Design, Construction, and Characterization of High-Performance

Artificial membrane fusion behavior between the synthetic devices in which pilot and ..... By taking into account the application of our devices in re...
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Design, Construction, and Characterization of High-Performance Membrane Fusion Devices with Target-Selectivity Ayumi Kashiwada,* Iori Yamane, Mana Tsuboi, Shun Ando, and Kiyomi Matsuda Department of Applied Molecular Chemistry, Graduate School of Industrial Technology, Nihon University, Narashino, Chiba 275-8575, Japan S Supporting Information *

ABSTRACT: Membrane fusion proteins such as the hemagglutinin glycoprotein have target recognition and fusion accelerative domains, where some synergistically working elements are essential for target-selective and highly effective native membrane fusion systems. In this work, novel membrane fusion devices bearing such domains were designed and constructed. We selected a phenylboronic acid derivative as a recognition domain for a sugar-like target and a transmembranepeptide (Leu-Ala sequence) domain interacting with the target membrane, forming a stable hydrophobic α-helix and accelerating the fusion process. Artificial membrane fusion behavior between the synthetic devices in which pilot and target liposomes were incorporated was characterized by lipid-mixing and innerleaflet lipid-mixing assays. Consequently, the devices bearing both the recognition and transmembrane domains brought about a remarkable increase in the initial rate for the membrane fusion compared with the devices containing the recognition domain alone. In addition, a weakly acidic pH-responsive device was also constructed by replacing three Leu residues in the transmembrane-peptide domain by Glu residues. The presence of Glu residues made the acidic pH-dependent hydrophobic αhelix formation possible as expected. The target-selective liposome−liposome fusion was accelerated in a weakly acidic pH range when the Glu-substituted device was incorporated in pilot liposomes. The use of this pH-responsive device seems to be a potential strategy for novel applications in a liposome-based delivery system.

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INTRODUCTION A membrane fusion phenomenon is one of the fundamental events in the life of eukaryotic cells. For example, fertilization involves a membrane fusion process of a sperm with an egg through cell division, after which the membranes are resealed to be useful as plasma membranes.1−3 In addition, other membrane fusion systems are observed in endocytosis, exocytosis, and cellular membrane traffic. 4−9 In living organisms, fusion reactions are mediated by many kinds of membrane fusion proteins to overcome large energetic barriers in biological membranes.10−13 Influenza virus hemagglutinin (HA), one of the best-known glycoproteins, plays a key role in both host cell recognition and membrane fusion. The HA glycoprotein consists of two chains, HA1 and HA2, respectively 329 and 175 residues long.14−18 For instance, the fusion event in influenza viruses is achieved as follows: The HA1 chain binds to sialic acid residues on the target membrane, and the HA2 chain is inserted in the target membrane through specific interactions. This well-programmed and controlled membrane fusion system is expected to be applicable to many fields of bioscience, biotechnology, and biomedicine. In particular, model studies on the construction of membrane fusion systems will open up new possibilities for advanced drug delivery and gene transfer. Artificial membrane fusion systems using functionalized liposomes provide an excellent resource to investigate these © 2011 American Chemical Society

processes in a quantitative manner by controlling the interaction at vesicular surfaces. There are two approaches to construct artificial membrane fusion systems. One is to use designed synthetic target recognition devices inspired by the natural fusion machinery such as the HA1 chain. These systems have a bioinspired molecular recognition function via supramolecular formation at the liposomal interfaces.19−27 Interactions between small molecules or ions at the surface of liposomes equipped with membrane binding devices can induce liposome−liposome binding and fusion. Our group has also constructed the one-way membrane fusion systems using phenylboronic acid derivatives or boronopeptides as the membrane fusion devices.28,29 Our fusion systems have used a boronic acid and a sugar-like cyclic cis-diol structure wellknown as a molecular recognition pair to form an interliposomal complex. The other approach is to use a kind of membrane affinitive polypeptide as “a warhead” to the target liposome. Some research groups have described the interactions and fusion of artificial liposomes induced by HA2 mimetic peptides and de novo-designed peptides.30−32 Although these polypeptides were not designed for any target-selective fusion, Special Issue: Bioinspired Assemblies and Interfaces Received: September 30, 2011 Revised: November 21, 2011 Published: December 28, 2011 2299

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stearic acid was coupled to the side chain amino group of Lys through TBTU/HOBt activation. This coupling reaction was also confirmed by the ninhydrin test. Then, deprotection of the Fmoc group by 20% piperidine in NMP was carried out. After confirmation of the removal of the Fmoc group, the second Fmoc-Lys(Mtt)-OH residue was coupled, and Mtt group was removed. Phenylboronic acid derivatives29 with different length of PEG spacers were coupled to the side chain amino group of the second Lys residue. The following amino acids were then coupled in order of the sequences of the transmembrane peptide domain with the standard Fmoc strategy. Deprotection and cleavage were carried out by treatment with 2 mL of a solution containing TFA/triisopropylsilane/water (89/10/1, v/v) for 2 h. Purifications of crude polypeptides were carried out by reversed-phase HPLC on a HITACHI D-7000 system equipped with a Inertsil ODS-3 column (10 μm, 250 mm ×10 mm i.d., GL-science, Japan), and the purity was followed by an analytical HPLC (Inertsil ODS-3 column, 5 μm, 250 mm ×4.6 mm i.d., GL-science, Japan). Synthesized devices were eluted with a linear gradient of water/acetonitrile/0.1% TFA. All products were identified by a high-resolution ESI-TOF MS using an Agilent 6210 ESI-TOF LC-MS spectrometer (Agilent Technologies Inc., Sanat Clara, CA, USA) and an analytical HPLC (Table S1 and Figure S1 in ESI). Preparation of the Pilot and Target Liposomes. Phospholipids for preparation of the pilot and target liposomes were purchased from Avanti Polar Lipids (Alabaster, AL). The pilot liposome consists of EggPC and the fusion device (97.5:2.5 molar ratio), and the target liposome consists of EggPC and PI (95.0:5.0 molar ratio). One hundred-nanometer-sized large unilamellar vesicles (LUVs) were prepared by evaporation of the chloroform solution of the lipid mixture in a round-bottom flask, followed by hydration in 10 mM buffer solutions (pH 5.0: acetic acid/sodium acetate; pH 7.4: tricine/ sodium hydroxide) containing 100 mM sodium chloride. The suspension was subjected to five freeze−thaw cycles for uniform dispersion. Then, LUVs for this experiment were then prepared by passage of the suspension through 100-nm unipore polycarbonate membranes (Whatman) 10 times with a Mini-Extruder Set (Avanti). The total lipid concentration was determined by an L-type Wako phospholipids kit (Wako) and adjusted to 2.0 mM. Lipid Mixing Assays.47 The target liposomes bearing NBD-PE and Rh-PE were prepared by the same procedures as described above. The concentrations of the NBD-PE and Rh-PE were 0.5 mol % against the lipid mixture. The mixing of phospholipids was followed by the fluorescence resonance energy transfer (FRET) method. We used equal concentrations of the unlabeled (pilot) and labeled (target) liposomes diluted into a buffer solution, and we monitored a fluorescence of 531 nm from NBD and 590 nm from Rh. The degree of fusion was estimated by the lipid mixing defined by I = (It − I0)/ (Imax − I0) × 100, where I0 and It are the fluorescence intensities at 531 nm at the initial time and arbitrary experimental time, respectively, and Imax is the fluorescence (at 531 nm) after disruption of the liposomes in 0.5% (w/v) Triton X-100. Experimental data were obtained on a HITACHI F-2500 spectrofluorometer at an excitation wavelength of 470 nm. Inner Leaflet Mixing Assays.48,49 Reduction of NBD-PE- and Rh-PE-labeled liposomes was carried out as follows: A 1:1 mixture of NBD-PE/Rh-PE-labeled liposomes (2.0 mM) and 100 mM sodium dithionite (in 10 mM tricine/sodium hydroxide buffer solution containing 100 mM sodium chloride) were incubated at 4 °C for 1 h. Free sodium dithionite was removed by gel filtration through Sephadex G-25 fine columns. The inner leaflet mixing assay was identical with the lipid mixing assay described above. CD Measurements. All CD measurements were performed on a Jasco J-820 spectropolarimeter using a 2-mm path-length cuvette at 20 °C (Jasco PTC-348WI peltier thermostat) at a device concentration of 10 μM in 10-mM buffer solutions (pH 5.0: acetic acid/sodium acetate; pH 7.4: tricine/sodium hydroxide). All buffers contained 100 mM sodium chloride. Concentrations of transmembrane peptide domains were measured by UV absorbance of the Trp residue at 280 nm. The CD spectra were the average of 10-scan data obtained from 240 to 190 nm with a 1-nm interval at 50 nm/min rate.

it was made clear that they contributed to various liposome− liposome fusion processes as an acceleration factor. Thus, it has been considered that an efficient combination of the target recognition and acceleration factors would lead to the development of a more useful membrane fusion device such as artificial HA. We are confident that this efficient combination will result in acceleration in the initial rate of membrane fusion in comparison with our past works.28,29 The improvement of increasing the initial rate of membrane fusion will contribute to constructing more effective target selective drug delivery and gene transfer systems. In this work, membrane fusion devices bearing both the target recognition and acceleration moieties are designed and constructed. We select poly(ethylene glycol) (PEG)-linked phenylboronic acid derivatives as a cis-diol recognition domain in the same way as our previous work.29 On the other hand, we select a designed transmembrane peptide domain as a fusion accelerator. It has been well-known that native or synthetic transmembrane peptides have a strong interaction (hydrophobic interaction) with membrane lipids. This interaction contributes membrane lysis, aggregation, and fusion. Native transmembrane (membrane-interactive) peptides such as magainins and melittins have a tranemembrane α-helical domain. These peptides are usually unstructured without any lipid and then folded into α-helices when exposed to a membrane environment.33−37 In order to investigate the relationship between the structure and function of transmembrane-peptides, de novo-designed peptides with lowcomplexity hydrophobic sequence are designed, constructed, and characterized.38,39 We employ a hydrophobic Leu-Ala repetitional sequence as a transmembrane-peptide domain of designed devices. Control of membrane fusion processes may be crucial in cytoplasmic delivery of bioactive molecules such as proteins and nucleic acids, and particularly pH-sensitive liposomes induce fusogenic activity under weakly acidic conditions as promising systems. Among the various methods to produce pH-sensitive liposomes,40−44 we have also realized the construction of pH-sensitive liposomal fusion systems with target selectivity.45,46 Membrane fusion devices bearing both the target recognition and acceleration moieties are attractive tools for membrane activation, and further addition of pH-sensitivity to the transmembrane-peptide domain as a fusion accelerator is expected to display a more effective function as a novel device for target-selective fusion systems. The transmembrane peptide domain in our original devices consists of a Leu-Ala repetitional sequence and forms hydrophobic α-helices in lipid bilayers of target liposomes in a wide range of pH. In this study, we also develop a pH-responsive fusion device really working in the weakly acidic range and characterize its pH-responsiveness. To this end, we design a transmembrane LEA peptide domain in which Leu residues are replaced by acidic Glu ones and examine whether the targe- selective membrane fusion system is controlled by the pH of the solution.

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EXPERIMENTAL SECTION

Synthesis and Purification of the Membrane Fusion Devices. The membrane fusion devices (phenylboronic acid/transmembrane peptide conjugates) were constructed on Fmoc-Lys(Mtt)-Wang resin (0.61 mmol/g). The Mtt group was removed by immersion in the solution of 1% TFA in CH2Cl2 two times for 30 min. After confirmation of the removal of the Mtt group by the ninhydrin test, 2300

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Characterization of pH-Responsive Behavior of Designed Membrane Fusion Conjugate in the Liposomal Membrane. The target liposomes bearing dansyl-PE were prepared by the same procedures as described above. The concentrations of the dansyl-PE were 5 mol % against the lipid mixture. We measured the fluorescence at 350 nm from Trp and 510 nm from dansyl. The distance between the surface of the pilot liposome (Trp residue at BA-11-(LEA)-St) and target liposome (dansyl) was estimated by FRET efficiency (I510/I350). Eight kinds of standard polypeptides with different lengths of Glycine spacers, W-Gn-St (n = 0, 1, 5, 10, 15, 18, 20, and 30), were used for obtaining information on the distance. Experimental data were obtained on a HITACHI F-2500 spectrofluorometer at an excitation wavelength of 280 nm.

Figure 2. FRET experiments for measuring total lipid mixing (A) and inner leaflet lipid mixing (B). The fluorophores containing target liposomes were mixed with pilot liposomes at pH 7.4. All the measurements were performed in 10 mM tricine/sodium hydroxide buffer solution containing 100 mM sodium chloride at 30 °C.

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RESULTS AND DISCUSSION Design of Membrane Fusion Devices. In our system, molecular recognition at the surfaces of pilot and target liposomes caused perturbing lipid packing, leading to membrane fusion.28,29 Not only our fusion system but also the other system has made use of the perturbation around the liposome surface as the driving force of membrane fusion.21 In these systems, the membrane fusion processes are passive in nature. Here, we aim to design and construct phenylboronic acid/transmembrane-peptide conjugates as novel membrane fusion devices (Figure 1). All devices have a stearoyl group to

effective membrane fusion devices, while the rate of lipid mixing was slower and less effective when BA-0-(LA)-St or LASt was incorporated in the pilot liposomes. We have reported that it is important for the spacer (PEG5) to have length suitable for target recognition of phenylboronic acid to overcome the repulsive hydration forces at the liposomal interfaces. The boronic acid moiety in the pilot liposomes incorporating BA-0-(LA)-St (without the PEG spacer) is buried into the hydration layer of its own liposome, and thus recognition against the target liposomes is interrupted (result of lipid mixing experiment for the control device, BA-0-St, is shown in Figure S2 in the Supporting Information).28 If the lipid mixing phenomena discussed above occur, the outer leaflet of each liposome will intermix without mixing of the inner leaflet, in other words, without the formation of any fusion pore or mixing of the contents. In order to examine the mixing of the inner leaflet, we monitored the inner leaflet mixing process through selective reduction of NBD fluorophores in the outer leaflet by sodium dithionite, a membraneimpermeable reductant.48,49 The results of the inner leaflet mixing experiments are shown in Figure 2B. Almost the same lipid mixing behavior was also kinetically observed for the dithionite-treated target liposomes blended with the various devices bearing pilot liposomes at pH 7.4. To obtain more information about the fusion events, we have probed mixing of the aqueous liposomal contents by employing a conventional 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS)/p-xylenebispyridinium bromide (DPX) quenching assay.52 A fluorescent ANTS was encapsulated in the target liposomes, and a quencher DPX was encapsulated in the pilot liposome. Mixing of liposomal contents brought about a fluorescent quenching of ANTS. The result of the contents mixing assay is shown in Figure S3 in the Supporting Information. This contents mixing behavior is in good agreement with the results of the lipid mixing and inner leaflet mixing shown in Figure 2. These results demonstrate that our devices induce the liposome−liposome full fusion. In order to gain further insight into the function of the designed membrane fusion devices, we have examined whether the membrane fusion can be accelerated by the synergism effect of the target recognition and membrane distortion. Figure 3A shows a contribution of the transmembrane LA domain of the device, BA-11-(LA)-St, to the efficiency of lipid mixing. The kinetics of lipid mixing reveals that BA-11-(LA)-St possessing the transmembrane LA domain functions as a very effective fusion device toward PI-decorated target liposomes. The phenylboronic acid moiety in the lower fusogenic variant,

Figure 1. Structures of phenylboronic acid (BA)/transmembrane− peptide (LA) conjugates as novel membrane fusion devices designed in this work.

anchor in the liposomes. By incorporating the transmembrane peptide sequence into the membrane fusion devices, we attempt to construct a more active fusion system toward phosphatidylinositol (PI) bearing target liposome. We also try to construct BA-5-St, BA-11-St, and LA-St as control devices (Figure 1). Membrane Fusion. The pilot and target liposomes prepared in this study were around 100 nm in diameter in terms of their biocompatibility and allowing for tumor-specific delivery.50,51 The pilot liposomes composed of EggPC contain 5 mol % of the novel membrane fusion devices. The membrane fusion behavior was examined by a probe dilution assay based on the FRET between the membrane-bound fluorophores NBD-PE and Rh-PE. 47 The FRET fluorophores were incorporated in the membranes of the target liposomes at an equimolar concentration (0.5 mol %). The efficiency of the membrane fusion was estimated from lipid mixing (%) calculated from the change in the intensity of NBD fluorescence emission. Figure 2A shows the lipid mixing behavior observed by the three different devices, BA-0-(LA)-St, BA-5-(LA)-St, and BA-11-(LA)-St incorporated in the pilot liposomes at pH 7.4. The fusion kinetics based on the lipid mixing indicates that BA-5-(LA)-St and BA-11-(LA)-St act as 2301

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liposomes. If the membrane fusion is accelerated by the synergism effect of phenylboronic acid and LA moieties, then the lack of LA will decrease the initial rate. This prediction is in good agreement with our results in Figure 3B. Endosomal pH-Triggered Membrane Fusion. We have also designed and constructed a weakly acidic pH-activated device on the basis of characterization of the original membrane fusion devices described above. The LA domain of the original devices bearing a helix-promoting hydrophobic Leu-Ala repetitional sequence might be inserted into the lipid bilayers of the target liposomes to form stable α-helices. This helix functionally plays an important role in the acceleration of packing and fusion of the perturbed lipid. By taking into account the application of our devices in response to the weakly acidic environment, we have replaced three Leu residues of the LA moiety by acidic Glu residues and investigated whether the fusogenicity of the mutated device depends on pH. Substituting Glu for Leu causes a structural change from a hydrophilic random coil at neutral pH to a hydrophobic α-helix in an acidic pH range. This structural change would have a crucial effect on the membrane distortion and fusion behavior induced by the designed device. Figure 5 shows the newly constructed model,

Figure 3. Results of total lipid mixing experiment for discussing the contribution of the transmembrane LA domains of designed devices BA-11-(LA)-St (A) and BA-5-(LA)-St (B). The inset of panel B shows the fusion kinetics of initial state. The fluorophores containing target liposomes were mixed with pilot liposomes at pH 7.4. All the measurements were performed in 10 mM tricine/sodium hydroxide buffer solution containing 100 mM sodium chloride at 30 °C.

BA-11-St, recognizes target liposomes, but does not lead to effective fusion because of its longer PEG11 spacer. It is wellknown that the distance between pilot and target liposomes plays a crucial role in the membrane fusion process.25,52 From these discussions, the investigation of the effect of the PEG11 spacer (ca. 8.0 nm) on the membrane fusion is reasonable and beneficial. We have demonstrated that addition of the LA domain to the imperfect BA-11-St device, leading to the BA-11(LA)-St device, renders the membrane fusogenic. A schematic illustration of the membrane fusion system by BA-11-(LA)-St as a fusion device is shown in Figure 4. The fusion event is considered to be caused as follows: (i) The phenylboronic acid domain recognizes PI on the target liposomes, (ii) The conformation of LA domain changes frpm random to α-helical (Figure S4),53 (iii) The moment conformational change occurs, the LA domain distorts the lipid bilayer of target liposomes located at a certain distance. Thus, the fusogenicity is improved by the synergism effect of the phenylboronic acid and LA domains. The lipid mixing behavior driven by BA-5-(LA)-St or BA-5St is also shown in Figure 3B. The final extent of lipid mixing after 30 min was approximately 20% in each case. By contrast, the inset of Figure 3B shows that the presence of LA domain leads to a significant increase in the initial rate of lipid mixing. The initial rate of lipid mixing (the mean rate for the first 30 s) driven by BA-5-(LA)-St and BA-5-St is 11.5 and 5.5%/min, respectively. Almost the same kinetics is observed in inner leaflet lipid mixing and liposomal contents mixing (Figure S5 in the Supporting Information). The initial rate is influenced by the frequency of the contact between the pilot and the target

Figure 5. Structure of membrane fusion device with weakly acidic-pHresponsiveness; BA-11-(LEA)-St designed based on BA-11-(LA)-St.

which is designed to be responsive in a weakly acidic pH range, and BA-11-(LEA)-St is incorporated in view of BA-11-(LA)-St with a remarkable acceleration effect by the transmembrane LA peptide domain on the target-selective membrane fusion. Figure 6A shows the lipid mixing behavior when the pilot liposomes are added to the target liposomes at pHs 7.4 and 5.0. The fusion kinetics based on the lipid mixing indicates that the designed device, BA-11-(LEA)-St, is not fusogenic at pH 7.4, while it acts as an effective membrane fusion device at pH 5.0. Almost the same pH-dependence is observed when the dithionite-treated target liposomes are blended with the pilot liposomes (Figure 6(B)). The contents mixing assay and DLS measurements are also carried out in this pH-sensitive system. The pilot liposomes bearing BA-11-(LEA)-St significantly

Figure 4. Schematic illustration of the membrane fusion system driven by the novel designed membrane fusion device BA-11-(LA)-St with target recognition and transmembrane−peptide domains. 2302

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Figure 6. FRET experiments for measuring total lipid mixing (A) and inner leaflet lipid mixing (B). The fluorophores containing target liposomes were mixed with pilot liposomes at pH 7.4 and 5.0. All the measurements were performed in 10 mM buffer solutions (buffer = pH 5.0: acetic acid/sodium acetate; pH 7.4: tricine/sodium hydroxide) containing 100 mM sodium chloride at 30 °C.

Figure 7. CD spectra of the mixture of BA-11-(LEA)-St incorporated pilot liposomes and target liposomes at pHs 7.4 and 5.0. The spectra were taken at 30 min after the pilot and target liposomes were mixed. The measurements were performed in 10 mM buffer solutions (buffer = pH 5.0: acetic acid/sodium acetate; pH 7.4: tricine/sodium hydroxide) containing 100 mM sodium chloride at 30 °C.

induced internal content mixing only at pH 5.0 (Figure S6 in ESI). The assay of contents mixing clearly revealed the membrane fusion process through dilution of fluorescent lipid probes. Moreover, the results obtained from the DLS measurements show the acidic pH-sensitivity of the fusion device, BA-11-(LEA)-St. When the pilot (displaying BA-11(LEA)-St) and target liposomes are mixed at pH 5.0, the mean diameter is increased sharply due to the turbulence of surfaces by the contact of the pilot with the target liposomes, and then decreases to around 140 nm (Figure S7 in ESI). This behavior is characteristic of membrane fusion rather than aggregation.21,28 On the other hand, the mixture of the pilot and the target liposomes at pH 7.4 shows the mean diameter around 250 nm. This scattering at neutral pH indicates the formation of low-level aggregation. In this case, the phenylboronic acid moiety in the device recognizes target liposomes, but does not lead to fusion because of the ineffective transmembrane LEA peptide domain and longer PEG11 spacer. On the basis of the results obtained here, the acidic pH-sensitive fusion events are achieved by the use of BA-11-(LEA)-St as the pilot device. In order to estimate whether the weakly acidic pH dependence of lipid mixing (or fusion) behavior is due to a secondary structural change of LEA domain in the liposome membrane, we have measured circular dichroism (CD) spectra of the designed devices during the membrane fusion. We took the CD spectra at 30 min after the BA-11-(LEA)-Stincorporated pilot and PI-incorporated target liposomes were mixed at pH 7.4 or 5.0 (Figure 7). The CD spectra show that the device assumes α-helical conformation under acidic conditions. This pH dependence is in good agreement with the lipid fusogenicity shown in Figure 6. To gain further information about the insertion of pH-responsive LEA domain into the target membrane, we have performed FRET measurements. The LEA domain of the pH-responsive device (BA-11-(LEA)-St) has a Trp residue, which serves as a fluorescent donor, whereas PI-bearing target liposomes contain the dansyl fluorophore as a fluorescent acceptor. No effective FRET was observed at physiological pH (pH 7.4) because of too long a distance (>10 nm) for interaction between the LEA unit and the target liposomes (Figure 8).25,54 On the other hand, excitation of the Trp in the LEA unit resulted in a remarkable increase in the emission of the dansyl at endosomal pH (pH 5.0) (Figure 8). The pH dependence of fusogenicity in the device, BA-11-(LEA)-St, has been assumed to be closely connected to the relation between the hydrophobical helix formation and target membrane distortion. The pH depend-

Figure 8. Fluorescence spectra of a mixture of the pilot liposome (2.5 mol % of BA-11-(LEA)-St in EggPC liposome) and the PI-bearing target liposome (containing 0.5% of dansyl-PE) with the excitation wavelength at 280 nm. The measurements were performed in 10 mM buffer solutions (buffer = pH 5.0: acetic acid/sodium acetate; pH 7.4: tricine/sodium hydroxide) containing 100 mM sodium chloride at 30 °C. The maximum around 350 nm is the emission of the tryptophan (Trp), and the emission around 510 nm originated in the dansyl group.

ence of structural change of the LEA domain from CD measurements and pH-dependent change of FRET efficiency strongly support the idea that the formation of hydrophobic αhelices allows the LEA domain to insert into the target liposomes and distort the liposome−liposome surfaces. These results corroborate the idea that the proposed mechanism of the fusion system shown in Figure 4 is a reasonable hypothesis.

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CONCLUSIONS The novel membrane fusion device, BA-11-(LA)-St or BA-5(LA)-St, with a transmembrane peptide domain as well as a target recognition domain, induces membrane fusion toward PI-containing target liposomes. The membrane fusion system induced by the designed device is as follows. The phenylboronic acid (BA) in the device forms a stable intervesicular complex through recognition of PI, followed by distorting of LA domain to the target membrane. That is to say, the transmembrane LA peptide takes an active part in perturbing lipid packing and plays a crucial role in acceleration of fusion 2303

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rate. A weakly acidic pH responsive fusion device, BA-11(LEA)-St, which is originated from the BA-11-(LA)-St, is also characterized. Only in a weakly acidic pH does the substituted LEA domain form α-helices and has the ability to distort target liposomes. This system was stable in the range above neutral pH, but showed the fusion activity around the endosomal pH. Additional studies are required to design elaborate devices for specific targets (such as a tumor-specific carbohydrate antigen), but our simple and effective strategy for membrane activation will be used to develop liposome-based versatile drug/gene delivery systems and contribute to the development of target-specific nonviral vectors.

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ASSOCIATED CONTENT

S Supporting Information *

Identification of designed membrane fusion devices; results of lipid mixing and inner leaflet lipid mixing experiments for control devices; results of liposomal contents mixing experiments by the use of designed devices; circular dichroism of the mixture of the BA-11-(LA)-St incorporated pilot liposomes and target liposomes; inner leaflet lipid mixing and liposomal contents mixing behavior driven by BA-5-(LA)-St; results of liposomal contents mixing experiments by the use of pHresponsive device; results of DLS measurements in pHresponsive system. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Mailing address: Department of Applied Molecular Chemistry, Graduate School of Industrial Technology, Nihon University, Narashino, Chiba 275-8575, Japan. TEL: +81-47474-2564. FAX: +81-47-474-2579. E-mail: kashiwada.ayumi@ nihon-u.ac.jp.

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ACKNOWLEDGMENTS The authors express their appreciation to Prof. M. Hirata for helpful comments on the preparation of the manuscript. REFERENCES

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