Formation of a Mimetic Biomembrane from the Hydrophobic Protein

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Formation of Mimetic Biomembrane from the Hydrophobic Protein Zein and Phospholipids: Structure and Application Liping Wang, Toshiaki Goto, Yuzhu Wang, Tsutomu Kouyama, and Jin-Ye Wang J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b04573 • Publication Date (Web): 18 Aug 2017 Downloaded from http://pubs.acs.org on August 24, 2017

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The Journal of Physical Chemistry

Formation of Mimetic Biomembrane from the Hydrophobic Protein Zein and Phospholipids: Structure and Application Liping Wang,a Toshiaki Gotoh,b YuzhuWang,c Tsutomu Kouyama,b*and Jin-Ye Wang a* a

School of Biomedical Engineering, Shanghai Jiaotong University, 800 Dongchuan Road,

Shanghai 200240, PR China b

Department of Physics, Graduate School of Science, Nagoya University, Nagoya, 464-8602,

Japan c

Shanghai Synchrotron Radiation Facility (Shanghai Institute of Applied Physics, Chinese

Academy of Science), 239 Zhangheng Road, Shanghai 201210, P.R. China * To whom correspondence should be addressed Tel: (86-21)34205824; Fax: (86-21)34205824; Email: [email protected] Tel:(81-52)4648602; Email: [email protected]

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ABSTRACT: α-zein, a storage protein in corn endosperm, could be purified easily and in large amounts. In this study, α-zein was incorporated into phospholipid-cholesterol (PC-Chol) liposomes. The maximal amount of α-zein incorporated in the liposome was 0.05% (mol/mol) and the PC/Zein molar ratio was near 2400. At this level of the zein insertion, the phase transition temperature of the lipid bilayer was little affected, but the leakage of doxorubicin (DOX) from the PC-Chol liposome became slower obviously when α-zein was added at a higher temperature than the phase transition temperature. Cryogenic transmission electron micrographs of the PC-Chol-Zein liposome showed that adjacent membranes in multilamellar vesicles were often aligned at a regular interval of about 7 nm. Data from synchrotron small angle X-ray scattering of the PC-Chol-Zein liposome indicated the formation of the multilamellar structure with an inter-membrane interval of 7.2 nm, whereas no homogenous membrane alignment was observed in the absence of zein. The present observation can be well explained by supposing that α-zein takes such an elongated conformation that it penetrates through two adjacent membrane layers. This feature seems to be compatible with a recently proposed super helical structural model of α-zein. Meanwhile, experiments with the fluorescent labelled α-zein showed that the PC-Chol-Zein liposome could be uptaken by an intact cell and localized in some specialized area (possibly endosomes) within the cell instead of diffuse distribution in the cell. Thus the PC-Chol-Zein liposome seems to act as an interesting biomembrane model and may be applicable as a drug delivery system.

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INTRODUCTION One approach to understanding biological systems is to unravel the complex components and hierarchical structures, and then bring together by incorporating many perspectives. Cell membranes act both as a barrier between the cytosol and the extracellular space and as the site for protein−lipid interactions, signal transduction, and endo- and exocytosis.1,

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Biomembranes have complex structures composed of various lipids and proteins and the lateral heterogeneity of lipids and proteins allows for various functions.3,

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Thus, many

researchers have recognized the necessity of an adequate model system that accurately mimics the native membrane environment, with both lipids and proteins. In 1985, Deisenhofer and Michel confirmed the presence of transmembrane α-helices and allowed an atomic-level interpretation of biophysical data for the first time.5 Now more than 180 membrane proteins with high-resolution structures have been reported. The clearest feature from them is that interactions between membrane proteins and the lipid bilayer are important, but no general principles about their interactions have yet emerged.6 Difficulties include the ability: to express membrane proteins in large quantities; to handle membrane proteins in vitro without inactivating them.7, 8 The study of biomimetic membrane has been hampered by the lack of well-defined, sufficient and suitable model membrane proteins.9,10 A desirable cell membrane mimetic system would provide enormous opportunities in developing products for biomedical research and applications.11 The storage proteins in corn endosperm are collectively known as zein. They are classified as α, β, γ, or δ depending on their solubility. Among them, α-zein is the most 3



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abundant and comprises 75-85% of total zeins. It is low-cost and easily purified on a large scale.12,13 This protein has been used widely, especially in the food industry. Recently, its promising area of commercialization has been recognized in the biomedical field, and increasing attention has been drawn to fabrication of zein-based drug delivery systems.14-16 α-zein is rich in hydrophobic amino acids (more than 50%), especially aliphatic amino acids (alanine, leucine, and proline). The low contents of charged amino acids in α-zein explain the insolubility in water and the tendency to aggregate. A previous study suggested that α-zein may have a transmembrane domain, and is primarily localized near the membrane enclosing the protein body in corn endosperm.17 Due to its hydrophobicity, α-zein may be easily integrated into lipid bilayers; in this case, it could be used as an ideal protein for studying membrane-bound proteins in mimetic biomembrane. We consider the system not only as an ideal biomembrane model in basic science, but also in biotechnology field such as drug delivery system.

EXPERIMENTAL SECTION Materials. Egg phosphatidylcholine (PC) was purchased from Sigma-Aldrich (dried egg yolk, minimum PC >60%, St. Louis, MO) and was used without further purification. Zein of biochemical purity was obtained from Wako Pure Chemical Industries, Ltd. (Japan). The small molecular drug doxorubicin (DOX; 99.0% purity), which was used as the water-soluble fluorescent marker in liposomes, was purchased from Melone Pharmaceutical Co., Ltd. (Dalian, China). The zein content was determined using a BCA protein assay 4


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(Pierce, USA; #23235). 1,6-diphenyl-1,3,5-hexatriene (DPH) was obtained from Molecular Probes (Sigma, USA). Cy5-NHS ester, a fluorescent dye reactive with amino groups, was purchased from Wuhan Sinaide Biotechnology Ltd. (Wuhan, China).

Preparation of Liposomes. Small unilamellar vesicles (SUV)and/or multilamellar vesicles (MLV) were prepared using a sonication method under nitrogen. The molar ratio of PC:Chol was 1:1 and that of Zein:PC was varied from 0 to 0.3. Briefly, appropriate amounts of egg PC and cholesterol in chloroform (for PC-Chol-Zein liposome zein was dissolved in 80% ethanol) were mixed in the flask and dried by evaporation under nitrogen. The samples were then placed in a vacuum dryer for over 1 h. The thin lipid film that formed on the wall of flask was hydrated with a 0.15 M phosphate buffered saline solution (PBS, pH 7.4) and sonicated under nitrogen for 25 min (50 s on and 10 s off for each cycle) with a probe sonicator (130 W, Sonics & Materials, USA) at 4 ℃ in ice water bath. Subsequent centrifugation at 10,000 g for 10 min was conducted to remove untrapped lipids and titanium. For preparation of DOX-encapsulated liposomes, a citrate buffered saline solution (pH 3.6) was added into the thin lipid film instead of the PBS solution. After sonication, DOX was added to the citrated lipid solution, and the pH of the solution was adjusted to 7.35. Then, centrifugation was performed (10000 g for 10 min). The untrapped DOX was separated by gel filtration on a Sephadex G-50 column using PBS buffer as the eluent.

Fluorescence Anisotropy Measurement. Steady-state fluorescence spectra of DPH were recorded using a Steady-State & Time-Resolved Fluorescence Spectrofluorometer (QM/TM/IM, PTI, USA). Excitation was at 360 nm, and emission was at 424 nm. The background spectra (from unlabeled liposomes treated in the same manner) were subtracted 5



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from those of the labeled liposomes. The fluorescence anisotropy (r) values were obtained using the expression r = (IVV – GIVH)/(IVV + 2GIVH), where IVV and IVH are the vertically and horizontally polarized components of probe emission with excitation by vertically polarized light at the respective wavelength, and G is the sensitivity factor of the detection system. Each intensity value used in this expression represents the computer-averaged values of 8 successive measurements. All spectral measurements were performed at 37 ℃ in freshly prepared solutions.

Dynamic Light Scattering (DLS). The mean diameter, size distribution and polydispersity index of liposomes diluted in PBS were measured using the DLS technique within 24 h after preparation (Zetasizer 3000 HAS, Malvern Instruments Ltd., U.K.). The PC concentration of liposomes tested was 0.5 mM. The scattering cuvette was immersed in a refractive index matching fluid, and the temperature was kept at 25 ℃. The scattered intensity was registered at a scattering angle of 90°.

Small Angle X-ray Scattering (SAXS). Synchrotron SAXS experiments were performed at beamline BL16B1 of the Shanghai Synchrotron Radiation Facility (SSRF) in China. The two-dimensional SAXS data were collected using a 2D MarCCD165 detector and were reduced to one-dimensional SAXS data. The wavelength was 1.24 Å, and the sample-to-detector distance (SDD) was set at 1.85 m. All experiments were performed at room temperature, and the average exposure time was 1000 s. Silver behenate (AgC22H43O2) was used as the standard material for calibrating the scattering vector. The PC concentration in the investigated samples was 50 mM. All scattering curves were corrected for the solvent

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contribution. The intensity profiles were output as a plot of the scattering intensity (I) versus the scattering vector (q=(4π/λ)sin(θ/2), where θ is the scattering angle).

Differential Scanning Calorimetry (DSC). DSC measurements were performed on a Pyris 1 Instrument (Perkin Elmer, Inc., USA) operated at a heating or cooling rate of 10 °C/min between 5 and 60 °C. The scans were recorded in the presence or absence of zein. All samples were degassed prior to use. Five scans were recorded for each sample, and their averaged scan profile was used for data analysis using the Microcal Origin 7.5 software to calculate T1/2 and Tm. The calorimetric enthalpies (∆Hcal) were calculated by integrating the peak areas.

Characterization of Zein. The prepared PC-Chol liposome and PC-Chol-Zein liposome were lyophilized. N-butyl alcohol was added to the lyophilized samples to dissolve PC and cholesterol, the samples were then centrifuged at 7000 rpm for 15 min, and the supernatant was removed. The residual zein was dried at 4 ℃. The dried zein was dissolved in 80% ethanol aqueous, and the concentration of zein was determined using a BCA protein assay. The electrophoresis of zein was conducted using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with Commassie brilliant blue.

Cryo-TEM Measurements. A sample solution was loaded onto a holey-carbon film-supported grid. A thin aqueous film was produced by blotting with a filter paper. The grids were immediately plunged into liquid propane before the thin sample began to evaporate. The frozen sample was stored in liquid nitrogen and transferred to a cryo-transfer

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stage (Oxford CT3500) kept at approximately -180 ℃. Cryo-TEM images were then acquired with a JEM2010 (JEOL, Japan) electron microscope operated at 200kV.

Anti-zein Immunolabeling. Anti-zein antibody, which was prepared by immunizing rabbit with zein, was obtained from Lingchao Biotechnology Co., Ltd. (Shanghai, China). 5 µl anti-zein antibody and immunogold (20 nm) were added to 100 µl liposome solution, and after incubation at room temperature for 2 h, the mixture was centrifuged at 2000 rpm for 10 min to remove the aggregates of immunogold. Then the supernatant was dropped on a 200-mesh copper grid coated with a carbon film, and the copper grid was rinsed three times with DDW, followed by the addition of 1 drop of staining solution (1 wt% uranyl acetate); after incubation for 30 s, the excess solution was removed using a filter paper, and the sample on the grid was dried under infrared light. Then the dried sample was observed using B-TEM (FEI Tecnai G2 Spirit Biotwin, U.S.A.).

DOX Leakage. DOX was chosen as a low-molecular-weight model drug to investigate drug leakage from PC-Chol and PC-Chol-Zein liposomes. DOX began to exhibit strong fluorescence when released from the liposomes and diluted. The time course of DOX leakage from the liposomes was determined by measuring the fluorescence intensity(F(t)) of DOX at time t using a spectrofluorometer (Hitachi F-2500, Japan). The wavelengths of excitation and emission were 505 nm and 559 nm, respectively. The percentage of DOX that leaked from the liposomes was calculated according to the following expression: DOX(%)=100*(F(t)-F(0)/(Ftotal-F(0)), where F(0) is the fluorescence intensity at time 0 and Ftotal is the fluorescence intensity when DOX leakage reached to 100%.

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Labelling of Zein with Cy5. For labeling of zein with Cyanine 5 monosuccinimidyl ester (Cy5-NHS), 10 mg of α-zein was dissolved in 800 µL of 80% aqueous ethanol containing sodium carbonate buffer (pH 8.3). After stirring at room temperature for 1 h, 1 mg Cy5-NHS dissolved in 500 µL N,N-dimethylformamide was added to the zein solution. Then the reaction mixture was stirred for 4 h at room temperature in the dark and unreacted dyes were removed by dialysis in 80% ethanol solution. After the fluorescence labelling was confirmed by a fluorescence spectrometer, the labelled zein was used for preparation of fluorescent labelled PC-Chol-Zein liposome. PC-Chol-Cy5 was prepared by adding 1 mg Cy5-NHS directly into PC-Chol solution in chloroform, and the liposome was prepared as described in the section of Preparation of Liposomes.

Cellular Uptake of PC-Chol-Cy5 and PC-Chol-Zein-Cy5 Liposomes. NIH3T3 cells were plated at a density of 1 ×104 cells/ml in a 35 mm cuvette for 24 h in DMEM media supplemented with 10% serum. NIH3T3 cells were then incubated with either PC-Chol-Cy5 liposome or PC-Chol-Zein-Cy5 liposome for 15 min, 30 min, 45 min and 60 min at 37 ℃ in DMEM media. After incubation with the liposomes, the medium was removed and the cells were washed three times with PBS. Then, 4% paraformaldehyde was added to the cells. Fluorescence images were taken using a confocal inverted fluorescence microscope (Nikon A1S, Japan).

RESULTS AND DISCUSSION Liposome Characterization. All liposome samples showed a similar narrow size 9



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distribution of approximately 150 nm as analyzed by dynamic light scattering (DLS, Fig. S1 in the supporting information). The amount of zein integrated into the liposomes reached a maximal amount value of 0.05% (mol/mol), which was far below the amount that was added during the preparation. The PC/Zein molar ratio was estimated to be near 2400 (Table 1). As α-zein is not soluble in aqueous solution, we supposed that the hydrophobic part of α-zein was inserted into the hydrophobic region of phospholipids. Menger et al. prepared giant vesicles with a high zein content using a mixture of palmitoyloleoyl

phosphatidylcholine

(POPC),

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and zein.18 In their giant vesicles, the molar ratio of PC/Zein could reach to 98:2, which was much higher than that observed in our system (100:0.05). At the PC/Zein molar ratio of 100:0.05 (mol/mol), the zein content is nearly 1.9% (wt/wt), which is near to the content of a typical transmembrane protein (2-21%, w/w).19, 20

Table 1. Main features of the investigated liposome samples. Samples

[zein] mg ml-1(b) 0

R (nm)(c)

A

[zein] mg ml-1(a) 0

R (nm)(d)

6.94

PC/Zein(mol ar ratio)(e) 151.07 ± 2.73 /

PC/Zein(mol ar ratio)(f) /

B

9

0.93

7.22

148.53 ± 3.36 300:1

2415

C

18

0.87

7.25

155.47 ± 2.00 200:1

2422

D

27

1.00

7.20

164.67 ± 3.09 100:1

2399

(a) The content of zein added in the liposome preparation. (b) The content of zein incorporated into liposomes (n=3). (c) The center to center distance between adjacent membranes in multilameller vesicles calculated from SAXS data. (d) The hydrodynamic radius of liposomes determined by DLS analysis. (e) The molar ratio of phospholipids to zein at the initial stage of liposome preparation. (f) The molar ratio of phospholipid to zein in the reconstituted liposome (n=3).

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The zein sample purchased from Wako Pure Chemical Industries contained two components of α-zein with apparent molecular weights of 23 and 26 kDa, which are called Z19 and Z22, respectively.21 The result of SDS-PAGE showed that the low-molecular-weight component (Z19) was preferentially incorporated into PC-Chol liposome (Figure 1). Under the solvent condition used for preparation of the PC-Chol-Zein liposome, the affinity of the high-molecular-weight component (Z22) to the liposome was suggested to be very low. Whether this affinity can be improved in a different lipid environment remains an interesting question.

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Figure 1. Coomassie brilliant blue stained SDS-PAGE of α-zein. Wells: (1) The standard protein ladder; (2) purchased zein; (3) α-zein incorporated into PC-Chol liposome (0.05% zein in molar ratio); (4) α-zein incorporated into the PC-Cholliposome (0.1% zein in molar ratio).

Cryo-TEM Images of the Reconstituted Liposomes. Cryo-TEM was used to investigate the structure of the PC-Chol liposome with and without α-zein (Figure 2). Both unilamellar vesicles and mutilamellar vesicles were observed in the PC-Chol and PC-Chol-Zein liposome solutions. Statistics analysis of the PC-Chol liposomes showed that there were 56 multimellar vesicles in 245 liposomes observed in cryo-TEM images; i.e., the formation percentage of multimellar vesicles was about 23%. In the presence of α-zein, there were 118 multilamellar vesicles in 380 liposomes in cryo-TEM images; i.e., the percent of multilamellar vesicles was about 31%, a little higher than that in PC-Chol liposome. An interesting feature of the PC-Chol-Zein liposome is seen in that adjacent membranes were often aligned at a regular interval of about 7 nm (Figure 2a and 2b). In the absence of α-zein on the other hand, there was no regular alignment of adjacent membranes (Figure 2c).

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Figure 2. TEM images of liposomes. (a, b) Cyro-TEM images of PC-Chol-Zein liposome, (c) Cryo-TEM image of PC-Chol liposome, (d) TEM image of the PC-Chol-Zein liposome with an immunogold labeled to zein (scale bar is 100 nm).

The incorporation of zein protein into the liposomal membrane was confirmed by immunogold labeling against the anti-zein antibody. TEM images as shown in Figure 2d revealed that one or two immunogolds were labeled on the single PC-Chol-Zein liposome. Since this labelling efficiency was considerably lower than the value that was expected from the zein content in the PC-Chol-Zein liposome, we cannot exclude the possibility that there was a preferential orientation of the zein molecule during the liposome reconstitution.

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Nonetheless, it can be argued that the hydrophobic protein zein could insert into the liposome bilayer membrane. SAXS Data. Except the cryo-TEM, synchrotron small angle X-ray scattering (SAXS) was often used to determine the protein-containing mimetic membrane structures.22, 23 Here, SAXS experiment was carried out to characterize the multilamellar structure of the PC-Chol-Zein liposome. SAXS curves from three independent preparations were measured to confirm reproducibility. The data shown in Figure 3 revealed a clear difference between the zein incorporated liposome and the zein-free liposome. In the 2D SAXS image of the PC-Chol liposome, a diffusive scattering ring could be observed at q = 0.9 nm-1 (Figure 3a). A broad peak in the SAXS intensity curve is indicative of non-regular alignment of adjacent membranes in the multilamellar vesicles. On the other hand, the SAXS intensity curve observed for the PC-Chol-Zein was characterized by a strong sharp peak at q = 0.88 nm-1 and a weaker peak at q = 1.76 nm-1 (Figure 3b) This profile is accountable by the formation of multilamellar vesicles with a homogeneous inter-membrane interval of 7.2 nm. Angelova et al. reported that the membrane architectures could be affected by the protein loading in the lipid phase,24 So it is not unusual to consider that the sharpening of the scattering ring observed in the presence of zein is due to the ordered arrangement of lipid bilayers. This result is consistent with the TEM images showing that the insertion of zein into the PC-Chol liposome enhanced the regular alignment of membrane layers within multilamellar vesicles. It has been reported that the presence of a transmembrane protein with a long hydrophobic length could bring about an increase in the lipid bilayer thickness.25 In our case, however, the length of the major axis of α-zein (12-13 nm) is much longer than the lipid 14


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bilayer. Later we will discuss the possibility that each zein molecule is able to penetrate through two adjacent membrane bilayers.

Figure 3. SAXS results of (a) PC-Chol liposome and (b) PC-Chol-Zein liposome. q is the scattering vector given by: q =(4π/λ)sin(θ/2), where θ is the scattering angle. The 2D SAXS images were inserted.

Effect of Zein on the Membrane Fluidity. Next, a fluorescence anisotropy experiment was performed to investigate the influence of the zein insertion on the membrane fluidity of the liposomes using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a fluorescence probe. The fluorescence anisotropy is a measure of lipid ordering and microviscosity of the bilayer.26 It is inversely related to the membrane fluidity; that is, lower anisotropy value indicates a higher membrane fluidity.27 The PC-Chol liposome showed a higher anisotropy value compared with the control PC liposomes at 37 °C (Figure 4). This observation is 15



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consistent with a previous report that cholesterol has an effect to increase the lipid ordering and decrease the membrane fluidity.28. 29 Interestingly, there was a significant difference in the fluorescence anisotropy for the PC-Chol liposome and PC-Chol-Zein liposomes, i.e., the presence of α-zein increased the membrane fluidity. This result suggests that some part of α-zein molecule penetrates into the hydrophobic regions of the lipid bilayer where DPH is inserted.30 It is worth nothing that the insertion of α-zein into PC liposomes has an effect opposite to that of the cholesterol insertion. It seems likely that the length of the membrane-spanning hydrophobic region of α-zein is not completely compatible with that of the lipid bilayer. An alternative possibility is that the inserted zein molecule attracts cholesterol molecules, causing an obvious decrease of the cholesterol occupation in other membrane area where DPH molecules are preferentially located. Indeed, we observed that α-zein was not well incorporated into the PC liposome without cholesterol.

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Figure 4. Fluorescence anisotrropy of DPH in PC, PC-Chol and PC-Chol-Zein liposomes (* indicates the significant difference at P < 0.05, n = 3).

The Phase Transition Temperature of PC-Chol-Zein Liposome. DSC was used to investigate the effect of α-zein on the phase transition of liposomes. Representative calorimetric traces observed for the PC-Chol and PC-Chol-Zein liposomes are shown in Figure 5. The DSC curve of the PC-Chol liposome showed an endothermic peak at 34.5 ℃ due to the phase transition of the lipid bilayer. The profile of phase transition changed only slightly in the presence of α-zein; i.e., the main transition temperature Tm shifted from 34.5 ℃ to 35 °C, and the calorimetric enthalpy slightly decreased from 0.78 kcal/mol to 0.73 17



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kcal/mol. The weak influence of the zein insertion on the lipid phase transition temperature seems to reflect the low protein content in the PC-Chol-Zein liposome.31

Figure 5. . Differential scanning calorimetry (DSC) of the PC-Chol and PC-Chol-Zein liposomes.

Structure of the Zein Molecule Incorporated into the PC-Chol Liposome. Matsushima et al. reported that α-zein dissolved in 70% ethanol has an elongated prism-like shape with an approximate axial ratio of 6:1. The structural model of 18


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α-zein deduced from SAXS analysis indicated a protein structure with a maximum length of approximately 12-13 nm and transverse dimensions ranging from 2 to 4 nm.21,

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More

recently, Momany et al. proposed a model using molecular dynamics simulation method for Z19 with three super helical structures, each of which is composed of three antiparallel helical segments linked by a glutamine-rich turn.33 Because the major axial of α-zein has been suggested to be much longer than the thickness of the lipid bilayer (approximately 4 nm), we consider the possibility that each α-zein molecule penetrates through two adjacent membranes, as shown in Figure 6. In this Figure, the model of three super helical structures is illustrated, because it is the most likely model that can explain the regular inter-membrane distance (d = 7.2 nm) observed in the multilamellar vesicles of the PC-Chol-Zein liposome.

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Figure 6. Plausible structure model of zein incorporated into the PC-Chol liposome.

Drug Leakage from the PC-Chol-Zein Liposome. To check whether α-zein affects the structural integrity of the phospholipid bilayer, we carried out a dye leakage assay with doxorubicin (DOX)-loaded liposomes. As shown in Figure 7, the leakage assay was performed at 4 °C (i.e., below the phase transition temperature) and 37 °C (i.e., above the phase transition temperature). At 4 °C, the leakage rate of DOX from the PC-Chol-Zein liposome seems slightly higher than that observed in the absence of zein, but was not significant. At 37 °C, on the other hand, the DOX leakage was apparently slowed after incubation more than 25 min in the presence of α-zein. Since the cryo-TEM results showed 20


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that multilamellar vesicles were more efficiently formed in presence of zein, it is possible that the slower leakage of DOX in the presence of zein is attributed to the high formation efficiency of multilamellar vesicles.

Figure 7. Leakage panel of PC-Chol and PC-Chol-Zein liposomes.

Cellular Uptake and Intracellular Trafficking of PC-Chol-Zein Liposome. Membrane proteins are involved in a wide range of cellular processes,34 and increasing number of researches have shown that the cellular processes are influenced by the interactions between membrane protein and phospholipids.35, 36 In this study, a fluorescent imaging tools was used to study the effect of α-zein on the cellular uptake and intracellular 21



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trafficking of the PC-Chol liposome. For this purpose, we labeled α-zein with a fluorescent probe Cy5 and monitored the cellular uptake of PC-Chol-Zein liposomes into NIH3T3 cells by confocal microscopy. The fluorescence images were recorded after different incubation periods (Figure 8). When NIH3T3 cells were incubated with the PC-Chol-Zein-Cy5 for 15 min, diffuse red emission was observed from the area near the cell surface. It was suggested that the PC-Chol-Zein liposomes were efficiently adhered to the cell surface. Meanwhile, incubation of NIH3T3 cells with the PC-Chol liposomes and encapsulated Cy5 resulted in a faint staining of the cell surface.

Figure 8. Confocal fluorescence images of PC-Chol (Cy5 loaded) and PC-Chol-Zein (zein was covalent linked with Cy5) liposomes (scale bar is 10 µm). When the incubation duration was increased to 30 min, bright red fluorescent spots were observed inside the cells. As compared with diffuse emission near the cell surface, these 22


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bright spots became more significant when the incubation duration was further increased. It seemed likely that PC-Chol-Zein liposomes that were rapidly adhered to the cell surface were slowly uptaken by endocytosis and then transported to the intracellular perinuclear area, probably late endosomes. These results indicated that zein might be an advantageous effect for the uptake of liposome by cells.

CONCLUSION The present study showed that when the hydrophobic protein α-zein was mixed with phospholipid vesicles, the zein content incorporated into the liposomes reached the maximal amount of 0.05% (mol/mol). At this content level, the property of the lipid bilayer was not largely affected. Nonetheless, the insertion of α-zein into the lipid bilayer resulted in the regular alignment of adjacent membranes within multilamellar vesicles. This observation suggests that each zein molecules takes such an elongated conformation that it penetrates through two adjacent membranes. It was also shown that the zein incorporated liposome was efficiently uptaken by an intact cell and localized in some specialized regions within the cell. This result suggests that the PC-Chol-Zein liposome could act as an interesting biomembrane model in physical, structural, biochemical, and biotechnological research.

Supporting Information Size distribution of reconstituted liposome, and fluorescence emission spectra of PC-Chol-Zein-Cy5 liposome.

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Author Information Corresponding Author * Tel: (86-21)34205824; Fax: (86-21)34205824; Email: [email protected] Tel:(81-52)4648602; Email: [email protected]

Notes

The authors declare no competing financial interest.

Acknowledgement This study is supported by the International S&T Cooperation Program of China (2014DFG02330, 2015DFG32730). We also thank the Shanghai Municipal Science and Technology Commission (13JC1403400 and 15540723900). We thank Prof. Shao Zhifeng, Fang Ke and Chen Xuecheng for providing help for confocal images. We appreciated Prof. Fujisawa Tetsuro for valuable discussion on the SAXS data.

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Table of Contents (TOC):

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Formation of a Mimetic Biomembrane from the Hydrophobic Protein

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