Adriana Cambón

Influence of Electrostatic Interactions on the Fibrillation Process of Human Serum Albumin

The fibrillation propensity of the multidomain protein human serum albumin (HSA) has been analyzed under physiological and acidic conditions at room and elevated temperatures with varying ionic strengths by different spectroscopic techniques. The kinetics of fibril formation under the different solution conditions and the structures of resulting fibrillar aggregates were also determined. In this way, we have observed that fibril formation is largely affected by electrostatic shielding: at physiological pH, fibrillation is progressively more efficient and faster in the presence of up to 50 mM NaCl; meanwhile, at larger salt concentrations, excessive shielding and further enhancement of the solution hydrophobicity might involve a change in the energy landscape of the aggregation process, which makes the fibrillation process difficult. In contrast, under acidic conditions, a continuous progressive enhancement of HSA fibrillation is observed as the electrolyte concentration in solution increases. Both the distinct ionization and initial structural states of the protein before incubation may be the origin of this behavior. CD, FT-IR, and tryptophan fluorescence spectra seem to confirm this picture by monitoring the structural changes in both protein tertiary and secondary structures along the fibrillation process. On the other hand, the fibrillation of HSA does not show a lag phase except at pH 3.0 in the absence of added salt. Finally, differences in the structure of the intermediates and resulting fibrils under the different conditions are also elucidated by TEM and FT-IR.

Doxorubicin-loaded micelles of reverse poly(butylene oxide)-poly(ethylene oxide)-poly(butylene oxide) block copolymers as efficient "active" chemotherapeutic agents.

Five reverse poly(butylene oxide)-poly(ethylene oxide)-poly(butylene oxide) block copolymers, BOnEOmBOn, with BO ranging from 8 to 21 units and EO from 90 to 411 were synthesized and evaluated as efficient chemotherapeutic drug delivery nanocarriers and inhibitors of the P-glycoprotein (P-gp) efflux pump in a multidrug resistant (MDR) cell line. The copolymers were obtained by reverse polymerization of poly(butylene oxide), which avoids transfer reaction and widening of the EO block distribution, commonly found in commercial poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamers). BOnEOmBOn copolymers formed spherical micelles of 10-40 nm diameter at lower concentrations (one order of magnitude) than those of equivalent poloxamers. The influence of copolymer block lengths and BO/EO ratios on the solubilization capacity and protective environment for doxorubicin (DOXO) was investigated. Micelles showed drug loading capacity ranging from ca. 0.04% to 1.5%, more than 150 times the aqueous solubility of DOXO, and protected the cargo from hydrolysis for more than a month due to their greater colloidal stability in solution. Drug release profiles at various pHs, and the cytocompatibility and cytotoxicity of the DOXO-loaded micelles were assessed in vitro. DOXO loaded in the polymeric micelles accumulated more slowly inside the cells than free DOXO due to its sustained release. All copolymers were found to be cytocompatible, with viability extents larger than 95%. In addition, the cytotoxicity of DOXO-loaded micelles was higher than that observed for free drug solutions in a MDR ovarian NCI-ADR-RES cell line which overexpressed P-gp. The inhibition of the P-gp efflux pump by some BOnEOmBOn copolymers, similar to that measured for the common P-gp inhibitor verapamil, favored the retention of DOXO inside the cell increasing its cytotoxic activity. Therefore, poly(butylene oxide)-poly(ethylene oxide) block copolymers offer interesting features as cell response modifiers to complement their role as efficient nanocarriers for cancer chemotherapy.

Modulation of Size and Shape of Au Nanoparticles Using Amino-X-Shaped Poly(ethylene oxide)−Poly(propylene oxide) Block Copolymers

In the present work, the formation and stabilization of gold nanoparticles in a one-pot water-based synthesis has been achieved in the presence of a four-arm, star-shaped polyoxyethyelene-polyoxypropylene (PEO-PPO) block copolymer, Tetronic T904, which acts as both reductant and stabilizer. The influence of several parameters such as copolymer and gold salt concentration, reaction temperature, and solution pH on both the size and shape of the resulting nanocrystals has been established. Low copolymer/gold salt molar ratios favor the formation of either triangular or hexagonal planar nanostructures due to a low reduction rate which turns the reaction into kinetic control. As the molar ratio increases, reduction becomes faster with the subsequent increase in the number of crystal seeds and, thus, the decrease in particle size. In addition, there is an increase in the reduction rate which causes the reduction reaction to be governed by thermodynamics, and consequently, spherical geometries are favored. A particle spherical shape can also be promoted as a consequence of the accumulation of block copolymer molecules on different crystallographic planes, homogenizing the metal surface structure and disabling the growth in different crystallographic directions. The same behavior was observed when the reaction temperature was raised. The size and shape of gold nanoparticles could also be controlled by varying the pH of the medium. As the pH becomes more acidic, protons prevent the oxyethylene part of the copolymer from the reduction of metal ions, and consequently, the number of nuclei decreases. This explains the overall increase in the particle size and the change in shape when the synthesis is carried out in acid medium. Finally, comparison with nanoparticles obtained in the presence of a structurally related linear block copolymer Pluronic P105, with a similar number of EO and PO units as T904, denoted an important incidence of the arrangement of PEO and PPO blocks on the reduction reaction rate and the size and shape of the resulting nanoparticles.

Hydration effects on the fibrillation process of a globular protein: the case of human serum albumin

In this work, we have studied the fibrillation process of human serum albumin (HSA) under different solution conditions. In particular, aggregation kinetics, fibril morphology, and composition structural changes were investigated under varying experimental conditions such as pH (2.0 and 7.4), temperature (at 25 and 65 °C), and solvent polarity (ethanol/water mixtures, 10–90% v/v). The characterization was carried out by means of static and dynamic light scattering (SLS and DLS), ThT fluorescence, circular dichroism (CD) and Fourier Transform Infrared (FT-IR) spectroscopy, and transmission electron microscopy (TEM). The aggregation process and the α-helix to β-sheet transitions were found to be favored by temperature and physiological pH. Also, pH was observed to influence both the fibrillation pathway and aggregation kinetics, changing from a classical fibrillation process with a lag phase under acidic conditions to a downhill polymerization process at physiological pH in the presence of the alcohol. Regarding protein structural composition, at room temperature and physiological pH ethanol was found to promote an α-helix to β-sheet conformational transition at intermediate alcohol concentrations, whereas at low and high ethanol contents α-helix prevailed as the predominant structure. Under acidic conditions, ethanol promotes an important fibrillation at high cosolvent concentrations due to screening of electric charges and a decrease in solvent polarity. On the other hand, important differences in the morphology of the resulting fibrils and aggregates are observed depending on the solution conditions. In particular, the formation of classical amyloid-like fibrils at physiological pH and high temperature is observed, in contrast to the usual curly morphology displayed by HSA fibrils under most of the solution conditions. Although the high temperature and pH are the main parameters influencing the protein structure destabilization and subsequent aggregation upon incubation, ethanol helps to regulate the hydrogen bonding, the attractive hydrophobic interactions, and the protein accessible surface area, thus, modifying the packing constraints and the resulting aggregate morphologies.

One-Dimensional Magnetic Nanowires Obtained by Protein Fibril Biotemplating

Magnetic nanowires were obtained through the in situ synthesis of magnetic material by Fe-controlled nanoprecipitation in the presence of two different protein (human serum albumin (HSA) and lysozyme (Lys)) fibrils as biotemplating agents. The structural characteristics of the biotemplates were transferred to the hybrid magnetic wires. They exhibited excellent magnetic properties as a consequence of the 1D assembly and fusion of magnetite nanoparticles as ascertained by SQUID magnetometry. Prompted by these findings, we also checked their potential applicability as MRI contrast agents. The magnetic wires exhibited large r(2)* relaxivities and sufficient contrast resolution even in the presence of an extremely small amount of Fe in the magnetic hybrids, which would potentially enable their use as T(2) contrast imaging agents.

Block copolymer-regulated synthesis of gold nanocrystals with sharp tips and edges

Gold decahedra and triangular plates of small size (30 and 50 nm, respectively) were grown by a green one-step synthesis process of HAuCl4 in the presence of citric acid and amphiphilic copolymer Tetronic T904® (from BASF), which both act as reducing and stabilizing agents. Tetronic T904® is a star-shaped copolymer composed of polyethylene oxide and propylene oxide units. Both compounds are key in the formation of decahedral particles due to their specific adsorption on certain crystallographic planes of the nanoparticles. Withdrawal or substitution of one of them involves the formation of irregular particles. On the other hand, by changing the reactions conditions the nanoparticle size and shape could be modified. In particular, by varying the molar ratio between the block copolymer a more efficient protection provided by the block copolymer is achieved, which allows the reaction to turn into kinetic control and, thus, a change from decahedral to triangular shape is observed. Due to the presence of acute tips and sharp edges, which sustain large electromagnetic fields upon excitation with light of appropriate energy, these nanoparticles demonstrated their utility as SERS substrates. Concentrations of up to ∼10−11 M of the Raman probe 4-nitrobenzenethiol displayed a readable SERS spectrum, with electromagnetic enhancement factors of up to ∼4.2 × 107.

Micellisation of triblock copolymers of ethylene oxide and 1,2-butylene oxide: Effect of B-block length

We have used pyrene fluorescence spectroscopy and isothermal titration calorimetry (ITC) to investigate the effect of hydrophobic-block length on values of the critical micelle concentration (cmc) for aqueous solutions of triblock poly(butylene oxide)-poly(ethylene oxide)-poly(butylene oxide) block copolymers (B(n)E(m)B(n), where m and n denote the respective block lengths) with hydrophobic block lengths in the range n=12-21. Combined with results from previous work on B(n)E(m)B(n) copolymers with shorter B blocks, plots of log(10)(cmc) (cmc in molar units and reduced to a common E-block length) against total number of B units (n(t)=n for diblock or n(t)=2n for triblock copolymers) display transitions in the slopes of the two plots, which indicate changes in the micellisation equilibrium. These occur at values of n(t)which can be assigned to the onset and completion of collapse of the hydrophobic B blocks, an effect not previously observed for reverse triblock copolymers. The results are compared with related data for diblock E(m)B(n) copolymers.

Poly(ethylene oxide)-poly(styrene oxide)-poly(ethylene oxide) copolymers: Micellization, drug solubilization, and gelling features

Two new poly(ethylene oxide)-poly(styrene oxide) triblock copolymers (PEO-PSO-PEO) with optimized block lengths selected on the basis of previous studies were synthesized with the aim of achieving a maximal solubilization ability and a suitable sustained release, while keeping very low material expense and excellent aqueous copolymer solubility. The self-assembling and gelling properties of these copolymers were characterized by means of light scattering, fluorescence spectroscopy, transmission electron microscopy, and rheometry. Both copolymers formed spherical micelles (12-14 nm) at very low concentrations. At larger concentration (>25 wt%), copolymer solutions showed a rich phase behavior, with the appearance of two types of rheologically active (more viscous) fluids and of physical gels depending on solution temperature and concentration. The copolymer behaved notably different despite their relatively similar block lengths. The ability of the polymeric micellar solutions to solubilize the antifungal drug griseofulvin was evaluated and compared to that reported for other structurally-related block copolymers. Drug solubilization values up to 55 mg g(-1) were achieved, which are greater than those obtained by previously analyzed poly(ethylene oxide)-poly(styrene oxide), poly(ethylene oxide)-poly(butylene oxide), and poly(ethylene oxide)-poly(propylene oxide) block copolymers. The results indicate that the selected SO/EO ratio and copolymer block lengths were optimal for simultaneously achieving low critical micelle concentrations (cmc) values and large drug encapsulation ability. The amount of drug released from the polymeric micelles was larger at pH 7.4 than at acidic conditions, although still sustained over 1 day.

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.