International Journal of Pharmaceutics 465 (2014) 77–82
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An apolipoprotein E modified liposomal nanoparticle: Ligand dependent efficiency as a siRNA delivery carrier for mouse-derived brain endothelial cells Mina Tamaru, Hidetaka Akita * , Kazuaki Kajimoto, Yusuke Sato, Hiroto Hatakeyama, Hideyoshi Harashima ** Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 September 2013 Received in revised form 21 December 2013 Accepted 8 February 2014 Available online 12 February 2014
A disorder in the brain endothelium is thought to be closely related to the pathophysiology of brain diseases. A method for delivering nucleic acids (i.e. short interference RNA; siRNA) to the brain endothelium should be an attractive strategy for curing brain disorders. A liposomal nanoparticle containing a proton-ionizable amino lipid was recently developed as a carrier of encapsulated siRNA. The aim of this study was to evaluate the utility of apolipoprotein E (ApoE) as a targeting ligand for mouse brain endothelial cells (MBEC4 cells). The cellular uptake of the ApoE-modified nanoparticles was gradually increased in an ApoE-density dependent manner. Furthermore, the ApoE-modified nanoparticles were taken up via both clathrin and caveolae mediated endocytosis, thus permitting them to avoid lysosomal degradation. Finally, endogenous gene silencing in MBEC4 cells was efficiently achieved depending on the ApoE-modification. Collectively, the ApoE-modified nanoparticle is a promising carrier for delivering nucleic acids to the brain endothelium. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Apolipoprotein E (ApoE) Brain endothelial cell Multifunctional envelope-type nano device (MEND) YSK05 siRNA delivery In vitro
1. Introduction The neurovascular unit (NVU), which is composed of vascular cells (i.e. endothelium and pericytes), glial cells (i.e. astrocytes, microglia, and oligodendroglia), and neurons, cooperatively plays a key role in the maintenance of homeostasis and normal brain function (Zlokovic 2011; Stanimirovic and Friedman 2012). Brain endothelial cells are the main structural components of the blood brain barrier (BBB).
Abbreviations: siRNA, short interference RNA; ApoE, apolipoprotein E; NVU, neurovascular unit; BBB, blood brain barrier; pDNA, plasmid DNA; MEND, multifunctional envelop-type nano device; LDLR, low-density lipoprotein receptor; LRP1, LDLR-related protein 1; VLDLR, very low density lipoprotein receptor; HAS, human serum albumin; PEG, polyethyleneglycol; LNP, liposomal nanoparticle; DiD, 1,10 dioctadecyl-3,30 -tetramethylindocarbocyamine perchorate; DiI, 1,10 -dioctadecyl3,30 -tetramethylindodicarbocyamine perchorate; DMEM, Dulbecco’s modified eagle’s medium; FBS, fetal bovine serum; DMG, dimyristoyl-sn-glycerol; t-BuOH, tertiary butanol; CLSM, confocal laser scanning microscopy; qRT-PCR, quantitative reverse transcriptase-polymerase chain reaction; HSPG, heparin sulfate proteoglycan; CNS, central nervous system; KPI, Kunitz protease inhibitor; APP, amyloid precursor protein. * Corresponding author. Tel.: +81 11 706 3735; fax: +81 11 706 4879. ** Corresponding author. Tel.: +81 11 706 3919; fax: +81 11 706 4879. E-mail addresses: [email protected] (H. Akita), [email protected] (H. Harashima). http://dx.doi.org/10.1016/j.ijpharm.2014.02.016 0378-5173/ã 2014 Elsevier B.V. All rights reserved.
Perturbation of the BBB is closely related to the progress of various diseases, since it regulates the entry of cells and molecules into the brain and functions as a pump to remove potentially neurotoxic molecules (Zlokovic 2008) including thrombin (Grammas 2011), fibrin (Paul et al., 2007), and hemosiderin (Zhong et al., 2009). The deliveryof nucleic acids, such as siRNA, is one of the rational strategies for repairing the dysfunction of cells. However, naked siRNA is susceptible to digestion by extracellular enzymes or rapid urinary exclusion. Moreover, the functional transport of native nucleic acids to the cytoplasm is intrinsically limited due to their poor cellular uptake, and, if they enter, to degradation in lysosomes. We previously developed a liposomal nanoparticle containing encapsulated plasmid DNA (pDNA) and siRNA that is referred to as a multi-functional envelop-type nano device (MEND). The particle was designed to protect the encapsulated cargos from enzymatic degradation, and to overcome the intracellular biomembranes (i.e. endosome) (Kogure et al., 2008; Nakamura et al., 2012). The particles are applied for targeting the liver and tumors via the systemic circulation since the fenestrae in liver and discontinuous neovascular vessels in cancer permit the particle to penetrate through the endothelium (Hatakeyama et al., 2011; Sakurai et al., 2011; Sakurai et al., 2013; Khalil et al., 2011). In contrast to these, the penetration of the particle through the continuous endothelium in a majority of the organs was found to be severely limited. Therefore,
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a ligand for a specific type of receptor on the endothelium would be required for tissue targeting. For the delivery of the therapeutic molecules to the brain, artificial carriers; liposomes and micelles, as well as endogenous lipoproteins and exosomes are used as a carrier (Alvarez-Erviti et al., 2011; Pardridge 2007). From the point of view of tissue targeting, an uncharged particle would be highly desirable, since when positively charged particles are administered, large aggregates with erythrocyte are produced, which are then distributed or trapped to the non-targeting tissues (i.e. lung) (Sakurai et al., 2001; Ogris et al., 1999). Recently, a proton-ionizable aminolipid, denoted as YSK05 was synthesized, whose incorporation into the lipid envelope structure (YSK-MEND) permits a neutral particle to be prepared, which, in turn, permits efficient membrane fusion to endosomes and subsequent delivery of the encapsulated siRNA into cytosol (Sato et al., 2012). Consequently, the intratumoral administration of the YSK-MEND conferred more efficient gene silencing compared with MENDs composed of conventional cationic lipids (Sato et al., 2012). Thus, in the present study, the YSK-MEND was used as a platform for a siRNA carrier to evaluate the function of the ligand. To date, a large variety of the ligands such as endogenous peptides derived from hormones (i.e. leptins), foreign peptides discovered by phage display methods and antibodies against the receptors on the BBB have been identified (van Rooy et al., 2011). In this report, we focused on ApoE as a targeting ligand for the brain endothelium. ApoE is a member of the family of soluble apolipoproteins and binds triglyseride-rich lipoproteins (Hatters et al., 2006; Peters-Libeu et al., 2006). The endogenous lipoproteins interact with members of the low-density lipoprotein receptor (LDLR) family through ApoE and are subsequently internalized via clathrin-mediated endocytosis (Nykjaer and Willnow 2002). In addition to hepatocytes or the sinusoidal endothelium, several studies have demonstrated that LDL receptor families such as LDLR, LDLR-related protein1 (LRP1), and very low density lipoprotein receptor (VLDLR) (Zlokovic 2013) are also expressed by brain endothelial cells. Very recently, Wagner et al. reported that the chemical conjugation of ApoE onto human serum albumin (HSA) nanoparticles via a polyethyleneglycol (PEG) spacer enhanced the uptake into the brain endothelium-derived in vitro cultured cells (Wagner et al., 2012). Furthermore, the conjugation of ApoEderived peptides (141–150), a highly cationic variety, was also used as a ligand for targeting brain endothelial cells (Sauer et al., 2005; Leupold et al., 2009). Furthermore, Kuwahara and co-workers demonstrated that cholesterol-conjugated siRNA conferred gene silencing in the brain capillary endothelial cells in vivo and in vitro mainly due to an accelerated cellular uptake mediated by the ApoEassociated formation of lipoproteins, followed by recognition by LDLRs (Kuwahara et al., 2011). Meanwhile, Akinc and co-workers reported that liposomal nanoparticles (LNP) composed of ionizable lipids accumulated at high levels in the liver via the LDLR, aided by endogenous ApoE (Akinc et al., 2010). This study prompted us to evaluate whether the simple incubation of liposomes formed by a
pH-ionizable lipid with ApoE might accelerate the uptake of lipid nanoparticles by the brain endothelium. In this study, we prepared the ApoE-modified YSK-MEND (ApoE/ YSK-MEND) to target brain endothelial cells, and then investigated its intracellular fate, and function as a siRNA carrier in mouse brain derived endothelial cells (MBEC4). 2. Materials and methods 2.1. Materials Anti-Gapdh siRNA was purchased from Hokkaido System Science Co., Ltd. (Sapporo, Japan). Cholesterol was purchased from Avanti Polar Lipid (Albaster, AL, USA). RiboGreen was purchased from Molecular Probes (Eugene, OR, USA). DiI (1,10 -dioctadecyl3,3,30 ,30 -tetramethylindocarbocyanine perchorate), DiD (1,10 -dioctadecyl-3,3,30 ,30 -tetramethylindodicarbocyanine perchorate), TRIzol reagent and LysoTracker Green DND-26 were purchased from Invitrogen (Carlsbad, CA, USA). Hoechst33342 was purchased from Dojindo Laboratories (Kumamoto, Japan). Recombinant Human ApoE3 was purchased from BioVision Incorporated (Milpitas Boulevard, Milpitas, CA, USA). 2.2. Cell cultures MBEC4 cells derived from mouse brain endothelial cells were generously supplied by Dr. T. Tsuruo and Dr. M Naito (Tokyo University, Japan). The cells were maintained with Dulbecco’s modified eagle’s medium (DMEM, SIGMA–Aldrich) supplemented with 10% fetal bovine serum (FBS), NaHCO3 (3.7 g/L), streptomycin (100 mg/L), penicillin (16.67 mg/L) at 37 C under an atmosphere of 5% CO2. 2.3. MEND formulations The MEND composed of YSK05/cholesterol/PEG-Dimyristoylsn-glycerol (DMG) (70/30/3) was prepared by the tertiary butanol (t-BuOH) dilution procedure as described previously (Sato et al., 2012). PEG-DMG was used to stabilize the lipid membrane during the formation process and for preservation. Briefly, 10 mM of total lipids were dissolved in a 90% t-BuOH solution. siRNA was dissolved in 20 mM citrate buffer (pH 4.0) and was added dropwise into the lipid solution under vigorous mixing to avoid a low local concentration of t-BuOH. Subsequently, the solution was quickly diluted with citrate buffer to a final concentration of 3 h at arbitrary concentrations. The average diameter and zeta-potential of MENDs were determined using a Zetasizer Nano ZS ZEN3600 (Malvern Instrument, Worcestershire, UK). DiD labeled
Table 1 Physicochemical properties of ApoE/YSK-MENDs. ApoE (mg/mL)
0
0.1
0.3
1
3
Size (nm) z-Potential (mV) PdI siRNA encapsulation (%)
84 11 0.52 1.35 0.22 0.1 88.6 2.1
86 3 1.2 0.74 0.17 0.02 90.0 0.4
94 3 0.02 1.73 0.19 0.04 88.1 0.5
104 10 3.42 0.56 0.21 0.08 82.4 2.3
94 9 4.83 0.77 0.19 0.08 74.6 5.4
Particle diameter, polydispersity (PdI), and zeta-potential were measured using Malvern Zetasizer. Percentage of siRNA encapsulation was determined by RiboGreen fluorescence assay to measure the amount of siRNA relative to total siRNA percent. DATA points are expressed as the mean SD (n = 3).
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2.5. Quantification of cellular uptake of ApoE/YSK-MENDs by flow cytometry
Fig. 1. Effect of ApoE-modification on the cellular uptake of YSK-MEND. MBEC4 cells were incubated with the DiD-labeled ApoE/YSK-MENDs at a concentration of 1 mM of MENDs for 1 h. Cellular uptake of the ApoE/YSK-MENDs was analyzed by means of the flow cytometry. The data are represented as the mean fluorescence intensity (MFI) SD (n = 3).
MENDs were prepared by adding a DiD (0.5 mol% of total lipid) in the lipid composition. 2.4. RiboGreen fluorescence assay To determine siRNA encapsulation and its concentration, RiboGreen fluorescence assay was performed. MENDs were diluted in 200 mL of 10 mM HEPES buffer (pH 7.4) containing 20 mg/mL dextran sulfate, and then 0.5 mL of RiboGreen was applied in the presence or absence of 0.1% (w/v) Triton X-100. Fluorescence was measured by EnSpire 2300 Multilabel Reader (PerkinElmer) with lex = 495 nm, lem = 525 nm. siRNA concentration was calculated from siRNA standard curve. siRNA encapsulation efficiency was calculated by comparing siRNA concentration in the presence and absence of Triton X-100.
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The cellular uptake of the ApoE/YSK-MENDs was assessed by flow cytometry. MBEC4 cells were seeded at a density of 7.5 104 cells per well in 6 well plates (Corning incorporated, Corning, NY, USA) in 2 mL of the culture medium at 37 C under an atmosphere with 5% CO2. After 24 h, the cells were incubated with DiD-labeled ApoE/YSK-MEND at a MEND concentration of 1 mM in serum free medium for 1 h. The cells were washed with PBS and then trypsinized. The cell suspension was centrifuged (700 g, 4 C, 3 min), and then the collected cells were suspended in 1 mL PBS containing 0.5% bovine serum albumin and 2 mM EDTA (MACS buffer). After the filteration through a nylon mesh to remove cell aggregates and dust, the cells were analyzed using a FACS Calibur flow cytometer (FACScan, Becton Dickinson). The cellular uptake of DiD-labeled MENDs were expressed as the mean fluorescence intensity, calculated using the CellQuest software (Becton Dickinson). 2.6. Investigation of the cellular uptake pathway of ApoE/YSK-MEND MBEC4 cells were seeded at a density of 1 105 cells per well in 6 well plates in 2 mL culture medium at 37 C under an atmosphere with 5% CO2. After 24 h, the cells were washed with 1 mL PBS and then pre-incubated with serum-free medium in the absence or presence of the chlorpromazine (3 mg/mL) and Filipin III (2.5 mg/ mL) for 30 min. The cells were incubated with ApoE/YSK-MEND at concentration of 1 mM of MENDs for 1 h. The cells were washed 3 times with PBS to remove the unbound MEND. The cellular uptake was quantified using flow cytometry analysis as described above. 2.7. Observation of intracellular localization of MENDs by confocal microscopy MBEC4 cells were seeded at a density of 1 105 cells on a 35 mm glass base dish (IWAKI, Osaka, Japan) in 2 mL culture medium at 37 C under an atmosphere with 5% CO2. After 24 h, the DiI-labeled MENDs were incubated with the cells in 1 mL of serum free medium for 1 h. To stain the nuclei and endosome/lysosome fractions, the cells were incubated with culture medium containing 1 mg/mL Hoechst33342 (Dojindo Laboratories, Kumamoto, Japan) for 10 min and 1 mM LysoTrackerGreen (Invitrogen, Carlsbad, CA, USA) for 30 min, respectively. After washing the cells 3 times with PBS to remove the unbound MENDs, the cells were fixed with 4% paraformaldehyde (Wako Chemicals, Osaka, Japan) for 10 min at room temperature. After several washes, the cells were observed by confocal laser scanning microscopy (Nikon A1; Nikon Co., Ltd., Tokyo, Japan). 2.8. Quantification of gene silencing effect of ApoE-MEND by quantitative reverse transcription-PCR
Fig. 2. Cellular uptake pathway of the ApoE/YSK-MEND. MBEC4 cells were preincubated in the absence or presence of chlorpromazine (3 mg/mL) or filipin III (2.5 mg/mL) for 30 min. The cells were incubated with the ApoE/YSK-MENDs at a MEND concentration of 1 mM for 1 h. Cellular uptake of the ApoE/YSK-MENDs was analyzed by means of the flow cytometry. The data are represented as a percent of control. Error bars represent S.D. for four different experiments. Double-asterisks represent a significant differences determined by one-way analysis of variance (ANOVA), followed by Dunnett’s multiple-comparison post hoc test (P < 0.01).
MBEC4 cells were seeded at a density of 7.5 104 cells in 6 well plates in 2 mL of culture medium at 37 C. After 24 h, ApoE/YSKMEND containing Gapdh siRNA were transfected with these cells in 1 mL of serum free medium at a concentration of 150 nM of siRNA. The medium was replaced with fresh culture medium after 1 h. Cells were harvested 24 h after transfection. Total RNA was isolated from the cells by TRIzol Reagent (Invitrogen, CA, USA) and reverse transcribed using a High Capacity RNA-to-cDNA kit (ABI) according to manufacturer’s protocol. A quantitative PCR analysis was performed using SYBR Green Master Mix (ABI) and Mx3500P Real-time QPCR system (Agilnet, Foster City, CA, USA). All reactions were performed at a final mixture volume of 25 mL. The primer for mouse Gapdh were (forward) 50 -AGCAAGGACACTGAGCAAG-30 and (reverse) 50 -TAGGCCCCTCCTGTTATTATG-30 and for mouse b-actin
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Fig. 3. Intracellular localization of the MENDs. MBEC4 cells were incubated with the DiI-labeled YSK-MENDs (A) or ApoE/YSK-MENDs (B) for 1 h. The localization of the intracellular MENDs was observed by means of the confocal laser scanning microscopy. Nuclei and endosome/lysosome fractions were stained by Hoechst33342 and Lysotracker Green, respectively. Scale bars = 25 mm.
were (forward) 50 -AGAGGGAAATCGTGCGTGAC-30 and (reverse) 50 CAATAGTGATGACCTGGCCGT-30 . The PCR parameters consisted of a primary denaturing at 95 C 10 min, followed by 40 cycles of PCR at 95 C 15 s, and 60 C 1 min. Specificity was preliminarily verified by melting curb analysis. 3. Results 3.1. Physical properties of the MENDs We compared the characteristics of ApoE/YSK-MENDs that had been modified with various densities of ApoE. The average diameter and zeta-potential of the MENDs were estimated using a Zetasizer Nano ZSZEN3600 instrument (Malvern Instruments, Worcestershire, UK). siRNA encapsulation efficiencies were determined by RiboGreen fluorescence assay (Table 1). While the sizes of the MENDs were comparable (80–100 nm), and the particles acquired a negative charge when incubated with a higher amount of ApoE.
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Regardless of the degree of ApoE-modification, the encapsulation efficiency of siRNA remained above 70%. 3.2. Effect of ApoE-modification on the cellular uptake of YSK-MEND To validate the ability of ApoE as a ligand for targeting brain endothelial cells, the cellular uptake of the DiD-labeled YSK-MENDs by MBEC4 were determined by flow cytometry analysis. In a previous study, Zensi and his colleagues demonstrated that the ApoE-dependent uptake of the nanoparticle into the b. End3 cells reached a plateau at 1 h after the incubation (Zensi et al., 2009). Thus, in this study, cellular uptake was also determined at 1 h after the transfection to minimize the situation where the fluorescence signal was decreased by the intracellular degradation of MENDs. As a result, MBEC4 cells treated with the ApoE-free YSK-MENDs were close to background value. On the other hand, the uptake of the ApoE/YSK-MEND was drastically increased and this increase was ApoE density-dependent (Fig. 1). Therefore, ApoE-modification appears to be a highly promising strategy for enhancing the cellular uptake of YSK-MEND to MBEC4 cells. Since the cellular uptake monotonically increased (at most, >39 fold against ApoE-free YSKMEND) depending on the ApoE concentration, the subsequent analysis of cellular uptake and intracellular trafficking clearly indicated that the use of ApoE/YSK-MEND modified with 1 mg/mL of ApoE represents a promising approach to this problem. 3.3. Cellular uptake pathway of the ApoE/YSK-MEND
Fig. 4. Effect of the ApoE-modification on the gene silencing activity of siRNAencapsulating YSK-MEND. MBEC4 cells were treated with ApoE/YSK-MEND at a dose of 150 nM siRNA for 1 h in the presence of serum. The gene silencing effect was then measured at 24 h after transfection. Error bars represent S.D. for three different experiments. N.T.; non treatment.
To examine whether the cellular uptake pathway for the ApoE/ YSK-MEND is shared with the endogenous lipoprotein, we investigated the effects of inhibitors of clathrin-mediated endocytosis (chlorpromazine) and caveolae-mediated endocytosis (filipin III) on the cellular uptake of ApoE/YSK-MEND by flow cytometry analysis (Fig. 2). The cellular uptake of the ApoE/YSK-MEND pretreated with chlorpromazine was diminished by 36.9%, compared to the control. Also, a comparable inhibition was observed when MBEC4 cells were treated with filipin III (30.3%). These results suggest that the ApoE/YSK-MEND was internalized via caveolae-mediated endocytosis, as well as via chrathrinmediated endocytosis, a pathway by which endogenous lipoprotein are taken up.
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3.4. Intracellular localiation of the ApoE/YSK-MEND To verify the subsequent intracellular fate of the ApoE/YSKMENDs, their sub-cellular localization was observed by confocal laser scanning microscopy (CLSM). The extensive cellular uptake of ApoE/YSK-MEND in preference to the YSK-MEND was also reproduced (Fig. 3). Furthermore, the major fraction of the ApoE/ YSK-MENDs was not co-localized with endosome/lysosome markers. These results indicate that the ApoE/YSK-MEND is capable of efficiently escaping from lysosomal degradation. 3.5. ApoE-dependent gene silencing effect of siRNA-encapsulating ApoE/YSK-MEND Finally, the potential of the ApoE/YSK-MEND as a siRNA delivery carrier for targeting brain endothelial cells was evaluated. siRNAs targeting mouse Gapdh were encapsulated in the MENDs, and gene knockdown was then evaluated using the quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The gene silencing effect of the MENDs increased as a function of the extent of ApoE-modification (Fig. 4). The results indicate that ApoE enhanced the gene silencing effects of siRNAs by promoting the cellular uptake of the MENDs. 4. Discussion The objective of this study was to construct a carrier to deliver a nucleic acid to the brain endothelium. To realize this, we prepared an ApoE/YSK-MEND and evaluated its utility using mouse brain endothelial cells (MBEC4). The results indicated that the cellular uptake and gene silencing effect of this carrier were enhanced depending on the extent of modification of ApoE (Figs. 1 and 4). The enhancement in the gene silencing effect was accompanied by an increase in the level of internalized ApoE/YSK-MEND. This indicates that the cellular uptake might be the rate-limiting step for their effective gene silencing (Figs. 1 and 4). ApoE is a ligand for members of the LDLR family and heparin sulfate proteoglycans (HSPGs). (Hatters et al., 2006; MacArthur et al., 2007; Peters-Libeu et al., 2006) The most plausible receptor for the ApoE/YSK-MEND is LDLR that recognizes ApoE-containing lipoproteins destined for the clathrin-mediated endocytosis (Nykjaer and Willnow 2002). In addition, Candela and co-workers indicated that the endogenous LDL passed through their BBB model via the caveolae-caveosome transcellular pathway (Candela et al., 2008). These previous data are consistent with our results showing that the ApoE/YSK-MEND was taken up by MBEC4 cells via clathrinand caveolae-mediated endocytosis (Fig. 2). Meanwhile, Wagner and co-workers demonstrated that ApoE-modified human albumin nanoparticles were internalized into b.End3 cells via LRP1 and entered the central nervous system (CNS) by transcytosis in vivo (Wagner et al., 2012; Zensi et al., 2009). While the mechanism for the LRP1-mediated uptake of ApoE-modified particles remains elusive, Ke and co-workers reported that the pDNA compacted with polyamidoamine dendrimer (PAMAM) that are modified with angiopep-2, a LRP1-targeting peptide derived from the Kunitz protease inhibitor (KPI) domain in Aprotinin, Bikunin, and amyloid precursor protein (APP) was internalized by brain capillary endothelial cells (BCECs) through both clathrin- and caveolaemediated endocytosis. These data indicate that the ApoE/YSKMEND might be alternatively taken up via LRP1-mediated endocytosis (Ke et al., 2009). Regarding the uptake of triglyceride-rich lipoproteins, HSPG is known to be another type of cellular surface molecules that contributes to LDLR-independent cellular uptake (MacArthur et al., 2007). It is noteworthy that Sauer and co-workers developed an ApoE-derived peptide, referred to as A2, that are tandem dimers of
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highly cationic ApoE-residues (141–150) (Sauer et al., 2005). In their study, the liposomes were chemically conjugated with the A2 peptide via a PEG spacer and were internalized by cells via a HSPGdependent pathway. However, the liposomes were highly cationic. Therefore, the electrostatic interactions between the A2-modified liposomes and HSPGs might be a crucial driving force. If this hypothesis is valid, the HSPGs might not function as a receptor for the ApoE/YSK-MEND since the j-potentials of the particles become more negatively charged depending on the extent of ApoE modification (Table 1). Since LDL receptor family members are expressed in various cells including endothelial cells in many organs and hepatocytes, the ApoE/YSK-MEND might be rapidly excluded from the blood circulation after intravenous administration. Therefore, injection of the carrier into the arteria carotis might be the most plausible strategy for delivering the ApoE/YSK-MEND to maximize its accumulation in the brain. Brain endothelial cells constitute the NVU and function cooperatively to maintain the normal brain function with glial cells and neurons. While many researchers proposed the existence of a relationship between the abnormality of the NVU and neurodegenerative diseases, the molecular mechanisms of cellto-cell interaction remain unexplained. We predict that the ApoE/ YSK-MEND may become a useful tool for developing a better understanding of these mechanisms by silencing the gene of interest. Acknowledgements This work was supported in part by Funding Program for Next Generation World-Leading Researchers (NEXT Program), and partially by a Grant for Industrial Technology Research from New Energy and Industrial Technology Development Organization (NEDO). H.A. is also supported by the Uehara Memorial Foundation. The authors would also like to thank Dr. M.S. Feather for his helpful advice in writing the English manuscript. References Akinc, A., Querbes, W., De, S., et al., 2010. Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Molecular Therapy 18, 1357–1364. Alvarez-Erviti, L., Seow, Y., Yin, H., Betts, C., Lakhal, S., Wood, M.J., 2011. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nature Biotechnology 29, 341–345. Candela, P., Gosselet, F., Miller, F., Buee-Scherrer, V., Torpier, G., Cecchelli, R., Fenart, L., 2008. Physiological pathway for low-density lipoproteins across the bloodbrain barrier: transcytosis through brain capillary endothelial cells in vitro. Endothelium 15, 254–264. Grammas, P., 2011. Neurovascular dysfunction, inflammation and endothelial activation: implications for the pathogenesis of Alzheimer’s disease. Journal of Neuroinflammation 8, 26. Hatakeyama, H., Akita, H., Ito, E., et al., 2011. Systemic delivery of siRNA to tumors using a lipid nanoparticle containing a tumor-specific cleavable PEG-lipid. Biomaterials 32, 4306–4316. Hatters, D.M., Peters-Libeu, C.A., Weisgraber, K.H., 2006. Apolipoprotein E structure: insights into function. Trends in Biochemical Sciences 31, 445–454. Ke, W., Shao, K., Huang, R., et al., 2009. Gene delivery targeted to the brain using an Angiopep-conjugated polyethyleneglycol-modified polyamidoamine dendrimer. Biomaterials 30, 6976–6985. Khalil, I.A., Hayashi, Y., Mizuno, R., Harashima, H., 2011. Octaarginine- and pH sensitive fusogenic peptide-modified nanoparticles for liver gene delivery. Journal of Controlled Release 156, 374–380. Kogure, K., Akita, H., Yamada, Y., Harashima, H., 2008. Multifunctional envelope-type nano device (MEND) as a non-viral gene delivery system. Advanced Drug Delivery Reviews 60, 559–571. Kuwahara, H., Nishina, K., Yoshida, K., et al., 2011. Efficient in vivo delivery of siRNA into brain capillary endothelial cells along with endogenous lipoprotein. Molecular Therapy 19, 2213–2221. Leupold, E., Nikolenko, H., Dathe, M., 2009. Apolipoprotein E peptide-modified colloidal carriers: the design determines the mechanism of uptake in vascular endothelial cells. Biochimica et Biophysica Acta 1788, 442–449. Arthur, Mac, Bishop, J.M., Stanford, J.R., Wang, K.I., Bensadoun, L., Witztum, A., Esko, J. L., J. D, 2007. Liver heparan sulfate proteoglycans mediate clearance of
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Sato, Y., Hatakeyama, H., Sakurai, Y., Hyodo, M., Akita, H., Harashima, H., 2012. A pHsensitive cationic lipid facilitates the delivery of liposomal siRNA and gene silencing activity in vitro and in vivo. Journal of Controlled Release 163, 267–276. Sauer, I., Dunay, I.R., Weisgraber, K., Bienert, M., Dathe, M., 2005. An apolipoprotein Ederived peptide mediates uptake of sterically stabilized liposomes into brain capillary endothelial cells. Biochemistry 44, 2021–2029. Stanimirovic, D.B., Friedman, A., 2012. Pathophysiology of the neurovascular unit: disease cause or consequence? Journal of Cerebral Blood Flow and Metabolism 32, 1207–1221. van Rooy, I., Mastrobattista, E., Storm, G., Hennink, W.E., Schiffelers, R.M., 2011. Comparison of five different targeting ligands to enhance accumulation of liposomes into the brain. Journal of Controlled Release 150, 30–36. Wagner, S., Zensi, A., Wien, S.L., et al., 2012. Uptake mechanism of ApoE-modified nanoparticles on brain capillary endothelial cells as a blood-brain barrier model. Public Library of Science 7, e32568. Zensi, A., Begley, D., Pontikis, C., Legros, C., Mihoreanu, L., Wagner, S., Büchel, C., von Briesen, H., Kreuter, J., 2009. Albumin nanoparticles targeted with Apo E enter the CNS by transcytosis and are delivered to neurones. Journal of Controlled Release 137, 78–86. Zhong, Z., Ilieva, H., Hallagan, L., et al., 2009. Activated protein C therapy slows ALSlike disease in mice by transcriptionally inhibiting SOD1 in motor neurons and microglia cells. The Journal of Clinical Investigation 119, 3437–3449. Zlokovic, B.V., 2008. The blood–brain barrier in health and chronic neurodegenerative disorders. Neuron 57, 178–201. Zlokovic, B.V., 2011. Neurovascular pathways to neurodegeneration in Alzheimer’s disease and other disorders. Nature Reviews. Neuroscience 12, 723–738. Zlokovic, B.V., 2013. Cerebrovascular effects of apolipoprotein E: implications for Alzheimer disease. JAMA Neurology 70, 440–444.
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
An apolipoprotein E modified liposomal nanoparticle: Ligand dependent efficiency as a siRNA delivery carrier for mouse-derived brain endothelial cells Mina Tamaru, Hidetaka Akita * , Kazuaki Kajimoto, Yusuke Sato, Hiroto Hatakeyama, Hideyoshi Harashima ** Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 September 2013 Received in revised form 21 December 2013 Accepted 8 February 2014 Available online 12 February 2014
A disorder in the brain endothelium is thought to be closely related to the pathophysiology of brain diseases. A method for delivering nucleic acids (i.e. short interference RNA; siRNA) to the brain endothelium should be an attractive strategy for curing brain disorders. A liposomal nanoparticle containing a proton-ionizable amino lipid was recently developed as a carrier of encapsulated siRNA. The aim of this study was to evaluate the utility of apolipoprotein E (ApoE) as a targeting ligand for mouse brain endothelial cells (MBEC4 cells). The cellular uptake of the ApoE-modified nanoparticles was gradually increased in an ApoE-density dependent manner. Furthermore, the ApoE-modified nanoparticles were taken up via both clathrin and caveolae mediated endocytosis, thus permitting them to avoid lysosomal degradation. Finally, endogenous gene silencing in MBEC4 cells was efficiently achieved depending on the ApoE-modification. Collectively, the ApoE-modified nanoparticle is a promising carrier for delivering nucleic acids to the brain endothelium. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Apolipoprotein E (ApoE) Brain endothelial cell Multifunctional envelope-type nano device (MEND) YSK05 siRNA delivery In vitro
1. Introduction The neurovascular unit (NVU), which is composed of vascular cells (i.e. endothelium and pericytes), glial cells (i.e. astrocytes, microglia, and oligodendroglia), and neurons, cooperatively plays a key role in the maintenance of homeostasis and normal brain function (Zlokovic 2011; Stanimirovic and Friedman 2012). Brain endothelial cells are the main structural components of the blood brain barrier (BBB).
Abbreviations: siRNA, short interference RNA; ApoE, apolipoprotein E; NVU, neurovascular unit; BBB, blood brain barrier; pDNA, plasmid DNA; MEND, multifunctional envelop-type nano device; LDLR, low-density lipoprotein receptor; LRP1, LDLR-related protein 1; VLDLR, very low density lipoprotein receptor; HAS, human serum albumin; PEG, polyethyleneglycol; LNP, liposomal nanoparticle; DiD, 1,10 dioctadecyl-3,30 -tetramethylindocarbocyamine perchorate; DiI, 1,10 -dioctadecyl3,30 -tetramethylindodicarbocyamine perchorate; DMEM, Dulbecco’s modified eagle’s medium; FBS, fetal bovine serum; DMG, dimyristoyl-sn-glycerol; t-BuOH, tertiary butanol; CLSM, confocal laser scanning microscopy; qRT-PCR, quantitative reverse transcriptase-polymerase chain reaction; HSPG, heparin sulfate proteoglycan; CNS, central nervous system; KPI, Kunitz protease inhibitor; APP, amyloid precursor protein. * Corresponding author. Tel.: +81 11 706 3735; fax: +81 11 706 4879. ** Corresponding author. Tel.: +81 11 706 3919; fax: +81 11 706 4879. E-mail addresses: [email protected] (H. Akita), [email protected] (H. Harashima). http://dx.doi.org/10.1016/j.ijpharm.2014.02.016 0378-5173/ã 2014 Elsevier B.V. All rights reserved.
Perturbation of the BBB is closely related to the progress of various diseases, since it regulates the entry of cells and molecules into the brain and functions as a pump to remove potentially neurotoxic molecules (Zlokovic 2008) including thrombin (Grammas 2011), fibrin (Paul et al., 2007), and hemosiderin (Zhong et al., 2009). The deliveryof nucleic acids, such as siRNA, is one of the rational strategies for repairing the dysfunction of cells. However, naked siRNA is susceptible to digestion by extracellular enzymes or rapid urinary exclusion. Moreover, the functional transport of native nucleic acids to the cytoplasm is intrinsically limited due to their poor cellular uptake, and, if they enter, to degradation in lysosomes. We previously developed a liposomal nanoparticle containing encapsulated plasmid DNA (pDNA) and siRNA that is referred to as a multi-functional envelop-type nano device (MEND). The particle was designed to protect the encapsulated cargos from enzymatic degradation, and to overcome the intracellular biomembranes (i.e. endosome) (Kogure et al., 2008; Nakamura et al., 2012). The particles are applied for targeting the liver and tumors via the systemic circulation since the fenestrae in liver and discontinuous neovascular vessels in cancer permit the particle to penetrate through the endothelium (Hatakeyama et al., 2011; Sakurai et al., 2011; Sakurai et al., 2013; Khalil et al., 2011). In contrast to these, the penetration of the particle through the continuous endothelium in a majority of the organs was found to be severely limited. Therefore,
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a ligand for a specific type of receptor on the endothelium would be required for tissue targeting. For the delivery of the therapeutic molecules to the brain, artificial carriers; liposomes and micelles, as well as endogenous lipoproteins and exosomes are used as a carrier (Alvarez-Erviti et al., 2011; Pardridge 2007). From the point of view of tissue targeting, an uncharged particle would be highly desirable, since when positively charged particles are administered, large aggregates with erythrocyte are produced, which are then distributed or trapped to the non-targeting tissues (i.e. lung) (Sakurai et al., 2001; Ogris et al., 1999). Recently, a proton-ionizable aminolipid, denoted as YSK05 was synthesized, whose incorporation into the lipid envelope structure (YSK-MEND) permits a neutral particle to be prepared, which, in turn, permits efficient membrane fusion to endosomes and subsequent delivery of the encapsulated siRNA into cytosol (Sato et al., 2012). Consequently, the intratumoral administration of the YSK-MEND conferred more efficient gene silencing compared with MENDs composed of conventional cationic lipids (Sato et al., 2012). Thus, in the present study, the YSK-MEND was used as a platform for a siRNA carrier to evaluate the function of the ligand. To date, a large variety of the ligands such as endogenous peptides derived from hormones (i.e. leptins), foreign peptides discovered by phage display methods and antibodies against the receptors on the BBB have been identified (van Rooy et al., 2011). In this report, we focused on ApoE as a targeting ligand for the brain endothelium. ApoE is a member of the family of soluble apolipoproteins and binds triglyseride-rich lipoproteins (Hatters et al., 2006; Peters-Libeu et al., 2006). The endogenous lipoproteins interact with members of the low-density lipoprotein receptor (LDLR) family through ApoE and are subsequently internalized via clathrin-mediated endocytosis (Nykjaer and Willnow 2002). In addition to hepatocytes or the sinusoidal endothelium, several studies have demonstrated that LDL receptor families such as LDLR, LDLR-related protein1 (LRP1), and very low density lipoprotein receptor (VLDLR) (Zlokovic 2013) are also expressed by brain endothelial cells. Very recently, Wagner et al. reported that the chemical conjugation of ApoE onto human serum albumin (HSA) nanoparticles via a polyethyleneglycol (PEG) spacer enhanced the uptake into the brain endothelium-derived in vitro cultured cells (Wagner et al., 2012). Furthermore, the conjugation of ApoEderived peptides (141–150), a highly cationic variety, was also used as a ligand for targeting brain endothelial cells (Sauer et al., 2005; Leupold et al., 2009). Furthermore, Kuwahara and co-workers demonstrated that cholesterol-conjugated siRNA conferred gene silencing in the brain capillary endothelial cells in vivo and in vitro mainly due to an accelerated cellular uptake mediated by the ApoEassociated formation of lipoproteins, followed by recognition by LDLRs (Kuwahara et al., 2011). Meanwhile, Akinc and co-workers reported that liposomal nanoparticles (LNP) composed of ionizable lipids accumulated at high levels in the liver via the LDLR, aided by endogenous ApoE (Akinc et al., 2010). This study prompted us to evaluate whether the simple incubation of liposomes formed by a
pH-ionizable lipid with ApoE might accelerate the uptake of lipid nanoparticles by the brain endothelium. In this study, we prepared the ApoE-modified YSK-MEND (ApoE/ YSK-MEND) to target brain endothelial cells, and then investigated its intracellular fate, and function as a siRNA carrier in mouse brain derived endothelial cells (MBEC4). 2. Materials and methods 2.1. Materials Anti-Gapdh siRNA was purchased from Hokkaido System Science Co., Ltd. (Sapporo, Japan). Cholesterol was purchased from Avanti Polar Lipid (Albaster, AL, USA). RiboGreen was purchased from Molecular Probes (Eugene, OR, USA). DiI (1,10 -dioctadecyl3,3,30 ,30 -tetramethylindocarbocyanine perchorate), DiD (1,10 -dioctadecyl-3,3,30 ,30 -tetramethylindodicarbocyanine perchorate), TRIzol reagent and LysoTracker Green DND-26 were purchased from Invitrogen (Carlsbad, CA, USA). Hoechst33342 was purchased from Dojindo Laboratories (Kumamoto, Japan). Recombinant Human ApoE3 was purchased from BioVision Incorporated (Milpitas Boulevard, Milpitas, CA, USA). 2.2. Cell cultures MBEC4 cells derived from mouse brain endothelial cells were generously supplied by Dr. T. Tsuruo and Dr. M Naito (Tokyo University, Japan). The cells were maintained with Dulbecco’s modified eagle’s medium (DMEM, SIGMA–Aldrich) supplemented with 10% fetal bovine serum (FBS), NaHCO3 (3.7 g/L), streptomycin (100 mg/L), penicillin (16.67 mg/L) at 37 C under an atmosphere of 5% CO2. 2.3. MEND formulations The MEND composed of YSK05/cholesterol/PEG-Dimyristoylsn-glycerol (DMG) (70/30/3) was prepared by the tertiary butanol (t-BuOH) dilution procedure as described previously (Sato et al., 2012). PEG-DMG was used to stabilize the lipid membrane during the formation process and for preservation. Briefly, 10 mM of total lipids were dissolved in a 90% t-BuOH solution. siRNA was dissolved in 20 mM citrate buffer (pH 4.0) and was added dropwise into the lipid solution under vigorous mixing to avoid a low local concentration of t-BuOH. Subsequently, the solution was quickly diluted with citrate buffer to a final concentration of 3 h at arbitrary concentrations. The average diameter and zeta-potential of MENDs were determined using a Zetasizer Nano ZS ZEN3600 (Malvern Instrument, Worcestershire, UK). DiD labeled
Table 1 Physicochemical properties of ApoE/YSK-MENDs. ApoE (mg/mL)
0
0.1
0.3
1
3
Size (nm) z-Potential (mV) PdI siRNA encapsulation (%)
84 11 0.52 1.35 0.22 0.1 88.6 2.1
86 3 1.2 0.74 0.17 0.02 90.0 0.4
94 3 0.02 1.73 0.19 0.04 88.1 0.5
104 10 3.42 0.56 0.21 0.08 82.4 2.3
94 9 4.83 0.77 0.19 0.08 74.6 5.4
Particle diameter, polydispersity (PdI), and zeta-potential were measured using Malvern Zetasizer. Percentage of siRNA encapsulation was determined by RiboGreen fluorescence assay to measure the amount of siRNA relative to total siRNA percent. DATA points are expressed as the mean SD (n = 3).
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2.5. Quantification of cellular uptake of ApoE/YSK-MENDs by flow cytometry
Fig. 1. Effect of ApoE-modification on the cellular uptake of YSK-MEND. MBEC4 cells were incubated with the DiD-labeled ApoE/YSK-MENDs at a concentration of 1 mM of MENDs for 1 h. Cellular uptake of the ApoE/YSK-MENDs was analyzed by means of the flow cytometry. The data are represented as the mean fluorescence intensity (MFI) SD (n = 3).
MENDs were prepared by adding a DiD (0.5 mol% of total lipid) in the lipid composition. 2.4. RiboGreen fluorescence assay To determine siRNA encapsulation and its concentration, RiboGreen fluorescence assay was performed. MENDs were diluted in 200 mL of 10 mM HEPES buffer (pH 7.4) containing 20 mg/mL dextran sulfate, and then 0.5 mL of RiboGreen was applied in the presence or absence of 0.1% (w/v) Triton X-100. Fluorescence was measured by EnSpire 2300 Multilabel Reader (PerkinElmer) with lex = 495 nm, lem = 525 nm. siRNA concentration was calculated from siRNA standard curve. siRNA encapsulation efficiency was calculated by comparing siRNA concentration in the presence and absence of Triton X-100.
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The cellular uptake of the ApoE/YSK-MENDs was assessed by flow cytometry. MBEC4 cells were seeded at a density of 7.5 104 cells per well in 6 well plates (Corning incorporated, Corning, NY, USA) in 2 mL of the culture medium at 37 C under an atmosphere with 5% CO2. After 24 h, the cells were incubated with DiD-labeled ApoE/YSK-MEND at a MEND concentration of 1 mM in serum free medium for 1 h. The cells were washed with PBS and then trypsinized. The cell suspension was centrifuged (700 g, 4 C, 3 min), and then the collected cells were suspended in 1 mL PBS containing 0.5% bovine serum albumin and 2 mM EDTA (MACS buffer). After the filteration through a nylon mesh to remove cell aggregates and dust, the cells were analyzed using a FACS Calibur flow cytometer (FACScan, Becton Dickinson). The cellular uptake of DiD-labeled MENDs were expressed as the mean fluorescence intensity, calculated using the CellQuest software (Becton Dickinson). 2.6. Investigation of the cellular uptake pathway of ApoE/YSK-MEND MBEC4 cells were seeded at a density of 1 105 cells per well in 6 well plates in 2 mL culture medium at 37 C under an atmosphere with 5% CO2. After 24 h, the cells were washed with 1 mL PBS and then pre-incubated with serum-free medium in the absence or presence of the chlorpromazine (3 mg/mL) and Filipin III (2.5 mg/ mL) for 30 min. The cells were incubated with ApoE/YSK-MEND at concentration of 1 mM of MENDs for 1 h. The cells were washed 3 times with PBS to remove the unbound MEND. The cellular uptake was quantified using flow cytometry analysis as described above. 2.7. Observation of intracellular localization of MENDs by confocal microscopy MBEC4 cells were seeded at a density of 1 105 cells on a 35 mm glass base dish (IWAKI, Osaka, Japan) in 2 mL culture medium at 37 C under an atmosphere with 5% CO2. After 24 h, the DiI-labeled MENDs were incubated with the cells in 1 mL of serum free medium for 1 h. To stain the nuclei and endosome/lysosome fractions, the cells were incubated with culture medium containing 1 mg/mL Hoechst33342 (Dojindo Laboratories, Kumamoto, Japan) for 10 min and 1 mM LysoTrackerGreen (Invitrogen, Carlsbad, CA, USA) for 30 min, respectively. After washing the cells 3 times with PBS to remove the unbound MENDs, the cells were fixed with 4% paraformaldehyde (Wako Chemicals, Osaka, Japan) for 10 min at room temperature. After several washes, the cells were observed by confocal laser scanning microscopy (Nikon A1; Nikon Co., Ltd., Tokyo, Japan). 2.8. Quantification of gene silencing effect of ApoE-MEND by quantitative reverse transcription-PCR
Fig. 2. Cellular uptake pathway of the ApoE/YSK-MEND. MBEC4 cells were preincubated in the absence or presence of chlorpromazine (3 mg/mL) or filipin III (2.5 mg/mL) for 30 min. The cells were incubated with the ApoE/YSK-MENDs at a MEND concentration of 1 mM for 1 h. Cellular uptake of the ApoE/YSK-MENDs was analyzed by means of the flow cytometry. The data are represented as a percent of control. Error bars represent S.D. for four different experiments. Double-asterisks represent a significant differences determined by one-way analysis of variance (ANOVA), followed by Dunnett’s multiple-comparison post hoc test (P < 0.01).
MBEC4 cells were seeded at a density of 7.5 104 cells in 6 well plates in 2 mL of culture medium at 37 C. After 24 h, ApoE/YSKMEND containing Gapdh siRNA were transfected with these cells in 1 mL of serum free medium at a concentration of 150 nM of siRNA. The medium was replaced with fresh culture medium after 1 h. Cells were harvested 24 h after transfection. Total RNA was isolated from the cells by TRIzol Reagent (Invitrogen, CA, USA) and reverse transcribed using a High Capacity RNA-to-cDNA kit (ABI) according to manufacturer’s protocol. A quantitative PCR analysis was performed using SYBR Green Master Mix (ABI) and Mx3500P Real-time QPCR system (Agilnet, Foster City, CA, USA). All reactions were performed at a final mixture volume of 25 mL. The primer for mouse Gapdh were (forward) 50 -AGCAAGGACACTGAGCAAG-30 and (reverse) 50 -TAGGCCCCTCCTGTTATTATG-30 and for mouse b-actin
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Fig. 3. Intracellular localization of the MENDs. MBEC4 cells were incubated with the DiI-labeled YSK-MENDs (A) or ApoE/YSK-MENDs (B) for 1 h. The localization of the intracellular MENDs was observed by means of the confocal laser scanning microscopy. Nuclei and endosome/lysosome fractions were stained by Hoechst33342 and Lysotracker Green, respectively. Scale bars = 25 mm.
were (forward) 50 -AGAGGGAAATCGTGCGTGAC-30 and (reverse) 50 CAATAGTGATGACCTGGCCGT-30 . The PCR parameters consisted of a primary denaturing at 95 C 10 min, followed by 40 cycles of PCR at 95 C 15 s, and 60 C 1 min. Specificity was preliminarily verified by melting curb analysis. 3. Results 3.1. Physical properties of the MENDs We compared the characteristics of ApoE/YSK-MENDs that had been modified with various densities of ApoE. The average diameter and zeta-potential of the MENDs were estimated using a Zetasizer Nano ZSZEN3600 instrument (Malvern Instruments, Worcestershire, UK). siRNA encapsulation efficiencies were determined by RiboGreen fluorescence assay (Table 1). While the sizes of the MENDs were comparable (80–100 nm), and the particles acquired a negative charge when incubated with a higher amount of ApoE.
[(Fig._4)TD$IG]
Regardless of the degree of ApoE-modification, the encapsulation efficiency of siRNA remained above 70%. 3.2. Effect of ApoE-modification on the cellular uptake of YSK-MEND To validate the ability of ApoE as a ligand for targeting brain endothelial cells, the cellular uptake of the DiD-labeled YSK-MENDs by MBEC4 were determined by flow cytometry analysis. In a previous study, Zensi and his colleagues demonstrated that the ApoE-dependent uptake of the nanoparticle into the b. End3 cells reached a plateau at 1 h after the incubation (Zensi et al., 2009). Thus, in this study, cellular uptake was also determined at 1 h after the transfection to minimize the situation where the fluorescence signal was decreased by the intracellular degradation of MENDs. As a result, MBEC4 cells treated with the ApoE-free YSK-MENDs were close to background value. On the other hand, the uptake of the ApoE/YSK-MEND was drastically increased and this increase was ApoE density-dependent (Fig. 1). Therefore, ApoE-modification appears to be a highly promising strategy for enhancing the cellular uptake of YSK-MEND to MBEC4 cells. Since the cellular uptake monotonically increased (at most, >39 fold against ApoE-free YSKMEND) depending on the ApoE concentration, the subsequent analysis of cellular uptake and intracellular trafficking clearly indicated that the use of ApoE/YSK-MEND modified with 1 mg/mL of ApoE represents a promising approach to this problem. 3.3. Cellular uptake pathway of the ApoE/YSK-MEND
Fig. 4. Effect of the ApoE-modification on the gene silencing activity of siRNAencapsulating YSK-MEND. MBEC4 cells were treated with ApoE/YSK-MEND at a dose of 150 nM siRNA for 1 h in the presence of serum. The gene silencing effect was then measured at 24 h after transfection. Error bars represent S.D. for three different experiments. N.T.; non treatment.
To examine whether the cellular uptake pathway for the ApoE/ YSK-MEND is shared with the endogenous lipoprotein, we investigated the effects of inhibitors of clathrin-mediated endocytosis (chlorpromazine) and caveolae-mediated endocytosis (filipin III) on the cellular uptake of ApoE/YSK-MEND by flow cytometry analysis (Fig. 2). The cellular uptake of the ApoE/YSK-MEND pretreated with chlorpromazine was diminished by 36.9%, compared to the control. Also, a comparable inhibition was observed when MBEC4 cells were treated with filipin III (30.3%). These results suggest that the ApoE/YSK-MEND was internalized via caveolae-mediated endocytosis, as well as via chrathrinmediated endocytosis, a pathway by which endogenous lipoprotein are taken up.
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3.4. Intracellular localiation of the ApoE/YSK-MEND To verify the subsequent intracellular fate of the ApoE/YSKMENDs, their sub-cellular localization was observed by confocal laser scanning microscopy (CLSM). The extensive cellular uptake of ApoE/YSK-MEND in preference to the YSK-MEND was also reproduced (Fig. 3). Furthermore, the major fraction of the ApoE/ YSK-MENDs was not co-localized with endosome/lysosome markers. These results indicate that the ApoE/YSK-MEND is capable of efficiently escaping from lysosomal degradation. 3.5. ApoE-dependent gene silencing effect of siRNA-encapsulating ApoE/YSK-MEND Finally, the potential of the ApoE/YSK-MEND as a siRNA delivery carrier for targeting brain endothelial cells was evaluated. siRNAs targeting mouse Gapdh were encapsulated in the MENDs, and gene knockdown was then evaluated using the quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The gene silencing effect of the MENDs increased as a function of the extent of ApoE-modification (Fig. 4). The results indicate that ApoE enhanced the gene silencing effects of siRNAs by promoting the cellular uptake of the MENDs. 4. Discussion The objective of this study was to construct a carrier to deliver a nucleic acid to the brain endothelium. To realize this, we prepared an ApoE/YSK-MEND and evaluated its utility using mouse brain endothelial cells (MBEC4). The results indicated that the cellular uptake and gene silencing effect of this carrier were enhanced depending on the extent of modification of ApoE (Figs. 1 and 4). The enhancement in the gene silencing effect was accompanied by an increase in the level of internalized ApoE/YSK-MEND. This indicates that the cellular uptake might be the rate-limiting step for their effective gene silencing (Figs. 1 and 4). ApoE is a ligand for members of the LDLR family and heparin sulfate proteoglycans (HSPGs). (Hatters et al., 2006; MacArthur et al., 2007; Peters-Libeu et al., 2006) The most plausible receptor for the ApoE/YSK-MEND is LDLR that recognizes ApoE-containing lipoproteins destined for the clathrin-mediated endocytosis (Nykjaer and Willnow 2002). In addition, Candela and co-workers indicated that the endogenous LDL passed through their BBB model via the caveolae-caveosome transcellular pathway (Candela et al., 2008). These previous data are consistent with our results showing that the ApoE/YSK-MEND was taken up by MBEC4 cells via clathrinand caveolae-mediated endocytosis (Fig. 2). Meanwhile, Wagner and co-workers demonstrated that ApoE-modified human albumin nanoparticles were internalized into b.End3 cells via LRP1 and entered the central nervous system (CNS) by transcytosis in vivo (Wagner et al., 2012; Zensi et al., 2009). While the mechanism for the LRP1-mediated uptake of ApoE-modified particles remains elusive, Ke and co-workers reported that the pDNA compacted with polyamidoamine dendrimer (PAMAM) that are modified with angiopep-2, a LRP1-targeting peptide derived from the Kunitz protease inhibitor (KPI) domain in Aprotinin, Bikunin, and amyloid precursor protein (APP) was internalized by brain capillary endothelial cells (BCECs) through both clathrin- and caveolaemediated endocytosis. These data indicate that the ApoE/YSKMEND might be alternatively taken up via LRP1-mediated endocytosis (Ke et al., 2009). Regarding the uptake of triglyceride-rich lipoproteins, HSPG is known to be another type of cellular surface molecules that contributes to LDLR-independent cellular uptake (MacArthur et al., 2007). It is noteworthy that Sauer and co-workers developed an ApoE-derived peptide, referred to as A2, that are tandem dimers of
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highly cationic ApoE-residues (141–150) (Sauer et al., 2005). In their study, the liposomes were chemically conjugated with the A2 peptide via a PEG spacer and were internalized by cells via a HSPGdependent pathway. However, the liposomes were highly cationic. Therefore, the electrostatic interactions between the A2-modified liposomes and HSPGs might be a crucial driving force. If this hypothesis is valid, the HSPGs might not function as a receptor for the ApoE/YSK-MEND since the j-potentials of the particles become more negatively charged depending on the extent of ApoE modification (Table 1). Since LDL receptor family members are expressed in various cells including endothelial cells in many organs and hepatocytes, the ApoE/YSK-MEND might be rapidly excluded from the blood circulation after intravenous administration. Therefore, injection of the carrier into the arteria carotis might be the most plausible strategy for delivering the ApoE/YSK-MEND to maximize its accumulation in the brain. Brain endothelial cells constitute the NVU and function cooperatively to maintain the normal brain function with glial cells and neurons. While many researchers proposed the existence of a relationship between the abnormality of the NVU and neurodegenerative diseases, the molecular mechanisms of cellto-cell interaction remain unexplained. We predict that the ApoE/ YSK-MEND may become a useful tool for developing a better understanding of these mechanisms by silencing the gene of interest. Acknowledgements This work was supported in part by Funding Program for Next Generation World-Leading Researchers (NEXT Program), and partially by a Grant for Industrial Technology Research from New Energy and Industrial Technology Development Organization (NEDO). H.A. is also supported by the Uehara Memorial Foundation. The authors would also like to thank Dr. M.S. Feather for his helpful advice in writing the English manuscript. References Akinc, A., Querbes, W., De, S., et al., 2010. Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Molecular Therapy 18, 1357–1364. Alvarez-Erviti, L., Seow, Y., Yin, H., Betts, C., Lakhal, S., Wood, M.J., 2011. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nature Biotechnology 29, 341–345. Candela, P., Gosselet, F., Miller, F., Buee-Scherrer, V., Torpier, G., Cecchelli, R., Fenart, L., 2008. Physiological pathway for low-density lipoproteins across the bloodbrain barrier: transcytosis through brain capillary endothelial cells in vitro. Endothelium 15, 254–264. Grammas, P., 2011. Neurovascular dysfunction, inflammation and endothelial activation: implications for the pathogenesis of Alzheimer’s disease. Journal of Neuroinflammation 8, 26. Hatakeyama, H., Akita, H., Ito, E., et al., 2011. Systemic delivery of siRNA to tumors using a lipid nanoparticle containing a tumor-specific cleavable PEG-lipid. Biomaterials 32, 4306–4316. Hatters, D.M., Peters-Libeu, C.A., Weisgraber, K.H., 2006. Apolipoprotein E structure: insights into function. Trends in Biochemical Sciences 31, 445–454. Ke, W., Shao, K., Huang, R., et al., 2009. Gene delivery targeted to the brain using an Angiopep-conjugated polyethyleneglycol-modified polyamidoamine dendrimer. Biomaterials 30, 6976–6985. Khalil, I.A., Hayashi, Y., Mizuno, R., Harashima, H., 2011. Octaarginine- and pH sensitive fusogenic peptide-modified nanoparticles for liver gene delivery. Journal of Controlled Release 156, 374–380. Kogure, K., Akita, H., Yamada, Y., Harashima, H., 2008. Multifunctional envelope-type nano device (MEND) as a non-viral gene delivery system. Advanced Drug Delivery Reviews 60, 559–571. Kuwahara, H., Nishina, K., Yoshida, K., et al., 2011. Efficient in vivo delivery of siRNA into brain capillary endothelial cells along with endogenous lipoprotein. Molecular Therapy 19, 2213–2221. Leupold, E., Nikolenko, H., Dathe, M., 2009. Apolipoprotein E peptide-modified colloidal carriers: the design determines the mechanism of uptake in vascular endothelial cells. Biochimica et Biophysica Acta 1788, 442–449. Arthur, Mac, Bishop, J.M., Stanford, J.R., Wang, K.I., Bensadoun, L., Witztum, A., Esko, J. L., J. D, 2007. Liver heparan sulfate proteoglycans mediate clearance of
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