R. Campardelli

Au-PLA nanocomposites for photothermally controlled drug delivery

Stimuli-responsive drug delivery systems were obtained by encapsulating near-infrared (NIR) sensitive hollow gold nanoshells (HGNs) together with the molecule to be released into biodegradable poly-lactic acid (PLA) sub-micron particles. The rapid heating of the PLA particles caused by NIR radiation enabled use of the PLA-HGN composites as a photo-triggered drug release system. Rhodamine was used as a test molecule to obtain release profiles under different irradiation conditions. HGNs (32 nm diameter, 4.5 nm shell thickness) were synthesized via galvanic replacement of cobalt nanoparticles, using poly(vinylpyrrolidone) (PVP) as a stabilizer. PLA-HGN sub-micron particles (with mean diameters around 200 nm) encapsulating rhodamine were obtained using the supercritical emulsion extraction (SEE) technique. A good gold dispersion and a loading efficiency around 50% in the polymeric matrix were obtained for different HGN loadings. The release rate could be tuned by controlling the intensity of NIR exposition. Rhodamine release was completed in less than 10 hours when applying intense NIR irradiation for a few minutes, whereas 12 days of release was necessary in its absence. The system also allowed rhodamine release in a pulsed pattern.

PLGA Microdevices for Retinoids Sustained Release Produced by Supercritical Emulsion Extraction: Continuous Versus Batch Operation Layouts

Retinyl acetate (RA) was selected as a model compound to be entrapped in poly(lactic-co-glycolic)acid (PLGA) microspheres using supercritical emulsion extraction (SEE). Several oil-in-water emulsions prepared using acetone and aqueous glycerol (80% glycerol, 20% water) were processed using supercritical carbon dioxide (SC-CO2 ) to extract the oily phase and to induce microspheres formation. The characteristics of the microspheres obtained by conventional liquid emulsion extraction and SEE were also compared: SEE produced spherical and free flowing microspheres, whereas the conventional liquid-liquid extraction showed large intraparticles aggregation. Emulsion extraction by SC-CO2 technology was tested using two different operation layouts: batch (SEE-B) and continuous (SEE-C). SEE-C was performed using a packed tower to produce emulsion/SC-CO2 contact in countercurrent mode, allowing higher microsphere recovery and process efficiencies. Operating at 80 bar and 36°C, SEE-C produced PLGA/RA microspheres with mean sizes between 3.3 and 4.5 μm with an excellent encapsulation efficiency of 80%-90%. Almost all the drug was released in about 6 days when charged at 2.7% (w/w), whereas only 40% and 10% of RA were released in the same period of time when the charge was 5.2% and 8.8% (w/w), respectively. Release kinetics constants calculated from the experimental data, using a mathematical model, were also proposed and discussed.

Synergistic effect of sustained release of growth factors and dynamic culture on osteoblastic differentiation of mesenchymal stem cells.

Microparticles have been utilized as delivery vehicles of soluble factors to modify cellular behavior and therefore enhance tissue engineering regeneration. When incorporated into three-dimensional systems, microparticles can provide geometrical and temporal controlled release of bioactive agents, such as growth factors (GFs) to surrounding cells. This study investigates the effect of GFs release from biopolymer microparticles on osteoblastic differentiation of human mesenchymal stem cells (hMSCs) encapsulated in calcium (Ca)-alginate scaffolds while cultured in a tubular perfusion system bioreactor system. Empirical and deterministic models were used to demonstrate that poly(D,L-lactic-co-glycolic acid)-encapsulated GFs would result in a delayed release profile compared to GFs encapsulated into scaffolds directly. We hypothesized that the dual delivery of human bone-morphogenetic protein 2 (hBMP2) and human vascular endothelial growth factor to cells in dynamic culture would provide molecular and physical cues to promote differentiation. Results indicated that the exposures of hBMP2 and dynamic flow are sufficient in enhancing the osteoblastic differentiation pathway compared to no GF addition and static culture. The GF delivery system in a dynamic flow environment resulted in a synergistic effect on osteoblastic differentiation of hMSCs.

Liposomes Size Engineering by Combination of Ethanol Injection and Supercritical Processing

Supercritical fluid extraction using a high-pressure packed tower is proposed not only to remove the ethanol residue from liposome suspensions but also to affect their size and distribution leading the production of nanosomes. Different operating pressures, temperatures, and gas to liquid ratios were explored and ethanol was successfully extracted up to a value of 400 ppm; liposome size and distribution were also reduced by the supercritical processing preserving their integrity, as confirmed by Z-potential data and Trasmission Electron Microscopy observations. Operating at 120 bar and 38°C, nanosomes with a mean diameter of about 180 ± 40 nm and good storage stability were obtained. The supercritical processing did not interfere on drug encapsulation, and no loss of entrapped drug was observed when the water-soluble fluorescein was loaded as a model compound. Fluorescein encapsulation efficiency was 30% if pure water was used during the supercritical extraction as processing fluid; whereas an encapsulation efficiency of 90% was obtained if the liposome suspension was processed in water/fluorescein solution. The described technology is easy to scale up to an industrial production and merge in one step the solvent extraction, liposome size engineering, and an excellent drug encapsulation in a single operation unit.

An engineered multiphase 3D microenvironment to ensure the controlled delivery of cyclic strain and hGDF-5 for the tenogenic commitment of hBMSCs

At present, injuries or rupture of tendons are treated by surgical repair or conservative approaches with unpredictable clinical outcome. Alternative strategies to repair tendon defects without the undesirable side effects associated with the current options are needed. With this in mind, a tissue engineering approach has gained considerable attention as a promising strategy. Here we investigated a synthetic three-dimensional (3D) microenvironment able to interact with stem cells and inducing, via coupled biochemical and physical signals, their early commitment toward the tenogenic lineage. This multiphase 3D construct consisted of a braided hyaluronate elastic band merged with human bone marrow mesenchymal stem cells (hBMSCs) and poly-lactic-co-glycolic acid microcarriers loaded with human growth differentiation factor 5 (hGDF-5) by means of fibrin hydrogel. The multiphase structure allowed hBMSC culture under cyclic strain within a microenvironment where a controlled amount of hGDF-5 was regularly delivered. The cooperative biochemical and physical stimuli induced significantly increased expression of tenogenic markers, such as collagen type I and III, decorin, scleraxis, and tenascin-C, within only 3 days of dynamic hBMSC culture. This approach opens exciting perspectives for future development of engineered tendon tissue substitutes.

Encapsulation of Hydrophilic and Lipophilic Compounds in Nanosomes Produced with a Supercritical Based Process

Liposomes are created when phospholipids self-assemble in an aqueous medium creating spherical closed structures. These vesicles can be loaded with hydrophilic active principles (AP) into the aqueous inner core or with lipophilic compounds in the lipidic double layer. In this work a new supercritical based process for the one-step continuous production of nanosomes is proposed for the encapsulation of hydrophilic and lipophilic compounds. This process is called Supercritical Assisted Liposome Formation (SuperLip). The innovation of this process consists in the inversion of the traditional phases of production of liposomes: water droplets are created by a spray atomization in a high pressure vessel, and then a double layer of phospholipids fast surrounds them. A systematic study on liposome size, morphology, encapsulation efficiency has been performed for several different hydrophilic AP (ampicillin, ofloxacin, bovine serum albumin, fluorescein, eugenol and theophylline). Some operative parameters were also optimized to achieve the production of nanometric liposomes with high encapsulation efficiencies. Operating in this way nanometric and monodispersed liposome suspensions were produced with EE up to 99%. To complete the study, other lipidic compounds were entrapped in the double lipidic layer, obtaining high entrapment efficiencies (TE), also in this case, up to 84.9%.