Microfluidic Drug Discovery 

Flow Control Performance and Enhanced Automation for Microfluidic Drug Discovery Applications

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A University College London (UCL) Paper 

Paper: Awwad, S.; Ibeanu, N.; Liu, T.; Velentza-Almpani, A.; Chouhan, N.; Vlatakis, S.; Khaw, P. T.; Brocchini, S.; Bouremel, Y. Real-Time Monitoring Platform for Ocular Drug Delivery. Pharmaceutics 2023, 15 (5), 1444. https://doi.org/10.3390/pharmaceutics15051444 

Part of the Faculty of Brain Sciences, the UCL Institute of Ophthalmology holds the top global ranking for ophthalmology studies (per CWUR Rankings by Subject 2017). Collaborating with Moorfields Eye Hospital, they form the largest co-located hub for eye research, education, and care worldwide. Through groundbreaking research in ophthalmology and eye health, their interdisciplinary approach unites scientists, clinicians, and patients, leading to tangible improvements in people’s lives. 

In collaboration with UCL, Optceutics Ltd.  manufactures advanced in vitro models of the human eye (PK-Eye™) that accelerate the development of longer-acting intraocular medicines. Optceutics Ltd. offers fee-for-service research and strategic collaborations, specializing in preclinical ophthalmic formulation optimization. 

PK-Eye™ Model: Example of Real-Time Monitoring Platform for Ocular Drug Development.  

The PK-Eye™ model, designed and manufactured by Optceutics Ltd., emerged as a robust and user-friendly tool, addressing the limitations of in vivo models for developing long-acting intraocular medicines. Combining this in vitro model with real-time monitoring systems will facilitate more efficient preclinical testing by increasing data collection while minimizing manual intervention.[8]  

In a paper published in Pharmaceutics (2023), a newly designed real-time monitoring platform was scaled and automated for ocular drug delivery. This platform aims to provide proof-of-concept for a fully automated and optimized PK-Eye™ model testing setup, to accelerate intraocular drug development by streamlining sample analysis and improving accuracy (figure 3). 

 Key features of the platform included monitoring flow, temperature, eye movements, and concentration evaluation of labeled protein molecules (figure 4). 

Figure 3: Schematic of the PK-Eye™ model.[8] 

Figure 4: Schematic of the PK-Eye™ platform including Fluigent’s FlowEZ.[7] 

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I. Droplet-based microfluidics, a microfluidic chip application

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 microfluidic chip application 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 

Microfluidic Single Emulsion Device

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Easy droplet generation chip

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With these characteristics, droplet microfluidics has a large value, including bio(chemical) analysis, and nano and microscale generation of materials⁷. Several microfluidic chip designs exist to generate droplets for a various field of applications. 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). 

Application of microfluidic chip using 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 Digital^TM 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 

Microfluidic chip application 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) 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 application chip 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 application of microfluidic chip method, PLGA microparticles ranging from 15 to 50 µm diameters have been successfully generated. 

Microfluidic Single Emulsion Device

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Microfluidic Double Emulsion Device

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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. 

A microfluidic chip application for 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 microfluidic chip applications an excellent approach 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 microfluidic chip application methods is Drop-Seq technology, which entraps a single cell and a single primer-barcoded bead in each droplet (figure 4). 

PDMS Drop-seq chip for Drop-seq experiments

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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 microfluidic chip application 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. 

III. Organ on a chip, a cutting edge application of microfluidic 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 application of microfluidic chip 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 application setup 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 model to capture organ-level functions of the human lung¹⁹.

Microfluidic chip applications enable 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 applications platforms overcome many drawbacks encountered with conventional tissue culture models. OOAC engineering microfluidic chip application 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 design to reconstitute 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 microfluidic chip application 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).

In fact, the device recreates physiological breathing movements (shown in Figure 8.B) by applying vacuum to the side chambers and causing mechanical stretching of the PDMS membrane forming the alveolar-capillary barrier. The device is made of 3 PDMS layers that are bonded to form two sets of three parallel microchannels. (Figure 8.C) PDMS etchant is flowed through the side channels in order to form the two large side chambers. (Figure 8.D). Figure 8.E represents an image of an actual lung-on-a-chip microfluidic device¹⁹.

To put it in a nutshell, air is subsequently introduced into the compartment to create an air-liquid interface to mimic the lining of the alveolar air space¹⁹ .

Air Liquid Interface Cell culture versatile chip 

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Using this microfluidic chip application platform, the authors demonstrated that breathing motions, simulated by the organ-on-chip 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. 

V. Micromixers an application using microfluidic chips

Micromixers play a crucial role in lab-on-chip devices for various microfluidic chip applications, including drug delivery, sequencing, amplification, and biochemical reactions. They can be broadly classified into two categories based on the actuation method: passive and active. 

Passive mixing relies on the microfluidic chip’s geometry and fluid properties without external sources. In laminar flow, typical in microfluidics, mixing primarily occurs through diffusion. This property allows for precise tuning of mixing by employing lamination, where two or more liquids flow in parallel, enabling diffusion to take place. For emproved and faster mixing, chaotic advection can be induced by modifying the microfluidic chip’s geometry, altering channel shapes for splitting, folding, stretching, or disrupting fluid flow. 

On the other hand, active mixing involves external perturbation. Various methods are employed in the microfluidic chip application field for active mixing. Dielectrophoresis mixing uses an electric field to move particles toward or away from an electrode, creating chaotic advection. Acoustic wave energy can also mix fluids by generating strong acoustic waves that interfere with each other, creating advection ²⁴. Additionally, adjusting the microfluidic chamber temperature can enhance mixing, as the diffusion coefficient of a liquid is temperature-dependent²⁵. 

Submillisecond organic synthesis using a serpentine-shaped microfluidic chip application 

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. The conceptual scheme of the 3D serpentine microchannel fabricated by lamination is represented in figure 10.A.

The figure 10.B represents a detailed scheme for a nanoliter reaction space of rectangular 3D serpentine channels with three inlets and the optical image showing from the top the nanoliter reaction space schematized in B. The optical images of respectively the chip reactor module and assembly are illustrated in Figure 10.D and E.  

The utilization of this platform for the application enabled submillisecond mixing. 

VII. Wearable microfluidics: an emerging application

An emerging application of microfluidic chip, 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 of the microfluidic chip, 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 

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. 

References

1. Seemann, R., Brinkmann, M., Pfohl, T. & Herminghaus, S. Droplet based microfluidics. Reports Prog. Phys. 75, (2012). 

2. Paquin, F., Rivnay, J., Salleo, A., Stingelin, N. & Silva, C. Droplet Control Technologies for Microfluidic High Throughput Screening (µHTS). Muhsincan Sesen,a Tuncay Alan,a and Adrian Neild∗a 10715–10722 (2017) doi:10.1039/b000000x. 

3. Galas, J. C., Bartolo, D. & Studer, V. Active connectors for microfluidic drops on demand. New J. Phys. 11, (2009). 

4. Jullien, M.-C., Tsang Mui Ching, M.-J., Cohen, C., Menetrier, L. & Tabeling, P. Droplet break in a low capillary T-junction. in 19th Mechanical French Congress (AFM, Maison de la Mécanique, 39/41 rue Louis Blanc-92400 Courbevoie, 2009). 

5. Yu, L., Chen, M. C. W. & Cheung, K. C. 2010 Droplet-based microfluidic system for multicellular tumor spheroid formation and anticancer drug testing. Lab Chip 10, 2424–2432 (2010). 

6. N.Shembekar, C.Chaipan, R. U. & C. A. M. 2016 Droplet-based microfluidics in drug discovery, transcriptomics and high-throughput molecular genetics. Lab Chip (2016) doi:10.1039/C6LC00249H. 

7. Shang, L., Cheng, Y. & Zhao, Y. Emerging Droplet Microfluidics. Chem. Rev. 117, 7964–8040 (2017). 

8. Suea-Ngam, A., Howes, P. D., Srisa-Art, M. & Demello, A. J. Droplet microfluidics: From proof-of-concept to real-world utility? Chem. Commun. 55, 9895–9903 (2019). 

9. Vladisavljević, G. T., Al Nuumani, R. & Nabavi, S. A. Microfluidic production of multiple emulsions. Micromachines 8, (2017). 

10. Soppimath, K. S. & Aminabhavi, T. M. Ethyl acetate as a dispersing solvent in the production of poly(DL-lactide-co-glycolide) microspheres: Effect of process parameters and polymer type. J. Microencapsul. 19, 281–292 (2002). 

11. Qi, F., Wu, J., Li, H. & Ma, G. Recent research and development of PLGA / PLA microspheres / nanoparticles : A review in scienti fi c and industrial aspects. (2018). 

12. Anderson, J. M. & Shive, M. S. Biodegradation and biocompatibility of PLA and PLGA microspheres. Adv. Drug Deliv. Rev. 64, 72–82 (2012). 

13. Macosko, E. Z. et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161, 1202–1214 (2015). 

14. Halldorsson, S., Lucumi, E., Gómez-Sjöberg, R. & Fleming, R. M. T. Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices. Biosens. Bioelectron. 63, 218–231 (2015). 

15. Yeo, L. Y., Chang, H. C., Chan, P. P. Y. & Friend, J. R. Microfluidic devices for bioapplications. Small 7, 12–48 (2011). 

16. Coluccio, M. L. et al. Microfluidic platforms for cell cultures and investigations. Microelectron. Eng. 208, 14–28 (2019). 

17. Ayuso, J. M. et al. Development and characterization of a microfluidic model of the tumour microenvironment. Sci. Rep. 6, 1–16 (2016). 

18. Caballero, D. et al. Organ-on-chip models of cancer metastasis for future personalized medicine: From chip to the patient. Biomaterials 149, 98–115 (2017). 

19. Huh, D. et al. Reconstituting organ-level lung functions on a chip. Science (80-. ). 328, 1662–1668 (2010). 

20. Bhise, N. S. et al. Organ-on-a-chip platforms for studying drug delivery systems. J. Control. Release 190, 82–93 (2014). 

21. Zhang, B., Korolj, A., Lai, B. F. L. & Radisic, M. Advances in organ-on-a-chip engineering. Nat. Rev. Mater. 3, 257–278 (2018). 

22. C. Wyatt Shields IV, Dr. Catherine D. Reyes, and P. G. P. L. Microfluidic Cell Sorting: A Review of the Advances in the Separation of Cells from Debulking to Rare Cell Isolation. Lab Chip (2015) doi:10.1039/c4lc01246a. 

23. Kuntaegowdanahalli, S. S., Bhagat, A. A. S., Kumar, G. & Papautsky, I. Inertial microfluidics for continuous particle separation in spiral microchannels. Lab Chip 9, 2973–2980 (2009). 

24. Vivek, V., Zeng, Y. & Kim, E. S. NOVEL ACOUSTIC-WAVE MICROMIXER. 

25. Ward, K. & Fan, Z. H. Mixing in microfluidic devices and enhancement methods. J. Micromechanics Microengineering 25, (2015). 

26. Kim, H. et al. Submillisecond organic synthesis: Outpacing Fries rearrangement through microfluidic rapid mixing. Science (80-. ). 352, 691–694 (2016). 

27. Thorsen, T., Maerkl, S. J. & Quake, S. R. Microfluidic large-scale integration. Science (80-. ). 298, 580–584 (2002). 

28. Aditya Aryasomayajula, Pouriya Bayat, Pouya Rezai, P. R. S. Microfluidic Devices and Their Applications. Springer vol. 50 (2017). 

29. Jensen, E. C., Bhat, B. P. & Mathies, R. A. A digital microfluidic platform for the automation of quantitative biomolecular assays. Lab Chip 10, 685–691 (2010). 

30. Vincent, M., Xu, Y. & Kong, H. Helicase-dependent isothermal DNA amplification. EMBO Rep. 5, 795–800 (2004). 

31. Erik C. Jensen, Yong Zeng, Jungkyu Kim, and R. A. M. Microvalve Enabled Digital Microfluidic Systems for High Performance Biochemical and Genetic Analysis. 15, 455–463 (2011). 

32. Kim, J., Jensen, E. C., Stockton, A. M. & Mathies, R. A. Universal microfluidic automaton for autonomous sample processing: Application to the mars organic analyzer. Anal. Chem. 85, 7682–7688 (2013). 

33. Oh, K. W. & Ahn, C. H. A review of microvalves. J. Micromechanics Microengineering 16, (2006). 

34. Kim, J., Stockton, A. M., Jensen, E. C. & Mathies, R. A. Pneumatically actuated microvalve circuits for programmable automation of chemical and biochemical analysis. Lab Chip 16, 812–819 (2016). 

35. Chin, V. I. et al. Microfabricated platform for studying stem cell fates. Biotechnol. Bioeng. 88, 399–415 (2004). 

36. Yeo, J. C., Kenry & Lim, C. T. Emergence of microfluidic wearable technologies. Lab Chip 16, 4082–4090 (2016). 

37. Ahyeon Koh, Daeshik Kang, Yeguang Xue, Seungmin Lee, Rafal M. Pielak, Jeonghyun Kim, Taehwan Hwang, Seunghwan Min,1 Anthony Banks, Philippe Bastien, Megan C. Manco, Liang Wang, Kaitlyn R. Ammann, Kyung-In Jang, Phillip Won Seungyong Han, Roozbeh Ghaffari, J. A. R. A soft, wearable microfluidic device for the capture, storage, and colorimetric sensing of sweat. Sci. Transl. Med. 26, 39–46 (2017). 

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 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 

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

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.

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Omi, the new automated organ-on-chip platform

Discover Omi, the new automated platform for OOAC applications developed by Fluigent. Equipped with state-of-the-art technologies, this platform will enable you to carry out any perfusion protocol. It has the ability to customize and automate any protocol, including simple perfusion, recirculation, sampling and injection. It meets the needs of beginners in organ-on-chip cell culture research and advanced organ-on-chip researchers looking for automation and reproducibility. 

This versatile, automated organ-on-a-chip platform can perform long-term OOAC cell cultures under flow to control shear stress conditions. Its two-hour battery life and WiFi connectivity ensure easy, intuitive control. It can be easily transported from incubator to microscope for live cell imaging while maintaining cell perfusion under battery power. You can also monitor your experiment from anywhere using the Omi app. 

Towards the next generation of organ-on-a-chip cell culture platforms 

Read our expertise page to learn more about the benefits of flow control in cell culture and about our products and those of our partners (Beonchip), and to reach out to our team of experts to find the solution that best meets your needs.