Efficient delivery of carotenoids to adipocytes with albumin.

Carotenoids are very effectively delivered by albumin to adipocytes. The uptake of carotenoids to the cells occurs in the form of self-aggregates that localize in the vicinity of the adipocyte membrane, as shown by high spatial resolution Raman spectroscopy. The binding of carotenoids to albumin and the mechanism of their transport were elucidated with the help of chiroptical spectroscopies, in tandem with molecular docking and molecular dynamics simulations. In particular, apart from the recognized high affinity pocket of albumin that binds a carotenoid monomer in domain I, we have identified a hydrophobic periphery area in domain IIIB that loosely bounds the self-aggregated carotenoid in aqueous media and enables its easy detachment in hydrophobic environments. This explains the effectiveness of albumins as nanocarriers of carotenoids to adipocytes in vitro.

Findings on the interaction of the antimicrobial peptide cecropin-melittin with a gold surface from molecular dynamics studies.

The immobilization of gold nanoparticles (AuNPs) with antimicrobial peptides (AMPs) is a new and promising way to enhance both the activity and targeting capabilities of AMPs. However, a full understanding of the adsorption process underlying these materials is still lacking. Cecropin-melittin is a peptide with a broad antimicrobial activity while displaying low hemolytic properties, whose conjugation with AuNPs has not been studied before. In this context, we report the investigation of the adsorption process of the cecropin-melittin peptide, with (CM-SH) and without (CM) cysteine at its C-terminus, onto a gold surface based on all-atom MD simulations. Our results show that the way the peptides approach the surface dictates the final conformation and the time required to achieve it in both CM-SH and CM cases. Most important, it is demonstrated that the presence of cysteine promotes a faster conformational stabilization during the lockdown regime of the CM-SH peptide, noticeably affecting this by acting as a preferential anchoring point. This investigation represents a first step in rationalizing, with atomistic detail, some experimentally observed features of CM-SH and CM immobilized gold nanoparticles.

Formulation and Characterization of Stimuli-Responsive Lecithin-Based Liposome Complexes with Poly(acrylic acid)/Poly(N,N-dimethylaminoethyl methacrylate) and Pluronic® Copolymers for Controlled Drug Delivery.

Polymer-liposome complexes (PLCs) can be efficiently applied for the treatment and/or diagnosis of several types of diseases, such as cancerous, dermatological, neurological, ophthalmic and orthopedic. In this work, temperature-/pH-sensitive PLC-based systems for controlled release were developed and characterized. The selected hydrophilic polymeric setup consists of copolymers of Pluronic®-poly(acrylic acid) (PLU-PAA) and Pluronic®-poly(N,N-dimethylaminoethyl methacrylate) (PLU-PD) synthesized by atom transfer radical polymerization (ATRP). The copolymers were incorporated into liposomes formulated from soybean lecithin, with different copolymer/phospholipid ratios (2.5, 5 and 10%). PLCs were characterized by evaluating their particle size, polydispersity, surface charge, capacity of release and encapsulation efficiency. Their cytotoxic potential was assessed by determining the viability of human epithelial cells exposed to them. The results showed that the incorporation of the synthesized copolymers positively contributed to the stabilization of the liposomes. The main accomplishments of this work were the innovative synthesis of PLU-PD and PLU-PAA by ATRP, and the liposome stabilization by their incorporation. The formulated PLCs exhibited relevant characteristics, notably stimuli-responsive attributes upon slight changes in pH and/or temperature, with proven absence of cellular toxicity, which could be of interest for the treatment or diagnosis of all diseases that cause some particular pH/temperature change in the target area.

Atomistic-Level Investigation of a LL37-Conjugated Gold Nanoparticle By Well-Tempered Metadynamics.

LL37 is a cathelicidin-derived antimicrobial peptide (AMP) with a broad spectrum of antimicrobial activity and wound-healing potential. The enhancement of these characteristics was recently demonstrated for a cysteine (CYS)-modified cathelicidin-derived LL37-SH conjugated with gold nanoparticles (AuNPs). Considering the potential of this peptide, we hereby report a computational study in which well-tempered metadynamics was applied to unveil the interaction of LL37-SH and LL37 with a AuNP with atomistic detail. A structural analysis combined with the free energy surface (FES) characterization allowed the assessment of the role of CYS residue during the formation of the conjugate, as well as to understand how the AuNP improves the antimicrobial activity of the peptide. It was found that CYS promotes a lower conformational entropy (before and after adsorption onto the AuNP) and a faster adsorption process when compared to the LL37 without CYS. The FES for LL37-SH is characterized by one global minimum, while for LL37 a potential metastable state was found. The presence of the AuNP leads to an elongation of the peptides along with the adsorption, which translates into the increase of the solvent-accessible surface area. This elongation combined with the greater availability of positively charged residues upon adsorption rationalizes the observed enhancement of the activity of the LL37-SH/AuNP conjugate.

Effect of different types of surfactants on the microstructure of methyltrimethoxysilane-derived silica aerogels: A combined experimental and computational approach.

HYPOTHESIS: Surfactants interfere with sol-gel particle/pore growth, influencing the structure and properties of silica aerogels. Their ability to induce microscopic changes in the aerogel's structure may be useful to improve/control the thermal insulation performance of aerogels. EXPERIMENTS: The influence of different types of surfactants (anionic, cationic and non-ionic) on the microstructural arrangement and macroscopic properties of methyltrimethoxysilane (MTMS)-based aerogels was evaluated for the first time, using an experimental and computational comparative approach. Molecular dynamics simulations were performed based on two representative silica molecular structures derived from MTMS, while the experimentally-obtained silica aerogels were characterized in terms of chemical/structural/mechanical/thermal insulation properties. FINDINGS: The use of both hexadecyltrimethylammonium bromide (CTAB) and sodium dodecylsulfate (SDS) led to a decrease in bulk density, thermal conductivity and average pore size of the aerogels, with notorious increase of their flexibility. The observed changes were due to microstructural arrangements, as evidenced by scanning electron microscopy (SEM). However, the non-ionic surfactant, Pluronic F-127, did not have a positive impact on the desired properties. Globally, the simulation results support the experimental findings, suggesting differentiated microstructural changes induced by the use of cationic or anionic surfactants. The addition of CTAB and SDS generally resulted in smaller or larger silica aggregates, respectively.

Antimicrobial peptide-gold nanoscale therapeutic formulation with high skin regenerative potential.

Chronic skin wounds affect ≈3% of persons aged >60years (Davies et al., 2007) [1]. These wounds are typically difficult to heal by conventional therapies and in many cases they get infected making even harder the regeneration process. The antimicrobial peptide (AMP) LL37 combines antimicrobial with pro-regenerative properties and thus represents a promising topical therapy to address both problems. Here, we investigated the wound healing potential of soluble and immobilized LL37 (LL37-conjugated gold nanoparticles, LL37-Au NPs), both in vitro (migration of keratinocytes) and in vivo (skin wound healing). Our results show that LL37-Au NPs, but not LL37 peptide, have the capacity to prolong the phosphorylation of EGFR and ERK1/2 and enhance the migratory properties of keratinocytes in a large in vitro wound model. We further report that both LL37 and LL37-Au NPs promote keratinocyte migration by the transactivation of EGFR, a process that seems to be initiated at the P2X7 receptor, as confirmed by chemical and genetic inhibition studies. Finally, we show in vivo that LL37-Au NPs have higher wound healing activity than LL37 peptide in a splinted mouse full thickness excisional model. Animal wounds treated by LL37-Au NPs have higher expression of collagen, IL6 and VEGF than the ones treated with LL37 peptide or NPs without LL37. Altogether, the conjugation of AMPs to NPs offers a promising platform to enhance their pro-regenerative properties.

One-step synthesis of high-density peptide-conjugated gold nanoparticles with antimicrobial efficacy in a systemic infection model.

The increase in antibiotic drug resistance and the low number of new antibacterial drugs approved in the last few decades requires the development of new antimicrobial strategies. Antimicrobial peptides (AMPs) are very promising molecules to fight microbial infection since they kill quickly bacteria and, in some cases, target bacterial membrane. Although some AMPs may be stable against proteolytic degradation by chemical modification, in general, low AMP activity and stability in the presence of serum and proteolytic enzymes as well as their cytotoxicity have impaired their clinical translation. Here, we describe a one-step methodology to generate AMP-conjugated gold nanoparticles (Au NPs), with a high concentration of AMPs (CM-SH) (≈240 AMPs per NP), controlled size (14 nm) and low polydispersity. AMP-conjugated Au NPs demonstrated higher antimicrobial activity and stability in serum and in the presence of non-physiological concentrations of proteolytic enzymes than soluble AMP, as well as low cytotoxicity against human cells. Moreover, the NPs demonstrated high antimicrobial activity after in vivo administration in a chronic wound and in an animal model of systemic infection.

Matrix-isolation FT-IR spectra and theoretical study of dimethyl sulfate.

The preferred conformations of dimethyl sulfate and their vibrational spectra were studied by matrix-isolation FT-IR spectroscopy and theoretical methods (DFT and MP2, with basis sets of different sizes, including the quadruple-zeta, aug-cc-pVQZ basis). Conformer GG (of C2 symmetry and exhibiting OSOC dihedral angles of 74.3 degrees ) was found to be the most stable conformer in both the gaseous phase and isolated in argon. Upon annealing of the matrix, the less stable observed conformer (GT; with C1 symmetry) quickly converts to the GG conformer, with the resulting species being embedded in a matrix-cage which corresponds to the most stable matrix-site for GG form. The highest energy TT conformer, which was assumed to be the most stable conformer in previous studies, is predicted by the calculations to have a relative energy of ca. 10 kJ mol-1 and was not observed in the spectra of the matrix-isolated compound.

3D printing of new biobased unsaturated polyesters by microstereo-thermallithography.

New micro three-dimensional (3D) scaffolds using biobased unsaturated polyesters (UPs) were prepared by microstereo-thermal-lithography (µSTLG). This advanced processing technique offers indubitable advantages over traditional printing methods. The accuracy and roughness of the 3D structures were evaluated by scanning electron microscopy and infinite focus microscopy, revealing a suitable roughness for cell attachment. UPs were synthesized by bulk polycondensation between biobased aliphatic diacids (succinic, adipic and sebacic acid) and two different glycols (propylene glycol and diethylene glycol) using fumaric acid as the source of double bonds. The chemical structures of the new oligomers were confirmed by proton nuclear magnetic resonance spectra, attenuated total reflectance Fourier transform infrared spectroscopy and matrix assisted laser desorption/ionization-time of flight mass spectrometry. The thermal and mechanical properties of the UPs were evaluated to determine the influence of the diacid/glycol ratio and the type of diacid in the polyester's properties. In addition an extensive thermal characterization of the polyesters is reported. The data presented in this work opens the possibility for the use of biobased polyesters in additive manufacturing technologies as a route to prepare biodegradable tailor made scaffolds that have potential applications in a tissue engineering area.

Poly(ester amide)s based on (L)-lactic acid oligomers and α-amino acids: influence of the α-amino acid side chain in the poly(ester amide)s properties.

Novel biodegradable and low cytotoxic poly(ester amide)s (PEAs) based on α-amino acids and (L)-lactic acid (L-LA) oligomers were successfully synthesized by interfacial polymerization. The chemical structure of the new polymers was confirmed by spectroscopic analyses. Further characterization suggests that the α-amino acid plays a critical role on the final properties of the PEA. L-phenylalanine provides PEAs with higher glass transition temperature, whereas glycine enhances the crystallinity. The hydrolytic degradation in PBS (pH = 7.4) at 37 °C also depends on the α-amino acid, being faster for glycine-based PEAs. The cytotoxic profiles using fibroblast human cells indicate that the PEAs did not elicit an acute cytotoxic effect. The strategy presented in this work opens the possibility of synthesizing biodegradable PEAs with low citotoxicity by an easy and fast method. It is worth to mention also that the properties of these materials can be fine-tuned only by changing the α-amino acid.

Phosphonium-based ionic liquids as modifiers for biomedical grade poly(vinyl chloride).

This work reports and discusses the influence of four phosphonium-based ionic liquids (PhILs), namely trihexyl(tetradecyl) phosphonium dicyanamide, [P(6,6,6,14)][dca]; trihexyl(tetradecyl) phosphonium bis(trifluoromethylsulfonyl)imide, [P(6,6,6,14)][Tf(2)N]; tetrabutyl phosphonium bromide, [P(4,4,4,4)][Br]; and tetrabutyl phosphonium chloride, [P(4,4,4,4)][Cl], on some of the chemical, physical and biological properties of a biomedical-grade suspension of poly(vinyl chloride) (PVC). The main goal of this work was to evaluate the capacity of these PhILs to modify some of the properties of neat PVC, in particular those that may allow their use as potential alternatives to traditional phthalate-based plasticizers in PVC biomedical applications. PVC films having different PhIL compositions (0, 5, 10 and 20 wt.%) were prepared (by solvent film casting) and characterised by Fourier transform infrared, thermogravimetric analysis, differential scanning calorimetry, dynamical mechanical thermal analysis, scanning electron microscopy/energy-dispersive X-ray/electron probe microanalysis, X-ray diffraction, transmittance, permeability towards oxygen and carbon dioxide, thermal degradation, contact angle measurement, water and vapour uptake, leachability and biocompatibility (haemolytic potential, thrombogenicity and cytotoxicity). A conventional organic plasticizer (di-isononyl phthalate) was used for comparison purposes. The results obtained showed that it was possible to change the neat PVC hydrophobicity, and consequently its water uptake capacity and plasticizer leachability, just by changing the PhIL employed and its composition. It was also possible to significantly change the thermal and mechanical properties of PVC films by choosing appropriate PhIL cation/anion combinations. However, a specific PhIL may not always be capable of simultaneously keeping and/or improving both physical properties. In addition, ionic halide salts were found to promote PVC dehydrochlorination. Finally, none of the prepared materials presented toxicity against Caco-2 cells, though pure [P(6,6,6,14)][dca] decreased HepG2 cells viability. Moreover, PVC films with [P(6,6,6,14)][dca] and [P(4,4,4,4)][Cl] were found to be haemolytic and thus these PhILs must be avoided as PVC modifiers if biomedical applications are envisaged. In conclusion, from all the PhILs tested, [P(6,6,6,14)][Tf(2)N] showed the most promising results regarding blood compatibility, leaching and permeability to gases of PVC films. The results presented are a strong indicator that adequate PhILs may be successfully employed as PVC multi-functional plasticizers for a wide range of potential applications, including those in the biomedical field.

Matrix isolation FTIR spectroscopic and theoretical study of dimethyl sulfite.

The preferred conformations of dimethyl sulfite and their vibrational spectra were studied by matrix-isolation Fourier transform infrared spectroscopy and theoretical methods (density functional theory (DFT) and Moller-Plesset (MP2), with basis sets of different sizes, including the quadruple-zeta, aug-cc-pVQZ basis). Five minima were found at these levels of theory. At the MP2/6-31++G(d,p) and DFT/B3LYP/aug-cc-pVQPZ levels, the GG conformer (where the O-S-O-C dihedral angles are 73.2 and 70.8 degrees ) resulted in the conformational ground state. At the highest level of theory used, the GT conformer (O-S-O-C = +68.5 and -173.2 degrees ) is 0.83 kJ mol(-1) higher in energy than the GG form, while conformer GG' (O-S-O-C = +85.7 and -85.7 degrees ) has a relative energy of 1.18 kJ mol(-1). The remaining two conformers (G'T and TT) are high-energy forms and not experimentally relevant. In consonance with the theoretical predictions, conformer GG was found to be the most stable conformer in the gaseous phase as well as in the low-temperature matrices. Annealing of the argon matrices first promotes the GG'-->GT isomerization, which is followed by conversion of GT into the most stable conformer. There is no evidence of occurrence of GG'-->GG direct conversion in the low-temperature matrices. On the other hand, during deposition of the xenon matrices conformer GG' totally converts to conformer GT. Two observations demonstrated this fact: no evidence of bands corresponding to GG' were observed in xenon matrices and the GG/GT intensity ratio became similar to the GG/(GT + GG') intensity ratio observed in argon matrices. All these results could be explained by taking into account the relative values of the theoretically predicted energy barriers for the different isomerization processes: GG'-->GT, 1.90 kJ mol(-1); GT-->GG, 9.64 kJ mol(-1); and GG'-->GG, 19.46 kJ mol(-1).