Journal: iScience

Article Title: Dendritic peptide-conjugated polymeric nanovectors for non-toxic delivery of plasmid DNA and enhanced non-viral transfection of immune cells

doi: 10.1016/j.isci.2022.104555

Figure Lengend Snippet: Cytotoxicity of PPDP/pDNA nanovector in RAW 264.7 macrophages RAW 264.7 macrophages were incubated with PPDP/pDNA nanocomplexes that were prepared with a different polymer to pDNA ratios (30:1, 60:1, 120:1) for 24 h at 37 °C. Untreated cells (Control), Lipo2K/pDNA complexes (Lipo2K), PEI/pDNA complexes (PEI with the molecular weight of 25 kDa), and dendritic peptide (DP)/pDNA complexes were included as benchmarks. The cell viability for (a) PPDP/S-pDNA complexes and (b) PPDP/L-pDNA complexes was then measured by the MTT assay. Asterisks indicate the experimentally determined optimal ratio for each formulation. Data are presented as the mean ± SD (n = 3).

Article Snippet: Figure 3 Cytotoxicity of PPDP/pDNA nanovector in RAW 264.7 macrophages RAW 264.7 macrophages were incubated with PPDP/pDNA nanocomplexes that were prepared with a different polymer to pDNA ratios (30:1, 60:1, 120:1) for 24 h at 37 °C.

Techniques: Incubation, Polymer, Control, Molecular Weight, MTT Assay, Formulation

Journal: iScience

Article Title: Dendritic peptide-conjugated polymeric nanovectors for non-toxic delivery of plasmid DNA and enhanced non-viral transfection of immune cells

doi: 10.1016/j.isci.2022.104555

Figure Lengend Snippet: Optimization of PPDP/pDNA nanovectors for enhanced DNA binding capability Electrophoretic mobility shift assay (EMSA) of PPDP/pDNA nanocomplexes with different polymer to pDNA ratios from 1:1 to 60:1. Naked pDNA, Lipofectamine 2000/pDNA complexes (Lipo2K), PEI/pDNA complexes (PEI with the molecular weight of 25 kDa), and dendritic peptide (DP1)/pDNA complexes were included as control groups. PPDP formulations were optimized by complexing with (a) S-pDNA and (b) L-pDNA. (red triangle points to PPDP/pDNA nanocomplexes that quenched the GelRed® nucleic acid stain). Effect of the mass ratio of (c) PPDP2:S-pDNA and PPDP5:S-pDNA and, (d) PPDP2:L-pDNA and PPDP5:L-pDNA on the nanocomplex particle size (red) and zeta potential (blue). Asterisks indicate the optimal PPDP:pDNA ratio. Data are presented as the mean ± SD (n = 3). e,f) Representative Cryo-TEM micrographs of the optimal PPDP/pDNA nanocomplexes. Scale bar = 100 nm.

Article Snippet: Figure 3 Cytotoxicity of PPDP/pDNA nanovector in RAW 264.7 macrophages RAW 264.7 macrophages were incubated with PPDP/pDNA nanocomplexes that were prepared with a different polymer to pDNA ratios (30:1, 60:1, 120:1) for 24 h at 37 °C.

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Polymer, Molecular Weight, Control, Staining, Zeta Potential Analyzer

Journal: iScience

Article Title: Dendritic peptide-conjugated polymeric nanovectors for non-toxic delivery of plasmid DNA and enhanced non-viral transfection of immune cells

doi: 10.1016/j.isci.2022.104555

Figure Lengend Snippet: Endosomal escape and cytosolic delivery of PPDP2/pDNA complexes in RAW 264.7 macrophages RAW 264.7 macrophages were incubated with PPDP2/AF488-labeled S-pDNA (pcDNA3.1, 5.4 kb) nanocomplexes formed using a PPDP2 to pDNA weight ratio of 60:1. Representative confocal images display PPDP2/S-pDNA complexes within cells after 1, 4, and 18 h incubation periods. LysoTracker red was used to label late endosomes/lysosomes. Nuclei were stained with DAPI (blue). Co-localization of green plasmid DNA and red endo/lysosomes appears as yellow in the images. Scale bar = 10 μm.

Article Snippet: Figure 3 Cytotoxicity of PPDP/pDNA nanovector in RAW 264.7 macrophages RAW 264.7 macrophages were incubated with PPDP/pDNA nanocomplexes that were prepared with a different polymer to pDNA ratios (30:1, 60:1, 120:1) for 24 h at 37 °C.

Techniques: Incubation, Labeling, Staining, Plasmid Preparation

Journal: iScience

Article Title: Dendritic peptide-conjugated polymeric nanovectors for non-toxic delivery of plasmid DNA and enhanced non-viral transfection of immune cells

doi: 10.1016/j.isci.2022.104555

Figure Lengend Snippet: Cytotoxicity of PPDP/pDNA nanovector in RAW 264.7 macrophages RAW 264.7 macrophages were incubated with PPDP/pDNA nanocomplexes that were prepared with a different polymer to pDNA ratios (30:1, 60:1, 120:1) for 24 h at 37 °C. Untreated cells (Control), Lipo2K/pDNA complexes (Lipo2K), PEI/pDNA complexes (PEI with the molecular weight of 25 kDa), and dendritic peptide (DP)/pDNA complexes were included as benchmarks. The cell viability for (a) PPDP/S-pDNA complexes and (b) PPDP/L-pDNA complexes was then measured by the MTT assay. Asterisks indicate the experimentally determined optimal ratio for each formulation. Data are presented as the mean ± SD (n = 3).

Article Snippet: The PPDP technology described herein is a stimuli-responsive polymeric nanovector that can be leveraged to meet diverse challenges in gene delivery.

Techniques: Incubation, Polymer, Control, Molecular Weight, MTT Assay, Formulation

Journal: iScience

Article Title: Dendritic peptide-conjugated polymeric nanovectors for non-toxic delivery of plasmid DNA and enhanced non-viral transfection of immune cells

doi: 10.1016/j.isci.2022.104555

Figure Lengend Snippet: Transfection of PPDP/L-pDNA nanovector in fibroblasts, dendritic cells, and T cells NIH 3T3, BMDC, and Jurkat T cells were transfected with L-pDNA (pL-CRISPR.EFS.tRFP, 11.7 kb) using PPDP2 and PPDP5 nanovectors with the PPDP to pDNA weight ratio of 60:1. (A) Representative confocal image of PPDP2/L-pDNA complexes demonstrated the transfection of L-pDNA after cellular uptake. Scale bar = 20 μm. (B) Transfection efficiency for NIH3T3 cells. (C and D) Flow cytometry histogram and (d) the percentage of transfected cells in BMDCs. (E and F) Flow cytometry histogram and (f) the percentage of transfected Jurkat T cells. Naked pDNA and Lipo2K/pDNA complexes (Lipo2K) were included as negative and positive controls, respectively. Data are presented as the mean ± SD ( n = 3–4). Significance was determined by ANOVA with post hoc Tukey’s multiple comparisons test (5% significance level). ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The PPDP technology described herein is a stimuli-responsive polymeric nanovector that can be leveraged to meet diverse challenges in gene delivery.

Techniques: Transfection, CRISPR, Flow Cytometry