Polycationic Adamantane-Based Dendrons of Different Generations Display High Cellular Uptake without Triggering Cytotoxicity

Dendrons used as synthetic carriers are promising nanostructures for biomedical applications. Some polycationic dendritic systems, such as the commercially available polyethylenimine (PEI), have the ability to deliver genetic material into cells. Nevertheless, polycationic vectors are often associated with potential cellular toxicity, which prevents their use in clinical development. In this context, our research focused on the design and synthesis of a novel type of polycationic dendrons that are able to penetrate into cells without triggering cytotoxic effects. We synthesized first- and second-generation polycationic adamantane-based dendrons via a combined protection/deprotection strategy starting from different adamantane scaffolds. The linker between the adamantane cores is constituted of short ethylene glycol chains, and the periphery consists of ammonium and guanidinium groups. None of these dendritic structures, which we previously called <i>HYDRAmers</i>, displayed significant cytotoxicity effects on two different cell lines (RAW 264.7 and HeLa). Conjugation of the fluorescent probe cyanine 5 at their focal point via click chemistry permitted the evaluation of their cellular internalization. All of the dendrons penetrated through the membrane with efficient cellular uptake depending of the dendron generation and the nature of the peripheral groups. These results suggest that the polycationic <i>HYDRAmers</i> are potentially interesting as new vectors in biomedical applications, including gene and drug delivery

Elucidation of the Cellular Uptake Mechanisms of Polycationic HYDRAmers

Dendrimers and dendrons appeared to potentially fulfill the requirements for being good and well-defined carriers in drug and gene delivery applications. We recently demonstrated that polycationic adamantane-based dendrons called <i>HYDRAmers</i> are easily internalized by both phagocytic and nonphagocytic cells in vitro. The aim of the present study was to investigate which of the different pathways of cellular internalization is involved in the cellular uptake of the first and second generation ammonium and guanidinium <i>HYDRAmers</i>. For this purpose, we have evaluated the internalization of fluorescently labeled <i>HYDRAmers</i> in both phagocytic murine macrophages and nonphagocytic human cervix epithelioid carcinoma cells in the presence of different well-known active uptake inhibitors. Our data revealed that the first and second generation <i>HYDRAmers</i> are internalized via different endocytic pathways based on the cellular type and on the type of functional groups present at the periphery of the dendrons. In particular, it was registered that the first generations were mainly internalized by clathrin-mediated endocytosis and macropinocytosis while the cellular internalization of the second generations was less affected by the inhibitory conditions of the endocytic pathways. These results suggest the possibility of addressing dendrimers toward specific subcellular compartments by tuning their structure properties and, in particular, the functional groups at their periphery

Structural Transformation of Coassembled Fmoc-Protected Aromatic Amino Acids to Nanoparticles

Materials made of assembled biomolecules such as amino acids have drawn much attention during the past decades. Nevertheless, research on the relationship between the chemical structure of building block molecules, supramolecular interactions, and self-assembled structures is still necessary. Herein, the self-assembly and the coassembly of fluorenylmethoxycarbonyl (Fmoc)-protected aromatic amino acids (tyrosine, tryptophan, and phenylalanine) were studied. The individual self-assembly of Fmoc-Tyr-OH and Fmoc-Phe-OH in water formed nanofibers, while Fmoc-Trp-OH self-assembled into nanoparticles. Moreover, when Fmoc-Tyr-OH or Fmoc-Phe-OH was coassembled with Fmoc-Trp-OH, the nanofibers were transformed into nanoparticles. UV–vis spectroscopy, Fourier transform infrared spectroscopy, and fluorescence spectroscopy were used to investigate the supramolecular interactions leading to the self-assembled architectures. π–π stacking and hydrogen bonding were the main driving forces leading to the self-assembly of Fmoc-Tyr-OH and Fmoc-Phe-OH forming nanofibers. Further, a mechanism involving a two-step coassembly process is proposed based on nucleation and elongation/growth to explain the structural transformation. Fmoc-Trp-OH acted as a fiber inhibitor to alter the molecular interactions in the Fmoc-Tyr-OH or Fmoc-Phe-OH self-assembled structures during the coassembly process, locking the coassembly in the nucleation step and preventing the formation of nanofibers. This structural transformation is useful for extending the application of amino acid self- or coassembled materials in different fields. For example, the amino acids forming nanofibers could be applied for tissue engineering, while they could be exploited as drug nanocarriers when they form nanoparticles

Uptake path of model (1) SWNT.

<p><b>a</b>, Internalization mechanism obtained from unconstrained MD simulations with closed and non functionalized SWNT displays a 3-step passive diffusion phenomenon. The lipid membrane head and tails sections are shown as red and blue surfaces, respectively. For clarity, water molecules and counterions are not shown. <b>b</b>, Voronoi tessellations of membrane surface present in average an inflation of the area per lipid but reveals also local contractions in the neighborhood of the tube penetrating the membrane. Red areas in Voronoi diagrams correspond to internalizing SWNT. <b>c</b>, Close examination of SWNT trajectory (black curve) and insertion angle (wine curve) show sudden penetration phase. Left ordinate scale refer to SWNT center of mass position (black curve), mean nitrogen position of lipid headgroups (red curve), mean phosphorous position of lipid headgroups (blue curve) and mean position of lipid glycerol backbone (green curve). Right ordinate scale refers to SWNT insertion angle (α) with respect to the normal of the membrane plane (wine curve). The angle curve is smoothed by averaging the angle value in 1 ns window.</p

Studied models of open ended <i>f</i>-CNTs.

<p>Different types of opened SWNTs have been investigated depending on their degree of functionalization. The SWNT edges have been passivated by H atoms. Amino derivatives were randomly distributed on the surface of the tubes.</p

Studied models of closed <i>f</i>-CNTs.

<p>Different types of closed SWNTs have been investigated depending on their degree of functionalization. Amino derivatives were randomly distributed on the surface of the tubes.</p

Uptake path of open ended <i>f</i>-CNTs.

<p>Results obtained from unconstrained MD simulations with: <b>a,</b> opened and non functionalized SWNT [model <b>(5)</b>]; <b>b,</b> low degree functionalized and opened SWNT [model <b>(6)</b>]; <b>c,</b> or highly functionalized and opened SWNT [model <b>(7)</b>]. The yellow surface represents the SWNT core. H atoms (red balls) are used to passivate the SWNT edges. The TEG-NH₃⁺ functional groups (red surfaces) are attached to the SWNT surface. The lipid membrane head and tails sections are shown as red and blue surfaces, respectively. Note that at the end of each trajectory, a single lipid molecule stays strongly anchored at the SWNT tips. This anchored lipid molecule is shown explicitly as blue (acyl chains), red (carboxyl group) and blue/white (phosphatidylcholine headgroup) balls. For clarity reasons, water molecules and counterions are not shown.</p

Free energy profile of model (1) SWNT insertion.

<p>The profile obtained using the ABF approach of a closed and pristine SWNT diffusing across a POPC bilayer shows two energy minima: One at the lipid/water interface and another, more attractive in the bilayer midplane.</p

Uptake path of closed <i>f</i>-CNTs.

<p>Results obtained from unconstrained MD simulations for: <b>a,</b> closed and low degree side functionalized SWNT [model <b>(2)</b>]; <b>b,</b> low degree side and tip functionalized SWNT [model <b>(3)</b>]; <b>c,</b> or highly side functionalized SWNT [model <b>(4)</b>]. Note that <i>f</i>-CNTs can be completely taken up only when the cationic functional groups are deprotonated (<i>cf.</i> text). The yellow surface represents the SWNT core while the amino functional groups attached to the latter are shown as red (charged form) or green (neutral form) atoms. The lipid membrane head and tails sections are shown as pale red and blue surfaces, respectively. For clarity, water molecules and counterions are not shown.</p

Self-Assembled Carbon Nanotube Honeycomb Networks Using a Butterfly Wing Template as a Multifunctional Nanobiohybrid

Insect wings have many unique and complex nano/microstructures that are presently beyond the capabilities of any current technology to reproduce them artificially. In particular, <i>Morpho</i> butterflies are an attractive type of insect because their multifunctional wings are composed of nano/microstructures. In this paper, we show that carbon nanotube-containing composite adopts honeycomb-shaped networks when simply self-assembled on <i>Morpho</i> butterfly wings used as a template. The unique nano/microstructure of the composites exhibits multifunctionalities such as laser-triggered remote-heating, high electrical conductivity, and repetitive DNA amplification. Our present study highlights the important progress that has been made toward the development of smart nanobiomaterials for various applications such as digital diagnosis, soft wearable electronic devices, photosensors, and photovoltaic cells