Matthew Leung

Chlorotoxin bound magnetic nanovector tailored for cancer cell targeting, imaging, and siRNA delivery

Ribonucleic acid interference (RNAi) is a powerful molecular tool that has potential to revolutionize the treatment of cancer. One major challenge of applying this technology for clinical application is the lack of site-specific carriers that can effectively deliver short interfering RNA (siRNA) to cancer cells. Here we report the development and assessment of a cancer-cell specific magnetic nanovector construct for efficient siRNA delivery and non-invasive monitoring through magnetic resonance imaging (MRI). The base of the nanovector construct is comprised of a superparamagnetic iron oxide nanoparticle core coated with polyethylene glycol (PEG)-grafted chitosan, and polyethylenimine (PEI). The construct was then further functionalized with siRNA and a tumor-targeting peptide, chlorotoxin (CTX), to improve tumor specificity and potency. Flow cytometry, quantitative RT-PCR, and fluorescence microscopy analyses confirmed receptor-mediated cellular internalization of nanovectors and enhanced gene knockdown through targeted siRNA delivery. The ability of this nanovector construct to generate specific contrast enhancement of glioblastoma cells was demonstrated through MR imaging. These findings suggest that this CTX enabled nanoparticle carrier may be well suited for delivery of RNAi therapeutics to brain cancer cells.

Chitosan-alginate 3D scaffolds as a mimic of the glioma tumor microenvironment

Despite recent advances in the understanding of its cell biology, glioma remains highly lethal. Development of effective therapies requires a cost-effective in vitro tumor model that more accurately resembles the in vivo tumor microenvironment as standard two-dimensional (2D) tissue culture conditions do so poorly. Here we report on the use of a three-dimensional (3D) chitosan-alginate (CA) scaffold to serve as an extracellular matrix that promotes the conversion of cultured cancer cells to a more malignant in vivo-like phenotype. Human U-87 MG and U-118 MG glioma cells and rat C6 glioma cells were chosen for the study. In vitro tumor cell proliferation and secretion of factors that promote tumor malignancy, including VEGF, MMP-2, fibronectin, and laminin, were assessed. The scaffolds pre-cultured with U-87 MG and C6 cells were then implanted into nude mice to evaluate tumor growth and blood vessel recruitment compared to the standard 2D cell culture and 3D Matrigel matrix xenograft controls. Our results indicate that while the behavior of C6 cells showed minimal differences due to their highly malignant and invasive nature, U-87 MG and U-118 MG cells exhibited notably higher malignancy when cultured in CA scaffolds. CA scaffolds provide a 3D microenvironment for glioma cells that is more representative of the in vivo tumor, thus can serve as a more effective platform for development and study of anticancer therapeutics. This unique CA scaffold platform may offer a valuable alternative strategy to the time-consuming and costly animal studies for a wide variety of experimental designs.

Feeder-free self-renewal of human embryonic stem cells in 3D porous natural polymer scaffolds.

Human embryonic stem cells (hESCs) are routinely cultured on fibroblast feeder layers or in fibroblast-conditioned medium, which requires continued supply of feeder cells and poses the risks of xenogenic contamination and other complications such as feeder-dependent outcome. Here, we demonstrate a strategy that supports sustained self-renewal of hESCs in a three-dimensional porous natural polymer scaffold, comprised of chitosan and alginate, without the support of feeder cells or conditioned medium. We assessed the pluripotency of the renewed hESCs both in vitro by evaluation of cellular proliferation, functionality, and gene activities for 21 days, and in vivo by implantation of the stem cell populated scaffolds in an immunodeficient mouse model to induce teratoma formation. The self-renewed stem cells can be easily recovered for subculture by decomposing the scaffold under a mild condition. We further subcultured recovered hESCs for 14 days and verified their pluripotency. In addition to providing a clean environment for stem cell renewal, this strategy, with the demonstrated biocompatibility and biodegradability of chitosan and alginate, may potentially allow for the direct implantation of stem cell populated scaffolds for a broad spectrum of applications in tissue engineering and regenerative medicine.

Cell transcytosing poly-arginine coated magnetic nanovector for safe and effective siRNA delivery

Lack of safe and effective carriers for delivery of RNA therapeutics remains a barrier to its broad clinical application. We report the development of a cell tanscytosing magnetic nanovector engineered as an siRNA carrier. Iron oxide nanoparticles were modified with poly(ethylene glycol) (PEG), small interfering RNA (siRNA), and a cationic polymer layer. Three nanovector formulations with cationic polymer coatings of poly-arginine (pArg), polylysine (pLys), and polyethylenimine (PEI), respectively, were prepared. The three nanovector formulations where evaluated for safety and ability to promote gene silencing in three types of cancer cells C6/GFP(+), MCF7/GFP(+), and TC2/GFP(+), mimicking human cancers of the brain, breast, and prostate, respectively. Cell viability and fluorescence quantification assays revealed that pArg-coated nanovectors were most effective in promoting gene knockdown and least toxic of the three nanovector formulations tested. Transmission electron microscopy (TEM) imaging of nanovector treated cells further demonstrated that pArg-coated nanovectors enter cells through cell transcytosis, while pLys and PEI coated nanovectors enter cells endocytosis. Our findings suggest that NPs engineered to exploit the cell transcytosis intracellular trafficking pathway may offer a more safe and efficient route for siRNA delivery.

Chitosan-Alginate Scaffold Culture System for Hepatocellular Carcinoma Increases Malignancy and Drug Resistance

ABSTRACTPurposeHepatocellular carcinoma (HCC) is a prevalent solid malignancy. Critically needed discovery of new therapeutics has been hindered by lack of an in vitro cell culture system that can effectively represent the in vivo tumor microenvironment. To address this need, a 3D in vitro HCC model was developed using a biocompatible, chitosan-alginate (CA) scaffold cultured with human HCC cell lines.MethodsThe correlation between the cell function, such as secretion of growth factors and production of ECM in vitro, and the tumor growth and blood vessel recruitment in vivo was investigated.ResultsHCC cells grown on 3D CA scaffolds demonstrated morphological characteristics and increased expression of markers of highly malignant cells. Implantation of CA scaffolds cultured with human HCC cells in mice showed accelerated tumor growth. Histology revealed marked differences in morphology and organization of newly formed blood vessels between tumors produced by different pre-cultured conditions. Resistance to doxorubicin was significantly pronounced in CA scaffold-cultured HCC cells compared to 2D or Matrigel cultured HCC cells.ConclusionsThis 3D model of HCC, with its ability to more closely mimic the in vivo tumor behavior, may serve as an invaluable model for study and application of novel anticancer therapeutics against HCC.

High-strength pristine porous chitosan scaffolds for tissue engineering

Chitosan, a biodegradable naturally occurring polymer, has drawn considerable attention in recent years as a scaffolding material in tissue engineering and regenerative medicine. Despite its favorable biological properties, the weak mechanical strength of scaffolds produced from chitosan has limited the scope of their application. Here we fabricated 3D pristine porous chitosan scaffolds with unprecedented mechanical strength and investigated the regulatory role of chitosan and acidic concentrations on the crystallinity and thus on the mechanical and biological properties of produced scaffolds. Chitosan scaffolds of varying mechanical properties were prepared from solutions with chitosan concentrations of 4–12 wt%. The produced scaffolds showed no apparent shape change after immersion in Dulbeccos Modified Eagle Medium (DMEM), phosphate buffered saline, and simulated body fluid for two weeks. We showed that the crystallinity of the scaffold increased with increasing chitosan concentration or decreasing solution acidity, and the maximum compressive mechanical strength and modulus of 1.74 ± 0.01 MPa and 17.99 ± 0.11 MPa, respectively, were achieved at a chitosan concentration of 12 wt%. MG-63 osteoblast cells demonstrated improved adhesion, proliferation and osteogenic activity on chitosan scaffolds of increased chitosan concentration or mechanical strength. The ability to produce high-strength chitosan scaffolds and engineer their mechanical properties can substantially expand the applicability of chitosan in tissue engineering as well as other engineering applications.

Evaluation of three-dimensional porous chitosan-alginate scaffolds in rat calvarial defects for bone regeneration applications.

This study investigated the use of three-dimensional porous chitosan-alginate (CA) scaffolds for critical size calvarial defect (diameter, 5.0 mm) repair in Sprague-Dawley rats. CA scaffolds have been used for in vitro culture of many cell types and demonstrated osteogenesis in ectopic locations in vivo, but have yet to be evaluated for functional bone tissue engineering applications. CA scaffolds demonstrated the ability to support undifferentiated mesenchymal stem cells (MSCs) in culture for 14 days in vitro and promoted spherical morphology. In vivo tests were performed using CA scaffolds and CA scaffolds with treatments including undifferentiated MSCs, bone marrow aspirate, and bone morphogenetic protein-2 (BMP-2) growth factor in comparison to unfilled bone defect used as a control. The samples were analyzed with MicroCT, histology, and immunohistochemical staining at 4 and 16 weeks. Partial defect closure was observed in all experimental groups at 16 weeks, with the greatest defect closure (71.56 ± 19.74%) in the animal group treated with CA scaffolds with BMP-2 (CA + BMP-2). The experimental samples demonstrated osteogenesis in histology and immunohistochemical staining, with the CA + BMP-2 group, showing the greatest level of osteogenesis. Tissue engineered CA scaffolds show promise in reconstruction of critical size bone defects.

Glypican-3 Targeting of Liver Cancer Cells Using Multifunctional Nanoparticles

Imaging is essential in accurately detecting, staging, and treating primary liver cancer (hepatocellular carcinoma [HCC]), one of the most prevalent and lethal malignancies. We developed a novel multifunctional nanoparticle (NP) specifically targeting glypican-3 (GPC3), a proteoglycan implicated in promotion of cell growth that is overexpressed in most HCCs. Quantitative real-time polymerase chain reaction was performed to confirm the differential GPC3 expression in two human HCC cells, Hep G2 (high) and HLF (negligible). These cells were treated with biotin-conjugated GPC3 monoclonal antibody (αGPC3) and subsequently targeted using superparamagnetic iron oxide NPs conjugated to streptavidin and Alexa Fluor 647. Flow cytometry demonstrated that only GPC3-expressing Hep G2 cells were specifically targeted using this αGPC3-NP conjugate (fourfold mean fluorescence over nontargeted NP), and magnetic resonance imaging (MRI) experiments showed similar findings (threefold R2 relaxivity). Confocal fluorescence microscopy localized the αGPC3 NPs only to the cell surface of GPC3-expressing Hep G2 cells. Further characterization of this construct demonstrated a negatively charged, monodisperse, 50 nm NP, ideally suited for tumor targeting. This GPC3-specific NP system, with dual-modality imaging capability, may enhance pretreatment MRI, enable refined intraoperative HCC visualization by near-infrared fluorescence, and be potentially used as a carrier for delivery of tumor-targeted therapies, improving patient outcomes.

Effect of nano- and micro-scale topological features on alignment of muscle cells and commitment of myogenic differentiation

Skeletal muscle injury can lead to severe motor deficits that adversely affect movement and quality of life. Current surgical treatments for skeletal muscle are hindered by the poor formation of organized myotube bundles at the wound site. Tissue-engineered skeletal muscle constructs to date have been unable to generate high degrees of myotube density and alignment. Generating a suitable in vitro tissue-engineered skeletal muscle construct requires the design of a scaffold that recapitulates the structural combination of nanoscale collagen fibrils and aligned microscale basal lamina tracks present in the native extracellular matrix (ECM). We hypothesized that a 3D aligned tubular porous scaffold containing aligned nanofibers inside the pores can mimic the native muscle tissue environment. We constructed a laminar section of the hypothesized scaffold with aligned chitosan-PCL nanofibers arranged co-axially with the aligned microscale chitosan scaffold bands to mimic the required myogenic environment. A 6-day study of C2C12 mouse myoblast cells cultured on this hybrid scaffold indicated that the nanofibers and scaffold bands in the scaffold played a synergetic role in directing cell orientation, interaction, migration and organization. Our results showed that aligned nanofibers mediated cell alignment and the aligned scaffold bands induced the formation of a more compact assembly of myotube cells as compared to various control substrates including chitosan films, nanofibers, and chitosan bands. The expression levels of both early and late-stage myogenic differentiation genes associated with myogenin and myosin heavy chain, respectively, were higher on the hybrid substrate than on control substrates. Our study suggests that the combination of nano and microscale topological features in the ECM can direct myogenic differentiation, and the hybrid material has the potential to improve the outcome of skeletal tissue engineering.

Nanofiber-based in vitro system for high myogenic differentiation of human embryonic stem cells.

Myogenic progenitor cells derived from human embryonic stem cells (hESCs) can provide unlimited sources of cells in muscle regeneration but their clinical uses are largely hindered by the lack of efficient methods to induce differentiation of stem cells into myogenic cells. We present a novel approach to effectively enhance myogenic differentiation of human embryonic stem cells using aligned chitosan-polycaprolactone (C-PCL) nanofibers constructed to resemble the microenvironment of the native muscle extracellular matrix (ECM) in concert with Wnt3a protein. The myogenic differentiation was assessed by cell morphology, gene activities, and protein expression. hESCs grown on C-PCL uniaxially aligned nanofibers in media containing Wnt3a displayed an elongated morphology uniformly aligned in the direction of fiber orientation, with increased expressions of marker genes and proteins associated with myogenic differentiation as compared to control substrates. The combination of Wnt3a signaling and aligned C-PCL nanofibers resulted in high percentages of myogenic-protein expressing cells over total treated hESCs (83% My5, 91% Myf6, 83% myogenin, and 63% MHC) after 2 days of cell culture. Significantly, this unprecedented high-level and fast myogenic differentiation of hESC was demonstrated in a culture medium containing no feeder cells. This study suggests that chitosan-based aligned nanofibers combined with Wnt3a can potentially act as a model system for embryonic myogenesis and muscle regeneration.