Endocytic Uptake Pathways Utilized by CPMV Nanoparticles

Cowpea mosaic virus (CPMV) has been used as a nanoparticle platform for biomedical applications including vaccine development, in vivo vascular imaging, and tissue-targeted delivery. A better understanding of the mechanisms of CPMV targeting and cell internalization would enable enhanced targeting and more effective delivery. Previous studies showed that, following binding and internalization by mammalian cells, CPMV localizes in a perinuclear late-endosome compartment where it remains for as long as several days. To further investigate endocytic trafficking of CPMV within the cell, we used multiple approaches including pharmacologic inhibition of pathways and colocalization with endocytic vesicle compartments. CPMV internalization was clathrin-independent and utilized a combination of caveolar endocytosis and macropinocytosis pathways for entry. CPMV particles colocalized with Rab5⁺ early endosomes to traffic ultimately to a lysosomal compartment. These studies facilitate the further development of effective intracellular drug-delivery strategies using CPMV

Summary of studies measuring RDI-eIgG1 protection from LeTx challenge in rats.

<p>Male 200 gram HSD were co-injected i.v. with LeTx (40 µg PA+12 µg LF) alone or in combination with RDI-eIgG1. Number of surviving animals in each study and the average time to death (in hours) is reported. Statistical analyses of survival curves (log-rank Mantel-Cox) and time-to-death (Student T-test) were performed using Prism (Graph Pad, Inc.).</p

Construction, expression and half-life of RDI and RDI-eIgG1.

<p>A. Schematic of RDI and RDI-eIgG1 constructs. VWA/I: vonWillebrand Factor A/Integrin-like I domain. Mutations T250Q and M428L in the Fc portion confer extended circulation time. B. SimplyBlue (Invitrogen) stained gel of RDI-eIgG1 separated on a SDS-containing10% polyacrylamide gel. Molecular weight markers are indicated. C. Detection of RDI (top panel) or RDI-eIgG1 (bottom panel) after intravenous injection in 2 rats by ELISA analysis. Curve-fitting and half-life (t_1/2) was calculated using Prism (Graph Pad, Inc.) D. Inhibition of LeTx activity in RAW264.7 cells by RDI or RDI-eIgG1.</p

RDI-eIgG1 protects rats from LeTx challenge in the short term but shows delayed toxicity.

<p>Male 200 gram HSD rats (5/group) were co-injected i.v. with LeTx (40 µg PA+12 µg LF) and RDI (19 µg) or RDI-eIgG1 (49 µg). Rats were monitored and time of death recorded.</p

Delayed toxicity of rats following LeTx challenge with RDI-IgG1 and RDI-IgG2 fusion proteins.

<p>Male 200 gram HSD rats (5/group) were co-injected i.v. with LeTx (40 µg PA+12 µg LF) and either 19 µg of RDI; or 49 µg of either RDI-eIgG1, RDI-IgG1, or RDI-IgG2. Rats were monitored and time of death recorded.</p

Association of PA₆₃ and LF with RDI-eIgG1 during circulation <i>in vivo</i>.

<p>Rats were injected through jugular vein cannulas with a total volume of 500 µl containing 40 µg PA, 12 µg LF, 122 µg of RDI-eIgG1 in PBS vehicle per rat. This corresponds to a 5∶1 molar ratio of the VWA/I domain∶PA. Serum was prepared from blood collected 15 hours after injection and protein G sepharose was used to precipitate RDI-eIgG1 and associated proteins from the rat serum. Samples were separated on a SDS-containing 4–12% polyacrylamide gel and analyzed by immunoblotting for the presence of RDI-eIgG1, PA, or LF.</p

RDI-eIgG1 alone is not toxic in rats.

<p>Male 200 gram HSD rats (Harlan Laboratories, Indianapolis, IN) (3/group) were co-injected i.v. with LeTx (40 µg PA+12 µg LF) alone or in combination with 49 ug RDI-eIgG1; or were dosed i.v. with 123 µg RDI-eIgG1 alone. Rats were monitored and time of death recorded.</p

Construction of RDI fusion proteins with IgG1 or IgG2 Fc portions.

<p>A. Schematic of the RDI and Fc portions of the fusion proteins. B. SimplyBlue stained gel of RDI-eIgG1, RDI-IgG1, or RDI-IgG2 separated on 10% SDS-PAGE. Molecular weight markers are indicated. C. Inhibition of LeTx activity in RAW264.7 cells by RDI-eIgG1, RDI-IgG1, and RDI-IgG2.</p

Encapsidated Atom-Transfer Radical Polymerization in Qβ Virus-like Nanoparticles

Virus-like particles (VLPs) are unique macromolecular structures that hold great promise in biomedical and biomaterial applications. The interior of the 30 nm-diameter Qβ VLP was functionalized by a three-step process: (1) hydrolytic removal of endogenously packaged RNA, (2) covalent attachment of initiator molecules to unnatural amino acid residues located on the interior capsid surface, and (3) atom-transfer radical polymerization of tertiary amine-bearing methacrylate monomers. The resulting polymer-containing particles were moderately expanded in size; however, biotin-derivatized polymer strands were only very weakly accessible to avidin, suggesting that most of the polymer was confined within the protein shell. The polymer-containing particles were also found to exhibit physical and chemical properties characteristic of positively charged nanostructures, including the ability to easily enter mammalian cells and deliver functional small interfering RNA

Lysine Addressability and Mammalian Cell Interactions of Bacteriophage λ Procapsids

Chemically or genetically modified virus particles, termed viral nanoparticles (VNPs), are being explored in applications such as drug delivery, vaccine development, and materials science. Each virus platform has inherent properties and advantages based on its structure, molecular composition, and biomolecular interactions. Bacteriophage λ was studied for its lysine addressability, stability, cellular uptake, and the ability to modify its cellular uptake. λ procapsids could be labeled primarily at a single residue on the gpE capsid protein as determined by tandem mass spectrometry, providing a unique attachment site for further capsid modification. Bioconjugation of transferrin to the procapsids mediated specific interaction with transferrin receptor-expressing cells. These studies demonstrate the utility of bacteriophage λ procapsids and their potential use as targeted drug delivery vehicles