Effect of charge and role of caveolae in dendrimer internalization.

<p>A) Cells were exposed for 12 h to 8 charged cationic, anionic and neutral capped dendrimers and internalization was assessed after fixation using fluorescence microscopy. Results are reported as % biotin-positive cells in five fields per slide as determined using Image J and shown as mean ± S.E.M. (n = 3 separate experiments), **<i>p</i><0.01; ***<i>p</i><0.001; ****<i>p</i><0.0001. B) WT and caveolin-1 KO iMEFs were exposed to 8 charged cationic dendrimer with either Arg, his or Lys head groups. Internalization was assessed using fluorescence microscopy, is reported as % biotin-positive cells and shown as mean ± S.E.M. (n = 3 separate experiments), *<i>p</i><0.05; ****<i>p</i><0.0001. C) WT and PTRF KO iMEFs were exposed to 8 charged cationic dendrimer with either Arg, his or Lys head groups. Internalization was assessed using fluorescence microscopy, is reported as % biotin-positive cells and shown as mean ± S.E.M. (n = 3 separate experiments), *<i>p</i><0.05; ***<i>p</i><0.001; ****<i>p</i><0.0001.</p

Arginine head groups promote entry of dendrimers in cells forming caveolae.

<p>WT and caveolin-1 KO MEFs were incubated for 12 h with (A) 4 charged (B) 8 charged and (C) 16 charged cationic dendrimers. Cells were imaged using fluorescence microscopy after fixation. A, B) Results are reported as % positive red cells and shown as mean ± S.E.M. (n = 3 separate experiments), ns; not significant, *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001; ****<i>p</i><0.0001. C) Results are reported as fluorescence intensity per cell as measured using Image J, expressed as arbitrary units (AU) and shown as mean ± S.E.M. (n = 3 separate experiments), ns; not significant, *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001; ****<i>p</i><0.0001.</p

Effect of positive charge density on dendrimer cellular entry.

<p>WT cells were incubated with 4, 8 and 16 charged cationic dendrimers for 12 h. Internalization was assessed using fluorescence microscopy. Results are reported as % biotin-positive red and shown as mean ± S.E.M. (n = 3 separate experiments), ns; not significant, **<i>p</i><0.01; ****<i>p</i><0.0001.</p

Dendrimers used in this study.

<p>A) General structure of 4 (G1 generation), 8 (G2 generation) and 16 (G3 generation) charged cationic dendrimers. B) ID of dendrimers and their ending head groups and charge. C) Average zeta potential of the biotinylated dendrimers at 1mg/mL in 10 mM NaCl solution. The mean ± S.E.M. (n = 3 separate measurements) is shown; A = Arginine, L = Lysine, H = Histidine, AA = Neutral (Amide) and SA = Anionic (carboxylic) head group.</p

Positive charge-specific entry is abolished in cells devoid of caveolae.

<p>A) Caveolin-1 KO cells were exposed for 12 h to 8 charged cationic, anionic and neutral capped dendrimers and internalization was assessed after fixation using fluorescence microscopy. Results are reported as % biotin-positive cells in five fields per slide as determined using Image J and shown as mean ± S.E.M. (n = 3 separate experiments), ns; not significant *<i>p</i><0.05; **<i>p</i><0.01; ****<i>p</i><0.0001. B) Cationic, anionic and neutral dendrimers internalization after 12 h was assessed in WT and caveolin-1 KO cells using fluorescence microscopy. Results are reported as % positive red cells and shown as mean ± S.E.M. (n = 3 separate experiments).</p

Characterization of dendrimer endocytosis.

<p>A) WT, caveolin-1 KO and PTRF- KO cells were incubated with 4, 8 and 16 charged cationic arginine dendrimer at concentrations 0, 50 and 100 μg/ml as indicated for 12 h. Cells were subjected to an acid wash (AW) to remove surface bound dendrimers, or washed with control buffer (No acid wash, NAW). Dendrimer endocytosis was assessed using fluorescence microscopy after fixation. For 4 and 8 charged dendrimers, results are reported as % biotin-positive red cells and shown as mean ± S.E.M. (n = 3 separate experiments), ns; not significant. For 16 charged dendrimers, results are reported as fluorescence intensity per cell expressed in arbitrary units (AU) and shown as mean ± S.E.M. (n = 3 separate experiments), ns; not significant. B) Kinetics of internalization of 16 charged cationic arginine dendrimer 50 μg/ml in WT, caveolin-1 KO and PTRF- KO cells was assessed using wild field fluorescence microscopy. Results are reported as fluorescence intensity per cell expressed in arbitrary units (AU) and shown as mean ± S.E.M. (n = 3 separate experiments), ns; not significant, *<i>p</i><0.05; **<i>p</i><0.01. C) Kinetics of internalization of 16 charged cationic arginine dendrimer 50 μg/ml in WT, caveolin-1 KO and PTRF- KO cells was assessed using confocal fluorescence microscopy. Results are reported as fluorescence intensity per cell expressed in arbitrary units (AU) and shown as mean ± S.E.M. (n = 3 separate experiments), ns; not significant, *<i>p</i><0.05; **<i>p</i><0.01; ****p<0.0001.</p

Express in Vitro Plasmid Transfection Achieved with 16⁺ Asymmetric Peptide Dendrimers

Asymmetric cationic amino acid-based dendrimers are highly branched chemically derived gene vectors developed to transport cargo such as plasmid DNA across the plasma membrane. We have previously demonstrated their propensity to enter cells that form caveolae, driven by positive charge density and promoted by arginine head groups. Caveolae are plasma membrane subdomains serving a number of cellular functions including endocytosis. Their formation requires membrane proteins (caveolins) and cytoplasmic proteins (cavins), so that gene disruption of either caveolin-1 or cavin-1 (also known as PTRF, i.e., polymerase I and transcript release factor) results in caveola deficiency. Here we evaluated the ability of a 16⁺ charged asymmetric arginine dendrimer to transfect plasmid DNA into cultured cells. We unveiled efficient transfection efficiencies (≥30%) 24–48 h after exposing the cells to dendrimer/pDNA complexes for only 5 min. Using wild type (WT) and caveolin-1 or PTRF gene-disrupted, i.e., caveola-deficient mouse embryo fibroblasts, we further show that caveolae promote pDNA transfection by 16⁺ charged asymmetric arginine dendrimers

Authentic standard comparison of putative compounds in leaf juice with available reference compounds.

<p>Authentic standard comparison of putative compounds in leaf juice with available reference compounds.</p

Multivariate analysis of positive ionisation LC-MS data from <i>Carica papaya</i> leaf extracts.

<p>(A) PCA scores plot, PC1 [t1] versus PC2 [t2] generated from three extracts–Leaf juice (LJ, red triangle), leaf decoction (LD, blue square) and brewed leaf juice (BLJ, green circle). Each symbol represents one replicate. (B) OPLS-DA scores plot of predictive component t[1] versus orthogonal component t₀[1]. The ellipse in (A) and (B) represents the Hotelling’s T² 95% confidence interval for the multivariate data. (C) Loadings plot derived from (B); the variables with higher abundance in LJ than BLJ/LD are highlighted in red with an arrow indicating mass [email protected] minutes. (D) Extracted ion chromatogram (EIC) of m/z 518.374 at retention time 16.559 mins. (E) Mass spectrum for EIC peak.</p

Comparison of feature [email protected] in leaf juice against authentic standard pheophorbide A.

<p>(A) BPC chromatogram of leaf juice. (B) BPC chromatogram of pheophorbide A standard. (C) EIC of m/z 593.2752 for leaf juice. (D) EIC of m/z 593.2752 for pheophorbide A standard. (E) Mass spectrum of peak X in leaf juice. (F) Mass spectrum of pheophorbide A standard peak. (G) MS/MS spectrum of peak X in leaf juice. (H) MS/MS spectrum of pheophorbide A standard peak.</p