Lanthanide oxide nanoparticles

Yttrium oxide is well-known for its photodurability and low phonon energy, which makes Ln³⁺-doped Y₂O₃ nanoparticles (Y₂O₃: Ln³⁺) very attractive for optical applications, including bioimaging.^{Reference Sotiriou, Schneider and Pratsinis12,Reference Kamimura, Kanayama, Tokuzen, Soga and Nagasaki13} However, prior to their application in biomedicine, any potential toxicity of these nanoparticles must be ruled out; three aspects are mainly addressed in the literature: (1) dosage, (2) surface modification, and (3) size and morphology. During the last decade, several studies have investigated upconverting and NIR-emitting nanophosphors with regard to these aspects.

For instance, concentration-dependent in vitro cytotoxicity studies on Y₂O₃:Er³⁺ nanoparticles revealed their uptake by macrophages (cells that consume foreign material), thus inducing increased cell death with increasing dosage (Figure 3a).^{Reference Venkatachalam, Hemmer, Yamano, Hyodo, Kishimoto and Soga14} For the same nanoparticles, no cytotoxic effect was found toward non-phagocytic B-cell hybridomas, which are fused hybrid cells—between B-cells (a type of lymphocyte) and myeloma tumor cells—that do not uptake foreign material. The cytotoxic effect toward macrophages was ascribed to the poor chemical stability of Y₂O₃ under acidic conditions, as found inside these cells. Consequently, careful surface modification of the nanophosphors providing chemical stability (as well as dispersion stability in biological fluids, biocompatibility, site-selectivity [toward cancerous cells], and controlled clearance from the organism) is a general and essential requirement. Herein, SiO₂ and polymer-based coatings are suitable to prevent cytotoxicity and unspecific interference with cells, improving the in vivo biodistribution.^{Reference Atabaev, Jin, Lee, Han, Vu, Hwang and Kim15–Reference Venkatachalam, Yamano, Hemmer, Hyodo, Kishimoto and Soga18} It was further shown that the functionalization of Y₂O₃:Er³⁺,Yb³⁺ with amine groups allows specific surface immobilization, and provides water-dispersibility, biocompatibility, and the ability for bioconjugation.^{Reference Lim, Riehn, Tung, Ryu, Zhuo, Dalland and Austin19}

Figure 3.

(a) Cytotoxic behavior of Y₂O₃:Er³⁺. The uptake of the nanoparticles by macrophages results in the decrease of their viability with increasing particle concentration due to the toxic effect of Y³⁺-ions leaking from the particles once uptaken by the macrophages. In contrast, no significant reduction in viability is observed for B-cell hybridomas, which generally show less or no tendency to uptake foreign material. (b) Cytotoxic behavior of Gd₂O₃:Er³⁺,Yb³⁺. The surface modification with PEG-b-PAAc results in increased viability when compared to bare nanostructures, which is ascribed to an improved chemical stability and therefore reduced formation of toxic Gd³⁺-ions upon uptake by the macrophages. (c) Over-1000 nm fluorescence bioimaging of a mouse. White arrows represent the 980 nm laser light used for excitation as well as the emitted near-infrared (NIR) light of 1.5 μm wavelength. The emission of 1.5 μm light can be seen through the skin of the mouse, revealing the high potential of NIR light for deep-tissue in vivo imaging. The bright 1.5 μm emission mainly originates from particles accumulated in the liver, as obvious from images taken of the opened mouse body (size of the liver in the range of 1 to 2 cm). (a) Reproduced with permission from Reference 3. © 2013 The Royal Society of Chemistry. (b) Adapted with permission from Reference 27. © 2013 Elsevier. (c) Reproduced with permission from Reference 9. © 2012 Springer. Note: PEG-b-PAAc, poly(ethylene glycol)-b-poly(acrylic acid) block co-polymer; λ_ex, excitation wavelength; λ_em, emission wavelength; t, incubation time; negative control: incubation in cell culture medium without nanostructures; positive control: anticancer drug carboplatin (100 μg mL^–1).

For reliable cancer imaging, Gd³⁺ ions are potential co-dopants to introduce magnetic properties, allowing for magnetic resonance imaging (MRI).^{Reference Setua, Menon, Asok, Nair and Koyakutty20} Aside from doping with Gd³⁺ ions, several groups investigated the suitability of Ln³⁺-doped magnetic host materials as multimodal biomarkers that combine excellent optical and magnetic properties, allowing for high spatial resolution imaging via MRI. Here, gadolinium oxide, Gd₂O₃, is a promising host material.^{Reference Luo, Tian, Yang, Xiao, Hu, Chen and Li21,Reference Azizian, Riyahi-Alam, Haghgoo, Saffari, Zohdiaghdam and Gorj22} Still, concerns may arise about leaching of free Gd³⁺ ions from the Gd₂O₃ nanophosphors causing toxicity. Yet, again, ${\rm{SiO}}_{\rm{2}}^ - $^{Reference Zhou, Gu, Liu, Yin, Tian, Yan, Jin, Ren, Xing, Li, Chang, Hu and Zhao23} or PEG-based^{Reference Ahrén, Selegard, Klasson, Söderlind, Abrikossova, Skoglund, Bengtsson, Engström, Käll and Uvdal24–Reference Hemmer, Venkatachalam, Hyodo and Soga26} (PEG, polyethylene glycol) surface modifications were shown to not only prevent aggregation, enhance blood retention, facilitate cellular uptake, and provide binding sites for targeting, but also prevent leaching and reduce cytotoxicity. Soga et al. evaluated the cytotoxic effects of PEG-b-PAAc (poly(ethylene glycol)-b-poly(acrylic acid) block co-polymer) -modified upconverting and NIR-emitting Gd₂O₃:Er³⁺,Yb³⁺ nanostructures, and in contrast to unmodified nanostructures, no toxic effect was observed (Figure 3b).^{Reference Hemmer, Yamano, Kishimoto, Venkatachalam, Hyodo and Soga27} Moreover, the NIR emission of 1.5 μm under 980 nm excitation is suitable for in vivo bioimaging, allowing for the observation of the biodistribution of such nanostructures in live mice (Figure 3c).

As previously mentioned, besides surface modification and dosage, particle size and morphology play a crucial role when it comes to risk assessment. In this context, in vivo MRI indicated the clearance of sub-10 nm lanthanide oxide particles through kidneys in mice, which is a prerequisite for clinical applications.^{Reference Kattel, Park, Xu, Kim, Lee, Bony, Heo, Jin, Baeck, Chang, Kim, Bae, Chae and Lee28} In contrast, larger nanoparticles and nanorods had been reported to accumulate in the liver, which may induce death of the mouse. Generally speaking, the toxicity of the nanostructures does not necessarily increase with increasing size. As observed by Venkatachalam et al.,^{Reference Venkatachalam, Hemmer, Yamano, Hyodo, Kishimoto and Soga14} a decrease in the size of Y₂O₃:Er³⁺ particles induced increasing toxicity toward macrophages. A size-comparison study by Lee et al. revealed a similar effect.^{Reference Lee, Kasuga and Kato29} Consequently, careful risk assessment is required for different probes under different conditions. Heng et al. in their study emphasize the importance of rethinking commonly used laboratory protocols for cytotoxic assessment of nanomaterials.^{Reference Heng, Das, Zhao, Ma, Tan, Ng and Loo30} The authors stress that, in particular, for mildly toxic nanomaterials, a longer duration cellular exposure is required for cytotoxic effects to become apparent, and most studies would not further investigate the post-exposure cell biology and physiology. This means not monitoring the cellular conditions after the nanoparticle exposure and subsequent reseeding of the cells onto new, nanoparticle-free culture plates.

It should be further mentioned that the interaction between biological matter and upconverting or NIR-emitting oxide nanostructures can be translated to other Ln³⁺ dopants or other Ln³⁺-based host materials not showing upconversion or NIR emission. For example, Eu³⁺ or Tb³⁺-doped Gd₂O₃ and Y₂O₃ emitting in the visible range under UV excitation were reported as non-toxic fluorescent labels.^{Reference Atabaev, Jin, Lee, Han, Vu, Hwang and Kim15,Reference Nichkova, Dosev, Gee, Hammock and Kennedy31–Reference Das and Tan33} Interestingly, cytotoxicity studies by Setua et al. revealed no reduction in the viability of normal and cancer cells when incubated with Y₂O₃ nanoparticles, nor was the formation of reactive oxygen species (ROS) observed; ROS are known to induce cell damage.^{Reference Setua, Menon, Asok, Nair and Koyakutty20} In fact, antioxidative properties that promote cell survival under conditions of oxidative stress have been reported for Y₂O₃.^{Reference Schubert, Dargusch, Raitano and Chan34} Y₂O₃ and Gd₂O₃ may be the most intensively investigated oxide host materials for Ln³⁺-doped nanophosphors, yet toxicity studies analyzing other lanthanide oxides, such as Eu₂O₃ and Dy₂O₃, indicate a similar non-toxic behavior, and they have been reported as promising candidates for bioimaging.^{Reference Kattel, Park, Xu, Kim, Lee, Bony, Heo, Jin, Baeck, Chang, Kim, Bae, Chae and Lee28,Reference Kattel, Park, Xu, Kim, Lee, Bony, Heo, Chang, Kim, Do, Chae, Kwak and Lee35}

Beyond oxides: Lanthanide-based fluorides

Biomedical applications often require ultra-small nanoparticles of 10 nm or less in size in order to avoid blockage of blood vessels, to allow cellular uptake, and to ensure clearance from the body. However, one must consider that oxide nanoparticles of such small sizes may suffer from quenching of their photoluminescence.^{Reference Hemmer, Venkatachalam, Hyodo, Hattori, Ebina, Kishimoto and Soga3} Yet, efficient emission in this size range is possible when Ln³⁺ ions are doped into a fluoride host matrix,^{Reference Chen, Ohulchanskyy, Kumar, Agren and Prasad36–Reference Cao, Yang, Gao, Yang, Hu and Li38} and upconverting non-toxic NaYF₄:Ln³⁺ nanoparticles have successfully been applied for in vitro cellular and in vivo animal imaging.^{Reference Xiong, Chen, Yu, Li, Liu and Huang39,Reference Xiong, Yang, Yang, Xu and Li40} Investigations of the in vivo biodistribution revealed rather slow clearance from the body. However, long-term studies indicated that mice survive the injection for at least 115 days without any evident toxic effect. Further, NaGdF₄ has been suggested as a non-toxic host material for lanthanide doping that provides NIR-to-NIR upconversion luminescence in combination with magnetic properties.^{Reference Zhou, Sun, Du, Xiong, Hua and Li41} Aiming for the fusion of information from several imaging techniques, GdDTPA (chelated gadolinium, diethylenetriamine pentaacetic acid)-complexed NaLuF₄: Tm³⁺, Yb³⁺ nanoparticles were presented as a trimodal marker for NIR-to-NIR imaging, x-ray computed tomography, and MRI, enabling optical, x-ray-based, and magnetic imaging with one single marker. Again, the presented nanophosphors showed low cytotoxicity as indicated by in vitro, in vivo, and ex vivo studies.^{Reference Xia, Chen, Gao, Wu, Feng and Li42}