3 research outputs found

    Enhanced siRNA Delivery Using a Combination of an Arginine-Grafted Bioreducible Polymer, Ultrasound, and Microbubbles in Cancer Cells

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    RNAi-based gene therapy for cancer treatment has not shown significant clinical impact due to poor siRNA delivery to the target site. Here, we design a nonviral siRNA gene carrier using a combination of an arginine-grafted bioreducible polymer (ABP), microbubbles (MB), and ultrasound (US), for targeting vascular endothelial growth factor (VEGF) in a human ovarian cancer cell line. Newly designed MBs with a perfluorocrownether gas core show higher stability compared to controls. Further, MBs in combination with polyplexes show a significant higher loading capacity compared to naked siRNA. Lastly, only siRNA-ABP-MB (SAM) complexes in combination with US show significant VEGF knock down in A2780 human ovarian cancer cell line compared to naked siRNA when incubated for a short time after sonication treatment

    A Nanoparticle Platform To Evaluate Bioconjugation and Receptor-Mediated Cell Uptake Using Cross-Linked Polyion Complex Micelles Bearing Antibody Fragments

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    Targeted nanomedicines are a promising technology for treatment of disease; however, preparation and characterization of well-defined protein-nanoparticle systems remain challenging. Here, we describe a platform technology to prepare antibody binding fragment (Fab)-bearing nanoparticles and an accompanying real-time cell-based assay to determine their cellular uptake compared to monoclonal antibodies (mAbs) and Fabs. The nanoparticle platform was composed of core-cross-linked polyion complex (PIC) micelles prepared from azide-functionalized PEG<i>-<i>b</i>-</i>poly­(amino acids), that is, azido-PEG<i>-<i>b</i>-</i>poly­(l-lysine) [N<sub>3</sub>–PEG<i>-<i>b</i>-</i>PLL] and azido-PEG<i>-<i>b</i>-</i>poly­(aspartic acid) [N<sub>3</sub>–PEG<i>-<i>b</i>-</i>PAsp]. These PIC micelles were 30 nm in size and contained approximately 10 polymers per construct. Fabs were derived from an antibody binding the EphA2 receptor expressed on cancer cells and further engineered to contain a reactive cysteine for site-specific attachment and a cleavable His tag for purification from cell culture expression systems. Azide-functionalized micelles and thiol-containing Fab were linked using a heterobifunctional cross-linker (FPM-PEG<sub>4</sub>-DBCO) that contained a fluorophenyl-maleimide for stable conjugation to Fabs thiols and a strained alkyne (DBCO) group for coupling to micelle azide groups. Analysis of Fab–PIC micelle conjugates by fluorescence correlation spectroscopy, size exclusion chromatography, and UV–vis absorbance determined that each nanoparticle contained 2–3 Fabs. Evaluation of cellular uptake in receptor positive cancer cells by real-time fluorescence microscopy revealed that targeted Fab–PIC micelles achieved higher cell uptake than mAbs and Fabs, demonstrating the utility of this approach to identify targeted nanoparticle constructs with unique cellular internalization properties
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