Emilio I. Alarcon

New Insights into Peptide–Silver Nanoparticle Interaction: Deciphering the Role of Cysteine and Lysine in the Peptide Sequence

We studied the interaction of four new pentapeptides with spherical silver nanoparticles. Our findings indicate that the combination of the thiol in Cys and amines in Lys/Arg residues is critical to providing stable protection for the silver surface. Molecular simulation reveals the atomic scale interactions that underlie the observed stabilizing effect of these peptides, while yielding qualitative agreement with experiment for ranking the affinity of the four pentapeptides for the silver surface.

The biocompatibility and antibacterial properties of collagen-stabilized, photochemically prepared silver nanoparticles.

Spherical 3.5 nm diameter silver nanoparticles (AgNP) stabilized in type I collagen (AgNP@collagen) were prepared in minutes (5-15 min) at room temperature by a photochemical method initiated by UVA irradiation of a water-soluble non-toxic benzoin. This biocomposite was examined to evaluate its biocompatibility and its anti-bacterial properties and showed remarkable properties. Thus, while keratinocytes and fibroblasts were not affected by AgNP@collagen, it was bactericidal against Bacillus megaterium and E. coli but only bacteriostatic against S. epidermidis. In particular, the bactericidal properties displayed by AgNP@collagen were proven to be due to AgNP in AgNP@collagen, rather than to released silver ions, since equimolar concentrations of Ag are about four times less active than AgNP@collagen based on total Ag content. This new biocomposite was stable over a remarkable range of NaCl, phosphate, and 2-(N-morpholino)ethanesulfonic acid concentrations and for over one month at 4 °C. Circular dichroism studies show that the conformation of collagen in AgNP@collagen remains intact. Finally, we have compared the properties of AgNP@collagen with a similar biocomposite prepared using α-poly-L-Lysine and also with citrate stabilized AgNP; neither of these materials showed comparable biocompatibility, stability, or anti-bacterial activity.

Surface Plasmons Control the Dynamics of Excited Triplet States in the Presence of Gold Nanoparticles

Aqueous gold nanoparticles (AuNPs) cause a large increase in the yield of methylene blue triplets ((3)MB*) obtained upon 650 nm laser excitation as a result of surface plasmon field interactions that can be described as transmitter-receiver antenna effects. Two distinct (3)MB* populations are observed; a fast decaying one (tau(T) approximately 25 ns) is believed to be due to molecules on the AuNP surface at the time of excitation and is described as static quenching. A longer lived (3)MB* population has lifetimes in the tens of microseconds but is subject to an anomalously high rate constant for a AuNP quenching of 6.4 x 10(13) M(-1) s(-1). This ultrafast quenching is attributed to a nonrandom distribution caused by the AuNP plasmon field that preferentially excites MB molecules located in the proximity of the AuNP where they are subject to antenna type interactions with the nanoparticle and are spatially predisposed for efficient quenching.

Tuning plasmon transitions and their applications in organic photochemistry

The ketone-photoinduced formation of Au, Ag, and Cu nanoparticles from their corresponding ions in solution has been carried out using benzoin photoinitiators. Ketones are good photosensitizers for nanoparticle synthesis not because of the energy they can absorb or deliver, but rather because of the reducing free radicals they can generate. Efficient photochemical nanoparticle generation thus requires a careful selection of substrates and experimental conditions such that free radical generation occurs with high quantum efficiency, where metal ion precursors do not inhibit radical formation. A key consideration to achieve nanoparticle synthesis with short exposure times is to minimize excited-state quenching by metal ions. Applications of nanostructures in catalysis require control of the nanoparticle characteristics, such as dimension, morphology, and surface properties. Part of this article describes the strategies to modify photochemically prepared particles. Finally, we illustrate some of the nanoparticle applications that interest us, with some emphasis on plasmon-mediated processes.

Safety and efficacy of composite collagen–silver nanoparticle hydrogels as tissue engineering scaffolds

The increasing number of multidrug resistant bacteria has revitalized interest in seeking alternative sources for controlling bacterial infection. Silver nanoparticles (AgNPs), are amongst the most promising candidates due to their wide microbial spectrum of action. In this work, we report on the safety and efficacy of the incorporation of collagen coated AgNPs into collagen hydrogels for tissue engineering. The resulting hybrid materials at [AgNPs] < 0.4 μM retained the mechanical properties and biocompatibility for primary human skin fibroblasts and keratinocytes of collagen hydrogels; they also displayed remarkable anti-infective properties against S. aureus, S. epidermidis, E. coli and P. aeruginosa at considerably lower concentrations than silver nitrate. Further, subcutaneous implants of materials containing 0.2 μM AgNPs in mice showed a reduction in the levels of IL-6 and other inflammation markers (CCL24, sTNFR-2, and TIMP1). Finally, an analysis of silver contents in implanted mice showed that silver accumulation primarily occurred within the tissue surrounding the implant.

Photobehavior of merocyanine 540 bound to human serum albumin

The photobehavior of merocyanine 540 (MC) was studied in homogeneous media (ethanol, buffer and glycerol), and in microheterogenous systems (Triton X-100 micelles and in the presence of human serum albumin) using stationary and time-resolved techniques. Merocyanine 540 in aqueous solution mostly forms aggregates, which in the presence of Triton X-100 or HSA are disaggregated. The extent of binding to HSA and its characteristics were estimated from dye absorption and fluorescence changes following protein addition; the Trp-214 fluorescence quenching was also employed to assess the extent of dye association, and physical separation was employed to evaluate the dyes apparent association constant. These results showed that dye adsorption on HSA takes place at both main protein-binding sites (I and II). This adsorption leads to dye monomerization, changing its photobehavior remarkably, with a noticeable increase in fluorescence and triplet lifetimes. These slower decays can be ascribed to a reduction of the dye photoisomerization rate. In addition, the adsorption of the dye partially protects it from the oxygen present in solution, thus reducing the apparent dye triplet-quenching rate constant. However, singlet oxygen and MC triplet quantum yields remain very low in all the systems tested. Finally, we found that the photoconsumption of merocyanine bound to HSA takes place predominantly by a type I mechanism, being more than seven times more efficient than that taking place in ethanol.

LL37 peptide@silver nanoparticles : combining the best of the two worlds for skin infection control

Capping silver nanoparticles with LL37 peptide eradicates the antiproliferative effect of silver on primary skin cells, but retains the bactericidal properties of silver nanoparticles with activities comparable to silver nitrate or silver sulfadiazine. In addition, LL37 capped silver nanoparticles have anti-biofilm formation activity.

Coloured cornea replacements with anti-infective properties: expanding the safe use of silver nanoparticles in regenerative medicine

Despite the broad anti-microbial and anti-inflammatory properties of silver nanoparticles (AgNPs), their use in bioengineered corneal replacements or bandage contact lenses has been hindered due to their intense yellow coloration. In this communication, we report the development of a new strategy to pre-stabilize and incorporate AgNPs with different colours into collagen matrices for fabrication of corneal implants and lenses, and assessed their in vitro and in vivo activity.

Evaluation of solute binding to proteins and intra-protein distances from steady state fluorescence measurements

Steady state fluorescence measurements, due to their relative simplicity and fast and easy implementation, are one of the most employed techniques for evaluating the non-covalent binding of small molecules to proteins. In the present review we discuss the main characteristics of solute binding and the experimental procedures that can be employed for evaluating both, the efficiency of the process and the number of binding sites. It is also discussed the possibility of determining the distance between endogenous fluorophores and non-covalently bound solutes. Albumins (human serum albumin and bovine serum albumin) are considered as model proteins due to their relevance as solute carriers, the extensive available data comprising binding of a large number of solutes, and the reduced number of intrinsic fluorophores which simplifies the data treatment. It is shown that, in spite of the apparent simplicity of the systems, extreme care must be exercised in data treatment and interpretation to avoid misleading results. This applies to the evaluation of binding constants, number of binding sites, and average distance between intrinsic fluorophores and non-covalently bound solutes associated to the proteins.

NIR excitation of upconversion nanohybrids containing a surface grafted Bodipy induces oxygen-mediated cancer cell death

We report the preparation of water-dispersible, ca. 30 nm-sized nanohybrids containing NaYF4:Er3+, Yb3+ up-conversion nanoparticles (UCNPs), capped with a polyethylene glycol (PEG) derivative and highly loaded with a singlet oxygen photosensitizer, specifically a diiodo-substituted Bodipy (IBDP). The photosensitizer, bearing a carboxylic group, was anchored to the UCNP surface and, at the same time, embedded in the PEG capping; the combined action of the UCNP surface and PEG facilitated the loading for an effective energy transfer and, additionally, avoided photosensitizer leaching from the nanohybrid (UCNP-IBDP@PEG). The effectiveness of the nanohybrids in generating singlet oxygen after near-infrared (NIR) excitation (975 nm) with a continuous wavelength (CW) laser was evidenced by using a probe molecule. In vitro assays demonstrated that the UCNP-IBDP@PEG nanohybrid was taken up by the SH-SY5Y human neuroblastoma-derived cells showing low cytotoxicity. Moreover, ca. 50% cancer cell death was observed after NIR irradiation (45 min, 239 mW).