Dario Carugo

Microfluidic and lab-on-a-chip preparation routes for organic nanoparticles and vesicular systems for nanomedicine applications.

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.

Liposome production by microfluidics: potential and limiting factors

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.

Nanoparticle‐Loaded Protein–Polymer Nanodroplets for Improved Stability and Conversion Efficiency in Ultrasound Imaging and Drug Delivery

A new formulation of volatile nanodroplets stabilized by a protein and polymer coating and loaded with magnetic nanoparticles is developed. The droplets show enhanced stability and phase conversion efficiency upon ultrasound exposure compared with existing formulations. Magnetic targeting, encapsulation, and release of an anticancer drug are demonstrated in vitro with a 40% improvement in cytotoxicity compared with free drug.

Contrast agent-free sonoporation: The use of an ultrasonic standing wave microfluidic system for the delivery of pharmaceutical agents

Sonoporation is a useful biophysical mechanism for facilitating the transmembrane delivery of therapeutic agents from the extracellular to the intracellular milieu. Conventionally, sonoporation is carried out in the presence of ultrasound contrast agents, which are known to greatly enhance transient poration of biological cell membranes. However, in vivo contrast agents have been observed to induce capillary rupture and haemorrhage due to endothelial cell damage and to greatly increase the potential for cell lysis in vitro. Here, we demonstrate sonoporation of cardiac myoblasts in the absence of contrast agent (CA-free sonoporation) using a low-cost ultrasound-microfluidic device. Within this device an ultrasonic standing wave was generated, allowing control over the position of the cells and the strength of the acoustic radiation forces. Real-time single-cell analysis and retrospective post-sonication analysis of insonated cardiac myoblasts showed that CA-free sonoporation induced transmembrane transfer of fluorescent probes (CMFDA and FITC-dextran) and that different mechanisms potentially contribute to membrane poration in the presence of an ultrasonic wave. Additionally, to the best of our knowledge, we have shown for the first time that sonoporation induces increased cell cytotoxicity as a consequence of CA-free ultrasound-facilitated uptake of pharmaceutical agents (doxorubicin, luteolin, and apigenin). The US-microfluidic device designed here provides an in vitro alternative to expensive and controversial in vivo models used for early stage drug discovery, and drug delivery programs and toxicity measurements.

Continuous-flow production of polymeric micelles in microreactors: experimental and computational analysis

We report the development of a microfluidic-based process for the production of polymeric micelles (PMs) in continuous-flow microreactors where Pluronic® tri-block copolymer is used as model polymeric biomaterial relating to drug delivery applications. A flow focusing configuration is used enabling a controllable, and fast mixing process to assist the formation of polymeric micelles through nanoprecipitation which is triggered by a solvent exchange process when organic solutions of the polymer mixed with a non-solvent. We experientially investigate the effect of polymer concentration, flow rate ratio and microreactor dimension on the PMs size characteristics. The mixing process within the microfluidic reactors is further analyzed by computational modeling in order to understand the hydrodynamic process and its implication for the polymeric micelles formation process. The results obtained show that besides the effect of the flow rate ratio, the chemical environment in which the aggregation takes place plays an important role in determining the dimensional characteristics of the produced polymeric micelles. It is demonstrated that microfluidic reactors provide a useful platform for the continuous-flow production of polymeric micelles with improved controllability, reproducibility, and homogeneity of the size characteristics.

A microfluidic device for the characterisation of embolisation with polyvinyl alcohol beads through biomimetic bifurcations

A microfluidic based device has been developed for the characterisation of embolisation behaviour with polyvinyl alcohol (PVA) hydrogel beads within a microchannel network with bifurcations which mimic the blood vessel network. Both distal and proximal embolisations were achieved within the PMMA-made microdevice exhibiting comparable embolisation characteristics with those observed in vivo. Results showed that small beads allowed more distal embolisations with a reduced control of the spatial location of occlusion sites. In contrast, large beads generated effective proximal embolisations with an improved reproducibility of embolisation performance. Embolic bead hydrodynamics, partitioning at bifurcations, penetration through microchannels and embolisation locations across the channel network were characterised by quantifying the effects of embolic bead size, bead concentration, channel geometry and fluidic conditions. This development provided further insights into the physical principles governing embolisation performances within the constructed microdevices allowing the improvement of the predictability and controllability of the clinical process outcomes. Furthermore, it can potentially provide a useful platform for preclinical research as an alternative to animal models, with an ultimate goal to reduce the amount of animal testing.

Removal of Interproximal Dental Biofilms by High-velocity Water Microdrops

The influence of the impact of a high-velocity water microdrop on the detachment of Streptococcus mutans UA159 biofilms from the interproximal (IP) space of teeth in a training typodont was studied experimentally and computationally. Twelve-day-old S. mutans biofilms in the IP space were exposed to a prototype AirFloss delivering 115 µL water at a maximum exit velocity of 60 m/sec in a 30-msec burst. Using confocal microscopy and image analysis, we obtained quantitative measurements of the percentage removal of biofilms from different locations in the IP space. The 3D geometry of the typodont and the IP spaces was obtained by micro-computed tomography (µ-CT) imaging. We performed computational fluid dynamics (CFD) simulations to calculate the wall shear stress (τw) distribution caused by the drops on the tooth surface. A qualitative agreement and a quantitative relationship between experiments and simulations were achieved. The wall shear stress (τw) generated by the prototype AirFloss and its spatial distribution on the teeth surface played a key role in dictating the efficacy of biofilm removal in the IP space.

Halbach arrays consisting of cubic elements optimised for high field gradients in magnetic drug targeting applications

A key challenge in the development of magnetic drug targeting (MDT) as a clinically relevant technique is designing systems that can apply sufficient magnetic force to actuate magnetic drug carriers at useful tissue depths. In this study an optimisation routine was developed to generate designs of Halbach arrays consisting of multiple layers of high grade, cubic, permanent magnet elements, configured to deliver the maximum pull or push force at a position of interest between 5 and 50 mm from the array, resulting in arrays capable of delivering useful magnetic forces to depths past 20 mm. The optimisation routine utilises a numerical model of the magnetic field and force generated by an arbitrary configuration of magnetic elements. Simulated field and force profiles of optimised arrays were evaluated, also taking into account the forces required for assembling the array in practice. The resultant selection for the array, consisting of two layers, was then constructed and characterised to verify the simulations. Finally the array was utilised in a set of in vitro experiments to demonstrate its capacity to separate and retain microbubbles loaded with magnetic nanoparticles against a constant flow. The optimised designs are presented as light-weight, inexpensive options for applying high-gradient, external magnetic fields in MDT applications.

Life under flow: a novel microfluidic device for the assessment of anti-biofilm technologies

In the current study, we have developed and fabricated a novel lab-on-a-chip device for the investigation of biofilm responses, such as attachment kinetics and initial biofilm formation, to different hydrodynamic conditions. The microfluidic flow channels are designed using computational fluid dynamic simulations so as to have a pre-defined, homogeneous wall shear stress in the channels, ranging from 0.03 to 4.30 Pa, which are relevant to in-service conditions on a ship hull, as well as other man-made marine platforms. Temporal variations of biofilm formation in the microfluidic device were assessed using time-lapse microscopy, nucleic acid staining, and confocal laser scanning microscopy (CLSM). Differences in attachment kinetics were observed with increasing shear stress, i.e., with increasing shear stress there appeared to be a delay in bacterial attachment, i.e., at 55, 120, 150, and 155 min for 0.03, 0.60, 2.15, and 4.30 Pa, respectively. CLSM confirmed marked variations in colony architecture, i.e.,: (i) lower shear stresses resulted in biofilms with distinctive morphologies mainly characterised by mushroom-like structures, interstitial channels, and internal voids, and (ii) for the higher shear stresses compact clusters with large interspaces between them were formed. The key advantage of the developed microfluidic device is the combination of three architectural features in one device, i.e., an open-system design, channel replication, and multiple fully developed shear stresses.

Mithramycin encapsulated in polymeric micelles by microfluidic technology as novel therapeutic protocol for beta-thalassemia

This report shows that the DNA-binding drug, mithramycin, can be efficiently encapsulated in polymeric micelles (PM-MTH), based on Pluronic® block copolymers, by a new microfluidic approach. The effect of different production parameters has been investigated for their effect on PM-MTH characteristics. The compared analysis of PM-MTH produced by microfluidic and conventional bulk mixing procedures revealed that microfluidics provides a useful platform for the production of PM-MTH with improved controllability, reproducibility, smaller size, and polydispersity. Finally, an investigation of the effects of PM-MTH, produced by microfluidic and conventional bulk mixing procedures, on the erythroid differentiation of both human erythroleukemia and human erythroid precursor cells is reported. It is demonstrated that PM-MTH exhibited a slightly lower toxicity and more pronounced differentiative activity when compared to the free drug. In addition, PM-MTH were able to upregulate preferentially γ-globin messenger ribonucleic acid production and to increase fetal hemoglobin (HbF) accumulation, the percentage of HbF-containing cells, and their HbF content without stimulating α-globin gene expression, which is responsible for the clinical symptoms of β-thalassemia. These results represent an important first step toward a potential clinical application, since an increase in HbF could alleviate the symptoms underlying β-thalassemia and sickle cell anemia. In conclusion, this report suggests that PM-MTH produced by microfluidic approach warrants further evaluation as a potential therapeutic protocol for β-thalassemia.