1. Introduction

Biological membranes are the boundaries that separate interiors of cells from their external environment. The composition of biological membranes is complex; they are made of lipid bilayers [1, 2] with a wide array of components depending on the type, function and age of the cell [3]. Carbohydrates are a key structural feature of cell membranes. In cell membranes, carbohydrates are mostly found covalently attached with other biomolecules, these pairs are termed glycoconjugates. Glycoconjugates are compounds in which one or more carbohydrate units are covalently linked to a biomolecule such as a protein or a lipid [4]. Depending upon the counterparts to which the carbohydrates are linked, glycoconjugates are classified as glycoproteins, glycolipids, glycosaminoglycans or proteoglycans. These glycoconjugates

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

are involved in regulating biological activities such as fertilization, host- pathogen recognition, immunity and immune response, and in cancers where changes in glycosylation are commonly observed [5].

cholesterol content of membranes will disrupt lipid rafts, alter lipid organization, and affect cell behavior [16]. Using two-photon microscopy, liquid-ordered and liquid-disordered (raftlike) domains were observed in living cells including macrophages [17]. The removal of 9.5% of the cholesterol from live RAW264.7 cells affects the cells morphology by removing membrane protrusions, while adding more cholesterol increases the number of cell-cell contact points. The fluorescence data show a shift toward a greater population of liquid-disordered domains. Two–photon fluorescence imaging has also been used to show that cholesterol depletion in live macrophages caused disappearance of lipid rafts and that restoration of cholesterol restored the raft organization [18]. Another example concerns the culturing of hippocampal neurons, in which depletion of cholesterol resulted in a number of directly observable effects including the loss of many synapses and dendritic spines and the internalization of AMPA receptors [19]. The glycolipid ganglioside GM1 was contained within these rafts. An opportunity exists for further study of the effect of perturbation of lipid constitution

Monolayers of Carbohydrate-Containing Lipids at the Water-Air Interface

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This chapter will give insights into methods used for designing and studying monolayer model systems in general along with some pertinent experimental results. The primary component of the monolayer chosen for discussion here are glycolipids. However, the methods and theories described are not limited to glycolipids or biological systems and in principle can

Lipids are amphiphilic molecules consisting of a hydrophilic head-group and a hydrophobic

Monolayers are assembled by depositing droplets of lipid solution onto the water surface and subsequently waiting for the solvent to evaporate. The molecules spread out while the solvent evaporates. An example of a good spreading solvent is chloroform, although not all lipids are soluble in chloroform and sometimes mixed solvents with an alcohol must be used. Once deposited on the water surface, the polar or charged head-group orients towards the water surface and the hydrophobic tail(s) aligns away from the water. The lipid molecules get spread uniformly over the water surface forming a monomolecular thick film called a Langmuir monolayer named after

tail made of one or more hydrocarbon chains that may be saturated or unsaturated.

Irving Langmuir [20], who pioneered this technique together with Katharine B. Blodgett.

Selection of solvent is critical for uniform spreading of monolayer. An ideal solvent should be volatile, chemically inert, relatively pure and with enough solubilization power to dissolve the solutes under study. Care must also be taken to make sure that the solvents are insoluble in the subphase [21]. Chloroform, cyclohexane, benzene, hexane, and mixtures with acetone, ethanol or methanol are some commonly used solvents. Water or buffer solutions of various composi-

The depositions are carried out in a Langmuir-Blodgett (LB) trough, depicted in Figure 2 where some of the main monolayer techniques are also schematically depicted. The basic

on lipid organization and hence on cell behavior in living cells in culture.

2. Fabrication of monolayer of lipids as membrane models

be adapted to many other interfacial systems.

tion and pH are used as the subphase.

The complex nature of biomembranes makes them challenging to study [6], see Figure 1. This complexity necessitated development of simpler model systems, which would mimic native membranes but at the same time would give control over parameters such as structure, composition, size and facilitate monitoring of molecules of interest [7]. The majority of model systems are tailored to incorporate the bilayer structure of biological membranes. These bilayer model systems can be arranged in two-dimensions on a solid support or can form threedimensional spherical structures (supported or free in solution) as in liposomes [7–9]. Liposomes may be small, large, or giant in size and can have either one (unilamellar) or multiple (multilamellar) lipid bilayers. Liposomes are the subject of intense clinical interest where they are being studied as a vehicle for drug delivery [10–12]. Alternatively, one can fabricate monolayers of biomolecules as mimetic model systems. The compositions of monolayers are chosen to mimic one of the two leaflets of a biological membrane. One can then study the changes these systems would go through when they interact with external stimuli, which could be pathogens such as bacteria or viruses, proteins, or changes in environmental factors as temperature, pressure or pH. The results of such studies may then be extrapolated to natural biological membrane systems.

The lateral organization of lipids and cholesterol in cell membranes is important for cellular functions, especially cell signaling activities. Lipid rafts [13, 14], which are aggregates of sphingolipids and cholesterols, are also known to incorporate glycolipids [15] and host key cell signaling proteins such as glycosylphosphatidylinositol (GPI)-anchored proteins. Studies in which the lipid organization is both perturbed and also observed in living cells under culture conditions are challenging. Exposure to agents such as methyl β-cyclodextrin that perturbs the

Figure 1. A representation of the complex nature of cell membranes.

cholesterol content of membranes will disrupt lipid rafts, alter lipid organization, and affect cell behavior [16]. Using two-photon microscopy, liquid-ordered and liquid-disordered (raftlike) domains were observed in living cells including macrophages [17]. The removal of 9.5% of the cholesterol from live RAW264.7 cells affects the cells morphology by removing membrane protrusions, while adding more cholesterol increases the number of cell-cell contact points. The fluorescence data show a shift toward a greater population of liquid-disordered domains. Two–photon fluorescence imaging has also been used to show that cholesterol depletion in live macrophages caused disappearance of lipid rafts and that restoration of cholesterol restored the raft organization [18]. Another example concerns the culturing of hippocampal neurons, in which depletion of cholesterol resulted in a number of directly observable effects including the loss of many synapses and dendritic spines and the internalization of AMPA receptors [19]. The glycolipid ganglioside GM1 was contained within these rafts. An opportunity exists for further study of the effect of perturbation of lipid constitution on lipid organization and hence on cell behavior in living cells in culture.

This chapter will give insights into methods used for designing and studying monolayer model systems in general along with some pertinent experimental results. The primary component of the monolayer chosen for discussion here are glycolipids. However, the methods and theories described are not limited to glycolipids or biological systems and in principle can be adapted to many other interfacial systems.
