Apolipoprotein (apo) A-I, a 243-residue, 28.1-kDa protein is a major mediator of the reverse cholesterol transport (RCT) pathway, a process that may reduce the risk of cardiovascular disease in humans. In plasma, a small fraction of lipid-free or lipid-poor apoA-I is likely a key player in the first step of RCT. Therefore, a basic understanding of the structural details of lipid-free apoA-I will be useful for elucidating the molecular details of the pathway. To address this issue, we applied the combined approach of cross-linking chemistry and high-resolution mass spectrometry (MS) to obtain distance constraints within the protein structure. The 21 lysine residues within apoA-I were treated with homo bifunctional chemical cross-linkers capable of covalently bridging two lysine residues residing within a defined spacer arm length. After trypsin digestion of the sample, individual peptide masses were identified by MS just after liquid chromatographic separation. With respect to the linear amino acid sequence, we identified 5 short-range and 12 long-range cross-links within the monomeric form of lipid-free apoA-I. Using the cross-linker spacer arm length as a constraint for identified Lys pairs, a molecular model was built for the lipid-free apoA-I monomer based on homology with proteins of similar sequence and known three-dimensional structures. The result is the first detailed model of lipid-free apoA-I. It depicts a helical bundle structure in which the N- and C-termini are in close proximity. Furthermore, our data suggest that the self-association of lipid-free apoA-I occurs via C- and N-termini of the protein based on the locations of six cross-links that are unique to the cross-linked dimeric form of apoA-I.
The recent synergy of sophisticated computer modeling techniques with hard experimental data has generated new models for apolipoprotein A-I in certain subclasses of HDL produced in vitro. The challenge now is to adapt and test these models in the more complex forms of HDL isolated directly from human plasma.
The interest in developing new sunscreens is increasing due to the harmful effects of UV radiation on the skin, such as erythema, accelerated skin ageing (photoageing) and the induction of skin cancer. However, many molecular sunscreens penetrate into the skin causing photoallergies, phototoxic reactions and skin irritation. Thus, the aim of this work was the preparation and characterization of polymeric and solid lipid nanoparticles to act carriers of benzophenone-3 (BZ3), aiming to improve the safety of sunscreen products by increasing the sun protection factor (SPF), decreasing BZ3 skin penetration and decreasing BZ3 concentration in sunscreen formulation. BZ3 was encapsulated in poly(epsilon-caprolactone) (PCL) nanoparticles by the nanoprecipitation method and in solid lipid nanoparticles (SLN) by the hot high pressure homogenization method. The particles were stable for 40 days. The BZ3 encapsulated in PCL nanoparticles was released faster than BZ3 encapsulated in SLN. The sun protection factor increased when BZ3 was encapsulated in both nanostructures. However, BZ3 encapsulated in PCL nanoparticles decreased its skin permeation more than SLN-BZ3. Furthermore, BZ3 encapsulated in SLN did not exhibit cytotoxic or phototoxic effects in human keratinocytes (HaCaT cells) and BABL/c 3T3 fibroblasts, whereas PCL nanoparticles with BZ3 showed phototoxic potential in HaCaT cells. Nevertheless, BZ3 free and encapsulated in PCL nanoparticles or in SLN did not show allergic reactions in mice. Our results suggest that these nanostructures are interesting carriers for sunscreen.
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