Zaire ebolavirus (ZEBOV), a highly pathogenic zoonotic virus, poses serious public health, ecological and potential bioterrorism threats. Currently no specific therapy or vaccine is available. Virus entry is an attractive target for therapeutic intervention. However, current knowledge of the ZEBOV entry mechanism is limited. While it is known that ZEBOV enters cells through endocytosis, which of the cellular endocytic mechanisms used remains unclear. Previous studies have produced differing outcomes, indicating potential involvement of multiple routes but many of these studies were performed using noninfectious surrogate systems such as pseudotyped retroviral particles, which may not accurately recapitulate the entry characteristics of the morphologically distinct wild type virus. Here we used replication-competent infectious ZEBOV as well as morphologically similar virus-like particles in specific infection and entry assays to demonstrate that in HEK293T and Vero cells internalization of ZEBOV is independent of clathrin, caveolae, and dynamin. Instead the uptake mechanism has features of macropinocytosis. The binding of virus to cells appears to directly stimulate fluid phase uptake as well as localized actin polymerization. Inhibition of key regulators of macropinocytosis including Pak1 and CtBP/BARS as well as treatment with the drug EIPA, which affects macropinosome formation, resulted in significant reduction in ZEBOV entry and infection. It is also shown that following internalization, the virus enters the endolysosomal pathway and is trafficked through early and late endosomes, but the exact site of membrane fusion and nucleocapsid penetration in the cytoplasm remains unclear. This study identifies the route for ZEBOV entry and identifies the key cellular factors required for the uptake of this filamentous virus. The findings greatly expand our understanding of the ZEBOV entry mechanism that can be applied to development of new therapeutics as well as provide potential insight into the trafficking and entry mechanism of other filoviruses.

The phosphoinositide-3 kinase (PI3K) pathway regulates diverse cellular activities related to cell growth, migration, survival, and vesicular trafficking. It is known that Ebola virus requires endocytosis to establish an infection. However, the cellular signals that mediate this uptake were unknown for Ebola virus as well as many other viruses. Here, the involvement of PI3K in Ebola virus entry was studied. A novel and critical role of the PI3K signaling pathway was demonstrated in cell entry of Zaire Ebola virus (ZEBOV). Inhibitors of PI3K and Akt significantly reduced infection by ZEBOV at an early step during the replication cycle. Furthermore, phosphorylation of Akt-1 was induced shortly after exposure of cells to radiation-inactivated ZEBOV, indicating that the virus actively induces the PI3K pathway and that replication was not required for this induction. Subsequent use of pseudotyped Ebola virus and/or Ebola virus-like particles, in a novel virus entry assay, provided evidence that activity of PI3K/Akt is required at the virus entry step. Class 1A PI3Ks appear to play a predominant role in regulating ZEBOV entry, and Rac1 is a key downstream effector in this regulatory cascade. Confocal imaging of fluorescently labeled ZEBOV indicated that inhibition of PI3K, Akt, or Rac1 disrupted normal uptake of virus particles into cells and resulted in aberrant accumulation of virus into a cytosolic compartment that was non-permissive for membrane fusion. We conclude that PI3K-mediated signaling plays an important role in regulating vesicular trafficking of ZEBOV necessary for cell entry. Disruption of this signaling leads to inappropriate trafficking within the cell and a block in steps leading to membrane fusion. These findings extend our current understanding of Ebola virus entry mechanism and may help in devising useful new strategies for treatment of Ebola virus infection.

We report on a novel type of triblock copolymer polymersomes with temperature-controlled permeability within the physiologically relevant temperature range of 37–42 °C for sustained delivery of anticancer drugs. These polymersomes combine characteristics of liposomes, such as biocompatibility, biodegradability, monodispersity, and stability at room temperature, with tunable size and thermal responsiveness provided by amphiphilic triblock copolymers. The temperature-sensitive poly­(N-vinylcaprolactam) n -poly­(dimethylsiloxane)65-poly­(N-vinylcaprolactam) n (PVCL n -PDMS65-PVCL n ) copolymers with n = 10, 15, 19, 29, and 50 and polydispersity indexes less than 1.17 are synthesized by controlled RAFT polymerization. The copolymers are assembled into stable vesicles at room temperature when the ratio of PVCL to the total polymer mass is 0.36 < f < 0.52 with the polymersome diameter decreasing from 530 to 40 nm as the length of PVCL is increased from 10 to 19 monomer units. Importantly, the permeability of polymersomes loaded with the anticancer drug doxorubicin can be precisely controlled by PVCL length in the temperature range of 37–42 °C. Increasing the temperature above the lower critical solution temperature of PVCL results in either gradual vesicle shrinkage (n = 10 and n = 15) or reversible formation of beadlike aggregates with no size change (n = 19), both leading to sustained drug release. All temperature-triggered morphological changes are reversible and do not compromise the structural stability of the vesicles. Finally, concentration- and time-dependent cytotoxicity of drug-loaded polymersomes to human alveolar adenocarcinoma cells is demonstrated. Considering the high loading capacity (∼40%) and temperature responsiveness in the physiological range, these polymer vesicles have considerable potential as novel types of stimuli-responsive drug nanocarriers.

We demonstrated a simple and facile approach to fabricate biocompatible monodisperse hollow microparticles of controlled geometry. The hemispherical, spherical, and cubical microparticles are obtained by drying multilayer capsules of hydrogen-bonded poly(N-vinylpyrrolidone)/tannic acid (PVPON/TA)n. Drying spherical capsules results in hemispherical particles if 15 < n < 20. This shape transformation is controlled by capsule stiffness, which is regulated by the layer number, capsule diameter, and PVPON molecular weight. Cubical and spherical hollow particles maintaining their three-dimensional shapes in the dry state are obtained if n ≥ 25.5. A 17-fold stiffness increase is required to lead from totally collapsed (PVPON/TA)5.5 to dried self-supporting (PVPON/TA)25.5 particles of 2 μm in dimensions. All hollow particles could be further resuspended in aqueous solutions while retaining their shapes upon rehydration. The cell growth and viability studies using human cancer cells revealed noncytotoxic properties of the (PVPON/TA) multilayer particles. Both spherical and hemispherical capsules were internalized by macrophages with the uptake of the hemispherical particles per cell two times more efficient. The method presented here allows for a robust preparation of biocompatible shaped particles whose shape and dimensions can be easily tuned by controlling capsule size and wall thickness. The reported structures can be potentially useful for biomedical applications such as shape-controlled cellular uptake and flow dynamics.

Oropouche (ORO) virus, a member of the Simbu serogroup, is one of the few human pathogens in the Orthobunyavirus genus in the family Bunyaviridae. Genetic analyses of ORO-like strains from Iquitos, Peru, identified a novel reassortant containing the S and L segments of ORO virus and the M segment of a novel Simbu serogroup virus. This new pathogen, which we named Iquitos (IQT) virus, was first isolated during 1999 from a febrile patient in Iquitos, an Amazonian city in Peru. Subsequently, the virus was identified as the cause of outbreaks of “Oropouche fever” during 2005 and 2006 in Iquitos. In addition to the identification of 17 isolates of IQT virus between 1999 and 2006, surveys for neutralizing antibody among Iquitos residents revealed prevalence rates of 14.9% for ORO virus and 15.4% for IQT virus. Limited studies indicate that prior infection with ORO virus does not seem to protect against disease caused with the IQT virus infection. Identification of a new Orthobunyavirus human pathogen in the Amazon region of Peru highlights the need for strengthening surveillance activities and laboratory capabilities, and investigating the emergence of new pathogens in tropical regions of South America.

Doxorubicin (DOX)-loaded poly(methacrylic acid) hydrogel cubes release the drug at pH <5. These hydrogels are developed for shape-directed cellular uptake for drug delivery.

Shape and responsiveness of nanoengineered delivery carriers are crucial characteristics for rapid and efficient delivery of therapeutics. We report on a novel type of micrometer-sized hydrogel particles of controlled shape with dual pH- and redox-sensitivity for intracellular delivery of anticancer drugs. The cubical and spherical poly(methacrylic acid) (PMAA) networks with disulfide links are obtained by cross-linking PMAA with cystamine within hydrogen-bonded multilayers of PMAA/poly(vinylpyrrolidone) (PMAA/PVPON) on sacrificial mesoporous templates. The pH-triggered hydrogel swelling/shrinkage not only affords effective doxorubicin entrapment but also efficient endosomal/lysosomal escape, and redox-triggered degradation provides drug release into the cytosolic space. The hydrogels degrade rapidly to low molecular weight chains in the presence of the typical intracellular concentration of glutathione, which should ensure a rapid renal clearance in vivo. Particle shape is found to affect internalization at the initial step of cell-particle interactions. Drug-loaded spherical particles are found to be 12% more cytotoxic than the corresponding cubes within the first 10 h of cell incubation suggesting more rapid internalization of spheres. Both doxorubicin-loaded hydrogel cubes and spheres demonstrate 50% and 90% cytotoxicity when incubated with HeLa cancer cells for 24 and 48 h, respectively. The presented approach integrates the advantages of pH-sensitivity, enzymatic degradation, and shape-regulated internalization for novel types of "intelligent" three-dimensional networks with programmable behavior for use in controlled delivery of therapeutics.

We report on novel diblock copolymers of poly(N‐vinylcaprolactam) (PVCL) and poly(N‐vinyl‐2‐pyrrolidone) (PVPON) (PVCL‐b‐PVPON) with well‐defined block lengths synthesized by the MADIX/reversible addition‐fragmentation chain transfer (RAFT) process. We show that the lower critical solution temperatures (LCST) of the block copolymers are controllable over the length of PVCL and PVPON segments. All of the diblock copolymers dissolve molecularly in aqueous solutions when the temperature is below the LCST and form spherical micellar or vesicular morphologies when temperature is raised above the LCST. The size of the self‐assembled structures is controlled by the molar ratio of PVCL and PVPON segments. The synthesized homopolymers and diblock copolymers are demonstrated to be nontoxic at 0.1–1 mg mL−1 concentrations when incubated with HeLa and HEK293 cancer cells for various incubation times and have potential as nanovehicles for drug delivery. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014, 52, 2725–2737