Membrane Microdomain Structures of Liposomes and Their

Dec 28, 2015 - We further performed self-organizing maps (SOM) clustering to simplify the complicated behavior of the domain formation to obtain its e...
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Membrane Microdomain Structures of Liposomes and Their Contribution to the Cellular Uptake Efficiency into HeLa Cells Yoshinori Onuki,*,† Yasuko Obata,‡ Kumi Kawano,§ Hiromu Sano,‡ Reina Matsumoto,‡ Yoshihiro Hayashi,† and Kozo Takayama‡ †

Department of Pharmaceutical Technology, Graduate School of Medical and Pharmaceutical Science, Unversity of Toyama, Sugitani 2630, Toyama-shi, Toyama 930-0194, Japan ‡ Department of Pharmaceutics and §Department of Drug Delivery Research, Hoshi University, Ebara 2-4-41, Shinagawa, Tokyo 142-8501, Japan S Supporting Information *

ABSTRACT: The purpose of this study is to obtain a comprehensive relationship between membrane microdomain structures of liposomes and their cellular uptake efficiency. Model liposomes consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol (Ch) were prepared with various lipid compositions. To detect distinct membrane microdomains in the liposomes, fluorescence-quenching assays were performed at temperatures ranging from 25 to 60 °C using 1,6-diphenyl-1,3,5-hexatriene-labeled liposomes and (2,2,6,6-tetramethylpiperidin-1-yl)oxyl. From the data analysis using the response surface method, we gained a better understanding of the conditions for forming distinct domains (Lo, Ld, and gel phase membranes) as a function of lipid composition. We further performed self-organizing maps (SOM) clustering to simplify the complicated behavior of the domain formation to obtain its essence. As a result, DPPC/ DOPC/Ch liposomes in any lipid composition were integrated into five distinct clusters in terms of similarity of the domain structure. In addition, the findings from synchrotron small-angle X-ray scattering analysis offered further insight into the domain structures. As a last phase of this study, an in vitro cellular uptake study using HeLa cells was conducted using SOM clusters’ liposomes with/without PEGylation. As a consequence of this study, higher cellular uptake was observed from liposomes having Ch-rich ordered domains. KEYWORDS: membrane microdomain, liposome, cellular uptake efficiency, Kohonen’s self-organizing maps, response surface method

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INTRODUCTION Liposomes, closed spherical vesicles consisting of selfassembled lipid bilayers enclosing an aqueous compartment, are considered to be the best candidates for drug nanocarriers.1 Liposomal drugs could offer great benefits compared with the free drugs. These include increased drug stability, extended circulation in the bloodstream, increased tumor accumulation, and reduced systemic toxicity.2−5 Liposome technology has been expanding to a wide range of applications, including nanodevices for cancer therapy and anti-inflammatory therapy, lipoplex vectors for gene therapy, and cancer imaging agents.6−8 To date, more than 10 liposome-based products have been approved by the US FDA, and many other liposomal drugs are undergoing clinical trials.7,8 Despite the great success in clinical © 2015 American Chemical Society

applications, formulation optimization of liposomal drugs is still a great challenge for further development of liposomal therapy. Cellular uptake efficiency is an essential element in designing liposomal drugs. It is well-known that different membrane properties substantially affect their cellular uptake.9 Membrane fluidity is one of the most popular properties used to characterize the liposome membrane. To date, several researchers have investigated the relationships between the membrane fluidity and the cellular uptake efficiency of Received: Revised: Accepted: Published: 369

August 5, 2015 November 7, 2015 December 28, 2015 December 28, 2015 DOI: 10.1021/acs.molpharmaceut.5b00601 Mol. Pharmaceutics 2016, 13, 369−378

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Molecular Pharmaceutics liposomes using various cell lines.10−13 However, despite their best endeavors, consensus has yet to be reached regarding this matter. For example, Orthmann et al. reported that higher membrane fluidity of liposome improved transcytosis into epithelial cells, MDCK cells,10 while Kawano et al. reported that folate-targeted liposomes with high membrane fluidity exhibited lower cellular uptake into folate receptor (+) KB cells.11 It is our opinion that a major reason for the lack of agreement is the complicated membrane structure of liposomes. As well as a material for medical nanodevices, liposomes have been widely used as model membranes to investigate the structure and functions of biological membranes. In particular, over the past decade, many researchers in the fields of membrane biology and biophysics have focused on the function and structure of membrane microdomains in plasma membranes, including lipid raft.14−19 With the further development of this research area, the concept of membrane microdomains in liposomes has become sophisticated.14,17,20 We note the liposomal membrane itself is no longer considered a uniform structure but rather a composite of distinct membrane microdomains. It is known that membrane domains possess at least four distinct phases: gel, liquid ordered (Lo), liquid crystalline, and liquid disordered (Ld).20 The membrane fluidity increases in the following order: Lo ≈ gel < Ld < liquid crystalline. Liposomes consisting of pure phospholipids undergo a gel-to-liquid crystalline phase transition at their phasetransition temperature. The gel phase membrane shows ordered and tight packing, whereas the liquid crystalline phase membrane is disordered and is a fluid, like a liquid. Cholesterol (Ch)-enriched membrane can exist as Lo and Ld phase membranes. With regard to the Lo phase membrane, its ordered packing is comparable to that of the gel phase, but its fast axial rotation and high lateral mobility are quite different from the gel phase. Ld is considered as a liquid crystalline phase having a considerable amount of Ch. In biomembranes, the lipid raft probably exists in the Lo phase and behaves like an island floating in a sea of loosely packed domains in an Ld state. The concept of membrane microdomains offers a profound insight into membrane fluidity of liposomes; based on this concept, membrane fluidity that has been used in liposome research can be considered as a complex property integrating those of distinct domains. We also note that the behavior of domain formation is very complicated and sensitive to the lipid composition. A number of research groups reported phase diagrams of liposomes composed of ternary lipids (i.e., saturated lipid, unsaturated lipid, and cholesterol);16,18,19 however, to the best of our knowledge, such a systematic research approach has never been applied to the design of liposomal drugs. Against this background, this study focused on the contribution of the domain structure of liposomes to their cellular uptake efficiency. First, this study was dedicated to a better understanding of the domain structure in liposomes. In our previous study, we devised a novel method to examine the complicated relationships between lipid composition and membrane domain structure.21,22 This method is established based on a response surface method (RSM) and Kohonen’s self-organizing maps (SOMs). The RSM with multivariate spline interpolation (RSM-S) is a nonlinear RSM previously developed by our group. A notable characteristic of the method is that it enables estimation of nonlinear relationships between factors and response variables with high accuracy. From our previous studies, the RSM-S is considered as an effective tool to

understand the complicated relationships between formulation factors and characteristics of pharmaceuticals.23−26 As for SOM, it is a feedforward-type neural network model.27 Recently, it has gained great attention as a promising tool for clustering data. For one thing, it enables the expression of relationships of multidimensional data as a two-dimensional surface. 27 Applications of SOM have been reported across a range of pharmaceutical and medical fields.28−30 We have been using SOM to understand the latent relationships concerning formulation design of pharmaceuticals.31−34 In this study, SOM clustering was used to sort the model membranes in terms of similarity of membrane structure. This study employed the ternary lipid system with different lipid compositions consisting of 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Ch as a model liposome. A fluorescencequenching assay15,35 was employed to investigate their domain structures. The conditions for forming the distinct domains as a function of lipid composition were expressed by response surfaces. Afterward, SOM clustering was performed to extract the essence of the complicated behavior of domain formation. We succeeded in classifying the DPPC/DOPC/Ch liposomes in any lipid composition into distinct clusters in terms of similarity of the domain structure. By using a liposome belonging to each SOM cluster, we performed synchrotron small-angle X-ray scattering (SAXS) analysis and a cellular uptake study using HeLa cells. As a result of the study, we gained further insight into the domain structure of the liposomes and a comprehensive understanding of the contribution of domain structure to cellular uptake efficiency.

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METHODS Materials. DPPC, DOPC, Ch, (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO), and lead(II) stearate were purchased from Wako (Osaka, Japan). 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) was purchased from Invitrogen (Carlsbad, CA, USA). 1,2-Distearoyl-sn-glycero-3phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] [DSPE-PEG(2000)] was purchased from Avanti Polar Lipids (Alabaster, AL, USA). 1,6-Diphenyl-1,3,5-hexatriene (DPH) was purchased from Aldrich (Milwaukee, WI, USA). All other chemicals were of analytical grade and commercially available. Liposome Preparation. DPH-labeled liposomes for fluorescence-quenching assay was prepared according to the protocol of Bakht et al., with minor modifications.15 In brief, 100 μL of desired lipid mixture in ethanol (5 mM) and 100 μL of DPH solution (0.025 mM) were dispersed in 9.8 mL of phosphate-buffered saline (PBS) at 70 °C. Final samples contained 50 μM lipid and 0.5 mol % DPH. Samples were cooled and stored in a dark bottle at room temperature until they were used in the experiments. DiI-labeled liposomes were prepared using the thin-film hydration method for cellular uptake studies. As well as naked liposomes, liposomes coated with PEG (PEGylated liposomes) were prepared by incorporating DSPE-PEG(2000) into the lipids (5 mol % to total lipids). In brief, designated amounts of lipids and DiI dissolved in chloroform were transferred to a flask and the chloroform was removed by evaporation at room temperature under a stream of nitrogen. This procedure resulted in the formation of a thin lipid film on the wall of the flask. The film was stored overnight in a vacuum desiccator to ensure complete evaporation of the chloroform. PBS (10 mL) was added to the flask, and the lipids were hydrated for 30 min. 370

DOI: 10.1021/acs.molpharmaceut.5b00601 Mol. Pharmaceutics 2016, 13, 369−378

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Molecular Pharmaceutics The suspension was sonicated for 10 min at about 60 °C using a bath-type sonicator. Final samples contained 15 mM lipid and 1.0 mol % DiI. The resultant samples were extruded using an extruder (mini-extruder; Avanti Polar Lipids) with 0.1 μm polycarbonate membrane. After the samples were cooled to room temperature, they were stored at room temperature for a maximum of 2 days before use in the experiments. This study also prepared naked liposomes without any label using the thinfilm hydration method, and they were then used for the SAXS study. Regarding the sample preparation, pure water was used as an aqueous phase instead of PBS. Fluorescence-Quenching Assay. TEMPO in ethanol (5 μL) was added to DPH-labeled liposome (1000 μL) as a quencher. The final concentration of TEMPO was fixed at 5 mM. After incubation for 10 min at 25 °C, the fluorescence intensity of DPH (F) was measured at temperatures ranging from 25 to 60 °C using a fluorescence spectrophotometer (F450; Hitachi, Tokyo, Japan) (Ex = 353 nm, Em = 430 nm). The rate of increasing temperature was maintained at 1 °C/min. In addition, liposomes were incubated with pure ethanol instead of TEMPO solution and then their fluorescence intensity (F0) was also measured as a reference. Then, the ratio of fluorescence intensity in the presence of quencher to that in its absence (F/F0) was calculated. Small-Angle X-ray Scattering Analysis. The measurement of SAXS profiles of liposomes was carried out at the Photon Factory BL6A at the High Energy Accelerator Research Organization (Ibaraki, Japan). The wavelength (λ) of the X-ray beam was about 0.151 nm, and the sample-to-detector distances were about 1600 mm (SAXS). SAXS profiles were recorded based on the delay line principle covering the q-ranges from qmin = 4.5 × 10−4 nm to qmax = 3.74 nm−1. The reciprocal spacing S = 1/d(2/λ) sin(2θ/2) was calibrated from the lattice spacing of a lead(II) stearate at room temperature (d-spacing of 4.91 nm), where 2θ is the scattering angle and d is the lattice distance. A sample cell containing the liposome suspension was sealed with a polyimide film and placed in the sample holder of the differential scanning calorimeter. The sample temperature ranged from 25 to 55 °C using a differential scanning calorimeter (FP-99; Mettler-Toledo, Tokyo, Japan). HeLa Cell Culture. Human cervical carcinoma (HeLa) cell line was obtained from the European Collection of Cell Culture (Wiltshire, U.K.). The cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/ mL streptomycin at 37 °C, 5% CO2. HeLa cell cultures were prepared by plating cells in a 35 mm culture dish 24 h prior to each experiment. Cellular Uptake. The cells were washed three times with 1 mL of serum-free DMEM. DiI-labeled liposomes (100 μL, 15 mM) were added to 10 mL of serum-free DMEM and then gently applied to the cells. In this study, all samples were incubated with cells for 2 h at 37 °C in serum-free DMEM. At the end of the incubation of liposomes with cells, the cells were washed three times with 1 mL of PBS and detached from the plate by incubating with 0.05% trypsin and EDTA solution at 37 °C for 3 min. The cells were centrifuged at 1500g, and the supernatant liquid was discarded. The cells were resuspended with PBS (pH 7.4) containing 0.1% BSA and 1 mM EDTA and directly introduced into a FACSVerse flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with a 488 nm argon ion laser. Data for 10000 fluorescent events were obtained by

recording forward scatter (FSC) and side scatter (SSC) with red (for DiI; 585/42 nm) fluorescence. Data Analysis. The data analysis was roughly divided into four distinct procedures as follows: (1) collecting experimental data, (2) constructing correlation models between lipid composition and responses, (3) predicting an enormous number of data sets, and (4) SOM clustering.21,22 First, model liposomes with different lipid compositions were prepared, and then the fluorescence-quenching assays were conducted as mentioned. In this study, 56 liposomes with different lipid compositions were tested (Figure 1). Based on

Figure 1. Lipid compositions of a model membrane. Maximum contents of DPPC, DOPC, and Ch were 100, 100, and 60 mol %, respectively. Each point represents the lipid composition of a model membrane.

the observed data, a correlation model between lipid compositions and responses was constructed using RSM. dataNESIA software, version 3.0 (Azbil Corp., Tokyo, Japan), was used for RSM-S. The observed F/F0 values at every 5 °C from 25 to 60 °C, and the differences in values from 25 to 60 °C were used as tutorial data for generating response surfaces using RSM-S. In accordance with the previous study,21 leaveone-out cross-validation (LOOCV) was implemented. In brief, every point was removed from the data set in turn as a testing datum, and then correlation models were constructed by RSMS using the n − 1 data sets. Afterward, the prediction accuracy of the correlation model thus constructed was assessed using the testing data. F/F0 values at each temperature for an enormous number of untested lipid compositions were predicted from the response surfaces for the following SOM clustering. The number of untested lipid compositions for the prediction was 4275. Lipid composition and their F/F0 values at different temperatures were regarded as an input data set. Namely, 4331 data sets (56 experimental data sets and 4275 predicted data sets) were used for SOM clustering. SOM clustering was performed using Viscovery software, SOMine version 4.0 (Eudaptics Software, Vienna, Austria). The number of nodes in the output was set at 2000. Viscovery software includes several clustering techniques such as SOM−Ward, Ward, and SOM−Single-Linkage. The SOM−Ward technique was employed for clustering. After the SOM clustering, the input data were rearranged to show the distribution of each cluster. 371

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RESULTS Detection of Membrane Microdomains in DPPC/ DOPC/Ch Liposomes by Fluorescence-Quenching Assay. Examples of fluorescence-quenching curves are presented in Figure 2; below the phase-transition temperature

model liposomes, correlation models between lipid compositions and F/F0 values at the designated temperatures (at every 5 °C from 25 to 60 °C) were constructed by RSM-S. To evaluate the prediction accuracy of the response surfaces, LOOCV was conducted (Supporting Information, see Figure S1). The correlation coefficient was very high (r = 0.928− 0.959), indicating that the RSM-S constructed a reliable model of the correlation between lipid composition and F/F0 values. We next prepared a large number of data sets of untested lipid compositions for SOM clustering; namely, F/F0 values for untested lipid compositions were predicted by reading points on the response surfaces. Subsequently, the experimental and predicted data sets were employed for the following SOM clustering. According to the SOM clustering, we intended to simplify the complicated behavior to get to its essence. As shown in Figure 3, the DPPC/DOPC/Ch liposomes with

Figure 2. TEMPO quenching of the fluorescence of DPH labeled in distinct phase membranes as a function of temperature. Whole membrane of DPPC, DPPC/Ch, and DOPC liposomes at 25 °C are regarded as being the gel, Lo, and liquid-crystalline phase membranes, respectively. The temperature was scanned at 1 °C/min.

(Tm) of DPPC, the entire membrane area of DPPC, DPPC/Ch (70/30), and DOPC liposomes can be regarded as gel, Lo, and liquid crystalline phase membranes, respectively. The fluorescence-quenching assay is based on the assumption that DPH (the fluorescence probe) partitions equally in ordered and disordered phase membranes, whereas TEMPO (the quencher) binds preferentially to disordered phase membranes.15,35 By incubation of DPH-labeled liposome with TEMPO, fluorescence of DPH distributed in disordered domains was quenched by TEMPO. Thus, the fluorescence intensity observed from the samples represents the presence of ordered domains in the liposomes. As shown, the F/F0 values of DPPC and DPPC/Ch liposomes at about 25 °C were much higher than that of DOPC liposome (liquid crystalline phase membranes), indicating that DPPC and DPPC/Ch liposomes exist in an ordered state. When the temperature was raised, the value of DPPC liposome substantially decreased, which reflects the phase transition of DPPC from the gel to the liquid crystalline phase. It is known that the Tm of DPPC is ca. 42 °C.36,37 In contrast to those of the DPPC liposome, F/F0 values of the DPPC/Ch liposome (Lo phase membrane) were very stable to a change in temperature over the range from 25 to 60 °C. The DOPC liposome (liquid crystal phase) was kept at a constant low value throughout the experiment. We note that, despite the different lipid compositions, the F/F0 values of DPPC liposome in the gel phase (at around 25 °C) and in the liquid crystalline phase (at around 60 °C) were almost the same as those of DPPC/Ch and DOPC liposomes at the corresponding temperatures. This indicates that the F/F0 values can be used as an index to estimate the extent of ordered domains in the liposomes. The same fluorescence-quenching assay was conducted on all model liposomes. Then, the fluorescence quenching curves were substantially changed as a function of lipid composition (experimental data was shown in Supporting Information, see Table S1). SOM Clustering of Lipid Compositions of DPPC/ DOPC/Ch Liposomes in Terms of Similar Membrane Structure. Based on the fluorescence-quenching assay of the

Figure 3. Distribution of SOM clusters of lipid compositions. SOM clustering was performed using F/F0 values at each temperature (25− 60 °C at every 5 °C) for experimental and untested lipid compositions as input data. Large circles represent the centroids of each SOM cluster.

different lipid compositions were classified into five distinct clusters in terms of the similarity of the fluorescence quenching curves. The centroid lipid composition of each cluster is listed in Table 1. Cluster 1 contained the highest proportion of Table 1. Lipid Compositions Corresponding to the Centroids of SOM Clusters

cluster 1 (DPPC-Ch-rich sample) cluster 2 (DPPC-rich sample) cluster 3 (Ch-rich sample) cluster 4 (DPPC-DOPC-rich sample) cluster 5 (DOPC-rich sample)

DPPC (mol %)

Ch (mol %)

DOPC (mol %)

42.4

42.5

15.1

63.1 24.6 33.5

13.2 47.2 24.1

23.7 28.1 42.4

27.1

10.5

62.4

ordered-domain components, DPPC and Ch (42.4 and 42.5 mol %, respectively), whereas cluster 5 was mostly composed of the disordered domain component, DOPC. Cluster 2 was abundant in DPPC (63.1 mol %). In clusters 3 and 4, a considerable amount of all lipids was observed. 372

DOI: 10.1021/acs.molpharmaceut.5b00601 Mol. Pharmaceutics 2016, 13, 369−378

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Figure 4. Response surfaces generated by RSM-S for F/F0 values at 25 °C (a) and 60 °C (b), and their delta values (c). The difference in F/F0 values between 25 and 60 °C was expressed as the “delta value”. Fluorescence-quenching curves (d) of each SOM cluster predicted by response surfaces. The centroid lipid compositions shown in Table 1 were regarded as being typical liposomes of each cluster. Points in (a)−(c) represent lipid compositions tested, while those in (d) are F/F0 values predicted by the response surfaces at the corresponding temperature.

(DPPC-rich sample) (Figure 4c). Therefore, their ordered domains mostly existed as the Lo phase. In addition, by considering Figure 4a,b, the proportion of ordered and disordered domains in the membranes is likely to depend on the content of Ch; higher Ch content resulted in a larger amount of ordered domains. Regarding cluster 5 (DOPC-rich liposomes), the values were kept at the lowest level throughout the temperatures ranging from 25 to 60 °C, and the curve was quite similar to that of the DOPC membrane shown in Figure 2; thus, most of this cluster’s membrane exists as Ld or liquid crystalline phase. SAXS Profiles of the Domain Structure of Each Cluster. To obtain further information concerning the domain structure of the SOM clusters, synchrotron SAXS analysis was conducted. This experiment was also conducted using the centroid lipid compositions (Table 1) as typical liposomes of each cluster. Figure 5 shows SAXS profiles of each cluster as a function of temperature. SAXS profiles of the five investigated clusters at 25 °C are shown in Supporting Information (see Figure S2). Regarding cluster 1, lamellar structure with 6.58 nm (S = 0.152 nm−1) was observed at 25 °C. This structure was relatively stable to the change in the temperature because the peak position was constant during this experiment. Regarding cluster 2, the repeat distance of the lamellar structure at 25 °C was 7.04 nm (S = 0.142 nm−1); at around 30−40 °C, the lamellar structure was substantially changed and eventually decreased to 6.56 nm (S = 0.152 nm−1). Regarding cluster 3, a single lamellar structure with 6.48 nm (S = 0.154 nm−1) was observed at 25 °C, and this structure was relatively stable to the

As examples of all response surfaces constructed, those at 25 and 60 °C are shown in Figure 4. The response surfaces are shown in color: higher regions at 25 and 60 °C correspond to regions enriched in the ordered domain (Lo + gel) and the Lo phase domain, respectively. Figure 4c shows a response surface of delta values; the difference in F/F0 values between 25 and 60 °C was calculated as the “delta value”, and then the response surface was generated by RSM-S. This surface helps us to make out the distribution of the gel phase domain. In addition, the fluorescence-quenching curves of each cluster (Figure 4d) were generated by predicting F/F0 values from response surfaces. In this study, the centroid lipid compositions shown in Table 1 were regarded as being typical liposomes of each cluster. Cluster 1 (DPPC-Ch rich sample) was completely consistent with the region having the high F/F0 values at 60 °C (Figure 4b). The F/F0 values of cluster 1 retained the highest values regardless of the temperature (Figure 4d). Thus, this cluster is mostly composed of Lo phase membrane. Regarding cluster 2 (DPPC-rich sample), the delta values show high levels (Figure 4c). In addition, from the fluorescence-quenching curve (Figure 4d), substantial decreases in the F/F0 values were observed from around 37.5 to 50 °C. The change in the F/F0 values represents the phase transition of DPPC-enriched domains from gel to liquid crystalline phase; therefore, this cluster is rich in the gel phase domain comprising DPPC. The curves of cluster 3 (Ch-rich sample) and cluster 4 (DPPC-DOPC-rich sample) were almost parallel to that of cluster 1 (DPPC-Chrich sample), and their fluorescence-quenching curves showed no substantial decrease in the F/F0 values, like cluster 2 373

DOI: 10.1021/acs.molpharmaceut.5b00601 Mol. Pharmaceutics 2016, 13, 369−378

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Figure 5. Contour map of SAXS profiles of SOM clusters as a function of temperature. Liposomes of each SOM cluster were prepared according to the centroid lipid compositions. The red area represents strong intensity, and the blue area represents weak intensity.

Figure 6. Cellular uptake efficiencies of liposomes of the SOM clusters into HeLa cells. Naked (a,b) and PEGylated (c,d) liposomes of each SOM cluster were prepared according to the centroid lipid compositions. Representative FACS histograms (a,c) and fluorescence changes from those of cluster 5 (b,d). Values are the means (±SD) of three independent experiments. *p < 0.05 (Dunnet test for comparisons with cluster 5).

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Molecular Pharmaceutics

liposomes at the corresponding temperatures. This allows us to use the value for estimation of the amount of the domain in the liposomes; a higher F/F0 value represents a larger amount of ordered domains in the sample. Based on the results of the fluorescence-quenching assay, we investigated the domain structure of DPPC/DOPC/Ch liposomes. The behavior of the domain formation is considered to be very complicated and sensitive to the lipid composition; thus, in general, huge data collection for a large number of lipid compositions is required to gain a full picture of this subject. For instance, Buboltz et al. tested 1294 different lipid mixtures using FRET and then built phase diagrams of DPPC/DOPC/ Ch (the range of lipid composition: DPPC, 0−100 mol %; DOPC, 0−100 mol %; Ch, 0−70 mol %).19 However, as it is, this procedure is not practical and feasible for general studies, including the fluorescence-quenching assay, because it is difficult to collect the huge amount of data by experiments. To overcome this problem, we applied the RSM. RSM-S is a nonlinear RSM developed in our laboratory. Multivariate spline interpolation (MVS) is integrated into RSM-S as a method of generating the response surface. The basic concept of MVS involves a boundary element method.45 It can estimate nonlinear relationships between factors and characteristics with high accuracy without any complicated operation. From the leave-one-out cross-validation, the correlation coefficients were very high (r = 0.928−0.959) (Figure S1), indicating that a reliable correlation model between lipid compositions and F/F0 values at different temperatures was constructed. The F/F0 values at 60 °C and the delta values between 25 and 60 °C can be regarded as quantities of Lo and gel phase domains in the liposomes, respectively. Owing to the response surfaces, we could fully understand the conditions for forming the distinct domains as a function of lipid composition. Furthermore, we performed SOM clustering to simplify the complicated behavior of the domain formation to get to its essence. As a result of the SOM clustering, DPPC/DOPC/Ch liposomes in any lipid composition were classified into five distinct clusters. Based on the response surfaces (Figure 4a−c) and the changed behaviors of F/F0 (Figure 4d), we characterized the membrane structures of the SOM clusters as follows: cluster 1 (DPPC-Ch-rich sample) was mostly composed of Lo phase membrane; cluster 2 (DPPC-rich sample) contained a considerable amount of gel phase membrane consisting of DPPC; cluster 3 (Ch-rich sample) and cluster 4 (DPPC-DOPC-rich sample) are a mixture of ordered and disordered domains, and their ordered domains are mostly composed of Lo phase membrane; cluster 5 is mostly composed of disordered phase membrane (liquid crystalline and Ld phases). For further information regarding the domain structure, Anchordoquy et al. recently reported that Chnanodomains existed in the liposomes composed of N-(1-(2,3dioleoloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP).46 According to their report,46 when the Ch content in the DOTAP liposome exceeds 66 mol %, anhydrous Ch crystallites exist in the liposome as a Ch-nanodomain. We think the Ch-nanodomains can be formed in the DPPC/DOPC/Ch liposomes. However, as far as the five investigated liposomes were concerned, we do not think they have Ch-nanodomains. For one thing, their Ch contents were much lower than the threshold (66 mol %) for the formation of Ch-nanodomains reported by Anchordoquy et al. Furthermore, we previously measured the differential scanning calorimetry (DSC) charts of liposomes composed of sphingomyelin (SM)/DOPC/Ch.21

change in the temperature. We note that cluster 4 showed two distinct peaks at 25 °C; the repeat distances of the lamellar structures were 6.69 and 6.34 nm (S = 0.149 and 0.158 nm−1). The longer repeat distance was closer to those of clusters 1 and 3, while the shorter one was closer to those of cluster 5 (6.37 nm, S = 0.157 nm−1 at 25 °C). These two peaks were integrated at around 30 °C, resulting in a broad single peak with the repeat distance of 6.43 nm (S = 0.155 nm−1). In Vitro Cellular Uptakes. In vitro cellular uptake experiments for each cluster were performed. In addition to the naked DiI-labeled liposomes, this study examined the cellular uptake of the PEGylated liposomes. As for the representative lipid compositions of the clusters, this study also used the centroid of the clusters shown in Table 1. The size of liposomes tested was adjusted to 100−200 nm using an extruder. The mean values of the fluorescence-activated cell sorting (FACS) histograms (Figure 6a,c) were recorded, and then these values were normalized by taking account of the labeling efficiency of DiI with liposomes; namely, the mean values were corrected by the fluorescence intensity of the intact liposomes (shown in Table S2). Afterward, the fluorescence changes from that of cluster 5 were calculated (Figure 6b,d). With regard to the naked liposomes, the highest cellular uptake efficiency was observed from cluster 1; the fluorescence change was nearly 10 times higher than that of cluster 5 (Figure 6b). In addition, the fluorescence changes of clusters 2 and 4 were obviously higher than those of cluster 5. By contrast, fluorescence changes of cluster 3 were very low and almost equal to those of cluster 5. The cellular uptakes of liposomes were substantially decreased by PEGylation (Figure 6c). With regard to the rank order of cellular uptake efficiencies, except for a substantial reduction of cluster 2, similar results were observed from the PEGylated liposomes; clusters 1 and 4 were still significantly higher than that of cluster 5 regardless of PEGylation.

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DISCUSSION Recently, the concept of membrane microdomains has gained a great deal of attention from many researchers, especially in the fields of membrane biology and biophysics. At present, it is well recognized that liposomes are composed of distinct domains having different membrane properties. To detect the membrane microdomains in liposomes, various methods have been applied. They include fluorescence microscopy, 16,18,38 FRET,19,39−42 2H NMR spectroscopy,43 ESR spectroscopy,42 and molecular dynamics.44 From the various methods, this study selected the fluorescence-quenching assay using TEMPO.15,35 As mentioned, this method is based on the fact that TEMPO prefers to bind to disordered membranes, while DPH can distribute proportionally between ordered and disordered membranes. From this principle, we can observe the presence of ordered domains as a fluorescence of DPH. A notable characteristic of the method is its high-definition monitoring; according to Suga et al., ordered domains of ca. 1.3 nm were detectable by this method.35 This experiment was performed under the condition that the experimental temperature range covers the Tm of DPPC (ca. 42 °C)36,37 to identify the gel, Lo, and disordered phase membranes (liquid crystalline and Ld phases). Figure 2 obviously shows that the F/F0 values depend not on the lipid compositions but on the state of the membrane; the F/F0 values of DPPC liposome in the gel phase (at around 25 °C) and in the liquid crystalline phase (at around 60 °C) were almost the same as those of DPPC/Ch and DOPC 375

DOI: 10.1021/acs.molpharmaceut.5b00601 Mol. Pharmaceutics 2016, 13, 369−378

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Molecular Pharmaceutics

uptake compared with cluster 5. As clarified above, the entire membrane of cluster 1 is Ch-rich ordered membrane. Cluster 4 consists of two distinct phase domains; the ordered Ch-rich domains are close to cluster 1 and clearly separated from the disordered domains. It is now well accepted that liposomes are internalized into the cells through endocytosis.56,57 Ch-rich domains (i.e., caveolae and rafts) in the plasma membrane serve as a platform for some endocytic pathways. Recently, there have been several reports showing that Ch-rich domains in liposomes can improve intracellular delivery.46,58−64 Pozzi et al. reported that Ch-containing liposomes can act as promoters of their fusion with plasma and endosomal membranes.59 Anchordoquy et al. found that the Ch-nanodomains formed in Ch-enriched liposomes enhanced serum stability and transfection efficiency.46,65−67 We speculate that the Ch-rich ordered domains in the liposomes exert a positive effect on their cellular uptake. In contrast to clusters 1 and 4, the cellular uptake of cluster 3 seemed to be low for the large amount of ordered domains; compared with cluster 4, cluster 3 had a larger amount of ordered domains (Figure 4), while its cellular uptake was very low. Although the precise reason for this remains unclear and needs further investigation, it may be related to the domain structure of liposomes. As mentioned, obvious differences in the SAXS profiles were observed between clusters 3 and 4; cluster 4 has two distinct domains (e.g., ordered and disordered membranes), while the entire membrane of cluster 3 seemed to be homogeneous. Compared with Ch-rich domains in clusters 1 and 4, those of cluster 3 are thought to be fluid because of the destitution of DOPC in the entire membrane. Such a difference in membrane rigidity of the Ch-rich domain may affect interaction with the plasma membrane, resulting in lower cellular uptake. Regarding cluster 2, the naked liposome showed high cellular uptake competitive with cluster 4, while that of the PEGylated liposome was substantially reduced and was almost equal to that of cluster 5. Although the detail remains unclear, we speculate that the membrane structure of cluster 2 was substantially affected by the insertion of DSPE-PEG(2000). We note that the naked liposome of cluster 2 contains a considerable amount of gel phase domain of DPPC. DPPC, a lipid with saturated acyl chains, can form lipid raft-like Lo phase membranes with Ch. The gel phase membrane of DPPC is known to be more rigid than that of the Lo phase membrane of DPPC/Ch because higher fluorescence anisotropy is observed from the membrane.36 As shown in Figure 2, the naked liposome can retain the rigid membrane structure during the cellular uptake experiments at 37 °C. Therefore, there is a possibility that the ordered domains also exerted a positive effect on the endocytosis pathway like the Ch-rich domains of clusters 1 and 4. As a consequence of this study, we consider that the domain structure of liposomes is worth considering when endeavoring to improve their cellular uptake efficiency.

From the DSC experiments, liposomes with 69.4 mol % of Ch obviously showed an endothermic peak at around 40 °C, which represents the phase transition of anhydrous cholesterol crystallites, indicating that Ch-nanodomains were formed in the liposome. By contrast, the liposome with 40.8 mol % of Ch (similar Ch contents to clusters 1 and 3) did not show an endothermic peak. To gain further understanding of the domain structure of the SOM clusters, we obtained the SAXS profiles of the SOM clusters. It was clarified that the lamellar spacing of the ordered domain of cluster 1 was 6.58 nm, and this periodicity was almost constant throughout the experiment. Regarding cluster 2, the peak position in SAXS profiles was obviously changed and substantial diminution of lamellar periodicity from 7.04 to 6.56 nm was observed. There is no doubt that the change is due to the phase transition of DPPC from the gel to the liquid crystalline phase. It is worth noting that cluster 4 clearly has two distinct domains of 6.69 and 6.34 nm of lamellar structures at 25 °C; we speculate that one was the membrane domain close to that of cluster 1, while the other is a disordered domain like cluster 5. Buboltz et al. investigated the phase behavior of DPPC/DOPC/Ch liposomes and identified the region in which gel and Lo coexisted.19 The coexistence region agreed well with the region of cluster 4. It is also worth noting that the SAXS profile of cluster 3 showed only single lamellar structure with 6.48 nm. This indicates that the membrane structure of cluster 3 is obviously different from that of cluster 4; the membrane structure is homogeneous rather than a coexistence of ordered and disordered membranes. As the last phase of this study, we investigated the cellular uptake efficiency of the SOM clusters using HeLa cells. The size and fluorescence intensity of the intact liposomes are listed in Supporting Information (see Table S2). It is well documented that the size of liposomes influences pharmacokinetics, tissue distribution, and clearance.47 In general, liposomes 50−200 nm in diameter are considered to retain blood capillaries for long periods in their intact form. The majority of clinically approved liposomes have diameters of 50−300 nm. Taking account of these issues, we prepared liposomes with a size distribution in the range from 100 to 200 nm. In this experiment, we tested PEGylated liposomes as well as the naked liposomes. PEGylation has become the standard procedure for preparing liposomal drugs. In general, naked liposomes are swiftly cleared from systemic circulation by the reticuloendothelial system, mainly in the liver and spleen.48 PEGylation is a common method of producing “stealth liposomes,” which can prolong the circulation and enhance accumulation in solid tumors based on the enhanced permeation and retention (EPR) effect.48−50 Moreover, PEG has also been used as a linker for lipid conjugation of targeting molecules, such as folate,11,51,52 transferrin,52 and peptide.53,54 As shown in Figure 6, the cellular uptakes of liposomes were substantially decreased by the PEGylation. This result was entirely predictable because PEGylation masks the liposomal surface and reduces interactions with cells, resulting in a negative effect on cellular uptake and intercellular trafficking. For instance, studies by Li et al. on siRNA delivery concluded that delivery efficiency of nanoparticles was significantly reduced to 1/10 compared with the naked nanoparticles.55 Our result is completely consistent with the report. Regarding cellular uptake efficiencies of the clusters, cluster 1 showed the highest values regardless of PEGylation. Cluster 4 also did not affect PEGylation and showed much higher cellular

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CONCLUSIONS To the best of our knowledge, this is the first report focusing on a comprehensive understanding of membrane microdomain structure of liposomes as drug nanocarriers. We suggest that the domain structure is one of the important factors affecting their cellular uptake efficiency; in particular, the Ch-rich ordered domain in the liposomes exerted a positive effect on the cellular uptake pathway. This issue has been overlooked to date; however, from now on, we believe the domain structure will become an essential element for development of liposomal 376

DOI: 10.1021/acs.molpharmaceut.5b00601 Mol. Pharmaceutics 2016, 13, 369−378

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Molecular Pharmaceutics

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drugs. Because there is a good possibility that the domain structure acts as a platform for the multiple functions of liposomal drugs, we predict that more sophisticated liposomal drugs can be designed by taking account of the membrane domain structure. The findings will be valuable information for the development of effective liposomal drugs.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.5b00601. Additional figures (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and the Research Foundation for Pharmaceutical Sciences. We thank Professor Takatsune Shimizu, Ms. Ayana Shiraiwa, and Mr. Yuto Isami at Hoshi University for their technical assistance. The synchrotron X-ray scattering experiments were performed in a BL6A at the Photon Factory under approval of the Photon Factory Advisory Committee (2012G055 and 2014G137).

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