Eva Villar-Alvarez

Fluorescent drug-loaded, polymeric-based, branched gold nanoshells for localized multimodal therapy and imaging of tumoral cells.

Here we report the synthesis of PLGA/DOXO-core Au-branched shell nanostructures (BGNSHs) functionalized with a human serum albumin/indocyanine green/folic acid complex (HSA-ICG-FA) to configure a multifunctional nanotheranostic platform. First, branched gold nanoshells (BGNSHs) were obtained through a seeded-growth surfactant-less method. These BGNSHs were loaded during the synthetic process with the chemotherapeutic drug doxorubicin, a DNA intercalating agent and topoisomerase II inhibitior. In parallel, the fluorescent near-infrared (NIR) dye indocyanine green (ICG) was conjugated to the protein human serum albumin (HSA) by electrostatic and hydrophobic interactions. Subsequently, folic acid was covalently attached to the HSA-ICG complex. In this way, we created a protein complex with targeting specificity and fluorescent imaging capability. The resulting HSA-ICG-FA complex was adsorbed to the gold nanostructures surface (BGNSH-HSA-ICG-FA) in a straightforward incubation process thanks to the high affinity of HSA to gold surface. In this manner, BGNSH-HSA-ICG-FA platforms were featured with multifunctional abilities: the possibility of fluorescence imaging for diagnosis and therapy monitoring by exploiting the inherent fluorescence of the dye, and a multimodal therapy approach consisting of the simultaneous combination of chemotherapy, provided by the loaded drug, and the potential cytotoxic effect of photodynamic and photothermal therapies provided by the dye and the gold nanolayer of the hybrid structure, respectively, upon NIR light irradiation of suitable wavelength. The combination of this trimodal approach was observed to exert a synergistic effect on the cytotoxicity of tumoral cells in vitro. Furthermore, FA was proved to enhance the internalization of nanoplatform. The ability of the nanoplatforms as fluorescence imaging contrast agents was tested by preliminary analyzing their biodistribution in vivo in a tumor-bearing mice model.

What can the kinetics of amyloid fibril formation tell about off-pathway aggregation?

Some of the most prevalent neurodegenerative diseases are characterized by the accumulation of amyloid fibrils in organs and tissues. Although the pathogenic role of these fibrils has not been completely established, increasing evidence suggests off-pathway aggregation as a source of toxic/detoxicating deposits that still remains to be targeted. The present work is a step toward the development of off-pathway modulators using the same amyloid-specific dyes as those conventionally employed to screen amyloid inhibitors. We identified a series of kinetic signatures revealing the quantitative importance of off-pathway aggregation relative to amyloid fibrillization; these include non-linear semilog plots of amyloid progress curves, highly variable end point signals, and half-life coordinates weakly influenced by concentration. Molecules that attenuate/intensify the magnitude of these signals are considered promising off-pathway inhibitors/promoters. An illustrative example shows that amyloid deposits of lysozyme are only the tip of an iceberg hiding a crowd of insoluble aggregates. Thoroughly validated using advanced microscopy techniques and complementary measurements of dynamic light scattering, CD, and soluble protein depletion, the new analytical tools are compatible with the high-throughput methods currently employed in drug discovery.

Submicron Patterning of Polymer Brushes: An Unexpected Discovery from Inkjet Printing of Polyelectrolyte Macroinitiators

Using an electrostatic-based super inkjet printer we report the high-resolution deposition of polyelectrolyte macroinitiators and subsequent polymer brush growth using SI-ARGET-ATRP. We go on to demonstrate for the first time a submicron patterning phenomenon through the addition of either a like charged polyelectrolyte homopolymer or through careful control of ionic strength. As a result patterning of polymer brushes down to ca. 300 nm is reported. We present a possible mechanistic model and consider how this may be applied to other polyelectrolyte-based systems as a general method for submicron patterning.

Lenghty reverse poly(butylene oxide)-poly(ethylene oxide)-poly(butylene oxide) polymeric micelles and gels for sustained release of antifungal drugs.

In this work, we present a detailed study of the potential application of polymeric micelles and gels of four different reverse triblock poly(butylene oxide)-poly(ethylene oxide)-poly(butylene oxide) copolymers (BOnEOmBOn, where n denotes the respective block lengths), specifically BO8EO90BO8, BO14EO378BO14, BO20EO411BO20 and BO21EO385BO21, as effective drug transport nanocarriers. In particular, we tested the use of this kind of polymeric nanostructures as reservoirs for the sustained delivery of the antifungals griseofulvin and fluconazole for oral and topical administration. Polymeric micelles and gels formed by these copolymers were shown to solubilize important amounts of these two drugs and to have a good stability in physiologically relevant conditions for oral or topical administration. These polymeric micellar nanocarriers were able to release drugs in a sustained manner, being the release rate slower as the copolymer chain hydrophobicity increased. Different sustained drug release profiles were observed depending on the medium conditions. Gel nanocarriers were shown to display longer sustained release rates than micellar formulations, with the existence of a pulsatile-like release mode under certain solution conditions as a result of their inner network structure. Certain bioadhesive properties were observed for the polymeric physical gels, being moderately tuned by the length and hydrophobicity of the polymeric chains. Furthermore, polymeric gels and micelles showed activity against the yeast Candida albicans and the mould demartophytes (Trichophyton rubrum and Microsporum canis) and, thus, may be useful for the treatment of different cutaneous fungal infections.

Complex Self-Assembly of Reverse Poly(butylene oxide)- Poly(ethylene oxide)-Poly(butylene oxide) Triblock Copolymers with Long Hydrophobic and Extremely Lengthy Hydrophilic Blocks

Amphiphilic block copolymers have emerged during last years as a fascinating substrate material to develop micellar nanocontainers able to solubilize, protect, transport, and release under external or internal stimuli different classes of cargos to diseased cells or tissues. However, this class of materials can also induce biologically relevant actions, which complement the therapeutic activity of their cargo molecules through their mutual interactions with biologically relevant entities (cellular membranes, proteins, organelles...); these interactions at the same time, are regulated by the nature, conformation, and state of the copolymeric chains. For these reasons, in this paper we investigated the self-assembly process and physico-chemcial properties of two reverse triblock poly(butylene oxide)-poly(ethylene oxide)-poly(butylene oxide) block copolymers, BO14EO378BO14 and BO21EO385BO21, which have been recently found to be very useful as drug delivery nanovehicles and biological response modifiers under certain conditions (A. Cambón et al. Int. J. Pharm. 2013, 445, 47-57) in order to obtain a clear picture of the solution behavior of this class or block copolymers and to understand their biological activity. These block copolymers are characterized by possessing long BO blocks and extremely lengthy central EO ones, which provide them with a rich rheological behavior characterized by the formation of flowerlike micelles with sizes ranging from 20 to 40 nm in aqueous solution and the presence of intermicellar bridging even at low copolymers concentrations as denoted by atomic force microscopy. Bridging is also clearly observed by analyzing the rheological response of these block copolymers both storage and loss moduli upon changes on time, temperature, and or concentration. Strikingly, the relatively wide Poisson distribution of the polymeric chains make the present copolymers behave rather distinctly to conventional associative thickeners. The observed rich rheological behavior and their tunability also make these copolymers promising materials to configure drug gelling depots.

Insoluble Off-Pathway Aggregates as Crowding Agents during Amyloid Fibril Formation

The study of drug candidates for the treatment of amyloidosis and neurodegenerative diseases frequently involves in vitro measurements of amyloid fibril formation. Macromolecular crowding and off-pathway aggregation (OPA) are, by different reasons, two important phenomena affecting the scalability of amyloid inhibitors and their successful application in vivo. On the one hand, the cellular milieu is crowded with macromolecules that drastically increase the effective (thermodynamic) concentration of the amyloidogenic protein. On the other hand, off-pathway aggregates, rather than amyloid fibrils, are increasingly appointed as causative agents of toxicity. The present contribution reveals that insoluble off-pathway aggregates of hen egg-white lysozyme (HEWL) are a peculiar type of crowding agents that, unlike classical macromolecular crowders, decrease the thermodynamic concentration of protein. Illustrating this effect, OPA is shown to resume after lowering the fraction of insoluble aggregates at a constant soluble HEWL concentration. Protein depletion and thioflavin-T fluorescence progress curves indicate that OPA rebirth is not accompanied by additional amyloid fibril formation. The crystallization-like model extended to account for OPA and time-dependent activity coefficients is able to fit multiple kinetic results using a single set of three parameters describing amyloid nucleation, autocatalytic growth, and off-pathway nucleation. The list of fitted results notably includes the cases of aggregation rebirth and all types of progress curves measured for different HEWL concentrations. The quantitative challenges posed by macromolecular crowding and OPA find here a unified response with broader implications for the development of on- and off-pathway inhibitors.

Characterization of the complexation phenomenon and biological activity in vitro of polyplexes based on Tetronic T901 and DNA

The complexation process and underlying mechanisms that rule the interaction of DNA with the cationic block copolymer Tetronic T901 to form polyplexes and their potential transfection efficiency have been studied under different solution conditions. We noted that T901 favors the formation of self-assembled structures with partially condensed DNA at smaller polymer concentrations than other Pluronic™/Tetronic™-type copolymers previously analysed. The observed polyplexes display sizes from the nano- to the micro- range as derived from DLS, electronic and optical microscopies. Also, copolymer micelles are observed at concentrations below the copolymer critical micellar concentration (cmc) induced by the presence of DNA. The complexation process is dependent on solution conditions, with electrostatic and ionic interactions being more important at acidic pH thanks to the predominant diprotonated form of the block copolymer which is less aggregation-prone, whilst dispersive forces are increasingly enhanced under basic conditions or when rising the solution temperature. Whatever the case, the complexation is mainly governed by entropic contributions, as denoted from ITC data. In vitro transfection experiments after complexing T901 with a pDNA encoding the expression of green fluorescein protein, GFP, show a relative successful fluorescence of transfected HeLa cells, which confirms the uptake, internalization and release of the genetic material within the cells at suitable [N]/[P] ratios with relatively low cytotoxicity. Despite the observed successful outcomes, the obtained transfection efficacies are slightly lower than those obtained with Lipofectamine2000, so further optimization of the polyplex formation conditions is envisaged in future studies.

Gold Nanorod-Based Nanohybrids for Combinatorial Therapeutics

In this work, multifunctional nanocarriers consisting of poly(sodium-4-styrenesulfonate) (PSS)/doxorubicin (DOXO)/poly-l-lysine hydrobromide (PLL)/hyaluronic acid (HA)-coated and (PSS/DOXO/PLL)2/HA-coated gold nanorods were assembled by the layer-by-layer technique with the aims of coupling the plasmonic photothermal properties of the metal nanoparticles for plasmonic hyperthermia and the chemoaction of drug DOXO for potential intended combinatorial cancer therapeutics in the future as well as providing different strategies for the controlled and sustained release of the cargo drug molecules. To do that, DOXO could be successfully loaded onto the hybrid nanoconstructs through electrostatic interactions with high efficiencies of up to ca. 78.3 ± 6.9% for the first formed drug layer and 56 ± 13% for the second one, with a total efficiency for the whole system [(PSS/DOXO/PLL)2/HA-coated NRs] of ca. 65.7 ± 1.4%. Nanohybrid internalization was observed to be enhanced by the outer HA layer, which is able to target the CD44 receptors widely overexpressed in some types of cancers as lung, breast, or ovarian ones. Hence, these nanohybrid systems might be versatile nanoplatforms to simultaneously deliver sufficient heat for therapeutic plasmonic hyperthermia and the anticancer drug. Two controlled mechanisms were proposed to modulate the release of the chemodrug, one by means of the enzymatic degradable character of the PLL layer and another by the modulation of the interactions between the polymeric layers through the exploitation of the optical properties of the hybrid particles under near infrared (NIR) laser irradiation. The combination of this bimodal therapeutic approach exerted a synergistic cytotoxic effect on both HeLa and MDA-MB-231 cancer cells in vitro. Cell death mechanisms were also analyzed, elucidating that plasmonic photothermal therapy induces cell necrosis, whereas DOXO activates the cell apoptotic pathway. Therefore, the present NIR laser-induced targeted cancer thermo/chemotherapy represents a novel targeted anticancer strategy with easy control on demand and suitable therapeutic efficacy.