Microfluidic Flow Focusing Research Articles

Nanoencapsulation of Bacteriophages in Liposomes Prepared Using Microfluidic Hydrodynamic Flow Focusing.

Increasing antibiotic resistance in pathogenic microorganisms has led to renewed interest in bacteriophage therapy in both humans and animals. A “Trojan Horse” approach utilizing liposome encapsulated phages may facilitate access to phagocytic cells infected with intracellular pathogens residing therein, e.g., to treat infections caused by Mycobacterium tuberculosis, Listeria, Salmonella, and Staphylococcus sp. Additionally, liposome encapsulated phages may adhere to and diffuse within mucosa harboring resistant bacteria which are challenges in treating respiratory and gastrointestinal infections. Orally delivered phages tend to have short residence times in the gastrointestinal tract due to clinical symptoms such as diarrhea; this may be addressed through mucoadhesion of liposomes. In the present study we have evaluated the use of a microfluidic based technique for the encapsulation of bacteriophages in liposomes having mean sizes between 100 and 300 nm. Encapsulation of two model phages was undertaken, an Escherichia coli T3 podovirus (size ~65 nm) and a myovirus Staphylococcus aureus phage K (capsid head ~80 nm and phage tail length ~200 nm). The yield of encapsulated T3 phages was 109 PFU/ml and for phage K was much lower at 105 PFU/ml. The encapsulation yield for E. coli T3 phages was affected by aggregation of T3 phages. S. aureus phage K was found to interact with the liposome lipid bilayer resulting in large numbers of phages bound to the outside of the formed liposomes instead of being trapped inside them. We were able to inactivate the liposome bound S. aureus K phages whilst retaining the activity of the encapsulated phages in order to estimate the yield of microfluidic encapsulation of large tailed phages. Previous published studies on phage encapsulation in liposomes may have overestimated the yield of encapsulated tailed phages. This overestimation may affect the efficacy of phage dose delivered at the site of infection. Externally bound phages would be inactivated in the stomach acid resulting in low doses of phages delivered at the site of infection further downstream in the gastrointestinal tract.

Microfluidic-assisted bacteriophage encapsulation into liposomes

Microfluidics has recently emerged as a new method of manufacturing liposomes, which allows reproducible mixing in miliseconds on the nanoliter scale. Here we investigated the feasibility of a microfluidic flow focusing setup built from commercially available fittings to encapsulate phages into liposomes. Two types of Pseudomonas phages, PEV2 (Podovirus, ∼65 nm) and PEV40 (Myovirus, ∼220 nm), were used as model phages. A mixture of soy phosphatidylcholine and cholesterol at a ratio of 4:1 dissolved in absolute ethanol with a total solid content of 17.5 mg/mL was injected through the center inlet channel of a cross mixer. Phage suspensions were injected into the cross mixer from the two side channels intersecting with the center channel. The total flow rate (TFR) varied 160–320 µL/min and the organic/aqueous flow rate ratio (FRR) varied 1:3–2:3. The size of liposomes and the encapsulation efficiency both increased with increasing FRR and slightly decreased with increasing TFR. Due to the different size of the two studied phages, the size of liposomes encapsulating PEV2 were smaller (135–218 nm) than those encapsulating the Myovirus PEV40 (261–448 nm). Highest encapsulation efficiency of PEV2 (59%) and PEV40 (50%) was achieved at a TFR of 160 µL/ml and a FRR of 2:3. Generally, the encapsulation efficiency was slightly higher than that obtained from the conventional thin film hydration followed by extrusion method. In summary, the proposed microfluidic technique was capable of encapsulating phages of different size into liposomes with reasonable encapsulation efficiency and minimal titer reduction.

Monodispersed silk fibroin microdroplets for protein stabilization

Low stability of globular protein droplets in emulsion significantly limits their applications in drug encapsulation, long-term storage, and controlled drug release. Here, a microfluidic flow-focusing device was utilized to synthesize horseradish peroxidase (HRP)-loaded silk fibroin microdroplets. The two immiscible streams of microfluidic flow-focusing were regenerated by silk fibroin solution and a mixture of 95 wt. % sunflower oil and 5 wt. % span 80 as the dispersed and continuous phases, respectively. In this study, the water-in-oil silk fibroin microdroplets were homogeneously produced by leveraging the discrete and periodic breakup of microdroplets and regulating the flow rates. Moreover, the result showed that the stability of encapsulated HRP in microdroplets was 25% higher than that of HRP after 6 weeks incubation. Thus, the microfluidic flow-focusing is a promising technique to form monodisperse microdroplets and maximize the stability of protein droplets.

Geometrically-mediated snap-off of water-in-oil emulsion droplets in microfluidic flow focusing devices.

Microfluidic channel networks allow the control of flowing fluids within structures with length scales on the order of single or tens of micrometers (μm). This affords the opportunity to mix and separate fluids with fine precision and, in the case of immiscible multiphase flows, generate stable emulsions with well-controlled sizes and size distributions. It is generally well understood that emulsion droplet size can be regulated by carefully balancing capillary-associated parameters, such as relative fluid velocity, with the interfacial tension of the immiscible phases. Channel size and geometry, particularly that of the junction where fluids merge in microfluidic flow focusing (or "pinch flow") devices, has been shown to scale droplet size and bound the lower droplet size. Channel constrictions or "nozzles" are commonly employed to amplify the extensional flow at channel junctions, but their function has not been quantified and is, therefore, not well understood. This paper describes the use of geometry as a tunable parameter in microfluidic droplet generator design by focusing upon the effect of nozzle geometry (relative width, length and depth) upon droplet snap off behavior. Our results show that nozzle geometry can dramatically influence droplet size by shifting its snap-off position, an effect that can be anticipated by Raleigh-Plateau theory.

Real-time size modulation and synchronization of a microfluidic dropmaker with pulsed surface acoustic waves (SAW)

We show that a microfluidic flow focusing drop maker can be synchronized to a surface acoustic waves (SAW) triggered by an external electric signal. In this way droplet rate and volume can be controlled over a wide range of values in real time. Using SAW, the drop formation rate of a regularly operating water in oil drop maker without SAW can be increased by acoustically enforcing the drop pinch-off and thereby reducing the volume. Drop makers of square cross-sections (w = h = 30 µm, with width w and height h) that produce large drops of length l = 10 w can be triggered to produce drops as short as l ~ 2w, approaching the geometical limit l = w without changing the flow rates. Unlike devices that adjust drop size by changing the flow rates the acoustic dropmaker has very short transients allowing to adjust the size of every single drop. This allows us to produce custom made emulsions with a defined size distribution as demonstrated here not only for a monodisperse emulsion but also for binary emulsions with drops of alternating size. Moreover, we show that the robustness and monodispersity of our devices is enhanced compared to purely flow driven drop makers in the absence of acoustic synchronization.

A novel approach to producing uniform 3-D tumor spheroid constructs using ultrasound treatment

Producing three-dimensional (3-D) multicellular tumor spheroids (TSs) is valuable for characterizing anticancer drugs since they provide a more representative model of the 3-D in vivo tumor than conventional two-dimensional (2-D) monolayer culture. The interaction of tumor cells with the extracellular matrix (ECM) in a 3-D culture environment is more similar to a tumor in vivo than in a 2-D environment; cell-cell and cell-ECM interaction can influence cell behaviour, such as in response to drug treatment. In vitro tumor spheroid models have been developed using microfluidic systems to generate 3-D hydrogel beads containing components of alginate and ECM protein, such as collagen, with high uniformity and throughput. Cell-laden hydrogel droplets are formed using a flow focusing process wherein the hydrogel precursors should be a homogeneous mixture. During gelation of the droplets into beads, the alginate acts as a fast gelling component helping to maintain the spherical shape of beads and preventing coalescence as the temperature-sensitive collagen I component gels more slowly. To produce uniform hydrogel droplets using the microfluidic flow focusing system, the mixtures must be homogeneous. However, collagen's sensitivity to temperature can lead to formation of chunks of collagen gel inside of the mixture, causing the mixture to become non-uniform and risking chip clogging. In order to overcome this limitation, previous approaches have used a cooling system during bead encapsulation while tumor cells were also present in the mixture, but this procedure can contribute to a delay in cell proliferation. Here a novel yet simple method is developed to prepare homogeneous pre-bead-encapsulation-mixtures containing collagen type I through ultrasonication. This method allows the cultivation of homogenous TS cultures with high uniformity and compact structure, and not only maintains cell viability but also the proliferation of cells in alginate/collagen hydrogel bead cultures. Depending on the sonication parameters, time and temperature, collagen can form small sized fibrils to thick fibers. Here, the mixtures containing collagen are assessed for morphology of collagen fibers/fibrils, cell viability, and proliferation. Human source Michigan Cancer Foundation-7 (MCF-7) breast cancer cells are successfully incorporated into alginate/collagen mixtures, followed by sonication, and then bead production. After bead gelation, the encapsulated MCF-7 cells remained viable and proliferated to form uniform TSs when the beads contained alginate and collagen. Results indicate that ultrasound treatment (UST) provides a powerful technique to change the structure of collagen from fiber to fibril, and to disperse collagen fibers in the mixture homogeneously for an application to generate uniform hydrogel beads and spheroids while not inhibiting cell proliferation.

Combined flow-focus and self-assembly routes for the formation of lipid stabilized oil-shelled microbubbles

Lipid and polymer stabilized microbubbles are used in medicine as contrast agents for ultrasound imaging and are being developed for the delivery of water soluble drugs to diseased areas of the body. However, many new therapeutics exhibit poor water solubility or stability, which has led to the requirement for the development of effective hydrophobic drug delivery systems. This study presents a new method to produce microbubbles coated with an oil layer capable of encapsulating hydrophobic drugs and suitable for targeted, triggered drug release. This new method utilizes highly controllable flow-focusing microfluidics with lipid oil nanodroplets self-assembling and spreading at gas–aqueous interfaces. Oil layer inside microbubbles were produced with diameters of 2.4±0.3 μm (s.d., 1.6 μm) and at concentrations up to 106 bubbles per milliliter. The mechanism of oil layer inside microbubble assembly and stability were characterized using methods including contact angle measurements, quartz crystal microbalance with dissipation monitoring and fluorescence resonance energy transfer imaging.

Biocompatible Amphiphilic Hydrogel-Solid Dimer Particles as Colloidal Surfactants.

Emulsions of two immiscible liquids can slowly coalesce over time when stabilized by surfactant molecules. Pickering emulsions stabilized by colloidal particles can be much more stable. Here, we fabricate biocompatible amphiphilic dimer particles using a hydrogel, a strongly hydrophilic material, and achieve large contrast in the wetting properties of the two bulbs, resulting in enhanced stabilization of emulsions. We generate monodisperse single emulsions of alginate and shellac solution in oil using a flow-focusing microfluidics device. Shellac precipitates from water and forms a solid bulb at the periphery of the droplet when the emulsion is exposed to acid. Molecular interactions result in amphiphilic dimer particles that consist of two joined bulbs: one hydrogel bulb of alginate in water and the other hydrophobic bulb of shellac. Alginate in the hydrogel compartment can be cross-linked using calcium cations to obtain stable particles. Analogous to surfactant molecules at the interface, the resultant amphiphilic particles stand at the water/oil interface with the hydrogel bulb submerged in water and the hydrophobic bulb in oil and are thus able to stabilize both water-in-oil and oil-in-water emulsions, making these amphiphilic hydrogel-solid particles ideal colloidal surfactants for various applications.

In Vitro Sonothrombolysis Enhancement by Transiently Stable Microbubbles Produced by a Flow-Focusing Microfluidic Device.

Therapeutic approaches that enhance thrombolysis by combining recombinant tissue plasminogen activator (rtPA), ultrasound, and/or microbubbles (MBs) are known as sonothrombolysis techniques. To date, sonothrombolysis approaches have primarily utilized commercially available MB formulations (or derivatives thereof) with diameters in the range 1-4µm and circulation lifetimes between 5 and 15min. The present study evaluated the in vitro sonothrombolysis efficacy of large diameter MBs (d MB≥10µm) with much shorter lifetimes that were produced on demand and in close proximity to the blood clot using a flow-focusing microfluidic device. MBs with a N2 gas core and a non-crosslinked bovine serum albumin shell were produced with diameters between 10 and 20µm at rates between 50 and 950×103 per second. Use of these large MBs resulted in approximately 4.0-8.8 fold increases in thrombolysis rates compared to a clinical rtPA dose and approximately 2.1-4.2 fold increases in thrombolysis rates compared to sonothrombolysis techniques using conventional MBs. The results of this study indicate that the large diameter microbubbles with transient stability are capable of significantly enhanced in vitro sonothrombolysis rates when delivered directly to the clot immediately following production by a flow focusing microfluidic device placed essentially in situ adjacent to the clot.

Solvent Effects on the Porosity and Size of Porous PLGA Microspheres Using Gelatin and PBS as Porogens in a Microfluidic Flow-Focusing Device

Solvent Effects on the Porosity and Size of Porous PLGA Microspheres Using Gelatin and PBS as Porogens in a Microfluidic Flow-Focusing Device

PEGylated crosslinked hyaluronic acid nanoparticles designed through a microfluidic platform for nanomedicine.

A high versatile microfluidic platform is proposed to design, in a one-step strategy, PEGylated crosslinked hyaluronic acid nanoparticles (cHANPs) entrapping a magnetic resonance imaging contrast agent and a dye for multimodal imaging applications. Clinically relevant biomaterials were shaped in the form of spherical NPs through a microfluidic flow focusing approach. A comparison between post processing and simultaneous PEGylation is reported to evaluate the potentiality of the chemical decoration of the cHANPs in microfluidics. An accurate control of the NPs in terms of size, PEGylation and loading was obtained. Furthermore, in vitro cell viability is reported and their ability to boost the magnetic resonance imaging signal is also confirmed. The proposed microfluidic approach reveals its ability to overcome several limitations of the traditional processes and to become an easy-to-use platform for theranostic applications.

Dynamics of high viscosity contrast confluent microfluidic flows

The laminar nature of microfluidic flows is most elegantly demonstrated via the confluence of two fluids forming two stable parallel flows within a single channel meeting at a highly stable interface. However, maintenance of laminar conditions can become complicated when there is a large viscosity contrast between the neighbouring flows leading to unique instability patterns along their interface. Here, we study the dynamics of high viscosity contrast confluent flows – specifically a core flow made of highly viscous glycerol confined by sheath flows made of water within a microfluidic flow focusing system. Our experiments indicate the formation of tapered core structures along the middle of the channel. Increasing the sheath flow rate shortens the tapered core, and importantly induces local instability patterns along the interface of core-sheath flows. The dynamics of such tapered core structures is governed by the intensity of instability patterns and the length of the core, according to which the core structure can experience stable, disturbed, broken or oscillated regimes. We have studied the dynamics of tapered core structures under these regimes. In particular, we have analysed the amplitude and frequency of core displacements during the broken core and oscillating core regimes, which have not been investigated before.

Pulsation of electrified jet in capillary microfluidics

In this work, we investigate the pulsation of an electrically charged jet surrounded by an immiscible dielectric liquid in flow-focusing capillary microfluidics. We have characterized a low-frequency large-amplitude pulsation and a high-frequency small-amplitude pulsation, respectively. The former, due to the unbalanced charge and fluid transportation is responsible for generating droplets with a broad size distribution. The latter is intrinsic and produces droplets with a relatively narrow size distribution. Moreover, the average size of the final droplets can be tuned via the intrinsic pulsating frequency through changing the diameter of the emitted liquid jet. Our results provide degree of control over the emulsion droplets with submicron sizes generated in microfluidic-electrospray platform.

High-throughput continuous production of liposomes using hydrodynamic flow-focusing microfluidic devices

The microfluidic hydrodynamic flow-focusing is a simple technique for nanoscale liposome formation that provides several advantages compared to the traditional manufacturing techniques. This work aimed to perform a systematic study of the liposome formation using planar microfluidic devices with different channel aspect-ratios, as an alternative to enhance the throughput of liposome synthesis. In general, liposomes with a low polydispersity and a precise control of the size were successfully produced from alteration of the flow rate ratio and channel aspect-ratio. The higher aspect-ratio enabled the most rapid generation of liposomes with similar diameter and significant lower polydispersity index than the obtained by other batch technique. Besides, β-carotene was successfully incorporated into liposomes with efficiency of approximately 60% and the incorporation ability was not specific to a choice of microfluidic device aspect-ratio. The results suggest that the use of microfluidic devices could be employed for liposome production with a possible advantage to minimize the degree of parallelization of processes. These results demonstrate the potential technical feasibility of microfluidic processes for future industrial applications.

Purely elastic instabilities in a microfluidic flow focusing device

Complex fluids generate different types of flow instabilities with increasing flow rate under inertialess flow conditions in a flow focusing device.

Fundamental studies on throughput capacities of hydrodynamic flow-focusing microfluidics for producing monodisperse polymer nanoparticles

Microfluidics enables the manipulation of liquids at the picoliter (or less) scale and proves to be superior over conventional bulk methods for mixing and reaction. The ability of microfluidic systems to rapidly mix reagents to provide homogeneous reaction environments, to vary the reaction conditions continuously, and to even allow reagent addition during the progress of a reaction, makes it attractive for nanoparticle synthesis. However, the low production rate limits its practical applications. Different approaches have been developed to achieve higher yield but most of them rely on the design of complex devices. Herein, we investigated fundamentally the throughput capacities of hydrodynamic flow-focusing microfluidics for producing poly (lactide-co-glycolide)-b-polyethylene glycol (PLGA-PEG) nanoparticles with uniform size ranging from 50–150nm. The effects of different factors of microfluidic design, including channel width, channel depth, channel structure and flow rate ratios, on particle size, size distribution, and production throughput were studied and compared. In contrast to the widely used microfluidic device which has a production rate of 1.8mg/h, our simple approach is capable of increasing the production rate of nanoparticles by more than two orders of magnitude up to 288mg/h using a single simple device. This study demonstrated the potentials of using simple 2D microfluidic devices for large-scale production of polymeric nanoparticles that could eliminate the need for designing and fabricating complex microfluidic devices.

Three dimensional phase-field investigation of droplet formation in microfluidic flow focusing devices with experimental validation

In this paper, the droplet formation process at a low capillary number in a flow focusing micro-channel is studied by performing a three-dimensional phase field benchmark based on the Cahn–Hilliard Navier–Stokes equations and the finite element method. Dynamic moving contact line and wetting condition are considered, and generalized Navier boundary condition (GNBC) is utilized to demonstrate the dynamic motion of the interface on wall surface. It is found that the mobility parameter plays a very critical role in the squeezing and breakup process to control the shape and size of droplets. We define the characteristic mobility Mc to represent the correct relaxation time of the interface. We also demonstrate that the characteristic mobility is associated with the physical process and should be kept as a constant as the product of the mobility tuning parameter χ and the square of interfacial thickness ε2. This criterion is applied for different interfacial thicknesses to correctly capture the physical process of droplet formation. Moreover, the size of the droplet, the velocity of the droplet along the downstream, and the period of droplet formation are compared between the numerical and experimental results which agree with each other both qualitatively and quantitatively. The presented model and criterion can be used to predict the dynamic behavior and movement of multiphase flows.

Preparation of lidocaine-loaded porous Poly (lactic-co-glycolic acid) microparticles using microfluidic flow focusing and phosphate buffer solution porogen

Local anesthesia is widely used in the treatment of postoperative pain. Long time duration and constant release of the local anesthetic is important for pain control. In this study, a preparation method of porous Poly (lactic-co-glycolic acid) (PLGA) microparticles loaded with local anesthetic, lidocaine was studied. Microfluidic flow-focusing device was used to prepare monodispersed microparticles. A phosphate buffer solution (PBS) was used as porogen. The geometry of microparticles was analyzed, and the drug release profile was determined by UV spectroscopy. Test results showed that porous microparticles are beneficial for sustained release of local anesthetic in long time duration over 40 days.

Negative Pressure Induced Droplet Generation in a Microfluidic Flow-Focusing Device.

We introduce an effective method to actively induce droplet generation using negative pressure. Droplets can be generated on demand using a series of periodic negative pressure pulses. Fluidic network models were developed using the analogy to electric networks to relate the pressure conditions for different flow regimes. Experimental results show that the droplet volume is correlated to the pressure ratio with a power law of 1.3. Using a pulsed negative pressure at the outlet, we are able to produce droplets in demand and with a volume proportional to the pulse width.

A single microfluidic chip with dual surface properties for protein drug delivery

Principles of double emulsion generation were incorporated in a glass microfluidic chip fabricated with two different surface properties in order to produce protein loaded polymer microspheres. The microspheres were produced by integrating two microfluidic flow focusing systems and a multi-step droplet splitting and mixing system into one chip. The chip consists of a hydrophobic and a hydrophilic section with two different heights, 12μm and 45μm, respectively. As a result, the protein is homogenously distributed throughout the polymer microsphere matrix, not just in its center (which has been studied before). In our work, the inner phase was bovine serum albumin (BSA) in phosphate buffered saline, the disperse phase was poly (lactic acid) in chloroform and the continuous phase was an aqueous solution of poly(vinyl alcohol). After solvent removal, BSA loaded microspheres with an encapsulation efficiency of up to 96% were obtained. Our results show the feasibility of producing microspheres loaded with a hydrophilic drug in a microfluidic system that integrates different microfluidic units into one chip.