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Molecular Scale Insights Into Activity of Polyoxometalate as Membrane Targeting Nanomedicine from Single Molecule Observations Aya Sakamoto, Kei Unoura, and Hideki Nabika J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b11251 • Publication Date (Web): 02 Jan 2018 Downloaded from http://pubs.acs.org on January 3, 2018

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The Journal of Physical Chemistry

Molecular Scale Insights Into Activity of Polyoxometalate as Membrane Targeting Nanomedicine from Single Molecule Observations Aya Sakamoto, Kei Unoura, Hideki Nabika* Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, 14-12 Kojirakawa, Yamagata 990-8560, Japan * [email protected]

ABSTRACT

Polyoxometalate (POM) is rapidly emerging as an attractive antimicrobial inorganic cluster that exhibits its antimicrobial activity by attacking the cell membrane. Precise understanding and control of the antimicrobial activity of POM can allow us to design novel functional nanomedicines with high stability, high selectivity, and low cost. Therefore, in this study, we investigated the interaction between POM and a model cell membrane through single molecule observation and found that the presence of POM causes macroscopic morphological changes in the microbial membrane, and these changes were detectable as modulations in the diffusivity of the membrane. Numerical analyses based on mean square displacement and diffusion length

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histogram revealed the reduction in the fluidity of the membrane in the presence of POM. Further analysis from single molecule tracking revealed the formation of pores in the membrane, along with the formation of POM-lipid assemblies. The pores were found to act as diffusion barriers and diffusion trap sites, and thus contributed to the reduction in the fluidity of the membrane. Furthermore, pore formation also led to the loss of important functions of the cell membrane. Based on this ability of POM to induce pore formation and form assemblies with membrane lipids, we believe that POM is a promising candidate for use as a membrane-targeting bioactive nanomedicine.

Introduction Artificial nanomaterials have become the focus of intense research in the field of nanomedicine, because they possess novel functionalities, stabilities, and synthesizabilities that cannot be achieved with biological molecules.1-5 Among the various biologically active nanomaterials, the ones that attack the cell membrane are highly expected to replace biological antibiotics, because cell membranes are less likely to acquire resistance to the medicinal materials. Thus, a number of nanomaterials such as metals,6-8 semiconductors,9-11 metal oxides,12,13 polymers,14,15 and carbon nanoparticles16-18 have been designed to have specific biological activities. One of the greatest advantages of nanoparticles is that their activity can be controlled by modifying their chemical and physical nature such as the shape,7,16 surface charge,6,12,14 hydrophobicity,8 and size.19 However, this advantage can also be a drawback in some cases. During the chemical or physical synthesis of nanoparticles, fluctuations in chemical reactions, nucleations, and crystallizations cause unavoidable distribution in the size, shape, surface charge, and hydrophobicity of the


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synthesized nanoparticles. As a result, nanoparticles synthesized in one batch include particles with different chemical and physical properties, which makes it difficult to tightly control the biological activity of the synthesized nanoparticles. Nanoclusters, which have well-defined atomic structures and specific molecular formulae, have uniform chemical and physical properties; this enables precise control of their biological activity at the cell membrane interface. Among the various nanoclusters, the biological activity of anionic clusters of polyoxometalate (POM) has long been discussed for application in nanomedicine.20,21 POMs with various sizes, shapes, and charges, such as Keggin-type POM [XM 12 O 40 ]n- and Dawson-type POM [X 2 M 18 O 62 ]n- (where M = W, Mo, etc. and X = P, Si, etc.), are independently synthesizable.22 These POMs exhibit activities toward biological macromolecules such as Aβ peptides,23 prions,24 human serum albumin,25, fibroblast growth factor,26 HIV-1 protease,27 and protein kinase CK2,28 and some of these activities are the result of a Coulombic interaction between the negatively charged POM and the positively charged moiety of the biological macromolecules.22-25 Furthermore, POM shows activity toward methicillin-resistant Staphylococcus aureus (MRSA), with a synergetic effect with β-lactam antibiotics.29 Although the synergetic mechanism between POM and antibiotics has been proposed as the depression of penicillin-binding protein formation in cell metabolism, the complexity of the chemical and biological actions of cells makes it difficult to elucidate the whole picture. Since POMs show activities against not only resistant bacteria but also cancer cells30 and gram-negative and gram-positive bacteria,31 they are expected to be used as novel membrane-targeting bioactive nanomedicines with well-defined and controllable structures and activities.


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For understanding and controlling the activities of POMs on various cells, it is important to study their activity on the surface of the lipid bilayer cell membrane, which is the first reaction site between the POM and a cell. Previous studies conducted using fluorescence microscopy and calorimetry have revealed that POM induces morphological changes in lipid bilayers or liposomes.32-34 Furthermore, the interaction between POM and lipid bilayers can be tuned by changing the charge and size of the POM.32,35 However, insights into how POM acts and causes morphological changes at the surface of the lipid bilayer from the molecular viewpoint cannot be obtained using only macroscopic observations; this has inhibited the design of novel POM-based clusters with well-defined action at the surface of biological cell membranes so far. A real-time single molecule imaging technique based on total internal reflection fluorescence microscopy (TIRFM) is a powerful tool that can provide molecular information about the action of nanomaterials at the lipid bilayer surface.36 However, there has been no report on the activity of POM that leads to the morphological changes in lipid bilayers determined using TIRFM observations. Therefore, in the present study, we carried out TIRFM observations of a dye-doped lipid bilayer in the presence of POM to elucidate the action of POM at the bilayer surface and to study the subsequent changes in the structure of the lipid bilayer upon interaction with POM. Our study sheds light on the origin of the antibacterial activity of POM and highlights the potential of POM clusters for use as novel membrane-targeting bioactive nanomedicines with well-defined and controllable structures and activities.


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METHODS A commercially available Keggin-type POM, H 4 [SiW 12 O 40 ] (Wako Pure Chemical Industries Ltd., referred to as POM), was used without further purification. POM was dissolved in 150 mM Tris HCl buffer (pH = 7.42) to achieve a final concentration of 2 mM and stored in the dark until use. The lipid bilayer was obtained from the self-spreading method to yield single bilayer. L-αphosphatidylcholine from eggs (egg-PC) and Texas Red 1,2-dihexadecanoyl-sn-glycero-3phosphoethanolamine (TR-DHPE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA) and Molecular Probes (Eugene, OR, USA), respectively, and used as received. Chloroform solution of a 10-5 mol % TR-DHPE/egg-PC mixture was prepared and stored until use. Three microliters of the 10-5 mol % TR-DHPE/egg-PC chloroform solution was placed onto a clean glass substrate. After removing the chloroform by drying, the substrate cast with the TRDHPE/egg-PC mixture was immersed in a 150 mM Tris HCl (pH 7.4) buffer and incubated at 20°C overnight. After the incubation, the bilayer reached equilibrium and spreading was no more observed during further experiments. Before the TIRFM observation, the sample was kept at 30°C for 30 min. An appropriate amount of 2 mM POM solution was added to the sample solution. After 10 min, the sample was observed using IX-73 (Olympus Co., Ltd., Japan) equipped with an EMCCD camera iXon Ultra (Andor Technology Ltd.,), where the reaction between POM and bilayer reached equilibrium for 10 min and further changes were not observed. An excitation laser at 532 nm was delivered through an oil-immersion objective lens (100× NA = 1.49). The molecular trajectories were obtained by recording the centers of masses of the bright spots using Image-Pro Plus (Media Cybernetics).


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RESULTS AND DISCUSSION TIRFM observations of a 10-5 mol % TR-DHPE/egg-PC bilayer on glass substrate yielded bright diffusing spots corresponding to TR-DHPE molecules diffusing in the egg-PC bilayer, which gave various quantitative information about the state of the egg-PC bilayer. One of the major quantitative analyses is the determination of the mean square displacement (MSD), where the diffusion length (r) of each bright spot is plotted as a function of time interval (τ), 〈𝑟𝑟 2 〉 = 4𝐷𝐷𝐷𝐷 𝛼𝛼

(1)

where r, D, τ, and α are the diffusion length, diffusion coefficient, time, and anomalous exponent, respectively.37 MSD plots of the 10-5 mol % TR-DHPE/egg-PC bilayer are shown in Figure S1, and the corresponding averaged MSD plots under various POM concentrations are shown in Figure 1a. Without POM, 〈𝑟𝑟 2 〉 reached 8 μm2 at τ = 0.5 s, where the relationship

between 〈𝑟𝑟 2 〉 and τ was almost linear. With the addition of POM, the value of 〈𝑟𝑟 2 〉 was lowered. 〈𝑟𝑟 2 〉 at τ = 0.5 s was reduced to 1.5 μm) disappeared in the presence of POM. Modulation in the diffusion length distribution became more evident from the differential histograms (Figure 2b). A difference histogram was obtained by subtracting the histogram of zero concentration from the histogram of each concentration. At 30 μM, the difference histogram was positive at r < 0.8 μm and negative at r > 0.8 μm, indicating that the population of lipids with r < 0.8 μm and r > 0.8 μm increased and decreased, respectively, compared to that observed without POM. The change in the abundance of lipids became more significant with an increase in POM concentration. Furthermore, the border in the r value between the increase and decrease in the abundance of lipids reduced from 0.8 μm to 0.4 μm with an increase in POM concentration. These observed changes in the histogram analysis answered the question as to whether all the molecules or only those molecules with a specific state contributed to the decrease in D. We also simulated the changes in the histograms on the above two systems. First was a system


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assuming that all molecules had the same diffusivity defined with a single distribution peak (single D model). In this system, the D and r values of all molecules decreased as the concentration of POM increased, which appeared as the shift of the distribution curve to the low r side (Figure 2c). The corresponding difference histograms revealed a clear shift in the border diffusion length to the low r side. On the other hand, the second system assuming the appearance of only molecules with a specific state with lower D, with original D for the remaining molecules, was characterized with two D values (double D model). In this system, the proportion of molecules with a specific state with low D increased as the POM concentration increased, and thus the proportion of molecules with original D decreased (Figure 2d). The difference histogram showed a change in the abundance ratio between molecules with lower and original diffusivities; however, no shift in the border diffusion length was observed. Thus, it became clear that the border diffusion length exhibited obviously different changes between single and double D models, indicating that border can be used as the ruler to determine whether all the molecules or only those molecules with a specific state contributed to the decrease in D. Since experimental results exhibited a gradual shift in the border, the decrease in D was explained with the single D model. This finding clearly revealed the ability of POM to modulate the nature of the membrane, which caused a homogeneous decrease in the diffusivity. To determine the origin of the observed decrease in the fluidity and diffusivity, imaging analysis on individual diffusing bright spots was carried out. Most of the spots in both the absence and presence of POM exhibited random Brownian motion, as shown in Figure 3a. However, some spots showed several diffusion modes other than Brownian motion in the presence of POM. For example, some spots stopped diffusing and remained at the same position (Figure 3b). Although optical fluctuation resulted in slight diffusivity, the position of the center


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of optical gravity remained below the spatial resolution of the optical microscope. The average number of immobile spots per observation increased with an increase in POM concentration (Figure 3h), which indicated that the interaction between POM and the egg-PC bilayer played a role in the appearance of the immobile species. It was also observed that some spots exhibited a transition between mobile and immobile states. For example, a spot in Figure 3c was mobile for the first five frames (33 ms × 5 = 165 ms), but it changed to the immobile state in the next 5 frames. Transition from the immobile to the mobile state can also be seen in Figure 3d. Since the mobile to immobile transition was not observed in the absence of POM (Figure 3i), we concluded that the interaction between POM and the egg-PC bilayer played a role in making the bright spot immobile. Since the D value of the spots with the immobile state was lower than the D value of the mobile spots, the appearance of the immobile state upon addition of POM was presumed to play a role in the observed decrease in D value. In addition to the transition between the mobile and immobile states of the individual molecules, tandem diffusion between two bright spots, where the two spots diffused while the others remained in close proximity, was observed. In the initial frame of Figure 3e, two bright spots depicted with red and blue arrows were separated at a distance of ca 1 μm. Although they diffused independently during the first four frames, the distance between these two spots remained almost constant at ca 1 μm. Since the fluorescence intensities of the two diffusing spots in this period were almost constant (Figure 3f), they were assigned as two independent fluorescent molecules. Then, they overlapped and formed a single spot that continued to diffuse for the next five frames. During this overlapping state, the fluorescence intensity was approximately the same as the sum of the original fluorescence intensities of the two independent spots. This supports our assumption that the two independently diffusing fluorophores


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approached closer to each other within the spatial resolution and formed a single diffusing spot. They then returned to a state of being two separated spots again and continued to diffuse while remaining in close proximity at ca 1 μm. The fact that the fluorescence intensity returned to the original intensity also suggested that the overlapped spots returned to the original individual two spots. From these successive images, it was clear that the two spots diffused while maintaining close proximity at a distance of ca 1 μm until ca 400 ms, during which they were in the mobile state and diffused within a few micrometers. However, since there was no attractive interaction between the TR-DHPE molecules to keep them at a constant distance for a certain period, it was very unlikely that the two observed spots were two TR-DHPE molecules. Furthermore, such tandem diffusion was observed only in the presence of POM (Figure 3j). These results suggested that the observed tandem diffusion was brought upon from two independent POM-based assemblies with a size of 1 μm, which interacted and contacted with each other. If the TR-DHPE molecules were contained in both assemblies, they could be observed as two independent bright spots that diffused together at a distance of 1 μm. The possible POM-based assembly formed on the egg-PC bilayer in the presence of POM is a POM-lipid assembly known as a surfactant encapsulated cluster (SEC).38,39 Although the size of the SEC formed in the present system could not be identified, a previous study has confirmed that a SEC a few nanometer to a few micrometers in size forms spontaneously.32,40,41 In the present study, POM diffused from the bulk solution and adsorbed on the egg-PC membrane surface, where POM interacted with lipid molecules and SECs grew with time. If the SEC contained TR-DHPE, the diffusion of SEC was also observed as a diffusing bright spot. During the growth and surface diffusion of two independent SECs, they occasionally collided and bound through attractive van der Waals, hydrophobic, and electrostatic interactions. Thus, the tandem diffusion of two bright spots seen


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in Figure 3e can be attributed to two SECs that were bound to each other and diffused together on the membrane surface. Depending on the relative position of TR-DHPE molecules in each SEC, the bright spots were not resolvable and were observed as a single overlapped spot with the sum of the fluorescence intensities of the two individual spots. Furthermore, we also observed a blinking mode that will be explained in the following paragraph from the viewpoint of the formation of SECs. In the initial frame, there was no observable spot. However, a bright spot appeared at the next frame and remained for four frames. This spot disappeared in the next frame, after which the other spot appeared at a different position. Although the disappearance of bright spots can be explained by optical bleaching of TR-DHPE by the laser excitation, bleached TR-DHPE cannot recover its fluorescence. Furthermore, since the solubility of TR-DHPE in water is extremely low, it is unlikely that the observed disappearance and reappearance was brought upon by the desorption and readsorption of TR-DHPE molecules from and to the membrane. One plausible explanation is the desorption and readsorption of SECs that formed and diffused on the egg-PC membrane surface, since both surfaces of the SEC and the membrane consist of lipid molecules that can attract each other through van der Waals, hydrophobic, and electrostatic interactions. Since the blinking behavior was also observed only when POM was added to the solution, the interaction between POM and the lipid that induced SEC formation could have given rise to the observed blinking mode. From our observations showing the formation of the SEC and its dynamic behavior, we hypothesized about the role of POM at the surface of the lipid membrane (Figure 4): First, POM is adsorbed on the surface of the lipid membrane through hydrophobic and electrostatic interactions depending on the density and charge of the lipid.35 This interaction between POM and the lipid results in the formation of POM-lipid assemblies such as SECs.38,39 Some of these


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assemblies were observed as bright spots when TR-DHPE was contained in the SECs. SECs could grow, bind, desorb, and readsorb on the surface of the lipid membrane (Figure 3e, 3g). Since the lipids required to form the SEC were supplied by the membrane, pores lacking lipid molecules were formed in the lipid membrane. Thus, formation of SEC a few nanometer to a few micrometers in size leaves pores with similar dimension. Although these microscopic pores cannot be observed under optical microscope, macroscopic pore that were formed by continuous fusion of microscopic pores were confirmed by TIRFM observations at relatively high TRDHPE concentrations (Figure S2). Since the edge of the lipid membrane including the fringe of micro- and macroscopic pores had defects with less organized molecular packing and alignment, the pore can act as the diffusion barrier. Although both micro- and macroscopic pores acts as diffusion barrier to reduce the diffusion length, presence of optically unresolvable pores, not pore in Figure S2, would be the dominant origin of the decrease in the diffusivity because the diffusion length per frame is less than 1 μm as shown in Figure 2. Also, these defects also acted as trap and pin sites to make the TR-DHPE molecules and SECs immobile. Thus, the TR-DHPE molecules and SECs that diffused and encountered the edge of the micro- and macroscopic pores were occasionally trapped there, which resulted in the transition from the mobile to the immobile state. The immobile state was observed as immobile bright spots (Figure 3b, 3c, 3d). However, thermal fluctuation can release the trapped molecules, which would result in the transition from the immobile back to the mobile state (Figure 3d). In addition, the pore can act as a barrier to the diffusion in the bilayer, due to which no molecules can diffuse into the pore region. Thus, it is proposed that the pore has two roles in the bilayer. One is as a diffusion trap and the other one is as a diffusion barrier, both of which reduce the diffusion length and diffusion coefficient. Thus, the observed decrease in the diffusion length and diffusion coefficient is attributable to the


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formation of micro- and macroscopic pores in the egg-PC bilayer. Although further statistical analysis on the observed various diffusion modes such as mobile, immobile, transition between the mobile and immobile states, tandem, and blinking can give further information on their diffusivity,42 the finding on the appearance of these anomalous diffusions by POM is the significant progress of this paper. It should be also noted here that we cannot rule out the effect of solid substrate on the diffusivity of lipids. However, since we have conducted all experiments on the same substrate, relative comparison before and after the addition of POM is considered to be adequately valid. From the viewpoint of the antimicrobial activity of POM, we propose that the formation of pores accompanying SEC growth and desorption plays a critical role to making POM a membrane-targeting bioactive nanomedicine. Since it is expected that the capability of SEC formation can be controlled by the shape and charge of POM, this finding indicates that it is possible to develop a new antibacterial active POM-based cluster material with well-defined and controllable structure and activity.

CONCLUSION We studied the origin of the antimicrobial activity of a Keggin-type POM cluster, which is one of the most studied antimicrobial POMs, through single molecule observation of the egg-PC bilayer formed on a glass substrate. From numerical analysis based on MSD plots and diffusion length histograms, the fluidity of the egg-PC bilayer was found to be reduced in the presence of POM. The origin of the observed decrease in the fluidity was identified as the formation of pores in the membrane owing to the formation of SECs; these pores acted as diffusion barriers and trap


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sites. Furthermore, TIRFM observations revealed that the bright spots underwent various diffusion modes such as mobile, immobile, transition between the mobile and immobile states, tandem, and blinking. Among them, the tandem and blinking modes were difficult to understand by TR-DHPE diffusion. Instead, these modes were explained by the dynamic behavior of SECs on the egg-PC bilayer, including the association between two SECs and their desorption and readsorption on the bilayer. These results implied that POM is capable of forming and releasing SECs from the membrane, which is accompanied by pore formation in the membrane. Since pore formation causes loss of important functions of cell membranes, the ability of POM to function as a membrane-targeting bioactive nanomedicine is proposed to have originated from the pore formation accompanying the formation and desorption of SECs from the lipid membranes.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: Raw data for the mean square displacement and image showing pore formation.

ACKNOWLEDGMENT This work was supported by Nippon Sheet Glass Foundation for Materials Science and Engineering and JSPS KAKENHI Grant Number 26600021 and 16H04092.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

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(30) Wang, X. H.; Liu, J. F.; Chen, Y. G.; Liu, Q.; Liu, J. T.; Pope, M. T. Synthesis, Characterization and Biological Activity of Organotitanium Substituted Heteropolytungstates. J. Chem. Soc., Dalton Trans. 2000, 1139-1141. (31) Barsukova-Stuckart, M.; Piedra-Garza, L. F.; Gautam, B.; Alfaro-Espinoza, G.; Izarova, N. V.; Banerjee, A.; Bassil, B. S.; Ullrich, M. S.; Breunig, H. J.; Silvestru, C. et al. Synthesis and Biological Activity of Organoantimony (III) – Containing Heteropolytungstates. Inorg. Chem. 2012, 51, 12015-12022. (32) Nabika, H.; Inomata, Y.; Itoh, E.; Unoura, K. Activity of Keggin and Dawson Polyoxometalates Toward Model Cell Membrane. RSC Adv. 2013, 3, 21271-21274. (33) Nabika, H.; Sakamoto, A.; Tero, R.; Motegi, T.; Yamaguchi, D.; Unoura, K. Imaging Characterization of Cluster-Induced Morphological Changes of a Model Cell Membrane. J. Phys. Chem. C 2016, 120, 15640-15647. (34) Jing, B.; Hutin, M.; Connor, E.; Cronin, L.; Zhu, Y. Polyoxometalate Macroion Induced Phase and Morphology Instability of Lipid Membrane. Chem. Sci. 2013, 4, 3818−3826. (35) Kobayashi, D.; Ouchi, Y.; Sadakane, M.; Unoura, K.; Nabika, H. Structural Dependence of the Effects of Polyoxometalates on Liposome Collapse Activity. Chem. Lett. 2017, 46, 533535. (36) Lee, Y. K.; Kim, S.; Oh, J. W.; Nam, J. M. Massively Parallel and Highly Quantitative Single-Particle Analysis on Interactions between Nanoparticles on Supported Lipid Bilayer. J. Am. Chem. Soc. 2014, 136, 4081-4088.


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(37) Saxton, M. J.; Jacobson, K. Single-Particle Tracking; Applications to Membrane Dynamics. Annu. Rev. Biophys. Struct. 1997, 26, 373-399. (38) Volkmer, D.; Du Chesne, A.; Kurth, D. G.; Schnablegger, H.; Lehmann, P.; Koop, M. J.; Müller, A. Toward Nanodevices: Synthesis and Characterization of the Nanoporous Surfactant-Encapsulated Keplerate (DODA) 40 (NH 4 ) 2 [(H 2 O) n ⊂ Mo 132 O 372 (CH 3 COO) 30 (H 2 O) 72 ]. J. Am. Chem. Soc. 2000, 122, 1995-1998. (39) Kurth, D. G.; Lehmann, P.; Volkmer, D.; Cölfen, H.; Koop, M. J.; Müller, A.; Du Chesne, A. Surfactant-Encapsulated Clusters (SECs): (DODA) 20 (NH 4 )[H 3 Mo 57 V 6 (NO) 6 O 183 (H 2 O) 18 ], a Case Study. Chem. Eur. J. 2000, 6, 385393. (40) Li, H.; Sun, H.; Xu, M.; Wu, L. Onionlike Hybrid Assemblies Based on SurfactantEncapsulated Polyoxometalates. Angew. Chem, Int. Ed. 2007, 46, 1300-1303. (41) Yan, Y.; Wang, H.; Li, B.; Hou,G.; Yin, Z.; Wu, L.; Yam, V. W. W. Smart Self-Assemblies Based on a Surfactant-Encapsulated Photoresponsive Polyoxometalate Complex. Angew. Chem. 2010, 133, 9419-9422. (42) Schütz, G. J.; Schindler, H.; Schmidt, T. Single-molecule Microscopy on Model Membranes Reveals Anomalous Diffusion. Biophys. J. 1997, 73, 1073–1080.


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(b)

(a) 0 μM

200 μM

(c)

(d)

Figure 1. (a) Mean square displacement (MSD) plots of 10-5 mol % TR-DHPE/egg-PC bilayer under various POM concentrations. Black: 0 μM, red: 30 μM, blue: 50 μM, green: 100 μM, and purple: 200 μM. (b) Double-logarithmic plots of (a). Dashed line depicts a straight line with slope = 1. The least squares fitting of the double-logarithmic plots yields the anomalous exponent α. The value of α as a function of POM concentration is shown in (c). The diffusion coefficients D were calculated with α = 1 and are shown in (d).


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(a) 0 μM

(b)

30 μM

0.8 μm

30 μM

(c) single D model

r (a.u.)

50 μM

0.6 μm

100 μM

0.5 μm

50 μM

100 μM

200 μM

0.4 μm

200 μM

(d) double D model

r (a.u.)

r (a.u.)

r (a.u.)

Figure 2. (a) Histogram of diffusion length per frame (0.033 s) of 10-5 mol % TR-DHPE/egg-PC bilayer under various POM concentrations. (b) A difference histogram was obtained by subtracting the histogram of zero concentration from the histogram of each concentration. Dashed purple line and its corresponding number indicate the diffusion length at which the switch between the increase and decrease in the difference histogram appears. (c) Single D model for the diffusion length histogram and difference histogram. Gray arrow indicates the shift in the diffusion length histogram with the decrease in D. (d) Double D model, in which the gray arrow indicates the change with the increase in the abundance for low D.


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(a) mobile start

end

start end

3 µm

(b) immobile start

end

10

3 µm

(c) mobile to immobile start

5 end

2 µm

end

immobile state

start

(d) immobile to mobile start

start

3

end

2 µm

end

immobile state

(f) Fluorescence intensity analysis

(e) tandem start

intensity (a.u.)

30

3 µm

overlapping state

end

20 10 overlapping state

0 (g) blinking end

start 2 µm

dark state

dark state

(i) mobile/immobile

2

0.2

1

0

0

30

50

100 200

[POM] (μM)

(k) blinking

(j) tandem

3

0.8 0.6

0.1

0

dark state

0

30

50

100 200

[POM] (μM)

frequency

0.3

frequency

3

frequency

(h) immobile

frequency

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.4 0.2 0

0

30

50

100 200

[POM] (μM)

2

1

0

0

30

50

100 200

[POM] (μM)

Figure 3. Successive TIRFM images of bright spots with different diffusion modes: (a) mobile, (b) immobile, (c) switch from mobile to immobile and (d) switch from immobile to mobile. (e) Successive TIRFM images showing tandem diffusion, where two bright spots diffuse together.


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Between the 5th and 9th frames, two spots overlap in the optical resolution. During this overlapping state, the fluorescence intensity is doubled as shown in (f). (g) Successive TIRFM images showing blinking mode, where the bright spot shows repetitive appearance/disappearance. The frequency of the spot per observation (5 s) of (h) immobile, (i) switch between mobile and immobile, (j) tandem, and (k) blinking modes.


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POM Desorbed POM-lipid assembly

Pore Trapped POM-lipid assembly (immobile state)

Adsorbed POM Tandem POM-lipid assembly

Figure 4. Schematic illustration of the proposed mechanism of action of POM (green sphere) on the surface of the lipid membrane. POM-lipid assembly undergoes dynamic behavior (not only diffusion but also desorption and re-adsorption). Pores are left in the lipid membrane due to the formation of the POM-lipid assemblies, which is the origin of the membrane-targeting antimicrobial activity of POM.


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TOC Graphic

desorption

immobile

pore

tandem


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