The proteomic and particle composition of human platelet lysate for cell therapy products.

Following health agencies warning, the use of animal origin supplements should be avoided in biological products proposed as therapy in humans. Platelet lysate and several other growth factors sources are alternatives to replace fetal calf serum, the current gold standard in clinical-grade cell culture. However, the platelet supplement's content lacks data due to different production methods. The principle behind these products relays on the lysis of platelets that release several proteins, some of which are contained in heterogeneous granules and coordinate biological functions. This study aims to analyze the composition and reproducibility of a platelet lysate produced with a standardized method, by describing several batches' protein and particle content using proteomics and dynamic light scattering. Proteomics data revealed a diversified protein content, with some related to essential cellular processes such as proliferation, morphogenesis, differentiation, biosynthesis, adhesion, and metabolism. It also detected proteins responsible for activation and binding of transforming growth factor beta, hepatocyte growth factor, and insulin-like growth factor. Total protein, biochemical, and growth factors quantitative data showed consistent and reproducible values across batches. Novel data on two major particle populations is presented, with high dispersion level at 231 ± 96 d.nm and at 30 ± 8 d.nm, possibly being an important way of protein trafficking through the cellular microenvironment. This experimental and descriptive analysis aims to support the content definition and quality criteria of a cell supplement for clinical applications.

HPLC-UV method for temozolomide determination in complex biological matrices: Application for in vitro, ex vivo and in vivo studies.

A high-performance liquid chromatography method for temozolomide (TMZ) determination in complex biological matrices was developed and validated for application in in vitro, ex vivo and in vivo studies of new nanotechnology-based systems for TMZ nasal delivery. The method was able to quantify TMZ in nanoemulsions, following cellular uptake, in the porcine nasal mucosa and in mouse plasma and brain. Analyses were performed on a C18 column at 35°C, under UV detection at 330 nm. The mobile phase was methanol-acetic acid 0.5% (30:70, v/v), eluted at an isocratic flow rate of 1.1 mL/min. The method was found to be specific, precise, accurate, robust and linear (0.05 to 5 µg/mL) for TMZ determination in all matrices. No interference of TMZ degradation products was found under various stress conditions such as acidic, alkaline, oxidative, light and thermal exposure, demonstrating stability. The method was applied for the quantification of TMZ in different matrices, i.e. the efficiency of nanoemulsions in vitro in increasing TMZ cellular uptake, ex vivo TMZ permeation and retention in the porcine nasal mucosa tissue, and for in vivo TMZ quantification in mouse brain following intranasal nanoemulsion administration compared with free TMZ.

Physicochemical properties of cationic nanoemulsions and liposomes obtained by microfluidization complexed with a single plasmid or along with an oligonucleotide: Implications for CRISPR/Cas technology.

In this study, we investigated the effects of the association of a single plasmid or its co-complexation along with an oligonucleotide on the physicochemical properties of cationic nanoemulsions and liposomes intended for gene editing. Formulations composed of DOPE, DOTAP, DSPE-PEG (liposomes), MCT (nanoemulsions), and water were obtained by microfluidization. DSPE-PEG was found to play a crucial role on the size and polydispersity index of nanocarriers. Nucleic acids were complexated by adsorption at different charge ratios. No significant differences were noticed in the physicochemical properties of nanocarriers (i.e. droplet size, polydispersity index, or zeta potential) when a single plasmid or both plasmid and oligonucleotide were adsorbed to the formulations. Transmission electron microscopy photomicrographs suggested round nanostructures with the nucleic acids and DSPE-PEG enfolding the surface. Complexes at +4/-1 charge ratio protected nucleic acids against DNase I degradation. The oligonucleotide seemed to be released from the liposomal complexes, while nanoemulsions only released the plasmid after 24 and 48 h of incubation in DMEM supplemented or not. In vitro experiments demonstrated that complexes were highly tolerable to human fibroblasts, Hep-G2, and HEK-293 cells, demonstrating also an uptake ability of about 30%, 30%, and 90%, respectively, no matter what the formulation or the combination of nucleic acids used. Transfection efficiency of the formulations was around 25% in human fibroblasts, 32% in HEK-293, and 15% in Hep-G2 cells. The overall results demonstrated the behavior of liposomes and nanoemulsions complexed with a plasmid or a mixture of a plasmid and an oligonucleotide, and demonstrated that the association with one or two nucleic acids sequences of different length does not seem to interfere in the physicochemical characteristics of complexes or in the uptake capacity by three different types of cells.

A novel, simplified and stability-indicating high-throughput ultra-fast liquid chromatography method for the determination of rosmarinic acid in nanoemulsions, porcine skin and nasal mucosa.

Currently, there is an increasing interest on the development of topical formulations containing rosmarinic acid (RA) due to its well-documented antioxidant activity. This study aimed to develop and validate a stability-indicating ultra-fast liquid chromatography (UFLC) method for the determination of RA in nanoemulsions, porcine skin and nasal mucosa intended to be applied in permeation/retention studies and for development of topical nanoemulsions. Chromatographic separation was carried out using a C18 column packed with 2.6 µm particle size in isocratic conditions using as mobile phase water:acetonitrile (83:17, v/v), acidified with 0.1% trifluoracetic acid (v/v), with a total time of analysis of 3.5 min and detection at 330 nm. RA analysis was specific in the presence of both non-biological (blank nanoemulsion and receptor fluid) and biological matrices (porcine ear skin and porcine nasal mucosa). No interference of degradation products of RA was verified after different stress conditions such as acidic, alkaline, oxidative, light exposure (UV-A and UV-C) and thermal demonstrating the method stability-indicating property. The analytical (0.1-10.0 µg·mL-1) and bioanalytical (0.5-10.0 µg·mL-1) linearity was proved by analysis of the calibration curves of RA and no matrix effect was observed. The method was sensitive, precise and accurate, and showed recovery higher than 85%. The method was considered robust as evaluated by a Plackett-Burman experimental design. In the validated conditions, the RA was determined in the nanoemulsions obtained by spontaneous emulsification procedure (1.007 ±â€¯0.040 mg·mL-1), porcine ear skin (1.13 ±â€¯0.19 µg·cm-2) and nasal mucosa (22.46 ±â€¯3.99 µg·cm-2) after retention/permeation studies. Thus, a highly sensitive, simple, fast and stability-indicating method was developed for RA analysis during the development of topical nanoemulsions and bioanalytical assays in complex matrices.