Membrane Microfluidics Research Articles

Liquid biopsy technologies based on membrane microfluidics: High‐yield purification and selective quantification of biomarkers in nanocarriers

Liquid biopsy, screening cancer non-invasively and frequently by detecting and quantifying molecular markers in physiological fluids, would significantly improve cancer survival rate but it remains a distant goal. The key obstacles presented by the highly heterogeneous samples are rapid/high-yield purification and precise/selective marker capture by their antibody and oligo probes. As irregular expressions of these molecular biomarkers are the key signals, quantifying only those from the cancer cells would greatly enhance the performance of the screening tests. The recent discovery that the biomarkers are carried by nanocarriers, such as exosomes, with cell-specific membrane proteins suggests that such selection may be possible, although a new suite of fractionation and quantification technologies would need to be developed. Although under-appreciated, membrane microfluidics has made considerable contributions to resolving these issues. We review the progress made so far, based on ion-selective, track-etched, and gel membranes and advanced electrophoretic and nano-filtration designs, in this perspective and suggest future directions.

Abstract 690: Selection of resistance by anticancer drugs is not responsible for alterations in membrane micro-fluidity: A study by Fluorescence Correlation Micro-Spectroscopy

Abstract Expression of P-glycoprotein (P-gp), the multidrug resistance (MDR) 1 gene product, can lead to MDR in tumors. Previous studies on multidrug-resistant cells have suggested changes in membrane fluidity associated with P-gp expression in drug-selected resistant cells. However, Aleman and co-workers have shown that, in MDR1 transfected cells, expression of P-gp has little effect on membrane fluidity and the changes in this parameter observed in drug-selected cells must reflect other host adaptations to drug selection. In this study and the other reported on drug-selected resistant cells, membrane fluidity has been measured using fluorescent membrane probes and fluorescence anisotropy or electron spin resonance on cell population samples (Aleman et al. Cancer Res. 2003 63:3084-91). Here, we propose to explore the plasma membrane microfluidity by using the fluorescent membrane probe DiA and fluorescence correlation micro-spectroscopy (FCM-S) on a single living cell and nanometer scale. By using the fluorescent membrane probe DiA and FCM-S to explore the plasma membrane micro-fluidity, we have previously shown a decrease in plasma membrane micro-fluidity in LR73R cells (the Chinese hamster ovarian LR73 cells transfected with MDR1 cDNA; Boutin et al., J Biomed Opt. 2009 14: 034030). Moreover, a strategy using flow cytometry and calcein-AM functional fluorescence-activated cell sorting permitted us to select two cell populations from LR73R cells expressing distinct levels of P-gp function. In this case, we were able to observe distinct level of membrane micro-fluidity in the two populations. Cells showing high level of calcein-AM uptake presented a higher membrane micro-fluidity. In contrast, the FCM-S yielded opposite data when we compared the membrane micro-fluidity of several parental cell lines (MCF7, KB3.1, LR73, ME-SSA) and their resistant counterparts which have been selected in the presence of anticancer drugs. Our data are not in agreement with previous published data and demonstrates clearly that alterations that can affect drug-selected cells do not induce any changes in membrane fluidity. Moreover, micro-analysis of cell membrane could allow to more precise measurements of membrane fluidity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 690. doi:10.1158/1538-7445.AM2011-690

An apolipoprotein E4 fragment can promote intracellular accumulation of amyloid peptide beta 42

Apolipoprotein E (apoE) plays a crucial role in lipid transport in circulation and the brain. The apoE4 isoform is a major risk factor for Alzheimer's disease (AD). ApoE4 is more susceptible to proteolysis than other apoE isoforms and apoE4 fragments have been found in brains of AD patients. These apoE4 fragments have been hypothesized to be involved in the pathogenesis of AD, although the mechanism is not clear. In this study we examined the effect of lipid-free apoE4 on amyloid precursor protein processing and 40-amino-acid Aβ variant and 42-amino-acid Aβ variant levels in human neuroblastoma SK-N-SH cells. We discovered that a specific apoE4 fragment, apoE4[Δ(166-299)], can promote the cellular uptake of extracellular 40-amino-acid Aβ variant and 42-amino-acid Aβ variant either generated after amyloid precursor protein transfection or added exogenously. A longer length fragment, apoE4[Δ(186-299)], or full-length apoE4 failed to elicit this effect. ApoE4[Δ(166-299)] effected a 20% reduction of cellular sphingomyelin levels, as well as changes in cellular membrane micro-fluidity. Following uptake, approximately 50% of 42-amino-acid Aβ variant remained within the cell for at least 24 h, and led to increased formation of reactive oxygen species. Overall, our findings suggest a direct link between two early events in the pathogenesis of AD, apoE4 proteolysis and intraneuronal presence of amyloid beta peptide.

Abstract 3537: P-glycoprotein expression level is responsible for alterations in plasma membrane microfluidity as probed by fluorescence correlation spectroscopy

Abstract Expression of P-glycoprotein (P-gp), the multidrug resistance (MDR) 1 gene product, leads to MDR in tumors. By using the fluorescent membrane probe DiA and Fluoresence Correlation Spectroscopy (FCS) to explore the plasma membrane microfluidity, we have previousely shown a showed a decrease in plasma membrane microfluidity in LR73R cells (the Chinese hamster ovarian LR73R cells transfected with MDR1 cDNA) (1). In the present study, and in order to study the relationship between membrane microfluidity and the level of P-gp expression, a strategy using flow cytometry and calcein-AM functional fluorescence-activated cell sorting permitted us to select two cell populations from LR73R cells expressing distinct levels of P-gp (P4 “lower calcein-AM uptake” and P5 “higher calcein-AM uptake”). Data obtained on the cytotoxic effects of doxorubicin (DOX) showed clearly that P4 cells were 10-fold resistant to DOX than P5 cells. A direct correlation between the sensitivity of the cells to DOX and the calcein-AM uptake was observed. In addition, intranuclear DOX measurements using microspectrofluorometry showed a decrease in nuclear accumulation of DOX in P4 cells when compared to P5 cells. This was in agreement with the functional calcein-AM uptake assay. By using the fluorescent membrane probe DiA FCS analysis, we explored the membrane microfluidity in P4 and P5 cells. We show that P5 cells presented an increase in membrane fluidity when compared to P4 cells. LR73 cells presented the highest microfluidity when compared to P4 and P5 cells. This was in agreement with the previous published data (1) and demonstrate also that the microfluidity of the plasma membrane is strongly related to the level of Pgp expression. (1) Boutin C, Roche Y, Millot C, Deturche R, Royer P, Manfait M, Plain JM, Jeannesson P, Millot JM, Jaffiol R. High heterogeneity of plasma membrane microfluidity in multidrug-resistant cancer cells. J Biomed Opt. 2009 14(3): 034030. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3537.

Control over micro-fluidity of liposomal membranes by hybridizing metal nanoparticles

This study introduces a facile method to hybridize metal nanoparticles with lipid vesicles, which allows us to control over their membrane micro-fluidity. We have fabricated these hybrid liposomes by directly hybridizing metal nanoparticles with lipid bilayers solely consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC). For this, we have used the dehydration and rehydration method. Characterizing their morphology and micro-fluidity, in which we have used electron microscopy and fluorescence anisotropy spectroscopy, enables us to demonstrate that metal nanoparticles with different surface properties create interactions with either phosphorus end groups or hydrophobic tails of DPPC, thereby resulting in decrease in micro-fluidity of the assembled lipid membranes, especially for the hydrophobic layers. Our approach to hybridize metal nanoparticles in between lipid layers offers a flexible means that allows us to obtain a liposome system with more controllable membrane properties.

Surfactin-Triggered Small Vesicle Formation of Negatively Charged Membranes: A Novel Membrane-Lysis Mechanism

The molecular mode of action of the lipopeptide SF with zwitterionic and negatively charged model membranes has been investigated with solid-state NMR, light scattering, and electron microscopy. It has been found that this acidic lipopeptide (negatively charged) induces a strong destabilization of negatively charged micrometer-scale liposomes, leading to the formation of small unilamellar vesicles of a few 10s of nanometers. This transformation is detected for very low doses of SF (Ri=200) and is complete for Ri=50. The phenomenon has been observed for several membrane mixtures containing phosphatidylglycerol or phosphatidylserine. The vesicularization is not observed when the lipid negative charges are neutralized and a cholesterol-like effect is then evidenced, i.e., increase of gel membrane dynamics and decrease of fluid membrane microfluidity. The mechanism for small vesicle formation thus appears to be linked to severe changes in membrane curvature and could be described by a two-step action: 1), peptide insertion into membranes because of favorable van der Waals forces between the rather rigid cyclic and lipophilic part of SF and lipid chains and 2), electrostatic repulsion between like charges borne by lipid headgroups and the negatively charged SF amino acids. This might provide the basis for a novel mode of action of negatively charged lipopeptides.

E-cadherin tethered to micropatterned supported lipid bilayers as a model for cell adhesion.

Cell-cell adhesion is a dynamic process requiring recruitment, binding, and reorganization of signaling proteins in the plane of the plasma membrane. Here, we describe a new system for investigating how this lateral mobility influences cadherin-based cell signaling. This model is based on tethering of a GPI-modified E-cadherin protein (hEFG) to a supported lipid bilayer. In this report, membrane microfluidics and micropatterning techniques are used to adopt this tethered protein system for studies with the anchorage-dependent cells. As directly formed from proteoliposomes, hEFG exhibits a diffusion coefficient of 0.6 +/- 0.3 microm(2)/s and mobile fraction of 30-60%. Lateral structuring of the supported lipid bilayer is used to isolate mobile proteins from this mixed mobile/immobile population, and should be widely applicable to other proteins. MCF-7 cells seeded onto hEFG-containing bilayers recognize and cluster this protein, but do not exhibit cell spreading required for survival. By micropatterning small anchors into the supported lipid bilayer, we have achieved cell spreading across the bilayer surface and concurrent interaction with mobile hEFG protein. Together, these techniques will allow more detailed analysis of the cellular dynamics involved in cadherin-dependent adhesion events.

Alterations in Membrane Lipid Dynamics of Leukemic Cells Undergoing Growth Arrest and Differentiation: Dependency on the Inducing Agent

The effect of various differentiation inducers on membrane cell dynamics was studied using HL-60 and K562 leukemic cell lines. Membrane lipid dynamics was measured by the steady-state fluorescence polarization (P) method utilizing either 1,6-diphenyl-1,3,5-hexatriene (DPH) or the trimethyl ammonium derivative of DPH (TMA-DPH), which ascertains anchorage of the label to the membrane–water–lipid interface. Decrease in membrane microfluidity was observed in HL-60 cells undergoing differentiation into macrophages by 1,25-dihydroxyvitamin D3and by K562 cells induced to differentiate by DMSO. Sodium butyrate caused an increase in membrane fluidity in K562 cells undergoing differentiation into erythroid-like cells while in HL-60 cells a dual effect was observed. At 0.4 mM concentration, in which the cells were induced to differentiate along the monocyte pathway, a decrease in membrane fluidity was observed, while at 1 mM concentration an increase in membrane fluidity occurred. Interferon-γ (IFN-γ) induced an increase in membrane fluidity in both cell lines. Using HL-60 cells fluorescently labeled by TMA-DPH, similar results indicating fluidization of the membrane following IFN-γ treatment were obtained. Advanced fluorescence lifetime measurements, evaluated either by phase modulation spectrofluorometry or by single photon correlation fluorometry confirmed that the decrease in fluorescence polarization by IFN-γ resulted from membrane fluidization and not from elongation of the probe's excited state lifetime. It is suggested that the inducer mode of action, and not the differentiation route, determine the outcome of changes in membrane microviscosity.