Light-Driven Directed Proton Transport across the Liposomal Membrane

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Light-driven directed proton transport across the liposomal membrane Romina Zappacosta, Gabriella Siani, Serena Silvi, Alberto Credi, and Antonella Fontana Langmuir, Just Accepted Manuscript • DOI: 10.1021/la503604e • Publication Date (Web): 24 Oct 2014 Downloaded from http://pubs.acs.org on October 29, 2014

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Light-driven directed proton transport across the liposomal membrane¥ Romina Zappacosta,† Gabriella Siani,† Serena Silvi,‡ Alberto Credi,‡ and Antonella Fontana†,* †

Dipartimento di Farmacia, Università “G. d’Annunzio”, Via dei Vestini, I-66100 Chieti, Italy

‡

Photochemical Nanosciences Laboratory, Dipartimento di Chimica “G. Ciamician”, Università

di Bologna, Via Selmi 2, I-40126 Bologna, Italy KEYWORDS: spiropyran, proton pump, lysosomes, liposomes, nano-devices

ABSTRACT. We have developed a simple artificial photoresponsive ion gating device by inserting molecular switches in the membrane of liposomes. A controlled and directed proton transport across the bilayer membrane can lower the internal pH of the liposomes from neutral to around 4 under combined light and chemical stimulation.

INTRODUCTION Lipid bilayer membranes are impermeable to small ions. Ion diffusion across cell membranes is enabled either by ion-selective carrier molecules or by ion channels [1-3]. These transport systems involve a concatenation of processes at the molecular level and require energy. The

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importance of such systems for living organisms is well represented by lysosomes, membraneenclosed aqueous compartments that break down cellular debris by means of enzymes called hydrolases [4]. As these enzymes are active at acidic pH, the lysosomal membrane contains a proton pump which uses the energy of ATP hydrolysis to maintain a 100-fold higher proton concentration inside the compartment. Inspired by these amazing biosystems we have developed a simple artificial photoresponsive membrane-based ion gating device by inserting acid-base and light responsive molecular switches in the membrane of POPC liposomes. We chose liposomes because they exhibit several of the properties of natural membranes and cells; in particular, they can incorporate guests in the bilayer and in the inner aqueous phase, and their membrane is capable of ion discrimination [57]. Our results indicate that a controlled and directional proton transport across the liposomal membrane is realized under combined light and chemical stimulation. Such a proton transfer can lower the internal pH of the liposomes from neutral to around 4, thus generating a pH gradient between the external environment and the vesicular core. This behavior reminds that of lysosomes, although our ion gating device - unlike lysosomes - couples proton translocation to anion transport down its imposed transmembrane concentration gradient to induce a directional proton transfer. Erokhina et al. have previously studied a proteic proton pump, i.e. bacteriorhodopsin, in order to obtain a light-driven pH variation of the internal volume of nanocapsules [8]. In the present study, we have selected for the experiments the molecular switch spiropyran SP (Chart), which is converted to the merocyanine form (ME or, in the presence of protons, MEH+) [9-11] upon light irradiation in solution. The behaviour of spiropyran photochromes inserted into phospholipidic bilayers was previously investigated [12] with the aim of estimating the bilayer viscosity, the


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free volume properties [13] and membrane interactions in the lamellar phase [14], studying the dynamics of fast photoreactions in the membrane [15], probing the membrane perturbation caused by the guest-induced lipid conformational change [16] and photocontrolling ion permeation through the bilayer membrane [17-20]. Spiropyran derivatives have been used to control transport properties of materials [9, 10], to create a pH-responsive fluorescent probe [21], to engineer light-driven supramolecular nanocapsules [22] and generate photocurrent by a lightdriven proton pump obtained upon incorporation of a spiropyran photochrome in an artificial liquid membrane [23]. However, the exploitation of the photo-acid/basic properties of SP in a nanocompartmentalized heterogeneous system such as liposomes is unprecedented.

Chart 1. Photo- and chemically-induced interconversion of SP and MEH+.

EXPERIMENTAL Material. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was purchased from Avanti Polar Lipids (Alabaster, AL). 1’,3’-Dihydro-1’,3’,3’-trimethyl-6-nitrospiro[2H-1benzopyran-2,2’-(2H)-indole] (98% purity), 5- and 6-carboxy-2’,7’-dichlorofluorescein, Sephadex G-25, N,N’-dimethyl-9,9’-biacridinium dinitrate, CH2Cl2, HCl, CF3COOH and acetonitrile were purchased from Fluka and Sigma-Aldrich. 4-(2-Pyridylazo)-N,Ndimethylaniline (98% purity) was purchased from Alfa Aesar.


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Instruments. The extrusion was performed on a nitrogen-driven Lipex Biomembranes (Vancouver, BC, Canada) apparatus operating at room temperature. Absorption spectra were performed at 25.0 ± 0.1 °C by using a Jasco V-550 UV/Vis spectrophotometer (path length = 5 mm) using quartz cells. Due to light scattering from the liposomes the spectra of liposomal solutions have been corrected by substraction of the corresponding blank liposomal suspension spectrum. Luminescence intensity measurements were performed with a Jasco FP-6200 spectrofluorimeter. Samples were irradiated in cuvettes with a LOT 200 W LSH303/303 27A lamp coupled to a band pass filter (254, 365 and 436 nm). Laser light scattering and ζ-potential experiments where performed by using a 90Plus/BI-MAS ZetaPlus multiangle particle size analyzer. POPC liposomes preparation. POPC liposomes were prepared by rehydration of a homogeneous thin film [24-28], obtained by evaporation of a chloroform solution of POPC (10 mg/mL) or a mixture of the guest in acetonitrile and POPC in chloroform in the case of guestcontaining liposomes (i.e. SP-loaded, AZ-loaded and SP/AZ-loaded liposomes). The initial phospholipid/spiropyran molar ratio was 50:1; the initial spiropyran/azopyridine molar ratio was 1:2. The phospholipid film was kept at 4 °C overnight before rehydration with milliQ water. The resulting multilamellar vesicle dispersion was extruded five times through polycarbonate filters with 100 nm pores. Determination of encapsulation efficiency. Encapsulation efficiency of SP-loaded liposomes and/or AZ-loaded liposomes were calculated by determining the amount of free guest recovered in the supernatant after liposomal extrusion. The extruded liposomal suspension was centrifuged for 10 min at 2000 rpm and subsequently the supernatant was collected and analyzed in order to carefully quantify the switches. Control spectra were obtained from corresponding solution in


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acetonitrile. In order to evaluate any loss of guest during extrusion, filters used in this step were analyzed by UV-vis spectrophotometry. Data collected revealed that the POPC bilayer allows to solubilize 1 SP out of 60 POPC molecules in SP-loaded POPC liposomes whereas in the case of SP/AZ-loaded POPC liposomes the ratios SP/POPC and AZ/POPC are 1:70 and 1:50, respectively (see Tables S1 and S2 in the Supporting Information). Determination of proton transfer through changes in AZH+ absorption. The amount of H+ transferred into the liposomes was calculated by considering: the total volume of a liposome with a diameter of 74.0±4.0 nm (as measured by DLS analyses) = 2.12×10–19 dm3; a thickness of the liposome bilayer = 3.7 nm [29]; liposome internal aqueous volume = 1.55×10–19 dm3; [POPC] = 1.32×10–3 M; 8.1×104 molecules of POPC per liposome [30]. Therefore 1 dm3 of bulk solution contains 9.81×1015 liposomes with a total internal volume of 1.52×10–3 dm3. Transport of chloride ions through the bilayer. SP-loaded liposomes and, for the sake of comparison, SP-free liposomes, were prepared by rehydration of a homogeneous thin film with a phosphate buffered solution (100 mM NaNO3, 10 mM sodium phosphate) of lucigenin dye (0.025 mM). The subsequent extrusion and gel filtration [31] afforded the liposomal samples that were added of 0.07 mM HCl. SP-loaded liposomes evidenced a lucigenin quenching of 3% and 13.3% with respect to the pure liposomes upon one or four UV/visible irradiation cycles, respectively (Figure S20; control experiments, Figures S15-S19).

RESULTS AND DISCUSSION


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Here we have examined the photoswitching of SP/MEH+ within POPC liposomes prepared by rehydration of a phospholipid thin film [24-28] (see Table S3 in the Supporting Information). The addition of CF3COOH (Figure 1) to the POPC/SP liposomal suspension followed by irradiation at 254 or 365 nm causes the conversion of up to 25% SP into MEH+ (Figure 1, black to red line). Subsequent irradiation at 436 nm leads to an almost complete transformation of MEH+ back to SP (Figure 1, green line), indicating that the SPMEH+ interconversion takes place inside the liposomal membrane. The SPMEH+ switching can also be triggered by addition of HCl and irradiation (Figure S8 in the Supporting Information). For the sake of comparison, the corresponding photoswitching in acetonitrile was also performed (Figures S1-S7 in the Supporting Information). Experimental evidences and encapsulation data highlight that the leakage of MEH+ from the liposomes is very limited within 24 h (Table S1 in the Supporting Information). Nevertheless, the formation of MEH+ alters the ability of the membrane to entrap the molecular switch, as shown by gel filtration on sephadex beads in which small molecules loosely bound to liposomes can diffuse and thus be delayed in their passage down the column. As a matter of fact, gel permeation performed immediately after the SPMEH+ switching strongly reduces the amount of guest dissolved in the membrane (Figure 1, blue line). Most of MEH+ is recovered in the gel column, indicating a high affinity of this species for the stationary sephadex phase. Such an observation reveals the tendency of photogenerated MEH+ to move from the inner lipophilic core of the membrane to the more hydrophilic interface with the aqueous solvent, eventually coming out of the bilayer [32]. This displacement of MEH+ is consistent with the rather poor affinity, compared with that of the corresponding SP form, of an analogous non-protonated merocyanine derivative for the POPC bilayer [12, 14, 17-19].


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Figure 1. Absorption spectra of SP in the POPC membrane (black line), upon addition of CF3COOH and irradiation at 254 nm (red line) immediately followed by irradiation at 436 nm (green line), or immediately filtered through a G-25 column (blue line) without visible light irradiation. Inset: absorption spectra of a liposomal suspension containing SP and AZ before (solid line) and after (dashed line) the addition of acid. See the text for details. In the switching cycle (Chart 1) a proton, provided by either HCl or CF3COOH, is taken from the solution to convert SP into MEH+ and is successively released back to the solution when the MEH+SP transformation occurs. In the present liposomal system, however, differently from a homogeneous solvent, the proton can be released in two different aqueous compartments, i.e. the


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internal core and the external bulk, the release in the bilayer being rather unlikely in the absence of a base. Hence, the SPMEH+ switching may be exploited, in principle, to accomplish a directed transfer of protons across the membrane from an aqueous compartment to another [33]. To confirm and quantify such a proton transfer we tried to measure the internal pH of the liposomes by entrapping a pH-sensitive luminescent probe in their interior. Besides having an appropriate pH window, such a probe must meet a number of requirements to be useful in the present context, namely a negligible escape from the intravesicular space, no spectral interference with the SP/MEH+ features, and photostability under UV and visible irradiation. Unfortunately, no species fulfilling all these conditions could be found. An additional problem comes from the fact that luminescent pH indicators are weak acids or bases which, at the concentrations required to detect their fluorescence, will alter significantly the inner pH of the liposomes. Hence, in order to investigate the effects of the SPMEH+ photoreaction on the nanocontainers, we incorporated an additional molecular acid-base switch, 4-(2-pyridylazo)N,N-dimethylaniline (AZ), in the bilayer. The pH-controlled reversible interconversion of AZ and AZH+ can be easily monitored by absorption spectroscopy (Figures S9-S11 in the Supporting Information). Moreover, the coupled operation of the AZ/AZH+ and SP/MEH+ switches by photoinduced proton transfer was thoroughly investigated in solution [34, 35]. Since both unprotonated and protonated forms of AZ are soluble in the bilayer,¶ and the visible absorption band of AZH+ (λmax = 554 nm; Figure 1, inset) [34, 35] does not overlap with those of SP and MEH+, this species is an ideal candidate for the role of a probe that (i) can exchange a proton with SP in the membrane and (ii) can be easily monitored as the SPMEH+ process takes place.


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Encapsulation efficiencies, calculated by measuring the amount of free guest after extrusion, indicate that under the adopted experimental conditions the concentration of AZ in the bilayer is ca. 70% of that of SP. Such a value is three times higher than the maximum MEH+ concentration that can be afforded by UV-induced conversion of SP (Table S2 in the Supporting Information). Besides, AZH+ (pKa = 2.64±0.05, Figure S12 in the Supporting Information) is more acidic than MEH+ (pKa = 4.04±0.02, Figure S13 in the Supporting Information) and therefore can behave as a proton donor in the bilayer compartment. The addition of 10 equivalents of CF3COOH to a suspension of liposomes containing SP and AZ in their membrane affords (Figure 1, inset) the quantitative formation of AZH+ inside the bilayer (process 1 in Figure 2). In order to ensure that, in the following irradiation step, MEH+ is generated by the proton released from AZH+ rather than by the excess acid outside the liposomes, the suspension was neutralized with NaOH. Indeed, the observed 12% decrease of AZH+ upon neutralization of excess acid is associated to the deprotonation of part of the AZH+ solubilised in the aqueous bulk; no evidence of deprotonation of the membrane-embedded AZH+ was found. This observation is confirmed by the fact that (i) the deprotonation of AZH+, obtained upon addition of 10 equivalents of NaOH, corresponds to the amount of AZ solubilized in the aqueous bulk, the residual AZH+ being equal to that solubilized in the membrane; (ii) control experiments in water show that complete deprotonation of AZH+ is afforded upon addition of 10 equivalents of NaOH (Figure S11 in the Supporting Information). These observations suggest that in the time scale of the experiment (i.e. 1 hour) the membraneembedded AZH+ species does not equilibrate with the bulk solution, most likely because of the inability of externally added OH– ions to penetrate the bilayer and the retention of AZH+ on the


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inner surface primarily due to the transmembrane potential generated by anions diffusion that would oppose outward cation migration (see below) [36]. UV Irradiation (254 nm) of the suspension causes absorption spectral changes fully consistent with the transformation of SP into MEH+ and the concomitant conversion of AZH+ into AZ (Figure S14 in the Supporting Information), thus pointing out that the proton transfer at the basis of the intermolecular communication between the two switches [34, 35] works within the bilayer as well (process 2 in Figure 2). Subsequent visible light irradiation (436 nm) induces the release of H+ ions from MEH+ which, however, are not transferred back to AZ to yield AZH+; in other words, differently from the homogeneous solution [34, 35], the intermolecular communication cycle is not closed. As a matter of fact, consecutive cycles of alternated ultraviolet and visible light irradiation cause a decrease in the concentration of AZH+ in the membrane (Figure 3).


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Figure 2. Schematics of the proton transfer across the POPC bilayer caused by the photoinduced SPMEH+ switching and the coupled AZH+AZ interconversion in the membrane. The scheme is not supposed to give hints on the location of the various species within the membrane.


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This result is ascribed to the relatively low basicity of AZ and the consequent need of a large excess of H+ to significantly protonate it (Figures S9-S11 in the Supporting Information). Indeed, the presence of 1 equivalent of H+ causes

Light-Driven Directed Proton Transport across the Liposomal Membrane

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