EDITORIAL

Ligand-mediated drug delivery systems have enormous potential for improving the efficacy of cancer treatment. In particular, Arg-Gly-Asp peptides are promising ligand molecules for targeting α_vβ₃/α_vβ₅ integrins, which are overexpressed in angiogenic sites and tumors, such as intractable human glioblastoma (U87MG). We here achieved highly efficient drug delivery to U87MG tumors by using a platinum anticancer drug-incorporating polymeric micelle (PM) with cyclic Arg-Gly-Asp (cRGD) ligand molecules. Intravital confocal laser scanning microscopy revealed that the cRGD-linked polymeric micelles (cRGD/m) accumulated rapidly and had high permeability from vessels into the tumor parenchyma compared with the PM having nontargeted ligand, “cyclic-Arg-Ala-Asp” (cRAD). As both cRGD/m- and cRAD-linked polymeric micelles have similar characteristics, including their size, surface charge, and the amount of incorporated drugs, it is likely that the selective and accelerated accumulation of cRGD/m into tumors occurred <i>via</i> an active internalization pathway, possibly transcytosis, thereby producing significant antitumor effects in an orthotopic mouse model of U87MG human glioblastoma

MOESM1 of Combined CatWalk Index: an improved method to measure mouse motor function using the automated gait analysis system

Additional file 1. Example spreadsheet with CCI coefficients. An annotated example spreadsheet containing CatWalk and BMS data from impact force optimization experiments. This spreadsheet shows how to calculate CCI coefficients and scores

Living Unimodal Growth of Polyion Complex Vesicles via Two-Dimensional Supramolecular Polymerization

Understanding the dynamic behavior of molecular self-assemblies with higher-dimensional structures remains a key challenge to obtaining well-controlled and monodispersed structures. Nonetheless, there exist few systems capable of realizing the mechanism of supramolecular polymerization at higher dimensions. Herein, we report the unique self-assembling behavior of polyion complexes (PICs) consisting of poly­(ethylene glycol)-polyelectrolyte block copolymer as an example of two-dimensional supramolecular living polymerization. Monodispersed and submicrometer unilamellar PIC vesicles (nano-PICsomes) displayed time-dependent growth while maintaining a narrow size distribution and a unilamellar structure. Detailed analysis of the system revealed that vesicle growth proceeded through the consumption of unit PICs (uPICs) composed of a single polycation/polyanion pair and was able to restart upon the further addition of isolated uPICs. Interestingly, the resulting vesicles underwent dissociation into uPICs in response to mechanical stress. These results clearly frame the growth as a two-dimensional supramolecular living polymerization of uPICs

Enzyme-Loaded Polyion Complex Vesicles as in Vivo Nanoreactors Working Sustainably under the Blood Circulation: Characterization and Functional Evaluation

Enzyme-loaded synthetic vesicles have attracted great attention for their feasibility to exert the efficient and prolonged functionality of loaded enzymes in harsh environments, such as in vivo. However, several issues remain regarding the optimization of their structures toward practical application. Herein, we fabricated polyion complex vesicles (PICsomes) loaded with l-asparaginase (ASNase@PICsomes) and conducted a detailed characterization to ensure their utility as nanoreactors functioning under the harsh in vivo environment of the bloodstream. ASNase@PICsomes showed 100 nm-sized monodispersed vesicular structures. Fluorescence cross-correlation spectroscopy revealed essentially no empty PICsome fraction in the product, indicating the quantitative formation of ASNase@PICsomes. Furthermore, fluorescence anisotropy measurement showed that the loaded enzymes were located essentially in the inner aqueous phase of PICsomes, being successfully segregated from the external environment. ASNase@PICsomes exhibited significantly prolonged enzymatic reaction compared with free ASNase after systemic injection into mice, corroborating their functionality as in vivo nanoreactors working under the blood circulation

Histopathological findings of AAAs after treatment with rapamycin nanoparticles.

<p>Low power field images (A-E, scale bar = 500 μm) and high power field images (F-O, scale bar = 100 μm) of the rat AAA at 7 days after elastase infusion. The sections were stained by hematoxylin-eosin (A-J) or via the Elastica van Gieson method (K-O). During the process of AAA development, the rats received intravenous injections of PBS (A,F,K), free/RAP-0.1 (B,G,L), free/RAP-1 (C,H,M), RAP/nano-1 (D,I,N), or RAP/nano-1 (E,J,O). The size of the AAA after injections of RAP/nano-0.1 (D) and RAP/nano-1 (E) are smaller than those after the other injections (A–C). Considerable numbers of inflammatory cells are observed in the AAA after injections of PBS (F), free/RAP-0.1 (G), and free/RAP-1 (H), with concomitant destruction of the medial elastic laminae (K–M). L indicates the lumen.</p

Microscopic distribution of Alexa647-labeled rapamycin nanoparticles in the rat AAA.

<p>(A) Confocal laser scanning micrograph of the rat AAA 7-days post induction and 24 hours after injection of the Alexa647-labeled rapamycin nanoparticles. Note the abundant accumulation of Alexa647-labeled rapamycin nanoparticles (red dots) distributed in the media and adventitia with progressive destruction of the wall structure. Nuclei are stained by Hoechst33342 (blue dots), and elastic laminae of the media are visualized by intrinsic fluorescence (green). L indicates the lumen, scale bar = 500 μm. (B) Micrograph of the cross sections stained for CD68 (green). Co-localization with Alexa647-labelled rapamycin nanoparticles (red dots) appears as a yellow color. The majority of nanoparticle dots were co-localized with CD68-positive cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157813#pone.0157813.s001" target="_blank">S1A Fig</a>). Scale bar = 10 μm. (C) Micrograph of the cross sections stained for αSMA (green). There is little co-localization with Alexa647-labeled rapamycin nanoparticles (red dots, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157813#pone.0157813.s001" target="_blank">S1B Fig</a>). Scale bar = 10 μm.</p

Polyplex Micelles with Double-Protective Compartments of Hydrophilic Shell and Thermoswitchable Palisade of Poly(oxazoline)-Based Block Copolymers for Promoted Gene Transfection

Improving the stability of polyplex micelles under physiological conditions is a critical issue for promoting gene transfection efficiencies. To this end, hydrophobic palisade was installed between the inner core of packaged plasmid DNA (pDNA) and the hydrophilic shell of polyplex micelles using a triblock copolymer consisting of hydrophilic poly­(2-ethyl-2-oxazoline), thermoswitchable amphiphilic poly­(2-<i>n</i>-propyl-2-oxazoline) (PnPrOx) and cationic poly­(l-lysine). The two-step preparation procedure, mixing the triblock copolymer with pDNA below the lower critical solution temperature (LCST) of PnPrOx, followed by incubation above the LCST to form a hydrophobic palisade of the collapsed PnPrOx segment, induced the formation of spatially aligned hydrophilic–hydrophobic double-protected polyplex micelles. The prepared polyplex micelles exhibited significant tolerance against attacks from nuclease and polyanions compared to those without hydrophobic palisades, thereby promoting gene transfection. These results corroborated the utility of amphiphilic poly­(oxazoline) as a molecular thermal switch to improve the stability of polyplex gene carriers relevant for physiological applications

Gelatinase activities and expression of inflammation-related factors in AAA homogenates after treatment of rapamycin nanoparticles.

<p>(A) Gelatin zymography of AAA lysates. The rats were subjected to elastase infusion, and 7 days later, AAA samples were collected. During the process of AAA formation, the rats received intravenous injections of PBS (<i>n</i> = 3), free/RAP-1 (<i>n</i> = 3), or RAP/nano-1 (<i>n</i> = 3). Gelatinase activities at 64, 72, and 92 kD represent matrix metalloproteinase-2 (MMP-2), latent form MMP-2 (pro-MMP-2), and latent form MMP-9 (pro-MMP-9), respectively. The MMP marker containing MMP-2, pro-MMP-2, and pro-MMP-9 was applied in the most right lane (M). (B–D) Gelatinase activities of MMP-9 (B), pro-MMP-2 (C), and pro-MMP-2 (D) were quantified by densitometry. In the expressions of pro-MMP-2 and MMP-2, the values after injections of RAP/nano-1 were significantly reduced as compared with those after injections of PBS and free/RAP-1. Contrarily, no differences were detected in the activities of pro-MMP-9 between the 3 treatments. Profiler array analyses of AAA at 7 days after elastase infusion revealed significant findings in expressions of interleukin (IL)-1α (E), IL-1β (F), and cytokine-induced neutrophil chemoattractant (CINC)-1 (G). The expression of IL-1α, IL-1β, and CINC-1 were significantly suppressed in the AAA after injections of RAP/nano-1 as compared with those after injections of PBS and free/RAP-1 (n = 3 for each). The experiments were repeated 3 times. In panels B–G, black dots represent the specific values in each group. Long and short bars represent mean and standard deviation, respectively. Error bars denote s.d. *<i>p</i> < 0.05, †<i>p</i> < 0.01 (unpaired Student’s <i>t</i>-test).</p

Structure and properties of rapamycin-incorporated nanoparticles.

<p>(A) Illustration showing that rapamycin-incorporated nanoparticles (rapamycin nanoparticles) are synthesized by mixing an equal mass of rapamycin and poly(ethylene glycol)-<i>b</i>-poly(γ-benzyl L-glutamate) (PEG-<i>b</i>-PBLG). Rapamycin is incorporated into the core of the PEG-shelled nanoparticle. (B) The diameter of nanoparticles measured by a Zetasizer. The size of the nanoparticles formed solely with PEG-<i>b</i>-PBLG was 42 nm (dotted line), whereas that of the rapamycin nanoparticles was 106 nm (solid line). (C) Time-course of the scattered light intensity of rapamycin nanoparticles under physiological conditions. Rapamycin nanoparticles are stable over the first 2 days. Data represent the means ± standard deviation (s.d.).</p

Accumulation of Alexa647-labeled rapamycin nanoparticles in the AAA rat model.

<p>(A) Representative images of macroscopic distribution of Alexa647 in the aorto-iliac specimens as imaged using the IVIS® imaging system. Abundant signals of Alexa647 were specifically distributed in the AAA at 4, 8, 16, and 24 hours after injection of Alexa647-labeled rapamycin nanoparticles (red-staining, at 1, 4, 8, and 24 hours, <i>n</i> = 4; at 16 hours, <i>n</i> = 3). (B) The plasma clearance of rapamycin nanoparticles in the rat model. The residual ratio of rapamycin nanoparticles in the plasma was high at 1 hour after injection. (C) Fluorescence intensities of the lysates of AAA and the thoracic aorta are shown as adjusted absorbance values. The values of AAA were significantly higher than those of the thoracic aorta at 8, 16, and 24 hours after injection. Data represent the means ± s.d. N.C. indicates negative control. *<i>p</i> < 0.05, †<i>p</i> < 0.01 (unpaired Student’s <i>t</i>-test).</p