Two-Photon Absorption of Cationic Conjugated Polyelectrolytes

Feb 22, 2017 - Effects of Aggregation and Application to 2‑Photon-Sensitized. Fluorescence from .... P-C-3 and P-O-3 exhibit moderately large σ2 va...
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Two-Photon Absorption of Cationic Conjugated Polyelectrolytes: Effects of Aggregation and Application to 2‑Photon-Sensitized Fluorescence from Green Fluorescent Protein Shanshan Wang,†,‡ Zhiliang Li,†,‡ Xinglei Liu,§ Samantha Phan,† Fengting Lv,⊥ Kevin D. Belfield,§ Shu Wang,*,⊥ and Kirk S. Schanze*,†,∥ †

Department of Chemistry and Center for Macromolecular Science and Engineering, University of Florida, Gainesville, Florida 32611-7200, United States ∥ Department of Chemistry, University of Texas at San Antonio, One UTSA Circle, San Antonio, Texas 78249, United States § Department of Chemistry and Environmental Science, New Jersey Institute of Technology, Newark, New Jersey 07102, Unites States ⊥ Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China S Supporting Information *

ABSTRACT: Two cationic conjugated polyelectrolytes (CPEs) based on poly(phenylene ethynylene) are found to exhibit efficient 2-photon excited fluorescence. In particular, the CPE aggregates display enhanced 2-photon absorption compared to the nonaggregated states. The CPEs are used as 2-photon absorption sensitizers to harvest light and amplify the fluorescence from green fluorescent protein (GFP) through Förster resonance energy transfer. Considerably enhanced GFP fluorescence under near-infrared 2-photon excitation is demonstrated both in solution and in HeLa cells. The results suggest applications in 2-photon fluorescence imaging of cells, tissues, and organs of living animals expressing GFP.

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INTRODUCTION

water solubility and biocompatibility in addition to exhibiting high σ2 in the near-infrared spectral region. Water-soluble conjugated polyelectrolytes (CPEs) have attracted much attention in the field of fluorescence sensing and more recently in mammalian cell imaging.14−16 The conjugated backbone of CPEs endows them with good light harvesting and fluorescence properties, and their generally good water solubility also makes them desirable as fluorescent probes in biological systems. 17−19 In addition, the amplified fluorescence quenching by charge transfer or induced aggregation,18,20 optical amplification through Förster resonance energy transfer (FRET),17,21−23 and their ability to generate reactive oxygen species (ROS)24,25 have augmented the number of applications for the materials to include biodetection and sensing, gene delivery, drug release, and antibacterial and anticancer therapy. The 2PA optical properties have not been explored much to date; in this regard, we recently reported that an anionic poly(phenylene ethynylene) sulfonate (PPE-SO3−) exhibits moderate σ2 in the near-infrared

Two-photon fluorescence microscopy (2PFM) has emerged as a rapidly expanding field in microscopy, becoming a powerful technique for biochemical/medical applications, owing to its numerous advantages including deep tissue penetration, low tissue autofluorescence/phototoxicity, reduced photobleaching/photodamage, and high three-dimensional spatial resolution.1,2 Two-photon absorption (2PA) fluorophores are key to the application of 2PFM, and a good 2PA fluorophore should possess large 2PA cross section (σ2), high fluorescence quantum yield (ϕ), low photodecomposition quantum yield, low toxicity, and low photobleaching.3,4 Over the years, efforts have been devoted to the design and synthesis of 2PA fluorophores with a variety of structures and functionality including but not limited to organic dyes, quantum dots, gold nanoparticles, and carbon-based materials (carbon dots and graphene quantum dots).5−10 Despite the large number of 2PA chromophores that have been developed, 2PA fluorophores exhibiting high σ2 are usually hydrophobic and most watersoluble 2PA materials have σ2 less than 100 GM (1 GM = 10−50 cm4 s photon−1).11−13 For biological applications of 2PFM, it is desirable to design and synthesize 2PA materials with good © 2017 American Chemical Society

Received: February 16, 2017 Revised: February 22, 2017 Published: February 22, 2017 3295

DOI: 10.1021/acs.chemmater.7b00676 Chem. Mater. 2017, 29, 3295−3303

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Chemistry of Materials

the one- and two-photon excited fluorescence of the polymers was investigated. Figure 1a presents the 1-photon excited

region and demonstrated that the CPE can be used for twophoton fluorescence cell imaging.26 Genetically encoded fluorescent proteins are a special class of fluorescent probes,27 which can be introduced into living systems using molecular genetics.28,29 Fluorescent proteins have been used in noninvasive optical imaging in cells and organisms,30 gene delivery and expression,31 protein trafficking,32 and dynamic protein interactions.33 Genetically modified fluorescent proteins are also used as cellular encoded fluorescent probes for chemical signals such as pH and ion concentration.34−36 The 2PA properties of fluorescent proteins have been explored extensively.37−41 However, the σ2 values of fluorescent proteins are generally quite low compared to synthetic 2PA chromophores.42 Given that the 2PEF intensity is proportional to the product σ2ϕf, where the latter is the fluorescence quantum yield, the 2PEF intensity of fluorescent proteins is typically not satisfactory for biological applications. In view of this, developing strategies to enhance the 2PEF of fluorescent proteins is of considerable interest. Here we report the considerable enhancement of 2PEF intensity from a green fluorescent protein (GFP) via a strategy which relies on cationic poly(phenylene ethynylene)-type CPEs, P-C-3 and P-O-3, as 2PA sensitizers to harvest light and transfer energy to GFP through FRET. P-C-3 and P-O-3 exhibit moderately large σ2 values and ϕf in water. The cationic CPEs and GFP appear to form a complex in aqueous buffer solution, likely driven by electrostatic and/or hydrophobic interactions, allowing for efficient FRET from CPEs to GFP under both one- and two-photon excitation. This effect allows for the observation of comparatively efficient florescence from the CPE-GFP complex under 2PA excitation using 100 fs pulsewidth near-infrared excitation. The utility of this interesting effect is demonstrated by utilizing it to image fluorescence from GFP within mammalian cells (HeLa) under two-photon excitation. To the best of our understanding, this is the first report to document enhancement of the 2PA excited fluorescence of GFP via a sensitizer-FRET mechanism.

Figure 1. 1-Photon and 2-photon excited fluorescence of P-C-3 and PO-3 in water. (a) One-photon excited fluorescence spectra; excitation wavelength is 335 nm. (b) Two-photon excited fluorescence; excitation wavelength is 700 nm. One-photon fluorescence spectra are area normalized relative to the quantum efficiencies, and 2-photon spectra were obtained under identical conditions (same sample concentrations and laser power).

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RESULTS AND DISCUSSION One- and Two-Photon Excited Fluorescence. P-C-3 and P-O-3 (shown in Chart 1) are cationic CPEs that emit blue-green fluorescence in water. In a first set of experiments,

fluorescence (1PEF) spectra in water, normalized relative to the polymer fluorescence quantum yields. P-C-3 features efficient fluorescence (ϕf ≈ 0.26) and a structurally defined emission spectrum with a clear 0−0 band at 415 nm, along with a vibronic band at 435 nm. In addition, a weak feature is seen at λ ≈ 510 nm which is due to a small fraction of emission from the aggregated state of the polymer.43 By contrast, P-O-3 has a significantly lower emission yield (ϕf ≈ 0.06), and the emission appears as broad band which is considerably red shifted with λmax ≈ 490 nm. The distinctly different fluorescence spectra and quantum yields of P-C-3 and P-O-3 suggest that P-C-3 is mostly molecularly dissolved in water, while P-O-3 is more aggregated in water.44,45 The different behavior is attributed to the effect of the −CH2− and −O− linkers to the ionic side units. This parallels effects reported earlier on structurally related CPEs,46 and several possible reasons for the linker-group effect on solution properties are discussed in the earlier report. The fluorescence spectra of the CPEs obtained under 2photon excitation at 700 nm (2PEF) are illustrated in Figure 1b. The 2PEF spectra of P-C-3 and P-O-3 feature similar shapes to the 1PEF spectra, except that the long-wavelength band attributed to aggregates at λ ≈ 510 nm is noticeably enhanced in the 2PEF spectrum of P-C-3. Furthermore,

Chart 1. Structures of P-C-3 and P-O-3

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DOI: 10.1021/acs.chemmater.7b00676 Chem. Mater. 2017, 29, 3295−3303

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Chemistry of Materials comparing the absolute fluorescence intensity of these two polymers under 2-photon excitation, it is observed that the relative fluorescence intensity of P-O-3 to P-C-3 is greater compared to 1PEF. Given that it is expected that the ϕf values should be the same for 1PA and 2PA and that the fluorescence intensity under 2PA is proportional to the product σ2ϕf, this result suggests that the cross section is enhanced for the polymer aggregate state. Similar enhancement in σ2 due to aggregation of a conjugated polymer has been reported and attributed to the interchain interactions within in the aggregates.47 In order to shed more light on the 2PA of the CPEs, the wavelength-dependent 2PA cross sections, σ2(λ), of P-C-3 and P-O-3 in water were measured by the 2PEF method by reference to 4,4′-diphenylaminostilbene as a 2PA standard (Figure S1).5,48 The 2PA spectral shapes are quite similar, exhibiting a relatively low-intensity band (∼200 GM) centered at ∼800 nm, with a stronger absorption which onsets at ∼775 nm and increases at shorter wavelengths. The maximum σ2 values are ∼700 GM at 700 nm for P-C-3 and ∼1400 GM at 720 nm for P-O-3. The general shape of the spectra is consistent with that of a centrosymmetric chromophore, where the more intense 2PA is blue shifted from the degenerate wavelength for the 1PA absorption (Figure S1). Although P-C3 has a lower maximum σ2 value than P-O-3, the higher ϕf of PC-3 results in a greater overall intensity in the 2PEF (Figure 1b). One and Two Photon Excited Fluorescence Quenching. In order to further explore the effect of aggregation on the 2PEF spectra of the CPEs, titration experiments were carried out using pyrophosphate ion (PPi). In previous work we have shown that PPi induces aggregation of cationic CPEs, presumably by inducing network formation among the molecularly dissolved cationic polymer chains by ionic complex formation between charged groups on different chains. The resulting PPi-induced aggregates typically exhibit a broad, redshifted emission with reduced ϕf.43 Previous studies also show that the extent of aggregation of cationic CPEs in aqueous solution is reduced at higher ionic strength and at lower pH.43,49 Thus, in the PPi titration studies, MES buffer (10 mM, pH 6.5) was used to reduce the extent of polymer aggregation. Compared to the pure aqueous solutions, the buffered CPE solutions exhibit more defined fluorescence spectra (Figures 2 and S2), signaling that there is less aggregation in the absence of PPi. Figure 2a presents the 1PEF spectra of P-C-3 as a function of PPi concentration. The intensity of the well-resolved 0−0 band with vibrational structure (415−435 nm) decreases continuously with PPi addition. Meanwhile, the broad band at λ ≈ 510 nm increases slightly when PPi concentration increases up to 6 μM. This band corresponds to a lower energy aggregate excited state of the polymer, resulting from the intermolecular exciton coupling among polymer chains that are brought into close proximity by PPi anions. After reaching the highest intensity at 6 μM PPi, this band decreases gradually from 6 to 12 μM PPi, which may be due to partial precipitation of the CPE. As seen in Figure 2b, the 2PEF spectra obtained under the same conditions reveal different features concomitant to addition of PPi. In particular, the fluorescence from the molecularly dissolved CPE at λ ≈ 435 nm decreases continuously from 0 to 12 μM PPi, similar to the 1PEF spectra. However, there is a significant increase in the intensity of the band due to the aggregated polymer with λ ≈ 510 nm. Interestingly, in contrast

Figure 2. Fluorescence of P-C-3 quenched by pyrophosphate under 1photon (a) and 2-photon excitation (b). Ten micromolar P-C-3 titrated with PPi in MES buffer (10 mM, pH 6.5); PPi concentration range from 1 to 12 μM. Spectra shown in b have been corrected by eliminating occasional “spikes” in intensity that occur due to large aggregates diffusing through the excitation beam.

to the spectra under 1PEF conditions, the aggregate emission dominates the spectrum for PPi concentration above 5 μM. PPi titration for P-O-3 in aqueous buffer is also performed under 2-photon excitation. Figure S2b shows the 2PEF spectra change during the PPi titration from 0 to 6 μM. The emission peak at ∼440 nm which corresponds to the 0−0 band of the nonaggregated polymer diminishes gradually during PPi addition, resulting from the PPi-induced polymer aggregation and fluorescence quenching. In contrast, the emission above 490 nm rises steadily, which is attributed to the induced polymer aggregation. Taken together, the studies which compare the fluorescence spectra of the CPEs obtained under 1PEF and 2PEF conditions clearly reveal that the overall efficiency of fluorescence from the aggregated state is enhanced under 2PEF. This result strongly suggests that the 2PA cross-section for excitation of the aggregate state is boosted relative to that of the molecularly dissolved state. It has been shown theoretically and experimentally that molecular aggregates can enhance the 2PA efficiency via intermolecular cooperative interactions.50,51 In the present CPE system, the polymer spontaneous selfassembly and/or PPi-induced association extends the πdelocalization52,53 and may induce three-dimensional (3D) conjugation. These interactions may induce a shift in the 2PA spectrum of the aggregated CPE relative to the molecularly 3297

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Figure 3. One-photon excited fluorescence of mixture solutions containing 10 μM (a) P-C-3 or (b) P-O-3 with different concentrations of GFP. Excitation wavelengths for P-C-3 and P-O-3 are 380 and 390 nm, respectively.

ent fluorescence lifetime data (see Supporting Information) also supports the CPE → GFP energy transfer mechanism. Having demonstrated the 1-photon excited FRET between the polymer and GFP, we further studied the enhanced GFP fluorescence upon 2-photon excitation. When excited with near-IR pulses (100 fs, 710 nm for P-C-3/GFP, and 720 nm for P-O-3/GFP mixtures), the CPE/GFP mixtures exhibit strong GFP fluorescence (Figure S6). However, virtually no fluorescence is observed when only GFP-containing solution is excited with the same excitation. Thus, the energy transfer from the polymers to GFP is also demonstrated by 2-photon excitation. Intracellular 2PE-FRET and Application in Imaging GFP with 2-Photon Excitation. GFP, as a noninvasive fluorescent probe, can be introduced into living organisms by gene transfection. However, the 2PA-related biological applications of GFP are limited by its low σ2. It is reported that the σ2 maximum value of wild-type green fluorescent protein (wtGFP) is about 12 GM at 810 nm,37 and the σ2 maximum value of the enhanced green fluorescent protein (EGFP) is less than 40 GM at 927 nm.38 The results presented above demonstrate the 2-photon excited FRET (2PE-FRET) approach to enhance the GFP fluorescence by using the CPEs effectively as “2-photon antenna” to harvest the near-IR photons and transferring energy to the fluorescent protein. Given this interesting effect, we decided that it would be of further interest to demonstrate its application to give rise to enhanced 2-photon excited fluorescence from GFP fluorescence within a living cell. Therefore, as a proof of concept demonstration, we used HeLa cells as the living model and realized the intracellular 2PE-FRET. The cell permeability and toxicity of P-C-3 and P-O-3 were inspected on breast cancer cells (MCF-7) in a separate investigation.58 In this work it was shown by using confocal laser scanning microscopy (CLSM) that the CPEs enter the cells after 8−10 h incubation, locating in lysosomes. The mechanism for cell penetration by the CPEs is likely via endocytosis. Methylthiazolyldiphenyltetrazolium (MTT) cell viability assays demonstrate that the CPEs exhibit relatively low cytotoxicity at concentrations < 20 μg·mL−1. In the current study, CPE→ GFP 2PE-FRET was investigated in HeLa cells that were genetically modified to express GFP (HeLa/GFP). The HeLa/GFP cells were incubated with solutions containing the CPEs for 10 h and then fixed for 2-photon excited confocal microscopy (2PFM)

dissolved chains, in addition to leading to 2−3-fold enhancement in σ2 at specific wavelengths.54 One- and Two-Photon Excited FRET from CPE to GFP. Given that the cationic CPEs exhibit moderate 2PA cross sections, we decided to explore whether the materials could be used to sensitize the emission of fluorescent proteins under 2PEF conditions. This is of interest because fluorescent proteins typically exhibit relatively low σ2 and thus are difficult to directly excite using near-IR light. As background for this concept, it was recently shown by Chen and co-workers that a cationic oligo(fluorene) is able to undergo FRET with GFP.55 A prerequisite for FRET is that there is spectral overlap between the absorption of the acceptor and the fluorescence of the donor. In keeping with this requirement, GFP has an absorption maximum with λ ≈ 488 nm,38 where both P-C-3 and P-O-3 emit strongly as shown in Figure 1. Another requirement for FRET is that the donor and acceptor need to be in close proximity (d < 100 Å). It is reported that GFP has a net negative surface charge at pH 6.5.56,57 Thus, we anticipated that electrostatic interactions between the cationic CPEs and GFP will bring them into close proximity, enabling energy transfer from CPEs to GFP. To explore FRET between the polymers and GFP, the fluorescence excitation and emission spectra of the CPEs were studied under 1PA excitation conditions as a function of added GFP in MES buffer (10 mM, pH 6.5).55 As shown in Figure 3, the fluorescence of both P-C-3 and P-O-3 decrease in intensity with increasing GFP concentration. Moreover, a new fluorescence band is clearly observed with λ ≈ 515 nm, which is due to the GFP. These results suggest that both CPEs are able to sensitize the fluorescence from GFP by a FRET mechanism. Additional support for CPE → GFP energy transfer comes from emission excitation studies (Figure S3), where the excitation was scanned over the 300−500 nm region while monitoring the GFP fluorescence at 515 nm. Here it is seen that the absorption of P-C-3 (or P-O-3) is clearly observed and dominates the excitation spectra. The energy transfer efficiencies (E) for CPE → GFP FRET ([GFP] = 30 μg/mL) were calculated from the data shown in Figure 3 using the equation E = 1 − (Fda/Fd), where Fda and Fd are the fluorescence (area) of the donor (CPE) in the presence and absence of acceptor (GFP), respectively. The calculated E for polymer/GFP (30 μg/mL) mixtures are 22% for P-C-3 and 38% for P-O-3, suggesting the energy transfer efficiencies for two polymers are comparable in solution. Wavelength-depend3298

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Figure 4. Two-photon excited fluorescence microscopy (2PFM) images. Excitation is 720 nm, 200 fs pulses, collected emission range is 502−538 nm. HeLa/GFP cells incubated with (a) 5 μM P-C-3 or (d) 5 μM P-O-3 for 10 h. HeLa cells (not expressing GFP) incubated with (b) 5 μM P-C-3 or (e) 5 μM P-O-3 for 10 h. HeLa/GFP cells without incubation with polymers (c and f).

absorption coefficient for GFP at 405 nm. Note that in the 1PFM images the intensity of the fluorescence from the CPEincubated HeLa/GFP cells (Figures S7a−c and S8a−c) is much stronger than that from the polymer-incubated HeLa cells (Figures S7d−e and S8d−e). These phenomena suggest that the sum of the fluorescence in the 425−510 nm range is increased by the contribution from the FRET-enhanced GFP fluorescence. The intracellular fluorescence imaging of fluorescent proteins is particularly important in developmental biology,59 immunology,60 neurobiology,61 transplantation,62 and tumor angiogenesis.63 2PFM of cells, tissues, organs, and whole animals expressing fluorescent proteins circumvents the light absorption and scattering of biological samples, which is the major problem of 1PFM.64 The enhanced GFP fluorescence in 2PFM that is demonstrated here could be useful for in vivo deep imaging in tissues and organs of living animals expressing green fluorescent proteins. The enhanced intracellular GFP fluorescence in 2PFM may also improve the optical performance of GFPs used as genetically encoded sensors to visualize and quantify enzymatic activities, ion concentrations, and various physiological events in vivo.64 Finally, it may be possible to extend the 2PE-FRET approach described here to other fluorescent proteins to enhance multicolor labeling applications.

studies. Imaging of the HeLa/GFP cells with illumination at 720 nm (200 fs pulses) with the P-C-3 or P-O-3 gives rise to a clearly observable green emission (filter range 502−538 nm) which can be assigned to the GFP (Figure 4a and 4d). The green emission is localized in lysozomes, consistent with the previous work that indicates that the CPEs are localized in this region of the cells. Several control experiments were carried out to confirm that the signal observed arises from the CPE→ GFP 2PE-FRET mechanism. First, as shown in Figure 4b and 4e, nearly dark images were obtained for HeLa cells (non-GFP expressing) containing only P-C-3 or P-O-3 when they were imaged using the same 2PFM conditions (720 nm excitation, emission range 502−538 nm). In this case little emission is observed because the fluorescence from the CPEs is centered below 500 nm. Second, as shown in Figure 4c and 4f, dark images are also observed when HeLa/GFP cells are imaged in the absence of added CPE. This result confirms that GFP is not efficiently excited by 720 nm light due to its comparatively low σ2 value. The intracellular FRET from polymers to GFP was also probed in 1-photon fluorescence microscopy (1PFM) on an inverted fluorescence microscope. In the 1PFM, the excitation band is limited to 405 ± 20 nm and the emission is collected in the range of 425−510 nm by using a filter cube. The 1PFM cellular images (Figures S7 and S8) illustrated that HeLa/GFP cells as well as HeLa cells incubated with the CPEs exhibit fluorescence in the region 425−510 nm, which coincides with the 1PA fluorescence spectra obtained in solution (Figure 3). Importantly, dark images are obtained for HeLa/GFP cells in the absence of the CPEs, consistent with the low 1-photon

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CONCLUSION In conclusion, we developed an effective approach to enhance the GFP 2PEF through a 2PE-FRET mechanism by using two cationic polymers (P-C-3 and P-O-3) as 2-photon absorbing 3299

DOI: 10.1021/acs.chemmater.7b00676 Chem. Mater. 2017, 29, 3295−3303

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Chemistry of Materials sensitizers. P-C-3 and P-O-3 demonstrate efficient 2PA properties in aqueous solutions. Besides, an interesting aggregation-enhanced 2PEF is discovered for both polymers. The energy transfer from cationic polymers to GFP is studied through 1-photon steady state and time-resolved fluorescence spectroscopy and 2-photon excited fluorescence spectroscopy. Moreover, we demonstrated the intracellular 2PFM on the enhanced GFP fluorescence through the 2-photon excited energy transfer from cationic polymers in HeLa cells. The enhanced 2PEF of GFP is of special practical significance for biological applications.

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σ2,s =

F2,s(λreg )Crηr (λreg ) F2,r(λreg )Csηs(λreg )

σ2,r

(1)

In eq 1, the subscripts r and s refer to the reference and sample, respectively. The variable σ2 is the two-photon cross section, F2(λreg) is the two-photon excited fluorescence intensity at a specific registration wavelength, Cr is the concentration, and ηr(λreg) is the absorbancecorrected differential quantum efficiency at a specific registration wavelength. The concentrations used for the calculation were obtained from Beer’s Law according to eq 2

C=

A εl

(2)

where A is the maximum absorbance, l is the optical length, and ε is the molar absorptivity. The absorbance-corrected differential quantum yield is calculated according to eq 3

EXPERIMENTAL SECTION

Materials. The synthesis of P-C-3 and P-O-3 will be described elsewhere.65 The molecular weights of the polymers were determined by GPC on Boc-protected precursors: P-C-3 12 000 (Đ = 2.2); P-O-3 21 600 (Đ = 1.9). Throughout the study all solution concentrations are based on the polymer repeat unit (PRU) concentration which is determined on the basis of the molecular weight of the repeating unit. The GFP for in vitro experiments was expressed in E. coli and then extracted, purified, and concentrated to a stock solution in PBS buffer according to procedures reported in the literature.55,66 Sodium pyrophosphate was purchased from Sigma-Aldrich. Dulbecco’s modified essential medium (DMEM) was purchased from Invitrogen. Fetal bovine serum (FBS) and penicillin−streptomycin (PS) were purchased from Atlanta Biologicals. Phosphate buffer saline (10× PBS) was purchased from Sigma-Aldrich. Lipofectamine 2000 reagent was purchased from Invitrogen. Plasmid DNA encoding GFP was used for transfection and in situ GFP expression in HeLa cells. Water purified by a Millipore purification system (Simplicity ultrapure water system from EMD Millipore) was used to prepare all aqueous solutions. Buffer solutions were prepared using reagent-grade materials from Fisher-Scientific or Sigma-Aldrich. All polymer concentrations are provided in terms of polymer repeat unit (PRU). Instrumentation. Corrected one-photon excited steady-state emission spectra were measured on a Photon Technology International (PTI) spectrophotometer. Time-resolved fluorescence measurements were carried out on a time-correlated single-photon counting system (TCSPC, Picoquant, Gmbh) and analyzed using FluoFit software in the global fitting mode. Two-photon excited emission spectra were collected on a home-built set up, composed of a Tsunami Ti:sapphire femtosecond laser and a Millennia eV pump laser (Spectra-Physics) and a fluorimeter (FluoroMax-3, Spex) with blocked light source. One centimeter light path quartz cuvettes are used for all measurements. General Methods. In the PPi or GFP addition experiments, small aliquots of a concentrated PPi stock solution in water or a GFP stock solution in PBS buffer were added to the 2 mL of CPE containing buffer in a quartz cuvette; then the mixture was mixed well on a vortexer before taking measurements. Determination of Two-Photon Cross Sections. The twophoton absorbance cross section (σ2) is evaluated based on the relative intensities of the two-photon excited fluorescence (2PEF) and onephoton excited fluorescence (1PEF).48 In order to calculate the absorbance of 10 μM CPE and reference standard of 10 μM TPV0-L was measured to determine the two-photon excitation wavelength. The excitation wavelength was determined as the wavelength in which the absorbance of the two solutions is close in value (335 nm). The 1PEF was then measured with the excitation wavelength determined from the absorbance measurement. The 1PEF spectrum is used to calibrate the fluorescence quantum yield of the sample and the efficiency of fluorescence detection. From the 1PEF spectra, a registration wavelength (λreg) is chosen where the reference solution and the CPE samples have matched emission intensities (565 nm for P-O-3 and 422 nm for P-C-3). The 2PEF was then measured at different wavelengths from 700 to 850 nm using a Ti:sapphire femtosecond laser. The 2PA σ2 was calculated using eq 1, as shown below.38,48

η(λreg ) =

F1(λreg ) A(λexc)

(3)

where A is the absorbance at the excitation wavelength and F1 is the one-photon fluorescence intensity at the registration wavelength. Cell Cultures. HeLa cells and HeLa/GFP cells were cultured in DMEM medium supplemented with 10% FBS and 100 units/mL PS and incubated at 37 °C in a humidified atmosphere containing 5% CO2. GFP Plasmid Transfection and GFP Expression in HeLa Cells. Before transfection, HeLa cells were seeded onto a 100 mm culture dish and reached 85% confluency after culturing for 36 h. Then one portion of 50 μL of Lipofectamine 2000 reagent and one portion of 20 μg of plasmid DNA encoding GFP were diluted in 500 μL of DMEM serum-free medium, respectively. The 500 μL of diluted DNA was added to the 500 μL of diluted Lipofectamine 2000 reagent and made a mixture of 1000 μL. The mixture was mixed well by pipetting and incubated at room temperature for 5 min. The 1000 μL of mixture was then added to HeLa cells at 85% confluency, mixing gently by rocking the dish back and forth. After incubation for 6 h, the medium was replaced with fresh DMEM medium with serum to terminate the transfection. The transfected cells were incubated for another 18 h before visualizing the green fluorescence under the fluorescence microscope. Cell Imaging. HeLa cells and HeLa/GFP cells were placed onto poly-D-lysine-coated coverslips in 24-well glass plates (5 × 104 cells per well) and incubated for 48 h before incubating with the CPEs. To investigate the efficiency and specification of these novel polymers, five comparative groups were set for the experiment: (1) HeLa/GFP cells incubated with 5 μM P-C-3; (2) HeLa cells incubated with 5 μM P-C3; (3) HeLa/GFP cells incubated with 5 μM P-O-3; (4) HeLa cells incubated with 5 μM P-O-3; (5) HeLa/GFP cells not incubated with CPE. For the CPE incubation process, polymer stock solutions are added to cell culture medium to reach the desirable concentration; the incubation time is 10 h before medium extraction. HeLa/GFP cells not incubated with CPE (group 5) are incubated in cell culture medium alone for the same amount of time. Then the culture medium was extracted, and the cells were washed abundantly with PBS buffer twice. Cells were then fixed with 3.7% formaldehyde solution in PBS buffer for 10 min. The fixing agent was extracted and washed twice with PBS. A fresh solution of NaBH4 (1 mg/mL) in PBS buffer was used to treat the fixed cells twice to reduce the autofluorescence. The coverslipped cells were then washed with PBS buffer (twice), followed by deionized water (once), and mounted on microscope slides using the Prolong Gold antifade reagent (Invitrogen, USA). Two-photon fluorescent microscopy (2PFM) was performed using a Bruker Fluorescence microscope equipped with a Coherent Mira 900 laser source (200 fs, 76 MHz). The fluorescence emission was excited at 720 nm and collected using an external nondescanned PMT detector (NDD). A 20×, 1.0 N.A. water immersion objective was employed for the 2PFM. One-photon fluorescence microscopy (1PFM) images were recorded on an Olympus IX-71 microscope equipped with a 3300

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Chemistry of Materials Qimaging-CCD camera. Fluorescence images of fixed cells were taken using two customized filter cubes (Ex 405/20 nm; DM 420 nm; Em 460/50 nm and Ex 377/50 nm; DM 509 nm; Em 536/40 nm). Statistical analysis of scanned images was performed with Fiji software.

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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/acs.chemmater.7b00676. Two-photon absorption cross sections of P-C-3 and P-O3, 2-photon excited fluorescence quenching of 10 μM PC-3 and P-O-3 with PPi in MES buffer, 1-photon excitation scan of P-C-3 or P-O-3 with different concentrations of GFP, fluorescence lifetimes of P-C-3 with different concentrations of GFP, fluorescence lifetimes of P-O-3 with different concentrations of GFP, 2-photon excited fluorescence of mixture solutions containing 10 μM P-C-3 or P-O-3 with different amounts of GFP, 1-photon excited fluorescence microscopy (1PFM) images of HeLa/GFP and HeLa cells incubated with 5 μM P-C-3 for 10 h, 1-photon excited fluorescence microscopy (1PFM) images of HeLa/GFP and HeLa cells incubated with 5 μM P-O-3 for 10 h, 1-photon excited fluorescence microscopy (2PFM) images of HeLa/GFP cells without incubation in CPE solution, discussion on the fluorescence lifetime studies (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]fl.edu; [email protected]. ORCID

Kevin D. Belfield: 0000-0002-7339-2813 Shu Wang: 0000-0001-8781-2535 Kirk S. Schanze: 0000-0003-3342-4080 Author Contributions ‡

S.W. and Z.L. contributed equally. Z.L. accomplished synthesis of the CPEs, and S.W. led the photophysical and biological work and manuscript preparation. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the U.S. Defense Threat Reduction Agency for support through grant HDTRA1-08-1-0053. REFERENCES

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