3-D PLLA scaffolds formation by a supercritical freeze extraction assisted process.

Various techniques have been reported in the literature for the fabrication of biodegradable scaffolds; but, it is very difficult to obtain in the same structure macro, micro and nanostructural characteristics. In this work we developed a supercritical freeze extraction process (SFEP) for the formation of poly(L-lactic acid) (PLLA) scaffolds, that combines the advantages of thermally induced phase separation with those of supercritical drying. We processed solutions in chloroform of two PLLA molecular weights and at different polymer concentrations ranging between 5 and 20 % w/w. Supercritical drying was performed at 35 °Cand pressures ranging between 100 and 250 bar. 3-D scaffolds characterized by high porosity (between 88 and 97.5 %), with coexisting micro and nanometric morphology were obtained. Structures generated were characterized by pores ranging between 10 and 30 µm and with a wrinkled nanostructure of about 200 nm, superimposed on the internal pore surface, that could be useful for biomedical applications. A solvent residue lower than 5 ppm was also measured.

3D PLLA/ibuprofen composite scaffolds obtained by a supercritical fluids assisted process.

The emerging next generation of engineered tissues is based on the development of loaded scaffolds containing bioactive molecules in order to control the cellular function or to interact on the surrounding tissues. Indeed, implantation of engineered biomaterials might cause local inflammation because of the host's immune response; thereby, the use of anti-inflammatory agents, whether steroidal or nonsteroidal is required. One of the most important stages of tissue engineering is the design and the generation of a porous 3D structure, with high porosity, high interconnectivity and homogenous morphology. Various techniques have been reported in the literature for the fabrication of biodegradable scaffolds, but they suffer several limitations. In this study, for the first time, the possibility of generating 3D polymeric scaffolds loaded with an active compound by supercritical freeze extraction process is evaluated; this innovative process combines the advantages of the thermally induced phase separation process and of the supercritical carbon dioxide drying. Poly-L-lactid acid/ibuprofen composite scaffolds characterized by a 3D geometry, micrometric cellular structures and wrinkled pores walls have been obtained; moreover, homogeneous drug distribution and controlled release of the active principle have been assured.

Continuous supercritical emulsions extraction: a new technology for biopolymer microparticles production.

Supercritical emulsion extraction (SEE) was recently proposed for the production of biopolymer microparticles starting from oil-in-water emulsions. This technology can improve the product quality because of the fast and selective extraction of the dispersed oily phase by using supercritical carbon dioxide (SC-CO(2) ). However, until now, SEE was proposed in batch configuration, sharing with the traditional processes an intrinsically discontinuous operation and problems of batches reproducibility and process yield. In this study, by using a countercurrent packed column, the SEE process was proposed in a continuous operating mode (SEE-CM) for the production of poly-lactic-co-glycolic acid (PLGA) microparticles. The new process design takes advantage of the large contact area between the SC-CO(2) and emulsion allowing the production of PLGA microparticles with controlled and narrow size distributions in only few minutes. SEE-CM operating parameters such as pressure, temperature, and flow rate ratios were analyzed and the process efficiency in terms of recovered material and its size distribution compared with SEE (batch mode operation) and conventional evaporation technology. PLGA microparticles showed a mean particle size between 1-3 µm (depending on the droplet sizes) with a SD that was always smaller than that associated with particles produced by discontinuous processes. Single and double emulsions were successfully treated and the microparticles physico-chemical properties showed no morphological and structural differences between the SEE-CM-produced microparticles and the ones obtained by conventional evaporation technology.

A bioavailability study on microbeads and nanoliposomes fabricated by dense carbon dioxide technologies using human-primary monocytes and flow cytometry assay.

Supercritical Emulsion Extraction (SEE) and Supercritical assisted Liposome formation (SuperLip), use dense gases such as carbon dioxide (dCO2) to fabricate advanced micro/nanocarriers. SEE uses dCO2 to extract solvent from the oily phase of an emulsion and obtain biopolymer microbead; For this study, poly-Lactic Acid (PLA) microbeads of 1 ±â€¯0.2 µm in mean size loaded at 1 µg/mgPLA with Rhodamine B (ROD) were prepared by SEE; the beads showed a solvent residue lower than 10 ppm and encapsulated the fluorochrome with an efficiency of 90%. SuperLip uses dCO2 to enhance lipid/ethanol/water mixing and to promote the ethanol extraction from liposome suspension. In this case, phosphatidyl-choline (PC) vesicles with a mean size of 0.2 ±â€¯0.05 µm and loaded with Fluorescein Iso-ThioCyanate (FITC) at 8 µg/mgPC were prepared; small unilamellar structure was observed for all the vesicles with FITC encapsulation efficiency of 80%. Ethanol residue of 50 ppm was measured in all the liposome suspensions. The bioavailability of microbeads and nanoliposomes was assessed through incubation with human monocytes previously isolated from healthy donors' blood. A specifically optimized protocol that allowed their quenching on the cell surface was developed to monitor by flow cytometer assay only the cell population that effectively internalized the carriers. When microbeads were tested, the percentage of alive internalizing monocytes was of about 30%. An internalization of 96.1 ±â€¯21% was, instead, obtained at dosage of 0.1 mg/mL for nanoliposomes. In this last case, monocytes showed a vitality of almost 100% after vesicles internalization at all the concentrations studied; on the other hand, cell apoptosis progressively increased in a dose/response manner, after polymer microbeads phagocytosis. The proposed data suggested that dCO2 technologies can be reliably used to fabricate intracellular carriers.

Biomaterials and Supercritical Fluid Technologies: Which Perspectives to Fabricate Artificial Extracellular Matrix?

The foundation of tissue engineering for either therapeutic or diagnostic applications is the ability to exploit living cells. Tissue engineering utilizes living cells as engineering materials implanted, seeded or bioplotted into an artificial structure capable of supporting three-dimensional tissue formation. These structures, typically called scaffolds, are critical, both ex vivo and in vivo, to influence their own microenvironments. Scaffolds can serve the following purposes: allow cell attachment and migration, deliver and retain cells and biochemical factors, enable diffusion of vital cell nutrients or expressed products, exert certain mechanical and biological influences to modify the behaviour of the cell phase. Traditional tissue engineering strategies typically employ a "top-down" approach, in which cells are seeded on a biodegradable three dimensional monolithic polymeric scaffold. More recently they have been updated by a "bottom- up" approach, also known as modular tissue engineering; it is aimed to address the challenge of recreating bio-mimetic structures by designing structural micro-features to build modular tissues, used as building blocks to re-create larger ones. These two different approaches will require scaffolds with given characteristics obtainable by choosing different fabrication technologies. Conventional and innovative supercritical technologies for monolithic scaffold production or biopolymer micro/nano devices will be discussed in this chapter. Some examples of bone and cartilage tissue engineering produced by using modular scaffold will be also discussed, as well as the fabrication of artificial extracellular matrix for spatio-temporally delivery of biological and mechanical signal to address cell fate.

"Leaching or not leaching": an alternative approach to antimicrobial materials via copolymers containing crown ethers as active groups.

In this work, new copolymers containing either MMA and 18C6 crown-ether pendants, or PEG, MMA and 18C6 crown-ether pendants were synthesized to test the idea that sequestering structural alkali-earth ions from the bacterial outer membrane (OM) may lead to bacterial death. The copolymers were obtained either via uncontrolled radical polymerization or ATRP; the latter approached allowed us to produce not only linear copolymers but also branched Y-like structures. After checking for the capability of complexing magnesium and calcium ions, the antimicrobial activity of all copolymers was tested placing their casted plaques in contact with pure water E. coli suspensions. All plaques adsorbed alkali-earth ions and killed bacteria, albeit with different antimicrobial efficiencies. Differences in the latter characteristic were attributed to different plaque roughness. The role of the 18C6 crown-ether pendants was elucidated by pre-saturating plaques with Mg/Ca ions, the marked reduction in antimicrobial efficiency indicating that losing the latter from OM due to surface complexation does play an important role in killing bacteria at short (<5 h) contact times. At longer times, the mode of action is instead related to the poly-cationic nature acquired by the plaques due to ion sequestering.

Corticosteroid microparticles produced by supercritical-assisted atomization: process optimization, product characterization, and "in vitro" performance.

In this work, the production of dexametasone and dexametasone acetate microparticles is proposed using supercritical-assisted atomization (SAA). This process is based on the solubilization of supercritical carbon dioxide in a liquid solution containing the drug; then, the ternary mixture is sprayed through a nozzle and submicroparticles are formed as a consequence of the enhanced atomization. Several process parameters such as different organic solvent (methanol and acetone), solute concentration and flow rate ratio between the liquid solution and carbon dioxide are investigated; their influence is evaluated on the morphology and size of precipitated particles. Spherical corticosteroid particles with mean diameters ranging from 0.5 to 1.2 microm are produced at the optimum operating conditions and narrow particle size distributions (PSDs) have also been obtained. No drug degradation was observed after SAA processing and solvent residues of 300 and 500 ppm for acetone and methanol, respectively, were measured. Drug microparticles produced by SAA can be semi-crystalline or amorphous depending on the process condition; a micronized drug surface area ranging from about 4 to 5 m2/g was also observed. The "in vitro" activity of both untreated and SAA processed glucocorticoids was tested on the release of pro-inflammatory cytokines from stimulated cells. The results shown that SAA-glucocorticoids have retained the activity of the parent untreated compounds and, in the case of dexamethasone, SAA processing improves drug performance.

FEM modeling of the reinforcement mechanism of Hydroxyapatite in PLLA scaffolds produced by supercritical drying, for Tissue Engineering applications.

Scaffolds have been produced by supercritical CO2 drying of Poly-L-Lactid Acid (PLLA) gels loaded with micrometric fructose particles used as porogens. These structures show a microporous architecture generated by the voids left in the solid material by porogen leaching, while they maintain the nanostructure of the gel, consisting of a network of nanofilaments. These scaffolds have also been loaded with Hydroxyapatite (HA) nanoparticles, from 10 to 50% w/w with respect to the polymer, to improve the mechanical properties of the PLLA structure. Based on miscroscopic and mechanical considerations, we propose a parametric Finite Element Method (FEM) model of PLLA-HA composites that describes the microporous structure as a close-packing of equal spheres and the nanoscale structure as a space frame of isotropic curved fibers. The effect of HA on the mechanical properties of the scaffolds has been modeled on the basis of SEM images and by taking into consideration the formation of concentric cylinders of HA nanoparticles around PLLA nanofibers. Modeling analysis confirms that mechanical properties of these scaffolds depend on nanofibrous network connections and that bending is the major factor causing deformation of the network. The FEM model also takes into account the formation of HA multi-layer coating on some areas in the nanofiber network and its increase in thickness with HA percentage. The Young modulus tends to a plateau for HA percentages larger than 30% w/w and when the coverage of the nanofibers produced by HA nanoparticles reaches a loaded surface index of 0.14 in the FEM model.

Terbutaline microparticles suitable for aerosol delivery produced by supercritical assisted atomization.

A new micronization technique called supercritical assisted atomization has been used to produce terbutaline microparticles with controlled particle size distribution in the range of drug particles deliverable by aerosol. The process is based on the solubilization of a fixed amount of supercritical carbon dioxide in a liquid solution; then, the ternary mixture is sprayed through a nozzle and atomized in order to produce microparticles. Water has been used as the liquid solvent; heated nitrogen has also been delivered into the precipitator to evaporate the liquid droplets. The process has been first optimized with respect to pressure and temperature (mixing temperature and pressure, precipitation temperature) and very mild operation conditions have been selected; then, the influence of the solute concentration in the liquid solution on particle size has been studied. The terbutaline produced powders were characterized with respect to morphologies and particle size. Spherical particles with very narrow volumetric particle size distributions were produced. Particularly, operating at 30 and 50mg of terbutaline per ml of water, more than 90% of the two distributions ranged between 1 and 3 microm; at 80 mg/ml more than 99% of the distribution ranged between 1 and 4 microm. HPLC analysis confirmed that no chemical degradation occurred in the drug as a consequence of the supercritical processing.

Ampicillin micronization by supercritical assisted atomization.

The micronization technique called supercritical assisted atomization (SAA) was used to produce ampicillin microparticles with controlled particle size and particle size distribution suitable for aerosol drug delivery. The process is based on the solubilization of supercritical CO2 in a liquid solution. The ternary mixture is then sprayed through a nozzle and, as a consequence of enhanced atomization, solid microparticles are formed. Water and organic solvents were tested with ampicillin to determine the influence of the solvent on the process mechanism. SAA process parameters were studied by testing different supercritical/liquid solvent flow ratios, ampicillin concentrations in the liquid solution and nozzle diameters. The effect of these parameters on morphology, particle size and particle size distribution of microparticles was analysed. Ampicillin particles suitable for aerosol delivery in the size range 1-5 microm were obtained using buffered water. Moreover, by varying the solute concentration, ampicillin particles in a narrower range (1-3 microm) than that usually suggested for aerosol deliverable drugs were obtained. This is an example of particle size tailoring by SAA.

Griseofulvin micronization and dissolution rate improvement by supercritical assisted atomization.

Supercritical assisted atomization (SAA) was used to micronize griseofulvin (GF), selected as a model compound, to verify the performance of this innovative process. SAA is based on the solubilization of supercritical carbon dioxide in a liquid solution containing the drug. The ternary mixture is then sprayed through a nozzle and microparticles are formed as a consequence of the enhanced atomization. Precipitation temperature and drug concentration in the liquid solution were studied to evaluate their influence on morphology and size of precipitated particles. A good particle size control was obtained and GF spherical particles with mean diameters ranging from 0.5 to 2.5 microm were produced with a narrow particle size distribution. Processed GF was characterized by high-performance liquid chromatography-UV/vis, headspace-gas chromatography-flame ionization detection, differential scanning calorimetry, BET and X-ray analyses. No drug degradation was observed and a solvent residue (acetone) less than 800 ppm was measured. GF microparticles showed good stability and surface areas ranging from about 4 to 6 m(2) g(-1); moreover, the micronized drug retained the crystalline habit. GF capsules were formulated with starch and used to compare the dissolution rate of SAA-processed and conventional jet-milled drug. A faster dissolution and a better reproducibility of the dissolution profile were observed for SAA-processed GF.

NSAID drugs release from injectable microspheres produced by supercritical fluid emulsion extraction.

Supercritical fluid emulsion extraction is an innovative technology that uses supercritical carbon dioxide (SC-CO(2)) to extract the dispersed oily phase of an emulsion. This technology was used to produce poly-lactic-co-glycolic acid (PLGA) microspheres charged with two common NSAIDs: piroxicam (PX) and diclophenac sodium (DF). Single (O/W) and double (W/O/W) emulsions were tested and a comparative study between the characteristics of the microspheres obtained by SC-CO(2) extraction and the ones produced by conventional solvent evaporation (SE) is proposed. Varying the droplet dimensions, microspheres with mean diameters (MDs) of 1, 2, and 3 microm were obtained; however, the microspheres produced by SC-CO(2) gave always a better reproduction of the MD of original droplets because aggregation phenomena often modify the mean size and distribution of the microparticles produced by SE. Moreover, very efficient drug loadings (88% w/w of DF in PLGA using W/O/W emulsion and 97% of PX w/w in PLGA starting from O/W emulsion) were measured in the products obtained by SC-CO(2), respectively; whereas, the SE produced a drug loading of 30% in the case of double emulsion and of 70% for single emulsion. Solvent residue of 10 ppm was also measured by SC-CO(2) technology against the 600 ppm of the SE products. The release profiles of the entrapped drugs were also monitored to check the structure of the microspheres produced by this new technology.

Albumin/gentamicin microspheres produced by supercritical assisted atomization: optimization of size, drug loading and release.

In this work, the supercritical assisted atomization (SAA) is proposed, for the first time, not only as a micronization technology but also as a thermal coagulation process for the production of bovine serum albumin (BSA) microspheres charged with Gentamicin sulfate (GS). Particularly, different water solutions of BSA/GS were processed by SAA to produce protein microspheres with different size and antibiotic content. SAA precipitation temperature was selected in the range 100-130 °C to generate protein coagulation and to recover micronized BSA in form of hydrophobic aggregates; GS loading was varied between 10% and 50% (w/w) with an encapsulation efficiency which often reached 100%. In all cases, spherical and noncoalescing particles were successfully produced with a mean particle size of 2 µm and with a standard deviation of about ±1 µm. The microspheres also showed a good stability and constant water content after 60 days of storage. The release profiles of the entrapped drug were monitored using Franz cells to evaluate the possible application of the produced microspheres in wound dressing formulations. Particularly, the microspheres with a BSA/GS ratio of 4:1 after the first burst effect (of 40% of GS loaded) were able to release the GS continuously over 10 days.

Supercritical gel drying: a powerful tool for tailoring symmetric porous PVDF-HFP membranes.

In this work, poly(vinylidene fluoride) copolymer with hexafluoropropylene (PVDF-HFP) membrane-like aerogels have been generated for the first time. PVDF-HFP gels have been prepared from polymer-acetone solutions by adding various amounts of ethanol. A series of supercritical drying experiments have been performed at different pressures (from 100 to 200 bar) and temperatures (from 35 to 45 degrees C) and at various polymer concentrations (from 5 to 12 wt %). The effects of the process conditions on the membrane morphology have been evaluated, and structure-property relationships have been found. In all cases, the membranes exhibit interconnected structures with nanosized pores and high porosity, leading to reduced resistance to the gas mass transfer and high hydrophobic character of the surfaces. These membrane-like aerogels promise to form a new class of highly hydrophobic porous interfaces, potentially suitable to be used in membrane operations based, for example, on the contactor technology.

Cyclodextrins micrometric powders obtained by supercritical fluid processing.

Supercritical fluid technology offers the possibility to produce dry powder formulations of biocompatible materials, overcoming the drawbacks of classical micronization processes. In this work, Supercritical Assisted Atomization (SAA) has been used to micronize alpha-cyclodextrin (alpha-CD) and hydroxypropyl-beta-cyclodextrin (HP-beta-CD). Some process parameters, such as precipitation temperature and solute concentration in the liquid solution, have been studied to evaluate their influence on morphology and size of precipitated particles. Cyclodextrins (CDs) micronization has been successful: well-defined spherical microparticles of alpha-CD and HP-beta-CD have been produced. Particle size analysis revealed that sharp distributions have been obtained: 95% of particles have diameters ranging between 0.1 and 5 microm for both CDs. X-ray and DSC analyses have been also performed to investigate CDs modifications induced by SAA processing: amorphous particles have been obtained in both cases, whereas raw alpha-CD was crystalline and raw HP-beta-CD was amorphous.

Micronization by means of supercritical fluids: possibility of application to pharmaceutical field.

The state of the art in drug micronization is briefly reviewed. The Authors propose and discuss the adoption of a new micronization technique based on supercritical fluids properties: high solvent power and selectivity, fast solute precipitation. A supercritical spray apparatus that has been designed to maximize micronization of pharmaceutical interest products is described. A preliminary scanning of supercritical solvents and cosolvents suitable to dissolve poorly soluble products is also proposed.