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Ca2Si5N8:Eu2+,Tm3+ presents outstanding long lasting luminescence at about 610 nm. However, to be useful for in vivo optical imaging, persistent luminescence materials should possess high optical performance combined with sizes in the nanoscale. With this aim, we investigated two different techniques for the preparation of nanoparticles from Ca2Si5N8:Eu2+,Tm3+ bulk powder. First, nanoparticles were successfully prepared with the pulsed laser ablation method in liquid (abbreviated as PLAL). Secondly, nanoparticles obtained by selective sedimentation from the bulk compound resulted in satisfactory yield and allowed to perform the first real-time in vivo imaging with Ca2Si5N8:Eu2+,Tm3+ host. Finally the influence of surface functionalization on the biodistribution of the probe after systemic injection is discussed.
©2012 Optical Society of America
1. Introduction
In vivo optical imaging using photons as direct information source, has been extensively developed in the last few years [1]. We have recently focused our attention on nanosized crystalline mixed oxides presenting persistent luminescence properties, mainly silicates and phosphates [2–6]. Such materials possess the ability to be excited under UV-light or X-rays and emit long-lasting luminescence during several hours after the excitation has ended. Focusing on the use of persistent luminescence for in vivo bioimaging, we recently reported the synthesis of persistent luminescence nanoparticles (PLNP), extracted from a silicate host, that could emit in the red/near-infrared range and could be used as sensitive optical probe for in vivo imaging, circumventing autofluorescence from animal tissues [2]. Biodistribution of these nanoparticles (NPs) was shown to be highly dependent on both core diameter and global surface charge [7]. Briefly, we reported that for a given surface coverage, the smaller the nanoparticle, the longer its residency time in the bloodstream. In particular, 80 nm nanoparticles with polyethylene glycol surface coverage, compared to nanoparticles with a 180 nm core, were shown to evade more easily the classical uptake by liver and spleen. Besides, we showed that these PLNP could successfully target cancer cells in vitro [8,9]. However, to permit longer observation, the luminescence from such nanophosphors still needs to be improved and new hosts are still required [10,11] in order to provide long-term monitoring of in vivo probes accumulation and improved optical imaging.
We have already shown that bulk Ca2Si5N8:Eu2+,Tm3+ has outstanding long lasting luminescence at about 610 nm [12–14]. However, solid state synthesis of this material produces powder with grain sizes in the micrometer range, thus limiting direct use in living animals. Conception of an efficient probe for in vivo imaging requires several critical conditions to be fulfilled: a long and intense persistent luminescence in suspension, an emission in the range of low tissue absorption, the ability to be functionalized, good chemical stability and the need for small particles with sizes in the nanometer range. The present paper investigates the formation of nanoparticles from rare earth doped nitridosilicate, Ca2Si5N8:Eu2+,Tm3+ (referred as CSN), and their use as potential nanoprobes for in-vivo optical imaging.
In order to obtain such nanoparticles, two ways are envisioned in this article: first, a laser ablation treatment of bulk powder in solution is explored, or a wet grinding of the same powder followed by selective sedimentation in alkaline solution. The yield and persistent luminescence of the recovered nanoparticles are investigated and discussed to provide the best result for in vivo optical bioimaging in small animal.
2. Synthesis and optical characteristics of CSN bulk powder
Ca2Si5N8:Eu2+ (1%),Tm3+ (1%) powder was prepared by mixing appropriate amounts of Ca3N2 (Alfa Aesar, 99%) and α-Si3N4 (Alfa Aesar, 99.85%) precursors for the host material and EuF3 and TmF3 (Alfa Aesar, 99.9%) for the dopants, under a protective nitrogen atmosphere. The powder mixture was then sintered during 1 hour at 1300°C under reducing atmosphere (90% N2, 10% H2) [12].
The Ca2Si5N8 host belongs to the monoclinic space group Cc [15]. Like the other nitrido-silicates, it is very resistant against heat and moisture, thanks to the interconnected SiN4 tetrahedrons. This is of crucial importance to retain the luminescent properties when used in solution. We found that the afterglow intensity of the bulk powder after two years storage in water has dropped by only 2%, compared to 25% for Sr2MgSi2O7:Eu2+,Dy3+ and 52% for SrAl2O4:Eu2+,Dy3+, which shows the excellent stability of Ca2Si5N8 in an aqueous medium.
The active divalent Eu2+ cation easily substitutes the Ca2+ cation while the trivalent Tm3+ presumably induces defects responsible for the very long afterglow (or persistent luminescence). The emission spectrum consists of a single broad band (FWHM of 100 nm) peaking at 610 nm, responsible for the orange colour of the luminescence. The excitation spectrum for the steady state photoluminescence extends well into the visible region, up to 550 nm [12,13].
The decay time of the material can be defined as the time between the end of the excitation and the moment when the afterglow intensity drops below 0.32 mcd/m2, about 100 times the sensitivity of the human eye. For CSN this is approximately 2500 seconds after 1 minute excitation with a Xe arc lamp at 1000 lux (Fig. 1 ). The persistent luminescence can be induced both by UV- and visible light up to 500 nm (Fig. 1). This was confirmed by thermoluminescence excitation spectroscopy [14].
Fig. 1 Decay of the afterglow intensity in CSN after 1 min excitation with 1000 lx of an unfiltered Xe arc lamp. The dotted line indicates the 0.32 mcd/m2 threshold level. Inset: Trap filling probability using various excitation wavelengths, as measured with thermoluminescence excitation spectroscopy [14].
3. Extraction of CSN nanoparticles by Pulsed laser ablation in solution (PLAL)
Laser ablation seems to combine many advantages compared to other extraction techniques, notably regarding size distributions of the resulting NPs [16,17]. Besides, various materials have already been obtained with this method such as metal NPs [18], semiconductors [19], dielectric compounds [20], but also nitride-based compounds such as C3N4 [21]. For this reason, we first focused on PLAL in order to obtain CSN nanoparticules. For all experiments, we used the third harmonic of a pulsed YAG:Nd3+ laser (355 nm, 10ns). The laser irradiation could vary from a few minutes to 3 hours.
As mentioned earlier, the nitridosilicate compound could be well excited in the UV range [12,13]. For PLAL experiments, the CSN targets (size 8mm) were placed at the bottom of a 50 ml beaker and covered with 35 ml of deionised water. The influence of irradiation time and power was investigated. During irradiation under focalized laser beam, the solution becomes milky and at the end the supernatant is collected and analyzed. The variation of the NPs number in the supernatant is determined by using dynamic light scattering measurements (Fig. 2 ).
Fig. 2 355 nm laser power effect on the particles concentration measured according to the intensity of the diffuse light and the irradiation time (1 hour or 2 hours).
As we can see in Fig. 2, the number of particles in the supernatant increases with the laser power. It seems that an increase of the laser ablation duration do not necessary increases the scattering centers number. The sedimentation of the bigger particles or aggregates could be faster than the particles formation. Formation of aggregates with this method has already been reported and to limit such formation, additives are currently used [19,20]. Without any additives, aggregates with size larger than 350 nm are obtained. Such NPs aggregation can be prevented by adding 5 mM NaOH in the beaker. In these conditions, negative charges appear at the surface of the nanoparticles, avoiding their aggregation. TEM measurements of the supernatant reveal that NPs obtained with this technique display relative small diameters in the range 3-5 nm (Fig. 3 -left) in agreement with published results [20]. Indeed, the point of zero charge (PZC) is about 3.7 for this compound and the pH of the solution is about 7-8. This gives a surface charge of about −40 mV, sufficient to issue efficient electronic repulsion between nanoparticles.
Fig. 3 Transmission electron micrographs (TEM) of the CSN NPs obtained from the PLAL technique. TEM measurements were performed on a EI Tecnai 120 Twin microscope operating at 120 kV and equipped with a high resolution Gatan Orius CCD 4 k 6 4 k numeric camera. Left: NPs in NaOH aqueous solution (scale bar: 5 nm). Right: NPs in NaOH with acetate as stabilizing agent. Inset: NPs magnification (scale bar: 10 nm).
The addition of acetate as stabilizing agent during the experiment also seems to prevent NPs’ aggregation, by the formation of a web-like structure (Fig. 3, right), without affecting size distribution of the obtained NPs (Insert in Fig. 3, right). These observations are in good agreement with the literature [10]. Results from dispersive X-rays spectroscopy (EDS) on the TEM apparatus, indicate that the Ca/Si ratio is 2/5, which is in good agreement with the Ca2Si5N8 material composition. Yet, due to the very small amount of NPs in solution, it was not possible to check their structure by conventional X-ray diffraction.
As presented in Fig. 4 , the optical properties of the NPs and bulk materials are very similar. A broad band due to the 5d → 4f Eu2+ emission is observed at 610 nm. For the parity allowed 5d → 4f transition, it is well known that crystal field variation and structure modifications strongly affect the emission profile and energetic position [22,23]. As the emission bands are located at the same position for the NPs and the bulk material, and even if the quantity of obtained NPs is too small to further control structural modification by X-ray diffraction, laser ablation in solution seems to be an efficient way to elaborate very small (3.5 nm) nanoparticles of rare earth doped CSN.
Fig. 4 Emission spectrum of bulk and nanoparticles made from CSN in solution under UV light excitation.
The duration of the persistent luminescence appears lower for the nanoparticles in comparison to the bulk powder. Such effect has already been witnessed with silicate-based PLNP [7] and could be the result of nonradiative recombination and quenching effects associated to the important variation in the surface to volume ratio when decreasing the size of the crystal to 3-5 nm [24,25]. In that latter case there is about 40% of the dopant close to the surface. In addition, the persistent luminescence process is related to the interplay between Eu2+ ions and traps in the vicinity of the Tm3+ co-dopant. For the given sizes of the NPs there is a lower probability to find the two doping cations in close proximity. Thus it is important to keep the nanoparticles at a sufficient size and to increase the NPs concentration in solution in order to perform in vivo imaging.
To go further and determine the potential of the rare earth doped nitridosilicate as material for optical imaging, a second method for the preparation of nanoparticles was used.
4. Extraction of CSN nanoparticles by selective sedimentation and surface functionalization
We have previously reported a general method based on selective centrifugation for the extraction of nanoparticles in basic solution (5 mM NaOH) after wet grinding of a polydisperse silicate bulk powder [2,7,8]. This technique was successfully translated to the present bulk nitridosilicate powder in order to obtain a narrow size distribution of negatively charged hydroxyl-terminated CSN nanoparticles with a mean diameter close to 200 nm and a 10% yield.
Indeed, due to rapid surface oxidation at room temperature after exposure to oxygen-containing atmosphere, air especially; silicon nitride-based compounds are known to display surface properties very close to silicon dioxide and silicate materials [26]. For this reason, we were able to transpose the reaction scheme optimized for the functionalization of silicate-based persistent luminescence nanoparticles [7] to the present CSN surface (Fig. 5 ).
First, 5 mg of hydroxyl-terminated CSN nanoparticles (Fig. 6A , 6B and 6C) were reacted with 3-aminopropyltriethoxysilane (40 µmol) in 2 mL DMF (dimethylformamide) to give positively charged CSN displaying primary amines on their surface (Fig. 6B). This intermediate was then reacted with polyethylene glycol (PEG 10 µmol) via classical coupling reaction using a slight excess of BOP (benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate) as activating agent (Fig. 5).
Fig. 6 Characterization of CSN nanoparticles before and after functionalization steps. A: Size distribution of hydroxyl-terminated CSN and PEGylated CSN in 150 mM NaCl; B: Zeta potential measurements after each functionalization step; C: Transmission electron micrography of CSN-OH in 5mM NaOH (scale bar: 200 nm).
Each step was characterized using zeta potential measurements in order to follow the evolution of global surface charge of the nanoparticle with surface coverage and functional groups decorating the probe. As observed with many other nanomaterials intended for biological applications, PEG grafting has the ability to mask the surface charge of the nanoparticle (zeta potential close to neutral value in Fig. 6B). This change in surface properties associated with PEG grafting also comes with a large increase of the hydrodynamic diameter (Fig. 6A). Such size distribution shift is certainly not the result of PEG chain length only. Indeed, a similar trend has already been described with silicate host and is very likely to be related to the first reaction with the aminosilanes, known to form a thick layer around the particle [27].
5. Real-time in vivo optical bioimaging in healthy mice
In order to investigate a possible application of nitridosilicate hosts for bioimaging in living animal, we compared the biodistribution of two different surface coverages (hydroxyl and PEG-terminated CSN) after systemic injection to BALB/c mice. For the experiment, mice were intravenously injected with 1012 nanoparticles of either CSN-OH or CSN-PEG after 5 minutes excitation of both suspensions under UV light (6 W lamp, 254 nm). Figure 7 displays the optical images obtained 15 minutes after intravenous injection. The signal, coming from persistent luminescence CSN-based nanoprobes, is detected with a photon-counting system (Photon-Imager, Biospace Lab, France).
Fig. 7 Biodistribution of CSN-OH and CSN-PEG, 15 minutes after tail vain injection. Luminescence intensity is expressed in false color unit (1 unit = 2800 photons per s.cm2.steradians).
First, we clearly observe the signal from the two types of CSN-based persistent luminescence nanoparticles through the animal no matter which type of surface coverage is used. We should notice that the signal coming from the probe is very clearly detectable through living tissues. The emission of the nitrido-silicate, around 610 nm puts this phosphor at the edge of the tissue transparency window. Due to lipids, blood and water the absorption is relatively high for wavelengths shorter than 600 nm [28]. However, the broad and intensive emission of CSN yields significant emission intensity in the long wavelength region. As observed in Fig. 7, hydroxyl-terminated CSN nanoparticles are almost instantly trapped within the liver. This behaviour of negatively charged nanoparticles has already been described with several carriers displaying similar surface coverage [1]. It is the result of a two-step recognition process initiated by opsonins from the bloodstream that bind negatively charged nanoparticles and trigger a global uptake of the resulting opsonised nanoprobes by Kupffer cells in the liver. The image from the other mouse shows a different behaviour of PEGylated CSN nanoparticles that evade instant trapping from the reticulo-endothelial system and circulate much longer in the bloodstream (Fig. 7).
6. Conclusion
We report the first use of orange-emitting Ca2Si5N8:Eu2+,Tm3+ persistent luminescence nanoparticles for real-time bioimaging in living animal. The preparation of the Ca2Si5N8:Eu2+,Tm3+ persistent luminescence nanoparticles by pulsed laser ablation in solution gives NPs with diameters in the range of 3-5 nm but the low quantity in solution prevents further use for bioimaging. However, extraction of nanoparticles after wet grinding gives enough NPs to realise the first proof that such nitridosilicate host can be used as optical probe for small animal in vivo imaging. Functionalization through simple surface chemistry could be used to change the behaviour of the probe in vivo. PEG grafting is responsible for a better distribution of the nanoparticles after systemic injection, delaying their uptake by the reticulo-endothelial system.
Acknowledgments
The authors acknowledge C. Rosticher and P. Legriel for the TEM measurements, D. Amans and D. Giaume for fruitful discussion for the pulsed laser ablation in liquid method and NPs characterization and P. Aschehoug for technical assistance. We thank also R. Lai Kuen, B. Saubaméa, and J. Seguin for their contribution and help to perform transmission electron micrographs, in vivo experiments, and image analysis with Image J software. This work has been supported by the French National Research agency (ANR) in the frame of its program in Nanosciences and Nanotechnologies (NATLURIM project n°ANR-08-NANO-025).
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