Amphipathic helical peptide-Nile Red probes for fluorescence probing of the lipid packing defects and their surrounding membranes on exosomes

Amphipathic helical peptide-Nile Red probes for fluorescence probing of the lipid packing defects and their surrounding membranes on exosomes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Amphipathic helical peptide-Nile Red probes for fluorescence probing of the lipid packing defects and their surrounding membranes on exosomes Yusuke Sato, Kazuya Segawa, Tomomi Sakamoto, Arihiro Narita, Kota Matsumoto, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5995359/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract Amphipathic helical (AH) peptide-based fluorescent probes were explored for analysis of lipid packing defects (LPDs) in the membrane surface of exosomes. Two kinds of AH peptide sequences, derived from the C-terminal sequence of Apolipoprotein A-I (ApoC) and from human α-synuclein (p2-23), were examined, where they differ in the hydrophobic face that can be inserted into LPDs. From the examination of the insertion depth of the AH peptides and the competitive binding using synthetic liposomes as exosome models, we found that ApoC peptide could serve as a binder for deep LPDs whereas p2-23 peptide preferentially recognize shallow LPDs. These peptides conjugated with an environment-sensitive dye Nile Red (NR) were demonstrated to be useful for assessing both the abundance of target LPDs by the fluorescent enhancement response and the membrane properties surrounding these LPDs by the emission wavelength of the probes, respectively. With these properties, our probes successfully characterized the LPDs of exosomes from three kinds of cancer cells (A549, Hela and MCF7 cells). We showed that exosomal membranes exhibited unique structural properties regarding deep and shallow LPDs and their surrounding membrane polarity. In addition, these properties significantly depended on the donor cells. Our probes would serve as powerful tools for LPD analysis with a view toward a better understanding of exosomal membranes. Biological sciences/Biological techniques/Sensors and probes Biological sciences/Biological techniques/Sensors and probes/Fluorescent dyes Fluorescent probe Exosome Membranes Lipid packing defect Polarity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Exosomes, a subgroup of extracellular vesicles that generally range from 30 to 150 nm, are secreted by almost all eukaryotic cells. 1 It is generally believed that exosomes originate from the endosomes formed by the inward budding of the plasma membrane. Exosomes shuttle diverse biomolecules like nucleic acids (microRNAs and DNAs), proteins and lipids from a donor cell to recipient cells. 2 More and more studies highlighted their pivotal roles in various physiological and pathological processes including cell-to-cell communication, cell growth and differentiation, and immune response. 3 It is of great importance to understand the fundamental properties characteristic of exosomes for unraveling their functions inside the cells. Recently, the significance and impact of exosomal membranes have been gradually given attention, while the number of reports is very limited in comparison with exosomal nucleic acids and proteins. 4 Exosomal membrane displays unique lipid expression and distribution patterns compared to cellular membranes, which indicates that lipids are integrated into the membranes through highly regulated processes. 5 There are growing reports on the critical roles of exosomal membranes’ properties on the intercellular communications of exosomes, including cellular uptake, mobility and biodistribution. 6–8 Fluorescent probes whose emission responses to the microenvironment of the membranes, such as Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) and DPH (1,6-diphenyl-1,3,5-hexatriene) and their derivatives (cf. Figure 1 ), offer a valuable means for characterizing lipid order of the exosomal membranes. 9–12 These probes report the average lipid order of the bulk of the exosomal membranes as they would uniformly distribute into the membranes. Previous research using these probes demonstrated that exosomal membranes exhibited high rigidity and stability comparable to the liquid-ordered phase of raft-mimicking model membranes. 9–12 It was also found that the polarity and fluidity of exosomal membranes were reported to vary depending on culture pH conditions 9 and the type of donor cells. 10 Of particular interest to us is lipid packing defect (LPD) found in highly curved membranes like exosomal membranes. LPD means the degree of exposure of membrane hydrophobic regions to the aqueous environment, which arises from the mismatch between the actual membrane curvature and the lipid geometry. 13–16 In the cells, LPDs are suggested to be responsible for the recruitment of many peripheral proteins to highly curved membranes. 17–19 LPD are important features of exosomal membranes, so that LPD analysis should advance our understanding of exosomes’ functions in biological events. However, LPD properties on exosomal membranes remained unclear due to the lack of useful analytical methods. In this work, we explored the use of amphipathic helical (AH) peptide-based fluorescent probes for the analysis of LPD properties on exosomal membranes (Fig. 1 A). AH peptides, one of the most common motifs in lipid-membrane-binding proteins, 20 are known to recognize LPDs on highly-curved membranes, 21,22 and they have been employed for the construction of molecular tools for biological membranes. 23–26 We recently developed a C-terminal of apolipoprotein A- I (ApoC)-derived AH peptide conjugated with an environment-sensitive fluorophore, Nile Red (NR), for the aim of exosome detection. 23 ApoC unit is capable of adopting an α-helix structure for binding to highly-curved membranes, followed by the insertion of the resulting hydrophobic face of α-helix into LPDs. 21,22 ApoC-NR probe exhibits the fluorescence enhancement for exosomes as the NR unit partitions into the membrane region upon binding of the ApoC unit. We demonstrated that ApoC-NR enabled the rapid and straightforward detection of exosomes in a mix and read fashion. We envision that this class of probes represents a unique analytical tool for LPDs of exosomal membranes. We focused on the abundance and depth (deep or shallow LPD: cf. Figure 1 A) of LPDs, which are significantly affected by the membrane properties including the lipid components and compositions, as revealed by computational analysis of model atomistic lipid membranes. 13–16 These LPD properties would be thus distinct depending on the type of exosomes, such as the exosomes derived from different cell lines. We also envision that the membrane polarity surrounding the LPDs can be assessed by means of the solvatochromic property of the NR unit of the probes. Recently, a series of fluorescent probes have been designed specifically for analyzing the local membrane property within the cells. 27–29 For example, the G protein-coupled receptor (GPCR) antagonist was covalently tethered to the NR, enabling the embedding of the NR unit within the membrane in close proximity to target GPCR through its interaction with the antagonist. 27 The wavelength-shifting emission of the probe offers valuable insights into the local properties of the membrane surrounding the GPCR. Encouraged by these successes, our AH peptide-NR probes are expected to be useful for probing the local membrane environment around the LPDs given the LPD-targeting property of the AH peptide (Fig. 1 A). Herein, we investigated two kinds of AH peptides, namely ApoC 23 and p2-23 derived from human α-synuclein, 30 as LPD-binding units conjugated with NR for the probing the exosomal membranes (Fig. 1 B). Previous studies have elucidated the significance of the inherent nature of LPDs for modulating the binding abilities of AH domains in highly-curved membrane-targeting proteins. 20,31 LPDs are qualitatively characterized as either “deep” or “shallow” depending on the relative depth of the defect site with respect to the nearest glycerol backbone. 16 Deep and shallow LPDs are suggested to accommodate distinct classes of AH peptides. ApoC peptide possesses a larger hydrophobic face within its α-helix structure compared to p2-23 peptide (Fig. 1 B), as shown in the helical wheel presentation determined by HELIQUEST. 32 This suggests a preferential binding to deep LPDs. Conversely, shallow LPD would be preferentially bound by the p2-23 peptide. Indeed, we found that these peptides were able to discriminate their target LPDs from others by the examination of a series of synthetic liposomes. Their NR conjugate probes were shown to be useful for assessing both the abundance of target LPDs by the fluorescent enhancement response and the membrane properties surrounding these LPDs by the emission wavelength of the probes. With these properties, our probes successfully characterized the LPDs of exosomes from three kinds of cancer cells. These features of AH peptide-NR probes are discussed as a basis for the advanced design of analytical tools for exosomal membranes. Experimental Reagents Fmoc-protected amino acids with L-configuration except glycine were purchased from Watanabe Chemical Industries (Hiroshima, Japan) or AAPPTec (Louisville, KY, U.S.A.). All phospholipids and cholesterol were purchased from Avanti Lipids (Alabaster, AL, USA, Figure S1 ). Nile Red derivatives (NR-C5-COOH (6-(9-diethylamino-5-oxo-5H-benzo[a]phenoxazin-2-yloxy)hexanoic acid) and NR-C1-COOH (2-((9-(diethylamino)-5-oxo-5H-benzo[a]phenoxazin-2-yl)oxy)acetic acid)) were synthesized according to the literature. 23,33 Other reagents were commercially available and used without further purification. Water was deionized (≥ 18.0 MΩ cm specific resistance) by an Elix 5 UV water purification system and a Milli-Q Synthesis A10 system (Millipore Corp., Bedford, MA), followed by filtration through a BioPak filter (Millipore Corp.). Unless otherwise mentioned, all measurements were performed at 25°C in 1×PBS buffer solution (pH 7.4). Errors are the standard deviations obtained from three independent experiments ( N = 3). Probe Synthesis All probes were synthesized using Biotage Initiator + Alstra peptide synthesizer (Biotage, Uppsala, Sweden) based on a Fmoc solid phase peptide chemistry on a Rink-Amide-ChemMatrix resin (Biotage) according to our previous report. 23, 25 The obtained crude product was purified by a reverse-phase HPLC system (pump, PU-2086 Plus ×2; mixer, MX 2080-32; column oven, CO-1565; detector, UV-2070 plus and UV-1570M (Japan Spectroscopic Co. Ltd., Tokyo, Japan)) equipped with a C18 column (Inertsil ODS3 (5.0 µm particle size, 250 mm × 20 mm column size); GL Sciences Inc., Tokyo, Japan) using a gradient of water/acetonitrile containing 0.1% TFA (Figure S2A). The probe was verified by MALDI-TOF-MS (Bruker Daltonics autoflex Speed-S1, Germany) (Figure S2B and Table S1 ). The concentration of Nile Red-carrying peptide probes was determined based on the molar absorption coefficient of NR-C5-COOH. 23 Preparation of synthetic liposomes Synthetic liposomes were prepared by extrusion according to the literature. 23 Chloroform- or methanol-suspended lipids were combined to form lipid mixtures of the desired molar ratio. The lipid film was obtained by drying under nitrogen gas and then hydrated overnight at 4°C in PBS buffer (pH 7.4). The lipid suspension was subjected to five freeze-thaw cycles. The vesicles were then prepared by manual extrusion through polycarbonate membranes (Whatman, NJ, USA) with a pore diameter of 100 nm using Avanti mini-extruder (Avanti lipids), which affords an average diameter of 115 nm. Liposome size was characterized by dynamic light scattering (DLS) measurements (Zetasizer Nano-ZS, Malvern, UK). Fluorescence measurements Fluorescence spectra and anisotropies were recorded on a JASCO model FP-6500 spectrofluorophotometer (Japan Spectroscopic Co. Ltd., Tokyo, Japan) with a thermoelectrically temperature-controlled cell holder. Measurements were done using a 3 × 3 mm quartz cuvette. The apparent dissociation constant ( K d ), which describes the lipid concentration where 50% of the peptide probe is bound, was determined from the fluorescence anisotropy titration experiments. 34 Evaluation of insertion depth of AH peptides using Laurdan-labelled liposomes 35 100% DOPC (dioleoyl-phosphatidylcholine) liposomes were mixed with Laurdan and incubated at 37°C for 1 h in the dark. 100 µM peptides were added, followed by incubation at 37 ℃ for 30 min in the dark. The final concentration is 250 µM for liposomes, 0.50 µM for Laurdan and 20 µM for peptides. The fluorescence spectrum was recorded with an excitation wavelength of 340 nm at 25°C. General polarization (GP) value for Laurdan was determined by the equation: GP = ( I 440 − I 490 ) / ( I 440 + I 490 ), where I 440 and I 490 are the emission intensities of Laurdan at 440 and 490 nm, respectively. The change of GP (ΔGP) upon addition of the peptides was determined by the equation: ΔGP = GP liposome − GP liposome + peptide , where GP liposome and GP liposome + peptide denote GP values in the absence and presence of peptides, respectively. Isolation of exosomes from cell culture supernatants All cell lines were obtained from Cell Resource Center for Biomedical Research, Tohoku University or ATCC (Rockville, MD, USA). All cells were grown in RPMI 1640 media supplemented with 10% fetal bovine serum and 2% penicillin/streptomycin at 37°C in a 5% CO 2 incubator. Cells were grown until about 80% confluence and then cultured in an EV-depleted medium for 48 h. Exosomes were collected and isolated from the cell culture supernatants using MagCapture Exosome Isolation Kit PS version 2 (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan). Results and discussion 22-meric ApoC and p2-23 peptides were selected in this study among various highly-curved membrane-binding AH peptides due to the following reasons. First, both peptides are reported to sense the LPDs on the lipid membranes. 23,30 Despite the same peptide length, these peptides differ in the hydrophobic face when adopting the α-helix structure for binding to membranes (Fig. 1 B). p2-23 peptide is shown to selectively recognize shallow LPDs due to its poorly developed hydrophobic face. 30,31 Meanwhile, ApoC is expected to recognize deep LPDs due to its multiple bulky residues in the hydrophobic face, akin to the well-characterized amphipathic lipid-packing sensor (ALPS) motifs such as ArfGAP1. 20–22 Binding characteristics of ApoC and p2-23 peptides We first investigated the insertion depth of two AH peptides for binding to synthetic liposomes in order to examine their binding characteristics to LPDs, in which DOPC liposomes (average diameters of 115 nm) labelled with Laurdan (Fig. 1 C), a well-characterized fluorophore for membrane analysis, 36,37 were used according to the literature. 35 Molecular dynamic simulation and the subsequent characterization of LPDs by Packmem algorithm showed that DOPC liposomes featured both deep and shallow LPDs on their membrane surface. 16 Laurdan can be inserted into the lipid bilayer at the depth of the lipid tails. Its emission spectrum undergoes significant changes based on the degree of its exposure to water, enabling to analyze the peptide insertion on the membranes. 35 Fig. 2 A shows the change of GP value (ΔGP) of Laurdan embedded in DOPC liposomes upon binding of AH peptides. ApoC binding resulted in a large increase in ΔGP of Laurdan, indicative of reduced exposure of Laurdan to the aqueous environment. This suggests that the lipid packing of the membrane is tightened by the deeper insertion of ApoC peptide into the membranes. In contrast, we observed a decrease in ΔGP value for p2-23 peptide. This means increased access of the water to Laurdan within the liposomes upon binding of p2-23 peptide, which is highly likely due to its shallower insertion into the membrane. It is noteworthy that the observed ΔGP for ApoC was comparable to that for M2 peptide (RLFFK SIYRF FEHGL KRG) that was shown to be deeply inserted into the membranes. 35 Combined with their preferential binding to LPDs, these results indicate that ApoC and p2-23 peptides are capable of recognizing deep and shallow LPDs on highly curved membranes, respectively. We next investigated whether these peptides could compete with each other for the binding to DOPC liposomes or not. If ApoC and p2-23 peptides selectively recognize deep and shallow LPDs, respectively, competitive binding for these peptides would not occur. Here, the peptides conjugated with NR units at the N-terminal through C5 linker (ApoC-NR and p2-23-NR, Figs. 1 B) were prepared. While these NR probes displayed weak emission in the buffer, the addition of DOPC liposomes caused enhancement of the emission as well as a blue shift of the emission peak (Figure S3). This originates from the partition of the NR unit into the membrane upon binding of AH peptide unit into the LPDs on the membrane surface. 23 The apparent dissociation constant ( K d ) was determined by the fluorescence anisotropy titration experiments. K d values of ApoC-NR and p2-23-NR were obtained as 45 ± 14 µM and 60 ± 12 µM, respectively (Inset, Figure S3), which shows comparable binding affinity for these probes. In the competitive binding experiments, p2-23 peptide was initially incubated with DOPC liposome to form the p2-23/liposome complex, after which ApoC-NR probe was added. In the absence of p2-23 peptides, ApoC-NR showed strong emission upon binding to the liposomes and the emission remained unaffected in the presence of 5–50 µM p2-23 peptide (Fig. 2 B). It shows that ApoC-NR is not displaced by p2-23 peptide on the liposomes, indicating that ApoC peptide does not compete with p2-23 peptide for the binding to the liposomes because of the difference in their binding sites. It should be noted that we observed a significant decrease in ApoC-NR’s emission upon the addition of M2 peptide (Figure S4). This is attributable to the competitive binding of ApoC with M2 peptides as both peptides target deep LPDs, which validates the present experiments for analysis of AH peptides’ binding preference toward LPDs. We also found negligible competition in the case of the addition of p2-23-NR into ApoC/liposome complex (Figure S5). Taken together with the results for insertion depth examination (cf. Figure 2 A), ApoC peptide can serve as a deep-LPD binder whereas p2-23 peptide preferentially recognizes shallow LPDs. Fluorescence response of AH peptide-NR probes for synthetic liposomes We next assessed the fluorescence enhancement response of ApoC-NR and p2-23-NR probes for synthetic DOPC liposomes or POPC (palmitoyl-oleoyl-phosphatidylcholine) liposomes. Following fluorescence experiments were done with excitation wavelength at 470 nm in order to discuss the solvatochromic response of NR unit in addition to the fluorescence enhancement response of the probes according to the previous literatures. 27–29 LPD abundance was largely influenced by the number of unsaturated acyl chains (two double bonds in DOPC vs one double bond in POPC; Figure S1 ) in the lipid composition of liposomes. 15 Both deep and shallow LPDs were reported to decrease when DOPC was replaced by POPC. 16 The emission of the probes enhanced as increasing the concentration of DOPC liposomes (Fig. 3 A), where ApoC-NR displayed a larger fluorescence response for the liposomes compared to p2-23-NR. The response of ApoC-NR to DOPC liposomes was 4.8-fold larger than that to POPC liposomes. Also, p2-23-NR showed 2.2-fold larger response for DOPC liposomes over POPC ones. These results are attributable to more favorable binding of the probes to DOPC liposomes because of the increased target LPDs caused by an increased number of unsaturation in the acyl chain. 15,16 It is thus highly likely that the fluorescence enhancement response of these probes can reflect the abundance of target LPDs on the membranes. We then turned to the solvatochromic property of NR units of the probes in order to assess whether they could report the local membrane polarity surrounding the LPDs or not. NR units in ApoC-NR and p2-23 probes were confirmed to show the strong solvatochromic characters by the spectroscopic measurements in a series of solvents, similar to NR itself (Figure S6). Here, we prepared synthetic DOPC liposomes or POPC liposomes with varying cholesterol (Chol) contents (0–50%). Prior to the use of our probes, the membrane polarity of these liposomes was estimated by means of GP values of Laurdan according to the literature. 11 Membranes of DOPC liposomes were found to be more polar (less ordered) than those of POPC ones and the increase in Chol content resulted in the decreased polarity (Figure S7), which is consistent with the results in the literature. 38 Figs. 3 B displays the normalized emission spectra of ApoC-NR in the presence of DOPC/Chol liposomes. ApoC-NR showed a strong emission with a maximum (λ em ) at 635 nm for DOPC liposomes. It was found that λ em value for POPC liposomes with lower membrane polarity was blue-shifted to 620 nm (Figure S8). In addition, a gradual blue-shift was observed with decreasing membrane polarity as the Chol content increased for both DOPC and POPC liposomes (Figs. 3 B and S8). Hence, combined with its binding preference, its emission is highly likely to reflect the local membrane polarity surrounding the deep LPDs. Likewise, p2-23-NR demonstrated the wavelength-shifting emission in response to changes in the membrane polarity (Figure S9). p2-23-NR would report differences in the membrane polarity around shallow LPDs. In order to obtain further gain into the local membrane property, we examined a ratiometry-based analysis for the probes’ response to liposomes. As the emission band position of NR unit describes the local polarity, we analyzed the short and long-wavelength parts of the NR’s emission (ApoC-NR: 590 nm and 650 nm, p2-23-NR: 590 nm and 660 nm), in line with a previously used methodology. 27,28 Stronger emission in the short wavelength indicates a more polar microenvironment whereas we could observe stronger emission in the long wavelength for a less polar microenvironment. Accordingly, the relative ratio of the emission intensity at this wavelength ( F 590 / F 650 and F 590 / F 660 for ApoC-NR and p2-23-NR, respectively) can be used for probing the polarity of the local membranes surrounding the LPDs. Indeed, we found that the ratio values for both ApoC-NR and p2-23-NR probes against the above liposomes are highly correlated with membrane polarity (GP value) determined by Laurdan assay (Fig. 4 ). These results verified the usefulness of the ratiometric analysis of local membranes’ polarity using our probes. It should be noted that the appropriate linker length between AH peptide and NR units plays a crucial role in the analysis of local membrane properties. We found a decrease or even lack of the solvatochromism of the NR units in response to the variation in the membrane polarity when AH peptide probes carrying NR unit through a short C1 linker were examined (Figure S10). This is attributable to that the NR unit did not partition effectively into the membrane when the short linker was used. Thus, ApoC-NR and p2-23-NR probes carrying a C5 linker (cf. Figure 1 B) were used for further studies. Exosomal membrane analysis based on the probes’ fluorescence response ApoC-NR and p2-23-NR were finally applied to the analysis of exosomal membrane property through their binding-induced fluorescence response. Exosomes were obtained from the supernatant media of A549 (human lung cancer), Hela (human cervical cancer) and MCF7 (human breast cancer) cells using MagCapture exosome isolation kit that utilizes magnetic beads immobilized with Tim4 protein capable of binding to phosphatidylserine (PS) displayed on exosome surface. 39 Nanotracking analysis revealed that the size distribution of all kinds of exosomes falls into the range of typical exosomes (Figure S11). 1,2 We first sought to examine the membrane polarity of the obtained exosomes (3.9 × 10 7 particles/µL) using Laurdan; however, we did not see any response of Laurdan for the exosomes under the present condition (data not shown), presumably due to weak partitioning of Laurdan into the exosomal membranes. Instead, DPH (Fig. 1 C) was examined for probing the membrane fluidity of the exosomes based on its fluorescence anisotropy. 11 Again, DPH would report the average property of exosomal membranes because it lacks the binding selectivity to specific local regions in the membranes. All exosomes had relatively lower fluidity, where the fluidity is close to that for 70%POPC/30%Chol liposomes (cf. Figure S7). We found negligible differences in membrane fluidity for these exosomes (Figure S12). This indicates average membrane property is almost comparable. In sharp contrast, we observed remarkable differences in local membrane properties for these exosomes by means of our probes. Figure 5 A depicts the fluorescence spectra of ApoC-NR in the absence and presence of 3.9 × 10 7 particles/µL exosomes. It was found that the response varied depending on the type of exosomes. The increase in the emission at 650 nm was in the order of ExoA549 < ExoHela < ExoMCF7. Considering the fluorescence enhancement property of ApoC-NR, deep LPDs are highly likely to be abundant for ExoMCF7 compared to ExoA549. We note that the response observed here was more moderate compared to our previous results, 23 presumably due to the difference in the isolation methods of exosomes, namely Tim4-immobilized magnetic beads or the combination of tangential flow filtration and size exclusion chromatography. Moreover, we observed the shoulder at 590 nm in the fluorescence spectra of ApoC-NR bound to the exosomes, which indicates the low polarity of the membranes surrounding the deep LPDs given the solvatochromism of the NR unit. The ratio of the emission intensities at 590 nm and 650 nm ( F 590 / F 650 ) was then calculated for the assessment of the exosomal membrane’s polarity (Fig. 5 C). F 590 / F 650 values for exosomes are significantly larger in comparison with that of ApoC-NR in the absence of exosomes. The comparison among the exosomes showed that ExoMCF7 and ExoHela showed much larger F 590 / F 650 values over ExoA549. These results indicate that the membranes around deep LPDs in ExoMCF7 and ExoHela are less polar than that in ExoA549. It is highly likely that deep LPD’s abundance on exosomal membranes and the surrounding membrane polarity change depending on the donor cells that produce the exosomes. Meanwhile, the response of p2-23-NR to exosomes is found to be relatively moderate compared to ApoC-NR (Fig. 5 B). This indicates that deep LPDs are more abundant relative to shallow LPDs. p2-23-NR showed a slight fluorescence enhancement at 660 nm for ExoHela. Accordingly, ExoHela possesses shallow LPDs to some extent. On the other hand, negligible fluorescence enhancement was observed for ExoA549 and ExoMCF7, which suggests that these exosomes have scarce shallow LPDs enough to accommodate p2-23-NR probe. We do not observe a significant blue-shift of the emission spectra of p2-23-NR in the presence of exosomes, unlike ApoC-NR. Indeed, the fluorescence emission ratio was almost unchanged in sharp contrast to ApoC-NR (Fig. 5 C). It seems to be hard to discuss the variation in membrane polarity near shallow LPDs among these exosomes based on the obtained spectra considering the small fluorescence response of p2-23-NR to exosomes. Taken together, these findings demonstrate that exosomal membranes exhibit unique structural properties regarding deep and shallow LPDs and their surrounding membrane polarity. Furthermore, these properties significantly depend on the donor cells. Given such local membrane information cannot be obtained by conventional fluorescence probes such as DPH and Laurdan (cf. Figure S12), our probes should serve as powerful tools for LPD analysis with a view toward better understanding of exosomal membranes and their correlation with biological functions. Conclusions In summary, we report that AH peptide-NR probes enable to probe the LPD properties on highly-curved membranes through their fluorescence response. ApoC and p2-23 peptides were demonstrated to serve as deep and shallow-LPD binders, respectively. Their NR probes exhibited binding-induced fluorescence enhancement and wavelength-shifting emission for synthetic liposomes, where the response significantly depends on the abundance of target LPDs and their surrounding membrane polarity, respectively. These unique properties allowed us to assess the local membrane property of exosomes. We demonstrated that the deep/shallow LPDs and the membrane polarity around them significantly varied depending on the exosome-producing donor cells. To the best of our knowledge, this work represents the first report on molecular probes to characterize LPD properties on exosomes. Combined with increasing knowledge of lipid molecules in the biological events and their regulatory mechanism, 4,5 the obtained results can contribute to furthering our understanding of exosomal membranes with a view toward the comprehensive analysis of exosome functions. LPD analysis by our probes might hold a potential in exosome-related diagnosis applications as observed for the analysis of lipids 40,41 and membrane viscosity 42 in exosomes. It is here noted that our probes can only analyze the exosomes purified from the supernatant media of the cells (Fig. 5 ). For the analysis in complex biological matrices, we need to further improve the probes’ functions such as the binding selectivity to target exosomes to suppress non-specific response to contaminants in the biological media. Following the present results with ApoC and p2-23 peptides, we expect that systematic studies on the NR conjugates with various AH peptide sequences having different hydrophobic faces or charges would enable the development of the improved probes for more sensitive and accurate analysis of exosomal membranes. Given the structural similarities between exosomes and enveloped virus particles (virions), we also envision that AH peptide-based probes hold great potential to serve as useful tools for enveloped virions. 43,44 We are continuing studies in this field of research. Declarations Data availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Acknowledgements We thank Prof. Hitoshi Kasai and Dr. Ryuju Suzuki for the DLS measurements. We also thank Biomedical Research Unit of Tohoku University Hospital for technical support. This work was supported by JST PRESTO (Grant No. JPMJPR19H4 to Y.S.). Author Contributions Y.S. conceived the study. K. S., T. S., A. N. and K. M. synthesized the probes and characterized their functions. All authors analyzed data. Y. S. and S. N. wrote the paper. Competing interests The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at… Correspondence and requests for materials should be addressed to Y.S. References van Niel, G., D’Angelo, G., & Raposo, G. Shedding light on the cell biology of extracellular vesicles. Nat. Rev. Mol. Cell Biol. 19 , 213−228. https://www.nature.com/articles/nrm.2017.125 (2018). Jeppesen, D. K. et al. Reassessment of exosome composition. Cell 177 , 428−445. e18 https://www.sciencedirect.com/science/article/pii/S0092867419302120?via%3Dihub (2019). Meldolesi, J. Exosomes and Ectosomes in Intercellular Communication. Curr. Biol. 28 , R435-R444. https://www.sciencedirect.com/science/article/pii/S0960982218300927?via%3Dihub (2018). Donoso-Quezada, J., Ayala-Mar, S. & Gonzalez-Valdez, J. The role of lipids in exosome biology and intercellular communication: Function, analytics and applications. Traffic 22 , 204-220. https://onlinelibrary.wiley.com/doi/10.1111/tra.12803 (2021). Susannah, H., Agota, T., Georges, E. G., Michael, E. B. & Alexander, K. L. Understanding the extracellular vesicle surface for clinical molecular biology. J. Extracell. Vesicles 11 , e12260. https://isevjournals.onlinelibrary.wiley.com/doi/full/10.1002/jev2.12260 (2022). Banquy, X. et al. Effect of mechanical properties of hydrogel nanoparticles on macrophage cell uptake. Soft Matter 5 , 3984–3991. https://pubs.rsc.org/en/content/articlelanding/2009/sm/b821583a (2009). Vorselen, D., Piontek, M.C., Roos W. H. & Wuite, G. J. L. Mechanical Characterization of Liposomes and Extracellular Vesicles, a Protocol. Front. Mol. Biosci. 7 , 139. https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2020.00139/full (2020). Skliar, M. et al. Membrane proteins significantly restrict exosome mobility. Biochem. Biophys. Res. Commun. 501 , 1055-1059. https://www.sciencedirect.com/science/article/pii/S0006291X18311707?via%3Dihub (2018). Parolini, I. et al. Microenvironmental pH Is a Key Factor for Exosome Traffic in Tumor Cells. J. Biol. Chem. 284 , 34211-34222. https://www.sciencedirect.com/science/article/pii/S0021925820377024?via%3Dihub (2009). Simbari, F. et al. Plasmalogen enrichment in exosomes secreted by a nematode parasite versus those derived from its mouse host: implications for exosome stability and biology. J. Extracell. Vesicles 5 , 30741. https://isevjournals.onlinelibrary.wiley.com/doi/10.3402/jev.v5.30741 (2016). Suga, K., Matsui, D., Watanabe, N., Okamoto, Y. & Umakoshi, H. Insights into the Exosomal Membrane: From Viewpoints of Membrane Fluidity and Polarity. Langmuir 37 , 11195-11202. https://pubs.acs.org/doi/full/10.1021/acs.langmuir.1c00687 (2021). Yasuda, T., Watanabe, H., Hirosawa, K. M., Suzuki, K. G. N., Suga, K. & Hanashima, S. Fluorescence Spectroscopic Analysis of Lateral and Transbilayer Fluidity of Exosome Membranes. Langmuir 38 , 14695-14703. https://pubs.acs.org/doi/10.1021/acs.langmuir.2c02258 (2022). Vanni, S. et al. Amphipathic Lipid Packing Sensor Motifs: Probing Bilayer Defects with Hydrophobic Residues. Biophys. J. 104 , 575-584. https://www.cell.com/biophysj/fulltext/S0006-3495(12)05143-0 (2013). Vamparys, L. et al. Conical Lipids in Flat Bilayers Induce Packing Defects Similar to that Induced by Positive Curvature. Biophys. J. 104 , 585-593. https://www.cell.com/biophysj/comments/S0006-3495(12)05142-9 (2013). Vanni, S., Hirose, H., Barelli, H., Antonny, B. & Gautier, R. A sub-nanometre view of how membrane curvature and composition modulate lipid packing and protein recruitment. Nat. Commun. 5 , 4916. https://www.nature.com/articles/ncomms5916 (2014). Gautier, R., Bacle, A., Tiberti, M. L., Fuchs, P. F. J., Vanni, S. & Antonny, B. PackMem: A Versatile Tool to Compare and Visualize Interfacial Packing Defects in Lipid Bilayers. Biophys. J. 115 , 436-444. https://www.sciencedirect.com/science/article/pii/S0006349518307628?via%3Dihub (2018). Nikos, S. H. et al. How curved membranes recruit amphipathic helices and protein anchoring motifs. Nat. Chem. Biol. 5 , 835-841. https://www.nature.com/articles/nchembio.213 (2009). Brigitte, N. et al. a-Synuclein Has a High Affinity for Packing Defects in a Bilayer Membrane: A THERMODYNAMICS STUDY. J. Biol. Chem. 21 , 21966-21975. https://www.sciencedirect.com/science/article/pii/S0021925820668053?via%3Dihub (2004). Anuj, T. et al. Interfacial of lipid head group and packing defects in driving amyloid-beta-mediated myelin-like model membrane deformation. J. Biol. Chem. 299 , 104653. https://www.jbc.org/article/S0021-9258(23)00295-8/fulltext (2023). Has, C. & Das, S. L. Recent developments in membrane curvature sensing and induction by proteins. Biochim. Biophsy. Acta Gen. Subj. 1865 , 129971. https://www.sciencedirect.com/science/article/pii/S0304416521001306?via%3Dihub (2021). Drin, G. & Antonny, B. Amphipathic helices and membrane curvature. FEBS Lett. 584 , 1840-1847. https://febs.onlinelibrary.wiley.com/doi/10.1016/j.febslet.2009.10.022 (2010). Giménez-Andrés, M., Čopič, A. & Antonny, B. Biomolecules , 8 , 45. The Many Faces of Amphipathic Helices. https://www.mdpi.com/2218-273X/8/3/45 (2018). Sato, Y., Kuwahara, K., Mogami, K., Takahashi, K. & Nishizawa, S. Amphipathic helical peptide-based fluorogenic probes for a marker-free analysis of exosomes based on membrane-curvature sensing. RSC Adv. 10 , 38323-38327. https://pubs.rsc.org/en/content/articlelanding/2020/ra/d0ra07763a (2020). Kawano, K. et al. Development of a Simple and Rapid Method for In Situ Vesicle Detection in Cultured Media. J. Mol. Biol. 432 , 5876-5888. https://www.sciencedirect.com/science/article/pii/S0022283620305416?via%3Dihub (2020). Ohira, K., Sato, Y. & Nishizawa, S. Self-Assembly and Disassembly of Membrane Curvature-Sensing Peptide-Based Deep-Red Fluorescent Probe for Highly Sensitive Sensing of Exosomes. ACS Sens. 8 , 522-526. https://pubs.acs.org/doi/10.1021/acssensors.2c02498 (2023). Murayama, T. et al. Loosening of Lipid Packing Promotes Oligoarginine Entry into Cells. Angew Chem Int Ed. 56, 7644-7647. https://onlinelibrary.wiley.com/doi/full/10.1002/anie.201703578 (2017). Hanser, F. et al. Nile Red-Based GPCR Ligands as Ultrasensitive Probes of the Local Lipid Microenvironment of the Receptor. ACS Chem. Biol. 16 , 651-660. https://pubs.acs.org/doi/full/10.1021/acschembio.0c00897 (2021). Danylchuck, D. I., Jouard, P-H. & Klymchenko, A. S. Targeted Solvatochromic Fluorescent Probes for Imaging Lipid Order in Organelles under Oxidative and Mechanical Stress. J. Am. Chem. Soc. 143 , 912-924. https://pubs.acs.org/doi/full/10.1021/jacs.0c10972 (2021). Jimenez-Sanchez, A., Lei, E. K. & Kelly, S. O. A Multifunctional Chemical Probe for the Measurement of Local Micropolarity and Microviscosity in Mitochondria. Angew. Chem. Int. Ed. 57 , 8891-8895. https://onlinelibrary.wiley.com/doi/full/10.1002/anie.201802796 (2018). Jensen, M. B. et al. Membrane Curvature Sensing by Amphipathic Helices: A SINGLE LIPOSOSOME STUDY USING a-SYNUCLEIN AND ANNEXIN B12. J. Biol. Chem. 286 , 42603-42614. https://www.sciencedirect.com/science/article/pii/S0021925820871488?via%3Dihub (2011). Pranke, I. M. et al. a-Synuclein and ALPS motifs are membrane curvature sensors whose contrasting chemistry mediates selective vesicle binding. J. Cell Biol. 194 , 89-103. https://rupress.org/jcb/article/194/1/89/36569/Synuclein-and-ALPS-motifs-are-membrane-curvature (2011). Gautier, R., Douguet, D., Antonny, B. & Drin, G. HELIQUEST: a web server to screen sequences with specific a-helical properties. Bioinformatics 24 , 2101-2102. https://academic.oup.com/bioinformatics/article/24/18/2101/192677?login=true (2008). Vurgun, N. & Nitz, M. Highly Functionalized b-Cyclodextrins by Solid-Supported Synthesis. Chem. Eur. J. 24 , 4459-4467, https://chemistry-europe.onlinelibrary.wiley.com/doi/full/10.1002/chem.201800028 (2018). Rusu, L., Gambhir, A., McLaughlin, S. & Radler, J. Fluorescence Correlation Spectroscopy Studies of Peptide and Protein Binding to Phospholipid Vesicles. Biophys. J. 87 , 1044-1053. https://www.sciencedirect.com/science/article/pii/S0006349504735869?via%3Dihub (2004). Martyna, A., Bahsoun, B., Badham, M. D., Srinivasan, H. M. J. & Rossman, J. S. Membrane remodeling by the M2 amphipathic helix drives influenza virus membrane scission. Sci. Rep. 7 , 44695. https://www.nature.com/articles/srep44695 (2017). De Vequi-Suplicy, C. C., Benatti, C. R. & Lamy, M. T. Laurdan in Fluid Bilayers: Position and Structural Sensitivity. J. Fluoresc. 16 , 431−439. https://link.springer.com/article/10.1007/s10895-005-0059-3?utm_source=acs&getft_integrator=acs (2006). Parasassi, T., De Stasio, G., Ravagnan, G., Rusch, R. M. & Gratton, E. Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of Laurdan fluorescence. Biophys. J. 60 , 179-189. https://www.sciencedirect.com/science/article/pii/S0006349591820410 (1991). Suga, K. & Umakoshi, H. Detection of Nanosized Ordered Domains in DOPC/DPPC and DOPC/Ch Binary Lipid Mixtures Systems of Large Unilamellar Vesicles Using a TEMPO Quenching Method. Langmuir 29 , 4830-4838. https://pubs.acs.org/doi/full/10.1021/la304768f (2013). Nakai, W. et al. A novel affinity-based method fopr the isolation of highly purified extracellular vesicles. Sci. Rep. 6 , 33935. https://www.nature.com/articles/srep33935 (2016). Kumari, S. & Singh, A. Urinary Exosomal Lipidomics Reveals Markers for Diabetic Nephropathy. Curr. Metabol . 6, 131-139. https://www.ingentaconnect.com/content/ben/cmb/2018/00000006/00000002/art00009 (2018). Fan, T. W. M. et al. Exosomal lipids for classifying early and late stage non-small cell lung cancer. Anal. Chim. Acta 1037 , 256-264. https://www.sciencedirect.com/science/article/pii/S0003267018302794?via%3Dihub (2018). Li, N. et al. Reflecting size differences of exosomes by using the combination of membrane-targeting viscosity probe and fluorescence lifetime imaging microscopy. Anal. Chem. 91 , 15308-15316. https://pubs.acs.org/doi/full/10.1021/acs.analchem.9b04587 (2019). Jackman, J. A. Antiviral peptide engineering for targeting membrane-enveloped viruses: Recent progress and future directions. Biochim. Biophys. Acta Biomembr. 1864 , 183821. https://www.sciencedirect.com/science/article/pii/S0005273621002698?via%3Dihub (2022). Sato, Y. et al. Viral membrane-targeting amphipathic helical peptide-based fluorogenic probes for the analysis of infectious titers of enveloped viruses. Anal. Chem. 97, 5480-5487. https://pubs.acs.org/doi/10.1021/acs.analchem.4c04852 (2025). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5995359","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":444341933,"identity":"6cbcbe71-61f6-4623-91dc-28e608eb6c94","order_by":0,"name":"Yusuke Sato","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFUlEQVRIie2QP2rDMBSHnzB4EtX6RGndI8gYAoUQX0VB4Cw9QKAlFQQy+QAuBHqF5AYuAmVx0zUlk5dO6d6hhAr3Dxnslm6F6hse0k/vQ08C8Hj+KER/rfZ9AITgY1v+oIRNyfDXijlU2hGb0RMvxpCy22kJL/Rhwo6nJqEwiIDct14jNhc9vqhgWNhQkhy3yOdWKQoq1rCWHUrI6xlIsFQAFVt0SWIoBK69Ep2D1XtII6eQV7n+VK6/UWSPLzWQhVMCWpaN4gYznQqvdsl5YXG4tJk0J1rxmyJT8Vys4lnHW45Wo/oxv+qnp8bc1c96wBgqg7vxZcSw/cfO3lNs6kGHGynEqs2ASLfGDSzvPvN4PJ7/xBt2BF2pfANLZAAAAABJRU5ErkJggg==","orcid":"","institution":"Tohoku University","correspondingAuthor":true,"prefix":"","firstName":"Yusuke","middleName":"","lastName":"Sato","suffix":""},{"id":444341934,"identity":"be7b9644-29a5-430f-abfe-1014762aa244","order_by":1,"name":"Kazuya Segawa","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Kazuya","middleName":"","lastName":"Segawa","suffix":""},{"id":444341935,"identity":"8ef6be50-18f1-4637-aa03-9be48f00a125","order_by":2,"name":"Tomomi Sakamoto","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Tomomi","middleName":"","lastName":"Sakamoto","suffix":""},{"id":444341936,"identity":"87859dda-2a7b-4cd5-9716-85cee5fc0b88","order_by":3,"name":"Arihiro Narita","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Arihiro","middleName":"","lastName":"Narita","suffix":""},{"id":444341937,"identity":"139cf854-b69f-4778-97d3-d9a39638aeb4","order_by":4,"name":"Kota Matsumoto","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Kota","middleName":"","lastName":"Matsumoto","suffix":""},{"id":444341938,"identity":"68299110-cef8-4191-bb77-5ab8fc23901c","order_by":5,"name":"Seiichi Nishizawa","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Seiichi","middleName":"","lastName":"Nishizawa","suffix":""}],"badges":[],"createdAt":"2025-02-10 03:53:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5995359/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5995359/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-08534-w","type":"published","date":"2025-07-03T15:58:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80914855,"identity":"52221983-b24b-4f75-ae2a-5fc56b13cae0","added_by":"auto","created_at":"2025-04-18 17:31:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":179014,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Schematic illustration of fluorescence probing of lipid packing defects (LPDs) and their surrounding membrane polarity by amphipathic helical (AH) peptide-Nile Red (NR) conjugate probes. (B) Structures of ApoC-NR and p2-23-NR probes used in this study. Helical wheel presentations of AH peptides obtained using HELIQUEST\u003csup\u003e16\u003c/sup\u003e were also shown. Arrows indicate the vector of hydrophobic moment for each peptide. Color: yellow, bulky and hydrophobic residues; grey, small nonpolar residues; blue, cationic residues; pink and purple, polar non-charged residues; green, proline. The projection for p2-23 peptide was calculated using the wild type sequence (DVFMK GLSKA KEGVV AAAEK TK).\u003csup\u003e30\u003c/sup\u003e (C) Chemical structures of Laurdan and DPH.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5995359/v1/3f30ecf659ed7fe76101df12.png"},{"id":80914853,"identity":"2542e834-db0a-4030-b6e4-5767017975d0","added_by":"auto","created_at":"2025-04-18 17:31:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":45598,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Changes in DGP values of Laurdan-labelled DOPC liposomes after the addition of 20 mM AH peptide (p2-23, ApoC and M2). [Laurdan] = 0.5 mM, [DOPC liposome (total lipid)] = 250 mM. (B) Fluorescence spectra of ApoC-NR in the presence of DOPC liposome or p2-23/liposome complex. [ApoC-NR] = 2.0 mM, [DOPC liposome (total lipid)] = 500 mM,[p2-23 peptide]= 0-50 mM. Excitation, 552 nm.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5995359/v1/bd72788c2a5cc3efb114daf7.png"},{"id":80914864,"identity":"76de8712-4ff9-45ed-bc49-47d1b88217bf","added_by":"auto","created_at":"2025-04-18 17:31:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":109542,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Fluorescence enhancement of the probes upon addition of DOPC or POPC liposomes. \u003cem\u003eF\u003c/em\u003e and \u003cem\u003eF\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e denote the fluorescence intensity at 650 nm (ApoC-NR) or 660 nm (p2-23-NR) in the presence and absence of liposomes, respectively. Excitation, 470 nm. (B) Normalized fluorescence spectra of ApoC-NR bound to the DOPC liposomes with varying Chol content (0-50%). [ApoC-NR] = 2.0 mM, [Liposome] = 500 mM. Excitation, 470 nm.\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5995359/v1/f87a54b02322fdbeb28359a5.png"},{"id":80914872,"identity":"638d1960-fbf0-410c-a315-7cef65153b09","added_by":"auto","created_at":"2025-04-18 17:31:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":51507,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence emission ratio of the probes for the liposomes with different membrane polarity (GP) determined by Laurdan assay.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5995359/v1/2e2356508b5c18dd83c5487e.png"},{"id":80915325,"identity":"67cba9bf-af59-46cd-ab8f-5c9695fec9f5","added_by":"auto","created_at":"2025-04-18 17:39:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":207134,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence spectra of the probe (2.0 mM: (A) ApoC-NR and (B) p2-23-NR) in the absence and presence of three kinds of exosomes (3.9 ´ 10\u003csup\u003e7\u003c/sup\u003e particles/mL). Excitation, 470 nm. (C) Fluorescence emission ratio of the probes in the absence and presence of exosomes. For the response of ApoC-NR, \u003cem\u003ep\u003c/em\u003e-values calculated by the Student’s t-test were shown. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, n.s.: not significant.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5995359/v1/f9c6f2db6254b4ca7b4f59b4.png"},{"id":86179527,"identity":"e93c7245-086c-4e2a-a885-b835d9c61543","added_by":"auto","created_at":"2025-07-07 16:17:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1382955,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5995359/v1/c0825ba5-4b96-471b-a1cc-bd431534e881.pdf"},{"id":80914861,"identity":"0191ff5b-261b-47ce-bb43-ca4d66007aaa","added_by":"auto","created_at":"2025-04-18 17:31:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":740455,"visible":true,"origin":"","legend":"","description":"","filename":"SegawaSIYS250414.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5995359/v1/af445ae432e8165bbe352153.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Amphipathic helical peptide-Nile Red probes for fluorescence probing of the lipid packing defects and their surrounding membranes on exosomes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eExosomes, a subgroup of extracellular vesicles that generally range from 30 to 150 nm, are secreted by almost all eukaryotic cells.\u003csup\u003e1\u003c/sup\u003e It is generally believed that exosomes originate from the endosomes formed by the inward budding of the plasma membrane. Exosomes shuttle diverse biomolecules like nucleic acids (microRNAs and DNAs), proteins and lipids from a donor cell to recipient cells.\u003csup\u003e2\u003c/sup\u003e More and more studies highlighted their pivotal roles in various physiological and pathological processes including cell-to-cell communication, cell growth and differentiation, and immune response.\u003csup\u003e3\u003c/sup\u003e It is of great importance to understand the fundamental properties characteristic of exosomes for unraveling their functions inside the cells.\u003c/p\u003e \u003cp\u003eRecently, the significance and impact of exosomal membranes have been gradually given attention, while the number of reports is very limited in comparison with exosomal nucleic acids and proteins.\u003csup\u003e4\u003c/sup\u003e Exosomal membrane displays unique lipid expression and distribution patterns compared to cellular membranes, which indicates that lipids are integrated into the membranes through highly regulated processes.\u003csup\u003e5\u003c/sup\u003e There are growing reports on the critical roles of exosomal membranes\u0026rsquo; properties on the intercellular communications of exosomes, including cellular uptake, mobility and biodistribution.\u003csup\u003e6\u0026ndash;8\u003c/sup\u003e Fluorescent probes whose emission responses to the microenvironment of the membranes, such as Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) and DPH (1,6-diphenyl-1,3,5-hexatriene) and their derivatives (cf. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), offer a valuable means for characterizing lipid order of the exosomal membranes.\u003csup\u003e9\u0026ndash;12\u003c/sup\u003e These probes report the average lipid order of the bulk of the exosomal membranes as they would uniformly distribute into the membranes. Previous research using these probes demonstrated that exosomal membranes exhibited high rigidity and stability comparable to the liquid-ordered phase of raft-mimicking model membranes.\u003csup\u003e9\u0026ndash;12\u003c/sup\u003e It was also found that the polarity and fluidity of exosomal membranes were reported to vary depending on culture pH conditions\u003csup\u003e9\u003c/sup\u003e and the type of donor cells.\u003csup\u003e10\u003c/sup\u003e Of particular interest to us is lipid packing defect (LPD) found in highly curved membranes like exosomal membranes. LPD means the degree of exposure of membrane hydrophobic regions to the aqueous environment, which arises from the mismatch between the actual membrane curvature and the lipid geometry.\u003csup\u003e13\u0026ndash;16\u003c/sup\u003e In the cells, LPDs are suggested to be responsible for the recruitment of many peripheral proteins to highly curved membranes.\u003csup\u003e17\u0026ndash;19\u003c/sup\u003e LPD are important features of exosomal membranes, so that LPD analysis should advance our understanding of exosomes\u0026rsquo; functions in biological events. However, LPD properties on exosomal membranes remained unclear due to the lack of useful analytical methods.\u003c/p\u003e \u003cp\u003eIn this work, we explored the use of amphipathic helical (AH) peptide-based fluorescent probes for the analysis of LPD properties on exosomal membranes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). AH peptides, one of the most common motifs in lipid-membrane-binding proteins,\u003csup\u003e20\u003c/sup\u003e are known to recognize LPDs on highly-curved membranes,\u003csup\u003e21,22\u003c/sup\u003e and they have been employed for the construction of molecular tools for biological membranes.\u003csup\u003e23\u0026ndash;26\u003c/sup\u003e We recently developed a C-terminal of apolipoprotein A- I (ApoC)-derived AH peptide conjugated with an environment-sensitive fluorophore, Nile Red (NR), for the aim of exosome detection.\u003csup\u003e23\u003c/sup\u003e ApoC unit is capable of adopting an α-helix structure for binding to highly-curved membranes, followed by the insertion of the resulting hydrophobic face of α-helix into LPDs.\u003csup\u003e21,22\u003c/sup\u003e ApoC-NR probe exhibits the fluorescence enhancement for exosomes as the NR unit partitions into the membrane region upon binding of the ApoC unit. We demonstrated that ApoC-NR enabled the rapid and straightforward detection of exosomes in a mix and read fashion. We envision that this class of probes represents a unique analytical tool for LPDs of exosomal membranes. We focused on the abundance and depth (deep or shallow LPD: cf. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) of LPDs, which are significantly affected by the membrane properties including the lipid components and compositions, as revealed by computational analysis of model atomistic lipid membranes.\u003csup\u003e13\u0026ndash;16\u003c/sup\u003e These LPD properties would be thus distinct depending on the type of exosomes, such as the exosomes derived from different cell lines. We also envision that the membrane polarity surrounding the LPDs can be assessed by means of the solvatochromic property of the NR unit of the probes. Recently, a series of fluorescent probes have been designed specifically for analyzing the local membrane property within the cells.\u003csup\u003e27\u0026ndash;29\u003c/sup\u003e For example, the G protein-coupled receptor (GPCR) antagonist was covalently tethered to the NR, enabling the embedding of the NR unit within the membrane in close proximity to target GPCR through its interaction with the antagonist.\u003csup\u003e27\u003c/sup\u003e The wavelength-shifting emission of the probe offers valuable insights into the local properties of the membrane surrounding the GPCR. Encouraged by these successes, our AH peptide-NR probes are expected to be useful for probing the local membrane environment around the LPDs given the LPD-targeting property of the AH peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eHerein, we investigated two kinds of AH peptides, namely ApoC\u003csup\u003e23\u003c/sup\u003e and p2-23 derived from human α-synuclein,\u003csup\u003e30\u003c/sup\u003e as LPD-binding units conjugated with NR for the probing the exosomal membranes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Previous studies have elucidated the significance of the inherent nature of LPDs for modulating the binding abilities of AH domains in highly-curved membrane-targeting proteins.\u003csup\u003e20,31\u003c/sup\u003e LPDs are qualitatively characterized as either \u0026ldquo;deep\u0026rdquo; or \u0026ldquo;shallow\u0026rdquo; depending on the relative depth of the defect site with respect to the nearest glycerol backbone.\u003csup\u003e16\u003c/sup\u003e Deep and shallow LPDs are suggested to accommodate distinct classes of AH peptides. ApoC peptide possesses a larger hydrophobic face within its α-helix structure compared to p2-23 peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), as shown in the helical wheel presentation determined by HELIQUEST.\u003csup\u003e32\u003c/sup\u003e This suggests a preferential binding to deep LPDs. Conversely, shallow LPD would be preferentially bound by the p2-23 peptide. Indeed, we found that these peptides were able to discriminate their target LPDs from others by the examination of a series of synthetic liposomes. Their NR conjugate probes were shown to be useful for assessing both the abundance of target LPDs by the fluorescent enhancement response and the membrane properties surrounding these LPDs by the emission wavelength of the probes. With these properties, our probes successfully characterized the LPDs of exosomes from three kinds of cancer cells. These features of AH peptide-NR probes are discussed as a basis for the advanced design of analytical tools for exosomal membranes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents\u003c/h2\u003e \u003cp\u003eFmoc-protected amino acids with L-configuration except glycine were purchased from Watanabe Chemical Industries (Hiroshima, Japan) or AAPPTec (Louisville, KY, U.S.A.). All phospholipids and cholesterol were purchased from Avanti Lipids (Alabaster, AL, USA, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Nile Red derivatives (NR-C5-COOH (6-(9-diethylamino-5-oxo-5H-benzo[a]phenoxazin-2-yloxy)hexanoic acid) and NR-C1-COOH (2-((9-(diethylamino)-5-oxo-5H-benzo[a]phenoxazin-2-yl)oxy)acetic acid)) were synthesized according to the literature.\u003csup\u003e23,33\u003c/sup\u003e Other reagents were commercially available and used without further purification. Water was deionized (\u0026ge;\u0026thinsp;18.0 MΩ cm specific resistance) by an Elix 5 UV water purification system and a Milli-Q Synthesis A10 system (Millipore Corp., Bedford, MA), followed by filtration through a BioPak filter (Millipore Corp.). Unless otherwise mentioned, all measurements were performed at 25\u0026deg;C in 1\u0026times;PBS buffer solution (pH 7.4). Errors are the standard deviations obtained from three independent experiments (\u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProbe Synthesis\u003c/h3\u003e\n\u003cp\u003eAll probes were synthesized using Biotage Initiator\u0026thinsp;+\u0026thinsp;Alstra peptide synthesizer (Biotage, Uppsala, Sweden) based on a Fmoc solid phase peptide chemistry on a Rink-Amide-ChemMatrix resin (Biotage) according to our previous report.\u003csup\u003e23, 25\u003c/sup\u003e The obtained crude product was purified by a reverse-phase HPLC system (pump, PU-2086 Plus \u0026times;2; mixer, MX 2080-32; column oven, CO-1565; detector, UV-2070 plus and UV-1570M (Japan Spectroscopic Co. Ltd., Tokyo, Japan)) equipped with a C18 column (Inertsil ODS3 (5.0 \u0026micro;m particle size, 250 mm \u0026times; 20 mm column size); GL Sciences Inc., Tokyo, Japan) using a gradient of water/acetonitrile containing 0.1% TFA (Figure S2A). The probe was verified by MALDI-TOF-MS (Bruker Daltonics autoflex Speed-S1, Germany) (Figure S2B and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The concentration of Nile Red-carrying peptide probes was determined based on the molar absorption coefficient of NR-C5-COOH.\u003csup\u003e23\u003c/sup\u003e\u003c/p\u003e\n\u003ch3\u003ePreparation of synthetic liposomes\u003c/h3\u003e\n\u003cp\u003eSynthetic liposomes were prepared by extrusion according to the literature.\u003csup\u003e23\u003c/sup\u003e Chloroform- or methanol-suspended lipids were combined to form lipid mixtures of the desired molar ratio. The lipid film was obtained by drying under nitrogen gas and then hydrated overnight at 4\u0026deg;C in PBS buffer (pH 7.4). The lipid suspension was subjected to five freeze-thaw cycles. The vesicles were then prepared by manual extrusion through polycarbonate membranes (Whatman, NJ, USA) with a pore diameter of 100 nm using Avanti mini-extruder (Avanti lipids), which affords an average diameter of 115 nm. Liposome size was characterized by dynamic light scattering (DLS) measurements (Zetasizer Nano-ZS, Malvern, UK).\u003c/p\u003e\n\u003ch3\u003eFluorescence measurements\u003c/h3\u003e\n\u003cp\u003eFluorescence spectra and anisotropies were recorded on a JASCO model FP-6500 spectrofluorophotometer (Japan Spectroscopic Co. Ltd., Tokyo, Japan) with a thermoelectrically temperature-controlled cell holder. Measurements were done using a 3 \u0026times; 3 mm quartz cuvette. The apparent dissociation constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e), which describes the lipid concentration where 50% of the peptide probe is bound, was determined from the fluorescence anisotropy titration experiments.\u003csup\u003e34\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eEvaluation of insertion depth of AH peptides using Laurdan-labelled liposomes\u003c/b\u003e \u003csup\u003e35\u003c/sup\u003e \u003c/p\u003e \u003cp\u003e100% DOPC (dioleoyl-phosphatidylcholine) liposomes were mixed with Laurdan and incubated at 37\u0026deg;C for 1 h in the dark. 100 \u0026micro;M peptides were added, followed by incubation at 37 ℃ for 30 min in the dark. The final concentration is 250 \u0026micro;M for liposomes, 0.50 \u0026micro;M for Laurdan and 20 \u0026micro;M for peptides. The fluorescence spectrum was recorded with an excitation wavelength of 340 nm at 25\u0026deg;C. General polarization (GP) value for Laurdan was determined by the equation: GP = (\u003cem\u003eI\u003c/em\u003e\u003csub\u003e440\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;\u003cem\u003eI\u003c/em\u003e\u003csub\u003e490\u003c/sub\u003e) / (\u003cem\u003eI\u003c/em\u003e\u003csub\u003e440\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eI\u003c/em\u003e\u003csub\u003e490\u003c/sub\u003e), where \u003cem\u003eI\u003c/em\u003e\u003csub\u003e440\u003c/sub\u003e and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e490\u003c/sub\u003e are the emission intensities of Laurdan at 440 and 490 nm, respectively. The change of GP (ΔGP) upon addition of the peptides was determined by the equation: ΔGP\u0026thinsp;=\u0026thinsp;GP\u003csub\u003eliposome\u003c/sub\u003e \u0026minus; GP\u003csub\u003eliposome + peptide\u003c/sub\u003e, where GP\u003csub\u003eliposome\u003c/sub\u003e and GP\u003csub\u003eliposome + peptide\u003c/sub\u003e denote GP values in the absence and presence of peptides, respectively.\u003c/p\u003e\n\u003ch3\u003eIsolation of exosomes from cell culture supernatants\u003c/h3\u003e\n\u003cp\u003eAll cell lines were obtained from Cell Resource Center for Biomedical Research, Tohoku University or ATCC (Rockville, MD, USA). All cells were grown in RPMI 1640 media supplemented with 10% fetal bovine serum and 2% penicillin/streptomycin at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. Cells were grown until about 80% confluence and then cultured in an EV-depleted medium for 48 h. Exosomes were collected and isolated from the cell culture supernatants using MagCapture Exosome Isolation Kit PS version 2 (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan).\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e22-meric ApoC and p2-23 peptides were selected in this study among various highly-curved membrane-binding AH peptides due to the following reasons. First, both peptides are reported to sense the LPDs on the lipid membranes.\u003csup\u003e23,30\u003c/sup\u003e Despite the same peptide length, these peptides differ in the hydrophobic face when adopting the α-helix structure for binding to membranes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). p2-23 peptide is shown to selectively recognize shallow LPDs due to its poorly developed hydrophobic face.\u003csup\u003e30,31\u003c/sup\u003e Meanwhile, ApoC is expected to recognize deep LPDs due to its multiple bulky residues in the hydrophobic face, akin to the well-characterized amphipathic lipid-packing sensor (ALPS) motifs such as ArfGAP1.\u003csup\u003e20\u0026ndash;22\u003c/sup\u003e\u003c/p\u003e\n\u003ch3\u003eBinding characteristics of ApoC and p2-23 peptides\u003c/h3\u003e\n\u003cp\u003eWe first investigated the insertion depth of two AH peptides for binding to synthetic liposomes in order to examine their binding characteristics to LPDs, in which DOPC liposomes (average diameters of 115 nm) labelled with Laurdan (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), a well-characterized fluorophore for membrane analysis,\u003csup\u003e36,37\u003c/sup\u003e were used according to the literature.\u003csup\u003e35\u003c/sup\u003e Molecular dynamic simulation and the subsequent characterization of LPDs by Packmem algorithm showed that DOPC liposomes featured both deep and shallow LPDs on their membrane surface.\u003csup\u003e16\u003c/sup\u003e Laurdan can be inserted into the lipid bilayer at the depth of the lipid tails. Its emission spectrum undergoes significant changes based on the degree of its exposure to water, enabling to analyze the peptide insertion on the membranes.\u003csup\u003e35\u003c/sup\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA shows the change of GP value (ΔGP) of Laurdan embedded in DOPC liposomes upon binding of AH peptides. ApoC binding resulted in a large increase in ΔGP of Laurdan, indicative of reduced exposure of Laurdan to the aqueous environment. This suggests that the lipid packing of the membrane is tightened by the deeper insertion of ApoC peptide into the membranes. In contrast, we observed a decrease in ΔGP value for p2-23 peptide. This means increased access of the water to Laurdan within the liposomes upon binding of p2-23 peptide, which is highly likely due to its shallower insertion into the membrane. It is noteworthy that the observed ΔGP for ApoC was comparable to that for M2 peptide (RLFFK SIYRF FEHGL KRG) that was shown to be deeply inserted into the membranes.\u003csup\u003e35\u003c/sup\u003e Combined with their preferential binding to LPDs, these results indicate that ApoC and p2-23 peptides are capable of recognizing deep and shallow LPDs on highly curved membranes, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next investigated whether these peptides could compete with each other for the binding to DOPC liposomes or not. If ApoC and p2-23 peptides selectively recognize deep and shallow LPDs, respectively, competitive binding for these peptides would not occur. Here, the peptides conjugated with NR units at the N-terminal through C5 linker (ApoC-NR and p2-23-NR, Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) were prepared. While these NR probes displayed weak emission in the buffer, the addition of DOPC liposomes caused enhancement of the emission as well as a blue shift of the emission peak (Figure S3). This originates from the partition of the NR unit into the membrane upon binding of AH peptide unit into the LPDs on the membrane surface.\u003csup\u003e23\u003c/sup\u003e The apparent dissociation constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e) was determined by the fluorescence anisotropy titration experiments. \u003cem\u003eK\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e values of ApoC-NR and p2-23-NR were obtained as 45 \u0026plusmn; 14 \u0026micro;M and 60 \u0026plusmn; 12 \u0026micro;M, respectively (Inset, Figure S3), which shows comparable binding affinity for these probes. In the competitive binding experiments, p2-23 peptide was initially incubated with DOPC liposome to form the p2-23/liposome complex, after which ApoC-NR probe was added. In the absence of p2-23 peptides, ApoC-NR showed strong emission upon binding to the liposomes and the emission remained unaffected in the presence of 5\u0026ndash;50 \u0026micro;M p2-23 peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). It shows that ApoC-NR is not displaced by p2-23 peptide on the liposomes, indicating that ApoC peptide does not compete with p2-23 peptide for the binding to the liposomes because of the difference in their binding sites. It should be noted that we observed a significant decrease in ApoC-NR\u0026rsquo;s emission upon the addition of M2 peptide (Figure S4). This is attributable to the competitive binding of ApoC with M2 peptides as both peptides target deep LPDs, which validates the present experiments for analysis of AH peptides\u0026rsquo; binding preference toward LPDs. We also found negligible competition in the case of the addition of p2-23-NR into ApoC/liposome complex (Figure S5). Taken together with the results for insertion depth examination (cf. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), ApoC peptide can serve as a deep-LPD binder whereas p2-23 peptide preferentially recognizes shallow LPDs.\u003c/p\u003e\n\u003ch3\u003eFluorescence response of AH peptide-NR probes for synthetic liposomes\u003c/h3\u003e\n\u003cp\u003eWe next assessed the fluorescence enhancement response of ApoC-NR and p2-23-NR probes for synthetic DOPC liposomes or POPC (palmitoyl-oleoyl-phosphatidylcholine) liposomes. Following fluorescence experiments were done with excitation wavelength at 470 nm in order to discuss the solvatochromic response of NR unit in addition to the fluorescence enhancement response of the probes according to the previous literatures.\u003csup\u003e27\u0026ndash;29\u003c/sup\u003e LPD abundance was largely influenced by the number of unsaturated acyl chains (two double bonds in DOPC vs one double bond in POPC; Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) in the lipid composition of liposomes.\u003csup\u003e15\u003c/sup\u003e Both deep and shallow LPDs were reported to decrease when DOPC was replaced by POPC.\u003csup\u003e16\u003c/sup\u003e The emission of the probes enhanced as increasing the concentration of DOPC liposomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), where ApoC-NR displayed a larger fluorescence response for the liposomes compared to p2-23-NR. The response of ApoC-NR to DOPC liposomes was 4.8-fold larger than that to POPC liposomes. Also, p2-23-NR showed 2.2-fold larger response for DOPC liposomes over POPC ones. These results are attributable to more favorable binding of the probes to DOPC liposomes because of the increased target LPDs caused by an increased number of unsaturation in the acyl chain.\u003csup\u003e15,16\u003c/sup\u003e It is thus highly likely that the fluorescence enhancement response of these probes can reflect the abundance of target LPDs on the membranes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then turned to the solvatochromic property of NR units of the probes in order to assess whether they could report the local membrane polarity surrounding the LPDs or not. NR units in ApoC-NR and p2-23 probes were confirmed to show the strong solvatochromic characters by the spectroscopic measurements in a series of solvents, similar to NR itself (Figure S6). Here, we prepared synthetic DOPC liposomes or POPC liposomes with varying cholesterol (Chol) contents (0\u0026ndash;50%). Prior to the use of our probes, the membrane polarity of these liposomes was estimated by means of GP values of Laurdan according to the literature.\u003csup\u003e11\u003c/sup\u003e Membranes of DOPC liposomes were found to be more polar (less ordered) than those of POPC ones and the increase in Chol content resulted in the decreased polarity (Figure S7), which is consistent with the results in the literature.\u003csup\u003e38\u003c/sup\u003e Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB displays the normalized emission spectra of ApoC-NR in the presence of DOPC/Chol liposomes. ApoC-NR showed a strong emission with a maximum (λ\u003csub\u003eem\u003c/sub\u003e) at 635 nm for DOPC liposomes. It was found that λ\u003csub\u003eem\u003c/sub\u003e value for POPC liposomes with lower membrane polarity was blue-shifted to 620 nm (Figure S8). In addition, a gradual blue-shift was observed with decreasing membrane polarity as the Chol content increased for both DOPC and POPC liposomes (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and S8). Hence, combined with its binding preference, its emission is highly likely to reflect the local membrane polarity surrounding the deep LPDs. Likewise, p2-23-NR demonstrated the wavelength-shifting emission in response to changes in the membrane polarity (Figure S9). p2-23-NR would report differences in the membrane polarity around shallow LPDs.\u003c/p\u003e \u003cp\u003eIn order to obtain further gain into the local membrane property, we examined a ratiometry-based analysis for the probes\u0026rsquo; response to liposomes. As the emission band position of NR unit describes the local polarity, we analyzed the short and long-wavelength parts of the NR\u0026rsquo;s emission (ApoC-NR: 590 nm and 650 nm, p2-23-NR: 590 nm and 660 nm), in line with a previously used methodology.\u003csup\u003e27,28\u003c/sup\u003e Stronger emission in the short wavelength indicates a more polar microenvironment whereas we could observe stronger emission in the long wavelength for a less polar microenvironment. Accordingly, the relative ratio of the emission intensity at this wavelength (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e590\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003e650\u003c/sub\u003e and \u003cem\u003eF\u003c/em\u003e\u003csub\u003e590\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003e660\u003c/sub\u003e for ApoC-NR and p2-23-NR, respectively) can be used for probing the polarity of the local membranes surrounding the LPDs. Indeed, we found that the ratio values for both ApoC-NR and p2-23-NR probes against the above liposomes are highly correlated with membrane polarity (GP value) determined by Laurdan assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results verified the usefulness of the ratiometric analysis of local membranes\u0026rsquo; polarity using our probes. It should be noted that the appropriate linker length between AH peptide and NR units plays a crucial role in the analysis of local membrane properties. We found a decrease or even lack of the solvatochromism of the NR units in response to the variation in the membrane polarity when AH peptide probes carrying NR unit through a short C1 linker were examined (Figure S10). This is attributable to that the NR unit did not partition effectively into the membrane when the short linker was used. Thus, ApoC-NR and p2-23-NR probes carrying a C5 linker (cf. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) were used for further studies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExosomal membrane analysis based on the probes\u0026rsquo; fluorescence response\u003c/h2\u003e \u003cp\u003eApoC-NR and p2-23-NR were finally applied to the analysis of exosomal membrane property through their binding-induced fluorescence response. Exosomes were obtained from the supernatant media of A549 (human lung cancer), Hela (human cervical cancer) and MCF7 (human breast cancer) cells using MagCapture exosome isolation kit that utilizes magnetic beads immobilized with Tim4 protein capable of binding to phosphatidylserine (PS) displayed on exosome surface.\u003csup\u003e39\u003c/sup\u003e Nanotracking analysis revealed that the size distribution of all kinds of exosomes falls into the range of typical exosomes (Figure S11).\u003csup\u003e1,2\u003c/sup\u003e We first sought to examine the membrane polarity of the obtained exosomes (3.9 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e particles/\u0026micro;L) using Laurdan; however, we did not see any response of Laurdan for the exosomes under the present condition (data not shown), presumably due to weak partitioning of Laurdan into the exosomal membranes. Instead, DPH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) was examined for probing the membrane fluidity of the exosomes based on its fluorescence anisotropy.\u003csup\u003e11\u003c/sup\u003e Again, DPH would report the average property of exosomal membranes because it lacks the binding selectivity to specific local regions in the membranes. All exosomes had relatively lower fluidity, where the fluidity is close to that for 70%POPC/30%Chol liposomes (cf. Figure S7). We found negligible differences in membrane fluidity for these exosomes (Figure S12). This indicates average membrane property is almost comparable. In sharp contrast, we observed remarkable differences in local membrane properties for these exosomes by means of our probes. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA depicts the fluorescence spectra of ApoC-NR in the absence and presence of 3.9 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e particles/\u0026micro;L exosomes. It was found that the response varied depending on the type of exosomes. The increase in the emission at 650 nm was in the order of ExoA549\u0026thinsp;\u0026lt;\u0026thinsp;ExoHela\u0026thinsp;\u0026lt;\u0026thinsp;ExoMCF7. Considering the fluorescence enhancement property of ApoC-NR, deep LPDs are highly likely to be abundant for ExoMCF7 compared to ExoA549. We note that the response observed here was more moderate compared to our previous results,\u003csup\u003e23\u003c/sup\u003e presumably due to the difference in the isolation methods of exosomes, namely Tim4-immobilized magnetic beads or the combination of tangential flow filtration and size exclusion chromatography. Moreover, we observed the shoulder at 590 nm in the fluorescence spectra of ApoC-NR bound to the exosomes, which indicates the low polarity of the membranes surrounding the deep LPDs given the solvatochromism of the NR unit. The ratio of the emission intensities at 590 nm and 650 nm (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e590\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003e650\u003c/sub\u003e) was then calculated for the assessment of the exosomal membrane\u0026rsquo;s polarity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). \u003cem\u003eF\u003c/em\u003e\u003csub\u003e590\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003e650\u003c/sub\u003e values for exosomes are significantly larger in comparison with that of ApoC-NR in the absence of exosomes. The comparison among the exosomes showed that ExoMCF7 and ExoHela showed much larger \u003cem\u003eF\u003c/em\u003e\u003csub\u003e590\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003e650\u003c/sub\u003e values over ExoA549. These results indicate that the membranes around deep LPDs in ExoMCF7 and ExoHela are less polar than that in ExoA549. It is highly likely that deep LPD\u0026rsquo;s abundance on exosomal membranes and the surrounding membrane polarity change depending on the donor cells that produce the exosomes. Meanwhile, the response of p2-23-NR to exosomes is found to be relatively moderate compared to ApoC-NR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). This indicates that deep LPDs are more abundant relative to shallow LPDs. p2-23-NR showed a slight fluorescence enhancement at 660 nm for ExoHela. Accordingly, ExoHela possesses shallow LPDs to some extent. On the other hand, negligible fluorescence enhancement was observed for ExoA549 and ExoMCF7, which suggests that these exosomes have scarce shallow LPDs enough to accommodate p2-23-NR probe. We do not observe a significant blue-shift of the emission spectra of p2-23-NR in the presence of exosomes, unlike ApoC-NR. Indeed, the fluorescence emission ratio was almost unchanged in sharp contrast to ApoC-NR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). It seems to be hard to discuss the variation in membrane polarity near shallow LPDs among these exosomes based on the obtained spectra considering the small fluorescence response of p2-23-NR to exosomes. Taken together, these findings demonstrate that exosomal membranes exhibit unique structural properties regarding deep and shallow LPDs and their surrounding membrane polarity. Furthermore, these properties significantly depend on the donor cells. Given such local membrane information cannot be obtained by conventional fluorescence probes such as DPH and Laurdan (cf. Figure S12), our probes should serve as powerful tools for LPD analysis with a view toward better understanding of exosomal membranes and their correlation with biological functions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, we report that AH peptide-NR probes enable to probe the LPD properties on highly-curved membranes through their fluorescence response. ApoC and p2-23 peptides were demonstrated to serve as deep and shallow-LPD binders, respectively. Their NR probes exhibited binding-induced fluorescence enhancement and wavelength-shifting emission for synthetic liposomes, where the response significantly depends on the abundance of target LPDs and their surrounding membrane polarity, respectively. These unique properties allowed us to assess the local membrane property of exosomes. We demonstrated that the deep/shallow LPDs and the membrane polarity around them significantly varied depending on the exosome-producing donor cells. To the best of our knowledge, this work represents the first report on molecular probes to characterize LPD properties on exosomes. Combined with increasing knowledge of lipid molecules in the biological events and their regulatory mechanism,\u003csup\u003e4,5\u003c/sup\u003e the obtained results can contribute to furthering our understanding of exosomal membranes with a view toward the comprehensive analysis of exosome functions. LPD analysis by our probes might hold a potential in exosome-related diagnosis applications as observed for the analysis of lipids\u003csup\u003e40,41\u003c/sup\u003e and membrane viscosity\u003csup\u003e42\u003c/sup\u003e in exosomes. It is here noted that our probes can only analyze the exosomes purified from the supernatant media of the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). For the analysis in complex biological matrices, we need to further improve the probes\u0026rsquo; functions such as the binding selectivity to target exosomes to suppress non-specific response to contaminants in the biological media. Following the present results with ApoC and p2-23 peptides, we expect that systematic studies on the NR conjugates with various AH peptide sequences having different hydrophobic faces or charges would enable the development of the improved probes for more sensitive and accurate analysis of exosomal membranes. Given the structural similarities between exosomes and enveloped virus particles (virions), we also envision that AH peptide-based probes hold great potential to serve as useful tools for enveloped virions.\u003csup\u003e43,44\u003c/sup\u003e We are continuing studies in this field of research.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe thank Prof. Hitoshi Kasai and Dr. Ryuju Suzuki for the DLS measurements. We also thank Biomedical Research Unit of Tohoku University Hospital for technical support. This work was supported by JST PRESTO (Grant No. JPMJPR19H4 to Y.S.).\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eY.S. conceived the study. K. S., T. S., A. N. and K. M. synthesized the probes and characterized their functions. All authors analyzed data. Y. S. and S. N. wrote the paper.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e The online version contains supplementary material available at\u0026hellip;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u0026nbsp;\u003c/strong\u003eand requests for materials should be addressed to Y.S.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003evan Niel, G., D\u0026rsquo;Angelo, G., \u0026amp; Raposo, G. Shedding light on the cell biology of extracellular vesicles. \u003cem\u003eNat. Rev. Mol. Cell Biol.\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 213\u0026minus;228. https://www.nature.com/articles/nrm.2017.125 (2018).\u003c/li\u003e\n\u003cli\u003eJeppesen, D. K. et al. Reassessment of exosome composition. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e177\u003c/strong\u003e, 428\u0026minus;445. e18 https://www.sciencedirect.com/science/article/pii/S0092867419302120?via%3Dihub (2019).\u003c/li\u003e\n\u003cli\u003eMeldolesi,\u003cem\u003e J. \u003c/em\u003eExosomes and Ectosomes in Intercellular Communication.\u003cem\u003e Curr. Biol.\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, R435-R444. https://www.sciencedirect.com/science/article/pii/S0960982218300927?via%3Dihub (2018).\u003c/li\u003e\n\u003cli\u003eDonoso-Quezada, J., Ayala-Mar, S. \u0026amp; Gonzalez-Valdez, J. The role of lipids in exosome biology and intercellular communication: Function, analytics and applications. \u003cem\u003eTraffic\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 204-220. https://onlinelibrary.wiley.com/doi/10.1111/tra.12803 (2021).\u003c/li\u003e\n\u003cli\u003eSusannah, H., Agota, T., Georges, E. G., Michael, E. B. \u0026amp; Alexander, K. L. Understanding the extracellular vesicle surface for clinical molecular biology. \u003cem\u003eJ. Extracell. Vesicles\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e12260. https://isevjournals.onlinelibrary.wiley.com/doi/full/10.1002/jev2.12260 (2022).\u003c/li\u003e\n\u003cli\u003eBanquy, X. et al. Effect of mechanical properties of hydrogel nanoparticles on macrophage cell uptake. \u003cem\u003eSoft Matter\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 3984\u0026ndash;3991. https://pubs.rsc.org/en/content/articlelanding/2009/sm/b821583a (2009).\u003c/li\u003e\n\u003cli\u003eVorselen, D., Piontek, M.C., Roos W. H. \u0026amp; Wuite, G. J. L. Mechanical Characterization of Liposomes and Extracellular Vesicles, a Protocol. \u003cem\u003eFront. Mol. Biosci.\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 139. https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2020.00139/full (2020). \u003c/li\u003e\n\u003cli\u003eSkliar, M. et al. Membrane proteins significantly restrict exosome mobility. \u003cem\u003eBiochem. Biophys. Res. Commun.\u003c/em\u003e \u003cstrong\u003e501\u003c/strong\u003e, 1055-1059. https://www.sciencedirect.com/science/article/pii/S0006291X18311707?via%3Dihub (2018). \u003c/li\u003e\n\u003cli\u003eParolini, I. et al. Microenvironmental pH Is a Key Factor for Exosome Traffic in Tumor Cells. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cstrong\u003e284\u003c/strong\u003e, 34211-34222. https://www.sciencedirect.com/science/article/pii/S0021925820377024?via%3Dihub (2009).\u003c/li\u003e\n\u003cli\u003eSimbari, F. et al. Plasmalogen enrichment in exosomes secreted by a nematode parasite versus those derived from its mouse host: implications for exosome stability and biology. \u003cem\u003eJ. Extracell. Vesicles\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 30741. https://isevjournals.onlinelibrary.wiley.com/doi/10.3402/jev.v5.30741 (2016). \u003c/li\u003e\n\u003cli\u003eSuga, K., Matsui, D., Watanabe, N., Okamoto, Y. \u0026amp; Umakoshi,\u003cem\u003e \u003c/em\u003eH. Insights into the Exosomal Membrane: From Viewpoints of Membrane Fluidity and Polarity. \u003cem\u003eLangmuir\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 11195-11202. https://pubs.acs.org/doi/full/10.1021/acs.langmuir.1c00687 (2021). \u003c/li\u003e\n\u003cli\u003eYasuda, T., Watanabe, H., Hirosawa, K. M., Suzuki, K. G. N., Suga, K. \u0026amp; Hanashima, S. Fluorescence Spectroscopic Analysis of Lateral and Transbilayer Fluidity of Exosome Membranes. \u003cem\u003eLangmuir\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 14695-14703. https://pubs.acs.org/doi/10.1021/acs.langmuir.2c02258 (2022).\u003c/li\u003e\n\u003cli\u003eVanni, S. et al. Amphipathic Lipid Packing Sensor Motifs: Probing Bilayer Defects with Hydrophobic Residues. \u003cem\u003eBiophys. J.\u003c/em\u003e \u003cstrong\u003e104\u003c/strong\u003e, 575-584. https://www.cell.com/biophysj/fulltext/S0006-3495(12)05143-0 (2013). \u003c/li\u003e\n\u003cli\u003eVamparys, L. et al. Conical Lipids in Flat Bilayers Induce Packing Defects Similar to that Induced by Positive Curvature. \u003cem\u003eBiophys. J.\u003c/em\u003e \u003cstrong\u003e104\u003c/strong\u003e, 585-593. https://www.cell.com/biophysj/comments/S0006-3495(12)05142-9 (2013). \u003c/li\u003e\n\u003cli\u003eVanni, S., Hirose, H., Barelli, H., Antonny, B. \u0026amp; Gautier, R. A sub-nanometre view of how membrane curvature and composition modulate lipid packing and protein recruitment. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 4916. https://www.nature.com/articles/ncomms5916 (2014). \u003c/li\u003e\n\u003cli\u003eGautier, R., Bacle, A., Tiberti, M. L., Fuchs, P. F. J., Vanni, S. \u0026amp; Antonny, B. PackMem: A Versatile Tool to Compare and Visualize Interfacial Packing Defects in Lipid Bilayers. \u003cem\u003eBiophys. J.\u003c/em\u003e \u003cstrong\u003e115\u003c/strong\u003e, 436-444. https://www.sciencedirect.com/science/article/pii/S0006349518307628?via%3Dihub (2018). \u003c/li\u003e\n\u003cli\u003eNikos, S. H. et al. How curved membranes recruit amphipathic helices and protein anchoring motifs. \u003cem\u003eNat. Chem. Biol.\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 835-841. https://www.nature.com/articles/nchembio.213 (2009). \u003c/li\u003e\n\u003cli\u003eBrigitte, N. et al. a-Synuclein Has a High Affinity for Packing Defects in a Bilayer Membrane: A THERMODYNAMICS STUDY. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 21966-21975. https://www.sciencedirect.com/science/article/pii/S0021925820668053?via%3Dihub (2004).\u003c/li\u003e\n\u003cli\u003eAnuj, T. et al. Interfacial of lipid head group and packing defects in driving amyloid-beta-mediated myelin-like model membrane deformation. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cstrong\u003e299\u003c/strong\u003e, 104653. https://www.jbc.org/article/S0021-9258(23)00295-8/fulltext (2023).\u003c/li\u003e\n\u003cli\u003eHas, C. \u0026amp; Das, S. L. Recent developments in membrane curvature sensing and induction by proteins. \u003cem\u003eBiochim. Biophsy. Acta Gen. Subj.\u003c/em\u003e \u003cstrong\u003e1865\u003c/strong\u003e, 129971. https://www.sciencedirect.com/science/article/pii/S0304416521001306?via%3Dihub (2021).\u003c/li\u003e\n\u003cli\u003eDrin, G. \u0026amp; Antonny, B. Amphipathic helices and membrane curvature. \u003cem\u003eFEBS Lett.\u003c/em\u003e \u003cstrong\u003e584\u003c/strong\u003e, 1840-1847. https://febs.onlinelibrary.wiley.com/doi/10.1016/j.febslet.2009.10.022 (2010). \u003c/li\u003e\n\u003cli\u003eGim\u0026eacute;nez-Andr\u0026eacute;s, M., Čopič, A. \u0026amp; Antonny, B. \u003cem\u003eBiomolecules\u003c/em\u003e, \u003cstrong\u003e8\u003c/strong\u003e, 45. The Many Faces of Amphipathic Helices. https://www.mdpi.com/2218-273X/8/3/45 (2018).\u003c/li\u003e\n\u003cli\u003eSato, Y., Kuwahara, K., Mogami, K., Takahashi, K. \u0026amp; Nishizawa, S. Amphipathic helical peptide-based fluorogenic probes for a marker-free analysis of exosomes based on membrane-curvature sensing. \u003cem\u003eRSC Adv.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 38323-38327. https://pubs.rsc.org/en/content/articlelanding/2020/ra/d0ra07763a (2020). \u003c/li\u003e\n\u003cli\u003eKawano, K. et al. Development of a Simple and Rapid Method for In Situ Vesicle Detection in Cultured Media. \u003cem\u003eJ. Mol. Biol.\u003c/em\u003e \u003cstrong\u003e432\u003c/strong\u003e, 5876-5888. https://www.sciencedirect.com/science/article/pii/S0022283620305416?via%3Dihub (2020). \u003c/li\u003e\n\u003cli\u003eOhira, K., Sato, Y. \u0026amp; Nishizawa, S. Self-Assembly and Disassembly of Membrane Curvature-Sensing Peptide-Based Deep-Red Fluorescent Probe for Highly Sensitive Sensing of Exosomes. \u003cem\u003eACS Sens.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 522-526. https://pubs.acs.org/doi/10.1021/acssensors.2c02498 (2023). \u003c/li\u003e\n\u003cli\u003eMurayama, T. et al. Loosening of Lipid Packing Promotes Oligoarginine Entry into Cells. \u003cem\u003eAngew Chem Int Ed.\u003c/em\u003e 56, 7644-7647. https://onlinelibrary.wiley.com/doi/full/10.1002/anie.201703578 (2017).\u003c/li\u003e\n\u003cli\u003eHanser, F. et al. Nile Red-Based GPCR Ligands as Ultrasensitive Probes of the Local Lipid Microenvironment of the Receptor. \u003cem\u003eACS Chem. Biol.\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 651-660. https://pubs.acs.org/doi/full/10.1021/acschembio.0c00897 (2021). \u003c/li\u003e\n\u003cli\u003eDanylchuck, D. I., Jouard, P-H. \u0026amp; Klymchenko, A. S. Targeted Solvatochromic Fluorescent Probes for Imaging Lipid Order in Organelles under Oxidative and Mechanical Stress. \u003cem\u003eJ. Am. Chem. Soc.\u003c/em\u003e \u003cstrong\u003e143\u003c/strong\u003e, 912-924. https://pubs.acs.org/doi/full/10.1021/jacs.0c10972 (2021). \u003c/li\u003e\n\u003cli\u003eJimenez-Sanchez, A., Lei, E. K. \u0026amp; Kelly, S. O. A Multifunctional Chemical Probe for the Measurement of Local Micropolarity and Microviscosity in Mitochondria. \u003cem\u003eAngew. Chem. Int. Ed.\u003c/em\u003e \u003cstrong\u003e57\u003c/strong\u003e, 8891-8895. https://onlinelibrary.wiley.com/doi/full/10.1002/anie.201802796 (2018).\u003c/li\u003e\n\u003cli\u003eJensen, M. B. et al. Membrane Curvature Sensing by Amphipathic Helices: A SINGLE LIPOSOSOME STUDY USING a-SYNUCLEIN AND ANNEXIN B12. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cstrong\u003e286\u003c/strong\u003e, 42603-42614. https://www.sciencedirect.com/science/article/pii/S0021925820871488?via%3Dihub (2011).\u003c/li\u003e\n\u003cli\u003ePranke, I. M. et al. a-Synuclein and ALPS motifs are membrane curvature sensors whose contrasting chemistry mediates selective vesicle binding. \u003cem\u003eJ. Cell Biol.\u003c/em\u003e \u003cstrong\u003e194\u003c/strong\u003e, 89-103. https://rupress.org/jcb/article/194/1/89/36569/Synuclein-and-ALPS-motifs-are-membrane-curvature (2011).\u003c/li\u003e\n\u003cli\u003eGautier, R., Douguet, D., Antonny, B. \u0026amp; Drin, G. HELIQUEST: a web server to screen sequences with specific a-helical properties. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 2101-2102. https://academic.oup.com/bioinformatics/article/24/18/2101/192677?login=true (2008).\u003c/li\u003e\n\u003cli\u003eVurgun, N. \u0026amp; Nitz, M. Highly Functionalized b-Cyclodextrins by Solid-Supported Synthesis. \u003cem\u003eChem. Eur. J.\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 4459-4467, https://chemistry-europe.onlinelibrary.wiley.com/doi/full/10.1002/chem.201800028 (2018).\u003c/li\u003e\n\u003cli\u003eRusu, L., Gambhir, A., McLaughlin, S. \u0026amp; Radler, J. Fluorescence Correlation Spectroscopy Studies of Peptide and Protein Binding to Phospholipid Vesicles. \u003cem\u003eBiophys. J.\u003c/em\u003e \u003cstrong\u003e87\u003c/strong\u003e, 1044-1053. https://www.sciencedirect.com/science/article/pii/S0006349504735869?via%3Dihub (2004).\u003c/li\u003e\n\u003cli\u003eMartyna, A., Bahsoun, B., Badham, M. D., Srinivasan, H. M. J. \u0026amp; Rossman, J. S. Membrane remodeling by the M2 amphipathic helix drives influenza virus membrane scission. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 44695. https://www.nature.com/articles/srep44695 (2017).\u003c/li\u003e\n\u003cli\u003eDe Vequi-Suplicy, C. C., Benatti, C. R. \u0026amp; Lamy, M. T. Laurdan in Fluid Bilayers: Position and Structural Sensitivity. \u003cem\u003eJ. Fluoresc.\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 431\u0026minus;439. https://link.springer.com/article/10.1007/s10895-005-0059-3?utm_source=acs\u0026amp;getft_integrator=acs (2006). \u003c/li\u003e\n\u003cli\u003eParasassi, T., De Stasio, G., Ravagnan, G., Rusch, R. M. \u0026amp; Gratton, E. Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of Laurdan fluorescence. \u003cem\u003eBiophys. J.\u003c/em\u003e\u003cstrong\u003e 60\u003c/strong\u003e, 179-189. https://www.sciencedirect.com/science/article/pii/S0006349591820410 (1991).\u003c/li\u003e\n\u003cli\u003eSuga, K. \u0026amp; Umakoshi, H. Detection of Nanosized Ordered Domains in DOPC/DPPC and DOPC/Ch Binary Lipid Mixtures Systems of Large Unilamellar Vesicles Using a TEMPO Quenching Method. \u003cem\u003eLangmuir\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 4830-4838. https://pubs.acs.org/doi/full/10.1021/la304768f (2013).\u003c/li\u003e\n\u003cli\u003eNakai, W. et al. A novel affinity-based method fopr the isolation of highly purified extracellular vesicles. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 33935. https://www.nature.com/articles/srep33935 (2016).\u003c/li\u003e\n\u003cli\u003eKumari, S. \u0026amp; Singh, A. Urinary Exosomal Lipidomics Reveals Markers for Diabetic Nephropathy. \u003cem\u003eCurr. Metabol\u003c/em\u003e. 6, 131-139. https://www.ingentaconnect.com/content/ben/cmb/2018/00000006/00000002/art00009 (2018). \u003c/li\u003e\n\u003cli\u003eFan, T. W. M. et al. Exosomal lipids for classifying early and late stage non-small cell lung cancer. \u003cem\u003eAnal. Chim. Acta\u003c/em\u003e \u003cstrong\u003e1037\u003c/strong\u003e, 256-264. https://www.sciencedirect.com/science/article/pii/S0003267018302794?via%3Dihub (2018).\u003c/li\u003e\n\u003cli\u003eLi, N. et al. Reflecting size differences of exosomes by using the combination of membrane-targeting viscosity probe and fluorescence lifetime imaging microscopy. \u003cem\u003eAnal. Chem.\u003c/em\u003e \u003cstrong\u003e91\u003c/strong\u003e, 15308-15316. https://pubs.acs.org/doi/full/10.1021/acs.analchem.9b04587 (2019).\u003c/li\u003e\n\u003cli\u003eJackman, J. A.\u003cem\u003e \u003c/em\u003eAntiviral peptide engineering for targeting membrane-enveloped viruses: Recent progress and future directions.\u003cem\u003e Biochim. Biophys. Acta Biomembr.\u003c/em\u003e \u003cstrong\u003e1864\u003c/strong\u003e, 183821. https://www.sciencedirect.com/science/article/pii/S0005273621002698?via%3Dihub (2022).\u003c/li\u003e\n\u003cli\u003eSato, Y. et al. Viral membrane-targeting amphipathic helical peptide-based fluorogenic probes for the analysis of infectious titers of enveloped viruses. \u003cem\u003eAnal. Chem.\u003c/em\u003e 97, 5480-5487. https://pubs.acs.org/doi/10.1021/acs.analchem.4c04852 (2025).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Fluorescent probe, Exosome, Membranes, Lipid packing defect, Polarity","lastPublishedDoi":"10.21203/rs.3.rs-5995359/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5995359/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAmphipathic helical (AH) peptide-based fluorescent probes were explored for analysis of lipid packing defects (LPDs) in the membrane surface of exosomes. Two kinds of AH peptide sequences, derived from the C-terminal sequence of Apolipoprotein A-I (ApoC) and from human α-synuclein (p2-23), were examined, where they differ in the hydrophobic face that can be inserted into LPDs. From the examination of the insertion depth of the AH peptides and the competitive binding using synthetic liposomes as exosome models, we found that ApoC peptide could serve as a binder for deep LPDs whereas p2-23 peptide preferentially recognize shallow LPDs. These peptides conjugated with an environment-sensitive dye Nile Red (NR) were demonstrated to be useful for assessing both the abundance of target LPDs by the fluorescent enhancement response and the membrane properties surrounding these LPDs by the emission wavelength of the probes, respectively. With these properties, our probes successfully characterized the LPDs of exosomes from three kinds of cancer cells (A549, Hela and MCF7 cells). We showed that exosomal membranes exhibited unique structural properties regarding deep and shallow LPDs and their surrounding membrane polarity. In addition, these properties significantly depended on the donor cells. Our probes would serve as powerful tools for LPD analysis with a view toward a better understanding of exosomal membranes.\u003c/p\u003e","manuscriptTitle":"Amphipathic helical peptide-Nile Red probes for fluorescence probing of the lipid packing defects and their surrounding membranes on exosomes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-18 17:31:17","doi":"10.21203/rs.3.rs-5995359/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-15T06:27:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-14T09:38:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-08T18:53:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"292423479133651213501718976859220897628","date":"2025-05-05T16:23:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"48346025836220460257195413998585554957","date":"2025-04-21T11:21:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-17T08:36:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-14T08:13:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-04-14T04:29:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"83606f5b-5b1c-4a53-a8ad-edd64a38d284","owner":[],"postedDate":"April 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47306628,"name":"Biological sciences/Biological techniques/Sensors and probes"},{"id":47306629,"name":"Biological sciences/Biological techniques/Sensors and probes/Fluorescent dyes"}],"tags":[],"updatedAt":"2025-07-07T16:08:09+00:00","versionOfRecord":{"articleIdentity":"rs-5995359","link":"https://doi.org/10.1038/s41598-025-08534-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-03 15:58:31","publishedOnDateReadable":"July 3rd, 2025"},"versionCreatedAt":"2025-04-18 17:31:17","video":"","vorDoi":"10.1038/s41598-025-08534-w","vorDoiUrl":"https://doi.org/10.1038/s41598-025-08534-w","workflowStages":[]},"version":"v1","identity":"rs-5995359","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5995359","identity":"rs-5995359","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}