Boronic Acid-Functionalized Conjugated Polymer for Controllable Cell

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Boronic Acid-Functionalized Conjugated Polymer for Controllable Cell Membrane Imaging Hao Zhao, Ke Peng, Fengting Lv, Libing Liu, and Shu Wang ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00212 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 9, 2019

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ACS Applied Bio Materials

Boronic Acid-Functionalized Conjugated Polymer for Controllable Cell Membrane Imaging Hao Zhao,a,b Ke Peng,a,b Fengting Lv,a Libing Liu,a and Shu Wanga,b* a Beijing

National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids,

Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, P. R. China b

College of Chemistry, University of Chinese Academy of Sciences, Beijing 100049, P. R. China Email: [email protected]

ACS Paragon Plus Environment

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KEYWORDS: conjugated polymers, boronic acid, dynamic covalent bond, controllable selfassembly, cell membrane imaging

ABSTRACT In this work, we designed and synthesized a new cationic conjugated polyfluorene tagging with phenylboronic acid groups (PFP-PBA) for controllable cell membrane imaging. By balancing the synergistic effect of dynamic covalent bonds and electrostatic interactions between positively charged PFP-PBA and negatively charged cell membrane, the controllable cell membrane imaging could be realized. These findings demonstrated that conjugated polymers could be used as effective materials for regulating interactions with cells to develop controllable self-assembly systems for various biological applications.


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Self-assembly is the process in which building blocks automatically organize into specific assembly structures by weak interactions such as host-guest, electrostatic, hydrophobic interactions and so on.1-4 In recent years, self-assembly systems have been widely developed and applied in molecular recognition,5-6 sensing,7 supramolecular chemotherapy,8-10 antibiotic switch11-13 and photothermal therapy.14 Thus, the development of controllable self-assembly systems is highly important for various biological applications. Recently, phenylboronic acid (PBA) as a cis-diols recognition group has been extensively used in molecular sensing,15 bacterial killing16 and other biomedical applications.17-18 PBA could bind to cell membrane by recognizing cis-diol units of glycoproteins, and the formed dynamic covalent bonds could be modulated by adjusting pH or adding competition molecules.19 Moreover, the charge of boron element in PBA could change from neutral to negative state20 accompanying by PBA/cis-diols interaction, indicating the possibility of tunable electrostatic interactions between negatively charged cell membrane and positively charged functional materials in a self-assembly manner. These formed the basis to construct PBA-based controllable functionalized self-assembly systems, especially for cell membrane imaging. Cationic conjugated polymers (CCPs), featured with -conjugated structure and strong light-harvesting ability, have attracted much attention for excellent bioimaging due to their high brightness, good photostability and tunable emission spectra.21 Combining the unique characteristics of CCPs with the PBA/cis-diols interaction, CCP-based controllable cell imaging system could be constructed in a self-assembly manner. In this work, a cationic conjugated polyfluorene functionalizing with PBA groups (PFP-PBA) was designed and


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prepared. As shown in Scheme 1a, PFP-PBA could bind to the cell surface to achieve controllable imaging through the dynamic covalent bonds of PBA/cis-diol units of membrane glycoproteins as well as the electrostatic interactions between PFP-PBA and cell membrane. As a competition molecule to cis-diol units, D-glucose could block the binding sites of PFPPBA to reduce its interaction with cells, thus inhibiting the cell membrane imaging ability of PFP-PBA. It should be noted that the charge change of PBA from neutral to negative state played a vital role in adjusting the electrostatic interactions between PFP-PBA and cell surface.

Scheme 1. (a) Graphical illustration of PFP-PBA for controllable cell membrane imaging. (b) Synthetic route for PFP-PBA.


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PFP-PBA was synthesized as the route illustrated in Scheme 1b. Compound 1 was prepared according to the literature.22 Polymer 2 was obtained by Suzuki coupling of compound 1 with 1,4benzenediboronic acid bis(pinacol) ester in the presence of Pd(dppf)Cl2 with a yield of 43 %. Polymer 2 reacted with 3-bromomethylphenylboronic acid in THF to afford PFP-PBA with a yield of 25 %. 1H-NMR spectrum of PFP-PBA as shown in Figure 1a demonstrated that the modification ratio of PBA on PFP-PBA was 85%. In addition, as shown in Figure 1b, the boron peaks ( ～ 18.5 and 27.9 ppm) in

11B-NMR

spectra of PFP-PBA and PBA were in the same

positions, confirming the attachment of PBA on conjugated polymer. Moreover, as shown in Figure 1c, the presence of boron peak at ～ 188 eV and oxygen peak at ～ 531 eV in XPS spectrum of PFP-PBA was another evident to prove the successful modification of PBA to conjugated polymer. The photophysical properties of PFP-PBA were studied in DMSO and water, respectively. As shown in Figure 1d, the maximum absorption peak of PFP-PBA in DMSO was located at ～370 nm while the emission spectrum showed a maximum peak at ～ 420 nm. The absolute fluorescence quantum yield of PFP-PBA was 56.8 %, and the extinction coefficient (ε) was calculated to be 5.87 × 104 M-1cm-1. In water, the maximum absorption peak of PFP-PBA was located at 375 nm with an extinction coefficient of 5.57 × 104 M-1 cm-1. PFPPBA exhibited a maximum emission peak at 422 nm with an absolute fluorescence quantum yield of 10.7 % in water. The slight red shift of maximum absorption and emission peaks together with the decrease of absolute fluorescence quantum yield could be explained by the aggregation of PFP-PBA in water rather than in DMSO.23


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Figure 1. (a) 1H-NMR spectrum of PFP-PBA in DMSO-d6. (b) 11B-NMR spectra of PFP-PBA and PBA in DMSO-d6. (c) XPS spectrum of PFP-PBA (inset: magnified spectrum). (d) Normalized absorption and emission spectra of PFP-PBA in DMSO and H2O, respectively. λex = 380 nm.

Alizarin Red S (ARS, chemical structure shown in Scheme S1a), a fluorescent boronic acid indicator containing cis-diol units, was used to verify the covalent linking of PBA on PFP-


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PBA. As shown in Figure 2a, the absorption peaks of ARS were located at 333 nm and 515 nm, respectively. Upon adding PFP-PBA, the absorption peak of ARS at 515 nm displayed significant blue shift to 463 nm, indicating the formation of a stable complex of PFP-PBA/ARS. Moreover, the peak at 383 nm correspond to the π−π∗ transition of PFP-PBA displayed increment in the absorbance upon adding PFP-PBA. PBA alone could also bind to ARS and the changes in absorption spectra of ARS were illustrated in Figure 2b. Upon adding PBA, the absorption peak of ARS located at 515 nm showed significant blue shift to 469 nm, indicating the formation of PBA/ARS stable complex. The similar changes in absorption spectra of ARS upon adding PFP-PBA and PBA confirmed the covalent linking of PBA on PFP-PBA. To demonstrate that spectral changes were due to the specific dynamic covalent reaction rather than electrostatic interactions between positively charged PFP-PBA and negatively charged ARS, cationic conjugated polyfluorene without PBA group (PFP, chemical structure shown in Scheme S1b) was applied as the control polymer. As shown in Figure 2c, the absorption peak of ARS located at 515 nm displayed no change after adding PFP, this confirmed that the specific dynamic covalent reactions between PFP-PBA and ARS played a vital role in ARS indicating processes, rather than the electrostatic interactions. Furthermore, as shown in Figure 2d, the color of ARS solution changed from pink to orange after adding PFP-PBA and PBA. While after adding PFP, the color of ARS solution did not change, which further confirmed the covalent linking of PBA on PFP-PBA.


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Figure 2. Absorption spectra of ARS upon adding (a) PFP-PBA, (b) PBA and (c) PFP. (d) Digital photograph of ARS solution in the presence of PBA, PFP and PFP-PBA. [ARS] = 25 μM, [PFPPBA] = [PFP] = 20 μM (in repeat units), [PBA] = 300 μM. All the measurements were performed in HEPES buffer (10 mM, pH = 7.98).

D-glucose was used as a model molecule to verify the specific reaction of PFP-PBA with bioactive cis-diols. Considering that after reacting with D-glucose, the PBA on PFP-PBA would change from neutral trigonal planar boronic acid to negative tetrahedral boronate anion, the charge variations of PFP-PBA could characterize the reactivity of PFP-PBA with


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D-glucose. Figure 3a showed the zeta potential changes of PFP-PBA before and after reacting with D-glucose. The zeta potential of +17.3 ±1.1 mV was observed for PFP-PBA because of the cationic side chains on PFP-PBA. After adding D-glucose, zeta potential of PFP-PBA was reduced to +13.7±0.3 mV. Besides, the hydrodynamic diameter of PFP-PBA (65.4 ± 4.5 nm) increased after addition of D-glucose (474.3 ± 25.5 nm) as shown in Figure 3b. In control studies, as shown in Figure 3c, the zeta potential of PFP before (+15.2 ± 0.2 mV) and after (+16.1 ± 0.2 mV) reacting with D-glucose exhibited no obvious change. Similarly, the hydrodynamic diameter of PFP (56.3 ± 2.5 nm) also remain unchanged after adding D-glucose (66.4 ± 3.3 nm) as shown in Figure 3d. The interactions of PFP-PBA with D-glucose by dynamic covalent bonds together with the charge changes of PFP-PBA formed the basis for achieving controllable cell membrane imaging.


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Figure 3. (a) Zeta potential and (b) hydrodynamic diameter of PFP-PBA in the absence and presence of D-glucose. (c) Zeta potential and (d) hydrodynamic diameter of PFP in the absence and presence of D-glucose. [PFP-PBA] = [PFP] = 20 μM (in repeat units), [D-glucose] = 10 mM. All the measurements were performed in HEPES buffer (10 mM, pH = 7.98). Finally, PFP-PBA based controllable cell membrane imaging was performed with PC12 cells since they expressed abundant glycoproteins.24 PFP was chosen as control polymer. As shown in Figure 4a, without D-glucose, PFP-PBA was mainly located on PC12 cell membrane. However, if PFP-PBA was preincubated with D-glucose, the cell membrane imaging ability of PFP-PBA was significantly reduced. This could be attributed to following reasons: (1) the cis-


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diols binding sites of PFP-PBA were blocked by D-glucose; (2) the PBA on PFP-PBA changed from neutral to negative state leading to the reduction of the net positive charge of PFP-PBA. For control polymer PFP, as shown in Figure 4b, the cell membrane imaging ability of PFP was not affected even after preincubated with D-glucose, since the electrostatic interactions between PFP and cell membrane played the dominant role in binding process. As PBA was the major difference between PFP-PBA and PFP, it could be concluded that PBA moiety occupied the dominant status to control the cell membrane imaging ability of PFP-PBA. Thus, by balancing the synergistic effect of dynamic covalent bonds and electrostatic interaction between positively charged PFP-PBA and negatively charged cell membrane, the controllable cell membrane imaging was realized.


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Figure 4. Confocal laser scanning microscope images of PC12 cells after incubation with (a) PFP-PBA and (b) PFP in the absence and presence of D-glucose. [PFP-PBA] = [PFP] = 20 μM (in repeat units), [D-glucose] = 10 mM.

In summary, we proposed a new strategy to achieve controllable cell membrane imaging based on boronic acid-functionalized conjugated polymer PFP-PBA. By balancing synergistic effect of dynamic covalent bonds and electrostatic interactions between positively charged PFP-PBA and negatively charged cell membrane, the controllable cell imaging was realized. These findings demonstrated that conjugated polymers could be used as effective materials for regulating interactions with cells to develop controllable self-assembly systems for various biological applications. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website: experimental procedures, chemical structure of PFP and Alizarin Red S (Scheme S1).

AUTHOR INFORMATION Corresponding Author


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*[email protected] Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT The work described herein was supported by the National Natural Science Foundation of China (Nos. 21473221, 91527306, and 21661132006), and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16020804), and the Youth Innovation Promotion Association CAS (No.2016029).

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SYNOPSIS


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Boronic Acid-Functionalized Conjugated Polymer for Controllable Cell

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