Leptin-derived peptide, a targeting ligand for mouse brain-derived endothelial cells via macropinocytosis

Biochemical and Biophysical Research Communications 394 (2010) 587–592

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Leptin-derived peptide, a targeting ligand for mouse brain-derived endothelial cells via macropinocytosis Mina Tamaru, Hidetaka Akita, Takahiro Fujiwara, Kazuaki Kajimoto, Hideyoshi Harashima * Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo, Hokkaido 060-0812, Japan

a r t i c l e

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Article history: Received 22 February 2010 Available online 7 March 2010 Keywords: Leptin Macropinocytosis Brain endothelial cell Cellular uptake Liposome

a b s t r a c t Leptin is an appetite regulatory hormone that is secreted into the blood circulation by adipose tissue, and functions in the central nerve system (i.e. hypothalamus) by crossing the blood brain barrier (BBB). In the present study, we investigated the function of a leptin-derived peptide (Lep70–89) as a ligand for mouse brain-derived endothelial cells (MBEC4). Lep70–89-modified liposomes, prepared with a polyethyleneglycol (PEG) spacer (Lep70–89-PEG-LPs) exhibited a significantly higher cellular uptake than peptide-unmodified liposomes (PEG-LPs). Furthermore, cellular uptake was inhibited by amiloride, while no significant inhibitory effect was observed by the presence of chlorpromazine and filipin III, suggesting that macropinocytosis largely contributed to the cellular uptake of Lep70–89-PEG-LPs. Imaging studies revealed that Lep70–89-PEG-LPs were not colocalized with endosome/lysosomes, whereas neutral dextran (70 kDa) was predominantly colocalized with these compartments. This indicates that Lep70–89-PEG-LPs are taken up via macropinocytosis and are subject to non-classical intracellular trafficking, resulting in the circumvention of lysosomal degradation in endothelial cells. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Central nervous system (CNS)-targeting delivery systems for low molecular drugs, peptides, proteins and nucleic acids is highly desirable in terms of curing various neurodegenerative disorders such as Alzheimer’s disease [1] and Parkinson’s disease [2]. However, the transfer of hydrophilic drugs and/or high molecular weight compounds (i.e. peptides) from circulating blood to the brain are severely restricted due to the blood brain barrier (BBB), which consists of a tight junction comprised of connecting cerebral capillary endothelial cells that are devoid of fenestrae [3]. The use of nanoparticles represents a promising strategy for overcoming the BBB [4]. Liposomes coated with polyethyleneglycol (PEG-LP) show potential for use as potent drug carriers, since they able to escape from the reticuloendothelial system (RES) and circulate in the blood for a long period of time [5,6]. In addition, various antibodies can be attached to PEG chains on the surface, thus permitting them to be actively taken up by the brain [7]. As well as an antibody, use of peptide ligands (i.e. the RGD motif for integrins, and NGR for endothelium-associated form of aminopeptidase N (CD13) [8,9]) are also promising and convenient strategies for targeting specific receptors. For the successful delivery to CNS, the recognition of receptors on capillary endothelial cells must be considered. Neuroactive factors which are biosynthesized by peripheral tissues represent * Corresponding author. Fax: +81 11 706 4879. E-mail address: [email protected] (H. Harashima). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.03.024

highly potent candidates in the use of CNS-targeting ligands since an active transport system from blood to CNS is a prerequisite for overcoming the BBB, as has been demonstrated for certain types of bioactive peptides and neuropeptides [10,11]. Leptin, a 146 amino acid polypeptide plays an important role in homeostasis of body weight by regulating a food intake and energy expense [12]. The hormone is mainly secreted by white adipose tissue to the blood circulation, and acts on leptin receptor (ObR)-expressing neurons located in the CNS, such as the hypothalamus. Since leptin has a molecular weight of 16 kDa, its size precludes it from passive diffusion, a selective transport system is considered to be involved in its delivery to the brain by crossing the BBB [10,13]. A number of groups have reported that, in rodents and primates, the uptake of leptin by the brain is mediated by a saturable transport system [14–17], while the molecular mechanism for this remains to be elucidated. Saturable leptin transport was observed even in the Koletsky rat, in which all subsets of functional ObR were mutated, indicating that an ObR-independent uptake pathway contributed to its uptake by the brain [15]. Meanwhile, recent studies have shown that various isoforms of ObR are also expressed in cerebral microvessels [18]. Furthermore, the administration of soluble type ObR receptors inhibited leptin transport across the BBB [19]. Collectively, these data indicate that leptin is taken up by the brain via transcytosis in a saturable manner via at least two distinct mechanisms, namely ObR-dependent and independent processes. A very recent study indicated that a leptin fragment (amino acid residues 61–90), a peptide overlapping a ObR-binding site IIb [20,21] is taken up by the brain to an extent comparable to

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wild-type leptin [22]. Moreover, peptides comprising amino acid residues 70–89 of leptin (Lep70–89) possessed strong cell proliferating activity, most likely due to its agonist activity against ObR [23]. In the present study, we describe the development of PEG-LPs that are modified with Lep70–89 (Lep70–89-PEG-LPs) to investigate whether Lep70–89 has the ability to function as a ligand for endothelial cells. Furthermore, the mechanism for its uptake and subsequent intracellular trafficking were examined.

2. Materials and methods 2.1. Reagents Cholesterol, distearoyl-sn-glycero-3-phoshoethanolamine-N[methoxy (polyethylene glycol)-2000] (PEG-DSPE) and rhodamine-labeled 1.2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (Rho-DOPE) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Egg york phosphatidylcholine (EPC) and DSPE-PEG with a functional maleimide moiety at the terminal end of PEG: N-[(3maleimide-1-oxopropyl) aminopropyl polyethyleneglycol-carbamyl] distearoyl-phosphatidyl-ethanolamine (Mal-PEG-DSPE) were purchased from Nippon Oil and Fat Co. (Tokyo, Japan). [cholesteryl1,2–3H(N)]-cholesteryl hexadecyl ether ([3H]CHE) was purchased from PerkinElmer Life Science Japan (Tokyo, Japan). The leptinderived peptide (Lep70–89) (87.9% purity, obsd. Mw = 2729.08 sequence: SRNVIQISNDLENLRDLLHVGGYC) was obtained from HOKKAIDO BIO SYSTEM Co, LTD (Hokkaido, Japan). Texas Redlabeled neutral dextran, 70,000 Mw (TXR-ND; D-1830) and Lysotracker Blue (DND-22) was purchased form Invitrogen (Carlsbad, CA, USA). Hoechst33342 was purchased from Dojindo Laboratories (Kumamoto, Japan). 2.2. Cell culture MBEC4 cells derived from mouse brain endothelial cells were generously supplied by Drs. T. Tsuruo and M. Naito (Tokyo University, Japan). The cells were maintained in DMEM supplemented with 10% fetal bovine serum and 0.5 lg/ml heparin sulfate under an atmosphere of 5% CO2/air at 37 °C. 2.3. RT-PCR studies Total RNA was extracted from MBEC4 cells with the TRIÒ Reagent (Sigma–Aldrich) according to the manufacturer’s procedure. The expression of ObR was confirmed by reverse transcription polymerase chain reaction (RT-PCR) analysis. The cDNA was synthesized using a High Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, USA) from 1 lg of total RNA. Amplification of the ObR fragment was performed with the forward primer (50 -CCATCTTTTATAT GATCTGCCTGAAGT-30 ) and the reverse primer (50 -TGCATTGGACA GTCTGAAAGCT-30 ) using GoTaqÒ Green Master Mix (Promega, Madison, WI, USA). The DNA was denaturized at 95 °C for 30 s, and annealed at 60 °C for 30 s. Extension was performed at 72 °C for 30 s. This process was repeated for 33 cycles. As an internal control, a GAPDH fragment was amplified with the forward primer (50 -TGTGTC CGTCGTGGATCTGA-30 ) and the reverse primer (50 -CCTGCTTCACCA CCTTCTTGA-30 ) following the same PCR protocols. 2.4. Preparation of liposomes Two types of liposomes, PEG-LPs and Lep70–89-PEG-LPs were prepared by the hydration method. For the preparation of PEGLPs, a lipid film was prepared in a glass test tube by evaporating a chloroform solution of lipids, containing EPC, cholesterol and PEG-DSPE (total lipid amount: 137.5 nmol). The prepared lipid film

then was hydrated with distilled water, and the tube was then sonicated for 1 min in a bath-type sonicator (AU-25C; Aiwa Co., Tokyo, Japan). Lep70–89-PEG-LPs were prepared by replacing the PEG-DSPE with Lep70–89-modified PEG-DSPE (Lep70–89-PEG-DSPE). Lep70–89-PEG-DSPE was synthesized in a single-step reaction of Mal-PEG-DSPE with Lep70–89 following the procedure used for the synthesis of demorphin-PEG-DSPE [24]. At this point, 3 mM of Lep70–89 (SRNVIQISNDLENLRDLLHVGGYC) in DMF was mixed with 3 mM of DSPE-PEG-Mal in DMF and 15 mM of triethylamine to permit the cysteine residue to react with the maleimide moiety via a Michael addition reaction. MALDI-TOF MS spectroscopy data were obtained on a Bruker MALDI-TOF-MS Reflex II instrument to determine the masses of DSPE-PEG-Mal and Lep70–89-PEG-DSPE using acetonitrile:water = 3:7 with 0.1% of trifluoroacetate as the matrix solution, supplied with 10 mg/ml solution of dihydroxybenzoic acid with a trace of NaCl, and 10 mg/ml of sinapinic acid, respectively. The amount of Lep70–89-PEG-DSPE was quantified by measuring the absorption of light at 280 nm derived from the tyrosine residues in the Lep70–89 peptide. The diameter and zeta potential of the Liposomes were determined using an electrophoretic lightscattering spectrophotometer (Zetasizer; Malvern Instruments Ltd., Malvern, WR, UK). For the preparation of rhodamine-labeled liposomes, Rho-DOPE was added to the lipid film preparation (0.5% of total lipid). For isotope labeling, 62.5 pmol (97.5 kBq) of [3H]CHE was added to the lipid composition in preparing the lipid film (total lipid amount: 137.5 nmol). 2.5. Cellular uptake study of radioisotope-labeled liposomes To examine the intracellular trafficking of liposomes, MBEC4 cells were seeded on a MULTIWELL 12 well (Falcon, Becton Dickinson Labware, NJ, USA) in 2 ml of culture medium until 100% confulency was attained. Uptake was initiated by adding the radiolabeled liposomes to the medium after washing the culture dishes three times, followed by preincubation with 1 HEPES buffer for about 3 min. The 1 HEPES buffer consisted of 135 mM NaCl, 5.4 mM KCl, 1.0 mM MgCl26H2O, 1.8 mM CaCl22H2O, 5.0 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid and 10 mM glucose adjusted to pH 7.3. The final concentration of the [3H]PEG-LPs and [3H]Lep70–89-PEG-LPs was 5.5 nM of total lipid. To examine the cellular uptake mechanism, cells were pre-incubated at 37 °C in 1 HEPES buffer in the absence or presence of amiloride (2.5 mM) for 30 min, or with chlorpromazine (14 lM) for 60 min, or with filipin III (5 or 15 lg/ml) for 1 h. [3H]Lep70–89-PEG-LPs were then added and the suspension incubated for 1 h at 37 °C in the presence or absence of inhibitors. The cells were then washed three times with 2 ml of 1 HEPES buffer and solubilized in 400 ll of 1 N NaOH. After adding 200 ll of 2 N HCl, 500 ll aliquots were transferred to scintillation vials. The radioactivity associated with the cells and medium was determined by liquid scintillation counting (LS 6000SE; Beckman Instruments, Inc., Fullerton, CA) after adding 4.5 ml of scintillation fluid (Hionic flow; Packard Instrument Co., Downers Grove, IL) to the scintillation vials. Uptake is given as the percent of the applied dose (%). 2.6. Analysis of lysosomal localization To examine the intracellular trafficking of liposomes, MBEC4 cells were seeded on a 3.5 cm glass base dish (IWAKI, Osaka, Japan) in 2 ml of culture medium until 70% confluency was attained before the examination. Rhodamine-labeled PEG-LPs and Lep70–89PEG-LPs (13.75nM of total lipid) were incubated with cells in 1 mL of 1 x HEPES buffer (135 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES, and 10 mM glucose). Images were

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acquired using a Nikon ECLIPSE TE-2000-U wild-field fluorescence microscope equipped with a Nikon PlanApo 100/1.4 oil immersion objective (Nikon Corporation, Tokyo, Japan). Control of the microscopy and the acquisition of digital images were performed with the NIS-Elements software program (Nikon). A mercury lamp was used for illumination. Blue (Lysotracler Blue DND-22, Hoexst33342) and red fluorophores (TXR-ND and rhodamine liposomes) were excited with light filtered through 350/50 and 580/ 20 excitation filters, respectively. Fluorescence was collected in the epi direction. The fluorescence was passed through a dichromatic mirror, reflecting at the exciting wavelength (82100v2bs; Chroma Technology Corp., Rockingham, VT, USA), and were further filtered from residual excitation light by bandpass filters (450/30 and 630/60, respectively). Image sequences were captured with an electron multiplier charge coupled device camera (ImagEM; Hamamatsu Photonics, Hamamatsu, Japan). A Z-series of images were acquired in approximately 0.4 lm steps, and were subjected to the Richardson–Lucy algorithm-based 3D-deconvolution thereafter to prevent out-of focus blur. 3. Results and discussion 3.1. Expression of ObR in MBEC4 cells The ObR gene encodes several spliced variants as a result of tissue-dependent differential splicing. The ObRa gene is considered to be a receptor that is involved in the transport of leptin across the BBB [25]. In fact, the transcellular transport of leptin was reported to be enhanced when the ObRa gene was expressed in the polarized Mardin–Darby Canine Kidney cells [26]. Meanwhile, recent studies indicate that other types of isoforms are also expressed on cerebral microvessels [18], and mediate leptin transport [27]. The isoforms of ObR differ from each other in their cytoplasmic domains [27]. As a result, the leptin-binding domains (extracellular domains) are conserved. Thus, the expression of total ObR in MBEC4 cells was confirmed by RT-PCR using a set of primers designed to hybridize to the sequences encoding the common region [18]. As shown in Fig. 1, the PCR fragments for ObR (Lane 3) and GAPDH (Lane 4) were amplified at the expected length (81 and 76 bps, respectively) while the expression level of ObR was less than GAPDH. Thus, MBEC4 can be used to investigate the utility of Lep70–89 as a targeting ligand for brain capillary endothelial cells. 3.2. Characterization of liposomes Lep70–89-PEG-DSPE was prepared by incubating a 1:1 (mol/mol) mixture of DSPE-PEG-Mal and Lep70–89. In MALDI-TOF MS spectroscopy, the Lep70–89-PEG-DSPE gave a unimodal peak at a high molecular position (obsd. Mw = 5662.706, Mw/Mn = 0.99, calcd. Mn = 5670.72), compared to DSPE-PEG-Mal (obsd. Mw = 3042.935, Mw/Mn = 1.02, calcd. Mn = 2941.64), indicating that the synthesis of Lep70–89-PEG-DSPE was successful (data not shown). The physicochemical characteristics were first compared between PEG-LPs and Lep70–89-PEG-LPs (Table 1). The sizes of both liposomes were comparable (approximately 180 nm), as were the f-potentials (approximately 20 mV). 3.3. Cellular uptake of liposomes To evaluate the cellular uptake of the liposomes, rhodamine-labeled liposomes were incubated with cells for 1 h, and visualized by wide-field fluorescence microscopy followed by 3D-deconvolution. The cellular uptake of PEG-LPs was slightly observed, as shown in Fig. 2A. It is generally considered that electrostatic interactions between particles and negatively charged heparin sulfate

Fig. 1. ObR expression in MBEC4 cells. Expression of total ObR in MBEC4 cells was confirmed by RT-PCR using a set of primers designed to hybridize to the common sequence. As an internal control, the GAPDH fragment was amplified. Lane 1, marker (100 bp ladder); Lane 2, negative control (without primers); Lane 3, ObR; Lane 4, GAPDH.

Table 1 Physicochemical properties of PEG-LPs and Lep70–89-PEG-LPs. Liposome

Size (nm)

f-Potential (mV)

PEG-LPs Lep70–89-PEG-LPs

187.5 ± 28.9 178.8 ± 19.2

22.1 ± 3.8 18.0 ± 1.1

Data are presented as means ± SD for three independent experiments.

proteoglycans (HSPGs) are a crucial driving force for cellular uptake [28,29]. The poor cellular uptake of PEG-LPs is most likely due to electrostatic repulsion between the negatively charged particles and HSPGs on the surface of cells (Table 1). Since Lep70–89 contains three positively charged amino acids (two arginines and one histidine) and three anionic charged residues (two asparagic acids and one glutamic acid), it is an apparently a neutral peptide at physiological conditions. Thus, modification of the peptide on the periphery of PEG had minor effect on the f-potential of the liposomes (Table 1). Nevertheless, the uptake of Lep70–89-PEG-LPs liposomes was enhanced (Fig. 2B), suggesting that Lep70–89 induced the binding of liposomes to the cellular surface via a specific interaction with its receptor, but not via an electrostatic interaction with plasma membranes. 3.4. Identification of cellular uptake pathway While previous reports demonstrated that ObRs expressed in HeLa cells are internalized by clathrin-mediated endocytosis under the control of ubiquitylation [30], information concerning the mechanism of cellular uptake by brain endothelial cells is limited. Thus, we further investigated the contribution of different endocytic pathways to the uptake of [3H]Lep70–89-PEG-LPs (Fig. 3) using inhibitors that specifically block macropinocytosis, clathrin-mediated endocytosis and caveolar endocytosis. Chlorpromazine was used to inhibit clathrin-mediated endocytosis via dissociation of the clathrin lattice [31]. Amiloride inhibits macropinocytosis by inhibiting the Na+/H+ exchange required for macropinocytosis [32]. Filipin inhibits caveolar uptake through cholesterol depletion [33]. Consistent with results in Fig. 2, the cellular uptake of [3H]Lep70–89-PEG-LPs was significantly higher than that of [3H]PEG-LPs. It is noteworthy that the uptake of [3H]Lep70–89-PEGLPs was decreased to levels comparable to that of [3H]PEG-LPs. In contrast, no significant inhibition was found in the cases of chlorpromazine and filipin III. Little toxicity was observed during at least

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Fig. 2. Cellular uptake of PEG-LPs and Lep70–89-PEG-LPs to MBEC4 cells. MBED4 cells were incubated with rhodamine-labeled PEG-LPs (A) and Lep70–89-PEG-LPs (B) for 1 h. Cellular uptake of the liposomes was analyzed by means of the wild-field microscopy. Nuclei were stained with Hoechst33342. Obtained images were processed by Richardson–Lucy algorithm-based 3D-deconvolution. Bar indicates 10 lm.

2.5

*

*

2 1.5 1 0.5

Inhibitors

(-) PEG-LPs

(-)

A m iro C hl rid or e pr om az in e Fi 5 lip µg in /m III L Fi 15 lip µg in I /m II L

0

Lep70-89-PEG-LPs Fig. 3. Contribution of different cellular uptake pathways. MBEC4 cells were preincubated at 37 °C in HEPES buffer in the absence or presence of amiloride (2.5 mM) for 30 min, or with chlorpromazine (14 lM) for 60 min, or with filipin III (5 or 15 lg/ml) for 1 h. [3H]Lep70–89-PEG-LPs were then added and the suspensions were incubated for additional 1 h at 37 °C in the presence or absence of inhibitors. The uptake of [3H]PEG-LPs was also determined as a control. Asterisks represent a significant differences determined by one-way analysis of variance (ANOVA), followed by Dunnett’s multiple-comparison post hoc test (P < 0.05).

a 2 h-exposure to these inhibitors, as evidenced by an MTT assay (data not shown). Collectively, these data suggest that Lep70–89PEG-LPs are predominantly taken up via macropinocytosis. 3.5. Analysis of intracellular trafficking We previously demonstrated that octaarginine (R8)-modified liposomes with a low R8-density (R8-LP-LD) are taken up via clathrin-mediated endocytosis, whereas those with a high surface level of R8 (R8-LP-HD) are taken up by NIH3T3 cells via macropinocytosis [34,35]. Furthermore, the cellular uptake pathway determines, in part, the intracellular fate of the liposomes. Confocal microscopy revealed that R8-LP-LD is highly colocalized with a lysosomal marker, whereas only partial colocalization was observed for the case of R8-LP-HD. This observation indicates that internalization through macropinocytosis may prevent lysosomal degradation [34,35]. These observations, therefore, prompted us to evaluate the intracellular trafficking of Lep70–89-PEG-LPs.

To analyze the subsequent intracellular fate of the Lep70–89PEG-LPs, their colocalization with the lysosomal compartment was evaluated (Fig. 4). Rhodamine-labeled Lep70–89-PEG-LPs were incubated with MBEC4 cells. The lysosomes were stained with Lysotracker Blue DND-22 after 30 min, and observed by wild-field fluorescence microscopy followed by 3D-deconvolution. TXR-ND (Mw; 70 kDa), a well known as a fluid phase marker that can trace internalization via macropinocytosis was applied to the cells as a reference [36]. Rhodamine/Texas Red and lysosomes are pseudocolored in red and green, respectively. Predominant colocalization with the lysosomal compartment was found in the case of TXR-ND at 1 h after the incubation (Fig. 4A), whereas the major portion of Lep70–89-PEG-LPs was not observed to be colocalized with lysosomes (Fig. 4B). Therefore, the sorting pathways for Lep70–89-PEGLPs may be different from that for TXR-ND while it is likely that both are taken up via macropinocytosis. The mechanism for the different sorting pathways remains to be clarified. A very recent study showed that the macropinocytosis pathway is induced by the leptin treatment of macrophages via the activation of AMP-activated protein kinase (AMPK) signaling [37]. Since the peptide Lep70–89 possessed agonist activity against ObR [23], Lep70–89-PEG-LPs may induce AMPK signaling-dependent macropinocytosis. In contrast, it can be easily imagined that a neutral dextran would not induce any type of signaling. Thus, cell signaling-induced macropinocytosis may be sorted by a different mechanism in endothelial cells. Alternatively, Lep70–89 may possess multi-functions; endosomedisruptive activity, as well as a ligand activity, and thereby endosomal escape of Lep70–89-PEG-LPs might be facilitated. Macropinocytosis is known to be associated with transcytosis in intestinal epithelial cells [38,39]. In contrast, there is an increasing body of evidence to indicate that caveolae-dependent uptake plays a crucial role in transcytosis in the endothelium [40,41], whereas the physiological role of macropinocytosis is unknown in endothelial cells. Since the uptake of Lep70–89-PEG-LPs was not prevented by filipin III, the liposome itself may lack the activity to permit it to pass through the capillary endothelium. Lep70–89 is generally useful as a target ligand of liposomes for brain endothelium-derived cells. Since Lep70–89-modified liposomes were taken up via a unique pathway; macropinocytosis and avoid sorting to lysosomes, it may be useful for the intracellular delivery of encapsulating cargos to capillary endothelial cells. Furthermore, dual ligand system of Lep70–89 and caveolae-targeting ligands may be an attractive strategy for using Lep70–89 for CNS delivery in the future.

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Fig. 4. Lysosomal localization of Lep70–89-PEG-LPs and TXR-ND. MBEC4 cells were incubated with the Texas Red-labeled neutral dextran (TXR-ND) (A) and rhodamine-labeled Lep70–89-PEG-LPs (B) for 1 h. Lysosomal compartments were stained with Lysotracker Blue DND-22. Cellular uptake of the liposomes was analyzed by means of wild-field microscopy. The resulting images were processed by Richardson–Lucy algorithm-based 3D-deconvolution.

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