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Chapter 4

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The Role of Langmuir Monolayers To Understand Biological Events Luciano Caseli,*,1 Thatyane Morimoto Nobre,2 Ana Paula Ramos,3 Douglas Santos Monteiro,4 and Maria Elisabete Darbello Zaniquelli3 1Institute

of Environmental, Chemical and Pharmaceutical Sciences, Federal University of Sao Paulo, Rua Sao Nicolau, 210, 2° andar, Centro, Diadema, SP, Brazil, 09913-030 2Physics Institute of Sao Carlos, University of Sao Paulo, Avenida Trabalhador Saocarlense, 400, Parque Arnold Schimidt, Sao Carlos, SP, Brazil, 13566-590 3Chemistry Department, Faculty of Philosophy, Sciences and Letters of Ribeirao Preto, Department of Chemistry, University of Sao Paulo, Avenida Bandeirantes, 3900, Monte Alegre, Ribeirao Preto, SP, Brazil, 14040-901 4Institute of Sciences, Engineering and Technology, Federal University of Jequitinhonha and Mucuri Valleys, Rua do Cruzeiro, 01, Jardim São Paulo, Teófilo Otoni, MG, Brazil, 39803-371 *E-mail: [email protected].

In this chapter, we present an overview about different roles of Langmuir monolayers as biomembrane models, contributing to understand biological phenomena or help develop biotechnological process. Beginning with experiments that allow the development of the concept of biomembrane organization, we then discuss the interaction of lipids with bioactive compounds, peptides (i.e., fragments of proteins) and proteins. The presence of nanoparticles at the lipidic interface was also discussed. Finally, this chapter also shows some aspects of Langmuir monolayers on the nucleation and growing of nanocrystals mimicking biomineralization processes.

© 2015 American Chemical Society In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Colloid and surface sciences have been developed for the preparation of new materials and surface characterization techniques. However, the colloidal systems self-assembled by biologically relevant lipids can be also used as biomimetic models, due to the resemblance with the organization found in biomembranes. In this context, colloids and surface sciences can provide information about specific processes or for the elucidation of diseases (1). These biomimetic systems can also improve or accelerate the development of new drugs or diagnostic protocols as discussed in the following (2, 3).

Langmuir Monolayer as Biomembrane Model System Micelles, liposomes (LP), planar bilayer membranes (BLM) and Langmuir monolayers (LM) are examples of colloidal self-assembled systems employed as biomimetic models. In common, these systems have an organized amphiphilic interface separating the hydrophilic (aqueous phase) from the hydrophobic (air or oil phases). Micelles are systems in equilibrium with no lateral pressure. In these systems, the area occupied by a lipid molecule at the interface can be easily accessed. Phospholipids with two alkyl chains can produce self-assembled planar or spherical bilayer systems. However, they cannot form spherical micelles due to geometrical constraints. When small amounts of lipids are deposited on the surface of water or of any aqueous solution, they can be spread along the air-water interface and a layer is produced, forming LMs, which consists of an organized monomolecular film (4, 5). In this case, the polar head of the lipid molecules is oriented toward the aqueous phase and the hydrophobic tail is oriented toward the air phase. The repulsive force between lipid molecules is responsible for their spreading and the origin of a positive surface pressure, π = γo - γ , defined as the difference between the surface tension of the aqueous subphase before (γo) and after the spreading of the monolayer (γ). In order to develop a model for the liquid state, Irving Langmuir upgraded an apparatus (developed by Agnes Pockels) that is able to measure changes in π while a moving barrier compressed the air-liquid interface, changing the available area per molecule, A(6). With these two sets of data, π and A, a surface pressure-area isotherm obtained at a constant temperature is plotted. The attractiveness in this simple and elegant experiment is the possibility to correlate macroscopic data with intermolecular forces acting between molecules forming the LM. The monolayer can be submitted to an external lateral pressure by means of mobile barriers forcing the monolayer to reach more condensed states so as to counterbalance the lateral pressure exerted by the lipid molecules (7). During the compression, we can study the properties that depend on the molecular area, such as lipid surface density, electrical charge density, surface compressibility (surface Young modulus), and dynamic surface elasticity. As shown in Table 1, the above-mentioned properties are difficult to measure by another technique. Also, temperature, pH, and electrolytic composition of the aqueous phase can be changed and controlled during the experiments. Additionally, these monolayers can be transferred to solid supports by the Langmuir-Blodgett (LB) approach, enlarging the number of techniques to 66 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


characterize the monolayer. A scheme for an LM as model for cell membranes is shown in Figure 1. Gorter and Grendel, who are pioneers in models describing membranes, proposed in 1925 that membranes of erythrocytes were composed of a bimolecular leaflet of lipid molecules (8). This model was proposed based on data obtained from a Langmuir trough experiment involving the spreading of lipids from erythrocyte membranes extracted with acetone. Ever since, lipid LMs as model for biological membranes have been used to study mechanisms of biological events. A recent review emphasises the growing interest in LMs to study interactions in biointerfaces, correlating topics with the structure of water, biophysics of peptides and enhanced inorganic-organic composites (9). LMs were also object of a review on interfacial chemical reactions catalyzed by enzymes, particularly those involving lipid molecules, and coupling of polyelectrolytes to oppositely charged monolayers (10). Another importance is the interaction of lipids organized in LM with peptides and enzymes as well as nucleating and growing of nanoparticles. Such subjects will be presented in this chapter.

Table 1. Comparison of Colloidal Biomembrane Model Systems Feature / Model System

Liposome

Micelle

BLM

LM

Typical Average Thickness (nm)

2

5

5

2.5

Lipid surface density change

No

No

No

Yes

Electrical charge density change

No

No

No

Yes

Surface elasticity access

No

Yes

Yes

Yes

Figure 1. Scheme for a monolayer of lipids and membrane proteins representing a model for cell membranes. Some of the properties that can be assessed through characterization techniques are represented in this sketch. 67 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Biomembrane Organization The lipid bilayer model of Gorter and Grendel obtained from LM experiments represented a major key point in the development of the biomembrane models (8, 11). Despite most proteins are not able to accommodate in half a bilayer, or many can denature at the lipid air-liquid interface, several experiments could be conducted to show the interaction of enzymes with lipids and the consequences of the catalytic activity of the enzyme, and these results will be discussed in details in the following sections of this chapter. Also, it is worth mentioning the importance of the values of surface density and surface elasticity of the biomembrane model for the enzyme activity (3, 12), as well as the increase in elasticity promoted by cardiolipin of a LM biomembrane model (13). Cardiolipin levels in mitochondria are associated with Parkinson´s disease. Also, the constitution and mechanism of ion channels in biomembranes is extremely relevant. After the establishment of the correspondence between the lipid-water interface in a lipid monolayer of a bilayer and those of a lipid monolayer formed on mercury electrodes (14), electrochemical experiments involving lipid monolayers containing gramicidin forming channels have also shown the importance of the lipidic environments and the composition of these environments for the channel activity (15). Another aspect of the membrane organization is the so-called raft domains (16). This corresponds to lipid microdomains induced by preferential packing of sphingolipids and cholesterol, where membrane-anchored proteins (through glycosylphosphatidylinositol anchor – GPI proteins) are preferentially located (17). The clustering of GPI proteins as well as lipid distribution in membrane models, LM or BLM, are mostly studied by means of AFM and fluorescence microscopy (18). These domains play an important role in signal transduction, enzyme activity and membrane transport.

Peptides and Proteins in Langmuir Monolayers Peptides are molecular sequences of amino acid units with molecular weight lower than 10,000. These structures have been intensively studied in the last decades since they are involved in several biological mechanisms. For instance, they can initiate an infectious process or be involved in the diagnostic of some diseases (19–21). Understanding the interaction of peptides with cell membranes can explain numerous biological events, including pathologies. Also, peptide-cell membrane model interactions have explored in order to elucidate the mechanism of action of peptides that act as drugs. Furthermore, some peptides can permeate cell membranes to deliver drugs (22). In some circumstances, peptides can also be the drug themselves (23–25). In all cases, LMs have been shown to be an appropriate tool to mimic a cell membrane, with easy control of the composition and the molecular architecture. Mimicking biomembranes using LM allows the study of specific events involving peptides separately from the numerous ones occurring at the same time on the cell surface. 68 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Peptides can be obtained from desired regions of proteins of interest to better understand the role of this specific region on protein-membrane interactions. In some cases, peptides corresponding to the C- or N-terminus (26) or containing specific binding domains of proteins can be synthesized (27), and their interaction with LMs can elucidate the mechanism about how proteins anchor to the membrane. Sometimes, it is necessary to screen the complete sequence of the protein to identify regions of interest, and then one can synthesize those different “pieces”, as peptides, to determine which region is related to a specific biological event (19, 27). This is how the Langmuir technique has been employed to study Alzheimer´s disease (AD). Despite the different hypothesis for the origin of AD, there is a consensus about the involvement of amyloid-β (Aβ) protein in this pathology since patients with AD present Aβ insoluble toxic fibrils in the brain (28, 29). In the literature, Aβ protein was deconstructed in peptides and the role of different regions of the protein on the fibrillation/aggregation process was studied (29–37). Since the conversion of the protein into its toxic form involves changes in secondary structure (from α-helix to β-sheet), polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) has been used to evaluate these systems (29). Also, microscopic techniques such as epifluorescence and Brewster angle microscopy (BAM) are useful to identify aggregates at the interface. In some cases, Aβ peptides were synthesized containing an aliphatic chain attached either to its C- or to its N- terminal region, as a strategy to keep the peptide at the air-water interface. The role of the membrane in the aggregation of Aβ-peptides was also investigated, focusing particularly on the membrane composition (36). Viral infection is also an important process studied by LMs (38–41). In general, after attaching to the host cells, a virus infects the host cells by entering into the cells and then introducing its genetic material. This attachment involves the interaction of proteins from the viral envelope with proteins and lipids on the cell membrane of the target cell. After that, viral entry occurs usually by fusion of the membranes. For the Human Immunodeficiency Virus (HIV), the literature shows that the N-terminus region of the gp41 protein in the viral envelope is a putative fusion peptide between HIV and target cells. The peptide containing 23 amino acid residues of the gp41 N-terminus was synthesized and its interfacial properties as well as its interaction with different lipids were characterized (39). The results indicated the conditions for peptide aggregation at the interface and showed that an oblique orientation of fusogenic peptides inserted into the membrane is necessary to cause membrane perturbations, which favours the initial steps of the fusion process. Antimicrobial peptides (AMPs) are a class of peptides widely studied by the Langmuir technique. The great interest in these compounds is due to their potential as antibiotics. AMPs can be isolated from plants, bacteria, amphibians and mammals, including humans. AMPs can be also synthesized or bioinspired by some peptides found in nature (42). It has been reported that some parameters play an important role in determining AMP activity: peptide primary sequence and secondary structure, charge, amphiphilicity, hydrophobicity (polar/nonpolar amino acid ratio), and the angle related to hydrophilic/hydrophobic helix surfaces (43–51). In order to address the mechanism about how the peptide works and to improve its antimicrobial activity, peptides with different sequences were 69 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


synthesized and their interaction with membranes was evaluated. In some cases, replacing a single residue can drastically affect the interaction of the peptide with the membrane and consequently this may affect its activity (45, 46). For example, increasing the positively charged residues can enhance the interaction between the peptide and the lipid monolayers, especially those built with anionic lipids, which are the major constituents of bacterial membranes. However, the presence of additional non-polar residues (such as tryptophan) also boosted the interaction of the peptide with the lipid monolayer. This indicates that hydrophobic interactions are also important to damage the membrane. Alteration in the primary sequence of the peptide can also change its secondary structure, resulting in a more (or less) rigid structure and the change of the conformational flexibility of the peptide, which drastically affects the depth of peptide incorporation into the lipid monolayer (44–46). A very important property of an AMP is its membrane selectivity: as more selective to bacterial membranes, as more potential this peptide will be able to act as an antibiotic. This selectivity means a major interaction with bacterial membranes and no interaction with mammalian cells. The membrane selectivity is widely investigated by using LMs. Considering that mammalian cells have a more neutral character, the phosphatidylcholine lipids (DPPC - dipalmytoylphosphatidylcholine, DOPC - dioleoylphosphatidylcholine, egg-PC, etc.) are usually preferred. As bacterial cells are negatively charged, it is possible to predict, by using different lipid compositions in the LM, whether an AMP is a good candidate as a bactericidal drug. In addition, the detailed mechanisms of the AMP interaction with biomembranes and the potential to improve its activity by modification of the peptide structure can be investigated by LM technique. As a result, studies on the interaction between proteins and lipids by using LMs are very useful to understand phenomena occurring at the cell membrane level. Because of the great advance in techniques that are able to characterize interfacial events, it is now possible to obtain more details to address the interaction between lipid membranes and peptides/proteins. LM technique is powerful because lots of parameters are variable and can be monitored. The following example concerns a LM formed for pathology study. With LM technique, it is probable not only to address which peptide (region of protein) is able to interact with the membrane, but also to elucidate how the membrane can modulate this interaction when the parameters (like pH and temperature) related to the subphase are changed. As for peptides, how the peptide inserts into membrane depends on the composition and the architecture of the membrane and this is relevant to understand the pathology of a certain disease. Thus, a considerable variety of proteins were studied by the LM technique. Depending on the properties of various proteins, different strategies for the LM film confection have been employed (19, 52–63). The first method is co-spreading, which consists in spreading protein and lipid from the same stock solution. It is useful for some hydrophobic proteins (and peptides). However, since the protein is solubilized in organic solvents (e.g., chloroform), protein denaturation is possible (63, 64) , although proteins can readapt their conformation when submitted to other conditions and have their active conformation restored. 70 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Regarding the proteins soluble in aqueous solution, the injection method (i.e., the second method) is popular. This method consists in injecting the protein in the aqueous subphase, underneath a pre-formed lipid monolayer. This method is similar to the biological environment and has the advantage of being able to obtain kinetic parameters. The ability of the protein to diffuse and adsorb at the lipid interface can be monitored in terms of surface pressure versus time, and parameters like diffusion coefficient (23) can be obtained. If the diffusion is very slow, the protein can be homogenized in the aqueous subphase prior to the lipid film formation, and the method (i.e., the third method) can be called “protein as subphase”. In these cases, diffusion from the bulk to the interface can be ignored. Besides the similarities between these two last methodologies, surface pressure-area isotherms involving the same protein can be significantly different depending on the strategy of incorporation. In fact, there is a debate in the literature about proteins incorporated at lipid monolayers by using the subphase method: it was reported that proteins at the bare interface can denature, at least partially, due to the exposure of hydrophobic regions towards the air. However, considering that proteins are generally stored in the solid state, the exposure of hydrophobic regions towards the air should not be the only factor responsible for the protein denaturation, but the overall changes of the polypeptide at the interface including from those protein groups in contact with the aqueous phase. Although some authors observed that after lipid spreading at the interface the original conformation of the protein can be restored (63), there is still no agreement about this fact. The fourth strategy is spreading the protein on the top of the interface after lipid spreading. This strategy can be called “top methodology” (i.e., the fourth method); the idea is to avoid the contact of the protein with the organic solvent. In this strategy, the problem is that the protein solution can carry some lipid molecules to the subphase by aggregation. The last strategy (i.e., the fifth method) to incorporate a protein into a lipid film is to increase the ionic strength of the subphase. Then the protein is forced to adsorb at the lipid interface due to the salting out effect (65). Again, this methodology can result in protein unfolding due to the high salt concentration. Considering all the limitations and peculiarities mentioned above, LMs is a suitable strategy to mimic the cell membrane environment. In this way, interactions of several proteins with lipids were investigated successfully, such as the previously mentioned interaction between lipids and proteins related to fibril formation in neurodegenerative diseases, mainly Alzheimer´s disease (AD) (19).

Enzymes Adsorbed in Langmuir and Langmuir−Blodgett films Enzymes are biological compounds able to catalyze chemical reactions. Except for catalytic RNA molecules, enzymes are polypeptides containing special structures to interact specifically with the substrates and can promote a new mechanism for a given reaction. This new mechanism will result in a relative shorter time of reaction because the activation energies of the reaction are lowered in the new mechanism and the conversion rate of the substrates is increased to 71 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


millions of times faster. Many metabolic processes in cells require enzymes to occur in rates able to sustain life. Enzymes are usually specific for certain substrates due to their threedimensional structures. This specificity may be useful for biotechnology since enzymes can be used as a component of a sensor for a specific substance. In the literature, enzymes have been immobilized in solid matrices to serve as biosensors (66, 67). For Langmuir-Blodgett (LB) films, the first reports are related to the spreading of pure enzymes at the air-water interface and their subsequent transfer to solid supports (68–70). The advantage of the LB methodology relies on the facts that such films can be produced in several kinds of solid supports, producing molecularly ordered arrays and with high control of the molecular architecture. These facts open the possibility to construct devices with fast response with a low quantity of enzyme. Also, the films can be constructed in controlled environments, such as temperature, pH, and pressure. Furthermore, the sensitivity and detection limit may be pre-determined by changing some aspects of the molecular architecture, such as number of layers, and substances co-adsorbed. These aspects are interesting for the fabrication of biosensors based on the direct transduction of biological signals, which make these films as materials called “bio-inspired”. Enzymes are usually sensitive to vicinity conditions, and it is a challenge to find environments able to conserve the structure of the biomacromolecules and maintain at least part of their biological activity. Enzymes adsorbed at a clean airwater interface are reported. (67, 68, 70). If a higher amount of enzyme needs to be adsorbed, a high concentration of ions in the aqueous subphase favours enzymes to migrate from the bulk to the surface because of the salting out effect (71) as discussed as the fifth method in the previous section. However, an excess of salt may cause tough effects on the structure of the enzyme (72). A current approach to avoid the loss of enzyme activity is to form mixed lipid-enzyme monolayers at the air-water interface to be subsequently transferred to solid supports by using the LB methodology (73–76). The main idea is that lipids, being amphiphilic, contain hydrophobic and hydrophilic groups that help the accommodation of the enzyme into the lipid layer in such way that the major structures of the macromolecule are kept. In this case, the method of insertion of proteins can be done by using the second method in the previous section. For that, the ability of the enzyme to penetrate into the lipid monolayer can be investigated by varying the initial surface pressure prior to the enzyme insertion. Usually, high surface pressures decrease the ability of enzyme adsorption because the enzyme penetration may be inhibited when the surface pressure increases. Plots of increase of surface pressure owing to enzyme penetration versus initial surface pressures usually give the so-called exclusion surface pressure, πe (77), obtained by the extrapolation of the straight line to the x-axis (Figure 2). For initial surface pressures equal to or higher than πe, the enzyme is reported to be unable to penetrate into the lipid monolayer. However, this fact may not be considered as definite evidence that the enzyme cannot interact with the monolayer since adsorption of the enzyme on the lipid polar heads may still occur, and this would not lead necessarily to increase the surface pressure of the monolayer (78). Also, some hydrophobic enzymes may provide a sudden increase of the surface pressure 72 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


due to a fast enzyme penetration, in an effect called “piston” by some authors (79, 80). Further accommodation of the enzyme may provide a slow decrease of the surface pressure until equilibrium is reached.

Figure 2. Example for the determination of exclusion surface pressure of a protein inserted below a lipid monolayer.

Another point to be discussed is how an enzyme incorporated in a lipid monolayer in a high initial surface pressure can be compared to an enzyme incorporated in low initial surface pressures and then subjected to compression until a desired surface pressure is reached. Although thermodynamic equilibrium is expected for both, the process may be considered to be in a low dynamic state. Also, as the enzyme must be injected in specific points below the lipid monolayer, it is experimentally difficult to distribute the enzyme homogeneously, and equilibrium must be achieved after complete lateral diffusion of the enzyme, which is commonly slower than enzyme diffusion from the aqueous subphase to the air-water interface. A strategy to avoid the concentration of enzymes in specific points on the surface has been to spread the enzyme and the lipid together from the same solution (81–84). However, the organic solvents usually employed 73 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


to dissolve the lipid may denature the enzyme, and dispersion of lipids in water may make the spreading on the interface difficult. Another strategy is to immobilize the enzyme on a pre-formed lipid LB film. The solid substrate is inserted into the enzyme solution allowing for enzyme adsorption (70, 85–87), which promotes specific interactions of the enzyme with hydrophilic or hydrophobic surfaces. However, the weak association of the enzyme with the lipid surface may be a major drawback since the enzyme can be desorbed when re-inserted in aqueous solutions (88). A recent study compares the enzyme activity of uricase immobilized in several architectures of stearic acid (SA) as LB films (78). Uricase was adsorbed in pre-formed SA LB films, in which the LB film exposed the hydrophobic or the hydrophilic part. In other architectures, uricase was transferred with SA from a mixed enzyme-lipid LM. For that 1 or 4 monolayers were transferred. Enzyme activity for 1-layer LB films was higher than for 4-layer LB films, demonstrating that the catalytic activity is more relevant for enzymes incorporated in the outmost layer. Also, enzyme activity is better when the enzyme is adsorbed on hydrophilic layers, rather than on a hydrophobic layer. Since enzymes are immobilized in solid matrices together lipids, this functionalized biomimetic membrane has been shown to be structurally stable and able to preserve the enzyme activity for long periods of time (89). Enzyme activities in solid matrices are usually partially retained in comparison to that in solution (17), owing to restrictions of the biomacromolecule in terms of mobility and possibility to conformational adaptations. Higher enzyme activities in comparison with enzyme solutions are usually reported with heme-enzymes, for which the hydrophobic environments provided by the lipids favor the accessibility of the catalytic substrate to the catalytic site of the enzyme (64, 90). Typical behaviors of the enzyme retained at the surface of the biomimetic membrane have demonstrated potential usefulness of such assemblies for investigations in biomimetic environments, with favourable orientation of recognition sites. Also, self-molecular assembly of biomolecules allows for the insertion of the enzyme at a specific geometry, improving the recognition properties (17, 64, 90, 91). Figure 3 shows a scheme for enzymes being incorporated in LMs of lipids and the subsequent transfer to solid supports as an LB film with the purpose of using it as a matrix for molecular recognition. Regarding this scheme, it must be considered that proteins may adsorb on the solid support immersed in the aqueous subphase prior to the step of removal of the support. For this reason, some tests must be performed in order to check if the immobilized enzyme comes only from the film at the air-water interface. A possible test is inserting the solid support in the aqueous subphase with the enzyme dissolved, but with no lipid monolayer present. The solid support is then removed and the possible adsorption of the enzyme analyzed.Another possible experiment regarding this fact is to transfer the monolayer with the enzyme adsorbed to another compartment with a pure water subphase by means of a shallow lateral slot communicating the two compartments. After the mixed monolayer is transferred, the solid support is removed from the pure water subphase. In this sense, Table 2 shows some recent reports of enzymes adsorbed in Langmuir and LB films. 74 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 3. Scheme for obtaining mixed enzyme-lipid LB film.

Table 2. Enzymes Incorporated in LMs and/or LB Films Enzyme Organophosphorus Acid Anhydrolase

Main Finding/Reaction Product

Architecture

Ref.

Pure Enzyme On The Air-Water Interface

Enzyme Activity Could Be Measured

Ref. (92)

Lysozyme

Pure Enzyme On The Air-Water Interface

Salt Ions Minimize The Water-Accessible Surface Area Of The Protein, Enhancing Protein Dehydration And Assisting In Protein Refolding And Association.

Ref. (65)

Glutamate Dehydrogenase

Behenic Acid

Glutamate

Ref. (88)

Laccase

HeptylBis(Thiophene) Carbazole And Tricosenoic Acid

2,2′-Azino-Bis(3Ethylbenzthiazoline6-Sulphonate) Abts

Ref. (93)

Phosphatase Alkaline

Dimyristoyl Phosphatidic Acid (Dmpa)

P-Nitrophenolphosphate

Ref. (87)

Urease

Dipalmitoyl Phosphatidyl Glycerol (Dppg)

Urea

Ref. (94)

Tyrosinase

Arachidic Acid

Pyrogallol

Ref. (95)

Alcohol Dehydrogenase (Adh)


Ethanol

Ref. (96) Continued on next page.

75 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Table 2. (Continued). Enzymes Incorporated in LMs and/or LB Films


Enzyme

Main Finding/Reaction Product

Architecture

Ref.

Horseradish Peroxidase

Dppg

Hydrogen Peroxide

Ref. (64)

Catalase


Hydrogen Peroxide

Ref. (90)

Uricase

Stearic Acid

Uric Acid

Ref. (78)

Sucrose Phosphorylase

Dmpa

Sucrose

Ref. (54)

Penicillinase

Dmpa

Penicillin

Ref. (97)

Cellulase And Adh

Dppc

Detecting Ethanol

Ref. (98)

Hyaluronidase

Dppc

Hyaluronic Acid

Ref. (61)

God

Phospholipid Analogous Vinyl Polymer

Signal Intensity Increasing With The Number Of Deposited Layers.

Ref. (99)

Also, it is important to mention that several studies use lipases inserted in the aqueous subphase and employ the lipid monolayer as catalytic substrate (99–103). Usually, the action of the enzyme changes the properties of the monolayer, which can be accompanied by analyzing how surface pressure varies with time or how infrared spectra are changed (104).

Bioactive Compounds in Langmuir Films as Biomembrane Models There are several reports in the literature studying the interaction between bioactive drugs and LMs. These studies have the purpose not only to understand how membrane interacts with active drugs, but also to increase its selectivity. For some drugs, the membrane is not the primary target of bioactive compounds, as they usually bind to a receptor (e.g., a DNA or an enzyme) inside the cell. However, the interaction with the membrane is crucial for the drug incorporation and to determine its selectivity. Also, the great interest in investigating the interaction between drugs and lipids is due to the possibility of incorporating such molecules in liposomes for drug delivery, especially aiming at the reduction of the toxicity. Many reports use LMs to investigate the interaction of membrane with antimicrobials (105–116), antiparasitic (117, 118), antitumor (119, 120), anti-inflammatory (121, 122), antipsychotic (123–126), and coronary vasodilator drugs (127). A suitable strategy to investigate drug selectivity is to build LMs of a specific compound found in a certain organism or organelle. Dynarowicz-Latka and co-workers verified that the antimycotic amphotericin B (AmB) is more toxic for 76 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


fungi than for host cells by comparing the results for the Langmuir films built with different sterols: ergosterols from fungi, and cholesterols from animals (128). For antibiotics, researchers have proven the specificity of the molecules and attested its low toxicity by using the fact that bacteria membranes are negatively charged, while mammalian cell membranes are, in general, composed of a great percentage of zwitterionic lipids (128). Moreover, for some bacteria, the specific phospholipid composition can be studied to evaluate the interaction with some antibiotics. Also, the interaction of drugs with Gram-negative bacteria has been evaluated by using LMs of lipopolysaccharides (129), the major constituent of the outer leaflet of Gram-negative bacteria. Total lipid extracts can also produce Langmuir films, and this strategy has been used mainly for bacteria (130). Cerebrosides and gangliosides are also interesting molecules to be taken into account since they are found mainly in nerves and brain tissue membranes. In addition, it has been found that gangliosides play an important role in tumor progression (131), so it could be applied as a molecular target for antitumor molecules that can easily be assessed by LMs. A difficulty in establishing a common mechanism of action for the drugs arises from the fact that they present very different chemical structures, and there are few connections between structure and activity involving drugs and lipids. In this case, an alternative is to synthesize molecules inspired by pre-existent drugs and verify their effects in the membrane. Alkyllysophospholipids are synthetic analogs of lysophosphatidylcholines (LPC) with anti-tumor properties (132) that are synthesized replacing the acyl group with an alkyl group. In order to increase the metabolic stability of LPC, the analogs edelfosine, milefosine and erucylphosphocholine were synthesized, and their effects were evaluated in terms of their interaction with LMs (119, 120, 133–137). Some examples of compounds and their derivatives that were also studied by the Langmuir technique are the antipsychotic phenothiazine (derivatives studied: chlorpromazine and trifluoperazine) (124), the antibiotics tetracycline and oxytetracycline (138, 139) and the fluoroquinolones antibiotics ciprofloxacin and moxifloxacin (140, 141).

Incorporation of Nanoparticles in LM and LB Films Acting as Cellular Membrane Understanding the mechanism of interaction between nanoparticles (NPs) and cell membranes is a major topic for the development of new therapeutic agents. In this way, systems organized in nanometric scale are a potent strategy in many technological fields since it is possible to evaluate the influence of NPs in living organisms using artificial models. The importance of such studies lies in the fact that many NPs are potentially toxic and can cause hazardous effects (142, 143). In this sense, a possible way to elucidate how nanoparticles act in living organisms is to work with theoretical and experimental membrane models able to mimic living cells (9, 144). Such models include LMs as shown in Figure 4. The studies involving NPs and LM can be divided into two approaches. The first involves systems in which nanoparticles interact with biomimetic systems, i.e., monolayers of lipids at the air-water interface above aqueous dispersions of NPs 77 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


(145). The second includes NPs that form stable structures at liquid-air interfaces forming interfacial aggregates that resemble LMs. Both systems can be employed in several areas such as materials, medical and biological sciences. NPs can promote significant changes in the lipid monolayer structure by means of ionic (145) or hydrophobic (146) interactions, changing lipid surface packing and being inserted in the interfacial layer (147), or altering phase behavior (148–150). However, the exact mechanism of interaction of NPs and LMs remains poorly understood. In the literature NPs are reported to be inserted under the lipid monolayer as pre-formed dispersions (145) or injected into the aqueous subphase beneath a lipid monolayer that has been already spread (151, 152) , which sometimes complicates the comparison owing to different effects resulted from different ways of incorporation. Nevertheless, there have been efforts to describe in general some aspects related to NPs. Some properties intrinsic to NPs must be considered such as flotation forces (driven by gravity) and immersion forces (driven by wetting). Important about these capillary forces, which are not often considered, is that the former acts on large particles (

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