Microfluidics Drug Discovery
Microfluidic automated fluidic workflows as valuable tools for drug discovery
- Increased throughput and reproducibility
- Minimal reagent consumption
- Fast reaction times
- Automated fluidic workflow
Main applications in microfluidics drug discovery
Target selection and validation (drug synthesis)
When developing a drug target, protein structural and affinity studies are needed for target selection and validation, and protein interactions within cells must be studied. Single-cell protein quantitation, protein analysis in nanoliter droplets, and high-sensitivity ligand-binding interactions can be performed using microfluidic devices.
Hit identification and optimization (drug screening)
The potential pool size of drug candidates is estimated to be of the order of 1063. Microfluidic drug screening devices, including high-throughput microfluidic multiplexed systems that contain thousands of micrometer chambers, microwell arrays, or microvalves perform high-throughput screening studies with higher sensitivity and shorter reaction times while decreasing reagents volumes and costs.
Preclinical studies (drug evaluation)
In recent years, alternatives to animal experimentation have become a research hotspot. Organs on chips and organoids are widely used as alternatives as they emulate the architectural and functional complexity of native organs. These systems also enable exploration of facets of human disease and development that are not accurately recapitulated by animal models.
Resources
Flow Control Performance and Enhanced Automation for Microfluidics Drug Discovery Applications
- Drug synthesis and droplet microfluidics: When it comes to droplet or particle generation, having control of the fluid delivery system is important. During droplet or particle production, the flow rate of each phase must remain constant and stable to allow the production of monodisperse droplets. As experts in fluid control for droplet microfluidics, Fluigent provides droplet generation packages and platforms for researchers. These seamlessly generate micrometer droplets for a wide range of applications. Custom systems are also available to integrate technological and industrial devices.
- Multiplexing for drug screening: More than twenty reagents can be tested simultaneously when performing multiplexing applications on microfluidic devices. Automated fluidic workflows are a prerequisite. Fluigent developed the Aria to automate multiple fluid deliveries. Reproducibility is improved through complete automation of the fluidic protocol. Fluigent is also able to develop a custom system to fit user specifications and requirements.
- Optimized shear stress and recirculation for organ on a chip studies and cell culture: When performing drug evaluation on 2D/3D cell culture and organs on chips, cell culture should be optimized through constant perfusion to enable nutrients/oxygen renewal and by inducing adequate passive stimulation through shear flow as the effect is substantial on cell properties. Sterility on the fluidic path is also a prerequisite. Fluigent develops fluid recirculating systems that perform unidirectional fluid recirculation ensuring improved and more stable flow rates compared to peristaltic pumps. The system can run for 10 days. Custom systems are also available for technology integration into industrial devices.
Fluigent Solutions as an Alternative to Syringe Pumps for Better Performance and Automation Capabilities in Microfluidics Drug discovery
Flow rate stability and responsiveness is critical for the applications listed above. Syringe pumps are commonly used during this process. Depending on the model in use, syringe pumps show limited flow control, and the actual flow rate cannot be monitored. In addition, injection volumes are limited by the syringe, and automation can thus be difficult. An alternative to syringe pumps is pressure-based flow controllers. These show high-precision flow control, fast reaction time, and flow monitoring.
The benefits of choosing Fluigent for your flow control system:
- Best in class stability: < 0.5% thanks to our field-proven, patented FASTAB™ technology allowing optimal flow control with the robustness required in demanding industrial environments
- Workflow automation becomes straightforward with the Fluigent SDK and software included in the system
- Our engineering team members are experts in microfluidic design, mechanical and software integration, and biology applications
I. Droplet-based microfluidics
Microfluidic generation of droplets has attracted a lot of interest, enabling high throughput experiments by generating millions of micro-reactors and chambers in a few seconds. This method produces highly monodispersed droplets of very small volumes (μL to fL) of fluids with high frequency (up to hundreds of kHz), providing better control of processes like mixing, encapsulation, sorting, and sensing. Microfluidic-based droplets have many diverse and varied applications, such as particle synthesis3 and chemical analysis4. Highly controlled droplet production also enables single-cell analysis or drug testing 5,6.
Microfluidic-based droplet generation and control allow for:
- Highly monodispersed (<2% size variation) droplet production, with potentially high generation rate (up to hundreds of kHz)
- Highly reproducible complex structures (water-in-oil-in-water emulsions, multiple encapsulations…)
- Single droplet manipulation as an individual pL scale biochemical reactor
- Miniaturization of production and bioanalytical devices
With these characteristics, droplet microfluidics has a large value, including bio(chemical) analysis, and nano and microscale generation of materials7. Several chip designs exist to generate droplets. A common design is the T-junction, where the dispersed phase is injected from a channel that is perpendicular to the channel carrying the continuous phase (figure 1).
Droplet microfluidic concepts, components, and processes are now being adopted and leveraged by end-users to enable new science and innovation. Real-world success is now evidenced through a range of mainstream commercial products that are applied to key biological and healthcare-related problems (e.g., 10X Genomics, Drop-seq, and nucleic acid quantification via Droplet DigitalTM PCR systems)8.
Today, it is possible to produce multiple emulsions with complex droplet morphologies. Production of multi-cored droplets (droplets that contain a controlled number of inner droplets at one or more hierarchical levels), Janus droplets (i.e. biphasic or triphasic droplets with two or three physically and chemically distinct surface domains) is now possible using droplet microfluidics9 (figure 2).
Figure 2: Types of multiple emulsions from the simple encapsulation (a), double emulsion (b), and so on (c,d) to bi- and tri-phasic structures (e to h), multiple encapsulations (I to k), and hybrid microjet achievable using droplet-based microfluidics9
A microfluidic chip for the production of highly reproducible PLGA microparticles
In recent years, biodegradable microspheres/microparticles have gained widespread importance in the delivery of bioactive agents10. The copolymers of Poly (D, L-lactic-co-glycolic acid) (called PLGA)/poly (lactic acid) (called PLA) microparticles are one of the most successful new drug delivery systems (DDS) in labs and clinics. Because of their biocompatibility and biodegradability, they can be used in various areas, such as long-term release systems, vaccine adjuvant, and tissue engineering11.
In PLGA microparticle production for drug release and delivery, microparticle size is a key parameter as it is directly related to the microparticles degradation rate and the drug release rate12. Although PLGA microparticle synthesis appears to be a successful drug delivery system, the current processes and tools to produce PLGA microparticles have many limitations, such as wide microparticle size distribution, poor repeatability, and aggressive chemical preparation conditions11. To solve these problems, droplet-based microfluidics offers an efficient method for improvement.
Fluigent provides the Raydrop: a new breakthrough technology leading to outstanding particle size monodispersity and production flexibility. The Raydrop works as a co-flow focusing principle (figure 3). The nozzle and outlet capillary are aligned in a continuous phase chamber, the dispersed phase comes through the nozzle to create the microparticles into the continuous phase and exits by the outlet insert. Using this system, PLGA microparticles ranging from 15 to 50 µm diameters have been successfully generated.
The PLGA microparticle production station allows excellent reproducibility and significantly improved monodispersity (CV < 2%) as compared to other methods on the market. It allows one to continuously produce PLGA microparticles (up to 10 000 droplets/s) without unwanted interruption for long-term experiments.
Single-cell mRNA-seq sequencing using droplets: Drop-seq technology
The production of highly monodispersed emulsions or more complex structures (water-in-oil-in-water emulsions, multiple encapsulations…) makes microfluidics an excellent tool for single-cell analysis or single-cell culture. The technique allows for droplet-based single-cell RNA-sequencing, such that one can characterize complex tissues with many cell types and states under diverse conditions. One of the pioneering methods is Drop-Seq technology, which entraps a single cell and a single primer-barcoded bead in each droplet (figure 4).
Cells are thus separated into nanoliter-sized aqueous droplets, with a different barcode associated with each cell’s mRNAs. The primers on beads contain a barcode consisting of three sequences. One sequence is for PCR amplification and is common to all the beads. The second sequence consists of hundreds of individual primers that also share the same ‘‘cell barcode’’. Finally, the third part has different unique molecular identifiers (UMI), enabling mRNA transcripts to be digitally counted13 (figure 5). The droplets are sequenced altogether, allowing quick profiling of thousands of individual cells from a heterogeneous population. The power of this technology resides in the fact that during sequencing, one can distinguish where the original information came on a cell to cell basis. This allows one to make a gene expression map of the cell, or even to distinguish cell populations within a tissue.
II. Microfluidic cell culture
Microfluidic cell culture has significant advantages over macroscopic culture in flasks, dishes, and well-plates14 (figure 6). The microfluidic chip fabrication process allows great flexibility in the design of microfluidic devices, permitting one to understand and control interactions between cells, substrates, and the surrounding medium, physically as well as biochemically15.
This technology offers new possibilities to accurately reproduce the cellular environment and enables the analysis of biological processes that were not accessible before. Morphology-wise, chips can be structured at the cell scale to reproduce the mechanical constraints experienced by cells. Biochemically, stable gradients can be implemented with a high spatial resolution (typically, micrometer resolution).
Finally, constant perfusion enables the continuous renewal of nutrients and oxygen to promote cell growth and maintain optimal activity during long-term cell culture. Cost reduction due to volume reduction is also a major benefit15.
Development and characterization of a microfluidic model of the tumor microenvironment
The physical microenvironment of tumors is characterized by heterotypic cell interactions and physiological gradients of nutrients, waste products, and oxygen. This tumor microenvironment has a major impact on the biology of cancer cells and their response to chemotherapeutic agents. Despite this, most in vitro cancer research still relies primarily on cells grown in 2D and in isolation in nutrient and oxygen-rich conditions. Ayuso et al. presented an easy to use microfluidic device that can mimic the three-dimensional architecture of multicellular spheroids, while at the same time generating a visible, live “tumor slice” that allows easy monitoring of cells in different regions of the microenvironment in real-time as well as their response to different drugs17 (figure 7).
In this study, tumor cell behavior in different regions of the microdevice was studied and analyzed in conjugation with measurements of hypoxia and glucose concentrations across the device. The differential cellular response to several well-known drugs in different parts of the microdevice emphasizes the potential of this technology for analyzing the impact of microenvironmental parameters on drug response.
Figure 7: Microdevice operation of the tumor microenvironment. (A) 3 microdevices in a Petri dish containing a central culture chamber (detailed in C) and 6 channels. (B) One microdevice is filled with (yellowish) collagen hydrogel flowing to the microchamber from the right middle channel and blue-colored water perfused through the two lateral microchannels. (C) Culture medium perfused through the lateral microchannels provides nutrients and oxygen creating physiological gradients across the device. Cells near the ‘surrogate’ blood vessels are viable, whereas oxygen-poor cells in the center of device start to die creating a ‘necrotic core’ similar to the necrotic regions of tumors. (D) Cellular monitoring with fluorescent dye in the microdevice17.
III. Organ on a chip
Many efforts are devoted to the development of cancer metastasis models that can help in understanding the disease and the development of innovative therapeutic strategies. Current in vitro and in vivo cancer models are incapable of satisfactorily predicting the outcome of various clinical treatments on patients18. Therefore, new methods and approaches for drug discovery and health research are being developed. The concept of mimicking the organ-level function of human physiology or disease using cells inside a microfluidic chip was first published in 2004. In 2010 that the term organ-on-a-chip (OOAC) was invented by Ingber, et. al., who developed a microfluidic chip to capture organ-level functions of the human lung19. Microfluidics enables one the unique ability to control the cellular microenvironment with high spatiotemporal precision and to present cells with mechanical and biochemical signals in a more physiologically relevant context19. The manipulation of the micro-liter volumes of liquids has made these models a platform where scaling, and dynamic crosstalk between cells can be achieved. Microfluidic chips can now use geometries and structures to permit the use of physiological length scales, concentration gradients, and the mechanical forces generated by fluid flow to mimic the in vivo microenvironment experienced by cells20. These biomimetic platforms overcome many drawbacks encountered with conventional tissue culture models. OOAC engineering has attracted enormous interest and attention from the pharmaceutical industry, regulatory agencies, and even national defense agencies. This is demonstrated by the increase of OOAC research papers and by the emergence of at least 28 organ-on-a-chip companies in less than seven years21.
A microfluidic chip to reconstitue organ-level lung functions
To demonstrate that it is possible to engineer a microsystem that replicates the complex physiological functionality of living organs, Huh et al. developed a multifunctional microdevice that reproduces key structural, functional, and mechanical properties of the human alveolar-capillary interface, which is the fundamental functional unit of the living lung19. The device consisted of compartmentalized PDMS microchannels to form an alveolar-capillary barrier on a thin, porous, flexible PDMS membrane coated with the extracellular matrix and human alveolar epithelial cells and human pulmonary microvascular endothelial cells is cultured on opposite sides of the membrane (figure 8). Air is subsequently introduced into the compartment to create an air-liquid interface to mimic the lining of the alveolar air space19
Using this organ on a chip platform, the authors demonstrated that breathing motions, simulated by the platform, might greatly accentuate the proinflammatory activities of silica nanoparticles and contribute substantially to the development of acute lung inflammation19. This behavior could not be determined using existing in vitro models.
IV. Particle/cell sorting
Isolating and sorting cells from complex, heterogeneous mixtures represents a critical task in many areas of biology, biotechnology, and medicine. Cell sorting is often used to enrich or purify cell samples into well-defined populations to enhance efficiency in research and development applications22. Cell detection is generally performed using optical methods such as FACS (Fluorescent Activated Cell Sorting), where cells pass through a laser beam and the light scattered is characteristic to the cells and their components. Cells are subsequently sorted according to the scattered emission. It is an automated and robust solution for cell sorting and was considered a gold standard for cell sorting. The immense contributions of current commercial cell sorting platforms using FACS are tempered by several significant and persistent limitations, including limited sample throughput and processing speeds that make the generation of clinical-scale samples very difficult.
As opposed to conventional instrumentation, microfluidic chips are affordable, easy to use, and smaller. These chips make use of a wide range of techniques to sort cells, with specific speeds and efficiencies. The dimensions of the chip also allow a wide range of cell sorters to exist, from large volume cell sorters to precise single-cell sorters. Moreover, microfluidic cell sorting can be combined with additional fluidic operations within a single chip for complete lab-on-a-chip applications, diagnostic and therapeutic purposes. Microfluidic cell sorting is a promising application both in academic and industrial labs. Cell sorting is based on determining a specific cell parameter that can differ from one cell type to another, such as cell size, shape, density, surface markers. In general, a heterogeneous cell solution is injected through a microfluidic chip.
The chip is designed such that cells with different properties experience a different amount of force (that could originate from inertia, channel geometry, external sources …) while similar cells undergo equal forces. The differentiated cells are subsequently pushed into different streamlines and exit the chip from different outlets. Microfluidic cell sorting can be categorized into three distinct groups: fluorescent label-based, bead-based, and label-free cell sorting22. Label-free cell sorting in microfluidic devices is perhaps the most studied of the three approaches as it encompasses active systems (i.e., systems that rely on the use of external fields cells for sorting), and passive systems that do not rely on fluorescent labels or beads. Instead, these methods rely on the inherent differences in cellular morphology between cell groups22.
Inertial microfluidics for continuous particle separation in spiral microchannels
Kuntaegowdanahalli et al. developed a simple inertial microfluidic device that achieves continuous multi-particle separation using the principle of Dean-coupled inertial migration in spiral microchannels.
The dominant inertial forces coupled with the Dean rotational force due to the curvilinear microchannel geometry, cause particles to occupy a single equilibrium position near the inner microchannel wall. The position at which particles equilibrate is dependent on the ratio of the inertial lift to Dean drag forces.
Using this concept, they demonstrated a spiral lab-on-a-chip for size-dependent focusing of particles at distinct equilibrium positions across the microchannel cross-section from a multi-particle mixture23.
A major benefit of this system is its high throughput (e.g., 1.5 mL/min) without sheath flow or sequential cell manipulation, which is useful for processing native biological fluids and flow cytometry.
V. Micromixers
Micromixers are important components of lab-on-chip devices for applications in drug delivery, sequencing, amplification, and biochemical reactions. Based on the actuation method, micromixers can be broadly classified as passive and active. In passive mixing, no external sources are used. Thus mixing typically relies on the microfluidic chip geometry and on fluid properties. Under laminar flow, which is the typical fluid regime in microfluidics, mixing mostly happens through diffusion. This property allows performing mixing using lamination: two or more liquids are flowing in parallel, allowing for diffusion to happen and thus permitting to precisely tune mixing between liquids. For improved and faster mixing, chaotic advection can be generated by modifying the microfluidic chip geometry, such as by altering the shape of the channel for splitting, folding, stretching, or breaking the flow of fluid.
Active mixing occurs by using an external perturbation. Many active mixing methods exist. Dielectrophoresis mixing makes use of an electric field to move particles toward or away from an electrode, creating a chaotic advection. Acoustic wave energy can also be used to mix fluids: strong acoustic waves can be produced, and interfere with each other as they propagate in the fluid, generating advection24. As the diffusion coefficient of a liquid depends on the temperature, mixing can also be enhanced by increasing the microfluidic chamber temperature25.
Submillisecond organic synthesis using a serpentine-shaped microfluidic chip
In chemical synthesis, it is important to explore the synthetic pathways of an intermediate. To fully observe these pathways, control over its lifetime and mixing time is required.
The reaction mixture had to be well-mixed within the lifetime of the reactive intermediate. An efficient means of prolonging the lifetime is to lower the reaction temperature (-78°C to -100 °C), above the melting point of many organic solvents. Using microfluidic devices, mixing can be extremely fast, with mixing times unattainable by batches26. However, mixing time is increased at low temperatures, as the solvent viscosity exponentially increases.
To circumvent this issue, the authors used a three-dimensional serpentine-shaped microfluidic chip, allowing improved mixing by chaotic advection. Using this microfluidic chip, submillisecond mixing was performed.
VI. Microvalves
In the past 10 years, efforts have been devoted to the development of microfluidic platforms capable of performing several assays using programmable fluidic operations within an array of microvalves. Similar to programmable logic circuits where multiple electronic computing routines are executed on a single microdevice, programmable microfluidic platforms have been implemented27, allowing one to perform fluidic operations such as mixing, sampling, washing, and reacting automatically within a single chip by modifying the sequence/order of fluidic operations using the software. The primary advantage of microvalves over their macroscale counterparts is the significantly reduced dead volume, which is important in many applications that require precise flow control at small flow rates28. They are useful in biological and chemical applications, such as quantitative metabolic biomarker and genetic analysis29,30, protein-based biomarker detection31, or small molecule chemical and environmental analysis32. These platforms usually consist of a 2D array of microvalves that permit flow regulation, on/off switching and sealing of liquids, gases, or vacuums33. Several microvalves have been developed using pneumatic, electrokinetic and electrochemical actuators. Among these mechanisms, pneumatic actuation is often recognized as the most reliable method due to the simplicity of fabrication, ease of use, scalability, reliability, and a high degree of accuracy. Pneumatically actuated microvalves utilize the deflection of an elastomer (typically PDMS) membrane to control fluid flow34.
A fully integrated multilayer microfluidic chemical analyze for automated sample processing, labelling, and analysis
Capillary zone electrophoresis (CZE), is a powerful tool for chemical analysis and is widely used for environmental monitoring, astrobiology, and biosensing32. CZE assays usually require complex and manual sample processing.
Commercial platforms for automated CZE have been implemented to address this concern, but are expensive, and large, thus hardly field deployable in challenging environments. Microfluidic systems allow miniaturization, automation, and reduction in sample volume requirements for chemical and biochemical sensing.
Using membrane microvalve technology, it is possible to automate metering, transporting, routing, and mixing operations. Kim et al. introduced a microfluidic platform consisting of pneumatically actuated “lifting gate” microvalves integrated with a glass CZE microchip, providing extremely low dead volumes between components.
All the procedures, including buffer filling, labeling, and dilution, can be automated. The microchip was used to analyze diverse compound classes, such as amino acids, and oxidized biomarker compounds, like aldehydes/ketones and carboxylic acids in less than 30 min.
VII. Wearable microfluidics: an emerging application
An emerging field is the use of microfluidic concepts for wearable device applications36. Here, microfluidics presents several key value propositions. The microstructures store or handle fluids and are the core of the sensing device. Using microchannels, precise liquid amounts can be manipulated, allowing for highly accurate and reliable devices and being useful for bodily fluids that are often secreted or extracted in limited quantities. Also, wearable microfluidic devices could also stock a specific drug for precise dispensing at controlled intervals. Innovations in flexible microfluidics and electronics have led to numerous applications. Typically, a wearable microfluidic device will collect a fluid, transfer it to a localized site where detection or measurement is performed. Blood and sweat are common analytes as they provide insights into physiological states such as temperature, pH, and hydration36. Wearable microfluidics finds applications in the pharmaceutical, food, sportswear, and cosmetic fields.
A wearable microfluidic device for the capture, storage and colorimetric sensing of sweat
As mentioned previously, sweat is an analyte of interest because of its rich content of important biomarkers. It is easy to collect compared to blood. In situ quantitative analysis of sweat is of great interest for monitoring physiologic health status (for example, hydration state) and for the diagnosis of disease37.
Existing systems for sweat collection and analysis are confined to laboratories, where standard analytical technologies can be performed. Though highly precise, the analysis is time-consuming and costly.
To address this issue, Kho et al. developed a soft wearable microfluidic system than can directly harvest sweat from pores on the surface of the skin37.
The device routes the sample to different channels and reservoirs for multiparametric sensing of markers of interest, with options for wireless interfaces to external devices for image capture and analysis.
The device can measure total sweat loss, pH, lactate, chloride, and glucose concentrations by colorimetric detection using wireless data transmission. As it is a simple, low-cost, and fast analysis point of care device, it could be used to accumulate data from individual users over time, and this could serve as an analytical approach for interpreting trends in marker concentrations, potentially providing warning signs when performing physical activity.
Conclusion
Since the introduction of microfluidics, the scope of applications has kept extending over the years. The first applications were focused on analytical chemistry, but today the field of life science and specifically point of care is in the core of microfluidics. We have presented applications where microfluidics show great advantages compared to conventional systems. The importance of these applications was illustrated by showing research papers related to these applications. Some important applications were introduced here. Microfluidics covers a wide range of applications such as microreactors, bioprinting, or fuel cells, and many more.
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The Lamprou Lab (Queen’s University Belfast)
The Lamprou lab, affiliated with Queen’s University Belfast, specializes in three main areas: nanoparticles for imaging and therapy, lab-on-a-chip technology, and therapeutic implants. Their interdisciplinary approach has driven innovation in healthcare since 2012, developing emerging technologies and novel drug delivery devices. The lab is led by Professor Dimitrios Lamprou, a leading expert in pharmaceutical technologies known for his significant contributions to 3D printing, microfluidics, and nanofibers, with over 150 peer-reviewed publications.
What are engineered nanoparticles?
Nanotechnology gained prominence after Richard P. Feynman’s 1959 famous lecture, „There’s plenty of room at the bottom“.1 However, the use of nanoparticles can be traced back to ancient times, as seen in the Romans’ incorporation of nanoparticles into glass manufacturing in the fourth century AD. The Lycurgus cup, an artifact from this period, notably displayed distinctive color changes under various lighting conditions due to the integration of nano-glass particles.2
In modern times, nanotechnology has developed into a comprehensive scientific discipline with diverse applications spanning multiple industries. From water purification and information technologies to drug development, environmental solutions, and the creation of robust yet lightweight materials, nanotechnology has emerged as a pivotal player.3,4
The fundamental units of nanotechnology are nanoparticles, defined as small particles ranging from 10 nm to 1000 nm in size. Engineered nanoparticles, specifically designed with dimensions under 100 nm, play a crucial role in manipulating materials at the molecular and atomic levels, demonstrating significant chemical, structural, electrical, biological and mechanical characteristics. They are classified into categories including ceramic, carbon-based, semiconductor, metal, lipid-based and polymeric nanoparticles.
What makes engineered nanoparticles promising in nanomedicine?
Engineered nanoparticles hold promise for various nanotechnology applications in medicine, including in-vivo and in-vitro diagnosis, drug delivery, and production of biocompatible materials. They are characterized by their high mass-to-surface area ratio, ability to adsorb and carry compounds, and quantum properties.5,6
These characteristics can benefit the drug delivery field, as its primary objective is to enhance the specificity of drug targeting, improve safety and biocompatibility, and reduce toxicity while maintaining therapeutic effects. Nanoparticles with dimensions less than 100 nm are considered excellent drug carriers due to their unique biological and physiological properties, allowing them to cross tissue and cell barriers effectively.7
Engineered nanoparticles also exhibit a higher likelihood of cellular uptake due to their larger surface area, enabling increased protein loading. The interaction of NPs with biological substances results in the formation of a „protein corona,“ enhancing nanocarriers’ uptake by the reticuloendothelial system. This protein corona can serve as a functional carrier for targeted drug delivery, improving poorly soluble drug uptake, directing drugs to specific locations, and enhancing drug bioavailability.8
Overall, engineered nanoparticle applications in nanomedicine have been progressing recently, particularly in controlled drug delivery, nucleic acid-based treatment, cancer cell targeting, angiogenesis inhibition, and inflammation control.
How are engineered nanoparticles traditionally produced
The success of nanoparticles as drug carriers and in nanomedicine applications depends on a number of crucial factors, including NP fabrication strategies, physical properties, drug loading efficiencies, drug release potential, and especially the carrier’s toxicity.
Lipid-based nanoparticles exhibit low toxicity in in-vivo experiments. They can carry both hydrophilic and hydrophobic molecules, leading to prolonged half-life and controlled drug release. Here, we focus on two main classes of lipid-based nanoparticles: liposomes and solid lipid nanoparticles.
Liposomes
A liposome is a microsphere lipid constructed from one or multiple phospholipid bilayers, closely mirroring the structure of cell membranes. The liposome preparation process consists of three main stages: preparing aqueous and lipid phases, primary processing with lipids, and optional secondary processing steps. Most methods involve dissolving phospholipids in an organic solvent, with subsequent removal of the solvent through evaporation—a critical step in liposome formation. Two straightforward methods of liposome synthesis are film hydration and solvent injection.
Liposomes generated through film hydration tend to be polydisperse. Parameters like the duration of rotary evaporation, mixing speed, and temperature after hydration affect liposome quality, emphasizing the need for careful monitoring.10,11
The major factors to consider for use of the solvent injection method are the temperature during injection and the injection rate. These factors will affect the size, shape, and polydispersity of the liposomes produced. The solvent injection method involves certain challenges, with continuous exposure of therapeutic substances to high temperatures and organic solvents affecting liposomal product stability and safety.
This results in high polydispersity and a non-homogeneous formulation.12
Solid Lipid Nanoparticles
Solid lipid nanoparticles (SLN) have emerged as a promising alternative to other lipid formulations like liposomes or polymeric nanoparticles. These spherical colloidal particles, ranging from 10 to 1000 nm in size, offer controlled drug release due to limited drug mobility in the solid lipid. SLNs provide advantages such as targeted drug delivery, controlled release, increased stability, scalability in production, and avoidance of organic solvents. Comprising a solid lipid core in an aqueous medium with a surfactant, SLNs use different lipids (steroids, fatty acids, triglycerides, etc.) and require stabilizing agents like surfactants or emulsifiers. The drug insertion process depends on drug hydrophobicity, solid lipid category, and polymeric alterations in the lipid. Production techniques include high-pressure homogenization, solvent evaporation, ultrasonication, hot homogenization, microemulsion, and others.13,14
Table: comparison of the different manufacturing methods used for polymeric NPs and lipid-based NPs
Nanoparticles Type | Manufacturing Method | Advantages | Disadvantages |
---|---|---|---|
Lipid formulation | Film hydration | – Established method – Understood method | – High consuming of the organic solvents – High PDI – Lack of reproducibility – Need for additional downsizing step – Difficulties in scaling-up |
Lipid formulation | Solvent injection | – Simple and fast – Scaling-up possibility | – Exposing to organic solvent – High PDI – Stability problems |
Lipid formulation | Extrusion | – Uniform and homogenous formulation | – Possible clogging of the membrane pores. – Difficulties in scaling-up |
Lipid formulation | High pressure homogenization | – Scaling-up possibility – Uniform formulation | – High energy consumption – Multiple steps – Bulky system |
Lipid formulation | Microemulsion | – Small particle size – Homogenous formulation | – Difficulty in removing the excess water – Use high concentration of surfactants |
Nanoparticles Type | Manufacturing Method | Advantages | Disadvantages |
---|---|---|---|
Polymeric | Emulsification-salting out | – Avoids surfactants and chlorinated solvents | – Need for purification steps – Encapsulate lipophilic drugs only |
Polymeric | Emulsification solvent diffusion | – Scaling-up possibility – Batch-to-batch reproducibility | – The possible diffusion of the hydrophilic drug into the aqueous phase – The need to eliminate high volume of aqueous phase from the colloidal dispersion |
Polymeric | Emulsification- evaporation | – Simple and versatile | – Risk of nanodroplets coalescence during the evaporation process – Time consuming |
Polymeric | Dialysis | – Effective and simple method – Produce polymeric nanoparticles with narrow distribution | – Time consuming – Use of high amount of dialyzing medium, which stimulate the premature release of NPs content |
Polymeric | Nonparticipation | – Simple and established method – Use low concentrations of surfactant | – Restricted for lipophilic drugs – Low polymer concentration obtained |
How does microfluidics enhance the properties of engineered nanoparticles
The principal innovation of microfluidics is the ability to transfer the traditional bulk technique to microscale fluidic chips. Solvents can be mixed within microchannels by a pumping system with continuous laminar flow. This type of flow offers high mixing quality and enhances the performance of microscale devices. The ability to adjust the flow rate ratio (FRR) and total flow rate (TFR) allows for continuous production of monodisperse and homogenous engineered nanoparticles.
Fluigent’s pressure-driven controllers and flow sensors enable this high stability and fast response in fluid flow, which is highly challenging to achieve with traditional pumping methods like peristaltic or syringe pumps. Overall, this method offers high reproducibility and low batch-to-batch variations. In addition, the method’s versatility makes the encapsulation process faster while keeping encapsulation efficiency high.
Microfluidics offers a fast, simple, single-step technique for liposome manufacturing, but challenges for large-scale production currently hinder its wide-scale implementation due to high costs.15,16
Most microfluidics-based solid lipid nanoparticle manufacturing follows a similar procedure involving dissolving lipids and drugs in an organic solvent, which is then introduced into the microfluidic device alongside an aqueous phase with a surfactant. Control over lipid-to-drug concentration, flow rate, and velocity influences the final nanocarrier characteristics.
Solid lipid nanoparticles produced by microfluidic processes have smaller particle sizes, better homogeneity, and higher encapsulation efficiency compared to bulk methods. In addition, cytotoxic studies demonstrate potent anti-proliferative effects of microfluidic SLNs in cancer cell lines.
However, research on microfluidic-produced SLNs is limited, and challenges exist, particularly with regard to the material used for microfluidic chips, with PDMS-based chips being sensitive to organic solutions.17
Conclusion
In this paper highlight, we presented the promise and challenges of microfluidics technology for the design and formulation of nanomedicines. While traditional methods face limitations in producing small engineered nanoparticles with desirable characteristics, microfluidic systems offer a one-step, controllable process with improved outcomes.
Comparative studies have shown that nanocarriers produced by microfluidics exhibit superior properties. Future advancements in microfluidic systems, combined with complementary tools like process analytical technology and molecular imaging technologies, are expected to optimize NP production and expand their medical applications.
Microfluidics holds promise for shaping the future of engineered nanoparticle research and development.
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References
(1) Shirai, Y.; Osgood, A.J.; Zhao, Y.; Yao, Y.; Saudan, L.; Yang, H.; Yu-Hung, C.; Alemany, L.B.; Sasaki, T.; Morin, J.-F. Surface-rolling molecules. J. Am. Chem. Soc. 2006, 128, 4854–4864.
(2) Bayda, S.; Adeel, M.; Tuccinardi, T.; Cordani, M.; Rizzolio, F. The History of Nanoscience and Nanotechnology: From Chemical-Physical Applications to Nanomedicine. Molecules 2019, 25, 112. Schoenmaker L, et al. mRNA-lipid nanoparticle COVID-19 vaccines: structure and stability. Int Pharm. 2021;601: 120586 .
(3) Grobert, N.; Hutton, D. Nanoscience and nanotechnologies: Opportunities and uncertainties. Lond. R. Soc. R. Acad. Eng. Rep.2004, 46, 618.
(4) Thiruvengadam, M.; Rajakumar, G.; Chung, I.M. Nanotechnology: Current uses and future applications in the food industry.3 Biotech 2018, 8, 74.
(5) Duncan, R. The dawning era of polymer therapeutics. Nat. Rev. Drug Discov. 2003, 2, 347–360.
(6) De Jong, W.H.; Borm, P.J. Drug delivery and nanoparticles:applications and hazards. Int. J. Nanomed. 2008, 3, 133–149.
(7) LaVan, D.A.; McGuire, T.; Langer, R. Small-scale systems for in vivo drug delivery. Nat. Biotechnol. 2003, 21, 1184–1191.
(8) Nel, A.E.; Mädler, L.; Velegol, D.; Xia, T.; Hoek, E.M.; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. Understanding biophysicochemical interactions at the nano-bio interface. Nat. Mater. 2009, 8, 543–557.
(9) Mitchell, M.J., Billingsley, M.M., Haley, R.M. et al. Engineering precision nanoparticles for drug delivery. Nat Rev Drug Discov 20, 101–124 (2021).
(10) Jaradat, E.; Weaver, E.; Meziane, A.; Lamprou, D. A. Microfluidics Technology for the Design and Formulation of Nanomedicines. Nanomaterials 2021, 11 (12), 3440.
(11) Alam, S.; Mattern-Schain, S.; Best, M. Targeting and triggered release using lipid-based supramolecular assemblies as medicinal nanocarriers. In Comprehensive Supramolecular Chemistry II; Elsevier: Oxford, UK, 2017; pp. 329–364.
(12) Maherani, B.; Arab-Tehrany, E.; Mozafari, M.R.; Gaiani, C.; Linder, M. Liposomes: A review of manufacturing techniques and targeting strategies. Curr. Nanosci. 2011, 7, 436–452.
(13) Mehnert, W.; Mäder, K. Solid lipid nanoparticles: Production, characterization and applications. Adv. Drug Deliv. Rev. 2001, 47,165–196.
(14) Uner, M.; Yener, G. Importance of solid lipid nanoparticles (SLN) in various administration routes and future perspectives. Int. J.Nanomed. 2007, 2, 289–300.
(15) Weaver, E.; Uddin, S.; Cole, D.K.; Hooker, A.; Lamprou, D.A. The Present and Future Role of Microfluidics for Protein and Peptide-Based Therapeutics and Diagnostics. Appl. Sci. 2021, 11, 4109.
(16) Guimarães Sá Correia, M.; Briuglia, M.L.; Niosi, F.; Lamprou, D.A. Microfluidic manufacturing of phospholipid nanoparticles: Stability, encapsulation efficacy, and drug release. Int. J. Pharm. 2017, 516, 91–99.
(17) Arduino, I.; Liu, Z.; Rahikkala, A.; Figueiredo, P.; Correia, A.; Cutrignelli, A.; Denora, N.; Santos, H.A. Preparation of cetylpalmitate-based PEGylated solid lipid nanoparticles by microfluidic technique. Acta Biomater. 2021, 121, 566–578.
Features of the cell encapsulation platform for flow cytometry
➤ Fluigent Precision and Flexibility
Produce robust and highly monodisperse emulsions using Fluigent’s pressure-based flow controllers and the Raydrop while accurately controlling the size of your droplets and shell thickness.
➤ Start emulsions production right away
The system is a fully equipped, mounted and controlled tool to create double emulsions with little setup time.
➤ Complete and easy-to-use engineered system
Our organized system requires easy priming and cleaning processes for better performance. It includes dedicated optics for optimized droplet visualization at a high frequency.
➤ Innovative and much sought-after application for cell analysis
The system is an easy-to-use platform for creating single-cell encapsulations into double emulsion droplets compatible with high-throughput screening and FACS experiments.
The encapsulation platform for FACS allows:
- Elimination of the risk of cross-contamination.
- Fast and efficient mixing of the reagents that occurs inside droplets.
- Ability to work with cells of limited availability.
Description
The cell encapsulation platform includes an organized flow path with pressure controllers, filters, flowmeters, and valves to facilitate the start-up, shut-down, and cleaning of the system between runs. Dedicated optics are included to optimize the visualization of droplet production at high generation frequency.
What is FACS technology?
In the field of biology, FACS stands for Fluorescence-Activated Cell Sorting. It is a technology aimed at separating and isolating certain cells from a heterogeneous population based on their fluorescent properties. This fluorescent signal allows users to translate the production of certain molecules of interest or the expression of specific phenotypes. The process of this technique is to label the cells with fluorescent markers such as antibodies or dyes which have the role of binding to certain structures or molecules of the cell in a specific way. These labeled cells can then be passed through the flow cytometer, an instrument allowing for the detection and measurement of the fluorescence emitted by the cells. This fluorescence detection will allow the flow cytometer to sort the cells into different populations, allowing the researcher to collect and analyze these different groups of cells separately.
Using FACS enables the scientist to perform an efficient sorting with a limited number of doublets or multiple cells being sorted together and from there, the characterization of cellular heterogenicity and the rare cell subpopulation.
Through our microfluidic technology, the encapsulation platform for FACS allows for the generation of samples, which can be processed in flow cytometry.
How to use the platform for FACS experiments?
Microfluidics can be associated with FACS technology to improve results. Working with microliter volumes allows for a considerable reduction in cost since the volume of samples required is greatly reduced. For instance, it minimizes the need for large cell cultures.
In addition, the use of encapsulation methods in droplets allows users to confine the fluorescence signal, avoiding the use of chemicals, and to perform bioassays at the single cell scale. Single cell or bacteria growth in drops can also be considered.
As the encapsulation platform for droplet sorting produces double emulsions to encapsulate cells (from 5 µm to 40 µm), the limitations of the FACS are overcome. The first limitation is that the secreted molecules are generally detectable only if a binding protocol has been performed. In the opposite case, they remain dissolved in the surrounding medium. The second is that the secreted cells significantly impact each other. Combining droplet microfluidics and FACS technology then appears to be a major advance for research and allows users to obtain more reliable and reproducible data.
Our selection of webinars on double emulsion for FACS
Encapsulation of fluorescent bacteria in double emulsions for FACS
Learn how to use our double emulsion platform to encapsulate bacteria in W/O/W DE and discover the DE sorting efficiency using FACS.
Webinar summary:
- Introduction to FACS sorting: technology, advantages, and drawbacks
- Presentation of the platform: encapsulation of single bacteria in DE
- Microscopy characterization and FACS sorting
- Interactive Q&A session
Single cell encapsulations compatible with FACS sorting, API encapsulations in biocompatible polymers, and more.
Until now, performing high efficiency encapsulation processes continuously has been challenging, as existing technologies have limitations (low reproducibility, the need for multiple surface coatings, poor size distribution, etc). The Raydrop encapsulation platform from Secoya solves these issues by providing an easy-to-use system that produces highly monodisperse W/O/W or O/W/O single or double emulsion from 15 µm to 400 µm.
Webinar Summary:
- Theoretical presentation of the Raydrop’s encapsulation process
- Case study: encapsulation of cells, yeast, and bacteria in a small double emulsion for FACS sorting
- Case study encapsulation of API in microbeads and microcapsules
- Q&A
Cell encapsulation platform: Examples of use
Standard cell encapsulation platform
The standard cell encapsulation platform allows for the encapsulation of single cells for high throughput screening applications involving drug discovery, toxicity testing, various ‘omics’ studies and rare cell analysis1,2.
How to inject a small sample?
The encapsulation platform for flow cytometry with an injection loop is a tool capable of greatly improving working conditions as it handles fluid volumes, as low as a few dozen microliters. Adding the L-Switch is advantageous in many scientific and medical applications, making it possible to work with rarer cells.
For instance, researchers working on stem cells are often challenged by their low availability. Indeed, because of their limited distribution in time (available only during a period of human life, difficulties to self-renew into stem cells after differentiation) and space (often nested in environments favorable to their functioning, protection and regulation, which limit their dispersion in the organism) and their delicate and expensive conditions of culture, their large-scale production is not possible. Thus, to be able to work with small volumes of culture and thus a low number of cells is advantageous.
Another example is the use of primary cells, directly collected from the patient and useful for personalized medicine. Acquiring this type of cells can be logistically challenging and the available volume can also be limited. The cell encapsulation platform’s injection loop solves this predicament by enabling researchers to work with small volumes of patient-derived cells.
Finally, using this platform for FACS also allows users to limit the use of expensive reagents and to limit wastes, unlocking new possibilities in the field of biomedical research and personalized medicine.
How to add a reagent?
In many microfluidic procedures, an integrated tooling for mixing is essential for appropriate functionality in a broad range of applications.
The usual encapsulation platform for drop allowing users to integrate a new phase for optimal agent mixing. It is recommended to mix two solutions in a fluidic path before encapsulation. After the precise formation of self‐ assembled lipid or polymeric particles by microfluidic mixing, the encapsulation of active molecules is loaded into synthesized particles.
The efficiency of many biosensors and the possibility of investigating reaction kinetics with quick time resolution depends on controlled mixing. It can be also used to encapsulate cells with barcoded beads for Next generation sequencing (DropSeq), for enzymatic tests, or phenotyping5.
Which applications can use the encapsulation platform for FACS?
High-throughput screening: Cell sorting
Cell encapsulation can improve high-throughput screening outcomes since the highly monodisperse droplets provide homogeneous reaction conditions. It can be combined with cell sorting tools such as Fluorescence Activated Cell Sorting (FACS), to achieve higher speed and efficiency compared to conventional approaches. Benefits include:
- Lower costs while increasing sequencing accuracy and depth:
- Sorting and sequencing only cells containing droplets could vastly reduce reagent costs and increase accuracy and depth for downstream Next Generation Sequencing, including eliminating common issues with single-cell droplet sequencing such as reads from empty droplets due to the encapsulation of free-floating transcripts.
- Increased range of measurement:
- Encapsulated cells may be sorted based on phenotypes not currently measurable with standard cell sorting, such as enzymatic turnover, presence of secreted molecules, or quantification of proteins lacking cell surface markers.
- Multi-omics profiling:
- In contrast with conventional methods, cell sorting using microfluidics provides a unique tool to link genotypes with phenotypes through compartmentalization.
- Our cell encapsulation platform allows for the combination of single-cell phenotyping and genome-wide sequencing, enabling multiomic measurements and directly linking cellular phenotypes to their underlying genetic mechanism2.
Drug delivery
Cell or drug encapsulation for drug delivery consists of the immobilization of bioactive materials, mainly cells, within a double emulsion, generally surrounded by a polymeric membrane. The latter permits the free passage of nutrients and oxygen and the egress of therapeutic protein products. Encapsulation allows for the protection of the cell content from mechanical stress and in the case of allogeneic tissue also from the host’s immune response. Microcapsules are commonly used in pharmaceutical or medical processes as drug carriers or for the encapsulation of organic cells.
Locally-implanted droplets may represent a minimally invasive manner of delivering therapeutic proteins such as growth factors and cytokines. It could also allow for mosaic injections containing several droplets to provide complex release profiles or multi-protein deliveries3.
Transplants
The microencapsulation of living cells may serve as an alternative therapy for patients requiring organ transplants. Researchers can control droplets size to mitigate the immune infiltration of transplanted cells while keeping oxygen and waste products transported. Droplets can also immobilize cells at the desired transplant size, which is essential as many cell therapies rely on systemic cell administration.
Cell encapsulation for cell-based therapy is emerging as a promising strategy for treating a wide range of human diseases, such as diabetes, blood disorders, acute liver failure, spinal cord injury, and several types of cancer through transplants. Pancreatic islets, blood cells, hepatocytes, and stem cells are among the many cell types currently used for this strategy. The encapsulation of these “therapeutic” cells is intended to prevent immune rejection, to provide a controlled and supportive environment, and to enable a complete retrieval of the graft in the case of an adverse body reaction4.
Specifications
We propose 4 different platforms to target different droplet sizes.
Description | Product | P.N |
Cell encapsulation device | Cell encapsulation platform | O-FACS1-PTF |
Double Emulsion production device | – RayDrop Double Emulsion 30-70-45 | – O-DE-RDRPC05- EUP |
Fluid handling system | – LINK Module – Flow EZ 7 bars for all 3 phases | – LU-LNK-0002 – LU-FEZ-7000PCK |
Reservoirs | – Continuous phase: 50 mL Pcap with 50 mL Falcon tube Tubing: 500 µm – Shell phase: 15 mL Pcap with 15 mL Falcon tube Tubing: 125 µm – Core phase: 15 mL Pcap with 15 mL Falcon tube Tubing: 125 µm | – P-CAP50-HP-PCK – P-CAP15-HP-PCK – P-CAP15-HP-PCK |
Flow Meters | – Continuous phase: Flow Unit L – Shell phase: Flow Unit M – Core phase: Flow Unit M | – FLU-L-D – FLU-M-D – FLU-M-D |
Optical System | – Light source – Microscope objective (x10) – Specific colour camera (up to 400 fps, 1µs integration time) – XYZ translation stages | N/A |
Tubing & Fittings | – Tubing: OD: 1/16 and 1/32 OD ID: 250 µm & 500 µm Materials: PFA – Manual valves: 4 way valves 2 way valves – Filters: 2 µm filter for continuous phase 2 µm filter for dispersed phase | N/A |
Wetted materials | – Platform: PEEK, PFA, PCTFE, PTFE, SS316L, GLASS – Sealing: FFKM | N/A |
Unit dimensions | – 61 x 46 x 43cm 3 (L x W x H) | N/A |
Weight | – 4 kg without the protective wood – 21 kg with the protective wood | N/A |
Droplet Size Range* | 25-45 µm | N/A |
Description | Product | P.N |
Cell encapsulation device | Cell encapsulation platform | O-FACS2-PTF |
Double Emulsion production device | – RayDrop Double Emulsion 30-70-60 | – O-DE-RDRPC06- EUP |
Fluid handling system | – LINK Module – Flow EZ 7 bars for all 3 phases | – LU-LNK-0002 – LU-FEZ-7000PCK |
Reservoirs | – Continuous phase: 50 mL Pcap with 50 mL Falcon tube Tubing: 500 µm – Shell phase: 15 mL Pcap with 15 mL Falcon tube Tubing: 125 µm – Core phase: 15 mL Pcap with 15 mL Falcon tube Tubing: 125 µm | – P-CAP50-HP-PCK – P-CAP15-HP-PCK – P-CAP15-HP-PCK |
Flow Meters | – Continuous phase: Flow Unit L – Shell phase: Flow Unit M – Core phase: Flow Unit M | – FLU-L-D – FLU-M-D – FLU-M-D |
Optical System | – Light source – Microscope objective (x10) – Specific colour camera (up to 400 fps, 1µs integration time) – XYZ translation stages | N/A |
Tubing & Fittings | – Tubing: OD: 1/16 and 1/32 OD ID: 250 µm & 500 µm Materials: PFA – Manual valves: 4 way valves 2 way valves – Filters: 2 µm filter for continuous phase 2 µm filter for dispersed phase | N/A |
Wetted materials | – Platform: PEEK, PFA, PCTFE, PTFE, SS316L, GLASS – Sealing: FFKM | N/A |
Unit dimensions | – 61 x 46 x 43cm 3 (L x W x H) | N/A |
Weight | – 4 kg without the protective wood – 21 kg with the protective wood | N/A |
Droplet Size Range | 45-60 µm | N/A |
Description | Product | P.N |
Cell encapsulation device | Cell encapsulation platform | O-FACS3-PTF |
Double Emulsion production device | – RayDrop Double Emulsion 60-120-60 | – O-DE-RDRPC07- EUP |
Fluid handling system | – LINK Module – Flow EZ 7 bars for all 3 phases | – LU-LNK-0002 – LU-FEZ-7000PCK |
Reservoirs | – Continuous phase: 50 mL Pcap with 50 mL Falcon tube Tubing: 500 µm – Shell phase: 15 mL Pcap with 15 mL Falcon tube Tubing: 125 µm – Core phase: 15 mL Pcap with 15 mL Falcon tube Tubing: 125 µm | – P-CAP50-HP-PCK – P-CAP15-HP-PCK – P-CAP15-HP-PCK |
Flow Meters | – Continuous phase: Flow Unit L – Shell phase: Flow Unit M – Core phase: Flow Unit M | – FLU-L-D – FLU-M-D – FLU-M-D |
Optical System | – Light source – Microscope objective (x5) – Specific colour camera (up to 400 fps, 1µs integration time) – XYZ translation stages | N/A |
Tubing & Fittings | – Tubing: OD: 1/16 and 1/32 OD ID: 250 µm & 500 µm Materials: PFA – Manual valves: 4 way valves 2 way valves – Filters: 2 µm filter for continuous phase 2 µm filter for dispersed phase | N/A |
Wetted materials | – Platform: PEEK, PFA, PCTFE, PTFE, SS316L, GLASS – Sealing: FFKM | N/A |
Unit dimensions | – 61 x 46 x 43cm 3 (L x W x H) | N/A |
Weight | – 4 kg without the protective wood – 21 kg with the protective wood | N/A |
Droplet Size Range | 50-60 µm | N/A |
Description | Product | P.N |
Cell encapsulation device | Cell encapsulation platform | O-FACS4-PTF |
Double Emulsion production device | – RayDrop Double Emulsion 60-120-90 | – O-DE-RDRPC08- EUP |
Fluid handling system | – LINK Module – Flow EZ 7 bars for all 3 phases | – LU-LNK-0002 – LU-FEZ-7000PCK |
Reservoirs | – Continuous phase: 50 mL Pcap with 50 mL Falcon tube Tubing: 500 µm – Shell phase: 15 mL Pcap with 15 mL Falcon tube Tubing: 125 µm – Core phase: 15 mL Pcap with 15 mL Falcon tube Tubing: 125 µm | – P-CAP50-HP-PCK – P-CAP15-HP-PCK – P-CAP15-HP-PCK |
Flow Meters | – Continuous phase: Flow Unit L – Shell phase: Flow Unit M – Core phase: Flow Unit M | – FLU-L-D – FLU-M-D – FLU-M-D |
Optical System | – Light source – Microscope objective (x5) – Specific colour camera (up to 400 fps, 1µs integration time) – XYZ translation stages | N/A |
Tubing & Fittings | – Tubing: OD: 1/16 and 1/32 OD ID: 250 µm & 500 µm Materials: PFA – Manual valves: 4 way valves 2 way valves – Filters: 2 µm filter for continuous phase 2 µm filter for dispersed phase | N/A |
Wetted materials | – Platform: PEEK, PFA, PCTFE, PTFE, SS316L, GLASS – Sealing: FFKM | N/A |
Unit dimensions | – 61 x 46 x 43cm 3 (L x W x H) | N/A |
Weight | – 4 kg without the protective wood – 21 kg with the protective wood | N/A |
Droplet Size Range | 70-90 µm | N/A |
OxyGEN
Control in real-time, protocol automation, data record and export |
ver. 2.2.0.0 or more recent |
Software Development Kit
Custom software application |
ver. 22.2.0.0 or more recent |
References
- Steele, J.A.M. et al. (2014) “Therapeutic cell encapsulation techniques and applications in diabetes,” Advanced Drug Delivery Reviews, 67-68, pp. 74–83. Available at: https://doi.org/10.1016/j.addr.2013.09.015.
Collins, D.J. et al. (2015) “The Poisson distribution and beyond: Methods for microfluidic droplet production and single cell encapsulation,” Lab on a Chip, 15(17), pp. 3439–3459. Available at: https://doi.org/10.1039/c5lc00614g.
Headen, D.M., García, J.R. and García, A.J. (2018) “Parallel droplet microfluidics for high throughput cell encapsulation and synthetic microgel generation,” Microsystems & Nanoengineering, 4(1). Available at: https://doi.org/10.1038/micronano.2017.76.
Farina, M. et al. (2019) “Cell encapsulation: Overcoming barriers in cell transplantation in diabetes and beyond,” Advanced Drug Delivery Reviews, 139, pp. 92–115. Available at: https://doi.org/10.1016/j.addr.2018.04.018.
- Damiati, S. et al. (2018) “Microfluidic devices for drug delivery systems and drug screening,” Genes, 9(2), p. 103. Available at: https://doi.org/10.3390/genes9020103.
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Expertise & resources
Introduction
Despite considerable progress in recent years, various disease diagnostics and treatments continue to present constraints such as low sensitivity or specificity, drug toxicity, and severe side effects [1]. Cancer represents one of the best examples of a disease where localized delivery of therapeutics is of great importance, as the potent yet toxic mechanisms of action of such compounds can lead to either an effective response or side effects. Today, most drug formulations are not capable of targeting specific sites of interest. Nanoparticle-based drug delivery platforms have emerged as suitable vehicles for overcoming these limitations [2]. Nanoparticles, such as liposomes, have proven advantageous at preserving therapeutic material and allowing for extended half-lives of drugs within the body [3].
Liposome nanoparticles are specialized delivery vehicles that serve multiple roles in enhancing the capabilities of active pharmaceutical ingredients (APIs). They can shield a drug from detection by the body’s immune system, and they serve to help solubilize highly lipophilic drug molecules or modulate the pharmacokinetics and biodistribution of the API.
What are liposome nanoparticles?
Liposome nanoparticles were discovered in the 1960s. These hollow nanoparticles are phospholipid vesicles consisting of at least one lipid bilayer (figure 1). This bilayer is usually composed of amphiphilic phospholipids that have a hydrophilic phosphate head and a hydrophobic tail consisting of two fatty acid chains. This structural feature has facilitated liposome applications, including their use as artificial cell membranes, carriers for drug delivery systems, encapsulating agents for food ingredients, and analytical tools [4–8].
Recent state-of-the-art applications
During the COVID-19 pandemic, the first vaccines to reach clinical trials were based on viral vector and nucleic acid technologies. One of the most promising vaccine candidates was based on nucleoside-modified mRNA and encapsulated within lipid nanoparticles (LNP) [9]. This confirms the need for lipidic nanoparticles for present and future drug delivery applications.
In recent years, liposome nanoparticles have attracted significant attention as a trusted class of drug delivery vehicles. Their self-closed structures can encapsulate multiple drugs at once, protecting the enclosed cargo from hydrolysis and breakdown. Additionally, targeting proteins and surface functional ligands on the outer shell of the lipid bilayer can add novel functionality—enabling targeted entry of liposomes into cells, either via antibodies or receptor-targeted ligands. These ligands attach to cell receptors that are over-expressed in certain diseased cells, allowing entry of the drug through the cell membrane.
What is the difference between Liposome and Lipid Nanoparticles?
Liposome and lipid nanoparticles are lipid-based structures used for drug delivery. Liposomes are spherical vesicles with lipid bilayers surrounding an aqueous core, while lipid nanoparticles are solid particles composed of solid lipids or a mixture of solid and liquid lipids. Liposomes have a hydrophilic outer layer and can encapsulate both hydrophilic and hydrophobic molecules, offering versatility but potentially reduced stability.
In contrast, lipid nanoparticles provide improved stability and are relatively easier to manufacture at scale. They are especially suitable for encapsulating poorly water-soluble drugs or nucleic acids. Liposomes have been widely used, while lipid nanoparticles are gaining in popularity, particularly in mRNA-based vaccines like those for COVID-19. Both lipid-based systems contribute to advanced drug delivery strategies, with liposomes offering flexibility and lipid nanoparticles providing enhanced stability and ease of production.
Comparison with Another Production Method
Batch method | Fluigent microfluidic method | |
Particle size distribution | Low | High |
Reproducibility | Low | High |
Live particle size control | No | Precise |
Range of particle size | Limited size range | Wide size range |
Continuous (/in line) production | No | Yes |
How to produce liposome nanoparticles
Equipment
Liposome nanoparticle generation
A stream of lipid in alcohol solution is surrounded by an aqueous phase within a glass capillary. The alcohol solution containing lipids diffuses into the aqueous solution (and reciprocally the water diffuses into the alcohol), until the alcohol concentration decreases to the solubility limit of the lipids. Consequently, this diffusion triggers the formation of liposomes by a mechanism described as “self-assembly”, where lipids assemble into a more energetically favorable structure.
The liposome nanoparticle production system is illustrated in figure 2.
Partial results
Conclusion
Liposome nanoparticles have proven advantageous for solubilizing therapeutic substances. Macroscale batch methods widely employed for liposome production lack control over liposome morphology, size, and distribution. Microfluidic systems allow for the production of highly monodisperse liposome nanoparticles. We have demonstrated the production of liposomes using a microfluidic system consisting of pressure-based flow controllers and the Raydrop™ microfluidic device with standard configuration. Liposomes ranging from 30 to 150 nm were generated. Sizes can be adjusted by controlling the device flow input parameters, particularly the flow rate ratio (FRR). The polydispersity index (PDI) ranges from 0.07 to 0.15. This system enables synthesis of liposomes for drug delivery applications, as encapsulating agents for food ingredients, or for other applications requiring nano-sized and spherical liposomes.
A complete, cost-effective and commercially-available platform for on-demand production of monodisperse liposome nanoparticles is now available. This allows for control of liposome size and frequency by adjusting flow parameters.
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References
- Bozzuto, G. & Molinari, A. Liposomes as nanomedical devices. Int. J. Nanomedicine 10, 975–999 (2015).
- Blanco, E., Shen, H. & Ferrari, M. Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat. Biotechnol. 33, 941–951 (2015).
- Torchilin, V. P. Recent advances with liposomes as pharmaceutical carriers. Nat. Rev. Drug Discov. 4, 145–160 (2005).
- Bally, M. et al. Liposome and lipid bilayer arrays towards biosensing applications. Small 6, 2481–2497 (2010).
- Fathi, M., Mozafari, M. R. & Mohebbi, M. Nanoencapsulation of food ingredients using lipid based delivery systems. Trends Food Sci. Technol. 23, 13–27 (2012).
- Grimaldi, N. et al. Lipid-based nanovesicles for nanomedicine. Chem. Soc. Rev. 45, 6520–6545 (2016).
- Andrew Pohorille & David Deamer. Artificial cells: prospects for biotechnology. Trends Biotechnol. Biotechnol. 31- (2002).
- Rongen, H. A. H., Bult, A. & Van Bennekom, W. P. Liposomes and immunoassays. J. Immunol. Methods 204, 105–133 (1997).
- Vogel, A. B. et al. A prefusion SARS-CoV-2 spike RNA vaccine is highly immunogenic and prevents lung infection in non-human primates. bioRxiv 2020.09.08.280818 (2020).
- Pattni, B. S., Chupin, V. V. & Torchilin, V. P. New Developments in Liposomal Drug Delivery. Chem. Rev. 115, 10938–10966 (2015).
- Mui, B., Chow, L. & Hope, M. J. Extrusion Technique to Generate Liposomes of Defined Size. Methods Enzymol. 367, 3–14 (2003).
- Carugo, D., Bottaro, E., Owen, J., Stride, E. & Nastruzzi, C. Liposome production by microfluidics: Potential and limiting factors. Sci. Rep. 6, 1–15 (2016).
- Hood, R. R., Devoe, D. L., Atencia, J., Vreeland, W. N. & Omiatek, D. M. A facile route to the synthesis of monodisperse nanoscale liposomes using 3D microfluidic hydrodynamic focusing in a concentric capillary array. Lab Chip 14, 2403–2409 (2014).
- Jahn, A., Vreeland, W. N., Devoe, D. L., Locascio, L. E. & Gaitan, M. Microfluidic directed formation of liposomes of controlled size. Langmuir 23, 6289–6293 (2007).
- Jahn, A. et al. Microfluidic mixing and the formation of nanoscale lipid vesicles. ACS Nano 4, 2077–2087 (2010).
What are the advantages of microfluidics in biology?
The development of microfluidics has brought many advantages for biological research. Indeed, due to the miniaturization and the automation of the setups, the consumption of sample and reagent is largely decreased, the experiment times are considerably reduced and the global costs of the applications are minimized. Moreover, working with small quantities allows better carry out separations and detection with high resolution and sensitivity at reduced costs.
Finally, thanks to this new generation of techniques and equipments, new capabilities are offered for biology research since microfluidics allows to obtain more accurate results, to reduce detection limits and even to perform multiple analyses simultaneously.
To learn more about it, feel free to reed our white paper about microfluidic.
Main applications of microfluidics in cell biology
RNA and DNA Hybridization
For decades, in situ hybridization has been used with greater frequency to capture the localization, structure and expression of specific DNA and RNA sequences within tissues. The high sensitivity and specificity of this technique relies on the hybridization of labelled oligonucleotide probes to targeted DNA or RNA sequences within tissues. Chromosomal microdeletion, amplification, structure, translocation and expression can be easily detected.
Compared to PCR analysis, Northern Blotting or a DNA microarray performed on lysed cells, hybridization provides spatial information as specific RNA and DNA are detected within tissues. It has significantly improved gene mapping, cytogenetics and various diagnostic techniques (oncogenic, prenatal, viral infection, etc.).
Recently, with the use of microfluidic for cell biology, a temporal dimension was added to the technology, as it further evolved to be performed on living cells. The spatio-temporal expression, degradation and storage of RNA molecules can now be thoroughly investigated by real time imaging of the hybridization of labelled oligonucleotides in living cells.
Drug Combination and Long-Term Perfusion
In various therapeutic areas (glaucoma, vascular, HIV, oncology), a combination of drugs is typically found to be more effective for disease treatment. Oncology, in particular, has paved the way for this approach. The emergence of next-generation sequencing technology profiling has revealed the heterogeneity in many cancers at the origin of the differential response to treatment. As a result, therapeutic strategies are evolving towards multi-targeted drug combinations that effectively inhibit the cancer cells and block the emergence of drug resistance while selectively incurring minimal side effects on healthy cells.
Drug combination therapy is not straight forward as in many cases the results do not equal the sum of the parts. Cross-reactions are observed and fall into 5 categories: low risk & synergy, low risk & no synergy, caution, unsafe, and dangerous. The contribution and dosage of each active molecule should be closely investigated in terms of dose, frequency and duration to evaluate the efficacy of the drug combination.
Therefore, microfluidics for cell biology holds great promise in cancer diagnosis and also serves as an emerging tool for understanding cancer biology. Microfluidics can be a valuable tool in cancer investigation due to its high sensitivity, high throughput, less material-consumption, low cost, and enhanced spatio-temporal control.
Immunostaining
Immunostaining experiments are mult-istep protocols to detect specific antigens in biological samples. The sample is successively incubated or exposed to fixation agents, washing buffers, and probes.
In conventional protocols on petri dishes or well plates, fluid deliveries are performed manually using pipettes. Solutions are directly added to the sample.
Transferring protocols from a Petri dish to a microfluidic format usually requires some minor adjustments, as cells or tissue samples are exposed to solutions differently. Therefore, the use of microfluidic for cell biology provides many advantages by replacing the use of Petri dishes with microfluidic chambers (closed systems). Consequently, solutions cannot be deposited directly on the top of the cells but are perfused over the sample at a controlled flow rate, reaching all cells homogeneously. The use of a rotary valve with a perfusion system enables one to perfuse different solutions at given time points in the chip or chamber. In addition, automating the sequential delivery of solutions saves time and avoids creating bubbles inside the chips by disconnecting and reconnecting the traditional pump to the chip.
Resources
How important is the microlfluidic flow control in cell biology?
In drug discovery, it‘s important to control the flow rate of the sample used. As multiple formulations are tested, experimenters must be able to control flow rate while keeping the same level control of fluid handling at each stage of the experiment. DNA and RNA hybridization are often performed under a microscope. Manual injection over a microscope can be risky for the following reasons:
- A touch of pipette cone can misplace the dish and misrecord the position
- Samples can be flushed away during pipetting
- Liquid can be spilled over the microscope
Flow Controllers Available on the Market
Syringe Pumps
Multiple flow control technologies are available for sub-millimeter range fluid management. Syringe pumps are commonly used for fluid control. However, in microfluidics, particularly in microfluidics for cell biology, the use of syringe pumps is often problematic because the flow is not constant. This discontinuity is due to the mechanical system that creates an oscillating flow.
Manual Injection
Current methods for multiple fluid delivery, where microscopy techniques are simultaneously required and mostly made by manual pipetting action include , Immunolabeling, and DNA and RNA hybridization.. Manual injection can lead to uncontrolled and non-homogeneous fluid velocity, which can damage samples.
Pipette | Aria | |
---|---|---|
Type of injections | Abrupt injections (up to 1mL in few seconds) Disparate injections Turbulent flow | Smooth & controled injections Identical injections Laminar flow |
Geometry at injecion tip | Conic shape: Important shear strain Unhomogeneous fluid velocity | Straight shape: No modification at the injection tip |
Fluigent Pressure-Based Flow Control
As the demand for microfluidic pumps with higher flow stability, fast response time, versatility, and automation capabilities has increased, pressure controllers have become the device of choice for many users.
The working principle of such pumps is to pressurize the sample reservoirs to control the pressure drop between the inlet and the outlet of the microfluidic system. The responsiveness of the generated flow rate depends on the responsiveness of the pressure pump.
To overcome these issues, Fluigent’s Aria automates multiple fluid deliveries. The sample is preserved as the flow rate is controlled. Reproducibility increases as inter and intra operator variability are eliminated.
MIMLIVER ON CHIP
MIMLIVER on CHIP project coordinated by the laboratory of Biomechanics and Bioengineering from UTC/CNRS aims at developing a functional liver-on-chip system to evaluate the toxicity of drugs. It is motivated by the established observation that 90% of potential drug candidates fail in clinical trials due to the lack of relevant models in the early drug development stages. The Liver is a central organ for toxicology assessment as it metabolizes the drugs into compounds that can be more active than the parent drug itself. Prior to being metabolized by hepatocytes, drugs are first filtered across an endothelial barrier which is typical of the liver. This selective barrier loses its properties in pathological conditions.
MIMLIVER on CHIP project Kick Off
- October 18, 2019
Fluigent and our collaborators are pleased to announce that the 1.5 M€ project: MIMLIVER on CHIP has been funded by the French National Research Agency.
This project coordinated by the laboratory of Biomechanics and Bioengineering from UTC/CNRS aims at developing a functional liver-on-chip system to evaluate the toxicity of drugs.
This project is motivated by the established observation that 90% of potential drug candidates fail in clinical trials due to the lack of relevant models in the early drug development stages. The Liver is a central organ for toxicology assessment as it metabolizes the drugs into compounds that can be more active than the parent drug itself. Prior to being metabolized by hepatocytes, drugs are first filtered across an endothelial barrier which is typical of the liver. This selective barrier loses its properties in pathological conditions.
In order to produce more reliable results in preclinical development, the MIMLIVERonCHIP project aims at developing the first in vitro model that reproduces both the liver and its associated vascular system.
Participating in this project are 4 partners with complementary expertise:
- The laboratory of Biomechanics and Bioengineering who have already developed and validated a liver on chip model. They will lead the development the endothelial cell chip and its connection to the liver chip.
- The Laboratory of Integrated Micro Mechatronic Systems who have strong expertise in endothelial cells, angiogenesis and vascular networks. They will grow primary endothelial cells and drive their growth into a relevant cell layer representative of the liver’s vascularisation.
- HCS Pharma is a CRO specialized in the development of innovative 2D and 3D cellular models for pharmacology and toxicology. They will validate the final liver-on-chip model by evaluating the effect of known substances and benchmark it with existing techniques.
- Fluigent will develop an automated instrument that will meet pharma standards in terms of throughput, automation, connection, compactness and ease of use.
The kick-off meeting took place on the 3rd of October. This project is very innovative and we expect to find significant results on the co-culture of the hepatocytes and LSEC (Liver Specific Endothelial Cells) on our way to the final report.
MYOCHIP FETOPEN PROJECT
The aim of the MyoChip project is to build a 3D human skeletal muscle irrigated by vasculature and innervated by neurons. The reconstituted 3D muscle will mirror the architecture and function found in vivo, namely in shape, contractility and microenvironment, while irrigation by a vascular network and innervation by human motor neurons will bring additional physiologic pertinence to it. This organ-on-a-chip technology will have numerous applications including but not limited to research on muscle building and aging, drug testing and screening, as well as prosthetics and biorobotics. The feasibility of the project relies on the interdisciplinary approach which joins a team of cell biologists, material engineers, experts in microfluidics and mathematical modellers. The architecture of skeletal muscle and its regenerative capabilities make muscle a prime candidate to push the 3D tissue engineering field. As such the project will lay the technical, material and methodological foundations to tackle the next generation of complex organ-on-a-chip systems that the MyoChip consortium can exploit for the generation of highly complex 3D in vitro systems of many organs.
BIO-ART LUNG 2020
The BIOART-LUNG 2020 project aims to develop a long term, autonomous, portable, artificial lung for patients suffering from acute respiratory distress. This innovative therapeutic approach will provide 2 major advantages compared to existing extracorporeal membrane oxygenation:
- Portable device that will increase mobility of patients while waiting for lung transplantation surgery
- Physiologically relevant device which would reduce blood activation and extended blood oxygenation above the current three weeks limitation.
To validate intermediate steps, Fluigent has conceived, developed and made a customized fluidic platform connected to the device that integrates all the functions the final system will have. This fully automated platform allows for testing of the devices under realistic conditions.
HOLIFAB
Microfluidics is currently faced with 2 main difficulties:
- A considerable hindrance to fast prototyping and industrialisation, because system performances rely on complex selection and assembly of numerous fluidic, mechanic, optical, electronic (etc…) components,
- A complex process for setting the system requirements, because constraints and specifications of microfluidic systems cover a huge range in terms of complexity level, materials, dimensions, acceptable costs, without any standardisation and rationalisation of design and production.
The HoliFAB project aims at overcoming these difficulties with a holistic system-oriented and problem-solving approach, starting from the customer’s need, optimising the chip and addressing the question of its environment.
HoliFAB change of paradigm implies:
- A holistic approach at the instrument level: Providing new methods and production tools to instruments manufacturers and new generation of applications to users,
- A holistic approach for the chip production: Using a technology-agnostic strategy integrating both 3D printing and injection moulding technologies for the design of the microfluidic chip.
INDEX
The aim of Project INDEX is to isolate and characterize nanoparticles available in bodily fluids through development and integration of novel technological breakthroughs. The technology will enable the analysis of clinically valuable nanoparticles called exosomes towards new generation diagnostics. Exosomes are known to mediate communication between cells and their effective utilization holds a great promise of revolutionizing the standard of clinical care. However, their detection and molecular profiling is technically challenging. The proposed technology will isolate exosomes that are as small as 30nm in diameter from human plasma with high purity, and provide in-depth, multi-parameter characterization of the particles through digital counting, size determination, and biological phenotyping.
Towards this goal: (1) Novel microfluidics will be developed and used for efficient magnetic enrichment; (2) Isolated particles will be detected and analyzed with a novel biological nanoparticle (BNP) sensor (3) Immune-capture and release chemistries as well as phenotyping assays will be developed; (4) Critically, complete on-chip integration of isolation, detection and analysis will be accomplished; (5) Utility of in-depth exosome characterization will be demonstrated with clinical samples for lung cancer.
Project INDEX requires successful integration of multiple sub-units and assays that each represents technological frontiers, which is extremely challenging. However, the breadth of information on exosomes that will be available with the integrated system is unmatched. Although, the clinical utility of exosomes is still developing, the uncertainty can only be clarified through automated technologies that provide latitude of information. Once completed, Project INDEX can demonstrate a new paradigm in cancer diagnostics, and also present a potential future technology for other applications involving nanoparticles.
MFMANUFACTURING
The objective of the MFManufacturing project is to bring the manufacturing of microfluidic devices to the same level of maturity and industrialisation of electronic devices, enabling them to address more widely in the healthcare needs. Electronic devices, which have been on the market for many years, have benefited from the long going standardization of electronic components, and were therefore easily integrated in the production process of the major foundries.
The anticipated standardization in the microfluidics field – first of all aimed at strengthening Europe’s position will focus on increasing maturity of both functional and fabrication process aspects:
- Gain of maturity in MF functions focusing both on novel functional modules and their interoperability.
- Gain of maturity in manufacturing process: focusing on a distributed pilot line, on novel hybrid integration processes and on increasing maturity of some selected manufacturing processes, which have a good short term commercial perspective.
This will have an effect on availability, reliability as well as accessibility and will result in device cost reduction and improved time-to market. These conditions will enable large scale uptake of microfluidic devices in the markets identified.
About The Keyser lab – University of Cambridge
The Keyser Lab is a group of researchers at the Cavendish Laboratory, University of Cambridge, UK. Since its founding in 1874, the Cavendish Laboratory has been at the forefront of discovery in physics, with a core focus on experimental physics supported by excellence in theory. The department promotes world-leading experimental and theoretical physics in all its diversity. Scientists from the Keyser Lab study the physics of ions, macromolecules and particles, with a particular focus on particles in confined geometries at the single molecule/particle level. To exert maximum control over all parameters for their experiments, they make use of several cutting-edge techniques such as DNA self-assembly (origami), optical trapping, electrophysiology, and microfluidics and nanofluidics.
The team includes researchers with expertise in physics, engineering, physical chemistry, biochemistry/biology, and micro- and nanofabrication.
What are Giant Unilamellar Vesicles (GUVs)?
Giant Unilamellar Vesicles (GUVs) are micron-sized compartments composed of lipid bilayers, serving as models for cell membranes or as encapsulating structures for biological materials within cell-like environments. These vesicles have dimensions ranging from 1 to 100 µm, mimicking the size of cells. Like natural cell membranes, GUVs are constructed from lipids, predominantly phospholipids and cholesterol. The amphiphilic properties of these lipids enable them to spontaneously arrange into spherical compartments when immersed in an aqueous solution.
They offer the advantages of having well-defined lipid compositions, being easy to image, and being controlled systems for studying transport processes. These characteristics open the door to a variety of applications in biology and biomedicine, especially membrane biophysics and synthetic biology.1,2
Working principle of GUV production
In 2016, the Dekker Laboratory from the Delft University of Technology developed a novel microfluidics-based method called Octanol-Assisted Liposome Assembly (OLA) to create uniform, cell-sized (5–20 mm) GUVs with high encapsulation efficiency. Using Fluigent’s pressure-driven MFCS-EZ controller with the Oxygen software tool, an inner aqueous phase (IA) and an outer lipid-carrying 1-octanol phase (LO) were combined, resulting in double-emulsion droplets via hydrodynamic flow focusing. These droplets developed a side-connected 1-octanol pocket, which, due to interfacial energy minimization, separated to rapidly form fully assembled solvent-free liposomes. Microfluidic GUV production addresses the persistent issue of residual oil in vesicle bilayers.
This method allows researchers to generate GUVs with higher monodispersity and greater size control compared to traditional methods, and with a faster process than alternative microfluidic methods.3
Keyser Lab: Microfluidics for GUV generation for antimicrobial efficacy and biomimetic vesicle membrane testing
Scientists from the University of Cambridge integrated octanol-assisted liposome assembly into their microfluidic platforms (“lab on a chip” devices) for quantifying drug permeation and antimicrobial efficacy on biomimetic vesicle membranes.
In a first paper published in Lab on a Chip (2019), they reported on a microfluidic platform for testing antimicrobial peptides on artificial vesicle membranes. This platform produced vesicles with an encapsulated dye to assess the efficacy of antimicrobial peptides by measuring the time it takes for vesicles to lyse.4
This Giant Unilamellar Vesicle production technique also utilized Fluigent‘s MFCS-EZ and its accompanying software (Oxygen) for fluid control. Vesicle formation and perfusion inlets were connected, and the microfluidic chip was loaded with an inner aqueous phase base stock. The outlet was connected to Fluigent’s 2-Switch, which can switch between open or closed configurations for waste removal. Vesicles flowed through a channel to the connector chip, and 1-octanol pockets pinched off to form droplets. Density-based separation removed vesicle production waste, and the vesicles were distributed to trapping chambers. Peptide doses were controlled, replacing the inner aqueous phase buffer, while maintaining a constant input pressure. The entire process was monitored under an inverted microscope.
Validated with cecropin B on bacterial-mimicking membranes, the platform enabled the researchers to study over 1000 vesicles simultaneously. The results showed dose-dependent disruption of vesicle membranes, demonstrating the platform’s potential for controlled, quantitative assessments of the efficacy and selectivity of antimicrobial peptides. This microfluidic approach to GUV production is suggested as a new standard for pre-clinical development of membrane-active antimicrobials, offering advantages in cost efficiency and parallelization.
In another paper published in Biomembranes (2020), scientists from the University of Cambridge and the University of Exeter produced GUVs with tunable binary lipid mixtures to determine lipid diffusion in OLA vesicles.5
The focus was on expanding the capabilities of this microfluidic approach to form GUVs with tunable binary lipid mixtures. An MFCS-EZ equipped with a Fluiwell-4C reservoir kit with OLA solutions was connected to the microfluidic chip. The microfluidic GUV production method allowed for a high degree of control over vesicle sizes by adjusting pressures in different channels. This was possible due to the high responsiveness, stability and repeatability of the pressure generated by the MFCS-EZ. In addition, flow speed was successfully matched to the outlet channel length to prevent residual octanol attachment to vesicles.
This study employed fluorescence recovery after photobleaching to investigate lipid lateral diffusion coefficients in GUVs produced by the microfluidic approach, finding values within the expected range. Comparisons with electroformed vesicles indicated quantitative similarity in lipid diffusion coefficients. The results served as a quantitative biophysical validation of OLA-derived GUVs, enhancing the potential application of this versatile platform in drug discovery, artificial cell production, and lipid membrane studies.
Conclusion
To produce Giant Unilamellar Vesicles, fluids were controlled using Fluigent pressure-based flow controllers. Researchers from the Keyser Lab (University of Cambridge) typically operated the chip with input pressures of 40 mbar for the inner aqueous phase and dissolved-lipid phases and 100 mbar for the outer aqueous phase. For precise flow measurements, Fluigent Flow Units can be added on the fluidic path. Microfluidic GUV production also allowed researchers to adjust the sizes of the generated vesicles by adjusting the microfluidic pressures of the phases. This degree of control is difficult to achieve using standard methods such as electroformation or traditional syringe pumps.
Testimonials
“Microfluidics presents various advantages to researchers who need small volumes and high throughput in answering their scientific questions. In our lab, we use microfluidic devices for standardization and control of experimental parameters like concentration and timing. In the complex (biological) systems we are working on, the mentioned characteristics are fundamental in collecting reliable meaningful statistics, and microfluidics in combination with light microscopy offers just that. We also heavily rely on the ability to rapidly prototype devices, as we can design bespoke solutions at minimal production cost and time.”
“We use the pressure-based pumps from Fluigent for experiments that require swift responsiveness when manipulating fluids, and fine tuning at low flow rates. We use the Fluigent systems during fabrication and running of the microfluidic chips. The ability to pump in air at high precision makes the Fluigent pressure-based systems ideally suited to selectively coat and functionalize micro-channels within a microfluidic network. After coating we then fill the devices with the experimental solutions and use the pressure controls to move fluids around, open and close valves and carefully time the introduction of small molecules in the experiments.“
Kareem Al Nahas, University of Cambridge
Expertises & Resources
References
- Naziris, N., Demetzos, C. (2021). Liposomes: Production Methods and Application in Alzheimer’s Disease. In: Vlamos, P. (eds) GeNeDis 2020. Advances in Experimental Medicine and Biology, vol 1339. Springer, Cham.
- Pereira, David & Valentão, Patrícia & Andrade, Paula. (2014). Nano- and Microdelivery Systems for Marine Bioactive Lipids. Marine Drugs. 12. 6014. 10.3390/md12126014.
- Deshpande, S.; Caspi, Y.; Meijering, A. E. C.; Dekker, C. Octanol-Assisted Liposome Assembly on Chip. Nat Commun 2016, 7 (1), 10447.
- Al Nahas, K.; Cama, J.; Schaich, M.; Hammond, K.; Deshpande, S.; Dekker, C.; Ryadnov, M. G.; Keyser, U. F. A Microfluidic Platform for the Characterisation of Membrane Active Antimicrobials. Lab Chip 2019, 19 (5), 837–844.
- Schaich, M.; Sobota, D.; Sleath, H.; Cama, J.; Keyser, U. F. Characterization of Lipid Composition and Diffusivity in OLA Generated Vesicles. Biochimica et Biophysica Acta (BBA) – Biomembranes 2020, 1862 (9), 183359.
Introduction
Microcapsules are becoming commonly used drug delivery systems as they can be easily administered and can be engineered with different structures and functions for keeping drug stability, delivering drugs to a desired location, and releasing drugs at a predetermined rate in a well-controlled manner [3].
Droplet-based microfluidics has been used as a tool for small scale single-cell or cell culture analysis, chemical synthesis as well as high-throughput screening [4]. Achieved by dispersions of stabilized liquids within continuous immiscible fluids, thousands of particles are generated within minutes. These can be used as microcompartments suitable for such experiments [6]. Gaining in usage are water-in-oil-in-water (w/o/w) and oil-in-water-in-oil (o/w/o) emulsions, which can be employed for applications such as drug delivery vehicles, cell carriers, barcoding of droplets, microscale sensors, and more [5].
For these applications, the use of agarose microcapsules is expanding due to the advantages of this polymer. Agarose is a natural polysaccharide obtained from red seaweed and in aqueous solution can form a hydro-gel at low temperatures.
Agarose microcapsules are a well-established for a variety of applications such as protein detection, DNA hybridization and as a mean to immobilize biomolecules in order to support reactions and to permit a faster molecular detection with higher sensitivity and lower reagent consumption. This makes them an ideal choice for microscaled lab-on-chip devices. More specifically, microcapsule-based microfluidic platforms enhance planned chemical or biochemical reactions, increasing the degree of interaction between the biomolecules and the functionalized surfaces [15].
Agarose microcapsules production: Materials and methods
Materials: Reagents
Producing a double emulsion requires 3 phases: the continuous phase, shell phase and core phase. The shell phase must be immiscible with both other phases.
Continuous phase:
- MCT 2: MCT with 2% w/w PGPR (E576)
Shell phase:
- Ag1: MiliQ water with 1% w/w TWEEN 80 and 1%w/w Agarose
- Ag2: MiliQ water with 1% w/w TWEEN 80 and 2%w/w Agarose
- Ag3: MiliQ water with 1% w/w TWEEN 80 and 3%w/w Agarose
Core phase:
- MCT 1: MCT oil only
Materials: Products
Double emulsion production method: How to synthetize agarose microcapsules?
Oil-core agarose shell emulsions are manufactured using a RayDrop 90-160-450 due to its flexibility and stability.
Agarose microcapsules production is performed in several steps:
- Preparation:
It’s recommended to filter all liquids to avoid clogging and to degas solutions to minimize the apparition of air bubbles inside the system.
All fluidic lines of the setup have to be sequentially filled with liquid (continuous phase, core phase and shell phase) to prime the system and ensure that it’s wet and free of air bubbles.
- Shell Phase Simple Emulsion:
Production of a single emulsion of the shell phase in the continuous phase, increasing the pressure until reaching a jetting mode.
- Double Emulsion:
Production of a double emulsion adjusting the core flow, due to the shearing of this phase with the previous phase (single emulsion droplet already formed).
- Droplet collection:
After gelation is complete, the microcapsules are collected on a filter/sieve, washed with excess water and resuspended in MCT oil working solution.
- Production run:
Oil core – hydrogel shell microcapsules are produced at gram quantities. The system was left running for at least 30 minutes to determine the long-term stability and ability to withstand clogging.
Production of Agarose microcapsules: RESULTS
Ag1 (1% agarose solution) and Ag2 (2% agarose solution) have resulted in the successful formation of oil core–agarose shell microcapsules.
Ag3 (3% agarose solutions) did not result in stable droplet formation due to the to the high viscosity of the solution.
Premium quality agarose microcapsules were successfully obtained. Droplet formation was stable enough to enable long-term production of the oil core – agarose shell sample over the course of 180 minutes.
For a configuration with the nozzle and output capillaries (respectively 90µm and 450µm as presented in this note), adjusting the flow rates of the fluids allows for fine control of the capsule dimensions (see Figure 2). With this setup, agarose microcapsules from 215µm to 300µm are easily produced. The shell thickness of microcapsules can also be varied by changing the ratio of flow rates of the shell and core phases, as shown in Figure 3. Here core thickness varies from 80µm to 170µm.
Conclusion
Using Fluigent’s double emulsion production station it is possible to generate agarose microcapsules over the outside diameter range of 215 and 300 µm with standard RayDrop configuration (Nozzle of 90µm and outlet capillary 150µm) using agarose solution at 1% in water. Other concentrations of agarose such as 2%, which is widely used in biological applications, have also been successfully tested by following the same process.
This demonstrates that the RayDrop double emulsion and Fluigent liquid control technology can be successfully used together to produce double emulsions. The ability to produce highly monodisperse agarose microcapsules has also been demonstrated.
Variation of the continuous phase flow does not produce a significant change in droplet formation rate or size – this further adds to the stability of the system. Long-term production of agarose microcapsules (over 180 minutes) was successfully undertaken.
The droplet size variation can be successfully controlled by altering the flow rates of the droplet phases and/or the size of the Raydrop chips. This also shows that core-shell ratio can be successfully adjusted by altering the relative flow rates of core and shell phases.
This confirms that the Raydrop™, in combination with Fluigent pumping technology, are one of the most competitive double emulsion systems available due to the flexibility (changing capillary size can be easily done to target different droplet sizes), the ease of use, the breadth of accessible chemical systems, the long-term performance stability and the lack of coatings (e.g. sigma coat)
References
1. Bae, Y., & Park, K. (2011). Targeted drug delivery to tumors: Myths, reality and possibility. Journal Of Controlled Release, 153(3), 198-205. doi: 10.1016/j.jconrel.2011.06.001
2. Delcea, M., Möhwald, H., & Skirtach, A. (2011). Stimuli-responsive LbL capsules and nanoshells for drug delivery. Advanced Drug Delivery Reviews, 63(9), 730-747. doi: 10.1016/j.addr.2011.03.010.
3. Ma, Z., Li, B., Peng, J., & Gao, D. (2022). Recent Development of Drug Delivery Systems through Microfluidics: From Synthesis to Evaluation. Pharmaceutics, 14(2), 434. doi: 10.3390/pharmaceutics14020434U.
4. Khan, I., Serra, C., Anton, N., & Vandamme, T. (2014). Production of nanoparticle drug delivery systems with microfluidics tools. Expert Opinion On Drug Delivery, 12(4), 547-562. doi: 10.1517/17425247.2015.974547
5. He, F., Zhang, M., Wang, W., Cai, Q., Su, Y., & Liu, Z. et al. (2019). Designable Polymeric Microparticles from Droplet Microfluidics for Controlled Drug Release. Advanced Materials Technologies, 4(6), 1800687. doi: 10.1002/admt.201800687
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7. Guo, J., Hou, L., Hou, J., Yu, J., & Hu, Q. (2020). Generation of Ultra-Thin-Shell Microcapsules Using Osmolarity-Controlled Swelling Method. Micromachines, 11(4), 444. doi: 10.3390/mi11040444
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Introduction
Polymers were first introduced three decades ago as bioresorbable surgical devices. Since then, polymer-based nanoparticles have been extensively studied. Nanocarriers formulated with biocompatible and biodegradable polymers approved by the US FDA (Food and Drug Administration) and EMA (European Medicines Agency) are being studied for the controlled delivery of various therapeutic agents (1).
Among the various polymers synthesized for formulating polymeric nanoparticles, poly(lactic-co-glycolic acid) (PLGA) is the most popular. PLGA nanoparticles have several beneficial properties such as controlled and sustained release, low cytotoxicity, long-standing biomedical applications, biocompatibility with tissues and cells, prolonged residence time and targeted delivery (6). These characteristics have accelerated the PLGA nanoparticle synthesis for use as nano-drug delivery systems (nanoDDS) in a wide variety of diseases, including cardiovascular, neurodegenerative and inflammatory and immune system diseases, infection, cancer, regenerative medicine and the fields of theragnostic and vaccines (4).
When PLGA is used as an active pharmaceutical ingredient carrier, it’s important to produce highly monodispersed particles for drug release reproducibility. PLGA nanoparticle synthesis with different characteristics (size, size distribution, morphology, zeta potential) is also possible by controlling the parameters specific to the synthesis method employed (3).
Current methods of particle synthesis rely largely on batch stirred homogenizers (single emulsion, double emulsion, etc.). However, they generally tend to have low reproducibility and are not well controlled. Some have low encapsulation efficiency and low drug loading. As narrow distributions, small particle size, and controllable synthesis are required in the field of smart drug delivery, these do not provide a highly effective solution for the pharmaceutical industry (4).
Microfluidic methods and, especially the 3D hydrodynamic flow-mediated nanoparticle production strategy of the RayDropTM make it possible to obtain a continuous PLGA nanoparticle synthesis with high monodispersity, high reproducibility and a wide range of nanoparticle size.
PLGA nanoparticle synthesis: Materials and methods
PLGA nanoparticle production has been performed with Fluigent’s Nanoparticle Production Station, a robust and complete system for precise and long-term production of nanoparticles with flexible particle size range.
PLGA Nanoparticle production setup
Reagents
Continuous phase: deionized water and 1 % Polyvinyl alcohol Mw 9000 – 10000 80% hydrolyzed (Sigma Aldrich).
Inner phase: technical acetone, and PLGA Resomer 756 1 % (Sigma Aldrich).
Inner phase to initiate and clean : technical acetone.
Figure 1: Scheme of the fluidic setup
Figure 2: Picture of the Fluigent equipment
PLGA nanoparticle synthesis
In the microfluidic solvent diffusion method, nanoparticles are synthesized in a microchannel after mixing between PLGA-acetone solution and water, following a three-dimensional hydrodynamic flow focusing (3D MHF) strategy.
In this approach, flow focusing squeezes the PLGA in acetone stream between water streams fully surrounding the PLGA phase and resulting in rapid solvent exchange via diffusion and PLGA nanoparticles precipitation (9). Particle formation takes place spontaneously at the nucleation spots that are distributed through the mixture Figure 1 (10).
The reagents and precipitating NPs are isolated from the channel walls, minimizing aggregation and/or clogging. In addition, by constraining the sample stream in the center of the microchannel- where flow velocity reaches the maximum with less variation- the 3D focused sample stream is expected to have a uniform width and thus improve the uniformity of the solvent/non-solvent ratio. This allows a robust and predictable nanoparticle synthesis, and facilitates the production of highly uniform nanoscale PLGA nanoparticles (12-14).
PLGA nanoparticle synthesis: Partial results
Fluigent has generated PLGA nanoparticles of different sizes by varying parameters related to our microfluidic system, thus establishing a relation between the diameter of the nanoparticle, the stream diameter, the flow rate ratio and the total flow rate.
Conclusion
PLGA nanoparticles as biocompatible nanocarriers represent one of the most innovative, non-invasive approaches for drug delivery applications. However, their targeting functions are largely affected by size. In the case of tumor targeting and drug delivery, currently the commonly recognized size range for PLGA nanoparticles is 100-300 nm, as it allows for the correct targeting of nanoparticles to the desired tissue (18).
PLGA nanoparticle size may be controlled by tuning the synthesis method and parameters of operation.
In this application note, we have demonstrated the PLGA nanoparticle synthesis using a microfluidic system (3D microfluidic hydrodynamic flow) consisting of pressure-based flow controllers and the RayDrop™ microfluidic device with standard configuration.
PLGA nanoparticles ranging from 110 to 250 nm were generated. This size range is optimal for various biological applications, such as tumor targeting, as it falls within the compatible size range. The Polydispersity Index (PDI) ranges from 0.05 to 0.1. Sizes can be adjusted by controlling the device flow input parameters, particularly the flow rate ratio (FRR). In this way, the ability to synthesize PLGA nanoparticles in a more controllable and reproducible way creates possibilities for custom tuning surface properties.
A full-featured, cost-effective and readily available platform for the on-demand production of monodisperse PLGA nanoparticles is now available. This allows for control of nanoparticle size and frequency by adjusting flow parameters.
Resources & Expertises
References
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