Introduction

Exosomes, a subgroup of extracellular vesicles that generally range from 30 to 150 nm, are secreted by almost all eukaryotic cells1. 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 cells2. Numerous studies highlighted their pivotal roles in various physiological and pathological processes including cell-to-cell communication, cell growth and differentiation, and immune response3. 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 gradually receieved attention, while the number of reports is very limited compared to exosomal nucleic acids and proteins4. Exosomal membranes display unique lipid expression and distribution patterns compared to cellular membranes, which indicates that lipids are integrated into the membranes through highly regulated processes5. There are growing reports on the critical roles of exosomal membranes’ properties in the intercellular communications of exosomes, including cellular uptake, mobility and biodistribution6,7,8. Fluorescent probes whose emissions respond 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 the lipid order of the exosomal membranes9,10,11,12. 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 membranes9,10,11,12. It was also found that the polarity and fluidity of exosomal membranes varied depending on culture pH conditions9 and the type of donor cells10. 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 geometry13,14,15,16. In the cells, LPDs are suggested to be responsible for the recruitment of many peripheral proteins to highly curved membranes17,18,19. LPDs 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. 1A). AH peptides, one of the most common motifs in lipid-membrane-binding proteins20, are known to recognize LPDs on highly-curved membranes21,22, and they have been employed for the construction of molecular tools for biological membranes23,24,25,26. We recently developed a C-terminal sequence of apolipoprotein A- I (ApoC)-derived AH peptide conjugated with an environment-sensitive fluorophore, Nile Red (NR), for the purpose of exosome detection23. The 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 the α-helix into LPDs21,22. ApoC-NR probe exhibits 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 1A) 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 membranes13,14,15,16. 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 properties within the cells27,28,29. 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 antagonist27. 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. 1A).

Herein, we investigated two kinds of AH peptides, namely ApoC23 and p2-23 derived from human α-synuclein30, as LPD-binding units conjugated with NR for probing the exosomal membranes (Fig. 1B). 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 proteins20,31. LPDs are qualitatively characterized as either “deep” or “shallow” depending on the relative depth of the defect site with respect to the nearest glycerol backbone16. 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 the p2-23 peptide (Fig. 1B), as shown in the helical wheel presentation determined by HELIQUEST32. This suggests a preferential binding to deep LPDs. Conversely, shallow LPDs 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.

Fig. 1
figure 1

(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 HELIQUEST16 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)30. (C) Chemical structures of Laurdan and DPH.

Full size image

Experimental

Reagents

Fmoc-protected amino acids with L-configuration except for 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 literature23,33. 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 report23,25. 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-1570 M (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-COOH23.

Preparation of synthetic liposomes

Synthetic liposomes were prepared by extrusion according to the literature23. 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 (Kd), which describes the lipid concentration where 50% of the peptide probe is bound, was determined from the fluorescence anisotropy titration experiments34.

Evaluation of insertion depth of AH peptides using Laurdan-labelled liposomes35

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 was 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 = (I440 − I490) / (I440 + I490), where I440 and I490 are the emission intensities of Laurdan at 440 and 490 nm, respectively. The change in GP (ΔGP) upon addition of the peptides was determined by the equation: ΔGP = GPliposome − GPliposome + peptide, where GPliposome and GPliposome + 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% CO2 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 membranes23,30. Despite the same peptide length, these peptides differ in the hydrophobic face when adopting the α-helix structure for binding to membranes (Fig. 1B). p2-23 peptide is shown to selectively recognize shallow LPDs due to its poorly developed hydrophobic face30,31. 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 ArfGAP120,21,22.

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 using DOPC liposomes (average diameters of 115 nm) labelled with Laurdan (Fig. 1C), a well-characterized fluorophore for membrane analysis36,37, according to the literature35. 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 surface16. 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 membranes35. Fig. 2A shows the change in 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 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 membranes35. 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.

Fig. 2
figure 2

(A) Changes in ΔGP values of Laurdan-labelled DOPC liposomes after the addition of 20 µM AH peptide (p2-23, ApoC and M2). [Laurdan] = 0.5 µM, [DOPC liposome (total lipid)] = 250 µM. (B) Fluorescence spectra of ApoC-NR in the presence of DOPC liposome or p2-23/liposome complex. [ApoC-NR] = 2.0 µM, [DOPC liposome (total lipid)] = 500 µM, [p2-23 peptide] = 0–50 µM. Excitation, 552 nm.

Full size image

We next investigated whether these peptides could compete with each other for the binding to DOPC liposomes. If ApoC and p2-23 peptides selectively recognize deep and shallow LPDs, respectively, competitive binding between these peptides would not occur. Here, the peptides conjugated with NR units at the N-terminus through C5 linker (ApoC-NR and p2-23-NR, Figs. 1B) were prepared. While these NR probes displayed weak emission in the buffer, the addition of DOPC liposomes caused enhancement of emission as well as a blue shift of the emission peak (Figure S3). This originates from the partitioning of the NR unit into the membrane upon binding of AH peptide unit into the LPDs on the membrane surface23. The apparent dissociation constant (Kd) was determined by the fluorescence anisotropy titration experiments. Kd 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 liposomes 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. 2B). 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 between ApoC and 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 of insertion depth examination (cf. Figure 2A), 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. The 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 literature27,28,29. 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 liposomes15. Both deep and shallow LPDs were reported to decrease when DOPC was replaced by POPC16. The emission of the probes was enhanced with increasing the concentration of DOPC liposomes (Fig. 3A), 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 chain15,16. It is thus highly likely that the fluorescence enhancement response of these probes can reflect the abundance of target LPDs on the membranes.

Fig. 3
figure 3

(A) Fluorescence enhancement of the probes upon addition of DOPC or POPC liposomes. F and F0 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 µM, [Liposome] = 500 µM. Excitation, 470 nm.

Full size image

We then turned to the solvatochromic property of NR units in 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 literature11. 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 literature38. Figure 3B displays the normalized emission spectra of ApoC-NR in the presence of DOPC/Chol liposomes. ApoC-NR showed a strong emission with a maximum (λem) at 635 nm for DOPC liposomes. It was found that λem 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. 3B 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 insight 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 methodology27,28. 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 these wavelengths (F590/F650 and F590/F660 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 membrane 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 1B) were used for further studies.

Fig. 4
figure 4

Fluorescence emission ratio of the probes for the liposomes with different membrane polarity (GP) determined by Laurdan assay.

Full size image

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 surfaces39. 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 × 107 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. 1C) was examined for probing the membrane fluidity of the exosomes based on its fluorescence anisotropy11. Again, DPH reports 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 among 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 5A depicts the fluorescence spectra of ApoC-NR in the absence and presence of 3.9 × 107 particles/µL exosomes. It was found that the response varied depending on the type of exosomes. The increase in 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 in ExoMCF7 compared to ExoA549. We note that the response observed here was more moderate compared to our previous results23, 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 (F590/F650) was then calculated for the assessment of the exosomal membrane’s polarity (Fig. 5C). F590/F650 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 F590/F650 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. 5B). 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. 5C). 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 that 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.

Fig. 5
figure 5

Fluorescence spectra of the probe (2.0 µM: (A) ApoC-NR and (B) p2-23-NR) in the absence and presence of three kinds of exosomes (3.9 × 107 particles/µL). Excitation, 470 nm. (C) Fluorescence emission ratio of the probes in the absence and presence of exosomes. For the response of ApoC-NR, p-values calculated by the Student’s t-test were shown. *p < 0.05, n.s.: not significant.

Full size image

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 properties 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 mechanism4,5, 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 potential for exosome-related diagnosis applications as observed for the analysis of lipids40,41 and membrane viscosity42 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 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 could 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 virions43,44. We are continuing our studies in this field of research.