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.

Acute Radiation Dermatitis Evaluation with Reflectance Confocal Microscopy: A Prospective Study.

BACKGROUND: During radiotherapy (RT), most breast cancer patients experience ionizing radiation (IR)-induced skin injury-acute radiation dermatitis (ARD). The severity of ARD is determined by a physician according to CTCAE or RTOG scales, which are subjective. Reflectance confocal microscopy (RCM) is a noninvasive skin imaging technique offering cellular resolution. Digital dermoscopy (DD) performed in conjugation with RCM can provide more information regarding skin toxicity. The purpose of this study is to create an RCM and DD features-based ARD assessment scale, to assess the association with CTCAE scale and possible predictive value. METHODS: One hundred and three breast cancer patients during RT were recruited; every week, clinical symptoms of ARD (CTCAE scale) were evaluated and RCM, together with digital dermoscopy (DD), was performed. RESULTS: According to RCM; after 2 RT weeks, exocytosis and/or spongiosis were present in 94% of patients; after 3 weeks, mild contrast cells (MMCs) were detected in 45%; disarrayed epidermis (DE) was present in 66% of patients after 4 weeks and in 93% after 5 weeks; abnormal dermal papillae (ADP) were present in 68% of patients after 5 weeks. The coefficients of RCM features (RCMcoef) alone and together with dermoscopically determined erythema (RCM-ERYcoef) were significantly associated with ARD severity grade. RCMcoef is a significant predictive factor for the clinical manifestation of ARD. CONCLUSIONS: RCM features of irradiated skin appear earlier than clinical symptoms, have a characteristic course, and allow the severity of ARD to be predicted.

Hitchhiking Nanoparticles: Mesenchymal Stem Cell-Mediated Delivery of Theranostic Nanoparticles.

Nanotechnology has emerged as a promising solution to permanent elimination of cancer. However, nanoparticles themselves lack specificity to tumors. Due to enhanced migration to tumors, mesenchymal stem cells (MSCs) were suggested as cell-mediated delivery vehicles of nanoparticles. In this study, we have constructed a complex composed of photoluminescent quantum dots (QDs) and a photosensitizer chlorin e6 (Ce6) to obtain multifunctional nanoparticles, combining cancer diagnostic and therapeutic properties. QDs serve as energy donors-excited QDs transfer energy to the attached Ce6 via Förster resonance energy transfer, which in turn generates reactive oxygen species. Here, the physicochemical properties of the QD-Ce6 complex and singlet oxygen generation were measured, and the stability in protein-rich media was evaluated, showing that the complex remains the most stable in protein-free medium. In vitro studies on MSC and cancer cell response to the QD-Ce6 complex revealed the complex-loaded MSCs' potential to transport theranostic nanoparticles and induce cancer cell death. In vivo studies proved the therapeutic efficacy, as the survival of tumor-bearing mice was statistically significantly increased, while tumor progression and metastases were slowed down.

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.

Individual Radiosensitivity as a Risk Factor for the Radiation-Induced Acute Radiodermatitis.

BACKGROUND: Up to 95% of irradiated patients suffer from ionizing radiation (IR) induced early skin reaction, acute radiation dermatitis (ARD). Some experts think that additional skin hydration can reduce acute skin reactions. Individual radiosensitivity (IRS) determined from lymphocytes may help to predict acute radiation toxicity. The purpose of this study is to evaluate the clinical manifestation of ARD in different skincare groups during whole breast radiotherapy depending on IRS and other risk factors. METHODS: A total of 108 early-stage breast cancer patients were randomized into best supportive care (BSC) and additional skincare (ASC) groups. IRS was evaluated using a G2 assay modified with caffeine-induced G2 checkpoint arrest. All patients received a 50 Gy dose to the breast planning target volume (PTV). Clinical assessment of ARD symptoms according to the CTCAE grading scale was performed once a week. RESULTS: IRS was successfully determined for 91 out of 108 patients. A total of 10 patients (11%) had normal IRS, 47 patients (52%) were categorized as radiosensitive, and 34 (37%) as highly radiosensitive. There was no significant difference in the manifestation of ARD between patient groups by skincare or IRS. According to logistic regression, patients with bigger breasts were prone to more severe ARD (p = 0.002). CONCLUSIONS: The additional skincare did not improve skin condition during RT. A total of 89% of patients had increased radiosensitivity. IRS determined before RT did not show the predictive value for the manifestation of ARD. Logistic regression revealed that breast volume was the most significant risk factor for the manifestation of ARD.

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.

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.

Impact of Quantum Dot Surface on Complex Formation with Chlorin e6 and Photodynamic Therapy.

Nanomaterials have permeated various fields of scientific research, including that of biomedicine, as alternatives for disease diagnosis and therapy. Among different structures, quantum dots (QDs) have distinctive physico-chemical properties sought after in cancer research and eradication. Within the context of cancer therapy, QDs serve the role of transporters and energy donors to photodynamic therapy (PDT) drugs, extending the applicability and efficiency of classic PDT. In contrast to conventional PDT agents, QDs' surface can be designed to promote cellular targeting and internalization, while their spectral properties enable better light harvesting and deep-tissue use. Here, we investigate the possibility of complex formation between different amphiphilic coating bearing QDs and photosensitizer chlorin e6 (Ce6). We show that complex formation dynamics are dependent on the type of coating-phospholipids or amphiphilic polymers-as well as on the surface charge of QDs. Förster's resonant energy transfer occurred in every complex studied, confirming the possibility of indirect Ce6 excitation. Nonetheless, in vitro PDT activity was restricted only to negative charge bearing QD-Ce6 complexes, correlating with better accumulation in cancer cells. Overall, these findings help to better design such and similar complexes, as gained insights can be straightforwardly translated to other types of nanostructures-expanding the palette of possible therapeutic agents for cancer therapy.

Nanoparticle delivery to metastatic breast cancer cells by nanoengineered mesenchymal stem cells.

We created a 3D cell co-culture model by combining nanoengineered mesenchymal stem cells (MSCs) with the metastatic breast cancer cell line MDA-MD-231 and primary breast cancer cell line MCF7 to explore the transfer of quantum dots (QDs) to cancer cells. First, the optimal conditions for high-content QD loading in MSCs were established. Then, QD uptake in breast cancer cells was assessed after 24 h in a 3D co-culture with nanoengineered MSCs. We found that incubation of MSCs with QDs in a serum-free medium provided the best accumulation results. It was found that 24 h post-labelling QDs were eliminated from MSCs. Our results demonstrate that breast cancer cells efficiently uptake QDs that are released from nanoengineered MSCs in a 3D co-culture. Moreover, the uptake is considerably enhanced in metastatic MDA-MB-231 cells compared with MCF7 primary breast cancer cells. Our findings suggest that nanoengineered MSCs could serve as a vehicle for targeted drug delivery to metastatic cancer.

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.

Skin-derived mesenchymal stem cells as quantum dot vehicles to tumors.

PURPOSE: Cell-mediated delivery of nanoparticles is emerging as a new method of cancer diagnostics and treatment. Due to their inherent regenerative properties, adult mesenchymal stem cells (MSCs) are naturally attracted to wounds and sites of inflammation, as well as tumors. Such characteristics enable MSCs to be used in cellular hitchhiking of nanoparticles. In this study, MSCs extracted from the skin connective tissue were investigated as transporters of semiconductor nanocrystals quantum dots (QDs). MATERIALS AND METHODS: Cytotoxicity of carboxylated CdSe/ZnS QDs was assessed by lactate dehydrogenase cell viability assay. Quantitative uptake of QDs was determined by flow cytometry; their intracellular localization was evaluated by confocal microscopy. In vitro tumor-tropic migration of skin-derived MSCs was verified by Transwell migration assay. For in vivo migration studies of QD-loaded MSCs, human breast tumor-bearing immunodeficient mice were used. RESULTS: QDs were found to be nontoxic to MSCs in concentrations no more than 16 nM. The uptake studies showed a rapid QD endocytosis followed by saturating effects after 6 h of incubation and intracellular localization in the perinuclear region. In vitro migration of MSCs toward MDA-MB-231 breast cancer cells and their conditioned medium was up to nine times greater than the migration toward noncancerous breast epithelial cells MCF-10A. In vivo, systemically administered QD-labeled MSCs were mainly located in the tumor and metastatic tissues, evading most healthy organs with the exception being blood clearance organs (spleen, kidneys, liver). CONCLUSION: Skin-derived MSCs demonstrate applicability in cell-mediated delivery of nanoparticles. The findings presented in this study promise further development of a cell therapy and nanotechnology-based tool for early cancer diagnostics and therapy.

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.

Nano-engineered skin mesenchymal stem cells: potential vehicles for tumour-targeted quantum-dot delivery.

Nanotechnology-based drug design offers new possibilities for the use of nanoparticles in imaging and targeted therapy of tumours. Due to their tumour-homing ability, nano-engineered mesenchymal stem cells (MSCs) could be utilized as vectors to deliver diagnostic and therapeutic nanoparticles into a tumour. In the present study, uptake and functional effects of carboxyl-coated quantum dots QD655 were studied in human skin MSCs. The effect of QD on MSCs was examined using a cell viability assay, Ki67 expression analysis, and tri-lineage differentiation assay. The optimal conditions for QD uptake in MSCs were determined using flow cytometry. The QD uptake route in MSCs was examined via fluorescence imaging using endocytosis inhibitors for the micropinocytosis, phagocytosis, lipid-raft, clathrin- and caveolin-dependent endocytosis pathways. These data showed that QDs were efficiently accumulated in the cytoplasm of MSCs after incubation for 6 h. The main uptake route of QDs in skin MSCs was clathrin-mediated endocytosis. QDs were mainly localized in early endosomes after 6 h as well as in late endosomes and lysosomes after 24 h. QDs in concentrations ranging from 0.5 to 64 nM had no effect on cell viability and proliferation. The expression of MSC markers, CD73 and CD90, and hematopoietic markers, CD34 and CD45, as well as the ability to differentiate into adipocytes, chondrocytes, and osteocytes, were not altered in the presence of QDs. We observed a decrease in the QD signal from labelled MSCs over time that could partly reflect QD excretion. Altogether, these data suggest that QD-labelled MSCs could be used for targeted drug delivery studies.

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.

Suppression of a Specific Intracellular Uptake Pathway by a Saturating Accumulation of Quantum Dots.

Quantum dots (QDs) play an active role in triggering biological effects and should not be viewed as ordinary carriers for biomedical applications; therefore, the aim of this study was to investigate the molecular mechanisms involved in the saturating accumulation of non-targeted, carboxylated QDs, the related specific internalization pathways and the induced changes in the endocytotic cycle in NIH3T3 cells. We determined that the saturating accumulation of QDs suppressed the internalization of subsequently introduced QDs that had an identical chemical composition. However, the reinitiation of uptake was detected in the NIH3T3 cells after 8 h of incubation in medium without QDs. A very small suppressive effect of accumulated QDs was observed on uptake via the clathrin-mediated endocytosis pathway and macropinocytosis. In contrast, uptake via the caveolin-mediated pathway was almost completely prevented. Deeper insight into the suppression mechanism was obtained by transiently transfecting NIH3T3 cells with the plasmid pEGFP-C1-Caveolin-1. In these transfected cells, the usual intracellular presence of Caveolin-1 near the plasma membrane was not observed after long-term incubation with QDs. The putative application of QDs for diagnostic visualization in combination with certain anticancer substances was also evaluated. The QDs did not affect the intracellular photosensitization pattern of the amphiphilic molecule chlorin e6 in the case of photodynamic therapy. However, the saturating accumulation of QDs increased the resistance of NIH3T3 cells to the widely used anticancer drug cisplatin.