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: The design and structure of the PEG m - b -PPS n -ss-DP (PPDP) gene delivery system The general strategy for enhancing the intracellular release of plasmid DNA (pDNA) is illustrated.

Article Snippet: The size distribution and zeta potential of the PPDP nanostructures were measured using a Zetasizer Nano instrument (Malvern Instruments).

Techniques: 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: PPDP2 and PPDP5 achieve higher plasmid DNA transfection efficiencies and reporter expression levels than Lipo2K in a macrophage-based screen RAW 264.7 macrophages were incubated with the specified materials for 48 h. (A) Schematic of the model small size plasmid DNA (pCMV-DsRed, 4.6 kb), containing a CMV promoter and a DsRed reporter gene. (B) Transfection efficiency of S-pDNA using PPDP nanovectors (PPDP2-PPDP7) quantified as the percentage of cells expressing fluorescent reporter proteins. (C) Transfection efficiency of S-pDNA plotted with the mean fluorescence intensity (MFI) of cells expressing the DsRed reporter. (D) Schematic of the model large size plasmid DNA (pL-CRISPR.EFS.tRFP, 11.7 kb) used in this study, containing an EFS promoter with NSL, Cas9, and RFP reporter genes. (E) Transfection efficiency of L-pDNA by PPDP nanovectors. (F) Transfection efficiency and MFI of cells expressing the RFP reporter. (G) Fold difference in the PPDP-mediated plasmid transfection efficiency (left) and reporter expression level (right) compared to Lipo2K. The rank of PPDP2 and PPDP5 is annotated below each heatmap. In all cases, a 60:1 PPDP:pDNA weight ratio was used. Data are presented as the mean ± SD (n = 3). Transfections using were performed per manufacturer instructions. For panels (b, e), significant differences were determined by ANOVA with post hoc Tukey’s multiple comparisons test (5% significance level). ∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Comparisons to control and pDNA (gray bars), Lipo2k (red bars), and within PPDP treatment groups (black bars) are presented in the specified colors.

Article Snippet: The size distribution and zeta potential of the PPDP nanostructures were measured using a Zetasizer Nano instrument (Malvern Instruments).

Techniques: Plasmid Preparation, Transfection, Expressing, Incubation, Fluorescence, CRISPR, Control

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 size distribution and zeta potential of the PPDP nanostructures were measured using a Zetasizer Nano instrument (Malvern Instruments).

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: The size distribution and zeta potential of the PPDP nanostructures were measured using a Zetasizer Nano instrument (Malvern Instruments).

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: The 60:1 PPDP:p-DNA ratio was optimal for the transfection of macrophages with both small and large plasmids The transfection efficiency in RAW 264.7 macrophages was determined by flow cytometry using PPDP2 (purple bar plots) or PPDP5 (red bar plots) complexed with (a) the small model plasmid (S-pDNA; pCMV-DsRed, 4.6 kb), or (b) the large model plasmid (L-pDNA; pL-CRISPR.EFS.tRFP, 11.7 kb) at the specified PPDP:pDNA ratios. Data are presented as the mean ± SD (n = 3). Significant differences were determined by ANOVA with post hoc Tukey’s multiple comparisons test (5% significance level). ∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (c) Representative confocal image of reporter gene expression by macrophages transfected with PPDP2/L-pDNA (60:1) or Lipo2K/L-pDNA. Cellular background (control) and cells transfected with naked L-pDNA are also presented. Scale bar = 20 μm.

Article Snippet: The size distribution and zeta potential of the PPDP nanostructures were measured using a Zetasizer Nano instrument (Malvern Instruments).

Techniques: Transfection, Flow Cytometry, Plasmid Preparation, CRISPR, Gene Expression, Control

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: Structural characterization of PPDP (A) Schematic illustration depicting the amino acid composition of the PPDP polymer (PEG m - b -PPS n -ss-DP). (B and C) Representative Cryo-TEM micrograph (scale bar = 100 nm) and (c) size distribution of PPDP2 determined by DLS. (D) FT-IR spectra of PEG- b -PPS (green), dendritic peptide (black), and PPDP2 (pink). The regions highlighted in blue correspond to the amide I (1700–1600 cm −1 ) and amide II (1590–1520 cm −1 ) bands. (E and F) PPDP2 spectra with PEG- b -PPS contributions subtracted. Circular dichroism (CD) spectroscopy analysis of DP secondary structure in (f) assembled PPDP2 nanostructures or in (g) free form (i.e. unconjugated peptide control). Suspensions were adjusted to pH 7.5, 6.5, or 5.5, as specified in the plot legends.

Article Snippet: The size distribution and zeta potential of the PPDP nanostructures were measured using a Zetasizer Nano instrument (Malvern Instruments).

Techniques: Polymer, Circular Dichroism, Control

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 size distribution and zeta potential of the PPDP nanostructures were measured using a Zetasizer Nano instrument (Malvern Instruments).

Techniques: Transfection, CRISPR, Flow Cytometry

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