Effective Light Directed Assembly of Building Blocks with Microscale Control.

Light-directed forces have been widely used to pattern micro/nanoscale objects with precise control, forming functional assemblies. However, a substantial laser intensity is required to generate sufficient optical gradient forces to move a small object in a certain direction, causing limited throughput for applications. A high-throughput light-directed assembly is demonstrated as a printing technology by introducing gold nanorods to induce thermal convection flows that move microparticles (diameter = 40 µm to several hundreds of micrometers) to specific light-guided locations, forming desired patterns. With the advantage of effective light-directed assembly, the microfluidic-fabricated monodispersed biocompatible microparticles are used as building blocks to construct a structured assembly (≈10 cm scale) in ≈2 min. The control with microscale precision is approached by changing the size of the laser light spot. After crosslinking assembly of building blocks, a novel soft material with wanted pattern is approached. To demonstrate its application, the mesenchymal stem-cell-seeded hydrogel microparticles are prepared as functional building blocks to construct scaffold-free tissues with desired structures. This light-directed fabrication method can be applied to integrate different building units, enabling the bottom-up formation of materials with precise control over their internal structure for bioprinting, tissue engineering, and advanced manufacturing.

Near-infrared light responsive multi-compartmental hydrogel particles synthesized through droplets assembly induced by superhydrophobic surface.

Light-responsive hydrogel particles with multi-compartmental structure are useful for applications in microreactors, drug delivery and tissue engineering because of their remotely-triggerable releasing ability and combinational functionalities. The current methods of synthesizing multi-compartmental hydrogel particles typically involve multi-step interrupted gelation of polysaccharides or complicated microfluidic procedures with limited throughput. In this study, a two-step sequential gelation process is developed to produce agarose/alginate double network multi-compartmental hydrogel particles using droplets assemblies induced by superhydrophobic surface as templates. The agarose/alginate double network multi-compartmental hydrogel particles can be formed with diverse hierarchical structures showing combinational functionalities. The synthesized hydrogel particles, when loaded with polypyrrole (PPy) nanoparticles that act as photothermal nanotransducers, are demonstrated to function as near-infrared (NIR) light triggerable and deformation-free hydrogel materials. Periodic NIR laser switching is applied to stimulate these hydrogel particles, and pulsatile release profiles are collected. Compared with massive reagents released from single-compartmental hydrogel particles, more regulated release profiles of the multi-compartmental hydrogel particles are observed.

Layer-by-layer polyelectrolyte-polyester hybrid microcapsules for encapsulation and delivery of hydrophobic drugs.

A two-step process is developed to form layer-by-layer (LbL) polyelectrolyte microcapsules, which are able to encapsulate and deliver hydrophobic drugs. Spherical porous calcium carbonate (CaCO3) microparticles were used as templates and coated with a poly(lactic acid-co-glycolic acid) (PLGA) layer containing hydrophobic compounds via an in situ precipitation gelling process. PLGA layers that precipitated from N-methyl-2-pyrrolidone (NMP) had a lower loading and smoother surface than those precipitated from acetone. The difference may be due to different viscosities and solvent exchange dynamics. In the second step, the successful coating of multilayer polyelectrolytes poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS) onto the PLGA coated CaCO3 microparticles was confirmed with AFM and ζ-potential studies. The release of a model hydrophobic drug, ibuprofen, from these hybrid microcapsules with different numbers of PAH/PSS layers was investigated. It was found that the release of ibuprofen decreases with increasing layer numbers demonstrating the possibility to control the release of ibuprofen with these novel hybrid microcapsules. Besides loading of hydrophobic drugs, the interior of these microcapsules can also be loaded with hydrophilic compounds and functional nanoparticles as demonstrated by loading with Fe3O4 nanoparticles, forming magnetically responsive dual drug releasing carriers.

Surface functionalization of nanoparticles to control cell interactions and drug release.

Nanoparticles made from poly(dl-lactide-co-glycolide) (PLGA) are used to deliver a wide range of bioactive molecules, due to their biocompatibility and biodegradability. This study investigates the surface modification of PLGA nanoparticles via the layer-by-layer (LbL) deposition of polyelectrolytes, and the effects of these coatings on the release behavior, cytotoxicity, hemolytic activity, and cellular uptake efficiency. PLGA nanoparticles are modified via LbL adsorption of two polyelectrolyte pairs: 1) poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS) and 2) poly(L-lysine hydrobromide) (PLL) and dextran sulfate (DES). It is demonstrated that both PAH/PSS and PLL/DES coatings suppress the burst release usually observed for unmodified PLGA nanoparticles and that the release behavior can be adjusted by changing the layer numbers, layer materials, or by crosslinking the layer constituents. Neither bare nor polyelectrolyte-modified PLGA nanoparticles show any signs of cytotoxicity. However, nanoparticles with a positively charged polyelectrolyte as the outermost layer induce hemolysis, whereas uncoated particles or particles with a negatively charged polyelectrolyte as the outermost layer show no hemolytic activity. Furthermore, particles with either PAH or PLL as the outermost layer also demonstrate a higher uptake efficiency by L929 fibroblast cells, due to a higher cell-particle affinity. This study suggests that LbL coating of PLGA nanoparticles can control the release behavior of bioactive molecules as well as the surface activity, therefore providing a promising strategy to enhance the efficiency of nanoparticulate drug-delivery systems.

Engineering of erythrocyte-based drug carriers: control of protein release and bioactivity.

This work reports the fabrication of layer-by-layer (LbL) polyelectrolyte coated erythrocyte carriers that provide a simple means for controlling the burst and subsequent release of lysozyme. Erythrocytes were loaded with RITC-lysozyme as model compound via the hypotonic dialysis method. An encapsulation efficiency of 41.6% and a loading amount of 12.7 pg/cell was achieved. It is demonstrated that these carriers maintain their shape and integrity similar to natural erythrocytes after the encapsulation procedures, and achieve a uniform distribution of the encapsulated lysozyme. The erythrocyte carriers were fixed with glutaraldehyde and then successfully coated with biocompatible polyelectrolytes, poly-L: -lysine hydrobromide and dextran sulfate, using the LbL method. It is demonstrated that the release profile of the encapsulated macromolecule can be regulated by adjusting the number of polyelectrolyte layers. Furthermore by adjusting the concentrations of the cross linking agent the activity of the encapsulated lysozyme can be well preserved. These core-shell microcapsules, consisting of erythrocytes loaded with bioactive substances and coated with a polyelectrolyte multilayer shell, hold promise for a new type of biocompatible and biodegradable drug delivery system.

Application of (R)-3-hydroxyalkanoate methyl esters derived from microbial polyhydroxyalkanoates as novel biofuels.

Microbial polyhydroxyalkanoates (PHA) were proposed for the first time as a new type of biofuel. In this paper, poly-R-3-hydroxybutyrate (PHB) and medium chain length PHA (mcl PHA) were, respectively, esterified to become R-3-hydroxybutyrate methyl ester (3HBME) and medium chain length hydroxyalkanoate methyl ester (3HAME) via acid-catalyzed hydrolysis. The recovery percentages of 3HBME and 3HAME were 52 and 65%, respectively. The purities of 3HBME and 3HAME were 97 and 96%, respectively. Combustion heats of 3HBME, 3HAME, ethanol, n-propanol, n-butanol, 0(#) diesel, 90(#) gasoline, and 3HBME-based and 3HAME-based blended fuels were investigated and compared, respectively. It was found that 3HBME and 3HAME had combustion heats valuing 20 and 30 KJ/g, respectively. Ethanol has a combustion heat of 27 KJ/g, while addition of 10% 3HBME or 3HAME enhanced the combustion heat of ethanol to 30 and 35 KJ/g, respectively. The addition of 3HBME or 3HAME into n-propanol and n-butanol led to a slight reduction of their combustion heats. Combustion heats of blended fuels 3HBME/diesel or 3HBME/gasoline and of 3HAME/diesel or 3HAME/gasoline were lower than that of the pure diesel or gasoline. It was roughly estimated that the production cost of PHA-based biofuels should be around US$1200 per ton.

Fast-responsive hydrogel as an injectable pump for rapid on-demand fluidic flow control.

Chemically synthesized functional hydrogels have been recognized as optimized soft pumps for on-demand fluidic regulation in micro-systems. However, the challenges regarding the slow responses of hydrogels have very much limited their application in effective fluidic flow control. In this study, a heterobifunctional crosslinker (4-hydroxybutyl acrylate)-enabled two-step hydrothermal phase separation process for preparing a highly porous hydrogel with fast response dynamics was investigated for the fabrication of novel microfluidic functional units, such as injectable valves and pumps. The cylinder-shaped hydrogel, with a diameter of 9 cm and a height of 2.5 cm at 25 °C, achieved a size reduction of approximately 70% in less than 30 s after the hydrogels were heated at 40 °C. By incorporating polypyrrole nanoparticles as photothermal transducers, a photo-responsive composite hydrogel was approached and exhibited a remotely triggerable fluidic regulation and pumping ability to generate significant flows, showing on-demand water-in-oil droplet generation by laser switching, whereby the droplet size could be tuned by adjusting the laser intensity and irradiation period with programmable manipulation.

Heterogeneous multi-compartmental hydrogel particles as synthetic cells for incompatible tandem reactions.

In nature, individual cells contain multiple isolated compartments in which cascade enzymatic reactions occur to form essential biological products with high efficiency. Here, we report a cell-inspired design of functional hydrogel particles with multiple compartments, in which different enzymes are spatially immobilized in distinct domains that enable engineered, one-pot, tandem reactions. The dense packing of different compartments in the hydrogel particle enables effective transportation of reactants to ensure that the products are generated with high efficiency. To demonstrate the advantages of micro-environmental modifications, we employ the copolymerization of acrylic acid, which leads to the formation of heterogeneous multi-compartmental hydrogel particles with different pH microenvironments. Upon the positional assembly of glucose oxidase and magnetic nanoparticles, these hydrogel particles are able to process a glucose-triggered, incompatible, multistep tandem reaction in one pot. Furthermore, based on the high cytotoxicity of hydroxyl radicals, a glucose-powered therapeutic strategy to kill cancer cells was approached.Cells contain isolated compartments where cascade enzymatic biochemical reactions occur to form essential biological products with high efficiency. Here the authors produce functional hydrogel particles with multiple compartments via microfluidics that contain spatially immobilized natural enzymes in distinct domains for one-pot, tandem reactions.

Production of Hollow Bacterial Cellulose Microspheres Using Microfluidics to Form an Injectable Porous Scaffold for Wound Healing.

Bacterial cellulose (BC) is a biocompatible material with high purity and robust mechanical strength used to fabricate desirable scaffolds for 3D cell culture and wound healing. However, the chemical resistance of BC and its insolubility in the majority of solutions make it difficult to manipulate using standard chemical methods. In this study, a microfluidic process is developed to produce hollow BC microspheres with desirable internal structures and morphology. Microfluidics is used to generate a core-shell structured microparticle with an alginate core and agarose shell as a template to encapsulate Gluconacetobacter xylinus for long-term static culture. G. xylinus then secretes BC, which becomes entangled within the shell of the structured hydrogel microparticles and forms BC microspheres. The removal of the hydrogel template via thermal-chemical treatments yields robust BC microspheres exhibiting a hollow morphology. These hollow microspheres spontaneously assemble as functional units to form a novel injectable scaffold. In vitro, a highly porous scaffold is created to enable effective 3D cell culture with a high cell proliferation rate and better depth distribution. In vivo, this injectable scaffold facilitates tissue regeneration, resulting in rapid wound-healing in a Sprague Dawley rat skin model.

A one-step hydrothermal route to programmable stimuli-responsive hydrogels.

A hydrothermal route to regulate the swelling and responsiveness properties of poly(N-isopropylacrylamide) (PNIPAM) hydrogels is reported. During the process, water is the only reactive medium used, and the hydrogel properties can be programmed effectively.

Sustained release of hydrophobic drugs by the microfluidic assembly of multistage microgel/poly (lactic-co-glycolic acid) nanoparticle composites.

The poor solubility of many newly discovered drugs has resulted in numerous challenges for the time-controlled release of therapeutics. In this study, an advanced drug delivery platform to encapsulate and deliver hydrophobic drugs, consisting of poly (lactic-co-glycolic acid) (PLGA) nanoparticles incorporated within poly (ethylene glycol) (PEG) microgels, was developed. PLGA nanoparticles were used as the hydrophobic drug carrier, while the PEG matrix functioned to slow down the drug release. Encapsulation of the hydrophobic agents was characterized by fluorescence detection of the hydrophobic dye Nile Red within the microgels. In addition, the microcomposites prepared via the droplet-based microfluidic technology showed size tunability and a monodisperse size distribution, along with improved release kinetics of the loaded cargo compared with bare PLGA nanoparticles. This composite system has potential as a universal delivery platform for a variety of hydrophobic molecules.

Remote modulation of neural activities via near-infrared triggered release of biomolecules.

The capability to remotely control the release of biomolecules provides an unique opportunity to monitor and regulate neural signaling, which spans extraordinary spatial and temporal scales. While various strategies, including local perfusion, molecular "uncaging", or photosensitive polymeric materials, have been applied to achieve controlled releasing of neuro-active substances, it is still challenging to adopt these technologies in many experimental contexts that require a straightforward but versatile loading-releasing mechanism. Here, we develop a synthetic strategy for remotely controllable releasing of neuro-modulating molecules. This platform is based on microscale composite hydrogels that incorporate polypyrrole (PPy) nanoparticles as photo-thermal transducers and is triggered by near-infrared-light (NIR) irradiation. Specifically, we first demonstrate the utility of our technology by recapitulating the "turning assay" and "collapse assay", which involve localized treatment of chemotactic factors (e.g. Netrin or Semaphorin 3A) to subcellular neural elements and have been extensively used in studying axonal pathfinding. On a network scale, the photo-sensitive microgels are also validated for light-controlled releasing of neurotransmitters (e.g. glutamate). A single NIR-triggered release is sufficient to change the dynamics of a cultured hippocampal neuron network. Taking the advantage of NIR's capability to penetrate deep into live tissue, this technology is further shown to work similarly well in vivo, which is evidenced by synchronized spiking activity in response to NIR-triggered delivery of glutamate in rat auditory cortex, demonstrating remote control of brain activity without any genetic modifications. Notably, our nano-composite microgels are capable of delivering various molecules, ranging from small chemicals to large proteins, without involving any crosslinking chemistry. Such great versatility and ease-of-use will likely make our optically-controlled delivery technology a general and important tool in cell biology research.

Photoresponsive microvalve for remote actuation and flow control in microfluidic devices.

Microvalves with different actuation methods offer great integrability and flexibility in operation of lab-on-chip devices. In this work, we demonstrate a hydrogel-based and optically controlled modular microvalve that can be easily integrated within a microfluidic device and actuated by an off-chip laser source. The microvalve is based on in-channel trapping of microgel particles, which are composed of poly(N-isopropylacrylamide) and polypyrrole nanoparticles. Upon irradiation by a near-infrared (NIR) laser, the microgel undergoes volumetric change and enables precisely localized fluid on/off switching. The response rate and the "open" duration of the microvalve can be simply controlled by adjusting the laser power and exposure time. We showed that the trapped microgel can be triggered to shrink sufficiently to open a channel within as low as ∼1-2 s; while the microgel swells to re-seal the channel within ∼6-8 s. This is so far one of the fastest optically controlled and hydrogel-based microvalves, thus permitting speedy fluidic switching applications. In this study, we successfully employed this technique to control fluidic interface between laminar flow streams within a Y-junction device. The optically triggered microvalve permits flexible and remote fluidic handling, and enables pulsatile in situ chemical treatment to cell culture in an automatic and programmed manner, which is exemplified by studies of chemotherapeutic drug induced cell apoptosis under different drug treatment strategies. We find that cisplatin induced apoptosis is significantly higher in cancer cells treated with a pulsed dose, as compared to continuous flow with a sustained dose. It is expected that our NIR-controlled valving strategy will provide a simple, versatile, and powerful alternative for liquid handling in microfluidic devices.

NeuroArray: a universal interface for patterning and interrogating neural circuitry with single cell resolution.

Recreation of neural network in vitro with designed topology is a valuable tool to decipher how neurons behave when interacting in hierarchical networks. In this study, we developed a simple and effective platform to pattern primary neurons in array formats for interrogation of neural circuitry with single cell resolution. Unlike many surface-chemistry-based patterning methods, our NeuroArray technique is specially designed to accommodate neuron's polarized morphologies to make regular arrays of cells without restricting their neurite outgrowth, and thus allows formation of freely designed, well-connected, and spontaneously active neural network. The NeuroArray device was based on a stencil design fabricated using a novel sacrificial-layer-protected PDMS molding method that enables production of through-structures in a thin layer of PDMS with feature sizes as small as 3 µm. Using the NeuroArray along with calcium imaging, we have successfully demonstrated large-scale tracking and recording of neuronal activities, and used such data to characterize the spiking dynamics and transmission within a diode-like neural network. Essentially, the NeuroArray is a universal patterning platform designed for, but not limited to neuron cells. With little adaption, it can be readily interfaced with other interrogation modalities for high-throughput drug testing, and for building neuron culture based live computational devices.

Near-infrared light triggerable deformation-free polysaccharide double network hydrogels.

To prepare a hydrogel with robust mechanical properties and programmable remotely-controlled releasing ability, we synthesized an agarose/alginate double network hydrogel incorporating polypyrrole (PPy) nanoparticles as a near-infrared (NIR) laser responsive releasing system. This hydrogel exhibited pulsatile releasing behaviours according to the laser switching while maintaining its morphology and mechanical strength.

Near-infrared photothermal activation of microgels incorporating polypyrrole nanotransducers through droplet microfluidics.

Poly(N-isopropylacrylamide) (pNIPAM) composite microgels incorporating polypyrrole (PPy) nanoparticles were produced using droplet microfluidics. The composite microgels exhibited site-specific de-swelling-swelling properties that were activated by near-infrared light. Their applications for programmable drug release by pulsed-light control were also demonstrated.

Layer-by-layer microcapsules templated on erythrocyte ghost carriers.

This work reports the fabrication of layer-by-layer (LbL) microcapsules that provide a simple mean for controlling the burst and subsequent release of bioactive agents. Red blood cell (RBC) ghosts were loaded with fluorescently labeled dextran and lysozyme as model compounds via hypotonic dialysis with an encapsulation efficiency of 27-31%. It is demonstrated that these vesicles maintain their shape and integrity and that a uniform distribution of the encapsulated agents within these carriers is achieved. The loaded vesicles were then successfully coated with the biocompatible polyelectrolytes, poly-L-arginine hydrochloride and dextran sulfate. It is demonstrated that the release profiles of the encapsulated molecules can be regulated over a wide range by adjusting the number of polyelectrolyte layers. In addition, the LbL shell also protects the RBC ghost from decomposition thereby potentially preserving the bioactivity of encapsulated drugs or proteins. These microcapsules, consisting of an RBC ghost coated with a polyelectrolyte multilayer, provide a simple mean for the preparation of loaded LbL microcapsules eliminating the core dissolution and post-loading of bioactive agents, which are required for conventional LbL microcapsules.

Effects of oligo(3-hydroxyalkanoates) on the viability and insulin secretion of murine beta cells.

Biopolyesters of polyhydroxyalkanoates (PHAs), including poly-3-hydroxybutyrate (PHB), co-polyester of 3-hydroxybutyrate and 4-hydroxybutyrate (P3HB4HB), and co-polyester of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) have been well investigated for their biocompatibility. For in vivo application, it is very important that the degradation products of PHAs, especially the oligomers, are not harmful to the cells and surrounding tissues. In this study, in vitro effects of oligo(3-hydroxybutyrate) (OHB), oligo(3-hydroxybutyrate-co-4-hydroxybutyrate) (O3HB4HB) and oligo(3-hydroxybutyrate-co-3-hydroxyhexanoate) (OHBHHx) on growth and differentiation of the murine beta cell line NIT-1 were investigated. Among the three oligo-hydroxyalkanoates (Oligo-HAs), cells treated with OHBHHx displayed higher viability, as measured by CCK-8 assay. Flow cytometric analysis of NIT-1 cells indicated that Oligo-HAs had an inhibitory effect on cell apoptosis. The cytosolic Ca(2+) transient of NIT-1 cells increased when fed with 0.04 g/l Oligo-HAs. For gap junction intercellular communication of cells, the effect of OHBHHx was the best among all materials tested. More importantly, extracellular insulin secretion was up-regulated after growing in OHBHHx for 48 h. The results demonstrated that the degradation products of PHAs, especially OHBHHx from PHBHHx, were not harmful to the beta cells. Therefore, PHBHHx warrant further study for application as a pancreatic tissue engineering material.