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Purpose Solid lipid nanoparticles are largely used in biomedical research and are characterized by high stability and biocompatibility and are also able to improve the stability of various loaded molecules. In vitro studies demonstrated that these nanoparticles are low cytotoxic, while in vivo studies proved their efficiency as nanocarriers for molecules characterized by a low bioavailability. However, to our knowledge, no data on the systemic biodistribution and organ accumulation of solid lipid nanoparticles in itself are presently available. Methods In this view, we investigated the solid lipid nanoparticles biodistribution by a multimodal imaging approach correlating in vivo and ex vivo analyses. We loaded solid lipid nanoparticles with two different fluorophores (cardiogreen and rhodamine) to observe them with an optical imager in the whole organism and in the excised organs, and with fluorescence microscopy in tissue sections. Light and transmission electron microscopy analyses were also performed to evaluate possible structural modification or damage due to nanoparticle administration. Results Solid lipid nanoparticles loaded with the two fluorochromes showed good optic characteristics and stable polydispersity. After in vivo administration, they were clearly detectable in the organism. Four hours after the injection, the fluorescent signal occurred in anatomical districts corresponding to the liver and this was confirmed by the ex vivo acquisitions of excised organs. Brightfield, fluorescence and transmission electron microscopy confirmed solid lipid nanoparticles accumulation in hepatocytes without structural damage. Conclusion Our results support the systemic biocompatibility of solid lipid nanoparticles and demonstrate their detailed biodistribution from the whole organism to organs until the cells.

This investigation is a study of new lipid nanoparticles for cutaneous antioxidant delivery. Several molecules, such as α-tocopherol and retinoic acid, have been shown to improve skin condition and even counteract the effects of exogenous stress factors such as smoking on skin aging. This work describes the design and development of lipid nanoparticles containing antioxidant agents (α-tocopherol or retinoic acid) to protect human skin against pollutants. Namely, solid lipid nanoparticles and nanostructured lipid carriers were prepared using different lipids (tristearin, compritol, precirol or suppocire) in the presence or absence of caprylic/capric triglycerides. The formulations were characterized by particle size analysis, cryogenic transmission electron microscopy, small-angle X-ray diffraction, encapsulation efficiency, preliminary stability, in vitro cytotoxicity and protection against cigarette smoke. Nanostructured lipid carriers were found to reduce agglomerate formation and provided better dimensional stability, as compared to solid lipid nanoparticles, suggesting their suitability for antioxidant loading. Based on the preformulation study, tristearin-based nanostructured lipid carriers loaded with α-tocopherol were selected for ex vivo studies since they displayed superior physico-chemical properties as compared to the other nanostructured lipid carriers compositions. Human skin explants were treated with α-tocopherol-loaded nanostructured lipid carriers and then exposed to cigarette smoke, and the protein levels of the stress-induced enzyme heme oxygenase were analyzed in skin homogenates. Interestingly, it was found that pretreatment with the nanoformulation resulted in significantly reduced heme oxygenase upregulation as compared to control samples, suggesting a protective effect provided by the nanoparticles.

The main cause of low back pain is the intervertebral disc (IVD) degeneration. Designing an effective disc regeneration strategy still remains a major challenge, especially for the lack of effective self-healing capacity. Understanding the properties of IVD cells in the degenerate microenvironment could help to develop in situ regeneration strategies. The objective of the present study was to investigate the ability of degenerate cells to respond to conditions they experience physiologically in their niche in vivo, namely the presence of the hypoxic environment and trophic factors.Degenerate cells from IVD of patients operated for herniated disc were exposed to hypoxic priming and decellularized Wharton's jelly matrix (DWJM) as scaffold and trophic factors source for 48 h in culture. Cell response was evaluated in terms of cell viability, proliferation, cytoskeletal organization, migratory ability and expression of discogenic transcription factors (SOX9, TRPS1), hypoxia-inducible factor 1α (HIF-1α) and longevity transcription factor FOXO3a. The recruitment of HIF-1α at FOXO3a and SOX9 gene promoters was analyzed by Chromatin immunoprecipitation.Degenerate IVD cells were able to re-acquire the discogenic phenotype, and to re-adapt to hypoxia after exposure to hypoxic priming and DWJM. We demonstrated for the first time that HIF-1α is specifically recruited at the promoter of SOX9 and FOXO3a which are crucial for IVD homeostasis and repair.These results open new avenues to engineer IVD by demonstrating that appropriate stimuli are able to dampen the degenerated IVD cell phenotype and to promote anabolic activity in cells which are constitutively characterized by poor reparative capacity.

Infections acquired in public spaces (i.e., transports, restaurants, and bars, hospitals) present a serious burden for the entire health systems. In this respect, appropriate preventative and control measures in order to eliminate or reduce the negative effects of surface-transmitted infections appear highly desirable. Alongside recommendations for treatment and hygiene, antimicrobial material surfaces can offer indeed an important contribution to the prevention of infections. The aim of the current paper is therefore to describe the preparation and characterization of a new material obtained by an innovative anodic oxidation, defined as golden hard anodizing GHA. The anodic oxide surface thanks to the nanoporous structure acts as reservoir of silver ions (Ag+) which in turn confer antimicrobial properties to the material surface. Specifically, the manuscript presents a thorough preparation and characterization of a new material obtained by an innovative anodic oxidation treatment applied on commercially available aluminum alloys including the microscopic analysis and the description of the antimicrobial performances against a number of microorganisms, including among the others, Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. More specifically, the current article describes some of the properties of GHA materials. The tribological properties of GHA were evaluated through experimental tests performed with a pin-on-disk tribometer. The morphology of the wear surfaces was studied by means of a scanning electron microscope (SEM) analysis and profilometry investigations. Furthermore, in order to evaluate the possible anticorrosive properties of GHA, tests in neutral salt spray are in addition described.

Continuous-flow production of liposomes using microfluidic reactors has demonstrated advantages compared to batch methods, including greater control over liposome size and size distribution and reduced reliance on post-production processing steps. However, the use of microfluidic technology for the production of nanoscale vesicular systems (such as liposomes) has not been fully translated to industrial scale yet. This may be due to limitations of microfluidic-based reactors, such as low production rates, limited lifetimes, and high manufacturing costs. In this study, we investigated the potential of millimeter-scale flow reactors (or millireactors) with a serpentine-like architecture, as a scalable and cost-effective route to the production of nanoscale liposomes. The effects on liposome size of varying inlet flow rates, lipid type and concentration, storage conditions, and temperature were investigated. Liposome size (i.e., mean diameter) and size dispersity were characterised by dynamic light scattering (DLS), z-potential measurements and TEM imaging were also carried out on selected liposome batches. It was found that the lipid type and concentration, together with the inlet flow settings, had significant effects on the properties of the resultant liposome dispersion. Notably, the millifluidic reactor was able to generate liposomes with size and dispersity ranging from 54 to 272 nm, and from 0.04 to 0.52 respectively, at operating flow rates between 1 and 10 mL/min. Moreover, when compared to a batch ethanol-injection method, the millireactor generated liposomes with a more therapeutically relevant size and size dispersity.

The skin and mucous membranes are subjected to many disorders and pathological conditions. Nature offers a wide range of molecules with antioxidant activity able to neutralize, at least in part, the formation of free radicals and therefore to counteract the phenomena of cellular aging. Since synthetic drugs for the treatment of skin diseases can induce resistance, it is particularly interesting to use compounds of plant origin, transporting them in pharmaceutical forms capable of controlling their release and absorption. This review provides an overview of new findings about the use of lipid-based nanosystems for the delivery of natural molecules useful on the topical treatment of skin disorders. Several natural molecules encapsulated in lipid nanosystems have been considered in the treatment of some skin pathologies or diseases. Particularly, the use of rosemary and eucalyptus essential oil, saffron derivatives, curcumin, eugenol, capsaicin, thymol and lycopene has been reported. The molecules have been alternatively encapsulated in viscous systems, such as the organogels, or in liquid systems, such as ethosomes, transferosomes, solid lipid nanoparticles and monoolein based dispersions thickened by inclusion in carbomer gels. The nanostructured forms have been in vitro and in vivo investigated for the treatment of skin disorders due to dehydration, inflammation, melanoma, wound healing, fungal infections or psoriasis. The data reported in the different studies have suggested that the cutaneous application of lipid nanosystems allows a deep interaction between lipid matrix and skin strata, promoting a prolonged release and efficacy of the loaded natural molecules. This review suggests that the application of natural molecules onto the skin by lipid-based nanosystems can provide numerous clinician benefits in dermatology and cosmetics.

International audience; Traditional two-dimensional (2D) cell culture models are limited in their ability to reproduce human structures and functions. On the contrary, three-dimensional (3D) microtissues have the potential to permit the development of new cell-based assays as advanced in vitro models to test new drugs. Here, we report the use of a dehydrated gelatin film to promote tumor cells aggregation and 3D microtissue formation. The simple and stable gelatin coating represents an alternative to conventional and expensive materials like type I collagen, hyaluronic acid, or matrigel. The gelatin coating is biocompatible with several culture formats including microfluidic chips, as well as standard micro-well plates. It also enables long-term 3D cell culture and in situ monitoring of live/dead assays.

International audience; The development of novel bonding techniques could enable new applications and uses of plastics in microfluidic cell culture, complementing the omnipresent polydimethylsiloxane (PDMS). In this respect, the present paper describes a reversible gelatin-based method (named GEL-D) for bonding microfluidic chip parts, constituted of different materials. The herein introduced method enables the bonding of the most of the commonly used materials in microfluidics such as PMMA, PDMS, glass and NOA. Notably, the fabricated chips resist to pressure up to 0.7 MPa, to organic solvent exposure and temperature up to 70 C. To show the versatility of the described method, microchips with different sizes, materials, and geometries were bonded, including microchannel down to 200mm (width x depth) and round microstructures. The bonded chips are suitable to microfluidic cell culture procedures, including formation of microtissues, cells viability analysis and confocal microscopy. Therefore, the room-temperature bonding method appear to be highly efficient for cell culture on plastic chips, where in situ analysis of the seeded cells is required after microchip de-bonding.

International audience; The fabrication of microfluidic chips remains a complex and expensive process requiring specific equipment and protocols, often if not always limited to the most privileged laboratories. As an alternative to the most sophisticated methods, the present paper describes the fabrication of microfluidic chips by an approach that uses coins as positive master for the rapid production of multigeometry chips. All steps of chip production were carried out using inexpensive approaches by low-cost chemicals and equipment. The chips were validated by different "classic" microfluidic tasks, such as hydrodynamic focusing, droplets generation, micromixing, and on-chip cell culture. The use of coins is not only an efficient method for rapid prototyping but also represents an inspiring possibility for the design of new microfluidic chips. Finally, coin-inspired chips could represent a laboratory experiment doable at a high school level.

Cell encapsulation is a technological procedure with the potential to treat a wide range of human diseases by replacing damaged or diseased cells. For this reason, in recent years, cell encapsulation and delivery for cellular therapy has been proposed for the treatment of different pathologies, including degenerative diseases, tumors, autoimmune diseases and trauma. Cellular therapy is generally based on two alternative concepts: the first one is based on the administration of free cells or aggregates, while in the second approach, cells are usually embedded or encapsulated in scaffolds. Generally, the first approach (defined as scaffold-free method) has the main inconvenience of likely evoking immune rejection of the implanted cells by the recipient. In addition, once implanted, the cells are not preserved by external exogenous stimuli, such as mechanical stimulation, and not surrounded by an appropriate environment promoting the regeneration of tissues and organs. On the contrary, the use of cell encapsulation methods allows immunoisolation of implanted cells, which increases their long-term in vivo survivability opening new avenues for targeted cell delivery. Moreover, encapsulation in specific scaffolds, typically made of polymers, provides the “appropriate” mechanical support for cell growth and tissue development.

Cutaneous administration represents a good strategy to treat skin diseases, avoiding side effects related to systemic administration. Apart from conventional therapy, based on the use of semi-solid formulation such as gel, ointments and creams, recently the use of specialized delivery systems based on lipid has been taken hold. This review provides an overview about the use of cubic phases, cubosomes and ethosomes, as lipid systems recently proposed to treat skin pathologies. In addition in the final part of the review cubic phases, cubosomes and ethosomes are compared to solid lipid nanoparticles and lecithin organogel with respect to their potential as delivery systems for cutaneous application. It has been reported that lipid nanosystems are able to dissolve and deliver active molecules in a controlled fashion, thereby improving their bioavailability and reducing side-effects. Particularly lipid matrixes are characterized by skin affinity and biocompatibility allowing their application on skin. Indeed, after cutaneous administration, the lipid matrix of cubic phases and cubosomes coalesces with the lipids of the stratum comeum and leads to the formation of a lipid depot from which the drug associated to the nanosystem can be released in the deeper skin strata in a controlled manner. Ethosomes are characterized by a malleable structure that promotes their interaction with skin, improving their potential as skin delivery systems with respect to liposomes. Also in the case of solid lipid nanoparticles it has been suggested a deep interaction between lipid matrix and skin strata that endorses sustained and prolonged drug release. Concerning lecithin organogel, the peculiar structure of this system, where lecithin exerts a penetration enhancer role, allows a deep interaction with skin strata, promoting the transdermal absorption of the encapsulated drugs.

An "off-the-shelf" microfluidic chip approach, utilizing lowcost, commercially available components, for liposome production, is presented. Microfluidic devices with different geometries have been conveniently designed and assembled, allowing the production of narrowly dispersed unilamellar and very reproducible liposomes. The presented results indicate that off-the-shelf microfluidic devices can hold great promises for the efficient preparation of different lipid based colloidal systems for biomedical applications.

This study describes the development and optimization of a method to encapsulate the potent and expensive cannabinoids drugs in nanostructured lipid carriers; namely, URB597, AM251 and rimonabant have been considered. NLC production by melt and ultrasonication protocol has been specifically designed to optimize nanoparticle recovery and drug encapsulation efficiency. Special care has been devoted to the modality of oil and water phase emulsification and the entire production has been studied and discussed. NLC recovery, morphology, dimensional distribution and encapsulation efficiency are presented.

RNA virus infections can lead to the onset of severe diseases such as fever with haemorrhage, multiorgan failure, and mortality. The emergence and reemergence of RNA viruses continue to pose a significant public health threat worldwide with particular attention to the increasing incidence of flaviviruses, among others Dengue, West Nile Virus, and Yellow Fever viruses. Development of new and potent antivirals is thus urgently needed. Ivermectin, an already known antihelminthic drug, has shown potent effectsin vitroonFlavivirushelicase, with EC50values in the subnanomolar range for Yellow Fever and submicromolar EC50for Dengue Fever, Japanese encephalitis, and tick-borne encephalitis viruses. However ivermectin is hampered in its application by pharmacokinetic problems (little solubility and high cytotoxicity). To overcome such problems we engineered different compositions of liposomes as ivermectin carriers characterizing and testing them on several cell lines for cytotoxicity. The engineered liposomes were less cytotoxic than ivermectin alone and they showed a significant increase of the antiviral activity in all the Dengue stains tested (1, 2, and S221). In the current study ivermectin is confirmed to be an effective potential antiviral and liposomes, as drug carriers, are shown to modulate the drug activity. All together the results represent a promising starting point for future improvement of ivermectin as antiviral and its delivery.

This study describes the design and characterization of a liposphere gel containing clotrimazole for the treatment of Candida albicans. Lipospheres were produced by the melt-dispersion technique, using a lipid phase constituted of stearic triglyceride in a mixture with caprylic/capric triglyceride or an alkyl lactate derivative. The latter component was added to improve the action of clotrimazole against candida. The liposphere morphology and dimensional distribution were evaluated by scanning electron microscopy. Clotrimazole release kinetics was investigated by an in vitro dialysis method. An anticandidal activity study was conducted on the lipospheres. To obtain formulations with suitable viscosity for vaginal application, the lipospheres were added to a xanthan gum gel. The rheological properties, spreadability, leakage, and adhesion of the liposphere gel were investigated. Clotrimazole encapsulation was always over 85% w/w. The anticandidal study demonstrated that the encapsulation of clotrimazole in lipospheres increased its activity against Candida albicans, especially in the presence of the alkyl lactate derivative in the liposphere matrix. A dialysis method demonstrated that clotrimazole was slowly released from the liposphere gel and that the alkyl lactate derivative further controlled clotrimazole release. Adhesion and leakage tests indicated a prolonged adhesion of the liposphere gel, suggesting its suitability for vaginal application.

Background Wound healing is a biological process that can get in a state of pathologic inflammation, requiring the use of specific medications able to promote proper tissue repair. Objective The study describes the production and characterization of nanoparticle based gel for wound healing treatment designed to deliver hyaluronic acid and retinyl palmitate onto the skin. Methods Tristearin solid lipid nanoparticles and nanostructured lipid carriers based on a tristearin and caprylic/capric triglyceride mixture were produced and characterized. Gel spreadability and viscosity were investigated. Drug diffusion and "in vitro" wound healing were assessed by Franz cell and scratch wound assay in keratinocytes. Results Cryogenic transmission electron microscopy evidenced flat discoid nanoparticles. Photon correlation spectroscopy analysis indicated homogeneous dimensional distribution and mean diameter 132±46 nm. X-ray evidenced a lamellar inner structure of lipid nanoparticles. Nanostructured lipid carriers, being based on a heterogeneous solid/ liquid lipid mixture, could better solubilize retinyl palmitate and control its stability. The hyaluronic acid directly added into nanoparticles' dispersion enabled to obtain a shear-thinning gel suitable for cutaneous administration. Retynil palmitate diffusion was slower from the nanoparticulate gel with respect to the plain nanoparticle dispersion. The "wound healing" effect of nanoparticulate gel containing retinyl palmitate and hyaluronic acid, analyzed in HaCaT cells, showed significant differences in wounded areas between treated and control cells during the first 24 h postwounding suggesting a synergic effect of retinyl palmitate and hyaluronic acid in "in vitro" wound healing. Conclusions This study suggests that a nanoparticle based hyaluronate gel containing retinyl palmitate can be efficiently used for wound healing.

Background: Porcine Sertoli cells (pSCs) have been employed for cell therapy in pre-clinical studies for several chronic/immune dis- eases as they deliver molecules associated with trophic and anti-inflam- matory effects. To be employed for human xenografts, pSCs products need to comply with safety and stability. To fulfill such requirements, we employed a microencapsulation technology to increase pre-trans- plant storage stability of specific pathogen-free pSCs (SPF-pSCs) and evaluated the in vivo long-term viability and safety of grafts. Methods: Specific pathogen free neonatal pigs underwent testis excision under sterility. pSCs were isolated, characterized by immunofluores- cence (IF) and cytofluorimetric analysis (CA) and examined in terms of viability and function (namely, production of anti-m€ hormone (AMH), inhibin B, and transforming growth factor beta-1 (TFGb-1)). After microencapsulation in barium alginate microcapsules (Ba-MC), long-term SPF-pSCs (Ba-MCpSCs) viability and barium concentrations were evaluated at 1, 24 throughout 40 h to establish pre-transplant stor- age conditions. Results: The purity of isolated pSCs was about 95% with negligible contaminating cells. Cultured pSCs monolayers, both prior to and after microencapsulation, maintained high function and full viability up to 24 h of storage. At 40 h post-encapsulation, pSCs viability decreased to 80%. Barium concentration in Ba-MCpSCs lagged below the normal maximum daily allowance and was stable for 4 months in mice with no evident side effects. Conclusions: Such results suggest that this protocol for the isolation and microencapsulation of pSCs is compatible with long-haul transpor- tation and that Ba-MCpSCs could be potentially employable for xeno- transplantation.

This study describes the preparation, characterization, and in vivo evaluation in rats of nanostructured lipid carriers (NLCs) encapsulating rimonabant (RMN) as prototypical cannabinoid antagonist. A study was conducted in order to optimize NLC production by melt and ultrasonication method. NLCs were prepared by alternatively adding the lipid phase into the aqueous one (direct protocol) or the aqueous phase into the lipid one (reverse protocol). RMN-NLCs have been characterized by cryogenic transmission electron microscopy (cryo-TEM), X-ray, photon correlation spectroscopy (PCS) and sedimentation field flow fractionation (SdFFF). Reverse NLCs were treated with polysorbate 80. RMN release kinetics have been determined in vitro by dialysis method. In vivo RMN biodistribution in rats was evaluated after intranasal (i.n.) administration of reverse RMN-NLC. The reverse protocol enabled to prevent the lost of lipid phase and to achieve higher RMN encapsulation efficacy (EE) with respect to the direct protocol (98% w/w versus 67% w/w). The use of different protocols did not affect NLC morphology and dimensional distribution. An in vitro dissolutive release rate of RMN was calculated. The in vivo data indicate that i.n. administration of RMN by reverse NLC treated with polysorbate 80 increased RMN concentration in the brain with respect to the drug in solution. The nanoencapsulation protocol presented here appears as an optimal strategy to improve the low solubility of cannabinoid compounds in an aqueous system suitable for in vivo administration.

This study describes the preparation, characterization, and biodistribution of radiolabelled nanostructured lipid carriers (NLCs) especially designed for in vivo tomographic study. A preliminary formulative study was conducted in order to incorporate (99m)Tc based tracer in NLCs. At this aim a (99m)Tc complex containing a terminal (99m)Tc ≡ N multiple bond ([(99m)Tc]N-DBODC2) has been synthesized and included in NLCs produced by a stirring and ultrasonication method. The morphological and dimensional characteristics of the produced NLCs have been accurately investigated by a number of specific techniques, including: cryogenic transmission electron microscopy, X-ray, photon correlation spectroscopy and sedimentation field flow fractionation. The obtained NLCs were employed for achieving in vivo tomographic images of the rat body by small-animal SPECT scanner that enabled the investigation of NLC biodistribution after intraperitoneal, intravenous, intranasal and oral administration. NLC production protocol allowed to firmly encapsulate the radiotracer within the nanoparticles. In vivo studies evidenced that NLC remained stable in vivo, suggesting their suitability as controlled release system for drugs and radiochemical for therapeutic and diagnostic purposes. Moreover the high resolution images obtained by the SPECT technique allowed to detect NLC presence in brown fat tissue, suggesting NLC therapeutic application for treating human obesity and related metabolic disorders.

Spermatogenesis is a highly complicated biological process that occurs in the epithelium of the seminiferous tubules. It is regulated by a complex network of endocrine and paracrine factors and by juxtacrine testicular cross-talk. Sertoli cells (SC) play a key role in spermatogenesis due to their production of trophic, differentiation and immune-modulating factors, but many of the molecular pathways of SC action remain controversial and unclear. Over the last two decades, research has focused on extracellular vesicles as an important mechanism of intercellular communication. The aim of this study was to investigate the presence of extracellular vesicles (EVs) in SC and the modulation of their content in SC after FSH and testosterone stimulation. Highly purified porcine pre-pubertal Sertoli cells were isolated according to previously established methods. After 48 h of culture with FSH or FSH + testosterone stimulation, we identified sertolian EVs containing specific mRNAs. Proteomic analysis of EVs content identified 29 proteins under non-stimulatory conditions, most of which were related to receptor binding activity. FSH stimulation induced increases in inhibin-alpha, inhibin-beta, plakoglobin, haptoglobin, D-3-phosphoglycerate dehydrogenase and sodium/potassium-transporting ATPase in sertolian EVs. Testosterone stimulation enhanced the abundance of inhibin-alpha, inhibin-beta, tissue-type plasminogen activator, epidermal growth factor-like protein 8, elongating factor 1-gamma and D-3-phosphoglycerate dehydrogenase. These results are likely to help determine the unknown molecular secretion of Sertoli cells.

Environmental pollution is one of the main factors responsible for reducing fertility in males. Lead is one of the major heavy metal contaminants that impairs several organs; it preferentially accumulates in male reproductive organs and alters sperm quality both in vivo and in vitro. However, the underlying mechanisms remain unclear. Sertoli cells (SCs) provide structural and physiological support to spermatogenic cells within seminiferous tubules. Therefore, changes in SCs affect the developing germ cells and alter spermatogenesis. This study aimed to assess whether exposure to subtoxic doses of adversely affects SC functioning in higher mammals. Purified and functional porcine neonatal SCs were exposed to lead acetate at three different concentrations. Lead exposure decreased the mRNA expression and protein levels of inhibin B and anti-Mullerian hormone (AMH) compared to control, indicating loss of FSH-r integrity in terms of 17-β-oestradiol production under FSH stimulation. In addition, we observed an increase in the mRNA levels of Akt and mTOR, and the phosphorylation of p38 and Akt in SCs exposed to lead at all concentrations compared to unexposed control SCs. In conclusion, lead is toxic to SCs, even at low concentrations, and is expected to alter spermatogenesis.

In recent years, the development of nano- and micro-particles has attracted considerable interest from researchers and enterprises, because of the potential utility of such particles as drug delivery vehicles. Amongst the different techniques employed for the production of nanoparticles, microfluidic-based methods have proven to be the most effective for controlling particle size and dispersity, and for achieving high encapsulation efficiency of bioactive compounds. In this study, we specifically focus on the production of liposomes, spherical vesicles formed by a lipid bilayer encapsulating an aqueous core. The formation of liposomes in microfluidic devices is often governed by diffusive mass transfer of chemical species at the liquid interface between a solvent (i.e., alcohol) and a non-solvent (i.e., water). In this work, we developed a new approach for the analysis of mixing processes within microfluidic devices. The method relies on the use of a pH indicator, and we demonstrate its utility by characterizing the transfer of ethanol and water within two different microfluidic architectures. Our approach represents an effective route to experimentally characterize diffusion and advection processes governing the formation of vesicular/micellar systems in microfluidics, and can also be employed to validate the results of numerical modelling.

This study describes the potential of solid lipid nanoparticles and nanostructured lipid carriers as nano-formulations to administer to the central nervous system poorly water soluble drugs. Different neuroactive drugs, i.e. dimethylfumarate, retinyl palmitate, progesterone and the endocannabinoid hydrolysis inhibitor URB597 have been studied. Lipid nanoparticles constituted of tristearin or tristearin in association with gliceryl monoolein were produced. The nanoencapsulation strategy allowed to obtain biocompatible and non-toxic vehicles, able to increase the solubility of the considered neuroactive drugs. To improve URB597 targeting to the brain, stealth nanoparticles were produced modifying the SLN surface with polysorbate 80. A behavioural study was conducted in rats to test the ability of SLN containing URB597 given by intranasal administration to alter behaviours relevant to psychiatric disorders. URB597 maintained its activity after nanoencapsulation, suggesting the possibility to propose this kind of vehicle as alternative to unphysiological mixtures usually employed for animal and clinical studies.

Tissue engineering approaches using biomaterials have gain important roles in the regeneration of cartilage. This paper describes the production by microfluidics of alginate based microfibers containing both extracellular matrix (ECM)-derived biomaterials and chondrocytes. As ECM components gelatin or decellularized urinary bladder matrix (UBM) were investigated. The effectiveness of the composite microfibers has been tested to modulate the behavior and re-differentiation of de-differentiated chondrocytes. The complete re-differentiation, at the single cell level, of the chondrocytes, without cell aggregate formation, was observed after 14 days of cell culture. Specific chondrogenic markers and high cellular secretory activity was observed in embedded cells. Notably, no sign of collagen type 10 deposition was determined. The obtained data suggest that de-differentiated chondrocytes regain a functional chondrocyte phenotype when embedded in appropriate 3D scaffold based on alginate plus gelatin or UBM. The proposed scaffolds are indeed valuable to form a cellular microenvironment mimicking the in vivo ECM, opening the way to their use in cartilage tissue engineering.

This investigation describes a scaling up study aimed at producing progesterone containing nanoparticles in a pilot scale. Particularly hot homogenization techniques based on ultrasound homogenization or high pressure homogenization have been employed to produce lipid nanoparticles constituted of tristearin or tristearin in association with caprylic-capric triglyceride. It was found that the high pressure homogenization method enabled to obtain nanoparticles without agglomerates and smaller mean diameters with respect to ultrasound homogenization method. X-ray characterization suggested a lamellar structural organization of both type of nanoparticles. Progesterone encapsulation efficiency was almost 100% in the case of high pressure homogenization method. Shelf life study indicated a double fold stability of progesterone when encapsulated in nanoparticles produced by the high pressure homogenization method. Dialysis and Franz cell methods were performed to mimic subcutaneous and skin administration. Nanoparticles constituted of tristearin in mixture with caprylic/capric triglyceride display a slower release of progesterone with respect to nanoparticles constituted of pure tristearin. Franz cell evidenced a higher progesterone skin uptake in the case of pure tristearin nanoparticles. A human in vivo study, based on tape stripping, was conducted to investigate the performance of nanoparticles as progesterone skin delivery systems. Tape stripping results indicated a decrease of progesterone concentration in stratum corneum within six hours, suggesting an interaction between nanoparticle material and skin lipids.

Progesterone containing nanoparticles constituted of tristearin or tristearin in association with caprylic-capric triglyceride were produced in a lab scale by ultrasound homogenization and in a pilot scale by high pressure homogenization. A study was conducted to select the pressure to be used in order to obtain homogenously sized nanoparticles. The Dialysis method was performed to mimic subcutaneous administration of lipid nanoparticles. Mathematical analyses of the results were conducted to understand and compare the drug release mechanisms. A human in vivo study, based on tape stripping, was conducted to investigate the performance of nanoparticles as progesterone skin delivery systems. Tape stripped stratum corneum was analyzed by light microscopy.

In recent years, advancements in the fields of microfluidic and lab-on-a-chip technologies have provided unique opportunities for the implementation of nanomaterial production processes owing to the miniaturisation of the fluidic environment. It has been demonstrated that microfluidic reactors offer a range of advantages compared to conventional batch reactors, including improved controllability and uniformity of nanomaterial characteristics. In addition, the fast mixing achieved within microchannels, and the predictability of the laminar flow conditions, can be leveraged to investigate the nanomaterial formation dynamics. In this article recent developments in the field of microfluidic production of nanomaterials for drug delivery applications are reviewed. The features that make microfluidic reactors a suitable technological platform are discussed in terms of controllability of nanomaterials production. An overview of the various strategies developed for the production of organic nanoparticles and colloidal assemblies is presented, focusing on those nanomaterials that could have an impact on nanomedicine field such as drug nanoparticles, polymeric micelles, liposomes, polymersomes, polyplexes and hybrid nanoparticles. The effect of microfluidic environment on nanomaterials formation dynamics, as well as the use of microdevices as tools for nanomaterial investigation is also discussed.

In the present study, a nasal powder of the antidiuretic peptide desmopressin (DDAVP) formulated as chimera agglomerates was studied to improve drug bioavailability and provide a flexible drug product. Firstly, DDAVP was spray-dried along with mannitol and lecithin to produce primary microparticles capable of instantaneous dissolution in water. The chimera agglomerates were spontaneously formed by mechanically vibrating the microparticles on two stacked sieves. Agglomerate formation and strength were favored by the presence of lecithin. Drug content and dissolution rate remained unmodified after agglomeration. However, owing to the agglomerate larger size, powder flowability was greatly improved in comparison with the original microparticles, allowing accurate powder dosing into the nasal delivery device. DDAVP in vitro permeation across excised rabbit nasal mucosa from the agglomerates was significantly higher than that obtained from a commercial liquid nasal spray. In rats, intranasal DDAVP agglomerates allowed for efficient administration with almost 80% of the loaded powder emitted from the device into the animal nose. The administration of DDAVP agglomerates induced a significant reduction in urine production. Moreover, the antidiuretic effect of agglomerates did not significantly differ from the one induced by an intravenous injection of DDAVP at a ten-fold lower dose. © 2012 Elsevier B.V.

The current paper reports the production of polymeric micelles (PMs), based on pluronic block-copolymers, as drug carriers, precisely controlling the cellular delivery of drugs with various physico-chemical characteristics. PMs were produced with a microfluidic platform to exploit further control on the size characteristic of the PMs. PMs were designed for the co-delivery of dexamethasone (Dex) and ascorbyl-palmitate (AP) to in vitro cultured human periodontal ligament mesenchymal stem cells (hPDLSCs) for the combined induction of osteogenic differentiation. Mixtures of block-copolymers and drugs in organic, water miscible solvent, were conveniently converted in PMs within microfluidic channel leveraging the fast mixing at the microscale. Our results demonstrated that the drugs can be efficiently co-encapsulated in PMs and that different production parameters can be adjusted in order to modulate the PM characteristics. The comparative analysis of PM produced by microfluidic and conventional procedures confirmed that the use of microfluidics platforms allowed the production of PMs in a robust manner with improved controllability, reproducibility, smaller size and polydispersity. Finally, the analysis of the effect of PMs, containing Dex and AP, on the osteogenic differentiation of hPDLSCs is reported. The data demonstrated the effectiveness and safety of PM treatment on hPDLSC. In conclusion, this report indicates that microfluidic approach represents an innovative and useful method for PM controlled preparation, warrant further evaluation as general methodology for the production of colloidal systems for the simultaneous drug delivery.

This paper provides an analysis of microfluidic techniques for the production of nanoscale lipid-based vesicular systems. In particular we focus on the key issues associated with the microfluidic production of liposomes. These include, but are not limited to, the role of lipid formulation, lipid concentration, residual amount of solvent, production method (including microchannel architecture), and drug loading in determining liposome characteristics. Furthermore, we propose microfluidic architectures for the mass production of liposomes with a view to potential industrial translation of this technology.

Dimethyl fumarate has been demonstrated useful in relapsing remitting multiple sclerosis treatment (Tecfidera®). Nevertheless, since Tecfidera® capsules induce flushing, gastro-intestinal events and other more serious drawbacks, in this investigation a nanoparticle based system to be administered by an alternative way is proposed. In particular this study describes the preparation and characterization of dimethyl fumarate-containing solid lipid nanoparticles (SLN). Namely SLN based on tristearin, tristearin SLN treated with polysorbate 80 and cationic SLN constituted of tristearin in mixture with dimethyldioctadecylammonium chloride were investigated. The effect of the presence of dimethyl fumarate, functionalization by polysorbate 80 and dimethyldioctadecylammonium chloride was studied on morphology and dimensional distribution of SLN, by photon correlation spectroscopy and cryogenic transmission electron microscopy. Dimethyl fumarate release from SLN, studied by Franz cell, evidenced a Fickian dissolutive type kinetic in the case of SLN treated by polysorbate 80. Moreover fluorescent SLN were produced and characterized in order to investigate their in vitro permeability and in vivo biodistribution in mice. An in vitro study of fluorescent SLN permeability performed through a model of mouse brain microvascular endothelial cells, indicated that cationic SLN displayed higher permeability values with respect to neutral SLN and SLN treated by polysorbate 80. Biodistribution of polysorbate 80 treated SLN was studied by fluorescent imaging after intraperitoneal or intranasal administration in mice. The in vivo images indicate that polysorbate 80 treated SLN were able to reach the brain, even if they prevalently accumulated in liver and spleen, especially by intraperitoneal route.

We describe a cost-effective and simple method to fabricate PDMS-based microfluidic devices by combining micromilling with replica moulding technology. It relies on the following steps: (i) microchannels are milled in a block of acrylic; (ii) low-cost epoxy adhesive resin is poured over the milled acrylic block and allowed to cure; (iii) the solidified resin layer is peeled off the acrylic block and used as a mould for transferring the microchannel architecture onto a PDMS layer; finally (iv) the PDMS layer is plasma bonded to a glass surface. With this method, microscale architectures can be fabricated without the need for advanced technological equipment or laborious and time-consuming intermediate procedures. In this manuscript, we describe and validate the microfabrication procedure, and we illustrate its applicability to emulsion and microbubble production. Electronic supplementary material The online version of this article (doi:10.1007/s10544-015-0027-x) contains supplementary material, which is available to authorized users.

This study describes the use of lecithin organogels (LO) as versatile vehicles for percutaneous administration of methyl nicotinate (MN), fenretinide (4HPR) and curcumin (CUR). LO are able to increase the solubility of poor water-soluble molecules probably due to the formation of a short-range hexagonal organization of the lecithin cylindrical reverse micelles. After six months from production, no phase separation and an almost absence of aggregates was observed. LO exhibited similar viscosities and rheological behaviour both in the absence and in the presence of drug. The in vitro drug diffusion from LO studied by Franz cell showed that higher viscous vehicles result in lower diffusion coefficient. The microscopic investigation of human skin displayed no significant alterations after treatment with LO and with PBS. In vivo topical activity on erythema after cutaneous application of LO showed that LO-MN induce a strong erythema, while LO-CUR inhibit skin erythema due to CUR anti-inflammatory activity. Tape-stripping experiments performed on skin after topical administration of LO showed a decrease in the amount of CUR in the stratum corneum after 1 h from the occlusion. Shelf life stability studies demonstrated the higher stability of LO-HPR as compared to the others drugs.

This article describes the preparation of starch particles, by spray drying, for possible application to a dry powder coating process. Dry powder coating consists of spraying a fine powder and a plasticizer on particles. The efficiency of the coating is linked to the powder morphological and dimensional characteristics. Different experimental parameters of the spray-drying process were analyzed, including type of solvent, starch concentration, rate of polymer feeding, pressure of the atomizing air, drying air flow, and temperature of drying air. An optimization and screening of the experimental parameters by a design of the experiment (DOE) approach have been done. Finally, the produced spray-dried starch particles were conveniently tested in a dry coating process, in comparison to the commercial initial starch. The obtained results, in terms of coating efficiency, demonstrated that the spray-dried particles led to a sharp increase of coating efficiency value.

The description of a microencapsulation procedure for Wharton's jelly mesenchymal stem cells (WJMSCs) is reported. The applied method is based on the generation of monodisperse droplets by a vibrational nozzle. An ionic alginate encapsulation procedure was utilized for the microbeads hardening. Different experimental parameters were analyzed, including frequency and amplitude of vibration, polymer pumping rate, and distance between the nozzle and the gelling bath. The produced barium-alginate microbeads were characterized by excellent morphological characteristics as well as a very narrow size distribution. The microencapsulation procedure did not alter the morphology and viability of the encapsulated WJMSCs. In addition, the current paper reports the functional properties in terms of secretive profiles of both free and encapsulated WJMSCs. The analyzed factors were members of the family of interleukins, chemokines, growth factors, and soluble forms of adhesion molecules. These experiments showed that despite encapsulation, most of the proteins analyzed were secreted both by the free and encapsulated cells, even if in a different extent. In conclusion, the described encapsulation procedure represents a promising strategy to utilize WJMSCs for possible in vivo applications in tissue engineering and biomedicine.