Wharton's jelly-derived multifunctional hydrogels: New tools to promote intervertebral disc regeneration in vitro and ex vivo.

The degeneration of intervertebral disc (IVD) is a disease of the entire joint between two vertebrae in the spine caused by loss of extracellular matrix (ECM) integrity, to date with no cure. The various regenerative approaches proposed so far have led to very limited successes. An emerging opportunity arises from the use of decellularized ECM as a scaffolding material that, directly or in combination with other materials, has greatly facilitated the advancement of tissue engineering. Here we focused on the decellularized matrix obtained from human umbilical cord Wharton's jelly (DWJ) which retains several structural and bioactive molecules very similar to those of the IVD ECM. However, being a viscous gel, DWJ has limited ability to retain ordered structural features when considered as architecture scaffold. To overcome this limitation, we produced DWJ-based multifunctional hydrogels, in the form of 3D millicylinders containing different percentages of alginate, a seaweed-derived polysaccharide, and gelatin, denatured collagen, which may impart mechanical integrity to the biologically active DWJ. The developed protocol, based on a freezing step, leads to the consolidation of the entire polymeric dispersion mixture, followed by an ionic gelation step and a freeze-drying process. Finally, a porous, stable, easily storable, and suitable matrix for ex vivo experiments was obtained. The properties of the millicylinders (Wharton's jelly millicylinders [WJMs]) were then tested in culture of degenerated IVD cells isolated from disc tissues of patients undergoing surgical discectomy. We found that WJMs with the highest percentage of DWJ were effective in supporting cell migration, restoration of the IVD phenotype (increased expression of Collagen type 2, aggrecan, Sox9 and FOXO3a), anti-inflammatory action, and stem cell activity of resident progenitor/notochordal cells (increased number of CD24 positive cells). We are confident that the DWJ-based formulations proposed here can provide adequate stimuli to the cells present in the degenerated IVD to restart the anabolic machinery.

Decellularized extracellular matrix-based scaffold and hypoxic priming: A promising combination to improve the phenotype of degenerate intervertebral disc cells.

AIMS: 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. MAIN METHODS: 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. KEY FINDINGS: 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. SIGNIFICANCE: 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.

Gold hard anodized (GHA) materials with antimicrobial surface properties: mechanical, tribological, and microbiological characterization.

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.

DoE Analysis of Approaches for Hydrogel Microbeads' Preparation by Millifluidic Methods.

Hydrogel microbeads hold great promise for immune-protective cell transplants and in vitro studies. Millifluidic generation of hydrogel microbeads is a highly efficient and reproducible approach enabling a mass production. This paper illustrates the preparation and characterization of highly controlled and reproducible microbeads made by different types of hydrogel using millifluidic approaches. The optimization of the process was made by a design of experiments (DoE) approach. The microbeads' large-scale production can be potentially used for single cells or clusters encapsulation.

Continuous-Flow Production of Liposomes with a Millireactor under Varying Fluidic Conditions.

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.

A Correlative Imaging Study of in vivo and ex vivo Biodistribution of Solid Lipid Nanoparticles.

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.

Nanomedicines to Treat Skin Pathologies with Natural Molecules.

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.

Lipid nanostructures for antioxidant delivery: a comparative preformulation study.

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.

Coins in microfluidics: From mere scale objects to font of inspiration for microchannel circuits.

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.

Gelatin-Coated Microfluidic Channels for 3D Microtissue Formation: On-Chip Production and Characterization.

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.

Production and Characterization of Nanoparticle Based Hyaluronate Gel Containing Retinyl Palmitate for Wound Healing.

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.

Testosterone and FSH modulate Sertoli cell extracellular secretion: Proteomic analysis.

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.

Production and Characterization of a Clotrimazole Liposphere Gel for Candidiasis Treatment.

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.

Acute effects of lead on porcine neonatal Sertoli cells in vitro.

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-ß-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.

Data on scaling up and in vivo human study of progesterone lipid nanoparticles.

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.

Progesterone lipid nanoparticles: Scaling up and in vivo human study.

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.

Dedifferentiated Chondrocytes in Composite Microfibers As Tool for Cartilage Repair.

Tissue engineering (TE) 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 redifferentiation of dedifferentiated chondrocytes. The complete redifferentiation, 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 dedifferentiated 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 TE.

Lipid nanoparticles for administration of poorly water soluble neuroactive drugs.

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.

Nanoformulations for dimethyl fumarate: Physicochemical characterization and in vitro/in vivo behavior.

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.

Analysis of the Diffusion Process by pH Indicator in Microfluidic Chips for Liposome Production.

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.