Polarimetric second harmonic generation microscopy of partially oriented fibers II: Imaging study.

Polarimetric second harmonic generation (SHG) microscopy imaging is employed to investigate the ultrastructural organization of biological and biomimetic partially oriented fibrillar structures. The linear polarization-in polarization-out SHG microscopy measurements are conducted with rat tail tendon, rabbit cornea, pig cartilage, and biomimetic meso-tetra(4-sulfonatophenyl)porphine (TPPS4) cylindrical aggregates, which represent different two- and three-dimensional (2D and 3D) configurations of C6 symmetry fibril structures in the focal volume (voxel) of the microscope. The polarization-in polarization-out imaging of rat tail tendon reveals that SHG intensity is affected by parallel/antiparallel arrangements of the fibers, and achiral (R) and chiral (C) susceptibility component ratio values change by tilting the tendon fibers out of image plane. The R ratio changes for the 2D crossing fibers observed in cornea tissue. The 3D crossing of fibers also affects R ratio in cartilage tissue. The distinctly different dependence of R on crossing and tilting of fibers is demonstrated in collagen and TPPS4 aggregates, due to the achiral molecular susceptibility ratio having values below and above 3, respectively. The polarimetric microscopy results correspond well with the analytical expressions of amplitude and R and C ratios dependence on the crossing angle of the fibers. The experimentally measured SHG intensity and R and C ratio maps are consistent with the computational modeling of various fiber configurations presented in the preceding article. The demonstrated SHG intensity and R and C ratio dependencies on fibril configurations provide the basis for interpreting polarimetric SHG microscopy images in terms of 3D ultrastructural organization of fibers in each voxel of the samples.

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.

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.

Uptake and distribution of carboxylated quantum dots in human mesenchymal stem cells: cell growing density matters.

BACKGROUND: Human mesenchymal stem cells (MSCs) have drawn much attention in the field of regenerative medicine for their immunomodulatory and anti-inflammatory effects. MSCs possess specific tumor-oriented migration and incorporation highlighting the potential for MSCs to be used as an ideal carrier for anticancer agents. Bone marrow is the main source of MSCs for clinical applications. MSCs tracking in vivo is a critical component of the safety and efficacy evaluation of therapeutic cell products; therefore, cells must be labeled with contrast agents to enable visualization of the MSCs migration in vivo. Due to their unique properties, quantum dots (QDs) are emerging as optimal tools in long-term MSC optical imaging applications. The aim of this study was to investigate the uptake dynamics, cytotoxity, subcellular and extracellular distribution of non-targeted carboxylated quantum dots in human bone marrow MSCs at different cell growing densities. RESULTS: QDs had no negative impact on MSC viability throughout the experiment and accumulated in all observed cells efficiently; however, in some MSCs QDs induced formation of lipid droplets. At low cell growing densities QDs distribute within MSCs cytoplasm already after 1 h of incubation reaching saturation after 6 h. After 24 h QDs localize mainly in the perinuclear region of the cells in endosomes. Interestingly, in more confluent culture QDs localize mostly outside MSCs. QDs abundantly mark MSC long filopodia-like structures attaching neighboring cells. At high cell density cultivation, we for the first time demonstrated that carboxylated QDs localize in human bone marrow MSC extracellular matrix. Moreover, we observed that average photoluminescence lifetime of QDs distributed in extracellular matrix are longer than lifetimes of QDs entrapped in endocytic vesicles; thus, for the first time showing the possibility to identify and distinguish localization of QDs in various extracellular and intracellular structures using fluorescence-lifetime imaging microscopy without additional staining assays. CONCLUSION: Carboxylated QDs can be used as nonspecific and effective dye for staining of human bone marrow MSCs and their specific extracellular structures. These results are promising in fundamental stem cell biology as well as in cellular therapy, anticancer drug delivery and tissue engineering.

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.

Embryotoxicity of Quantum Dots in Rainbow Trout Oncorhynchus mykiss During the Hatching Period.

Due to the active development and application of nanotechnology, nanoparticles have emerged as a new class of environmental pollutants. The aim of the study was to investigate quantum dots (QDs) access routes and distribution in embryos and larvae of rainbow trout Oncorhynchus mykiss and to determine the toxicity of QDs to rainbow trout larvae depending on the duration of exposure. CdSe/ZnS-COOH QDs at sublethal concentration was used during the toxicity test (1, 4 and 14 days). The results showed that QDs could get from the solutions into the larvae after hatching. QDs induced a significant increase in mortality, gill ventilation frequency and behavioral responses and a decrease in relative body mass in larvae at the end of the test. Larvae exposed to QDs were found to possess developmental malformations (blood clots). It was found that biological responses of larvae significantly depended on the duration of exposure to QDs.

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.

Gene and miRNA expression signature of Lewis lung carcinoma LLC1 cells in extracellular matrix enriched microenvironment.

BACKGROUND: The extracellular matrix (ECM), one of the key components of tumor microenvironment, has a tremendous impact on cancer development and highly influences tumor cell features. ECM affects vital cellular functions such as cell differentiation, migration, survival and proliferation. Gene and protein expression levels are regulated in cell-ECM interaction dependent manner as well. The rate of unsuccessful clinical trials, based on cell culture research models lacking the ECM microenvironment, indicates the need for alternative models and determines the shift to three-dimensional (3D) laminin rich ECM models, better simulating tissue organization. Recognized advantages of 3D models suggest the development of new anticancer treatment strategies. This is among the most promising directions of 3D cell cultures application. However, detailed analysis at the molecular level of 2D/3D cell cultures and tumors in vivo is still needed to elucidate cellular pathways most promising for the development of targeted therapies. In order to elucidate which biological pathways are altered during microenvironmental shift we have analyzed whole genome mRNA and miRNA expression differences in LLC1 cells cultured in 2D or 3D culture conditions. METHODS: In our study we used DNA microarrays for whole genome analysis of mRNA and miRNA expression differences in LLC1 cells cultivated in 2D or 3D culture conditions. Next, we indicated the most common enriched functional categories using KEGG pathway enrichment analysis. Finally, we validated the microarray data by quantitative PCR in LLC1 cells cultured under 2D or 3D conditions or LLC1 tumors implanted in experimental animals. RESULTS: Microarray gene expression analysis revealed that 1884 genes and 77 miRNAs were significantly altered in LLC1 cells after 48 h cell growth under 2D and ECM based 3D cell growth conditions. Pathway enrichment results indicated metabolic pathway, MAP kinase, cell adhesion and immune response as the most significantly altered functional categories in LLC1 cells due to the microenvironmental shift from 2D to 3D. Comparison of the expression levels of selected genes and miRNA between LLC1 cells grown in 3D cell culture and LLC1 tumors implanted in the mouse model indicated correspondence between both model systems. CONCLUSIONS: Global gene and miRNA expression analysis in LLC1 cells under ECM microenvironment indicated altered immune response, adhesion and MAP kinase pathways. All these processes are related to tumor development, progression and treatment response, suggesting the most promising directions for the development of targeted therapies using the 3D cell culture models.

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.

5-aminolevulinic-acid-based fluorescence spectroscopy and conventional colposcopy for in vivo detection of cervical pre-malignancy.

BACKGROUND: Sensitized fluorescence diagnostics are based on selective accumulation of photosensitizer in the tissue where carcinogenesis has started. The present study compared topical 5-aminolevulinic acid (5-ALA)-based fluorescence spectroscopy (FS) in vivo with conventional colposcopy for cervical intraepithelial neoplasia (CIN) detection. METHODS: We enrolled 48 patients who were referred for colposcopy because of high-grade changes in cervical cytology. Every inspected cervix was divided in to quadrants, and there were 174 quadrants included in the study. Each patient had a cytological smear, colposcopy, FS and histopathological analysis. For FS, 3% 5-ALA cream was used topically and after an average 135 min incubation, fluorescence spectra were recorded from the cervix in vivo. FS and colposcopy results were correlated with histopathology. RESULTS: All spectra were evaluated by a ratio of the protoporphyrin IX fluorescence intensity at 634 nm and autofluorescence intensity at 510 nm. For proper grouping of low-risk and high-risk cases, a threshold of 3.87 was calculated. Data per quadrant showed that FS had higher sensitivity than colposcopy (71.7% vs 67.4%) but specificity was greater for colposcopy (86.6% vs 75.6%). Combination of the methods showed higher sensitivity (88.0% vs 67.4%) but reduced specificity (88.0% and 69.5%), but it had the highest number of correctly identified high-risk changes and the highest (79.3%) accuracy. Data for each patient showed FS sensitivity of 91.2%, which was greater than for colposcopy (88.2%). Higher overdiagnosis resulted in decreased specificity for fluorescence methodology-71.4% versus 78.6% for colposcopy. In both cases, accuracy was 85.4% and effectiveness was >80%, which means that both methods can be used to determine high-risk cervical intraepithelial neoplasia. The diagnostic sensitivity of 97.1% for this complementary diagnosis indicates that it could be the best choice for detection of high-risk changes. CONCLUSIONS: 5-ALA-based FS is an objective method, requiring short-term administration for appropriate fluorescence measurements. FS is a promising diagnostic tool with similar accuracy as colposcopy but with the potential advantage of providing objective results.

Nevomelanocytic atypia detection by in vivo reflectance confocal microscopy.

BACKGROUND AND OBJECTIVE: In vivo reflectance confocal microscopy (RCM) is a promising novel technology for non-invasive early diagnostics of cutaneous melanoma. However, the possibility to detect melanocytic atypia in nevi by means of in vivo RCM remains unknown. The aim of the study was to evaluate the significance of in vivo RCM features of melanocytic atypia for the diagnosis of melanocytic nevi, dysplastic nevi and cutaneous melanoma. MATERIALS AND METHODS: A total of 138 melanocytic skin lesions comprising 25 melanocytic nevi, 69 dysplastic nevi and 44 melanomas were analyzed by means of dermoscopy, in vivo RCM and routine histopathology. In vivo RCM images were analyzed for the arrangement of keratinocytes in epidermis, pagetoid cells and junctional melanocytic nests and correlated refractivity aspects of nests with histopathology. RESULTS: Separately and all together taken the in vivo RCM features of melanocytic atypia were significant in differential diagnosis of benign and malignant melanocytic skin lesions, though none of the features was significant in discriminating nevi without cytologic atypia of dysplastic nevi. In vivo RCM feature of dense cell clusters corresponded with melanin containing nevomelanocytes on histopathology though exact correspondence of non-homogeneous and atypical sparse cell clusters remained questionable. CONCLUSIONS: Nevus with histopathologically confirmed nevomelanocytic atypia (dysplastic nevus) could not be distinguished from nevus without atypia using analyzed in vivo RCM features of melanocytic atypia. More accurate diagnostics by means of in vivo RCM needs further investigation on reflectance of single and nested cutaneous melanocytes in benign and malignant skin lesions.

Accumulation and biological effects of cobalt ferrite nanoparticles in human pancreatic and ovarian cancer cells.

BACKGROUND AND OBJECTIVE: Superparamagnetic iron oxide nanoparticles (SPIONs) emerge as a promising tool for early cancer diagnostics and targeted therapy. However, both toxicity and biological activity of SPIONs should be evaluated in detail. The aim of this study was to synthesize superparamagnetic cobalt ferrite nanoparticles (Co-SPIONs), and to investigate their uptake, toxicity and effects on cancer stem-like properties in human pancreatic cancer cell line MiaPaCa2 and human ovarian cancer cell line A2780. MATERIALS AND METHODS: Co-SPIONs were produced by Massart's co-precipitation method. The cells were treated with Co-SPIONs at three different concentrations (0.095, 0.48, and 0.95µg/mL) for 24 and 48h. Cell viability and proliferation were analyzed after treatment. The stem-like properties of cells were assessed by investigating the cell clonogenicity and expression of cancer stem cell-associated markers, including CD24/ESA in A2780 cell line and CD44/ALDH1 in MiaPaCa2 cell line. Magnetically activated cell sorting was used for the separation of magnetically labeled and unlabeled cells. RESULTS: Both cancer cell lines accumulated Co-SPIONs, however differences in response to nanoparticles were observed between MiaPaCa2 and A2780 cell. In particular, A2780 cells were more sensitive to exposition to Co-SPIONs than MiaPaCa2 cells, indicating that a safe concentration of nanoparticles must be estimated individually for a particular cell type. Higher doses of Co-SPIONs decreased both the clonogenicity and ESA marker expression in A2780 cells. CONCLUSIONS: Co-SPIONs are not cytotoxic to cancer cells, at least when used at a concentration of up to 0.95µg/mL. Co-SPIONs have a dose-dependent effect on the clonogenic potential and ESA marker expression in A2780 cells. Magnetic detection of low concentrations of Co-SPIONS in cancer cells is a promising tool for further applications of these nanoparticles in cancer diagnosis and treatment; however, extensive research in this field is needed.

Distribution of polyethylene glycol coated quantum dots in mice skin.

The distribution of nanoparticles (NP) in an organism is an important issue for developing NP-based drug delivery systems and for general nanotoxicology. The knowledge of NP localisation in the skin is crucial for the optimisation of NP behaviour in vivo. Therefore, we have used semiconductor quantum dots (QD) to investigate their biodistribution in the skin by means of confocal fluorescence microscopy after subcutaneous injection. The results obtained showed that the diffusion of QD in the dermis is limited by basement membrane and dense connective tissue fibres, which resulted in negligible QD penetration into the epidermis, hair follicles, sebaceous and sweat glands, nerves and blood vessels. Low permeation of QD through the tissues results in slow clearance and raises the risks of potential immune, inflammatory and cytotoxic responses. The study reveals the significance of the tissue architecture for the interstitial and intracellular migration patterns of non-functionalised QD.

High resolution imaging of the human cardiac conduction system using reflectance confocal microscopy.

Rhythmical contraction of the heart is controlled by the cardiac conduction system (CCS) that consists of the three main parts: the sino-atrial node, the atrioventricular node and the His-Purkinje system. A heartbeat signal, originated from CCS, spreads through its branches to the different parts of the heart, initiating depolarization of the ventricles. However, this highly important system could not be distinguished visually from the surrounding heart tissues: myocardium (MC) and connective tissue (CT). Thus, during surgical procedures, CCS could be easily damaged; namely, the reliable method for identification of CCS either in vivo or ex vivo does not exist. Accordingly, there is a definite need for developing a CCS imaging method. Reflection confocal microscopy (RCM) offers non-destructive imaging of the tissue at depths of up to 0.35 mm with the capability of identification of a single cell. During the visualization procedure, a given tissue is illuminated with infrared laser light and the image is obtained because of different reflections from the tissue structures. However, the reflective structures in the heart tissues are still not identified. In the present study, for the first time we investigated cardiac tissues by RCM. The resolution of the method allowed us to distinguish MC cells and CCS cells. The method also allowed us to distinguish the network-like structures that are main components of CT. The ability to visualize different tissue components indicates a great potential for RCM to be used in non-destructive cardiac investigations and for imaging CCS.

The effect of nanoparticles in rats during critical periods of pregnancy.

BACKGROUND AND OBJECTIVE. Nanotechnology works with substances at a nanometer scale, and it offers many solutions for biomedicine. Nanoparticles (NPs) have been shown as effective agents for imaging, drug delivery, pathogen detection, etc. However, to date, NP toxicity is poorly known. The aim of our study was to investigate the embryotoxicity and teratogenicity of quantum dots (QDs) at the different stages of rat embryogenesis. MATERIALS AND METHODS. Wistar rats were injected with CdSe/ZnS or CdTe QDs on the 6th, 13th, and 18th days of embryogenesis. Cyclophosphamide was chosen as a positive control of embryotoxicity. On the 21st day, the number of resorptions, weight, length, and external malformations of the embryos were estimated. Fluorescence spectroscopy and microscopy analysis were used to determine the accumulation of QDs in the tissues. RESULTS. Exposure to cyclophosphamide during the pregnancy decreased the embryonic weight and length when compared with the control group and produced numerous malformations. The effects depended on the stage of embryogenesis. Meanwhile, QDs did not cause any embryotoxic or teratogenic effects. However, CdTe QDs induced necrosis in the tissues of the peritoneal cavity. The necrotic tissues contained QDs with altered spectroscopic properties. Spectroscopic and microscopic tissue examination revealed that QDs accumulated in the placenta, but no penetration to the embryonic tissues was observed. CONCLUSIONS. QDs did not cause any direct embryotoxic or teratogenic effects, but they had adverse effects on the maternal organism. The observed QD effects and the long-term accumulation of QDs in the maternal organism may increase the risk of adverse effects on embryo development.

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.

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.

Transport of nanoparticles through the placental barrier.

Nanoparticles (NP) are organic or inorganic substances, the size of which ranges from 1 to 100 nm, and they possess specific properties which are different from those of the bulk materials in the macroscopic scale. In a recent decade, NP were widely applied in biomedicine as potential probes for imaging, drug-delivery systems and regenerative medicine. However, rapid development of nanotechnologies and their applications in clinical research have raised concerns about the adverse effects of NP on human health and environment. In the present review, special attention is paid to the fetal exposure to NP during the period of pregnancy. The ability to control the beneficial effects of NP and to avoid toxicity during treatment requires comprehensive knowledge about the distribution of NP in maternal body and possible penetration through the maternal-fetal barrier that might impair the embryogenesis. The initial in vivo and ex vivo studies imply that NP are able to cross the placental barrier, but the passage to the fetus depends on the size and the surface coating of NP as well as on the experimental model. The toxicity assays indicate that NP might induce adverse physiological effects and impede embryogenesis. The molecular transport mechanisms which are responsible for the transport of nanomaterials across the placental barrier are still poorly understood, and there is a high need for further studies in order to resolve the NP distribution patterns in the organism and to control the beneficial effects of NP applications during pregnancy without impeding the embryogenesis.