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[Shiho Tsutsumi](https://orcid.org/0009-0006-8089-6477), [Yuki Takechi-Haraya](https://orcid.org/0000-0002-8754-6457), [Yasuhiro Abe](https://orcid.org/0000-0002-5931-6590), [Kohsaku Kawakami](https://orcid.org/0000-0002-3466-9365)

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[Impact of Lipid Bilayer Composition and Physicochemical Properties on Constitution of a Transmembrane Helical Peptide into Exosome-Mimetic Vesicles](https://mdr.nims.go.jp/datasets/376b5259-abe5-40e0-9132-a46392260f80)

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Impact of Lipid Bilayer Composition and Physicochemical Properties on Constitution of a Transmembrane Helical Peptide into Exosome-Mimetic VesiclesImpact of Lipid Bilayer Composition and Physicochemical Propertieson Constitution of a Transmembrane Helical Peptide into Exosome-Mimetic VesiclesShiho Tsutsumi, Yuki Takechi-Haraya, Yasuhiro Abe, and Kohsaku Kawakami*Cite This: Mol. Pharmaceutics 2025, 22, 6874−6886 Read OnlineACCESS Metrics & More Article Recommendations *sı Supporting InformationABSTRACT: Exosomes are expected to efficiently deliver drugs, such as microRNAs and proteins, to targeted organs. However,using natural exosomes presents many difficulties in terms of safety, quality control, and manufacturing; therefore, developingexosome-mimetic artificial materials is desirable. In this study, we elucidated how sphingomyelin (SM) and cholesterol (CH), themain constituents of the exosome membrane, in addition to phosphatidylcholine (PC), influence the physicochemical properties ofPC vesicles. Then, the relevance of these properties to the secondary structure and insertion efficiency of a helical peptide, thetransmembrane domain of integrin α, was investigated. The constitution of this peptide was most successful with exosome-mimeticvesicles (EMV) bearing 15 mol % SM and 40 mol % CH, and the exclusion of SM or CH resulted in low dispersion stability orunsuccessful peptide constitution. Physicochemical analysis of the membrane properties revealed that successful peptideincorporation into the lipid membrane relied on the membrane softness induced by CH and the appearance of a highly mobileboundary phase induced by SM, which together created a favorable environment for the peptide. These results provide importantinsights that serve as a foundation for developing EMV as drug carriers.KEYWORDS: exosomes, exosome-mimetic vesicles, lipid membrane, helical peptide■ INTRODUCTIONAs our understanding of exosome functions has deepened,research on exosome-based drug delivery has grown.1Exosomes are cell-secreted, small vesicles of approximately50−150 nm in diameter and are known to deliver signalingmolecules, such as microRNAs and proteins, to distant targetorgans.2 This function makes exosomes attractive as a drugdelivery vehicle. The molecular mechanism of their organo-tropism has been partially revealed. The combination ofintegrin α and β subunits is involved in the determination oftarget organs;3 at least 18 α and eight β subunits are known inthe human integrin family, whose principal structural featuresare well conserved.4However, the use of exosomes as pharmaceutical productspresents some problems. First, only autologous exosomes arefeasible for administration due to safety concerns.1 Themanufacturing cost is high even if sufficient amounts ofdrug-encapsulated donor-derived exosomes are produced.Second, quality control of exosomes is challenging, as theirheterogeneity and functions are not yet well-understood.2Thus, fully designed exosome-mimetic vesicles (EMV) withthe desired functions are preferable.Preferential accumulation of fully designed EMV decoratedwith Integrin α6β4 in the lung of mice has been reported,together with the successful delivery of encapsulated micro-RNAs to the recipient cells.5 Lipids used for this EMV wereegg phosphatidylcholine (PC), egg sphingomyelin (SM),cholesterol (CH), and C16 ceramide. This study validatednot only the function of integrin but also the design andReceived: June 3, 2025Revised: September 24, 2025Accepted: September 24, 2025Published: October 8, 2025Articlepubs.acs.org/molecularpharmaceutics© 2025 The Authors. Published byAmerican Chemical Society6874https://doi.org/10.1021/acs.molpharmaceut.5c00825Mol. Pharmaceutics 2025, 22, 6874−6886This article is licensed under CC-BY 4.0Downloaded via NATL INST FOR MATLS SCIENCE (NIMS) on November 12, 2025 at 20:53:05 (UTC).See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Shiho+Tsutsumi"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Yuki+Takechi-Haraya"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Yasuhiro+Abe"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Kohsaku+Kawakami"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/showCitFormats?doi=10.1021/acs.molpharmaceut.5c00825&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?goto=articleMetrics&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?goto=recommendations&?ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?goto=supporting-info&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=tgr1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=tgr1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=tgr1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=tgr1&ref=pdfhttps://pubs.acs.org/toc/mpohbp/22/11?ref=pdfhttps://pubs.acs.org/toc/mpohbp/22/11?ref=pdfhttps://pubs.acs.org/toc/mpohbp/22/11?ref=pdfhttps://pubs.acs.org/toc/mpohbp/22/11?ref=pdfpubs.acs.org/molecularpharmaceutics?ref=pdfhttps://pubs.acs.org?ref=pdfhttps://pubs.acs.org?ref=pdfhttps://doi.org/10.1021/acs.molpharmaceut.5c00825?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://pubs.acs.org/molecularpharmaceutics?ref=pdfhttps://pubs.acs.org/molecularpharmaceutics?ref=pdfhttps://acsopenscience.org/researchers/open-access/https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/concept of exosome-mimetic vesicles. Another report showedhigher cellular uptake of EMV than PC/CH liposomes by theA549 cell line and successful gene silencing by encapsulatedsmall-interfering RNA (siRNA).6 This EMV was formulatedwith a mixture of unsaturated phospholipids and CH withoutany targeting ligand. This report highlights the advantages ofexosome-like lipid compositions over simple PC/CH for-mulations.To develop EMV as a pharmaceutical product, itsphysicochemical characteristics must be understood andcontrolled. For example, thermodynamic properties, mem-brane polarity, phase separation, particle size, ζ potential, andthe ability to retain functional proteins on the membrane likelyinfluence EMV pharmacokinetics and storage capability. Animportant consideration here is that the physicochemicalproperties of the membrane are likely to influence both thesecondary structure of the integrin transmembrane domain andits structural stability within the membrane, as well as theloading efficiency of integrins. However, physicochemicalstudies on exosome membranes or exosome-mimetic mem-branes are limited, and it remains unclear whether themembrane properties affect the secondary structure andstability of the integrin transmembrane domain, despitenumerous reports on the reconstitution of integrins into lipidmembranes and despite numerous studies on the lipid raft. Thelipid raft is a membrane-protein domain on the cell membrane,enriched in CH and high-melting temperature (Tm) lipids suchas sphingolipids,7 forming the liquid-ordered (lo) phase that ishard to solubilize.8 The lipid raft is phase-separated from theliquid-disordered (ld) phase consisting of low-Tm lipids,9−12and it is widely recognized that some membrane proteinslocalize at the lipid raft. Therefore, the addition of both thesphingolipid and the CH might be important for the stablereconstitution of integrin to the EMV. From a practical pointof view, the PC species used in marketed drug formulations aretypically saturated lipids such as dipalmitoylphosphatidylcho-line (DPPC) or distearoylphosphatidylcholine (DSPC),because the pharmaceutical products generally require long-term stability over several years. The optimal design of EMVwould not be an accurate mimic of exosomes using biorelevantlipids, which are typically unsaturated lipids, but would includethe acquirement of vesicles with similar functions using thesepharmaceutically relevant PC species.Previous studies of EMV did not confirm whether thetransmembrane domain of the reconstituted integrin forms itsexpected α-helical structure stably within the membrane. Thisis likely because it is technically difficult to selectively analyzethe structure of the transmembrane domain within the full-length protein. Also, the physicochemical evaluations werelimited in the previous studies on EMVs, as they generallyfocused more on efficacy and biodistribution. In this study, weinvestigated the effects of SM and CH on the physicochemicalproperties of the PC membrane. This combination of lipidswas selected based on a lipidomic study of exosomes, whichreported a larger proportion of SM (approximately 15 mol %)and CH (approximately 40 mol %) in the exosome membranethan in the cell membrane of its origin.13 For PC, DPPC wasused in this study because it is chemically stable and its acylchain length is identical to most of the egg SM. Use of anunsaturated lipid such as 1-palmitoyl-2-oleoyl-snT-glycero-3-phosphocholine (POPC) was not considered, as it is not usedin approved liposomal formulations due to their oxidativeinstability. Phosphatidylserine is also an important exosomalcomponent. However, this study did not consider its inclusionin EMV, as it accelerates clearance of the vesicle from blood bypromoting macrophage phagocytosis.14 Then, we investigatedthe impact of the membrane properties on the constitution of apeptide corresponding to the transmembrane domain ofintegrin α using circular dichroism (CD) spectroscopy and ζpotential measurements in order to find the optimal design ofexosome-mimetic vesicles.■ METHODSMaterials. DPPC and CH were purchased from NipponOil and Fat (Tokyo, Japan) and Sigma-Aldrich (St. Louis, MO,USA), respectively. Egg SM and dipalmitoylphosphatidylserine(DPPS) were obtained from Avanti Polar Lipids (Alabaster,AL, USA). 1-[6-(Dimethylamino)naphthalen-2-yl]dodecan-1-one (laurdan) and sucrose were supplied by Cayman Chemical(Ann Arbor, MI, USA) and FUJIFILM Wako Pure Chemical(Osaka, Japan), respectively. 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) was pur-chased from Nacalai Tesque (Kyoto, Japan). A peptide withan amino acid sequence of the transmembrane domain ofi n t eg r in α (ERAIPIWWVLVGVLGGLLLLTILV-LAMWKVGFFKRNRPP) was chemically synthesized byHokkaido System Science (Sapporo, Japan). All reagentsused in this study were of reagent grade and used as supplied.The molecular weight of the synthesized peptide wasconfirmed using a TripleTOF 6600 plus time-of-flight massspectrometer (Sciex, Framingham, MA, USA) by directinfusion of the peptide solubilized in a methanol−chloroformmixture. Monoisotopic peaks corresponding to triply tohexaply charged ions were observed at m/z = 1491.2181,1118.6656, 895.1341, and 746.1114. The major impuritiesobserved were monooxidized species, proline adducts, andglycine adducts, which accounted for 13.9, 4.5, and 1.5% of theintensity of the quadruply charged ion of the peptide,respectively. It should be noted that oxidation may haveoccurred during electrospray ionization.Preparation of Vesicles. PC, CH, and DPPS weredissolved in chloroform, and SM was dissolved in ethanol ata concentration of 10 mM. The solutions were mixed atvarious ratios (Table 1) in glass tubes. The mixed solutionswere then dried under a flow of nitrogen gas, followed byhydration using a 285 mM sucrose solution at lipidconcentrations ranging from 0.1 to 15 mg/mL. Sucrose wasused to prevent aggregation and to stabilize both theTable 1. Lipid Compositions of the Vesiclessample name DPPC % CH % SM % DPPS %DPPC 100 0 0 0CH10 90 10 0 0CH20 80 20 0 0CH30 70 30 0 0CH40 60 40 0 0SM5 95 0 5 0SM10 90 0 10 0SM15 85 0 15 0SM15CH10 75 10 15 0SM15CH20 65 20 15 0SM15CH30 55 30 15 0SM15CH40 45 40 15 0SM15CH40DPPS5 40 40 15 5Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics Articlehttps://doi.org/10.1021/acs.molpharmaceut.5c00825Mol. Pharmaceutics 2025, 22, 6874−68866875pubs.acs.org/molecularpharmaceutics?ref=pdfhttps://doi.org/10.1021/acs.molpharmaceut.5c00825?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asmembranes and the peptide. This hydration step was carriedout at 60 °C using a water bath with a minute of sonication.Subsequently, the suspensions were extruded through a 100nm pore polycarbonate membrane (Cytiva, Marlborough, MA,USA) at 60 °C five times using a high-pressure extruder(Northern Lipids, Burnaby, Canada).Preparation of Peptide-Loaded Vesicles by theIncubation-Extrusion Method. A peptide with the integrinα transmembrane domain sequence was dissolved in achloroform/methanol (9:1, v/v) solution at 1 mg/mL. Thesolution was then mixed with the lipids in organic solvent at apeptide concentration of 2 wt % relative to the lipids, followedby solvent removal, hydration, and extrusion as describedabove, except that the suspension was incubated at 25 °C for24 h prior to extrusion.Preparation of Peptide-Loaded Vesicles by theCHAPS Method. A zwitterionic detergent, CHAPS, wasused to load the peptide into the SM15CH40 membrane as analternative method.15−18 The peptide was dissolved at 0.1 mg/mL in a solution containing 1 vol % CHAPS and 285 mMsucrose, which was then added to hydrated, but not yetextruded, SM15CH40, at 10 vol %, at a peptide concentrationof 2 wt % relative to the amount of lipids. The suspension wasincubated at 25 °C for 24 h, followed by five extrusion cycles at60 °C. CHAPS was removed by diafiltration using an AmiconUltra Centrifugal Filter with a molecular weight cutoff of10,000 Da (Merck Millipore, Burlington, MA, USA). A controlsample without the peptide was prepared by using the sameprocedure. The vesicle concentration was adjusted to 0.23 mg/mL based on the UV absorption at 215 nm.Dynamic Light Scattering (DLS). DLS analysis wascarried out using a Stunner system (Unchained Laboratories,Pleasanton, CA, USA) equipped with a 660 nm laser diode anda detector at 142°. 2 μL portion of vesicles diluted to 0.1 mg/mL with the sucrose solution was applied to a microfluidiccircuit on a Stunner plate. Each measurement was repeatedfour times in duplicate or in triplicate for different samples toconfirm the reproducibility. The Lunatic Client software(Unchained Laboratories) was used to analyze the data andprovide Z-average diameters based on cumulant analysis. Theviscosity and refractive index of the sucrose solution used forthe calculation were 1.07 cP and 1.34, respectively.19,20Nanoparticle Tracking Analysis (NTA). A NanoSightNS-300 system (Malvern Panalytical, Worcestershire, U.K.)equipped with a 488 nm laser was used to analyze the particlesize distribution. For the measurements, vesicles prepared at0.1 mg/mL were diluted 1000-fold with 285 mM sucrosesolution, which was filtered through an Amicon UltraCentrifugal Filter with a cutoff molecular weight of 10,000Da beforehand. This filtration step was crucial for the analysis,as sucrose-derived particles were otherwise observed, asdescribed in the Supporting Information (Figure S1). Themeasurements were carried out at 25 °C.Lipid Concentration Measurements. Lipid concentra-tions of the vesicles were measured by high-performance liquidchromatography (HPLC) on a Nexera XR system (Shimadzu,Kyoto, Japan), using an Inertsil ODS-4 as a separation column(250 × 4.6 mm2 ID, 3 μm particle size, GL Sciences, Tokyo,Japan). An isocratic flow with a mobile phase consisting ofmethanol, tetrahydrofuran, and 170 mM ammonium acetatesolution (94:5:1, v/v/v) was used at a flow rate of 0.5 mL/min,where the column temperature was maintained at 35 °C. Thelipids were detected using a photodiode array detector at awavelength of 215 nm. Vesicles prepared at 1 mg/mL werediluted 5-fold with ethanol, and 10 μL was injected for analysis.A standard solution containing 1 mg/mL of DPPC, SM, andCH in 80 vol % ethanol was sequentially diluted with the samesolvent to obtain a standard curve ranging from 0.0125 to 1mg/mL, where linearity was confirmed.Differential Scanning Calorimetry (DSC). DSC meas-urements were performed on a MicroCal PEAQ-DSC system(Malvern Panalytical) at a heating rate of 1.5 °C/min, with 5min of isothermal stabilization before every run, and in high-feedback mode. A sucrose solution was used as the reference.Samples ranging from 5 to 15 mg/mL were subjected tomeasurements after vacuum treatment for approximately 5min. The baseline of the signal was corrected using a splinecurve to determine the peak-top phase-transition temperature(Tc) and enthalpy (ΔH).Fluorescence Measurements to Determine Mem-brane Polarity. Laurdan, a fluorescent polarity probe, wasdissolved in chloroform at a concentration of 1 mM and addedto the lipid solutions at a final concentration of 1 mol %relative to the total lipid content. Vesicles were prepared underprotection from light using the method described above. Theprobe was excited at 340 nm, and fluorescence spectra from360 to 600 nm were acquired using an FP-6500 spectro-fluorometer (Jasco Corp., Tokyo, Japan). The measurementswere performed at 25 and 37 °C. Generalized polarization(GP) values21,22 were calculated from the fluorescentintensities at 440 and 490 nm using the following equationto quantify the relative polarity of the membranes:I I I IGP ( )/( )440nm 490nm 440nm 490nm= + (1)Fluorescence Lifetime Measurements to EvaluateMembrane Heterogeneity. Fluorescence lifetime of laurdanincorporated in 0.1 mg/mL vesicles at 1 mol % was measuredby a FluoroCube time-correlated single-photon counting(TCSPC) system equipped with a PicoBrite pulse laser at375 nm (HORIBA, Kyoto, Japan), with a suspension ofLUDOX-HS-30 colloidal silica (Sigma-Aldrich) as a reference.The lifetime measurement was carried out at 25 and 37 °C, atreverse TCSPC mode, with a coaxial delay time of 85 ns, asynchronization delay time of 0 ns, and a repetition rate of 16MHz. The measurement continued until the photon countreached 3000. Decay profile simulations were performed usinga nonlinear least-squares method on the DAS6 fluorescencedecay analysis software (HORIBA). The data was first fitted toa single-component decay function, F(t), as follows:F t A B( ) e t T/= + (2)where t and T represent the time and the lifetime of the probe,respectively. A and B are constants. The number ofexponentials (or decay components) was increased until F(t)fitted the data, and the χ2 value was below 1.2. For instance,the fluorescence decay of the two components is given byF t A B B( ) e et T t T1/2/1 2= + + (3)where T1 and T2 are the lifetimes of the decay components,and B1 and B2 provide the relative amplitudes (percentages ofphotons coming from different decays) calculated by BiTi/∑BiTi. The fluorescence decay curve obtained from ahomogeneous membrane is linear on a semilogarithmic plot,whereas that in a phase-separated membrane shows a nonlinearcurve due to multiple exponential components. FluorescenceMolecular Pharmaceutics pubs.acs.org/molecularpharmaceutics Articlehttps://doi.org/10.1021/acs.molpharmaceut.5c00825Mol. Pharmaceutics 2025, 22, 6874−68866876https://pubs.acs.org/doi/suppl/10.1021/acs.molpharmaceut.5c00825/suppl_file/mp5c00825_si_001.pdfpubs.acs.org/molecularpharmaceutics?ref=pdfhttps://doi.org/10.1021/acs.molpharmaceut.5c00825?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asdecay curves for DPPC, CH10, CH40, SM15, SM15CH10,and SM15CH40 membranes were obtained at 10 nm intervalsin the range from 420 to 500 nm, at 25 and 37 °C.Atomic Force Microscopy (AFM). The bending stiffnessof the lipid membranes was determined by quantitativeimaging using a NanoWizard ULTRA Speed atomic forcemicroscope system (JPK, Berlin, Germany), where thecantilever was pressed at the center of a vesicle and the stresswas measured.23−25 Bending stiffness of CH10, CH40, SM15,SM15CH10, and SM15CH40 was evaluated at room temper-ature. DPPC was excluded from the evaluation because of itsaggregation. Aminopropylated mica substrates (AP-mica) wereprepared by immersing mica substrates (SPI-Chem Mica gradeV-1 12 mm D × 0.15 mm thickness, SPI Supplies, WestChester, PA, USA) in 1% 3-aminopropyltriethoxysilane for 20min at room temperature, followed by rinsing with deionizedwater. Prior to the experiment, the mica substrate was gluedonto a glass slide with an acrylamide ring as a barrier, allowingsurface modification and a subsequent sample fixation processto be performed without direct contact with the mica substrate.Vesicles were fixed on the mica substrate by leaving 100 μL of100 μM vesicles for 10 min at room temperature, followed bythe addition of 1.4 mL of sucrose solution. A commercialsilicon-based cantilever BioLever mini (BL-AC40TS-02,Olympus, Tokyo, Japan) of nominal spring constant 0.09 N/m was calibrated via the thermal-noise method prior to theimaging.26 The quantitative imaging was conducted over a 1μm × 1 μm area at 128 pixel × 128 pixel (<8 nm/pixel), with aset point of 150−250 pN, cantilever speed of 15 μm/s, and z-resolution of 20 points/nm. Approximately 10 images wereacquired per sample to capture 80−120 vesicles. Vesiclestiffness was analyzed as described previously.27ζ Potential Measurement. The ζ potentials of the vesicleswere measured by using a Zetasizer Nano-ZS system (MalvernPanalytical). Samples were diluted to 0.01 mg/mL with 285mM sucrose solution, which was filtered through an Amiconultra centrifugal filter with a cutoff molecular weight of 10,000Da (Merck Millipore) prior to use. A folded capillary ζ cell(Malvern Panalytical) was used for the measurements, and avoltage of 150 V was applied. Each measurement wasperformed with ten repeats, and data were analyzed usingthe Smoluchowski model with the refractive index andviscosity of the sucrose solution described earlier. Particlesize measurements were also performed on the same systemfor making comparisons with the data obtained by NTA. Themeasurement was repeated four times, with duplicates persample to confirm reproducibility. The detection angle was173°.CD Spectroscopy. CD spectra of the peptides incorpo-rated into the membranes were obtained by using a J-815 CDspectrometer equipped with a temperature controller PTC-423S (Jasco, Tokyo, Japan). The samples were diluted to 0.23mg/mL with a 285 mM sucrose solution. A quartz cell with anoptical path length of 10 mm was used for the measurements.CD spectra between 200 and 260 nm were measured at 25 °C,at a scan speed of 50 nm/min, with a response speed of 4 s atstandard sensitivity. The spectral data were averaged over 12times to eliminate noise and obtain smooth curves. Nitrogengas was introduced into the sample room at a flow rate of 10L/min. A baseline curve was first recorded using lipid vesicleswithout the peptide. This baseline was then subtracted fromthe spectrum of vesicles containing the peptide.■ RESULTSEffect of Extrusion Process on Lipid Composition andConcentration of Vesicles. The lipid concentrations ofSM15CH40 and SM15CH10 before and after extrusion weremeasured by using HPLC to verify that slight clogging duringextrusion did not affect the final lipid composition andconcentration. After five extrusion cycles, the total peak area ofSM15CH40 decreased by 7% and that of SM15CH10decreased by 9% (data not shown), which would notsignificantly affect the results of subsequent experiments. Inaddition, the lipid compositions of both samples remainedmostly unchanged (Figure S2A−C).Figure 1. Size distribution of 0.1 mg/mL vesicles analyzed by NTA. (A) CH10, (B) CH40, (C) SM15, (D) SM15CH10, (E) SM15CH40, and (F)SM15CH40DPPS5.Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics Articlehttps://doi.org/10.1021/acs.molpharmaceut.5c00825Mol. Pharmaceutics 2025, 22, 6874−68866877https://pubs.acs.org/doi/suppl/10.1021/acs.molpharmaceut.5c00825/suppl_file/mp5c00825_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig1&ref=pdfpubs.acs.org/molecularpharmaceutics?ref=pdfhttps://doi.org/10.1021/acs.molpharmaceut.5c00825?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asSize Distribution of Phospholipid Vesicles. Theinfluence of SM, CH, and negatively charged PS on the sizedistribution of the phospholipid vesicles was investigated byusing NTA. DPPC vesicles were not evaluated because theyaggregated immediately after preparation. All samples hadmean diameters of approximately 90−100 nm, with slightlylarger diameters observed for those containing 40 mol % CH(Figure 1A−F). The addition of charged DPPS did not affectthe vesicle size distribution (Figure 1F). As the vesicle size wascontrolled during the extrusion, the difference in size originatesfrom the difference in the membrane stiffness and possiblefusion of vesicles after the extrusion. DLS was also employedfor particle size measurements to confirm whether differencesin the measurement principle influenced the results. Whenanalyzed by DLS, all vesicles exhibited larger average sizes thanthe mean and mode diameters obtained by NTA (Table 2).NTA is a number-based evaluation method that determinesthe velocity of Brownian motion for each particle, whereasDLS calculates the average diameter, an intensity-basedaverage particle size, from the fluctuation in the scatteredlight intensity of the entire sample solution. The averagediameter tends to be biased toward larger particle sizes. Theresult is also easily influenced by the presence of largerparticles, because the scattered light intensity is proportional tothe sixth power of the particle diameter. When the vesicleswere visualized by AFM, the mean diameters were closer to theresults of NTA than to those of DLS (Table 2 and FigureS2A−E).Effect of SM and CH on Thermodynamic Behaviors ofthe Membrane. The DSC measurements revealed that theaddition of SM to the DPPC membrane decreased the phase-transition temperature (Tc) and increased the phase-transitionenthalpy (ΔH), whereas the addition of CH broadened thetransition peak to reduce ΔH (Table 3 and Figure 2A−D).The addition of 40 mol % CH resulted in a 90% loss of ΔH,indicating that the change in the physicochemical properties atthe transition temperature became less clear, which agrees withthe previous reports.28 The addition of SM to DPPC at 15 mol% increased ΔH by 37%, which was also in agreement withprior literature on DPPC/palmitoyl sphingomyelin (PSM)mixture,29 where a slight increase in ΔH was observed as thePSM ratio increased up to 40%. The reduction in ΔH by CHwas partially recovered by the addition of SM, indicating thatthese molecules have opposite roles in the phase-transitionproperties of the membrane.The vesicles were prepared at higher concentrations for DSCmeasurement than those for other evaluations, as it wasrequired for assuring sensitivity. This likely influenced the DSCcurves of some samples to exhibit split peaks, suggesting thecoexistence of unilamellar and multilamellar vesicles. Never-theless, the overall enthalpy of the DPPC vesicles was similarto the one reported for large unilamellar vesicles.28Effect of SM and CH on Membrane Polarity. Thefluorescence spectrum of laurdan incorporated into the DPPCmembrane is known to peak at approximately 440 nm belowTc, which is weakened and shifts to 490 nm with increasingtemperature as the membrane undergoes a phase transition.30The red shift in the laurdan spectrum indicates an increase inpolarity,31 which may be explained by the diffusion of watermolecules into the membrane.22 Fluorescence intensities at440 and 490 nm were used to calculate the GP values, asdescribed in the Methods. A higher GP value indicates a lowerpolarity that originated from tighter packaging of the lipidmembrane. Figure 3A−F shows the impact of CH and/or SMaddition on the fluorescence spectra of laurdan, at 25 and 37°C. The polarity at 37 °C was higher than that at 25 °C,regardless of the membrane composition (Figure 3G). Theeffects of CH and/or SM addition on the membrane polaritywere clearer at 37 °C than at 25 °C, as it was closer to Tc. At37 °C, CH and SM decreased and increased the polarity,respectively, while they both decreased the polarity at 25 °C.The polarity change between 25 and 37 °C was suppressed inthe presence of CH but enhanced in the presence of SM,which is consistent with the trend of ΔH. The membranepolarity depends on the lipid packing, which is influenced bymembrane fluidity. In the presence of CH, the contrast aboveand below the transition is smaller than that for pure DPPCmembrane because of the increase in fluidity in the gel stateand its decrease in the liquid state. The contribution of SM islikely to be the opposite. As the membrane assembly isdominated by hydrophobic interaction, the ordering of thehydrophilic part, including hydrated water molecules, shouldmake a significant contribution to ΔH. As this is alsoinfluenced by the change in membrane fluidity at the transitiontemperature, it seems to be natural for the polarity and ΔH toexhibit the same trend.Dynamic Heterogeneity of the Lipid MembraneRevealed by Fluorescence Lifetime Analysis. Figure 4shows the fluorescence lifetime of laurdan and the proportionTable 2. Comparison of NTA, DLS, and AFM Data on the Size Distribution of the VesiclesNTA DLS AFM AFMmean (nm) mode (nm) SD average diameter (nm) PDI mean diameter (nm) SD mean height (nm) SDCH10 93.6 85.4 29.1 109 0.10 82.4 21.4 69.3 17.8CH40 106 91.8 36 129 0.14 113 27 75.6 18.1SM15 92.7 73.2 32.3 111 0.11 116 27 72.2 18.0SM15CH10 89.0 83.1 29.8 109 0.15 75.7 17.4 73.1 19.0SM15CH40 114 97.5 37 125 0.14 113 27 69.8 19.5Table 3. Phase-Transition Enthalpy and Temperaturesample ΔH (kJ/mol) Tc (peak top) (°C)DPPC 29.4 41.0CH10 23.7 41.0CH20 20.3 40.5CH30 7.60 45.0CH40 3.00 45.0SM5 23.1 41.0SM10 39.8 40.0SM15 40.5 40.0SM15CH10 26.5 39.2SM15CH20 24.4 39.2SM15CH30 12.0 38.0SM15CH40 3.91 44.0Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics Articlehttps://doi.org/10.1021/acs.molpharmaceut.5c00825Mol. Pharmaceutics 2025, 22, 6874−68866878https://pubs.acs.org/doi/suppl/10.1021/acs.molpharmaceut.5c00825/suppl_file/mp5c00825_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.molpharmaceut.5c00825/suppl_file/mp5c00825_si_001.pdfpubs.acs.org/molecularpharmaceutics?ref=pdfhttps://doi.org/10.1021/acs.molpharmaceut.5c00825?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asof lifetime components in different membranes at 25 and 37°C. Only one component with 6.0−7.3 ns lifetime representinggel-like phase (gel or lo with lower mobility) was detected forDPPC, CH10, and CH40 membranes at any wavelength at 25°C, whereas a second component with a short fluorescencelifetime of 3.0−3.3 ns appeared at 460−470 nm for SM15,SM15CH10, and SM15CH40 (Figure 4A and Tables S1 andS2). The short fluorescence lifetime of the second componentindicates loose packing and high molecular mobility of thesurrounding lipids that should facilitate quick energy transferfrom the excited fluorophore to the ground state. Theproportions of the second component were estimated to be0.8% for SM15, 0.6% for SM15CH10, and 1.2% forSM15CH40 (Figure 4B), relative to the area under the curve(AUC) of the fluorescent spectra of laurdan within the 420−500 nm range.At 37 °C, the presence of three components was confirmedfor all membranes tested (Figure 4C). These components hadthe fluorescence lifetimes of 5.5−7.3, 3.2−4.5, and 4.9−6.2 ns,and were observed in a relatively shorter, intermediate, andlonger wavelength ranges, respectively (Tables S3−S5). Basedon the polarities indicated by the wavelength ranges and themolecular packing indicated by the fluorescence lifetime, thefirst and the third components were understood to be the gel-like and liquid-crystalline (lc)-like (lc or lo with higher mobility)phases, respectively. Addition of 40% CH diminished thedifference in average fluorescence lifetimes between the gel-likephase and the lc-like phases, while 15% SM enhanced it, whichwas consistent with the tendencies observed for changes in ΔHand GP. The second component, which had the shortestlifetime of laurdan and was detected at the intermediatewavelength range, was considered to reflect the boundarybetween the gel-like and the lc-like phases, since the interfacebetween two distinct phases is inherently unstable, and suchinstability is associated with loose molecular packing andincreased molecular mobility. The second component of 3.0−3.3 ns lifetime observed at 25 °C was also regarded as theboundary, although the lc-like phase was undetected, likelybecause its abundance was below the detection limit. Figure4D shows the proportion of these three components calculatedrelative to the AUC of the fluorescence spectra of laurdanwithin the 420−450 nm range. SM was revealed to induce theboundary under both temperature conditions.Bending Stiffness of Lipid Vesicles Determined byAFM. Figure 5 shows the bending stiffness of the vesiclesevaluated by AFM. The addition of 40% CH reduced thestiffness, which is consistent with the literature reporting theformation of a moderately fluid lo phase at higher CHlevels.32,33 On the other hand, the impact of the addition ofSM on the bending stiffness was obscure. Although thebending stiffness of DPPC vesicles was not available because oftheir poor dispersion stability, it is expected to be betweenCH10 and SM15, considering other physical properties,including thermodynamic transition behavior and polarity.Figure 2. (A) Phase-transition enthalpy (bar graph) and temperatures (dots) of lipid membranes with different compositions. (B−D) DSC curvesshowing the impact of (B) CH addition, (C) SM addition, and (D) both CH and SM addition to DPPC. CH30, CH40, and SM15CH30 wereprepared and measured at 10 mg/mL, and SM15CH40 was measured at 15 mg/mL. Other vesicles were prepared and measured at 5 mg/mL.Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics Articlehttps://doi.org/10.1021/acs.molpharmaceut.5c00825Mol. Pharmaceutics 2025, 22, 6874−68866879https://pubs.acs.org/doi/suppl/10.1021/acs.molpharmaceut.5c00825/suppl_file/mp5c00825_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.molpharmaceut.5c00825/suppl_file/mp5c00825_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.molpharmaceut.5c00825/suppl_file/mp5c00825_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig2&ref=pdfpubs.acs.org/molecularpharmaceutics?ref=pdfhttps://doi.org/10.1021/acs.molpharmaceut.5c00825?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asCH40 and SM15CH40 were found to have significantly softermembranes compared to those of other vesicles.Peptide Constitution to the Vesicles. The trans-membrane helical peptide of integrin α was loaded into thelipid membranes simply by mixing it with lipids in an organicsolvent, followed by solvent removal, hydration, and extrusion.Figure 6A shows the effect of incubation before high-pressureextrusion on the secondary structure of the peptide, where anα-helical CD spectrum was observed in the incubated sample.The formation of the α-helical structure indicated successfulconstitution of the peptide in the lipid membranes. Whenextruded without incubation, the CD spectrum did not exhibita 209 nm local minimum or a 222 nm local minimum, whichare the characteristics of the α-helical structure.34 A successfulα-helix formation of the peptide was achieved when thepeptide/SM15CH40 mixture was incubated for 24 h at 25 orat 4 °C prior to extrusion but not when incubated at 40 °C(Figure 6B). When the peptide/SM15CH40 mixture wasextruded without incubation and stored at 4 °C for 6 days, theCD spectrum did not change from the initial state (Figure 6C),suggesting that the mechanical stress after peptide-membraneinteraction enhances peptide insertion into the membrane. Thepeptide concentration was set at 2 wt % to the vesicles in thisinvestigation because the sensitivity of CD measurement wasinsufficient at concentrations lower than 2%, and the additionof 4% peptide caused aggregation of vesicles (data not shown).Surfactants such as Triton X-100 or CHAPS are commonlyused when peptides or proteins are incorporated intomembranes. Consequently, we also tested a method in whichthe peptide solubilized in the CHAPS solution was added tothe SM15CH40 vesicle solution, followed by incubation,extrusion, and diafiltration. The diafiltration step wasperformed to remove the remaining CHAPS and unincorpo-rated peptide molecules. However, the sample obtained by thismethod did not exhibit an α-helical CD spectrum (Figure S4).Lipid composition also affected the secondary structure ofthe peptide. In vesicles with less SM or CH, the CD spectraexhibited similar but different patterns that cannot beelucidated as that of α-helical structure, at the initial timepoint (Figures 6D−I and S5A−C). Interestingly, CD spectraobtained after 2 weeks of storage at 4 °C revealed that thesecondary structure of the peptide was stable only inSM15CH40, not in the other vesicles.Figure 7 and Table 4 describe the ζ potential of the peptide-loaded vesicles and the peptide-free vesicles evaluated after 2weeks of storage at 4 °C. Proper insertion of the peptideshould not cause a significant change in the ζ potential becausemost region of the molecule is entrapped in the lipidmembrane. Peptide-bearing CH10 and SM15 exhibitedpositive charges, indicating that the cationic peptide was notproperly entrapped in the membrane but was either adsorbedon the surface or protruding from the vesicles. CH40 with thepeptide exhibited a more negative charge than peptide-freeCH40, suggesting that the terminal anionic amino acidprotruded from the membrane.Figure 3. Effect of CH and SM on the fluorescent spectra of laurdan and the membrane polarity. Fluorescence spectra of laurdan showing theimpact of (A) CH addition at 25 °C, (B) SM addition at 25 °C, (C) SM and CH addition at 25 °C, and (D−F) their counterparts at 37 °C. (G)GP values of the membranes at 25 and 37 °C.Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics Articlehttps://doi.org/10.1021/acs.molpharmaceut.5c00825Mol. Pharmaceutics 2025, 22, 6874−68866880https://pubs.acs.org/doi/suppl/10.1021/acs.molpharmaceut.5c00825/suppl_file/mp5c00825_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.molpharmaceut.5c00825/suppl_file/mp5c00825_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig3&ref=pdfpubs.acs.org/molecularpharmaceutics?ref=pdfhttps://doi.org/10.1021/acs.molpharmaceut.5c00825?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asAlthough the peptide constitution did not significantly affectthe average particle size of the vesicles (Table 4), aggregationwas observed for most peptide-loaded vesicles except for SM15and SM15CH40 after 2 weeks of storage at 4 °C. These resultssuggest that both SM and CH are essential for the successfuland stable constitution of this helical peptide. The poly-dispersity index (PDI) values for the peptide-loaded vesicleswere higher than those of the peptide-free vesicles, indicating astronger attractive interaction between the peptide-loadedvesicles.■ DISCUSSIONEffect of CH and SM on the PhysicochemicalProperties of Lipid Membrane. DPPC membrane under-goes phase transition from gel (or solid-ordered (so) phase) toliquid crystalline (or liquid-disordered (ld) phase) at its maintransition at 41 °C. Rippled phase, which is the coexistence ofso and ld aligned periodically, is formed at a temperaturebetween the pretransition at 37 °C and the maintransition.32,35,36 Addition of 10−30 mol % CH to DPPCinduces coexistence of so and lo phase below Tc,26 and furtheraddition of CH (30−50 mol %) provides a homogeneous lophase that does not exhibit significant state change upontemperature increase.37,38 In simpler words, CH renders thegel-phase more fluid and the liquid-crystalline phase morerigid.22 Thus, CH addition reduces ΔH,39−41 which isconsistent with our observation. It should be noted that theΔH of the DPPC membrane is influenced by lamellarity,28,42and our enthalpy data are similar to those reported for 100 nmlarge unilamellar vesicles,28 although split peaks indicatingheterogeneous lamellarity were observed for some samples.Although the effects of CH addition on PC membranes havebeen widely investigated, knowledge of the effects of SMaddition is limited. The addition of 40 mol % CH to egg SMmembrane was reported to decrease ΔH from 5.8 kcal/mol(24.3 kJ/mol) to 0.5 kcal/mol (2.1 kJ/mol), where thetransition peak broadened and shifted toward higher temper-ature.43 A similar observation was reported for CH addition tothe PSM membrane.29 These observations suggest that theeffect of adding CH to the SM membrane is similar to that ofits addition to the PC membrane. Meanwhile, the addition ofFigure 4. Fluorescence lifetime of laurdan and its proportion in different membranes at 25 and 37 °C. (A) Average fluorescence lifetimes of the gel-like and the boundary phase at 25 °C and (B) their percentages. (C) Average fluorescence lifetimes of the gel-like phase, the boundary phase, andthe lc-like phase at 37 °C in different membranes and (D) their percentages.Figure 5. Bending stiffness of vesicles measured by AFM. Data arepresented as mean ± standard deviation (SD). The statisticalsignificance was determined by Tukey’s multiple comparison test.*p < 0.05, ****p < 0.0001, n.s.: not significant (p > 0.05).Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics Articlehttps://doi.org/10.1021/acs.molpharmaceut.5c00825Mol. Pharmaceutics 2025, 22, 6874−68866881https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig5&ref=pdfpubs.acs.org/molecularpharmaceutics?ref=pdfhttps://doi.org/10.1021/acs.molpharmaceut.5c00825?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asPSM to the DPPC membrane slightly increased ΔH below40% PSM.29 The phase diagram of DPPC/SM/CH ternarymixture has not been reported, while that of palmitoyloleoyl-phosphatidylcholine (POPC)/PSM/CH is available44 Accord-ing to the phase diagram, so and lo phases appeared uponaddition of PSM and CH to POPC, respectively, under thetemperature region where pure POPC membrane forms the ldphase. At 15% PSM and 40% CH, the coexistence of ld and lo isdescribed at both 25 and 37 °C. The DPPC membrane formsthe so phase at room temperature. Given the structural andphysicochemical similarities between DPPC and SM, weexpected the phase behavior of the DPPC/SM/CH mixtureto resemble that of the DPPC−CH mixture. Our phys-icochemical investigations indicated that SM15CH40 wasindeed similar to CH40 in many aspects, including thethermodynamic behavior, the polarity, and the stiffness. Thefluorescence lifetimes of the gel- and lc-like phases of CH40and SM15CH40 were also similar, showing little temperature-dependent change. Therefore, the majority of SM15CH40 isconsidered to be in the lo phase. The only difference betweenCH40 and SM15CH40 was the appearance of a small fractionof the boundary phase at 25 °C for SM15CH40.The difference in the GP values between 25 and 37 °C wassmaller and larger in the presence of CH and SM, respectively,than that for the pure DPPC membrane (Figure 3G). A similartrend was observed for the change in the Gibbs energycalculated from the DSC curves (Figure 8), which suggested arelevance between the polarity and thermodynamic stability ofthe lipid membranes. This may be a natural finding, as both areaffected by the molecular packing of the lipid membrane.Figure 6. CD spetra of integrin transmembrane peptide in EMV (A) incubated or not incubated before extrusion, (B) incubated at differenttemperatures before extrusion, and (C) without incubation on the first day and after 6 days of storage at 4 °C. (D−I) CD spectra of constitutedpeptide in different membranes at initial time point and after 2 weeks of storage at 4 °C.Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics Articlehttps://doi.org/10.1021/acs.molpharmaceut.5c00825Mol. Pharmaceutics 2025, 22, 6874−68866882https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig6&ref=pdfpubs.acs.org/molecularpharmaceutics?ref=pdfhttps://doi.org/10.1021/acs.molpharmaceut.5c00825?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asDecrease in the membrane polarity by CH addition at both25 and 37 °C is consistent with a previous report,22 in whichthe effect was explained by (i) the displacement of watermolecules by CH and (ii) tight packing of phospholipids as aconsequence of a restriction of their molecular motions. Theeffect of the SM on the membrane polarity can be explained inthe same manner. Importantly, the fluorescence spectra oflaurdan incorporated in several cell membranes are reported tobe similar to those in CH-rich DPPC/CH membranes,indicating a dominant role for CH in influencing membranepolarity.45,46 Thus, our exosome-mimetic membrane(SM15CH40) is expected to offer an environment similar tothat of exosome membranes.Fluorescence lifetime analysis revealed the presence of threecomponents at 37 °C for all of the vesicles. Two of them couldbe easily assigned to the gel-like and lc-like phases based on theemission wavelength of laurdan. The lifetime of laurdan inDPPC membrane in its gel-phase is reported to be 5.9 ns,while that for 1,2-dilauroyl-sn-glycero-3-phosphocholine(DLPC) membrane in the lc-phase is 4.0 ns.47 In our data,the gel-like and lc-like phases had fluorescent lifetimes of 6−7and 5−6 ns, respectively, showing a trend consistent with theprevious report. The lifetimes of the gel-like phase and the lc-like phase in the CH-rich membranes were intermediatebetween those of the DPPC membrane (Figure 4C), becauseCH rendered the gel-like phase more fluid and the lc-like phasemore solid. The impact of 15% SM is likely the opposite ofCH, as assumed from the fluorescence lifetimes and the ΔH.The short-lifetime component, which was detected at anintermediate wavelength, suggested high mobility and might beinterpreted as the boundary between the gel-like and the lc-likephases. Such an intermediate fluorescence spectrum has beenobserved in membranes composed of gel and lc, althoughdetailed discussion has not been available.47,48Impact of the Physicochemical Properties on theConstitution of Helical Peptide. The presence of the highlymobile boundary phase in the SM15CH40 membrane at roomtemperature is likely one of the key factors for the successfulconstitution of the peptide, as it was the major differencebetween CH40 and SM15CH40. Peptide loading had nosignificant impact on the particle size or dispersion stability ofSM15CH40, but induced aggregation of CH40, indicating theimportance of the boundary phase induced by SM. However,the presence of the boundary phase alone was insufficient forthe successful constitution of the peptide. CH was alsorequired to maintain the peptide helicity within the membrane.AFM study revealed that the addition of CH increased thesoftness of the membrane. CH-induced fluidity/softness alsoseems to play an important role in tolerating and maintainingthe α-helix structure within the membrane. Importance of CHon peptide insertion is also reported for amyloid-β, wheremolecular dynamics simulation analysis revealed that theincorporation of CH into the phospholipid bilayer increasessurface hydrophobicity and alters lipid packing to enhanceamyloid-β monomer binding to the CH-rich region of themembrane.49Proper loading of the integrin transmembrane peptide intothe SM15CH40 membrane required incubation beforeextrusion. This indicates that the insertion process of thepeptide into the lipid membrane is slow, which is betterassisted by mechanical stress to complete the proper foldinginto the α-helix in the membrane. Such time-dependentpeptide insertion has also been reported for some amphiphilicpeptides, including magainin 2, where the positive charge ofthe peptide enhances the electrostatic interaction with thephospholipid headgroup, followed by hydrophobic interactionwith the acyl chains.50,51 Although the peptide was mixed withthe lipids in the organic solvent in this study, it seems that thepeptide was not immediately and fully integrated into themembrane in an α-helical conformation upon hydration of thedry film of the lipid-peptide mixture, as indicated by theimportance of the incubation procedure prior to extrusion. Thepeptide constituted into the SM15CH40 membrane with theFigure 7. ζ potential of vesicles with or without the peptide.Table 4. ζ Potential, the Average Diameter, and the PDI ofthe Vesicles with or without the Peptideζ potential (mV) average diameter (nm) PDIDPPC −12.0 124 0.13DPPC-peptide −10.2 116 0.32CH10 −10.0 112 0.15CH10-peptide 7.11 118 0.22CH40 −17.0 123 0.12CH40-peptide −25.1 131 0.22SM15 −4.15 106 0.17SM15-peptide 21.8 86.8 0.23SM15CH10 −3.31 120 0.14SM15CH10-peptide −4.39 121 0.27SM15CH40 −12.1 125 0.11SM15CH40-peptide −8.85 133 0.21Figure 8. Comparison of the differences in GP (bar graph) and ΔGvalues (dots) obtained at 25 and 37 °C.Molecular Pharmaceutics pubs.acs.org/molecularpharmaceutics Articlehttps://doi.org/10.1021/acs.molpharmaceut.5c00825Mol. Pharmaceutics 2025, 22, 6874−68866883https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig7&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig7&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig7&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig7&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig8&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig8&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig8&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00825?fig=fig8&ref=pdfpubs.acs.org/molecularpharmaceutics?ref=pdfhttps://doi.org/10.1021/acs.molpharmaceut.5c00825?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashelp of zwitterionic surfactant CHAPS did not exhibit an α-helical CD spectrum. This could be due to the residualsurfactant, which likely altered the physicochemical propertiesof the membrane. Thus, our surfactant-free procedure appearsto be more promising for the constitution of helical peptidesand should also be applicable to integrin loading.■ CONCLUSIONSThe effects of SM and CH on the physicochemical propertiesof DPPC-based EMV were investigated, and their relevance tothe constitution efficiency of a transmembrane helical peptidewas examined. While SM enhanced temperature-dependentchanges in the physicochemical properties and induced a smallfraction of the highly mobile boundary phase at roomtemperature, CH suppressed temperature-dependent changesin the physicochemical properties and increased the softness ofthe vesicles. The constitution of the peptide was mostsuccessful with vesicles bearing 15% SM and 40% CH, andthe exclusion of SM or CH resulted in low dispersion stabilityor unsuccessful peptide constitution. This was likely due to therequirement of both CH-induced membrane softness and thepresence of the SM-induced boundary phase. Also, the α-helixstructure formed efficiently by incubating the peptide-vesiclesuspension before extrusion, which indicated that thespontaneous interaction between the membrane and thepeptide, followed by mechanical assistance at high temper-ature, was important. These results provide important insightsthat serve as a foundation for developing EMVs as drugcarriers.■ ASSOCIATED CONTENT*sı Supporting InformationThe Supporting Information is available free of charge atht tps ://pubs .acs .org/doi/10 .1021/acs .molpharma-ceut.5c00825.Images of the impurity particles in sucrose solutionobserved by a nanoparticle tracking analysis system(Figure S1); chromatograms and a bar plot of peak areasfrom the quantification of lipids by liquid chromatog-raphy (Figure S2); images of the lipid vesicles obtainedby AFM (Figure S3); fluorescence spectra of polarityprobe laurdan incorporated in various lipid membranes(Figure S4); and CD spectra of a peptide incorporatedin the lipid membranes at various conditions (Figures S5and S6) (PDF)■ AUTHOR INFORMATIONCorresponding AuthorKohsaku Kawakami − Research Center for Macromoleculesand Biomaterials, National Institute for Materials Science,Tsukuba, Ibaraki 305-0044, Japan; Graduate School ofScience and Technology, University of Tsukuba, Tsukuba,Ibaraki 305-8577, Japan; orcid.org/0000-0002-3466-9365; Phone: +81-29-860-4424;Email: kawakami.kohsaku@nims.go.jpAuthorsShiho Tsutsumi − Research Center for Macromolecules andBiomaterials, National Institute for Materials Science,Tsukuba, Ibaraki 305-0044, Japan; Graduate School ofScience and Technology, University of Tsukuba, Tsukuba,Ibaraki 305-8577, Japan; Analytical Research Laboratory,Eisai Co. Ltd., Tsukuba, Ibaraki 300-2635, Japan;orcid.org/0009-0006-8089-6477Yuki Takechi-Haraya − Division of Biochemistry, NationalInstitute of Health Sciences, Kawasaki, Kanagawa 210-9501,Japan; orcid.org/0000-0002-8754-6457Yasuhiro Abe − Division of Drugs, National Institute ofHealth Sciences, Kawasaki, Kanagawa 210-9501, Japan;orcid.org/0000-0002-5931-6590Complete contact information is available at:https://pubs.acs.org/10.1021/acs.molpharmaceut.5c00825FundingS.T. and K.K. are paid employees of Eisai and the NationalInstitute for Materials Science, respectively. Y.T.-H. and Y.A.are paid employees of the National Institute of HealthSciences. This work was funded by each author’s affiliation.This study was partially supported by the Japan Society for thePromotion of Science KAKENHI (grant number JP23K06092to Y.T.-H.).NotesThe authors declare no competing financial interest.■ REFERENCES(1) Antimisiaris, S. G.; Mourtas, S.; Marazioti, A. 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