Gema Marcelo

A V-shaped cationic dye for nonlinear optical bioimaging

A symmetric cationic molecule with D-π-A(+)-π-D architecture was synthesized with high two-photon absorption cross-section (σ(2) ≈ 1140 GM). Application as a marker in fluorescence microscopy of living cells revealed its presence inside the cell staining vesicular shape organelles in the cytoplasm. Fluorescence lifetime imaging microscopy shows that it is also able to penetrate within the nucleus.

Host-Guest-Mediated DNA Templation of Polycationic Supramolecules for Hierarchical Nanocondensation and the Delivery of Gene Material.

Only a few examples of monodisperse molecular entities that can compact exogenous nucleic acids into nanocomplexes, protect the cargo from the biological environment, facilitate cell internalization, and promote safe transfection have been reported up to date. Although these species open new venues for fundamental studies on the structural requirements that govern the intervening processes and their application in nonviral gene-vector design, the synthesis of these moieties generally requires a relatively sophisticated chemistry, which hampers further development in gene therapy. Herein, we report an original strategy for the reversible complexation and delivery of DNA based on the supramolecular preorganization of a β-cyclodextrin-scaffolded polycationic cluster facilitated by bisadamantane guests. The resulting gemini-type, dual-cluster supramolecules can then undergo DNA-templated self-assembly at neutral pH value by bridging parallel DNA oligonucleotide fragments. This hierarchical DNA condensation mechanism affords transfectious nanoparticles with buffering capabilities, thus facilitating endosomal escape following cell internalization. Protonation also destabilizes the supramolecular dimers and consequently the whole supramolecular edifice, thus assisting DNA release. Our advanced hypotheses are supported by isothermal titration calorimetry, NMR and circular dichroism spectroscopic analysis, gel electrophoresis, dynamic light scattering, TEM, molecular mechanics, molecular dynamics, and transfection studies conducted in vitro and in vivo.

Nonlinear emission of quinolizinium-based dyes with application in fluorescence lifetime imaging.

Charged molecules based on the quinolizinum cation have potential applications as labels in fluorescence imaging in biological media under nonlinear excitation. A systematic study of the linear and nonlinear photophysics of derivatives of the quinolizinum cation substituted by either dimethylaniline or methoxyphenyl electron donors is performed. The effects of donor strength, conjugation length, and symmetry in the two-photon emission efficiency are analyzed in detail. The best performing nonlinear fluorophore, with two-photon absorption cross sections of 1140 GM and an emission quantum yield of 0.22, is characterized by a symmetric D-π-A(+)-π-D architecture based on the methoxyphenyl substituent. Application of this molecule as a fluorescent marker in optical microscopy of living cells revealed that, under favorable conditions, the fluorophore can be localized in the cytoplasmatic compartment of the cell, staining vesicular shape organelles. At higher dye concentrations and longer staining times, the fluorophore can also penetrate into the nucleus. The nonlinearly excited fluorescence lifetime imaging shows that the fluorophore lifetime is sensitive to its location in the different cell compartments. Using fluorescence lifetime microscopy, a multicolor map of the cell is drafted with a single dye.

Polymer-Coated Nanoparticles by Adsorption of Hydrophobically Modified Poly(N,N-dimethylacrylamide)

We prepared a reactive random copolymer of N-acryloxysuccinimide and N,N-dimethylacrylamide (DMA) by reversible addition-fragmentation chain transfer polymerization, with Mn ≈ 50k and 23 mol % reactive NAS groups. This copolymer was subsequently modified with hydrophobic (dodecyl) and fluorescent (pyrene, PY; phenanthrene, PHE; or anthracene, AN) side groups, to obtain fluorescent amphiphilic polymers with the same backbone and different substituents. These polymers were adsorbed onto model (ca. 130 nm diameter) poly(butyl methacrylate) nanoparticles, and the size and structure of the adsorbed layer were evaluated using a combination of fluorescence techniques and light scattering. The total diameter increases very fast with polymer concentration up to ca. 140 nm, and then more slowly to 154 nm, stabilizing at this value which corresponds to a polymer shell thickness of ca. 12 nm. In order to evaluate the distribution of hydrophobic groups on the adsorbed polymer layer, we used Förster resonance energy transfer between PHE- and AN-labeled poly(DMA) chains. The obtained concentration profile of the adsorbed polymer corresponds to a coated particle radius which is only slightly smaller than the hydrodynamic radius measured in the same conditions, indicating that the dyes are not located at the particle interface but mostly distributed across the adsorbed layer. Finally, we observed that hydrophobically modified PHE-labeled poly(DMA) chains adsorbed to the nanoparticles were very efficiently displaced by identical hydrophobically modified chains with five times their molecular weight (Mn ≈ 250k) but labeled with PY.

Interaction of gold nanoparticles with Doxorubicin mediated by supramolecular chemistry

A copolymer containing β-cyclodextrin, catechol and polyethylene glycol groups in its side chain was designed for the in situ synthesis and coating of gold nanoparticles (Au@PEG-CD NPs). These platforms were designed as a smart carrier and traceable delivery probe of the chemotherapeutic Doxorubicin drug (Dox). The coated polymer forms stable complexes with Dox in water with a high binding constant (K=2.3×10(4) M(-1) at 25°C), which is one hundred times greater than those reported for its complexation with native βCD. Therefore, Au@PEG-CD NPs were able to load 0.01 mg of the drug per mg of NP and to release up to 60% of it in 48 h at 37°C. In addition, Au@PEG-CD NPs had the capacity to act as a quencher of Dox fluorescence when it was complexed with βCD in the NP organic shell. This feature allows the Dox release to be tracked by monitoring the recovery of its fluorescence in real time. Therefore, the Dox release kinetics and the influence of temperature on the thermal stability of Dox/CD complexes on Au@PEG-CD NP were investigated. The increase in temperature favors the dissociation of the complexes and subsequent Dox release from the NP. The first order rate constant for drug releasing was 1.1×10(-2) min(-1) with a half-life time of 63 min at 37°C. Finally, the great potential of the carrier/probe double nature of Au@PEG-CD NPs was demonstrated in real time inside HeLa cells.

Versatile Tetrablock Copolymer Scaffold for Hierarchical Colloidal Nanoparticle Assemblies: Synthesis, Characterization, and Molecular Dynamics Simulation

A unique combination of molecular dynamics (MD) simulation and detailed size exclusion chromatography-multiangle light scattering (SEC-MALS) analysis is used to provide important a priori insights into the solution self-assembly of a well-defined and symmetric tetrablock copolymer with two acrylic acid (AA) outer blocks, two polystyrene (PS) inner blocks, and a trithiocarbonate (TTC) central group, prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization. SEC-MALS experiments show that the copolymer forms aggregates in both tetrahydrofuran and N,N-dimethylformamide (DMF), even in the presence of different salts, but not in 1,4-dioxane (dioxane). Combined with MD simulations, these results indicate that the AA units are the main cause of aggregation through intermolecular hydrogen bonding, with additional stabilization by the central TTC. The block copolymer chains self-assemble in dioxane by adding cadmium acetate, originating flowerlike inverse micelles with a cadmium acrylate core and the TTC groups in the outer surface of the PS corona. The micelles were used as nanoreactors in the templated synthesis of a single cadmium selenide (CdSe) quantum dot (QD) in the core of each micelle, whereas the shell TTC groups can be converted into thiol functions for further use of these units in hierarchical nanostructures. Only in dioxane where simulations and SEC-MALS suggest an absence of copolymer aggregates prior to cadmium acetate addition do well-dispersed and highly luminescent CdSe QDs form by templated synthesis. These results provide valuable insights into the self-assembly of RAFT copolymers in different solvent systems as it relates to the preparation of emissive QDs with polymer-spaced thiol functionality for binding to gold nanostructures.

Contribution of Size Exclusion Chromatography, Light Scattering, Viscometry, and Conductimetry to the Study of Solution and Gel Properties of a Cationic Polyelectrolyte

Abstract Solution properties of a cationic polyelectrolyte poly (acrylamide-co-diallyldimethylammonium) were studied by size exclusion chromatography with double detection (differential refractive index and light scattering), viscometry, and electrical conductimetry, in water containing different ionic salts. These techniques allow not only the determination of molecular weights, molecular dimensions, and scaling law coefficients, but also study the influence of the counterion on the unperturbed dimensions of the chain. Moreover, swelling properties of crosslinked gel samples of this copolymer, both in pure water and water containing ionic salts, were also studied. The swelling degree is also sensitive to the nature of the anion of the salt and there is a direct correlation between the solution properties of the linear samples and the swelling behaviour of their crosslinked counterparts. Thus, measurements of the polymer in aqueous solution can be used to anticipate the swelling behaviour of the corresponding hydrogel.

Correlation between solution and gel properties of copolymers of acrylamide and diallyldimethylammonium chloride

Abstract Several copolymers formed by acrylamide and diallyldimethyl ammonium chloride containing different fractions of charged diallyl residues, x, were synthesized. Solution properties of linear samples of these copolymers were studied by size exclusion chromatography coupled to differential refractive index and light scattering detectors, which allowed the determination of molecular weight averages, molecular dimensions, scaling law coefficients, and unperturbed dimensions. The results were compared with those of the corresponding homopolymers i.e., polyacrylamide and poly(diallyldimethylammonium chloride). On the other hand, crosslinked samples of the copolymers were prepared and their swelling in water was studied. The correlation between the solution properties of the linear samples and the swelling behaviour of their crosslinked counterparts can be illustrated through the relationship between the characteristic ratio of the unperturbed dimensions and the equilibrium degree of swelling since both magnitudes exhibit a similar dependence on x. Moreover, this dependence is not linear and it passes through a maximum at ca. x ≈ 0.5.