Red-emitting, EtTP-5-based organic nanoprobes for two-photon imaging in 3D multicellular biological models

Abstract

Biocompatible and biostable EtTP-5-loaded organic core–shell nanoparticles have been successfully evaluated for their potential as red-emitting fluorescent nanoprobes for two-photon imaging. Readily formed by Flash NanoPrecipitation, EtTP-5-based nanoprobes proved to penetrate well into multicellular spheroids and were easily imaged through several cell layers within these complex, 3D tissue models.

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Fluorescence microscopy is currently among the most powerful imaging tools for visualizing and understanding biological processes in a noninvasive and spatiotemporal manner.¹ Since the late nineties, two-photon excited microscopy (TPEM) has emerged as a preferred light imaging technique, offering a number of significant advantages over conventional one-photon confocal microscopy, including increased in-depth tissue penetration, higher spatiotemporal resolution, reduced scattering/phototoxic effects and negligible background fluorescence.² Thus, there is an ever-increasing demand for more sensitive and versatile two-photon (2P) fluorescent probes that enable imaging deep within complex three-dimensional (3D) biological structures. In recent years, much work has been focused on the development of small molecule probes for TPEM.³ As 2P probes for biological imaging must meet strict fluorescent and biological requirements, the molecular design space of such probes remains strongly constrained.

An alternative strategy, consisting of using nanoprobes (NPs) that encapsulate 2P active fluorophores, decouples the fluorescence and biological requirements.⁴ The decoupling gives more flexibility in the design and optimization of the organic fluorophores, while adding all of the benefits of a nanoprobe including improved fluorescence stability and potential for specific targeting. In terms of fluorescent requirements, an ideal 2P fluorescent NP should be excitable with a 2P absorption cross-section (σ₂) greater than 50 GM and should emit in the biological imaging window (650–900 nm).⁵ This minimizes light absorption and scattering by tissues and improves the imaging depth and sensitivity, which is particularly important when working with complex, three-dimensional tissue models.⁶ To date, only a handful of such NPs emit within this window and fewer still meet the σ₂ requirement.³ Additionally, NPs should be photostable to enable long-term imaging or tracking.^3b In terms of biological and NP requirements, a NP should be biocompatible, stable in biological media, and be made by a simple process allowing for the control of particle size and surface functionality, which are critical in determining the interaction with biological samples.⁷ In general, NPs under 200 nm in diameter are of interest,⁸ but the specific size is dictated by the application. The NP dispersibility and stability in biological media are strongly influenced by the surface functionality, with non-fouling surfaces being preferred.⁹ Additionally, the NP surface charge plays a significant role in the interaction with cells and penetration of the probes within the tissue models.^7a

Herein, we will explore the potential of core–shell nanoparticles loaded with EtTP-5, a bulky pentacene-based dye with a suitable photophysical and hydrophobicity profile (Fig. 1a),¹⁰ as novel 2P fluorescent nanoprobes for imaging deep inside 3D tissue models. We will show that (1) EtTP-5 can be excited in 2P mode with an appropriate σ₂, (2) by encapsulating EtTP-5 into NPs, the photophysical properties of the dye are globally maintained, (3) EtTP-5 NPs are stable and biocompatible, and (4) that NPs can be imaged through several cell layers within 3D tissue models via TPEM.

Fig. 1 Characterization of EtTP-5 in solution. (a) Structure of EtTP-5. (b) Absorbance (dashed line) and emission (solid line) spectra in toluene. (c) 2P excitation spectrum in toluene.

EtTP-5 – photophysical evaluation in solution

The one-photon photophysical properties of EtTP-5 were investigated in solution in toluene by means of UV-vis and fluorescence spectroscopies. The corresponding absorption and emission spectra are depicted in Fig. 1b. Table 1 summarizes the resulting photophysical parameters. The EtTP-5 dye exhibits three strong and sharp absorption bands at 457, 574 and 624 nm and two strong emission bands at 632 and 686 nm (Fig. 1b). The dye shows a red fluorescence around 700 nm, matching the desired biological imaging window.

Table 1 One- and two-photon photophysical properties of EtTP-5 in solution and nanoprobe form

Form	λ^absmax^a (ε × 10^−4)^b	λ^emmax^c	ϕ^d	σ2^e
a Maximum absorption wavelengths in nm.b Molar extinction coefficients in M^−1 cm^−1.c Maximum emission wavelengths in nm.d Fluorescence quantum yield at 457 nm in %.e 2P absorption cross section at 820 nm in GM.f Non determined.g All EtTP-5 solution tests were performed in toluene.h All EtTP-5 nanoprobe tests were performed in ultra-pure water.
Solution^g	457 (2.3), 574 (1.1), 624 (2.1)	632, 686	35	182
Nanoprobe^h	460 (4.4), 578 (1.9), 630 (3.3)	636, 694	21	nd^f

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For the first time, the two-photon performance of the EtTP-5 fluorophore was studied by the way of the 2P excited fluorescence method.^2e,11 As shown in the 2P excitation spectrum depicted in Fig. 1c, EtTP-5 exhibits a strong and relatively sharp two-photon absorption band in the near-IR 800–830 nm range, with a maximum σ₂ of about 180 GM at 820 nm (1 GM = 10⁻⁵⁰ (cm⁴ s⁻¹ per photon) in toluene. The σ₂ value is nearly four times larger than the required 50 GM for an efficient 2P probe. Importantly, EtTP-5 also presents its 2P absorption maximum in the wavelength range of most standard laser sources used in TPEM. Together, these photophysical data suggest that EtTP-5 is a promising fluorophore for 2P imaging applications.

EtTP-5 nanoprobe – formation, characterization and photophysical evaluation

Due to its highly hydrophobic nature (i.e., log P close to 10), EtTP-5 is well-suited to be encapsulated in core–shell organic nanoparticles.¹² As previously mentioned, the biological application determines the required NP characteristics. Hence, a simple, rapid and flexible synthesis method is necessary for the formation of application-specific NPs. For that purpose, we selected the Flash NanoPrecipitation (FNP) method, a well established one-step controlled precipitation process.¹³ Via the FNP process, the NP size, fluorophore loading and surface functionalization can be easily tuned for a desired application.^7c FNP is a kinetically-controlled process producing NPs using amphiphilic block copolymers (BCPs) to direct self-assembly.^7c,8,14 Thus, FNP allows the formation of uniform particles with tunable sizes from 40 to 400 nm, while the loading and surface properties can be controlled by the formulation and choice of stabilizing BCP.^7c,8

For our particular application, size and surface charge have been shown to be key parameters regarding the penetration of NPs within multicellular spheroids (MCS).^7a,b,15 NPs smaller than 100 nm with neutral or negative surface charge have been found to penetrate within MCS more effectively than large and positively charged NPs.^7a,b

By using the FNP process, the NP size and surface charge were easily controlled via the NP formulation.^7c As shown in Fig. 2a, the resulting NPs had a volume average size of 50 nm (PDI = 0.18) as determined via DLS, thus appropriate for MCS penetration. A TEM image of the NPs (Fig. 2b) shows spherical particles with a narrow size distribution ranging from 20 to 60 nm. A negative stain was used to visualize the polymeric particles due to their low electron density contrast. To create a neutral, non-fouling NP surface, poly(styrene)-b-poly(ethylene glycol) (1.6k-b-5.0k) was used as the stabilizing BCP. The NPs had a zeta potential of −1.5 mV, indicating a neutral surface charge (Fig. 2a inset). Using the Baleux assay, the PEG NP surface density was determined to be 1.1 ± 0.4 PEG chains per nm², which is within the brush regime.¹⁶ Because dense PEG layers are known to minimize protein adsorption in solution and confer NP stability, we evaluated the NP stability in a protein rich solution.⁹ NPs incubated in cell culture media with 10% fetal bovine serum at 37 °C exhibited a minimal change in size (a 7% increase in diameter) over 24 hours, demonstrating their stability in biological media (Fig. S1 in the ESI†).

Fig. 2 Characterization of EtTP-5 in nanoprobe form. (a) Volume average nanoprobe diameter plotted along with the nanoprobe zeta potential (inset). (b) TEM image. (c) Absorbance (dashed line) and emission (solid line) spectra in water. (d) Percent change in fluorescence of EtTP-5 NPs exposed to 633 nm light (diamond) and 850 nm 2PT light (square) over a 10 minute continuous exposure time (n = 2) (more details are given in the ESI†).

We next examined the photophysical profile of the NP loaded with EtTP-5 because changes in photophysical behavior, particularly the loss of fluorescence due to quenching, can occur when encapsulating fluorescent dyes in NP form. Previous work has shown that for a maximum fluorescence per NP, the optimal EtTP-5 concentration within the poly(styrene) (PS) matrices is 2.3 wt%, which results in a 3.9 nm lattice spacing between dye molecules.¹² Above this concentration, molecules are within a Förster radii and quenching is observed. As such, the 2.3 wt% EtTP-5 relative to the PS content was used in the NP formulation. The corresponding absorption and fluorescence spectra of the dye in NP form in mono-photon mode are plotted in Fig. 2c. A slight red shift of 3 nm at the apex of the emission peaks of the dye in NP form is observed due to the encapsulation in the polymer matrix. More notably, a near-doubling of the molar absorptivity is observed between the dye in solution and the encapsulated dye, increasing from 23 000 M⁻¹ cm⁻¹ in toluene to 42 000 M⁻¹ cm⁻¹ in NP form (Table 1). In contrast, the quantum yield decreases from 35% for the free dye in toluene to 21% for the encapsulated dye. Such changes in molar absorptivity and quantum yield due to encapsulation effects have already been observed for comparable systems in the literature.¹⁷ Of significance here is that the encapsulation of the EtTP-5 dye does not affect its native brightness value (Table 1).

We further investigated the photobleaching behavior of the fluorescent nanoprobe, a key parameter for long term monitoring of biological samples.³ The present encapsulated form was observed to be very resistant in both one-photon (633 nm) and 2P (850 nm) excitation modes (Fig. 2d). Less than a 10% loss in fluorescence signal was observed after 10 minutes of constant illumination using standard imaging conditions. The fluorophore is stabilized within the core of the NP in a solid state, which reduces photo-induced degradation.^14,18

EtTP-5 nanoprobe – application for 2P imaging in complex multicellular spheroids

In the present work, we have chosen the MCS¹⁵ as a 3D tissue model to evaluate the potential of EtTP-5-loaded NPs for 2P tissue imaging. Recently, MCSs have garnered significant attention as tissue models for a wide range of biological applications ranging from cell migration and adhesion studies to tissue engineering.^15,19 As in vitro models, MCSs reproduce cell–cell interactions, cell–extra cellular matrix interactions, and 3D cellular organization found in in vivo tissues.^15,19

We focused our attention on human mammary MCF7 adenocarcinoma cancer cells and first evaluated the cytotoxicity of EtTP5-based NPs against this human cell line. Using an Annexin V conjugation assay, no cytotoxicity (<5%) in monolayer-cultured MCF7 cells was observed after 24 hours of incubation up to the concentration of 1 mg mL⁻¹ NP.

As shown in Fig. 2a, EtTP-5 NPs fulfill the size (<100 nm) and charge requirements for penetration inside tissue such as MCS.^7a,b One- and two-photon imaging of NP interaction with cells was first performed in monolayer culture to demonstrate the ability of EtTP-5-based NPs to be imaged using TPEM. MCF7 cells were incubated with NPs for 3 hours and then incubated either with the cell-permeant Hoechst to stain nuclei in live cells or with cytoplasmic live CellTracker™ Green CMFDA fluorescent dye prior to imaging. As shown in Fig. 3a–f, the NPs are detected within cells as demonstrated by 3D live imaging (Fig. S3 in the ESI and ESI Movie 1†) and the NP fluorescence signals show the same localization under both one-photon (633 nm, yellow) and 2P (850 nm, red) excitation.

Fig. 3 (a–f) Images of MCF7 cells incubated with EtTP-5 nanoprobes (NPs) and then stained with the DNA cell permeant dye Hoechst 33342 ((a–c), in blue) or the CellTracker Green CMFDA ((d–f), in green). (a and d) EtTP-5 NPs (yellow) fluorescence under 633 nm excitation, (b and e) EtTP-5 NPs (red) fluorescence under 850 nm light in 2P mode. (c and f). The overlaid signals from EtTP-5 NPs excited in mono (yellow) and 2P (red) mode show a co-localization of fluorescence signals. (g–i) Images from a z-stack of a 400 μm MCF7 H2B GFP multi-cellular spheroid (green = GFP labeled histones) incubated with EtTP-5 NPs (red) acquired under 2PT excitation. The images shown in (g–i) correspond respectively to z = 20 μm, 50 μm and 70 μm inside the spheroid. The EtTP-5 NPs (red) penetrate through several cell layers within the MCS and are easily imaged via TPEM.

To demonstrate the ability of the EtTP-5 NPs to act as probes for imaging deep within complex 3D biological samples, 400 μm in diameter MCS of MCF7 cells engineered to stably express the histone H2B nuclear protein in fusion with GFP were incubated with EtTP-5 NPs. In these models, the histone H2B-GFP expression enables the visualization of the nuclei (green) in every cell within the whole volume of the MCS. After 24 hours, the distribution of NPs within the MCS was investigated using TPEM, exciting both the GFP and the probe at 850 nm. The probes penetrated well into the spheroids and were easily visualized via TPEM as shown in Fig. 3g–i. The probe signal (red) was observed several cell layers deep within the spheroids as shown on the 1 μm-step z-stack and the corresponding 3D reconstruction (ESI Movie 2†). We have further demonstrated that these results are not cell line-specific and that EtTP-5 NPs also penetrate and can be imaged in MCS from human colon HCT116 (ESI Movie 3†) and mammary metastatic MDA-MB231 cells (ESI Movie 4†). On the whole these results indicate both the appropriateness of the EtTP-5 dye and the NP vector for imaging deep within complex 3D biological structures.

Conclusions

The red-emitting EtTP-5 fluorophore has been shown for the first time to be amenable to two-photon excitation with a photophysical profile suited to biological imaging. After encapsulation of highly hydrophobic EtTP-5 into organic core–shell nanoparticles via Flash NanoPrecipitation, the resulting EtTP-5-loaded nanoparticles further proved biocompatible and biostable, as well as photostable for long-term imaging. Finally, these easy to prepare materials were successfully implemented as two-photon imaging nanoprobes, capable of (i) penetrating deep into complex 3D multicellular spheroids in a non cell-line specific manner and capable of (ii) being imaged through several cell layers within the spheroids. These nanoprobes are currently being used to investigate the interactions between nanoparticles and a variety of microtumors.

Acknowledgements

This work was supported by the Fondation RITC, the Fondation Toulouse Cancer Santé, the Agence Nationale de la Recherche (ANR-13-JS07-0003-01 CiTrON-Fluo) and the National Institutes of Health (Award No. 1RO1CA155061-1). B. K. W would like to acknowledge support from the National Science Foundation through the NSF Graduate Research Fellowship Program (NSF GRFP). M. J. B. acknowledges a fellowship from the Research Challenge Trust Fund of the University of Kentucky. F. B. thanks the Agence Nationale de la Recherche (ANR-13-NANO-0004) for financial support. We also thank Bruno Payer (CMEAM, Toulouse) for help with the TEM imaging, Karidia Konate (CRBM-CNRS, Montpellier) for help with the zeta potential measurements, Stéphanie Seyrac (LCC-CNRS, Toulouse) for help with the TGA measurements and Odile Mondésert (ITAV-CNRS, Toulouse) for help with the H2B GFP transfection. The GDR MIV 2588 group is acknowledged for discussions. Finally, N. M. P. and S. C. thank Bernard Ducommun (ITAV-CNRS, Toulouse) for scientific discussions.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra09954h

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