COORDINADOR(A) DE LA UGAM

As discussed in chapter 1.6, the mechanism of action for most antimicrobial peptides is electrostatically driven by their cationic nature [150]. Bacteria exhibit an over- all anionic charge, since they contain an anionic cell membrane, cell wall, and for Gram-negative bacteria, lipopolysaccharide coat [273]. Bacterial cell membranes con- tain 20-25% of negatively charged lipids including phosphatidylglycerol and cardiolipin [274]. Bacterial membranes contain on average 30% PG, an anionic lipid, although this varies greatly between bacteria. The remaining lipid composition is formed mainly of uncharged lipids, principally phosphatidylethanolamine (PE) [275]. In contrast eukary- otic cells contain 20-50% phosphatidylcholine (PC).

Supported lipid bilayers (SLBs), also known as model membranes, are model systems used to study the structure and function of membrane bound and membrane active biomolecules at the nanoscale using AFM [5], [59], [144], [160], [179], [234], [276]–[278]. Here we form supported lipid bilayers to mimic bacterial cell membranes, as described in chapter 3.1.3. Model membranes are used to obtain the flat featureless surfaces required to observe membrane disruption by AMPs at the nanoscale, allowing for the observations of pores which may be only a few nanometres wide.

In these studies bilayers were formed of composition DLPC:DLPG in a 3:1 (w/w) ratio as described in chapter 3.1.3 to maintain a similar level of anionic charge to bacterial membranes. The vesicle fusion method was used as described in 3.1.3.2 to form a single bilayer on the mica [176]. The use of DLPC in place of the more commonly occurring PE in bacterial cells was chosen due to its fluid form at room temperature, the fact that it has the same chain length and therefore height as its DLPG counterpart and also to complement other studies. DLPG and DLPC lipids both form bilayers in the fluid phase at room temperature due to their gel to fluid transition temperatures which are significantly lower than room temperature at -1◦_{C and -3}◦_{C respectively. As these}

lipids have equal chain length, they will form a flat bilayer of one continuous height for AFM studies. In addition other studies such as molecular dynamics simulations, NMR,

and nano-sims, were carried out on the same peptide using this lipid composition as described in [236].

AFM imaging is carried out in contact mode on a JPK Nanowizard 1 using MSNL-C and D cantilevers (k = 0.03− 0.05) in buffer solution to determine the presence of a bilayer on the mica surface. The AFM image of the surface in figure 6.5D shows a flat featureless substrate, identical to a flat featureless mica surface, shown in figure 6.5B, and as such further experiments must be done to prove the presence of a bilayer on the surface prior to the addition of peptides.

Figure 6.5: Verification of the presence of a DLPC:DLPG (3:1) lipid bilayer on a mica surface by AFM. A) A force curve taken on a mica surface. B) A contact mode AFM image of a mica surface. C) A force curve taken on the DLPC:DLPG (3:1) lipid bilayer surface with a breakthrough event (marked by the red circle) confirming the presence of the lipid bilayer. D) A contact mode AFM image of a supported lipid bilayer. Force spectroscopy can be used to measure the mechanical properties of a surface by indenting or deforming the surface with the AFM tip. For force spectroscopy, the tip is approached to and retracted from the surface, and while measuring the force exerted by the sample surface. To measure this force, the deflection of the cantilever is monitored and converted into a force using the spring constant of the cantilever. Figure 6.5A shows a force curve taken on mica, versus the motion of the sample surface (‘piezo height’), while the AFM topography images of the surface is shown in figure 6.5B. The region of zero force is where the tip is not interacting with the sample. The section of increasing force is where the tip is pushing on the mica substrate and the cantilever is bending as a result of the interaction. This can be directly compared to figure 6.5C, where after the zero force region, the tip pushes on the lipids, elastically deforming them before breaking through the bilayer as the force exerted by the tip ruptures the membrane and pushes on the mica substrate [234]. The presence of a breakthrough in the force curve allows us to confirm the presence of a bilayer on the mica surface,

while the topography appears similarly flat (figure 6.5D). This breakthrough occurs in the low nN regime as expected for a fluid phase bilayer of short chain length [278]. Having confirmed the presence of a bilayer on the surface, we must now confirm that we can image the surface under buffer conditions with no damage to the sample, to ensure that any effects observed on the membranes are a result of the membrane disruption by the antimicrobial peptides and not due to the tip-surface interaction. A DLPC:DLPG (3:1) bilayer was again prepared as outlined in section 3.1.3. The lipid bilayer was then imaged in contact mode on a JPK Nanowizard 1 using MSNL-C and D cantilevers (k = 0.03− 0.05) in a buffer solution for an hour. Figure 6.6 shows no disruption to the bilayer occurring over a period of 50 minutes of imaging under these conditions. The use of calcium and magnesium cations in solution promotes the adsorption of the lipids to the mica in a more stable conformation with greater surface coverage by screening the electrostatic repulsion between the negatively charged mica and the negatively charged lipids [242]. The use of divalent and monovalent cations can also affect the antimicrobial peptides, reducing their efficacy. However, the action of antimicrobial peptides in the presence of salt is anyway important, as this represents more physiologically relevant conditions (e.g., 1-2 mM Ca2+_{, 1-2 mM Mg}2+ _{and 100-150 mM NaCl) [150].}

Figure 6.6: AFM images of a DLPC:DLPG (3:1) lipid bilayer surface imaged in buffer solution over the course of an hour.

This flat lipid substrate must be both reproducible and unaffected by the movement of the cantilever during AFM imaging. Having confirmed by control experiments that the bilayer is not disrupted by AFM imaging, we can inject antimicrobial peptides into the fluid cell to examine their effect on the lipid bilayer and evaluate the mechanism

of disruption by AFM.