Letter www.acsami.org
Conjugated Polyelectrolyte Nanoparticles for Apoptotic Cell Imaging Yu Liu,†,‡ Pan Wu,†,‡ Jianhua Jiang,† Jiatao Wu,†,‡ Yan Chen,‡,§ Ying Tan,*,†,‡ Chunyan Tan,*,†,‡ and Yuyang Jiang‡,∥ †
Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China The Ministry-Province Jointly Constructed Base for State Key Lab - Shenzhen Key Laboratory of Chemical Biology, the Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, P. R. China § Shenzhen Technology and Engineering Laboratory for Personalized Cancer Diagnostics and Therapeutics, Shenzhen Kivita Innovative Drug Discovery Institute, Shenzhen 518055, P. R. China ∥ Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University, Beijing 100084, P. R. China ‡
S Supporting Information *
ABSTRACT: Three anionic conjugated polyelectrolytes (CPEs) with poly(p-phenylene ethynylene thiophene) backbones were designed and synthesized, among which PPET3-CO2Na showed greater molar extinction coefficient with red-shifted bands in both absorption and emission spectra compared to the well-studied PPECO2Na polymer. PPET3-CO2Na was thus chosen to construct CPE-based nanoparticles (CPNs) with cationic octaarginine (R8) peptide through electrostatic-interaction-induced self-assembly. Due to plasma membrane permeabilization and mitochondrial outer membrane permeabilization (MOMP) in early apoptotic cells, PPET3/R8 CPNs demonstrated excellent colocalization with MitoTracker Red in apoptotic cells instead of normal cells, which had potential application in cell imaging for early apoptosis recognition. KEYWORDS: conjugated polyelectrolytes (CPEs), apoptosis, cell imaging, conjugated polymer nanoparticles (CPNs), self-assemble
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CPE/multicharged acid25−27 has been used to form selfassembled CPNs. Herein, we designed and synthesized a series of anionic conjugated polyelectrolytes with carboxylic acid group side chains and alternating phenylene ethynylene and thiophene units as the backbone structure. Among the polymers, PPET3CO2Na was further chosen to assemble with a cationic octaarginine (R8) peptide to form CPNs and its application in apoptotic cell imaging was demonstrated. CPEs with a poly(p-phenylene ethynylene) (PPE) backbone possess excellent optical properties and can amplify fluorescence quenching response signals effectively, which have thus promoted their use as a novel biosensing platform.11,20 In this work, we used a Pd-catalyzed Sonogashira coupling reaction to obtain new poly(arylene ethynylene)s (PAEs) backbones of alternating phenylene ethynylene units with thiophene (PPET1), bithiophene (PPET2), and terthiophene (PPET3), as shown in Scheme 1. This synthetic strategy was successfully used to obtain similar CPEs with cationic side chains in our previous study.30 In the current study, ester polymer precursors were synthesized first and characterized by 1 H NMR and GPC, due to their solubility in organic solvents,
poptosis, known as the programmed cell death pathway, plays a significant role in life systems.1,2 Deregulation of apoptosis is related to several human diseases, including cancer, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, etc.3−6 Thus, the development of a convenient and sensitive method to observe apoptotic cells is of great importance in biological research, clinical diagnosis, and drug screening. Currently, flow cytometry,7 Western blot,8 enzyme linked immunosorbent assay (ELISA),9 and cell imaging10 are widely utilized to study apoptosis, among which cell imaging techniques have attracted lots of research interests by providing quantitative and real-time observation of cells. Expanded fluorescent probes are still in need to support the development of cell imaging techniques. Water-soluble conjugated polyelectrolytes (CPEs), which contain π-conjugated backbones with ionic side groups, have demonstrated potential application in chemical and biological sensing.11−20 In recent years, conjugated polymer nanoparticles (CPNs) have emerged as a promising approach toward cell imaging due to their prominent properties, such as easy preparation and separation, low cytotoxicity, excellent photostability, and good biocompatibility.21−25 Several methods have been reported to construct CPNs, including self-assembly,24−27 nanoprecipitation,28 and mini-emulsion.29 By taking advantage of the electrostatic interaction between CPEs and oppositely charged molecules, complexation of CPE/RGD peptide24 and © XXXX American Chemical Society
Received: July 28, 2016 Accepted: August 15, 2016
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DOI: 10.1021/acsami.6b09347 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces Scheme 1. General Synthetic Route of the Polymers and Their Molecular Weight Informationa
Reagents and conditions: (i) Pb(PPh3)4Cl2 (0.03 equiv), CuI (0.06 equiv), THF/Et3N (v/v = 3/1), 55 °C, 18 h; (ii) (n-Bu)4NOH (1 mol/L in CH3OH, 6.67 equiv), dioxane/THF (v/v = 5/1), rt, 24 h, then add NaClO4 (3 equiv) in H2O. a
such as THF and chloroform. The average molecular weights of the polymers ranged from 8.12 to 15.91 kDa, corresponding to about 13−19 repeated units. The polydispersity indices (PDI) were calculated to be from 1.68 to 1.87, which are in close agreement with the theoretical value of 2.0 for ideal stepgrowth polycondensation.31 Corresponding carboxylate polyelectrolytes, including PPET1-CO2Na, PPET2-CO2Na, and PPET3-CO2Na, were then obtained through hydrolysis of their ester precursors and analyzed further in a photophysical study (see the Supporting Information for details on synthesis and characterization). PPET1-CO2Na, PPET2-CO2Na, and PPET3-CO2Na are soluble in some polar solvents, including ethanol, methanol, and water. Photophysical properties of these three polymers were investigated and compared to the well-studied polymer PPE-CO2Na in aqueous solution. Table 1 lists the character-
Figure 1. Normalized UV−visible absorption (left) and fluorescence emission spectra (right) of PPET1-CO2Na (red), PPET2-CO2Na (black), PPET3-CO2Na (green), and PPE-CO2Na (blue) in aqueous solution. Excitation wavelength was 400 nm.
Table 1. Photophysical Parameters of PPET1-CO2Na, PPET2-CO2Na, and PPET3-CO2Na sample
λmaxabs (nm)
εmax (104 M−1 cm−1)
λmaxem (nm)
Φa
PPET1-CO2Na PPET2-CO2Na PPET3-CO2Na PPE-CO2Na
410 416 438 426
4.2 5.3 7.6 2.2
473 480 505 463
0.06 0.19 0.05 0.07
PPET3-CO2Na red-shifted about 42 nm compared to PPECO2Na. Therefore, PPET3-CO2Na was chosen for further cell imaging study based on its improved photophysical performance, including stronger light absorption, as well red-shifted absorption and emission band. Unlike polymer dots (PDots) that consist of primarily hydrophobic π-conjugated polymers with high packing density, CPNs were reported to be formed from charged CPEs in the presence of salts, acids, or oppositely charged species.24−27,33 Due to the negative charges on PPET3-CO2Na in physiological condition, it could be expected that the polymer itself had difficulty in permeating the cell membrane. Octaarginine (R8) peptide has been reported as a cell-penetrating peptide34,35 and is widely used to carry a variety of biological macromolecules into cells, including drugs,36 plasmids,37 proteins,38 and liposomes.39 CPNs were then prepared from PPET3-CO2Na and R8 peptide using the self-assembly method. Briefly, CPNs were made by mixing PPET3-CO2Na and R8 peptide solution at different molar ratios under vigorous stirring for 20 h in dark and were then collected by centrifugation. The zeta (ζ) potentials of CPNs were measured on a Zetasizer Nano ZS +MPT2 analyzer, and the results are listed in Table S1. The decrease in ζ-potential from −13.18 to −4.21 mV along with a decreasing molar ratio (PPET3:R8) from 16:1 to 1:10 indicated the incorporation of R8 into PPET3-CO2Na. It is interesting to
a
Quantum yields were measured according to the protocol of relative quantum yield determination.32 Coumarin 6 in ethanol solution was used as a standard, which has a reported quantum yield at 0.78 when excited at 400 nm.
ization data, whereas Figure 1 shows the absorption and fluorescence spectra of these polymers. Generally speaking, all three newly synthesized polymers showed typical characteristic spectra of PPE-type CPEs with a wide absorption band ranging from 350 to 550 nm. Compared to PPE-CO2Na, PPET3CO2Na exhibited a red-shifted absorption band with a maximum molar extinction coefficient (εmax) of 7.6 × 104 M−1 cm−1 in H2O (Table 1), as a result of a lower energy gap between the valence and conduction bands caused by introduction of terthiophene units.30 The wavelength maxima of fluorescence spectra systematically red-shifted in the following order: PPET3-CO2Na > PPET2-CO2Na > PPET1-CO2Na > PPE-CO2Na, among which B
DOI: 10.1021/acsami.6b09347 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces point out that the CPNs still appeared to have negative surface charge even when the cationic peptide was in excess. This might suggest that the formation of the CPNs involved an encapsulation process, which was similar to Wang’s report on using cationic CPE and negatively charged disodium salt 3,3′dithiodipropionic acid (SDPA) to form CPNs.26 The morphology of CPNs was characterized by transmission electron microscopy (TEM). As shown in Figure 2, the
Figure 3. Cell viability results of HeLa cells treated by various concentrations of PPET3-CO2Na and PPET3/R8 CPNs (PPET3:R8 = 1:10), respectively. The concentrations were 10, 50, 100, and 200 μM.
PPET3/R8 CPNs increased as the ζ-potential of CPNs increased. Cell imaging applications of PPET3/R8 CPNs (PPET3:R8 = 1:10) were further investigated. As suggested by some cell biological study, plasma membrane permeabilization41,42 and mitochondrial outer membrane permeabilization (MOMP)43 are crucial events in early stage of apoptosis. Normal cells and apoptotic cells treated by apoptosis inducer for 12 h were stained by AnnexinV-mFluor Violet and propidium iodide (PI), and then evaluated by flow cytometry. As shown in the flow cytometry results in Figure 5a, 89.43% cells are normal cells and about 6.07% and 3.94% cells are in early and late apoptotic cells, respectively, for nontreated HeLa cells. For apoptotic cells, there are 23.63% and 8.29% in early and late apoptotic stages, respectively. HeLa cells under these two conditions (nondrug treated vs apoptosis inducer treated for 12 h) were subject to further cell imaging comparison study using confocal fluorescence microscopy. Cell imaging results are shown in Figure 5b,c. Annexin V-mFluor Violet (blue channel) was used to indicate the state of the cell since it stains the cell membrane by specifically binding with the externalized phosphatidylserine (PS). As expected, the blue channel showed distinctions between normal cells and apoptotic cells, characterized by subtle blue emission from normal cells vs strong blue fluorescence along cell membrane in apoptotic cells. This result further confirmed the two different cell states in addition to the flow cytometry result. MitoTracker Red (red channel) was used for colocalization to confirm the cellular location of CPNs (green channel). Photostability of CPNs and MitoTracker Red was compared by continuous scan of cells every 5 min. As shown in Figure S7, about 73% of the initial fluorescent signal intensity was remained for CPNs after 70 min, whereas only 40% of the initial signal intensity remained for MitoTracker Red. CPNs showed the better photostability than MitoTracker Red. As shown in Figure 5b,c, CPNs showed different colocalization with MitoTracker Red in different cell states. Specifically, poor colocalization between the green and red channel can be observed in the merged image in Figure 5b for normal HeLa cells. The Pearson’s coefficient was calculated to be 0.04 in the magnified region. Quite differently, a good overlap of fluorescence emission from CPNs and MitoTracker Red in apoptotic HeLa cells can be seen in Figure 5c and the Pearson’s coefficient of the green and red channels was 0.78 in
Figure 2. TEM image of PPET3/R8 CPNs at the molar ratio of PPET3:R8 = 1:10.
complex of PPET3/R8 formed uniform nanoparticles with an average size of 30−40 nm. This size is bigger than PDots made of hydrophobic conjugated polymers, which had typical size about 20−30 nm.33 Photophysical properties of PPET3/R8 CPNs (PPET3:R8 = 1:10) were compared to PPET3-CO2Na in aqueous solution. As shown in Figure S6, PPET3/R8 CPNs demonstrated redshifted bands in both absorption and emission spectra compared to the polymer PPET3-CO2Na itself. These results suggested the formation of polymer aggregates induced by the electrostatic attraction between the polymer and the peptide. It has been reported that biocompatibility is one of the advantages of CPEs over some small organic fluorophores in biological applications.40 To test the potential application of the polymer PPET3-CO2Na and PPET3/R8 CPNs in cell imaging, cell viability of HeLa cells treated with different concentrations of the polymer and CPNs, respectively, was evaluated using MTT assay. As shown in Figure 3, both polymer and CPNs treated HeLa cells demonstrated excellent cell viability. For example, at the highest concentration of 200 μM, the polymer and CPNs treated HeLa cells showed cell viability about 95% and 91%, respectively. This result suggests that PPET3-CO2Na based polymer and CPNs had good biocompatibility. The fluorescence imaging of HeLa cells stained with PPET3CO2Na and PPET3/R8 CPNs at 4 different molar ratios were compared using confocal laser scanning fluorescence microscopy. As shown in Figure 4, minimal fluorescence was observed in PPET3-CO2Na stained HeLa cells (Figure 4a), whereas increasing fluorescence intensity was seen in PPET3/R8 CPNs stained cells with increasing R8 peptide ratio (Figures 4b−e). It is also noteworthy to pointing out that the CPNs with higher R8 peptide ratio demonstrated better cell membrane permeability characterized by the fluorescence signal both on the cell membrane and in the cytoplasm in HeLa cells. These results suggest that the cell membrane penetration ability of C
DOI: 10.1021/acsami.6b09347 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
Figure 4. Confocal micrographs of HeLa cells stained with 25 μM (a) PPET3-CO2Na, (b) PPET3/R8 CPNs (PPET3:R8 = 16:1), (c) PPET3/R8 CPNs (PPET3:R8 = 4:1), (d) PPET3/R8 CPNs (PPET3:R8 = 1:1), (e) PPET3/R8 CPNs (PPET3:R8 = 1:10). Scale bars: 10 μm.
Figure 5. (a) Flow cytometry results of normal Hela cells and apoptotic HeLa cells stained by AnnexinV-mFluor Violet and PI. Panels b and c are confocal micrographs of normal HeLa cells (b) vs apoptotic HeLa cells (c). Green channel, stained with 25 μM PPET3/R8 CPNs; red channel, stained with MitoTracker Red (90 nM) for 15 min; blue channel, Annexin V-mfluor violet (1×) for 40 min. Scale bars: 5 μm. Apoptotic cells were obtained by treating HeLa cells with apoptosis inducer A (1:2000) in culture medium for incubation of 12 h.
the backbone structure resulted in red-shifted absorption and fluorescence emission spectra in aqueous solution. Moreover, PPET3/R8 CPNs were constructed with anionic PPET3CO2Na and cationic octaarginine R8 peptide at different molar ratios using a self-assembly method. The introduction of R8 peptide enabled the cell membrane permeability of PPET3/R8 CPNs. Due to plasma membrane permeabilization and MOMP,
the magnified region. The significant difference between normal and apoptotic cells highly suggested that PPET3/R8 CPNs could be of potential use in recognizing early apoptotic cells by observing the colocalization of CPNs with MitoTracker Red. In conclusion, we synthesized three anionic CPEs with poly(p-phenylene ethynylene-thiophene) backbone and carboxylate side groups. The introduction of terthiophene unit into D
DOI: 10.1021/acsami.6b09347 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
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PPET3/R8 CPNs localized on mitochondria in apoptotic cells instead of normal cells, which can be used as a cell imaging method for early apoptosis recognition.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b09347. Detailed experimental, NMR spectra, photophysical characterization, photostability comparison (PDF).
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AUTHOR INFORMATION
Corresponding Authors
*Y. Tan. Email:
[email protected]. Tel: 86-75526036035. *C. Tan. Email:
[email protected]. Tel: 86-75526036533. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is supported by grants from Natural Science Foundation of China (No. 21572115 and 21302108), Shenzhen Municipal Government (CXB201104210014A and 20150113A0410006), and Shenzhen Reform Commission (Disciplinary Development Program for Chemical Biology).
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