Peggy P. Y. Chan

Microfluidic devices for bioapplications.

Harnessing the ability to precisely and reproducibly actuate fluids and manipulate bioparticles such as DNA, cells, and molecules at the microscale, microfluidics is a powerful tool that is currently revolutionizing chemical and biological analysis by replicating laboratory bench-top technology on a miniature chip-scale device, thus allowing assays to be carried out at a fraction of the time and cost while affording portability and field-use capability. Emerging from a decade of research and development in microfluidic technology are a wide range of promising laboratory and consumer biotechnological applications from microscale genetic and proteomic analysis kits, cell culture and manipulation platforms, biosensors, and pathogen detection systems to point-of-care diagnostic devices, high-throughput combinatorial drug screening platforms, schemes for targeted drug delivery and advanced therapeutics, and novel biomaterials synthesis for tissue engineering. The developments associated with these technological advances along with their respective applications to date are reviewed from a broad perspective and possible future directions that could arise from the current state of the art are discussed.

Template-free synthesis and encapsulation technique for layer-by-layer polymer nanocarrier fabrication

The encapsulation of therapeutic molecules within multiple layers of biocompatible and biodegradable polymeric excipients allows exquisite design of their release profile, to the extent the drug can be selectively delivered to a specific target location in vivo. Here, we develop a novel technique for the assembly of multilayer polyelectrolyte nanocarriers based on surface acoustic wave atomization as a rapid and efficient alternative to conventional layer-by-layer assembly, which requires the use of a sacrificial colloidal template over which consecutive polyelectrolyte layers are deposited. Polymer nanocarriers are synthesized by atomizing a polymer solution and suspending them within a complementary polymer solution of opposite charge subsequent to their solidification in-flight as the solvent evaporates; reatomizing this suspension produces nanocarriers with a layer of the second polymer deposited over the initial polymer core. Successive atomization-suspension layering steps can then be repeated to produce as many additional layers as desired. Specifically, we synthesize nanocarriers comprising two and three, and up to eight, alternating layers of chitosan (or polyethyleneimine) and carboxymethyl cellulose within which plasmid DNA is encapsulated and show in vitro DNA release profiles over several days. Evidence that the plasmids viability is preserved and hence the potential of the technique for gene delivery is illustrated through efficient in vitro transfection of the encapsulated plasmid in human mesenchymal progenitor and COS-7 cells.

Tumor inside a pearl drop

The confined internal space of a liquid marble, as well as its porous and non-adhesive shell, offers an attractive application possibility - accommodating living cells inside liquid marbles. Cancer cells in suspension may aggregate to form three dimensional structures, also known as cancer cell spheroids (CCS). In this study, CCS formation inside liquid marble is investigated. This liquid marble application opens significant and novel avenues for biomedical applications and cancer research.

Production of monodisperse epigallocatechin gallate (EGCG) microparticles by spray drying for high antioxidant activity retention.

Epigallocatechin gallate (EGCG) originated from green tea is well-known for its pharmaceutical potential and antiproliferating effect on carcinoma cells. For drug delivery, EGCG in a micro-/nanoparticle form is desirable for their optimized chemopreventive effect. In this study, first time reports that EGCG microparticles produced by low temperature spray drying can maintain high antioxidant activity. A monodisperse droplet generation system was used to realize the production of EGCG microparticles. EGCG microparticles were obtained with narrow size distribution and diameter of 30.24 ± 1.88 μM and 43.39 ± 0.69 μM for pure EGCG and lactose-added EGCG, respectively. The EC50 value (the amount of EGCG necessary to scavenge 50% of free radical in the medium) of spray dried pure EGCG particles obtained from different temperature is in the range of 3.029-3.075 μM compared to untreated EGCG with EC50 value of 3.028 μM. Varying the drying temperatures from 70°C and 130°C showed little detrimental effect on EGCG antioxidant activity. NMR spectrum demonstrated the EGCG did not undergo chemical structural change after spray drying. The major protective mechanism was considered to be: (1) the use of low temperature and (2) the heat loss from water evaporation that kept the particle temperature at low level. With further drier optimization, this monodisperse spray drying technique can be used as an efficient and economic approach to produce EGCG micro-/nanoparticles.

Organosilane deposition for microfluidic applications

Treatment of surfaces to change the interaction of fluids with them is a critical step in constructing useful microfluidics devices, especially those used in biological applications. Silanization, the generic term applied to the formation of organosilane monolayers on substrates, is both widely reported in the literature and troublesome in actual application for the uninitiated. These monolayers can be subsequently modified to produce a surface of a specific functionality. Here various organosilane deposition protocols and some application notes are provided as a basis for the novice reader to construct their own silanization procedures, and as a practical resource to a broader range of techniques even for the experienced user.

In vitro cytotoxicity evaluation of biomedical nanoparticles and their extracts

The present study presents a new approach for evaluating in vitro cytotoxicity of nanoparticles. The approach is based on American National Standard ISO 10993-5. Hepatoma HepG2 and fibroblast NIH3T3 cell lines were incubated with nanoparticles, and their associated extracts were derived at 70 and 121 degrees C. Nanoparticles proposed as potential biomedical imaging probes were evaluated on the basis of the detection of metabolic activities and cell-morphology changes. In general, nanoparticles incubated directly with cells showed higher cytotoxicity than their associated extracts. CdSe and core-shell CdSe@ZnS quantum dots resulted in low cell viability for both cell lines. The cytotoxicity of the quantum dots was attributed to the Cd ion and the presence of the nanoparticle itself. A statistically significant (p < 0.05) decrease in cell viability was found in higher dosage concentrations. Rare earth nanoparticles and their extracts appear to affect NIH3T3 cells only, with cell viability as low as 71.4% +/- 4.8%. Magnetic nanoparticles have no observable effects on the cell viabilities for both cell lines. In summary, we found the following: (1) both direct incubation and extracts of nanoparticles are required for complete assessment of nanoparticle cytotoxicity, (2) the rare earth oxide nanoparticles are less cytotoxic than the Cd-based quantum dots, and (3) the extent of cytotoxicity is dependent upon the cell line.

Preparation of a soft and interconnected macroporous hydroxypropyl cellulose methacrylate scaffold for adipose tissue engineering

This study describes the preparation and characterization of a biodegradable 3D hydrogel constructed from hydroxypropyl cellulose (HPC), modified with bifunctional methacrylic anhydride (MA) to form hydroxypropyl cellulose methacrylate (HPC-MA), for adipose tissue engineering applications. The hydrogels were prepared from three different concentrations (10 wt%, 15 wt% and 20 wt%) of HPC-MA with 0.35 degree of substitution. HPC-MA hydrogel scaffolds with open biphasic features were prepared by exploiting the thermal responsive phase behavior of HPC and temperature mediated phase separation of HPC-MA. The resulting scaffolds exhibited pore sizes ranging from 30 to 300 μm and an interconnected porosity of ∼90%. The swelling ratio (SR) and storage modulus of HPC-MA scaffolds were in the range of 12.94 to 35.83 and 0.75 to 4.28 kPa, respectively. The swelling ratio and storage modulus suggested that the scaffold exhibits high water retention, allowing medium exchange during cell culturing and that it is suitable for adipose tissue regeneration. The HPC-MA scaffolds were found to be biocompatible to human adipose-derived stem cells (ASCs). ASCs were successfully differentiated into the adipocytes inside the scaffolds, and therefore demonstrated the potential application of these HPC-MA scaffolds for adipose tissue engineering.

Three‐dimensional nanocharacterization of porous hydrogel with ion and electron beams

Porous hydrogels provide an excellent environment for cell growth and tissue regeneration, with high permeability for oxygen, nutrients, and other water‐soluble metabolites through their high water‐content matrix. The ability to image three‐dimensional (3D) cell growth is crucial for understanding and studying various cellular activities in 3D context, particularly for designing new tissue engineering scaffold, but it is still challenging to study cell‐biomaterial interfaces with high resolution imaging. We demonstrate using focused ion beam (FIB) milling, electron imaging, and associated microanalysis techniques that novel 3D characterizations can be performed effectively on cells growing inside 3D hydrogel scaffold. With FIB‐tomography, the porous microstructures were revealed at nanometer resolution, and the cells grown inside. The results provide a unique 3D measurement of hydrogel porosity, as compared with those from porosimetry, and offer crucial insights into material factors affecting cell proliferation at specific regions within the scaffold. We also proved that high throughput correlative imaging of cell growth is viable through a silicon membrane based environment. The proposed approaches, together with the protocols developed, provide a unique platform for analysis of the microstructures of novel biomaterials, and for exploration of their interactions with the cells as well. Biotechnol. Bioeng. 2013; 110: 318–326.

Injectable 3D Hydrogel Scaffold with Tailorable Porosity Post-Implantation

Since rates of tissue growth vary significantly between tissue types, and also between individuals due to differences in age, dietary intake, and lifestyle-related factors, engineering a scaffold system that is appropriate for personalized tissue engineering remains a significant challenge. In this study, a gelatin-hydroxyphenylpropionic acid/carboxylmethylcellulose-tyramine (Gtn-HPA/CMC-Tyr) porous hydrogel system that allows the pore structure of scaffolds to be altered in vivo after implantation is developed. Cross-linking of Gtn-HPA/CMC-Tyr hydrogels via horseradish peroxidase oxidative coupling is examined both in vitro and in vivo. Post-implantation, further alteration of the hydrogel structure is achieved by injecting cellulase enzyme to digest the CMC component of the scaffold; this treatment yields a structure with larger pores and higher porosity than hydrogels without cellulase injection. Using this approach, the pore sizes of scaffolds are altered in vivo from 32-87 μm to 74-181 μm in a user-controled manner. The hydrogel is biocompatible to COS-7 cells and has mechanical properties similar to those of soft tissues. The new hydrogel system developed in this work provides clinicians with the ability to tailor the structure of scaffolds post-implantation depending on the growth rate of a tissue or an individuals recovery rate, and could thus be ideal for personalized tissue engineering.

Rotational microfluidic motor for on-chip microcentrifugation

We report on the design of a surface acoustic wave (SAW) driven fluid-coupled micromotor which runs at high rotational velocities. A pair of opposing SAWs generated on a lithium niobate substrate are each obliquely passed into either side of a fluid drop to drive rotation of the fluid, and the thin circular disk set on the drop. Using water for the drop, a 5 mm diameter disk was driven with rotation speeds and start-up torques up to 2250 rpm and 60 nN m, respectively. Most importantly for lab-on-a-chip applications, radial accelerations of 172 m/s2 was obtained, presenting possibilities for microcentrifugation, flow sequencing, assays, and cell culturing in truly microscale lab-on-a-chip devices.We report on the design of a surface acoustic wave (SAW) driven fluid-coupled micromotor which runs at high rotational velocities. A pair of opposing SAWs generated on a lithium niobate substrate are each obliquely passed into either side of a fluid drop to drive rotation of the fluid, and the thin circular disk set on the drop. Using water for the drop, a 5 mm diameter disk was driven with rotation speeds and start-up torques up to 2250 rpm and 60 nN m, respectively. Most importantly for lab-on-a-chip applications, radial accelerations of 172 m/s2 was obtained, presenting possibilities for microcentrifugation, flow sequencing, assays, and cell culturing in truly microscale lab-on-a-chip devices.