Microfluidic encapsulation of mesenchymal stem cells in alginate hydrogels: Mesenchymal stem cells (MSCs) are a type of stem cells derived from bone marrow, umbilical cord and a few other tissues. Due to their wide therapeutic potential in tissue regeneration and immunoregulation, MSCs are currently regarded as the most promising candidate for new stem cell therapy. Some clinical experiments have shown promising results when using MSCs to treat systemic immune diseases. Despite their outstanding performance in some studies, consistent efficacy of MSC therapy is still hard to achieve. One of the biggest challenges that hinders the approval of MSC therapy is the short persistence time of MSCs in the host body. After administration in the host body, MSCs are usually lost within 2-3 days. This early cell loss is due to the lack of proper mechanical signaling during the transplantation process, as well as the attack of the host’s immune system. To overcome these problems, we develop microfluidics-based live cell encapsulation strategies to provide the injected cells with an extracellular substrate which at the same time can serve as a layer of protection against the host’s immune system. According to the required administration route, we can perform single- or multi-cell encapsulation in hydrogels of various structures and mechanical properties. Subcutaneous injection of MSC-encapsulating alginate microcapsules made with our method have shown promising persistence time in mouse experiments. Anqi Chen

Photodegradable hydrogel capsules for cell sorting: Hydrogel capsules are core-shell structures that have a liquid core encased in a solid shell. The shell is made from a semi-permeable hydrogel, allowing for the separation of the encapsulated targets while still permitting the exchange of small molecules with the surrounding environment. These capsules are excellent for isolating cells within picoliter- or nanoliter-sized compartments, which facilitates culturing and selection at the individual cell level. Using microfluidic techniques, these capsules can be produced in the millions, making them ideal for high-throughput screening. The durability of the hydrogel shell also supports cell sorting using standard commercial cell sorters. Once sorted, cells within the capsule can be safely released through brief UV exposure without any damage. Wentao Xu

Project abstract: Nanoparticles for improved delivery efficiency and enhanced therapy effects:  Drug delivery vehicles, such as nanoparticles, micelle, and vesicles play an important role in protecting the drugs and achieving the best treatment effect. Our research mainly focuses on designing nanoparticle carriers for improving drug delivery efficiency. There are many factors that influence the transfection efficiency of the drug, such as the nanoparticle’s morphology, composition, shape, and stiffness. We use microfluidic nano participation to design nanoparticles with uniform size and shape, such as the sphere, dimer, and trimer nanoparticles, The morphology of nanoparticles is modified by antibody, protein, and polysaccharide to improve the cell uptake efficiency. We investigate the relationship between the transfection efficiency of drugs and the design of nanoparticles. These nanoparticles with tunable shapes will provide an important platform to load diverse drugs for improved drug delivery and enhanced clinical therapy. Chenjing Yang, Chunhuan Liu, Tiffany Chen

Cell Behavior at Bone-to-Tendon interface in 3D Cell Culture: At the enthesis, because tendon cells have high alignment in a single direction, while bone cells have no specific orientation, the shared cellular environment is naturally anisotropic.  In this work, a hydrogel scaffold with tunable stiffness is created using UV and ionically curable polymer solutions.  Using cellulose fibers to implement alignment, crosslinked polymer networks with modulus and alignment gradients can be formed to simulate the complexity of the tendon-bone interface. The scaffolds are seeded with tendon and bone cells to monitor the formation of the natural ECM in response to mechanical and structural changes.  By studying this system in a 3D matrix as opposed to the commonly used 2D model, it is possible to probe cell proliferation, migration, and viability as a function of hydrogel mechanical properties in an environment with a closer resemblance to the native extracellular matrix.  This work aids in determination of the critical factors (matrix modulus, pore size, anisotropy, growth factor localization) that affect cellular response and subsequent bone regeneration at the tendon-to-bone multicellular junction. Bobby Haney

Microparticles for oral and injectable drug delivery: Microcapsules are spherical structures composed of a semi-permeable shell encompassing a liquid core, typically ranging in diameter from a few microns to 1 mm. This core-shell architecture makes microcapsules an ideal carrier for a wide range of biologically active materials, including peptides, enzymes, RNA, cells, and other substances, with high loading capacity. The protective shell serves as an effective barrier, shielding the encapsulated active agents from harsh external environments. Moreover, the separation of the core and shell facilitates precise control over the release rate of the enclosed drug, enabling modulation over periods spanning from hours to months. Yang Wang

Diné tea extract-encapsulating microgels: microfluidics-based fabrication and their antibacterial effects：Navajo teas or herbs have been used by Native American people to treat a variety of ailments, however, there are currently no standardized extraction processes to obtain bioactives from the teas/herbs. Additionally, the unprotected tea/herb extracts have short shelf-stability and are susceptible to degradation under harsh environmental conditions, which limits the application of Navajo teas/herbs in pharmaceutical scenarios. Here we test different extraction conditions to prepare extracts from four types of teas/herbs, including Navajo tea, Yucca root, Juniper and Bear root and use their antibacterial effects as the selection criteria to determine the best extraction conditions. We then encapsulate the tea/herb extracts in microgels made from pectin, a biopolymer extracted from orange peels to test the stability of extracts. We show that all the tested teas and herbs inhibit the growth of Escherichia coli (E.coli). Teas/herbs boiled for a shorter time of 5 minutes have higher antibacterial effects compared to those boiled for 15 or 30 minutes. The encapsulation of teas/herbs in pectin microgels enhanced the antibacterial properties of bear root tea extracts and further effects on stability is undergoing. This work is in collaboration with students from Navajo Technical University. Yan Liu

Activity-Based Gelation of Microdroplets for Ultrahigh-throughput Directed Enzyme Evolution : Droplet microfluidics has revolutionized the field of directed evolution for enzyme optimization. By encapsulating single cells or molecules within picoliter droplets, high-throughput screening of vast libraries becomes feasible. In this system, each droplet acts as an isolated microreactor, allowing for the precise control of reaction conditions. This technology greatly accelerates the identification of improved enzyme variants, facilitating rapid enzyme engineering and the discovery of novel biocatalysts. Fluorescent assays are commonly used to link enzyme activity to fluorescence intensity, enabling the sorting of droplets containing mutants with desired properties. However, the rate of fluorescence-activated cell sorting (FACS), which is typically in the range of 0.1-1 kHz, is unable to match the rate of droplet formation which can readily exceed 100 kHz. In other words, an hour of droplet formation at 100 kHz can create up to 360 million microdroplets and it will take FACS more than four days to sort the desired droplets for the next round of enzyme evolution. This mismatch in speed results in a bottleneck in the throughput of droplet microfluidics-based directed enzyme evolution, especially when a large mutant library is needed. We are addressing this challenge by developing a selective gelation method based on enzymatic activities. Instead of using fluorescent assays, we take advantage of the chemical reporters resulting from the enzymatic activities which can be utilized as initiators for the polymerization and crosslinking of added monomers to form polymeric shells over the desired microdroplets. The remaining undesired droplets can be broken and removed from the mixture with bulk processing, essentially removing the speed limits imposed by sequential droplet sorting. Lennon Luo

Model System for Producing Extracellular Vesicles for Drug Delivery: Small membrane bound vesicles, typically smaller than a micron, are naturally secreted by cells in our body. These vesicles, known as extracellular vesicles, play a crucial role in intercellular communication by transporting, protecting, and releasing cargo into the cytoplasm of recipient cells. Their native composition and biological function have inspired their use in drug delivery.

Resembling cells themselves, they possess an asymmetrical bilipid membrane enclosing an aqueous core. This compositional asymmetry confers them more versatility and mechanical stability compared to commonly used vesicles for drug delivery that have a symmetrical lipid membrane. Their asymmetric structure, small size, and biocompatibility make them unique candidates in drug delivery systems that require efficient cellular uptake and release for therapeutic impact. However, their main challenge is to produce these structures efficiently and at a scale small enough to achieve therapeutic effects.

In our lab we use droplet microfluidics to produce these asymmetric structures. Droplet microfluidics can generate multilayered asymmetrical structures resembling vesicles through the creation of multilayered emulsions as templates. This method offers precise control over the size, layer thickness, and composition of each emulsion. By manipulating the composition of each layer, we can generate asymmetrical structures. Currently the field of microfluidics is well established at the micron-scale. The main challenge is to produce these structures on the sub-micron scale. Our lab has developed a new microfluidic device, that enables us to produce sub-micron emulsions that can be used as templates to produce asymmetrical structures that resemble extracellular vesicles. If you are interested in learning more, please feel free to reach out to me at any time. Ryan Garry

Development of biomimetic liposomes with cell membrane proteins for targeted delivery: Liposomes offer a popular platform for drug delivery. Conventional liposomes have a range of attractive properties, including having tunable surface properties and size. How to further endow liposomes more efficient and target?  Cell membrane protein-based biomimetic liposomes are designed to mimic natural cells properties and functions and replicate their behavior in vivo. We incorporated the cell membrane protein in the nano-size liposomes by using microfluidic chips. Biomimetic liposomes can retain the unique biological functions of the source cells. Thereby, they can overcome biological roadblocks and to enhance targeting specificity and efficient. We will study the effects of cell membrane protein on the formation and structure of biomimetic liposomes, especially surface, and their interactions with cells. Then we will evaluate the performance and targeting of biomimetic liposomes in drug delivery. Our project will give a meaningful perspective for targeted drug delivery model. Chunhuan Liu, Kevin Jahnke, Chenjing Yang, Tiffany Chen

Chip Design and Automation of Lipid Nanoparticle Synthesis for mRNA Vaccines: Lipid nanoparticles (LiNPs) provided a breakthrough for mRNA COVID-19 vaccine development due to their exceptional properties as a drug carrier, with the added potential for a highly tunable membrane depending on functionality and synthesis. However current lipid formations in commercial uses suffer from a poorly controlled drug release rate and is limited to select formulations, which can prevent small molecules or cell-therapeutics from reaching target areas. Libraries of theoretical composition are available which offer significant improvements, such as Schrödinger, but there is no efficient method to screen thousands of combinatorics of LiNPs in microfluidic chips and simultaneously manufacture droplets to test with other diagnostics such as dynamic light scattering. This project introduced a novel setup to interface between well-plates and microfluidic chips, automating the rapid mixing and synthesis of LiNP that would be cumbersome to test at small research scales. Chip designs for micromixers were optimized using COMSOL to efficiently mix at high throughputs. Beyond PDMS-glass substrate chips, additional work involving 2-photon lithography was conducted to create three dimensional PDMS structures, to utilize this new dimension to rapidly mix and scale droplet production. Suvin Sundararajan

Quadruple Emulsion Templated Multilamellar Phospholipid Vesicles: Multilamellar vesicles are promising delivery vehicles for multiple components of drugs or enzymes for pharmaceuticals and cosmetics. There have been reports of multiple polymersomes for encapsulation and programmed release. However, phospholipids as the natural component of biological membranes have not been successfully used for fabrication of multilamellar vesicles, mainly because of their fragility and small size. In this project we want to develop a microfluidic method to prepare liposomes with double bilayers through quadruple emulsion templates. The resultant biocompatible vehicle will have potential application for separated storage and controlled release of multiple components. Moreover, we also look forward to providing a platform to study physical properties of biomembranes with multilamellar liposome structures. Anqi Chen

Fatty amine encapsulation: Fatty amine is a widely used nonionic surfactant for water in oil emulsion. It’s also been added to the marine engine lubricant as the neutralizing active. It’s a type of very effective acid remover but it is instable in high temperature, like working temperature in the engine. We are interested in protecting the fatty amine from early degradation and controllably releasing it in acidic environment. By encapsulating fatty amine in pH-responsive microcapsules, it is possible to use the capsule shell as the physical barrier to separate the air and the inside fatty amine, thus delay the oxidative degradation. Hao Pei

Smart Janus capsules. Microscopic materials with anisotropic structures or compositions are of great interesting and potentials for various applications. My project focus on fabrication of Janus microcapsules with anisotropic structures or compositions by microfluidics, which could be applied to stimulating responsive, drug delivery and microactuators. Yong Zhao

Electrically-Controlled Programmable Microfluidics System: You need to prepare for 3 hours and you collect the droplets for 15 minutes, leave along the time designing in AutoCAD, making wafer, and if some are blocked you need to debug and do it again. Have you ever thought about “make it once, run it immediately”?  You can do it if you have valves that controllable and connect the elements you want it to. Unlike programmable microfluidics project in Stanford, I use electrically controllable valves that largely facilitate the portability of the device, when study today are all using magnetically controlled or vacuum controlled valves which need abundant hardware and limit its usage; Now we have a 3CM*3CM chip that connect to microfluidics chips and control 3*3 grid of valves by computer, as well as a activator grid for valves comprising 64 addressable electrodes served as valves actuators. The goal of it is to demonstrate an electrically controlled “generous purpose” system comprising elements such as cell trap, channels, mixer, and detection mechanism and connected by valve grid via ipad coding. Yanzhe Qin

Multilayer Polyelectrolyte Capsule Assembly in Microfluidic Device. In the classical Layer-by-Layer (LbL) technique, polyelectrolyte multi-layer capsules are fabricated by alternatively deposited charged polyelectrolytes onto an oppositely bulk or colloidal template followed by dissolved the template which is high consuming. Droplet-based microfluidics which involves the generation of producing high mono-dispersity drops offers a platform for miniaturizing LbL technique by imparting benefits of time and reagent reduction.  We present a novel deposition method by designing a new microfluidic coating device which utilizes “Z” shape channel to guide discrete droplets. Similar to the game of pinball in which the templates guided and diverted to the downstream direction smoothly by repeated unit rows of fabricated channels. Right now, we achieved four layers of polyelectrolyte deposition on a template in less than 2 minutes by guiding discrete templates through coating and washing solution of flushing and two polyelectrolytes. Liyuan Zhang

Fabrication of multilayered microfluidic devices and its application for double emulsions: Photolithography is an accurate, reproducible and easy method for fabricating micron scale devices. The basic outcome of Photolithography is a single layer, and it is possible to repeat the exposure process and end up with multilayered landscapes. However, some topologies are impossible to achieve using multiple exposures and require a complementary method of stacking up devices after fabrication. We use a simple method for aligning stacks of micron-scale devices that relies on matching locks and keys that are an inherent part of the device. Applications of multilayered devices are numerous. We focus here on the generation of double emulsions that are useful for instance in encapsulation (drug delivery) and particle synthesis. The formation of double emulsions in single layered microfluidic devices demands precise spatial control over surface properties of the device channels. However in the case of axial symmetric microfluidic devices such as capillaries, this is no longer a constraint since there is no direct contact between the double emulsion and the device surface. We show that forming double emulsions in multilayered microfluidic device is similar to their formation in capillaries, alleviating the need for precise spatial control of wettability. Improving the robustness of double emulsion formation in photolithographic devices is important for large scale uses of double emulsions. Assaf Rotem

Spatially Segregated Wettability in Microfluidic Devices and Scale-up: In order to make water-in-oil-in-water double emulsions with microfluidic devices, the channels of the device must have spatially segregated wettability – in other words the wettability should be such that the first junction of the device wets the shell phase, and the second junction wets the oil phase. We use tandem emulsification to achieve spatially segregated wettability, and also explore methods of achieving segregated wettability in thermoplastic devices through careful engineering of device geometry and functionalization techniques. We also scale-up the production of double emulsions with sizes close to the millimeter-range and with viscous shells, shown here. Saraf Nawar

Encapsulation of high-viscosity biologics and on-demand release. The goal of this project is to encapsulate highly viscous antibody for subcutaneous injection and to realize triggered release in human body environment. To do this, we develop a microfluidics approach to fabricate microcapsules based on water-in-oil-in-water double emulsion template. The biologics is encapsulated in biocompatible and photocurable polymer, which transforms into crosslinked polymeric shells upon UV illumination. By tuning the flowrate of middle phase and inner phase, we are able to fabricate microcapsules with non-uniform shell thickness. The structural inhomogeneities lead to inhomogeneous deformation in shell upon external osmotic pressure change, enabling capsule rupture at the thinnest part of the shell. Liangliang Qu

Encapsulation of neutralizing agents for marine lubricants. Fatty amines (FAs) are used as effective acid neutralizing agents in marine lubricants, however they suffer thermal degradation in marine engine. The goal of this project is to protect FAs from early oxidation and thermal degradation and release them when entering into contact with organic acid. The challenge of encapsulating FAs is that FAs prefer to stay at interface due to very low surface tension. Our strategy is to trap FAs in the crosslinked polymer network as a core and generate protective shells by using pH-responsive polymer. Liangliang Qu

Reversibly responsive and selective microcapsules: Microcapsules have been established as efficient carrier vehicles for sensitive actives. Capsules with radii of 10s-100s of microns and thin shells offer a large cargo space while utilizing only a small amount of encapsulant. Many encapsulation systems are designed for single use and one way applications only, however. We develop microcapsule based encapsulation systems that allow for repeated uptake and release of cargo utilizing responsive and non-destructive permeability change of the capsule shell. We achieve this by using reversibly stimuli-responsive functional polymer shells, such as pH-responsive block copolymers, hydrogels, etc. The responsive nature of the polymers is complemented with selectivity by morphological and chemical design, allowing for discrimination of permeates by charge, size, or chemical functionality, leading to encapsulation systems with application areas far beyond the traditional ones, including purification and separation. Joerg Werner

OEG-based thermoresponsive microparticles by microfluidics: Polymeric microgels and microcapsules with temperature triggered property changes have promising applications in the fields of controlled release, cell laden, cosmetics, etc. Till now, PNiPAM is the mostly used polymer for thermoresponsive microparticles fabrication by microfluidics. However, the irreversible phase transition and the ambiguous biocompatibility due to the presence of amide groups in its structures limit its applications. As an alternative, oligoethylene glycol (OEG)-based dendronized polymers have several superior characteristics, including good biocompatibility, excellent antifouling property, fully reversible thermoresponsiveness, tunable LCSTs and switchable shielding effect, which will be used for microparticles fabrication in this project. We aim to develop novel thermoresponsive particles with excellent properties for materials applications. Wen Li

Ultrahigh drug loaded carriers: An advanced approach that can precisely control over the preparation process to generate homogeneously size distributed particles with ultrahigh mass fraction of therapeutics, controlled payload release profiles, as well as high throughput productivity is strongly desirable. With the help of microfluidics, I am aiming to develop a versatile and robust approach to efficiently synthesize optimal core/shell structured vectors. After characterization, the obtained optimal vectors will be utilized for cancer therapy or spinal cord injury treatment. Dongfei Liu