Endocytic Pathway and Resistance to Cholesterol Depletion of

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Article pubs.acs.org/molecularpharmaceutics

Endocytic Pathway and Resistance to Cholesterol Depletion of Cholesterol Derived Cationic Lipids for Gene Delivery Yun-Ui Bae,† Bieong-Kil Kim,‡ Jong-Won Park,§ Young-Bae Seu,‡ and Kyung-Oh Doh*,† †

Department of Physiology and §Department of Internal Medicine College of Medicine, Yeungnam University, Daegu 705-717, Korea ‡ School of Life Sciences and Biotechnology, Kyungpook National University, Daegu 702-701, Korea S Supporting Information *

ABSTRACT: Cholesterol-based cationic lipids have been widely used because of biocompatibility and serum resistance. However, the reason for the effectiveness of cholesterol-based cationic lipids remains unclear. We compared the transfection route of CHOL-E, a cholesterol-based cationic lipid having an amine head and an ether linker, with that of DOTAP. The luciferase assay with chemical inhibitors and microscopic observation of pathway markers revealed that clathrin mediated endocytosis is the main pathway for CHOL-E and DOTAP. However, CHOL-E showed resistance to cholesterol depletion by methyl-βcyclodextrin. Furthermore, CHOL-E recovered the transfection efficiency of DOTAP from cholesterol depletion. These results suggested that superior transfection of CHOL-E might be partly derived from effects on the cell membrane.

KEYWORDS: endocytic pathway, gene delivery, cholesterol, nonviral vector

1. INTRODUCTION Gene therapy has been expected to lead to new, powerful approaches for curing diseases such as cancer1 and heart failure.2 One of the primary objects of gene therapy is to develop efficient and nontoxic gene carriers that can effectively deliver foreign genetic materials such as plasmid, antisense, decoy, oligonucleotide, and siRNA into the target cells.3 There are two main gene delivery systems, viral and nonviral vector systems. Due to safety concerns and the difficulty of large-scale production, the utility of viral vectors is limited, so an easy-to-prepare nonviral delivery system without immunogenicity and other safety problems has been given more attention.4,5 Nonviral vectors can be grouped into three categories: cationic lipids, cationic polymers, and peptides. The structure of cationic lipid molecules generally includes a hydrophobic component determining the fluidity and the stability of the liposome, a hydrophilic cationic domain, allowing interactions with negatively charged DNA, and a linker connecting these two.5 There are two major types of hydrophobic components: aliphatic chains and cholesterolbased derivatives.6 Cholesterol-based cationic lipids have been well characterized as a delivery system because of biocompatibility and less toxicity than other cationic lipids.7,8 Numerous preclinical studies have utilized cholesterol-based cationic lipid for the delivery of plasmid DNA, antisense oligonucleotides, and siRNA.9 Conjugation of cholesterol derivatives with siRNA has been shown to enhance cellular uptake of siRNA10 and improve gene silencing in the liver.11 Furthermore, cholesterolbased cationic lipids exhibited enhanced transfection in vitro and resistance to serum-induced decrease in efficiency.12 Serum © 2012 American Chemical Society

has been reported to exert its inhibitory effect by binding serum proteins to the complex of cationic lipids and DNA (lipoplexes), which leads to structural reorganization, aggregation, and dissociation of the complexes. Thus, it is of interest to develop delivery systems that can resist serum-induced aggregation and transfer genes in the presence of serum efficiently.4,13 Recently, we also developed a novel cholesterol derived cationic lipid (CHOL-E) having an amine head, ether linker, and cholesterol tail.14 It showed low toxicity and efficient delivery of plasmid DNA irrespective of the presence of serum. However, the mechanism of effectiveness and serum-resistance of these cholesterol-based cationic lipids remains unclear. Several studies have suggested that transfection efficiency depends on endocytosis pathway.15 Indeed, some efforts have aimed at avoiding processing of the carriers along the degradative clathrin-mediated pathway toward lysosomes.16−18 Plasma membrane cholesterol is also known to play a critical role in the transfection efficiency of lipoplexes, and depletion of membrane cholesterol decreased transfection efficiency.19−21 Since the design of the successful nonviral gene vector requires an understanding of the mechanism by which it interacts with the target cells,22 we aimed to investigate the exact mechanism of gene transfection by cholesterol-based cationic lipids. We used several inhibitors of intracellular Received: Revised: Accepted: Published: 3579

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trafficking processes and additional cholesterol to study their effects on the transfection efficiency and roles in the transfection. Furthermore, we found that CHOL-E was resistant to cholesterol depletion and could have some role in the plasma membrane during transfection.

onto 24-well plates 2 days prior to the experiment. CHOL-E (3.5 μg) or DT (3.5 μg) was diluted to 50 μL of transfection optimizing medium (TOM, Welgene) and mixed with pCMVTnT-GFP or pcDNA-Luc plasmid DNA (0.7 μg) in 50 μL of TOM. The lipoplexes, consisting of liposomes and DNA, were incubated for 10 min at room temperature and then added to each well. Lipoplexes were removed 4 h after transfection, and the transfection media were changed with fresh media containing 10% FBS. The transfection efficiency was determined by means of an LMax II 384 luminometer (Molecular Devices, California, USA) using a luciferase assay kit (Promega, Wisconsin, USA) and a PIERCE BCA protein assay kit (Rockford, Illinois, USA). GFP expression was observed on a Nikon ECLIPSE TE300 fluorescence microscope 24 h after the transfection (Nikon, Tokyo, Japan). 2.5. Colocalization Studies. COS-7 cells were seeded onto 12 mm coverslips at a density of 6 × 104 cells in 24 well plates for 24 h. Alexa Fluor 488 transferrin (5 μg/mL, Molecular probe, Leiden, Netherlands) and Cy3 labeled CHOL-E and DT lipoplexes were added to the cells for 1 h to allow simultaneous uptake. For the study of trafficking through endosome, COS-7 cells were transfected for 3 h with Cy3 labeled CHOL-E. And then cells were fixed with 500 μL of 4% paraformaldehyde for 10 min at room temperature. Fixed cells were stained using primary antibodies against EEA1 and LAMP1 (molecular probe) followed by secondary antibodies conjugated to Alexa Fluor 488. Nuclei were visualized with TOPRO 3 (Molecular probe). Fluorescence images were taken via confocal microscopy (Leica TCS SP2, Wetzlar, Germany) in sequential mode to eliminate emission cross-talk between the various dyes. 2.6. Viability Assay. Trypan blue exclusion test and MTT assay were done to measure cell viability. Viable cells were determined with 0.2% trypan blue using a hemocytometer under phase microscopy. The medium added was 400 μL per well, and 20 μL of 1.25 mg/mL solution of MTT (SigmaAldrich) in PBS was added to each well. After incubation for 2 h at 37 °C, the medium was removed, and 250 μL of DMSO (Sigma-Aldrich) was added. Absorbance was measured at 550 nm using a microplate reader (Tecan, Austria), and cell viability was expressed as percentage relative to untreated control cells. 2.7. Size Measurement. Lipoplex size at a charge ratio of 1:5 = DNA/liposome in 100 μL of TOM was measured using dynamic light scattering analysis by a Zetasizer Nano ZS (Malvern, WR, U.K.). 2.8. Statistical Analyses. Results were presented as the mean ± SD of more than three experiments. The statistical significance of the difference between groups was evaluated by one way ANOVA and Tukey’s posthoc test. Asterisks (p < 0.05) indicate statistically significant differences.

2. MATERIALS AND METHODS 2.1. Cationic Lipid Preparation. CHOL-E, having an amine head, ether linker, and cholesterol tail, was synthesized, and its liposome preparation was carried out according to a method previously reported.14 DOTAP (DT) was purchased from Roche (Mannheim, Germany), Lipofectamine (LFA) from Invitrogen (New York, USA), and 1,2-dioleoyl-sn-glycero3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) ammonium salt (NBD-DOPE) from Avanti Polar Lipids (Alabaster, USA). For cellular internalization and uptake experiments, a 1% weight ratio of NBD-DOPE in total lipid concentration was added to the CHOL-E (NBD-CHOL-E) and DOTAP (NBD-DT) liposome formulation. 2.2. Inhibition of the Endocytic Pathway and Depletion of Cell Membrane Cholesterol. African green monkey kidney (COS-7) cells (Korean Cell Line Bank, Seoul, Korea) were incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 10 mg/mL penicillin/streptomycin (Gibco, New York, USA). COS-7 cells were seeded 6 × 104 onto 24-well plates 2 days prior to the experiment. Cells were treated with endocytic pathway inhibitors for each time point before lipoplexs were added, unless stated otherwise: chlorpromazine (8 μg/mL) or filipin III (1 μg/mL) or genistein (150 μM) for 1 h, sucrose (800 μM) or amiloride (200 μM) or chloroquine (250 μM) or H89 (50 μM) or bafilomycin (25 μM) for 30 min, and methyl-β-cyclodextrin (MBC, 9 mM) for 15 min at 37 °C in fresh serum free medium. For cholesterol depletion, cells were pretreated with MBC (9 mM) for 15 min at 37 °C. After the depletion, the cells were incubated with exogeneous addition of free cholesterol (cholesterol: MBC complexes) or CHOL-E in the presence of lovastatin (1 μg/mL). Lovastatin, an inhibitor of 3-hydroxy3-methylglutaryl-CoA reductase, was added, during both cholesterol depletion and subsequent transfection, to prevent refill of the depleted plasma membrane cholesterol pool by newly synthesized cholesterol. All reagents were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). 2.3. Cellular Internalization and Uptake of CHOL-E and DT Lipoplexes. To observe cellular internalization of lipoplexes, COS-7 cells were transfected for 2 h with Cy3labeled DNA using CHOL-E and DT in 4 or 37 °C. The plasmid was covalently labeled with the Label IT Cy3 nucleic acid labeling kit (Mirus, New Jersey, USA). For cellular uptake, COS-7 cells were transfected with NBD-CHOL-E and NBDDT lipoplex for 1 h. Fluorescence images were taken via confocal microscopy (Leica TCS SP2, Wetzlar, Germany). For flow cytometry, cells were then harvested by treatment with trypsin/EDTA solution (Gibco) containing 75 mM sodium azide. Analysis was performed using a Canto II flow cytometry instrument (Becton-Dickinson, New Jersey, USA). 2.4. Gene Transfection Efficiency of Complexes. The pcDNA-Luc and pCMVTnT-GFP were obtained from Welgene (Daegu, Korea). These plasmids were amplified with Escherichia coli DH-5α strain and were purified by the Plasmid Maxi prep Kit (Qiagen, Hilden, Germany) according to the manufacture’s instructions. COS-7 cells were seeded 6 × 104

3. RESULTS 3.1. Transfection Efficiency of CHOL-E. The transfection efficiency of CHOL-E was compared with commercial cationic lipids using GFP expression and luciferase assay 24 h after the transfection. CHOL-E showed 11 times enhancement of transfection efficiency as compared to DT and 1.8 times compared to LFA in COS-7 cells. Even in medium containing 10% FBS, CHOL-E had significantly higher transfection efficiency than others (Figure S1 in the Supporting Information). 3.2. Endocytosis Pathway of CHOL-E-and DT Lipoplex. To demonstrate the endocytosis of lipoplex, COS-7 cells were 3580

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Figure 1. Cellular uptake of CHOL-E and DT lipoplex. (A) COS-7 cells were pretreated with endocytosis inhibitor and transfected with NBDCHOL-E and NBD-DT lipoplex for 1 h. After the transfection, intracellular uptake of lipoplexe was analyzed by flow cytometry. Values are given as means ± SD of triplicates. * p < 0.05 compared with control. (B) Colocalization of CHOL-E lipoplex with transferrin, a known clathrin pathway marker. COS-7 cells were incubated with Alexa-Fluor 488 labeled transferrin (green) and CHOL-E and DT lipoplex containing Cy3-labeled DNA (red). Pictures were taken by confocal microscope 1 h after the transfection. Scale bar: CHOL-E 5 um, DT 10 μm.

Figure 2. Effect of endocytosis inhibitors on the transfection efficiency of CHOL-E and DT lipoplexes in the absence of serum. Luciferase activity was determined 24 h after the transfection, and relative expression efficiencies compared to control were displayed. Values are given as means ± SD of triplicates (Am, amiloride; MBC, methyl-β-cyclodextrin; F, filipin III; G, genistein; Su, sucrose; Cp, chlorpromazine; Con, without inhibitor). * p < 0.05 compared with control.

after 1 h transfection. However, chlorpromazine, a clathrin pathway inhibitor, caused significant inhibition of the uptake of NBD-CHOL-E and NBD-DT lipoplexes (Figure 1A). COS-7 cells were incubated for 1 h with CHOL-E and DT lipoplex containing Cy3-labeled plasmid DNA (red) and transferrin labeled with AlexaFluor 488 (green) (Figure 1B). The majority of CHOL-E and DT lipoplex were colocalized with transferrin (yellow orange), a clathrin pathway marker. In the analysis of transfection efficiency using the luciferase assay, amiloride had no effect on the gene expression with

transfected for 2 h with Cy3-labeled DNA using CHOL-E and DT at 4 or 37 °C. Internalization of CHOL-E and DT was dramatically reduced following incubation at 4 °C compared to 37 °C on a confocal microscope and flow cytometry. The data suggested that cellular internalization of CHOL-E and DT occurred mainly via energy dependent endocytosis (Figure S2 in the Supporting Information). Pretreatment of COS-7 cells with filipin III, a caveolae pathway inhibitor, or amiloride, a macropinocytosis inhibitor, showed no effect on NBD-CHOL-E and NBD-DT lipoplexes 3581

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3.3. Endosome−Lysosome Trafficking of the CHOL-E Lipoplex. Since endosome−lysosome trafficking is important for the transfection efficiency of the lipoplex, lysosome associated markers were used to see the intracellular maturation of the CHOL-E lipoplex. After a 2 h treatment with Cy3labeled CHOL-E lipoplex, cells were stained by EEA1 (early endosome marker) or LAMP1 (late endosome/lysosome marker) antibody. The CHOL-E lipoplex was colocalized with EEA1 and LAMP1 (Figure 3). These confocal microscopic images were consistent with the above results from chemical inhibitor tests and pathway marker experiments, suggesting clathrin-mediated endocytosis. Because lysosomal degradation is the reason for the decreasing transfection efficiency in the clathrin-mediated endocytosis, lysosomotrophic agents were used to see the blocking of the lysosomal degradation. But the lysosomotrophic agents H89, chloroquine, and bafilomycin decreased the gene expression efficiency of CHOL-E lipoplex to 25%, 50%, and 63%, respectively (Figure 4). Destabilization of endosome by

CHOL-E and DT lipoplex. Cholesterol depletion by MBC, known to inhibit both clathrin- and caveolae-mediated endocytosis, decreased the expression of DT lipoplex and CHOL-E lipoplex (Figure 2). In order to distinguish clathrin and caveolae-mediated endocytosis, cells were pretreated with filipin III and genistein which were known to block caveolae mediated endocytosis. However, there were no significant effects on the expression of transfected plasmids. On the contrary, sucrose and chlorpromazine, inhibitors of clathrinmediated endocytosis, dramatically inhibited both CHOL-E and DT lipoplex. Consequently, the data confirms that CHOLE and DT lipoplex were internalized through clathrin-mediated endocytosis. Because CHOL-E has significant transfection efficiency in the presence of the serum, the lipoplex size was measured to analyze the effect of serum on the size and the relation between size and transfection efficiency. When the serum was added to the CHOL-E lipoplex, the particle size was increased, but DT was just slightly decreased (Table 1). Table 1. Size of Liposomes and Lipoplexes in the Absence and in the Presence of Seruma

a

liposome formulation

particle diam (nm)

liposome formulation

particle diam (nm)

CHOL-E CHOL-E/DNA CHOL-E/DNA/FBS

214 ± 7 421 ± 49 844 ± 108

DT DT/DNA DT/DNA/FBS

160 ± 23 285 ± 20 260 ± 7

Values are given as means ± SD of triplicates. Figure 4. Effect of lysosomotrophic agents on the CHOL-E lipoplex. COS-7 cells were pretreated with H89, chloroquine (Cq), or bafilomycin (Bf) and transfected with CHOL-E lipoplex. Luciferase activity was determined 24 h after the transfection, and relative expression efficiencies compared to control were displayed. Values are given as means ± SD of triplicates. * p < 0.05 compared with control.

To see the effects of serum on the lipoplex endocytosis, chemical inhibitor experiments were done in the serumcontaining medium. Amiloride and filipin III, genistein, decreased by approximately 15% in gene expression efficiency, and MBC inhibited CHOL-E transfection efficiency to a greater extent as compared to the result with the absence of the serum. Therefore, the CHOL-E lipoplex seemed internalized mainly through clathrin-mediated endocytosis, but also partially caveolae-mediated endocytosis or macropinocytosis in the presence of the serum (Figure S3 in the Supporting Information).

lysomotrophic agents did not enhance the transfection efficiency of CHOL-E, and transfection with CHOL-E did not seem to be interfered with by endosome-lysosome trafficking. 3.4. Resistance to Cholesterol Depletion and Recovery of Transfection. The transfection efficiency of DT was

Figure 3. Intracellular processing of CHOL-E lipoplex. COS-7 cells were transfected for 2 h with Cy3-labeled DNA (red) using CHOL-E, and fixed and stained with antibodies to EEA1 and LAMP1, followed by secondary antibodies conjugated to Alexa-Fluor 488 (green). Nuclei were visualized with TO-PRO 3 (blue). After immunostaining, pictures were taken with a confocal microscope. Scale bar: 20 μm. 3582

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4. DISCUSSION In this study, we investigated the mechanism of CHOL-E from the various aspects. Several mechanisms of endocytosis are involved in DNA delivery.16 Clathrin-mediated endocytosis is the major and best characterized endocytosis pathway.23 Clathrin-mediated endocytosis is initiated by the formation of clathrin-coated pits, yielding clathrin-coated vesicles and leading to the formation of early and late endosomes, which ultimately fuse with lysosomes. Caveolae are smooth invaginations of the plasma membrane associated with caveolin-1 and enriched with cholesterol and sphingolipids. The endosome, taken up by the caveolae-mediated pathway, is known to avoid dropping in pH, and most pathogens that are internalized by caveolae can be directly transported to the Golgi and/or endoplasmic reticulum, thus avoiding normal lysosomal degradation.22,24 Macropinocytosis refers to the formation of large endocytic vesicles of irregular size and shape, generated by actin-driven invagination of the plasma membrane.25 Our results from luciferase assay, flow cytometry, and confocal microscopic observation using chemical inhibitors of these pathways clearly showed that the main transfection route of CHOL-E and DT in COS-7 is the clathrin-mediated endocytosis. However, these treatments should be analyzed with caution because chemical inhibitors usually show cell type variations and sometimes have toxicity. 17,26 To avoid misunderstanding of the effects of inhibitors, the optimal concentration and incubation time of each inhibitor were determined by MTT assay at several concentrations. All results were from the inhibition effect of chemicals, not from cytotoxicity. Although our results are consistent with the report of DT shown in A549 cells,15 there are some different results showing the macropinocytosis as the main pathway for lipoplexes.2728 This discrepancy could be caused by the different chemical compositions of carriers and the difference in cells used in observation of endocytosis.29,30 Although our results about the macropinocytosis at the aspect of cellular uptake and transfection efficiency using chemical inhibitor showed no definite involvement of macropinocytosis in CHOLE lipoplex internalization, we did not have colocalization data of CHOL-E with a macropinocytosis marker such as 70 kDa dextran. In addition, cellular uptake of macropinocytosis was most remarkable at 3 h after transfection in other reports compared with our result of 1 h after transfection.28 Therefore, further investigations about the role of macropinocytosis will clarify the precise mechanism of lipoplex endocytosis in future studies. To understand the difference in transfection efficiency despite the same endocytic pathway, we then investigated the intracellular trafficking. Since blocking of lysosomal degration, the addition of lysosomotropic agents such as H89, chloroquine, and bafilomycin during transfection usually favors transfection. This effect may be due to the destabilization of the endosomal/lysosomal membranes and/or to a slowdown in the translocation of DNA to the lysosomes. In this study, it seems that endosome−lysosome trafficking is not a hurdle for efficient gene delivery using CHOL-E because pretreatment of lysosomotropic agents decreased transfection efficiency. Serikawa et al. also showed blocking of lysosomal degradation may decrease the transfection because it is necessary for lipoplex to be degraded in the endosome or lysosome and then released to the cytoplasm.31 Since endosome−lysosome trafficking is inevitable during clathrin-mediated endocytosis, noninterfer-

much more inhibited by MBC pretreatment compared to CHOL-E in the chemical inhibitor experiment (Figure 2). Since cholesterol is a component of the cell membrane, the effects of free cholesterol and CHOL-E on transfection efficiency were compared to elucidate the effects of CHOL-E on the cell membrane. We used MBC to deplete the membrane cholesterol and lovastatin to inhibit the synthesis of cholesterol in the cell. Under this condition, CHOL-E was resistant to cholesterol depletion compared to DT (Figure 5A). Free

Figure 5. CHOL-E showed resistance to cholesterol depletion, and addition of free cholesterol and CHOL-E recovered the transfection efficiency of DT from cholesterol depletion. For cholesterol depletion, COS-7 cells were pretreated with 9 mM MBC and lovastatin (1 μg/ μL) for 15 min. Transfection efficiencies with CHOL-E and DT were measured by luciferase assay (A). After cholesterol depletion, the cells were incubated with exogeneous addition of free cholesterol from 25 to 200 μg/μL or CHOL-E from 12.5 to 50 μg/μL and transfected with DT lipoplex (B). Luciferase activity was determined 24 h after the transfection, and relative expression efficiencies compared to control were displayed. Values are given as means ± SD of triplicates. * p < 0.05 compared with lovastatin treated control.

cholesterol addition after cholesterol depletion enhanced the transfection efficiency of the DT lipoplex in a dose dependent manner (Figure 5B). Upon the addition of 200 μg/μL free cholesterol, there was a more than two times enhancement of transfection efficiency as compared to the control group. The addition of 12.5−50 μg/μL CHOL-E, the range without cellular toxicity (data known shown), prior to DT lipoplex transfection also showed the increase of transfection efficiency, suggesting the role of CHOL-E on the cell membrane (Figure 5B). 3583

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mediated endocytosis.43−45 The addition of serum increased the size of the CHOL-E lipoplex, over 500 nm, but the size of DT did not change much. Therefore, CHOL-E may use more than one pathway in the presence of serum, taking advantage of alternative trafficking processes. In conclusion, this study showed that CHOL-E is internalized mainly through clathrin-mediated endocytosis with no interference from endosome-lysosome trafficking and may change the membrane composition more optimally for transfection. CHOL-E showed good transfection efficiency in the presence of serum because of increasing size, which allows taking advantage of multiple endocytosis pathways.

ence from endosome−lysosome action may be the one reason for the superior transfection efficiency of CHOL-E compared to other lipids. There are several studies showing that the decreased transfection efficiency by MBC was caused by the depletion of cholesterol from the plasma membrane, and the addition of free cholesterol restored the transfection efficiency.19−21 Furthermore, cholesterol is related to the rigidity and stability of the bilayer structure of the cell membrane, as well as its endogenous biodegradability and fusion activity.5,32 To investigate the contribution of cholesterol and CHOL-E to gene transfection efficiency, cells were pretreated with free cholesterol or CHOL-E prior to the transfection experiment. The DT lipoplex showed significant enhancement of transfection efficiency compared to the control group by pretreatment of free cholesterol and CHOL-E. This result implies that the CHOL-E may influence the status of the cell membrane cholesterol composition like free cholesterol, which can be one cause of the superior ability of this material. If CHOL-E had similar effects with free cholesterol on the cell membrane, it can be an explanation for the little effects of free cholesterol on the CHOL-E lipoplex transfection efficiency. Although our results showed that the main mechanism of endocytosis in the CHOL-E lipopolex is the temperature dependent clathrin pathway, a recent report33,34 demonstrated that temperature independent membrane fusion is important for cellular uptake and transfection efficiency in a cholesterolbased liposome compared with a noncholesterol based liposome. Furthermore, gradual replacing of DOPE with cholesterol in DC-Chol-DOPE increased the transfection efficiency by increasing the fusion of lipoplex with the cell membrane.34 Our result using CHOL-E also showed higher temperature independent uptake compared with DT (Figure S2 in the Supporting Information),33 and this might be one explanation for the enhanced transfection efficiency of CHOLE. CHOL-E has shown high transfection efficiency even in the presence of serum as compared to LFA and DT. This is consistent with the previous report that DC-Chol, a well-known cholesterol derived cationic lipid, has better transfection efficiency in the presence of the serum.13,18 The complete mechanism of the serum compatibility of CHOL-E is not entirely explained; however, the slight responses to amiloride, filipin III, and genistein in the serum containing medium suggest that CHOL-E lipoplex may enter cells by caveolaemediated endocytosis and macropinocytosis in addition to the clathrin-mediated endocytosis in the presence of serum. Several studies have reported that the nanoparticle associated “protein corona” from plasma or other bodily fluids is important in the cell−nanoparticle interaction.35−37 It is also important for understanding the circulation, clearance rates, blood half-life, stability, immunogenicity, and organ biodistribution of nanoparticles.38−40 The majority of proteins surrounding nanoparticles were apolipoproteins, immune response-related proteins, immunoglobulins, acute-phase proteins, coagulationrelated proteins, and cell adhesion proteins.35,36 Serum proteins such as albumin and heparin are also known to bind to lipid membranes, causing aggregation of lipoplexes and an increase of the size.41,42 The change in endocytosis pathway may be attributable to these changes of particles. Lipoplexes approximately of 300 nm or less were supposed to enter cells basically via the clathrin-mediated endocytosis, and lipoplexes larger than 500 nm were reported to be internalized via caveolae-

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ASSOCIATED CONTENT

S Supporting Information *

Figures showing a comparison of transfection efficiency with commercial cationic lipids; the energy dependent and independent internalization of CHOL-E and DT lipoplex; and the effect of endocytosis inhibitors on the transfection efficiency of CHOL-E lipoplexes in the presence of serum. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +82-53-620-4335. Fax: +8253-651-3651. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by a National Research Foundation of Korea (NRF) grant to the Medical Research Center at Yeungnam University funded by the Korea government (MEST) (2011-0006185) and by a Basic Science Research Program funded by the Ministry of Education, Science and Technology (No. 2011-0026282). We would like to thank to Dr. Yoon Yeo and Johann H. Weidle III for critical reading of the manuscript.

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REFERENCES

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dx.doi.org/10.1021/mp300458h | Mol. Pharmaceutics 2012, 9, 3579−3585