In vivo tumour imaging employing regional delivery of novel gallium radiolabelled polymer composites.

BACKGROUND: Understanding the regional vascular delivery of particles to tumour sites is a prerequisite for developing new diagnostic and therapeutic composites for treatment of oncology patients. We describe a novel imageable 67Ga-radiolabelled polymer composite that is biocompatible in an animal tumour model and can be used for preclinical imaging investigations of the transit of different sized particles through arterial networks of normal and tumour-bearing organs. RESULTS: Radiolabelling of polymer microspheres with 67Ga was achieved using a simple mix and wash method, with tannic acid as an immobilising agent. Final in vitro binding yields after autoclaving averaged 94.7%. In vivo stability of the composite was demonstrated in New Zealand white rabbits by intravenous administration, and intrahepatic artery instillations were made in normal and VX2 tumour implanted rabbit livers. Stability of radiolabel was sufficient for rabbit lung and liver imaging over at least 3 hours and 1 hour respectively, with lung retention of radiolabel over 91%, and retention in both normal and VX2 implanted livers of over 95%. SPECT-CT imaging of anaesthetised animals and planar imaging of excised livers showed visible accumulation of radiolabel in tumours. Importantly, microsphere administration and complete liver dispersal was more easily achieved with 8 µm diameter MS than with 30 µm MS, and the smaller microspheres provided more distinct and localised tumour imaging. CONCLUSION: This method of producing 67Ga-radiolabelled polymer microspheres is suitable for SPECT-CT imaging of the regional vascular delivery of microspheres to tumour sites in animal models. Sharper distinction of model tumours from normal liver was obtained with smaller MS, and tumour resolution may be further improved by the use of 68Ga instead of 67Ga, to enable PET imaging.

99mTc-radiolabeled composites enabling in vivo imaging of arterial dispersal and retention of microspheres in the vascular network of rabbit lungs, liver, and liver tumors.

PURPOSE: Selective internal radiation therapy (SIRT) is an effective treatment option for liver tumors, using Y-90-loaded polymer microspheres that are delivered via catheterization of the hepatic artery. Since Y-90 is a beta emitter and not conveniently imaged by standard clinical instrumentation, dosimetry is currently evaluated in each patient using a surrogate particle, 99mTechnetium-labeled macroaggregated albumin (99mTc-MAA). We report a new composite consisting of 99mTc-labeled nanoparticles attached to the same polymer microspheres as used for SIRT, which can be imaged with standard SPECT. METHODS: Carbon nanoparticles with an encapsulated core of 99mTc were coated with the polycation protamine sulfate to provide electrostatic attachment to anionic polystyrene sulfonate microspheres of different sizes (30, 12, and 8 µm). The in vivo stability of these composites was determined via intravenous injection and entrapment in the capillary network of normal rabbit lungs for up to 3 hours. Furthermore, we evaluated their biodistribution in normal rabbit livers, and livers implanted with VX2 tumors, following intrahepatic artery instillation. RESULTS: We report distribution tests for three different sizes of radiolabeled microspheres and compare the results with those obtained using 99mTc-MAA. Lung retention of the radiolabeled microspheres ranged from 72.8% to 92.9%, with the smaller diameter microspheres showing the lowest retention. Liver retention of the microspheres was higher, with retention in normal livers ranging from 99.2% to 99.8%, and in livers with VX2 tumors from 98.2% to 99.2%. The radiolabeled microspheres clearly demonstrated preferential uptake at tumor sites due to the increased arterial perfusion produced by angiogenesis. CONCLUSION: We describe a novel use of radiolabeled carbon nanoparticles to generate an imageable microsphere that is stable in vivo under the shear stress conditions of arterial networks. Following intra-arterial instillation in the normal rabbit liver, they distribute in a distinct segmented pattern, with the smaller microspheres extending throughout the organ in finer detail, while still being well retained within the liver. Furthermore, in livers hosting an implanted VX2 tumor, they reveal the increased arterial perfusion of tumor tissue resulting from angiogenesis. These novel composites may have potential as a more representative mimic of the vascular distribution of therapeutic microspheres in patients undergoing SIRT.

First shark from the Late Devonian (Frasnian) Gogo Formation, Western Australia sheds new light on the development of tessellated calcified cartilage.

BACKGROUND: Living gnathostomes (jawed vertebrates) comprise two divisions, Chondrichthyes (cartilaginous fishes, including euchondrichthyans with prismatic calcified cartilage, and extinct stem chondrichthyans) and Osteichthyes (bony fishes including tetrapods). Most of the early chondrichthyan ('shark') record is based upon isolated teeth, spines, and scales, with the oldest articulated sharks that exhibit major diagnostic characters of the group--prismatic calcified cartilage and pelvic claspers in males--being from the latest Devonian, c. 360 Mya. This paucity of information about early chondrichthyan anatomy is mainly due to their lack of endoskeletal bone and consequent low preservation potential. METHODOLOGY/PRINCIPAL FINDINGS: Here we present new data from the first well-preserved chondrichthyan fossil from the early Late Devonian (ca. 380-384 Mya) Gogo Formation Lägerstatte of Western Australia. The specimen is the first Devonian shark body fossil to be acid-prepared, revealing the endoskeletal elements as three-dimensional undistorted units: Meckel's cartilages, nasal, ceratohyal, basibranchial and possible epibranchial cartilages, plus left and right scapulocoracoids, as well as teeth and scales. This unique specimen is assigned to Gogoselachus lynnbeazleyae n. gen. n. sp. CONCLUSIONS/SIGNIFICANCE: The Meckel's cartilages show a jaw articulation surface dominated by an expansive cotylus, and a small mandibular knob, an unusual condition for chondrichthyans. The scapulocoracoid of the new specimen shows evidence of two pectoral fin basal articulation facets, differing from the standard condition for early gnathostomes which have either one or three articulations. The tooth structure is intermediate between the 'primitive' ctenacanthiform and symmoriiform condition, and more derived forms with a euselachian-type base. Of special interest is the highly distinctive type of calcified cartilage forming the endoskeleton, comprising multiple layers of nonprismatic subpolygonal tesserae separated by a cellular matrix, interpreted as a transitional step toward the tessellated prismatic calcified cartilage that is recognized as the main diagnostic character of the chondrichthyans.

The uptake of soluble and nanoparticulate imaging isotope in model liver tumours after intra-venous and intra-arterial administration.

Delivery of chemotherapeutic drugs to tumours by reformulation as nanoparticles has often been proposed as a means of facilitating increased selective uptake, exploiting the increased permeability of the tumour vasculature. However realisation of this improvement in drug delivery in cancer patients has met with limited success. We have compared tumour uptake of soluble Tc99m-pertechnetate and a colloid of nanoparticles with a Tc99m core, using both intra-venous and intra-arterial routes of administration in a rabbit liver VX2 tumour model. The radiolabelled nanoparticles were tested both in untreated and cationised form. The results from this tumour model in an internal organ show a marked advantage in intra-arterial administration over the intra-venous route, even for the soluble isotope. Tumour accumulation of nanoparticles from arterial administration was augmented by cationisation of the nanoparticle surface with histone proteins, which consistently facilitated selective accumulation within microvessels at the periphery of tumours.

Laser actuation of cantilevers for picometre amplitude dynamic force microscopy.

As nanoscale and molecular devices become reality, the ability to probe materials on these scales is increasing in importance. To address this, we have developed a dynamic force microscopy technique where the flexure of the microcantilever is excited using an intensity modulated laser beam to achieve modulation on the picoscale. The flexure arises from thermally induced bending through differential expansion and the conservation of momentum when the photons are reflected and absorbed by the cantilever. In this study, we investigated the photothermal and photon pressure responses of monolithic and layered cantilevers using a modulated laser in air and immersed in water. The developed photon actuation technique is applied to the stretching of single polymer chains.

The accumulation of circulating histones on heparan sulphate in the capillary glycocalyx of the lungs.

Recent findings on the role of circulating histone proteins in mediating acute lung injury prompted us to investigate whether there is a specific mechanism for accumulation of histones in the lungs. Binding sites for polycations are already known in the vasculature of the lungs, and we postulated that these could also be involved in histone accumulation, since histones have a high content of positively charged amino acids. Using a histone-coated colloid of a radiolabelled nanocomposite to track histone biodistribution with imaging techniques, it was found that histones bind avidly in the lungs of rabbits after intravenous injection. Blocking experiments with competing polycations in vivo characterised histone lung binding as dependent on a charge interaction with microvessel polyanions. Pretreatment of rabbits with a specific heparinase confirmed that the lung binding sites consist of heparan sulphate in the endothelial glycocalyx. A range of heparan sulphate analogues was accordingly shown to prevent histone accumulation in the lungs by neutralising histones in blood. These findings provide a rational basis for the design of polyanions that can prevent accumulation of cytotoxic histones in the lungs and thereby intervene at an early key step in the development of acute lung injury.

Cationised radiolabelled nanoparticles for perfusion imaging of the lungs.

Diagnostic imaging of the blood perfusion of the lungs is currently performed using particles of macro-aggregated albumin, which are mechanically arrested at limiting diameters of the capillary bed. While the proportion of blood flow obstructed is typically very low and temporary, it would seem more desirable to image lung perfusion in patients using a non-obstructive method, and using materials that avoid biological hazards. We have characterised the in vitro and in vivo properties of a colloid of a cationised radiolabelled nanocomposite. The nanoparticles comprise a Technetium-99m core encapsulated in graphitic carbon, and coated with low molecular weight poly-lysine to provide a strong charge-based affinity for the endothelial glycocalyx of the lung. Following intravenous injection in rabbits and cats, the nanoparticles rapidly distribute and localise in the lungs, thus enabling gamma camera imaging of lung perfusion. Repeat administration of this colloid in both species over several weeks indicates favourable biocompatibility.

The earliest known stem-tetrapod from the Lower Devonian of China.

Recent discoveries of advanced fish-like stem-tetrapods (for example, Panderichthys and Tiktaalik) have greatly improved our knowledge of the fin-to-limb transition. However, a paucity of fossil data from primitive finned tetrapods prevents profound understanding of the acquisition sequence of tetrapod characters. Here we report a new stem-tetrapod (Tungsenia paradoxa gen. et sp. nov.) from the Lower Devonian (Pragian, ∼409 million years ago) of China, which extends the earliest record of tetrapods by some 10 million years. Sharing many primitive features with stem-lungfishes, the new taxon further fills in the morphological gap between tetrapods and lungfishes. The X-ray tomography study of the skull depicts the plesiomorphic condition of the brain in the tetrapods. The enlargement of the cerebral hemispheres and the possible presence of the pars tuberalis in this stem-tetrapod indicate that some important brain modifications related to terrestrial life had occurred at the beginning of the tetrapod evolution, much earlier than previously thought.

Dilatancy in slow granular flows.

When walking on wet sand, each footstep leaves behind a temporarily dry impression. This counterintuitive observation is the most common illustration of the Reynolds principle of dilatancy: that is, a granular packing tends to expand as it is deformed, therefore increasing the amount of porous space. Although widely called upon in areas such as soil mechanics and geotechnics, a deeper understanding of this principle is constrained by the lack of analytical tools to study this behavior. Using x-ray radiography, we track a broad variety of granular flow profiles and quantify their intrinsic dilatancy behavior. These measurements frame Reynolds dilatancy as a kinematic process. Closer inspection demonstrates, however, the practical importance of flow induced compaction which competes with dilatancy, leading more complex flow properties than expected.

Onset of mechanical stability in random packings of frictional spheres.

Using sedimentation to obtain precisely controlled packings of noncohesive spheres, we find that the volume fraction phiRLP of the loosest mechanically stable packing is in an operational sense well defined by a limit process. This random loose packing volume fraction decreases with decreasing pressure p and increasing interparticle friction coefficient mu. Using x-ray tomography to correct for a container boundary effect that depends on particle size, we find for rough particles in the limit p-->0 a new lower bound, phiRLP=0.550+/-0.001.

Imaging honey bee brain anatomy with micro-X-ray-computed tomography.

Technologies for imaging in three dimensions are greatly desired by researchers in many biological disciplines. However, when imaging small animals such as invertebrates, the achievement of satisfactory spatial resolution and adequate contrast between tissues often requires the use of expensive and time-consuming procedures. Micro-X-ray-computed tomography (muCT) is a convenient technique which is finding greater use alongside conventional microscopies. Staining with heavy metal salts, such as osmium tetroxide improves imaging in muCT, and allows visualization of the 3D structure of the honey bee brain undistorted within the intact head capsule. We obtained detailed information about the morphology of the different brain compartments and were able to show their orientations, relative to each other, within the head capsule. This technique offers a significant improvement in resolution, time, and expense for the quantitative, three-dimensional analysis of developing bee brain centers. In this article, we introduce a rapid, high-resolution, and inexpensive technique for the three-dimensional visualization of different compartments of the honey bee brain. A detailed discussion of the honey bee brain anatomy is provided, demonstrating that muCT, with osmium staining, can indeed visualise these structures. Hence, our results show that muCT is ideally suited for researchers who are interested in the 3D visualization of small invertebrate brains.

An exceptional Devonian fish from Australia sheds light on tetrapod origins.

The transition from fishes to tetrapods was one of the most dramatic events in the evolution of vertebrates, but many pivotal fossils are incomplete, resulting in gaps in the data that are used for phylogenetic reconstruction. Here we present new observations from the most complete, acid-prepared Devonian tetrapodomorph fish yet discovered, Gogonasus, which was previously placed just crownward of Kenichthys and rhizodontids, the most primitive taxa on the tetrapod lineage. Unexpectedly, Gogonasus shows a mosaic of plesiomorphic and derived tetrapod-like features. Whereas the braincase and dermal cranial skeleton exhibit generalized morphologies with respect to Eusthenopteron or Panderichthys, taxa that are traditionally considered to be phyletically close to tetrapods, the presence of a deeply invaginated, wide spiracle, advanced internal spiracular architecture and near-horizontal hyomandibula are specialized features that are absent from Eusthenopteron. Furthermore, the pectoral fin skeleton of Gogonasus shares several features with that of Tiktaalik, the most tetrapod-like fish. A new phylogenetic analysis places Gogonasus crownward of Eusthenopteron as the sister taxon to the Elpistostegalia. Aspects of the basic tetrapod limb skeleton and middle ear architecture can now be traced further back within the tetrapodomorph radiation.

Structure and properties of clinical coralline implants measured via 3D imaging and analysis.

The development and design of advanced porous materials for biomedical applications requires a thorough understanding of how material structure impacts on mechanical and transport properties. This paper illustrates a 3D imaging and analysis study of two clinically proven coral bone graft samples (Porites and Goniopora). Images are obtained from X-ray micro-computed tomography (micro-CT) at a resolution of 16.8 microm. A visual comparison of the two images shows very different structure; Porites has a homogeneous structure and consistent pore size while Goniopora has a bimodal pore size and a strongly disordered structure. A number of 3D structural characteristics are measured directly on the images including pore volume-to-surface-area, pore and solid size distributions, chord length measurements and tortuosity. Computational results made directly on the digitized tomographic images are presented for the permeability, diffusivity and elastic modulus of the coral samples. The results allow one to quantify differences between the two samples. 3D digital analysis can provide a more thorough assessment of biomaterial structure including the pore wall thickness, local flow, mechanical properties and diffusion pathways. We discuss the implications of these results to the development of optimal scaffold design for tissue ingrowth.

Analysis of 3D bone ingrowth into polymer scaffolds via micro-computed tomography imaging.

This paper illustrates the utility of micro-computed tomography (micro-CT) to study the process of tissue engineered bone growth. A micro-CT facility for imaging and visualising biomaterials in three dimensions (3D) is described. The facility is capable of acquiring 3D images made up of 2000(3) voxels on specimens up to 60mm in extent with resolutions down to 2 microm. This allows the 3D structure of tissue engineered materials to be imaged across three orders of magnitude of detail. The capabilities of micro-CT are demonstrated by imaging the Haversian network within human femoral cortical bone (distal diaphysis) and bone ingrowth into a porous scaffold at varying resolutions. Phase identification combined with 3D visualisation enables one to observe the complex topology of the canalicular system of the cortical bone. Imaging of the tissue engineered bone at a scale of 1cm and resolutions of 10 microm allows visualisation of the complex ingrowth of bone into the polymer scaffold. Further imaging at 2 microm resolution allows observation of bone ultra-structure. These observations illustrate the benefits of tomography over traditional techniques for the characterisation of bone morphology and interconnectivity and performs a complimentary role to current histomorphometric techniques.

Micromanipulation of phospholipid bilayers by atomic force microscopy.

The molecular details of adhesion mechanics in phospholipid bilayers have been studied using atomic force microscopy (AFM). Under tension fused bilayers of dipalmitoylphosphatidylcholine (DPPC) yield to give non-distance dependent and discrete force plateaux of 45.4, 81.6 and 113+/-3.5 pN. This behaviour may persist over distances as great as 400 nm and suggests the stable formation of a cylindrical tube which bridges the bilayers on the two surfaces. The stability of this connective structure may have implications for the formation of pili and hence for the initial stage of bacterial conjugation. Dimyristoylphosphatidylcholine (DMPC) bilayers also exhibit force plateaux but with a much less pronounced quantization. Bilayers composed of egg PC, sterylamine and cholesterol stressed in a similar way show complex behaviour which can in part be explained using the models demonstrated in the pure lipids.