Albumin-based nanoparticles as contrast medium for MRI: vascular imaging, tissue and cell interactions, and pharmacokinetics of second-generation nanoparticles.

This multidisciplinary study examined the pharmacokinetics of nanoparticles based on albumin-DTPA-gadolinium chelates, testing the hypothesis that these nanoparticles create a stronger vessel signal than conventional gadolinium-based contrast agents and exploring if they are safe for clinical use. Nanoparticles based on human serum albumin, bearing gadolinium and designed for use in magnetic resonance imaging, were used to generate magnet resonance images (MRI) of the vascular system in rats ("blood pool imaging"). At the low nanoparticle doses used for radionuclide imaging, nanoparticle-associated metals were cleared from the blood into the liver during the first 4 h after nanoparticle application. At the higher doses required for MRI, the liver became saturated and kidney and spleen acted as additional sinks for the metals, and accounted for most processing of the nanoparticles. The multiple components of the nanoparticles were cleared independently of one another. Albumin was detected in liver, spleen, and kidneys for up to 2 days after intravenous injection. Gadolinium was retained in the liver, kidneys, and spleen in significant concentrations for much longer. Gadolinium was present as significant fractions of initial dose for longer than 2 weeks after application, and gadolinium clearance was only complete after 6 weeks. Our analysis could not account quantitatively for the full dose of gadolinium that was applied, but numerous organs were found to contain gadolinium in the collagen of their connective tissues. Multiple lines of evidence indicated intracellular processing opening the DTPA chelates and leading to gadolinium long-term storage, in particular inside lysosomes. Turnover of the stored gadolinium was found to occur in soluble form in the kidneys, the liver, and the colon for up to 3 weeks after application. Gadolinium overload poses a significant hazard due to the high toxicity of free gadolinium ions. We discuss the relevance of our findings to gadolinium-deposition diseases.

Rac1 as a potential therapeutic target for chemo-radioresistant head and neck squamous cell carcinomas (HNSCC).

BACKGROUND: In order to improve therapy for HNSCC patients, novel methods to predict and combat local and/or distant tumour relapses are urgently needed. This study has been dedicated to the hypothesis that Rac1, a Rho GTPase, is implicated in HNSCC insensitivity to chemo-radiotherapy resulting in tumour recurrence development. METHODS: Parental and radiation-resistant (IRR) HNSCC cells were used to support this hypothesis. All cells were investigated for their sensitivity to ionising radiation and cisplatin, Rac1 activity, its intracellular expression and subcellular localisation. Additionally, tumour tissues obtained from 60 HNSCC patients showing different therapy response were evaluated for intratumoral Rac1 expression. RESULTS: Radiation-resistant IRR cells also revealed resistance to cisplatin accompanied by increased expression, activity and trend towards nuclear translocation of Rac1 protein. Chemical inhibition of Rac1 expression and activity resulted in significant improvement of HNSCC sensitivity to ionising radiation and cisplatin. Preclinical results were confirmed in clinical samples. Although Rac1 was poorly presented in normal mucosa, tumour tissues revealed increased Rac1 expression. The most pronounced Rac1 presence was observed in HNSCC patients with poor early or late responses to chemo-radiotherapy. Tissues taken at recurrence were characterised not only by enhanced Rac1 expression but also increased nuclear Rac1 content. CONCLUSIONS: Increased expression, activity and subcellular localisation of Rac1 could be associated with lower early response rate and higher risk of tumour recurrences in HNSCC patients and warrants further validation in larger independent studies. Inhibition of Rac1 activity can be useful in overcoming treatment resistance and could be proposed for HNSCC patients with primary or secondary chemo-radioresistance.

Albumin-based nanoparticles as magnetic resonance contrast agents: II. Physicochemical characterisation of purified and standardised nanoparticles.

We are developing a nanoparticulate histochemical reagent designed for histochemistry in living animals (molecular imaging), which should finally be useful in clinical imaging applications. The iterative development procedure employed involves conceptual design of the reagent, synthesis and testing of the reagent, then redesign based on data from the testing; each cycle of testing and development generates a new generation of nanoparticles, and this report describes the synthesis and testing of the third generation. The nanoparticles are based on human serum albumin and the imaging modality selected is magnetic resonance imaging (MRI). Testing the second particle generation with newly introduced techniques revealed the presence of impurities in the final product, therefore we replaced dialysis with diafiltration. We introduced further testing methods including thin layer chromatography, arsenazo III as chromogenic assay for gadolinium, and several versions of polyacrylamide gel electrophoresis, for physicochemical characterisation of the nanoparticles and intermediate synthesis compounds. The high grade of chemical purity achieved by combined application of these methodologies allowed standardised particle sizes to be achieved (low dispersities), and accurate measurement of critical physicochemical parameters influencing particle size and imaging properties. Regression plots confirmed the high purity and standardisation. The good degree of quantitative physicochemical characterisation aided our understanding of the nanoparticles and allowed a conceptual model of them to be prepared. Toxicological screening demonstrated the extremely low toxicity of the particles. The high magnetic resonance relaxivities and enhanced mechanical stability of the particles make them an excellent platform for the further development of MRI molecular imaging.

Albumin-based nanoparticles as magnetic resonance contrast agents: I. Concept, first syntheses and characterisation.

To develop a platform for molecular magnetic resonance imaging, we prepared gadolinium-bearing albumin-polylactic acid nanoparticles in the size range 20-40 nm diameter. Iterative cycles of design and testing upscaled the synthesis procedures to gram amounts for physicochemical characterisation and for pharmacokinetic testing. Morphological analyses showed that the nanoparticles were spheroidal with rough surfaces. Particle sizes were measured by direct transmission electron microscopical measurements from negatively contrasted preparations, and by use of photon correlation spectroscopy; the two methods each documented nanoparticle sizes less than 100 nm and generally 10-40 nm diameter, though with significant intrabatch and interbatch variability. The particles' charge sufficed to hold them in suspension. HSA retained its tertiary structure in the particles. The nanoparticles were stable against turbulent flow conditions and against heat, though not against detergents. MRI imaging of liquid columns was possible at nanoparticle concentrations below 10 mg/ml. The particles were non-cytotoxic, non-thrombogenic and non-immunogenic in a range of assay systems developed for toxicity testing of nanoparticles. They were micellar prior to lyophilisation, but loosely structured aggregated masses after lyophilisation and subsequent resuspension. These nanoparticles provide a platform for further development, based on non-toxic materials of low immunogenicity already in clinical use, not expensive, and synthesized using methods which can be upscaled for industrial production.

In vivo investigation of thiomer-polyvinylpyrrolidon nanoparticles using magnetic resonance imaging.

This study focused on the investigation of the permeation enhancing effects of a stomach targeted, nanoparticulate drug delivery system. The polyacrylic acid-cysteine/polyvinylpyrrolidon nanoparticles were loaded with the magnetic resonance imaging (MRI) contrast agent diethylenetriaminepentaacetic acid gadolinium(III)dihydrogen salt (Gd-DTPA). Average particle size was determined to be 130 nm and the optimum for stability was found to be below a pH of 4.5. In vitro permeation studies were performed on rat gastric mucosa and revealed an eightfold increase in Gd-DTPA uptake when incorporated in the nanoparticles compared to evaluation in the presence of unformulated polyacrylic acid-cysteine. In vivo investigations with rats were performed via the noninvasive MRI method in order to track the nanoparticles way through the gastrointestinal tract. When Gd-DTPA was administered orally as nanoparticulate suspension, an increased MRI signal in the urinary bladder was detected after 34 min, providing evidence for systemic uptake and renal elimination of the contrast agent. As control experiments with Gd-DTPA only or in combination with unformulated polyacrylic acid-cysteine revealed no MRI signal increase at all, the significant permeation enhancing effect could be identified based on the nanoparticulate formulation.

Comparative in vivo mucoadhesion studies of thiomer formulations using magnetic resonance imaging and fluorescence detection.

The aim of this study was to compare different oral delivery systems based on the thiolated polymer polycarbophil-cysteine (PCP-Cys) and to provide evidence for the validity of the hypothesis that unhydrated polymers provide better mucoadhesion in vivo. To achieve dry polymer application, a new, experimental dosage form named Eutex (made of Eudragit L100-55 and latex) capsule has been developed. Magnetic resonance imaging was used to localize the point of release of the thiolated polymer from the application forms via the positive magnetic resonance signal from a gadolinium complex (Gd-DTPA). In vivo mucoadhesion was determined by ascertaining the residence time of the fluorescence-tagged thiomer on intestinal mucosa after 3 h. Results showed that in comparison to conventional application forms the Eutex capsules led to 1.9-fold higher mucoadhesive properties of PCP-Cys when compared to application with a conventional enteric-coated capsule, and to 1.4-fold higher mucoadhesion when compared to administration with an enteric-coated tablet of the thiomer. The findings of this study should contribute to the understanding of mucoadhesion and mucoadhesion influencing parameters in vivo and should therefore be of considerable interest for the development of future mucoadhesive oral drug delivery dosage forms.

Human triglyceride-rich lipoproteins impair glucose metabolism and insulin signalling in L6 skeletal muscle cells independently of non-esterified fatty acid levels.

AIMS/HYPOTHESIS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS/INTERPRETATION: 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.

Hyperphosphorylation of histone H2A.X and dephosphorylation of histone H1 subtypes in the course of apoptosis.

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.

Intravital lectin perfusion analysis of vascular permeability in human micro- and macro- blood vessels.

We previously applied intravital lectin perfusion in mouse models to elucidate mechanisms underlying vascular permeability. The present work transfers this technique to human models, analysing vascular permeability in macro- and microvessels. Human vascular endothelial surface carbohydrate biochemistry differs significantly from its murine counterpart, lacking alpha-galactosyl epitopes and expressing the L-fucose moiety in the glycocalyx; the poly-N-lactosamine glycan backbone is common to all mammals. We examined extensively lectin binding specificities in sections and in vivo, and then applied the poly-N-lactosamine-specific lectin LEA and the L-fucose-specific lectin UEA-I in human intravital perfusions. Transendothelial transport differed in macrovessels and microvessels. In microvessels of adult human fat tissue, rectal wall and rectal carcinomas, slow transendothelial transport by vesicles was followed by significant retention at the subendothelial basement membrane; paracellular passage was not observed. Passage time exceeded 1 h. Thus we found barrier mechanisms resembling those we described previously in murine tissues. In both adult and fetal macrovessels, the vena saphena magna and the umbilical vein, respectively, rapid passage across the endothelial lining was observed, the tracer localising completely in the subendothelial tissues within 15 min; vesicular transport was more rapid than in microvessels, and retention at the subendothelial basement membrane briefer.

Ossification in the human calcaneus: a model for spatial bone development and ossification.

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.

Vascular permeability and hyperpermeability in a murine adenocarcinoma after fractionated radiotherapy: an ultrastructural tracer study.

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.

Monitoring of tumor microcirculation during fractionated radiation therapy in patients with rectal carcinoma: preliminary results and implications for therapy.

PURPOSE: To measure microcirculatory changes during chemoirradiation and to correlate perfusion index (PI) values with therapy outcome. MATERIALS AND METHODS: Perfusion data in 11 patients with cT3 (clinical staging, tumor invaded the perirectal tissue) rectal carcinoma who underwent preoperative chemoirradiation were analyzed. Perfusion data were acquired by using a T1 mapping sequence with a whole-body magnetic resonance (MR) imager. After contrast medium was intravenously infused at a constant rate, concentration-and-time curves were evaluated for arterial blood and tumor. All patients underwent MR imaging before and at constant intervals during chemoirradiation. Clinical stages before therapy were compared with surgical stages after therapy. RESULTS: Spatial and temporal resolution on dynamic T1 maps were sufficient to reveal changes in contrast medium accumulation in the tumor. Comparison of PI values and radiation dose showed a significant increase in the 1st (P: =.003) and 2nd weeks (P: =.01) of treatment; values subsequently returned to pretreatment levels or showed a renewed increase. High initial PI values correlated with greater lymph node downstaging (P: =.042). CONCLUSION: Dynamic T1 mapping provides a suitable tool for monitoring tumor microcirculation during chemoirradiation and offers the potential for individual optimization of therapeutic procedures. Furthermore, these results indicate that the PI map may serve as a prognostic factor.

Cryopreparation provides new insight into the effects of brefeldin A on the structure of the HepG2 Golgi apparatus.

High-pressure freezing and freeze-substitution were used to study Golgi ultrastructure and its brefeldin A-induced transformations in HepG2 human hepatoma cells. Cryoimmobilization arrested subcellular dynamics within milliseconds, thus considerably improving the temporal resolution in monitoring the very early effects of high brefeldin concentrations at the ultrastructural level (i.e., 20 microg/ml brefeldin applied for 35 s to 8 min). Moreover, this approach ruled out possible cumulative and/or synergistic effects of the drug and fixatives. Several findings differed from studies based on chemical fixation. In particular, Golgi breakdown did not proceed gradually but occurred in distinct steps. We found a conspicuous lag between the absence of nonclathrin coats on Golgi membranes after 30 s of brefeldin treatment and the disassembly of the stacks, which did not start until after 90 to 120 s. At this time, domains at the trans and cis faces separated from the stacks, starting tubulation and fragmentation. After 3-5 min the Golgi apparatus was completely replaced by loose meshworks of straight tubules of different sizes and staining properties; also frequent were bent tubules and vesicles forming glomerule-like structures. After 8 min all kinds of Golgi-derived structures had aggregated within huge clusters. The morphologically highly distinct structures found after brefeldin treatment could in part be correlated with particular Golgi domains in the control cells.

[Development and application of dynamic MR imaging in the evaluation of perfusion changes in rectal carcinoma during radiotherapy in clinical routine. Preliminary results]. / Entwicklung und Anwendung dynamischer MRT-Messungen zur Evaluierung von Perfusionsveränderungen bei Rektumkarzinomen unter Bestrahlung in der klinischen Routine. Erste Ergebnisse.

PURPOSE: This study was aimed at measuring microcirculatory parameters and contrast medium accumulation within the rectal carcinoma during fractionated radiotherapy in the clinical setting. MATERIAL AND METHODS: Perfusion data were observed in patients with rectal carcinoma (n = 8) who underwent a pre-operative combined chemo/radiotherapy. To acquire perfusion data, an ultrafast T1 mapping sequence was carried out on a 1.5-Tesla whole body imager to obtain T1 maps at intervals of 14 or 120 seconds. The overall measurement time was 40 minutes. The transaxial slice thickness (5 mm) was chosen in such a way that both arterial vessels and the tumor could be clearly identified. The gadolinium-DTPA (Gd-DTPA) concentration time curve was evaluated for arterial blood and tumor after intravenous constant rate infusion. The method allows a spatial resolution of 2 x 2 x 5 mm and a temporal resolution of 14 seconds. Patients underwent MR imaging before and at constant intervals during fractionated radiotherapy. RESULTS: Spatial and temporal resolution of dynamic T1 mapping was sufficient to reveal varying CM accumulation levels within the tumor and to identify the great arteries in the pelvis. In 6 patients Gd-DTPA concentration-time-curves were evaluated within the tumor during radiation. Pi index of Gd-DTPA versus radiation dose showed a significant increase in the first or second week of treatment, then either returned slowly to pretreatment level or a renewed increase was observed. The average Pi-value at the beginning was 0.16 (+/- 0.049), reaching highest level of 0.23 (+/- 0.058). In all groups the rise from the Pi-value to the Pi-maximum was statistically significant. The relative increase in perfusion ranged between 20 to 83%. CONCLUSION: The results show, that the ultrafast MR-technique described above provide a suitable tool for monitoring tumor microcirculation during therapeutic interventions and offers the potential for an individualized optimization of therapeutic procedures.

Endocytic routes to the Golgi apparatus.

The endocytic routes of labelled lectins as well as cationic ferritin were studied in cells with a regulated secretion, i.e. pancreatic beta cells, and in constitutively secreting cells, i.e. fibroblasts and HepG2 hepatoma cells, paying particular attention to routes into the Golgi apparatus. Considerable amounts of internalised molecules were taken up into the trans Golgi network (TGN) and into Golgi subcompartments in all three cell types as well as in secretory granules of the pancreatic beta cells. The internalised materials did not pass rapidly the TGN and Golgi stacks, but were still present hours after internalisation, being then particularly concentrated in TGN-elements and in the transmost Golgi cisterna. Endocytosed materials reached forming secretory granules present in the TGN. Further, direct fusion between endocytotic vesicles and mature secretory granules was observed. Golgi subcompartments as well as endocytic TGN containing endocytosed materials were in close apposition to specialised regions of the endoplasmic reticulum. The Golgi apparatus including its parts containing endocytosed materials were transformed into a tubular reticulum upon treatment with the fungal metabolite Brefeldin A. Rarely, internalised material was observed in the lumen of the endoplasmic reticulum, thus providing evidence for an endocytic plasma membrane to endoplasmic reticulum route.

Lectin intravital perfusion studies in tumor-bearing mice: micrometer-resolution, wide-area mapping of microvascular labeling, distinguishing efficiently and inefficiently perfused microregions in the tumor.

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 micron and optical resolution 1.5 micron) 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.