Conjugation to the Cell-Penetrating Peptide TAT Potentiates the Photodynamic Effect of Carboxytetramethylrhodamine

Cell-penetrating peptides (CPPs) can transport macromolecular cargos into live cells. However, the cellular delivery efficiency of these reagents is often suboptimal because CPP-cargo conjugates typically remain trapped inside endosomes. Interestingly, irradiation of fluorescently labeled CPPs with light increases the release of the peptide and its cargos into the cytosol. However, the mechanism of this phenomenon is not clear. Here we investigate the molecular basis of the photo-induced endosomolytic activity of the prototypical CPPs TAT labeled to the fluorophore 5(6)-carboxytetramethylrhodamine (TMR).We report that TMR-TAT acts as a photosensitizer that can destroy membranes. TMR-TAT escapes from endosomes after exposure to moderate light doses. However, this is also accompanied by loss of plasma membrane integrity, membrane blebbing, and cell-death. In addition, the peptide causes the destruction of cells when applied extracellularly and also triggers the photohemolysis of red blood cells. These photolytic and photocytotoxic effects were inhibited by hydrophobic singlet oxygen quenchers but not by hydrophilic quenchers.Together, these results suggest that TAT can convert an innocuous fluorophore such as TMR into a potent photolytic agent. This effect involves the targeting of the fluorophore to cellular membranes and the production of singlet oxygen within the hydrophobic environment of the membranes. Our findings may be relevant for the design of reagents with photo-induced endosomolytic activity. The photocytotoxicity exhibited by TMR-TAT also suggests that CPP-chromophore conjugates could aid the development of novel Photodynamic Therapy agents

Experimental and Computational Evidence for an Inversion in Guest Capacity in High-Generation Triazine Dendrimer Hosts

The synthesis, characterization, and host–guest chemistry of high-generation triazine dendrimers are described. With pyrene and camptothecin as guests, experiments revealed that the guest capacity of odd-generation triazine dendrimers increased until generation 7 but decreased at generation 9. Molecular dynamics simulations conducted in explicit solvent showed a useful fingerprint for this behavior in radial distribution functions of water molecules penetrating the interior of the dendrimers. A linear relationship between the guest capacity of dendrimers measured experimentally and the number of water molecules within the interior determined computationally was observed

Antitumor Activity and Molecular Dynamics Simulations of Paclitaxel-Laden Triazine Dendrimers

The antitumor activities of triazine dendrimers bearing paclitaxel, a well-known mitotic inhibitor, are evaluated in SCID mice bearing human prostate cancer xenografts. To increase the activity of a first generation prodrug <b>1</b> that contained twelve paclitaxel molecules tethered via an ester linkage, the new construct described here, prodrug <b>2</b>, tethers paclitaxel with linkers containing both an ester and disulfide. While PEGylation is necessary for solubility, and may improve biocompatibility and increase plasma half-life, it increases the heterogeneity of the sample with an average of eight to nine PEG chains (2 kDa each) incorporated. The heterogeneous population of PEGylated materials was used without fractionation based on models obtained from molecular dynamics simulations. Three models were examined; hexaPEGylated, nonaPEGylated, and dodecaPEGylated constructs. Intravenous delivery of prodrug <b>2</b> was performed by single, double or triple dosing regimes with doses spaced by one week. The doses varied from 50 mg of paclitaxel/kg to 200 mg of paclitaxel/kg. Tumor growth arrest and regression was observed over the 10-week treatment period without mortality for mice treated with the 50 mg of paclitaxel/kg treated three times

Light-Fuelled Transport of Large Dendrimers and Proteins

This work presents a facile water-based supramolecular approach for light-induced surface patterning. The method is based upon azobenzene-functionalized high-molecular weight triazine dendrimers up to generation 9, demonstrating that even very large globular supramolecular complexes can be made to move in response to light. We also demonstrate light-fuelled macroscopic movements in native biomolecules, showing that complexes of apoferritin protein and azobenzene can effectively form light-induced surface patterns. Fundamentally, the results establish that thin films comprising both flexible and rigid globular particles of large diameter can be moved with light, whereas the presented material concepts offer new possibilities for the yet marginally explored biological applications of azobenzene surface patterning

Synthesis of Large Dendrimers with the Dimensions of Small Viruses

The dendrimer chemistry reported offers a route to synthetic target molecules with spherical shape, well-defined surface chemistries, and dimensions that match the size of virus particles. The largest target, a generation-13 dendrimer comprising triazines linked by diamines, is stable across ranges of concentration, pH, temperature, solvent polarity and in the presence of additives. This dendrimer theoretically presents 16 384 surface groups and has a molecular weight exceeding 8.4 MDa. Transmission electron and atomic force microscopies, dynamic light scattering, and computations reveal a diameter of ∼30 nm. The target was synthesized through an iterative divergent approach using a monochlorotriazine macromonomer providing two generations of growth per synthetic cycle. Fidelity in the synthesis is supported by evidence from NMR spectroscopy, mass spectrometry, and high-pressure liquid chromatography

Gadolinium MRI Contrast Agents Based on Triazine Dendrimers: Relaxivity and In Vivo Pharmacokinetics

Four gadolinium (Gd)-based macromolecular contrast agents, <b>G3-(Gd-DOTA)</b>_<b>24</b>, <b>G5-(Gd-DOTA)</b>_<b>96</b>, <b>G3-(Gd-DTPA)</b>_<b>24</b>, and <b>G5-(Gd-DTPA)</b>_<b>96</b>, were prepared that varied in the size of dendrimer (generation three and five), the type of chelate group (DTPA or DOTA), and the theoretical number of metalated chelates (24 and 96). Synthesis relied on a dichlorotriazine derivatized with a DOTA or DTPA ligand that was incorporated into the dendrimer and ultimately metalated with Gd ions. Paramagnetic characteristics and in vivo pharmacokinetics of all four contrast agents were investigated. The DOTA-containing agents, <b>G3-(Gd-DOTA)</b>_<b>24</b> and <b>G5-(Gd-DOTA)</b>_<b>96</b>, demonstrated exceptionally high <i>r</i>1 relaxivity values at off-peak magnetic fields. Additionally, <b>G5-(Gd-DOTA)</b>_<b>96</b> showed increased <i>r</i>1 relaxivity in serum compared to that in PBS, which was consistent with in vivo images. While <b>G3-(Gd-DOTA)</b>_<b>24</b> and <b>G3-(Gd-DTPA)</b>_<b>24</b> were rapidly excreted into the urine, <b>G5-(Gd-DOTA)</b>_<b>96</b> and <b>G5-(Gd-DTPA)</b>_<b>96</b> did not clear as quickly through the kidneys. Molecular simulation of the DOTA-containing dendrimers suggests that a majority of the metalated ligands are accessible to water. These triazine dendrimer-based MRI contrast agents exhibit several promising features such as high in vivo <i>r</i>1 relaxivity, desirable pharmacokinetics, and well-defined structure

Gadolinium MRI Contrast Agents Based on Triazine Dendrimers: Relaxivity and In Vivo Pharmacokinetics

Four gadolinium (Gd)-based macromolecular contrast agents, <b>G3-(Gd-DOTA)</b>_<b>24</b>, <b>G5-(Gd-DOTA)</b>_<b>96</b>, <b>G3-(Gd-DTPA)</b>_<b>24</b>, and <b>G5-(Gd-DTPA)</b>_<b>96</b>, were prepared that varied in the size of dendrimer (generation three and five), the type of chelate group (DTPA or DOTA), and the theoretical number of metalated chelates (24 and 96). Synthesis relied on a dichlorotriazine derivatized with a DOTA or DTPA ligand that was incorporated into the dendrimer and ultimately metalated with Gd ions. Paramagnetic characteristics and in vivo pharmacokinetics of all four contrast agents were investigated. The DOTA-containing agents, <b>G3-(Gd-DOTA)</b>_<b>24</b> and <b>G5-(Gd-DOTA)</b>_<b>96</b>, demonstrated exceptionally high <i>r</i>1 relaxivity values at off-peak magnetic fields. Additionally, <b>G5-(Gd-DOTA)</b>_<b>96</b> showed increased <i>r</i>1 relaxivity in serum compared to that in PBS, which was consistent with in vivo images. While <b>G3-(Gd-DOTA)</b>_<b>24</b> and <b>G3-(Gd-DTPA)</b>_<b>24</b> were rapidly excreted into the urine, <b>G5-(Gd-DOTA)</b>_<b>96</b> and <b>G5-(Gd-DTPA)</b>_<b>96</b> did not clear as quickly through the kidneys. Molecular simulation of the DOTA-containing dendrimers suggests that a majority of the metalated ligands are accessible to water. These triazine dendrimer-based MRI contrast agents exhibit several promising features such as high in vivo <i>r</i>1 relaxivity, desirable pharmacokinetics, and well-defined structure

Gadolinium MRI Contrast Agents Based on Triazine Dendrimers: Relaxivity and In Vivo Pharmacokinetics

Four gadolinium (Gd)-based macromolecular contrast agents, <b>G3-(Gd-DOTA)</b>_<b>24</b>, <b>G5-(Gd-DOTA)</b>_<b>96</b>, <b>G3-(Gd-DTPA)</b>_<b>24</b>, and <b>G5-(Gd-DTPA)</b>_<b>96</b>, were prepared that varied in the size of dendrimer (generation three and five), the type of chelate group (DTPA or DOTA), and the theoretical number of metalated chelates (24 and 96). Synthesis relied on a dichlorotriazine derivatized with a DOTA or DTPA ligand that was incorporated into the dendrimer and ultimately metalated with Gd ions. Paramagnetic characteristics and in vivo pharmacokinetics of all four contrast agents were investigated. The DOTA-containing agents, <b>G3-(Gd-DOTA)</b>_<b>24</b> and <b>G5-(Gd-DOTA)</b>_<b>96</b>, demonstrated exceptionally high <i>r</i>1 relaxivity values at off-peak magnetic fields. Additionally, <b>G5-(Gd-DOTA)</b>_<b>96</b> showed increased <i>r</i>1 relaxivity in serum compared to that in PBS, which was consistent with in vivo images. While <b>G3-(Gd-DOTA)</b>_<b>24</b> and <b>G3-(Gd-DTPA)</b>_<b>24</b> were rapidly excreted into the urine, <b>G5-(Gd-DOTA)</b>_<b>96</b> and <b>G5-(Gd-DTPA)</b>_<b>96</b> did not clear as quickly through the kidneys. Molecular simulation of the DOTA-containing dendrimers suggests that a majority of the metalated ligands are accessible to water. These triazine dendrimer-based MRI contrast agents exhibit several promising features such as high in vivo <i>r</i>1 relaxivity, desirable pharmacokinetics, and well-defined structure