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SAXS measurements of azobenzene lipid vesicles reveal buffer-dependent photoswitching and quantitative Z->E isomerisation by X-rays

Photoresponsive materials feature properties that can be adjusted near- instantaneously, reversibly, and with high spatiotemporal precision, by light. There is considerable interest in maximising the degree of switching, and in measuring this degree during illumination in complex environments. We study the switching of photoresponsive lipid membranes that allow precise and reversible manipulation of membrane shape, permeability, and fluidity. Though these macroscopic photoresponses are clear, it is unclear how large the changes of trans/cis ratio are, and whether they can be improved. Here, we used small-angle X-ray scattering to measure the thickness of photoswitchable lipid membranes, and correlate thickness to trans/cis ratios. This revealed an unexpected dependency of photoswitching upon aqueous phase composition, highlighting smaller-than-expected photoswitching with deionized water, and showed thickness variations twice as large as previously observed. Soft X-rays can quantitatively isomerise photolipid membranes to the all- trans state: both enabling more powerful X-ray-based membrane control and underlining the role of high energy X-rays for observation-only soft matter experiments.


Introduction
Azobenzene photoswitches are molecular units, which can be switched between trans and cis isomer states at the single-molecule level by light. By embedding photoswitches into polymers or supramolecular assemblies containing many individual photoswitch units, materials with mechanical, electrical or optical properties that depend on the trans/cis ratio of the photoswitch population can be created: i.e., photoresponsive materials where these properties are adjustable by light. [1][2][3][4][5][6] Supramolecular assemblies can also provide further degrees of complexity, for example the populations of the two isomers may phase separate, 7 or a highly ordered assembly process may select for or stabilise one of the isomer states. 8 As a photoresponsive material's properties depend on the isomer ratio, there is considerable interest in measuring and maximising the degree of switching between mostly-trans and mostly-cis populations. 9 Typically, it is not possible to perform quantitative photoisomerisations to all-cis or to all-trans azobenzene populations, due to absorption overlaps. 8,[10][11][12][13] Hence, illuminations lead to mixed photostationary state (PSS) populations, with trans/cis ratios that depend on the absorption coefficients of the two isomers and the quantum yields of their photoisomerisation. However, a rare example of quantitative switching in one direction (to all-trans) was pioneered by Hecht et al., using electrocatalytic pathways that transiently oxidise or reduce the azobenzenes. [14][15][16] Here we study the switching of photoresponsive lipid membranes assembled from azobenzene-containing phosphatidylcholine (azo-PC), a synthetic lipid with a lightresponsive azobenzene group in one of its two hydrophobic tails. Optical control of membranes constructed from pure azo-PC allows precise and reversible manipulation of many mechanical properties including membrane shape, permeability, fluidity, and domain formation, and influence membrane protein function. 4,[17][18][19][20] So far, it remains unclear how large the changes of trans/cis ratio are that cause these property changes; and whether these ratio are close to the theoretical maxima of pure states, or whether they could be substantially improved by tailored conditions and switching stimuli -with associated improvements to switching of biophysical properties. For example, ultrastructure studies based on X-ray experiments have shown photostimulated membrane thickness changes of ca. 4 Å for ca. 42 Å azo-PC membranes: 4 but it was not known if this 10% change is already maximal, nor was it known what population-level of trans/cis ratios were responsible for this change. This partly derives from a technical challenge: while it is straightforward to measure trans/cis ratios in molecular solutions by a variety of methods, it is not straightforward to measure them in the pure azo-PC membranes, which are ordered anisotropic assemblies.
In this work, we develop small-angle X-ray scattering (SAXS) as a powerful method to measure the thickness of photoswitchable lipid membranes, and we use orthogonal measurements to relate SAXS-measured thickness to population-level trans/cis ratios.
This revealed an unexpected sensitivity of the photoswitches inside the lipid membranes to the aqueous buffer conditions outside them, particularly with a smallerthan-expected photoswitching yield under low ionic strength. Furthermore, by mixing pre-switched monomers, we show that membrane variations of about 8 Å are experimentally possible when photoswitching efficiency is unimpeded. This is twice as large as the thickness variation observed in previous vesicle photoswitching experiments, 4 and is very much larger than the membrane thickness variation achievable by temperature variations with conventional lipids. [21][22][23][24] We also discovered unexpectedly that while hard X-rays do not switch the membranes under study, soft X-rays (8 keV) efficiently and quantitatively isomerise photolipid membranes to the all-trans state within seconds, which we attribute to radical redox reactions following X-ray dose deposition in the medium. This enables soft X-rays to enforce a higher degree of membrane property control than photoswitching alone can achieve, while emphasising the role of high energy X-rays as low dose probes in soft matter experiments.

Synthesis of azo-PC and reference photoswitch FAzoM
Azo-PC was purchased from Avanti Polar Lipids, Inc. (Alabama, United States). Novel reference photoswitch FAzoM was synthesised by standard reactions (see Supporting Information).

Synthesis of azo-PC and reference photoswitches for benchmarking
In our hands, standard techniques to measure trans:cis ratios in molecular solutions (H-NMR, HPLC) were not reproducible when applied in situ to lipid membranes (ordered assemblies). Destructive readouts (e.g. adding cosolvents to homogenise membrane/water mixtures before HPLC measurement) were also tested but were also not reproducible, which we attributed to difficulties arising from the surfactant nature of the AzoPC. Finally, we developed a method to relate photolipid membrane trans/cis isomer ratio to the thickness determined by SAXS by using a calibration series for lipid membranes of known trans/cis composition. This series was created by mixing preconditioned all-trans and mostly-cis stocks (see below). The trans/cis ratios in the preconditioned AzoPC stocks were in turn determined by comparison of their UV-Vis spectra with that of the isoelectronic reference photoswitch FAzoM in the same conditions, since the trans/cis ratio of the apolar FAzoM can be reliably quantified by HPLC (see Supporting Information section S1 for details).

Preconditioning and mixing of azo-PC
Azo-PC was dissolved in chloroform (25 mg/mL) and stored at -20 °C until further use.
Azo-PC stocks have been stored in dark for several days to reach the all-trans state.
After illumination of molecularly-dissolved azo-PC by UV-A LED (Roschwege Star-UV365-03-00-00, λ = 365 nm, 9 nm FWHM, Conrad Electronic SE, Germany) the photostationary state ( ) has a cis fraction of 83% (see Supporting Notes for further detail). To adjust the cis fraction in the assembly, we mix azo-PC in the all-trans state with azo-PCs with cis fraction of 83%, in appropriate proportions. After mixing, we follow the protocol for vesicle preparation. All preparation steps were made in the dark.

Small unilamellar vesicle (SUV) preparation
The Azo-PC chloroform stock solution was evaporated under a nitrogen stream and stored under vacuum for 12 h. The resulting dry lipid film was dissolved in cyclohexane, and exposed to a vacuum of 6 x 10 -3 mbar at a temperature of -60 °C yielding a fluffy lipid powder. Immediately after lyophilisation, the azo-PC powder was hydrated with

SAXS measurements at 17.4 keV
X-ray data from azo-PC SUVs with preconditioned trans:cis ratios were recorded at a Mo-sourced small angle X-ray scattering (SAXS) setup. 25 The Mo anode delivers a beam with an energy of 17.4 keV, a beam size of 1.0 mm², and a flux of 2 • 10 6 cts•s -1 mm -2 . X-ray data were recorded by a Dectris Pilatus 3R Detector with 487 x 619 pixels of size 172 x 172 µm². All in-house SAXS measurements were performed in darkness.

Results and discussion
Azo-PC populations are driven towards cis-rich photostationary state isomer mixes by UV-A light at 365 nm wavelength, and towards trans-rich isomer mixes by blue light of 465 nm (Fig. 1a). The absorption spectra of these mixed states, either in molecular solution or in assemblies, have been reported by us and others 4, 17-18 and substantial differences between them have been noted. 18  cis azo-PC (see methods and SI), using calibrants at 0% ,10%, 19%, 39%, 58% and 83% cis isomers to cover the full range of ratios accessible during membrane photoswitching, with an estimated error margin of ±5% of the given value to account for mixing precision as well as cis fraction uncertainties in the preconditioned samples.
The referencing method developed to determine the stock cis percentage is, as far as we know, novel in this field; and it can prove generally useful for analysis of systems where measurements in bulk (e.g. membrane UV-Vis spectra) cannot be interpreted with reference to more easily characterised molecular measurements (e.g. UV-Vis spectra in monomer solution), see Methods and Supporting Information.
The SAXS measurements for these calibrant ratios are summarized in Fig. 1b. The Xray intensity distributions are typical for lipid bilayer samples. [27][28] The distributions vary strongly with the trans-to-cis ratio, e.g. the intensity dip around = 0.05 Å −1 shifts consecutively to higher q-values with increasing cis content. This indicates that the membrane thickness decreases with increasing cis isomer fraction. To extract the head-to-head distances , we model the SAXS intensities in Fig. 1b by an established lipid bilayer electron density profile, using the software SasView 29 (see supporting information section S5). The results of this analysis are shown in Fig. 1c.
The dark-adapted state shows a head-to-head-distance of (0 ± 5% ) = 40.9 ± 0.6 Å. The analysis reveals that the thickness of the azo-PC membrane depends almost linearly on the trans-to-cis ratio over a broad range. Increasing the percentage of cis azo-PC thins the membrane down to (83 ± 5% ) = 33.0 ± 0.5 Å for 83% cis. Thus, the membrane thickness changes by 8 Å.
In the following measurements, we us this relation between membrane thickness and cis fraction to infer the cis fraction of various photostationary states in response to illumination and buffer conditions (Fig. 1) Fig. 2, the full data set is shown in Fig. S3 and Fig. S4a.  Fig. S4) indicates cis contents around 64%. This is not unexpected, given the blueshifting of azobenzene absorption spectra seen upon going from molecular solutions to assembled systems. 18 However, when using blue light to photoswitch from a cis-to a trans-enriched state, the outcomes depend greatly on whether the system is buffered or not. In DI water, the maximal blue light photostationary state membrane thickness is = 39.0 ± 0.3 Å (Fig 2b, squares, dotted line), i.e. 30% cis isomer still remains. Instead, in PBS or 1x TE buffer, the membrane thickness increases to = 42.3 ± 0.4 Å (Fig 2a, squares, dotted line), i.e. only 3% cis isomer remains. Optical control of vesicles is therefore highly efficient in these buffered aqueous solutions. Experiments with simple NaCl solutions are ongoing, with results so far indicating that ionic strength is key to efficient photoswitching towards trans-enriched states in lipid vesicles: an effect that is not precedented for these molecules in molecular solutions.
Next, we explored methods to improve the limited optical control in DI water. For this purpose, we used X-ray exposures to deposit a certain X-ray dose into the vesicle solution, i.e. a certain energy of absorbed X-rays per mass of the exposed sample. 30 The X-ray dosing experiment was conducted as follows. First, we prepared the vesicles in a high cis fraction by UV-A illumination. We exposed the sample to a rather high Xray energy of 54 keV for 60 s. Five such consecutive exposures yield an identical SAXS signal, i.e. there is no sign of any hard X-ray induced effects after 5 min in total, even though this experiment was performed at a high brilliance Petra III synchrotron beamline with full beam on the sample, cf. Fig 3a. This finding is in agreement with the fact that the cross section for photoabsorption is dramatically reduced for high X-ray energies.
Depositing high doses in water requires soft X-rays (discussion in the SI section S6).
Such soft X-rays give rise to radiolysis of water, and oxidising and reducing radicals and reactive species, 31-32 which may provide pathways for catalytic redox-based unidirectional switching towards the thermodynamic groundstate (all-trans). 15 Therefore, we tested also the change of membrane thickness during six consecutive 8 keV X-ray exposures each of 2 s (Fig. 3a). After each X-ray exposure, a shift of the SAXS pattern was visible. To quantify the thickness change of the photomembrane, we modelled to extract the head-to-head distances, which increased dramatically after each soft X-ray exposure, depending on the total delivered X-ray dose. Remarkably, the final mean membrane thickness of = 42.5 ± 0.3 Å (Fig. 3b, star and square datapoints at high dose, dashed line; Fig. S4b) matches the thickness of the darkadapted all-trans state = 41.9 ± 0.9 Å (Fig. 2b, circular data points from repeated measurements, solid line). This is a direct indication of quantitative switching obtained by soft X-ray exposure.
These soft X-ray induced changes are fully reversible (isomer state of the azobenzene photoswitch) and should not be mistaken as irreversible radiation damage. They can however be employed as a reversible X-ray dose readout. To demonstrate this, we plot the membrane thickness as observed by hard X-ray SAXS in response to soft X-ray exposure with dose expressed in kGray [kGy] in Fig. 3b. The calculation of the dose, a routine calculation in radiation protection, is explained in the SI. Here, quantitative switching starts above 100 kGy, and saturates for an X-ray dose above 700 kGy. We propose that the measurement of azo-PC cis to trans transition for SUVs in DI water can therefore be used to read out the effective X-ray dose in a regime of up to 700 kGy. This range may help to calibrate critical doses for biological SAXS experiments which can range from 51 kGy, 33 to 400 kGy, 34 to 284 -7056 kGy. 31

Conclusions and summary
SAXS with hard X-rays provides a direct read out of the membrane bilayer thickness, which we show has a linear correlation to photolipid isomerization fraction.
The 8 Å thickness change accessible to photolipid membranes under photoswitching in buffered systems (20% of membrane thickness) is massive, and far beyond the effects achievable by temperature or temperature jumps in conventional lipids. 21-22, 24, 35 The huge membrane thickness change observed here may benefit from ordering effects in the trans state related to H-aggregate formation as observed in spectroscopic studies. 18 Hydrophobic matching of membrane thickness with trans-membrane proteins is a wide research field that could benefit enormously from the large thickness change effects observed here. 36 Due to the rapid development of high power UV-A LEDs, and the availability of pulsed UV-A lasers at synchrotron sources, 37 we expect that future experiments with pulsed illumination may allow membrane transition times of few ms and below. At these short timescales, the hydrodynamic coupling of the intercalated water to the photolipids may give rise to interesting transient phenomena. 21 We also show that it is possible to explore a still broader range of membrane bilayer thicknesses by mixing preconditioned photolipids before assembling membranes, rather than relying only on in situ photoswitching, with a correspondingly greater expected range of properties.
In buffered solutions, the blue light photostationary states (3% residual cis) closely approach the dark-adapted state (0% residual cis); i.e. photoswitching experiments in physiologically stabilized environments are efficient. However, the blue light photostationary states of vesicles in DI water (30% residual cis) do not approach the dark-adapted state. For this purpose, quantitative cis-to-trans switching schemes are needed. Here, we show that soft X-rays of 8 keV can induce quantitative switching for these studies, which has not been realised before. For higher X-ray energies such as 17.4 keV and 54 keV, we do not observe such catalytic backswitching, which also highlight the versatility of high energy X-rays as low-dose probes. The energy dependent X-ray dose for samples with high water fraction has a minimum at 36 keV ( Fig. S6), whereas the achievable SAXS signal is almost independent of the chosen beam energy (Fig. S6, inset). This suggests a favourable energy regime of 30 to 42 keV for SAXS experiments on radiation damage sensitive samples (further discussion in section S6 of the supporting information). Our experiments demonstrate that high quality SAXS data may be obtained even for weakly scattering biological samples.

Author Contributions
MFO performed vesicle preparation, and SAXS experiments and X-ray data analysis, and wrote the manuscript; AMD performed synthesis of azo-PC and FAzoM, and spectroscopic and HPLC analyses of cis/trans isomer ratios; AB developed a protocol for photolipid SUV preparation; HA enabled the SAXS experiments at ELETTRA and provided in depth expertise on SAXS calibration and data conversion; OTS designed cis/trans ratio measurements and wrote the manuscript; BN designed the study, joined the X-ray experiments, discussed data interpretation, and wrote the manuscript.