Albumin-based nanoparticles: small, uniform and reproducible.

Nanomedicine carries the hope of precisely identifying and healing lesion sites in vivo. However, the reproducible synthesis of monodisperse protein nanoparticles smaller than 50 nm in diameter and up-scalable to industrial production still poses challenges to researchers. In this report, we describe nanoparticles, so called Absicles, based on an albumin matrix and prepared by a procedure developed by the authors. These Absicles are monodisperse with tunable diameters ranging from 15 nm to 70 nm respectively. They exhibit long term stability against decomposition and aggregation, exceeding many months. The synthesis of Absicles shows exceptionally high reproducibility concerning size, and is simple and cost-effective for up-scaling. Absicles, bearing appropriate targeting groups, bind with high specificity to colon carcinoma tissue ex vivo; they present an attractive platform for further development towards drug delivery applications.

Targeting Drug Delivery in the Elderly: Are Nanoparticles an Option for Treating Osteoporosis?

Nanoparticles bearing specific targeting groups can, in principle, accumulate exclusively at lesion sites bearing target molecules, and release therapeutic agents there. However, practical application of targeted nanoparticles in the living organism presents challenges. In particular, intravasally applied nanoparticles encounter physical and physiological barriers located in blood vessel walls, blocking passage from the blood into tissue compartments. Whereas small molecules can pass out of the blood, nanoparticles are too large and need to utilize physiological carriers enabling passage across endothelial walls. The issues associated with crossing blood-tissue barriers have limited the usefulness of nanoparticles in clinical applications. However, nanoparticles do not encounter blood-tissue barriers if their targets are directly accessible from the blood. This review focuses on osteoporosis, a disabling and common disease for which therapeutic strategies are limited. The target sites for therapeutic agents in osteoporosis are located in bone resorption pits, and these are in immediate contact with the blood. There are specific targetable biomarkers within bone resorption pits. These present nanomedicine with the opportunity to treat a major disease by use of simple nanoparticles loaded with any of several available effective therapeutics that, at present, cannot be used due to their associated side effects.

Molecular imaging with nanoparticles: the dwarf actors revisited 10 years later.

We explore present-day trends and challenges in nanomedicine. Creativity in the laboratories continues: the published literature on novel nanoparticles is now vast. Nanoagents are discussed here which are composed entirely of strongly photoluminescent materials, tunable to desired optical properties and of inherently low toxicity. We focus on "quantum nanoparticles" prepared from allotropes of carbon. The principles behind strong, tunable photoluminescence are quantum mechanical: we present them in simple outline. The major industries racing to develop these materials can offer significant technical guidance to nanomedicine, which could help to custom-design strongly signalling nanoagents specifically for stated clinical applications. Since such agents are small, they can be targeted easily, making active targeting possible. We consider it timely now to study the interactions nanoparticles undergo with tissue components in living animals and to learn to understand and overcome the numerous barriers the organism interposes between the blood and targets in or on parenchymal cells. As the near infra-red spectrum opens up, detection of glowing nanoparticles several centimeters deep in a living human subject becomes calculable and we present a simple way to do this. Finally, we discuss the slow-fuse and resource-inefficient entry of nanoparticles into clinical application. A first possible reason is failure to target across the body's barriers, see above. Second, in the sparse translational landscape funding and support gaps yawn widely between academic research and subsequent development. We consider the agendas of the numerous "stakeholders" participating in this sad landscape and point to some faint glimmers of hope for the future.

Screening and identification of molecular targets for cancer therapy.

In recent decades, targeted therapeutics have significantly improved therapy results in patients with malignant tumors of different origins. However, malignant diseases characterized by aggressiveness and increased capacity for metastatic spread still require basic researchers and clinicians to direct enormous efforts toward the development of novel therapeutic targets. Potential targets should be selected with the clinical endpoint in view; targeted therapeutics can be developed: for use in combination with currently existing therapeutic approaches in order to improve their efficacy; to overcome the treatment resistance of tumor cells and thus protect the patient from recurrence; to repress molecular mechanisms related to immune escape of cancer cells; and to combat the metastatic dissemination of carcinoma cells. Taking into account the specific clinical aim that should be achieved, different strategies and techniques can be proposed to identify the most promising candidate molecules for further development as therapeutic targets. Since cellular membranes contain a large number of druggable molecules, evaluation of the membrane protein profiles of carcinoma cells having different properties can provide a basis for further development of therapeutic targets. This review considers how cellular membranes obtained from different pre-clinical and clinical samples can be used in screening and to identify targets for cancer therapy.

Radiation resistance: Cancer stem cells (CSCs) and their enigmatic pro-survival signaling.

Despite the fact that radiation therapy is a highly effective therapeutic approach, a small intratumoral cell subpopulation known as "cancer stem cells" (CSCs) is radiation-resistant and possesses specific molecular properties protecting it against radiation-induced damage. The exact mechanisms of this radioresistance are still not fully elucidated, but they relate to these cells' enhanced DNA repair capacities and their low intracellular ROS concentrations, resulting from their up-regulation of ROS scavengers. The low ROS content is accompanied by disturbances in cell cycle regulation, so it can be assumed that either CSCs are quiescent or dormant themselves, or that this cell population consists of at least two cell subpopulations: the normally and the slowly proliferating cells (quiescent or dormant cells). Slowly dividing CSCs show concomitant dysregulation of the signaling molecules mediating both cell cycle progression and maintenance of cell stemness. Despite a massive accumulation of data concerning the mechanisms underlying DNA damage response in CSCs, it represents a challenge to researchers in the era of personalized medicine to elucidate the role of intracellular ROS and of signaling pathways associated with the radiation resistance of these cells; there is a clear need to understand the molecular mechanisms helping CSCs to survive radiation exposure.

Proteomic approach to understand metastatic spread.

The majority of tumor-related deaths are due to metastasis. Despite the clinical importance of understanding metastasis, we lack knowledge of the molecular mechanisms underlying tumor cell spreading and cell survival far from the primary tumor. Elucidating the molecular characteristics of highly metastatic carcinoma cells would help identify biomarkers or therapeutic targets relevant to predicting or combatting metastasis, and for this the phenotype of metastatic cells could be much more important than their genotype. Hence, proteomic approaches have wide potential utility. This review discusses possibilities of analyzing metastasis-specific protein patterns in a range of sample types, including in vitro and in vivo cancer models, and tissues and biological fluids from patients. Proteome approaches can identify proteins involved in regulating the metastatic capacities of tumors.

Crosstalk between DNA repair and cancer stem cell (CSC) associated intracellular pathways.

DNA damaging agents (ionizing radiation and chemotherapeutics) are considered as most effective in cancer treatment. However, there is a subpopulation of carcinoma cells within the tumour demonstrating resistance to DNA damaging treatment approaches. It is suggested that limited tumour response to this kind of therapy can be associated with specific molecular properties of carcinoma stem cells (CSCs) representing the most refractory cell subpopulation. This review article presents novel data about molecular features of CSCs underlying DNA damage response and related intracellular signalling.

Putative biomarkers and therapeutic targets associated with radiation resistance.

Radiation therapy plays an important role in the management of malignant tumors, however, the problem of radiation resistance resulting in tumor recurrences after treatment is still unsolved. The emergence of novel biomarkers to predict cancer cell insensitivity to ionizing radiation could help to improve therapy results in cancer patients receiving radiation therapy. The proteomic approach could be effectively used to identify proteins associated with cancer radiation resistance. It is generally believed that radiation resistance could be associated with cancer stem cell persistence within the tumor. Therefore, determination of the molecular characteristics of cancer stem cells could provide additional possibilities to discover novel biomarkers to predict radiation resistance in cancer patients. This review addresses proteome-based findings that could be used for further biomarker identification and preclinical and clinical validation.

Proteomics of cancer stem cells.

PURPOSE: New understanding of cancer stem cell (CSC) biology continues to emerge due to development of novel methods in genomics and proteomics. Analysis of nucleic acids (RNA, DNA) is widely used to elucidate molecular perturbations in malignant tumors and carcinoma cells, however genome data do not reflect the functional activities of encoded proteins. Therefore proteome-based methods could enhance knowledge about deregulation of pathways as a result of altered expression and activities of proteins in CSC. METHODS AND RESULTS: A sufficient number of CSC for proteomic analyses can be obtained in a variety of ways: Fluorescence (FACS) and magnetic (MACS) activated cell sorting, laser cell capture microdissection, and three-dimensional spheroid/organoid cell culture. These methods to enrich and isolate CSC can be performed either with or without staining using antibodies against currently known CSC-specific cell surface molecules, such as clusters of differentiation 44, 24, 133 (CD44, CD24, CD133), epithelial cell adhesion molecule (EpCAM), aldehyde-dehydrogenase-1 (ALDH1), etc. The most important limitation on using antibody-based staining of CSC is that we still do not possess definitive CSC surface markers. This review article discusses methods that could be used to study protein profiling of CSC and to identify novel CSC-specific biomarkers and therapeutic targets. CONCLUSION: Despite an opinion that the proteomic approach is time-consuming, laborious and difficult, this method can be used effectively to clarify which pathways are involved in regulating various intratumoral processes, including activation of CSC. Based on this point of view, searching and identification of single molecules as biomarkers or therapeutic targets could become possible when CSC-associated pathways are well described and clearly understood due to detailed investigation of the protein patterns in pre-clinical models and clinical samples.

Rac1 as a multifunctional therapeutic target to prevent and combat cancer metastasis.

Metastatic progression of malignant tumors resistant to conventional therapeutic approaches is an ultimate challenge in clinical oncology. Despite the efforts of basic and clinical researchers, there is still no effective treatment schedule to prevent or combat metastatic spread of malignant tumors. This report presents recent findings that could help in the development of targeted therapeutics directed against the most aggressive and treatment-resistant carcinoma cells. It was demonstrated that HNSCC carcinoma cell lines with acquired treatment resistance possessed increased number of cells with carcinoma stem cell (CSC) properties. Furthermore, resistant cells were characterized by increased expression of Rac1, enhanced cell migration, and accelerated release of proangio- and vasculogenic factors (VEGF-A) and influence on endothelial cell (HMEC-1) migration. Inhibition of Rac1 signaling in the treatment-resistant carcinoma cells can interrupt metastatic process due to anoikis restoration and decrease of cell migration. It is also suggested that carcinoma cells with repressed survival capacities will be characterized by reduced release of proangiogenic factors, resulting in the decrease of endothelial cell migration. Therefore targeting of Rac1-related pathways may be considered as a promising therapeutic approach to prevent or combat metastatic lesions.

Nanomaterial interference with early human placenta: Sophisticated matter meets sophisticated tissues.

Next to nothing is known about nanoparticle and nanofiber trafficking at the feto-maternal interface in early human pregnancy. As the first trimester is thought to be crucial for the further placental and fetal development, it will be important to assess the possible risks of nanomaterial exposures during this period. There are some intriguing observations in nanotoxicology, however, indicating certain differences between classical toxicology and nanotoxicology. To understand nanomaterial-biokinetics and placental toxicity in early gestation, the special architecture, the hypoxic condition, the bilayer of villous trophoblast, the plugging of spiral arteries and the contribution of intrauterine glands to nutrition, as well as the delicate immunologic situation at the implantation site, will have to be considered. Unless nano-specific biokinetics are properly understood, it will be difficult to ensure identification of potential "nano-thalidomides" among all the newly engineered nanoparticles and fibers, based on the models available in reproductive toxicology.

Radioresistant head and neck squamous cell carcinoma cells: intracellular signaling, putative biomarkers for tumor recurrences and possible therapeutic targets.

PURPOSE: Treatment of local and distant head and neck cancer recurrences after radiotherapy remains an unsolved problem. In order to identify potential targets for use in effective therapy of recurrent tumors, we have investigated protein patterns in radioresistant (FaDu-IRR and SCC25-IRR, "IRR cells") as compared to parental (FaDu and SCC25) head and neck carcinoma cells. METHODS AND MATERIALS: Radiation resistant IRR cells were derived from parental cells after repeated exposure to ionizing radiation 10 times every two weeks at a single dose of 10 Gy, resulting in a total dose of 100 Gy. Protein profiling in parental and IRR cells was carried out using two-dimensional differential gel electrophoresis (2D-DIGE) followed by MALDI-TOF/TOF mass spectrometry. Cell viability, cell migration assays and Western blot analysis were used to confirm results obtained using the proteome approach. RESULTS: Forty-five proteins that were similarly modulated in FaDu-IRR and SCC25-IRR cells compared to parental cells were selected to analyze their common targets. It was found that these either up- or down-regulated proteins are closely related to the enhancement of cell migration which is regulated by Rac1 protein. Further investigations confirmed that Rac1 is up-regulated in IRR cells, and inhibiting its action reduces the migratory abilities of these cells. Additionally, the Rac1 inhibitor exerts cytostatic effects in HNSCC cells, mostly in migratory cells. CONCLUSIONS: Based on these results, we conclude that radioresistant HNSCC cells possess enhanced metastatic abilities that are regulated by a network of migration-related proteins. Rac1 protein may be considered as a putative biomarker of HNSCC radiation resistance, and as a potential therapeutic target for treating local and distant HNSCC recurrences.

Lectin conjugates as biospecific contrast agents for MRI. Coupling of Lycopersicon esculentum agglutinin to linear water-soluble DTPA-loaded oligomers.

Magnetic resonance imaging (MRI) requires synthesis of contrast media bearing targeting groups and numerous gadolinium chelating groups generating high relaxivity. This paper explores the results of linking the gadolinium chelates to the targeting group, a protein molecule, via various types of linkers. Polycondensates of diethylenetriaminepentaacetic acid (DTPA) with either diols or diamines were synthesised and coupled to the targeting group, a lectin (Lycopersicon esculentum agglutinin, tomato lectin) which binds with high affinity to specific oligosaccharide configurations in the endothelial glycocalyx. The polycondensates bear up to four carboxylic groups per constitutive unit. Gd-chelate bonds are created through dative interactions with the unshared pair of electrons on each oxygen and nitrogen atom on DTPA. This is mandatory for complexation of Gd(III) and avoidance of the severe toxicity of free gadolinium ions. The polymer-DTPA compounds were characterised by (1)H NMR and mass spectrometry. The final lectin-DTPA-polycondensate conjugates were purified by fast protein liquid chromatography (FPLC). The capacity for specific binding was assessed, and the MRI properties were examined in order to evaluate the use of these oligomers as components of selective perfusional contrast agents.

Lectin-Gd-loaded chitosan hydrogel nanoparticles: a new biospecific contrast agent for MRI.

PURPOSE: Non-specific extracellular contrast agents have been on the market for more than 15 years. Here, we report on the synthesis of new selective lectin-gadolinium (Gd)-loaded chitosan nanoparticles with a prolonged clearance time and a much higher relaxivity in comparison to other preparations. PROCEDURES: Chitosan nanoparticles were prepared from 85% deacetylated chitin by glutaraldehyde cross-linking of an aqueous acetic acid dispersion of chitosan in a mixture of n-hexane using sodium bis(ethylhexyl)sulfosuccinate as a surfactant. RESULTS: Several crucial parameters, namely, the Gd and protein content of the nanoparticles, their size and dispersity were determined. Magnetic resonance measurements were carried out by intravenous perfusion of mono-disperse suspensions of the nanoparticles into mice. CONCLUSIONS: Chitosan nanoparticles can be used as contrast agents in magnetic resonance imaging (MRI). They are excellent candidates for controlled delivery of bioactive compounds to molecular targets and as biospecific diagnostic tools in MRI.

Lead contributes to arterial intimal hyperplasia through nuclear factor erythroid 2-related factor-mediated endothelial interleukin 8 synthesis and subsequent invasion of smooth muscle cells.

OBJECTIVE: To validate the hypothesis that the toxic heavy metal lead (Pb) may be linked to cardiovascular diseases via the initiation of atherosclerosis, in vivo and in vitro studies were conducted. METHODS AND RESULTS: During the human study part of this project, serum Pb levels of healthy young women were correlated to carotid intima-media thickness. Multivariate logistic regression analyses showed that increased serum Pb levels were significantly associated with an increased intima-media thickness (P=0.01; odds ratio per SD unit, 1.6 [95% CI, 1.1 to 2.4]). In vitro, Pb induced an increase in interleukin 8 production and secretion by vascular endothelial cells. Nuclear factor erythroid 2-related factor-2 is the crucial transcription factor involved in Pb-induced upregulation of interleukin 8. Endothelial cell-secreted interleukin 8 triggered intimal invasion of smooth muscle cells and enhanced intimal thickening in an arterial organ culture model. This phenomenon was further enhanced by Pb-increased elastin synthesis of smooth muscle cells. CONCLUSIONS: Our data support the hypothesis that Pb is a novel, independent, and significant risk factor for intimal hyperplasia.

Epithelial-to-mesenchymal transition and c-myc expression are the determinants of cetuximab-induced enhancement of squamous cell carcinoma radioresponse.

PURPOSE: Radiation therapy cures malignant tumors of the head and neck region more effectively when it is combined with application of the anti-EGFR monoclonal antibody cetuximab. Despite the successes achieved, we still do not know how to select patients who will respond to this combination of anti-EGFR monoclonal antibody and radiation. This study was conducted to elucidate possible mechanisms which cause the combined treatment with cetuximab and irradiation to fail in some cases of squamous cell carcinomas. METHODS AND MATERIALS: Mice bearing FaDu and A431 squamous cell carcinoma xenograft tumors were treated with cetuximab (total dose 3 mg, intraperitoneally), irradiation (10 Gy) or their combination at the same doses. Treatment was applied when tumors reached 8mm in size. To collect samples for further protein analysis (two-dimensional differential gel electrophoresis (2-D DIGE), mass spectrometry MALDI-TOF/TOF, Western blot analysis, and ELISA), mice from each group were sacrificed on the 8th day after the first injection of cetuximab. Other mice were subjected to tumor growth delay assay. RESULTS: In FaDu xenografts, treatment with cetuximab alone was nearly as effective as cetuximab combined with ionizing radiation, whereas A431 tumors responded to the combined treatment with significantly enhanced delay in tumor growth. Tumors extracted from the untreated FaDu and A431 xenografts were analysed for protein expression, and 34 proteins that were differently expressed in the two tumor types were identified. The majority of these proteins are closely related to intratumoral angiogenesis, cell adhesion, motility, differentiation, epithelial-to-mesenchymal transition (EMT), c-myc signaling and DNA repair. CONCLUSIONS: The failure of cetuximab to enhance radiation response in FaDu xenografts was associated with the initiation of the program of EMT and with c-myc up-regulation in the carcinoma cells. For this reason, c-myc and EMT-related proteins (E-cadherin, vimentin) may be considered as potential biomarkers to predict squamous cell carcinoma response after treatment with cetuximab in combination with radiation.

Imaging of placental transport mechanisms: a review.

Functional analysis of material transfers requires precise statement of residence times in each tissue compartment. For the placenta, neither extractive biochemistry, isotope partitioning, nor mass-based quantitative assays provide adequate spatial resolution to allow the necessary precision. Dual-perfusion assays of material transfer in isolated placental cotyledons provide time-series data for two compartments, the maternal and fetal blood, but fail to distinguish the two cellular compartments (syncytiotrophoblast, fetal endothelium) which actively regulate rates of transfer in each direction for essentially every important molecule type. At present, no definitive technology exists for functional analysis of placental transfer functions. The challenge in developing such a technology lies in the exquisitely small and delicate structures involved, which are scaled at cellular and subcellular sizes (between 50 nm and 50 microm). The only available technologies attaining this high spatial resolution are imaging technologies, primarily light and electron microscopy. To achieve the high-quality images necessary, confocal laser scanning microscopy (CLSM) is required, to provide a uniform optical sectioning plane. In turn, this requires relatively high fluorescence intensities. Design of an adequate technology therefore bases on CLSM imaging fluorochrome-tagged tracers. The temporal resolution necessary to analyse placental material transfers is expected to be of the order of a few seconds, so that conventional wet-fixation protocols are too slow. For adequately rapid fixation, snap-freezing is required. As part of this review we report results obtained from an appropriately designed experimental protocol, analysed by CLSM and transmission electron microscopy (TEM). The images acquired were tested for uniformity of illumination and fluorescence emission strength. Relevant data was encoded in the green channel of the trichrome images obtained, and this was thresholded by application of strict quantitative criteria. The thresholding procedure is suitable for automation and produces reproducible, objectifiable results. Thresholded images were subjected to image calculation procedures designed to highlight image elements (pixels) containing (green) fluorescence associated with the tracer protein; all other sources of fluorescence were visualised in the final images only if no green fluorescence was detectable in that pixel. The resulting images were maps, showing the distribution of tracer molecules at a predefined time interval after perfusion of the tracer into the vital (term) cotyledon. Spatial resolution was routinely better than 1 microm and temporal resolution was approximately 5s. At timepoints up to 10 min after intravital application into the fetal vascular circulation, tracer was associated with capillaries in the villous structures, and no tracer was observed in the syncytiotrophoblast. Clear distinction was achieved between the four tissue compartments relevant to placental transfers, thus providing a novel technology capable of generating high-quality data concerning the regulation of transfers of any molecule that can bear a fluorescent tag. The potential applications of this methodology lie in analyses of factors influencing the rates of fetomaternal and maternofetal exchanges (for example, drugs), and of functional responses of the placental regulation to pathophysiological conditions such as hypoxia.