Liju Yang

Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors

Three-dimensional (3D) cell culture systems have gained increasing interest in drug discovery and tissue engineering due to their evident advantages in providing more physiologically relevant information and more predictive data for in vivo tests. In this review, we discuss the characteristics of 3D cell culture systems in comparison to the two-dimensional (2D) monolayer culture, focusing on cell growth conditions, cell proliferation, population, and gene and protein expression profiles. The innovations and development in 3D culture systems for drug discovery over the past 5 years are also reviewed in the article, emphasizing the cellular response to different classes of anticancer drugs, focusing particularly on similarities and differences between 3D and 2D models across the field. The progression and advancement in the application of 3D cell cultures in cell-based biosensors is another focal point of this review.

Effects of Dielectrophoresis on Growth, Viability and Immuno-reactivity of Listeria monocytogenes.

Dielectrophoresis (DEP) has been regarded as a useful tool for manipulating biological cells prior to the detection of cells. Since DEP uses high AC electrical fields, it is important to examine whether these electrical fields in any way damage cells or affect their characteristics in subsequent analytical procedures. In this study, we investigated the effects of DEP manipulation on the characteristics of Listeria monocytogenes cells, including the immuno-reactivity to several Listeria- specific antibodies, the cell growth profile in liquid medium, and the cell viability on selective agar plates. It was found that a 1-h DEP treatment increased the cell immuno-reactivity to the commercial Listeria species-specific polyclonal antibodies (from KPL) by ~31.8% and to the C11E9 monoclonal antibodies by ~82.9%, whereas no significant changes were observed with either anti-InlB or anti-ActA antibodies. A 1-h DEP treatment did not cause any change in the growth profile of Listeria in the low conductive growth medium (LCGM); however, prolonged treatments (4 h or greater) caused significant delays in cell growth. The results of plating methods showed that a 4-h DEP treatment (5 MHz, 20 Vpp) reduced the viable cell numbers by 56.8–89.7 %. These results indicated that DEP manipulation may or may not affect the final detection signal in immuno-based detection depending on the type of antigen-antibody reaction involved. However, prolonged DEP treatment for manipulating bacterial cells could produce negative effects on the cell detection by growth-based methods. Careful selection of DEP operation conditions could avoid or minimize negative effects on subsequent cell detection performance.

Inactivation of Bacillus anthracis spores by single-walled carbon nanotubes coupled with oxidizing antimicrobial chemicals.

In this study, we investigated the sporicidal effects of single-walled carbon nanotubes (SWCNTs) and SWCNTs combined with oxidizing antimicrobial chemicals, H₂O₂ and NaOCl, on B. anthracis spores. The results indicated that treatment with SWCNTs alone exhibited little sporicidal effect on B. anthracis spores, while treatment with H₂O₂ or NaOCl alone showed moderate sporicidal effect. The combination treatment with SWCNTs (100 μg/mL) and H₂O₂ (1.5%) or NaOCl (0.25%) exhibited much stronger sporicidal effect on the spores, compared to treatment with H₂O₂ or NaOCl alone at the same concentrations, doubling the log reduction of viable spore number (∼3.3 log vs ∼1.6 log). Such enhanced sporicidal efficiency was due to the synergistic effect contributed by the two individual antimicrobial mechanisms of SWCNTs and the oxidizing antimicrobial chemicals. The ordered sequential treatment with SWCNTs and H₂O₂ or NaOCl revealed that SWCNTs played the key role in making the spores more permeable/susceptible to chemicals. This study demonstrated the potential of combination treatment with SWCNTs and oxidizing antimicrobial agents in developing highly effective sporicidal agents/methods.

Three-Dimensional (3D) Cell Cultures in Cell-based Assays for in-vitro Evaluation of Anticancer Drugs

This study systematically investigated the cell proliferation rates, spheroid structures, cellular responses to different anti-cancer drugs, the expression of drug action-related proteins, and the possible correlations among these properties of 3D spheroids on Matrigel in comparison to 2D monolayer cells, using two cancer cell lines-the prostate cancer cell line, DU145, and the oral cancer cell line, CAL27. Compared to the traditional 2D-cultured cells, 3D-cultured CAL27 cells had enhanced proliferation by approximately 50-70% at various seeding cell densities, whereas 3D-cultured DU145 cells showed reduced proliferation at all tested seeding cell densities by 20-40%. In drug tests, the sensitivity of 3D-cultured DU145 cells relative to 2D-cultured cells showed an obvious drug action mechanism dependency in response to three anticancer drugs, Rapamycin, Docetaxel, and Camptothecin, whereas 3D-cultured CAL27 cells responded more sensitively than 2D-cultured cells to all three tested drugs, Docetaxel, Bleomycin, and Erlotinib, indicating the relative proliferation rate between 3D and 2D cultured cells may be a dominating factor in this case and mitigated the factor of drug action mechanism. The elevated expression of EGFR in 3D-cultured CAL27 was correlated with its more sensitive response to Erlotinib (acting through binding to EGRF) compared to 2D-cultured cells; Similarly, the expression of βIII tubulin in 3D-cultured DU145 cells was found to be increased and correlated with their higher resistance to Doxetaxel compared to 2D-cultured cells.

Inhibitory effects of nisin-coated multi-walled carbon nanotube sheet on biofilm formation from Bacillus anthracis spores.

Multi-walled carbon nanotube (MWCNT) sheet was fabricated from a drawable MWCNT forest and then deposited on poly(methyl methacrylate) film. The film was further coated with a natural antimicrobial peptide nisin. We studied the effects of nisin coating on the attachment of Bacillus anthracis spores, the germination of attached spores, and the subsequent biofilm formation from attached spores. It was found that the strong adsorptivity and the super hydrophobicity of MWCNTs provided an ideal platform for nisin coating. Nisin coating on MWCNT sheets decreased surface hydrophobicity, reduced spore attachment, and reduced the germination of attached spores by 3.5 fold, and further inhibited the subsequent biofilm formation by 94.6% compared to that on uncoated MWCNT sheet. Nisin also changed the morphology of vegetative cells in the formed biofilm. The results of this study demonstrated that the anti-adhesion and antimicrobial effect of nisin in combination with the physical properties of carbon nanotubes had the potential in producing effective anti-biofilm formation surfaces.

Influence of Matrices on 3D-Cultured Prostate Cancer Cells' Drug Response and Expression of Drug-Action Associated Proteins

This study investigated the effects of matrix on the behaviors of 3D-cultured cells of two prostate cancer cell lines, LNCaP and DU145. Two biologically-derived matrices, Matrigel and Cultrex BME, and one synthetic matrix, the Alvetex scaffold, were used to culture the cells. The cell proliferation rate, cellular response to anti-cancer drugs, and expression levels of proteins associated with drug sensitivity/resistance were examined and compared amongst the 3D-cultured cells on the three matrices and 2D-cultured cells. The cellular responses upon treatment with two common anti-cancer drugs, Docetaxel and Rapamycin, were examined. The expressions of epidermal growth factor receptor (EGFR) and β-III tubulin in DU145 cells and p53 in LNCaP cells were examined. The results showed that the proliferation rates of cells cultured on the three matrices varied, especially between the synthetic matrix and the biologically-derived matrices. The drug responses and the expressions of drug sensitivity-associated proteins differed between cells on various matrices as well. Among the 3D cultures on the three matrices, increased expression of β-III tubulin in DU145 cells was correlated with increased resistance to Docetaxel, and decreased expression of EGFR in DU145 cells was correlated with increased sensitivity to Rapamycin. Increased expression of a p53 dimer in 3D-cultured LNCaP cells was correlated with increased resistance to Docetaxel. Collectively, the results showed that the matrix of 3D cell culture models strongly influences cellular behaviors, which highlights the imperative need to achieve standardization of 3D cell culture technology in order to be used in drug screening and cell biology studies.

Antiviral Activity of Gold/Copper Sulfide Core/Shell Nanoparticles against Human Norovirus Virus-Like Particles

Human norovirus is a leading cause of acute gastroenteritis worldwide in a plethora of residential and commercial settings, including restaurants, schools, and hospitals. Methods for easily detecting the virus and for treating and preventing infection are critical to stopping norovirus outbreaks, and inactivation via nanoparticles (NPs) is a more universal and attractive alternative to other physical and chemical approaches. Using norovirus GI.1 (Norwalk) virus-like particles (VLPs) as a model viral system, this study characterized the antiviral activity of Au/CuS core/shell nanoparticles (NPs) against GI.1 VLPs for the rapid inactivation of HuNoV. Inactivation of VLPs (GI.1) by Au/CuS NPs evaluated using an absorbance-based ELISA indicated that treatment with 0.083 μM NPs for 10 min inactivated ~50% VLPs in a 0.37 μg/ml VLP solution and 0.83 μM NPs for 10 min completely inactivated the VLPs. Increasing nanoparticle concentration and/or VLP-NP contact time significantly increased the virucidal efficacy of Au/CuS NPs. Changes to the VLP particle morphology, size, and capsid protein were characterized using dynamic light scattering, transmission electron microscopy, and Western blot analysis. The strategy reported here provides the first reported proof-of-concept Au/CuS NPs-based virucide for rapidly inactivating human norovirus.

Carbon Dots’ Antiviral Functions Against Noroviruses

This study reported the first assessment of carbon dots’ (CDots) antiviral activity to human norovirus virus-like-particles (VLPs), GI.1 and GII.4 VLPs. CDots with different surface passivation molecules, 2,2′-(ethylenedioxy)bis(ethylamine) (EDA)-CDots and 3-ethoxypropylamine (EPA)-CDots, were synthesized and evaluated. The results indicated both EDA- and EPA- CDots were highly effective to inhibit both strains of VLPs’ bindings to histo-blood group antigens (HBGA) receptors on human cells at CDots concentration of 5 µg/mL, with EDA-CDots achieving 100% inhibition and EPA CDots achieving 85–99% inhibition. At low CDots concentration (2 µg/mL), positively charged EDA-CDots exhibited higher inhibitory effect (~82%) than non-charged EPA-CDots (~60%), suggesting the surface charge status of CDots played a role in the interactions between CDots and the negatively charged VLPs. Both types of CDots also exhibited inhibitory effect on VLP’s binding to their respective antibodies, but much less effective than those to HBGA binding. After CDots treatments, VLPs remained intact, and no degradation was observed on VLPs’ capsid proteins. Taken together, the observed antiviral effects of CDots on noroviruses were mainly through the effective inhibition of VLPs’ binding to HBGA receptors and moderate inhibition of VLPs’ binding to their antibodies, without affecting the integrity of viral capsid protein and the viral particle.

Antibacterial effects of carbon dots in combination with other antimicrobial reagents

This study was designed to investigate the antimicrobial effects of CDots in combination with other antimicrobial reagents, including H2O2, Na2CO3, and AcOH (acetic acid). CDots were synthesized and passivated with 2,2’-(ethylenedioxy)bis(ethylamine) (EDA). The minimal inhibitory concentration (MIC) of CDots was 64 μg/mL on both Gram negative bacteria E.coli cells and Gram positive bacteria Bacillus subtilis cells. When CDots were combined with H2O2, antibacterial synergistic effects were observed based on the fractional inhibitory concentration (FIC) index, and further confirmed by an isobologram analysis and viable cell number counting methods. With the combination treatment of 10 μg/mL CDots with 8.82 mM H2O2, the viable E.coli cell numbers decreased 2.46 log, which was significant lower than the log reduction from 8.82 mM H2O2 (1.57 log) or 10 μg/mL CDots (0.14 log) treatment alone. However, the combination of CDots with Na2CO3 or AcOH did not show synergistic effects, instead, exhibiting indifference effects according to the FIC index. This study indicated that the combination of CDots with their synergistic antimicrobial reagents, such as H2O2, could reach the goal of inhibiting bacteria growth by using lower concentration of each individual chemical in the combination than using one chemical treatment alone, reduce the risks imposed on environmental health and the possibilities of the development of microbial resistances.

Design and Evaluation of Three Immuno-based Assays for Rapid Detection of Human Norovirus Virus-like Particles

This study designed and evaluated three versatile immuno-based assays for the rapid detection of GI.1 norovirus virus-like particles at low (0-3.0 μg/mL) levels: 1) enzymatic absorbance-based ELISAs, 2) a fluorescentbased immunoassay, and 3) a “signal-down” capture ELISA. Variables including controllable variations in assay format (indirect or sandwich), assay time, binding sequence, and reporter molecule (fluorophore or enzyme) were thoroughly investigated and optimized in all three assays. Selectivity tests in the three-hour absorbance-based ELISA using VLPs representing two GI and two GII strains indicated the assays were selective to GI strains over GII strains. The three-hour enzymatic absorbance-based assay turned out to be a robust and rapid method capable of detecting GI.I VLPs in the range of 0.037 to 0.555 μg/mL, and the three-hour fluorescent immunoassay was capable of detecting VLPs in a high concentration range of 0.5-2.0 μg/mL under optimized conditions. The “signal-down” capture ELISA was conceptually demonstrated for the detection of VLPs at concentrations in excess of 1.0 μg/ mL, but did not appear to be suitable for quantifying VLPs under its current conditions. The methods reported here provide proof-of-concept that various ELISA-type approaches could be further developed to provide robust norovirus detection assays having various detection ranges, limits, and linearity.