2016 Volume 64 Issue 5 Pages 432-438
Liposomes are closed-membrane vesicles comprised of lipid bilayers, in which the inside of the vesicles is isolated from the external environment. Liposomes are therefore often used as models for biomembranes and as drug delivery carriers. However, materials encapsulated within liposomes often cannot respond to changes in the external environment. The ability of enclosed materials to maintain their responsiveness to changes in the external environment following encapsulation into liposomes would greatly expand the applicability of such systems. We hypothesize that embedding pore-like “access points” into the liposomal membrane could allow for the transmission of information between the internal and external liposomal environments and thus overcome this inherent limitation of conventional liposomes. To investigate this, we evaluated whether a change in the pH of an external solution could be transmitted to the inside of liposomes through the pore-forming protein, yeast voltage-dependent anion channel (VDAC). Transmission of a pH change via VDAC was evaluated using a polyglutamic acid/doxorubicin complex (PGA/Dox) as an internal pH sensor. Upon encapsulation into conventional liposomes, PGA/Dox exhibits no pH sensitivity due to isolation from the external environment. On the other hand, PGA/Dox was found to retain its pH sensitivity upon encapsulation into VDAC-reconstituted liposomes, suggesting that VDAC facilitated the transmission of information on the pH of the external environment to the inside of the liposomes. In conclusion, we successfully demonstrated the transmission of information between the external and internal liposomal environments by a stable pore-like structure embedded into the liposomal membranes, which serve as access points.
Liposomes are closed-membrane vesicles comprised of phospholipid bilayers, in which the aqueous phases of the vesicles are separated from the external environment.1) Owing to their structural characteristics, liposomes are often utilized as models of bio-membranes for constructing artificial cells,2,3) and as drug delivery carriers for stable delivery of unstable drugs to target sites.4,5) An inherent limitation of liposomes is that they cannot transmit information about the external environment (e.g. ion concentration) to the inner core. By overcoming this limitation, the utilities of liposomes would be expanded. Such improved liposomes could potentially be utilized as artificial cellular systems for analysis of intracellular response to changes in the external environment. Or additional functionalities derived from encapsulated materials could be given to liposomal drug delivery carriers modified their surface with antibodies and polyethylene glycol.6,7) Biological cells exhibit systems for transmitting information between the internal and external environments on their membrane, such as receptors and ion channels,8–10) which allows them to respond to extracellular information. Based on this fact, we hypothesized that inclusion of specific access points in the liposomal membrane could allow for the transmission of information about the external environment to the inside of liposomes.
To prove this hypothesis, we evaluated whether a change in the pH of an external solution could be transmitted to the inside of liposomes through pore-forming proteins embedded within the liposomal membrane, which serve as “access points.” To this end, we employed yeast voltage-dependent anion channel (VDAC), which is a β-barrel-type membrane protein found on the outer mitochondrial membrane that exhibits the ability to form stable membrane pores. Ions and small molecules (molecular weight (MW)<5000; e.g. ATP) can easily permeate through VDAC pores by passive diffusion, while larger molecules (MW>5000; e.g. protein) are unable to be transported.11,12) Thus, depending on the concentration gradient, protons can easily permeate through the VDAC pores via passive diffusion. We used a polyglutamic acid/doxorubicin complex (PGA/Dox) as a sensor for the internal pH of the liposomes.13–15) Once encapsulated into VDAC-reconstituted liposomes, PGA/Dox cannot leak from the liposomes owing to their large sizes, which prevent them from being transported via VDAC. PGA/Dox is formed by electrostatic interactions between PGA and Dox. Upon formation of the complex, the fluorescence of Dox is quenched. However, when the pH of the system is decreased to <5.5, Dox fluorescence is recovered by disruption of the PGA/Dox complex.14,15) Thus, a pH-change inside liposomes encapsulating PGA/Dox can be detected by measuring the fluorescence of the system. We herein employed this strategy, and evaluated whether the pH-sensitivity of PGA/Dox encapsulated in liposomes could be recovered upon fusion with VDAC-reconstituted liposomes.
Dioleoyl phosphatidyl choline (DOPC), dioleoyl phosphatidyl ethanol amine (DOPE), and 1,2-bis(oleoyloxy)-3-(trimethylammonio) propane (DOTAP) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, U.S.A.). Dihexadecyl phosphate (dicetyl phosphate; DCP) was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Poly-D-glutamic acid sodium salt (MW 15000–25000; PGA) and doxorubicin hydrochloride were obtained from Sigma-Aldrich. Yeast extract was obtained from Becton, Dickinson and Co. (Mountain View, CA, U.S.A.). Polypeptone was purchased from Nihon Pharmaceutical Co., Ltd. (Tokyo, Japan). Galactose, mannitol, and sorbitol were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals were of reagent grade and used without further purification.
Purification of VDAC from Yeast MitochondriaYeast was grown in yeast extract peptone dextrose (YPD) medium (1% yeast extract, 2% polypeptone, and 2% galactose). Cells were pre-cultured in 10 mL of YPD medium at 30°C. Thereafter, the pre-culture (OD600=1.0) was added to 1.5 L of YPD medium at 30°C, and the cells were incubated with rotation at 200 rpm for 36 h.
Yeast mitochondria were isolated as described previously,16) with the following modification to purify the mitochondria. Briefly, yeast cells were washed three times with cold distilled water and then incubated in 10 mM dithiothreitol (DTT) and 0.1 M Tris–SO4 (pH 8.0) at 30°C, with rotation at 100 rpm for 15 min. The DTT-treated cells were re-suspended in KPi buffer (1.2 M sorbitol and 20 mM KPi, pH 7.4) and then incubated (30°C, 100 rpm, 15 min) with 11.39 mg of zymolyase (Nacalai Tesque, Inc., Kyoto, Japan) in KPi buffer, per gram of yeast cells, to form spheroplasts. The spheroplasts were washed three times with KPi buffer, and subsequently ground in mannitol buffer (10 mM Tris–HCl, pH 7.4, containing 0.6 M mannitol, 0.1% bovine serum albumin (BSA), 0.1 mM ethylenediaminetetraacetic acid (EDTA), and 10 µM 4-amidinophenylmethanesulfonyl fluoride hydrochloride (p-APMSF)) at low speed in a chilled Potter–Elvehjem homogenizer. The homogenate was centrifuged for 5 min at 600×g. The pellet was discarded, and the supernatant was centrifuged for 10 min at 5400×g. The resulting mitochondrial pellet was re-suspended in mannitol buffer. The protein concentration of the mitochondrial suspension was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, U.S.A.) with BSA used as a standard.
The purified mitochondrial suspension was mixed with 1 mL of 6% hydroxyapatite (HA) buffer (6% Triton X-100, 20 mM KCl, 1 mM EDTA, and 10 mM KPi, pH 7.4) containing 1 mM p-APMSF, and incubated on ice for 10 min. The mixture was applied to a HA column (Bio-Rad, Hercules, CA, U.S.A.) equilibrated with 1% HA buffer (1% Triton X-100, 20 mM KCl, 1 mM EDTA, and 10 mM KPi, pH 7.4), and the flow-through was collected in 100 µL aliquots. All aliquots were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and the fractions containing VDAC were collected.
Construction of VDAC-Reconstituted LiposomesThe VDAC-reconstituted liposomes were prepared as previously described.17,18) Briefly, thin lipid films comprised of DOPC, DOPE, and DOTAP (at a molar ratio of DOPC–DOPE–DOTAP=6 : 3 : 2) with 1 mol% phosphoethanolamine-N-lissamine rhodamine B sulfonyl, [1,2-dioleoyl-sn-glycero-3-], ammonium salt (Rho-PE; Avanti Polar Lipids, Inc., Alabaster, AL, U.S.A.) were hydrated in liposomal buffer (50 mM NaCl and 10 mM KPi, pH 7.4) for 10 min at room temperature, followed by sonication for 5 min in a bath-type sonicator (total lipid concentration=10 mM). Then, 100 µL of the liposomal suspension was mixed with 100 µL of VDAC solution (final concentration=100 µg/mL) and 0.8% Triton X-100, and incubated on a rotator for 1 h at room temperature. Following incubation, 16 mg of Bio-Beads SM2 (Bio-Rad) was added, and the mixture was incubated on a rotator for 90 min; this step was repeated three times, so that a total of 48 mg of Bio-Beads SM2 was added. The supernatant containing VDAC-reconstituted liposomes was collected by centrifugation (21500×g for 5 min), and the sizes and ζ-potentials of the liposomes were measured using a Zeta Sizer Nano (Malvern Ins. Ltd., Worcestershire, U.K.). The VDAC-reconstituted liposome suspension was then fractionated using a Sephadex G-50 gel filtration column (GE-Healthcare, Little Chalfont, U.K.) into a total of twenty 1-mL fractions. The VDAC and liposomes in each fraction were evaluated by silver-stained SDS-PAGE analysis and rhodamine B fluorescence measurements, respectively.
Evaluation of Calcein/Dextran 70 Leakage from VDAC-Reconstituted LiposomesVDAC-reconstituted liposomes were prepared as described above. Additionally, liposomal buffer containing 1 mM calcein (Sigma-Aldrich) and 1 mg/mL TexasRed labeled-dextran 70 (Sigma-Aldrich) was added during the hydration step, while calcein and dextran 70 were added at similar concentrations during the reconstitution step. Following reconstitution, the liposomes and non-encapsulated calcein/dextran 70 were separated using Sepharose CL-6B (GE-Healthcare). Liposomal fractions were collected using fluorescence of rhodamine B as an indicator. The fluorescence intensities of calcein (Ex/Em=488/517 nm), dextran 70 (Ex/Em=585/625 nm), rhodamine B (Ex/Em=555/580 nm), and 7-nitro-2-1,3-benzoxadiazol (NBD; Ex/Em=465/535 nm) in the liposomal fractions were measured with an Infinite M200 microplate reader (Tecan Group Ltd., Männedorf, Switzerland), and the fluorescence intensities of calcein and dextran were corrected using that of rhodamine B and NBD, respectively.
Construction of PGA/Dox Complex-Encapsulated LiposomesPGA/Dox was embedded into liposomal membranes by fusion of small unilamellar vesicles (SUVs) around PGA/Dox, as previously reported.19) PGA/Dox was prepared by mixing 1 mg/mL (200 µg) PGA and 0.1 mg/mL (20 µg) Dox in liposomal buffer for 30 min at room temperature. SUVs were prepared as follows. Thin lipid films comprised of DOPE and DCP (at a molar ratio of DOPE–DCP=9 : 2) were hydrated in liposomal buffer for 10 min at room temperature, followed by sonication for 2 min in a bath-type sonicator (14B Ultrasonik, NEY), and subsequently sonicated for an additional 15 min using a probe-type sonicator (Q125 Sonicator, QSONICA) (total lipid concentration=10 mM). Aliquots (100 µL each) of the prepared PGA/Dox and SUV suspensions were mixed well, and subsequently incubated for 30 min at room temperature. The sizes and ζ-potentials of PGA/Dox-encapsulated liposomes were measured using a Zeta Sizer Nano (Malvern Ins., Ltd.).
Evaluation of Formation and Dissociation of PGA/Dox by Dox FluorescenceThe formation and dissociation of PGA/Dox were evaluated by measuring the fluorescence intensity of doxorubicin (Ex/Em=460/585 nm) using an Infinite M200 microplate reader (Tecan Group Ltd.). The fluorescence intensity was corrected using a 40 µg/mL doxorubicin solution.
Construction of PGA/Dox-Encapsulated Liposomes Containing VDACPGA/Dox-encapsulated liposomes and VDAC-reconstituted liposomes were mixed at a ratio of PGA/Dox-liposome–VDAC-liposome=4 : 1, and then incubated for 30 min at room temperature. Following incubation, the pH of the outer solution was adjusted to 7.0 or 5.5, and the dissociation of PGA/Dox was evaluated as described above. The formation of PGA/Dox-encapsulated liposomes containing VDAC was evaluated by Förster resonance energy transfer (FRET) analysis as follows. The PGA/Dox-encapsulated liposomal membrane was stained with 1 mol% Rho-PE, and VDAC-reconstituted liposomes were stained with 1 mol% 7-nitro-2-1,3-benzoxadiazol (NBD)-PE. Fluorescence spectra of the mixture were measured using an Infinite M200 microplate reader (Tecan Group Ltd.), at an excitation wavelength of 460 nm.
Statistical AnalysisAll data are presented as the mean±standard deviation (S.D.). Statistical analysis was performed using GraphPad Prism version 6.0 for Windows (GraphPad Software, San Diego, CA, U.S.A.). Statistical significance (* p<0.05, ** p<0.01, and *** p<0.001) was determined by t-test or ANOVA.
VDAC was purified from yeast mitochondrial outer membrane as previously described16–18) (Supplementary Fig. S1). Purified VDAC was mixed with Rho-PE-labeled liposomes in the presence of Triton X-100, reconstituted into the liposomal membrane by gradual removal of Triton X-100 using Bio-Beads, and fractionated by gel filtration chromatography. VDAC and liposomes were detected by SDS-PAGE analysis and fluorescence measurements, respectively. As shown in Fig. 1, the VDAC fluorescence peak was found to overlap with that of the rhodamine B fluorescence, and the peak (No. 11) was shifted forward to that of “VDAC only” solution (No. 14). Indeed, it was suggested that VDAC is reconstituted into the liposomal membrane (Table 1).
The VDAC-reconstituted liposomes and VDAC solution were fractionated into 20–100-µL fractions using a Sephadex-G-70 gel filtration column. The presence of VDAC was confirmed by silver-stained SDS-PAGE analysis. The band intensities of VDAC (29 kDa) were digitized using ImageJ software. The graph shows the relative intensity, with the peak intensity (Fraction No. 11 [VDAC-liposomes] or No. 14 [VDAC only]) normalized to 1.0. Rho-PE-labeled liposomes were evaluated by measuring the fluorescence intensity of rhodamine B. The size and ζ potential of liposomes used in this experiment was 106.6± 7.24 nm (PDI=0.234±0.033) and +29.8±2.72 mV, respectively. The graph shows the relative intensity, with the peak intensity (Fraction No. 11) normalized to 1.0.
| VDAC (µg/mL) | Particle size (nm) [PDI] | ζ-Potential (mV) |
|---|---|---|
| 0 | 90.14±8 [0.123±0.057] | 6.86±0.47 |
| 20 | 95.43±4 [0.120±0.017] | 13.73±2.65 |
| 100 | 105.57±6 [0.218±0.018] | 11.81±2.11 |
| 200 | 115.1±3 [0.241±0.026] | 33.77±3.14 |
The average diameter and ζ-potential of liposomes used in the experiments of Fig. 1 were measured using a Zeta Sizer Nano. Data represent average±S.D. (n=3). The solvent used for the measurement was phosphate buffered saline (PBS).
We evaluated whether VDAC embedded into the liposomal membrane retained its pore-forming activity. When VDAC was reconstituted into calcein-encapsulating liposomes, the amount of calcein in the liposomes was reduced with increasing VDAC (Fig. 2A). Moreover, Fig. 2B shows that the large dextran 70 species were unable to permeate through VDAC embedded in the liposomal membrane, while calcein was able to penetrate through VDAC, owing to its smaller size (Fig. 2B). These results confirm that the above method allowed for VDAC reconstitution into the liposomes, whilst retaining its pore-forming activity.
(A) Leakage of calcein from VDAC-reconstituted liposomes. VDAC was reconstituted into calcein-encapsulated Rho-PE-labeled liposomes at various concentrations. Following reconstitution, the liposomes were isolated by gel filtration chromatography, and the fluorescence intensity of calcein was measured (Ex/Em=488/517 nm). The fluorescence intensities were corrected using that of rhodamine B (Ex/Em=555/580 nm). The graph shows the relative intensity, with the value of non-VDAC-reconstituted liposomes (0 µg/mL) normalized to 1.0. The size and ζ potential of VDAC-liposomes used in this experiment were shown in Table 1. Data represent average±S.D. (n=3). The data was statistically analyzed by one-way ANOVA. F(3, 8)=164.8, p<0.0001. (B) Size-dependent permeability of VDAC-reconstituted liposomes. VDAC (100 µg/mL) was reconstituted into calcein- or dextran 70-coencapsulated liposomes labeled with Rho-PE and NBD-PE, respectively. Following reconstitution, the liposomes were isolated by gel filtration chromatography, and the fluorescence intensities of calcein (Ex/Em=488/517 nm) and dextran 70 (Ex/Em=585/625 nm) were measured. The fluorescence intensities of calcein and dextran were corrected using that of rhodamine B (Ex/Em=555/580 nm) and NBD (Ex/Em=465/535 nm). The graph shows the relative intensity with the fluorescence value of non-VDAC-reconstituted liposomes (0 µg/mL) normalized to 1.0. Data represent average±S.D. (n=3). The data was statistically analyzed by t-test. VDAC-liposomes released Calcein significantly (p<0.01), although Dextran 70 not significantly (p>0.05), compared to liposomes without VDAC.
The PGA/Dox complex was used as an internal liposomal pH sensor. Stable PGA/Dox complexes are formed by electrostatic interactions between PGA and Dox at neutral pH, resulting in quenching of Dox fluorescence. At acidic pH (<5.5), PGA/Dox complexes are disrupted by protonation of PGA, which results in subsequent recovery of Dox fluorescence.13–15) Therefore, pH changes inside of PGA/Dox-encapsulating liposomes can be monitored via fluorescence.
As the PGA/Dox complexes need to be small to allow for encapsulation into liposomes, we first evaluated the size distribution of PGA/Dox complexes prepared at various PGA : Dox ratios. As shown in Table 2, the sizes of the PGA/Dox complexes became larger with increasing amounts of PGA (Table 2). At a PGA : Dox ratio of 5 : 1, Dox was found to aggregate, inhibiting the formation of PGA/Dox complexes. Previously, it was reported that the increase of γ-PGA ratio improved the formation efficiency of γ-PGA/Dox ionic complex and solubilized them to water by concealing hydrophobic part of Dox into deeper side of the complex.19) Therefore, increasing the size of PGA : Dox complexes along to PGA ratio shown in Table 2 might be resulted from that each Dox molecules interacted with more PGA chain along to PGA ratio. When PGA ratio is too low (like PGA : Dox=5 : 1), hydrophobic part of Dox might be exposed to the surface of complex, hereby the complexes were aggregated by hydrophobic interaction. Based on these findings, we prepared PGA : Dox complexes using a PGA : Dox ratio of 10 : 1, which resulted in the smallest PGA/Dox complexes. We also confirmed that the formation and dissociation of PGA/Dox complexes can be detected by measuring Dox fluorescence. As previously reported, when PGA and Dox are mixed at pH 7.0, the Dox fluorescence was nearly 50% quenched, while Dox fluorescence was completely recovered at pH 5.5 (Fig. 3). These results suggest that pH changes can, indeed, be detected by measuring Dox fluorescence in the PGA/Dox complex.
| PGA : Dox (w/w) | Particle size (nm) [PDI] | ζ-Potential (mV) |
|---|---|---|
| 100 : 1 | 777±59 [0.612±0.144] | −65±3.1 |
| 50 : 1 | 563±83 [0.371±0.077] | −76.5±3.5 |
| 10 : 1 | 309±34 [0.447±0.243] | −83.6±2.8 |
| 5 : 1 | Aggregated | |
The average diameter and ζ-potential of PGA/Dox were measured using a Zeta Sizer Nano. Data represent average±S.D. (n=3). The solvent used for the measurement was PBS.
The formation and dissociation of PGA/Dox complexes were evaluated by measuring the fluorescence intensity of Dox. PGA/Dox complexes were formed by mixing PGA and Dox for 30 min. After formation, the fluorescence intensity of Dox (Ex/Em=460/585 nm) was measured at pH 7.0 and 5.5. The graph shows the relative intensity, with the fluorescence of Dox (without PGA) normalized to 1.0. Data represent average±S.D. (n=3). The data was statistically analyzed by two-way ANOVA followed by Holm–Sidak analysis. * Indicates differences of “PGA/Dox complex” from “Dox only” (*** p<0.001, N.S.=no significant difference) within each pH.
Based on the pH-sensitive fluorescence properties of the PGA/Dox complex, we encapsulated PGA/Dox in liposomes to serve as an internal pH sensor. We first attempted encapsulation of PGA/Dox by the lipid film hydration method. However, when the PGA/Dox suspension was added to the lipid film (10 mM DOPE : EPC=9 : 2) and subsequently sonicated, aggregates were formed (data not shown). It is suggested that the aggregates may result from hydrophobic interactions between the lipids and Dox, due to the high hydrophobicity of Dox.
Owing to the difficulty and limitations associated with encapsulation of PGA/Dox via the lipid film hydration method, we attempted to prepare PGA/Dox-encapsulating liposomes by fusion of SUVs around PGA/Dox.20) In this method, PGA/Dox is mixed with SUVs, and encapsulation of PGA/Dox results from membrane fusion of SUVs around the PGA/Dox complexes. When PGA/Dox was mixed with SUVs (10 mM DOPE : DCP=9 : 2), the particle sizes of the resulting liposomes were found to be smaller than that of PGA/Dox alone (Table 3), which may be due to tight packing of PGA/Dox by the lipid membrane.
| Particle size (nm) [PDI] | ζ-Potential (mV) | |
|---|---|---|
| PGA/Dox | 266±19 [0.364±0.226] | −51.2±5 |
| SUV | 86.4±3 [0.339±0.078] | −16.3±9 |
| PGA/Dox liposome | 217±21 [0.291±0.146] | −44.3±15 |
The average diameters and ζ-potentials of PGA/Dox, SUVs, and PGA/Dox-encapsulated liposomes were measured using a Zeta Sizer Nano. Data represent average±S.D. (n=3). The solvent used for the measurement was PBS.
We evaluated the formation and dissociation of PGA/Dox in liposomes by measuring the fluorescence of Dox. The fluorescence of Dox in the liposomes was quenched at pH 5.5, in contrast to that of non-encapsulated PGA/Dox (Fig. 4A), although the pH sensitivity of PGA/Dox was recovered following treatment with a detergent (Fig. 4B). Taken together, these results suggest that PGA/Dox was encapsulated into liposomes by the above method.
PGA/Dox-encapsulated liposomes were prepared by mixing PGA/Dox and SUVs for 30 min. Following incubation, the fluorescence intensity of Dox (Ex/Em=460/585 nm) was measured at pH 7.0 and 5.5 (A). Then, 1% sodium dodecyl sulfate (SDS) was added to solubilize the liposomal membrane, and the fluorescence intensities of Dox at pH 7.0 and 5.5 were measured again (B). The graph shows the relative intensity, with the fluorescence of Dox (without PGA) normalized to 1.0. Data represent average±S.D. (n=3). The data was statistically analyzed by two-way ANOVA followed by Holm-Sidak analysis. * Indicates differences of “PGA/Dox-liposome” from “Dox only” (** p<0.01, *** p<0.001, N.S.=no significant difference) within each pH. #“Indicates differences between “pH=7.0” and “pH=5.5” of PGA/Dox-liposomes (# p<0.05, N.S.=no significant difference).
As shown in Fig. 4, PGA/Dox does not exhibit pH-sensitivity following encapsulation into liposomes. We evaluated whether inclusion of VDAC pore structures, serving as proton access points within the liposomal membrane, could recover the pH-sensitivity of PGA/Dox.
To this end, we fused PGA/Dox-encapsulated liposomes and VDAC-reconstituted liposomes, and measured the Dox fluorescence. Liposomal fusions were evaluated by Förster resonance energy transfer (FRET) experiment. FRET was found to occur with high efficiency upon mixing PGA/Dox-encapsulated liposomes and VDAC-reconstituted liposomes (Fig. 5). Moreover, the sizes of the resulting liposomes expanded upon mixing (Table 4), suggesting that PGA/Dox-encapsulated liposomes and VDAC-reconstituted liposomes were fused. Next, we evaluated whether the pH-sensitivity of PGA/Dox inside the liposomes was recovered upon fusion with VDAC-reconstituted liposomes. Indeed, Dox fluorescence associated with the PGA/Dox-encapsulated liposomes increased at acidic pH following fusion with VDAC-reconstituted liposomes, while no change in fluorescence was observed in the absence of VDAC-reconstituted liposomes (Fig. 6). Further, we confirmed that liposomal fusion between PGA/Dox-liposomes and liposomes without VDAC cannot recover the pH-sensitivity. Taken together, these results suggest that the external pH change was transmitted to the inner core of the liposomes via the VDAC pore structures.
The fusion of PGA/Dox-encapsulated liposomes and VDAC-reconstituted liposomes was evaluated by FRET. The membranes of PGA/Dox-encapsulated liposomes and VDAC-reconstituted liposomes were labeled by NBD-PE and Rho-PE, respectively. The liposomes were mixed and incubated for 30 min. Following incubation, the fluorescence spectrum (Ex=460 nm, Em=500–700 nm) was measured. The fluorescence spectra of NBD-labeled PGA/Dox-encapsulated liposomes or Rho-PE-labeled VDAC-reconstituted liposomes were also measured as controls.
| Particle size (nm) [PDI] | ζ-Potential (mV) | |
|---|---|---|
| PGA/Dox-liposome | 258±17 [0.318±0.043] | −48.6±5 |
| VDAC-liposome | 114±9 [0.418±0.016] | +31.3±3 |
| PGA/Dox-liposome+VDAC-liposome | 353±23 [0.493±0.200] | −24.5±6 |
The average diameters and ζ-potentials of PGA/Dox-encapsulated liposomes, VDAC-reconstituted liposomes, and their resulting fusion product were measured using a Zeta Sizer Nano. Data represent average±S.D. (n=3). The solvent used for the measurement was PBS.
PGA/Dox-encapsulated liposomes and VDAC-reconstituted liposomes were mixed and incubated for 30 min. Following fusion, the fluorescence intensity of Dox (Ex/Em=460/585 nm) was measured at pH 7.0 and 5.5. The graph shows the relative intensity, with the fluorescence of Dox (without PGA) normalized to 1.0. Data represent average±S.D. (n=3). The data was statistically analyzed by two-way ANOVA followed by Holm–Sidak analysis. * Indicates differences of “PGA/Dox-liposome” or “PGA/Dox-liposome+VDAC-liposome” from “Dox only” (** p<0.01, *** p<0.001) within each pH. # Indicates differences between “pH=7.0” and “pH=5.5” of “PGA/Dox-liposomes” or “PGA/Dox-liposome+VDAC-liposome” (## p<0.01, N.S.=no significant difference).
In this study, we found that external pH changes could be transmitted into liposomes through the pore-forming membrane protein VDAC. Results of this study suggest that stable pore-like structures embedded within liposomal membranes can serve as “access points” and effectively facilitate the transmission of information between the external and internal liposomal environments. Direct transmission of information between the external and internal liposomal environments may allow for the construction of liposome-based artificial cells with responsivities to external environmental changes, or liposomal drug-carriers with additional functionalities in their inner phase. Thus, the findings reported herein could contribute to expanding the utility of liposomes.
The authors declare no conflict of interest.
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