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Mice and mouse platelets are major experimental models for hemostasis and thrombosis; however, important physiological data from this model has received little to no quantitative, 3D ultrastructural analysis. We used state-of-the-art, serial block imaging scanning electron microscopy (SBF-SEM, nominal Z-step size was 35 nm) to image resting platelets from C57BL/6 mice. α-Granules were identified morphologically and rendered in 3D space. The quantitative analysis revealed that mouse α-granules typically had a variable, elongated, rod shape, different from the round/ovoid shape of human α–granules. This variation in length was confirmed qualitatively by higher-resolution, focused ion beam (FIB) SEM at a nominal 5 nm Z-step size. The unexpected α-granule shape raises novel questions regarding α-granule biogenesis and dynamics. Does the variation arise at the level of the megakaryocyte and α-granule biogenesis or from differences in α-granule dynamics and organelle fusion/fission events within circulating platelets? Further quantitative analysis revealed that the two major organelles in circulating platelets, α-granules and mitochondria, displayed a stronger linear relationship between organelle number/volume and platelet size, i.e., a scaling in number and volume to platelet size, than found in human platelets suggestive of a tighter mechanistic regulation of their inclusion during platelet biogenesis. In conclusion, the overall spatial arrangement of organelles within mouse platelets was similar to that of resting human platelets, with mouse α-granules clustered closely together with little space for interdigitation of other organelles. [ABSTRACT FROM AUTHOR]

Abstract Background The 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. Methods Generation 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. Results The 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. Conclusion The 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.

The canalicular system (CS) has been defined as: 1) an inward, invaginated membrane connector that supports entry into and exit from the platelet; 2) a static structure stable during platelet isolation; and 3) the major source of plasma membrane (PM) for surface area expansion during activation. Recent analysis from STEM tomography and serial block face electron microscopy has challenged the relative importance of CS as the route for granule secretion. Here, We used 3D ultrastructural imaging to reexamine the CS in mouse platelets by generating high-resolution 3D reconstructions to test assumptions 2 and 3. Qualitative and quantitative analysis of whole platelet reconstructions, obtained from immediately fixed or washed platelets fixed post-washing, indicated that CS, even in the presence of activation inhibitors, reorganized during platelet isolation to generate a more interconnected network. Further, CS redistribution into the PM at different times, post-activation, appeared to account for only about half the PM expansion seen in thrombin-activated platelets, in vitro, suggesting that CS reorganization is not sufficient to serve as a dominant membrane reservoir for activated platelets. In sum, our analysis highlights the need to revisit past assumptions about the platelet CS to better understand how this membrane system contributes to platelet function. [ABSTRACT FROM AUTHOR]

Abstract Background Effective 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. Methods Magnetic 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. Results We 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. Conclusion The 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.