Synthesis and Functionalization of Monodisperse Near-ultraviolet and Visible Excitable Multifunctional Eu3+, Bi3+:REVO4 Nanophosphors for Bioimaging and Biosensing Applications

Nanoscale PAPER Cite this: DOI: 10.1039/c6nr03369e Received 25th April 2016, Accepted 9th May 2016 DOI: 10.1039/c6nr03369e www.rsc.org/nanoscale Synthesis and functionalization of monodisperse near-ultraviolet and visible excitable multifunctional Eu 3+ ,Bi 3+ :REVO 4 nanophosphors for bioimaging and biosensing applications† Alberto Escudero,* a,b Carolina Carrillo-Carrión, a,c Mikhail V. Zyuzin, a Sumaira Ashraf, a Raimo Hartmann, a Nuria O. Núñez, b Manuel Ocaña b and Wolfgang J. Parak a Near-ultraviolet and visible excitable Eu- and Bi-doped NPs based on rare earth vanadates (REVO 4 ,RE= Y, Gd) have been synthesized by a facile route from appropriate RE precursors, europium and bismuth nitrate, and sodium orthovanadate, by homogeneous precipitation in an ethylene glycol/water mixture at 120 °C. The NPs can be functionalized either by a one-pot synthesis with polyacrylic acid (PAA) or by a Layer-by-Layer approach with poly(allylamine hydrochloride) (PAH) and PAA. In the first case, the particle size can also be tuned by adjusting the amount of PAA. The Eu- Bi-doped REVO 4 based nanophosphors show the typical red luminescence of Eu(III), which can be excited through an energy transfer process from the vanadate anions, resulting in a much higher luminescence intensity in comparison to the direct excitation of the europium cations. The incorporation of Bi into the REVO 4 structure shifts the original absorption band of the vanadate anions towards longer wavelengths, giving rise to nanophosphors with an excitation maximum at 342 nm, which can also be excited in the visible range. The suitability of such nanophosphors for bioimaging and biosensing applications, as well as their colloidal stability in different buffer media of biological interest, their cytotoxicity, their degradability at low pH, and their uptake by HeLa cells have been evaluated. Their suitability for bioimaging and biosensing applications is also demonstrated. 1. Introduction Nanoparticles (NPs) are currently attracting wide research interest due to their potential use in medical 1 or energyconversion/storage applications. In the case of biomedically motivated research, luminescent NPs are interesting because of their optical properties, and can be used as optical biolabels for imaging of tissues or intracellular structures, as sensors to detect biological molecules, and as tracking devices. 2–7 For biomedical applications usually uniform NPs with controlled size, shape, composition, surface chemistry and other physicochemical properties are desired, 8 given the strong effect that the morphology, size, and structure of the particles may have on many physicochemical properties of these materials. 9–12 Among the different luminescent nanomaterials available in the literature, 13 including quantum dots 14 and nanostructures functionalized with organic dyes, 6 Rare Earth (RE) based nanophosphors exhibit important advantages due to their lower toxicity, photostability, high thermal and chemical stabilities, high luminescence quantum yields, and sharp emission bands. 15–17 These nanomaterials usually consist of an inorganic host matrix doped with luminescent lanthanide (Ln) cations. Red-emitting Eu 3+ is frequently used as a luminescent cation, as the autofluorescence of tissues is minimized in this wavelength range. 18 The main disadvantage of Lndoped nanophosphors is their relatively low global intensity luminescence, caused by the low absorptions of the parity forbidden Ln 3+ 4f–4f transitions, and constituting a serious limitation for their use for imaging applications. Different energy transfers schemes from the host materials to the Ln 3+ cations are employed to enhance the global luminescence of lanthanide-doped nanophosphors, including the use of vanadate or †Electronic supplementary information (ESI) available: Additional details of experiments and results (NP characterization, NPs’uptake and imaging). See DOI: 10.1039/c6nr03369e a AG Biophotonik, Fachbereich Physik, Philipps-Universität Marburg, Renthof 7. D-35037, Marburg, Germany b Instituto de Ciencia de Materiales de Sevilla, CSIC –Universidad de Sevilla. C. Américo Vespucio 49. E-41092, Seville, Spain. E-mail: [email protected] c CIC biomaGUNE. Paseo Miramón 182. E-20009, San Sebastian, Spain This journal is © The Royal Society of Chemistry 2016 Nanoscale Open Access Article. Published on 13 May 2016. Downloaded on 15/06/2016 09:36:03. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal oxyfluoride matrices. 19–23 However, ultraviolet excitation radiations with wavelengths below 300 nm are normally required, resulting in limitations for the use of such materials for bioimaging. It has been reported that the Eu 3+ :REVO 4 excitation peak can be shifted toward longer wavelengths upon doping with Bi 3+ , resulting in phosphors excitable by near-ultraviolet light. 24–29 Eu 3+ :Bi 3+ doped YVO 4 micro- and NPs of different morphologies (sphere-, granular-, and rod-like) have been synthesized by various wet-chemical processes including coprecipitation, 30 and hydrothermal 31–33 methods, and thin films have been produced by sol–gel processes. 34 Micrometric Eu 3+ :Bi 3+ :GdVO 4 particles have been produced by a hydrolysed colloid reaction (HCR) technique 35 and by a combustion process. 36 Gd bearing NPs are also of special interest due to their use as a relaxation agent in magnetic resonance imaging (MRI), owing to the magnetic moment and nanosecond time scale electronic relaxation time of Gd 3+ . 37 A functionalization process is normally required for the biomedical use of NPs. This process not only increases the colloidal stability of the NPs by introducing electrostatic and/or steric repulsions, 38 but also provides anchors for adding functional ligands of biomedical interest such as antibodies, peptides, proteins, and some anticancer drugs. 39 Functionalization of Eu 3+ :Bi 3+ :YVO 4 nanophosphors with additives such as sodium citrate 40 and branched polyethylenimine (BPEI) 32,33 have been reported. Alternatively, NPs can also be coated with polymers. 41 Poly- (acrylic acid) (PAA) is a frequently used polymeric ligand for the biofunctionalization of NPs, because of its negligible toxicity, 42 its hydrophilic character, 43 and its high affinity for cellular membranes. 44 Usually, the PAA surface modification of the NPs is carried out through the adsorption or ionic interaction process. 41 Another common functionalization strategy consists of the encapsulation of the NPs by multiple layers of polymers, leading to hybrid core/(polymer-)shell NPs. 45 The Layer-by-Layer (LbL) approach, which is based on the electrostatic deposition of layers of polyelectrolytes with alternating charge on the surface of the particles, 46 has been widely used to functionalize different kinds of nanostructured materials, such as gold NPs 47 and, especially, for the synthesis of polyelectrolyte capsules. 48 The application for RE-based NPs is however not common so far. 41,49 We report here a facile and fast synthesis method of Eu 3+ : Bi 3+ :REVO 4 (RE = Y and Gd) nanophosphors, based on a homogeneous precipitation process from solutions of appropriate RE and orthovanadate precursors in ethylene glycol (EG)/water mixtures at 120 °C. Both kinds of nanophosphors are functionalized with poly(acrylic acid) (PAA) by a one-pot synthesis strategy (i.e. PAA is added during the synthesis process), and also by a Layer-by-Layer approach with poly(allylamine hydrochloride) (PAH) and PAA. The effect of the level of Bi 3+ doping on the luminescence properties of both kinds of nanophosphors has also been studied, as well as their colloidal stability in different physiological buffers, their degradability at low pH, and their cell viability. Finally, we show how the europium and bismuth doped rare earth orthovanadate NPs can be used to image human cervical carcinoma (HeLa) cells by using nearultraviolet and visible radiation as excitation sources, and as pH sensors, after their conjugation with a pH-sensitive dye such as Nile Blue. The influence of the morphology and chemical composition of the NPs on the cellular uptake is also shown. 2. Results and discussion 2.1 Synthesis, PAA functionalization and physicochemical characterization of the NPs A homogeneous process with constant reaction kinetics is normally required to obtain uniform NPs by precipitation. This can be achieved by a slow and controlled release of the precipitating anions or cations within the solution. 50 In our case, a homogeneous precipitation is produced by using Y(acac) 3 as the yttrium precursor for the synthesis of YVO 4 -based particles. This compound decomposes on heating, and gives rise to a slow release of the Y cations. 51 It has also been described that the presence of additives during the synthesis process, such as PAA, can affect the final morphology of the particles. 52 Moreover, a specific reaction kinetics is also demanded, which is established after an analysis of the effects of the experimental reaction parameters on the morphological features of the precipitated particles. 50 The synthesis protocol herein reported for the synthesis of europium and bismuth doped YVO 4 produces NPs with similar morphological characteristics, irrespective of the Bi content. A representative transmission electron microscopy (TEM) micrograph of the Bi20YPAA2 sample (see Table 1 for sample information) is shown in Fig. 1A. As it can be observed, NPs with a quasi-spherical morphology and a mean size of 93 nm ± 7 nm were obtained. The NPs are single crystals, as shown by the SAED pattern obtained from a single particle, which is compatible with the tetragonal symmetry of YVO 4 (space group I4 1 /amd) and could be indexed along the [001] zone axis (inset in Fig. 1A). The high-angle annular darkfield (HAADF) image of the sample Bi20YPAA2 indicates a homogeneous distribution of bismuth in the NPs (Fig. 1D). The mean hydrodynamic diameter of such nanoparticles obtained by dynamic light scattering (DLS) was 108 ± 10 nm, which is expected for well-dispersed particles free of aggregation. The analogous sample synthesized in the absence of PAA (sample Bi20Y) consisted of NPs with slightly sharper edges and a mean size of (107 ± 14) nm (Fig. S1A†). Also in this case, the mean hydrodynamic diameter of the Bi20Y NPs obtained by DLS (138 ± 7 nm) indicated that they were free of aggregation. It should be noted that the presence of bismuth in the europiumdoped YVO 4 NPs had almost no effect on the particle morphology, since similar NPs with a mean size of 88 ± 14 nm were observed in the absence of Bi (sample Bi0YPAA2, Fig. S1B†). By increasing the concentration of PAA from 2 to 8 mg mL −1 (sample Bi20YPAA8), a significant decrease in the mean particle size to 51 ± 5 nm was observed (Table 2) without alteration of the morphology (Fig. 1B). This difference in the particle size produced Paper Nanoscale Nanoscale This journal is © The Royal Society of Chemistry 2016 Open Access Article. Published on 13 May 2016. Downloaded on 15/06/2016 09:36:03. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online on the addition of PAA to the solutions containing the precursors may be attributed to the adsorption of these molecules on the NP surface, which could block the growth of the NPs. Similar results were observed for the Eu- Bi-doped GdVO 4 samples. A representative TEM micrograph of the Bi20GdPAA sample is shown in Fig. 1C. As it can be seen, non-aggregated NPs with a smaller size and slightly sharper edges when compared with those based on YVO 4 (TEM size 40 ± 8 nm, DLS 41 ± 2 nm) were produced after the synthesis in the presence of PAA. Such NPs were also single crystals, as inferred from the SAED pattern of a single particle, which is compatible with tetragonal GdVO 4 (space group I4 1 /amd), zone axis [001], and from the high resolution TEM images (insets of Fig. 1C and F). Analogous to the yttrium vanadate samples, a homogeneous distribution of Bi in the NPs was indicated by the HAADF-TEM images (Fig. 1E). Much more polydisperse and bigger NPs with anotsowelldefinedshape(TEMsize51±15nm,DLS58±4 nm, Fig. S1C†) were obtained in the absence of PAA (sample Bi0Gd). Similar to the YVO 4 -based NPs, the presence of Bi did not affect the particle morphology, since NPs with a mean size of 39 ± 7 nm were obtained in the absence of Bi (sample Bi0GdPAA, Fig. S1D†). A summary of the data corresponding to all the synthesized samples is shown in Table S2.† The presence of PAA on the surface of both Bi20YPAA2 and Bi20GdPAA NPs was confirmed by Fourier transform infrared spectroscopy (FTIR) analysis. The FTIR spectra of the Bi20Y, Bi20Gd, Bi20YPAA2, and Bi20GdPAA samples are shown in Fig. 2. In all the samples, the characteristic bands of vanadate anions (<1000 cm −1 ) 53 and adsorbed water (broad bands at Fig. 1 TEM micrographs of the Bi20YPAA2 (A, scale bar 100 nm), Bi20YPAA8 (B, scale bar 100 nm), and Bi20GdPAA (C, scale bar 200 nm) samples. HAADF images of the Bi20YPAA2 (D, scale bar 50 nm), and Bi20GdPAA (E, scale bar 50 nm) samples. (F, scale bar 5 nm) HRTEM image of a particle of the Bi20GdPAA2 sample. Fig. 2 Fourier transform infrared spectroscopy (FTIR) spectra of (A) the Bi20Y (blue) and Bi20YPAA2 (red) samples; and (B) of the Bi20Gd (blue) and Bi20GdPAA (red) samples. Nanoscale Paper This journal is © The Royal Society of Chemistry 2016 Nanoscale Open Access Article. Published on 13 May 2016. Downloaded on 15/06/2016 09:36:03. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online 3400 and 1600 cm −1 ) can be observed, as well as some much weaker features in the 1500–1300 cm −1 region, which can be attributed to the presence of carbonate anions from the adsorption of atmospheric CO 2 . 54 Samples Bi20YPAA2 and Bi20GdPAA show extra signals around 1458 and 1550 cm −1 (marked with asterisks in Fig. 2), which can be ascribed to the symmetric and antisymmetric stretching frequencies of the carboxylate ion (COO − ), respectively. 52 Taking into account that the carbonyl stretching (CvO) vibration appears at 1717 cm −1 for pure PAA, the shift towards lower frequencies indicates that most of the carboxylate functional groups of the polymer are fully ionized 55 and, therefore, that the PAA molecules are bonded to the NP surface though the carboxylate groups. Further evidence of the PAA functionalization of the particles was obtained by comparing the ζ-potential of aqueous suspensions of the non-functionalized and PAA-functionalized particles. Bi20Y particles showed a ζ-potential of −19 mV in water at pH 7, whereas the value of this magnitude for the NPs synthesized in the presence of PAA was −42 mV at the same pH. Analogously, the ζ-potential of the Bi20GdPAA NPs was −41 mV, to be compared to the one of −25 mV as observed for the non-functionalized Bi20Gd particles. Thus, NPs synthesized in the presence of PAA are already functionalized with carbonyl groups. An estimation of the amount of PAA on the surface of the PAA-functionalized NPs was obtained from thermogravimetry (TG) analysis (Fig. S2†). The weight loss observed at temperatures below 250 °C can be associated with the elimination of absorbed water. At higher temperatures (up to 600 °C), the loss most likely is connected with the decomposition of the adsorbed PAA molecules, which was higher for the sample prepared with more PAA (ca. 2% for sample Bi20YPAA2, ca. 15% for sample Bi20YPAA8 –including more adsorbed water-, and ca. 4% for sample Bi20GdPAA). The successful incorporation of europium and bismuth into the NPs was evidenced from the energy dispersive X-ray spectra (EDX) obtained for single particles, which displayed europium and bismuth peaks, in addition to those of yttrium, vanadium, gadolinium and oxygen (Fig. S3†). Moreover, no extra particles with different morphologies or with a higher Bi or Eu content were observed. Induced coupled plasma atomic emission spectroscopy (ICP-AEP) measurements indicated that the experimental molar ratios corresponding to the doped particles were in good agreement with the nominal values (Table S3†). The X-ray diffraction (XRD) diagrams obtained for the Bi0YPAA2 and Bi20YPAA2 samples (Fig. 3A) consisted of peaks that are very similar to the standard pattern of tetragonal YVO 4 (PDF 01-070-1281). Similar results were observed for the Bi0GdPAA and Bi20GdPAA samples, whose XRD diagrams (Fig. 3B) were also very similar to the standard pattern of tetragonal GdVO 4 (PDF 01-086-0996). Based on these data, the possible formation of slightly non-stoichiometric phases of rare earth orthovanadates can however not be fully discarded. 56 It should also be pointed that for similar orthovanadate systems, an excess of [Na 3 VO 4 ] in the starting solutions was used to adjust the precipitation kinetic, yielding NPs with smaller size. 19,21,23 The slight differences in peak positions and intensities in both Y and Gd bearing samples when increasing the Bi content (see peaks under the vertical guide lines) indicate changes in the unit cell dimensions and composition. This was confirmed by the measurements of the unit cell parameters of all the doped samples synthesized in the presence of PAA, which showed in both cases a linear increase of the unit cell volume as the Bi doping level increased (Table 2, Fig. S4†). Such results are in agreement with the higher ionic radius of the Bi 3+ cations (1.17 Å) when compared to those of Y 3+ (1.019 Å) and Gd 3+ (1.053 Å), all of them in eightfold coordination. 57 The average crystal size (Table 2) estimated by using the Scherrer formula from several XRD peaks (101, 200, 112, 220, and 321) for all the particles was very similar to the size observed by TEM and DLS, also suggesting that the particles were single crystals. 2.2 Layer-by-Layer functionalization The LbL functionalization of NPs usually requires an optimization of the experimental parameters in order to avoid particle agglomeration. Several variables are known to play an important role in this process, including the polyelectrolyte stoichio- Fig. 3 XRD diagrams of (A) the Bi0YPAA2 and Bi20YPAA2 samples; and (B) of the Bi0GdPAA and Bi20GdPAA samples. Vertical guides are only intended as a guide to the eye. Paper Nanoscale Nanoscale This journal is © The Royal Society of Chemistry 2016 Open Access Article. Published on 13 May 2016. Downloaded on 15/06/2016 09:36:03. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online metric excess, the NP concentration, the size and degree of polymerization of the polyelectrolyte chains, and the salt concentration. 58 In general terms, an excess of polyelectrolyte, low NP concentrations, and short polyelectrolyte chains help obtain NPs free of aggregation. Regarding the salt concentration, low concentrations prevent agglomeration, but high concentrations normally increase the polyelectrolyte amount that is deposited onto the NP surface. 59 Functionalized particles free of aggregation (as concluded from the fact that hydrodynamic diameters as determined by DLS were only slightly bigger than inorganic core diameters as determined by TEM) were obtained for both kinds of bare NPs (samples Bi20Y and Bi20Gd) by using 10 mg per mL polyelectrolyte solutions in 0.05 M NaCl aqueous solutions at pH 6.5, as described in the Experimental section. The success of functionalization and the absence of a significant particle agglomeration were confirmed by the surface charge reversal observed after the deposition of every polyelectrolyte layer, and by the evolution of the hydrodynamic diameter of the particles, respectively (Fig. 4). The selection of PAH and PAA as polymers for the Layer-by-Layer functionalization of the NPs also provides reactive –COOH and –NH 2 groups, and increases the possibility of further conjugation with different molecules of biomedical interest. 2.3 Colloidal stability The colloidal stability of both, PAA- and LbL PAH/PAA/PAH functionalized NPs, was analyzed by studying the mean hydrodynamic diameter (d h ) obtained by DLS in water and in different buffer media of biological interest, containing salt and proteins. 60 The evolution of the hydrodynamic diameter with increasing time was also studied. The hydrodynamic diameters measured for freshly prepared suspensions of PAA-functionalized NPs in different buffers are shown in Fig. 5A and B, and in Table S4.†For both PAA-func- tionalized NPs similar hydrodynamic diameters were observed in water at pH 7 and in MES buffer at pH 6.5. Such values are very close to the size determined by TEM for both samples, indicating the absence of significant aggregation and, therefore, colloidal stability in both media. Bi20YPAA2 NPs also showed colloidal stability in PBS buffer at pH 7.4, whereas some aggregation was observed for the Bi20GdPAA NPs in this same buffer. A strong level of aggregation was observed for both PAA-functionalized NPs in cell medium, which was minimized in serum-supplemented cell medium. Proteins in solution are well-known to attach to the surface of the NPs, forming the so-called protein corona, 61 and provide the NPs with additional colloidal stability. No changes were observed in the mean hydrodynamic diameter of the YVO 4 -based PAA- functionalized NPs (sample Bi20YPAA2) with increasing aging time in water at pH 7, MES buffer at pH 6.5, PBS at pH 7.4, and serum-supplemented cell medium (Fig. S5A–D†), and similar results were observed for the Bi20GdPAA sample with increasing aging time in water at pH 7, in MES at pH 6.5, and serum-containing cell medium, with only some aggregation when aging in PBS at pH 7.4 (Fig. S5F–J†), manifesting that no particle aggregation took place upon aging in these buffer media at the studied pHs. Data corresponding to the PAH/PAA/PAH LbL functionalized YVO 4 NPs are shown in Fig. 5C and Table S3.†No significant aggregation was observed in MES buffer at pH 6.5. It should be mentioned that at least two polyelectrolyte layers were required to achieve colloidal stability in MES buffer at pH 6.5 (Fig. S6†). A decrease of the colloidal stability was observed for the LbL functionalized particles in both PBS at pH 7.4 and Fig. 4 ζ-Potential of the Bi20Y (A) and Bi20Gd (B) nanoparticles functionalized with PAH and PAA by the LbL technique after the deposition of different polyelectrolyte layers, measured in water at pH 7. (C) Evolution of the hydrodynamic diameter of the Bi20Y (blue) and Bi20Gd (red) functionalized nanoparticles with the number of deposited polyelectrolyte layers. Nanoscale Paper This journal is © The Royal Society of Chemistry 2016 Nanoscale Open Access Article. Published on 13 May 2016. Downloaded on 15/06/2016 09:36:03. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online in cell medium. Similar to the observation for the PAA-functio- nalized NPs, a considerable reduction in the agglomeration level was observed for the LbL functionalized particles in serum-supplemented cell medium, which was however not so strong as in the previous example. At this point, it worth pointing out that a similar level of aggregation (d h = 240 nm, Fig. S7A†) was observed for the non-functionalized bare particles (sample Bi20Y) in serum-supplemented cell medium, which indicates the strong effect of the protein absorption on the final colloidal stability of the NPs. Slightly worse colloidal stability was observed for the LbL functionalized GdVO 4 -based NPs (Fig. 5D, Table S4†). Very slight agglomeration was also observed for the bare Bi20Gd NPs in serum-supplemented cell medium (Fig. S7B†). The level of aggregation of both kinds of LbL functionalized NPs increased with aging in MES, PBS, and cell medium. No changes were observed with aging time in water at pH 7 and in serum-supplemented cell medium (Fig. S5†). These results indicate that the colloidal stability of the one pot PAA synthesized and functionalized NPs is higher than the one shown by the NPs functionalized in a second step by the LbL approach. The incorporation of either functionalizing agents or adsorbed organic molecules onto the NP surface during the synthesis process seems to play a role in the final colloidal stability of the particles, at least when dealing with polymeric functionalizing agents. In particular, incorporation of multiple particles into one polymer shell may lead to aggregation. However, the LbL strategy offers a useful functionalization alternative for systems in which no functionalizing agents attach to the NP surface during the synthesis process. The colloidal stability of the NPs functionalized by the LbL strategy increases with the number of deposited layers. 2.4 Optical properties Excitation spectra of both, 8% Eu- 20% Bi-doped YVO 4 and GdVO 4 PAA functionalized NPs (samples Bi20YPAA2 and Bi20GdPAA, Fig. 6A and B, respectively) were recorded by monitoring at a wavelength of 622 nm, which corresponds to the main emission line of Eu 3+ cations in rare earth vanadate matrices. Both spectra show a broad band with maxima at 342 nm, associated with the indirect excitation of the Eu 3+ cations. For both, Y- and Gd-bearing nanophosphors (i.e. Bifree samples), a broad excitation band with a maximum centered at around 280 nm can be observed (Fig. S8†). 19,21 This band has been ascribed to the optical excitation of the vanadate anions (originated from the O 2− →V 5+ charge transfer within the vanadate groups), followed by an energy transfer to the Eu 3+ cations. 62 The incorporation of Bi into the REVO 4 Fig. 5 Hydrodynamic diameter of the Bi20YPAA2 (A), Bi20GdPAA (B), Bi20Y@PAH@PAA@PAH LbL-functionalized (C), and Bi20Gd@PAH@PAA@PAH LbL-functionalized (D) nanoparticles in water at pH 7 (black lines), MES 50 mM at pH 6.5 (red lines), PBS at pH 7.4 (blue lines), serum-supplemented cell medium (green lines), and cell medium (purple lines). Paper Nanoscale Nanoscale This journal is © The Royal Society of Chemistry 2016 Open Access Article. Published on 13 May 2016. Downloaded on 15/06/2016 09:36:03. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online lattices produces changes in both the shape and the position of the maximum of the broad absorption band. Increasing the Bi content produces wider absorption bands with the maxima shifting toward longer wavelengths. (Fig. S8†). The observed changes can be explained by the appearance of an extra charge transfer band from the 6s level of Bi 3+ to the 3d levels of V 5+ , which is centered around 340 nm. 24 Fig. 6C shows the emission spectra of the suspensions of the Bi20YPAA2 (black line), Bi20YPAA8 (red line), and Bi20GdPAA (blue line) samples, recorded under a UV excitation of 342 nm. The spectra display the typical emissions corresponding to the 5 D 0 → 7 F J (J= 1, 2, 3, and 4) electronic transitions expected for the Eu 3+ cation. 62 The most intense emission ( 5 D 0 → 7 F 2 transitions) is split into two bands at 618 and 622 nm owing to the crystal field. 62 It can also be observed that the relative intensity of the 5 D 0 → 7 F 2 emission band is in all cases higher than that associated with the 5 D 0 → 7 F 1 transition, which is expected for Eu 3+ cations located in crystallographic sites without an inversion center. This is compatible with Eu 3+ cations replacing Y 3+ or Gd 3+ on the normal yttrium sites of the tetragonal REVO 4 structure, in which the RE sites show a local symmetry of D 2d . 62 The different emission intensities observed for the different samples should be connected with their different luminescence quantum yields, since the spectra were recorded from NP suspensions owing to the same NP concentration. At least qualitatively, the GdVO 4 host material seems to show a higher luminescence quantum yield than YVO 4 . The differences observed for the YVO 4 -based NPs of different sizes show that bigger particles are more efficient. This should be connected with their lower surface area per particle volume. As the size of the NPs decreases, there is a significant increase of the surface area per particle volume, as well as an increase in the number of surface defects and higher number of capping ligand molecules, causing a decrease in the luminescence efficiency. 63,64 It should also be mentioned that it has been reported that Eu, Bi-doped YVO 4 might exhibit photobleaching under continuous UV irradiation. 65,66 In our case, Bi20YPAA2 NPs were continuously irradiated with UV light (Fig. S9†). After 10 minutes of irradiation, around 80% of the initial intensity was still observed, being reduced to about 70% after 25 minutes of irradiation. 2.5 Cellular uptake and imaging Human cervical carcinoma cell line (HeLa) cells were used as a system to show the suitability of both Eu- Bi-doped YVO 4 and GdVO 4 NPs for bioimaging. Two different microscopes were used to image the NPs. On one hand, the broad absorption band of the NPs with a maximum at around 342 nm permits their excitation with near-ultraviolet light in a fluorescence microscope. On the other hand, the shift of the absorption band of the Bi-doped vanadate matrices toward longer wavelengths makes also possible their excitation with a visible radiation by using a laser as the excitation source in a confocal laser scanning microscope (cLSM). The study of the NP internalization by the cells was performed by observing the co-local- ization of the fluorescence of the NPs with those produced by different endocytic fluorescent markers that specifically attach to the cell membranes and organelles. 67 A strong red luminescence in the interior of the cells could be observed after irradiation at (340 ± 24) nm after 24 h of incubation (illustrated in Fig. 7 for the Bi20YPAA2 sample). This fluorescence was clearly co-localized with the yellow one produced by Fig. 6 Excitation spectra (emission recorded at λ em = 622 nm) of the Bi20YPAA2 (A) and Bi20GdPAA (B) samples. (C) Emission spectra of the Bi20GdPAA (blue line), Bi20YPAA2 (black line), and Bi20YPAA8 (red line) samples (excitation at λ exc = 342 nm). The photograph shows the red luminescence of water suspensions (0.05 mg mL −1 ) of the Bi20YPAA8 (left), Bi20YPAA2 (center), and Bi20GdPAA (right) samples when irradiated with a UV lamp. Nanoscale Paper This journal is © The Royal Society of Chemistry 2016 Nanoscale Open Access Article. Published on 13 May 2016. Downloaded on 15/06/2016 09:36:03. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online LAMP1, a marker for the membrane of lysosomes, indicating that the NPs are eventually located in the cell’s lysosomes after being internalized. Such red fluorescence was not observed under UV light excitation in the control experiments, in which HeLa cells were incubated without NPs (Fig. S10†). PAA-func- tionalized Eu- Bi-doped GdVO 4 NPs, as well as both Eu- and Bi-doped YVO 4 and GdVO 4 NPs functionalized by LbL could also be observed in the cell interior (Fig. S11–S14†). As it was previously mentioned, it is also possible to image both, YVO 4 - and GdVO 4 -based NPs with a confocal laser scanning microscope (cLSM) using visible light as the excitation source (wavelength of 405 nm, laser power 20%). The red luminescence of the NPs can clearly be observed to be associated with HeLa cells after 24 hours of incubation (Fig. 8, illustrated for sample Bi20YPAA2). Images of the control sample and of HeLa cells incubated with samples Bi20YPAA8 and Bi20GdPAA are shown in Fig. S15.† 2.6 Biosensing applications The presence of free carboxyl groups on the NP surface not only provides colloidal stability but also facilitates the conjugation of the NPs with various biomolecules, which paves way for further bio-applications. In this work, Nile Blue (NB) was chosen as the fluorescent pH indicator, since it has an amino group that is capable of forming a covalent bond with the carboxyl groups of the PAA layer onto the NP surface. Samples Bi20YPAA2 and Bi20GdPAA were chosen for conjugation with Nile Blue. Changes of the fluorescence response of the NB-PAA-NPs towards pH were evaluated over a wide pH range (pH = 3–11) recording independently the emission of the Eu 3+ ions from the NP cores (excitation wavelength 342 nm) and the emission of the NB from the NP shells (excitation wavelength 600 nm). The maximum emission of the Eu 3+ ions at 622 nm was found to be insensitive to the pH value and therefore can be used as reference. The emission of the NB attached to the NPs was recorded at 675 nm, and showed a clear dependence with the pH (illustrated in Fig. 9A for the Bi20YPAA2 NPs conjugated with Nile Blue at different pH values). Such behaviour allowed the use of the ratiometric intensity at these two different emission wavelengths as analytical signal, which is advantageous in terms of accuracy and reliability, because errors associated with environmental variations, probe distribution, and instrumental performance are diminished. The sigmoidal response obtained from the calibration curve of the ratio between the pH and the emission intensities at 622 and 675 nm (Fig. 9B) demonstrates that such particles can be used as pH sensors at pH between 4 and 11. A similar behaviour was observed for the GdVO 4 based nanophosphors (sample Bi20GdPAA) conjugated with NB (Fig. 9C). 2.7 Biocompatibility, cell viabilities and degradation of the particles Biocompatibility studies of both, Eu- Bi-doped YVO 4 - and GdVO 4 -based nanophosphors were undertaken by evaluating the cell viability of HeLa cells on the basis of the metabolic activity of living cells using a Resazurin based assay. 68 PAA- functionalized YVO 4 based NPs (sample Bi20YPAA2, Fig. 10A) showed under the used exposure conditions negligible toxicity effects, with viability percentages around 75–80% for concen- Fig. 7 Fluorescence images of HeLa cells incubated with Bi20YPAA2 nanoparticles for 24 h. (A) Red channel, nanoparticles; (B) yellow channel, lysosomes; (C) blue channel, cell nuclei; (D) green channel, cell membranes; (E) transmission image; and (F) merged all channels. Paper Nanoscale Nanoscale This journal is © The Royal Society of Chemistry 2016 Open Access Article. Published on 13 May 2016. Downloaded on 15/06/2016 09:36:03. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online trations up 1 mg mL −1 , and a LD 50 value measured from the sigmoidal curve (logistic dose response fit) of 1.52 ± 0.61 mg mL −1 . These toxicity data show low toxicity in comparison to similar particles, 33 which is probably associated with the low toxicity of the PAA. 42 However, an increase of the cytotoxicity as the particle size decreases is observed (sample Bi20YPAA8, Fig. 10A), with a measured LD 50 value of 0.16 ± 0.09 mg mL −1 . The PAA-functionalized GdVO 4 based NPs (sample Bi20GdPAA, Fig. 10B) showed much less toxic effects than their YVO 4 -based counterparts of similar size, with a measured LD 50 value of 1.10 ± 0.29 mg mL −1 . Such results indicate that the incorporation of Bi into the GdVO 4 particles had no considerable effects on the final cytotoxicity of the NPs, since similar values have been reported for the same Bi-free particles. 21 The LbL functionalization of both kinds of nanophoshors with PAH and PAH layers reduced their cytotoxicity effects (Fig. 10A and B), as it has been observed in similar systems. 69,70 These cytotoxicity results indicate the potential suitability of such nanophosphors for sensing and cell imaging applications, even more when taken into account that the NP concentrations used in cell imaging and FACS experiments were between 1 or 2 orders of magnitude lower than the measured LD 50 values. However, it clearly needs to be stated that concerning future in vivo use extended toxicity studies would be required, involving, in particular, the possible effects of long-term exposure. The dissolution of the particles at low pH was carried out under conditions mimicking the lysosome, namely acidic pH, by analysing the evolution of the hydrodynamic diameter measured by DLS with increase in the aging time in MES 50 mM buffer at pH 3.5 (Fig. 11). Both, Bi20YPAA2 and Bi20GdPAA NPs aggregate in MES buffer at pH 3.5, forming agglomerates of around 1 µm. In both cases the size of the agglomerates decreased with increase in the aging time, and finally clear solutions were observed (Fig. S16†). This shows how both, YVO 4 - and GdVO 4 -based NPs can be degraded in the cell lysosomes, where eventually the NPs are located, resulting in an efficient method for the NP excretion. 2.8 Flow cytometry study To reveal the influence of both, the chemical composition and the size of the NPs on the cellular uptake, flow cytometry studies with HeLa cells were performed for samples Bi20YPAA2, Bi20YPAA8, and Bi20GdPAA. Fig. 12A shows the mean red intensity fluorescence associated with every cell with increase in the incubation time. In general terms, this value increases with increase in the incubation time for the three analysed samples, indicating that the amount of NPs internalized by HeLa cells increases with time. After 24 hours of incubation, the highest red fluorescence was observed for the Bi20YPAA2 sample (black bar), followed by Bi20GdPAA (blue bar) and Bi20YPAA8 (red bar). The 2D density plots of the control measurements are shown in Fig. S17.†Such results, which are indeed consistent with the LSM images taken for HeLa cells after NP incubation (Fig. 8 and Fig. S14,†at least qualitatively), should however not be related to the amount of NPs associated with the cells, since the emission of the analyzed samples upon excitation at 355 nm depends on both chemical composition and the NP size (Fig. S10†). These different emission intensities were taken into account in Fig. 12B, whose results can then be related to the amount of Fig. 8 Confocal microscopy images of HeLa cells incubated with Bi20YPAA2 nanoparticles for 24 hours. (A) Transmission channel, (B) fluorescence channel, (C) merged image. Nanoscale Paper This journal is © The Royal Society of Chemistry 2016 Nanoscale Open Access Article. Published on 13 May 2016. Downloaded on 15/06/2016 09:36:03. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. 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