Dry Dosage Forms of Add-Value Bioactive Phenolic Compounds by Supercritical CO2-Assisted Spray-Drying.

Every year, grapevine pruning produces huge amounts of residue, 90% of which are from vine shoots. These are a rich source of natural antioxidants, mostly phenolic compounds, which, when properly extracted, can give rise to added-value products. However, their lack of solubility in aqueous media and high susceptibility to thermal and oxidative degradation highly limit their bioavailability. Encapsulation in suitable carriers may have a positive impact on their bioavailability and bioactivity. Previous data on vine-shoot extraction have identified gallic acid (GA) and resveratrol (RSV) as the main phenolic compounds. In this work, model dry powder formulations (DPFs) of GA and RSV using hydroxypropyl cellulose (HPC) as carriers were developed using Supercritical CO2-Assisted Spray Drying (SASD). A 32 full factorial Design of Experiments investigated the solid and ethanol contents to ascertain process yield, particle size, span, and encapsulation efficiency. Amorphous powder yields above 60%, and encapsulation efficiencies up to 100% were achieved, representing excellent performances. SASD has proven to be an efficient encapsulation technique for these phenolic compounds, preserving their antioxidation potential after three months in storage with average EC50 values of 30.6 µg/mL for GA-DPFs and 149.4 µg/mL for RSV-DPF as assessed by the scavenging capacity of the DPPH radical.

Solid Dosage Forms of Biopharmaceuticals in Drug Delivery Systems Using Sustainable Strategies.

Drug delivery systems (DDS) often comprise biopharmaceuticals in aqueous form, making them susceptible to physical and chemical degradation, and therefore requiring low temperature storage in cold supply and distribution chains. Freeze-drying, spray-drying, and spray-freeze-drying are some of the techniques used to convert biopharmaceuticals-loaded DDS from aqueous to solid dosage forms. However, the risk exists that shear and heat stress during processing may provoke DDS damage and efficacy loss. Supercritical fluids (SCF), specifically, supercritical carbon dioxide (scCO2), is a sustainable alternative to common techniques. Due to its moderately critical and tunable properties and thermodynamic behavior, scCO2 has aroused scientific and industrial interest. Therefore, this article reviews scCO2-based techniques used over the year in the production of solid biopharmaceutical dosage forms. Looking particularly at the use of scCO2 in each of its potential roles-as a solvent, co-solvent, anti-solvent, or co-solute. It ends with a comparison between the compound's stability using supercritical CO2-assisted atomization/spray-drying and conventional drying.

One-step microfluidics production of enzyme-loaded liposomes for the treatment of inflammatory diseases.

The biopharmaceuticals market is constantly growing. Despite their advantages over the conventional drugs, biopharmaceuticals have short biological half-lifes, which can be increased using liposomes. However, the common bulk methods to produce biopharmaceuticals-loaded liposomes result in lost of encapsulation efficiency (E.E.), resulting in an expensive process. Herein, the encapsulation of a therapeutic enzyme in liposomes is proposed, using a glass-capillary microfluidic technique. Cu,Zn- Superoxide dismutase (SOD) is successfully encapsulated into liposomes (SOD@Liposomes). SOD@Liposomes with a mean size of 135 ± 41 nm, a polydispersity index of 0.13 ± 0.01, an E.E. of 59 ± 6 % and an enzyme activity of 82 ± 3 % are obtained. in vivo experiments show, through an ear edema model, that SOD@Liposomes administered by the intravenous route enable an edema inhibition of 65 % ± 8 %, over the 20 % ± 13 % of SOD in its free form. The histopathological analyses show a higher inflammatory cell accumulation on the ear treated with SOD in its free form, than treated with SOD@Liposomes. Overall, this work highlights the potential of microfluidics for the production of enzyme-loaded liposomes with high encapsulation efficiency, with the intrinsic advantages of the low time-consuming and easily upscaling microfluidic assembly method.

Rational design of multistage drug delivery vehicles for pulmonary RNA interference therapy.

Small interfering RNA (siRNA) therapy has significant potential for the treatment of myriad diseases, including cancer. While intravenous routes of delivery have been found to be effective for efficient targeting to the liver, achieving high accumulations selectively in other organs, including lung tissues, can be a challenge. We demonstrate the rational design and engineering of a layer-by-layer (LbL) nanoparticle-containing aerosol that is able to achieve efficient, multistage delivery of siRNA in vitro. For the purpose, LbL nanoparticles were, for the first time, encapsulated in composite porous micro scale particles using a supercritical CO2-assisted spray drying (SASD) apparatus using chitosan as an excipient. Such particles exhibited aerodynamic properties highly favorable for pulmonary administration, and effective silencing of mutant KRAS in lung cancer cells derived from tumors of a non-small cell lung cancer (NSCLC) autochthonous model. Furthermore, efficient alveolar accumulation following inhalation in healthy mice was also observed, corroborating in vitro aerodynamic results, and opening new perspectives for further studies of effective lung therapies These results show that multistage aerosols assembled by supercritical CO2-assisted spray drying can enable efficient RNA interference therapy of pulmonary diseases including lung cancer.

All-in-one microfluidic assembly of insulin-loaded pH-responsive nano-in-microparticles for oral insulin delivery.

Here, a continuous two-step glass-capillary microfluidic technique to produce a multistage oral delivery system is reported. Insulin is successfully encapsulated into liposomes, which are coated with chitosan to improve their mucoadhesion. The encapsulation in an enteric polymer offers protection from the harsh gastric conditions. Insulin permeability is enhanced across an intestinal monolayer.

Cyclodextrin solubilization and complexation of antiretroviral drug lopinavir: In silico prediction; Effects of derivatization, molar ratio and preparation method.

Lopinavir (LPV) is currently used in combination with ritonavir for the clinical management of HIV infections due to its limited oral bioavailability. Herein, we report the application of an in silico method to study cyclodextrin (CyD) host-guest molecular interaction with LPV for the rational selection of the best CyD for developing a CyD based LPV delivery system. The predicted CyD, a (2-hydroxy)propyl-gamma derivative with high degree of substitution (HP17-γ-CyD) was synthesized and comparatively evaluated with γ-CyD and the commercially available HP-γ-CyD. All complexes were prepared by supercritical assisted spray drying (SASD) and co-evaporation (CoEva) at molar ratios (1:1 and 1:2); and afterwards fully characterized. Results indicate a higher LPV amorphization and solubilization ability of HP17-γ-CyD. The SASD processing technology also enhanced LPV solubilization and release from complexes. The application of in silico methodologies is a feasible approach for the rational and/or deductive development of CyD drug delivery systems.

Electrospun asymmetric membranes for wound dressing applications.

To accomplish a rapid wound healing it is necessary to develop an asymmetric membrane with interconnected pores consisting of a top layer that prevents rapid dehydration of the wound and bacteria penetration and a sub-layer with high absorption capacity and bactericidal properties. Polycaprolactone (PCL)/polyvinyl acetate (PVAc) asymmetric membranes loaded with the bactericidal monoterpene carvacrol (CRV) were synthesized and characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. Mechanical properties in dry and wet conditions and fluid handling behavior were also assessed. In addition, biological studies regarding their bactericidal effects, cytocompatibility and wound closure properties were also developed. Loading efficiencies of 40-50% were achieved in the prepared samples and 85-100% of the loaded CRV was released in simulated wound pH evolution medium. The significant inhibition of Gram negative (Escherichia coli S17) and Gram positive (Staphylococcus aureus ATCC 25923) bacteria growth clearly showed the suitability of the fabricated membranes for wound healing applications. Furthermore, cytocompatibility of the loaded membranes was demonstrated both in 2D and 3D human dermal fibroblast cultures, as well as cell migration was not impaired by released carvacrol from the membranes. These results highlight the potential of these polymeric electrospun membranes for wound healing.

POxylated Dendrimer-Based Nano-in-Micro Dry Powder Formulations for Inhalation Chemotherapy.

POxylated polyurea dendrimer (PUREG4OOx48)-based nanoparticles were loaded with paclitaxel (PTX) and doxorubicin (DOX) and micronized with chitosan (CHT) by using supercritical CO2-assisted spray drying (SASD). Respirable, biocompatible, and biodegradable dry powder formulations (DPFs) were produced to effectively transport and deliver the chemotherapeutics with a controlled rate to the deep lung. In vitro studies performed with the use of the lung adenocarcinoma cell line showed that DOX@PUREG4OOx48 nanoparticles were much more cytotoxic than the free drug. Additionally, the DPFs did not show higher cytotoxicity than the respective nanoparticles, and DOX-DPFs showed a higher chemotherapeutic effect than PTX formulations in adenocarcinoma cells.

Supercritical CO2-Assisted Spray Drying of Strawberry-Like Gold-Coated Magnetite Nanocomposites in Chitosan Powders for Inhalation.

Lung cancer is one of the leading causes of death worldwide. Therefore, it is of extreme importance to develop new systems that can deliver anticancer drugs into the site of action when initiating a treatment. Recently, the use of nanotechnology and particle engineering has enabled the development of new drug delivery platforms for pulmonary delivery. In this work, POXylated strawberry-like gold-coated magnetite nanocomposites and ibuprofen (IBP) were encapsulated into a chitosan matrix using Supercritical Assisted Spray Drying (SASD). The dry powder formulations showed adequate morphology and aerodynamic performances (fine particle fraction 48%-55% and aerodynamic diameter of 2.6-2.8 µm) for deep lung deposition through the pulmonary route. Moreover, the release kinetics of IBP was also investigated showing a faster release of the drug at pH 6.8, the pH of lung cancer. POXylated strawberry-like gold-coated magnetite nanocomposites proved to have suitable sizes for cellular internalization and their fluorescent capabilities enable their future use in in vitro cell based assays. As a proof-of-concept, the reported results show that these nano-in-micro formulations could be potential drug vehicles for pulmonary administration.

Ibuprofen loaded PVA/chitosan membranes: A highly efficient strategy towards an improved skin wound healing.

During wound healing, an early inflammation can cause an increase of the wound size and the healing process can be considerably belated if a disproportionate inflammatory response occurs. (S)-ibuprofen (IBP), a non-steroidal anti-inflammatory agent, has been used for muscle healing and to treat venous leg ulcers, but its effect in skin wound healing has not been thoroughly studied thus far. Herein, IBP-ß-cyclodextrins carriers were designed to customise the release profile of IBP from poly(vinyl alcohol)/chitosan (PVA/CS) dressings in order to promote a faster skin regeneration. The dressings were produced using supercritical carbon dioxide (scCO2)-assisted technique. In vitro IBP release studies showed that ß-cyclodextrins allowed a controlled drug release from the hydrogels which is crucial for their application in wound management. Moreover, the in vivo assays revealed that the presence of PVA/CS membranes containing IBP-ß-cyclodextrins carriers avoided scab formation and an excessive inflammation, enabling an earlier skin healing.

Aerosolizable gold nano-in-micro dry powder formulations for theragnosis and lung delivery.

Functionalized gold nanoparticles (AuNPs) have been widely investigated as promising multifunctional nanosystems for the theragnosis of lung cancer, the most common and prominent cause of cancer death worldwide. Nevertheless, nanoparticles are not in appropriate sizes for an accurate deep lung delivery and the lack of locally and effective delivery of therapeutic biomolecules to the deep lungs is, in fact, the major cause of low therapeutic outcome. Herein we incorporate, for the first time, AuNPs into respirable microparticles. AuNPs were functionalized with biocompatible oligo(2-oxazoline)-based optically stable fluorescent coatings, and conjugated with a laminin peptide (YIGSR) for targeted lung cancer delivery. These POxylated AuNPs were then incorporated into a chitosan matrix by a clean process, supercritical CO2-assisted spray drying (SASD), yielding nano-in-micro clean ultrafine dry powder formulations. The engineered formulations present the adequate morphology and flowability to reach the deep lung, with aerodynamic sizes ranging 3.2-3.8µm, and excellent fine particle fraction (FPF) (FPF of 47% for CHT-bearing targeted AuNPs). The optimal biodegradation and release profiles enabled a sustained and controlled release of the embedded nanoparticles, with enhanced cellular uptake, opening new prospects for future lung theragnosis.

Three-dimensional multilayered fibrous constructs for wound healing applications.

Electrospun materials are promising scaffolds due to their light-weight, high surface-area and low-cost fabrication, however, such scaffolds are commonly obtained as ultrathin two-dimensional non-woven meshes, lacking on topographical specificity and surface side-dependent properties. Herein, it is reported the production of three-dimensional fibrous materials with an asymmetrical inner structure and engineered surfaces. The manufactured constructs evidence fibrous-based microsized conical protrusions [length: (10 ± 3) × 10(2) µm; width: (3.8 ± 0.8) × 10(2) µm] at their top side, with a median peak density of 73 peaks per cm(2), while their bottom side resembles to a non-woven mesh commonly observed in the fabrication of two-dimensional electrospun materials. Regarding their thickness (3.7 ± 0.1 mm) and asymmetric fibrous inner architecture, such materials avoid external liquid absorption while promoting internal liquid uptake. Nevertheless, such constructs also observed the high porosity (89.9%) and surface area (1.44 m(2) g(-1)) characteristic of traditional electrospun mats. Spray layer-by-layer assembly is used to effectively coat the structurally complex materials, allowing to complementary tailor features such as water vapor transmission, swelling ratio and bioactive agent release. Tested as wound dressings, the novel constructs are capable of withstanding (11.0 ± 0.3) × 10(4) kg m(-2) even after 14 days of hydration, while actively promote wound healing (90 ± 0.5% of wound closure within 48 hours) although avoiding cell adhesion on the dressings for a painless removal.

Antimicrobial Contact-Active Oligo(2-oxazoline)s-Grafted Surfaces for Fast Water Disinfection at the Point-of-Use.

Water is one of the most valuable resources today and its purity is crucial to health and society well-being. The access to safe drinking water is decreasing in the world, which can have a huge socio-economic impact especially in developing countries, more prone to water-associated diseases. The goal of this work was to develop an innovative, fast, and cost-effective 3D material capable of decontaminating water. We have used an eco-friendly strategy, combining plasma surface activation and supercritical fluid technology to produce, for the first time, a 2-oxazoline-grafted 3D surface with broad-spectrum contact-active antimicrobial properties. Oligo(2-methyl-2-oxazoline) quaternized with N,N-dimethyldodecylamine and grafted to a chitosan (CHT) scaffold (CHT-OMetOx-DDA) efficiently and quickly (<3 min) killed >99.999% of Staphylococcus aureus and Escherichia coli cells upon direct contact and avoided bacterial adhesion to the materials surface, which is important for the prevention of biofilm formation. As a proof of concept, CHT-OMetOx-DDA scaffold was demonstrated to be suitable for water purification efficiently killing the microorganisms present in different water samples within minutes of contact and without leaching to the water. Additionally, we report for the first time a new method to clearly distinguish two mechanisms of action of bioactive surfaces: contact-active and releasing systems.

Supercritical carbon dioxide design strategies: from drug carriers to soft killers.

The integrated use of supercritical carbon dioxide (scCO(2)) and micro- and nanotechnologies has enabled new sustainable strategies for the manufacturing of new medications. 'Green' scCO(2)-based methodologies are well suited to improve either the synthesis or materials processing leading to the assembly of three-dimensional multifunctional constructs. By using scCO(2) either as C1 feedstock or as solvent, simple, economic, efficient and clean routes can be designed to synthesize materials with unique properties such as polyurea dendrimers and oxazoline-based polymers/oligomers. These new biocompatible, biodegradable and water-soluble polymeric materials can be engineered into multifunctional constructs with antimicrobial activity, targeting moieties, labelling units and/or efficiently loaded with therapeutics. This mini-review highlights the particular features exhibited by these materials resulting directly from the followed supercritical routes.

Antimicrobial activity of new green-functionalized oxazoline-based oligomers against clinical isolates.

BACKGROUND: The search for new antimicrobial compounds able to overcome the global issue of microbial resistance to antibiotics is a priority worldwide. Moreover, several commensal microorganisms have been increasingly associated to opportunistic microbial infections. Having previously disclosed the green synthesis and preliminary characterization of the oligomers [linear oligo(ethylenimine) hydrochloride and oligo(2-methyl-2-oxazoline) quaternized with N,N-dimethyldodecylamine] we herein report on the screening of these oligomers against a battery of 69 clinical isolates of Aerococcus spp., Candida spp., Staphylococcus spp. and Streptococcus spp. FINDINGS: The isolates' susceptibility to both oligomers was evaluated by determining their minimal inhibitory concentration (MIC) and the biocidal effectiveness of each compound was further confirmed through spectrophotometric measurements and fluorescence microscopy. The MIC values of the 69 isolates were highly variable, yet favourably comparable with those of other antimicrobial polymers. The viability assays resulted in 100% of microbial killing rate after only 5 min, highlighting the promising antimicrobial action of these oligomers. CONCLUSIONS: Though further studies are required, evidence suggests that a strong effort should be done in order to confirm these compounds as valid alternatives for several clinical applications. This is reinforced by their well described biocompatibility with human tissues and by their proposed mechanism of action which difficult the development of microbial resistance to these compounds.

POxylated Polyurea Dendrimers: Smart Core-Shell Vectors with IC50 Lowering Capacity.

The design and preparation of highly efficient drug delivery platforms using green methodologies is at the forefront of nanotherapeutics research. POxylated polyurea dendrimers are efficiently synthesized using a supercritical-assisted polymerization in carbon dioxide. These fluorescent, pH-responsive and water-soluble core-shell smart nanocarriers show low toxicity in terms of cell viability and absence of glutathione depletion, two of the major side effect limitations of current vectors. The materials are also found to act as good transfection agents, through a mechanism involving an endosomal pathway, being able to reduce 100-fold the IC50 of paclitaxel.

Biobased monoliths for adenovirus purification.

Adenoviruses are important platforms for vaccine development and vectors for gene therapy, increasing the demand for high titers of purified viral preparations. Monoliths are macroporous supports regarded as ideal for the purification of macromolecular complexes, including viral particles. Although common monoliths are based on synthetic polymers as methacrylates, we explored the potential of biopolymers processed by clean technologies to produce monoliths for adenovirus purification. Such an approach enables the development of disposable and biodegradable matrices for bioprocessing. A total of 20 monoliths were produced from different biopolymers (chitosan, agarose, and dextran), employing two distinct temperatures during the freezing process (-20 °C and -80 °C). The morphological and physical properties of the structures were thoroughly characterized. The monoliths presenting higher robustness and permeability rates were further analyzed for the nonspecific binding of Adenovirus serotype 5 (Ad5) preparations. The matrices presenting lower nonspecific Ad5 binding were further functionalized with quaternary amine anion-exchange ligand glycidyltrimethylammonium chloride hydrochloride by two distinct methods, and their performance toward Ad5 purification was assessed. The monolith composed of chitosan and poly(vinyl) alcohol (50:50) prepared at -80 °C allowed 100% recovery of Ad5 particles bound to the support. This is the first report of the successful purification of adenovirus using monoliths obtained from biopolymers processed by clean technologies.

Natural melanin: a potential pH-responsive drug release device.

This work proposes melanin as a new nanocarrier for pH-responsive drug release. Melanin is an abundant natural polymer that can be easily extracted from cuttlefish as nanoparticles with a suitable size range for drug delivery. However, despite its high potentiality, the application of this biopolymer in the pharmaceutical and biomedical fields is yet to be explored. Herein, melanin nanoparticles were impregnated with metronidazole, chosen as model antibiotic drug, using supercritical carbon dioxide. The drug release profile was investigated at acidic and physiologic pH, and the dominant mechanism was found to follow a non-Fickian transport. Drug release from melanin shows a strong pH dependency, which allied to its biocompatibility and lack of cytotoxicity envisages its potential application as nanocarrier in formulations for colon and intestine targeted drug delivery.

Structural evaluation of an alternative Protein A biomimetic ligand for antibody purification.

Affinity chromatography is one of the most common techniques employed at the industrial-scale for antibody purification. In particular, the purification of human immunoglobulin G (hIgG) has gained relevance with the immobilization of its natural binding counterpart-Staphylococcus aureus Protein A (SpA) or with the recent development of biomimetic affinity ligands, namely triazine-based ligands. These ligands have been developed in order to overcome economic and leaching issues associated to SpA. The most recent triazine-based ligand-TPN-BM, came up as an analogue of 2-(3-amino-phenol)-6-(4-amino-1-naphthol)-4-chloro-sym-triazine ligand also known as ligand 22/8 with improved physico-chemical properties and a greener synthetic route. This work intends to evaluate the potential of TPN-BM as an alternative affinity ligand towards antibody recognition and binding, namely IgG, at an atomic level, since it has already been tested, after immobilization onto chitosan-based monoliths and demonstrated interesting affinity behaviour for this purpose. Herein, combining automated molecular docking and molecular dynamics simulations it was predicted that TPN-BM has high propensity to bind IgG through the same binding site found in the crystallographic structure of SpA_IgG complex, as well as theoretically predicted for ligand 22/8_IgG complex. Furthermore, it was found that TPN-BM established preferential interactions with aromatic residues at the Fab domain (Trp 50, Tyr 53, Tyr 98 and Trp 100), while in the Fc domain the main interactions are based on hydrogen bonds with pH sensitive residues at operational regime for binding and elution like histidines (His 460, His 464, His 466). Moreover, the pH dependence of TPN-BM_IgG complex formation was more evident for the Fc domain, where at pH 3 the protonation state and consequently the charge alteration of histidine residues located at the IgG binding site induced ligand detachment which explains the optimal elution condition at this pH observed experimentally.

A green approach toward antibody purification: a sustainable biomimetic ligand for direct immobilization on (bio)polymeric supports.

This paper presents a sustainable strategy for improving the capture of antibodies by affinity chromatography. A novel biomimetic ligand (4-((4-chloro-6-(3-hydroxyphenoxy)-1,3,5-triazin-2-yl)oxy)naphthalen-1-ol) (TPN-BM) was synthesized using a greener and simple protocol to overcome solubility limitations associated with ligand 22/8, known as artificial protein A. Furthermore, its subsequent immobilization on chitosan-based monoliths induced by plasma surface activation allowed the design of a fast and efficient chromatographic platform for immunoglobulin G (IgG) purification. The TPN-BM functionalized monoliths exhibited high-binding capacity (160 ± 10 mg IgG per gram of support), and a selective capture of monoclonal antibodies directly from mammalian crude extracts in 85 ± 5% yield and 98% of purity. The synthesis of ligand TPN-BM and the routes followed for monoliths preparation and functionalization were inspired in the green chemistry principles allowing the reduction of processing time, solvents and purification steps involved, turning the integrated system attractive from an economical and chemical point of view.