Synergistic Antitumor Activity from Two-Stage Delivery of Targeted Toxins and Endosome-Disrupting Nanoparticles

Plant-derived Type I toxins are candidate anticancer therapeutics requiring cytosolic delivery into tumor cells. We tested a concept for two-stage delivery, whereby tumor cells precoated with an antibody-targeted gelonin toxin were killed by exposure to endosome-disrupting polymer nanoparticles. Co-internalization of particles and tumor cell-bound gelonin led to cytosolic delivery and >50-fold enhancement of toxin efficacy. This approach allows the extreme potency of gelonin to be focused on tumors with significantly reduced potential for off-target toxicity

Enhancing Radiotherapy by Lipid Nanocapsule-Mediated Delivery of Amphiphilic Gold Nanoparticles to Intracellular Membranes

Amphiphilic gold nanoparticles (amph-NPs), composed of gold cores surrounded by an amphiphilic mixed organic ligand shell, are capable of embedding within and traversing lipid membranes. Here we describe a strategy using crosslink-stabilized lipid nanocapsules (NCs) as carriers to transport such membrane-penetrating particles into tumor cells and promote their transfer to intracellular membranes for enhanced radiotherapy of cancer. We synthesized and characterized interbilayer-crosslinked multilamellar lipid vesicles (ICMVs) carrying amph-NPs embedded in the capsule walls, forming Au-NCs. Confocal and electron microscopies revealed that the intracellular distribution of amph-NPs within melanoma and breast tumor cells following uptake of free particles <i>vs</i> Au-NCs was quite distinct and that amph-NPs initially delivered into endosomes by Au-NCs transferred over a period of hours to intracellular membranes through tumor cells, with greater intracellular spread in melanoma cells than breast carcinoma cells. Clonogenic assays revealed that Au-NCs enhanced radiotherapeutic killing of melanoma cells. Thus, multilamellar lipid capsules may serve as an effective carrier to deliver amphiphilic gold nanoparticles to tumors, where the membrane-penetrating properties of these materials can significantly enhance the efficacy of frontline radiotherapy treatments

Freely Suspended Cellular “Backpacks” Lead to Cell Aggregate Self-Assembly

Cellular “backpacks” are a new type of anisotropic, nanoscale thickness microparticle that may be attached to the surface of living cells creating a “bio-hybrid” material. Previous work has shown that these backpacks do not impair cell viability or native functions such as migration in a B and T cell line, respectively. In the current work, we show that backpacks, when added to a cell suspension, assemble cells into aggregates of reproducible size. We investigate the efficiency of backpack−cell binding using flow cytometry and laser diffraction, examine the influence of backpack diameter on aggregate size, and show that even when cell−backpack complexes are forced through small pores, backpacks are not removed from the surfaces of cells

Synthesis of PLGA NPs with surface-conjugated VMP001 (VMP001-NPs).

<p>(A) Schematic illustration of synthesis of lipid-enveloped PLGA NPs with surface-conjugated VMP001. PLGA NPs were incubated with thiolated VMP001, conjugating the antigen to maleimide-functionalized lipids displayed on the particle membranes. Particles were then PEGylated in a reaction with PEG-thiol. (B) A scanning electron microcopy image of VMP001-NPs. Scale bar, 2 µm. (C) Confocal microscopy image of VMP001-NPs incubated with anti-his-tag and fluorescent secondary antibodies to detect particle surface-conjugated VMP001. Scale bar, 10 µm.</p

VMP001-NP immunization elicits high avidity antibodies capable of agglutinating live sporozoites.

<p>(A) Avidity indices of anti-VMP001 IgG sera obtained from mice immunized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031472#pone-0031472-g003" target="_blank">Fig. 3</a> with soluble VMP001+MPLA (red circles) or VMP001-NP+MPLA (blue circles) were characterized over 6 months following vaccination. *, <i>p</i><0.01 and **, <i>p</i><0.001, analyzed by two-way ANOVA, followed by a Bonferroni post-test. (B) Anti-VMP001 IgG antibodies elicited with soluble VMP001+MPLA (red circles) or VMP001-NP+MPLA (blue circles) were further examined for their affinities against key fragments of VMP001, including peptides representing the Type I repeat, AGDR motif, Region I, Region II, C-terminus, and scrambled negative peptide control. Sera from non-immunized mice were also included as controls (black squares). (C,D) Sera obtain from mice on day 63 post-immunizations with 2.5 µg VMP001 and 25 µg MPLA in either (C) soluble or (D) VMP001-NP formulations were incubated with live VK210 sporozoites, and immunoflurescence assay was performed to assess recognition of native CSP present on the surface of live sporozoites by anti-VMP001 IgG sera. Mice immunized with VMP001-NP vaccines raised sera that agglutinated live VK210 subtype of <i>P. vivax</i>.</p

Immunization with VMP001-NPs elicits Th1/Th2 balanced antibody responses.

<p>(A) C57Bl/6 mice were immunized <i>s.c.</i> on days 0 and 21 with 25 µg of MPLA and 1 µg of VMP001 in either soluble or VMP001-NP formulations, and anti-VMP001 IgG sera were characterized on days 35 and 120 for (A) IgG, (B) IgG₁, (C) IgG_2b, (D) IgG_2c, and (E) IgG₃ titers.</p

Block Copolymer Micelles as Nanocontainers for Controlled Release of Proteins from Biocompatible Oil Phases

Biocompatible oils are used in a variety of medical applications ranging from vaccine adjuvants to vehicles for oral drug delivery. To enable such nonpolar organic phases to serve as reservoirs for delivery of hydrophilic compounds, we explored the ability of block copolymer micelles in organic solvents to sequester proteins for sustained release across an oil−water interface. Self-assembly of the block copolymer, poly(ϵ-caprolactone)-<i>block</i>-poly(2-vinyl pyridine) (PCL-<i>b</i>-P2VP), was investigated in toluene and oleic acid, a biocompatible naturally occurring fatty acid. Micelle formation in toluene was characterized by dynamic light scattering (DLS) and atomic force microscopy (AFM) imaging of micelles cast onto silicon substrates. Cryogenic transmission electron microscopy confirmed a spherical morphology in oleic acid. Studies of homopolymer solubility implied that micelles in oleic acid consist of a P2VP corona and a PCL core, while P2VP formed the core of micelles assembled in toluene. The loading of two model proteins (ovalbumin (ova) and bovine serum albumin (BSA)) into micelles was demonstrated with loadings as high as 7.8% wt of protein per wt of P2VP in oleic acid. Characterization of block copolymer morphology in the two solvents after protein loading revealed spherical particles with similar size distributions to the as-assembled micelles. Release of ova from micelles in oleic acid was sustained for 12−30 h upon placing the oil phase in contact with an aqueous bath. Unique to the situation of micelle assembly in an oily phase, the data suggest protein is sequestered in the P2VP <i>corona</i> block of PCL-<i>b</i>-P2VP micelles in oleic acid. More conventionally, protein loading occurs in the P2VP <i>core</i> of micelles assembled in toluene

Target cell motility directly impacts CTL function.

<p>(<b>A–C</b>) CD4⁺ target cells (controls or pulsed with KK10 gag peptide) were spun to the bottom of collagen matrices prior to gelation to allow binding to the underlying glass substrate, which was coated with anti-CD4 antibody and ICAM or ICAM alone. E501 CTLs were added to the matrix (E:T ratio 1∶2) and target/CTL dynamics were recorded by videomicroscopy for 10 hr. (<b>A</b>) Wind-rose plots of target cell migration over a period of 20 min in the absence or presence of immobilizing anti-CD4. (<b>B</b>) Target cell death was assessed by sytox fluorescence after 10 hr. (<b>C</b>) Engagement histories were recorded for motile or immobilized targets that were killed. (<b>D, E</b>) A14 CTLs were loaded with FURA-2 AM and Ca²⁺ signaling was monitored for CTLs engaging peptide-pulsed CD4⁺ targets (2 nM SL9, 1 hr time-lapse). (<b>D</b>) Shown are representative Ca²⁺ traces (blue) and instantaneous cell velocities (CTL in red, target in black) over time on the x-axis. The period of CTL-target engagement is denoted on the time axis (live target in pink, killed in gray). (<b>E</b>) The presence or absence of Ca²⁺ signals was scored for CTL-target engagements concluding with target death (<i>n</i> = 89), a failed tether (<i>n</i> = 112), or a brush (<i>n</i> = 95). Data pooled from 6 independent experiments. Bars indicate mean ± SEM.</p