Adapted from: Ortuso, R. D.; Cataldi, U.; Sugihara, K., Mechanosensitivity of polydiacetylene with a phosphocholine head group. Soft Matter 2017, 13, 1728-1736.
Introduction to chapter
Figure 24: The chemical structures of DC(8,9)PC, TRCDA monomers and polydiacetylene
In recent decades the use of membrane-lysing peptides has gained much attention as a viable antibiotic substitution due to the increasing microbial resistance to medicines224,225. To study the interactions between the peptides and the cellular membrane that are encountered in vivo many simulated studies are done that use bilayers, vesicles or similar substrates that are able to mimic the cellular membrane108. To mimic faithfully the substrate that the membrane-lysing peptides encounter in vivo conditions, scientists recur mainly to two different techniques (for further details please refer to “Use of PDA for bio-detection” on page 66).
112 | 212
Having noticed the limitations of both approaches we demonstrated the colorimetric and fluorescence detection of one type of membrane-lysing peptide, melittin (a pore-forming peptide), with PDA made of 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC(8,9)PC).
The lipid head group is known to play a key role in the function of such pore-forming peptides. Especially the net electric charge of the lipid head group completely alters the peptide behaviour. For example, bilayers made of 100 % negatively charged lipids promote the adhesion of a cationic peptide melittin, but suppress pore formation226,227,228,229. In previous works diacetylene monomers such as 10,12-Tricosadiynoic acid (TRCDA), which is an amphiphilic molecule with a carboxyl head group and a single polymerizable tail (Figure 24), are self-assembled into vesicles and then UV-crosslinked to fabricate PDA vesicles. The colour change upon peptide addition is characterised by the absorption spectra and is calibrated against the peptide concentration108. However adhesion behaviour of this membrane-lysing peptide also depends on salt concentrations in the solutions. It implies that the change in the zeta potential of lipid head groups and peptides, due to the ion screening effect, influences the melittin properties230. The main component of cell membranes, phospholipids, are zwitterionic, while conventional PDAs with carboxyl head groups are completely negatively charged in physiological buffer solution (pH = 7.4). Although lipid mixing is often recurred used to provide a cushion around PDAs, the PDA still has a carboxyl head group, thus it is likely to detect the peptide-carboxyl group interactions108,122. Replacing the diacetylene monomers with the ones with a phospholipid head group will make the system even more attractive, as it will better mimic the cell membrane-peptide interactions.
In this work, we study the interaction between PDA made of DC(8,9)PC and the antimicrobial peptide melittin. DC(8,9)PC is a diacetylene monomer with a phosphocholine head group that can be cross-linked into PDA by UV irradiation (Figure 24). It has been previously used for bilayer micropatterning231 and for stabilising free-standing lipid bilayers232. However, its sensitivity against peptides has never been reported. The PDA vesicles fabricated with DC(8,9)PC expose phosphocholine to the aqueous solution and
113 | 212
therefore mimic cell membranes. Apart from the head group, another difference from the conventional TRCDA is that it has two tails where both of them have a polymerizable diacetylene moiety. This influences the way they polymerise.
The objective of the work is to test whether we can replace TRCDA with DC(8,9)PC for making the PDA assay more biologically relevant without sacrificing the sensitivity towards melittin.
114 | 212 Results and discussion
Figure 25: UV/VIS spectra of PDA vesicle suspension made of 75% DC(8,9)PC and 25% DOPC for different UV doses.
Figure 25 shows the absorption spectra of the PDA vesicle suspension made of 75 % DC(8,9)PC and 25 % DOPC during the polymerisation process at different UV irradiation doses (for further details refer to “Appendix D: materials and methods referred to chapter 3” on page 177). The peak at 600 nm, which represents the “blue state”, appears after 1.5 J/cm2 UV irradiation. It indicates crosslinking of DC(8,9)PC into blue-state PDA, where the conjugated backbone has an energy band gap that corresponds to the wavelength of 600 nm. Note that the word “blue state” originates from the appearance of the PDA in this state, where the light at around 600 nm wavelength is absorbed. Although for DC(8,9)PC, this colour rather appears to be violet to naked eyes, we use the word “blue” commonly used for PDA. Further polymerisation (2.0 - 3.0 J/cm2) diminishes the peak at 600 nm, while the other peaks at 495 nm and 525 nm (corresponding to the “red state”) develop. It is due to the structural change of PDA that broadened the band gap, causing a blue shift in the absorption spectra. The typical behaviour of PDA blue-to-red transition231 is well reproduced with PDA composed of DC(8,9)PC.
115 | 212
Figure 26: (A) Photos showing the appearance of the vesicle suspension before and after the addition of melittin. UV/VIS spectra of blue-state PDA vesicle suspension made of (B) 75% DC(8,9)PC + 25% DOPC and (C) 75% TRCDA + 25% DOPC upon addition of melittin at 0.285 mg/ml.
116 | 212
Next we tested the sensitivity of DC(8,9)PC-PDA towards melittin. Melittin is a pore-forming antimicrobial peptide abundant in bee venom and has been used as a model peptide for studying lipid-peptide interactions233. It is a bar-shaped peptide that inserts into lipid bilayers directly from solution229 and forms pores234 as an oligomer. Its atomic structure has been well studied by X-ray diffraction technique235,236 and nuclear magnetic resonance (NMR)237,238. The interaction between melittin and lipid bilayers and pore formation by melittin have been studied by combining oriented circular dichroism (OCD) for detecting the orientation of the melittin helix and neutron scattering for detecting transmembrane pores234. These experimental data were interpreted by the toroidal model, in which a leaflet of the lipid bilayer bends continuously through the pore so that the water core is lined by both the peptides and the lipid head groups. Melittin was selected as a model peptide because it is known to induce colorimetric response in PDA108. Blue state DC(8,9)PC-PDA vesicles were fabricated with the UV dose of 1.5 J/cm2 and melittin was added using a pipette to the vesicle suspension, followed by gentle mixing, reaching the final peptide concentration of 0.285 mg/ml (corresponding to a lipid to melittin molar ratio of 10:1). Figure 26B shows the absorption spectra of PDA vesicles made of 75 % DC(8,9)PC + 25 % DOPC taken at different time points since melittin was added to the sample. PDA made of DC(8,9)PC displayed sensitivity towards melittin similarly to what had been reported with PDAs with other head groups108. However we observed distinguished slow transition kinetics. The blue peak at 600 nm diminished and the red peak at 495 nm and 525 nm increased over a few hours after the addition of melittin. The appearance of the vesicle suspension is shown in Figure 26A. A similar time-dependent peptide-PDA interaction has been previously observed with TRCDA but over the time scale of a couple of minutes108. In our case, the PDA made of TRCDA reacted to melittin instantaneously after the addition of the peptide (Figure 26A, C).
117 | 212
Figure 27: Colorimetric response (CR) versus incubation time after the addition of melittin for PDAs made of 75% DC(8,9)PC + 25% DOPC (red) and 75% TRCDA + 25% DOPC (black). Melittin concentration in the samples is 0.285 mg/ml.
To study the transition kinetics in more detail, the absorption spectra were analysed in terms of colorimetric response119 (CR, for further details refer to “Appendix D: materials and methods referred to chapter 3” on page 177) and plotted against the duration of the melittin incubation in Figure 27. The CR characterises the ratio of the two peaks that correspond to the blue and the red states. The CR of blue PDAs is 0 % by definition, while CR
> 0 % suggests the transition of blue PDA into the red state PDA. In case of PDA vesicles made of DC(8,9)PC, colorimetric response increases gradually over the first hour and plateaus after a couple of hours (see the red plot in Figure 27). For the PDA vesicles made of TRCDA, the colorimetric response increases immediately after the addition of melittin and remains relatively stable over several hours (see the black plot in Figure 27). Although a time-dependent CR change over 100 s has been previously observed with TRCDA-PDA vesicles108, it was not reproduced in our experiments. It is also interesting to note that the maximum colorimetric response after the saturation is 80 % for the DC(8,9)PC-PDA, while we measured only a 30 % increase for TRCDA-PDA vesicles. This suggests that DC(8,9)PC-PDA has a higher sensitivity towards melittin compared to the conventional TRCDA-PDAs, if analysed in terms of CR. This result is somewhat surprising because we observed a larger absolute value change
118 | 212
for TRCDA spectra compared to DC(8,9)PC (the y axis for TRCDA in Figure 26C is one order of magnitude larger than that of DC(8,9)PC in Figure 26B). The colorimetric response enables one to analyse the change in the ratio of blue and red peaks. The large change in absolute values but small change in the peak ratio for TRCDA may imply that the PDA made of TRCDA contains a large amount of conjugated polymers (clear absorption peaks), though the structural change upon melittin addition is limited (small peak ratio change). On the other hand, for DC(8,9)PC, the absolute value of the absorption spectra is probably smaller because of an inefficient crosslink due to its two-tail configuration. Nevertheless, when melittin is added, the peak ratio alters significantly. It suggests that a large fraction of PDA reacted to melittin. For DC(8,9)PC-PDA, we reproducibly observed a sudden drop of CR at 15 min (see the red plot in Figure 27), which we will discuss later.
119 | 212
Figure 28: Fluorescence spectra of PDA suspension made of (A) 75% DC(8,9)PC + 25% DOPC and (B) 75% TRCDA +25% DOPC at different time points after the addition of melittin. These spectra were obtained at the excitation and emission wavelength of (A) 530nm and 545 nm for DC(8,9)PC-PDA, (B) 500 nm and 620 nm for TRCDA-PDA. The final melittin concentration is 0.285 mg/ml.
120 | 212
PDA presents fluorescence only when it is in the “red state”; the fluorescence pathway is forbidden in the blue state119. This characteristic has allowed PDAs to be used in fluorescence assays239. For our experiment we monitored the time evolution of fluorescent signals after the addition of melittin. With our samples, PDAs made of DC(8,9)PC, the fluorescence signal increased 15 min after the addition of melittin, followed by an unexpected decrease (Figure 28A). In the case of TRCDA-PDA vesicles, the fluorescence signal increased immediately after the addition of melittin and remained relatively stable for one hour (Figure 28B).
Figure 29: The time evolution of the fluorescent excitation/emission peak heights with 75% DC(8,9)PC + 25% DOPC PDA vesicles after the addition of melittin at 0.285 mg/ml.
The DC(8,9)PC-PDA excitation peak height at 530 nm and the emission peak height at 545 nm in Figure 29A were plotted against time in Figure 29 (for further details refer to
“Appendix D: materials and methods referred to chapter 3” on page 177). Both the excitation and the emission peak heights increased after the addition of melittin, reaching a maximum at 15 min, and then decreased, stabilising after approximately 1 hour. Even if we reduced the sampling rate significantly, we observed the same trend. This confirms that this fluorescence
121 | 212
decrease is not due to photobleaching. The fluorescence intensity maximum at 15 min coincides with the CR drop we mentioned previously. This CR drop is probably due to the over-estimation of the transmission light intensity (under-over-estimation of absorption) because of the strong fluorescence emission at 15 min (though we do not have an explanation for the CR drop at 1.5 h). Figure 27 and Figure 29 show that the both absorption and fluorescence spectra changed extensively over the first one hour and stabilised for the next few hours. To understand the origin of these unexpected time-dependent spectral changes we studied the vesicle particle size using dynamic light scattering (DLS).
Figure 30: The particle size estimated by dynamic light scattering (DLS) over time. The PDA vesicles were made of 75%
DC(8,9)PC + 25% DOPC. Melittin was added at the concentration of 0.285 mg/ml at 0 h.
Figure 30 shows the PDA vesicle diameter () estimated by DLS plotted against melittin incubation time. Although we probe-sonicated the lipids, the original blue PDA vesicles showed a particle size larger than > 2 μm (see Ref. in Figure 30). This is probably due to the aggregation of vesicles while cooling the samples in the fridge after sonication (this process is necessary for the optimised UV crosslink240). As soon as melittin was added to the PDA vesicles, the particle size became half (see the plot at 0 h incubation in Figure 29; note
122 | 212
that the scale is shown on the right axis). A similar reduction in the size of vesicles after the addition of peptides has been previously observed108. The particle size kept decreasing over the next hour and reached a plateau with a size of around a few hundred nanometres. This dramatic reduction in the particle size must be linked to the time evolution observed in the UV/VIS spectrometer and in the fluorescence spectra, because all of them presented a change over the first one hour.
Figure 31: A scheme showing the interaction between DC(8,9)PC-PDA suspension and melittin.
First, the CR increase can be explained by a picture as follows. Blue PDA samples are the aggregation of vesicles as the particle size is larger than 2 µm. Upon melittin addition, melittin peptides adhere on the surface of the vesicle aggregates, converting the blue PDAs on the surface into red PDAs (Figure 31). While this is happening, however, melittin cannot access the inner part of the PDA aggregates. Thus the inner part remains in the blue state. At the same time, melittin is breaking down the vesicle aggregates into smaller ones due to its amphiphilic property227 as we saw by DLS (Figure 30). Melittin is highly cationic, thus the electrostatic repulsion between melittin-adsorbed vesicles could also contribute to the reduction of the vesicle size. As the PDA size becomes smaller, new PDA surface area is available for melittin to interact with and induce the blue-to-red transition. Therefore, the decrease in the particle size over the first hour coincides with the increase in the CR. Similarly,
123 | 212
the fluorescence signal (see Figure 29) also kept increasing after the addition of melittin until it reaches a maximum at 15 min, after which it started decreasing. It may be that the decrease in the particle size (thus an increase in the particle density inside the suspension) made the suspension more opaque, which reduced the signal due to the shadowing effect. The transparency of a vesicle solution is known to be the product of many parameters such as the number of the bilayers241 and the polydispersity of the sample242.
Table 7: Dynamic light scattering (DLS) determined particle sizes estimates at different preparation steps.
Table 8: Zeta potential measurements of different samples after sonication.
(nm) DC(8,9)PC + DOPC DC(8,9)PC + DOPS TRCDA + DOPC
After Sonication 103 ± 3 53 ± 6 256 ± 38
Overnight in Fridge 2765 ± 362 54 ± 3 278 ± 7
After Polymerisation to
Blue State
2040 ± 12 52 ± 2 327 ± 11
(mV) DC(8,9)PC + DOPC DC(8,9)PC + DOPS TRCDA + DOPC
After Sonication -9.03 ± 0.54 -61.90 ± 3.00 -86.00 ± 2.90
124 | 212
Figure 32: UV/VIS spectra of blue-state PDA vesicle suspension made of 75% DC(8,9)PC + 25% DOPS upon addition of melittin at 0.285 mg/ml.
If the aggregation of vesicles is truly the origin of the slow PDA-melittin interaction, we should be able to speed up the reaction by dispersing the PDA vesicles better. We cannot simply probe-sonicate in order to break down the vesicle size after the polymerisation, because the temperature increase during the probe sonication will induce the blue-to-red transition and the PDA will lose its sensitivity towards melittin. To test this idea we fabricated PDA vesicles with 75 % DC(8,9)PC + 25 % DOPS with exactly the same sample preparation procedure. DOPS is a negatively charged lipid, and thus may help to disperse PDA vesicles due to the electrostatic repulsion. Table 7 shows the particle size studied by DLS for DC(8,9)PC + DOPC, DC(8,9)PC + DOPS and TRCDA + DOPC immediately after the probe-sonication, after leaving the samples in a fridge overnight and after the polymerisation. The addition of DOPS helped to suppress the aggregation. The particle size stayed the same (~ 50 nm) even after leaving the sample overnight. Melittin was added to this PDA suspension and we observed an immediate colour change as expected (Figure 32). This result confirms that the slow reaction observed in PDA vesicles made of DC(8,9)PC + DOPC was due to the aggregation of vesicles.
A significant increase in the particle size (beyond the error) was not observed in PDA vesicles with TRCDA + DOPC (Table 7). This is probably because TRCDA is also negatively charged.
125 | 212
These results suggest that the true origin of the difference in the interaction kinetics with melittin between DC(8,9)PC + DOPC and TRCDA + DOPC was their aggregation behaviour stemmed from the different net charge of the lipid head group. Zeta potential measurements indeed confirmed that the both TRCDA + DOPC and DC(8,9)PC + DOPS are much more negatively charged than DC(8,9)PC + DOPC (Table 8).
126 | 212
Figure 33: (A) UV/VIS spectra of 75% DC(8,9)PC + 25% DOPC at different melittin concentrations. (B) Colorimetric Response (CR) versus melittin concentration (dose curve). All data were taken after 5h incubation to assure saturation of the signal.
Finally, the colorimetric response of PDA, made of DC(8,9)PC + DOPC, was calibrated against melittin concentrations. Due to the slow increase in the UV/VIS signal, PDA vesicles
127 | 212
were incubated with melittin for 5 hours at each melittin concentration, assuring the saturation of the spectral signal. Figure 33A shows that the blue-to-red transition is clearly melittin-concentration dependent. We analysed these spectra and plotted the colorimetric response against the melittin concentration (Figure 33B). At low melittin concentrations (10 - 9 mg/ml < concentrations < 10 -3 mg/ml) a non-reproducible colorimetric response increase was observed. Since the error bars in this range are large, it was not clear if the change in CR was due to the response from PDA-melittin interactions or other factors (e.g. agitation by pipette, sample variance). The data was fitted with a sigmoidal function, with the half maximum response (EC50) and Hill coefficient (αHill) being the open parameters (for further details please refer to section “Appendix D: materials and methods referred to chapter 3” on page 177). The sigmoidal function is commonly used to fit dose curves based on simple binding models243. The poor fitting in the low concentration range may indicate that the PDA-melittin interaction cannot be represented by the standard binding model. Nevertheless, the half maximum response was obtained at a melittin concentration of 0.1 mg/ml. It is in the same range as the value from the conventional TRCDA-PDA244. Therefore, we can conclude that the head group of PDA was successfully replaced with phosphocholine without changing the sensitivity towards melittin. Surface plasmon resonance (SPR) has been previously used to study the binding of melittin on phospholipid bilayers244. The dose curve in this study showed a half maximum response at a melittin concentration of 0.4 mg/ml. Other researchers have investigated melittin pore formation using the fluorescence leakage assay227. In the leakage assay, a dye molecule is encapsulated inside phospholipid vesicles and the leakage of the dye upon melittin addition is measured by a fluorimeter. The dose curve from the leakage assay displayed a half maximum response at 0.2 mg/ml. The half maximum response in the present work (0.1 mg/ml) is in the same melittin concentration range as that in both previous reports using phospholipids. At this stage we are not certain whether the PDAs are detecting either the adhesion or the pore-formation of melittin. The fitting yielded a Hill coefficient of αHill = 2.1, which indicates positive cooperativity. Antimicrobial peptides such as melittin are well-known to present positive cooperativity245. This has been explained with a model that is linked
were incubated with melittin for 5 hours at each melittin concentration, assuring the saturation of the spectral signal. Figure 33A shows that the blue-to-red transition is clearly melittin-concentration dependent. We analysed these spectra and plotted the colorimetric response against the melittin concentration (Figure 33B). At low melittin concentrations (10 - 9 mg/ml < concentrations < 10 -3 mg/ml) a non-reproducible colorimetric response increase was observed. Since the error bars in this range are large, it was not clear if the change in CR was due to the response from PDA-melittin interactions or other factors (e.g. agitation by pipette, sample variance). The data was fitted with a sigmoidal function, with the half maximum response (EC50) and Hill coefficient (αHill) being the open parameters (for further details please refer to section “Appendix D: materials and methods referred to chapter 3” on page 177). The sigmoidal function is commonly used to fit dose curves based on simple binding models243. The poor fitting in the low concentration range may indicate that the PDA-melittin interaction cannot be represented by the standard binding model. Nevertheless, the half maximum response was obtained at a melittin concentration of 0.1 mg/ml. It is in the same range as the value from the conventional TRCDA-PDA244. Therefore, we can conclude that the head group of PDA was successfully replaced with phosphocholine without changing the sensitivity towards melittin. Surface plasmon resonance (SPR) has been previously used to study the binding of melittin on phospholipid bilayers244. The dose curve in this study showed a half maximum response at a melittin concentration of 0.4 mg/ml. Other researchers have investigated melittin pore formation using the fluorescence leakage assay227. In the leakage assay, a dye molecule is encapsulated inside phospholipid vesicles and the leakage of the dye upon melittin addition is measured by a fluorimeter. The dose curve from the leakage assay displayed a half maximum response at 0.2 mg/ml. The half maximum response in the present work (0.1 mg/ml) is in the same melittin concentration range as that in both previous reports using phospholipids. At this stage we are not certain whether the PDAs are detecting either the adhesion or the pore-formation of melittin. The fitting yielded a Hill coefficient of αHill = 2.1, which indicates positive cooperativity. Antimicrobial peptides such as melittin are well-known to present positive cooperativity245. This has been explained with a model that is linked