Changes in BBB permeability as revealed by MRI.

<p>Mice were pretreated with saline (Group A), 1.6 µg VEGF (Group B), or 3.0 µg VEGF (Group C) by venous injection 8 hours prior to administration of the contrast agent (Gd-DTPA). Mice from the three treatment groups were scanned before (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086407#pone-0086407-g002" target="_blank">Figure 2</a>.1 A1, B1 and C1) and immediately after Gd-DTPA injection (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086407#pone-0086407-g002" target="_blank">Figure 2</a>.1 A2, B2 and C2). Regions of interest (ROIs) was manually defined in both cerebral hemispheres and basal ganglia. ROI1 and ROI2 are located over regions of cerebral cortex and ROI3 and ROI4 over the basal ganglia. ROI5 is over the water tube. Compared to the saline-treated control group (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086407#pone-0086407-g002" target="_blank">Figure 2</a>.1 A2), signal intensity enhancement was observed after treatment with 1.6 µg VEGF, particularly around cerebral ventricles (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086407#pone-0086407-g002" target="_blank">Figure 2</a>.1 B2, arrow). Pretreatment with 3.0 µg VEGF also resulted in an obvious signal intensity enhancement in both cerebral cortex and basal ganglia (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086407#pone-0086407-g002" target="_blank">Figure 2</a>.1 C2, arrows) comparable with saline treatment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086407#pone-0086407-g002" target="_blank">Figure 2</a>.1 A2). Arrow head indicates the water tube. (2.2, 2.3) Statistical analysis of signal intensity changes from the cerebrum and basal ganglia. Signal intensity values of each animal were calculated as follows: ROIa = (ROI1/ROI5+ROI2/ROI5)/2, ROIb =  (ROI3/ROI5+ROI4/ROI5)/2. ROIa and ROIb were used for the statistical analysis of the three groups. ROIa, signal intensity from the cerebral hemisphere. ROIb, signal intensity from the basal ganglia.</p

Receptor-Mediated Delivery of Magnetic Nanoparticles across the Blood–Brain Barrier

A brain delivery probe was prepared by covalently conjugating lactoferrin (Lf) to a poly(ethylene glycol) (PEG)-coated Fe₃O₄ nanoparticle in order to facilitate the transport of the nanoparticles across the blood–brain barrier (BBB) by receptor-mediated transcytosis <i>via</i> the Lf receptor present on cerebral endothelial cells. The efficacy of the Fe₃O₄-Lf conjugate to cross the BBB was evaluated <i>in vitro</i> using a cell culture model for the blood–brain barrier as well as <i>in vivo</i> in SD rats. For an <i>in vitro</i> experiment, a well-established porcine BBB model was used based on the primary culture of cerebral capillary endothelial cells grown on filter supports, thus allowing one to follow the transfer of nanoparticles from the apical (blood) to the basolateral (brain) side. For <i>in vivo</i> experiments, SD rats were used as animal model to detect the passage of the nanoparticles through the BBB by MRI techniques. The results of both <i>in vitro</i> and <i>in vivo</i> experiments revealed that the Fe₃O₄-Lf probe exhibited an enhanced ability to cross the BBB in comparison to the PEG-coated Fe₃O₄ nanoparticles and further suggested that the Lf-receptor-mediated transcytosis was an effective measure for delivering the nanoparticles across the BBB

Synergistic Dual-Ligand Doxorubicin Liposomes Improve Targeting and Therapeutic Efficacy of Brain Glioma in Animals

Therapeutic outcome for the treatment of glioma was often limited due to low permeability of delivery systems across the blood–brain barrier (BBB) and poor penetration into the tumor tissue. In order to overcome these hurdles, we developed the dual-targeting doxorubicin liposomes conjugated with cell-penetrating peptide (TAT) and transferrin (T7) (DOX-T7-TAT-LIP) for transporting drugs across the BBB, then targeting brain glioma, and penetrating into the tumor. The dual-targeting effects were evaluated by both <i>in vitro</i> and <i>in vivo</i> experiments. <i>In vitro</i> cellular uptake and three-dimensional tumor spheroid penetration studies demonstrated that the system could not only target endothelial and tumor monolayer cells but also penetrate tumor to reach the core of the tumor spheroids and inhibit the growth of the tumor spheroids. <i>In vivo</i> imaging further demonstrated that T7-TAT-LIP provided the highest tumor distribution. The median survival time of tumor-bearing mice after administering DOX-T7-TAT-LIP was significantly longer than those of the single-ligand doxorubicin liposomes and free doxorubicin. In conclusion, the dual-ligand liposomes comodified with T7 and TAT possessed strong capability of synergistic targeted delivery of payload into tumor cells both <i>in vitro</i> and <i>in vivo</i>, and they were able to improve the therapeutic efficacy of brain glioma in animals

The sensitivity analysis of BiLC Rho GTPase biosensors to GEFs and GAPs.

<p>(A) The results of optical imaging among different upstream regulatory proteins. The luminescent signals were normalized using cotransfection of renilla luciferase plasmid (pRL-tk) and represented by the ratio of luminescent intensity of firefly luciferase (FL) at 600 nm to that of renilla luciferase (RL) at 500 nm. The final results were normalized by the luminescence ratio of the control vehicles, which were designated with “1”. Data is reported as the fold increase in luminescence ratio (FL/RL) relative to control. Error bars denote standard deviations. Asterisks (*) denotes samples that show a difference from the control vector with statistical significance by analysis of variance (ANONA) (<i>p</i>≤0.01). The data shown was obtained by three separate experiments performed with quadruplicate culture wells. The results highly accord with the well-known experimental data, indicating that the BiLC biosensors can response to the upstream regulatory molecules. And this ability of BiLC GTPase sensors can be used to examine the substrate selectivity of GEFs and GAPs and quantify their catalytic activities in intact living cells. (B and C) The western blots carried out in parallel to demonstrate protein expression among different GEFs and GAPs. The figure only shows the results of the CDC42 biosensors as a representative.</p

Optimizing the appropriate configuration (or domain arrangement) for BiLC Rho GTPase biosensor.

<p>(A) The optical results of eight different configurations. Relative luciferase activities were detected in living 293 cells cotransfected with the different configurations of N- and C-terminal FL fragments with interacting proteins CDC42 and WASP constructed with different orientations. Correct configuration is very critical to achieving efficient reconstruction of luciferase activity with PCA strategy. In each configuration, luciferase activity was compared among CDC42 WT, G12V, T17N and F37A mutants, which represent different levels of CDC42 activity and different degrees of the interaction. WT, G12V, T17N and F37A indicate wild type, constitutively active mutant, dominant negative mutant and effector mutant, respectively. The results were normalized using cotransfection of RL and represented by the ratio of luminescent intensity of FL at 600 nm to that of RL at 500 nm. The data shown are representative of three separate experiments performed with quadruplicate culture wells. The results show that the combinations containing Nfluc416-WASP/CDC42-Cfluc398 and Nfluc416-WASP/Cfluc398-CDC42 produced a greater level of luminescent signal and wider dynamic rang for different levels of CDC42 activity. (B) The western blots carried out in parallel to demonstrate the protein expression among CDC42 WT, G12V, T17N and F37A biosensors. This figure only shows the results of Nfluc416-WASP/Cfluc398-CDC42 as representative.</p

The application of BiLC strategy to image the three main members of Rho GTPases.

<p>(A) The results of optical imaging of three kinds of BiLC RhoGTPase biosensors. The relative luminescence was calculated by the ratio of luminescent intensity of firefly luciferase (FL) at 600 nm to that of renilla luciferase (RL) at 500 nm (n = 4, representative of 4 independent experiments). Error bars denote standard deviations. Asterisks (*) denotes samples that show a difference from the nonspecific complementation (the non-interactive GTPase-effector pairs or the effector loop mutants) with statistical significance by analysis of variance (ANOVA) (<i>p</i>≤0.01). This result indicates that the nonspecific complementation does not impede the correct interpretation of effective interactions induced by GTPase activation. WYJH (#) denotes samples that show a difference from the wild-type biosensor with statistical significance by ANOVA (<i>p</i>≤0.01). This result indicates that the BiLC sensors possess the discriminatory power among different GTPase activity states. (B) The results of coimmunoprecipitation. The results show that the expressions of the BiLC biosensors had no significant discrimination among different alleles of Rho biosensor, but the intensities of the PPIs displayed obvious diversities, which were in accordance with the results obtained by our optical imaging. (C) In vivo optical CCD imaging of BiLC Rho GTPase biosensors. The pseudotumors in living mice were generated by engrafting with transiently transfected 293 cells. The pRL-tk plasmid was cotransfected and RL activity was detected to normalize the planted cell number. 24 h after implantation, the mice were imaged using IVIS spectrum. A significant discrimination of luciferase activity was detected among different alleles of Rho GTPase. (I: the dominant active mutants; II: the wild-type Rho GTPases; III: the effector-loop mutants; IV: the dominant negative mutants).</p

Optimizing appropriate dissection site of firefly luciferase for BiLC Rho GTPase biosensor.

<p>(A) The optical imaging results among different split-sites of firefly luciferase. Relative luciferase activities were detected in living 293 cells cotransfected with the five different combinations of split firefly luciferase fragments (Nfluc416-WASP/Cfluc398-CDC42, Nfluc416-WASP/Cfluc417-CDC42, Nfluc437-WASP/Cfluc438-CDC42, Nfluc398-WASP/Cfluc384-CDC42, Nfluc445-WASP/Cfluc446-CDC42), respectively. In each luciferase fragments combination, luciferase activity was compared among CDC42 WT, G12V, and F37A mutants. WT, G12V, and F37A indicate wild type, the constitutively active mutant, and the effector-loop mutant, respectively. The results were normalized using cotransfection of renilla luciferase plasmid (pRL-tk) and represented by the ratio of luminescent intensity of firefly luciferase (FL) at 600 nm to that of renilla luciferase (RL) at 500 nm (FL/RL). The data shown are representative of four separate experiments performed with quadruplicate culture wells. The result shows that the combination (Nfluc416/Cfluc398) had the widest dynamic range and the highest luciferase activity restoration. (B) The western blots carried out in parallel to demonstrate the protein expression among CDC42 WT, G12V, and F37A biosensors. This figure only shows the results of Nfluc416-WASP/CDC42-Cfluc398 as representative.</p

The sensitivity analysis of BiLC Rho GTPase sensors to extracellular ligands.

<p>(A) The temporal response of BiLC Rho GTPase sensors stimulated by extracellular ligands. After being serum-starved in serum-free DMEM medium for 6 h, mouse fibroblast NIH3T3 cells were detected the luminescent signals by adding D-luciferin until the intensities became steady, then stimulated with insulin (2 mg/mL), lysophosphatidic acid (40 ng/mL) and bradykinin (100 ng/mL), which are the known activator of Rac1, RhoA and Cdc42 respectively, and then immediately acquired the sequence image (1-min exposure; emission filter, open; f-stop, 1; binning, 8; field of view, 15 cm) for 30 min using IVIS spectrum. The data shown were obtained by three separate experiments performed with quadruplicate culture wells. The result shows that not only the activation signals of Rho GTPases from upstream pathways but also the subsequent decrease following the hydrolysis of GTP can be displayed and quantified by the BiLC-based biosensors. And the optical results (left) accord with that of ‘pull-down’ in our previous work (right). (B) The responses of BiLC Rho GTPase sensors to different concentration of extracellular ligands. The cells were stimulated with different concentrations of the stimulators and the luciferase activity was acquired after 3 min by IVIS spectrum (1-min exposure; emission filter, open; f-stop, 1; binning, 8; field of view, 15 cm). The data shown was obtained by three separate experiments performed with quadruplicate culture wells. The optical results (upper) were in accordance with that of our previous ‘pull-down’ (under).</p

The schematic diagrams of the constructs and the mechanism for BiLC-based Rho GTPase biosensors.

<p>(A) The graphic schemes of the constructs used in process of optimizing the appropriate dissection sites of firefly luciferase. For the construct of CDC42-Cfluc, CDC42 (AA 1–176) was used and the carboxy-terminal region of CDC42 (AA171–191) was added to the downstream of the fusion protein, making sure the correct localization to the plasma membrane and the regulation of GDIs. (B) The graphic schemes of the constructs used in process of optimizing the appropriate orientation of the reporter fragments (FN and FC) and the interacting proteins (WASP and CDC42). If CDC42 was fused to the N-terminal of the reporter fragment, CDC42 (AA 1–176) was used and the carboxy-terminal region of CDC42 (AA 171–191) was added downstream of the reporter fragment. (C) The schematic diagram of the optimal configuration and the mechanism for BiLC-based Rho GTPase biosensors. We use the optimal configuration as representative to describe the mechanism of the biosensors. In this strategy, two non-functional luciferase fragments are respectively fused, in fame and with a short flexible linker (G₂S)_2∼4 or (G₄S)_1∼2, to Rho GTPase and the GBD of the specific effector. Once Rho GTPase is activated by upstream stimulating factors, the two luciferase fragments (luc1 and luc2) are brought into close proximity by the active Rho GTPase binding to the GBD of the effector, leading to the restoration of luciferase activity and photon production in presence of the substrate.</p

A preclinical evaluation of alternative site for islet allotransplantation

<div><p>The bone marrow cavity (BMC) has recently been identified as an alternative site to the liver for islet transplantation. This study aimed to compare the BMC with the liver as an islet allotransplantation site in diabetic monkeys. Diabetes was induced in Rhesus monkeys using streptozocin, and the monkeys were then divided into the following three groups: Group1 (islets transplanted in the liver with immunosuppressant), Group 2 (islets transplanted in the tibial BMC), and Group 3 (islets transplanted in the tibial BMC with immunosuppressant). The C-peptide and blood glucose levels were preoperatively measured. An intravenous glucose tolerance test (IVGTT) was conducted to assess graft function, and complete blood cell counts were performed to assess cell population changes. Cytokine expression was measured using an enzyme-linked immune sorbent assay (ELISA) and MILLIPLEX. Five monkeys in Group 3 exhibited a significantly increased insulin-independent time compared with the other groups (Group 1: 78.2 ± 19.0 days; Group 2: 58.8 ± 17.0 days; Group 3: 189.6 ± 26.2 days) and demonstrated increases in plasma C-peptide 4 months after transplantation. The infusion procedure was not associated with adverse effects. Functional islets in the BMC were observed 225 days after transplantation using the dithizone (DTZ) and insulin/glucagon stains. Our results showed that allogeneic islets transplanted in the BMC of diabetic Rhesus monkeys remained alive and functional for a longer time than those transplanted in the liver. This study was the first successful demonstration of allogeneic islet engraftment in the BMC of non-human primates (NHPs).</p></div