Exploring New Mitochondria-Targetable Theragnostic styrylBODIPYs †

: Two analogues of a previous mitochondria-targetable theragnostic styrylBODIPY have been designed, synthesized and submitted to preliminary studies in order to explore their capability to act as new theragnostic agents with absorption in the red region. The new dyes are obtained through a straightforward and low-cost synthetic protocol, and have styryl moieties bearing non-delocalized cations (trimethylammonium) attached to the BODIPY through a spacer, or directly linked. Preliminary results over of them in cell bioimaging showed internalization into HeLa cells and accumulation into mitochondria. Photodynamic therapy was also analyzed, demonstrating the viability of this red styrylBODIPY as mitochondria-targetable phototheragnostic agent.


Introduction
Boron dipyrromethenes (4-bora-3a,4a-diaza-s-indacene; abbreviated as BODIPY) constitute a family of organic dyes widely used in photonics due to their excellent physical and chemical properties, such as high fluorescence quantum yield, significant solubility in a wide range of organic solvents, and a great chemical versatility (known as BODIPYs Chemistry), which allows finely adjusting the photophysical, chemical and biological features of these dyes [1]. An interesting modification that can be obtained through this chemistry of BODIPYs is the extension of the π-conjugation of the chromophoric core, thereby causing a bathochromic shift of the absorption and emission wavelengths towards the red edge of the visible spectrum [2].
Red-to-NIR BODIPYs are interesting due to the advantages of the red-to-NIR region (specifically, the biological window region) for biological and medical applications [3]. Thus, dyes acting in this spectral region allow using red-to-NIR light, with a deeper penetration into the tissues, among other advantages [4].
A simple way to achieve the extension of the conjugation in BODIPYs is by Knoevenagel-like condensation of BODIPYs having methyl groups at the 3/5 BODIPY positions (acidic methyls) with aromatic aldehydes, to generate the corresponding 3-styrylBODIPYs or 3,5-distyrylBODIPYs [5]. By this method, the use of functionalized aromatic aldehydes can endow the dye with additional properties such as the specific recognition of biological systems (e.g., cell organelles) [6]. In this context, red-to-NIR styrylbased BODIPYs have been successfully developed as fluorescent bioprobes and ROS (reactive oxygen species) photosensitizers for photodynamic therapy (PDT) [7,8]. Interesting PDT photosensitizers can be also tuned for conducting photo-theragnosis, which consists in performing PDT and diagnosis (by luminescence-based bioimaging) by using a single fluorescent-enough photosensitizer [9][10][11][12].
Our research group has recently demonstrated that some highly fluorescent BODIPY dyes (poor ROS photosensitizers) are able to trigger an efficient PDT action when efficiently accumulated into "sensible-to-PDT" cell organelles, such a lipid droplets (e.g., I and II in Figure 1) [13,14], or mitochondria (III and IV in Figure 1) [15]. The case of the mitochondria-targetable dyes III and IV results especially interesting, due to both chromophoric π-extension and easy synthesis (styryl-based BODIPY dyes), on the basis of using triphenylphosphonium cations to promote the accumulation into mitochondria. In order to cast light on the possible extension of this Knoevenagel/cation approach for the development of new, highly-fluorescent photo-theragnostic agents based on selective accumulation into mitochondria, this communication describes preliminary results on the use of different cations (specifically, trimethylammonium) and spacers.

Synthetic Development
For our purposse, we have selected dyes 1 and 2, which are shown in Figure 2. Compound 1 is an analogue of previous IV, but involving trimethylammonium cations intead of triphenylphosphonium ones. On the other hand, compound 2 is an analogue of 1, but directly linking the key cation moiety to the chromophore, and involving meso-(p-tolyl) instead of meso-mesityl. BODIPY 1 was synthesized from meso-mesitylBODIPY 3 [16] according to the synthetic route shown in Scheme 1. Thus, Knoevenagel-like condensation of 3 with dimethylamino-based benzaldehyde 4 [17] under microwave irradiation generatates intermediate 5 (21% yield). Finally, standard methylation of 5 with methyl iodide leads to desired 1 (78% yield). Scheme 1. Synthesis of 1.

Photophysical Properties
The grafting of the styryl moieties at the BODIPY chromophoric positions 3 and 5 push the spectral bands of the dye deep to the red edge of the visible (around 630 and 620 nm, in ethanol for 1 and 2, respectively, Figure 3). Indeed, the long wavelength tail of the absorption profile falls within the biological window ( Figure 3). Further bathochromic shift is induced by the additional presense of the alcoxyl moiety at the styryls in the case of 1 (Table 1). These π-extended BODIPYs provide high molar absorption coefficients (around 70,000 M −1 cm −1 for 1, Table 1), guaranteeing an optimal harvesting of the incoming red light for biomaging or photoinduced therapy. The obtained probes were characterized photophysically in EtOH solution. Accordingly, both dyes display a strong red emission (placed at around 645 and 630 nm for 1 and 2, respectively, Figure 3), owing to their constrained geometry (reflected in low Stokes shift, lower than 300 cm −1 , Table 1), which minimizes undesirable non-radiative channels related with internal conversion. Specifically, dye 2 outstands by a high fluorescence efficiency (fluorescence quantum yield up to 86%, Table 1) and hence bright fluorescence images should be expected under the fluorescence microscope for this probe. Dye 1 shows a lower fluorescence efficiency (36%), although high enough for bioimaging purposses. Such different fluorescence performance could be attributed to the non-radiative deactivation channels in dye 1 related with photoinduced charge separation from the electron-rich alcoxyl-based rests to the dipyrrin, usually described as electron deficient.  Although both dyes bear cationic moieties, only the smaller one (dye 2) is soluble in water. It is noteworthy the high fluorescence signal of dye 2 in water (57%, Table 1), and hence a similar trend should be expected in physiological media, thus avoiding the typical problems of low solubility and aggregation of the BODIPYs in aqueous media. Therefore, both dyes absorb and emit light in a suitable region to render eficient and long lasting bioimaging. Particularly, probe 2 should behave as a better fluorescence biomarker, even in aqueous media.

Preliminary Biological Studies
Preliminary biological studies using HeLa cells and dye 1 were conducted in order to know about the possible photo-theragnostic activity of the new dyes.

Microscopy Studies
The conducted staining experiments, in combination with confocal microscopy, show that dye 1 is able to probe mitochondria when using an incubation time of 24 h, giving place to red or green fluorescence-based bioimages depending on the used excitation/recording microscope channels (Figure 4). On the one hand, the red bioimages display high specificity and brightness, whereas the green one is diffuse and weak. On the other hand, the specificity of the red labelling is lost when decreasing the incubation time and diffuse cytoplasmic staining can be seen in both the red and green bioimages, likely due to lack of time to complete the accumulation into the mitochondria ( Figure 4A1-D1). Interestingly, phase contrast optical microscopy does not show alteration of the cell morphology, which supports lack of citotoxicity under the bioimaging-experiment conditions.

Photodynamic Therapy Studies
A preliminary PDT experiment using 1 as PDT agent (5 μM; 3 h incubation; light dosis = 10.3 J/cm 2 ), based on the observation of key morphological changes upon the photodynamic treatment, shows that this dye is able of promoting apoptotic cell death upon ligh irradiation ( Figure 5).

Conclusions
The obtained results show that highly-accesible dye 1, based on ammonium cation instead of triphenylphosphonium one, could serve as a new platform for the rapid development of highly-fluorescent photo-theragnostic agents based on selective accumulation into mitochondria. The results also support the capability of mitochondria to serve as sensible target for performing efficient PDT (even by using highly fluorescent photosensitizers), and envisage a promising photo-theragnostic activity for water-soluble analogue 2. Futher experiments have been started to test this expected behavior.

Synthetic Procedures
General. Common solvents were dried and distilled by standard procedures. All starting materials and reagents were obtained commercially and used without further purifications. Elution flash chromatography's were conducted on silica gel (230 to 400 mesh ASTM or neutral alumina 70-290). Thin layer chromatography (TLC) was performed on silica gel plates (silica gel 60 F254, supported on aluminum). The NMR spectra were recorded at 20 °C, and the residual solvent peaks were used as internal standards. The NMR signals are given in ppm. The DEPT-135 NMR experiments were used for the assignation of the type of carbon nucleus (C, CH, CH2, and CH3). The FTIR spectra were recorded from neat samples using ATR technique and IR bands are given in cm −1 . High-resolution mass spectrometry (HRMS) was performed using ESI or MALDI-TOF.

Photophysical Properties
Diluted solutions of the BODIPY probes (around 5 × 10 −6 M) were prepared by adding the corresponding solvent (spectroscopic grade) to the residue from the adequate amount of a concentrated stock solution in ethanol, after vacuum evaporation of this solvent. UV-Vis absorption and steady-state fluorescence were recorded on a SPECORD S600 (Analytikjena) spectrophotometer and a spectrofluorimeter Fluoromax-4 (HORIBA Jobin Ybon), respectively, using 1 cm path length quartz cuvettes. Fluorescence quantum yields (φ) were calculated using commercial Nile Blue (φ = 0.27 in ethanol) as the reference.

Fluorescence Microscopy
Fluorescence microscopy images were acquired using an Olympus BX63 automated fluorescence microscope equipped with a CoolLED pE-300 light source (CoolLed Ltd., Andover, UK) and an Olympus DP74 digital camera (Olympus, Center Valley, PA, USA).

Cell Cultures
HeLa cells (ATCC, CCL-2) were cultured as monolayers in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) fetal calf serum 50 U/mL penicillin and 50 µ g/mL streptomycin. All products were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and sterilized using 0.22 µ m filters (Merck Milipore, Billerica, MA, USA). Cell culture was carried out in 5% CO2, plus 95% air atmosphere at 37 °C, and kept in a SteriCult 200 incubator (Hucoa-Erloss, Madrid, Spain). Subconfluent cell cultures seeded in 24-well plates (with or without coverslips, depending on the experiment) were used. All sterile plastics were purchased from Corning (New York, NY, USA).

Localization Experiments
The cells, seeded in 24-well plates with coverslips, were incubated for 30 min, and 24 h with the compound. After incubation, cells were washed in sterile PBS and the coverslip, still wet, was placed on a slide. This preparation was observed under the fluorescence microscope immediately.

Photodynamic Therapy Protocols
The cells were incubated for 3 h with the compounds and washed three times with sterile PBS (phosphate buffer saline). Subsequently, the cells were irradiated in complete culture medium with a Par 64 Short LED lamp (Showtec, Burgebrach Germany) with green light (λ = 520 ± 20 nm) with a light dose of 10.3 J/cm 2 . After irradiation, the cells were kept in the incubator for observation.