Supramolecular Association of Halochromic Switches and Halloysite Nanotubes in Fluorescent Nanoprobes for Tumor Detection

1 Supramolecular association of halochromic switches and halloysite nanotubes into fluorescent nanoprobes for intracellular detection Marina Massaro,a,1 Monica Notarbartolo,a,1 Françisco M. Raymo,b,* Giuseppe Cavallaro,c,d Giuseppe Lazzara,c,d Mercedes M. A. Mazza,b Cesar Viseras-Iborrae,f and Serena Rielaa,* aDipartimento di Scienze Biologiche, Chimiche e Farmaceutiche (STEBICEF), University of Palermo Viale delle Scienze, 90128 Palermo, Italy. dLaboratory for Molecular Photonics, Department of Chemistry, University of Miami,1301 Memorial Drive, Coral Gables, FL 33146-0431. cDipartimento di Fisica e Chimica “E. Segrè (DiFC), University of Palermo, Viale delle Scienze, 90128 Palermo, Italy. dConsorzio Interuniversitario Nazionale per la Scienza e Tecnologia dei Materiali, INSTM, I50121 Firenze, Italy. eDepartment of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Granada, Campus of Cartuja, 18071 s/n, Granada, Spain. fAndalusian Institute of Earth Sciences, CSIC-UGR, Avenida de las Palmeras 4, 18100, Armilla, Granada, Spain. 1Contributed equally 2 KEYWORDS. Halloysite nanotubes, halochromic switches, fluorescent probes, intracellular detection. ABSTRACT. Fluorescence imaging has become an indispensable tool in the biomedical laboratory to elucidate the fundamental dynamic and structural factors regulating cellular processes. The development of fluorescent nanoprobes represent a challenge to detect any cellular process under the microscope. Herein, a novel fluorescent nanomaterial was synthetized by exploiting the supramolecular interaction between a halochromic switch (1Cl) and halloysite nanotubes (HNTs). The obtained HNTs/1Cl nanomaterial was thoroughly studied by FT-IR and thermogravimetric analyses and the aqueous mobility was investigated by dynamic light scattering (DLS) and potential measurements. Furthermore, the morphology was imaged by transmission electron microscopy (TEM) and the interaction of the clay with the 1Cl was also studied by kinetic adsorption measurements. In addition, the spectroscopy properties of the resulting nanomaterial were studied in solution and in the solid state by UV-vis and fluorescence measurements. Finally, the ability of our nanomaterial to detect cancer cells was assessed with confocal laser-scanning microscopy measurements on normal (hTERT) and/or tumoral cell lines (MCF-7 and HL-60R). INTRODUCTION. The early detection of cancer is a decisive factor to fight this disease. Nanotechnology has been employed in the design of nanomaterials able to overcome biological barriers that also possess intrinsic properties suitable for cancer diagnosis. Fluorescence imaging has become an indispensable tool in the biomedical laboratory to elucidate the fundamental dynamic and structural factors regulating cellular processes.1 Indeed, the fast response and high sensitivity of fluorescence measurements,2 together with the spatial resolution 3 of optical microscopes,3 permit the visualization of subcellular structures in real time. A given sample of interest, however, must first be labeled with appropriate emissive probes for a fluorescence image to be captured. Excitation wavelengths causing minimal autofluorescence from and photodamage to the biological sample, small physical dimensions ensuring negligible structural perturbation to subcellular structures as well as sufficient aqueous solubility for administration to the extracellular matrix are generally the main characteristics expected from fluorescent probes for cellular imaging.4 Nonetheless, most organic chromophores are relatively hydrophobic and the latter requirement (i.e., aqueous compatibility) demands the covalent incorporation of hydrophilic groups with appropriate synthetic modifications. Alternatively, noncovalent encapsulation of hydrophobic fluorophores in supramolecular carriers can be exploited to solubilize the emissive probes in aqueous media, transport them across the plasma membrane of target cells and deliver them into selected intracellular compartments.5 Halloysite nanotubes (HNTs), a natural clay belonging to the kaolin group, have attracted attention for application in bioimaging.6 HNTs, an aluminosilicate with a chemical formula of Al2Si2O5(OH)4·nH2O, consist of rolled kaolinite sheets, where siloxane groups are localized at the external surface and aluminum hydroxide ones are present in the lumen. Generally, the inner and outer diameters of the tubes are in the ranges of 10–30 nm and 40–70 nm, respectively, while their length is in the range of 0.2–1.5 μm. Halloysite possesses different charged surfaces: positive in the inner lumen; negative in the external one, resulting in a significant influence on the aggregation and dispersion of HNTs in aqueous media. The modification of the inner and/or outer surfaces opens different strategies to improve HNTs properties expanding the application fields. 4 Halloysite biological safety was reported both by in vitro and in vivo studies.7 HNTs can penetrate the cellular membrane surrounding the cell nuclei.8 In addition, it was demonstrated that the modification of the tubes surfaces makes hybrid nanomaterials that penetrate the nucleus membrane, as well.9 Recently, it was reported the loading of fluorescent dyes into HNTs lumen or on the external surface and it was demonstrated that the presence of halloysite increases the dyes solubility in physiological conditions and improves its physico-chemical properties. Therefore nanomaterials based on HNTs could be useful for application in bio-imaging field.8b, 10 Herein, we report a study of the interaction between a halochromic switch and HNTs as probe for tumor detection. To reach this goal, a fluorescent coumarin chromophore combined with a switchable oxazine (compound 1Cl) was chosen as model.11 Upon acidification, the oxazine ring opens to generate 1OpH (Figure 1).12 This structural transformation brings the coumarin chromophore in electronic conjugation with the 3H-indolium cation to shift bathochromically its absorption and emission bands. As a result, intense fluorescence in the red region of the electromagnetic spectrum appears only after stimulation of 1Cl with acid inputs. On the basis of this behavior, we were able to acquire images of fixed cells with subdiffraction resolution13 as well as highlight acidic organelles of live cells.14 However, this relatively hydrophobic and water-insoluble compound (i.e., 1Cl) had to be encapsulated noncovalently within polymer nanoparticles13a or connected covalently to amphiphilic polymers14 or secondary antibodies13b to be delivered to intracellular targets. 5 Figure 1. Reversible interconversion of 1Cl and 1OpH. On the basis of these considerations, we envisaged the possibility of solubilizing 1Cl in aqueous media, under the supramolecular assistance of HNTs, with the ultimate objective of transporting this pH sensitive fluorophore into the intracellular environment. The obtained HNTs/1Cl nanomaterial was thoroughly studied by FT-IR and thermogravimetric investigations and the aqueous mobility was investigated by dynamic light scattering (DLS) and potential measurements. Furthermore, the morphology was imaged by transmission electron microscopy (TEM) and the interaction of the clay with the halochromic switch was also studied by kinetic adsorption measurements. In addition, the spectroscopy properties of the resulting nanomaterial were studied in solution and in the solid state by UV-vis and fluorescence measurements. Finally, the ability of our nanomaterial to detect cancer cells was assessed with confocal laserscanning microscopy measurements on normal (hTERT) and/or tumoral cell lines (MCF-7 and HL-60R). MATERIALS AND METHODS. Halloysite nanotubes used in this study were obtained from Merck and used as received. 1Cl was synthetized as reported elsewhere.14 6 Thermogravimetric analyses were performed on a Q5000 IR apparatus (TA Instruments) under a nitrogen flow of 25 cm3 min-1 for the sample and 10 cm3 min-1 for the balance. The weight of each sample was ca. 10 mg. Measurements were carried out by heating the sample from room temperature up to 900 °C at a rate of 10 °C min-1. FTIR spectra (KBr) were recorded with an Agilent Technologies Cary 630 FT-IR spectrometer. Specimens for these measurements were prepared by mixing 5 mg of the sample powder with 100 mg of KBr. UV-vis measurements were performed using a Beckmann DU 650 spectrometer. Steady-state fluorescence spectra were acquired using a JASCO FP-777W spectrofluorometer. Excitation and emission slits were 1.5 and 3 nm, respectively, with an emission interval ranging between 400 and 800 nm. Dynamic light scattering (DLS) measurements were carried out by means of a Zetasizer NANOZS (Malvern Instruments). The field-time autocorrelation functions were fitted by Laplace transformation, which provided an intensity-weighted apparent hydrodynamic radius (Rh). In detail, the fitting of the field-time autocorrelation functions provided the decay rate (Γ) of the diffusive mode. For the translational motion, the collective diffusion coefficient at a given concentration is Dt = Γ/q2 where q is the scattering vector given by 4πnλ−1 sin(θ/2), with n being the water refractive index, λ the wavelength (632.8 nm), and θ the scattering angle (173°). The apparent hydrodynamic radii were calculated by using the Stokes−Einstein equation as Rh = kbT/(6πη), kb being the Boltzmann constant, T the absolute temperature, and η the water viscosity. potential measurements were carried out by means of a Zetasizer NANO-ZS (Malvern Instruments) at 25.0 ± 0.1 °C. The concentration of the dispersions was 10-3 wt%. 7 Transmission electron microscopy (TEM) was performed by means of a FEI Titan G2 60–300 ultra-high-resolution transmission electron microscope (FEI, Lausanne, Switzerland) coupled with analytical electron microscopy (AEM) performed with a SUPERX silicon drift windowless energy dispersive X-ray spectroscopy (XEDS) detector. AEM spectra were saved in mode STEM (scanning transmission electron microscopy) with a HAADF (high angle annular dark field) detector. Adsorption kinetics The batch experiments were carried out for investigating the adsorption kinetics of 1 onto HNTs at room temperature in water. For the experiment, HNTs (1 ± 0.5 mg) were added into 2 mL of water and after, 0.5 mL of 1 solution in acetonitrile (1 × 10-4 M) were added. The amount of 1 adsorbed at time t (Qt, mol g-1) was calculated by the following equation: M VCC Qt t   )( 0 (Eq. 1) where C0 and Ct are initial and t time concentrations of 1 (M), respectively, M is the weight of HNTs (g) and V is the volume of 1 solution (L). Kinetic Release The release of 1 from the HNT was done as follows: 10 mg of the sample were dispersed in 0.5 mL of dissolution medium and transferred into a dialysis membrane (Medicell International Ltd MWCO 12-14000 with a diameter of 21.5 mm). Phosphate buffer (0.05 M, pH 7.4) was used as the release medium. Subsequently the membrane was put in a round bottom flask containing 5 mL of the release medium at 37 °C and stirred. At fixed time, 1 mL of the release medium has been withdrawn and analyzed by UV-vis measurements. To keep constant the volume of the 8 release medium, 1 mL of fresh solution has been used to replace the collected one. Total amounts of drug released (Ft) were calculated as follows:     1 0 t i iatmt CVCVF (Eq. 2) where Vm and Ct are the volume and the concentration of the drug at time t. Va is the volume of the sample withdrawn and Ci is the drug concentration at time i (i < t). Confocal images were acquired with an Olympus FluoView10i confocal laser scanning microscope (Olympus, Japan) equipped with humidity control and CO2 using a 10 × 0.3 NA objective. Aliquots of HNTs/1 dispersed in water were deposited on a cover glass (BRAND #1) using a disposable pipette. Measurement was acquired using laser excitation at 405 nm or 595 nm. Emitted fluorescence was acquired in photon-counting mode. Spectral detection has been performed using a bandwidth of 5 nm and a step size of 3 nm in the range 420–740 nm. The scan area was 256 × 256 pixels and the scan speed was 12 μs per pixel. RESULTS AND DISCUSSION The loading of 1Cl into halloysite was carried out by mixing an aqueous dispersion of halloysite (5 mL) with a concentrated 1Cl solution in ACN (10-2 M, 1 mL) (Scheme 1). Then, the obtained suspension was stirred and maintained under vacuum for 3 to 5 min, resulting in light fizzling, which indicated that air was released from the tubes. 9 Scheme 1. Schematic representation of the synthesis of HNTs/1Cl nanomaterial. Once the vacuum was removed, the solution entered the lumen and the loaded compound adsorbed within the tubes. This procedure was repeated 2 to 3 times to improve the loading efficiency. The dye loading of HNTs/1Cl was estimated by UV-vis spectroscopy. The amount of 1Cl loaded in the HNTs, expressed as the percent amount of dye in the final nanomaterial, was ca. 6 wt% with an entrapment efficiency of 95%. The HNTs/1Cl nanomaterial was characterized by FT-IR spectroscopy, thermogravimetric analysis and the colloidal properties were estimated by dynamic light scattering and potential measurements. Furthermore, the morphology of the nanomaterial was imaged by transmission electron microscopy (TEM) and High Angle Annular Dark Field Scanning TEM (HAADFSTEM). In Figure 2a, the FT-IR spectra of HNTs/1Cl nanomaterial and the pristine components HNTs and 1Cl are reported. The assignments for the bands of the pristine halloysite can be done on the basis of literature data. 15 The FT-IR spectrum of 1Cl shows the bands at 2966, 2927 and 2848 cm -1 corresponding to asymmetric and symmetric stretching of the methyl and methylene groups, the characteristic band 1714 cm -1 ascribed to the C=O group and the bands between 1620 and 900 cm -1 derive from stretching and bending of aromatic ring and C—O, C—N stretching 16 compartments of cells on a relatively short time scale (1 h), surrounding cell nuclei. A slight aggregation of nanomaterial indeed was observed in the cytoplasm of the cells, which indicated the heterogeneity of the distribution of nanomaterial in the cytoplasm, in accordance with previous studies.19 In particular, it is possible to observe a heterogeneous distribution of HNTs/1 in the cytoplasm, a change in the cellular organization with an increase in the number of cytoplasmic vacuoles and vesicles and a localization of the probe in the perinuclear region. Our results are in according to Liu et al.19 who show that the HNTs signals are partially co-localized with the Golgi and lysosomes apparatus in living cells suggesting that the HNTs have been transported via both organelles. Furthermore, the cytoskeletons, including microtubules and actin filaments, are also involved in the transport pathway of HNTs in tumor cells. In fact, in Figure 5A after 1, 6 and 24 h it is possible to observe the strong red fluorescence of the cytoskeletal structures, especially noticeable in hTERT cells and suggesting that microtubules also participate in cellular trafficking of this nanomaterial. Interestingly, the hTERT cells incubated with HNTs/1 can be imaged in two detection channels (Figure 6A) with resolved spectral regions (blue and red emission), indicating that the ringclosed (1Cl) and -open (1OpH) forms of 1 coexist in aqueous dispersion of the nanomaterial as previously highlighted by spectroscopic measurements. Moreover after 24 h of incubation, the adsorption of nanomaterials on the membranes and cell uptake did not result in membrane damage. This suggests that HNTs/1 can be useful as a visualization tool for cancer diagnostics, as already reported elsewhere.10 Compared with normal cells, the images of MCF-7 and HL-60R incubated with HNTs/1 are different (Figure 6B-C). In these cases, indeed, a decrease of blue fluorescence and increase of red one was observed after 6 h of treatment. Furthermore, the observed blue fluorescence does 17 not always colocalize with the red one, indicating a different distribution inside the same cell which depends on the type of cells used (in adhesion or floating, MCF-7 and HL-60R, respectively; tumoral or normal). Figure 6. Merged confocal images of hTERT, MCF-7 and HL60-R cell lines incubated with HNTs/1 (10 M of 1 corresponding to 90 g mL -1 ) at different times. Images were recorded under excitation at 405 nm and 595 nm. Scale bar 10 m. 18 CONCLUSIONS Herein we report the synthesis and characterization of a novel nanomaterial based on the supramolecular assembly of halloysite nanotubes (HNTs) and a switchable halochromic dye for intracellular detection. The dye chosen for this purpose was a fluorescent coumarin chromophore combined with a switchable oxazine (compound 1Cl) which suffers of insolubility in physiological fluids that limits its application in biological field. After loading of 1Cl into HNTs, the obtained nanomaterial was thoroughly characterized by several techniques. Morphological investigation by HAADF/STEM measurements coupled with EDX analysis showed that the organic molecules interact with both HNTs surface. The interaction was also investigated by means of some kinetic adsorption experiments. Spectroscopic studies by UV-vis and fluorescence measurements highlighted that the intrinsic acidic nature of HNTs allows the opening of the oxazine ring in 1Cl to give the protonated form 1OpH and that the two species co-existed in the HNTs based nanomaterial. Since kinetic release experiments showed that no leaching of the dye from HNTs occurs for at least 24 h, the system is promising as biological probe for tumor detection. Confocal microscopy experiments performed on normal (hTERT) cell lines highlighted the coexistence of the two forms of 1 (the closed 1Cl and open 1OpH) as proved by the imaging in two detection channels with resolved spectral regions (blue and red emission). Conversely, the incubation of tumoral cell lines, namely MCF-7 and HL-60R chosen as in adhesion or floating, respectively, model cells with HNTs/1 showed a decrease of blue fluorescence and increase of red one after 6 h of treatment indicating an opening of the 1Cl. In conclusion, the nanomaterial reported could be a promising probe for the detection of tumor environment and therefore for application in the diagnostic field. 19 ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. UV-vis spectra of 1 with or without HNTs, Kinetic adsorption of 1Cl on HNTs aqueous dispersion, UV-vis, and fluorescence spectra of 1, UV-vis spectra of MB with and without HNTs (word) AUTHOR INFORMATION Corresponding Author *[email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT The work was carried out in the frame of the PON “AIM: Attrazione e Mobilità Internazionale” No. 1808223-1 project. Confocal measurements were performed at ATeN Center – University of Palermo. REFERENCES 1. Pawley, J. B., Handbook of biological confocal microscopy: Third edition. 2006; p 1-985. 2. Lakowicz, J. R., Principles of fluorescence spectroscopy. 2006; p 1-954. 3. Murphy, D. B.; Davidson, M. W., Fundamentals of Light Microscopy and Electronic Imaging: Second Edition. 2012. 4. (a) Kobayashi, H.; Ogawa, M.; Alford, R.; Choyke, P. 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