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Simple and up-scalable synthesis method for human serum albumin nanoparticles with narrow size distribution, tunable size range, stable and reproducible.

Molecular imaging using magnetic resonance imaging (MRI) is expected to play a crucial future role in oncological diagnosis and in monitoring of therapeutic progress. Targeted nanoparticle contrast media (CM) with high relaxivities are required in order to obtain adequate signal-to-noise ratios as well as visualization of a desired pathologic area of the human body. The aims of this study were to synthesize and define certain physicochemical and enhancement properties of new doubly derivatized polylactic acid–bovine serum albumin (PLA-BSA) nanoparticles (NPs) modified by the covalent coupling of glutaraldehyde as a crosslinking agent. An additional functionalization with endothelial cells (ECs) targeting groups (tomato lectins; LEA) and signal-emitting moieties (DTPA-Gd) enables its use as a macromolecular, biodegradable contrast agent for MRI. The NPs were characterized by different spectroscopies, size exclusion chromatography, and scanning and transmission electron microscopy. In a human vein model, the dynamics of the nanoparticle interactions with the vein wall were examined in MRI, with correlative imaging in electron microscopy. In vitro studies were conducted to show endothelial binding and persistent enhancement at the apical EC surface. NPs with a diameter between 55 and 75 nm, able to carry simultaneous signal emitting, and targeting motifs on a single construct were successfully prepared. A high Gd payload and endothelial binding to blood vessel walls were observed. The binding affinity and specificity of LEA was preserved, and a strong enhancement at the endothelium was achieved. The stabilized core–shell structure of PLA-NP might allow for further encapsulation of lipophilic drugs or for attachment of other targeting molecules, such as antibodies. Graphical abstract

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.

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.

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.

Elevated fasting and postprandial plasma levels of triglyceride-rich lipoproteins (TGRLs), i.e. VLDL/remnants and chylomicrons/remnants, are a characteristic feature of insulin resistance and are considered a consequence of this state. The aim of this study was to investigate whether intact TGRL particles are capable of inducing insulin resistance. We studied the effect of highly purified TGRLs on glycogen synthesis, glycogen synthase activity, glucose uptake, insulin signalling and intramyocellular lipid (IMCL) content using fully differentiated L6 skeletal muscle cells. Incubation with TGRLs diminished insulin-stimulated glycogen synthesis, glycogen synthase activity, glucose uptake and insulin-stimulated phosphorylation of Akt and glycogen synthase kinase 3. Insulin-stimulated tyrosine phosphorylation of IRS-1, and IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase (PI3K) activity were not impaired by TGRLs, suggesting that these steps were not involved in the lipoprotein-induced effects on glucose metabolism. The overall observed effects were time- and dose-dependent and paralleled IMCL accumulation. NEFA concentration in the incubation media did not increase in the presence of TGRLs indicating that the effects observed were solely due to intact lipoprotein particles. Moreover, co-incubation of TGRLs with orlistat, a potent active-site inhibitor of various lipases, did not alter TGRL-induced effects, whereas co-incubation with receptor-associated protein (RAP), which inhibits interaction of TGRL particles with members of the LDL receptor family, reversed the TGRL-induced effects on glycogen synthesis and insulin signalling. Our data suggest that the accumulation of TGRLs in the blood stream of insulin-resistant patients may not only be a consequence of insulin resistance but could also be a cause for it.

Perichondral bone, the circumferential grooves of Ranvier and cartilage canals are features of endochondral bone development. Cartilage canals containing connective tissue and blood vessels are found in the epiphysis of long bones and in cartilaginous anlagen of small and irregular bones. The pattern of cartilage canals seems to be integral to bone development and ossification. The canals may be concerned with the nourishment of large masses of cartilage, but neither their role in the formation of ossification centres nor their interaction with the circumferential grooves of Ranvier has been established. The relationships between cartilage canals, perichondral bone and the ossification centre were studied in the calcaneus of 9 to 38-wk-old human fetuses, by use of epoxy resin embedding, three-dimensional computer reconstructions and immunhistochemistry on paraffin sections. We found that cartilage canals are regularly arranged in shells surrounding the ossification centre. Whereas most of the shell canals might be involved in the nourishment of the cartilage, the inner shell is directly connected with the perichondral ossification groove of Ranvier and with large vessels from outside. In this way the inner shell canal imports extracellular matrix, cells and vessels into the cartilage. With the so-called communicating canals it is also connected to the endochondral ossification centre to which it delivers extracellular matrix, cells and vessels. The communicating canals can be considered as inverted ‘internal’ ossification grooves. They seem to be responsible for both build up intramembranous osteoid and for the direction of growth and thereby for orientation of the ossication centre.

Intravital lectin perfusion was combined with computer-guided scanning digital microscopy to map the perfused elements of the vasculature in tumor-bearing mice. High-precision composite images (spatial precision 1.3 μm and optical resolution 1.5 μm) were generated to permit exact positioning, reconstruction, analysis, and mapping of entire tumor cross-sections (c. 1 cm in diameter). Collation of these mosaics with nuclear magnetic resonance maps in the same tumor plane identified sites of rapid contrast medium uptake as tumor blood vessels. Digitized imaging after intravital double labeling allowed polychromatic visualization of two different types of mismatched staining. First, simultaneous application of two lectins, each bearing a different fluorochrome, revealed organ-specific differential processing in the microvascular wall. Second, sequential application of two boluses of one lectin, bearing different fluorochromes successively, distinguished between double-labeled microvessels, representing efficiently perfused vascular segments, and single-labeled microvessels, with inefficient or intermittent perfusion. Intravital lectin perfusion images of blood vessels in the vital functional state thus highlighted biologically significant differences in vessel function and served as high-resolution adjuncts to MR imaging.

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. Conclusion— Our data support the hypothesis that Pb is a novel, independent, and significant risk factor for intimal hyperplasia.

We demonstrate bio-medical imaging using a Terahertz quantum cascade laser. This new optoelectronic source of coherent Terahertz radiation allows building a compact imaging system with a large dynamic range and high spatial resolution. We obtain images of a rat brain section at 3.4 THz. Distinct regions of brain tissue rich in fat, proteins, and fluid-filled cavities are resolved showing the high contrast of Terahertz radiation for biological tissue. These results suggest that continuous-wave Terahertz imaging with a carefully chosen wavelength can provide valuable data on samples of biological origin; these data appear complementary to those obtained from white-light images.

Molecular imaging, first developed to localise antigens in light microscopy, now encompasses all imaging modalities including those used in clinical care: optical imaging, nuclear medical imaging, ultrasound imaging, CT, MRI, and photoacoustic imaging. Molecular imaging always requires accumulation of contrast agent in the target site, often achieved most efficiently by steering nanoparticles containing contrast agent into the target. This entails accessing target molecules hidden behind tissue barriers, necessitating the use of targeting groups. For imaging modalities with low sensitivity, nanoparticles bearing multiple contrast groups provide signal amplification. The same nanoparticles can in principle deliver both contrast medium and drug, allowing monitoring of biodistribution and therapeutic activity simultaneously (theranostics). Nanoparticles with multiple bioadhesive sites for target recognition and binding will be larger than 20 nm diameter. They share functionalities with many subcellular organelles (ribosomes, proteasomes, ion channels, and transport vesicles) and are of similar sizes. The materials used to synthesise nanoparticles include natural proteins and polymers, artificial polymers, dendrimers, fullerenes and other carbon-based structures, lipid-water micelles, viral capsids, metals, metal oxides, and ceramics. Signal generators incorporated into nanoparticles include iron oxide, gadolinium, fluorine, iodine, bismuth, radionuclides, quantum dots, and metal nanoclusters. Diagnostic imaging applications, now appearing, include sentinal node localisation and stem cell tracking.

A polymer-grafted liposomal formulation that has the potential to be developed for aerosolic pulmonary delivery of vasoactive intestinal peptide (VIP), a potent vasodilatory neuropeptide, is described. As VIP is prone to rapid proteolytic degradation in the microenvironment of the lung a proper delivery system is required to increase the half-life and bioavailability of the peptide. Here we investigate structural parameters of unilamellar liposomes composed of palmitoyl-oleoyl-phosphatidylcholine, lyso-stearyl-phosphatidylglycerol and distearyl-phosphatidyl-ethanolamine covalently linked to polyethylene glycol 2000, and report on VIP–lipid interaction mechanisms. We found that the cationic VIP is efficiently entrapped by the negatively charged spherical liposomes and becomes converted to an amphipathic α-helix. By fluorescence spectroscopy using single Trp-modified VIP we could show that VIP is closely associated to the membrane. Our data suggest that the N-terminal random-coiled domain is embedded in the interfacial headgroup region of the phospholipid bilayer. By doing so, neither the bilayer thickness of the lipid membrane nor the mobility of the phospholipid acyl chains are affected as shown by small angle X-ray scattering and electron spin resonance spectroscopy. Finally, in an ex vivo lung arterial model system we found that liposomal-associated VIP is recognized by its receptors to induce vasodilatory effects with comparable high relaxation efficiency as free VIP but with a significantly retarded dilatation kinetics. In conclusion, we have designed and characterized a liposomal formulation that is qualified to entrap biologically active VIP and displays structural features to be considered for delivery of VIP to the lung.

‘‘Intermediate Filaments in Health and Disease’’ were the focus of the scientific program of the 55th Symposium of the Society for Histochemistry in Prague, Czech Republic. Various aspects of intermediate filaments (IF) and associated proteins were presented and discussed with emphasis on imaging and sample preparation methods including both light and electron microscopy and specific labeling techniques. The Symposium was organized in cooperation with BMBS COST Action Nanomechanics of Intermediate Filaments Networks (NANONET) and took place in the Conference Center of the Institute of the Molecular Genetics, Academy of Science of the Czech Republic. Pavel Hozak as the local organizer together with his dedicated team did put together a very exciting and representative program. Robert D. Goldman (Northwestern University Medical School, Chicago, USA) presented the ‘‘Robert Feulgen Lecture’’ sponsored by the Society of Histochemistry and communicated ‘‘Recent Insights into Cytoskeletal and Nucleoskeletal Intermediate Filaments Networks.’’ Ueli Aebi (M.E. Muller Institute for Structural Biology, University of Basel, Switzerland) presented the ‘‘EMBO Lecture’’ and gave a critical appraisal of ‘‘IF Research—Quo Vadis?’’ Nam-On Ku (Yonsei University, Seoul, South Korea) contributed the ‘‘Histochemistry and Cell Biology Lecture 2013’’ and reviewed ‘‘Keratin Function in Simple Type Epithelia’’ (Yi and Ku 2013). In the Session on ‘‘Regulation of IF Assembly and Dynamics in Vivo and in Vitro,’’ Harish C. Pant (Laboratory of Neuronal Cytoskeletal Protein Regulation Section, NINDS, NIH Bethesda, USA) presented an update on the topographic regulation of neuronal IF proteins in health and disease (Binukumar et al. 2013). In the Session on ‘‘IF Structure, Mechanical Properties and Mechanotransduction,’’ Ohad Medalia (Ben-Gurion University, Beer-Sheva, Israel) reviewed structural aspects of lamins and of the nuclear lamina organization as they classically relate to the shape of the nuclear envelope and the nucleus as well as the more recently recognized involvement of lamins in various nuclear functions such as DNA replication and repair, regulation of gene expression, and signaling. A comprehensive and critical appraisal of laminopathies caused by mutations of lamin A, and associated proteins were also presented (Zwerger and Medalia 2013). In the Session on ‘‘IF Associated Proteins and Regulatory Crosstalk in a Cell,’’ Gerhard Wiche (Max F. Perutz Laboratories, University of Vienna, Austria) presented a most comprehensive perspective on plectin-intermediate filament partnership in skin, skeletal muscle, and peripheral nerve. He gave an overview of the structural features and functional properties of plectin and discussed most recent progress in defining the role of its isoforms in stress-prone tissues and the implicated diseases (Castanon et al. 2013). In the same session, Yassemi Capetanaki (Biomedical J. Roth (&) Department of Integrated OMICS for Biomedical Science, WCU Program of Graduate School, Yonsei University, Seoul 120-749, Korea e-mail: jurgen.roth@yonsei.ac.kr

Local application of rapamycin (sirolimus) by drug-eluting stents prevents lumen obliteration after angioplasty by inhibition of neointimal hyperplasia. The effects of rapamycin on neointimal smooth muscle cells (niSMC) which are responsible for the occurrence of restenosis have not been investigated so far.Rat niSMC and medial SMC (mSMC) were obtained from balloon catheter-injured arteries. The niSMC exhibited higher basal NF-kappaB activity and TNF-alpha mRNA levels. Nuclear protein binding to NF-kappaB-DNA was attenuated in niSMC by incubation with rapamycin (0.1 and 1 microg/ml) for 24 and 48 h. In contrast in mSMC, 0.1 microg/ml rapamycin had no effect and at 1 microg/ml even increased nuclear protein binding to NF-kappaB-DNA. After 12 h incubation, rapamycin (0.001-10 microg/ml) induced IkappaB-alpha protein in niSMC, whereas in mSMC it stimulated IkappaB-alpha at much lower levels. Prolonged rapamycin treatment (1 microg/ml for 72 h) had no effect on TNF-alpha mRNA level and NF-kappaB activity in niSMC, whereas it led to their increase in mSMC. Vascular endothelial growth factor (VEGF) secretion was higher in mSMC than in niSMC; rapamycin decreased VEGF levels in both cell types. Ultrastructural analysis suggested that rapamycin caused early signs of degeneration in niSMC, but enhanced protein synthesis in mSMC.This study shows that rapamycin influences the inflammatory phenotypes of SMC in opposite directions: it reduces the high basal NF-kappaB activity in niSMC and enhances NF-kappaB activity and TNF-alpha expression in mSMC. In addition, rapamycin inhibits VEGF production regardless of the phenotype of SMC. These findings shed light on molecular mechanisms and structural changes underlying therapeutic applications of rapamycin in prevention of restenosis, inhibition of chronic transplant arteriosclerosis and reduction of secondary malignoma formation due to immunosuppression.

Large radiation doses cause postradiation vascular hyperpermeability by disrupting endothelia. The cumulative sequences of small doses (fractionated radiotherapy) standard in clinical practice cause it too, but not by endothelial disruption: the mechanisms are unknown. In this study, correlated fluorescent and ultrastructural localisation of a tracer revealed the architecture, fine structure and function of microvessels in mouse AT17 tumours, before and after 42 Gy fractionated radiation. Before irradiation, tumour vascular permeability lay in the normophysiological range defined by the gut and cerebral cortex. A double barrier regulated permeability: vesicular transport through the endothelial wall required approximately 2 h and then the basement membrane charge barrier trapped tracer for 2 h longer. Irradiation abolished the double barrier: tracer passed instantly through both endothelial wall and underlying basement membrane, forming diffusion haloes around microvessels within 2–5 min. Structurally, irradiated tumour microvessels were lined by a continuous and vital endothelium with closed interendothelial junctions; endothelial basement membranes were intact, though loosened. Irradiated endothelia exhibited extremely active membrane motility and intracellular vesicle trafficking. Radiation treatment raised vascular permeability by enhancing transendothelial transcytosis, and by altering the passive filter properties of the subendothelial basement membrane. This type of vascular hyperpermeability should be susceptible to pharmacological modulation.

Chromatin condensation paralleled by DNA fragmentation is one of the most important nuclear events occurring during apoptosis. Histone modifications, and in particular phosphorylation, have been suggested to affect chromatin function and structure during both cell cycle and cell death. We report here that phosphate incorporation into all H1 subtypes decreased rapidly after induction of apoptosis, evidently causing a strong reduction in phosphorylated forms of main H1 histone subtypes. H1 dephosphorylation is accompanied by chromatin condensation preceding the onset of typical chromatin oligonucleosomal fragmentation, whereas H2A.X hyperphosphorylation is strongly correlated to apoptotic chromatin fragmentation. Using various kinase inhibitors we were able to exclude some of the possible kinases which can be involved directly or indirectly in phosphorylation of histone H2A.X. Neither DNA-dependent protein kinase, protein kinase A, protein kinase G, nor the kinases driven by the mitogen-activated protein kinase (MAP) pathway appear to be responsible for H2A.X phosphorylation. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA), however, markedly reduced the induction of apoptosis in TNFalpha-treated cells with a simultaneous change in the phosphorylation pattern of histone H2A.X. Hyperphosphorylation of H2A.X in apoptotic cells depends indirectly on activation of caspases and nuclear scaffold proteases as shown in zVAD-(OMe)-fmk- or zAPF-cmk-treated cells, whereas the dephosphorylation of H1 subtypes seems to be influenced solely by caspase inhibitors. Together, these results illustrate that H1 dephosphorylation and H2A.X hyperphosphorylation are necessary steps on the apoptotic pathway.

We are indebted to W. Platzer, O. Gaber and K.H. Kunzel (Innsbruck) for tissue sampling, B. Afzelius and B. Fundin (Stockholm), H. Lassmann and R. Valenta (Vienna), G. Rutter (Hamburg), M. Pavelka, W. Pfaller, W. Schmidt and G. Sperk (Innsbruck) for helpful discussions, and C. Huckhagel, K. Gutleben and R. Haring for technical assistance.