Liposomes, transfersomes and niosomes: production methods and their applications in the vaccinal field.

One of the most effective strategies to fight viruses and handle health diseases is vaccination. Recent studies and current applications are moving on antigen, DNA and RNA-based vaccines to overcome the limitations related to the conventional vaccination strategies, such as low safety, necessity of multiple injection, and side effects. However, due to the instability of pristine antigen, RNA and DNA molecules, the use of nanocarriers is required. Among the different nanocarriers proposed for vaccinal applications, three types of nanovesicles were selected and analysed in this review: liposomes, transfersomes and niosomes. PubMed, Scopus and Google Scholar databases were used for searching recent papers on the most frequently used conventional and innovative methods of production of these nanovesicles. Weaknesses and limitations of conventional methods (i.e., multiple post-processing, solvent residue, batch-mode processes) can be overcome using innovative methods, in particular, the ones assisted by supercritical carbon dioxide. SuperSomes process emerged as a promising production technique of solvent-free nanovesicles, since it can be easily scaled-up, works in continuous-mode, and does not require further post-processing steps to obtain the desired products. As a result of the literature analysis, supercritical carbon dioxide assisted methods attracted a lot of interest for nanovesicles production in the vaccinal field. However, despite their numerous advantages, supercritical processes require further studies for the production of liposomes, transfersomes and niosomes with the aim of reaching well-defined technologies suitable for industrial applications and mass production of vaccines.

Production of Agarose-Hydroxyapatite Composites via Supercritical Gel Drying, for Bone Tissue Engineering.

Bone tissue engineering (BTE) is the most promising strategy to repair bones injuries and defects. It relies on the utilization of a temporary support to host the cells and promote nutrient exchange (i.e., the scaffold). Supercritical CO2 assisted drying can preserve scaffold nanostructure, crucial for cell attachment and proliferation. In this work, agarose aerogels, loaded with hydroxyapatite were produced in view of BTE applications. Different combinations of agarose concentration and hydroxyapatite loadings were tested. FESEM and EDX analyses showed that scaffold structure suffered from partial closure when increasing filler concentration; hydroxyapatite distribution was homogenous, and Young's modulus improved. Looking at BTE applications, the optimal combination of agarose and hydroxyapatite resulted to be 1% w/w and 10% w/v, respectively. Mechanical properties showed that the produced composites could be eligible as starting scaffold for BTE, with a Young's Modulus larger than 100 kPa for every blend.

Supercritical Fluid Extraction of Essential Oil and Sclareol from a Clary Sage Concrete.

Clary Sage extracts are of industrial interest: in particular, sclareol shows a strong pharmaceutical potential. Supercritical fluid extraction was used to recover compounds of interest from a Salvia sclarea L. waxy n-hexane extract ("concrete"), using semi-continuous fractionation and a multi-step extraction strategy. Multi-step extraction experiments were carried out in two phases: the first one operated at 90 bar and 50 °C; the second one at 100 bar and 40 °C. GC-MS traces showed that during the first extraction step, only lighter compounds (e.g., monoterpenes, sesquiterpenes, and derivatives) were collected, whereas, in the second step, only sclareol and related compounds were recovered. By adjusting operating conditions (temperature and pressure), selective extraction of different families of compounds was accomplished, with no further need for post-processing of the products. Moreover, using two separators in series, the compounds of interest were fractionated from paraffins and, by changing the operating conditions, the extraction yield increased from about 6.0% to 9.3% w/w as CO2 density increased.

Liposomes for Intra-Articular Analgesic Drug Delivery in Orthopedics: State-of-Art and Future Perspectives. Insights from a Systematic Mini-Review of the Literature.

Background and objectives: Liposomal structures are artificial vesicles composed of one or several lamellae of phospholipids which surround an inner aqueous core. Given the amphoteric nature of phospholipids, liposomes are promising systems for drug delivery. The present review provides an updated synthesis of the main techniques for the production of liposomes for orthopedic applications, focusing on the drawbacks of the conventional methods and on the advantages of high pressure techniques. Materials and Methods: Articles published in any language were systematically retrieved from two major electronic scholarly databases (PubMed/MEDLINE and Scopus) up to March 2020. Nine articles were retained based on the "Preferred Reporting Items for Systematic Reviews and Meta-Analyses" (PRISMA) guidelines. Results: Liposome vesicles decrease the rate of inflammatory reactions after local injections, and significantly enhance the clinical effectiveness of anti-inflammatory agents providing controlled drug release, reducing toxic side effects. Conclusions: This review presents an update on the improvement in musculoskeletal ailments using liposome treatment.

Growth factor sustained delivery from poly-lactic-co-glycolic acid microcarriers and its mass transfer modeling by finite element in a dynamic and static three-dimensional environment bioengineered with stem cells.

Poly-lactic-co-glycolic acid (PLGA) microcarriers (0.8 ± 0.2 µm) have been fabricated with a load of 20 µg/gPLGA by an emulsion-based-proprietary technology to sustained deliver human bone morphogenetic protein 2 (hBMP2), a growth factor largely used for osteogenic induction. hBMP2 release profile, measured in vitro, showed a moderate "burst" release of 20% of the load in first 3 days, followed by a sustained release of 3% of the load along the following 21 days. PLGA microbeads loaded with fluorescent marker (8 mg/gPLGA ) and hydroxyapatite (30 mg/gPLGA ) were also fabricated and successfully dispersed within three-dimensional (3D) alginate scaffold (Ca-alginate 2% wt/wt) in a range between 50 and 200 mg/cm3 ; the presence of microcarriers within the scaffold induced a variation of its stiffness between 0.03 and 0.06 MPa; whereas the scaffold surface area was monitored always in the range of 190-200 m2 /g. Uniform microcarriers dispersion was obtained up to 200 mg/cm3 ; higher loading values in the 3D scaffold produced large aggregates. The release data and the surface area were, then, used to simulate by finite element modeling the hBMP2 mass transfer within the 3D hydrogel bioengineered with stem cells, in dynamic and static cultivations. The simulation was developed with COMSOL Multiphysics® giving a good representation of hBMP2 mass balances along microbeads (bulk eroded) and on cell surface (cell binding). hBMP2 degradation rate was also taken into account in the simulations. hBMP2 concentration of 20 ng/cm3 was set as a target because it has been described as the minimum effective value for stem cells stimulation versus the osteogenic phenotype. The sensitivity analysis suggested the best microbeads/cells ratio in the 3D microenvironment, along 21 days of cultivations in both static and dynamic cultivation (perfusion) conditions. The simulated formulation was so assembled experimentally using human mesenchymal stem cells and an improved scaffold stiffness up to 0.09 MPa (n = 3; p ≤ 0.01) was monitored after 21 days of cultivation; moreover a uniform extracellular matrix deposition within the 3D system was detected by Von Kossa staining, especially in dynamic conditions. The results indicated that the described tool can be useful for the design of 3D bioengineered microarchitecture by quantitative understanding.

SC-CO2-assisted process for a high energy density aerogel supercapacitor: the effect of GO loading.

Energy density, safety, and simple and environmentally friendly preparation methods are very significant aspects in the realization of a compact supercapacitor. Herein we report the use of a supercritical CO2-assisted gel drying process (SC-CO2) for the preparation of porous electrodes containing dispersed graphene in a poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) binder membrane to sandwich in a new portable supercapacitor based on graphene oxide (GO). A GO loading of 60 wt.% was found to give the best combination of factors (porosity, wettability, mechanical and electrochemical properties). Cycling voltammetry and charge/discharge studies showed an excellent capacitance behaviour and stability in an ionic liquid electrolyte, suggesting SC-CO2 processing as a promising platform to produce highly bulky and porous films for supercapacitors. The supercapacitor device delivers a very high energy density of 79.2 Wh kg-1 at a power density of 0.23 KW kg-1 (current density 0.5 A g-1, specific capacitance 36.2 F g-1) while that of steel remains at 50.3 Wh kg-1 at a power density of 2.8 KW kg-1 (current density 6 A g-1, specific capacitance 23.5 F g-1).

Regeneration techniques for bone-to-tendon and muscle-to-tendon interfaces reconstruction.

INTRODUCTION: The complex structure of the bone-tendon and muscle-tendon junctions makes their reproduction for tissue engineering applications very difficult. Relatively few studies have investigated the characteristics of these regions from a tissue engineering view point. SOURCES OF DATA: PubMed, Thomson Reuters, Scopus and Google Scholar databases were searched using various combinations of the keywords 'Tendon', 'Myotendinous junction', 'Osteotendinous junction', 'Tissue engineering' and 'Scaffold'. AREAS OF AGREEMENT: The available studies can be divided according to whether the objective is to build an entire composite tissue unit or to assist the recreation of interfaces, such as improving integration of autografts with the surrounding bone or with the muscle. The most used techniques are based on the electrospinning and the self-reorganized constructs process, which were applied to both bone-to-tendon junction (BTJ) and muscle-to-tendon junction (MTJ) regeneration. The use of nanofibers that mimic the hierarchical structure of the extracellular matrix (ECM), eventually functionalized by encapsulation of bioactive components, allowed cell attachment and differentiation. AREAS OF CONTROVERSY: There have been no translational investigations. GROWING POINTS: There is a need to devise suitable techniques that allow suitable tissue engineering of BTJ and MTJ. AREAS TIMELY FOR DEVELOPING RESEARCH: Appropriately planned studies are needed to translate tissue engineering from a scientific challenge to a clinically applicable technique.

Different insight into amphiphilic PEG-PLA copolymers: influence of macromolecular architecture on the micelle formation and cellular uptake.

One constrain in the use of micellar carriers as drug delivery systems (DDSs) is their low stability in aqueous solution. In this study "tree-shaped" copolymers of general formula mPEG-(PLA)n (n = 1, 2 or 4; mPEG = poly(ethylene glycol) monomethylether 2K or 5K Da; PLA = atactic or isotactic poly(lactide)) were synthesized to evaluate the architecture and chemical composition effect on the micelles formation and stability. Copolymers with mPEG/PLA ratio of about 1:1 wt/wt were obtained using a "core-first" synthetic route. Dynamic Light Scattering (DLS), Field Emission Scanning Electron Microscopy (FESEM), and Zeta Potential measurements showed that mPEG2K-(PD,LLA)2 copolymer, characterized by mPEG chain of 2000 Da and two blocks of atactic PLA, was able to form monodisperse and stable micelles. To analyze the interaction among micelles and tumor cells, FITC conjugated mPEG-(PLA)n were synthesized. The derived micelles were tested on two, histological different, tumor cell lines: HEK293t and HeLa cells. Fluorescence Activated Cells Sorter (FACS) analysis showed that the FITC conjugated mPEG2K-(PD,LLA)2 copolymer stain tumor cells with high efficiency. Our data demonstrate that both PEG size and PLA structure control the biological interaction between the micelles and biological systems. Moreover, using confocal microscopy analysis, the staining of tumor cells obtained after incubation with mPEG2K-(PD,LLA)2 was shown to be localized inside the tumor cells. Indeed, the mPEG2K-(PD,LLA)2 paclitaxel-loaded micelles mediate a potent antitumor cytotoxicity effect.

Production of PEGylated Vancomycin-Loaded Niosomes by a Continuous Supercritical CO2 Assisted Process.

Niosomes are arousing significant interest thanks to their low cost, high biocompatibility, and negligible toxicity. In this work, a supercritical CO2-assisted process was performed at 100 bar and 40 °C to produce niosomes at different Span 80/Tween 80 weight ratios. The formulation of cholesterol and 80:20 Span 80/Tween 80 was selected to encapsulate vancomycin, used as a model active compound, to perform a drug release rate comparison between PEGylated and non-PEGylated niosomes. In both cases, nanometric vesicles were obtained, i.e., 214 ± 59 nm and 254 ± 73 nm for non-PEGylated and PEGylated niosomes, respectively, that were characterized by a high drug encapsulation efficiency (95% for non-PEGylated and 98% for PEGylated niosomes). However, only PEGylated niosomes were able to prolong the vancomycin release time up to 20-fold with respect to untreated drug powder, resulting in a powerful strategy to control the drug release rate.

Monolithic nanoporous crystalline aerogels.

Monolithic aerogels can be easily obtained by drying physical gels formed by linear uncross-linked polymers. Preparation methods, structure, and properties of these physically cross-linked polymeric aerogels are reviewed, with particular emphasis to those whose cross-linking knots are crystallites and, more in particular, crystallites exhibiting nanoporous-crystalline forms. The latter aerogels present beside disordered amorphous micropores (typical of all aerogels) also all identical nanopores of the crystalline phases. Their outstanding guest transport properties combined with low material cost, robustness, durability, and ease of handling and recycle make these aerogels suitable for applications in chemical separations, purification, and storage as well as in biomedicine. Scientific, technological, and industrial perspectives for monolithic nanoporous-crystalline polymeric aerogels are also discussed.

Production of Antioxidant Transfersomes by a Supercritical CO2 Assisted Process for Transdermal Delivery Applications.

Transfersomes are deformable vesicles that can transport drugs across difficult-to-permeate barriers in human tissues. In this work, nano-transfersomes were produced for the first time by a supercritical CO2 assisted process. Operating at 100 bar and 40 °C, different amounts of phosphatidylcholine (2000 and 3000 mg), kinds of edge activators (Span® 80 and Tween® 80), and phosphatidylcholine to edge activator weight ratio (95:5, 90:10, 80:20) were tested. Formulations prepared using Span® 80 and phosphatidylcholine at an 80:20 weight ratio produced stable transfersomes (-30.4 ± 2.4 mV ζ-potential) that were characterized by a mean diameter of 138 ± 55 nm. A prolonged ascorbic acid release of up to 5 h was recorded when the largest amount of phosphatidylcholine (3000 mg) was used. Moreover, a 96% ascorbic acid encapsulation efficiency and a quasi-100% DPPH radical scavenging activity of transfersomes were measured after supercritical processing.

Production of Exopolysaccharide-Based Porous Structures for Biomedical Applications: A Review.

Exopolysaccharides, obtained from microorganisms as fermentation products, are interesting candidates for biomedical applications as scaffolds: they are biocompatible, nontoxic, antimicrobial, antitumor materials. To produce exopolysaccharide-based scaffolds, sol-gel technology could be used, which ends with the removal of the liquid phase from the polymeric network (i.e., the drying step). The aim of this review is to point out the most relevant strengths and weaknesses of the different drying techniques, focusing attention on the production of exopolysaccharide-based porous structures. Among these drying processes, supercritical carbon dioxide-assisted drying is the most promising strategy to obtain dried gels to use in the biomedical field: it produces highly porous and lightweight devices with outstanding surface areas and regular microstructure and nanostructure (i.e., aerogels). As a result of the analysis carried out in the present work, it emerged that supercritical technologies should be further explored and applied to the production of exopolysaccharide-based nanostructured scaffolds. Moving research towards this direction, exopolysaccharide utilization could be intensified and extended to the production of high added-value devices.

Current Applications of Liposomes for the Delivery of Vitamins: A Systematic Review.

Liposomes have been used for several decades for the encapsulation of drugs and bioactives in cosmetics and cosmeceuticals. On the other hand, the use of these phospholipid vesicles in food applications is more recent and is increasing significantly in the last ten years. Although in different stages of technological maturity-in the case of cosmetics, many products are on the market-processes to obtain liposomes suitable for the encapsulation and delivery of bioactives are highly expensive, especially those aiming at scaling up. Among the bioactives proposed for cosmetics and food applications, vitamins are the most frequently used. Despite the differences between the administration routes (oral for food and mainly dermal for cosmetics), some challenges are very similar (e.g., stability, bioactive load, average size, increase in drug bioaccessibility and bioavailability). In the present work, a systematic review of the technological advancements in the nanoencapsulation of vitamins using liposomes and related processes was performed; challenges and future perspectives were also discussed in order to underline the advantages of these drug-loaded biocompatible nanocarriers for cosmetics and food applications.

Validation of a compartmental model to predict drug release from porous structures produced by ScCO2 techniques.

A global release model is proposed to study the drug release from porous materials for pharmaceutical applications. This model is defined by implementing a compartmental model where the release profile could be explained as the combination of mass transfer phenomena through three compartments as well as a desorption process or dissolution process from the support. This model was validated with five different systems produced with supercritical CO2 (aerogels, membranes, and fibers), showing different release processes. Numerical results indicate that this compartmental approach can be useful to determine adsorption and desorption constants as well as mass transfer resistances within the material. Likewise, this model can predict lag phases and imbibition phenomena. Therefore, the development of compartmental models can be an alternative to traditional models to successfully predict the drug profile of porous materials, achieving a complete understanding of the involved phenomena regardless of the material characteristics.

Chemical composition and biological activities of the essential oils from three Melaleuca species grown in Tunisia.

The chemical composition of the essential oils of Melaleuca armillaris Sm., Melaleuca styphelioides Sm. and Melaleuca acuminata F. Muell., collected in Tunisia, was studied by means of GC and GC-MS analysis. In all, 46 compounds were identified, 38 for M. armillaris, 20 for M. acuminata and eight for M. styphelioides, respectively. The presence of a sesquiterpenic fraction (52.2%) characterized the oil from M. armillaris; M. sthypheliodes oil was rich in methyl eugenol, a phenolic compound (91.1%), while M. acuminata oil is mainly constituted by oxygenated monoterpenoids (95.6%). The essential oils were evaluated for their in vitro potentially phytotoxic activity against germination and initial radicle growth of Raphanus sativus L., Lepidium sativum L., Sinapis arvensis L., Triticum durum L. and Phalaris canariensis L. seeds. The radicle elongation of five seeds was inhibited at the highest doses tested, while germination of all seeds was not affected. Moreover, the essential oils showed low antimicrobial activity against eight selected microorganisms.

Salicylic Acid Co-Precipitation with Alginate via Supercritical Atomization for Cosmetic Applications.

Alginate-based microparticles were produced via supercritical assisted atomization (SAA) with the aim of obtaining a biocompatible and low-cost carrier for the delivery of active compounds in cosmetic applications. Salicylic acid was selected as an active model compound, and it was co-precipitated with alginate via SAA, operating at 82 bar and 80 °C. In particular, the drug-to-polymer weight ratio was fixed at 1/4, whereas polymer concentration was varied from 5 to 20 mg/mL in the starting aqueous solution. Operating in this way, alginate-salicylic acid microparticles were characterized by a mean diameter of 0.72 ± 0.25 µm, and the active compound became amorphous after processing. A salicylic acid encapsulation efficiency close to 100% was reached, and the drug release time from the biopolymeric microparticles was prolonged up to nine times with respect to untreated salicylic acid powder.

The Nanostructure of Polymer-Active Principle Microparticles Produced by Supercritical CO2 Assisted Processing.

Traditional and supercritical CO2 assisted processes are frequently used to produce microparticles formed by a biopolymer containing an active principle to improve the bioavailability of the active principle. However, information about the internal organization of these microparticles is still scarce. In this work, a suspension of dextran + Fe3O4 nanoparticles (model system) and a solution of polyvinylpyrrolidone (PVP) + curcumin were used to produce spherical microparticles by supercritical CO2 processing. Periodic dynamic light scattering measurements were used to analyze the evolution of the microparticles dissolution, size, and size distribution of the guest active principle in the polymeric matrix. It was found that curcumin was dispersed in the form of nanoparticles in the PVP microparticles, whose size largely depended on its relative concentration. These results were validated by transmission electron microscopy and scanning electron microscopy of the PVP microparticles and curcumin nanoparticles, before and after the dissolution tests.

Supercritical Phase Inversion to Produce Photocatalytic Active PVDF-coHFP_TiO2 Composites for the Degradation of Sudan Blue II Dye.

TiO2-loaded poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-coHFP) membranes were produced by supercritical CO2-assisted phase inversion. Three different TiO2 loadings were tested: 10, 20, and 30 wt% with respect to the polymer. Increasing the TiO2 amount from 10 wt% to 20 wt% in the starting solution, the transition from leafy-like to leafy-cellular morphology was observed in the section of the membrane. When 30 wt% TiO2 was used, the entire membrane section showed agglomerates of TiO2 nanoparticles. These polymeric membranes were tested to remove Sudan Blue II (SB) dye from aqueous solutions. The adsorption/photocatalytic processes revealed that membrane morphology and TiO2 cluster size were the parameters that mainly affected the dye removal efficiency. Moreover, after five cycles of exposure of these membranes to UV light, SB removal was higher than 85%.

Supercritical Antisolvent Technique for the Production of Breathable Naringin Powder.

Flavonoids are polyphenolic compounds largely present in fruits and vegetables possessing antioxidant properties, anti-inflammatory and antibacterial activities. Their use in clinical practice is very poor due to their low bioavailability, susceptibility to oxidation and degradation. Moreover, their slight solubility in biological fluids and a consequent low dissolution rate leads to an irregular absorption from solid dosage forms, even though, anti-inflammatory formulations could be used as support for several disease treatment, i.e. the COVID-19 syndrome. To improve flavonoid bioavailability particle size of the powder can be reduced to make it breathable and to promote the absorption in the lung tissues. Supercritical fluid based antisolvent technique has been used to produce naringin particles, with size, shape and density as well as free flowing properties able to fit inhalation needs. The dried particles are produced with the removal of the solvent at lower temperatures compared to the most used traditional micronization processes, such as spray drying. The best breathable fraction for naringin particles is obtained for particles with a d50~7 µm manufactured at 35 °C-150 bar and at 60 °C-130 bar, corresponding to 32.6% and 36.7% respectively. The powder is produced using a high CO2 molar fraction (0.99) that assure a better removal of the solvent. NuLi-1 cell line of immortalised bronchial epithelial cells adopted to evaluate powder cytotoxicity indicated after 24 h absence of toxicity at concentration of 25 µM.

Polysaccharide-Based Aerogel Production for Biomedical Applications: A Comparative Review.

A comparative analysis concerning bio-based gels production, to be used for tissue regeneration, has been performed in this review. These gels are generally applied as scaffolds in the biomedical field, thanks to their morphology, low cytotoxicity, and high biocompatibility. Focusing on the time interval 2015-2020, the production of 3D scaffolds of alginate, chitosan and agarose, for skin and bone regeneration, has mainly been investigated. Traditional techniques are critically reviewed to understand their limitations and how supercritical CO2-assisted processes could overcome these drawbacks. In particular, even if freeze-drying represents the most widespread drying technique used to produce polysaccharide-based cryogels, supercritical CO2-assisted drying effectively allows preservation of the nanoporous aerogel structure and removes the organic solvent used for gel preparation. These characteristics are essential for cell adhesion and proliferation.