Induction by TNF- α

We have developed a microencapsulation procedure for the entrapment and manipulation of IB3-1 cystic fibrosis cells. The applied method is based on generation of monodisperse droplets by a vibrational nozzle. Different experimental parameters were analyzed, including frequency and amplitude of vibration, polymer pumping rate and distance between the nozzle and the gelling bath. We have found that the microencapsulation procedure does not alter the viability of the encapsulated IB3-1 cells. The encapsulated IB3-1 cells were characterized in term of secretomic profile, analyzing the culture medium by Bio-Plex strategy. The experiments demonstrated that most of the analyzed proteins, were secreted both by the free and encapsulated cells, even if in a different extent. In order to determine the biotechnological applications of this procedure, we determined whether encapsulated IB3-1 cells could be induced to pro-inflammatory responses, after treatment with TNF-α. In this experimental set-up, encapsulated and free IB3-1 cells were treated with TNF-α, thereafter the culture media from both cell populations were collected. As expected, TNF-α induced a sharp increase in the secretion of interleukins, chemokines and growth factors. Of great interest was the evidence that induction of interleukin-6 and interleukin-8 occurs also by encapsulated IB3-1 cells

Mithramycin encapsulated in polymeric micelles by microfluidic technology as novel therapeutic protocol for beta-thalassemia

This report shows that the DNA-binding drug, mithramycin, can be efficiently encapsulated in polymeric micelles (PM-MTH), based on Pluronic® block copolymers, by a new microfluidic approach. The effect of different production parameters has been investigated for their effect on PM-MTH characteristics. The compared analysis of PM-MTH produced by microfluidic and conventional bulk mixing procedures revealed that microfluidics provides a useful platform for the production of PM-MTH with improved controllability, reproducibility, smaller size, and polydispersity. Finally, an investigation of the effects of PM-MTH, produced by microfluidic and conventional bulk mixing procedures, on the erythroid differentiation of both human erythroleukemia and human erythroid precursor cells is reported. It is demonstrated that PM-MTH exhibited a slightly lower toxicity and more pronounced differentiative activity when compared to the free drug. In addition, PM-MTH were able to upregulate preferentially ?-globin messenger ribonucleic acid production and to increase fetal hemoglobin (HbF) accumulation, the percentage of HbF-containing cells, and their HbF content without stimulating ?-globin gene expression, which is responsible for the clinical symptoms of ß-thalassemia. These results represent an important first step toward a potential clinical application, since an increase in HbF could alleviate the symptoms underlying ß-thalassemia and sickle cell anemia. In conclusion, this report suggests that PM-MTH produced by microfluidic approach warrants further evaluation as a potential therapeutic protocol for ß-thalassemia.<br/

Liposome production by microfluidics: potential and limiting factors

This paper provides an analysis of microfluidic techniques for the production of nanoscale lipid-based vesicular systems. In particular we focus on the key issues associated with the microfluidic production of liposomes. These include, but are not limited to, the role of lipid formulation, lipid concentration, residual amount of solvent, production method (including microchannel architecture), and drug loading in determining liposome characteristics. Furthermore, we propose microfluidic architectures for the mass production of liposomes with a view to potential industrial translation of this technology

A Correlative Imaging Study of in vivo and ex vivo Biodistribution of Solid Lipid Nanoparticles

Purpose: Solid lipid nanoparticles are largely used in biomedical research and are characterized by high stability and biocompatibility and are also able to improve the stability of various loaded molecules. In vitro studies demonstrated that these nanoparticles are low cytotoxic, while in vivo studies proved their efficiency as nanocarriers for molecules characterized by a low bioavailability. However, to our knowledge, no data on the systemic biodistribution and organ accumulation of solid lipid nanoparticles in itself are presently available. Methods: In this view, we investigated the solid lipid nanoparticles biodistribution by a multimodal imaging approach correlating in vivo and ex vivo analyses. We loaded solid lipid nanoparticles with two different fluorophores (cardiogreen and rhodamine) to observe them with an optical imager in the whole organism and in the excised organs, and with fluorescence microscopy in tissue sections. Light and transmission electron microscopy analyses were also performed to evaluate possible structural modification or damage due to nanoparticle administration. Results: Solid lipid nanoparticles loaded with the two fluorochromes showed good optic characteristics and stable polydispersity. After in vivo administration, they were clearly detectable in the organism. Four hours after the injection, the fluorescent signal occurred in anatomical districts corresponding to the liver and this was confirmed by the ex vivo acquisitions of excised organs. Brightfield, fluorescence and transmission electron microscopy confirmed solid lipid nanoparticles accumulation in hepatocytes without structural damage. Conclusion: Our results support the systemic biocompatibility of solid lipid nanoparticles and demonstrate their detailed biodistribution from the whole organism to organs until the cells

In vivo and in vitro biodistribution of solid lipid nanoparticles

Solid lipid nanoparticles (SLN) are a versatile tool with a high potential of applications. They are constituted by biocompatible and biodegradable matrix with well-established safety profiles. Their matrix can improve the stability and bioavailability of labile molecules, assuring restrained release profile. Moreover, due to the physicochemical properties of lipids (i.e. the low melting temperature), SLN can be easily obtained by direct emulsification of the molten lipids and subsequent recrystallization, thus avoiding the use of potentially toxic solvents that are commonly required for the preparation of polymeric nanoparticles (1). SLN used for this study were produced by a protocol based on emulsification of the molten lipids in water by melt and ultra-sonication method (2), and were functionalized with polysorbate 80 (SLN-P80) for in vivo administration. In fact, it has been reported that this surfactant extend the nanoparticle circulation time in the blood, avoiding opsonization with the complement activation and uptake by the reticulo-endothelial system. The influence of nanoparticle composition and functionalization was investigated on morphology, dimension and inner structure by mean of cryogenic transmission electron microscopy (cryo-TEM), X-ray diffraction measurements, photon correlation spectroscopy (PCS) and sedimentation field flow fractionation (SdFFF) (2). SLN-P80 were labeled with cardiogreen fluorophore to allow their visualization in living organisms, and the in vivo biodistribution was evaluated with Fluorescent Luminescent Imaging (FLI) after intraperitoneal (i.p.) administration in a mice model. After the in vivo acquisition, mice were perfused and the excised organs (liver, kidney, spleen, brain, lung) were acquired to confirm SLN-P80 accumulation in specific districts (3). Organ tissues were also analyzed at light microscopy (LM) to evaluate SLN-P80 localization. After 4 h from systemic administration, SLN-P80 specifically accumulated in liver (Fig. 1a). LM revealed that no histological alteration is induced by SLN-P80 administration; however, a marked increase in lipid content (red oil staining) was found in hepatocytes, especially close to the centrilobular venula (Fig. 1b,c). To clarify this phenomenon, we investigated in vitro the uptake and intracellular fate of SLN-P80 in a murine cell line. 3T3 cells were incubated with SLN-P80 for 1, 4 and 24h, and then processed for both LM (red oil staining) and transmission electron microscopy (TEM). SLN-P80 administration induced accumulation of lipid droplets already after 1 h (Fig. 2a,b). At TEM scarce SLN-P80 were observed inside the cells: they occurred free in the cytoplasm, often in close proximity to lipid droplets showing a peripheral electron dense material (Fig. 2c). This suggests that SLN-P80 enter the cells by fusion with the plasma membrane, undergo rapid degradation and their components migrate, probably due to chemical affinity, into the lipid deposits

Acceleration of Functional Maturation and Differentiation of Neonatal Porcine Islet Cell Monolayers Shortly In Vitro Cocultured with Microencapsulated Sertoli Cells

The limited availability of cadaveric human donor pancreata as well as the incomplete success of the Edmonton protocol for human islet allografts fasten search for new sources of insulin the producing cells for substitution cell therapy of insulin-dependent diabetes mellitus (T1DM). Starting from isolated neonatal porcine pancreatic islets (NPIs), we have obtained cell monolayers that were exposed to microencapsulated monolayered Sertoli cells (ESCs) for different time periods (7, 14, 21 days). To assess the development of the cocultured cell monolayers, we have studied either endocrine cell phenotype differentiation markers or c-kit, a hematopoietic stem cell marker, has recently been involved with growth and differentiation of β-cell subpopulations in human as well as rodent animal models. ESC which were found to either accelerate maturation and differentiation of the NPIs β-cell phenotype or identify an islet cell subpopulation that was marked positively for c-kit. The insulin/c-kit positive cells might represent a new, still unknown functionally immature β-cell like element in the porcine pancreas. Acceleration of maturation and differentiation of our NPI cell monolayers might generate a potential new opportunity to develop insulin-producing cells that may suite experimental trials for cell therapy of T1DM

Pb effects on an experimental model of porcine prepubertal Sertoli cells

The environmental pollution is one of the main factors implicated in the world’s fertility decline. Lead (Pb) is one of the major heavy metal contaminants that impairs several organs but preferentially accumulates in male reproductive organs and alters in vivo and in vitro sperm quality [1]. Nowadays, the underlying mechanisms remain unclear. Sertoli cells (SC) provides structural and metabolic support to the spermatogenic cells within the seminiferous tubules, therefore, metabolic and structural changes in SC affect the developing germ cells and consequently alter spermatogenesis. This study aimed to assess whether exposure to subtoxic doses of Pb would adversely affect superior mammalian SC function. Highly purified and functional porcine pre-pubertal SC were isolated [2] and treated with three different Pb acetate concentrations. Parameters of SC functionality, such as inhibin B and anti-Müllerian hormone (AMH) mRNAs and proteins were decreased by Pb exposure respect to the control, such as the FSH-r integrity in terms of 17-β-estradiol production, under FSH stimulation. In addition, we observed an increase of AKT and mTOR mRNAs, p38 phosphorylation ratio and Akt phosphorylation ratio in all experimental conditions, respect to the control. In conclusion, the Pb-related toxicity on SC, even at low concentrations, is expected to alter spermatogenesis

Application of a new, simple and economic colorimetric method for the determination of non-esterified fatty acids in vegetable oils

The amount of non-esterified fatty acids in eight different vegetable oil samples has been determined by two different methods: (a) with a standard titration procedure, and (b) with a new colorimetric method which utilizes phenol red solubilized in reverse micelles. The results obtained by the two methods are in good agreement, although the standard deviation in the case of the phenol red method is significantly higher compared with the conventional titration. The main advantage of the new colorimetric method is an economic one: only small amounts of oil samples (less than 0·1 ml) are required and a comparably small amount of organic solvent (less than 5 ml isooctane) is needed