<i>In Vivo</i> Compatibility of Graphene Oxide with Differing Oxidation States

Graphene oxide (GO) is suggested to have great potential as a component of biomedical devices. Although this nanomaterial has been demonstrated to be cytocompatible <i>in vitro</i>, its compatibility <i>in vivo</i> in tissue sites relevant for biomedical device application is yet to be fully understood. Here, we evaluate the compatibility of GO with two different oxidation levels following implantation in subcutaneous and intraperitoneal tissue sites, which are of broad relevance for application to medical devices. We demonstrate GO to be moderately compatible <i>in vivo</i> in both tissue sites, with the inflammatory reaction in response to implantation consistent with a typical foreign body reaction. A reduction in the degree of GO oxidation results in faster immune cell infiltration, uptake, and clearance following both subcutaneous and peritoneal implantation. Future work toward surface modification or coating strategies could be useful to reduce the inflammatory response and improve compatibility of GO as a component of medical devices

Rational Design of a Biomimetic Cell Penetrating Peptide Library

Cell penetrating peptides have demonstrated potential to facilitate the cellular delivery of therapeutic molecules. Here we develop a set of 50 cell penetrating peptide based formulations with potential to deliver small interfering RNAs intercellularly. The transfection efficacy of siRNA containing lipid-like nanoparticles decorated with different peptides was evaluated both <i>in vitro</i> and <i>in vivo</i> and correlated with the peptide physical and chemical properties. <i>In vitro</i>, these particles were internalized primarily through macropinocytosis. When the peptides were presented to bone marrow-derived dendritic cells, they induce low immunoactivation relative to control cell penetrating peptides including the antennapedia homeodomain and TAT, as quantified by the expression of activation specific surface proteins like CD80, CD86, and major histocompatibility complex class II. <i>In vivo</i>, peptide decorated nanoparticles primarily accumulated in the lungs and the liver. Three human peptides derived from surfactant protein B (a lung surfactant protein), orexin (a neuropeptide hormone, and lactoferricin (a globular glycoprotein) that exist in many physiological fluids facilitated the <i>in vivo</i> delivery of siRNA and induce significant knock down (90%) of a hepatocyte expressed protein, coagulation Factor VII

Dendrimer-Inspired Nanomaterials for the <i>in Vivo</i> Delivery of siRNA to Lung Vasculature

Targeted RNA delivery to lung endothelial cells has the potential to treat conditions that involve inflammation, such as chronic asthma and obstructive pulmonary disease. To this end, chemically modified dendrimer nanomaterials were synthesized and optimized for targeted small interfering RNA (siRNA) delivery to lung vasculature. Using a combinatorial approach, the free amines on multigenerational poly­(amido amine) and poly­(propylenimine) dendrimers were substituted with alkyl chains of increasing length. The top performing materials from <i>in vivo</i> screens were found to primarily target Tie2-expressing lung endothelial cells. At high doses, the dendrimer–lipid derivatives did not cause chronic increases in proinflammatory cytokines, and animals did not suffer weight loss due to toxicity. We believe these materials have potential as agents for the pulmonary delivery of RNA therapeutics

Dendrimer-Inspired Nanomaterials for the <i>in Vivo</i> Delivery of siRNA to Lung Vasculature

Targeted RNA delivery to lung endothelial cells has the potential to treat conditions that involve inflammation, such as chronic asthma and obstructive pulmonary disease. To this end, chemically modified dendrimer nanomaterials were synthesized and optimized for targeted small interfering RNA (siRNA) delivery to lung vasculature. Using a combinatorial approach, the free amines on multigenerational poly­(amido amine) and poly­(propylenimine) dendrimers were substituted with alkyl chains of increasing length. The top performing materials from <i>in vivo</i> screens were found to primarily target Tie2-expressing lung endothelial cells. At high doses, the dendrimer–lipid derivatives did not cause chronic increases in proinflammatory cytokines, and animals did not suffer weight loss due to toxicity. We believe these materials have potential as agents for the pulmonary delivery of RNA therapeutics

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Delivery to human immune cells.

<p><b>A)</b> Human T cells and MDDCs were tested for delivery of cascade blue labeled 3kDa dextran, fluorescein labeled 70kDa dextran, and APC labeled IgG1. The representative histograms for a 30–4 (T cells) and 10–7 (MDDCs) device (left) and replicates across device designs (right) are displayed. <b>B)</b> SiRNA mediated knockdown of CD4 and DC-SIGN protein levels in CD4⁺ T cells and MDDCs respectively. <b>C)</b> Knockdown of CD4 expression in human regulatory T cells in response to treatment by a 30–4 device. Dead cells were excluded for delivery or knockdown analysis. <b>D)</b> Comparison of device performance in T cells to nucleofection by Amaxa. Protein expression 72hrs after siRNA delivery and cell viability after treatment are shown. <b>E)</b> Intracellular staining for the p24 antigen was used as an indicator of HIV infection level in treated human CD4⁺ T cells 24hrs after infection. In these studies, vif and/or gag, siRNA was delivered 24hrs prior to infection while CD4 siRNA was delivered 48hrs prior to infection. <b>F)</b> Median fluorescence intensity of the p24 antigen stain across repeats (min. N = 4) of the experimental conditions. Data are represented as mean + 1 standard error.</p

Delivery methodology and performance in mouse cells.

<p><b>A)</b> Illustration of device design and delivery mechanism. <b>B)</b> Illustration of the system setup and delivery procedure. <b>C)</b> Representative histograms of T cells, B cells and myeloid cells (CD11b⁺) treated by the CellSqueeze device to deliver APC-labeled IgG1. <b>D)</b> Delivery efficiency of Cascade blue-labeled 3 kDa dextran, fluorescein-labeled 70 kDa dextran, and APC-labeled IgG1. All results were measured by flow cytometry within an hour of treatment. Dead cells were excluded by propidium iodide staining. Viability is shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118803#pone.0118803.s002" target="_blank">S2 Fig</a></b>. Data in <b>D)</b> (mean ± SD) are from 3 independent experiments. Untreated cells were not put through the device or exposed to the biomolecules. The ‘no device’ samples were incubated with the biomolecules, but were not treated by the device. This control is meant to account for surface binding, endocytosis and other background effects.</p

Neutrophil extracellular traps.

<p>Representative z-stacked immunofluorescence images showing neutrophil elastase and DNA/histone-H1 on the surface of microcapsules. Alginate microcapsules were retrieved 1–2 weeks following implantation, while Polystyrene and PMMA microcapsules were retrieved 3 days following implantation. Images are representative of at least 2 independent experiments with total n ≥ 5 mice, and imaging of multiple retrieved microcapsules from each mouse. Scale bar = 100 μm.</p

Neutrophil Function.

<p>(A)–Confirmation of neutrophil phagocytic capacity. Fluorescent nanoparticles (~190 nm polystyrene nanoparticles) were injected intraperitoneally, 1 week following alginate microcapsule implantation. <i>Left</i>–Representative flow cytometry histograms generated following gating on Ly6G⁺ cells showing nanoparticles (NP) associated with neutrophils. Grey histograms are fluorescence intensities in control mice that have not been injected with nanoparticles. <i>Right</i>–Quantification of the NP uptake histograms, showing a large increase in NP MFI 3 hours post NP injection that drops over time. Data are representative of at least 1 independent experiment with total n ≥ 4. (B)–Multiplex luminex assay to measure chemokines and cytokines secreted by neutrophils. Neutrophils were isolated using a magnetic bead based negative selection technique, followed by <i>ex vivo</i> overnight culture. Higher amounts of key inflammatory cytokines and chemokines are secreted by peritoneal cavity but not bone marrow neutrophils. B.D.L. = below detectable levels. ** and *** indicate p<0.01 and p<0.001, respectively, using a two-tailed Student's t test with Welch's correction (for samples where the levels of cytokine/chemokine are above detectable levels). # indicates p<0.01 using a two-tailed Fisher's exact test, for samples where the levels of cytokine/chemokine were below detectable levels. Data presented are based on n = 6.</p

Flow cytometry schematics.

<p>Representative flow cytometry contour plots describing the gating scheme used to identify different immune cell subsets (isolated 2 weeks followed alginate microcapsule implantation) in the peritoneal cavity. All single cells retrieved from the peritoneal cavity were run through flow cytometry.</p