Article Cite This: Langmuir 2019, 35, 7242−7248
pubs.acs.org/Langmuir
Reversible Aggregation of Chlorophyll Derivative Induced by Phase Transition of Lipid Noriaki Nishimura,† Soichi Nakayama,† Ayu Horiuchi,† Masaki Kumoda,† and Tomohiro Miyatake*,†,‡ †
Department of Materials Chemistry and ‡Innovative Materials and Processing Research Center, Ryukoku University, 1-5 Yokotani, Seta Oe-cho, Otsu, Shiga 520-2194, Japan
Downloaded via NOTTINGHAM TRENT UNIV on August 13, 2019 at 10:18:47 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
S Supporting Information *
ABSTRACT: Controlling the supramolecular organization of pigment molecules will provide innovative materials that exhibit variable optical properties. In nature, photosynthetic systems employ chlorophyllous supramolecules in which each pigment molecule is suitably organized in proteins, and their properties are adequately optimized by changing the structures of the surrounding amino acid residues. Here, we report a strategy for varying the aggregation behavior of a chlorophyll derivative by using a phase-transition phenomenon of lipid bilayers. Methyl pyropheophorbide a (MPP) was employed as a chlorophyllous pigment in our artificial system, and synthetic phosphatidylcholines with saturated acyl chain(s) were also used. The MPP molecules successfully accumulated within the lipid bilayer of liposomes without changing the vesicular structure. When the lipid bilayer was in a gel form (under the phase-transition temperature, Tm), the embedded MPP aggregated to yield a dimeric form showing red-shifted absorption bands and circular dichroism signals. When the solutions of MPP-containing liposomes were heated to higher temperatures than their Tm, MPP disaggregated to monomeric form as the absorption spectrum changed into its original fashion in dichloromethane. The reversible thermochromic (dis)aggregation of the MPP molecules had good cyclability. Additional careful examination of the phase transition in the MPP−lipid co-assemblies clarified that the critical temperatures of the MPP (dis)aggregation were in good agreement with the phase-transition temperatures of the pigment-containing bilayers. The reversible MPP aggregation in the lipid bilayers occurred in a wide range of temperatures (around 10−55 °C) by changing the length of the diacyl side chains of phospholipids. The reversible thermochromism of the chlorophyllous system was established by varying the nature of the surrounding lipid bilayer. This study can provide a useful strategy for making variable tetrapyrrolic aggregate systems induced by mild extrinsic stimuli.
■
Tetrapyrrolic π-conjugated pigments are promising building blocks for fabricating soft crystals because they provide effective π−π interactions and specific optical properties.2,5,12,13 Furthermore, structural modifications at the peripheral positions of the tetrapyrrolic macrocycles can be used to produce a diversity of functionalities.14−17 Natural photosynthetic systems employ chlorophylls to harvest the sunlight and generate charge-separated states in photosynthetic membranes. Chlorophylls have a tetrapyrrole framework with some peripheral side groups and chiral centers.17 Generally, natural chlorophylls are complexed with proteins to form photosynthetic apparatuses such as light-harvesting antennas and reaction centers.18,19 Chlorophyll molecules in nature are not arranged in a crystalline form, but the tetrapyrrolic molecules are suitably organized in the protein matrix to achieve efficient intermolecular excitation energy transfer and electron transfer.20 Physical properties and functionalities of the natural chlorophylls are adequately regulated by changing
INTRODUCTION
Highly ordered supramolecular assemblies of functional molecules often provide unique properties that are not observed in their monomeric forms. In particular, π-conjugated pigments readily assemble to form aggregates while showing remarkable changes in optical or electrochemical properties.1−3 The nature of the molecular assemblies is strongly dependent on their intermolecular interactions. Therefore, if the molecular arrangement of the functional molecules could be changed by gentle stimuli, the properties of the supramolecular assemblies could be readily altered. Recently, the new material concept of “Soft Crystals” has been proposed.4 This idea contains flexible and ordered molecular systems that respond to gentle stimuli such as weak mechanical force,5,6 vapor exposure,7,8 light irradiation,9,10 and heating at around room temperature.11 Many kinds of crystalline aggregates of metal complexes or organic molecules have yielded soft crystals that show remarkable changes in color, luminescence, and nanostructure due to gentle stimuli. Controlling the strength and orientation of intermolecular interactions will be a key technology for creating supramolecular soft crystals. © 2019 American Chemical Society
Received: February 27, 2019 Revised: April 20, 2019 Published: May 7, 2019 7242
DOI: 10.1021/acs.langmuir.9b00586 Langmuir 2019, 35, 7242−7248
Article
Langmuir
Figure 1. Structures of methyl pyropheophorbide a (MPP) and phosphatidylcholines.
the structures of the surrounding amino acid residues of the protein matrixes.21 For example, in many types of lightharvesting complexes, the absorption wavelength of each chlorophyll molecule is slightly different, and the gradation of the excitation energy levels generates directional energy transfer toward a reaction center complex.22 Additionally, the protein matrixes of the photosynthetic apparatuses provide relatively rigid molecular environments to reduce vibrational and/or rotational relaxations of the chlorophyll molecules, which reduces the nonradiative decay of the pigments. The structures of the natural photosynthetic systems show us the importance of regulating the surrounding molecular environments to control the properties of the chlorophyll assemblies.23 This idea would appear to complement the theory of soft crystals if the molecular environments could be tuned by gentle stimuli. Lipid bilayers can provide specific nanoenvironments, and many kinds of organic molecules have been incorporated in the cores of lipid bilayer membranes. In nature, photosynthetic pigment−protein complexes are located in the lipid bilayers, while many kinds of artificial bilayers have been prepared for making artificial photosynthetic systems,24,25 photodynamic therapy systems,26−28 ion channels,29 biosensors,30,31 etc. Pioneering work on artificial photosynthetic membranes was pursued by T. A. Moore and A. L. Moore’s group,24 where they introduced a carotenoid−porphyrin−quinone triad into a bilayer of liposome as a reaction center model. Photoirradiation of the membrane produced a charge-separated state across the liposomal membrane, which successfully activated ATP synthetase incorporated in the lipid bilayer. In natural photosynthetic membranes, roughly 100 to 300 antenna chlorophyll molecules per reaction center are introduced to collect sunlight efficiently. Loading the natural tetrapyrrolic pigments at such a high density can be problematic, since the large number of incorporated chlorophyll molecules may destabilize the artificial lipid membranes. Despite such concerns about their preparation, many types of chlorophyll-containing lipid assemblies have been reported.26,32−38 Naturally occurring chlorophyll molecules are hydrophobic and barely soluble in aqueous media, and the photosynthetic pigments are incorporated in the core of the lipid bilayers. In some cases, chlorophyllous pigments are covalently linked with phospholipids, and such a modification has resulted in an increase in the compatibility of the π-conjugated pigments with lipid assemblies.33,34 Zheng’s group reported a large amount of pyropheophorbide a (chlorophyll a derivative possessing a carboxy group) successfully incorporated in a liposomal membrane consisting of a mixture of phosphatidylcholine (PC) and cholesterol.35
They found that the chlorophyll derivative aggregated while showing a red-shifted Qy absorption band and split circular dichroism (CD) signals in the lipid membrane. The lipid scaffold afforded a specific environment to stabilize a wellorganized chlorophyllous aggregate that exhibits a unique nonlinear optical property. Hoshina reported that the aggregation behavior of natural chlorophyll a in a lipid assembly was temperature-dependent and could be related to the phase transition of the lipid bilayer.36 Here, we report the reversible self-aggregation of methyl pyropheophorbide a (MPP, Figure 1 left) in lipid bilayers. MPP is readily prepared from the naturally occurring chlorophyll a and possesses a stable methyl ester at the side chain.39 Different types of MPP-containing lipid assemblies (liposomes, bicelles, and micelles) were prepared by using phosphatidylcholines (PCs), i.e., 1,2-diacyl-sn-glycero-3-phosphocholines: dihexanoylphosphatidylcholine (DHPC) (C6:0), dimyristoylphosphatidylcholine (DMPC) (C14:0), dipalmitoylphosphatidylcholine (DPPC) (C16:0), and distearoylphosphatidylcholine (DSPC) (C18:0); and 1-palmitoyl-2-hydroxysn-glycero-3-phosphocholine: lysopalmitoylphosphatidylcholine (LPPC) (C16:0) (Figure 1 right). The optical properties of MPP and the phase transition of the liposomal membranes were carefully examined. We found that the aggregation of MPP was obviously dependent on the phase transition of the lipid bilayer, i.e., the gel phase of a lipid membrane provided a suitable environment for chlorophyll aggregation. The results shown here allow us to propose an effective methodology for making tetrapyrrolic soft crystals whose structures and properties can be varied by changing their molecular environments.40
■
EXPERIMENTAL SECTION
Materials. Methyl pyropheophorbide a (MPP) was prepared from chlorophyll a extracted from the dried powders of cyanobacterium according to the reported procedures.39 Phosphatidylcholines (DHPC, DMPC, DPPC, DSPC, and LPPC) and a Mini-Extruder with a polycarbonate membrane (pore size 100 nm) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). 1,3-Di-(2-pyrenyl)propane was obtained from Toronto Research Chemicals, Inc. (Toronto, Canada), and 5(6)-carboxyfluorescein, Sephadex G-50, buffers, and salts were purchased from Sigma-Aldrich. All of the organic solvents and surfactants were purchased from Nacalai Tesque (Kyoto, Japan) and used without any further purification. Equipment. Visible absorption, CD, and fluorescence spectra were recorded with a Shimadzu UV-2700 spectrophotometer, a Jasco J-820W spectropolarimeter, and a Jasco FP-8600 fluorescence spectrophotometer, respectively, and all of these spectrometers were equipped with a stirrer and a temperature controller. Dynamic light scattering (DLS) measurements were carried out with a particle-size analyzer (Otsuka Electronics ELSZ-1000). The measurements were 7243
DOI: 10.1021/acs.langmuir.9b00586 Langmuir 2019, 35, 7242−7248
Article
Langmuir performed at 25 °C, and the mean diameter of the particles was evaluated by cumulant analysis, with their size distributions recorded using the Marquart algorithm. Preparation of MPP-Containing Liposomes and Micelles.41 Solutions of MPP (0.34−3.4 μmol) and phospholipid (34 μmol) in 2 mL of CHCl3/CH3OH (1:1, v/v) were dried using a rotaryevaporator and a dry vacuum pump (>2 h) to form thin films of pigment−lipid mixtures. The resulting films were hydrated with 1 mL buffer (10 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES), 107 mM KCl, pH 7.4) for more than 30 min and subjected to freeze (−78 °C)−thaw (60 °C) cycles (5×) and extrusions (15×, Mini-Extruder with two stacked polycarbonate membranes, pore size 100 nm). The resulting liposome (DMPC, DPPC or DSPC) or micelle (LPPC) was purified by gel filtration chromatography (Sephadex G-50) with buffer (10 mM HEPES, 107 mM KCl, pH 7.4). The collected pigment-containing fractions were appropriately diluted with the same buffer for optical measurements (O.D. = ca. 1.0 at Qy band). Preparation of MPP-Containing Bicelle.42 A dried film of MPP (2.9 μmol) and mixed phosphatidylcholines (DHPC 8.3 μmol/DPPC 29 μmol) was prepared by the gentle evaporation of their mixed CHCl3 solution (2 mL). A buffer solution (1 mL 10 mM HEPES, 107 mM KCl, pH 7.4) was added to the pigment−lipid film, and the hydrated solution was subjected to sonication (2 min) and freeze (−78 °C) cycles (10×). Subsequent centrifuge treatment (1800g for 30 min) collected excess MPP as black precipitates, and an MPPcontaining bicelle solution was obtained as the supernatant.
Supporting Information Table S1), which indicates that the density of MPP in the membrane was 1 × 10−15 g/μm2. The chlorophyllous density of the artificial membrane was close to that of a natural thylakoid membrane of cyanobacteria (1.5 × 10−15 g/μm2).43 When the MPP−DPPC liposomes were prepared with large amounts of MPP (more than 10 mol % of DPPC), the yields of the MPP−DPPC liposome decreased and black precipitates of excess MPP were found in the buffer suspension. Therefore, the chlorophyllous pigment in the artificial membrane was saturated in the MPP−DPPC liposome prepared with 10 mol % pigment content. Generally, the vesicles of the phospholipid assemblies are readily destroyed and the added chlorophyllous pigment possibly destabilizes the liposomal envelopes. The stabilities of the MPP−DPPC liposomes were estimated by the leakage of the entrapped CF.41 Figure S2B shows the lifetimes of the MPP−DPPC liposome⊃CF with different levels of MPP content. The amounts of “unbroken” vesicles stored at 4 °C gradually decreased over several days, and their half-lives were estimated in a single exponential curve fitting. The half-life of the “plain” DPPC liposome (not containing MPP) was estimated at 900 days, and this half-life gradually decreased with increasing MPP content. However, the MPP−DPPC liposome prepared with 10 mol % pigment still had a long halflife (160 days), which is long enough to handle the sample and to investigate the optical properties of the MPP liposome. Self-Aggregation and Optical Properties of MPP in Lipid Assemblies. Visible absorption and CD spectra of the MPP−DPPC liposomes prepared with 10 mol % MPP are shown in Figure 2B,F. Intense Soret and Qy absorption bands
■
RESULTS AND DISCUSSION Preparation of MPP-Containing Liposomes. MPPcontaining liposomal membranes were prepared using a slightly modified procedure to make a large unilamellar vesicle.41 A mixed solution of MPP and PC in chloroform/ methanol (1:1) was gently evaporated in a round-bottomed flask to yield a dried film of the MPP/PC mixture. The pigment−lipid film was hydrated with an aqueous buffer (pH 7.4), and the obtained pigment−lipid assembly was purified with size-exclusion chromatography. The aqueous solution of the MPP-containing liposome was collected as the first blackcolored fraction, and no precipitate of MPP was found in the solution. The DLS measurement of the MPP−DPPC liposome particles showed nearly monodisperse signals, and their average diameter was determined to be 73 nm, which is consistent with the formation of vesicular structure (Figure S1A,D). Therefore, the water-insoluble MPP molecules were successfully embedded in the liposomal bilayer membrane. The formation of a vesicular envelope was verified by preparing MPP−DPPC liposome⊃CF (MPP-containing DPPC liposomes loaded with 5(6)-carboxyfluorescein (CF), a watersoluble fluorescent compound).41 The fluorescence emission of the entrapped CF in the MPP−DPPC liposome selfquenched due to the highly concentrated condition ([CF] = 50 mM). When Triton X-100, a nonionic surfactant, was added to the MPP−DPPC liposome⊃CF, the average size of the pigment−lipid assembly decreased to 35 nm (Figure S1G) and a strong fluorescence emission of CF was observed at 517 nm (Figure S2A). The addition of the surfactant broke the liposomal membrane, and the released and diluted CF showed intense fluorescence emission. Therefore, the MPP−DPPC assembly entrapped the water-soluble CF probe in the inner aqueous phase of the liposomal envelope, and the added 10 mol % MPP did not disrupt the formation of the DPPC vesicle. The number of MPP molecules in a liposomal vesicle prepared with 10 mol % MPP−DPPC was roughly estimated at 2 × 104 by a pigment analysis of the MPP−DPPC liposome⊃CF (see
Figure 2. Visible absorption (A−D) and CD (E−H) spectra of MPP in CH2Cl2 (A, E), liposome (MPP/DPPC = 1:10, (B, F)), bicelle (MPP/DHPC/DPPC = 1:2.2:7.8, (C, G)), and micelle (MPP/LPPC = 1:10, (D, H)).
of the chlorophyll derivative were observed at 443 and 704 nm, respectively, which were red-shifted from those of monomeric MPP in a CH2Cl2 solution (414 and 667 nm, Figure 2A). The MPP solution of CH2Cl2 showed only weak CD signals (Figure 2E), while MPP in the liposomal membrane provided intense CD signals in the Soret and Qy regions. These redshifted absorption bands and the accompanying CD signals 7244
DOI: 10.1021/acs.langmuir.9b00586 Langmuir 2019, 35, 7242−7248
Article
Langmuir
Generally, LPPC does not form a bilayer but gives a micellelike assembly in aqueous media.46 A mixture of MPP and LPPC (MPP/LPPC = 0.1) was hydrated with an aqueous buffer to form a pigment−lipid assembly. The hydrophobic MPP was successfully solubilized in the LPPC assembly, but the lipid assembly did not entrap the water-soluble CF. The size of the pigment−lipid assembly was estimated at 10 nm by DLS measurement (Figure S1F), which indicates that the micelle-like aggregate was formed and the size was slightly enlarged from the plain LPPC micelle (9 nm) by the pigment incorporation. The resulting MPP-containing micelle showed a 673 nm band without a red-shifted band at 704 nm (Figure 2D). MPP in the LPPC micelle showed some weak CD signals, but the spectral feature was different from that of the MPP dimers found in liposomes and bicelles (Figure 2H). These experiments on different types of MPP-containing lipid assemblies (liposome, bicelle, and micelle) indicate that a specific environment of the lipid bilayers induces the dimerization of MPP. Thermochromic Reversible Aggregation of MPP in Lipid Bilayers. Heating the MPP−DPPC liposome solution prepared with 10 mol % MPP induced remarkable changes in the absorption spectra. Figure 4 (middle) shows the temper-
increased with increasing MPP/DPPC ratios from 0.01 to 0.1 (Figure 3A−D). Therefore, the red-shifted absorption bands
Figure 3. Visible absorption (A−D) and CD (F−H) spectra of MPP−DPPC liposomes prepared with different pigment/lipid ratios. MPP/DPPC = 0.01 (A, E), 0.02 (B, F), 0.05 (C, G), and 0.1 (D, H).
and the intense CD signals are presumably due to pigment− pigment interactions, i.e., MPP molecules preferentially selfaggregated in the lipid membrane. The red shift in the Qy absorption maxima observed in the liposomal membrane, 674 → 704 nm (Δν = 630 cm−1), would seem to act for dimerization, since the obtained visible and CD spectra were similar to those of dimeric MPP reported previously.44 A liposome solution prepared with a small pigment content (MPP/DPPC = 0.01) provided monomeric MPP with a Qy absorption maximum at 674 nm (Figure 3A). The 674 nm Qy band was slightly red-shifted (Δν = 160 cm−1) from that of MPP in CH2Cl2 (λmax = 667 nm in Figure 2A). A similar small red shift in the Qy absorption band has often been observed in monomeric chlorophylls and their derivatives in the lipid bilayer environment.45 An MPP−lipid assembly was prepared with a lipid mixture of DPPC and DHPC. These lipids have different lengths of fatty acid chains, and the lipid mixture yields a disk-like bilayer assembly, the so-called “bicelle”.42 A chlorophyll−lipid assembly was prepared with 8 mol % MPP with the mixed lipids (DPPC/DHPC = 3.5:1). The obtained aqueous solution was homogeneous and no precipitate of the chlorophyllous pigment was found. Consequently, MPP molecules were incorporated in the bicelle membrane. DLS measurement indicated an average particle size of the MPP-containing bicelle as 17 nm, which is smaller than that of liposomal vesicles but twice a large as that of the plain bicelle prepared without MPP (Figure S2B,E). The incorporation of the pigment influenced the size of the small fragment of the lipid bilayer. In addition, the water-soluble CF probe was not entrapped in the bicelle samples, showing that no liposomal envelope was formed in the lipid mixture. The MPP-containing bicelle solution showed a red-shifted Qy band at 704 nm with intense CD signals as well as the MPP−DPPC liposomes (Figure 2C,G). Another MPP−lipid assembly was prepared with lysophosphatidylcholine (LPPC; Figure 1), possessing a single fatty acid chain.
Figure 4. Temperature-dependent visible absorption spectra of MPP−PC liposomes prepared with MPP/PC = 0.1 mixtures. MPPcontaining DMPC, DPPC, and DSPC liposome solutions were heated (left) or cooled (right). Spectra were recorded at 5, 10, 15, 20, and 25 °C for DMPC liposome; 25, 30, 35, 40, and 45 °C for DPPC liposome; and 25, 45, 50, 55, and 60 °C for DSPC liposome. Each sample was stored for 10 min (in heating process) or 15 min (in cooling process) at the desired temperature before measurement. Arrows indicate the directions of the spectral changes during heating or cooling processes.
ature-dependent absorption spectra of the MPP−DPPC liposome. The 704 nm band of dimeric MPP decreased with an increase in the monomeric 674 nm band, showing an isosbestic point at 682 nm, when the solution was heated to 45 °C. Afterward, when the heated solution was cooled to 25 °C, the recovery of the dimeric MPP was observed in the liposomal solution. The reversible disaggregation−aggregation cycles were successfully repeated many times (Figure S3). The CD 7245
DOI: 10.1021/acs.langmuir.9b00586 Langmuir 2019, 35, 7242−7248
Article
Langmuir
small amount of DPyP (0.1 mol % of PC) was added in the preparation of the MPP-containing liposomes, and the fluorescence intensity ratios of the pyrenyl excimer (Iexcimer/ λem = 495 nm) and monomeric pyrene (Imonomer/λem = 377 nm) were plotted against the temperatures (Figure 5B). The phase-transition measurements of plain liposomes of DMPC, DPPC, and DSPC measured with the excimer probe were in good agreement with the Tm values in the references,48 and the phase-transition probe is applicable to our liposomal membranes (Figure S5). DPyP measurements of the MPPcontaining membranes showed slightly lower Tm values: 17 °C for DMPC, 38 °C for DPPC, and 52 °C for DSPC membranes. These results clearly show that there is a high correlation between the phase transition of the lipid bilayers and the aggregation of MPP in the membranes. The specific aggregation behavior of MPP in the lipid assemblies was observed only in the gel phase of the lipid bilayers. The chlorophyllous pigment was monomeric in different kinds of organic solvents, such as methanol, diethyl ether, dichloromethane, n-hexane, etc., while showing a Qy maximum at around 666−668 nm at relatively higher pigment concentrations (ca. 3 mM). Even in a dried film, MPP did not show the red-shifted band at around 700 nm (Table S2). Therefore, solvent polarity might not be the crucial factor for the dimerization of MPP. The dimerization of the chlorophyllous pigment appears to be related to the fluidity of the lipid molecules in the bilayer environment, and the same idea was suggested in the previous work by Zheng’s group.35 In our MPP−PC membranes, the optical properties of the MPP dimers were slightly affected by the length of the fatty acid chain of the PC bilayer; the observed Qy absorption maxima were 701, 704, and 707 nm for DMPC, DPPC, and DSPC liposomal membranes, respectively (Figure 4). The CD spectra of MPP in membranes also varied depending on the structure of the lipids; the intensities of spectra decreased with increase in the length of the hydrocarbon chain of the lipids (Figure S4). When the temperatures are lower than Tm, the nature of the PC bilayers strongly depends on the length of the fatty acid chains. In contrast, above the phase-transition temperature, the microfluidities of the membranes are independent of the acyl chains.47 Actually, the monomeric MPPs in the liquid crystalline phase (>Tm) exhibited the same Qy absorption maxima at 674 nm. Although the precise supramolecular structure of the chlorophyllous dimer has not yet been clarified, the inverse-S shaped CD signals observed in the MPP dimers predicted an anticlockwise arrangement of the Qy transition moment of the chlorophyllous pigments.
spectra of the heated and cooled samples also showed reversible disaggregation−aggregation of MPP (Figure S4 middle). The temperature-dependent disaggregation−aggregation processes were also observed in MPP-containing liposomes prepared with other diacyl lipids. Heating and cooling treatment of MPP−DMPC (C14:0) and MPP−DSPC (C18:0) liposome solutions showed reversible spectral changes: 701 ↔ 674 nm (for DMPC liposome, Figure 4 upper) and 707 ↔ 674 nm (for DSPC liposome, Figure 4 lower). The reversible aggregation phenomena of MPP were common in these liposomal membranes, but the transition temperatures were largely different in the structures of the lipids. Spectral changes were observed at around 17 °C for DMPC, 38 °C for DPPC, and 52 °C for DSPC membranes (Figure 5A); therefore, the (dis)aggregation of the MPP dimer might be related to the phase transition of the lipid bilayers.
Figure 5. Temperature-dependent MPP aggregation (A) and bilayer phase-transition (B) measurements in MPP−PC liposomes prepared with MPP/PC = 0.1 mixtures: (A) absorbance ratios of monomeric and dimeric MPPs (Amonomer/Adimer) in DMPC (blue solid squares), DPPC (red solid circles), and DSPC (green solid triangles) liposomes; (B) fluorescence−intensity ratios of excimeric and monomeric DPyP (Iexcimer/Imonomer) in MPP−PC liposomes prepared with MPP/PC = 0.1 mixtures: DMPC (blue open squares), DPPC (red open circles), and DSPC (green open triangles). The phasetransition temperature of each lipid membrane was detected as a sharp change in Iexcimer/Imonomer.
Phase transition between the liquid crystalline form and the gel form accompanies large changes in the fluidity of the lipid molecules.47,48 The phase-transition temperatures (Tm) of the lipid bilayers were reported as 23 °C for DMPC, 41 °C for DPPC, and 55 °C for DSPC membranes.48 The observed critical temperatures of MPP aggregations (Figure 5A) were slightly lower than these Tm, so the presence of MPP could have interfered with the phase transition of the lipid bilayers. The phase-transition temperatures of these MPP-containing membranes were estimated using 1,3-di-(2-pyrenyl)propane (DPyP) as a membrane fluidity probe.47 DPyP has two pyrenyl groups connected with a 1,3-propanyl linker and shows intramolecular excimer formation between the pyrene chromophores. The formation of pyrenyl excimer strongly depends on the fluidity of the surrounding medium, and it is used for detecting the phase transition of the lipid membrane, i.e., in the highly fluid conditions, the intramolecular excimers are preferentially formed to exhibit a strong fluorescence. A
■
CONCLUSIONS Chlorophyllous π-conjugated pigments successfully accumulated and organized themselves in the lipid bilayers of liposomes. Highly concentrated pigment contents were obtained in the liposomal vesicular systems while maintaining their stability. The organization of the MPP molecules in the membranes was controlled by changing the temperature, and the critical temperatures of MPP aggregation were modulated over a wide range (roughly 10−55 °C) by changing the molecular structure of the lipids. The reversible and cyclable aggregation of the chlorophyll derivative in membranes provided a thermochromic supramolecular system. In many cases, tetrapyrrolic conjugates readily form tightly stacked large aggregates due to their planer structures and relatively strong π−π interactions. Therefore, control of the π-conjugated 7246
DOI: 10.1021/acs.langmuir.9b00586 Langmuir 2019, 35, 7242−7248
Article
Langmuir
(2) Lu, H.; Kobayashi, N. Optically Active Porphyrin and Phthalocyanine Systems. Chem. Rev. 2016, 116, 6184−6261. (3) Hoeben, F. J. M.; Jonkheijm, P.; Meijer, E. W.; Schenning, A. P. H. J. About supramolecular assemblies of π-conjugated systems. Chem. Rev. 2005, 105, 1491−1546. (4) Kato, M.; Ito, H.; Hasegawa, M.; Ishii, K. Soft Crystals -Flexible Response Systems with High Structural Order. Chem. - Eur. J. 2019, 25, 5105−5112. (5) Hattori, S.; Vandendriessche, S.; Koeckelberghs, G.; Verbiest, T.; Ishii, K. Evaporation rate-based selection of supramolecular chirality. Chem. Commun. 2017, 53, 3066−3069. (6) Seki, T.; Ito, H. Molecular-Level Understanding of Structural Changes of Organic Crystals Induced by Macroscopic Mechanical Stimulation. Chem. - Eur. J. 2016, 22, 4322−4329. (7) Wenger, O. S. Vapochromism in organometallic and coordination complexes: Chemical sensors for volatile organic compounds. Chem. Rev. 2013, 113, 3686−3733. (8) Kato, M. Luminescent platinum complexes having sensing functionalities. Bull. Chem. Soc. Jpn. 2007, 80, 287−294. (9) Irie, M.; Fukaminato, T.; Matsuda, K.; Kobatake, S. Photochromism of Diarylethene Molecules and Crystals: Memories, Switches, and Actuators. Chem. Rev. 2014, 114, 12174−12277. (10) Miyatake, T.; Takamori, Y.; Yamaguchi, K. Synthesis of zinc chlorin−spiropyran dyads and their self-aggregation properties. J. Photochem. Photobiol., A 2015, 313, 36−43. (11) Seeboth, A.; Lö tzsch, D.; Ruhmann, R.; Muehling, O. Thermochromic polymers - Function by design. Chem. Rev. 2014, 114, 3037−3068. (12) Balaban, T. S. Tailoring porphyrins and chlorins for selfassembly in biomimetic artificial antenna systems. Acc. Chem. Res. 2005, 38, 612−623. (13) Miyatake, T.; Tamiaki, H. Self-aggregates of natural chlorophylls and their synthetic analogues in aqueous media for making light-harvesting systems. Coord. Chem. Rev. 2010, 254, 2593− 2602. (14) Lindsey, J. S. De Novo Synthesis of Gem-Dialkyl Chlorophyll Analogues for Probing and Emulating Our Green World. Chem. Rev. 2015, 115, 6534−6620. (15) Ethirajan, M.; Chen, Y.; Joshi, P.; Pandey, R. K. The role of porphyrin chemistry in tumor imaging and photodynamic therapy. Chem. Soc. Rev. 2011, 40, 340−362. (16) Ishihara, S.; Labuta, J.; Van Rossom, W.; Ishikawa, D.; Minami, K.; Hill, J. P.; Ariga, K. Porphyrin-based sensor nanoarchitectonics in diverse physical detection modes. Phys. Chem. Chem. Phys. 2014, 16, 9713−9746. (17) Senge, M. O.; Ryan, A. A.; Letchford, K. A.; MacGowan, S. A.; Mielke, T. Chlorophylls, symmetry, chirality, and photosynthesis. Symmetry 2014, 6, 781−843. (18) Chlorophylls and Bacteriochlorophylls: Biochemistry, Biophysics, Functions and Applications; Grimm, B.; Porra, R. J.; Rüdiger, W.; Scheer, H., Eds.; Springer: Dordrecht, 2006. (19) Bryant, D. A.; Canniffe, D. P. How nature designs lightharvesting antenna systems: Design principles and functional realization in chlorophototrophic prokaryotes. J. Phys. B: At., Mol. Opt. Phys. 2018, 51, No. 033001. (20) Mirkovic, T.; Ostroumov, E. E.; Anna, J. M.; Van Grondelle, R.; Govindjee; Scholes, G. D. Light absorption and energy transfer in the antenna complexes of photosynthetic organisms. Chem. Rev. 2017, 117, 249−293. (21) Saito, K.; Ishida, T.; Sugiura, M.; Kawakami, K.; Umena, Y.; Kamiya, N.; Shen, J. R.; Ishikita, H. Distribution of the cationic state over the chlorophyll pair of the photosystem II reaction center. J. Am. Chem. Soc. 2011, 133, 14379−14388. (22) Casazza, A. P.; Szczepaniak, M.; Müller, M. G.; Zucchelli, G.; Holzwarth, A. R. Energy transfer processes in the isolated core antenna complexes CP43 and CP47 of photosystem II. Biochim. Biophys. Acta, Bioenerg. 2010, 1797, 1606−1616.
pigments assembly is often problematic. Ishii and his colleague found an effective strategy to control the aggregation of the πconjugated tetrapyrroles by changing the nature of the surrounding molecular environments.40 They conjugated silicon phthalocyanine pigment and many kinds of media including liquid crystals, synthetic and biological polymers, ionic liquids, and natural lipid assemblies that exhibit phasetransition phenomena at around room temperature. In each case, the tetrapyrrolic pigment aggregated at lower temperatures than their Tm, which provides thermochromic supramolecular materials. Here, the molecular assembly of tetrapyrrolic chlorophyll derivative was successfully regulated by changing the nature of the lipid bilayers, and we clarified that the phase transition of the lipid assemblies of phospholipids served as an effective matrix for organizing the chlorophyllous pigment. Debnath et al. performed modeling studies of chlorophyll−lipid bilayer complexes and proposed that tetrapyrrolic pigments are preferentially localized in the vicinity of lipid polar heads.49 In such a specific environment, the tetrapyrrolic pigment would exhibit unique self-aggregation phenomena. Supramolecular thermochromism was observed with a mild stimulus of changed temperature in the MPP−lipid bilayer. The concept of matrix-induced reversible aggregation of the tetrapyrroles can extend the functionalities of the pigments and provide a useful strategy for making soft crystals with large π-conjugated frameworks.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.9b00586. Pigment analysis of MPP-containing liposomes; dynamic light scattering measurements of MPP−lipid assemblies; fluorescence measurements of CF in MPP-containing liposomes; thermal reversibility of MPP aggregation in a DPPC liposome; temperature-dependent CD spectra of MPP−PC liposomes; phase-transition measurements on MPP-free liposomes; absorption maxima of MPP in different solvents (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Tomohiro Miyatake: 0000-0002-6747-8004 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI Grant Number JP17H06375 and MEXT-Supported Program for Strategic Research Foundation at Private Universities. The authors are grateful to Prof. Stefan Matile and Dr. Naomi Sakai for their technical assistance for liposome preparation and to Ryoto Horiuchi and Ryo Inoue for their experimental assistance.
■
REFERENCES
(1) Würthner, F.; Kaiser, T. E.; Saha-Möller, C. R. J-aggregates: From serendipitous discovery to supramolecular engineering of functional dye materials. Angew. Chem., Int. Ed. 2011, 50, 3376−3410. 7247
DOI: 10.1021/acs.langmuir.9b00586 Langmuir 2019, 35, 7242−7248
Article
Langmuir (23) Miyatake, T.; Hasunuma, Y.; Mukai, Y.; Oki, H.; Watanabe, M.; Yamazaki, S. Assemblies of ionic zinc chlorins assisted by watersoluble polypeptides. Bioorg. Med. Chem. 2016, 24, 1155−1161. (24) Steinberg-Yfrach, G.; Rigaud, J.-L.; Durantini, E. N.; Moore, A. L.; Gust, D.; Moore, T. A. Light-driven production of ATP catalysed by F0F1-ATP synthase in an artificial photosynthetic membrane. Nature 1998, 392, 479−482. (25) Sumino, A.; Dewa, T.; Kondo, M.; Morii, T.; Hashimoto, H.; Gardiner, A. T.; Cogdell, R. J.; Nango, M. Selective assembly of photosynthetic antenna proteins into a domain-structured lipid bilayer for the construction of artificial photosynthetic antenna systems: Structural analysis of the assembly using surface plasmon resonance and atomic force microscopy. Langmuir 2011, 27, 1092−1099. (26) Rajora, M. A.; Lou, J. W. H.; Zheng, G. Advancing porphyrin’s biomedical utility: Via supramolecular chemistry. Chem. Soc. Rev. 2017, 46, 6433−6469. (27) Zhou, Y.; Liang, X.; Dai, Z. Porphyrin-loaded nanoparticles for cancer theranostics. Nanoscale 2016, 8, 12394−12405. (28) Viard, M.; Reichard, H.; Shapiro, B. A.; Durrani, F. A.; Marko, A. J.; Watson, R. M.; Pandey, R. K.; Puri, A. Design and biological activity of novel stealth polymeric lipid nanoparticles for enhanced delivery of hydrophobic photodynamic therapy drugs. Nanomedicine 2018, 14, 2295−2305. (29) Sakai, N.; Matile, S. Synthetic ion channels. Langmuir 2013, 29, 9031−9040. (30) Mazur, F.; Bally, M.; Städler, B.; Chandrawati, R. Liposomes and lipid bilayers in biosensors. Adv. Colloid Interface Sci. 2017, 249, 88−99. (31) Litvinchuk, S.; Tanaka, H.; Miyatake, T.; Pasini, D.; Tanaka, T.; Bollot, G.; Mareda, J.; Matile, S. Synthetic pores with reactive signal amplifiers as artificial tongues. Nat. Mater. 2007, 6, 576−580. (32) Shao, S.; Rajendiran, V.; Lovell, J. F. Metalloporphyrin nanoparticles: Coordinating diverse theranostic functions. Coord. Chem. Rev. 2019, 379, 99−120. (33) Massiot, J.; Rosilio, V.; Ibrahim, N.; Yamamoto, A.; Nicolas, V.; Konovalov, O.; Tanaka, M.; Makky, A. Newly Synthesized Lipid− Porphyrin Conjugates: Evaluation of Their Self-Assembling Properties, Their Miscibility with Phospholipids and Their Photodynamic Activity In Vitro. Chem. - Eur. J. 2018, 24, 19179−19194. (34) Ng, K. K.; Takada, M.; Harmatys, K.; Chen, J.; Zheng, G. Chlorosome-Inspired Synthesis of Templated Metallochlorin-Lipid Nanoassemblies for Biomedical Applications. ACS Nano 2016, 10, 4092−4101. (35) Cui, L.; Tokarz, D.; Cisek, R.; Ng, K. K.; Wang, F.; Chen, J.; Barzda, V.; Zheng, G. Organized Aggregation of Porphyrins in Lipid Bilayers for Third Harmonic Generation Microscopy. Angew. Chem., Int. Ed. 2015, 54, 13928−13932. (36) Hoshina, S. Temperature dependence of optical properties of chlorophyll a incorporated into phosphatidylcholine liposomes. Biochim. Biophys. Acta, Bioenerg. 1981, 638, 334−340. (37) Milenkovic, S. M.; Bǎrbînţa-̌ Pǎtraşcu, M. E.; Baranga, G.; Markovic, D. Z.; Ţ ugulea, L. Comparative spectroscopic studies on liposomes containing chlorophyll a and chlorophyllide a. Gen. Physiol. Biophys. 2013, 32, 559−567. (38) Taguchi, S.; Suga, K.; Hayashi, K.; Yoshimoto, M.; Okamoto, Y.; Nakamura, H.; Umakoshi, H. Aggregation of chlorophyll a induced in self-assembled membranes composed of DMPC and DHPC. Colloids Surf., B 2019, 175, 403−408. (39) Tamiaki, H.; Machida, S.; Mizutani, K. Modification of 3substituents in (bacterio)chlorophyll derivatives to prepare 3ethylated, methylated, and unsubstituted (nickel) pyropheophorbides and their optical properties. J. Org. Chem. 2012, 77, 4751−4758. (40) Ishii, K.; Ishita, K. Temperature-Responsive Colorant. Patent Application WO2018/143473A1, 2018. (41) Miyatake, T.; Nishihara, M.; Matile, S. A cost-effective method for the optical transduction of chemical reactions. Application to hyaluronidase inhibitor screening with polyarginine-counteranion complexes in lipid bilayers. J. Am. Chem. Soc. 2006, 128, 12420− 12421.
(42) Barbosa-Barros, L.; Barba, C.; Rodríguez, G.; Cócera, M.; Coderch, L.; López-Iglesias, C.; de la Maza, A.; López, O. Lipid Nanostructures: Self-Assembly and Effect on Skin Properties. Mol. Pharmaceutics 2009, 6, 1237−1245. (43) Utkilen, H. C.; Briseid, T.; Eriksson, B. Variation in photosynthetic membrane and pigment content with light intensity for Anacystis nidulans grown in continuous cultures. Microbiology 1983, 129, 1717−1720. (44) Korth, O.; Hanke, T.; Röder, B. Photophysical investigations of Langmuir-Blodgett mono- and multilayer films of pheophorbide-a. Thin Solid Films 1998, 320, 305−315. (45) Goc, J.; Hara, M.; Tateishi, T.; Miyake, J.; Planner, A.; Fra̧ckowiak, D. Spectral properties of the photosynthetic reaction units reconstituted from bacterial reaction centres and antenna pigments located in liposomes suspended in buffer or ordered in Langmuir-Blodgett films. J. Photochem. Photobiol., A 1997, 104, 123− 131. (46) Vitiello, G.; Ciccarelli, D.; Ortona, O.; D’Errico, G. Microstructural characterization of lysophosphatidylcholine micellar aggregates: The structural basis for their use as biomembrane mimics. J. Colloid Interface Sci. 2009, 336, 827−833. (47) Jurado, A. S.; Almeida, L. M.; Madeira, V. M. C. Fluidity of bacterial membrane lipids monitored by intramolecular excimerization of 1,3-di(2-pyrenyl)propane. Biochem. Biophys. Res. Commun. 1991, 176, 356−363. (48) Lentz, B. R.; Barenholz, Y.; Thompson, T. E. Fluorescence Depolarization Studies of Phase Transitions and Fluidity in Phospholipid Bilayers. 1. Single Component Phosphatidylcholine Liposomes. Biochemistry 1976, 15, 4521−4528. (49) Debnath, A.; Wiegand, S.; Paulsen, H.; Kremer, K.; Peter, C. Derivation of coarse-grained simulation models of chlorophyll molecules in lipid bilayers for applications in light harvesting systems. Phys. Chem. Chem. Phys. 2015, 17, 22054−22063.
7248
DOI: 10.1021/acs.langmuir.9b00586 Langmuir 2019, 35, 7242−7248
