Lipid nanoparticles containing coenzyme Q10 for topical applications: An overview of their characterization.

The coenzyme Q10 is a compound widely used in pharmaceutical and cosmetic formulations because it is a potent eliminator of free radicals, giving it antioxidant and anti-aging properties. It is naturally synthesized by the human body, but its production wanes with age, leading to the formation of wrinkles. The efficacy of topical application of the coenzyme to counteract this process is subject to several difficulties, due to its instability in the presence of light, low solubility in water and high lipophilicity. Because of these drawbacks, many studies have been conducted of release systems. Lipid nanoparticles stand out in this sense due to the advantages of skin compatibility, protection of the active ingredient against degradation in the external medium, capacity to increase penetration of that ingredient in the skin, and its controlled and prolonged release. In this context, this article presents a review of the main studies of the coenzyme Q10 encapsulated in lipid nanoparticles for topical use, focusing on the analytic methods used to characterize the systems regarding morphology, zeta potential, release profile, Q10 content, encapsulation efficiency, crystalline organization and structure of the lipid matrix, rheology, antioxidant activity, skin penetration and efficacy, among other aspects. We also describe the main results of the different studies and discuss the critical aspects - the simplest, most reproducible, best, and most relevant - that characterize lipid nanoparticles with encapsulated Q10 for topical use.

Multiple Response Optimization of Beeswax-Based Nanostructured Lipid Carriers for the Controlled Release of Vitamin E.

Consumer demand for cosmetics is growing, causing a need to develop new systems to release active ingredients. Among these, nanostructured lipid carriers (NLCs) have certain advantages regarding penetration of active compounds in the skin. The study reported here aimed to develop an NLC system for controlled release of vitamin E, a substance that has antioxidant and photoprotective properties. The NLCs containing vitamin E (NLC-VE) were prepared by the melting-emulsionsolidification method, using beeswax as the solid lipid, medium-chain triglycerides (MCTs), coconut oil or avocado oil as liquid lipids and three different nonionic surfactants. The composition of the system was defined by studying the effect of various experimental factors on the size distribution, average diameter and physical stability of the nanoparticles. The optimization of these characteristics, achieved by a Box-Behnken statistical design, showed that 8% w/w of the nonionic surfactant Tween 80, 24% ultrasound amplitude and processing time of 2 minutes and 16 seconds generated nanoparticles with homogeneous size (PDI = 0.11±0.02), average diameter of 180±20 nm and physical stability of 12 weeks. The NLC-VE systems prepared under the optimal conditions, containing Tween 80, beeswax and MCTs, were formulated as viscous suspensions by adding Pluronic F-127, a poly(ethylene oxide)-poly(propylene oxide) block copolymer, at a concentration of 10% w/w. The colloidal nanosuspension obtained had a viscosity of 222 mPa·s and released 70% of the active substance in 6 hours, indicating it is a promising candidate for controlled release of vitamin E.

Extraction of PLGA-microencapsulated proteins using a two-immiscible liquid phases system containing surfactants.

PURPOSE: The extraction of proteins from PLGA/PLA microspheres by a two-immiscible liquid phases system with the addition of surfactants was investigated. METHODS: First, the extraction without surfactants and the interaction between proteins (IFN-α2b and EGF) and empty microspheres (PLGA or PLA) was studied. Next, proteins stability in presence of different surfactants was evaluated by: (1) bicinchoninic acid protein assay, (2) reversed phase-high performance liquid chromatography, and (3) enzyme-linked immunosorbent assay. Then, proteins were extracted with PBS/dichloromethane including selected surfactants and characterized by the above mentioned techniques, biological activity tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization mass spectrometry. RESULTS: Without surfactants, protein recovery was only 27-43% for IFN-α2b and 58-73% for EGF. Protein content in solutions incubated with blank microspheres decreased to 66% for IFN-α2b and 86% for EGF. It was only possible to quantify the EGF and IFN-α2b in the same manner as in PBS alone when the surfactant added was Pluronic F-68 and SDS, respectively. Addition of these surfactants allowed the complete isolation of both biomolecules from the microspheres. The extraction procedure did not affect the encapsulated proteins. CONCLUSION: Proteins can be quantitatively extracted, without changes, from PLGA/PLA microspheres using PBS/dichloromethane system that include an appropriate surfactant.

Microencapsulation of alpha interferons in biodegradable microspheres.

The immunomodulatory, antiproliferative, and antiviral properties of interferons alpha (IFN-α) have made these cytokines attractive for numerous clinical applications. However, most of the current available IFN pharmaceutical dosage forms need to be injected frequently and may provoke adverse reactions in patients. This problem might be overcome by using biodegradable microspheres loaded with IFN. The encapsulation of IFN-α in microspheres and the current status of this technology are the main subjects reviewed here. To this end, we describe (i) the main methods and experimental parameters used to obtain IFN-loaded microspheres and (ii) characterization of these microspheres in terms of morphology, particle size, loading/encapsulation efficiency, residual water content, residual solvent content, release profile, and sterility. Also, we discuss both the characterization of the encapsulated IFN and the stabilization/protection of IFN during microencapsulation. Finally, a brief overview of preclinical and clinical studies using IFN-containing microspheres is given.

Detection of PEGylated proteins in polyacrylamide gels by reverse staining with zinc and imidazole salts.

The reverse staining, with imidazole-SDS-zinc, of PEG-linked proteins separated by SDS-PAGE was studied. Using model conjugates (interferon-alpha 2b (IFN-alpha2b) reacted with either a branched-chain (40,000) PEG (PEG2,40) or a linear monomethoxy PEG polymer (Mr of 12,000) and chromatographically purified monoPEG2,40-IFN-alpha2b), conventional small-format analytical gels (<1 mm thick) showed typical detection patterns (i.e., transparent, colorless bands clearly discernible against a zinc imidazolate-generated white gel background), in less than 20 min. Nonreacted (free) PEG was almost undetected, as expected. The reverse-stained PEGylated IFN-alpha2b patterns were qualitatively indistinguishable from those of parallel gels stained with iodine (I2). The LOD was estimated in the low nanogram range (e.g., at about 7 ng for mono- or bi-PEG2,40 IFN-alpha2b per lane on gradient (4-17%) gels). Also, this stain allowed the visualization of Coomassie blue-undetected PEG-IFN bands, and could be restained with I2. PEGylated species of lysozyme, a low-molecular-weight peptide, ovalbumin, and chymotrypsin were used to demonstrate the generality of this stain. We also show (i) how to counteract the adverse effect of some parameters (e.g., gel thickness above 1 mm, long gel length, low (e.g., 4-6%) acrylamide concentration) on the reverse staining process and (ii) that the properties of the reverse-stained PEGylated proteins remain unchanged, as judged by analyzing both the ion exchange chromatography-based positional isomer separation profile and enzyme-linked immunosorbent response of PEG-IFN recovered from gels. Consequently, this technique may be useful for the rapid analysis or the small-scale preparation of PEGylated proteins.

PEGylated interferon-alpha2b: a branched 40K polyethylene glycol derivative.

PURPOSE: The conjugation of interferon-alpha2b (IFN-alpha2b) to a branched-chain (40,000) polyethylene glycol (PEG2,40K) was studied. METHODS: We studied the conjugation of IFN-alpha2b at different pH values (6.5, 7, and 8), using the PEG2,40K reagent in either solution or solid state. MonoPEGylated interferon was isolated by ion-exchange chromatography and characterized using (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (2) cation exchange high-performance liquid chromatography, (3) bicinchoninic acid protein assay, (4) enzyme-linked immunosorbent assay, (5) cell-based bioassays, (6) thermal stability (at 60 degrees C), (7) tryptic digestion, and (8) pharmacokinetics in rats. RESULTS: PEGylation reaction gave 30-55% PEG2,40K-IFN-alpha2b, 1-10% polyPEGylated interferon, and 35-70% unmodified IFN-alpha2b. Compared to the polyPEGylated IFN-alpha2b species, the pure (96%) monoPEGylated conjugate retained a significantly higher bioactivity (IU/mg): 1.7x10(4)+/-8.5x10(3) vs. 2.8x10(6)+/-1.4x10(6) for antiviral and 1.9x10(4)+/-9.5x10(3) vs. 3.1x10(6)+/-1.6x10(6) for antiproliferative activity. Immunorecognition against IFN was reduced by the PEG2,40K moiety in the conjugate. This monoPEGylated IFN-alpha2b, which migrated as a single band in gel electrophoresis, was found to be a heterogeneous, complex mixture of different positional isomers. PEGylation markedly enhanced both the resistance to tryptic degradation and the thermal stability of IFN-alpha2b. The serum half-life of 40K PEG-IFN was 330-fold longer, while plasma residence time was increased 708 times compared to native IFN. CONCLUSION: The PEG2,40K conjugate of IFN-alpha2b has increased in vitroand in vivo stability as compared to the native cytokine.