancer cells is more than it in normal cells, while LDs’ polarity is lower than normal cells. Hence, few things are more suitable than monitoring the numbers and polarity variation of LDs to diagnose cancer. A wide variety of methods such as nuclear magnetic hydrogen spectrum [17], histochemical detection [18], and immunoelectron microscopic investigation [19] have been developed to monitor the polarity of LDs so far. Compared with the above method, fluorescence sensing is one advantageous detection technology due to high sensitivity and the capability of the nondestructive and realtime [20,21]. The fluorescent probes with aggregation-induced emission (AIE) properties can amplify fluorescent signals during aggregation, rather than quenching like traditional fluorescent probes [22e26]. AIE, some propeller-shaped fluorophores emit intensively in aggregated states through a mechanism of the restriction of intramolecular motions (RIM), was first proposed by Tang et al. [27] The AIE molecules solved the aggregation-caused quenching (ACQ) problem of traditional organic luminophores, which facilitates the progress in biosensors, optoelectronics and chemosensors fields. By contrast with other fluorescent materials, such as carbon dots [28e32], AIE fluorophores have some merits of high fluorescence intensity, easy modification, special selectiviy and so on. Thus, AIE fluorophores, in combination with polymer probe, offers an attractive technique to study biomoleucles of interest in a noninvasive manner with high spatial and temporal resolution. As a kind of functional polymers, polysiloxane have low toxicity, temperature resistance and excellent stability, due to the special physical properties of Si atom and SieO bond [33]. Moreover, selecting polysiloxane as the basement of the fluorescent probe can increase the local concentration and amplify the fluorescent signal. According this, we conjectured the polysiloxane-based fluorescent probe with AIE property exhibit higher fluorescence intensity when accumulate in LDs. However, to the best of our knowledge, the reported polysiloxane fluorescent probes for monitoring LDs do almost not exhibit AIE characteristic. Herein, we designed and synthesized a novel polysiloxanebased polymer fluorescent probe TR-1 with AIE characteristic to discriminate cancer cells from normal cells by detecting LDs numbers and polarity changes. The probe TR-1 exhibited notable solvatochromic effect because of the intramolecular charge transfer (ICT) characteristic. Furthermore, owing to more LDs numbers and lower LDs polarity, the probe TR-1 displayed the strongest green fluorescence in cancer cells (4T-1 and HepG2) and almost no fluorescence in normal cells (3T3 and HL-7702). Especially, the AIE property of TR-1 would make it express stronger fluorescent signals when probe aggregated in cancer cells.

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Scheme 1. The design concept of the probe TR-1.

various polar solvents. The high polarity environment would cause larger charge separation and lower excited state energy of the probe TR-1, thus leading to bathochromic shift of fluorescence maximum emission peak. Meanwhile, the hydrophobic nature of TR-1 was contributed with localization in LDs [35]. Moreover, the polarity of LDs in living cells would not exhibited too extreme level [36]. Based on this, the cancer cells would exhibit higher fluorescence intensity, due to the more quantity and lower polarity of LDs than in normal cells. The synthesis progress of probe TR-1 was shown in Scheme 2. We selected polysiloxane as the polymer basement, because of its excellent stability and biocompatible. P0 was consist of the aminomodified polysiloxane and 1,8- naphthalimide. Naphthalimide is an excellent chromophore group with good fluorescence quantum yield, large Stokes shift. Then, the addition of polar sensitive group triphenylamine (TPA) through react with 4-(diphenylamino)phenylboronic, and novel polysiloxane-based fluorescent probe TR-1 was obtained. The new polymer probe TR-1 was fully characterized by standard 1H NMR (Fig. S1), 13C NMR (Fig. S2) and Gel Permeation Chromatography(GPC) (Table S1). Moreover, the molecular weight (Mn) of TR-1 was about 5610 g/mol, which was higher than that of mid product P0 (3928 g/mol), and the polymer dispersity index (PDI) was about 1.01 (Table S1). The increase of the molecular weight also showed the success of the functionalization reaction. The reaction mechanism was confirmed by the infrared spectrum (Fig. S3). As shown in Fig. S3a, the sign peak of the amino group was originally located at 3312 cm1. However, with the reaction between the amino group on polysiloxane and the anhydridegroup in the naphthalic anhydride, the characteristic peak of the amino group disappeared and the peak of the bromine group appeared at 598 cm1(Fig. S3b). As shown in Fig. S3c, the disappeared peak of the bromine group illustrated the successful synthesis of the novel polymeric fluorescent probe TR-1. Meanwhile the peaks of SieO and benzene ring had no significant changes, which indicated that the polymer chain was not involved in the reaction process.

2. Result and discussion 2.1. Design and synthesis

2.2. UVevis and fluorescence spectra

The intramolecular charge transfer (ICT) structure consist with strong electron donoreacceptor (DeA) and p-conjugation system. In this work, the new polymer probe TR-1 (Scheme 1) with ICT character was comprised of electron-withdrawing group (A) and electron-donating group (D). According D-p-A structure, the triphenylamine (TPA) was selected as the D group and the amide group was chosen as accepting group. Meanwhile, the addition of polysiloxane can increase the stability of probe TR-1 in complex detection environment [34]. Due to owning the rigid structure, naphthalene ring was selected as the connector, which contributes to keep non radiative energy. Moreover, TR-1 with the donorepconjugationeacceptor (DepeA) structure exhibited sensitivity in

With the probe TR-1 in hand, we evaluated the optical property in various polarity solvents including toluene, dioxane, tetrahydrofuran, dichloromethane. As shown in Fig. 1a, the largest absorption wavelength of TR-1 in various polarity solvents were similar, which indicated the dipole moment of TR-1 in the ground state had almost no change [37]. By contrast, upon the solvent polarity increasing from toluene to dichloromethane, the corresponding emission maximum peak was red-shifted from 566 to 662 nm (Fig. 1b). Generally speaking, dipoles in solvents reorient around the fluorescent group of the excited state/reduced the energy of the excited state. With the increase of solvent polarity, the excitation energy of fluorescent groups decreased, resulting in red

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Scheme 2. Synthetic Routes of TR-1.

Fig. 1. (a) The absorption spectra of TR-1 in various solvents. (b) Normalized FL spectra of TR-1 in various polarity solvents. (c) Fluorescence spectra of TR-1 in PE/THF mixtures with different PE fractions (fp). (d) The maximum fluorescence intensities of TR-1 in PE/THF mixtures with different PE fractions (fp).

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Fig. 2. Images of 4T-1 cells incubated with TR-1 (10 mM) and Nile red (2 mM). Scale bar: 10 mM. Green channel: lex ¼ 405 nm, collected 500e550 nm. Red channel: lex ¼ 561 nm, collected 570e620 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3. Imaging of LDs polarity in normal cells 3T3 (a), HL-7702 (c) and cancer cells 4T-1 (b), HepG2 (d). The contrast fluorescence intensities of normal cells 3T3, HL-7702 and cancer cells 4T-1, HepG2 (eef). lex ¼ 405 nm; lem ¼ 500e550 nm. Scale bar ¼ 10 mM.

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Fig. 4. Photostability test of the probe TR-1 in 4T-1 cells. (a) The in situ imaging of 4T-1 cells during 20 min (b) The in situ fluorescence intensities of TR-1 in 4T-1 cells during 20 min.

shift of fluorescence spectrums and the obvious decline of fluorescence intensity (Fig. S4). Meanwhile, the color change of probe TR-1 in various polarity solvents could observe clearly under a 365 nm UV lamp (Fig. S5). In addition, the increasement of Stoke's shift of probe TR-1 in high polar solvent also corresponded to the above situation (Table S2). 2.3. AIE characteristic Under aggregate state, the intramolecular torsion restricted and the polar of local environment decreased, so that ICT state was limited and AIE effect appeared [38]. Petroleum ether (PE)/Tetrahydrofuran (THF) mixture with different PE fractions were utilized to evaluate the AIE properties of TR-1. As revealed in Fig. 1c, with increasing the fraction of PE in the solution mixture, the fluorescence intensities gradually increased, resulting from intermolecular interactions between the molecules within the aggregates. Meanwhile, due to the decline polarity of the solution environment upon aggregation formation, the maximum emission peak was blueshifted and the emission intensity was achieved 6-fold enhancement (Fig. 1d). To evaluate the AIE characteristic of TR-1, the linearly relation between maximum fluorescence emission variation of the probe TR-1 and different PE percentage solvents were plotted (Fig. S6), which undoubtedly demonstrating AIE characteristics. 2.4. pH and selectivity experiment Excellent selectivity is a crucial standard to evaluate the application of fluorescence probe. As shown in Fig. S7, addition of the 2  representative anions (Cl, Br, F, I, CN, CO2 3 ,SO4 , NO2 ), cationic (Ca2þ, Mg2þ, Fe2þ, Al3þ, Kþ, Naþ, Zn2þ, Fe3þ,Cu2þ, Hg2þ,Agþ), reactive oxygen (H2O2, OH, CH3COOONa, ClO), and 2 reducing agents (S2O2 3 , SO3 ) led no marked fluorescence response to the probe. Furthermore, the fluorescent probe with suitable physiological range of pH has potential application in biological system. Thus, the pH-dependence on the TR-1 was assessed in the pH 4e10 range, which the fluorescence intensity almost had no change, indicated the possibility of detection in living systems. Moreover, we then proceeded to investigate the feasibility of the probe TR-1 for imaging in living cell lines (HepG2, HL-7702, 4T-1

and 3T3). The standard MTT assayindicated that the probe TR-1 had low cytotoxicity and high biocompatibility (Fig. S9). 2.5. Cell colocalization experiment Subsequently, the confocal laser scan microscope(CLSM) imaging studies used Nile Red and probe TR-1 for subcellular localization in 4T-1 cells. Nile Red was a proveded commercial dye, which can be used to locate LDs [39,40]. Upon excitation at 405 nm, TR-1 accumulated in LDs of 4T-1 cell and show bright green fluorescence (Fig. 2a), which are similar to the fluorescent emissions from their aggregates in Petroleum ether. And the Nile Red exhibited red dot fluorescence in 4T-1 cells under excitation at 561 nm. Furthermore, the merged image exhibited yellow fluorescence (Fig. 2c) with the correlation of the intensity scatter plot achieved 0.95 (Fig. 2d), which indicated the specific LDs localization of TR-1. The intensity of TR-1 and Nile Red in 4T-1 cells had same change trend (Fig. 2e). The result suggested that TR-1 could be used as the fluorescence probe to visualize LDs in living cells. 2.6. Cancer cell diagnosis To further confirm the possibility of the probe TR-1 for cancer diagnosis in vivo, the cancer cells (4T-1 and HepG2) and the normal cells (3T3 and HL-7702) were incubated with TR-1 in a controlled trial. As anticipated, the green fluorescence spots in cancer cells (Fig. 3b and d) were significantly brighter than in normal cells (Fig. 3a and c). Compared with the normal cells (3T3 and HL-7702), TR-1 in the cancer cells (4T-1 and HepG2) exhibited about 8.0 times of fluorescence intensity (Fig. 3e and f), which suggested that the probe could be used to mark cancer cells. Due to the LDs' polarity in cancer cells is lower than normal cells, TR-1 with ICT character displayed stronger green fluorescent intensity. Meanwhile, LDs’ number in cancer cells is more than it in normal cells. Hence, the cancer cells exhibited brighter greeen fluorescence, because of the large aggregation of probe TR-1 with AIE characteristics. The above phenomenon could ascribe to the difference of LDs between the cancer cells and the normal cells, which mentioned before.Taken together, the results indicated the potential utility of probe TR-1 in diagnosis of cancer.

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2.7. Cell photostability Photostability was also one of the most important elements for constructing novel fluorescence probes [41]. The fluorescent probe with excellent stability contribute to fluorescence signal expression. The main chain of polysiloxane greatly strengthened the photostability of the probe TR-1. As illustrated in Fig. 4, the fluorescence signals remained 90% of original strength after continuous irradiating for 20 min, indicating the excellent photostability of probe TR-1 in 4T-1 cells.

[11] [12] [13] [14] [15]

[16]

3. Conclusion [17]

In summary, we have described the design, optical characteristic and living cell imaging applications of TR-1, a new polysiloxanebased polar fluorescent probe that can be used in potential cancer diagnosis. TR-1 exhibits fluorescence signal color changes influenced by environmental polarity due to its D-p-A structure. Moreover, Co-localization experiment suggested that the probe TR1 accurately located in intracellular LDs and exhibits excellent photostability as well as low cytotoxicity. And the aggregation of TR-1 in cancer cells could exhibit stronger fluorescence signal because of its AIE property. Importantly, we expect TR-1 will be as a smart molecular tool for cancer diagnosis.

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Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Acknowledgements This work was financially supported by NSFC (21472067, 21672083, 21877048), Natural Science Foundation of Shandong Province (ZR2018BB022), Taishan Scholar Foundation (TS201511041), and the startup fund of University of Jinan (309e10004, 1009428). Appendix A. Supplementary data

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