Preparation of Stable Aqueous Nanodispersions of β-Carotene by Supercritical Assisted Injection in a Liquid Antisolvent

Abstract In food industry, nanodispersions of water-insoluble active compounds are attractive food ingredients as they improve food formulations and the bioavailability of the active compounds. β-carotene, is a natural colorant used in food industry and, besides its excellent colorant properties, it has been employed as precursor of retinol and retinoic acid which have an important role in human health as vitamin A precursor and as cellular regulatory signal, respectively. Carotenoids are practically insoluble in water; it is possible to enhance their dissolution rate by lowering the particle size. For these reasons, precipitation processes are needed for the production of stabilized nanodispersion of β-carotene, thaking into account that carotenoids are unstable compounds, very sensitive to oxidation processes. Supercritical fluid precipitation techniques are exceptionally suitable for labile products. The precipitation process can be performed at mild temperature conditions, on an inert atmosphere, particle sizes in the micron and sub-micron range can be obtained. Recently a new supercritical assisted process was developed for the direct production of stabilized nanoparticles suspensions. The process, named Supercritical Assisted Injection in Liquid Antisolvent (SAILA), produces stable nanoparticle suspensions in water. In this work SAILA process was successfully applied for the production of β-carotene nanoparticle water suspensions. Nanoparticles in the range 50-150 nm were produced varying the process conditions. The produced nanodispersions produced were characterized by high Zeta-potential values and maintain their stabily over months

Supercritical assisted processes for the production of biopolymeric micro and nanocarriers

2011 - 2012This work provides an innovative point of view on obtaining nanoparticles by supercritical fluids. Supercritical fluids have already been exploited in the production and processing of micro or sub-micro particles, but the production of nanoparticles is still more ambitious, requiring a deep understanding of the thermodynamic of multiphase systems involved and of the fluid dynamics and mass transfer of the process. This thesis focused on the development of supercritical assisted process for the production of biopolymeric micro and nanocarrier for pharmaceutical and biomedical applications. After a wide study of the state of the, two different processes have been focused on the production of different kind of devices: 1) Supercrititical Emulsion Extraction process (SEE) for the production of multifunctional nanodevices; 2) Supercritical Assisted Injection in Liquid Antisolvent process (SAILA) for the production of stabilized nanoparticle water suspensions. Supercritical Emulsion Extraction (SEE) has been recently proposed in the literature for the production of drug/polymer microspheres with controlled size and distribution, starting from oil-in-water (o-w) and water-in-oil-in-water (w-o-w) emulsions. This process uses supercritical carbon dioxide (SC-CO) to extract the “oil” phase of emulsions, leading to near solvent-free microparticles. SEE offers the advantage of being a one-step process and is superior to other conventional techniques for the better particle size control, higher product purity and shorter processing times; the SEE process has been proposed in the continuous layout, using a high pressure packed tower. However, monodisperse sub-micro, nanoparticles of biopolymer suitable for pharmaceutical formulations have not been produced until now by SEE. Therefore, this part of the work was performed on the production of monodisperse biopolymer nanoparticles. Particularly, emulsion formulation parameters have been tested, such as, different surfactant concentrations and biopolymer percentages in the oily phase, and several emulsification techniques (ultrasound or high speed emulsification) and their interactions with SEE processing have been tested to obtain small droplets dimensions from the micro size to the nano size range. Poly-lactic-co-glycolic acid (PLGA), poly-lactic acid (PLA) and poly-caprolactone (PCL) were the polymers selected to produce micro and nano devices, relative results are shown in Chapter 7. Then, after the optimization of the process conditions for the production of monodisperse sub-micro and nanoparticles encapsulation of drugs, proteins, peptides and metals has been carried out. For the encapsulation study the SEE technique starting from single or double emulsions was used for the production of nanospheres. The SEE technology has a great potential regarding the field of protein encapsulation thanks to the mild extraction condition and the short process time, and mainly thanks to the possibility to use multiple emulsions. For this reason, the SEE has been applied to the production of biopolymeric micro and nanoparticles of PLA and PLGA encapsulating peptides and proteins starting from double emulsion. Results are shown in Chapter 8. Bovine serum albumin (BSA) has been selected as protecting protein for growth factor, such as Vascular Endothelial Growth Factor (VEGF) and Bone Morphogenetic Proteins (BMP). Both micro and nanoparticles were produced; the effect of size of particles on protein loading and release has been evaluated. PLGA microspheres loaded with GFs were charged inside a bioactive alginate scaffold to monitor the effect of the local release of these biosignals on cells differentiation. Human Mesenchimal Steam Cells (h-MSC), where used in view of the fact that they are a promising cell source for bone tissue engineering. Good cells differentiation indicates that the SEE process was successful in the encapsulation of proteins and peptides, preserving the functional structure of the proteins, thanks to the mild operating conditions used. Another important challenge that has been managed is also the production of nanocomposite biopolymeric particles encapsulating metal nanoparticles for the production of light sensitive drug delivery devices. The encapsulation of TiO2 and Au nanoparticles has been performed for different biomedical applications (Chapter 9 and 10). In this work PLA/nano-TiO2 microparticles have been produced using SEE for photodynamic therapy of cells and bacteria. Anatase type nano-TiO2 ethanol stabilized suspension has been synthesized by precipitation from solutions of titanium alcoxides and directly used as the water internal phase a of double emulsion water in oil in water and also TiO2 fine powder was produced and used for encapsulation experiments using a solid in oil in water emulsion. Both micro and sub-microparticles have been produced. [edited by Author]XI n.s

Liposomes: From Bangham to Supercritical Fluids

Liposomes are spherical vesicles made up of an aqueous core surrounded by phospholipids. These delivery systems (DS) are largely employed as drug carriers in several industrial fields, such as pharmaceutical and nutraceutical fields. The aim of this short review is to provide a fast overview on the main fundamentals of liposomes, thought as a compact guide for researchers and students that want to approach this topic for the first time. The mini-review will focus on the definitions, production methods and characterization protocols of the liposomes produced, making a critical comparison of the main conventional and supercritical based manufacturing methods available. The literature was analyzed deeply from the first works by Dr. Bangham in 1965 to the most recent supercritical fluid applications. The advantages and disadvantages of conventional and high-pressure processes will be described in terms of solvent elimination, production at the nanometric (50–300 nm) and micrometric level (1–100 μm) and encapsulation efficiency (20–90%). The first proposed methods were characterized by a low encapsulation efficiency (20–40%), resulting in drug loss, a high solvent residue and high operating cost. The repeatability of conventional processes was also low, due to the prevalent batch mode. Supercritical-assisted methods were developed in semi-continuous layouts, resulting in an easy process scale-up, better control of liposome dimensions (polydispersity index, PDI) and also higher encapsulation efficiencies (up to 90%)

Optimization of PCL Polymeric Films as Potential Matrices for the Loading of Alpha-Tocopherol by a Combination of Innovative Green Processes

Active food packaging represents an innovative way to conceive food packages. The innovation lies in using natural-based and biodegradable materials to produce a system intended to interact with the food product to preserve its quality and shelf-life. Compared to traditional plastics, active packaging is designed and regulated to release substances in a controlled manner, mainly antimicrobial and antioxidant compounds. Conventional technologies are not suitable for treating these natural substances; therefore, the research for innovative and green techniques represents a challenge in this field. The aim of this work is to compare two different polymeric structures: nanofibrous films obtained by electrospinning and continuous films obtained by solvent casting, to identify the best solution and process conditions for subjecting the samples to the supercritical fluids impregnation process (SFI). The supports optimized were functionalized by impregnating alpha-tocopherol using the SFI process. In particular, the different morphologies of the samples both before and after the supercritical impregnation process were initially studied, identifying the limits and possible solutions to obtain an optimization of the constructs to be impregnated with this innovative green technology in the packaging field

Zein and Spent Coffee Grounds Extract as a Green Combination for Sustainable Food Active Packaging Production: An Investigation on the Effects of the Production Processes

In this work, the effect of different production techniques was evaluated on the physical and antioxidant properties of bio-based packaging intended to prevent the premature oxidation of packaged foods. Spent coffee ground extract, rich in antioxidant molecules, obtained through high pressure and temperature extraction, was loaded on zein polymeric matrices. The techniques adopted in this work are particularly suitable due to their mild conditions to produce active packaging completely based on natural compounds: electrospinning, solvent casting, and spin coating. The novelty of this work lay in the investigation of the dependance of the properties of active packaging on the adopted production techniques; the results clearly indicated a strong dependence of the features of the films obtained by different production processes. Indeed, spin coated samples exhibited the best oxygen barrier properties, while a higher tensile strength was obtained for the casted samples, and the fastest release of active compounds was provided by electrospun mats. The films produced with different methods had different physical properties and the release of extract bioactive compounds can be tunable by varying the production technique, dependent on the variable to be considered. The products developed offer an alternative to traditional packaging solutions, being more eco-sustainable and promoting waste valorization

Operating Parameters Optimization for the Production of Liposomes Loaded with Antibodies Using a Supercritical Fluid-Assisted Process

Encapsulation of antibodies represents a significant advance to protect and deliver these therapeutics in a controlled manner, increasing the stability requested to cover the temporal gap between particle production and their administration. Furthermore, using encapsulation, extracellular, cell surface, and intracellular targets can be reached. This work examines the feasibility of encapsulating mouse IgG isotype control antibodies within phosphatidylcholine-based liposomes using a supercritical fluid-based process called SuperLip (Supercritical-assisted Liposome formation). This process allows a continuous production of both nano- and micrometric liposomes with high encapsulation efficiency working under mild operative conditions. The effect of some operative parameters has been studied on liposome mean diameter, particle size distribution, and antibody entrapment efficiency, comparing these data with those collected working with liposomes obtained by the thin-layer hydration technique. In particular, the effect of water flow rate and of the antibody loading were studied. Antibody-loaded liposomes with mean diameters in the range between 205 and 501 nm have been obtained by using a supercritical fluid-assisted process. High entrapment efficiencies up to 94% have been calculated

Operative parameters optimization production of liposomes for the encapsulation of hydrophilic compounds using a new supercritical process

Liposomes are spherical vesicles formed by a inner aqueous core and a double lipidic layer around it. Conventional techniques for the production of liposomes are characterized by several drawbacks, like the production of micrometric vesicles, a difficult control of the Particle Size Distribution (PSD) and low encapsulation efficiencies (EE) of hydrophilic compounds. Many supercritical semi-continuous techniques were proposed in literature. They are successful in the intent of producing liposomes of smaller diameter, but the EE of hydrophilic compounds and the reproducibility are still a challenge. For this reason, it was recently proposed a new supercritical process whose aim is to invert the steps of production of liposomes, by first creating water droplets and then to fast surround them by phospholipids. We discovered that the high diffusion coefficient of phospholipids in supercritical carbon dioxide allows a fast coverage of water droplets preserving the drug content into the liposome core. In this work, hydrophilic compounds were encapsulated in the vesicles produced using SuperLip, such as Fluorescein, Bovine Serum Albumin (BSA) and Ampicillin, obtaining monodispersed spherical vesicles with a mean size from 100 to 300 nm. Operative parameters like water flow rate and lipid to water mass ratio were optimized. The EEs were evaluated with UV-Vis spectroscopy according to methods reported in literature, and obtaining high values up to 99 % for the three investigated compounds

Potential Use of PLA-Based Films Loaded with Antioxidant Agents from Spent Coffee Grounds for Preservation of Refrigerated Foods

: The aim of this work concerned the production of an active food packaging suitable for refrigerated foods. Polylactic-acid-based films were produced by optimizing the solvent casting technique and testing different loadings of extracts obtained from spent coffee grounds. Indeed, an extract obtained by high-pressure and -temperature extraction (HPTE) and a further purified extract by liquid-liquid extraction (LLE) were separately used as active agents, and the effects on packaging features and active compounds migration were analyzed. The selected active agents showed antioxidant and lipid peroxidation inhibition effects on food simulants (peroxide values of 9.2 ÷ 12.0 meqO2/kg extra virgin olive oil), demonstrating the possibility of enhancing food shelf life. In addition, significant effects on the packaging structure due to the presence of the extract were observed, since it can enhance gas barrier properties of the polymer (O2 permeability of 1.6 ÷ 1.3 × 10-9 cm2/s) and confer better processability. In general, the HPTE extract exhibited better performances than the further purified extract, which was due to the presence of a complex pool of antioxidants and the browning effect on the film but a limited loading capacity on the polymer (840 μg caffeine/g PLA), while higher loading capabilities were enabled using LLE extract

Preparation of Stable Aqueous Nanodispersions of \u3b2-Carotene by Supercritical Assisted Injection in a Liquid Antisolvent

AbstractIn food industry, nanodispersions of water-insoluble active compounds are attractive food ingredients as they improve food formulations and the bioavailability of the active compounds. β-carotene, is a natural colorant used in food industry and, besides its excellent colorant properties, it has been employed as precursor of retinol and retinoic acid which have an important role in human health as vitamin A precursor and as cellular regulatory signal, respectively. Carotenoids are practically insoluble in water; it is possible to enhance their dissolution rate by lowering the particle size. For these reasons, precipitation processes are needed for the production of stabilized nanodispersion of β-carotene, thaking into account that carotenoids are unstable compounds, very sensitive to oxidation processes. Supercritical fluid precipitation techniques are exceptionally suitable for labile products. The precipitation process can be performed at mild temperature conditions, on an inert atmosphere, particle sizes in the micron and sub-micron range can be obtained. Recently a new supercritical assisted process was developed for the direct production of stabilized nanoparticles suspensions. The process, named Supercritical Assisted Injection in Liquid Antisolvent (SAILA), produces stable nanoparticle suspensions in water. In this work SAILA process was successfully applied for the production of β-carotene nanoparticle water suspensions. Nanoparticles in the range 50-150nm were produced varying the process conditions. The produced nanodispersions produced were characterized by high Zeta-potential values and maintain their stabily over months