Conjugated-Polymer-Based Red-Emitting Nanoparticles for Two

Jun 24, 2014 - Achilefu , S. The Insatiable Quest for Near-Infrared Fluorescent Probes for Molecular Imaging Angew. Chem., Int. Ed. 2010, 49, 9816– ...
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Conjugated-Polymer-Based Red-Emitting Nanoparticles for TwoPhoton Excitation Cell Imaging with High Contrast Shuang Li, Xiaoqin Shen, Lin Li, Peiyan Yuan, Zhenping Guan, Shao Q. Yao, and Qing-Hua Xu* Department of Chemistry, National University of Singapore, 3 Science Drive 3, Republic of Singapore 117543 S Supporting Information *

ABSTRACT: Two-photon fluorescence microscopy is a widely used noninvasive bioimaging technique because of unique advantages such as a large penetration depth and 3D mapping capability. Ideal two-photon fluorophores require large two-photon absorption cross sections and red emission with high quantum yields. Here we report red-emitting-dyedoped conjugated polymer nanoparticles that display high two-photon excitation brightness. In these nanoparticles, conjugated polymer (PFV) was chosen as a two-photon light-harvesting material, and red-emitting dyes (MgPc and Nile red) were chosen as the energy acceptors and redemitting materials. Two-photon excitation fluorescence of MgPc and Nile red was enhanced by up to ∼53 and ∼240 times, respectively. We have successfully demonstrated the application of these conjugated polymerbased nanoparticles in two-photon excitation cancer cell imaging with an excellent contrast ratio. This concept could become a general approach to the preparation of two-photon excitation red-emitting materials for deep-tissue live-cell imaging with high contrast.

1. INTRODUCTION Optical microscopy is one of the most widely used noninvasive bioimaging tools for analyzing biological structures and functions.1−3 However, the limited penetration depth of conventional one-photon excitation (1PE) techniques compromises the 3D capability of confocal microscopy. Twophoton excitation (2PE) techniques are advantageous over the traditional one-photon counterparts in a few aspects. 2PE renders intrinsic 3D-selective excitation and 3D mapping capability with significantly reduced bleaching effects (a major problem in 1PE-based fluorescence imaging).4 2PE fluorescence microscopy has been widely used in cell imaging owing to these unique advantages and high signal-to-background ratio fluorescence detection.4−8 Key requirements for two-photon imaging fluorophores include large two-photon absorption (TPA) cross sections and high emission quantum yields.9,10 In addition to the deep penetration of excitation light, the effective collection of fluorescence signals from deep tissues is also essential for achieving deep tissue imaging, which requires fluorophores that emit in the red or near-infrared regions.5,11 Significant efforts have been devoted to developing red-emitting fluorophores such as organic dyes for in vivo measurements and optical imaging.12−15 However, many red-emitting organic dyes suffer from a small Stokes shift, limited TPA cross sections, low emission quantum yields, and easy aggregation in physiological environments. Conjugated polymers have delocalized π-conjugated backbones and display large one- and two-photon absorption coefficients, high fluorescence quantum yields, good photo© 2014 American Chemical Society

stability, and excellent biocompatibility, which make them attractive imaging agents.16−20 However, most conjugated polymers generally emit in the visible range. Fluorescence resonance energy transfer (FRET) could be utilized to further extend the red shift between the excitation and emission wavelengths. FRET from conjugated polymers results in fluorescence amplification, which has been widely utilized for various biological applications with enhanced efficiency.21−24 2PE-based FRET (2PE-FRET) from conjugated polymers with large TPA cross sections11,25,26 to red-emitting dyes with high emission quantum yields could potentially realize near-infrared excitation and red emission for sensitive detection to enable highly efficient 3D deep tissue imaging. Here we report conjugated polymer nanoparticles doped with red-emitting dyes that emit in the far-red region (∼680 nm) with high two-photon excitation brightness. In these nanoparticles, poly[9,9′-bis(6″-bromohexyl)fluorene-2,7-ylenevinylene-co-alt-1,4-phenylene] (PFV) was chosen as the twophoton light-harvesting material, and magnesium phthalocyanine (MgPc, λem ≈ 680 nm) was chosen as the energy acceptor and red-emitting material. PFV is a conjugated polymer with a large two-photon absorption (TPA) cross section at 800 nm. MgPc is a red-emitting dye with a reasonable quantum yield (∼30%).27 These PFV- and MgPc-based nanoparticles display an effective TPA cross section of up to 4300 GM (per MgPc) at 800 nm. We have further prepared another red-emitting Received: May 12, 2014 Revised: June 20, 2014 Published: June 24, 2014 7623

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Figure 1. (a) Schematic preparation procedures of MgPc/PFV NPs. (b) Normalized absorption and emission spectra of PFV NPs and MgPc NPs. (c) Hydrodynamic diameter of MgPc/PFV NPs measured by DLS and their TEM images (inset). 2.4. Two-Photon Excitation (2PE) Fluorescence Measurements. 2PE fluorescence measurements were performed by using a femtosecond (fs) Ti:sapphire oscillator (Avesta TiF-100M) as the excitation source. The output laser pulses have a central wavelength of 800 nm with pulse duration of ∼80 fs and a repetition rate of 84.5 MHz. The laser beam was focused onto the samples that were contained in a cuvette with a path length of 1 cm. Emission was collected at the direction perpendicular to the excitation beam by a pair of lenses and an optical fiber that was connected to a monochromator (Acton Spectra Pro 2300i) coupled to a CCD (Princeton Instruments Pixis 100B) system. A short-pass filter with a cutoff wavelength of 750 nm was placed before the spectrometer to minimize the scattering from the pump beam. The concentration of nanoparticles in the water dispersion was about 10 μM in PFV repeat units. 2.5. Cell Culture and Viability. Human hepatocellular carcinoma cell line HepG2 was cultured in growth media (DMEM supplemented with 10% fetal bovine serum, 100.0 mg/L streptomycin, and 100 IU/ mL penicillin). Cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C. The cytotoxicity of these nanoparticles was evaluated by monitoring the cell viability of HepG2 cells after incubation with MgPc(0.75%)/PFV NPs of different concentrations (in terms of PFV repeat units) and MgPc NPs containing the same amount of MgPc, respectively. The cell viability was determined by using the XTT colorimetric cell proliferation kit (Roche) following the manufacturer’s guidelines. Briefly, cells were grown to 20−30% confluence (since they will reach 80−90% confluence within 48 to 72 h) in 96-well plates under the conditions described above. The medium was aspirated, washed with PBS, and then treated with 0.1 mL of the medium containing different amounts of the MgPc(0.75%)/ PFV NPs. After incubation for 24 h, cells were washed four times using PBS to remove excess nanoparticles. Proliferation was assayed by using the XTT colorimetric cell proliferation kit (read at A460 nm − A650 nm). A total of three replicas were performed. 2.6. Two-Photon Cell Imaging. 2PE cancer cell imaging experiments were conducted on a two-photon laser scanning confocal microscope by using femtosecond laser pulses at 800 nm. HepG2 cells were first incubated with 2.0 μM MgPc(0.75%)/PFV NPs in the growth medium (DMEM supplemented with 10% fetal bovine serum, 100 mg·L−1 streptomycin, and 100 IU·mL−1 penicillin) for 8 h. 2PE fluorescent images of HepG2 cells was taken with an excitation power

conjugated-polymer-based nanoparticle by using Nile red as the doping material. These conjugated-polymer-based nanoparticles were found to be capable of serving as excellent contrast agents for 2PE bioimaging applications.

2. EXPERIMENTAL SECTION 2.1. Materials. Magnesium phthalocyanine (MgPc), polyoxyethylene nonylphenylether (CO-520), and tetrahydrofuran (THF) were purchased from Sigma-Aldrich. Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum, streptomycin, penicillin, and phosphate-buffered saline (PBS) buffer solution were purchased from Invitrogen. Poly[9,9′-bis(6″-bromohexyl)fluorene-2,7-ylene-vinyleneco-alt-1,4-phenylene] (PFV) was synthesized as described in the literature.11 The molecular weight (Mw) was measured to be 30 965 with a PDI (Mw/Mn) of 1.81. 2.2. Preparation of Nanoparticles. MgPc-doped PFV NPs, PFV NPs, and MgPc NPs were prepared by using a reprecipitation method that has been described in detail previously.26,28 Briefly, MgPc, PFV, and a nonionic surfactant, polyoxyethylene nonylphenylether (CO520), were dissolved in THF first to act as the stock solution. Different amounts of MgPc (0.2 mM) were mixed with 4.0 μL of CO-520 (2.0 mM) and 1.0 mL of PFV (40.0 μM) in THF to prepare MgPc/PFV NPs with different molar ratios (based on starting materials). The mixtures were quickly added to 4.0 mL of deionized water under sonication. A nanoparticle dispersion with a yellow-green color was obtained after THF was removed by vacuum evaporation at room temperature. PFV NPs were prepared by the same method without MgPc. MgPc NPs were prepared by mixing different amounts of MgPc and 24.0 μL of a CO-520 stock solution in 1.0 mL of THF before being added to water. All of the concentrations of MgPc/PFV NPs and PFV NPs are in terms of the PFV repeat unit. 2.3. Characterizations. TEM images were taken on a JEOL 2010 transmission electron microscope (TEM) operating at an acceleration voltage of 200 kV. The size distribution of the nanoparticles was measured by dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern). UV−vis absorption and one-photon excitation (1PE) fluorescence spectra were measured by using a Shimadzu UV 2550 spectrometer and a Horiba Jobin Yvon FluoroMax-4 spectrofluorometer, respectively. 7624

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of 50 mW (before entering microscope) over a detection emission range of 650−700 nm.

3. RESULTS AND DISCUSSION MgPc-doped PFV nanoparticles were prepared by using a reprecipitation method that has been described in detail previously.26,28 The obtained nanoparticles have an averaged hydrodynamic diameter of 27 nm based on DLS measurements, consistent with the TEM imaging results (26 nm). In these composite nanoparticles, PFV acts as the two-photon lightharvesting materials, energy donor, and doping matrix, while the encapsulated MgPc acts as the energy acceptor. MgPc molecules are doped into the PFV matrix and are in direct contact with the PFV molecules to meet the distance requirement for efficient FRET. Upon absorption of two photons by PFV, the excitation energy can migrate along the polymer chain to transfer the energy to nearby acceptor (MgPc) molecules. As both donor and acceptor molecules are encapsulated into the nanoparticles, the donor (PFV) and acceptor (MgPc) are in 3D contact to allow 3D energy-transfer to further promote the energy-transfer efficiency. The emission intensity of MgPc is expected to be amplified via 2PE-FRET compared to the direct excitation of MgPc due to a much larger TPA cross section of PFV compared to that of MgPc.25,29 PFV NPs dispersed in water have a TPA cross section of 254 GM per repeat unit at 800 nm (Figure S1 in the Supporting Information) and emit fluorescence from 450 to 550 nm (Figure 1b) with a quantum yield of ∼10%. MgPc is a redemitting dye (λem,max ≈ 670 nm) with a quantum yield of ∼30% in 2-propanol.27 The emission spectrum of PFV and the absorption spectrum of MgPc have some overlap to ensure efficient energy transfer. CO-520 was introduced to modify the surfaces of the nanoparticles, thus reducing cytotoxicity and facilitating cell uptake. Figure 2 shows 1PE and 2PE fluorescence spectra of MgPc/ PFV NPs with different molar ratios of MgPc in H2O. 1PE fluorescence (1PEF) spectra were measured under excitation at the absorption maximum of PFV at 437 nm. 1PE fluorescence spectra of MgPc/PFV NPs are composed of two parts: the 450−550 nm range with distinct vibronic structures originating from PFV and a narrow emission band centered at ∼680 nm originating from MgPc. As MgPc molecules have little absorption at 437 nm, the observed MgPc emission primarily arises from energy transfer from the excited PFV to MgPc instead of direct excitation of MgPc. Compared to undoped PFV NPs (without MgPc), the intensity of PFV emission in MgPc/PFV NPs was found to be quenched due to energy transfer from PFV to MgPc (Figure 2a). The extent of quenching increased as the MgPc concentration increased, consistent with the trend as predicted by the Stern−Volmer law.30 The energy transfer efficiency (ET) can be calculated from ET = (1 − IDA/ID) × 100%, where IDA and ID are the fluorescence intensities of the donor (PFV) with and without the acceptor (MgPc), respectively. It is interesting to note that the energy-transfer efficiency from PFV to MgPc is quite high despite relatively small spectral overlap between the emission of PFV and the absorption of MgPc. The energy-transfer efficiency is up to 71.5% in the presence of 2% MgPc. The reason for such a high energy-transfer efficiency is that the small spectra overlap was compensated by the large absorption coefficient of MgPc and the 3D contact between the donor and acceptor molecules, which allows 3D energy-transfer processes between multiple donor and acceptor molecules, which further

Figure 2. 1PE (a) and 2PE (b) fluorescence spectra of PFV NPs and MgPc/PFV NPs. Excitation wavelengths for 1PE and 2PE are 437 and 800 nm, respectively.

promotes the energy-transfer efficiency. As the MgPc concentration increased, the emission intensity of MgPc increased first, reached an optimum, and then decreased. At low MgPc concentrations, one MgPc molecule is surrounded by many PFV polymer chains as the light-harvesting material, which results in a large fluorescence enhancement of MgPc emission via energy transfer compared to the direct excitation of MgPc. As the concentration of MgPc increases, the number of PFV molecules (donor) surrounding each MgPc molecule decreases and the fluorescence enhancement factors will decrease. At a very high concentration of MgPc, the distribution of MgPc molecules in the polymer matrix will become more inhomogeneous and may even form aggregates, which will result in a decrease in the emission intensities as observed at very high MgPc concentration (Figure 2a). MgPc/PFV NPs display the strongest MgPc emission when 0.75% of MgPc is introduced into the nanoparticles. Compared to MgPc nanoparticles in a water dispersion with the same amount of MgPc and CO-520 under excitation at its absorption maximum of 676 nm, the MgPc-doped PFV nanoparticles showed an optimal enhancement of 3.4-fold in 1PEF of MgPc for MgPc(0.125%)/PFV NPs. The 2PE fluorescence (2PEF) spectra of various MgPc/PFV nanoparticle dispersions are similar to those under 1PE: emission signals from both PFV and MgPc were observed. The 2PE fluorescence spectra exhibit a trend similar to 1PE fluorescence spectra: as the concentration of the doped MgPc increased, the PFV emission intensity decreased continuously due to energy transfer, while the MgPc emission increased first, reached an optimum, and then decreased. Under excitation at 800 nm, both PFV and MgPc could be directly excited. However, the MgPc emission intensities in MgPc/PFV NPs were significantly enhanced compared to those of MgPc NPs 7625

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Figure 3. (a) Cell viability assay of HepG2 cancer cells treated with MgPc(0.75%)/PFV NPs and MgPc NPs containing the same amount of MgPc for 8 h. (b−d) 2PE fluorescent images (b), bright-field images (c), and overlaid images (d) of HepG2 cancer cells after incubation with 2 μM MgPc(0.75%)/PFV NPs. The nanoparticle concentration in (a) is in terms of the PFV repeat unit.

containing the same amount of MgPc. Enhanced 2PEF of MgPc is due to efficient energy transfer from PFV to MgPc within the nanoparticles, in which PFV has a much larger TPA cross section than MgPc. The largest 2PEF enhancement of 53fold was obtained for MgPc(0.125%)/PFV NPs. The effective TPA cross section of MgPc(0.125%)/PFV NPs reached 4300 GM per MgPc at 800 nm. MgPc(0.75%)/PFV NPs displayed the strongest 2PE fluorescence intensity of MgPc due to a compromise between a higher MgPc concentration in the NPs and a slightly smaller 2PEF enhancement factor. MgPc(0.75%)/PFV NPs were chosen as the contrast agents because they give the largest 2PE fluorescence intensity at 686 nm per nanoparticle. Good 2PE fluorophores require good biocompatibility and photostability. Figure 3a shows that the cell viability of HepG2 cancer cells remained >85% after incubation with the MgPc/PFV NPs with a concentration as high as 10 μM. The cytotoxicity of MgPc/PFV NPs is clearly lower than that of the corresponding MgPc NPs. This result suggested that the cytotoxicity of MgPc became reduced upon encapsulation into PFV. The photostability of the MgPc/PFV nanoparticles was evaluated by monitoring their emission spectra at 2 min interval under continuous irradiation by femtosecond laser pulses at 800 nm with an average power of 50 mW. The results (Figure S2) showed that the emission spectra remained nearly unchanged under continuous laser irradiation for >10 min, suggesting that these nanoparticles were very stable against photobleaching. 2PE cancer cell imaging experiments were conducted by using 2.0 μM MgPc(0.75%)/PFV NPs. Under this condition, cell viability remained >98% after incubation with the nanoparticles. Figure 3b shows the 2PE fluorescence image of HepG2 cancer cells after incubation with MgPc(0.75%)/PFV NPs for 8 h. Intense red fluorescence emission signals were observed from the cells under excitation at 800 nm. The overlaid images (Figure 3d) of the 2PE fluorescence image (Figure 3b) and the bright-field image (Figure 3c) indicated that MgPc/PFV NPs were mainly accumulated around the

nuclei region of the HepG2 cells, confirming that these nanoparticles were actively taken up and internalized into the cells by endocytosis. This concept can be extended to a general approach for the preparation of 2PE red-emitting materials as 2PE imaging contrast agents. We have also prepared conjugated-polymerbased nanoparticles by using another red-emitting dye, Nile red, as a doping material and energy acceptor using the same reprecipitation method (Figure S3). The 1PE and 2PE fluorescence of Nile red was enhanced by factors of 9 and 240, respectively (Figure S4 in the Supporting Information). The effective TPA cross section of Nile red/PFV NPs reached 1400 GM per Nile red at 800 nm. Their applications in 2PE cell imaging have also been demonstrated (Figure S5 in the Supporting Information).

4. CONCLUSIONS Red-emitting-dye-doped conjugated-polymer nanoparticles have been prepared for two-photon cancer cell imaging. In these nanoparticles, PFV with a TPA cross section of 254 GM per repeat unit was utilized as a two-photon light -harvesting complex to enhance the 2PE fluorescence of red-emitting dyes, MgPc (emission at ∼680 nm) and Nile red (emission at ∼610 nm), by up to 53 and 240 times, respectively. These nanoparticles display excellent biocompatibility, good photostability, and large two-photon brightness in the red region with an effective TPA cross section of up to 4300 GM per MgPc and 1400 GM per Nile red at 800 nm. The application of these nanoparticles to the two-photon imaging of HepG2 cancer cells gave strong red emission signals with excellent contrast. This concept could be extended to a general approach to the preparation of two-photon-excitation red-emitting materials for deep-tissue live-cell imaging with high contrast. 7626

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ASSOCIATED CONTENT

S Supporting Information *

Fluorescence quantum yield and TPA cross-section measurements, two-photon excitation spectrum of PFV NPs, photostability of the MgPc/PFV NPs, and results of Nile red-doped PFV NPs. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +65 6516 2847. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the financial support of this work by the Faculty of Science, National University of Singapore (AcRF Tier 1 Grant R-143-000-403-112).

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REFERENCES

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