Mei-Hwa Lee

Synthesis of Magnetic Molecularly Imprinted Poly(ethylene-co-vinyl alcohol) Nanoparticles and Their Uses in the Extraction and Sensing of Target Molecules in Urine

Superparamagnetic nanoparticles are of great current interest for biomedical applications in both diagnostics and treatment. Magnetic nanoparticles (MNP) can be manipulated by magnetic fields, so that when functionalized, they can be used for the purification and separation of biomolecules and even whole cells. Here we report combining the separation capabilities of MNPs with the functional (binding) capability of molecularly imprinted polymers. Albumin- creatinine-, lysozyme-, and urea-imprinted polymer nanoparticles were synthesized from poly(ethylene-co-ethylene alcohol) via phase inversion, with both target molecules and hydrophobic magnetic nanoparticles mixed within the polymer solution. Several ethylene:ethylene alcohol mole ratios were studied. The rebinding capacities for those three target molecules varied from 0.76 +/- 0.02 to 5.97 +/- 0.04 mg/g of molecularly imprinted magnetic nanoparticles. Lastly, the composite nanoparticles were used for separation and sensing of template molecules (e.g., human serum albumin) in real samples (urine) and results were compared with a commercial ARCHITECT ci 8200 system.

Instant formation of molecularly imprinted poly(ethylene-co-vinyl alcohol)/quantum dot composite nanoparticles and their use in one-pot urinalysis.

The high stability of quantum dots (QDots) with photoluminescence has led to their increased use as imaging approaches in biological systems to replace conventional fluorescence labels. The antibodies are generally coated on the surface of QDots to the targeting site, and molecular imprinting polymers are designed to mimic the antibodies. Hence, quantum dots can be incorporated into molecularly imprinted polymers, which provide shape and selectivity, and then respond to template rebinding by emitting quenched photoluminescence. In this study, poly(ethylene-co-ethylene alcohol) creatinine-, albumin- and lysozyme-imprinted polymers nanoparticles are synthesized via phase inversion of poly(ethylene-co-ethylene alcohol) with various ethylene mole ratios when target molecules and hydrophobic quantum dots are mixed within the polymer solution. Finally, those particles were prepared for the detection of creatinine, human serum albumin and lysozyme in real sample (urine) and compared with commercial ARCHITECT ci 8200 system.

Ultrasound Mediates the Release of Curcumin from Microemulsions

Ultrasound is a powerful noninvasive modality for biomedical imaging, and holds much promise for noninvasive drug delivery enhancement and targeting. However, the optimal design of sound sensitive carriers is still poorly understood. In this study, curcumin, an important natural antioxidant and anticancer compound, was stably entrapped into microemulsion droplets with average size 20-35 nm. To release curcumin, low frequency (40 kHz) ultrasound at an intensity of 3.8 or 9.8 W/cm2 was applied to the microemulsions, using a probe sonicator. On insonation, much of the curcumin was released from the microemulsions and formed insoluble aggregates, as evidenced by decreased UV-vis absorption at 420 nm. The initial release rate (assayed by the rate of change of absorption) was as high as 0.11 microg/s (1.87%/sec) in phosphate buffered saline solution at neutral pH, but decreased at acidic pH. Interestingly, lower curcumin loading led to a more rapid release under insonation. Measurements of emulsion droplet size implicate droplet reorganization (fusion or fission) as an important contributing mechanism for the ultrasonic release of this compound. Although cargo in microemulsions is partitioned, rather than encapsulated (as in, for example, liposomes), these new results demonstrate that microemulsion carriers are feasible for some ultrasonic drug delivery applications.

Preparation of curcumin microemulsions with food-grade soybean oil/lecithin and their cytotoxicity on the HepG2 cell line

The choice of surfactants and cosurfactants for preparation of oral formulation in microemulsions is limited. In this report, a curcumin-encapsulated phospholipids-based microemulsion (ME) using food-grade ingredients soybean oil and soybean lecithin to replace ethyl oleate and purified lecithin from our previous study was established and compared. The results indicated soybean oil is superior to ethyl oleate as the oil phase in curcumin microemulsion, as proven by the broadened microemulsion region with increasing range of surfactant/soybean oil ratio (approx. 1:1-12:1). Further preparation of two formula with different particle sizes of formula A (30nm) and B (80nm) exhibited differential effects on the cytotoxicity of hepatocellular HepG2 cell lines. At 15μM of concentration, curcumin-ME in formula A with smaller particle size resulted in the lowest viability (approx. 5%), which might be explained by increasing intake of curcumin, as observed by fluorescence microscopy. In addition, the cytotoxic effect of curcumin-ME is exclusively prominent on HepG2, not on HEK293, which showed over 80% of viability at 15μM. The results from this study might provide an innovative applied technique in the area of nutraceuticals and functional foods.

Hydrolysis of magnetic amylase-imprinted poly(ethylene-co-vinyl alcohol) composite nanoparticles.

Molecularly imprinted polymers (MIPs) have frequently been employed as recognition elements in sensing applications, or for the controlled delivery of small molecule drugs. An equally important but less well studied application is the use of MIPs in the binding and immobilization of active enzymes. In this study, magnetic MIPs (MMIPs) recognizing the enzyme amylase were prepared using phase inversion of poly(ethylene-co-vinyl alcohol) (EVAL) solutions with 27-44 mol % ethylene in the presence of amylase. The size distribution, specific surface area, magnetization, and composition were characterized by dynamic light scattering (DLS), Brunauer-Emmett-Teller (BET) analysis, superconducting quantum interference devices (SQUID), and X-ray diffraction (XRD), respectively. The mean size of MMIPs was ~100 nm and the magnetization was 14.8 emu/g. The activities of both bound template and rebound enzyme was established by measuring glucose production via starch hydrolysis, at different temperatures, for MIPs with different compositions (wt % EVALs and mol % ethylene). The highest hydrolysis activity of MMIPs (obtained with 32 mol % ethylene) was found to be 1545.2 U/g. Finally, compared to the conventional catalysis process, MMIPs have the advantages of high surface area, suspension, easy removal from reaction, and rapid reload of enzyme. The good activity of amylase MMIPs persists after 50 cycles of starch hydrolysis.

In vitro suppression of oral squamous cell carcinoma growth by ultrasound-mediated delivery of curcumin microemulsions.

There is increasing interest in using natural products as anticancer agents, as many have antioxidative properties that may help to prevent cellular damage that can lead to cancer. In addition, there is the expectation that many natural products will have low toxicity and few side effects. However, most anticancer and antioxidative agents are hydrophobic, reducing their bioavailability in vivo and making them problematic to deliver. Curcumin provides a good model system for study. In low doses it shows both anticancer and antioxidation effects, whereas in high doses and delivered locally it could be cytotoxic for cancer cells. In this paper, curcumin microemulsions were formed with food-grade chemicals, including soybean lecithin, soybean oil, and Tween 80, a Food and Drug Administration-approved surfactant. The optimized composition formed curcumin microemulsions with a mean size of 40–50 nm, carrying a concentration of curcumin as high as 15 μM. The stability of curcumin microemulsions refrigerated at 5°C over at least 968 days was assessed by size distribution and zeta potential. The effects of low-frequency ultrasound on two oral squamous cell carcinoma cell lines (OSCC-4 and OSCC-25), and the synergy between treatment with curcumin microemulsions and low-frequency sonic stimulation, were tested. Finally, microscopic imaging of the cells confirmed the toxic effects of the curcumin microemulsions, showing damaged and ruptured cells after treatment. Brief exposure to the curcumin-containing microemulsions did have cytotoxic effects, but the addition of ultrasound strongly enhanced those effects, especially on OSCC-25 cells.

Comparison of the antioxidant and transmembrane permeative activities of the different Polygonum cuspidatum extracts in phospholipid-based microemulsions.

The main purpose of this study was to investigate the transmembrane permeability of polyphenol-containing Polygonum cuspidatum extracts (PCE) encapsulated in phospholipid-based o/w microemulsion system. First, preparations of several PCEs using solid- or liquid-phase extraction or a combination of both, as well as evaluation of their antioxidant activities, were conducted and compared. In the antioxidant study, results indicated that PC-1 with the least extraction process exhibited the best antioxidant activity. By comparing the permeability coefficient (K(p)) among all tested PCEs in microemulsions (ME-PCs), ME-PC1 also possessed the largest permeability coefficients of both resveratrol and emodin. In addition, comparison of the transmembrane permeability of several polyphenol-encapsulated microemulsions showed that resveratrol had the most competitive advantage in the microemulsion formula for the control-release process. Taken together, it can be concluded that the matrix removed from the solid-phase extraction in PC-1 not only possesses antioxidant activity but also acts as an enhancer in transmembrane permeation. The structure specificity of the polyphenol plays important roles in the mechanism of the transmembrane permeation process. These findings might provide scientific evidence for the value of developing polyphenol-containing PCEs as nutraceuticals and cosmoceutical products.

Integrated potentiostat for electrochemical sensing of urinary 3-hydroxyanthranilic acid with molecularly imprinted poly(ethylene-co-vinyl alcohol)

Changing demographics, the rise of personalized medicine and increased identification of biomarkers for diagnosis and management of chronic disease have increased the demand for portable bioanalytical instrumentation and point-of-care. The recent development of molecularly imprinted polymers enables production of low cost and highly stable sensing chips; however, the commercially available and full functional instruments employed for electrochemical analysis have shortcomings in actual homecare applications. In this work, integrated circuits (ICs) for monolithic implementation of voltammeter potentiostat with a large dynamic current range (5 nA to 1.2 mA) and short conversion time (10 ms) were fabricated in a 0.35 μm complementary metal-oxide-semiconductor (CMOS) process. The new instrumentation was tested with molecular imprinted sensors for 3-hydroxyanthranilic acid (3HAA) in urine. The sensor consisted of molecular imprinted of poly(ethylene-co-vinyl alcohol)s (abbreviated as EVALs) for implementation in a flow injection analysis system. The EVAL containing 32 ethylene mol% had the highest imprinting effectiveness for the target molecules. Fit-for-purpose figures of merit were achieved with a limit-of-detection (LOD) of 3.06 pg/mL. The measurements obtained in real undiluted urine samples fell within the reference concentration range of 50-550 ng/mL.

Optical sensing of urinary melatonin with molecularly imprinted poly(ethylene-co-vinyl alcohol) coated zinc oxide nanorod arrays.

In this work, aligned zinc oxide (ZnO) nanorods were selectively hydrothermally grown on acetate-seeded spots on a gold substrate; the nanorods had an average length and diameter of 1.7μm and 240nm, respectively. Melatonin was imprinted into poly(ethylene-co-vinyl alcohol), EVAL, which was coated onto ZnO nanorod arrays. The ZnO nanorods not only increased the surface area for sensing target molecules, but also constituted an optical sensing element, as the ZnO fluorescence decreases when targets bind to the imprinted EVAL film; the fluorescence decrease, as a function of melatonin concentration, is well fit by a Langmuir adsorption isotherm. Poly(ethylene-co-vinyl alcohol) with 44mol% ethylene showed the best imprinting effectiveness (ratio of the fluorescence decrease on binding melatonin to imprinted vs. non-imprinted EVAL-coated ZnO nanorod arrays) among the several compositions studied. In real urine analysis, the MIP films responded linearly to added (exogenous) melatonin, even in the presence of many possible interfering compounds in urine. This demonstrates the feasibility of using these MIPs as part of a total urinalysis MIP system.

Microcontact Imprinting of Algae on Poly(ethylene-co-vinyl alcohol) for Biofuel Cells

Hydrogen can be produced using microorganisms (e.g., bacteria and algae); algal production has the additional ecological benefit of carbon dioxide fixation. The conversion of hydrogen to electricity via fuel cells may be more efficient compared to other energy sources of electricity. However, the anode of biofuel cells requires the immobilization of microorganisms or enzymes. In this work, poly(ethylene-co-vinyl alcohol) (EVAL), was coated on the electrode, and green algae was microcontact imprinted onto the EVAL film. The readsorption of algae onto algae-imprinted EVAL thin films was compared to determine the ethylene content that gave highest imprinting effectiveness and algal binding. Scanning electron microscopy and fluorescence spectrometry were employed to characterize the surface morphology, recognition capacity, and reusability of the algae-imprinted cavities. The recognition of an individual algal cell by binding to the imprinted cavities was directly observed by video microscopy. Finally, the power and current density of the algal biofuel cell using the algae-imprinted EVAL-coated electrode were measured at about 2-fold higher than electrode sputtered platinum on poly(ethylene terephthalate).