18 Clusters in Biomimetic Membranes: Role of Size, Charge and

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Au (SCH ) Clusters in Biomimetic Membranes: Role of Size, Charge and Transmembrane Potential in Direct Membrane Permeation Lucia Becucci, Tiziano Dainese, and Rolando Guidelli ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.9b00306 • Publication Date (Web): 29 Mar 2019 Downloaded from http://pubs.acs.org on March 29, 2019

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ACS Applied Nano Materials

Au25(SCnH2n+1)18 Clusters in Biomimetic Membranes: Role of Size, Charge and Transmembrane Potential in Direct Membrane Permeation Lucia Becucci,†Tiziano Dainese,§ and Rolando Guidelli†,* † Department

of Chemistry, Florence University, Via della Lastruccia 3, 50019 Sesto Fiorentino (Firenze), Italy § Department of Chemistry, University of Padova, via Marzolo 1, 35131 Padova, Italy Keywords: gold nanoclusters, Au25, tethered bilayer lipid membranes, lipid self-assembled monolayers, transmembrane potential, mercury electrode ABSTRACT Gold nanoclusters and nanoparticles are promising materials for applications in nanomedicine and, therefore, understanding their interaction with cell membranes is of particular importance. A series of neutral and anionic Au25(SCnH2n+1)18 monolayer protected clusters (MPCs) (briefly, Cn0 and Cn– clusters), was embedded into two types of biomimetic membranes supported by mercury electrodes. The first was a dioleoylphosphatidylcholine (DOPC) self-assembled monolayer (SAM), whereas the second was a tethered bilayer lipid membrane (tBLM) obtained by first anchoring a thiolipid monolayer to the mercury surface and then self-assembling a DOPC monolayer on top of it. The diameter of these clusters, from 1.7 to 2.7 nm depending on the thiolate ligand, is smaller than the thickness of biomembranes and biomimetic membranes. Both neutral and anionic Au25(SCnH2n+1)18 MPCs can penetrate the lipid bilayer moiety of the tBLM, without disrupting it; in particular, anionic Au25 clusters require positive transmembrane potentials to do so. Neutral Au25 clusters exchange one electron with mercury in a DOPC SAM, where they can come in contact with the mercury surface, whereas they are prevented from doing so at the tBLM because of their inability to cross the hydrophilic chain separating the lipid bilayer moiety from the mercury surface. The potential of these Au25 clusters to penetrate directly the plasma membrane is particularly convenient for targeted drug delivery. They are highly stable, biocompatible and catalytic, and their uniform size is of importance in nanomedicine. Moreover, they may induce an efficient energy transfer to 3O2, allowing applications in radiotherapy and antimicrobial activity.

*Corresponding

author; retired professor

Telephone: (+39) 055-457-3105; E-mail: [email protected]

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INTRODUCTION Gold nanoparticles (AuNPs) display unique size-dependent optical, magnetic, electrochemical, and electronic properties that make them very interesting materials for both fundamental and applied purposes.1-4 In most biomedicine applications,3,4 AuNPs are required to cross the membrane by endocytosis or direct penetration.5-7 The mechanisms of interaction of AuNPs with cell membranes, however, is affected by several parameters, such as the NP size and shape, and the functional groups decorating the capping monolayer.5,8,9 Moreover, the presence of a surface charge and its sign can dramatically influence the cellular uptake of AuNPs.10,11 The interaction between NPs and cells may also be influenced by hardly controllable endogenous factors. In this regard, the use of biomimetic membranes12,13 has proved to be expedient to remove or minimize them. This is the case of vesicles, which have been extensively employed to investigate AuNP internalization.1416 It is worth noticing, however, that as a rule the transmembrane potential in vesicles is not controlled, although this factor plays an important role in the direct incorporation of AuNPs, particularly those carrying charges. Despite numerous important observations, the mechanisms of AuNP interactions with cellular and biomimetic membranes are still poorly understood and sometimes controversial. Gold nanoclusters are a special type of ultra-small AuNPs with a core diameter of less than 2 nm, coated with an organic monolayer. The ultra-small size of these nanoclusters induces distinctive quantum conﬁnement effects, which result in discrete electronic structure and molecular-like properties, such as HOMO–LUMO electronic transition. These ultra-small AuNPs, differently from their larger counterparts, can be synthesized with atomic precision; their stoichiometry and structure can be assessed by means of mass spectroscopy and single crystal X-ray crystallography.17,18 The characteristics of gold-nanoclusters are fundamentally different from those of larger AuNPs (also called nanocrystals), in which the optical properties are dominated by plasmon excitation, as opposed to the single-electron transition in gold nanoclusters. Information concerning the internalization of small gold nanoclusters stabilized by a protecting layer of surfactants, usually thiolates (monolayer protected clusters, MPCs), is especially limited for metal cores of diameter of less than 2 nm. We recently described the interaction of Au144 protected by 60 thiolated ligands (SR) with two types of biomimetic membranes assembled on mercury electrodes, which allowed full control of the transmembrane potential;19 with a core diameter of only 1.6 nm, this was the smallest cluster ever used for these studies. The first membrane consisted of a self-assembled monolayer (SAM) of dioleoylphosphatidylcholine (DOPC) on a hanging mercury-drop electrode (HMDE). This is an approach devised by Nelson20 and extensively employed by his group21 and by us.22-26 We focused on the hydrophobic Au144(SC2H4Ph)60 cluster and two clusters obtained by exchanging a part of the ligands with either 8-mercaptooctanoic acid or a thiolated channel-forming peptide (trichogin GA IV). The exchanged MPCs were found to interact significantly with the lipid monolayer.19 The second biomimetic membrane employed was obtained by first tethering a thiolipid monolayer to the surface of the HMDE;19 this thiolipid, called DPTL, consists of a tetraethyleneoxy hydrophilic chain terminated at one end with a lipoic acid residue for anchoring to the mercury surface and covalently linked at the other end to two phytanyl chains mimicking the hydrocarbon tails of a phospholipid.27 Selfassembling a DOPC monolayer on top of the DPTL SAM gives rise to a lipid bilayer interposed between the aqueous solution and the hydrophilic chain region, which acts as an ionic reservoir between the actual electrode surface and the bilayer moiety. This DPTL/DOPC tethered bilayer lipid membrane (tBLM) has been extensively used by us to investigate the functional activity of channel-forming peptides and small proteins.23-26,28 Gordillo et al. used the mercury-supported DOPC SAM to study the behavior of larger AuNPs (diameter of 2-10 nm) capped by 12 ACS Paragon Plus Environment

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mercaptoundecane-11-tetra(ethylene glycol).29 Vakurov et al. showed that the activity of TiO2 nanoparticles toward this biomimetic membrane is related to their charge-holding capacitance and particle size,30 and that close packing of SiO2 nanoparticles is manifested as a complete suppression of the capacitance current peaks in rapid cyclic voltammograms.31 It is noteworthy that the MPCs so far employed had a diameter generally larger than the thickness of membranes. An even smaller cluster is Au25(SR)18, which has a core diameter of only 1 nm and an X-ray crystal structure shown in Scheme S1. Its small number of Au-atoms makes it display molecular properties,32,33 which are nicely highlighted by its charge-dependent UV-vis absorption and NMR spectra.34,35 Distinct molecular features are especially evident in its electrochemical behavior,2 which is characterized by voltammetric peaks corresponding to the stepwise charging of the gold core. For Au25(SR)18 in organic solvents, the 0/–1 and +1/0 redox couples display reversible voltammetric behavior, whereas more highly charged states have limited lifetimes.35,36 As to the properties of the capping monolayer in Au25(SCnH2n+1)180 clusters in low-polarity solvents, we found that whereas for n < 12 the alkyl chains form a quite fluid monolayer of folded ligands, longer chains self-organize into bundles.37 Very few reports describe biological properties or activity of molecular clusters. Suitably functionalized Au25 clusters were found to penetrate efficiently the membrane of tumor cells.38 Gluthathione-capped Au25 was found to preferentially accumulate in tumor cells, with potential use in radiotherapy.39 Data suggest photodynamic activity of Au25 toward cancer cells.40 Clusters of similar size display antimicrobial activity, whereas slightly larger clusters show no activity.41 Small variations in size may indeed significantly affect the biointeractions of ultrasmall AuNPs. For example, whereas in biological media gluthathione-protected 2-3 nm AuNPs aggregate, those smaller than 2 nm (presumably Au144) are very stable.42 Importantly, whereas clusters with a number of Au atoms from 25 to  200 are readily eliminated through the kidneys, renal clearance efficiency drops rapidly for either larger or smaller clusters.43 In another very recent report, the role of the molecular size on antitumor effects is demonstrated with atomically precise subnanometer Au clusters.44 Overall, these very interesting examples point to the importance of understanding the interaction of molecular MPCs with membranes, with a special attention to Au25. Here, we report the first study of the interaction of a large series of hydrophobic Au25(SCnH2n+1)18 clusters with biomimetic membranes organized on mercury electrodes. To avoid secondary effects related to the aforementioned interligand interactions,37 we focused on using those Au25 clusters known to have a fluid structure of the capping monolayer. In addition to the ligand length, we varied the charge state by using both neutral and anionic Au25(SCnH2n+1)18 MPCs. The former had a number of carbon atoms n = 3, 8, and 10, and the latter n = 3, 5, 6, 8; henceforth, they will be denoted simply as Cn0 and Cn–. In organic solvents, these clusters have hydrodynamic diameters ranging from 1.7 to 2.7 nm,45 which are smaller than the thickness of the hydrophobic region of a membrane bilayer, and are comparable with that of a DOPC monolayer, generally considered to be around 2.3 nm.46 The particularly small size of the selected systems was also expected to minimize possible deformations of the bilayer. The membrane-Au25 interaction was interrogated using the two aforementioned mercury-supported biomimetic membranes and electrochemical methods sensitive to changes in the properties of the membranes and/or charge-transfer phenomena. The outcome of the experiments at DOPC SAMs and DPTL/DOPC tBLMs and their analyses and simulations indicate that both neutral and anionic Au25(SCnH2n+1)18 MPCs can penetrate lipid bilayers; in particular, anionic Au25 clusters require positive transmembrane potentials to do so. Neutral Au25 clusters exchange one electron with mercury in a DOPC SAM, where they can come in contact with the mercury surface, whereas they are prevented from doing so at a DPTL/DOPC tBLM because of their inability to cross the tetraethyleneoxy hydrophilic chain separating the lipid bilayer moiety of the tBLM from the mercury surface. These and further results provide 3 ACS Paragon Plus Environment


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quantitative information on the effect of charge state and ligand length on the properties of Au25 clusters smaller than the membrane thickness. The crucial importance of the transmembrane potential is clearly pointed out. The potential of these Au25 clusters to penetrate directly the plasma membrane is of particular importance for targeted drug delivery. Au25 clusters are highly stable and do not tend to aggregate, favoring their use in nanomedicine.42 They are highly biocompatible and catalytic,41 and their large optical gap is expected to induce an efficient energy transfer to 3O240 for applications in radiotherapy39 and antimicrobial activity.41 RESULTS AND DISCUSSION The description of the Au25 results is organized in three parts: (i) Behavior of Au25 clusters at the DOPC SAM; (ii) Behavior of Au25 clusters at the DPTL/DOPC tBLM; (iii) Adsorption of the C5– cluster on bare mercury. Au25 Clusters at the DOPC SAM Here following, the applied potential E is referred to the Ag/AgCl/(0.1 M KCl) reference electrode. Mercury-supported lipid mono- and bilayers have fluidity and lateral mobility comparable with those of conventional bilayers interposed between two bulk aqueous phases, thanks to the liquid state of mercury. In particular, DOPC forms a well-defined and tightly packed monolayer on mercury, which excludes nonspecific ion leakage through monolayer defects over the potential range from –0.20 to –0.80 V, in the absence of interacting molecules.21,25 In fact, any hypothetical surface defect would be immediately healed, thanks to the phospholipid lateral mobility. In the absence of exogenous species, the AC voltammogram at a mercury-supported DOPC SAM is characterized by a region of low and almost constant capacitance C (ca. 1.8 F cm– 2), followed by a sharp pseudocapacitance peak at −1.02 V, and by two further peaks at ca. −1.10 and −1.35 V (dashed curve in Figure 1).47 At potentials positive of the first peak, the DOPC polar heads establish a network of dipole-dipole interactions between the trimethylammonium ion of each lipid molecule and the phosphate ion of an adjacent one, imparting to the polar heads an orientation almost parallel to the monolayer plane.22 The first pseudocapacitance peak is ascribed to a reorientation of the polar heads. As soon as the electric field forces any polar head to reorient, the reorientation is rapidly transmitted to all other polar heads, triggering a domino effect that is responsible for the sharp shape of the peak. This, in turn, allows the penetration of ions and water molecules into the lipid film, causing the capacitance at more negative potentials to attain values close to those on bare mercury. The second peak, controlled by kinetics of nucleation and growth, marks the formation of bilayer patches on the electrode surface, whereas the third peak is determined by partial desorption of the bilayer patches, with possible formation of semivesicular structures.48 Finally, the capacitance plateau at potentials negative of the third peak is considered to be characterized by a compact layer of counterions, with a compressed DOPC layer on top.48 Total DOPC desorption in 0.1 M KCl takes place when the differential capacitance of the DOPC SAM comes to coincide with that at the bare electrode, at about –1.84 V. 49 On the basis of mild extrathermodynamic assumptions capable of interpreting a number of pieces of experimental evidence, the transmembrane potential m along the flat capacitance region of the DOPC SAM, with 1.8 F cm–2 capacitance, is approximately estimated at m = E+ 190 mV in the absence of exogenous species.28,50 4 ACS Paragon Plus Environment

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Anionic Au25 Clusters. Figure 1 shows the AC voltammograms at a mercury-supported DOPC SAM in the absence (dashed gray curve) and in the presence of 6 mol% C3– (blue curve) or 3 mol% C5– (red curve). Here and in what follows, the molar concentrations of the different Au25 clusters were selected so as to produce appreciable and possibly comparable effects on the electrochemical response. In several cases, the molar concentrations adopted corresponded to saturation of the lipid film, in that their further increase did not produce a detectable increase in the response. The presence of the anionic cluster C3– or C5– in the SAM generates a small bell-shaped peak at ca. – 0.50 V, which is enlarged in the inset of Figure 1 (scale on the right).

Figure 1. Curves of the differential capacitance C at 1 Hz against the applied potential E at a mercurysupported DOPC monolayer in 0.1 M KCl in the absence (dashed gray curve) and in the presence of 6 mol% C3– (blue curve) or 3 mol% C5– (red curve). The right-hand scale refers to the enlargement of the curves at E > –0.80 V.

A bell-shaped peak with an almost identical electric potential value and full width at halfmaximum is also exhibited by the other anionic clusters investigated, C6– and C8–(Figure S1 for C8–). It is also worth mentioning that a small hump at ca. –0.50V is also shown by a plot of the inphase component (Y’) of the admittance against the applied potential, as recorded by electrochemical impedance spectroscopy (EIS) at regular intervals of the bias potential between – 0.30 and –0.80 V upon superimposing a 10 mV peak-to-peak AC voltage (Figure S2). Figure 1 shows that the presence of C3– causes a small positive potential shift and depresses both the first and second pseudocapacitance peaks. These effects become more evident as the ligand length increases. This behavior is ascribed to the intercalation of the Cn– clusters between the lipid molecules hindering their sharp cooperative reorientation. The third peak is slightly shifted to more negative potentials by the anionic MPCs. The charge transients recorded by the chronocoulometric technique at a pure DOPC SAM, by stepping the potential from –0.35 V to progressively more negative final values (Ef) over the flat capacitance region, attain well-defined charge values (Q0) within a few milliseconds and then level off (Figure S3). The plot of Q0 against Ef is linear and exhibits a slope that yields a differential capacitance of ca. 1.8 F cm–2, in agreement with AC voltammetry. Addition of Cn– clusters has a negligible effect on the Q0 values; it merely induces a slight nonspecific ion leakage across the SAM, which is revealed by a slightly negative slope of the charge transients, after the rapid attainment of a Q0 value almost identical with that recorded at a pure DOPC SAM (Figure S4). 5 ACS Paragon Plus Environment


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This leakage is not due to disorganization of the well-compacted DOPC SAM, but rather to the cross-section of the Au25 clusters being larger than that, ca.0.6 nm2, of the vertically oriented DOPC molecules. This creates small voids around the clusters, into which K+ ions may sneak, approaching the metal surface. The cyclic voltammogram (CV) obtained by scanning E between –0.20 and – 0.90 V is affected by the presence of a Cn– cluster only at potentials more negative than –0.50 V, where it shows a slight increase with respect to the CV in its absence (Figure S5); this increase is, again, ascribable to nonspecific leakage of K+ ions across the SAM. We also checked the possibility that the entrapped Cn– clusters could mediate electron transfer (ET) from the electrode to a freely diffusing redox probe, Eu(H2O)63+. At a bare mercury electrode in aqueous 0.1 M KCl, the electrochemical reaction of the Eu(III)/Eu(II) redox couple is detected as a quasi-reversible CV with a formal potential of –0.657 V.51 However, addition of 110–4 M Eu(III) has no detectable effect on the CV recorded at a mercury-supported DOPC SAM, no matter if in the absence or presence of Cn–. This indicates that the SAM inhibits Eu(III) electroreduction and that the Cn– clusters are incapable of mediating ET across it. The bell-shaped peak in the AC voltammogram of Figure 1 exhibited by the anionic clusters at ca. –0.50 V can be interpreted on the basis of a quasi-reversible movement of these charged molecules between an inner and an outer location within the lipid SAM, following the AC voltage signal. An analogous movement of cationic counterions is to be excluded, because they do not dispose of an equilibrium location well inside the DOPC monolayer, but only one just outside it, or, at most, in the polar head region (see, e.g.,49). The presence of two distinct locations for the Cn– clusters stems from their dual nature of hydrophobic and negatively charged particles. Under equilibrium conditions, the electrochemical potential of the MPCs is the same at the two locations:

 in  in  kT ln N in  ein   out  out  kT ln N out  eout ,

(1)

where  in , in , Nin and in denote the electrochemical potential, the standard chemical potential, the MPC concentration and the absolute electric potential at the inner location, respectively,  whereas  out , out , Nout, and out denote the corresponding quantities at the outer location; e is the proton charge and k is the Boltzmann constant. In writing Eq. 1, the activities of MPCs were replaced by the corresponding concentrations as a first approximation. For convenience, the absolute electric potential  will be referred to its value in the bulk solution, taken to be zero; by this choice, the electrostatic energy of the charged MPCs vanishes outside the SAM, whereas its absolute value increases when the MPCs get closer to the electrode surface. The electric field in the SAM is mainly focused in the almost nonpolar and structurally homogeneous hydrocarbon tail region. Hence, it is reasonable to assume that the electric potential across this region varies linearly with the distance x from the metal surface. We can then write: in = m(d–xin)/dand out = m(d–xout)/d, where d is the thickness of the hydrocarbon tail region of the SAM, xin and xout are the distances of the inner and outer locations from the electrode surface, and m is the absolute potential difference across the SAM, i.e., the ‘transmembrane potential’. Replacing in and out into Eq. 1 yields:



N out N in  exp    qm



kT  ,

(2)


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with q = –e(xout – xin)/d and  = in–out. The latter quantity accounts for the change in the purely chemical interactions established by these particles in passing from the outer to the inner location. Since the selected Cn– clusters are hydrophobic and the inner location is more deeply embedded into the lipid environment,  is negative (see Scheme 1 for the schematic picture of an anionic MPC moving from the xin to the xout location in the DOPC SAM). The probability p of the Cn– clusters occupying the inner location is given by the Nin/(Nin+Nout) ratio. From Eq. 2, it follows:





p  1 exp    qm



kT 

  1 exp q  1

m

 m,1/2



kT 



1

,

(3)

where m,1/2 = –/q; m,1/2 is the transmembrane-potential value at which p = 0.5. This equation is quite similar to the expression for the probability of an ion channel being open, often referred to as the “one-sided Boltzmann function”.52,53

Scheme 1. The DOPC SAM is depicted as a group of four DOPC molecules anchored to mercury, and the tBLM as a group of four DPTL/DOPC constructs tethered to mercury via their lipoic acid residues. In the DOPC SAM, the arrow marks the movement of an anionic Au25 cluster from the xin to the xout location following a negative-going potential scan. In the DPTL/DOPC tBLM, the arrow marks the movement of an Au25 cluster from the (hydrophilic spacer)/(lipid bilayer) boundary to the polar head region following a negative-going potential scan. Both anionic and neutral clusters are considered to move across the lipid bilayer moiety of the tBLM, but only the anionic clusters can cross the hydrophilic spacer and release one electron to the metal during a positive-going potential scan.

The bell-shaped peak, obtained by subtracting from the AC voltammogram the almost constant capacitance of ca. 1.8 F cm–2 on the two sides of this hump, can be envisaged as a measure of the probability of the Cn– clusters occupying simultaneously the inner and outer locations as a function of the transmembrane potential. This probability is given by the product of the probability p of the Cn– clusters occupying the inner location by their probability (1–p) of occupying the outer location. The probability p(1–p) attains a maximum of 0.25 for m = m,1/2, and can fit the experimental 7 ACS Paragon Plus Environment


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bell-shaped peak by normalizing its maximum to that of the latter, as shown in Figure 2. The full width at half-maximum for the probability p(1–p) decreases with increasing q. A nearly perfect fit to the experimental bell-shaped peak is achieved for q/(kT) = –e(xout–xin)/(dkT) = –0.022 mV–1, which yields (xout–xin)/d = 0.56. This thickness ratio points to a xout value of ca. d and a xin value of ca. 0.5 d.

Figure 2. Plot of the difference, C, between the differential capacitance around the bell-shaped peak in the AC voltammogram of Figure 1 in the presence of 3 mol% C5– and the capacitance contribution of 1.80 F cm-2 (red curve). The other bell-shaped peaks are probabilities, p(1–p), calculated as described in the text for different q/(kT) values, reported in mV–1 in the inset of the figure, after normalizing them to the experimental ΔC vs. E maximum.

It is indeed conceivable that, at the less negative potentials, optimization of the van der Waals interactions between capping and lipid molecules keeps the center of the Au25 core at an inner location xin, in the middle of the hydrophobic part of the lipid SAM, i.e., at about 1.15 nm. This is very reasonable, also because the Stokes diameters of the MPCs investigated are only 1.72 (C3), 2.06 (C5), 2.20 (C6), and 2.42 nm (C8).45 This conclusion indicates that, as the applied potential becomes sufficiently negative, the cluster core is electrostatically pushed toward the solution side of the SAM by about 1.2 nm. Even at xout, however, nearly one half of the Au25 capping monolayer is still embedded in the polar head region of the DOPC SAM, due to attractive hydrophobic interactions that prevent further movement of the cluster toward the solution. The maximum of the bell-shaped peak in Figure 2 lies at a potential Emax of ca. –0.51 V. This corresponds to the transmembrane potential, m,1/2 = –/q, at which the anionic MPCs are equidistributed over the two different locations. Noting that m,1/2 is approximately given by Emax+0.190 V = –0.32 V,28,50 we obtain a standard chemical potential change, , of ca. –m,1/2q = –320 mV  0.022 mV–1kT  –7kT. The fact that chronocoulometry charge transients (Figure S4) or CVs (Figure S5) are not appreciably affected by the Cn– clusters indicates that the passage of these MPCs from the center of the DOPC SAM to its outer boundary upon changing E from –0.30 V to –0.80 V, as revealed by the quasi-equilibrium AC voltammetry measurements, is either too slow to keep up with the potential scan rate or involves too small an amount of nanoclusters. The appreciable depression of the AC voltammetry pseudocapacitance peaks induced by the presence of the anionic Au25 clusters in Figure 1 lends support to the former interpretation. In fact, AC voltammetry measurements, carried out with 10 mV peak-to-peak amplitude, 1 Hz frequency and ca. 50 mV min–1 scan rate of the bias potential, involve a 10 mV excursion straddling each bias potential and require ca. 1 min to cover a 50 mV potential range; conversely, cyclic voltammetry measurements at 50 mV s–1 scan 8 ACS Paragon Plus Environment

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rate cover the same potential range in 1 s. In this respect, AC voltammetry measurements can be regarded as in quasi equilibrium with respect to cyclic voltammetry measurements. Neutral Au25Clusters. The neutral MPCs incorporated in DOPC SAMs behave quite differently from their anionic counterparts. First, they do not show the bell-shaped peak typical of Cn– clusters, even though they depress the first two pseudocapacitance peaks similarly to their anionic counterparts, including a more marked effect as the ligand length increases. Second, addition of C80 or C100 clusters to the DOPC SAM slightly decreases the differential capacitance along the intermediate portion of the flat capacitance region, as shown in Figure 3 for C100 (ca.1.5 F cm–2) in comparison with C30(ca. 1.75 F cm–2). A decrease in capacitance is a typical effect of bulky exogenous surfactants having a low polarizability, comparable with that of the lipid molecules;54 it is ascribed to an increase in the thickness of the adsorbed film. The very fact that this effect is clearly detectable points to a non-negligible amount of Cn0 clusters in the SAM.

Figure 3. Curves of the differential capacitance C at 1 Hz against the applied potential E at a mercurysupported DOPC monolayer in 0.1 M KCl in the presence of 4 mol% C30 (blue curve) and 10 mol% C100 (red curve). The right-hand scale refers to the enlargement of the curves at E > –0.80 V.

This interpretation is supported by the EIS spectrum at a DOPC SAM in the presence of C100 at –0.40 V, as displayed on a Z’ vs. –Z” plot (called M or modulus plot), where  is the angular frequency and Z’, Z” are the in-phase and quadrature components of the electrochemical impedance (Figure 4, red curve). The figure also shows the M plot of the DOPC SAM both in the presence of C30 (blue curve) and in its absence (green curve) at the same bias potential. Whereas in the presence of C30 the spectrum shows a single partial semicircle, the C100 spectrum shows two partially fused semicircles.



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Figure 4. Plot of Z’ against –Z” at –0.40 V at a mercury-supported DOPC SAM in 0.1 M KCl in the presence of 4 mol% C30 (blue curve) or 10 mol% C0 (red curve), as well as in their absence (green curve).

It is worth recalling that the impedance spectrum of an adsorbed film consisting of a series of slabs with different dielectric properties can be simulated by an equivalent circuit consisting of a series of ‘RC meshes’ (i.e., parallel combinations of a capacitance C and a resistance R), one for each slab. On an M plot, a single RC mesh yields a semicircle of diameter 1/C, whose maximum lies at a frequency whose reciprocal equals the time constant, RC, of the given mesh.55 If some of the RC meshes simulating the film have similar time constants, the corresponding semicircles partially overlap in the M plot. Incidentally, the almost vertical, slightly curved portion of the three M plots lying at higher –Z” values (and, hence, at higher frequencies) is just the initial portion of a further large semicircle ascribable to the aqueous solution bathing the SAM; its small time constant depends exclusively upon the electrolyte concentration and takes practically the same value of about 10–7s for the three M plots in aqueous 0.1 M KCl. The two partially overlapping semicircles in the red curve of Figure 4 are indicative of two dielectric slabs of different composition, in series with each other. The hydrophobic C100 cluster is likely to be well imbedded in the DOPC SAM at –0.40 V. Hence, if it is present in sufficient amount, it is expected to push a number of lipid molecules away from direct contact with the mercury surface, giving rise to an overlapping lipid submonolayer. Comparison of the M plot of the DOPC SAM in the presence of 4 mol% C30 with that in its absence denotes only a slight effect of the smaller Au25 cluster on the SAM. Another remarkable feature that distinguishes the C80 and C100 clusters from Cn– clusters is represented by their CVs, which exhibit both a positive and a negative peak around –0.50 V. In the presence of 4 mol% C80, the peak-to-peak separation amounts to ca. 55 mV at a potential scan rate (v) of 50 mV s-1 (blue curve in Figure5) and decreases to ca. 30 mV at v = 10 mV s-1 (red curve). The negative peak is broader than the positive one, due to a tendency to splitting, which is more evident at the lower scan rate. Figure 5 also shows the CV obtained in the presence of 10 mol% C100 at 50 mV s-1, plotted against the enlarged right-hand scale to highlight the much smaller peaks. The midpoint potential, E1/2, between the two peaks amounts to –0.50 and –0.47 V for C80 and C100, respectively. These potential values are roughly consistent with those of the 0/–1 redox couple of the same clusters in dichloromethane (DCM).45 The CVs recorded at DOPC SAMs incorporating C80 and C100 can thus be reasonably ascribed to the 0/–1 redox couple. In DCM solution and independently of the ligand length, this process is characterized by a fully reversible electron transfer (ET) and is exclusively controlled by diffusion, giving rise to the expected peak-to-peak separation of 59 mV.37 On the other hand, in the present case the MPCs are confined inside a lipid 10 ACS Paragon Plus Environment

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monolayer, in the absence of diffusion, and the positive and negative peak potentials would be predicted to coincide if the ET were fully reversible.56 In the present situation the ET is quasireversible, probably due to the very small dielectric constant of the medium and the absence of electrolytes and, therefore, the peak-to-peak separation increases slightly with an increase in the potential scan rate.57 The modest splitting of the negative peak for the C80cluster (cf. Figure 5) at 50 mV s–1 scan rate can be justified upon assuming a slight detachment of the nascent anionic counterparts from direct contact with the metal surface. This would cause a slight electron flow to the mercury surface along the external circuit to maintain the applied potential constant. This interpretation is supported by the observation that the spitting is more pronounced at the lower scan rate of 10 mV s–1, when the nascent anionic clusters have more time for moving away from direct contact with the mercury surface.

Figure 5. Cyclic voltammograms with error bars at a mercury-supported DOPC SAM in 0.1 M KCl in the presence of 4 mol% C80 at a scan rate of 50 mV s-1 (blue curve) and 10 mV s-1 (red curve), and in the presence of 10 mol% C100 at a scan rate of 50 mV s–1 (gray curve). The left-hand scale refers to the blue and red curves, the right-hand one to the gray curve.

Despite the evident electron exchange between the electrode and the C80 and C100 clusters incorporated in the DOPC SAM, addition of 110–4 M Eu(III) to the aqueous solution has no detectable effect on the corresponding CVs, thus excluding mediation of ET across the SAM by these clusters. Neutral MPCs do not yield a bell-shaped peak along the flat capacitance region of AC voltammograms simply because their location within the lipid SAM is not expected to be appreciably influenced by a change in the transmembrane potential. In contrast to their neutral counterparts, anionic MPCs do not exhibit CVs with a negative and a positive peak over the flat capacitance region of the DOPC SAM, as shown in Figure S5. This points to their inability to exchange electrons with the metal over this broad potential range, at least at the usual potential scan rates of CVs. More significantly, the AC voltammograms in Figure 1 were recorded moving from –0.30 V toward more negative potentials. Hence, the anionic Au25 clusters remained for a relatively long time at the more positive accessible potentials; if they had been able to be completely oxidized to their neutral counterparts there, they would have yielded AC voltammogramms such as those in Figure 3, without the bell-shaped peak around –0.50 V. The different behavior between neutral and anionic Au25 clusters can be rationalized on the basis of the observation that the transmembrane potential along the whole flat capacitance region is negative, being approximately equal to m = E + 190 mV.28,50 Anticipating the results at the DPTL/DOPC tBLM, where the negative Au25 clusters start releasing an electron to the metal when 11 ACS Paragon Plus Environment


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m becomes positive, they are prevented from doing so at the DOPC SAM, where m is constantly negative. Conversely, neutral clusters are favored to accept an electron from the metal by the negative transmembrane potential values at the DOPC SAM, as the redox potential of the 0/–1 couple in the lipid monolayer is approached. The fact that the nascent anionic Cn– clusters release an electron to the metal during the positive-going potential scans in Figure 5 strongly suggests a metastable conformation somewhat different from that of the analogous negative clusters obtained by synthesis, which are not electro-oxidized to Cn0 even at –0.20 V, as appears from Figure 1. Au25Clusters at the DPTL/DOPC tBLM A significant distinguishing feature of mercury-supported DPTL/DOPC tBLMs with respect to mercury-supported DOPC SAMs is the presence of a hydrophilic ionic reservoir interposed between the lipid film and the mercury surface. Interpretation of several pieces of experimental evidence on the basis of mild extra-thermodynamic assumptions points to a transmembrane potential across the lipid bilayer moiety of a DPTL/DOPC tBLM expressed by the equation m = 0.72  (E + 0.520 V), in the absence of ions in the tBLM.19,25,50 Chronocoulometry current transients at tBLMs incorporating anionic or neutral Au25 MPCs reveal a slow but continuous flow of conduction electrons away from the metal surface along the external circuit, as the applied potential is stepped from an initial value of –0.30 V to final potentials positive of –0.40 V. The resulting curves of the positive charge (Q) on the metal surface against time are approximately linear, and their positive slope decreases with a negative shift in the final potential, approaching zero around –0.40 V. This general behavior is exemplified in Figure 6 for the C80 cluster. Progressively more negative final potentials induce an increasing flow of conduction electrons toward the mercury surface. The resulting charge transients show a negative slope whose absolute value increases gradually with a negative shift in the final potential during the first 10 s, and then decreases at longer times tending to a constant limiting value, which is almost independent of the final potential.

Figure 6. Charge vs. time curves at a mercury-supported DPTL/DOPC tBLM in 0.1 M KCl in the presence of 6 mol% C80 clusters, as recorded by stepping the applied potential from a constant initial value of –0.30 V to different final potentials, reported in mV in the inset.

This behavior can be explained by a slight nonspecific permeabilization of the lipid bilayer moiety of the tBLM toward inorganic ions, as elicited by the incorporation of the Au25 MPCs. The positive charge flowing at applied potentials positive of –0.40 V is due to a slow inflow of chloride ions into the hydrophilic spacer, which moves conduction electrons away from the electrode 12 ACS Paragon Plus Environment

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surface to maintain the electroneutrality of the whole electrified interface. Around the zero m value, the nonspecific ion leakage across the lipid bilayer is small, as expected. As the final potential is made progressively more negative, the outflow of chloride ions initially present in the spacer at – 0.30 V and the concomitant inflow of potassium ions determine an initial increase of the negative charge due to the movement of conduction electrons toward the mercury surface; at longer times, the negative slope of the charge transients decreases in magnitude attaining an almost constant limiting value, being exclusively determined by the maximum limiting inflow rate of potassium ions. A nonspecific permeabilization of the lipid film due to MPCs incorporation also takes place at DOPC SAMs, although it determines a much smaller ion flow across the lipid film because of the lack of an ionic reservoir capable of collecting an appreciable amount of permeant ions. At any rate, the regular arrangement of the lipid molecules does not appear to be appreciably disturbed by the MPCs, as indicated by EIS measurements. Thus, incorporation of the Cn– clusters in a DPTL/DOPC tBLM affects the differential capacitance only slightly over the potential range from –0.30 to –0.90 V at a frequency of 1 Hz, as shown in Figure S6 for C5–. On the other hand, Cn– clusters induce a rounded maximum at about –0.45 V in the curve of the in-phase component of the admittance (Y’) against potential, denoting a movement of the clusters within the lipid bilayer moiety with varying potential. A similar behavior is exhibited by the neutral C80 and C100 clusters, which, however, affect Y’ to a lesser extant than the anionic ones. (Figure S7 for C80). Incorporation of neutral Cn0 clusters in a DPTL/DOPC tBLM has a negligible effect on the corresponding CVs. Conversely, incorporation of the anionic C5– and C6– clusters in freshly formed DPTL/DOPC tBLMs yields CVs characterized by a negative and a corresponding positive peak, as shown in Figure 7. Instead, no peaks are observed with C8–.

Figure 7. Cyclic voltammograms with error bars at a mercury-supported DPTL/DOPC tBLM in 0.1 M KCl in the presence of 6 mol% C5– (blue curve), 6 mol% C6– (red curve) and 5 mol% C8– (green curve), at a scan rate of 50 mV s–1.

As expected, the peak-to-peak separation (Ep) decreases with decreasing potential scan rate, with a midpoint potential (E1/2) of ca.–0.38 V, corresponding to a transmembrane potential m of ca.+100 mV. The charge under these peaks, albeit small, is clearly detectable and significant, in view of the low charge-to-volume ratio of the anionic Au25 clusters. An increase in the molar concentration of C5– or C6– does not cause a further increase in this charge, pointing to the attainment of a maximum limiting value in the amount of these MPCs incorporated in the tBLM. Addition of 110–4 M Eu(III) to the solution bathing the tBLM does not affect the CVs, denoting both a blocking effect exerted by the tBLM on the Eu(III)/Eu(II) redox couple and no mediation of ET by the MPCs. 13 ACS Paragon Plus Environment


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To justify the appreciable movement of the C5– clusters inside the tBLM induced by voltage cycling and to make an approximate estimate of their surface concentration, we will adopt a variant of a modelistic treatment of cyclic voltammetry at tBLMs that some of us recently developed and successfully applied to ionic flow across the lipid bilayer moiety of DPTL/phospholipid tBLMs, promoted by different ion channels.58,59 We will assume that the C5–and C6– clusters incorporated in the DPTL/DOPC tBLM are approximately located at the boundary between the hydrophilic spacer and the lipid bilayer moiety of the tBLM at the more positive applied potentials E, where the transmembrane potential m is positive, while moving gradually away from it across the lipid bilayer moiety as m becomes progressively more negative (see their predicted locations in Scheme 1). The CVs can now be calculated by a computational approach similar to that employed to account for the translocation of potassium ions across an identical DPTL/DOPC tBLM incorporating gramicidin,58 melittin and syringopeptin 25A ion channels. 59 The tBLM can be regarded as consisting of the region of the lipoic acid residues, the tetraethyleneoxy spacer, and the lipid bilayer moiety. For the sake of simplicity, in the present case the polar head region will be disregarded, since its inclusion in similar treatments bears a small contribution at constant pH.58,59 The absolute potential differences across the above three regions can be approximately expressed, in the order, by Eq. 4:58

 

 qM  qM  F i    sp   m Cla  Csp 

with : m 

qM  F i . Cm

(4)

Here,  and  m are the extra-thermodynamic absolute potential differences across the whole mercury/water electrified interface and across the lipid bilayer moiety, respectively, qM is the charge density on the metal experienced by diffuse layer ions, Γi is the surface concentration of C5– clusters located at the boundary between the spacer and the lipid bilayer, and χsp is the surface dipole potential of the spacer. Cla, Csp, and Cm are the differential capacitances of the lipoic acid region, the spacer, and the lipid bilayer moiety, respectively. Several pieces of experimental evidence interpreted on the basis of a modelistic approach concur in estimating χsp at about –0.250 V.50 In the absence of exogenous species, Cla and Csp are about equal to 5 and 7 μF cm–2, whereas Cm equals 1 μF cm–2.58,60 In the presence of ions in the spacer, all capacitances increase to some extent, but Cm remains the smallest. Upon extracting qM from Eq. 4 as a function of , substituting the resulting expression into the equation, m = (qM+FΓ)/Cm, for the extrathermodynamic transmembrane potential across the lipid bilayer moiety and considering that the reciprocals of Cla and Csp are much smaller than the reciprocal of Cm, the expression for m takes the simplified form of Eq. 5:58

m    F  i C la   sp .

(5)

In the presence of the small charge density due to the incorporation of the anionic MPCs, Cla can be safely ascribed the value of 5 μF cm–2, as estimated in the absence of ions in the spacer.60 The current density j is obtained from the rate theory of ion transport across membranes, as applied to ion translocation across the potential energy barrier located in the lipid bilayer moiety of the tBLM:58,59,61 14 ACS Paragon Plus Environment

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



j  Fkt  i exp    Fm RT     tot   i  exp 1   Fm RT  ,

(6)

where kt is the translocation rate constant (in s–1), α is the charge-transfer coefficient, Γtot is the total surface concentration of the MPCs in the tBLM, and (Γtot –Γi) is their surface concentration located at the outer boundary of the lipid bilayer. CVs are determined numerically by subdividing the potential range  into 1106δ() steps.58 The current density j is calculated at each step, using Eq. 6 with  = 0.5, and is integrated by adding the corresponding charge contribution, δ(FΓi) = j δ()/v, to the sum of the preceding contributions, where v is the potential scan rate. The resulting FΓi values are used to continuously update m via Eq. 5, and the FΓi and m values are feedbacked into Eq. 6 for the current. An increase in the translocation rate constant kt decreases the peak-topeak separation of the calculated CVs, while leaving substantially unaltered their height, which increases linearly with the total charge density, Ftot, ascribed to the C5– clusters incorporated in the tBLM. Hence, the Ftot value estimated from the best fit to the experimental CVs is not affected by the choice of kt. Figure 8 shows the experimental CVs at a DPTL/DOPC tBLM in 0.1 M KCl in the presence of 6 mol% C5– recorded at potential scan rates of 50 mV s-1 (solid red curve) and 10 mV s-1 (solid blue curve), after subtraction of the capacitive contribution determined in the absence of the MPCs. The corresponding dashed curves are best fits calculated on the basis of the above approach by setting FΓtot= –0.85 μC cm–2, t = 0.2 s–1, and ascribing to the surface dipole potential χsp of the spacer the value of –0.250 V estimated by independent measurements.50 The calculated curves were first shifted by 0.195 V toward more negative potentials in order to pass from the scale of the absolute electric potential to that of the electric potential E relative to the Ag/AgCl/(0.1 M KCl) reference electrode,50,58 yielding the curves in the inset of Figure 8. Their superimposition on the experimental curves was then carried by adding to the calculated curves in the inset the resistive current responsible for the tilt of the experimental CVs. The corresponding resistance, which stems from the non-infinite resistance of the tBLM in the absence of exogenous species and is further enhanced by the presence of the C5–clusters (cf. Figure 6), is in parallel with the tBLM capacitance. Since the potential difference across this capacitance varies linearly with time (as a consequence of the constant potential scan rate), the current flowing along the tBLM resistance varies linearly with the applied potential.

Figure 8. Cyclic voltammograms at a mercury-supported DPTL/DOPC tBLM in 0.1 M KCl in the presence of 6 mol% C5– at 50 mV s-1 (solid red curve) and 10 mV s-1 (solid blue curve), after subtraction of the capacitive contribution determined in the absence of C5–. The corresponding dashed curves are the best fits



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to the experimental CVs, obtained as described in the text upon summing a resistive current linearly varying with potential to the calculated currents reported in the inset of the figure.

The C5– surface charge density of –0.85 μC cm–2 estimated from the fitting corresponds to a number density of 5.31012 molecules cm–2. Ascribing a thickness of 4.6 nm to the lipid bilayer moiety,46 this value corresponds to a volume concentration of ca.0.02 M. The above surface charge density can also be compared with the maximum number density of the C5– clusters in a closepacked hexagonal lattice, which is given by (0.866 l2)–1, where l is the diameter of the C5– clusters. Using the value l = 2.06 nm for these clusters,45 the degree of their surface coverage amounts to ca. 0.2. A similar surface coverage is also expected for their neutral counterparts, even though it cannot be determined experimentally. Figure 7 shows that the cyclic voltammetry peaks decrease in passing from C5– to C6– and are no longer present for C8–. This trend points to a greater difficulty for the bulkier clusters to be accommodated at the boundary between the hydrophilic spacer and the lipid bilayer moiety of the tBLM at the more positive potentials and/or to move following voltage cycling. Adsorption of the C5– Clusteron Bare Mercury Both neutral and anionic Au25 MPCs are soluble in organic solvents. However, thanks to their

negative charge, anionic Au25 clusters exhibit some solubility in polar solvents.62 We found that, when added to an aqueous solution, they tend to be preferentially adsorbed on hydrophobic surfaces, such as the bare mercury electrode and the Teflon coating of the stirrer. We, therefore, found it interesting to record the CV at a bare mercury electrode immersed in aqueous 0.1 M KCl, after addition of 0.27 μM C5–. Figure 9 shows that this CV is characterized by a positive and a negative peak lying at almost the same potential of ca. –0.49 V, in the proximity of the potential of zero charge.63 The almost coincidence of the negative and positive peak potentials denotes a reversible ET between C5– and mercury, which occurs in the adsorbed state.56 The vertical distance, Δj, between the negative and positive capacitive currents at potentials just positive of the two peaks yields a differential capacitance C of ca. 30 μF cm–2, upon applying the approximate expression Δj = Cv/2, where v = 50 mV s-1 is the potential scan rate. The same procedure applied to the Δj value at potentials negative of the two peaks yields a differential capacitance of ca. 16 F cm–2. The above C values are close to those at the mercury/water interface in the absence of exogenous species64 and indicate that the C5– clusters adsorbed on the mercury surface are immersed in a “sea” of water molecules and inorganic ions.


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Figure 9. Cyclic voltammogram with error bar at a bare mercury electrode immersed in aqueous 0.1 M KCl, after addition of 0.27 M C5–. Potential scan rate = 50 mV s-1.

This CV should be compared with that at a DOPC SAM incorporating C5–, which does not show peaks, as is apparent from Figure S5. Thus, this comparison confirms that it is the incorporation of anionic Cn– clusters into the lipid monolayer that prevents them from exchanging electrons with the metal. Such an exchange is possible with neutral Cn0 clusters, but neither neutral nor anionic clusters incorporated in a DOPC SAM or a DPTL/DOPC tBLM are capable of mediating ET from the Eu(III)/Eu(II) redox couple across the phospholipid monolayer. This is probably due to an effective ET barrier associated with the polar heads of the DOPC layer facing the aqueous solution. Adding 110–4 M Eu(III) to the same aqueous solution as in Figure 9 yields the CV in Figure 10 (red curve). For comparison, the blue curve shows the CV recorded on bare mercury in the absence of the C5– clusters. The presence of adsorbed C5– clusters induces a negative peak at –0.56 V for Eu(III) reduction and the corresponding positive peak at –0.47 V for Eu(II) oxidation. Their midpoint potential, E1/2 = –0.51 V, is only slightly negative of –0.49 V, which is the potential common to the two peaks in the CV of C5– in Figure 9. Conversely, it is appreciably shifted in the positive direction with respect to the midpoint potential value of –0.66 V for the Eu(III)/Eu(II) couple51 on bare mercury in the absence of C5–. Incidentally, the latter CV is quasi-reversible, in that the peak-to-peak separation amounts to 73 mV and is, therefore, close to the value, 59 mV, predicted for a Nernstian ET.

Figure 10. Cyclic voltammogram with error bars at a bare mercury electrode in aqueous solution of 0.1 M KCl and 110–4 M Eu(III) in the absence (blue curve) and in the presence of 0.27 M C5– (red curve). Potential scan rate = 50 mV s–1.

Clearly, the process generating the red curve in Figure 10 is an electrocatalytic process whereby adsorbed C5– releases one electron to the Eu(III) ion while accepting one from the metal along the negative peak, whereas a reverse electron movement occurs along the positive peak. As expected for a catalytic process, the current density j in the red CV of Figure 10 is about one order of magnitude higher than that for adsorbed C5– in Figure 9. Hence, these results confirm that negative Au25 clusters are indeed intrinsically capable of acting as redox mediators. This process is different in several respects from nanoparticle-mediated ET across organic SAMs with kinetics independent of their thickness.65 Thus, in the present case no intermediate SAM exists. Moreover, at an Au/SAM/nanoparticle construct, a redox couple such as Ru(NH3)63+/Ru(NH3)62+ yields a CV with exactly the same midpoint potential and peak-to-peak separation as at bare gold.66,67 A common feature is represented by the fact that, in both nanoparticle mediated processes, the mediation requires a direct contact of the redox couple partners with the nanoparticle.65 17 ACS Paragon Plus Environment


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CONCLUSIONS The behavior of monoanionic Au25(SCnH2n+1)18 clusters shown in Figure 8, quantitatively interpreted using model parameters estimated in previous works at the same mercury-supported DPTL/DOPC tBLM, demonstrates the ability of Cn– clusters to move across a lipid bilayer interposed between two hydrophilic media and to exchange one electron with the mercury electrode when the transmembrane potential m starts to become positive. This behavior is in keeping with their failure to release one electron to mercury at the DOPC SAM, where m is negative at all experimentally accessible applied potentials E. Nonetheless, at the DOPC SAM, Cn– clusters occupy the location xin closer to the mercury surface at less negative m values, where the electrostatic repulsion from the mercury surface is compensated for by their negative standard chemical potential difference , due to their hydrophobicity; a negative shift in the applied potential ultimately moves them to the outer location xout, generating the bell-shaped peak in the AC voltammogram of Figure 1. Neutral Au25 clusters exchange one electron with the metal along the flat capacitance region of the DOPC SAM, where m is negative, as appears from the CV in Figure 5. Conversely, they are electroinactive at the DPTL/DOPC tBLM, where m may assume both negative and positive values. This apparent discrepancy can be easily reconciled by considering that the neutral Cn0 clusters, being uncharged, are more hydrophobic than their negative counterparts. Consequently, after crossing the lipid bilayer moiety of the tBLM, their penetration into the hydrophilic tetraethyleneoxy moiety represents an insurmountable barrier that prevents them from approaching the mercury surface so as to exchange one electron with it. Their movement within the lipid bilayer moiety following the potential scan is confirmed by the Y’ vs. E peak around –0.45 V (Figure S7), similar to that exhibited by the Cn–clusters (Figure S6), albeit lower. Au25(SCnH2n+1)18 clusters can penetrate lipid bilayers without disrupting them, as demonstrated by the capacitance being practically identical with that at a pure mercury-supported lipid bilayer, as appears from Figures S6 and S7. In fact, bilayer disruption would increase the capacitance to a level typical of bare mercury. Any of the numerous methods for modulating the transmembrane potential m of plasma membranes may trigger the penetration of the anionic Au25 clusters; this is not required with neutral clusters, which can penetrate plasma membranes around their transmembrane redox potential. A direct penetration of Au25 clusters into the cytosol is particularly convenient for targeted drug delivery. Some advantages of Au25 clusters are as follows: (i) The 1 nm size of their core diameter imparts them an exceptional stability, differently from Au particles of core diameter between 2 and 3, which tend to readily aggregate in biological media, underscoring the importance of particle uniformity in nanomedicine;42 (ii) Au nanoclusters with core sizes less than 2 nm are highly biocompatible in mammalian cells, unlike their Ag counterparts, and are highly catalytic;41 (iii) the optical gap of Au25 clusters (ca. 1.3 eV) being larger than the energy of 1O2 (0.97 eV) is suggested to induce an efficient energy transfer to 3O2,40 permitting applications such as radiotherapy for cancer treatment39 and antimicrobial activity against both Gram-positive and Gram-negative bacteria.41 Many of the above applications can be realized by imparting a sufficient water solubility to the Au25 clusters upon coating their core with carboxylated compounds such as 6mercaptohexanoic acid,41 glutathione42 or captopril.40 EXPERIMENTAL SECTION 18 ACS Paragon Plus Environment

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The synthesis of the clusters with n = 3, 5, 6, 8, and 10 has been described previously (see Scheme S2 and a detailed description of the synthesis in the Supporting Information).60,87,93 Briefly, the method is based on the stepwise reduction of tetrachloroauric acid in tetrahydrofuran, first with the appropriate thiol and then with sodium borohydride, in the presence of tetraoctylammonium bromide. After 2-3 days, the so-prepared Au25(SR)18– cluster is purified from sodium borohydride byproducts, excess thiol, and electrolyte, and then stored as a powder under anaerobic conditions. Its quantitative one-electron oxidation to form the corresponding neutral Au25(SR)180 cluster is carried out by dissolving the cluster in DCM, followed by passage through a silica gel column under aerobic conditions.69 Water was obtained by an inverted osmosis unit, distilled once and then redistilled from alkaline permanganate. Suprapur® KCl (Merck, Darmstadt, Germany) was dried at 500 ˚C before use to remove any organic impurities. Eu(III) chloride hexahydrate was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as such. Dioleoylphosphatidylcholine (DOPC) was purchased in chloroform solution from Avanti Polar Lipids (Birmingham, AL, U.S.A.). The 2,3,di-O-phytanylsn-glycerol-1-tetraethylene-glycol-D,L- lipoic acid ester thiolipid (DPTL) was kindly provided by Prof. Adrian Schwan (Department of Chemistry, University of Guelph, Canada). Solutions of 0.2 mg/ml DPTL in ethanol were prepared from a 2 mg/ml solution of DPTL in ethanol. Stock solutions of this thiolipid were stored at –18 °C. Measurements were carried out with a homemade HMDE.70 A homemade glass capillary with a finely tapered tip, about 1 mm in outer diameter, was employed. To avoid any changes in drop area due to a change in temperature, capillary and mercury reservoir were thermostated at 25 ± 0.1 C in a water-jacketed box. The HMDE was used as the working electrode in a three-electrode system, with an Ag/AgCl/(0.1 M KCl) reference electrode and a platinum-coil counter electrode. The preparation of mercury-supported lipid SAMs and DPTL/DOPC tBLMs was described in detail in a previous paper.23 Briefly, the DOPC SAM was prepared by immersing the HMDE into the working aqueous solution of 0.1 M KCl, on whose surface a DOPC solution in pentane had been previously spread at a controlled rate, using an oleopneumatic system. The DPTL/DOPC tBLM was prepared by first keeping the HMDE in a 0.2 mg/ml DPTL solution in ethanol for 20 min, to anchor a DPTL monolayer to the mercury surface. Then, after allowing the ethanol to evaporate under nitrogen atmosphere, the same procedure adopted to form a DOPC SAM on bare mercury was employed to form a DOPC monolayer on top of the DPTL monolayer. Stock solutions of DOPC in pentane and of Au25 clusters in chloroform were mixed in the appropriate composition; the mixture was then diluted in pentane and spread on the surface of the working aqueous solution. Finally, a HMDE coated with either DOPC SAM or DPTL/DOPC tBLM was immersed across the previously spread pentane film. To exclude interferences from the small amount of chloroform, in a few cases the Au25 clusters were directly dissolved in pentane, without observing any difference with respect to the previous procedure. Impedance spectroscopy, AC voltammetry and cyclic voltammetry measurements were carried out with an Autolab instrument PGSTAT12 (Echo Chemie) supplied with FRA2 module for impedance measurements, SCAN-GEN scan generator and GPES 4.9007 software. Incidentally, AC voltammograms are plots of the quadrature component of the current against the applied potential (E) at constant frequency (f); the current was converted into a capacitance (C) by calibrating the instrument with a high precision capacitor. EIS measurements were carried out by superimposing an AC voltage of 10 mV peak-to-peak amplitude to the bias potential E and varying the frequency between 10–1 and 105 Hz. Potentials were measured vs. an Ag/AgCl electrode immersed in the 0.1 M KCl working solution, and are referred to this reference electrode. To 19 ACS Paragon Plus Environment


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facilitate the incorporation of Au25 clusters into the lipid film and to attain a stable electrochemical response, the DOPC SAM that had been previously passed across the pentane solution of the Au25 cluster was subjected to an EIS pretreatment over the frequency range from 105 to 0.1 Hz at bias potentials varying regularly from –0.30 to –0.80 V, by –50 mV increments. An analogous ‘EIS pretreatment’ was adopted for the DPTL/DOPC tBLM, upon varying the bias potential from –0.30 to –1.00 V. In a few cases, attainment of a stable electrochemical response required a second EIS pretreatment. All CVs reported herein were recorded starting from the most positive potential reported in the figure. Identical current peaks were obtained starting from the most negative potential, differently from the AC voltammograms in Figs. 1 and 3, where the disorganized lipid film at –1.5 V gives rise to a notably different positive-going scan.47 ASSOCIATED CONTENT Supporting Information Additional figures concerning electrochemical measurements, X-ray crystal structure of Au25(SC3H7)180, experimental details on the synthesis of Au25(SCnH2n+1)18 clusters. ACKNOWLEDGMENTS Thanks are due to Prof. Adrian Schwan (University of Guelph, Ontario, Canada) for providing us with the DPTL thiolipid. REFERENCES 1. Protected Metal Clusters: From Fundamentals to Applications. In Frontiers of Nanoscience; Tsukuda, T.; Häkkinen, H., Eds.; Elsevier, Amsterdam, 2015; Vol. 9. 2. Antonello, S.; Maran, F. Molecular Electrochemistry of Monolayer-Protected Clusters. Curr. Opin. Electrochem. 2017, 2, 18−25. 3. Zhang, Q.; Yang, M.; Zhu, Y.; Mao, C. Metallic Nanoclusters for Cancer Imaging and Therapy. Curr. Med. Chem. 2018, 25, 1379–1396. 4. Tao, Y.; Li, M., Ren, J.; Qu, X. Metal Nanoclusters: Novel Probes for Diagnostic and Therapeutic Applications. Chem. Soc. Rev. 2015, 44, 8636–8663. 5. Verma, A.; Uzun, O.; Hu, Y.; Hu, Y.; Han, H.-S.; Watson, N.; Chen, S.; Irvine, D. J.; Stellacci, F. Surface-Structure Regulated Cell-Membrane Penetration by MonolayerProtected Nanoparticles. Nat. Mater. 2008, 7, 588–595. 6. Cioran, A. M.; Musteti, A. D.; Teixidor, F.; Krpetic, Z.; Prior, I. A.; He, Q.; Kiely, C. J.; Brust, M.; Vinas, C. Mercaptocarborane-Capped Gold Nanoparticles: Electron Pools and Ion Traps with Switchable Hydrophilicity. J. Am. Chem. Soc. 2012, 134, 212–221. 7. Salvati, A.; Pitek, A. S.; Monopoli, M. P.; Prapainop, K.; Bombelli, F. B.; Hristov, D. R.; Kelly, P. M.; Aberg, C.; Mahon, E.; Dawson, K. A. Transferrin-Functionalized Nanoparticles Lose Their Targeting Capabilities When a Biomolecule Corona Adsorbs on the Surface. Nat. Nanotechnol. 2013, 8, 137–146. 8. Jiang, Y.; Huo, S.; Mizuhara, T.; Das, R.; Lee, Y.-W; Hou, S.; Moyano, D. F.; Duncan, B.; Liang, X.-J; Rotello, V. M. The Interplay of Size and Surface Functionality on the Cellular Uptake of Sub-10 nm Gold Nanoparticles. ACS Nano 2015, 10, 9986–9993. 20 ACS Paragon Plus Environment

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18 Clusters in Biomimetic Membranes: Role of Size, Charge and

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