Small molecular exogenous modulators of active forms of MMPs.

Matrix metalloproteinases (MMPs) are endopeptidases, and their activity depends on calcium and zinc metal ions. These enzymes are expressed originally in zymogenic form, where the active site of proteins is closed by a prodomain which is removed during activation. A homeostatic balance of their activity is primarily regulated by a 'cysteine switch' located on a consensus sequence of the prodomain and natural endogenous inhibitors, called tissue inhibitors of metalloproteinases (TIMPs). Breakage of this homeostasis may lead to various pathological conditions, which may require further activation and/or inhibition of these enzymes to regenerate that balance. Here, we report four modulators, more specifically, three inhibitors (I1, I2 and I3), and one exogenous activator (L) of the active form of human collagenase MMP-1 (without prodomain). The results were confirmed by binding studies using fluorescence-based enzyme assays.

Green Synthesis of Zein-Based Nanoparticles Encapsulating Lupulone: Antibacterial and Antiphotoaging Agents.

Prolonged skin exposure to UV radiation may result in sunburn, with possible inflammatory and oxidative stress to the skin, skin photoaging, photocarcinogenesis, even DNA damage, and apoptosis if sunscreen protection is not used. Due to the advantages that they offer, high encapsulation capability, increased stability of encapsulated bioactive agents, and release control, nanoparticulate materials have been used in sunscreens despite the hazard that they present: their capacity to penetrate the skin causing toxic side effects (especially the chemical sunscreens). The present study reports the preparation of nanoparticulate composites containing only GRAS substances and using an eco-friendly, inexpensive procedure. The ingredients used have properties that are beneficial to the skin. Zein (Z), a prolamin-rich protein from corn, is biodegradable and biocompatible, is a moisture attractor, and shows effective absorption by cells. Lupulone (L), extracted from hops, is an antibacterial and antioxidant agent that has a stimulating effect on the collagen production in the body due to its content of phytohormones. Gum arabic (GA) is a natural glycoprotein used in beverages and cosmetics as an emulsifier/stabilizer. Composite matrices containing Z/GA/L were prepared using a simple method (antisolvent), which replaces the flammable solvent ethanol with aqueous propylene glycol. The nanocomposites were characterized by FTIR, composition, encapsulation efficiency, and loading capacity for L, size, zeta potential, and morphology (SEM). Their biological activity was investigated as well. The zein-based nanoparticles showed antioxidant and antimicrobial effects (even some synergistic, unexpected behavior) and modulatory activity on the matrix metalloproteinase MMP-1. Due to their properties, the nanoparticles discussed herein show potential for use in formulations for the skin, especially for mature skin, replacing chemical substances with potential side effects used typically in topical delivery systems.

Impact of tannic acid on nisin encapsulation in chitosan particles.

This study investigates the effect of addition of tannic acid on nisin encapsulated in chitosan matrices. Composite materials were prepared using a mild, environmentally friendly procedure, ionotropic gelation of chitosan by sodium tripolyphosphate in the presence of nisin (N) at different concentrations. In two parallel sets of preparations, tannic acid (TA) was added at 10:1 and 5:1 N:TA, respectively. The obtained particles were characterized by FTIR, SEM, size, zeta potential, encapsulation efficiency, loading capacity, and ratio of residual free amino groups. The kinetics of nisin release from the particles was studied to assess the role of TA as a potential modulator thereof. Its addition resulted in enhanced release, higher at lower N:TA ratio. An additional benefit was that TA, a strong antioxidant, imparted antioxidant activity to the composites. Antimicrobial turbidimetric tests were performed against one gram-positive bacterium (Staphylococcus aureus) and two gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), all relevant for the food, pharmaceutical, and cosmetic industries. All the composites showed synergistic effects against all the bacteria tested. The positive coaction was stronger against the gram-negative species. This is remarkable since nisin by itself has not known activity against them.

Molecular "wiring" of plasma amine oxidase: Green and enzyme friendly approaches.

The study describes two approaches to enhance oxidoreductases. Both target molecular "wiring" of enzymes using green processes. The concepts were tested on plasma amine oxidase (PAO). In the first procedure PAO was transiently exposed to an ionic liquid (IL) in the presence of redox molecules, which resulted in partial unfolding. During subsequent dialysis, the enzyme refolded entrapping redox units and affording shorter distances for electron tunneling, hence a molecular "wire" to PAO's prosthetic groups. The other procedure described herein was totally reagentless, using high hydraulic pressure (HHP) to partially denature PAO (in the presence of redox molecules) followed by dialysis and refolding. The two approaches to enzyme "wiring" are discussed comparatively from the point of view of the parameters used during the procedure, residual enzyme activity, nature of the modifier, interaction between PAO and the redox molecules, and stability over time. The most active modified PAO (PAO-ME) from each series was tested in a biosensor for amine detection, toward applications in the food industry and clinical laboratory. Our approaches used "green" reagents (IL) and were made enzyme-friendly as well by the choice of "wires".

Antibacterial hop extracts encapsulated in nanochitosan matrices.

Hops and the components extracted from them are well known antibacterial agents used in beers and as food preservatives, in formulations for topical applications on their own or together with other antimicrobial agents, in hormone replacement therapy, as antioxidants, tumor development antagonists, and angiogenesis inhibitors. Their shortcomings: very low bioavailability, bitter taste, and susceptibility to oxidative decomposition have limited their applications. We propose nanosized chitosan, an inexpensive, readily available biopolymer with a broad spectrum of antibacterial activity, as carrier for lupulone (L) and xanthohumol (X), two components of hops. Chitosan nanoparticles (CNP) and chitosan-based nanocomposites encapsulating lupulone (CNL) and xanthohumol (CNX) were prepared by ionotropic gelation using sodium tripolyphosphate (TPP) as crosslinker. Different preparative ratios and conditions were investigated and the nanoparticles obtained were characterized by FTIR, colloidal titration, size, zeta potential, and antimicrobial activity. The kinetics of the release of L/X from composites was studied in vitro. All the nanoparticles were active against several Gram-positive, Gram-negative, and Candida strains. Synergistic interactions were observed in all cases, although hops are known mainly for their activity against Gram-positive bacteria. All nanoparticles showed good stability over several months.

Molecularly "wired" cholesterol oxidase for biosensing.

The influence of several factors on the activity of cholesterol oxidase (ChOx) transiently exposed to a room temperature ionic liquid (RTIL) was studied. Presence of flavin adenine dinucleotide (FAD, prosthetic group of ChOx) during exposure to RTIL makes the procedure enzyme-friendly, while the use of RTIL (green reagent) makes it environmentally-friendly. Following exposure to RTIL and its subsequent removal, FAD becomes part of the molecular structure of the refolded protein (a molecular "wire"). This makes the procedure used here a molecular one. The factors studied were: FAD presence in RTIL during modification, water presence during exposure to RTIL, and ratio FAD:RTIL during "wiring". Performance parameters monitored were: enzyme activity before and after "wiring" (expressed as (dA/dt)/mg enzyme, and measured spectrophotometrically), peak current in an amperometric biosensor for cholesterol detection, and linearity of the biosensor response depending on cholesterol concentration. After RTIL removal, the modified enzyme (ME) retained a high percentage of the added FAD, which supplemented that of the native enzyme (functioning as a "wire" and enhancing electron transfer kinetics), and a fraction of the initial activity. Used in an amperometric biosensor, ME showed catalytic activity, linear behavior as a function of cholesterol concentration, and stability.

Polymeric FAD used as enzyme-friendly mediator in lactate detection.

We present the fabrication and properties of lactate biosensors. The novel feature is the use of polymerized flavin adenine dinucleotide (FAD) as mediator for electron transfer. The biosensors were prepared using lactate dehydrogenase (LDH), lactate oxidase (LOX), or baker's yeast (BY) immobilized at the surface of the electrode. The sensors using purified enzymes showed good sensitivity, linearity, and stability. The sensitivity of the BY electrodes was slightly lower. The advantages of this type of sensors are discussed in connection with potential applications.