Alioscka A. Sousa

Targeted Killing of Cancer Cells in Vivo and in Vitro with EGF-Directed Carbon Nanotube-Based Drug Delivery

Carbon nanotube-based drug delivery holds great promise for cancer therapy. Herein we report the first targeted, in vivo killing of cancer cells using a drug-single wall carbon nanotube (SWNT) bioconjugate, and demonstrate efficacy superior to nontargeted bioconjugates. First line anticancer agent cisplatin and epidermal growth factor (EGF) were attached to SWNTs to specifically target squamous cancer, and the nontargeted control was SWNT-cisplatin without EGF. Initial in vitro imaging studies with head and neck squamous carcinoma cells (HNSCC) overexpressing EGF receptors (EGFR) using Qdot luminescence and confocal microscopy showed that SWNT-Qdot-EGF bioconjugates internalized rapidly into the cancer cells. Limited uptake occurred for control cells without EGF, and uptake was blocked by siRNA knockdown of EGFR in cancer cells, revealing the importance of EGF-EGFR binding. Three color, two-photon intravital video imaging in vivo showed that SWNT-Qdot-EGF injected into live mice was selectively taken up by HNSCC tumors, but SWNT-Qdot controls with no EGF were cleared from the tumor region in <20 min. HNSCC cells treated with SWNT-cisplatin-EGF were also killed selectively, while control systems that did not feature EGF-EGFR binding did not influence cell proliferation. Most significantly, regression of tumor growth was rapid in mice treated with targeted SWNT-cisplatin-EGF relative to nontargeted SWNT-cisplatin.

Effective transvascular delivery of nanoparticles across the blood-brain tumor barrier into malignant glioma cells

BackgroundEffective transvascular delivery of nanoparticle-based chemotherapeutics across the blood-brain tumor barrier of malignant gliomas remains a challenge. This is due to our limited understanding of nanoparticle properties in relation to the physiologic size of pores within the blood-brain tumor barrier. Polyamidoamine dendrimers are particularly small multigenerational nanoparticles with uniform sizes within each generation. Dendrimer sizes increase by only 1 to 2 nm with each successive generation. Using functionalized polyamidoamine dendrimer generations 1 through 8, we investigated how nanoparticle size influences particle accumulation within malignant glioma cells.MethodsMagnetic resonance and fluorescence imaging probes were conjugated to the dendrimer terminal amines. Functionalized dendrimers were administered intravenously to rodents with orthotopically grown malignant gliomas. Transvascular transport and accumulation of the nanoparticles in brain tumor tissue was measured in vivo with dynamic contrast-enhanced magnetic resonance imaging. Localization of the nanoparticles within glioma cells was confirmed ex vivo with fluorescence imaging.ResultsWe found that the intravenously administered functionalized dendrimers less than approximately 11.7 to 11.9 nm in diameter were able to traverse pores of the blood-brain tumor barrier of RG-2 malignant gliomas, while larger ones could not. Of the permeable functionalized dendrimer generations, those that possessed long blood half-lives could accumulate within glioma cells.ConclusionThe therapeutically relevant upper limit of blood-brain tumor barrier pore size is approximately 11.7 to 11.9 nm. Therefore, effective transvascular drug delivery into malignant glioma cells can be accomplished by using nanoparticles that are smaller than 11.7 to 11.9 nm in diameter and possess long blood half-lives.

PSD-95 Is Required to Sustain the Molecular Organization of the Postsynaptic Density

PSD-95, a membrane-associated guanylate kinase, is the major scaffolding protein in the excitatory postsynaptic density (PSD) and a potent regulator of synaptic strength. Here we show that PSD-95 is in an extended configuration and positioned into regular arrays of vertical filaments that contact both glutamate receptors and orthogonal horizontal elements layered deep inside the PSD in rat hippocampal spine synapses. RNA interference knockdown of PSD-95 leads to loss of entire patches of PSD material, and electron microscopy tomography shows that the patchy loss correlates with loss of PSD-95-containing vertical filaments, horizontal elements associated with the vertical filaments, and putative AMPA receptor-type, but not NMDA receptor-type, structures. These observations show that the orthogonal molecular scaffold constructed from PSD-95-containing vertical filaments and their associated horizontal elements is essential for sustaining the three-dimensional molecular organization of the PSD. Our findings provide a structural basis for understanding the functional role of PSD-95 at the PSD.

Distribution and clearance of PEG-single-walled carbon nanotube cancer drug delivery vehicles in mice

AIMS To study the distribution and clearance of polyethylene glycol (PEG)-ylated single-walled carbon nanotube (SWCNTs) as drug delivery vehicles for the anticancer drug cisplatin in mice. MATERIALS & METHODS PEG layers were attached to SWCNTs and dispersed in aqueous media and characterized using dynamic light scattering, scanning transmission electron microscopy and Raman spectroscopy. Cytotoxicity was assessed in vitro using Annexin-V assay, and the distribution and clearance pathways in mice were studied by histological staining and Raman spectroscopy. Efficacy of PEG-SWCNT-cisplatin for tumor growth inhibition was studied in mice. RESULTS & DISCUSSION PEG-SWCNTs were efficiently dispersed in aqueous media compared with controls, and did not induce apoptosis in vitro. Hematoxylin and eosin staining, and Raman bands for SWCNTs in tissues from several vital organs from mice injected intravenously with nanotube bioconjugates revealed that control SWCNTs were lodged in lung tissue as large aggregates compared with the PEG-SWCNTs, which showed little or no accumulation. Characteristic SWCNT Raman bands in feces revealed the presence of bilary or renal excretion routes. Attachment of cisplatin on bioconjugates was visualized with Z-contrast scanning transmission electron microscopy. PEG-SWCNT-cisplatin with the attached targeting ligand EGF successfully inhibited growth of head and neck tumor xenografts in mice. CONCLUSIONS PEG-SWCNTs, as opposed to control SWCNTs, form more highly dispersed delivery vehicles that, when loaded with both cisplatin and EGF, inhibit growth of squamous cell tumors.

Physiologic upper limit of pore size in the blood-tumor barrier of malignant solid tumors

BackgroundThe existence of large pores in the blood-tumor barrier (BTB) of malignant solid tumor microvasculature makes the blood-tumor barrier more permeable to macromolecules than the endothelial barrier of most normal tissue microvasculature. The BTB of malignant solid tumors growing outside the brain, in peripheral tissues, is more permeable than that of similar tumors growing inside the brain. This has been previously attributed to the larger anatomic sizes of the pores within the BTB of peripheral tumors. Since in the physiological state in vivo a fibrous glycocalyx layer coats the pores of the BTB, it is possible that the effective physiologic pore size in the BTB of brain tumors and peripheral tumors is similar. If this were the case, then the higher permeability of the BTB of peripheral tumor would be attributable to the presence of a greater number of pores in the BTB of peripheral tumors. In this study, we probed in vivo the upper limit of pore size in the BTB of rodent malignant gliomas grown inside the brain, the orthotopic site, as well as outside the brain in temporalis skeletal muscle, the ectopic site.MethodsGeneration 5 (G5) through generation 8 (G8) polyamidoamine dendrimers were labeled with gadolinium (Gd)-diethyltriaminepentaacetic acid, an anionic MRI contrast agent. The respective Gd-dendrimer generations were visualized in vitro by scanning transmission electron microscopy. Following intravenous infusion of the respective Gd-dendrimer generations (Gd-G5, N = 6; Gd-G6, N = 6; Gd-G7, N = 5; Gd-G8, N = 5) the blood and tumor tissue pharmacokinetics of the Gd-dendrimer generations were visualized in vivo over 600 to 700 minutes by dynamic contrast-enhanced MRI. One additional animal was imaged in each Gd-dendrimer generation group for 175 minutes under continuous anesthesia for the creation of voxel-by-voxel Gd concentration maps.ResultsThe estimated diameters of Gd-G7 dendrimers were 11 ± 1 nm and those of Gd-G8 dendrimers were 13 ± 1 nm. The BTB of ectopic RG-2 gliomas was more permeable than the BTB of orthotopic RG-2 gliomas to all Gd-dendrimer generations except for Gd-G8. The BTB of both ectopic RG-2 gliomas and orthotopic RG-2 gliomas was not permeable to Gd-G8 dendrimers.ConclusionThe physiologic upper limit of pore size in the BTB of malignant solid tumor microvasculature is approximately 12 nanometers. In the physiologic state in vivo the luminal fibrous glycocalyx of the BTB of malignant brain tumor and peripheral tumors is the primary impediment to the effective transvascular transport of particles across the BTB of malignant solid tumor microvasculature independent of tumor host site. The higher permeability of malignant peripheral tumor microvasculature to macromolecules smaller than approximately 12 nm in diameter is attributable to the presence of a greater number of pores underlying the glycocalyx of the BTB of malignant peripheral tumor microvasculature.

Nanoscale 3D cellular imaging by axial scanning transmission electron tomography

Electron tomography provides three-dimensional structural information about supramolecular assemblies and organelles in a cellular context, but image degradation, caused by scattering of transmitted electrons, limits applicability in specimens thicker than 300 nm. We found that scanning transmission electron tomography of 1,000-nm-thick samples using axial detection provided resolution comparable to that of conventional electron tomography. We demonstrated the method by reconstructing a human erythrocyte infected with the malaria parasite Plasmodium falciparum.

Effect of the Charge State (z = −1, 0, +1) on the Nuclear Magnetic Resonance of Monodisperse Au25[S(CH2)2Ph]18z Clusters

Monodisperse Au(25)L(18)(0) (L = S(CH(2))(2)Ph) and [n-Oct(4)N(+)][Au(25)L(18)(-)] clusters were synthesized in tetrahydrofuran. An original strategy was then devised to oxidize them: in the presence of bis(pentafluorobenzoyl) peroxide, the neutral or the negatively charged clusters react as efficient electron donors in a dissociative electron-transfer (ET) process, in the former case yielding [Au(25)L(18)(+)][C(6)F(5)CO(2)(-)]. As opposed to other reported redox methods, this dissociative ET approach is irreversible, easily controllable, and clean, particularly for NMR purposes, as no hydrogen atoms are introduced. By using this approach, the -1, 0, and +1 charge states of Au(25)L(18) could be fully characterized by (1)H and (13)C NMR spectroscopy, using one- and two-dimensional techniques, in various solvents, and as a function of temperature. For all charge states, the NMR results and analysis nicely match recent structural findings about the presence of two different ligand populations in the capping monolayer, each resonance of the two ligand families displaying distinct NMR patterns. The radical nature of Au(25)L(18)(0) is particularly evident in the (1)H and (13)C NMR patterns of the inner ligands. The NMR behavior of radical Au(25)L(18)(0) was also simulated by DFT calculations, and the interplay between theory and experiments revealed a fundamental paramagnetic contribution coming from Fermi contact shifts. Interestingly, the NMR patterns of Au(25)L(18)(-) and Au(25)L(18)(+) were found to be quite similar, pointing to the latter cluster form as a diamagnetic species.

Dual-axis electron tomography of biological specimens: Extending the limits of specimen thickness with bright-field STEM imaging

The absence of imaging lenses after the specimen in the scanning transmission electron microscope (STEM) enables electron tomography to be performed in the STEM mode on micrometer-thick plastic-embedded specimens without the deleterious effect of chromatic aberration, which limits spatial resolution and signal-to-noise ratio in conventional TEM. Using Monte Carlo calculations to simulate electron scattering from gold nanoparticles situated at the top and bottom surfaces of a plastic section, we assess the optimal acquisition strategy for axial bright-field STEM electron tomography at a beam-energy of 300keV. Dual tilt-axis STEM tomography with optimized axial bight-field detector geometry is demonstrated by application to micrometer-thick sections of beta cells from mouse pancreatic islet. The quality of the resulting three-dimensional reconstructions is comparable to that obtained from much thinner (0.3-micrometer) sections using conventional TEM tomography. The increased range of specimen thickness accessible to axial STEM tomography without the need for serial sectioning enables the 3-D visualization of more complex and larger subcellular structures.

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

Significance The postsynaptic density (PSD) at the glutamatergic excitatory synapse is a macromolecular machine that underlies synaptic transmission and information storage. Membrane-associated guanylate kinases (MAGUKs), the major scaffolding proteins at the PSD, are positively correlated with synaptic maturation and strength, but how MAGUKs sustain the strength of synaptic transmission remains unclear. Here, we remove three MAGUK proteins from neurons and find significant reductions in synaptic transmission by AMPARs and NMDARs with a concomitant reduction in PSD sizes and core scaffold and transmembrane structures. Our results show how MAGUKs anchor and organize both types of glutamate receptors, thereby regulating the strength of excitatory synapses. The postsynaptic density (PSD)-95 family of membrane-associated guanylate kinases (MAGUKs) are major scaffolding proteins at the PSD in glutamatergic excitatory synapses, where they maintain and modulate synaptic strength. How MAGUKs underlie synaptic strength at the molecular level is still not well understood. Here, we explore the structural and functional roles of MAGUKs at hippocampal excitatory synapses by simultaneous knocking down PSD-95, PSD-93, and synapse-associated protein (SAP)102 and combining electrophysiology and transmission electron microscopic (TEM) tomography imaging to analyze the resulting changes. Acute MAGUK knockdown greatly reduces synaptic transmission mediated by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-d-aspartate receptors (NMDARs). This knockdown leads to a significant rise in the number of silent synapses, diminishes the size of PSDs without changes in pre- or postsynaptic membrane, and depletes the number of membrane-associated PSD-95–like vertical filaments and transmembrane structures, identified as AMPARs and NMDARs by EM tomography. The differential distribution of these receptor-like structures and dependence of their abundance on PSD size matches that of AMPARs and NMDARs in the hippocampal synapses. The loss of these structures following MAGUK knockdown tracks the reduction in postsynaptic AMPAR and NMDAR transmission, confirming the structural identities of these two types of receptors. These results demonstrate that MAGUKs are required for anchoring both types of glutamate receptors at the PSD and are consistent with a structural model where MAGUKs, corresponding to membrane-associated vertical filaments, are the essential structural proteins that anchor and organize both types of glutamate receptors and govern the overall molecular organization of the PSD.

Monte Carlo electron-trajectory simulations in bright-field and dark-field STEM: Implications for tomography of thick biological sections

A Monte Carlo electron-trajectory calculation has been implemented to assess the optimal detector configuration for scanning transmission electron microscopy (STEM) tomography of thick biological sections. By modeling specimens containing 2 and 3 at% osmium in a carbon matrix, it was found that for 1-microm-thick samples the bright-field (BF) and annular dark-field (ADF) signals give similar contrast and signal-to-noise ratio provided the ADF inner angle and BF outer angle are chosen optimally. Spatial resolution in STEM imaging of thick sections is compromised by multiple elastic scattering which results in a spread of scattering angles and thus a spread in lateral distances of the electrons leaving the bottom surface. However, the simulations reveal that a large fraction of these multiply scattered electrons are excluded from the BF detector, which results in higher spatial resolution in BF than in high-angle ADF images for objects situated towards the bottom of the sample. The calculations imply that STEM electron tomography of thick sections should be performed using a BF rather than an ADF detector. This advantage was verified by recording simultaneous BF and high-angle ADF STEM tomographic tilt series from a stained 600-nm-thick section of C. elegans. It was found that loss of spatial resolution occurred markedly at the bottom surface of the specimen in the ADF STEM but significantly less in the BF STEM tomographic reconstruction. Our results indicate that it might be feasible to use BF STEM tomography to determine the 3D structure of whole eukaryotic microorganisms prepared by freeze-substitution, embedding, and sectioning.