Chih-Hsin Shih

Insight into Mechanisms of Cellular Uptake of Lipid Nanoparticles and Intracellular Release of Small RNAs

PurposeUnderstanding mechanisms of cellular uptake and intracellular release would enable better design of nanocarriers for delivery of nucleic acids such as siRNA and microRNA (miRNA).MethodIn this study, we investigated cellular pharmacokinetics of siRNA by co-encapsulating fluorescently labeled siRNA and molecular beacon (MB) in four different formulations of cationic lipid nanoparticles (LNPs). A miRNA mimic was also used as a probe for investigating cellular pharmacokinetics, which correlated well with RNAi activities.ResultsWe tried to find the best LNP formulation based on the combination of DOTMA and DODMA. When the DOTMA/DODMA ratio was at 5/40, the LNP containing a luciferase siRNA produced the highest gene silencing activity. The superior potency of DOTMA/DODMA could be attributed to higher uptake and improved ability to facilitate siRNA release from endosomes subsequent to uptake.ConclusionsOur findings may provide new insights into RNAi transfection pathways and have implications on cationic LNP design.

Tackification of Textile Fiber Preforms in Resin Transfer Molding

Tackified textile fiber preforms are used widely in resin transfer molding (RTM) to produce aerospace-grade composite parts. To optimize the preforming process, a thorough understanding of many important material and process issues, such as fabric formability, preform dimension control, preform permeability, fiber wetting, and tackifier compatibility with the matrix resin, is essential. These issues are investigated based on a commercial tackifier (PT 500 from 3M). The results show that tackifier concentration, tackifier powder size, and the application conditions all play important roles in governing the moldability of tackified fiber preforms.

Supernatant decanting on a centrifugal platform

This study presents a novel approach to decant supernatant on a centrifugal platform. By manipulating the centrifugally induced pressure and the elastic deformation of the plastic lids in the decanting chamber, fixed amounts of the supernatant can be decanted into the detection chamber at lower rotational speeds. The experimental results showed that decanted volume is affected by the volume of deformation and the operating parameters. Factors that influence the decanting ratio are also discussed. This approach has the advantages of simple design and low manufacturing cost; further, it has no need of surface modification. It has been applied to on-disk separation of plasma from whole blood, and the results showed good stability and repeatability.

A sample-to-result system for blood coagulation tests on a microfluidic disk analyzer

In this report, we describe in detail a microfluidic analyzer, which is able to conduct blood coagulation tests using whole blood samples. Sample preparation steps, such as whole blood aliquoting and metering, plasma separation, decanting, and mixing with reagents were performed in sequence through microfluidic functions integrated on a disk. Both prothrombin time (PT) and activated partial thromboplastin time (aPTT) were carried out on the same platform and the test results can be reported in 5 min. Fifty clinical samples were tested for both PT and aPTT utilizing the microfluidic disk analyzer and the instrument used in hospitals. The test results showed good correlation and agreement between the two instruments.

An enzyme-linked immunosorbent assay on a centrifugal platform using magnetic beads

An automated, disk-based, enzyme-linked immunosorbent assay (ELISA) system is presented in this work. Magnetic beads were used as the antibody carriers to improve the assay sensitivity and shorten the reaction time. The magnetic module integrated on the system is capable of controlling the magnetic beads to either move in the incubation stage or immobilize at a specific location during washing stage. This controlling mechanism utilizes a passive controlling approach so that it can be performed through disk spinning without the need of active control from external devices. The movement of the magnetic beads was investigated and the optimal rotational speed was found to be related to the ratio of the processing time to the cycle time of the magnetic beads. Comparing to ELISA conducted on microtiter plates, similar test results could be achieved by the disk-based ELISA but the entire protocol can be finished automatically within 45 min with much less reagent consumption.

Design and Analysis of Micromixers on a Centrifugal Platform

This work reports a novel mixing design that can do fast, batch-typed liquid mixing on a centrifugal platform. Mixing is an important procedure for many applications related to chemical reactors and biological assays, especially in the field of microfluidics. The extremely small sizes of microchannels make it difficult to achieve turbulence to assist mixing. To overcome this problem, a meandering fluidic design on the centrifugal platform is proposed. Centrifual force promotes mixing by inducing lateral flow movement in the circular section and flow focusing effect in the bending section. The degree of mixing was studied for solutions with various viscosities under different rotational speeds. The experimental results showed that this mixer can mix microliter or nanoliter volumes of aqueous solutions within one second.

Disk-based enzyme-linked immunosorbent assays using the liquid-aliquoting and siphoning-evacuation technique

A cost-effective way to carry out multiple enzyme-linked immunosorbent assays (ELISAs) on a centrifugal platform using the liquid-aliquoting and siphoning-evacuation (LASE) technique was developed in this paper. Instead of preloading all the reagents in the reservoirs before testing, each reagent was loaded only one time during testing. The reagent was distributed into equal aliquots and delivered into reaction chambers by the aliquoting fluidic function. In addition, a siphoning-evacuation technique was developed to improve the washing efficiency and simplify the assay protocol. Furthermore, the entire assay protocol can be conducted using a two-step spinning protocol, which greatly reduces the cost of the motor control system. With the LASE technique, a low-cost and user-friendly ELISA system can be achieved.

Robust Sequential Flow Controls on the Centrifugal Platform

The stabilities of the burst frequencies of the capillary valves have a strong impact on the robustness of sequential flow controls on the centrifugal platform. In this study, factors which influence the burst frequencies of the capillary valves were investigated. It was found that the burst frequencies of the capillary valves were affected by the flow retardation effects. Hydrophobic treatment on the surface of the capillary valve is necessary in order to reduce the retention time of the liquid. In addition, by increasing the depth of the reservoir and conducting hydrophilic treatment on the surface of the reservoir, variations in burst frequencies of the capillary valves can be greatly reduced.

Oscillating Micromixers on a Compact Disc

In this study, mixing efficiencies of oscillating micromixers based on a compact disc under various operating conditions and geometric parameters were analyzed. By alternating the direction of rotation, stirring of liquids inside the chamber can be used for mixing. The experimental results showed that under the same operating conditions, deeper chamber and lower liquid filling ratio resulted in better mixing efficiency. Under the same distance of motion, oscillating frequency had more impact on the mixing efficiency than the oscillating amplitude. In addition, the experimental results also demonstrated that this batch mixing mechanism can also be utilized to achieve satisfactory mixing of liquids with viscosity of 100 cp within 10 seconds.

Effect of Tetrafluoromethane Plasma Treatment of PMMA on MCF-7 Cell Proliferation

Polymethyl methacrylate (PMMA) was surface modified by tetrafluoromethane (CF4) plasma and its effect on the human breast cancer cells (MCF-7) adhesion and proliferation were investigated. The promotion of MCF-7 cell growth was positively and quantitatively related to the CF4 plasma modification as revealed by different characterizations. From ESCA analysis, it is revealed that CF4 plasma treatment chemically alters the capability of PMMA to adsorb proteins from cell culture medium, which is about four times comparing to that of pristine PMMA. In addition to chemical alteration, CF4 plasma also concurrently delivered physical modification by doubling the surface roughness, as observed by AFM analysis.