Temporary Membrane Permeabilization Via the Pore-Forming Toxin Lysenin

Boise State University ScholarWorks

Physics Faculty Publications and Presentations Department of Physics

5-2020

Temporary Membrane Permeabilization via the Pore-Forming Toxin Lysenin

Nisha Shrestha Boise State University

Christopher A. Thomas Boise State University

Devon Richtsmeier Boise State University

Andrew Bogard Boise State University

Rebecca Hermann Boise State University

See next page for additional authors Authors Nisha Shrestha, Christopher A. Thomas, Devon Richtsmeier, Andrew Bogard, Rebecca Hermann, Malyk Walker, Gamid Abatchev, Raquel J. Brown, and Daniel Fologea

This article is available at ScholarWorks: https://scholarworks.boisestate.edu/physics_facpubs/229 toxins

Article Temporary Membrane Permeabilization via the Pore-Forming Toxin Lysenin

Nisha Shrestha 1,2, Christopher A. Thomas 1 , Devon Richtsmeier 1, Andrew Bogard 1,2, Rebecca Hermann 3 , Malyk Walker 1, Gamid Abatchev 1,2, Raquel J. Brown 4 and Daniel Fologea 1,2,*

1 Department of Physics, Boise State University, Boise, ID 83725, USA; [email protected] (N.S.); [email protected] (C.A.T.); [email protected] (D.R.); [email protected] (A.B.); [email protected] (M.W.); [email protected] (G.A.) 2 Biomolecular Sciences Graduate Program, Boise State University, Boise, ID 83725, USA 3 Department of Biology, Boise State University, Boise, ID 83725, USA; [email protected] 4 Center of Biomedical Excellence in Matrix Biology, Biomolecular Research Center, Boise State University, Boise, ID 83725, USA; [email protected] * Correspondence: [email protected]

Received: 29 April 2020; Accepted: 20 May 2020; Published: 22 May 2020

Abstract: Pore-forming toxins are alluring tools for delivering biologically-active, impermeable cargoes to intracellular environments by introducing large conductance pathways into cell membranes. However, the lack of regulation often leads to the dissipation of electrical and chemical gradients, which might significantly affect the viability of cells under scrutiny. To mitigate these problems, we explored the use of lysenin channels to reversibly control the barrier function of natural and artificial lipid membrane systems by controlling the lysenin’s transport properties. We employed artificial membranes and electrophysiology measurements in order to identify the influence of labels and media on the lysenin channel’s conductance. Two cell culture models: Jurkat cells in suspension and adherent ATDC5 cells were utilized to demonstrate that lysenin channels may provide temporary cytosol access to membrane non-permeant propidium iodide and phalloidin. Permeability and cell viability were assessed by fluorescence spectroscopy and microscopy. Membrane resealing by chitosan or specific media addition proved to be an effective way of maintaining cellular viability. In addition, we loaded non-permeant dyes into liposomes via lysenin channels by controlling their conducting state with multivalent metal cations. The improved control over membrane permeability might prove fruitful for a large variety of biological or biomedical applications that require only temporary, non-destructive access to the inner environment enclosed by natural and artificial membranes.

Keywords: lysenin; permeabilization; pore forming toxins; chitosan

Key Contribution: We demonstrate that the pore-forming toxin lysenin may be used to achieve controlled permeabilization of natural and artiﬁcial lipid membranes, therefore enabling the transport of otherwise non-permeant molecules.

1. Introduction The ability to intracellularly deliver ions, nucleic acids, peptides, proteins, drugs, activators, inhibitors, and labeled compounds that interact with speciﬁc targets or modulate essential biological functionalities has enabled unprecedented advances in science, biotechnology, and medicine. The non-permeant nature of the plasma membrane, which often acts as a barrier that prevents access to the cytosol, frequently challenges the deployment of solutes to intracellular environments.

Toxins 2020, 12, 343; doi:10.3390/toxins12050343 www.mdpi.com/journal/toxins Toxins 2020, 12, 343 2 of 17

For well over a century, investigations focused on introducing non-permeant cargoes into cells while preventing irreversible damage or unintentional perturbations of cellular functionalities led to numerous strategies for mediating intracellular transport [1–13]. With this goal in mind, gaining access to the cytosol by using pore-forming proteins (PFPs) that disrupt the membrane barrier function and introduce conductive pathways that facilitate a diffusion-driven transport of solutes across membranes is a promising new mechanism [8–10,14,15]. In addition to allowing the passage of relatively large molecules [8–10,15,16], the potential modulation of the membrane permeability through intrinsic regulatory mechanisms might provide unique controls of the barrier function previously difficult to attain by alternative techniques. As an example, ion channels present high transport rate, selectivity, and regulation [17–19]; with a few notable exceptions [2], their narrow opening and high selectivity limit their applicability to intracellular delivery of specific, small-sized ions and compounds. Scientists have utilized pore-forming toxins (PFTs) as alternative pathways for intracellular delivery, which generally have a larger diameter and may allow passage of larger molecules, to address this shortcoming [8–10,14,16,20]. A concerning problem that is associated with the use of PFTs is a poor control over their conducting state, which might prove fatal for cells that are unable to engage internal repair mechanisms. Ultimately, such problems may lead to uncontrolled leakage, the dissipation of electrochemical gradients, and cellular death [21–27]. A widely used PFT for intracellular delivery purposes is Streptolysin O (SLO), a cholesterol-dependent cytolysin [8–10,16,28,29]. SLO is not endowed with clear regulatory mechanisms [30]; nonetheless, extensive purification followed by titration and further initiation of membrane repair mechanisms by Ca2+ addition allows for the delivery of large molecules while maintaining excellent cellular viability [8–10,16]. The recovery protocols vary greatly among reports, and careful optimization is recommended before application to any cell line [9]. Furthermore, this approach might not be applicable at all to artificial membrane systems, which lack internal repair systems. As an improvement with regard to endowing PFTs with regulatory mechanisms, the alpha-hemolysin channel was successfully engineered to function as a reversible switch that is responsive to Zn2+ ions [7,31], yet channel reopening might unintentionally occur if Zn2+ is depleted, and the rather narrow diameter of the conductive pathway might limit the passage of solutes larger than ~1 kDa in size [7]. Although it is recognized that achieving better control of the channels’ conducting state has the potential to improve PFT’s applicability to intracellular delivery, in most cases opening and closing conducting pathways in the cell membrane in a safe, robust, and well-controlled manner has yet to be achieved. We propose using lysenin channels as nano-valves that control the permeability of natural and artificial lipid membranes and enable free passage of non-permeant molecules to mitigate the challenge. Lysenin, a PFT that is found in the coelomic fluid of E. fetida, oligomerizes and introduces large nonameric channels (~3 nm diameter) in artificial and natural cell membranes containing sphingomyelin [32–36], a major component of mammalian cell membranes. The large channel enables the diffusion of non-permeant molecules that cannot pass through the narrow opening of other PFPs. In contrast to many other toxins, lysenin channels possess unique, intrinsic regulatory features [33,37,38] that make it a good candidate for intracellular delivery. For example, sub-micromolar concentration of trivalent metal cations closes the channel through a reversible ligand-gated mechanism [39,40]. Trivalent metal ions bind to a specific site on the lumen and induce conformational transitions from open to closed state, hence blocking the channel, as observed in prior experiments that employed single channel investigations [39,40]. Interestingly, divalent metals and more voluminous trivalent ions elicit conformational changes that result in the channels adopting a sub-conducting state (half-open) [39,40]. In most cases, multivalent ion removal by chelation or precipitation reinstates the original macroscopic conductance of lysenin channels reconstituted in artificial membrane systems [39,40]. In contrast, cationic polymers, such as polyethylenimine or chitosan, irreversibly block the channels and obliterate the membrane’s macroscopic conductance through a mechanism that might employ induced-gating and polymer trapping into the channel’s lumen [41]. Toxins 2020, 12, 343 3 of 17

Toxins 2020, 12, x FOR PEER REVIEW 3 of 18 This work demonstrates that lysenin channels may be used to control the permeability of both naturalmembrane and artificial systems membranes (planar lipid by bilayers modulating and their liposomes) conductance in electrophysiology, with chitosan, cell microscopy, culture media, and orspectroscopy multivalent metal assessments ions. We examinedto explore both this suspended hypothesis and. adherent Our results live cells, suggest andartificial that this membrane loading systemsmethodology (planar, with lipid the bilayers quick and and liposomes) well-controlled in electrophysiology, resealing of the microscopy, permeabilized and membranes spectroscopy, is assessmentsapplicable to to cells explore and thisartificial hypothesis. lipid membrane Our results systems suggest, and that it thisensures loading excellent methodology, post-treatment with the cell quickviability and. well-controlled resealing of the permeabilized membranes, is applicable to cells and artificial lipid membrane systems, and ensures excellent post-treatment cell viability. 2. Results and Discussion 2. Results and Discussion 2.1. Investigations of the Effect of Indicators and Culture Media on the Conductance of Lysenin Channels 2.1. Investigations of the Effect of Indicators and Culture Media on the Conductance of Lysenin Channels Reconstituted in Artificial Membranes Reconstituted in Artificial Membranes Our explorations on the temporary permeabilization of membranes via lysenin channels Our explorations on the temporary permeabilization of membranes via lysenin channels inserted inserted into the membrane of live cells rely on dyes and media more complex than the simple ionic into the membrane of live cells rely on dyes and media more complex than the simple ionic solutions that are traditionally used for assessments performed in artificial membrane systems. solutions that are traditionally used for assessments performed in artificial membrane systems. Multivalent cations significantly inhibit the macroscopic conductance of lysenin channels [39,40], Multivalent cations significantly inhibit the macroscopic conductance of lysenin channels [39,40], which affect their capability to create transmembrane conductive pathways that provide access to which affect their capability to create transmembrane conductive pathways that provide access to the the cytosol. Therefore, preliminary testing of the effects of media and dyes on lysenin’s conductance cytosol. Therefore, preliminary testing of the effects of media and dyes on lysenin’s conductance in in artificial membrane systems was performed to identify potential interferences with artificial membrane systems was performed to identify potential interferences with permeabilization. permeabilization. The reconstitution of lysenin channels in planar bilayer lipid membranes in The reconstitution of lysenin channels in planar bilayer lipid membranes in buffered KCl solutions buffered KCl solutions revealed the uniform, step-wise change of the ionic currents, indicative of revealed the uniform, step-wise change of the ionic currents, indicative of individual channel individual channel insertion [33,40] (Figure 1A). The completion of the insertion process was insertion [33,40] (Figure1A). The completion of the insertion process was indicated by the steady state indicated by the steady state of the ionic current that was achieved in ~1 h (Figure 1B), after which of the ionic current that was achieved in ~1 h (Figure1B), after which the lysenin still free in solution the lysenin still free in solution was removed by buffer exchange and the test solutions were added was removed by buffer exchange and the test solutions were added to the ground reservoir (to simulate to the ground reservoir (to simulate the extracellular environment). the extracellular environment).

Figure 1. Lysenin channels reconstitution in artificial lipid membranes. (A) Shortly after lysenin addition, theFigure ionic 1. currents Lysenin presented channels a reconstitution discrete, step-wise, in artificial and uniform lipid membranes. variation, indicative(A) Shortly of after individual lysenin channeladdition, insertion. the ionic (B ) currents The macroscopic presented current a discrete, reached step a- steady-statewise, and uniform in ~1 h, variation, indicative indicative of insertion of completion.individual channel Each plot insertion. represents (B) a The typical macroscopic trace representative current reached for the a steady given experimental-state in ~1 h, indicative conditions. of insertion completion. Each plot represents a typical trace representative for the given experimental Weconditions. recorded the ionic currents upon successively replacing small amounts of the buffered KCl solution in the ground reservoir with the above media (50–100 µL/step) to assess the influence of the Hanks’W Balancede recorded Salt the Solution ionic currents (HBSS) upon and cellsuccessive culturely media replac commonlying small amounts used for of culturing the buffered ATDC5 KCl andsolution Jurkat in cells the ground (DMEM reservoir/F12, and with RPMI the 1640—see above media the (50 Materials–100 µL/step) and Methods to assess section the influence for detailed of the descriptions)Hanks’ Balanced on the Salt macroscopic Solution (HBSS) conductance and cell of culture lysenin media channels. commonly The ionic used currents for culturing were converted ATDC5 intoand relative Jurkat macroscopiccells (DMEM/F12, conductance and RPMI (Gr) to1640 facilitate—see the a comparison Materials and between Methods experiments section comprisingfor detailed adescriptions) different number on of the inserted macroscopic channels. conductance Each step of replacement of lysenin withchannels DMEM. T/heF12 ionic elicited currents a significant were decreaseconverted in the into G r relative(Figure 2); macroscopic in contrast, conductancenegligible changes (Gr) in to conductance facilitate a werecomparison observed between upon experiments comprising a different number of inserted channels. Each step of replacement with DMEM/F12 elicited a significant decrease in the Gr (Figure 2); in contrast, negligible changes in conductance were observed upon successive replacement with HBSS (Figure 2). Similar to

Toxins 2020, 12, x FOR PEER REVIEW 4 of 18 Toxins 2020, 12, 343 4 of 17 DMEM/F12, a conductance decrease was also observed after replacing small amounts of buffered KCl with RPMI 1640 (Figure 2). Such inhibitory effects may be a result of the complex compositions, successive replacement with HBSS (Figure2). Similar to DMEM /F12, a conductance decrease was also which include multiple charged species that affected the conductance of lysenin channels. HBSS has observed after replacing small amounts of buffered KCl with RPMI 1640 (Figure2). Such inhibitory a much simpler composition, and multivalent charged species that would potentially affect the effects may be a result of the complex compositions, which include multiple charged species that affected channels’ conductance are absent. Therefore, we assumed that DMEM/F12 or RPMI 1640 can be the conductance of lysenin channels. HBSS has a much simpler composition, and multivalent charged further used to reinstate the barrier function of a membrane permeabilized with lysenin channels, species that would potentially affect the channels’ conductance are absent. Therefore, we assumed while HBSS would be suitable for achieving effective membrane permeabilization under the that DMEM/F12 or RPMI 1640 can be further used to reinstate the barrier function of a membrane provision that this medium does not affect the channel insertion itself. permeabilized with lysenin channels, while HBSS would be suitable for achieving effective membrane permeabilization under the provision that this medium does not affect the channel insertion itself.

Figure 2. Effects of culture media on the macroscopic conductance of lysenin channels. When the electrolyteFigure bu2. ffEffectser was of exchanged culture media with DMEM on the/ F12macroscopic (50 µL/step) conductance or RPMI 1640 of (100lyseninµL /step),channels. the relativeWhen the conductanceelectrolyte of buffer the membrane was exchanged decreased with significantly, DMEM/F12 indicating (50 µL/step) a strong or inhibitory RPMI 1640 eff ect.(100 In µL/step) contrast,, the similarrelative addition conductance of HBSS elicitedof the membrane only minor, decreased negligible significantly, changes in the indicating macroscopic a strong conductance. inhibitory Media effect. exchangeIn contrast, is indicated similar by addition arrows. of The HBSS plots elicited are constructed only minor, by using negligible experimental changes ionic in the current macroscopic data recordedconductance. from typical Media traces, exchange and the is symbols indicated were by added arrows. for guidance. The plots are constructed by using experimental ionic current data recorded from typical traces, and the symbols were added for Weguidance. asked whether HBSS would be the choice for inserting conducting lysenin channels into the membranes of live cells since DMEM/F12 and RPMI 1640 proved unsuitable for permeabilization experiments.We asked We whether reconstituted HBSS would lysenin be channels the choice in for planar inserting bilayer conducting lipid membranes lysenin channels exposed into to the HBSSmembranes to test the of potentiallive cells influencesince DMEM/F12 of HBSS and on channelRPMI 1640 insertion. proved Individualunsuitable channelfor permeabilization insertion wasexperiments monitored.from We reconstituted the variation lysenin of the ionicchannels currents in planar that werebilayer recorded lipid membrane at -80 mVs exposed bias potential to HBSS (Figureto test3A). the The potential uniform, influence stepwise ofvariation HBSS on of channel the current insertion resembled. Individual the insertion channel of insertion lysenin was channelsmonitored in planar from membranes the variation exposed of the to ionic buff ered currents KCl solutionsthat were (as recorded shownin at Figure -80 mV1A) bias [ 33 ,34 potential,40], leading(Figure to the 3A). conclusion The uniform, that HBSSstepwise did variation not affect of the the insertion current process. resembled Additionally, the insertion one of might lysenin observechannels that in no planar ionic membranes current was exposed recorded to atbuffered the beginning KCl solutions of the (as trace, shown which in Figure indicated 1A) that[33,34,40] no , changesleading in to the the membrane conclusion permeability that HBSS occurreddid not affect when the the ins membraneertion process. was exposed Additionally to HBSS., one Next, might weobserve extended that the no investigations ionic current towas include recorded the intercalatingat the beginning agents of the proposed trace, which for use indicated as indicators that no of membranechanges in the permeability, membrane i.e., permeability Ethidium occurred Bromide when homodimer the membrane (EtBr-HD) was and exposed Propidium to HBSS. Iodide Next, (PI),we which extended were the added investigations as small aliquots to include to thethe groundintercalating side ofagents the membrane.proposed for The use first as indicators addition of of EtBr-HDmembrane (final permeability, concentration i.e., 1.5EthidiumµM) induced Bromide a visiblehomodimer decrease (EtBr of-HD) the ionic and currentPropidium through Iodide the (PI), lysenin-permeabilizedwhich were added membrane,as small aliquots which to was the indicative ground side of a diminished of the membrane. macroscopic The fir conductancest addition of (FigureEtBr3-HDB). When (final the concentration bulk concentration 1.5 µM)of induced EtBr-HD a was visible raised decrease to 6.2 ofµ M, the the ionic ionic current current through quickly the decreasedlysenin- topermeabilized near zero. Amembrane, possible explanation which was forindicative this undesired of a diminished effect might macroscopic be provided conductance from the(Figure structural 3B). analysisWhen the of bulk the dye.concentration EtBr-HD of is EtBr a relatively-HD was longraised molecule to 6.2 µM that, the bears ionic four current positive quickly chargesdecreased that are to providednear zero. by A thepossible included explanation amine groups. for this These undesired positive effect charges might may be induce provided reversible from the or irreversiblestructural analysis conductance of the changesdye. EtBr by-HD either is a relatively gating [40 long] or a molecule gating and that trapping bears four mechanism positive charges [41]. that are provided by the included amine groups. These positive charges may induce reversible or

Toxins 2020, 12, x FOR PEER REVIEW 5 of 18

irreversible conductance changes by either gating [40] or a gating and trapping mechanism [41]. Although we did not investigate this hypothesis any further, we concluded that EtBr-HD is not suitable for use in experiments that are aimed at inducing controlled permeability via lysenin channels. We similarly tested PI for this purpose, which has a much smaller positive charge than the EtBr-HD, therefore a weaker interaction with the lysenin channels and a lower impact on the macroscopic conductance was expected. The addition of PI to the ground reservoir (1.5 µM final concentration) induced negligible changes in the open current, which suggests that PI does not modulate the channel’s conductance (Figure 3B). This observation was supported by a further increase of the PI concentration in the bulk (i.e., 4.5 µM and 7.5 µM), which also elicited insignificant changes of the macroscopic currents and confirmed PI as a suitable candidate for lysenin-induced permeability experiments. In the same line of preliminary explorations, we asked whether the PI in the bulk solutions might prevent channel blockage that is elicited in the presence of chitosan [41], intended to be added as an irreversible channel blocker. Our results (Figure 3B) show that chitosan addition (25 nM final concentration) to the HBSS bulk in the presence of 6 µM PI annihilated the macroscopic conductance of lysenin channels inserted into the planar lipid membrane. This result supported the feasibility of the proposed approach for obtaining controlled membrane permeability by irreversibly controlling the lysenin’s conducting state with chitosan. Toxins 2020Working, 12, 343 concentrations of calcein-AM and Alexa Fluor 546 Phalloidin (AF546-Phal) were5 ofalso 17 tested in similar electrophysiology experiments; none of them elicited a major influence on the Althoughlysenin channels’ we did not macroscopic investigate conductance, this hypothesis and any their further, effects we were concluded similar that to EtBr-HDthe HBSS is ( notas shown suitable in forFigure use in2). experiments that are aimed at inducing controlled permeability via lysenin channels.

Figure 3. Effects of intercalating agents and chitosan on the macroscopic conductance of lysenin channelsFigure 3. reconstituted Effects of intercalating in planar lipid agents membranes and chitosan with on Hanks’ the macroscopic Balanced Salt conductance Solution (HBSS) of lysenin as supportchannels electrolyte. reconstituted (A ) in The planar HBSS lipid bulk membranes electrolyte didwith not Hanks’ affect Balanced channel insertion. Salt Solution (B) ( EtBr-HDHBSS) as additionsupport inhibited electrolyte the. (A macroscopic) The HBSS conductance bulk electrolyte of lysenin did not channels. affect channel In contrast, insertion. no major (B) changes EtBr-HD inaddition macroscopic inhibit conductanceed the macroscopic were observed conductance upon of successive lysenin channels. addition In of contrast, Propidium no major Iodide changes (PI). The in lysenin-permeabilizedmacroscopic conductance membrane were bathedobserved by HBSS upon and successive PI underwent addition resealing of Propidium after addition Iodide of chitosan. (PI). The Eachlysenin graph-permeabilized represents a typicalmembrane trace ofbathed data recorded by HBSS in and the given PI under experimentalwent resealing conditions. after All addition the data of pointschitosan. in panel Each ( Bgraph) are experimentalrepresents a typical values, trace and theof data symbols recorded have beenin the added given toexperimental facilitate identification. conditions. TheAll arrows the data indicate points addition in panel of ( theB) dyesare experimental and chitosan, values, and their and concentrations. the symbols have been added to facilitate identification. The arrows indicate addition of the dyes and chitosan, and their Weconcentrations similarly tested. PI for this purpose, which has a much smaller positive charge than the EtBr-HD, therefore a weaker interaction with the lysenin channels and a lower impact on the macroscopic conductanceAll of the was data expected. presented The in addition Figure of 1 PIFigure to the 2 groundFigure 3 reservoir show typical (1.5 µ Mtraces final from concentration) individual inducedexperiments. negligible Nonetheless, changes in numerous the open current,independent which experiments suggests that performed PI does not in modulate identical theexperimental channel’s conductanceconditions qualitatively (Figure3B). This and observation quantitatively was replicated supported the by apresented further increase results. of The the PIchannel concentration insertion inprocess the bulk is a (i.e., statistical 4.5 µM one and; therefore 7.5 µM),, average which alsotraces elicited for the insignificant insertion events changes cannot of be the provided macroscopic since currentsthe insertions and confirmed occur atPI random as a suitable times. candidate In the same for lysenin-inducedline, the experimental permeability setup experiments.made it difficult In the to same line of preliminary explorations, we asked whether the PI in the bulk solutions might prevent channel blockage that is elicited in the presence of chitosan [41], intended to be added as an irreversible channel blocker. Our results (Figure3B) show that chitosan addition (25 nM final concentration) to the HBSS bulk in the presence of 6 µM PI annihilated the macroscopic conductance of lysenin channels inserted into the planar lipid membrane. This result supported the feasibility of the proposed approach for obtaining controlled membrane permeability by irreversibly controlling the lysenin’s conducting state with chitosan. Working concentrations of calcein-AM and Alexa Fluor 546 Phalloidin (AF546-Phal) were also tested in similar electrophysiology experiments; none of them elicited a major influence on the lysenin channels’ macroscopic conductance, and their effects were similar to the HBSS (as shown in Figure2). All of the data presented in Figures1–3 show typical traces from individual experiments. Nonetheless, numerous independent experiments performed in identical experimental conditions qualitatively and quantitatively replicated the presented results. The channel insertion process is a statistical one; therefore, average traces for the insertion events cannot be provided since the insertions occur at random times. In the same line, the experimental setup made it difficult to replicate the addition of media and dyes at identical time intervals for parallel experiments. Although all of the samples showed similar evolutions of the ionic currents, the time evolution of the changes differed between the experiments. This might be easily explained by taking into account that the mixing in Toxins 2020, 12, x FOR PEER REVIEW 6 of 18

replicate the addition of media and dyes at identical time intervals for parallel experiments. Although all of the samples showed similar evolutions of the ionic currents, the time evolution of the Toxinschanges2020, 12differed, 343 between the experiments. This might be easily explained by taking into account6 of 17 that the mixing in the chambers was not uniform and noise considerations often required adjustments of the rotational speed of the stir bars in the chambers, therefore influencing the mixing. theIn chambersspite of these was shortcomings, not uniform and the noise relative considerations changes in macroscopic often required conductance adjustments that of thewere rotational observed speedfor ide ofntical the stir conditions bars in the and chambers, after achieving therefore steady inﬂuencing state were the mixing.similar in In parallel spite of theseexperiments shortcomings,. the relative changes in macroscopic conductance that were observed for identical conditions and after achieving2.2. Investigations steady state on Jurkat were Cells: similar Viability in parallel and Permeabilization experiments. Assays

2.2.2.2.1. Investigations Viability A onssessments Jurkat Cells: Viability and Permeabilization Assays 2.2.1. ViabilityOur next Assessments explorations focused on testing the cytotoxic effects of chitosan, lysenin, and their combination on the viability of Jurkat cells (Figure 4) by comparison with negative controls (samples Our next explorations focused on testing the cytotoxic effects of chitosan, lysenin, and their that were incubated without lysenin and/or chitosan). Although chitosan is considered to be a combination on the viability of Jurkat cells (Figure4) by comparison with negative controls (samples that bio-inert molecule, there is not sufficient information in regard to its potential toxicity manifested were incubated without lysenin and/or chitosan). Although chitosan is considered to be a bio-inert against Jurkat cells. The Alamar Blue (resazurin) assay test [42] that was performed after 20 h molecule, there is not sufficient information in regard to its potential toxicity manifested against Jurkat incubation of Jurkat cells with increasing concentrations of chitosan showed no significant changes cells. The Alamar Blue (resazurin) assay test [42] that was performed after 20 h incubation of Jurkat in the cellular viability (Figure 4A), even at five times (up to 125 nM) the concentration needed to cells with increasing concentrations of chitosan showed no significant changes in the cellular viability block lysenin channels. (Figure4A), even at five times (up to 125 nM) the concentration needed to block lysenin channels.

FigureFigure 4. 4.Viability Viability tests tests on on Jurkat Jurkat cells cells exposed exposed to to chitosan chitosan and and lysenin. lysenin. (A (A) Exposure) Exposure of of Jurkat Jurkat cells cells toto chitosan chitosan concentrations concentrations up up to to 125 125 nM nM elicited elicited only only minor, minor, negligible negligible changes changes in in cellular cellular viability. viability. (B()B Jurkat) Jurkat cells cells exposed exposed to to only only lysenin lysenin (10 (10 nM nM final final concentration) concentration) suff sufferedered massive massive damage, damage leading, leading to ato reduced a reduced viability. viability. Chitosan Chitosan addition addition (25 (25 nM nM final final concentration) concentration) 20 20 min. min. after after lysenin lysenin exposure exposure protectedprotected the the cells cells and and led le tod to almost almost complete complete recovery. recovery. The The experimental experimen datatal data represents represents averages averages of three independent experiments (n = 3, SD). of three independent experiments (n =± 3, ± SD). We performed the resazurin viability test by employing 10 nM lysenin and 25 nM chitosan added We performed the resazurin viability test by employing 10 nM lysenin and 25 nM chitosan after 20 min. exposure to lysenin alone to assess the influence of lysenin channels on the viability of added after 20 min. exposure to lysenin alone to assess the influence of lysenin channels on the Jurkat cells with and without chitosan-induced channel blockage (Figure4B). This test did not include viability of Jurkat cells with and without chitosan-induced channel blockage (Figure 4B). This test PI addition, which might also affect the cellular viability. Exposure to lysenin alone led to a significantly did not include PI addition, which might also affect the cellular viability. Exposure to lysenin alone lower viability, estimated at ~12% by comparison to the negative control. However, the cells that were led to a significantly lower viability, estimated at ~12% by comparison to the negative control. treated with chitosan after lysenin-induced permeabilization did not suffer major damage, as inferred However, the cells that were treated with chitosan after lysenin-induced permeabilization did not from the high viability rate (~97%). This result suggests that the addition of chitosan prevented suffer major damage, as inferred from the high viability rate (~97%). This result suggests that the extensive membrane damage induced by lysenin and enabled remarkable recovery. addition of chitosan prevented extensive membrane damage induced by lysenin and enabled 2.2.2.remarkable Achievement recovery of. Controlled Permeability of Jurkat Cell Membranes

2.2.2Fluorescence. Achievement spectroscopy of Controlled was Permeability employed to of monitor Jurkat theCell cells Membranes by speciﬁcally tracking the uptake of dye into the nucleus of cells exposed to PI alone, PI and lysenin, and PI and lysenin subsequently blocked with chitosan in order demonstrate that lysenin and chitosan enable the achievement of controlled permeability of Jurkat cells membranes (Figure5). Upon continual exposure to PI only, the cells presented a steady, weak ﬂuorescence for the entire duration of the experiment (~50 min.), Toxins 2020, 12, x FOR PEER REVIEW 7 of 18

Fluorescence spectroscopy was employed to monitor the cells by specifically tracking the uptake of dye into the nucleus of cells exposed to PI alone, PI and lysenin, and PI and lysenin subsequently blocked with chitosan in order demonstrate that lysenin and chitosan enable the achievement of controlled permeability of Jurkat cells membranes (Figure 5). Upon continual exposureToxins 2020 ,to12 ,PI 343 only, the cells presented a steady, weak fluorescence for the entire duration of7 the of 17 experiment (~50 min.), suggesting that PI did not accumulate into the nucleus. The lack of intercalationsuggestingthat suggests PI did that not PI accumulate did not cross into the the plasma nucleus. membrane, The lack which of intercalation would be indicated suggests thatby an PI increasingdid not cross fluorescence the plasma signal. membrane, In contrast, which the would addition be indicated of lysenin by (10 an nM) increasing in the fluorescencepresence of PI signal. (1.5 µInM contrast,) showed the a gradual addition increase of lysenin of (10fluorescence, nM) in the which presence seemed of PI to (1.5 asymptoticallyµM) showed areach gradual saturation increase, thusof fluorescence, strongly suggesting which seemed that PI to crossed asymptotically the membrane reach saturation,and intercalated thus stronglyinto the suggestingnuclear DNA that for PI thecrossed entire the duration membrane of the and experiment intercalated (Fig intoure the 5). nuclear The samples DNA for containing the entire PI duration and lysenin of the channels experiment in the(Figure membrane5). The showed samples a containing very similar PI andpattern lysenin until channels the 20 min in the. time membrane mark, when showed chitosan a very ( similar25 nM final concentration) was added to induce a quick blockage of lysenin channels. pattern until the 20 min. time mark, when chitosan (25 nM final concentration) was added to induce a quick blockage of lysenin channels.

Figure 5. Chitosan controls the lysenin-mediated transport of PI across the membrane of Jurkat cells. FigurePI did 5. not Chitosan cross the contr cellols membrane the lysenin in- themediated absence transport of lysenin, of PI as across indicated the bymembrane the steady of fluorescenceJurkat cells. PIintensity. did not Incross contrast, the cell a continualmembrane increase in the absence in fluorescence, of lysenin, indicative as indicated of PI by intercalation, the steady wasfluorescence observed intensity.after lysenin In contrast, addition a (10continual nM final increase concentration). in fluorescence, For a similarlyindicative permeabilized of PI intercalation, sample, was addition observed of afterchitosan lysenin 20 min.addition after (10 the nM initiation final concentration) of permeabilization. For a similarly quickly permeabilized stabilized the fluorescentsample, addition intensity of chitosanand indicated 20 min. that after PI wasthe preventedinitiation of from permeabilization further crossing quickly the cell stabilized membrane. the The fluorescent symbols intensity represent andaverage indicated values that from PI was four prevented experiments from (n =further4, SD) crossing that comprised the cell membrane. individual The samples symbols made represent from the ± averagesame cell values culture from batch. four experiments (n = 4, ± SD) that comprised individual samples made from the same cell culture batch. The fluorescence intensity that was recorded a few minutes after chitosan addition (~25 min. fromThe the fluorescence beginning of intensity the experiment) that was showed recorded a decreased a few mi valuenutes when after comparedchitosan addition to the samples (~25 min. that fromincluded the beginning lysenin, but of no the added experiment) chitosan. showed This di aff erencedecreased accentuated value when over compared time and theto the samples samples that thatwere included blocked lysenin with chitosan, but no showed added chitosan. a rather stable,This difference steady state accentuated of the fluorescence over time signal.and the The samples slight thatincrease were that blocked was immediatelywith chitosan observed showed after a rather chitosan stable, addition steady was state interpreted of the fluorescence as originating signal. from The PI slightthat was increase still in that the was cytosol immediately (though not observed yet intercalated after chitosan into the addition nuclear was DNA interpreted before channel as originating blockage) fromor passed PI that at was a lower still in rate the into cytosol the cytosol (though before not yet complete intercalated membrane into the resealing. nuclear DNA The relatively before channel steady blockage)fluorescence or signal passed that at was a lower recorded rate after into chitosanthe cytosol addition before suggests complete that membrane the barrier resealing. function of The the relativelymembrane steady was reinstated. fluorescence This signal also confirmsthat was thatrecorded no major after changes chitosan in addition cell viability suggests occurred that after the barrierchitosan function addition, of whichthe membrane would lead was to rei a gradualnstated. increaseThis also of confirms the fluorescence that no intensity,major changes owing in to cell PI’s viabilitydiffusion occurred through after the compromised chitosan addition, membranes which would of dead lead cells to after a gradual channel increase blockage. of the fluorescence intensity, owing to PI’s diffusion through the compromised membranes of dead cells after channel blockage2.3. Investigations. on ATDC5 Cells: Permeabilization Assay Investigations on ATDC5 Cells 2.3. Investigations on ATDC5 Cells: Permeabilization Assay Our next investigations aimed at using lysenin channels to control the permeability of ATDC5 cell membranes to phalloidin, a bicyclic heptapeptide toxin that is extracted from the mushroom Amanita phalloides [43]. Phalloidin binds strongly to the polymeric, filamentous actin (F-actin), hence preventing its further depolymerization [44–46]. Phalloidin is freely permeant through the membrane of Toxins 2020, 12, x FOR PEER REVIEW 8 of 18

2.3.1. Investigations on ATDC5 Cells Our next investigations aimed at using lysenin channels to control the permeability of ATDC5 cellToxins membranes2020, 12, 343 to phalloidin, a bicyclic heptapeptide toxin that is extracted from the mushroom8 of 17 Amanita phalloides [43]. Phalloidin binds strongly to the polymeric, filamentous actin (F-actin), hence preventing its further depolymerization [44–46]. Phalloidin is freely permeant through the membranehepatocytes, of but,hepatocytes for other, but cells,, for access other tocells the, access cytosol to must the cytosol be provided must be by provided alternative by approaches, alternative approachessuch as microinjection,, such as microinjection, optoporation, optoporation, electroporation, electroporation, or others [1,9 or,16 others,47,48]. [1,9,16,47,48] Therefore, we. Therefore, explored wethe explored ability of the lysenin ability channels of lysenin to openchannels conductance to open conductance pathways that pathways facilitate that phalloidin’s facilitate passagephalloidin’s into passagethe cytosol into of the adherent cytosol ATDC5 of adherent cells. ATDC5 We used cells. the fluorescent We used the AF546-Phal fluorescent conjugate AF546- asPhal an conjugate indicator foras antransmembrane indicator for transmembrane transport through transpor lysenint channelsthrough lysenin to enable channels microscopy to enable imaging. microscopy Our experiments imaging. Ourcomprised experiments adherent comprised ATDC5 adherent cells that ATDC5 were exposed cells that to lysenin were exposed and phalloidin to lysenin conjugate and phalloidin as well conjugateas control as samples well as that control excluded samples the usethat of excluded lysenin. the After use cell of lysenin. identification After by cell microscopy identification under by microscotransmittedpy lightunder (Figure transmitted6A,C), the light presence (Figure of6A fluorescent, and 6C), the intracellular presence phalloidin of fluorescent, was identified intracellular by phalloidinconfocal microscopy. was identified by confocal microscopy.

FigureFigure 6. 6. ChannelsChannels enable enable the the transport transport of non-permeant of non-permeant AF546-Phal AF546 across-Phal across the membrane the membrane of adherent of adherentATDC5 cells. ATDC5 (A,C cells.) Transmitted (A), and light (C) Transmitted microscopy lightimaging microscopy shows the imaging presence shows of cells. the ( presenceB) Cells not of cells.exposed (B) toCells lysenin not exposed lack the redto lysenin ﬂuorescence, lack the suggesting red fluorescence that AF546-Phal, suggesting did not that cross AF546 the- membranePhal did not of crossnon-permeabilized the membrane cells. of non (D-)permeabilized AF546-Phal was cells. detected (D) AF546 in the-Phal cytosol was of detected cells exposed in the to cytosol lysenin of (10 cells nM exposedﬁnal concentration). to lysenin (10 nM final concentration).

TheThe cells cells that that were were exposed exposed to to AF546 AF546-Phal-Phal in in the the absence absence of of lysenin lysenin showed showed no no red red fluorescence fluorescence (Figure(Figure6 B). 6B) In. In contrast, contrast, the thecells cells exposed exposed to both to AF546-Phal both AF546 and-Phal lysenin and presented lysenin presented a strong fluorescent a strong fluorescentsignal (Figure signal6D), (Figure which was 6D), indicativewhich was of indicative binding to of F-actin binding after to F crossing-actin after the crossing cell membrane. the cell membrane.We concluded We thatconcluded the intact that cell the membranes intact cell membranes constituted constituted a barrier for a thebarrier passage for the of thepassage fluorescent of the fluorescentphalloidin conjugate, phalloidin and conjugate, lysenin addition and lwasysenin essential addition for rendering was essential the cell membranesfor rendering permeable the cell to membranesthe heptapeptide. permeable However, to the these heptapeptide. experiments However, were performed these experiments at short time were scales, performed and the viability at short of timethe cells scales, after and treatment the viability was not of assessed.the cells after Lysenin treatment and phalloidin was not both assessed. exert cytotoxicLysenin eandffects phalloidin by either bothdissipating exert thecytotoxic electrochemical effects by gradients either acrossdissipating the cell the membranes electrochemical or inhibiting gradients the depolymerization across the cell membranesof F-actin, respectively. or inhibiting We the added depolymerization calcein-AM as of a live-cellF-actin, respectively. indicator to examine We added the calcein cellular-AM viability as a liveafter-cell treatment indicator with to examine lysenin and the AF546-Phalcellular viability [49]. Theafter cells treatment were exposedwith lysenin to AF546-Phal and AF546 and-Phal lysenin [49]. similarly to the previous experiments described in this section. After 10 min. of incubation with lysenin and AF546-Phal, the samples were washed twice, and then incubated in DMEM/F12 (which also acted as channel blocker) for over five hours. The confocal microscopy analysis (Figure7) of the cells that Toxins 2020, 12, x FOR PEER REVIEW 9 of 18

ToxinsThe cells2020 ,were12, 343 exposed to AF546-Phal and lysenin similarly to the previous experiments described9 of in 17 this section. After 10 min. of incubation with lysenin and AF546-Phal, the samples were washed twice, and then incubated in DMEM/F12 (which also acted as channel blocker) for over five hours. wereThe confocal treated withmicroscopy lysenin, analysis AF546-Phal, (Figure and 7) thenof the blocked cells that with were DMEM treated/F12 with was lysenin, performed AF546 after-Phal, the andaddition then of blocked calcein-AM with asDMEM a viability/F12 was indicator performed and subsequent after the addition washing. of Image calcein analysis-AM as showed a viability the indicatorpresence ofand lysenin-permeabilized subsequent washing. cells Image (Figure analysis7A), whichshowed also the presented presence aof strong lysenin red-permeabilized fluorescence cells(Figure (Figure7B), suggesting 7A), which that also lysenin presented channels a strong altered red the fluo barrierrescence function (Figure of 7B), the membranesuggesting and that allowed lysenin channelscytosol access. altered The the same barrier samples function showed of the themembrane specific and green allowed fluorescence cytosol of access. the calcein The same dye thatsamples was showedreleased the from specific the AM-conjugate green fluorescence by the of catalytic the calcein activity dye ofthat esterases was released (Figure from7C), the hence AM indicating-conjugate bythe thecellular cataly viabilitytic activity of the of lysenin-exposed esterases (Figure cells. 7C), The hence dye distribution indicating insidethe cellular cells is presented viability of in the lyseninoverlap-exposed image (Figure cells. 7TheD). dye distribution inside cells is presented in the overlap image (Figure 7D).

Figure 7. CCellsells loaded loaded with with phalloidin phalloidin via via lysenin lysenin channels channels rema remainin viable viable after over five ﬁve hours incubation in in DMEM DMEM media. media. ( (AA)) Transmitted Transmitted-light-light image image of of ATDC ATDC cells cells upon upon exposure exposure to to lysenin, lysenin, phalloidin, andand calcein-AM.calcein-AM. ( B(B) AF546-Phal) AF546-Phal was was detected detected inside inside the the lysenin-permeabilized lysenin-permeabilized ATDC5 ATDC5 cells cells(red channel).(red channel) (C) The. (C lysenin-permeabilized) The lysenin-permeabilized cells further cells exposed further exposed to inhibitory to inhibitory DMEM/F12 DMEM remained/F12 remainedviable, as suggestedviable, as by suggested the green by ﬂuorescence the green presented fluorescence by calcein-AM presented by cleaved calcein by-AM esterases cleaved in the by esterasescytosol (green in the channel). cytosol (green (D) Overlap channel) of. transmitted,(D) Overlap red,of transmitted, and green channelred, and images. green channel images.

WWee prepared prepared identical identical cell cell samples samples that thatunderwent underwent an otherwise an otherwise identical identical procedure procedure but excluded but excludedlysenin addition lysenin to addition demonstrate to demonstrate that lysenin that is required lysenin is to required achieve permeabilization to achieve permeabilization at longer time at longerscales. time The cellsscales that. The were cells identified that were under identified transmitted under lighttransmitted (Figure 8lightA) showed (Figure no8A red) showe fluorescenced no red fluorescence(Figure8B), indicating (Figure 8B that), indicating lysenin addition that lysenin is essential addition for is theessential AF546-Phal for theto AF546 cross-Phal the membranes. to cross the membranes.However, strong However, fluorescence strong was fluorescence observed for was calcein observed (Figure for8C), calcein which (Figure was relatively 8C), which uniformly was relativelydistributed uniform into thely cytosol distributed (Figure into8D). the cytosol (Figure 8D).

Toxins 2020, 12, 343 10 of 17 Toxins 2020, 12, x FOR PEER REVIEW 10 of 18

Figure 8.8. AF546-PhalAF546-Phal does does not not cross cross the membranesthe membranes of ATDC5 of ATDC5 cells in thecells absence in the of absence lysenin. (ofA )lysenin. Transmitted (A) Transmittedlight image of light cells exposed image of to AF546-Phal cells exposed and to calcein-AM. AF546-Phal (B) Theand lack calcein of red-AM. fluorescence (B) The indicates lack of that red AF546-Phalfluorescence did indicates not cross that the membranesAF546-Phalnot did exposed not cross to lysenin.the membrane (C) Thes strong not exposed green fluorescence to lysenin. recorded(C) The strongafter over green five fluorescence hours indicates recorded that the after cells over remained five hours viable. indicates (D) The that overlap the cells of transmitted remained viable and green. (D) Thechannel overlap images of transmitted shows calcein and distribution green channel inside images cells. shows calcein distribution inside cells.

2.4. Reversible Liposome Liposome Loading and Unloading via L Lyseninysenin ChannelsChannels WWee conducted experiments using liposomes toto demonstrate that lysenin channels reconstituted into artificial artificial membranes membranes may may facilitate facilitate entrapment entrapment and releaseand release of non-permeant of non-permeant molecules. molecules We tracked. We trackedthe transmembrane the transmembrane transport oftransport non-permeant of non calcein-permeant to assess Calcein loading to assess into and loading release into from and liposomes release 3+ fromby microscopy. liposomes Additionally,by microscopy Al. Additionallycations were, Al utilized3+ cations in were these utilized experiments in these given experiments their ability given to quicklytheir ability and reversiblyto quickly modulateand reversibly lysenin modulate channel’s lysenin conductance channel’s by conductance a ligand-gating by mechanisma ligand-gating [39]. mechanismIt is worth noting [39]. It that is thisworth approach noting providedthat this an approach opportunity provided to simplify an opportunity the procedure to simplify of assessing the procedurelysenin-mediated of assessing loading. lysenin While-mediated unincorporated loading. While calcein unincorporated may be removed calcein by may classic be removed separation by classictechniques separation such as techniques dialysis, chromatography, such as dialysis, or chromatography, centrifugation, we or exploited centrifugation, a distinctive we exploited property a distinctiveof calcein, i.e.,property the fluorescence of calcein, i.e. quenching, the fluorescence by multivalent quenching metal by ionsmultivalent [50]. This metal property ions [50] enabled. This propertythe elimination enabled of the the elimination bulk fluorescence of the bulk to fluorescence render the loadedto render liposomes the loaded fluorescent liposomes againstfluorescent the 3+ againstbackground. the background. While a previous While report a previous shows that report Al is shows among that the most Al3+ potentis among multivalent the most ion potentfor the multivalentinhibition of ion the macroscopicfor the inhibition conductance of the ofmacroscopic lysenin [39 ], conductance no consistent of reports lysenin have [39] determined, no consistent the 3+ reporfluorescencets have quenching determined eff ects the of fluorescence Al on calcein. quenching Our fluorescence effects of spectroscopy Al3+ on calcein. exploration Our fluorescence indicated 3+ spectroscopythat successive exploration addition of Al indicatedcations that to a successive 50 µM calcein addition solution of strongly Al3+ cations quenched to a the 50 fluorescence µM calcein 3+ solutionin a concentration-dependent strongly quenched the manner fluorescence (Figure9 ),in and a concentration that 100 µM Al-dependereducednt manner the calcein (Figure fluorescence 9), and thatto negligible 100 µM values. Al3+ reduced In addition, the calcein fluorescence fluorescence quenching to negligible occurred at values concentrations. In addition, larger fluorescence than what quenchingis needed to occurred efficiently atinhibit concentrations the macroscopic larger conductance than what of lysenin is needed channels to efficiently [39]. The present inhibit work the macroscopicexploits this feature conductance for reducing of lysenin the background channels [39] fluorescence. The present by adding workthe exploit quenchings this multivalent feature for reduccationsing to the the background liposome mixture, fluorescence assuming by adding that these the ionsquenching would multivalent rapidly close cations the channels to the liposome and only quenchmixture, the assuming calcein in that the these external ions solution. would rapidly Once the close channels the channels were blocked, and only it was quench anticipated the calcein that the in the external solution. Once the channels were blocked, it was anticipated that the sealed membrane would prevent cation access to the inner environment and protect the entrapped dye from quenching.

Toxins 2020, 12, x FOR PEER REVIEW 11 of 18 Toxins 2020, 12, 343 11 of 17

Toxinssealed 2020 membrane, 12, x FOR PEER would REVIEW prevent cation access to the inner environment and protect the entrapped11 of 18 dye from quenching.

Figure 9. Calcein fluorescence is quenched by Al3+ ions in a concentration dependent manner. The relativeFigure 9. fluorescenceCalcein fluorescence of 50 µM is quenched calcein byprepared Al3+ ions in in 135 a concentration mM KCl and dependent 20 mM manner. HEPES The (pH relative 7.2) 3+ decreasefluorescenced significantly of 50 µM calcein upon prepared Al addition in 135; mM 100KCl µM and cation 20 mM concentration HEPES (pH effectively7.2) decreased quenched significantly the Figurecalcein’supon 9. Al Calcein3 fluorescence+ addition; fluorescence 100. µM cation is quenched concentration by Al3+ effectively ions in a quenchedconcentration the calcein’s dependent fluorescence. manner. The relative fluorescence of 50 µM calcein prepared in 135 mM KCl and 20 mM HEPES (pH 7.2) decreaseSphericalSphericald significantly membrane membrane upon permeabilizati permeabilization Al3+ additionon; 100was was µM performed performed cation concentration by by mixing mixing 50 effectively 50µLµ liposomesL liposomes quenched with with the 1 µL 1 µofL 30of nM 30calcein’s nM lysenin lysenin fluorescence at room at room. temperature. temperature. After After the theaddition addition of calcein of calcein (50 µM (50 µfinalM final concentrat concentration)ion) to the to solutionthe solution containing containing permeabilized permeabilized liposomes, liposomes, the sample the sample was left was to left equilibrate to equilibrate for six for hours six hoursat room at temperatureroomSpherical temperature membrane in a dark in a dark container.permeabilizati container. Wideon Wide-field-field was performed transmitted transmitted by light mixing light microscopy microscopy 50 µL liposomes imaging imaging with (Figure (Figure 1 µL 10 10ofAA) ) 30indicatedindicated nM lysenin the the at presence presence room temperature. of of uniform uniform Afterand and well well the dispe dispersedadditionrsed of liposomes calcein (50 in µM the final sample concentrat solution.ion) The The to samethe same solutionsamplesample containing thatthat was was analyzed permeabilizedanalyzed by by fluorescence fluorescence liposomes, microscopy the microscopy sample did was not did left show not to equilibrate show any individual any for individual six liposomes hours liposomesat room against temperatureagainstthe background the inbackground a (Figuredark container. 10(FigureB) and 10 Wide weB) concludedand-field we transmittedconcluded that the fluorescentthat light the microscopy fluorescent dye did not imagingdye accumulate did not (Figure accumulate either 10A) on indicatedeithertheir surfaces on the their presence orsurfa insidece ofs or their uniform inside membranes. theirand wellmembrane disperseds. liposomes in the sample solution. The same sample that was analyzed by fluorescence microscopy did not show any individual liposomes against the background (Figure 10B) and we concluded that the fluorescent dye did not accumulate either on their surfaces or inside their membranes.

FigureFigure 10. experimentsExperiments conducted conducted on on liposomes. liposomes. (A (A) )Transmitted Transmitted light light imaging imaging taken taken after after lysenin andand calcein calcein addition addition show show intact, intact, non non-aggregated-aggregated liposomes. ( (BB)) No No fluorescent fluorescent liposomes liposomes have have been been identifiedidentified against against the the background background after after calcein calcein addition addition to to the the solution solution of of liposomes liposomes containing containing open open Figurelyseninlysenin 10. channelschannels experiments in in the the conducted membrane. membrane. on Scale Scaleliposomes. bar bar == 1010 ( Aµµ)m. m.Transmitted light imaging taken after lysenin and calcein addition show intact, non-aggregated liposomes. (B) No fluorescent liposomes have been 3+ The open channels in the liposome membranes were blocked by the addition of 100 µM Al 3+ to identifiedThe open against channels the background in the liposome after calcein membranes addition were to the blockedsolution ofby liposomes the addition containing of 100 open µM Al to the mixture, which obliterated the transport through the lysenin channels, trapped the calcein inside thelysenin mixture, channels which in obliterated the membrane. the transportScale bar = through10 µm. the lysenin channels, trapped the calcein inside liposomes,liposomes, and and quenched quenched the the non non-loaded-loaded dye. dye. The The fluorescence fluorescence microscopy microscopy images images were were taken taken one one 3+ hour after Al3+ addition and revealed the presence of highly-fluorescent liposomes, indicative of hourThe after open Al channelsaddition in andthe liposomerevealed membranes the presence were of highlyblocked-fluorescent by the addition liposomes of 100, indicative µM Al3+ to of successful loading and membrane resealing (Figure 11A). We also observed that not all the liposomes thesuccessful mixture, which loading obliterated and membrane the transport resealing through (Figure the lysenin 11A). channels,We also trapped observed the that calcein not inside all the incorporated the dye by switching the illumination from fluorescence to visible-transmitted light liposomes,liposomes and incorporated quenched the thenon -loaded dye b ydye. switching The fluorescence the illumination microscopy images from were fluorescence taken one to conditions. Although such an effect could be a result of incomplete membrane resealing by channel hourvisible after-transmitted Al3+addition light and conditions revealed . the Although presence such of highlyan effect-fluorescent could be liposomes a result, indicative of incomplete of successful loading and membrane resealing (Figure 11A). We also observed that not all the liposomes incorporated the dye by switching the illumination from fluorescence to visible-transmitted light conditions. Although such an effect could be a result of incomplete

Toxins 20202020, 1212, 343x FOR PEER REVIEW 1212 of of 18 17 membrane resealing by channel blockage upon Al3+ addition and calcein leakage, the most 3+ reasonableblockage upon explanation Al addition relies andon the calcein fact that leakage, not all the of most the reasonablelarge liposomes explanation produced relies by onextrusion the fact werethat not unilamellar. all of the large Lysenin liposomes might produced interact with by extrusion multilamellar were unilamellar. structures, Lysenin but it cannot might interactbe assumed with thatmultilamellar the inserted structures, pores but will it cannot completely be assumed penetrate that thick, the inserted multilayered pores will structures completely to penetrate create a conductingthick, multilayered pathway structures between tothe create inner a water conducting-filled pathwaycavity and between the external the inner solution. water-filled We precipi cavitytated and 3+ thethe externalAl3+ cations solution. with We phosphate precipitated [39] the(10 Al mMcations final concentration) with phosphate added [39] (10 directly mM final to concentration) the liposome solutionadded directly to release to the liposome the incorporated solution to release calcein the. The incorporated fluorescence calcein. image The fluorescence taken 20 imagemin. takenafter phosphate20 min. after-induced phosphate-induced precipitation precipitation of the trivalent of the cations trivalent showed cations no showed fluorescent no fluorescent liposomes liposomes (Figure 1(Figure1B). However, 11B). However, transmitted transmitted-light-light imaging imaging showed showed intact intact liposomes liposomes,, like like those those in in Figure Figure 10A10A,, confirmingconfirming dye dye leakage leakage through through the the lysenin lysenin channels channels that that reopened reopened after after phosphate phosphate-removal-removal of of the the 3+ conductanceconductance-inhibiting-inhibiting Al Al3+ cations.cations.

FigureFigure 11. 11. AlAl3+3 ions+ ions reversibly reversibly control control the the lysenin lysenin-induced-induced permeability permeability in liposome in liposome membranes. membranes. (A) Addition(A) Addition of Al of3+ Al (1003+ (100 µM µfinalM final concentration) concentration) blocked blocked the the lysenin’s lysenin’s conducting conducting pathway, pathway, trapped trapped the the calceincalcein inside, quenchedquenched thethe non-entrappednon-entrapped calcein calcein and and rendered rendered the the liposomes liposomes fluorescent fluorescent against against the thebackground. background. (B) Addition(B) Addition of 10 mM of 10 phosphate mM phosphate buffer led buffer to Al 3le+ dprecipitation, to Al3+ precipitation, channel reopening, channel reopening,and a more and homogeneous a more homogeneous distribution distribution of the dye inside of the and dye outside inside ofand liposomes. outside of Scale lipos baromes.= 10 Scaleµm. bar = 10 µm. 3. Conclusions 3. ConclusionsOur work provides evidence that lysenin channels can mediate the controlled transport of non-permeantOur work ions provides and molecules evidence in that both lysenin natural channels and artificial can membrane mediate the systems. controlled Lysenin transport channels of nonwork-permeant as controlled ions transmembrane and molecules nano-valves in both natural for both and suspended artificial and membrane attached systems. cells. This Lysenin ability channelsto create conducting work as controlled pathways transmembrane that can be further nano occluded-valves for by both bio-inert suspended cationic and polymers, attached such cells. as Thchitosan,is ability is essential to create in maintainingconducting thepathways high viability that can of cells be further undergoing occluded temporary by bio permeabilization.-inert cationic polymersLysenin is, reportedsuch as aschitosan presenting, is essential weak cation in maintaining selectivity [34 the], which high might viability potentially of cells influence undergoing the temporarytransport of permeabilization. cationic solutes. However,Lysenin is this reported was not as the presenting case for anyweak of thecation employed selectivity dyes [34] and, which labels, mightalthough potentially it might influence manifest the for transport other compounds. of cationic Geneticsolutes. engineeringHowever, this might was adjustnot the the case channel’s for any ofselectivity, the employed but such dyes future and experiments labels, although must ensure it might that themanifest regulatory for otherfeatures, compounds. which are essential Genetic engineeringfor permeability might control, adjust the are channel’s preserved. selectivity, Controlling but such the transport future experiments in and out must of liposomes ensure that might the regulatoryfurther provide features, novel which avenues are essential for the design for permeability and development control, of are liposome-based preserved. Controlling nano-carriers the transportcapable of in delivering and out theirof liposomes cargo upon might exposure further to provide physical novel and chemical avenues stimuli. for the design While such and developmentapplications mayof liposome be easily-based developed nano- forcarriersin vitro capableexperiments, of delivering careful their consideration cargo upon must exposure be taken to physicalwith regards and to chemical the potential stimuli. toxicity While elicited such applicationsby lysenin for minay vivo be easilyapplications. developed We showed for in vitro that experiments,trivalent metal careful ions and consideration cationic polymers must be block taken the with lysenin regards channels, to the while potential other naturaltoxicity compoundselicited by lyseninmay prevent for in lyseninvivo application binding tos. We membranes showed that and /trivalentor oligomer metal formation ions and [ 51cationic]; given polymers the complexity block the of lyseninin vivo systems, channels, other while molecules other natural may interfere compounds with the may channel’s prevent transport lysenin binding properties. to membrane The limiteds and/orsize of theoligomer lysenin formation channel opening[51]; given in comparison the complexity to other of in toxins vivo (i.e., systems, SLO [ other16]) might molecules potentially may interfererestrict the with proposed the channel’s methodology. transport Nonetheless, properties. ions, The dyes,limited small size peptides,of the lysenin and even channel single opening stranded in comparisonnucleic acid molecules to other toxins diffuse (i.e., through SLO the[16] open) might lysenin potentially channel [restrict32,52], whichthe proposed is anticipated methodology to lead to. Nonetheless,numerous scientific, ions, dyes, biotechnological, small peptides and, and biomedical even single applications stranded that nucleic are based acid molecules on this controlled diffuse throughpermeabilization the open approach. lysenin channel [32,52], which is anticipated to lead to numerous scientific,

Toxins 2020, 12, 343 13 of 17

4. Materials and Methods

4.1. Bilayer Lipid Membrane Experiments Planar bilayer lipid membranes were formed from asolectin (Aso, Sigma–Aldrich, St. Louis, MO, USA), cholesterol (Chol, Sigma–Aldrich), and sphingomyelin (SM, Avanti Polar Lipids, Alabaster, AL, USA) dissolved in n-decane (Fisher Scientific, Hampton, NH, USA) at a weight ratio 10:5:4 with a final concentration of 10 mg/mL Aso in the solvent. The membranes were produced in a custom-made chamber while using the painting method [37,38] and the two reservoirs filled with either working electrolyte (135 mM KCl, 20 mM HEPES, pH 7.2) or other solutions as described in the main text. A 0.1 mg/mL stock solution of lysenin (Sigma–Aldrich) was obtained by solubilizing the lyophilized monomer in working electrolyte, which was further diluted as needed for the experiments. The bilayer membrane formation and its integrity were assessed by capacitance and conductance measurements that were aided by two Ag/AgCl electrodes embedded in the two reservoirs and wired to an Axopatch 200B amplifier (Molecular Devices, San Jose, CA, USA) that fed a Digidata 1440A digitizer for signal visualization and recording with the pClamp 10 software (Molecular Devices). The ionic currents were monitored and recorded at 80 mV bias potential (to avoid the voltage-induced gating that − manifests at positive voltages [37,38]), a sampling rate of one sample/s, a 1 kHz hardware filter, and a 0.1 kHz software filter. The solutions in the chamber were stirred at room temperature with a low noise magnetic stirrer (SPIN-2 bilayer stirplate, Warner Instruments, Hamden, CT, USA). After the bilayer membrane was formed and stabilized, lysenin channel insertion was initiated by addition of lysenin into the ground reservoir under continuous stirring. In most cases, a lysenin concentration of 1 pM sufficed for achieving a reasonable ionic current (in the nA range). The concentration of the lysenin in the reservoir was increased up to 100 pM by successive additions when needed. Once a stable population of inserted channels was achieved, the buffer was exchanged to remove the free lysenin and changes in ionic currents induced by addition of culture media or dyes were recorded. AF546-Phal, EtBr-HD, PI, calcein-AM, and calcein were purchased from ThermoFisher Scientific (Waltham, MA, USA). Medium molecular weight chitosan (200,000 average MW, Fisher Scientific, Pittsburgh, PA, USA) was prepared as a stock solution of 1% (w/v) in 0.1 M acetic acid (Sigma–Aldrich) and further diluted with deionized (DI) water. All the other common chemicals were purchased from various distributors and prepared in accordance with the manufacturer’s recommendations.

4.2. Cell Preparation and Analyses

4.2.1. Jurkat Cells Permeabilization and Viability Measurements

Jurkat cells (#TIB-152, ATCC, Manassas, VA, USA) were grown at 37 ◦C and 5% CO2 and passaged before reaching a density of ~106 cells/mL in RPMI 1640 medium (Sigma–Aldrich) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals, Minneapolis, MN, USA), 1% penicillin/streptomycin (PS), 1.5 g/L sodium bicarbonate, 4.5 g/L D-(+) glucose, 10 mM HEPES, 1 mM sodium pyruvate, and 2 mM l-glutamine. The cells were counted on the day of experimentation with a bright-line cytometer (Hausser Scientific, Horsham, PA, USA), pelleted by centrifugation, and washed twice with 1 mL of HBSS (Sigma–Aldrich, without added calcium chloride, magnesium sulfate, sodium bicarbonate, or phenol red). The cells were resuspended in HBSS containing PI (1.5 µM final concentration), from which we placed 100 µL in wells (four wells/experimental sample) of tissue culture treated plates (Fisher Scientific). The fluorescence spectroscopy assessment was performed on Jurkat cells that were only exposed to PI (control), Jurkat cells exposed to PI and lysenin (10 nM final concentration), and Jurkat cells exposed to PI and lysenin, but with the channels being blocked by the addition of chitosan (25 nM final concentration in the samples) 20 min. after fluorescence recording started. This amount of lysenin was much greater than what we used for the planar lipid membrane experiments, and it was required to achieve a significant permeabilization of a very high number of cell membranes within a short time Toxins 2020, 12, 343 14 of 17 frame (i.e., ~20 min.). The total surface area of the cells that underwent permeabilization was much larger than the surface area of the bilayer membrane and, consequently, the lysenin’s concentration was greater. For the permeabilization experiments, the time evolution of the PI’s fluorescence was monitored every five minutes at room temperature with a Biotek Synergy MX microplate reader (Biotek, Winooski, VT, USA) that was set for 535 nm excitation, 617 nm emission, and a sensitivity of 65 units. The fluorescence of all samples was measured for a total duration of 50 min. The viability of Jurkat cells was assessed by employing the Alamar Blue (resazurin) method [42]. For this test, we seeded 0.2 mL of ~5 105 cells/mL into flat-bottom 96-well plates in HBSS, rested the × samples for 30 min. in an incubator (37 ◦C, 5% CO2), treated them with chitosan (up to 125 nM final concentration), and continued the incubation for 20 h. After the addition of 10% v/v resazurin (Sigma–Aldrich), the samples were incubated for an additional four hours. The reduction of blue, non-fluorescent resazurin to fluorescent resorufin in viable cells was measured with the microplate reader (530 nm excitation, and 590 nm emission wavelengths) and the viability reported as percent relative to control samples not treated with chitosan. The viability test was similarly performed on Jurkat cells that were exposed to 10 nM lysenin, with and without the addition of chitosan (25 nM final concentration).

4.2.2. ATDC5 Cells Preparation and Experiments ATDC5 cells (#99072806, Sigma–Aldrich, kindly supplied by the Biomolecular Research Center at Boise State University) were grown in 75 cm2 flasks (VWR International, Radnor, PA, USA) containing 15 mL of DMEM/F12 (Gibco Dulbecco’s modified medium—nutrient mixture F12, supplemented with 5% FBS and 1% PS, ThermoFisher Scientific). After achieving 70–80% confluency, the cells were trypsinized for 7 min. with 0.25% trypsin-EDTA (Sigma–Aldrich), followed by enzyme deactivation by the addition of 7 mL medium. The cells were counted with a bench hemocytometer, centrifuged, washed, and resuspended in fresh medium before re-seeding into 75 cm2 tissue-culture treated flasks. The cells were grown and maintained in an incubator at 37 ◦C and 5% CO2. We monitored and characterized the permeabilization of cells with confocal microscopy. Glass-bottom (35 mm) culture dishes (MatTek, Ashland, MA, USA) were seeded with 2 mL of 5 cell solution (~10 cells/mL) and then incubated at 37 ◦C and 5% CO2 for 24 h. An AF546-Phal stock solution was prepared by mixing 300 units with 1.5 mL methanol, and a working solution was prepared by diluting 50 µL stock solution in 1 mL HBSS. The cells that were grown in glass-bottom dishes for 24 h were washed twice with 1mL of HBSS, followed by the addition of 100 µL of the same buffer. Each well was incubated with lysenin (10 nM final concentration) for ten minutes to induce permeabilization, washed twice with HBSS to remove the free lysenin, incubated with 100 µL of AF546-Phal for ten min., and then rinsed before imaging to reduce background signal. The control samples were prepared similarly, but without lysenin addition. Images were acquired with a Zeiss LSM 510 Meta confocal system paired with the Zeiss Axiovert Observer Z1 inverted microscope (Carl Zeiss, Inc., Thornwood, NY, USA), equipped with Ar 488 and HeNe 543 lasers, a band-pass emission filter (500–550 nm, green channel), and a long-pass emission filter (red channel). The collected images were processed with the ZEN 2009 imaging software (Carl Zeiss, Inc., Thornwood, NY, USA). The experiments aimed at estimating ATDC5 cells viability were conducted similarly, but the cells were incubated with 2 mL DMEM media (to block the lysenin channels) for 5 h at 37 ◦C and 5% CO2, washed, exposed to 100 µL of 2 µM calcein-AM, and then imaged by confocal microscopy for combined permeabilization/viability assessments.

4.3. Liposome Preparation and Analysis Liposomes were prepared by the extrusion method [53]. Chloroform solutions of 8 mg Aso, 2.5 mg SM, and 2.5 mg Chol were vacuum-dried overnight in a glass vial and the resulting lipid cake was slowly hydrated at 65 ◦C for five hours in 1 mL solution containing 135 mM KCl and 20 mM HEPES (pH 7.2). After each hour, the mixture was subjected to a freeze-thaw cycle by placing it Toxins 2020, 12, 343 15 of 17 in the freezer for twenty minutes, immediately followed by the immersion of the vial in hot water. Large aggregates that were visually observed in the solution after hydration were fragmented by short sonication (< 10 s) in a benchtop sonicator. After the completion of hydration, the lipid solution containing multilamellar liposomes was extruded 40 times through stacked polycarbonate filters (1 µm pore diameter, Avanti Polar Lipids) that were mounted in a Mini-Extruder (Avanti Polar Lipids) placed on a hotplate and kept at constant temperature (72 ◦C). After extrusion, the liposomes were cooled at room temperature and stored in a refrigerator for further experiments. A stock solution of calcein (50 µM) was prepared by dissolving it in 135 mM KCl, 20 mM HEPES, pH 7.2. Calcein fluorescence quenching by multivalent metal cations (Al3+, prepared as 10 mM solution in water from a stock AlCl3 solution—Fisher Scientific) was analyzed using a Fluoromax 4P fluorometer (Horiba Scientific, Piscataway, NJ, USA) set in emission mode (λex = 485 nm, λem = 490 nm–550 nm). The fluorescence spectrum of 50 µM calcein solution (prepared in the KCl buffer) was recorded before and after successive addition of AlCl3 solution to the sample in the cuvette under continuous stirring. The quenching curve was plotted as relative fluorescence intensity at 520 nm versus [Al3+]. Image recording and analysis was performed with a Nikon TS100 microscope (Nikon Instruments Inc., Melville, NY, USA) in transmission/epifluorescence mode, which was equipped with an Infinity camera (Lumenera, Ottawa, Canada) and appropriate filters. After lysenin addition, trivalent metal ion removal was performed by precipitation upon the addition of a sodium phosphate solution [39] that was obtained by mixing the monobasic dihydrogen phosphate and dibasic monohydrogen phosphate forms.

Author Contributions: Conceptualization, N.S. and D.F.; Formal analysis, D.F.; Investigation, N.S., C.A.T., D.R., A.B., R.H., M.W., G.A., R.J.B., and D.F.; Methodology, N.S., C.A.T., D.R., A.B., R.H., G.A., and D.F.; Project administration, D.F.; Supervision, D.F.; All authors have contributed to data analysis and interpretation, manuscript writing. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Science Foundation, grant number 1554166, and National Institute of Health (grant numbers P20GM103408 and P20GM109095). Acknowledgments: The ATDC5 cells (Sigma Aldrich #99072806) were kindly provided by the Biomolecular Research Center at Boise State University. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

1. Dhakal, K.; Black, B.; Mohanty, S. Introduction of impermeable actin-staining molecules to mammalian cells by optoporation. Sci. Rep. 2014, 4, 6553. [CrossRef][PubMed] 2. Doerner, J.F.; Febvay, S.; Clapham, D.E. Controlled delivery of bioactive molecules into live cells using the bacterial mechanosensitive channel MscL. Nat. Commun. 2012, 3, 990. [CrossRef][PubMed] 3. Escoﬀre, J.M.; Bouakaz, A. Minireview: Biophysical Mechanisms of Cell Membrane Sonopermeabilization. Knowns and Unknowns. Langmuir 2019, 35, 10151–10165. [CrossRef][PubMed] 4. Hapala, I. Breaking the Barrier: Methods for Reversible Permeabilization of Cellular Membranes. Crit. Rev. Biotechnol. 1997, 17, 105–122. [CrossRef][PubMed] 5. Murakami, M.; Kano, F.; Murata, M. LLO-mediated Cell Resealing System for Analyzing Intracellular Activity of Membrane-impermeable Biopharmaceuticals of Mid-sized Molecular Weight. Sci. Rep. 2018, 8, 1946. [CrossRef] 6. Neumann, E.; Schaefer-Ridder, M.; Wang, Y.; Hofschneider, P.H. Gene transfer into mouse lyoma cells by electroporation in high electric ﬁelds. The EMBO J. 1982, 1, 841–845. [CrossRef] 7. Russo, M.J.; Bayley, H.; Toner, M. Reversible permeabilization of plasma membranes with an engineered switchable pore. Nat. Biotechnol. 1997, 15, 278–282. [CrossRef] 8. Stewart, M.P.; Langer, R.; Jensen, K.F. Intracellular Delivery by Membrane Disruption: Mechanisms, Strategies, and Concepts. Chem. Rev. 2018, 118, 7409–7531. [CrossRef] Toxins 2020, 12, 343 16 of 17

9. Teng, K.W.; Ren, P.; Selvin, P.R. Delivery of Fluorescent Probes Using Streptolysin O for Fluorescence Microscopy of Living Cells. Curr. Protoc. Protein Sci. 2018, 93, e60. [CrossRef] 10. Walev, I.; Bhakdi, S.C.; Hofmann, F.; Djonder, N.; Valeva, A.; Aktories, K.; Bhakdi, S. Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O. Proc. Natl. Acad. Sci. USA 2001, 98, 3185–3190. [CrossRef] 11. Wong, F.K.; Haﬀner, C.; Huttner, W.B.; Taverna, E. Microinjection of membrane-impermeable molecules into single neural stem cells in brain tissue. Nat. Protoc. 2014, 9, 1170–1182. [CrossRef][PubMed] 12. Hennig, S.; van de Linde, S.; Lummer, M.; Simonis, M.; Huser, T.; Sauer, M. Instant Live-Cell Super-Resolution Imaging of Cellular Structures by Nanoinjection of Fluorescent Probes. Nano Lett. 2014, 15, 1374–1381. [CrossRef] 13. Leduc, M.; Guay, D.; Leask, R.L.; Coulombe, S. Cell permeabilization using a non-thermal plasma. New J. Phys. 2009, 11, 115021. [CrossRef] 14. Provoda, C.J.; Lee, K.-D. Bacterial pore-forming hemolysins and their use in the cytosolic delivery of macromolecules. Adv. Drug Deliv. Rev. 2000, 41, 209–221. [CrossRef] 15. Saito, G.; Amidon, G.L.; Lee, K.-D. Enhanced cytosolic delivery of plasmid DNA by a sulfhydryl-activatable listeriolysin O/protamine conjugate utilizing cellular reducing potential. Gene Ther. 2003, 10, 72–83. [CrossRef] [PubMed] 16. Teng, K.W.; Ishitsuka, Y.; Ren, P.; Youn, Y.; Deng, X.; Ge, P.; Lee, S.H.; Belmont, A.S.; Selvin, P.R. Labeling proteins inside living cells using external ﬂuorophores for microscopy. eLife 2016, 5, e20378. [CrossRef] 17. Hille, B. Ion Channels of Excitable Membranes, 3rd ed.; Sinauer Associates, Inc.: Sunderland, MA, USA, 2001; pp. 2–22. 18. Andersen, O.S.; Ingolfsson, H.I.; Lundbaek, J.A. Ion Channels. Wiley Encycl. Chem. Biol. 2008, 1–14. [CrossRef] 19. Bezanilla, F. Ion channels: From conductance to structure. Neuron 2008, 60, 456–468. [CrossRef] 20. Cabiaux, V. pH-sensitive toxins: Interactions with membrane bilayers and application to drug delivery. Adv. Drug Deliv. Rev. 2004, 56, 987–997. [CrossRef] 21. Gonzalez, M.R.; Bischofberger, M.; Pernot, L.; van der Goot, F.G.; Freche, B. Bacterial pore-forming toxins: The (w)hole story? Cell. Mol. Life Sci. 2008, 65, 493–507. [CrossRef] 22. Aroian, R.; van der Goot, F.G. Pore forming toxins and cellular non-immune defenses (CNIDs). Curr. Opin. Microbiol. 2007, 10, 57–61. [CrossRef][PubMed] 23. Bashford, C.L. Pore-Forming Toxins: Attack and Defence at the Cell Surface. Cell. Mol. Biol. Lett. 2001, 6, 328–333. 24. Gilbert, R.J.C. Pore-forming toxins. Cell. Mol. Life Sci. 2002, 59, 832–844. [CrossRef] 25. Parker, M.W.; Feil, S.C. Pore-forming protein toxins: From structure to function. Prog. Biophys. Mol. Biol. 2005, 88, 91–142. [CrossRef] 26. Mondal, A.K.; Sreekumar, A.; Kundu, N.; Kathuria, R.; Verma, P.; Gandhi, S.; Chattopadhyay, K. Structural Basis and Functional Implications of the Membrane Pore-Formation Mechanisms of Bacterial Pore-Forming Toxins. Adv. Exp. Med. Biol. 2018, 1112, 281–291. [CrossRef] 27. Mondal, A.K.; Chattopadhyay, K. Taking Toll on Membranes: Curious Cases of Bacterial β-Barrel Pore-Forming Toxins. Biochemistry 2020, 59, 163–170. [CrossRef] 28. Barry, E.L.; Gesek, F.A.; Friedman, P.A. Introduction of antisense oligonucleotides into cells by permeabilization with streptolysin O. Biotechniques 1993, 15, 1016–1020. 29. Spiller, D.G.; Tidd, D.M. Nuclear delivery of antisense oligodeoxynucleotides through reversible permeabilization of human leukemia cells with streptolysin O. Antisense Res. Dev. 1995, 5, 13–21. [CrossRef] 30. Keyel, P.A.; Loultcheva, L.; Roth, R.; Salter, R.D.; Watkins, S.C.; Yokoyama, W.M.; Heuser, J.E. Streptolysin O clearance through sequestration into blebs that bud passively from the plasma membrane. J. Cell Sci. 2011, 124, 2414–2423. [CrossRef][PubMed] 31. Acker, J.P.; Lu, X.M.; Young, V.; Cheley, S.; Bayley, H.; Fowler, A.; Toner, M. Measurement of trehalose loading of mammalian cells porated with a metal-actuated switchable pore. Biotechnol. Bioeng. 2003, 82, 525–532. [CrossRef][PubMed] 32. Bokori-Brown, M.; Martin, T.G.; Naylor, C.E.; Basak, A.K.; Titball, R.W.; Savva, C.G. Cryo-EM structure of lysenin pore elucidates membrane insertion by an aerolysin family protein. Nat. Commun. 2016, 7, 11293. [CrossRef][PubMed] Toxins 2020, 12, 343 17 of 17

33. Ide, T.; Aoki, T.; Takeuchi, Y.; Yanagida, T. Lysenin forms a voltage-dependent channel in artificial lipid bilayer membranes. Biochem. Biophys. Res. Commun. 2006, 346, 288–292. [CrossRef][PubMed] 34. Kwiatkowska, K.; Hordejuk, R.; Szymczyk, P.; Kulma, M.; Abdel-Shakor, A.B.; Płucienniczak, A.; Dołowy, K.; Szewczyk, A.; Sobota, A. Lysenin-His, a sphingomyelin-recognizing toxin, requires tryptophan 20 for cation-selective channel assembly but not for membrane binding. Mol. Membr. Biol. 2007, 24, 121–134. [CrossRef] 35. Podobnik, M.; Savory, P.; Rojko, N.; Kisovec, M.; Wood, N.; Hambley, R.; Pugh, J.; Wallace, E.J.; McNeill, L.; Bruce, M.; et al. Crystal structure of an invertebrate cytolysin pore reveals unique properties and mechanism of assembly. Nat. Commun. 2016, 7, 11598. [CrossRef] 36. Yilmaz, N.; Kobayashi, T. Visualization of Lipid Membrane Reorganization Induced by a Pore-Forming Toxin Using High-Speed Atomic Force Microscopy. ACS Nano 2015, 9, 7960–7967. [CrossRef][PubMed] 37. Fologea, D.; Krueger, E.; Lee, R.; Naglak, M.; Mazur, Y.; Henry, R.; Salamo, G. Controlled gating of lysenin pores. Biophys. Chem. 2010, 146, 25–29. [CrossRef] 38. Fologea, D.; Krueger, E.; Mazur, Y.I.; Stith, C.; Okuyama, Y.; Henry, R.; Salamo, G.J. Bi-stability, hysteresis, and memory of voltage-gated lysenin channels. Biochim. et Biophys. Acta. 2011, 1808, 2933–2939. [CrossRef] 39. Fologea, D.; Al Faori, R.; Krueger, E.; Mazur, Y.I.; Kern, M.; Williams, M.; Mortazavi, A.; Henry, R.; Salamo, G.J. Potential analytical applications of lysenin channels for detection of multivalent ions. Anal. Bioanal. Chem. 2011, 401, 1871–1879. [CrossRef] 40. Fologea, D.; Krueger, E.; Al Faori, R.; Lee, R.; Mazur, Y.I.; Henry, R.; Arnold, M.; Salamo, G.J. Multivalent ions control the transport through lysenin channels. Biophys. Chem. 2010, 152, 40–45. [CrossRef] 41. Fologea, D.; Krueger, E.; Rossland, S.; Bryant, S.; Foss, W.; Clark, T. Cationic polymers inhibit the conductance of lysenin channels. Sci. World J. 2013, 2013, 316758. [CrossRef] 42. Larson, E.M.; Doughman, D.J.; Gregerson, D.S.; Obritsch, W.F. A new, simple, nonradioactive, nontoxic in vitro assay to monitor corneal endothelial cell viability. Invest. Ophthalmol. Vis. Sci. 1997, 38, 1929–1933. [PubMed] 43. Vetter, J. Toxins of Amanita phalloides. Toxicon 1998, 36, 13–24. [CrossRef] 44. Lengsfeld, A.M.; L#xF6;w, I.; Wieland, T.; Dancker, P.; Hasselbach, W. Interaction of Phalloidin with Actin. Proc. Natl. Acad. Sci. USA 1974, 71, 2803–2807. [CrossRef][PubMed] 45. Le Bihan, T.; Gicquaud, C. Stabilization of actin by phalloidin: A differential scanning calorimetric study. Biochem. Biophys. Res. Commun. 1991, 181, 542–547. [CrossRef] 46. Dancker, P.; Löw, I.; Hasselbach, W.; Wieland, T. Interaction of actin with phalloidin: Polymerization and stabilization of F-actin. Biochim. et Biophys. Acta Protein Struct. 1975, 400, 407–414. [CrossRef] 47. Harkin, D.G.; Hay, E.D. Effects of electroporation on the tubulin cytoskeleton and directed migration of corneal fibroblasts cultured within collagen matrices. Cell Motil. Cytoskeleton 1996, 35, 345–357. [CrossRef] 48. Schmit, A.C.; Lambert, A.M. Microinjected fluorescent phalloidin in vivo reveals the F-actin dynamics and assembly in higher plant mitotic cells. Plant Cell 1990, 2, 129–138. [CrossRef] 49. Tawakoli, P.N.; Al-Ahmad, A.; Hoth-Hannig, W.; Hannig, M.; Hannig, C. Comparison of different live/dead stainings for detection and quantification of adherent microorganisms in the initial oral biofilm. Clin. Oral Investig. 2013, 17, 841–850. [CrossRef] 50. Brittain, H.G. Submicrogram determination of lanthanides through quenching of calcein blue fluorescence. Anal. Chem. 1987, 59, 1122–1125. [CrossRef] 51. Sukumwang, N.; Umawaza, K. Earthworm-Derived Pore-Forming Toxin Lysenin and Screening of Its Inhibitors. Toxins 2013, 5, 1392–1402. [CrossRef] 52. Shrestha, N.; Bryant, S.L.; Thomas, C.; Richtsmeier, D.; Pu, X.; Tinker, J.; Fologea, D. Stochastic sensing of Angiotensin II with lysenin channels. Scient. Rep. 2017, 7, 2448. [CrossRef][PubMed] 53. Pedersen, P.J.; Adolph, S.K.; Subramanian, A.K.; Arouri, A.; Andresen, T.L.; Mouritsen, O.G.; Madsen, R.; Madsen, M.W.; Peters, G.N.H.; Clausen, M.H. Liposomal Formulation of Retinoids Designed for Enzyme Triggered Release. J. Med. Chem. 2010, 53, 3782–3792. [CrossRef][PubMed]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).