Receptor-mediated transcytosis of macromolecules across the blood-brain barrier.

INTRODUCTION: The blood-brain barrier (BBB) restricts brain access of virtually all macromolecules. Receptor-mediated transcytosis (RMT) is one strategy toward their brain delivery. In this strategy, targeting ligands conjugated to therapeutic payload or decorating particles containing the payload interact with targets on brain capillary endothelial cells (BCEC), triggering internalization, trafficking, and release from BCEC. AREAS COVERED: RMT at the BBB has leveraged multiple formats of macromolecules and large particles. Interactions between those and BCEC have been studied primarily using antibodies, with findings applicable to the design of larger particles. BBB-penetrant constructs have also been identified in screening campaigns and directed evolution, and subsequently found to interact with RMT targets. In addition, BCEC targeted by constructs incorporating genomic payload can be made to produce therapeutic proteins. EXPERT OPINION: While targeting may not be strictly necessary to reach a therapeutic effect for all macromolecules, it can improve a molecule's BBB transport, exposing it to the entire brain parenchyma and enhancing its effect. Constructs with better BCEC transcytosis may be designed rationally, leveraging knowledge about BCEC trafficking, and found in screening campaigns, where this knowledge can reduce the search space and improve iterative refinement. Identification of new targets may also help generate BBB-crossing constructs.

Improved Drug Delivery to Brain Metastases by Peptide-Mediated Permeabilization of the Blood-Brain Barrier.

Patients with melanoma have a high risk of developing brain metastasis, which is associated with a dismal prognosis. During early stages of metastasis development, the blood-brain barrier (BBB) is likely intact, which inhibits sufficient drug delivery into the metastatic lesions. We investigated the ability of the peptide, K16ApoE, to permeabilize the BBB for improved treatment with targeted therapies preclinically. Dynamic contrast enhanced MRI (DCE-MRI) was carried out on NOD/SCID mice to study the therapeutic window of peptide-mediated BBB permeabilization. Further, both in vivo and in vitro assays were used to determine K16ApoE toxicity and to obtain mechanistic insight into its action on the BBB. The therapeutic impact of K16ApoE on metastases was evaluated combined with the mitogen-activated protein kinase pathway inhibitor dabrafenib, targeting BRAF mutated melanoma cells, which is otherwise known not to cross the intact BBB. Our results from the DCE-MRI experiments showed effective K16ApoE-mediated BBB permeabilization lasting for up to 1 hour. Mechanistic studies showed a dose-dependent effect of K16ApoE caused by induction of endocytosis. At concentrations above IC50, the peptide additionally showed nonspecific disturbances on plasma membranes. Combined treatment with K16ApoE and dabrafenib reduced the brain metastatic burden in mice and increased animal survival, and PET/CT showed that the peptide also facilitated the delivery of compounds with molecular weights as large as 150 kDa into the brain. To conclude, we demonstrate a transient permeabilization of the BBB, caused by K16ApoE, that facilitates enhanced drug delivery into the brain. This improves the efficacy of drugs that otherwise do not cross the intact BBB.

Ultrasound-mediated delivery and distribution of polymeric nanoparticles in the normal brain parenchyma of a metastatic brain tumour model.

The treatment of brain diseases is hindered by the blood-brain barrier (BBB) preventing most drugs from entering the brain. Focused ultrasound (FUS) with microbubbles can open the BBB safely and reversibly. Systemic drug injection might induce toxicity, but encapsulation into nanoparticles reduces accumulation in normal tissue. Here we used a novel platform based on poly(2-ethyl-butyl cyanoacrylate) nanoparticle-stabilized microbubbles to permeabilize the BBB in a melanoma brain metastasis model. With a dual-frequency ultrasound transducer generating FUS at 1.1 MHz and 7.8 MHz, we opened the BBB using nanoparticle-microbubbles and low-frequency FUS, and applied high-frequency FUS to generate acoustic radiation force and push nanoparticles through the extracellular matrix. Using confocal microscopy and image analysis, we quantified nanoparticle extravasation and distribution in the brain parenchyma. We also evaluated haemorrhage, as well as the expression of P-glycoprotein, a key BBB component. FUS and microbubbles distributed nanoparticles in the brain parenchyma, and the distribution depended on the extent of BBB opening. The results from acoustic radiation force were not conclusive, but in a few animals some effect could be detected. P-glycoprotein was not significantly altered immediately after sonication. In summary, FUS with our nanoparticle-stabilized microbubbles can achieve accumulation and displacement of nanoparticles in the brain parenchyma.

Quantification and Qualitative Effects of Different PEGylations on Poly(butyl cyanoacrylate) Nanoparticles.

Protein adsorption on nanoparticles (NPs) used in nanomedicine leads to opsonization and activation of the complement system in blood, which substantially reduces the blood circulation time of NPs. The most commonly used method to avoid protein adsorption is to coat the NPs with polyethylene glycol, so-called PEGylation. Although PEGylation is of utmost importance for designing the in vivo behavior of the NP, there is still a considerable lack of methods for characterization and fundamental understanding related to the PEGylation of NPs. In this work we have studied four different poly(butyl cyanoacrylate) (PBCA) NPs, PEGylated with different types of PEG-based nonionic surfactants-Jeffamine M-2070, Brij L23, Kolliphor HS 15, Pluronic F68-or combinations thereof. We evaluated the PEGylation, both quantitatively by nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and time-of-flight secondary ion mass spectrometry (ToF-SIMS) and qualitatively by studying ζ-potential, protein adsorption, diffusion, cellular interactions, and blood circulation half-life. We found that NMR and ToF-SIMS are complementary methods, while TGA is less suitable to quantitate PEG on polymeric NPs. It was found that longer PEG increases both blood circulation time and diffusion of NPs in collagen gels.

The effect of poly(ethylene glycol) coating and monomer type on poly(alkyl cyanoacrylate) nanoparticle interactions with lipid monolayers and cells.

The interaction of the promising drug carriers poly(alkyl cyanoacrylate) nanoparticles (PACA NPs) with lipid monolayers modeling the cell membrane and with RBE4 immortalized rat brain endothelial cells was compared to assess the relevance of lipid monolayer-based cell membrane models for PACA NP cellular uptake. NP properties such as size and charge of NPs and density of poly(ethylene glycol) coating (PEG) were kept in a narrow range to assess whether the type of PEG coating and the PACA monomer affected NP-monolayer and NP-cell interactions. The interaction with lipid monolayers was evaluated using surface pressure measurements and Brewster angle microscopy. NP association with and uptake by cells were assessed using flow cytometry and confocal laser scanning microscopy. The interaction between NPs and both lipid monolayers and the plasma membrane depended on the type of PEG. PEG density affected cellular uptake but not interaction with lipid monolayers. NP monomer, NPs size and charge had no effect on the interaction. This might be due to the fact that the size and charge distribution was kept rather narrow to study the effect of PACA monomer and PEG type. In conclusion, while modeling solely the passive aspect of NP-cell interactions, lipid monolayers nevertheless proved a valuable cell membrane model whose interaction with PACA NPs correlated well with NP-cell interaction. In addition, both NP-monolayer and NP-cell interactions were dependent on PEGylation type, which could be used in the design of NPs to either facilitate or hinder cellular uptake, depending on the intended purpose.

Labeling nanoparticles: Dye leakage and altered cellular uptake.

In vitro and in vivo behavior of nanoparticles (NPs) is often studied by tracing the NPs with fluorescent dyes. This requires stable incorporation of dyes within the NPs, as dye leakage may give a wrong interpretation of NP biodistribution, cellular uptake, and intracellular distribution. Furthermore, NP labeling with trace amounts of dye should not alter NP properties such as interactions with cells or tissues. To allow for versatile NP studies with a variety of fluorescence-based assays, labeling of NPs with different dyes is desirable. Hence, when new dyes are introduced, simple and fast screening methods to assess labeling stability and NP-cell interactions are needed. For this purpose, we have used a previously described generic flow cytometry assay; incubation of cells with NPs at 4 and 37°C. Cell-NP interaction is confirmed by cellular fluorescence after 37°C incubation, and NP-dye retention is confirmed when no cellular fluorescence is detected at 4°C. Three different NP-platforms labeled with six different dyes were screened, and a great variability in dye retention was observed. Surprisingly, incorporation of trace amounts of certain dyes was found to reduce or even inhibit NP uptake. This work highlights the importance of thoroughly evaluating every dye-NP combination before pursuing NP-based applications. © 2016 International Society for Advancement of Cytometry.

L-type calcium channels regulate filopodia stability and cancer cell invasion downstream of integrin signalling.

Mounting in vitro, in vivo and clinical evidence suggest an important role for filopodia in driving cancer cell invasion. Using a high-throughput microscopic-based drug screen, we identify FDA-approved calcium channel blockers (CCBs) as potent inhibitors of filopodia formation in cancer cells. Unexpectedly, we discover that L-type calcium channels are functional and frequently expressed in cancer cells suggesting a previously unappreciated role for these channels during tumorigenesis. We further demonstrate that, at filopodia, L-type calcium channels are activated by integrin inside-out signalling, integrin activation and Src. Moreover, L-type calcium channels promote filopodia stability and maturation into talin-rich adhesions through the spatially restricted regulation of calcium entry and subsequent activation of the protease calpain-1. Altogether we uncover a novel and clinically relevant signalling pathway that regulates filopodia formation in cancer cells and propose that cycles of filopodia stabilization, followed by maturation into focal adhesions, directs cancer cell migration and invasion.

Feasibility Study of the Permeability and Uptake of Mesoporous Silica Nanoparticles across the Blood-Brain Barrier.

Drug delivery into the brain is impeded by the blood-brain-barrier (BBB) that filters out the vast majority of drugs after systemic administration. In this work, we assessed the transport, uptake and cytotoxicity of promising drug nanocarriers, mesoporous silica nanoparticles (MSNs), in in vitro models of the BBB. RBE4 rat brain endothelial cells and Madin-Darby canine kidney epithelial cells, strain II, were used as BBB models. We studied spherical and rod-shaped MSNs with the following modifications: bare MSNs and MSNs coated with a poly(ethylene glycol)-poly(ethylene imine) (PEG-PEI) block copolymer. In transport studies, MSNs showed low permeability, whereas the results of the cellular uptake studies suggest robust uptake of PEG-PEI-coated MSNs. None of the MSNs showed significant toxic effects in the cell viability studies. While the shape effect was detectable but small, especially in the real-time surface plasmon resonance measurements, coating with PEG-PEI copolymers clearly facilitated the uptake of MSNs. Finally, we evaluated the in vivo detectability of one of the best candidates, i.e. the copolymer-coated rod-shaped MSNs, by two-photon in vivo imaging in the brain vasculature. The particles were clearly detectable after intravenous injection and caused no damage to the BBB. Thus, when properly designed, the uptake of MSNs could potentially be utilized for the delivery of drugs into the brain via transcellular transport.

Cellular uptake and intracellular degradation of poly(alkyl cyanoacrylate) nanoparticles.

BACKGROUND: Poly(alkyl cyanoacrylate) (PACA) nanoparticles have shown promise as drug carriers both to solid tumors and across the blood-brain barrier. Efficient drug delivery requires both high cellular uptake of the nanoparticles and release of the drug from the nanoparticles. Release of hydrophobic drugs from PACA nanoparticles is primarily governed by nanoparticle degradation, and this process has been poorly studied at the cellular level. Here we use the hydrophobic model drug Nile Red 668 (NR668) to investigate intracellular degradation of PACA nanoparticles by measuring changes in NR668 fluorescence emission and lifetime, as the spectral properties of NR668 depend on the hydrophobicity of the dye environment. We also assess the potential of poly(butyl cyanoacrylate) (PBCA) and poly(octyl cyanoacrylate) (POCA) nanoparticles for intracellular drug delivery in the prostate cancer cell line PC3 and rat brain endothelial cell line RBE4 and the role of endocytosis pathways in PACA nanoparticle uptake in those cell lines. RESULTS: Fluorescence lifetime imaging, emission spectra analysis and Förster resonance energy transfer indicated that the intracellular degradation was in line with the degradation found by direct methods such as gas chromatography and scanning electron microscopy, showing that PBCA has a faster degradation rate compared to POCA. The combined P(BCA/OCA) nanoparticles had an intermediate degradation rate. The uptake of POCA and PBCA nanoparticles was much higher in RBE4 than in PC3 cells. Endocytosis inhibition studies showed that both clathrin- and caveolin-mediated endocytosis were involved in PACA nanoparticle uptake, and that the former played a predominant role, particularly in PC3 cells. CONCLUSIONS: In the present study, we used three different optical techniques to show that within a 24-hour period PBCA nanoparticles degraded significantly inside cells, releasing their payload into the cytosol, while POCA nanoparticles remained intact. This indicates that it is possible to tune the intracellular drug release rate by choosing appropriate monomers from the PACA family or by using hybrid PACA nanoparticles containing different monomers. In addition, we showed that the uptake of PACA nanoparticles depends not only on the monomer material, but also on the cell type, and that different cell lines can use different internalization pathways.

Distinct c-Met activation mechanisms induce cell rounding or invasion through pathways involving integrins, RhoA and HIP1.

Many carcinomas have acquired oncogenic mechanisms for activating c-Met, including c-Met overexpression and excessive autocrine or paracrine stimulation with hepatocyte growth factor (HGF). However, the biological outcome of c-Met activation through these distinct modes remains ambiguous. Here, we report that HGF-mediated c-Met stimulation triggers a mesenchymal-type collective cell invasion. By contrast, the overexpression of c-Met promotes cell rounding. Moreover, in a high-throughput siRNA screen that was performed using a library of siRNAs against putative regulators of integrin activity, we identified RhoA and the clathrin-adapter protein HIP1 as crucial c-Met effectors in these morphological changes. Transient RhoA activation was necessary for the HGF-induced invasion, whereas sustained RhoA activity regulated c-Met-induced cell rounding. In addition, c-Met-induced cell rounding correlated with the phosphorylation of filamin A and the downregulation of active cell-surface integrins. By contrast, a HIP1-mediated increase in ß1-integrin turnover was required for the invasion triggered by HGF. Taken together, our results indicate that c-Met induces distinct cell morphology alterations depending on the stimulus that activates c-Met.