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Development of Smart Optical Gels with Highly Magnetically Responsive Bicelles Stéphane Isabettini, Sandro Stucki, Sarah Massabni, Mirjam Eva Baumgartner, Pernille Qwist Reckey, Joachim Kohlbrecher, Takashi Ishikawa, Erich Josef Windhab, Peter Fischer, and Simon Kuster ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17134 • Publication Date (Web): 20 Feb 2018 Downloaded from http://pubs.acs.org on February 20, 2018

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ACS Applied Materials & Interfaces

Development of Smart Optical Gels with Highly Magnetically Responsive Bicelles Stéphane Isabettini*,a, Sandro Stuckia, Sarah Massabnia, Mirjam E. Baumgartnera, Pernille Q. Reckeya, Joachim Kohlbrecherb, Takashi Ishikawac, Erich J. Windhaba, Peter Fischer*,a, and Simon Kustera a

Laboratory of Food Process Engineering, ETH Zürich, Schmelzbergstrasse 7, 8092 Zurich,

Switzerland. b

Laboratory for Neutron Scattering and Imaging, Paul Scherrer Institute, 5232 Villigen PSI,

Switzerland. c

Biomolecular Research Laboratory, Paul Scherrer Institute, 5232 Villigen PSI, Switzerland.

KEYWORDS Bicelle, hydrogel, gelatin, smart material, magnetically responsive, phospholipids, lanthanides, optical properties

ABSTRACT Hydrogels delivering on-demand tailorable optical properties are formidable smart materials with promising perspectives in numerous fields including the development of modern sensors and switches. The essential quality criteria being a defined and readily measured response to

ACS Paragon Plus Environment

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environmental changes. Lanthanide ion (Ln3+) chelating bicelles are interesting building blocks for such materials due to their magnetic-responsive nature. Imbedding these phospholipid-based nanodiscs in a magnetically aligned state in gelatin permits an orientation-dependent retardation of polarized light. The resulting tailorable anisotropy gives the gel a well-defined optical signature observed as a birefringence signal. These phenomena were only reported for a single bicelle-gelatin pair and required high magnetic field strengths of 8 T. Herein, we demonstrate the versatility and enhance the viability of this technology with a new generation of aminocholesterol (Chol-NH2) doped bicelles imbedded in two different types of gelatin. The highly magnetically responsive nature of the bicelles allowed to gel the anisotropy at commercially viable magnetic field strengths between 1 and 3 T. Thermoreversible gels with a unique optical signature were generated by exposing the system to various temperature conditions and external magnetic field strengths. The resulting optical properties were a signature of the gel’s environmental history, effectively acting as a sensor. Solutions containing bicelles simultaneously aligning parallel and perpendicular to the magnetic field directions were obtained by mixing samples chelating Tm3+ and Dy3+. These systems were successfully gelled, providing a material with two distinct temperature-dependent optical characteristics. The high degree of tunability in magnetic response of the bicelles enables encryption of the gel’s optical properties. The proposed gels are viable candidates for temperature tracking of sensitive goods and provide numerous perspectives for future development of tomorrow’s smart materials and technologies.

INTRODUCTION Smart hydrogels are soft materials that may be engineered to selectively respond to a trigger or stimuli, giving rise to a multitude of abrupt changes in physical properties and functionality.1,2,3


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Reported stimuli include temperature change, pH conditions, solvent nature, ionic composition, electric fields, magnetic fields, light intensity, and introduction of specific ions or molecules.4,5,6 The hydrogel’s response may originate from the polymer properties, the imbedded materials (such as particles), or a combination of both. Smart hydrogel systems provide countless engineering possibilities, interesting for a wide range of applications comprising nanoscale templates, biomedical devices, drug delivery, tissue engineering and sensor development.7,8,9 Magnetic fields are important external stimuli owing to their commercial availability and noninvasive nature. As a consequence, smart hydrogels have been engineered to respond to external magnetic fields by associating magnetic nanoparticles to the gel network.2,10-16 Polymolecular assemblies such as micelles, bicelles and liposomes are additional viable building blocks for the design of modern smart hydrogel systems.17-21

Bicelles are sub-micrometer sized disk-like polymolecular assemblies formed from amphiphiles in an aqueous solution. They are particularly interesting due to their ability of aligning in the presence of a magnetic field. This feature, combined with their versatility in design and viability as membrane model systems, have been at the source of their scientific success in the study of membrane bound and associated biomolecules.22 Recent efforts aim at expanding the field of applications of bicelles for dermal treatments, pharmaceutical drug delivery platforms, scaffolds for nanomaterial synthesis, or for the generation of smart optical gels.20,21,23-25 These technologies require the development of synthetic lipids specifically designed to deliver a defined physicochemical properties.26-29 For example, highly magnetically responsive bicelles composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC) and the corresponding lanthanide ion (Ln3+) chelating 1,2-


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dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid (DMPEDTPA) or 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid (DPPE-DTPA) synthetic phospholipid are required to deliver switchable anisotropy in gelatin-based optical hydrogels.20 The bicelle’s ability to associate with numerous paramagnetic Ln3+ is at the basis of their unprecedent magnetic response. The magnetic energy is described by

= −

where is the magnetic field strength, µ0 is the magnetic constant, n is the

aggregation number, is the Avogadro constant, and ∆χ is the magnetic susceptibility of the lipids within the bilayer.30,31 Therefore, for a given magnetic field strength B, the magnetic alignability of the bicelles may be enhanced by increasing their size (aggregate number n) and altering the magnetic susceptibility ∆χ of the phospholipids composing the bilayer. The former is readily achieved through the lipid composition or by including cholesterol (Chol-OH) into the bilayer.32-35 Fabrication procedures enable the formation of large and optimally alignable bicelles.36 The magnetic susceptibility ∆χ may be tuned to achieve a strong magnetic response by changing the chelated Ln3+ and introducing aminocholesterol (Chol-NH2) into the bilayer.37,38 The choice of the chelated Ln3+ allows for additional tailoring of the bicelle’s alignment direction.21,31,37,39,40 However, it is also possible to engineer the headgroup chemistry of the Ln3+chelating synthetic phospholipid to switch the sign of the magnetic susceptibility and inverse the alignment direction of the bicelles without altering the nature of the chelated Ln3+.41

Imbedding magnetically aligned bicelles in a porcine gelatin network was previously demonstrated by Liebi et al.20,21 These gels delivered unique optical properties with different spatial birefringence. The degree of anisotropy could be tuned by the magnetic field strength and the system’s thermal history, resulting in gels with highly tunable optical signatures. The


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potential application of these systems as temperature sensors was revealed as the anisotropic bicelles could be thermoreversibly set back to an isotropic state by cycling through the melting point of gelatin. Although promising, these features were only demonstrated with one bicellegelatin pair: DPPC/Chol-OH/DPPE-DTPA/Ln3+ (molar ratio 16:4:5:5) bicelles in porcine gelatin. This limits the versatility of the technology to the very defined magnetic properties of the imbedded bicelles and the gelling characteristics of porcine gelatin. Magnetically alignable bicelles must exist at temperatures above the gelling point of gelatin (22 °C in the case of a 10% (w/w) porcine gelatin solution cooled at 1 °C/min). Although DPPC/Chol-OH/DPPE-DTPA/Ln3+ (molar ratio 16:4:5:5) bicelles fulfill this requirement, their ability to deliver a high degree of anisotropy was bound to the depletion mechanism occurring in the vicinity of the gelatin aggregates, inducing bicelle stacking. High magnetic field strengths of 8 T were required to deliver gels with a reasonable optical signature. Recent developments with DMPC/DMPEDTPA/Ln3+ based systems resulted in polymolecular assemblies with unprecedented high magnetic response in the 1-2 T range, coupled with a high resistance to temperature.36,37 These novel systems are prime candidates for the future development of switchable optical gels obtained from magnetically responsive bicelles.

Herein, we aim to enhance the versatility and viability of this bicelle-gel technology with a fourstep investigation. In a first step, the DPPC/Chol-OH/DPPE-DTPA/Ln3+ (molar ratio 16:4:5:5) bicelles were replaced with DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles in 10% (w/w) porcine gelatin. The presence of Chol-NH2 in the bilayer significantly enhances the magnetic response of the bicelles by selectively altering the magnetic susceptibility ∆χ of the Ln3+-chelating phospholipids.37 Moreover, the steroid moiety generates a liquid-ordered state in


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the phospholipid bilayer, enabling the magnetically alignable bicelles to exist at temperatures as high as 35 °C instead of 21 °C observed in pure DMPC-based bicelles.21,36,38 The possibility of generating optical gels with this novel bicelle system was demonstrated and further investigated by SANS and birefringence measurements. In a second step, the magnetic response of the DMPC/Chol-NH2/DMPE-DTPA/Ln3+ (molar ratio 16:4:5:5) bicelles was greatly enhanced with optimized fabrication procedures.36 The largest and most magnetically responsive species were required to generate optically active gels at magnetic field strengths lower than 3 T. The alignment direction of the imbedded bicelles was tailored by altering the nature of the chelated Ln3+. Tm3+ was employed for perpendicular alignment with respect to the field direction and Dy3+ for parallel alignment. In a third step, the porcine gelatin was replaced by cold-water fish gelatin to ascertain the versatility of the proposed technology. The different amino acid profile of fish gelatin offers the possibility to work at lower temperatures before gelling occurs.42-44 Since the thermal energy of the solution acts against bicelle alignment, working at lower temperatures is beneficial to achieve the highest degree of alignment.21,30,35 In a fourth and final step, the additional degrees of freedom offered by the DMPC/Chol-NH2/DMPE-DTPA/Ln3+ (molar ratio 16:4:5:5) system in fish gelatin was employed to gel bicelles simultaneously aligning in two directions. The resulting optical characteristics of the gel were investigated as a function of the temperature history. We reveal systems with highly specific optical signatures, offering promising perspectives for the development of modern temperature sensors and switches.


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METHODS Materials 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and the hexa-ammonium salt of 1,2dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid (DMPEDTPA) phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL) in powdered form (99%). Anhydrous thulium(III) chloride (99.9%), anhydrous dysprosium(III) chloride (99.9%), porcine gelatin (type A), gelatin from cold-water fish skin, ethanol stabilized chloroform, and methanol (99.8%) were purchased from Sigma-Aldrich (Buchs, Switzerland). Sodium dihydrogen phosphate dihydrate and di-sodium hydrogen phosphate were purchased from Merk and employed to make a 50 mM phosphate buffer at a pH value of 7.4 in ultrapure water. D2O (99.9 atom %D) for SANS measurements was purchased from ARMAR Chemicals (Döttingen, Switzerland). Chol-NH2 was synthesized and characterized as described elsewhere.37,45 HR-MS(+): calculated for C27H48N [M+H]+: 386.3787. Found: 386.3781. ∆m/z: 1.5 ppm. 1H NMR (400 MHz, CDCl3) δ: 5.32 (d, 1H), 2.61 (m, 1H), 0.85-2.16 (m, 43H), 0.67 (s, 3H) ppm. 13C NMR (100 MHz, CDCl3/MeOD 4:1) δ: 140.69, 120.97, 56.53, 55.93, 51.25, 49.98, 42.03, 41.67, 39.54, 39.25 37.74, 36.21, 35.93, 35.56, 31.62, 31.57, 31.15, 27.96, 27.71, 23.98, 23.57, 22.39, 22.13, 20.73, 18.96, 18.34, 11.48 ppm.

Preparation of bicelles in gelatin solutions Bicelles were prepared following previously described procedures.20,36,46 10 mg/ml stock solutions of DMPC, DMPE-DTPA and Chol-NH2 were prepared in ethanol-stabilized chloroform. 10 mM stock solutions of thulium(III) chloride and dysprosium(III) chloride were prepared in methanol. The required quantities of each stock solution were mixed in a 25 ml


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round bottom flask to obtain DMPC/Chol-NH2/DMPE-DTPA/Ln3+ with a molar ratio 16:4:5:5 and a total lipid concentration of 15 mM. A dry lipid film was obtained by evaporating the organic solvents under vacuum at 40 °C. The required volume of a 50 mM phosphate buffer at a pH value of 7.4 was added to the dry lipid films. Samples for SANS experiments were prepared in an equivalent phosphate buffer in D2O. Five freeze thawing cycles were conducted in liquid nitrogen to 60 °C for hydration of the dry lipid film. The samples were extruded (Lipex Biomembranes, Vancouver, Canada) at 60 °C either 10 times through 200 nm pores and another 10 times through 100 nm pores, or 10 times through 800 nm pores using polycarbonate membranes (Sterico, Dietikon, Switzerland). Either 10 % (w/w) porcine gelatin or 30% (w/w) fish gelatin was subsequently added to the bicelle samples and dissolved by heating at 60 °C for several minutes. SANS Small angle neutron scattering SANS measurements were conducted on the SANS-I beamline at PSI, Villigen, Switzerland. Samples were prepared in a 50 mM D2O phosphate buffer with a pH value of 7.0. The neutron wavelength was fixed at 0.8 nm and a 2D 3He detector was placed at 6 m from the sample, covering a q-range of 0.1 - 0.8 nm-1. A superconducting magnet was employed, capable of supplying a 8 T magnetic field perpendicular to the neutron beam. The alignment factor Af was computed from the 2D scattering pattern of the sample in the q-range of 0.3 - 0.4 nm-1 to quantify the magnetic alignment of the samples as described previously.20,21,3538,40,41

The Af is the second cosine Fourier coefficient of the normalized azimuthal scattering

intensity (): A () =

∑ ()cos (2) ∑ ()


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The Af theoretically ranges from -1 (anisotropic neutron scattering pattern aligned parallel to the magnetic field direction, bicelles aligned perpendicular) to +1 (anisotropic neutron scattering pattern aligned perpendicular to the magnetic field direction, bicelles aligned parallel). Isotropic samples yield an Af value of 0. In practice, fully aligned DMPC/Chol-NH2/DMPE-DTPA/Ln3+ bicelles reach maximal Af values of ± 0.80 due to the intrinsic thickness of the resulting 2D neutron scattering patterns.37 Bicelles aligning parallel to the magnetic field direction have the freedom to rotate without experiencing any change in potential energy. Species aligning perpendicular to the field direction do not possess this additional degree of freedom.20,21,40

Birefringence Birefringence measurements were undertaken as a complementing means of evaluating the magnetic alignment of bicelles.20,21,29,35,40,46,47 The samples were loaded in a 10 mm thick temperature-controlled quartz cuvette (Hellma, Germany) and placed in the heart of a heliumcooled cryogenic magnet (Cryogenic Ltd, London, UK) capable of supplying a maximum magnetic field strength of 5.5 T. Monochromatic light from a diode laser at 635 nm was consecutively directed with non-polarizing mirrors through five elements: (1) a crossed linear polarizer (Newport, Irvine, USA), (2) a photoelastic modulator PEM-90 (Hinds Instruments, Hillsboro, USA), (3) the sample in the quartz cuvette, (4) a second crossed linear polarizer oriented at 90° of the first (Newport, Irvine, USA), and (5) a photo detector (Hinds Instruments, Hillboro USA). The photoelastic modulator operated at 50 kHz with an amplitude A0 set to 2.405 rad, making the DC component independent of birefringence. Two lock-in amplifiers SR830 (SRS, Sunnyvale, USA) recorded the first !" and second ! harmonic of the AC signal used to evaluate the degree of retardation # of the polarized light with


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# = arctan (

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! ) (*+ ) , !" )" (*+ )

where )" and ) are Bessel functions of the first kind, with )" (2.405) = 0.5191 and ) (2.405) = 0.4317. The retardation # was then converted into a birefringence signal ∆67 to quantify the degree of anisotropy in the material using 89

∆67 = −

:; where < was the wavelength of the laser at 635 nm and = is the thickness of the sample (10 mm). The birefringence signal ∆67 was normalized with the values obtained at 0 T and a 1 °C/min gradient was employed for temperature cycles.

Cryo-TEM Cryo transmission electron microscopy cryo-TEM is an invaluable method to characterize the architecture of polymolecular assemblies such as bicelles.21,29,45,48 Samples were prepared without gelatin as described previously.29,36,37 The lipid assemblies were suspended as thin aqueous films on holey carbon grids (Quantifoil, Jena, Germany) at 5 °C before being flashfrozen in liquid ethane. Samples were analysed with JEM2200FS (JEOL, Japan) equipped with a field emission gun and an in-column energy filter (JEOL) operated at 200 kV. A 4096 x 4096 CMOS camera (F416, TVIPS, Germany) with 5 - 10 µm under focus was used to increase the contrast. High contrast particles on the micrographs are ice crystals resulting from the freezing procedure. The carbon walls of the holey grid appear as high contrast areas on the micrographs.


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DLS Dynamic light scattering DLS measurements of the bicelle samples were performed on a Malvern Zetasizer Nano ZS with a He–Ne laser fixed at 901 operating at 633 nm. Samples were measured undiluted with non-invasive backscatter technology (NIBS) at 5 °C and in the absence of gelatin.

RESULTS AND DISCUSSION

Generating optical gels with DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles in 10% (w/w) porcine gelatin DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles are highly alignable in magnetic fields and deliver a strong thermal resistance.37,38 The high magnetic response originates from the altered magnetic susceptibility of the bilayer phospholipids in the presence of Chol-NH2.38 The liquid ordered phase induced by the presence of the steroid molecule gives rise to the thermal resistance of the bicelles.29,35,38,49,50 These systems are alignable above the gelling temperature of porcine gelatin of 22 °C on cooling at 1 °C/min. Therefore, the bicelles may be entrapped in an aligned state in the gelatin network, fulfilling the requirements for delivering optical properties to the gel. The highly tunable magnetic properties of DMPC-based systems make them promising candidates and viable alternatives to the DPPC/Chol-OH/DPPEDTPA/Tm3+ (molar ratio 16:4:5:5) bicelles proposed by Liebi et al. for delivering switchable anisotropy to the gelatin gels.20 This possibility was investigated by replacing the reference DPPC-based bicelle systems with DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5).


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The samples were prepared following the same protocols described by Liebi et al to guarantee comparability.20,21,35 After hydration of the dry lipid film with 50 mM phosphate buffer followed by freeze thawing cycles, the samples were consecutively extruded 10 times at 60 °C through membranes with a pore diameter of 200 and 100 nm, respectively. Bicelles with an average hydrodynamic diameter DH of 134 ± 23 nm were obtained, in line with previous findings.35,37,38 10% (w/w) porcine gelatin was subsequently dissolved in the bicelle solution at 60 °C and the sample was stored in a gelled state at 5 °C before measurement. The magnetic alignment of the bicelles as a function of temperature was evaluated through computation of alignment factors Af in SANS at 8 T and the resulting optical properties were monitored by birefringence at 5.5 T.


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Figure 1. Alignment factor Af as a function of temperature of DMPC/Chol-NH2/DMPEDTPA/Tm3+ (molar ratio 16:4:5:5) bicelles in 10% (w/w) porcine gelatin. The bicelles were prepared by extrusion through membranes with a pore diameter of 200 and 100 nm at 60 °C. The sample was subject to heating and cooling cycles at 1 °C/min either in the absence of a magnetic field or at 8 T. The Af were either negative or positive for bicelles aligning perpendicular or parallel to the magnetic field direction, respectively. The arrows in the legend represent the changing variable (either the temperature or the magnetic field strength) in the corresponding curve, which is either decreasing (downwards arrow) or increasing (upwards arrow). Triangular data points were recorded when ramping down to, or at 0 T. Circular data points were recorded when ramping up to, or at 8 T. Key positions along the temperature cycle are chronologically labelled from A to F and a corresponding schematic of the polymolecular assemblies is provided. Vesicles are represented with circles and the planar bicelles as lines. Bicelle alignment is shown assuming a vertical magnetic field direction. Light blue represents the liquid state of the surrounding solution and dark blue corresponds to the gelled state.

The Af obtained from the 2D neutron scattering patterns as a function of temperature for DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles in 10% (w/w) porcine gelatin are presented in Figure 1. The sample was first heated from 5 to 45 °C at 1 °C/min in the absence of a magnetic field (Figure 1, point A to B). The gelatin melted at 33 °C, freeing the bicelles into solution. Above 45 °C (Figure 1, point B), most of the bicelles transformed into non-alignable vesicles. The bicelle-to-vesicles transition is thermoreversible and originates from the disordered liquid state of the bilayer lipids.36 The magnetic field was ramped up to 8 T at 45 °C causing the absolute value of the Af to rise from 0 to - 0.30. The anisotropy arised from partial stacking of the bicelles as evidenced by the appearance of Bragg peaks in the 2D neutron scattering pattern in Figure 2A. An analogous phenomenon was reported in the reference DPPC/Chol-OH/DPPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles in gelatin.20 The stacking probably originates from the thermodynamic depletion interactions occurring between the bicelles and the gelatin molecules.20,21 Stacking promotes the magnetic aligning by enhancing the aggregation number n. The bicelle stacks have a larger cumulative magnetic orientation energy, allowing them to align at 45 °C.


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Figure 2. A) 2D neutron scattering patterns (0.1 - 0.8 nm-1) of DMPC/Chol-NH2/DMPEDTPA/Tm3+ (molar ratio 16:4:5:5) bicelles prepared by extrusion through 200 and 100 nm pore membranes in 10% (w/w) porcine gelatin upon cooling at 8 T from 45 to 26 °C. The solution is liquid under these conditions as gelling only occurs at 22 °C on cooling.20 The magnetic field direction B was horizontal as shown with a white arrow. Bragg peaks were apparent in the scattering patterns at 45, 35 and 30 °C and disappeared at 26 °C. B) Birefringence signal of the sample as a function of temperature. The solution was cooled from 58 to 4 °C at 1 °C/min under a 5.5 T magnetic field (blue line). The magnetic field was subsequently ramped down to 0 T at 5 °C and the sample was heated from 4 to 58 °C at 1 °C/min (red line).

The absolute value of the Af increased with decreasing temperature at a constant field strength of 8 T (Figure 1), highlighting an enhanced degree of alignment. This result is largely attributed to the regeneration of alignable bicelles from non-alignable vesicles upon cooling.21,35,36 The bicelle species remained partially stacked and the anisotropy strengthened upon cooling to 34 °C (Figure 1, point C). The absolute value of the Af rose at an increasing rate from 34 °C, reaching a peak


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value of -0.78 at 30 °C before decreasing again to -0.67 moments before gelling at 26 °C. This phenomenon should not be interpreted as a temporary enhanced state of alignment. This result is rather attributed to the disappearance of the Bragg peaks in this temperature window, causing a change in shape of the anisotropic 2D neutron scattering patters from which the Af values are computed, see Figure 2A. The bicelles were no longer stacked when gelled. This result is in sharp contrast to the reference DPPC/Chol-OH/DPPE-DTPA/Tm3+ (molar ratio 16:4:5:5) system that remained stacked in the gel.20 Although the stacking behavior enabled alignment at high temperatures, the naturally high magnetic alignability of DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles was responsible for delivering a high degree of anisotropy moments before gelling. This ultimately guarantees the gel’s optical properties and stresses the importance of working with intrinsically highly magnetically responsive polymolecular assemblies for generating switchable anisotropy in optical gels. The Af was stable as the anisotropic 2D neutron scattering pattern remained unchanged upon further cooling. The bicelles were fixed into position by the gel until the target temperature of 5 °C was reached (Figure 1, point E). The Af was also unchanged as the magnetic field was ramped down to 0 T at 5 °C, proving the successful gelling of the aligned bicelles. Although these systems did not benefit from an enhanced alignment due to stacking, a Af value of - 0.70 at 5 °C and 0 T was achieved. The anisotropic gel may be dismantled upon heating above 33 °C, where the bicelles were freed from the gelatin network and returned to an isotropic state in solution at 0 T (Figure 1, point E to F). The process was thermoreversible and the bicelles may be re-aligned and gelled starting from point B of Figure 1. The SANS experiments in Figure 1 demonstrated the possibility of imbedding DMPC/CholNH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles in 10% (w/w) porcine gelatin in a highly


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aligned state. However, the optical properties remained to be demonstrated. Analogously to what was done for the reference bicelle system by Liebi et al., the anisotropy for electromagnetic wave transmission was assessed by monitoring the birefringence signal as a function of temperature in Figure 2B.20 The birefringence signal is proportional to the degree of alignment of the bicelles, acting as a complementary means of quantifying anisotropy alongside the Af values computed from SANS.20,21,29,35-37,40,41,47 A cooling cycle from 58 to 4 °C at 1 °C/min was applied under a 5.5 T field to gel the bicelles in an aligned state. The high turbidity of the solution did not permit monitoring of the birefringence signal from 58 to 35 °C as evidenced by the noisy signal in Figure 2B and the spectrophotometry results in Figure S1. However, the system became transparent in the pre-gelling and gelled state below 35 °C and the birefringence signal could be monitored. The peak in birefringence signal between 35 and 25 °C was analogous to that observed in the Af values in SANS in Figure 1. It may be attributed to the disappearance of bicelle stacking before complete gelation. The birefringence signal is also influenced by molecular organization of the bilayer and by the geometry of the polymolecular assemblies.40 This is not the case for the Af values computed from 2D neutron scattering patterns. The changes in birefringence signal upon cooling from 25 to 4 °C and heating back to 25 °C in the gelled state in Figure 2B were caused by these intrinsic effects. For example, the peak in birefringence observed at 26 °C on heating could be caused by the appearance of a concentric hole in the bicelles, as previously reported in steroid-doped assemblies.21,35,36,38,40 The bicelle alignment was not altered in this temperature range as the Af values remained constant, see Figure 1. The birefringence signal remained unchanged when the magnetic field was ramped down to 0 T at 4 °C. Consequently, the bicelles were successfully gelled in an aligned state. The gel was optically active, supporting the affirmations made in the discussion of the Af results in Figure 1.


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Subsequent heating to 58 °C at 0 T removed the optical signature as the gel melted and the bicelles returned to an isotropic state above 33 °C. This was evidenced by a zeroing of the birefringence signal and the reappearance of a turbid solution, supported by spectrophotometry measurements in Figure S1. Gels with defined optical characteristics may be generated by working at various field strengths and gelling the bicelles at different degrees of alignment. The magnetic response and alignment direction may also be tailored with the chelated Ln3+.21,31,37,39,40 These possibilities will be demonstrated along with other variables, including the nature of the gelatin, which define the optical properties of the resulting gel. The multitude of available variables offers the possibility of generating gels with uniquely different optical signatures.

Enhancing the magnetic response of DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles in 10% (w/w) porcine gelatin The gels produced by DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles in 10% (w/w) porcine gelatin required a strong magnetic field strength of 8 T to obtain respectable optical properties. In order to envisage future applications, the magnetic response of these systems must be enhanced, aiming to achieve anisotropy at magnetic field strengths ranging from 1 T (permanent magnets) to 3 T (commercially available electromagnets). In a first step, we enhanced the magnetic response of the bicelles by increasing their size with an altered fabrication procedure. Larger bicelles contain more lipids capable of contributing to the cumulative magnetic energy Emag (increased aggregate number n) of the bilayer.32-36 The samples were extruded 10 times through membranes with a pore diameter of 800 nm at 60 °C instead of


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200 and 100 nm. The solution was composed of vesicles at 60 °C, whose size was tailored by the extrusion process.35,38 Bicelles were subsequently regenerated upon cooling to 5 °C and their dimensions were dictated by the vesicle precursors. We have demonstrated the viability of these fabrication techniques to deliver bicelles with unprecedent high magnetic alignments.36 In a second step, the lanthanide ion was changed to Dy3+, which has a larger and switched molar magnetic susceptibility ∆χ.21,31,37,39,40 The negative ∆χ of Dy3+ adds on to the naturally negative ∆χ of the bilayer phospholipids, enhancing the contribution of the individual lipids to the total Emag of the bilayer. A higher magnetic response is expected at lower magnetic field strengths.40 The switched ∆χ of Dy3+ resulted in bicelles aligning parallel to the magnetic field direction, instead of perpendicular with Tm3+. This tunable alignment direction offers additional orientation-dependent optical properties to the gels.21 The size and morphology of the DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles prepared by repeated extrusion through a membrane with a pore diameters 800 nm was assessed in the absence of gelatin at 5 °C by DLS and cryo-TEM. Species with an average hydrodynamic diameter DH of 442 ± 143 nm were obtained. The sample was considerably more polydisperse than the smaller bicelles obtained by extrusion through membranes with a pore diameter of 200 and 100 nm. The cryo-TEM micrographs of the sample confirmed the existence of large and polydisperse disk-like assemblies in Figure 3. The planar nature of the bicelles was highlighted by the weak contrast obtained when viewed from top-on and further confirmed from side-on view as the assembly appeared as a dark flat line.


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Figure 3. Cryo-TEM micrographs of a DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) sample prepared by repeated extrusion through a membrane with a pore diameter of 800 nm and flash-frozen at 5 °C in the absence of gelatin. Three different micrographs of the same sample are shown, separated with white lines. The scale bars represent 200 nm. Dark patches are water crystals resulting from the flash-freezing procedure. The wall of the microgrid appears as a dark line on the bottom-right of the lowest micrograph.


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Figure 4. Alignment factor Af as a function of temperature of A) DMPC/Chol-NH2/DMPEDTPA/Tm3+ and B) DMPC/Chol-NH2/DMPE-DTPA/Dy3+ (molar ratio 16:4:5:5) bicelles in 10% (w/w) porcine gelatin. The bicelles were prepared by extrusion through membranes with a pore diameter of 800 nm at 60 °C. The sample was subject to heating and cooling cycles at 1 °C/min either in the absence of a horizontal magnetic field (white arrow) or at 8 T. The Af were calculated from 2D neutron scattering patterns and were either negative or positive for bicelles aligning perpendicular or parallel to the magnetic field direction, respectively. Isotropic bicelles yield an Af value of zero. Af values approaching ± 0.80 correspond to full bicelle alignment.37,40 Key positions along the temperature cycle are chronologically labelled from A to F, analogously to Figure 1 and the same legend was employed to facilitate comparison. 2D neutron scattering patterns (0.1 - 0.8 nm-1) of the samples were recorded upon cooling at 8 T from 45 to 5 °C. The Tm3+ chelating bicelles aligned perpendicular to the field direction (parallel neutron scattering pattern), while the Dy3+ chelating bicelles aligned parallel (perpendicular neutron scattering pattern).

The Af of DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles in 10% (w/w) porcine gelatin fabricated by extrusion through pores with a diameter of 800 nm in Figure 4A evolved analogously with temperature to the corresponding sample extruded through 200 and


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100 nm pores (Figure 1). The larger bicelle species achieved an Af of -0.75 at 45 °C and 8 T, compared to -0.30 with the sample in Figure 1. The alignment was aided by bicelle stacking as evidenced by the appearance of Bragg peaks in the 2D neutron scattering pattern at 45 °C and 8 T in Figure 4A. However, the 2D neutron scattering pattern at 34 °C and 8 T in Figure 4A showed no evidence of Bragg peaks. Therefore, stacking was lost upon cooling from 34 °C onwards. This did not lead to any change in alignment as the Af was maintained at - 0.75, which is close to full alignment as described in the supplementary information. Upon further cooling, the absolute value of the Af gradually fell until gelling at a value of - 0.60 (see Figure 4A, point D). There was no minimum in Af between 34 and 26 °C, unlike what was observed in the sample composed of smaller bicelles in Figure 1. The loss of alignment on gelling could be attributed to the larger bicelle size, whose alignment is likely disturbed by the formation of the gelatin network.51-53 Nevertheless, the system was successfully gelled in an anisotropic state when ramping the magnetic field down to 0 T at 5 °C (see Figure 4A, point E), delivering optical properties to the gel. A high Af of + 0.70 was also achieved at 45 °C and 8 T when working with DMPC/CholNH2/DMPE-DTPA/Dy3+ (molar ratio 16:4:5:5) bicelles in 10% (w/w) porcine gelatin fabricated by extrusion through pores with a diameter of 800 nm in Figure 4B. The evolution of the Af with temperature was analogous to that of the Tm3+ chelating sample in Figure 4A. However, the Af was switched to positive values due to the opposite alignment direction of the Dy3+ chelating bicelles. The inversed alignment direction was confirmed by the perpendicular scattering of neutrons with respect to the magnetic field direction in Figure 4B. Unlike both Tm3+ chelating bicelles in Figure 1 and 4A, the Dy3+ chelating counterparts showed no sign of stacking along the studied temperature range. This was confirmed by the absence of Bragg peaks in the neutron


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scattering patterns, see Figure 4B. Polymolecular assemblies aligning parallel to the magnetic field direction are free to rotate around the axis defined by the magnet field direction, without experiencing any change in potential energy.39,40 This added degree of freedom could make it energetically unfavorable for the bicelles to stack, which could explain the absence of Bragg peaks in the Dy3+ chelating bicelles. The high degree of alignment achieved with DMPC/Chol-NH2/DMPE-DTPA/Dy3+ (molar ratio 16:4:5:5) bicelles in 10% (w/w) porcine gelatin was exploited to deliver anisotropic gels at lower magnetic field strengths. The gels were prepared following the same heating and cooling protocols under a 1 and 2 T field, delivering Af values of + 0.10 and + 0.25, respectively (see Figure S3). The bicelles successfully delivered optical anisotropy to the gel at commercially viable magnetic field strengths.

Gelling aligned DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles in 30% (w/w) fish gelatin Magnetically aligned DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles were imbedded in gelatin from cold-water fish skin instead of porcine gelatin. The samples were extruded 10 times through membranes with a pore diameter of 800 nm at 60 °C to deliver an optimal magnetic response. Fish gelatin has a lower concentration of imino acids (proline and hydroxyproline) than porcine gelatin, resulting in lower melting and gelling temperatures.43 The super-helix structure of gelatin gels is stabilized by steric restrictions imposed by pyrrolidine rings of the imino acids and by hydrogen bonding between amino acid residues. These physicochemical forces are responsible for stabilizing the ordered conformation of the gelatin


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gel.42-44 Fish gelatin does not gel as effectively as porcine gelatin, requiring lower temperatures and sufficient time to develop a stable network.54 Consequently, the gelatin content in the bicelle solutions was increased to 30 % (w/w), ensuring gel formation within 30 minutes at 5 °C. The lowered gelling temperature was strategic to achieve maximal alignment of the imbedded bicelles. A reduced thermal energy of the solution, coupled with phase changes occurring in the phospholipid bilayer, allowed the assemblies to reach higher degrees of alignment.21,30,35,38 The evolution of the Af with temperature of the DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles in 30% (w/w) fish gelatin in Figure 5A was very different than the corresponding system in 10% (w/w) porcine gelatin revealed in Figure 4A. The 2D neutron scattering patterns showed no evidence of Bragg peaks throughout the cooling process from 45 to 5 °C at 8 T. Consequently, the bicelles did not benefit from an enhanced alignment due to stacking, achieving a weak Af value of -0.10 at 45 °C and 8 T (see Figure 5A, point II). However, the slow gelling kinetics of the fish gelatin at 5 °C provided sufficient time for the regeneration of alignable bicelles from non-alignable vesicles upon cooling at 8 T. A strong regeneration occurred below 28 °C (see Figure 5A, point III), in line with previous results from SANS studies of DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles.38 An equivalent increase in birefringence signal occurred upon cooling (see Figure 5B, point III). An alignment factor of -0.48 and a birefringence signal of 2.5x10-5 was achieved at 5 °C after ramping down the magnetic field, delivering optical properties to the gel (see Figure 5A and 5B, point IV). These results prove the possibility of generating optical properties in different gelatin systems.


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Figure 5. A) Alignment factor Af as a function of temperature at 8 T of DMPC/CholNH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles in 30% (w/w) fish gelatin. B) Birefringence signal as a function of temperature at 5.5 T of DMPC/Chol-NH2/DMPEDTPA/Tm3+ (molar ratio 16:4:5:5) bicelles in 30% (w/w) fish gelatin. The bicelles were prepared by extrusion at 60 °C through membranes with a pore diameter of 800 nm. The samples were subject to heating and cooling cycles at 1 °C/min either with or without an external magnetic field. The Af were calculated from 2D neutron scattering patterns and were either negative or positive for bicelles aligning perpendicular or parallel to the magnetic field direction, respectively. The intensity of the birefringence signal increases with the degree of bicelle alignment, acting as a complementary means of quantifying anisotropy and monitoring the optical properties of the resulting gel. Key positions along the temperature cycle are chronologically labelled in roman numerals from I to V.

The anisotropy delivered by DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles entrapped in 30% (w/w) fish gelatin was lost upon heating to 15 °C at 0 T as the gel melted, see Figure 5. The optical properties were only lost above 33 °C in the analogous system made from porcine gelatin, see Figure 4. This temperature-induced loss of optical properties may be interesting for tracking the temperature history of sensitive goods. For example, a solid gel cube composed of DMPC/Chol-NH2/DMPE-DTPA/Tm3+ (molar ratio 16:4:5:5) bicelles aligned at 8 T and gelled at 5 °C in fish gelatin 30% (w/w) could be placed in the packing material of a product as it is being transported from one location to the other. If the gel cube is no longer


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birefringent upon arrival, the cold-chain was not respected during shipping. The optical properties of the gel effectively act as a temperature sensor. The thermoreversible nature of these materials make them reusable when regenerated with a heating and cooling cycle in the presence of an external magnetic field. Further tuning of the gel’s thermal-resistance with additives could increase the temperature window for applications.54 The melting point of the gel and the corresponding loss of optical properties could be tuned. However, the impact of additives on the bicelle alignment and the stability of the gel’s optical properties over time would need to be evaluated.

Gelling DMPC/Chol-NH2/DMPE-DTPA/Ln3+ (molar ratio 16:4:5:5) bicelles simultaneously aligned in two directions We conclude our investigation by evaluating the possibility of imbedding bicelles simultaneously aligning parallel and perpendicular to the magnetic field direction in a single solution, delivering gels with unique optical properties. This possibility was first demonstrated by mixing 50/50 (v/v) of DMPC/Chol-NH2/DMPE-DTPA/Ln3+ (molar ratio 16:4:5:5) bicelles chelating either Dy3+ or Tm3+ at 5 °C in the absence of gelatin in the supplementary information. The bicelles were prepared by extrusion through membranes with a pore diameter of 800 nm to form maximally alignable species. 30% (w/w) fish gelatin was subsequently added to the 50/50 (v/v) bicelle mixture at 19 °C and cooled down to 5 °C in a 2 mm thick quartz cuvette. The resulting gel was placed in the neutron beam for monitoring of the Af as a function of temperature as described in Figure 6.


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Figure 6. Alignment factor Af as a function of temperature of DMPC/Chol-NH2/DMPEDTPA/Ln3+ (molar ratio 16:4:5:5) bicelles chelating either Dy3+ or Tm3+ mixed 50/50 (v/v) with 30% (w/w) fish gelatin at 19 °C and cooled down to 5 °C (point A). The bicelles were prepared separately prior to mixing by extrusion through membranes with a pore diameter of 800 nm at 60 °C. The sample was subject to heating and cooling cycles at 1 °C/min either in the absence of a magnetic field or at 8 T. Key positions along the temperature cycle are labelled chronologically from A to F and the corresponding snapshots of the 2D neutron scattering patterns (0.1 - 0.8 nm1 ) are provided. Bicelles in solution are schematically represented as flat lines at points B and F and alignment is represented assuming a horizontal magnetic field direction. Species exclusively chelating Tm3+ are shown in red, while those chelating Dy3+ are black. Bicelles simultaneously chelating Tm3+ and Dy3+ are shown in green.

The bicelles aligned under a 8 T magnetic field when the fish gelatin melted at 15 °C in Figure 6 from point A to B. The sample was composed of bicelles simultaneously and strongly aligning parallel (species chelating Dy3+) and perpendicular (species chelating Tm3+) to the magnetic field


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direction. The neutron scattering patterns superimposed resulting in the cross observed in Figure 6. These results were analogous to those observed in the absence of gelatin in Figure S4A, point I. The low Af of -0.18 obtained in Figure 6 originated from the opposite alignment direction of the Dy3+ and Tm3+ chelating bicelles mixed in solution. The individual alignment factors act against each other and the obtained overall Af of the sample is low. This should not be misinterpreted as weak alignment. The Af calculation was chosen to best predict the gel’s birefringence signal. However, the calculation of the Af may be modified to stress the strong alignment of the bicelle mixture as shown in Figure S6. This simultaneous opposite aligned state of the bicelles was successfully gelled upon cooling to 4 °C and switching off the magnetic field in Figure 6, point C. The crossed 2D neutron scattering pattern in Figure 6 point B was stable in the time scale of this experiment. However, this bidirectional alignment is bound to deteriorate over time at 4 °C due to the exchange of DMPE-DTPA/Ln3+ lipids between the bicelles.55,56 The resulting time-resolved deterioration of the cross neutron scattering pattern could reveal important information on this mechanism. This phenomenon may also be quantified by monitoring the sample’s birefringence signal over time (see supplementary information), offering a viable alternative to reported small angle scattering methods or fluorescence-based studies.57-60 Unlike the other systems reported herein, the gel’s optical signature was not thermoreversible if heated above a certain temperature. The bilayer lipids diffused and irreversibly associated upon melting of the gel (and subsequent vesicle formation) on heating from 4 °C (Figure 6, point C) to 45 °C (Figure 6, point D). The bilayer lipids were in a liquid disordered state at 45 °C, giving them sufficient freedom to diffuse. Consequently, the regenerated bicelles upon cooling contained both DMPE-DTPA species chelating Dy3+ and Tm3+ in their bilayer. Since Dy3+ has an


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intrinsically larger molar magnetic susceptibility than Tm3+, the resulting bicelles aligned parallel to the magnetic field direction.40 The maximal Af of +0.38 at 5 °C in Figure 6, point F was smaller than that expected from bicelles of equivalent dimensions composed solely of Dy3+. Nevertheless, these results demonstrate the possibility of forming a smart hydrogel delivering two distinctly different optical properties using the same starting material. The optical properties were readily controlled by the combined action of temperature and magnetic fields.

CONCLUSION The combined high magnetic response and thermal stability of DMPC/Chol-NH2/DMPEDTPA/Ln3+ (molar ratio 16:4:5:5) bicelles allowed for the formation of novel optically active smart hydrogels. The optical properties originated from the aligned state of the bicelles imbedded in the gel, resulting in an anisotropic transfer of electromagnetic waves yielding different spatial birefringence.20 The interaction of the magnetically aligned Chol-NH2 doped bicelles with the gelatin network was markedly different to the previously reported systems.20,21 DMPC/CholNH2/DMPE-DTPA/Ln3+ (molar ratio 16:4:5:5) bicelles were not stacked in the gelatin matrix, providing a unique optical signature. The viability of these bicelles for the development of smart optical gels was demonstrated by enhancing their magnetic response, delivering anisotropy at commercially viable magnetic field strengths of 1 and 2 T. The enhanced magnetic alignability was achieved by increasing the bicellar size through optimized fabrication procedures.36 The versatility of the technology was extended by replacing the porcine gelatin with fish gelatin, demonstrating the possibility of employing different gels with defined gelling properties. The lower gelling temperature of fish gelatin allowed to gel a 50/50 (v/v) mixture of bicelles chelating either Tm3+ or Dy3+, which simultaneously aligned parallel and perpendicular to the magnetic field direction.


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The smart hydrogels presented herein could prove themselves useful in tracking of temperaturesensitive goods due to the following features: •

Every system offered a defined optical signature, tailored by the magnetic field strength or the nature of the gelatin-bicelle pair.

•

The temperature history of the gels influenced their optical properties.

•

The birefringence signal of an optically active gel cube transported alongside a product may be monitored before, during and upon arrival, guaranteeing a respected cold-chain.

•

The large panel of achievable and well-defined gel optical properties acts as a bar-code.

•

These materials are reusable owing to their thermoreversible nature. They are readily regenerated with a heating and cooling cycle in the presence of an external magnetic field.

•

The gels may contain more than one distinct optical property when imbedding a mixture of bicelles chelating different Ln3+.

•

The optical characteristics of the gels with bidirectionally aligned bicelles could be irreversibly switched upon heating above a given temperature if an added degree of security is required.

All these features enhance the degrees of freedom for engineering the properties of optical gels, providing a rich toolbox for future development of modern temperature sensors and switches.

Supporting Information. Figures S1 to S6 provide additional characterization of DMPC/CholNH2/DMPE-DTPA/Ln3+ (molar ratio 16:4:5:5) bicelles with SANS, birefringence and optical density measurements. The criteria for full alignment of the bicelles based on the Af values obtained from SANS 2D patterns are discussed. The mixture of bicelles simultaneously aligning parallel and perpendicular to the magnetic field direction is further investigated in the absence of gelatin. Alignment factor calculation considerations are further provided for this system. Corresponding Author


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*Stéphane Isabettini (E-mail: [email protected]), Peter Fischer (E-mail: [email protected]) Notes The authors declare no competing financial interests. Funding Sources This work was funded by the Swiss National Science Foundation. ACKNOWLEDGMENT The SANS experiments were performed at the Swiss spallation neutron source SINQ, Paul Scherrer Instute, Villigen, Switzerland. The authors acknowledge the Swiss National Science Foundation for funding this work (SMhardBi project number 200021_150088/1). REFERENCES 1) Bogue, R. Smart materials: A review of capabilities and applications. Assem. Autom. 2014, 34, 16–22. 2) Zrinyi, M. Intelligent polymer gels controlled by magnetic fields. Colloid Polym. Sci. 2000, 278, 98–103. 3) Ebara, M.; Kotsuchibashi, Y.; Uto K.; Aoyagi, T.; Kim, Y.-J.; Narain, R.; Idota, N.; Hoffman, J. M. In Smart Biomaterials; Springer: Tokyo, JP, 2014; Chapter 2, pp 9-65. 4) Xia, L.-W.; Xie, R.; Ju, X.-J.; Wang, W.; Chen, Q.; Chu, L.-Y. Nano-structured smart hydrogels with rapid response and high elasticity. Nat. Commun. 2013, 4, 2226. 5) Gil, E. S.; Hudson, S. M. Stimuli-responsive polymers and their bioconjugates. Prog. Polym. Sci. 2004, 29, 1173–1222.


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