L. James Lee

Growth and alignment of polyaniline nanofibres with superhydrophobic, superhydrophilic and other properties

Polyaniline nanofibres can be prepared by a number of methods based on chemical oxidative polymerization and in situ adsorption polymerization. However, the lack of alignment in these nanostructures makes them unsuitable for many applications. Here, we report a simple approach to chemical oxidative polymerization that can control the growth and simultaneous alignment of polyaniline nanofibres grown on a range of conducting and non-conducting substrates in a wide variety of sizes. The diameters of the tips of the nanofibres can be controlled within the range 10-40 nm, and the average length can be controlled within the range 70-360 nm. Moreover, the coatings display a range of properties including superhydrophilicity and superhydrophobicity. Such nanostructured coatings may be useful for applications such as anti-fog coatings, self-cleaning surfaces, DNA manipulation, transparent electrodes for low-voltage electronics, and chemical and biological sensors.

Design and Fabrication of CD-like Microfluidic Platforms for Diagnostics: Microfluidic Functions

In this paper, the design of a polymer based microfluidic compact disk (CD) platform is presented. Several microfluidic functions such as flow sequencing, cascade micro-mixing, and capillary metering can be integrated into the CD by balancing the centrifugal force and the capillary force. These functions are demonstrated experimentally. For flow sequencing, a two-point calibration design is used as an example to show how the release and flow of fluids can be precisely controlled by the rotation speed of the CD. For cascade micro-mixing, a typical application is reconstituting lyophilized protein. A simple metering technique based on bubble snap-off in the two-phase flow is also described.

Nanochannel electroporation delivers precise amounts of biomolecules into living cells

Many transfection techniques can deliver biomolecules into cells, but the dose cannot be controlled precisely. Delivering well-defined amounts of materials into cells is important for various biological studies and therapeutic applications. Here, we show that nanochannel electroporation can deliver precise amounts of a variety of transfection agents into living cells. The device consists of two microchannels connected by a nanochannel. The cell to be transfected is positioned in one microchannel using optical tweezers, and the transfection agent is located in the second microchannel. Delivering a voltage pulse between the microchannels produces an intense electric field over a very small area on the cell membrane, allowing a precise amount of transfection agent to be electrophoretically driven through the nanochannel, the cell membrane and into the cell cytoplasm, without affecting cell viability. Dose control is achieved by adjusting the duration and number of pulses. The nanochannel electroporation device is expected to have high-throughput delivery applications.

Receptor-targeted nanocarriers for therapeutic delivery to cancer

Abstract Efficient and site-specific delivery of therapeutic drugs is a critical challenge in clinical treatment of cancer. Nano-sized carriers such as liposomes, micelles, and polymeric nanoparticles have been investigated for improving bioavailability and pharmacokinetic properties of therapeutics via various mechanisms, for example, the enhanced permeability and retention (EPR) effect. Further improvement can potentially be achieved by conjugation of targeting ligands onto nanocarriers to achieve selective delivery to the tumour cell or the tumour vasculature. Indeed, receptor-targeted nanocarrier delivery has been shown to improve therapeutic responses both in vitro and in vivo. A variety of ligands have been investigated including folate, transferrin, antibodies, peptides and aptamers. Multiple functionalities can be incorporated into the design of nanoparticles, e.g., to enable imaging and triggered intracellular drug release. In this review, we mainly focus on recent advances on the development of targeted nanocarriers and will introduce novel concepts such as multi-targeting and multi-functional nanoparticles.

MicroRNA Delivery by Cationic Lipoplexes for Lung Cancer Therapy

Lung cancer is the leading cause of cancer deaths in western countries and carries a poor overall five year survival rate. Several studies demonstrate that microRNAs (miRNAs or miRs) are actively involved in tumor development by serving as tumor suppressors, oncogenes or both. In lung cancer, miRNAs may serve as both diagnostic and prognostic biomarkers as well as regulate in vitro and in vivo tumor progression. However, miRNA-based therapy is faced with several challenges including lack of tissue specificity, lack of optimal delivery systems, poor cellular uptake and risk of systemic toxicity. Here, we report a cationic lipid based miRNA delivery system to address some of these challenges. Among many lung cancer related miRNAs, miR-133b, a tumor suppressor, was selected as a therapeutic target because it directly targets the prosurvival gene MCL-1 thus regulating cell survival and sensitivity of lung cancer cells to chemotherapeutic agents. The efficacy of pre-miR-133b containing lipoplexes was evaluated in A549 non-small cell lung cancer (NSCLC) cells. Compared with siPORT NeoFX transfection agent, lipoplexes delivered pre-miR-133b in a more efficient manner with ~2.3-fold increase in mature miR-133b expression and ~1.8-fold difference in MCL-1 protein downregulation in vitro. In the in vivo biodistribution study, lipoplexes achieved ~30% accumulation in lung tissue, which was ~50-fold higher than siPORT NeoFX transfection agent. Mice treated with pre-miR-133b containing lipoplexes had mature miR-133b expression in lung ~52-fold higher than untreated mice. Our results demonstrated that cationic lipoplexes are a promising carrier system for the development of miRNA-based therapeutics in lung cancer treatment.

Lenalidomide down-regulates the CD20 antigen and antagonizes direct and antibody-dependent cellular cytotoxicity of rituximab on primary chronic lymphocytic leukemia cells

Lenalidomide, an immunomodulatory agent that enhances antibody-dependent cellular cytotoxicity (ADCC), is currently being investigated as a therapy for chronic lymphocytic leukemia (CLL). The anti-CD20 antibody rituximab is active in CLL and represents a rational agent to combine with lenalidomide. We therefore examined whether lenalidomide combined with rituximab enhances direct apoptosis and ADCC in CLL cells. In contrast to previous reports using CD20-positive lymphoma cell lines, lenalidomide down-regulated CD20 surface antigen expression in CLL patient cells via enhanced internalization, without influencing transcription. The CD20 surface antigen internalization enhanced delivery of an oligonucleotide incorporated into anti-CD20 immunoliposomes. In addition, CD20 surface antigen down-modulation by lenalidomide in CLL was accompanied by diminished rituximab-mediated apoptosis and ADCC. These observations suggest a need for alternative sequencing strategies to avoid antagonism between lenalidomide and rituximab therapy in CLL. In addition, they suggest that lenalidomide therapy might be useful to enhance targeted delivery of RNAi-based therapies using CD20 immunoliposomes in B-cell malignancies.

Mold filling and cure modeling of RTM and SRIM processes

Abstract This paper is to discuss the modeling and computer simulation techniques of liquid composite molding processes, i.e. resin transfer molding (RTM) and structural reaction injection molding (SRIM). Mold filling and curing are simulated using control volume/finite element methods. Several case studies are presented.

Therapeutic Delivery of MicroRNA-29b by Cationic Lipoplexes for Lung Cancer

MicroRNA-29b (miR-29b) expression has been shown to be reduced in non-small–cell lung cancer (NSCLC) tissues. Here, we have identified the oncogene cyclin-dependent protein kinase 6 (CDK6) as a direct target of miR-29b in lung cancer. We hypothesized that in vivo restoration of miR-29b and thus targeting of genes important to tumor initiation and progression may represent an option for lung cancer treatment. We developed a cationic lipoplexes (LPs)-based carrier that efficiently delivered miR-29b both in vitro and in vivo. LPs containing miR-29b (LP-miR-29b) efficiently delivered miR-29b to NSCLC A549 cells, reduced the expression of key targets CDK6, DNMT3B, and myeloid cell leukemia sequence 1 (MCL1), as well as cell growth and clonogenicity of A549 cells. In addition, the IC50 for cisplatin in the miR-29b–treated cells was effectively reduced. In a xenograft murine model, LPs efficiently accumulated at tumor sites. Systemic delivery of LP-miR-29b increased the tumor miR-29b expression by approximately fivefold, downregulated the tumor mRNA expression of CDK6, DNMT3B, and MCL1 by ~57.4, ~40.5, and ~52.4%, respectively, and significantly inhibited tumor growth by ~60% compared with LP-miR-NC (negative control). Our results demonstrate that cationic LPs represent an efficient delivery system that holds great potential in the development of miRNA-based therapeutics for lung cancer treatment.

Epigenetic regulation of connective tissue growth factor by MicroRNA‐214 delivery in exosomes from mouse or human hepatic stellate cells

Connective tissue growth factor (CCN2) drives fibrogenesis in hepatic stellate cells (HSC). Here we show that CCN2 up‐regulation in fibrotic or steatotic livers, or in culture‐activated or ethanol‐treated primary mouse HSC, is associated with a reciprocal down‐regulation of microRNA‐214 (miR‐214). By using protector or reporter assays to investigate the 3′‐untranslated region (UTR) of CCN2 mRNA, we found that induction of CCN2 expression in HSC by fibrosis‐inducing stimuli was due to reduced expression of miR‐214, which otherwise inhibited CCN2 expression by directly binding to the CCN2 3′‐UTR. Additionally, miR‐214 was present in HSC exosomes, which were bi‐membrane vesicles, 50‐150 nm in diameter, negatively charged (−26 mV), and positive for CD9. MiR‐214 levels in exosomes but not in cell lysates were reduced by pretreatment of the cells with the exosome inhibitor, GW4869. Coculture of either quiescent HSC or miR‐214‐transfected activated HSC with CCN2 3′‐UTR luciferase reporter‐transfected recipient HSC resulted in miR‐214‐ and exosome‐dependent regulation of a wild‐type CCN2 3′‐UTR reporter but not of a mutant CCN2 3′‐UTR reporter lacking the miR‐214 binding site. Exosomes from HSC were a conduit for uptake of miR‐214 by primary mouse hepatocytes. Down‐regulation of CCN2 expression by miR‐214 also occurred in human LX‐2 HSC, consistent with a conserved miR‐214 binding site in the human CCN2 3′‐UTR. MiR‐214 in LX‐2 cells was shuttled by way of exosomes to recipient LX‐2 cells or human HepG2 hepatocytes, resulting in suppression of CCN2 3′‐UTR activity or expression of CCN2 downstream targets, including alpha smooth muscle actin or collagen. Experimental fibrosis in mice was associated with reduced circulating miR‐214 levels. Conclusion: Exosomal transfer of miR‐214 is a paradigm for the regulation of CCN2‐dependent fibrogenesis and identifies fibrotic pathways as targets of intercellular regulation by exosomal miRs. (Hepatology 2014;59:1118–1129)

Targeted delivery systems for oligonucleotide therapeutics.

Oligonucleotides including antisense oligonucleotides and siRNA are emerging as promising therapeutic agents against a variety of diseases. Effective delivery of these molecules is critical to their successful clinical application. Targeted systems can greatly improve the efficiency and specificity of oligonucleotides delivery. Meanwhile, an effective delivery system must successfully overcome a multitude of biological barriers to enable the oligonucleotides to reach the site of action and access their biological targets. Several delivery strategies based on different platform technologies and different targeting ligands have been developed to achieve these objectives. This review aims at providing a summary and perspective on recent progress in this very active area of research.