Biodistribution of Multimodal Gold Nanoclusters Designed for Photoluminescence-SPECT/CT Imaging and Diagnostic.

Highly biocompatible nanostructures for multimodality imaging are critical for clinical diagnostics improvements in the future. Combining optical imaging with other techniques may lead to important advances in diagnostics. The purpose of such a system would be to combine the individual advantages of each imaging method to provide reliable and accurate information at the site of the disease bypassing the limitations of each. The aim of the presented study was to evaluate biodistribution of the biocompatible technetium-99m labelled bovine serum albumin-gold nanoclusters (99mTc-BSA-Au NCs) as photoluminescence-SPECT/CT agent in experimental animals. It was verified spectroscopically that radiolabelling with 99mTc does not influence the optical properties of BSA-Au NCs within the synthesized 99mTc-BSA-Au NCs bioconjugates. Biodistribution imaging of the 99mTc-BSA-Au NCs in Wistar rats was performed using a clinical SPECT/CT system. In vivo imaging of Wistar rats demonstrated intense cardiac blood pool activity, as well as rapid blood clearance and accumulation in the kidneys, liver, and urinary bladder. Confocal images of kidney, liver and spleen tissues revealed no visible uptake indicating that the circulation lifetime of 99mTc-BSA-Au NCs in the bloodstream might be too short for accumulation in these tissues. The cellular uptake of 99mTc-BSA-Au NCs in kidney cells was also delayed and substantial accumulation was observed only after 24-h incubation. Based on our experiments, it was concluded that 99mTc-BSA-Au NCs could be used as a contrast agent and shows promise as potential diagnostic agents for bloodstream imaging of the excretory organs in vivo.

Exploring BODIPY-Based Sensor for Imaging of Intracellular Microviscosity in Human Breast Cancer Cells.

BODIPY-based molecular rotors are highly attractive imaging tools for imaging intracellular microviscosity in living cells. In our study, we investigated the ability to detect the microviscosity of biological objects by using BDP-NO2 and BDP-H molecular rotors. We describe in detail the optical properties of BDP-NO2 and BDP-H molecular rotors in aqueous media with and without proteins, together with their accumulation dynamics and localization in live and fixed human breast cancer cells. Furthermore, we investigate the applicability of these molecules to monitor microviscosity in the organelles of human breast cancer cells by fluorescence lifetime imaging microscopy (FLIM). We demonstrate that the BDP-NO2 molecular rotor aggregates in aqueous media and is incompatible with live cell imaging. The opposite effect is observed with BDP-H which preserves its stability in aqueous media, diffuses through the plasma membrane and accumulates in lipid droplets (LDs) and the cytosol of both live and fixed MCF-7 and MDA-MB-231 cancer cells. Finally, by utilizing BDP-H we demonstrate that LD microviscosity is significantly elevated in more malignant MDA-MB-231 human breast cancer cells, as compared to MCF-7 breast cancer cells. Our findings demonstrate that BDP-H is a water-compatible probe that can be successfully applied to measure microviscosity in the LDs of living cells.

Blood Plasma Stabilized Gold Nanoclusters for Personalized Tumor Theranostics.

Personalized cancer theranostics has a potential to increase efficiency of early cancer diagnostics and treatment, and to reduce negative side-effects. Protein-stabilized gold nanoclusters may serve as theranostic agents. To make gold nanoclusters personalized and highly biocompatible, the clusters were stabilized with human plasma proteins. Optical properties of synthesized nanoclusters were investigated spectroscopically, and possible biomedical application was evaluated using standard cell biology methods. The spectroscopic investigations of human plasma proteins stabilized gold nanoclusters revealed that a wide photoluminescence band in the optical tissue window is suitable for cancer diagnostics. High-capacity generation of singlet oxygen and other reactive oxygen species was also observed. Furthermore, the cluster accumulation in cancer cells and the photodynamic effect were evaluated. The results demonstrate that plasma proteins stabilized gold nanoclusters that accumulate in breast cancer cells and are non-toxic in the dark, while appear phototoxic under irradiation with visible light. The results positively confirm the utility of plasma protein stabilized gold nanoclusters for the use in cancer diagnostics and treatment.

Polymer brush coated upconverting nanoparticles with improved colloidal stability and cellular labeling.

Upconverting nanoparticles (UCNPs) possess great potential for biomedical application. UCNPs absorb and convert near-infrared (NIR) radiation in the biological imaging window to visible (Vis) and even ultraviolet (UV) radiation. NIR excitation offers reduced scattering and diminished autofluorescence in biological samples, whereas the emitted UV-Vis and NIR photons can be used for cancer treatment and imaging, respectively. However, UCNPs are usually synthesized in organic solvents and are not readily suitable for biomedical application due to the hydrophobic nature of their surface. Herein, we have removed the hydrophobic ligands from the synthesized UCNPs and coated the bare UCNPs with two custom-made hydrophilic polyelectrolytes (synthesized via the reversible addition-fragmentation chain transfer (RAFT) polymerization method). Polymers containing different amounts of PEGylated and carboxylic groups were studied. Coating with both polymers increased the upconversion (UC) emission intensity and photoluminescence lifetime values of the UCNPs, which directly translates to more efficient cancer cell labeling nanoprobes. The polymer composition plays a crucial role in the modification of UCNPs, not only with respect to their colloidal stability, but also with respect to the cellular uptake. Colloidally unstable bare UCNPs aggregate in cell culture media and precipitate, rendering themselves unsuitable for any biomedical use. However, stabilization with polymers prevents UCNPs from aggregation, increases their uptake in cells, and improves the quality of cellular labeling. This investigation sheds light on the appropriate coating for UCNPs and provides relevant insights for the rational development of imaging and therapeutic tools.

Uptake of Upconverting Nanoparticles by Breast Cancer Cells: Surface Coating versus the Protein Corona.

Fluorophores with multifunctional properties known as rare-earth-doped nanoparticles (RENPs) are promising candidates for bioimaging, therapy, and drug delivery. When applied in vivo, these nanoparticles (NPs) have to retain long blood-circulation time, bypass elimination by phagocytic cells, and successfully arrive at the target area. Usually, NPs in a biological medium are exposed to proteins, which form the so-called "protein corona" (PC) around the NPs and influence their targeted delivery and accumulation in cells and tissues. Different surface coatings change the PC size and composition, subsequently deciding the fate of the NPs. Thus, detailed studies on the PC are of utmost importance to determine the most suitable NP surface modification for biomedical use. When it comes to RENPs, these studies are particularly scarce. Here, we investigate the PC composition and its impact on the cellular uptake of citrate-, SiO2-, and phospholipid micelle-coated RENPs (LiYF4:Yb3+,Tm3+). We observed that the PC of citrate- and phospholipid-coated RENPs is relatively stable and similar in the adsorbed protein composition, while the PC of SiO2-coated RENPs is larger and highly dynamic. Moreover, biocompatibility, accumulation, and cytotoxicity of various RENPs in cancer cells have been evaluated. On the basis of the cellular imaging, supported by the inhibition studies, it was revealed that RENPs are internalized by endocytosis and that specific endocytic routes are PC composition dependent. Overall, these results are essential to fill the gaps in the fundamental understanding of the nano-biointeractions of RENPs, pertinent for their envisioned application in biomedicine.

Multiplexed Nanobiosensors: Current Trends in Early Diagnostics.

The ever-growing demand for fast, cheap, and reliable diagnostic tools for personalised medicine is encouraging scientists to improve existing technology platforms and to create new methods for the detection and quantification of biomarkers of clinical significance. Simultaneous detection of multiple analytes allows more accurate assessment of changes in biomarker expression and offers the possibility of disease diagnosis at the earliest stages. The concept of multiplexing, where multiple analytes can be detected in a single sample, can be tackled using several types of nanomaterial-based biosensors. Quantum dots are widely used photoluminescent nanoparticles and represent one of the most frequent choices for different multiplex systems. However, nanoparticles that incorporate gold, silver, and rare earth metals with their unique optical properties are an emerging perspective in the multiplexing field. In this review, we summarise progress in various nanoparticle applications for multiplexed biomarkers.

Protein-stabilized gold nanoclusters for PDT: ROS and singlet oxygen generation.

Suitable properties as well as eco-friendly synthesis of photoluminescent Au nanoclusters (NCs) make them promising compounds for biomedical diagnostics and visualization applications. However, the potential photochemical activity of such agents on cancerous cells is largely unknown. The nanoclusters (BSA-Au NCs) were synthetized in the presence of BSA (an average hydrodynamic diameter was about 9.4 nm, while the size of the metal cluster was <1.3 nm according to atomic force microscopy measurements) and possessed a broad photoluminescence band at 680 nm in buffered (pH 7.2) aqueous medium. The photochemical activity was studied by adding two fluorescent probes (dihydrorhodamine or Singlet Oxygen Sensor Green) for detection of reactive oxygen species in samples irradiated at 405 nm to minimize direct excitation of the probes. The photoluminescence measurements evidenced the capability of BSA-Au NCs to generate reactive oxygen species upon light exposure, while the observed sensitivity of the photoluminescence properties might be used to indicate photooxidative processes in the medium. The viability test performed on breast cancer cells after incubation with BSA-Au NCs and subsequent irradiation revealed notable difference in induced phototoxicity between two cell lines, which was not the case after the corresponding treatment using the photosensitizer chlorin e6.

3D cellular spheroids as tools for understanding carboxylated quantum dot behavior in tumors.

BACKGROUND: Monolayer cell cultures have been considered the most suitable technique for in vivo cellular experiments. However, a lot of cellular functions and responses that are present in natural tissues are lost in two-dimensional cell cultures. In this context, nanoparticle accumulation data presented in literature are often not accurate enough to predict behavior of nanoparticles in vivo. Cellular spheroids show a higher degree of morphological and functional similarity to the tissues. METHODS: Accumulation and distribution of carboxylated CdSe/ZnS quantum dots (QDs), chosen as model nanoparticles, was investigated in cellular spheroids composed of different phenotype mammalian cells. The findings were compared with the results obtained in in vivo experiments with human tumor xenografts in immunodeficient mice. The diffusive transport model was used for theoretical nanoparticles distribution estimation. RESULTS: QDs were accumulated only in cells, which were localized in the periphery of cellular spheroids. CdSe/ZnS QDs were shown to be stable and inert; they did not have any side-effects for cellular spheroids formation. Penetration of QDs in both cellular spheroids and in vivo tumor model was limited. The mathematical model confirmed the experimental results: nanoparticles penetrated only 25µm into cellular spheroids after 24h of incubation. CONCLUSIONS: Penetration of negatively charged nanoparticles is limited not only in tumor tissue, but also in cellular spheroids. GENERAL SIGNIFICANCE: The results presented in this paper show the superior applicability of cellular spheroids to cell monolayers in the studies of the antitumor effect and penetration of nanomedicines.

Synthesis and functionalization of NaGdF4:Yb,Er@NaGdF4 core-shell nanoparticles for possible application as multimodal contrast agents.

Upconverting nanoparticles (UCNPs) are promising, new imaging probes capable of serving as multimodal contrast agents. In this study, monodisperse and ultrasmall core and core-shell UCNPs were synthesized via a thermal decomposition method. Furthermore, it was shown that the epitaxial growth of a NaGdF4 optical inert layer covering the NaGdF4:Yb,Er core effectively minimizes surface quenching due to the spatial isolation of the core from the surroundings. The mean diameter of the synthesized core and core-shell nanoparticles was ≈8 and ≈16 nm, respectively. Hydrophobic UCNPs were converted into hydrophilic ones using a nonionic surfactant Tween 80. The successful coating of the UCNPs by Tween 80 has been confirmed by Fourier transform infrared (FTIR) spectroscopy. Scanning electron microscopy (SEM), powder X-ray diffraction (XRD), photoluminescence (PL) spectra and magnetic resonance (MR) T1 relaxation measurements were used to characterize the size, crystal structure, optical and magnetic properties of the core and core-shell nanoparticles. Moreover, Tween 80-coated core-shell nanoparticles presented enhanced optical and MR signal intensity, good colloidal stability, low cytotoxicity and nonspecific internalization into two different breast cancer cell lines, which indicates that these nanoparticles could be applied as an efficient, dual-modal contrast probe for in vivo bioimaging.

Photoluminescent Gold Nanoclusters in Cancer Cells: Cellular Uptake, Toxicity, and Generation of Reactive Oxygen Species.

In recent years, photoluminescent gold nanoclusters have attracted considerable interest in both fundamental biomedical research and practical applications. Due to their ultrasmall size, unique molecule-like optical properties, and facile synthesis gold nanoclusters have been considered very promising photoluminescent agents for biosensing, bioimaging, and targeted therapy. Yet, interaction of such ultra-small nanoclusters with cells and other biological objects remains poorly understood. Therefore, the assessment of the biocompatibility and potential toxicity of gold nanoclusters is of major importance before their clinical application. In this study, the cellular uptake, cytotoxicity, and intracellular generation of reactive oxygen species (ROS) of bovine serum albumin-encapsulated (BSA-Au NCs) and 2-(N-morpholino) ethanesulfonic acid (MES)capped photoluminescent gold nanoclusters (Au-MES NCs) were investigated. The results showed that BSA-Au NCs accumulate in cells in a similar manner as BSA alone, indicating an endocytotic uptake mechanism while ultrasmall Au-MES NCs were distributed homogeneously throughout the whole cell volume including cell nucleus. The cytotoxicity of BSA-Au NCs was negligible, demonstrating good biocompatibility of such BSA-protected Au NCs. In contrast, possibly due to ultrasmall size and thin coating layer, Au-MES NCs exhibited exposure time-dependent high cytotoxicity and higher reactivity which led to highly increased generation of reactive oxygen species. The results demonstrate the importance of the coating layer to biocompatibility and toxicity of ultrasmall photoluminescent gold nanoclusters.

Accumulation and Toxicity of Superparamagnetic Iron Oxide Nanoparticles in Cells and Experimental Animals.

The uptake and distribution of negatively charged superparamagnetic iron oxide (Fe3O4) nanoparticles (SPIONs) in mouse embryonic fibroblasts NIH3T3, and magnetic resonance imaging (MRI) signal influenced by SPIONs injected into experimental animals, were visualized and investigated. Cellular uptake and distribution of the SPIONs in NIH3T3 after staining with Prussian Blue were investigated by a bright-field microscope equipped with digital color camera. SPIONs were localized in vesicles, mostly placed near the nucleus. Toxicity of SPION nanoparticles tested with cell viability assay (XTT) was estimated. The viability of NIH3T3 cells remains approximately 95% within 3-24 h of incubation, and only a slight decrease of viability was observed after 48 h of incubation. MRI studies on Wistar rats using a clinical 1.5 T MRI scanner were showing that SPIONs give a negative contrast in the MRI. The dynamic MRI measurements of the SPION clearance from the injection site shows that SPIONs slowly disappear from injection sites and only a low concentration of nanoparticles was completely eliminated within three weeks. No functionalized SPIONs accumulate in cells by endocytic mechanism, none accumulate in the nucleus, and none are toxic at a desirable concentration. Therefore, they could be used as a dual imaging agent: as contrast agents for MRI and for traditional optical biopsy by using Prussian Blue staining.