Influence of Hydrophobic Structures on the Plasma Membrane

May 13, 2010 - The cellular membrane permeability of compounds strongly depended on both the chemical structure and the cell-cycle phase. In the G1/S ...
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Influence of Hydrophobic Structures on the Plasma Membrane Permeability of Lipidlike Molecules Kenichi Niikura,*,† Katsuyuki Nambara,‡ Takaharu Okajima,§ Yasutaka Matsuo,† and Kuniharu Ijiro*,† † Research Institute for Electronic Science (RIES), Hokkaido University, Kita21, Nishi10, Kita-Ku, Sapporo 001-0021, Japan, ‡Division of Chemistry, Graduate School of Science, Hokkaido University, and § Graduate School of Information Science and Technology, Hokkaido University, Kita14, Nishi9, Kita-Ku, Sapporo 060-0814, Japan

Received March 15, 2010. Revised Manuscript Received April 26, 2010 A series of FITC-labeled hydrophobic molecules (1-8) were prepared, and their cellular uptakes have been investigated using cell-cycle-synchronized HeLa cells. The cellular membrane permeability of compounds strongly depended on both the chemical structure and the cell-cycle phase. In the G1/S phase, branched hydrocarbon-containing 3 and cis-olefin-containing 2 and 8 were efficiently internalized into cells by passive diffusion. In contrast, linear alkyl chain-containing 1 and 7 were retained on the membrane without rapid internalization. In the M phase, rapid permeation was suppressed for all molecules.

Introduction The cell membrane is the outermost layer of mammalian cells and has an important function as the first barrier to drug internalization into cells. With regard to cellular uptake, along with specific transporter protein-mediated transportation, drug hydrophobicitiy is one of the most important factors in determining cell membrane permeability.1-4 A commonly employed strategy for achieving good membrane permeability is the masking of the hydrophilic groups in an active compound,5,6 which is known as a prodrug approach. Liposome or lipophilic nanoparticle carriers have often been used to suspend these hydrophobic prodrugs in aqueous solutions, and the membrane partitioning of hydrophobic drugs between the plasma membrane and these carriers has been investigated.7 However, the relationship between the chemical structure of the hydrophobic moiety in prodrugs and their cellular membrane permeability is not yet fully understood. Our aim is to explore how the hydrophobic structure of compounds affects the membrane permeability of cells. These data can be applied to provide the molecular design that can regulate the cellular membrane permeability of drugs by the modification of their hydrophobic structure. Several model systems, such as liposomes and LB films, have been developed to estimate the membrane adsorption and/or permeability *Corresponding author. E-mail: [email protected]. Phone: þ81-11-706-9344. (1) Henin, Y.; Gouyette, C.; Schwartz, O.; Debouzy, J.-C.; Neumann, J.-M.; Huynh-Dinh, T. J. Med. Chem. 1991, 34, 1830–1837. (2) Ansell, S. M.; Johnstone, S. A.; Tardi, P. G.; Lo, L.; Xie, S.; Shu, Y.; Harasym, T. O.; Harasym, N. L.; William, L.; Bermudes, D.; Liboiron, B. D.; Saad, W.; Prud’homme, R. K.; Mayer, L. D. J. Med. Chem. 2008, 51, 3288–3296. (3) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev. 1997, 23, 3–25. (4) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev. 2001, 46, 3–26. (5) Ettmayer, P.; Amidon, C.; Schwartz, G. L.; Clement, B.; Testa, B. J. Med. Chem. 2004, 47, 2392–2404. (6) Zakharian, T. Y.; Seryshev, A.; Sitharaman, B.; Gilbert, B. E.; Knight, V.; Wilson, L. J. J. Am. Chem. Soc. 2005, 127, 12508–12509. (7) Fahr, A.; van Hoogevest, P.; May, S.; Bergstrand, N.; Leigh, M. L. S. Eur. J. Pharm. Sci. 2005, 26, 251–265. (8) Xiang, T.-X. J. Membr. Biol. 1994, 140, 111–122.

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of hydrophobic molecules.8-14 In such artificial cell membrane models, the influence of chain packing and permeant size on permeability has been systematically discussed. Human intestinal epithelial cell line Caco-2 cell monolayers have been used to predict intestinal drug absorption after oral administration.15-18 However, a few studies have sought to correlate the chemical structure of hydrophobic molecules and plasma membrane permeability in single cells. We examined the effect of the cell cycle on cellular uptake because some biophysical properties of the cell membrane vary during the cell cycle. The microviscosity of the cell membrane lipid layer reaches a maximum level in the M phase and decreases in the S phase of the cell cycle, and the diffusion coefficient of the membrane lipids in the M phase is smaller by a factor of 2 to 3 than those in other cell-cycle phases.19 It is also known that phospholipid metabolism (synthesis or degradation) is regulated by the cell cycle.20 On the basis of these previous data, the cellcycle phase of cells should be considered to study the permeability of target molecules across the membrane. In this letter, we evaluate the cellular uptake of lipidlike 1-9 into HeLa cells arrested in different cell-cycle stages (G1-S (9) Peetla, C.; Stine, A.; Labhasetwar, V. Mol. Pharmaceutics 2009, 6, 1264– 1276. (10) Xiang, T.-X.; Anderson, B. D. Biophys. J. 1998, 75, 2658–2671. (11) Aagaard, T. H.; Kristensen, M. N.; Westh, P. Biophys. Chem. 2006, 119, 61–68. (12) Seydel, J. K.; Wiese, M. Drug-Membrane Interactions; Wiley-VCH: Weinheim, Germany, 2002. (13) Rowe, E. S.; Zhang, F.; Leung, T. W.; Parr, J. S.; Guy, P. T. Biochemistry 1998, 37, 2430–2440.  Merino, S.; (14) Grancelli, A.; Morros, A.; Caba~nas, M. E.; Domenech, O.; Vazquez, J. L.; Montero, M. T.; Vi~nas, M.; Hernandez-Borrell, J. Langmuir 2002, 18, 9177–9182. (15) Ouyang, H.; Morris-Natschke, S. L.; Ishaq, K. S.; Ward, P.; Liu, D.; Leonard, S.; Thakker, D. R. J. Med. Chem. 2002, 45, 2857–2866. (16) Hidalgo, I. J.; Raub, T. J.; Borchardt, R. T. Gastroenterology 1989, 96, 736–749. (17) Artursson, P.; Palm, K.; Luthman, K. Adv. Drug Delivery Rev. 1996, 22, 67–84. (18) Liu, D.-Z.; Morris-Natschke, S. L.; Kucera, L. S.; Ishaq, K. S.; Thakker, D. R. J. Pharm. Sci. 1999, 88, 1169–1174. (19) de Laat, S. W.; van der Saag, P. T.; Shinitzky, M. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 4458–4461. (20) Jackowski, S. J. Biol. Chem. 1994, 269, 3858–3867.

Published on Web 05/13/2010

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Letter Scheme 1. Chemical Structures of FITC-Labeled 1-9

boundary (G1/S) and M phase). We found that the chemical structures of lipidlike molecules greatly affected their internalization into cells in the G1/S phase, whereas in the M phase all molecules were trapped on the plasma membrane without rapid internalization. Our data indicating that the cellular uptake of molecules depends on both the chemical structure and cell cycle phase may be of benefit in the design of novel prodrugs with hydrophobic moieties.

Experimental Section Materials and Methods. All reagents were used without further purification. Dulbecco’s modified Eagle’s culture medium (DMEM) was purchased from Sigma. Fetal bovine serum, penicillin, and streptomycin were purchased from Gibco. Fluorescence and differential interference contrast (DIC) images were obtained using an Olympus FV-300 confocal laser microscope (Olympus). Flow cytometry analyses were performed with a Langmuir 2010, 26(12), 9170–9175

FACSCanto flow cytometer (Becton Dickinson). The synthesis of lipidlike molecules is described in the Supporting Information. Synchronization of HeLa Cells. Cells were arrested in the G1/S phase by a double thymidine block. HeLa cells (1.0  105 cells) were seeded onto 35 mm tissue culture dishes and cultured for 1 day in humidified 5% CO2/95% air at 37 °C. The cells were washed twice with phosphate-buffered saline (PBS, 1.0 mL), and then DMEM (1.8 mL) was added. Thymidine was added from a 20 mM stock solution to give a final concentration of 2 mM thymidine. The cells were then incubated for 18 h in the presence of thymidine (first block). Thymidine was then removed by washing twice with PBS (1.0 mL), fresh DMEM (2.0 mL) was added to the HeLa cells in the culture dishes, and the cells were cultured for a further 10 h. After culturing, the DMEM was removed from the dishes. Fresh DMEM (1.8 mL) and 20 mM thymidine (200 μL) were then added over another 17 h (second block). HeLa cells were arrested in the M phase by a double thymidine block using the same method, followed by the removal of thymidine, washing twice with PBS (1.0 mL), and further DOI: 10.1021/la101039w

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Figure 1. Histograms of cellular DNA content in (A) nontreated, (B) G1/S-phase, and (C) M-phase cells. incubation of the cells for 10 h after the addition of fresh DMEM (2.0 mL). Analysis of Synchronization. Synchronization was confirmed by flow cytometric analysis of propidium iodide-stained cells. Synchronized HeLa cells were resuspended in 70% cold ethanol (500 μL), and the cold cell suspension was pipetted and stored at -20 °C for 1 day. After centrifugation (200g, 5 min), cells were resuspended in RNase A solution and incubated in a 37 °C water bath for 30 min. Propidium iodide (PI) from a stock solution of 1 mg/mL in distilled H2O was diluted (5 μL PI/200 μL PBS), and the cells were stained for 5 min. After further centrifugation (200g, 5 min), cells were resuspended in PBS (1.0 mL). This procedure was repeated twice. Finally, cells were resuspended in PBS (500 μL) and the DNA content of the cells was analyzed by flow cytometry.

Flow Cytometric Analysis of Synchronized HeLa Cells Treated with Lipidlike 1-9. Lipidlike 1-9 dissolved in DMSO

(10 μL) were added to HeLa cells arrested in the G1/S or M phase and incubated at 37 °C for 5 min in a serum-free culture medium (Opti-MEM, 2.0 mL). The final concentration of lipidlike molecules was 1.0 μM. The cells were then washed twice with PBS (1.0 mL) to remove the lipidlike molecules not incorporated into the cells. The cells were removed from the dishes by treatment with 0.05% trypsin/EDTA at 37 °C for 3 min. After centrifugation (200g, 5 min), the fluorescence of the cells was measured by flow cytometry.

Treatment of HeLa Cells Arrested in the G1/S or M Phase with Lipidlike 1-9 for CLSM (Confocal Laser Scanning Microscopy). HeLa cells (1.0  105 cells) were seeded onto 35 mm tissue culture dishes and arrested in the G1/S or the M phase using the procedures described above. The synchronized HeLa cells for application to CLSM were then treated with lipidlike 1-9.

Results and Discussion HeLa cells were synchronized in the G1/S phase by a conventional double thymidine block.21 Flow cytometry showed that more than 80% of cells were synchronized in the G1/S phase using the double thymidine block (Figure 1B). After releasing the HeLa cells from the double thymidine block for 10 h, the cells progressed to the M phase. The arresting of the cells in the M phase has often been performed using nocodazole or colcemid.22,23 (21) Whitfield, M. L.; Zheng, L.-X.; Baldwin, A.; Ohta, T.; Hurt, M. M.; Marzluff, W. F. Mol. Cell. Biol. 2000, 20, 4188–4198. (22) Shi, X.; Zhang, H.; Paddon, H.; Lee, G.; Cao, X.; Pelech, S. Biochemistry 2006, 45, 5857–5867. (23) Zieve, G. W.; Turnbull, D.; Mullins, J. M.; McIntosh, J. R. Exp. Cell Res. 1980, 126, 397–405.

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Figure 2. Flow cytometric analysis of (A) G1/S- and (B) M-phase HeLa cells treated with 1.0 μM lipidlike 1-9 for 5 min. Data are represented as mean values ( the standard deviation (SD) of three different experiments.

However, we were concerned that the high cytotoxicity of these reagents would affect the cellular uptake of the hydrophobic compounds. However, the thymidine block method has low toxicity and the effect of the reagent is minimal, thus this method provides a suitable assay system for the comparison of uptake in different cell phases. In this approach, the population of HeLa cells synchronized in the M phase was found to consist of cells phased from late G2 to early G1 as shown in Figure 1C, where the 2n and 4n DNA contents of the cells indicated that the cells were in the early G1 phase and M phase, respectively. We prepared FITC-labeled lipidlike 1-8 that contain hydrophobic chains. FITC-labeled hexa(ethylene oxide)-appended hydrophilic compound 9 was prepared as a control. Molecules 1-4 have two C10-based hydrophobic chains containing saturated hydrocarbons (C10-linear 1), unsaturated hydrocarbons (C10-cis 2), branched hydrocarbons (C10-branch 3) and fluorocarbon chains (C10-F linear 4). Molecules 5 and 6 have oligo(ethylene glycol)-attached hydrocarbon chains. Molecules 7 and 8 have two C18-based hydrophobic chains that contain saturated (C18-linear 7) and unsaturated (C18-cis 8) hydrocarbons. These compounds dissolved in DMSO were added to HeLa cells that were arrested in the G1/S or M phase. Dynamic light scattering (DLS) measurement of the culture medium after the addition of the lipidlike molecules (1 μM) did not indicate the presence of any aggregated structures such as micelles (data not shown). After incubation for 5 min, the cells were washed with buffer and the fluorescence intensity was analyzed by flow cytometry (Figure 2). In the G1/S phase, C18-linear 7 was the most readily incorporated into cells among the tested molecules. Subsequently, C18-cis 8 and C10-branch 3 were incorporated and their relative intensities in the G1/S phase were decreased by a factor of 3.7 compared to that of C18-linear 7. Interestingly, the incorporation of C10-branch 3 was almost the same as that of C18-cis 8 and 1.9 times larger than that of C10-linear 1. This means that the structure of the hydrophobic moiety and the length of the alkyl chains are important factors in determining their cellular uptakes. The cellular uptake of C10-F linear 4 (relative intensities 36.6 for G1/S and 15.2 for the M phase) was less efficient than that of its hydrocarbon counterpart, 1 (85.3 for G1/S and 25.6 for the M phase). The calculated logarithms of the 1-octanol/water partition coefficient (log P) for the lipid moieties of 1-4 were 6.6, 6.0, 6.3, and 10.1, respectively (Supporting Information). This means that the hydrophobicity of C10-F linear 4 is the highest among the C10-based molecules with the other three having almost the same log P value. However, the amounts taken up as revealed by flow cytometry analysis were not in proportion to their hydrophobicites: the uptake of C10-branch 3 was about 2-4 times higher Langmuir 2010, 26(12), 9170–9175

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Figure 3. CLSM images of HeLa cells superposed with differential interference contrast (DIC) images in the G1/S and M phases. HeLa cells were incubated with 1.0 μM 1-9 for 5 min.

those of the other three molecules. This result indicates that not only the hydrophobicity but also the chemical structure of the lipidlike molecules is an important determinant of cellular uptake. Molecule 6, which has a tetraethylene oxide group at the terminus of the alkyl chain and hydrophilic 9, was only sparingly incorporated into the cells, suggesting that the anchoring of a hydrophobic moiety to the cell membrane is the driving force for the incorporation of the lipidlike molecules in the HeLa cells. In Langmuir 2010, 26(12), 9170–9175

M-phase cells, the incorporation showed about a 3-fold reduction compared to that in G1/S-phase cells for each lipidlike molecule; however, the order of uptake of the molecules (7 . 3, 8 > 1 > 2, 4, 5 > 6, 9) was the same in both phases. This implies that the permeability of the cellular membrane to small molecules in the M phase is higher than that in the G1/S phase. To reinforce the flow cytometric analysis, we analyzed the changes in fluorescence intensity from the cell cultures DOI: 10.1021/la101039w

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(Supporting Information, Figure 1). The fluorescence from 3 and 7 dissolved in cell cultures was reduced to 48 and 13%, respectively, in the G1/S phase and 62 and 29%, respectively, in the M phase. The control, incubated in the absence of cells, showed no reduction in fluorescence intensity. These reductions were in good agreement with the relative fluorescence intensities of molecules incorporated into cells as revealed by flow cytometry analysis. The localization of 1-9 in the HeLa cells after incubation for 5 min was observed by CLSM (Figure 3). In the G1/S phase cell, 2, 3, 5, 6, and 8 were diffused into the cytosol whereas linear 1 and 7 and fluorinated 4 were localized on the cell surface (Figure 3A). The common structural feature in the rapidly diffusing molecules is the presence of a bulky alkyl chain, such as a cis-olefin (bent structure) or a branched methyl group, so they are energetically more unfavorable than linear molecules when inserted into the packed lipid bilayer. This would accelerate the diffusion across the membrane. We consider that the energetically favorable interaction between the cell membrane and linear-type lipids, such as 7, would lead to a higher accumulation in plasma membranes compared to that of cis-olefin-containing 8 (Figure 2). In particular, because the incorporated amount of C10-branch 3 was larger than that of C10-cis 2 (Figure 2), we concluded that a branched alkyl chain is an effective hydrophobic moiety for the promotion of molecular diffusion into cells over the cis-olefin structure. In the M-phase cells, all molecules were localized in the membrane. These results support the idea that the lipid membrane in the G1/S phase is loosely packed whereas that in the M phase is tightly packed and affords a stronger barrier to internalization. This conclusion is consistent with a previous report that the diffusion coefficient of lipids reaches a minimum in the M phase and then increases during the G1 phase.19 We focused on the high permeability of C10-branch 3 and monitored the time course of the cellular uptake of 3 for up to 1 h in both the G1/S and M phases (Figure 4). Although the rapid internalization of 3 was blocked by the surface membrane in the M phase, it was slowly internalized and distributed into the cytosol over a 1 h period (Figure 4A). The uptake ratio (slope of the time course) in the G1/S phase was about twice that in the M phase, and the level of uptake in the G1/S phase after 1 h was 3 times that in the M phase. A previous report indicates that endocytosis is shut down in the M phase;24 however, this data shows that the passive diffusion of 3 can occur even in the M phase. In fact, CLSM images support the passive diffusion of 3 into cells because it was observed to diffuse uniformly into cytosol and no endosomal dots in the both phases, which are often seen in association with the energy-dependent endosomal pathway,25,26 were observed (Figure 4B). Then, we examined the effects of temperature on the uptake of G1/S-phase permeable 2, 3, 5, 6, and 8 into the cells. It is known that endocytosis is temperaturedependent and is considerably inhibited at 4 °C compared to that at 37 °C. When HeLa cells in the G1/S and M phases were incubated with these molecules at 4 °C, no significant decrease in uptake was observed for any molecule (Figure 5). This means that the diffusion of the lipidlike molecules does not proceed via an energy-dependent, protein-mediated pathway, suggesting that passive diffusion is the predominant pathway for the internaliza(24) Warren, G. Annu. Rev. Biochem. 1993, 323–348. (25) Duan, H.; Nie, S. J. Am. Chem. Soc. 2007, 129, 3333–3338. (26) Negishi, Y.; Omata, D.; Iijima, H.; Takabayashi, Y.; Suzuki, K.; Endo, Y.; Suzuki, R.; Maruyama, K.; Nomizu, M.; Aramaki, Y. Mol. Pharmaceutics 2010, 7, 217–226. (27) Sakata, N.; Stoops, J. D.; Dixon, J. L. J. Biol. Chem. 1999, 274, 17068– 17074.

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Figure 4. (A) Time courses of the cellular uptake of 3 in the G1/S and M phases. Data are represented as mean values ( SD of three different experiments. (B) CLSM images of HeLa cells superposed with DIC images after incubation for 1 h. CLSM and DIC images of HeLa cells after the incubation of 1.0 μM 3 for 1 h in the G1/S and M phases.

Figure 5. Flow cytometric analysis of (A) G1/S- and (B) M-phase HeLa cells treated with 1.0 μM lipidlike 2, 3, 5, 6, and 8 for 5 min at 37 and 4 °C. Data are represented as mean values ( SD of three different experiments.

tion of these molecules. Finally, we checked the stability of C10branch 3 in rabbit reticulocyte lysate (RRL, Promega), which contains cellular components in the cytosol.27 MALDI-TOF mass spectrometry revealed that the incubation of C10-branch Langmuir 2010, 26(12), 9170–9175

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3 in RRL did not result in any degradation (Supporting Information, Figure 2). This means that the rapid diffusion of C10-branch 3 is not due to the specific degradation in the cytosol.

novel basis for the design of prodrugs to promote membrane permeability and also offer insights into the cell-cycle dependency of drug actions.

Conclusions

Acknowledgment. This work was supported by an Industrial Technology Research Grant Program in 2006 from the New Energy and Industrial Technology Development Organization (NEDO) of Japan and Basic Research Programs CREST Type, “Novel Engineering of Hierarchically Structured Biomimetic Surfaces” of JST (Japan Science and Technology Agency). The analysis of CLS microscopy was carried out at the OPEN FACILITY, Hokkaido University Sousei Hall and HINTS.

We have investigated the influence of the chemical structure on the cellular uptake of FITC-labeled hydrophobic molecules. C10branch 3 and cis-olefin-containing 2 and 8 were rapidly taken up into cells through passive diffusion in the G1/S phase, whereas linear alkyl-chain-containing 1 and 7 were distributed on the cell surface and fluorinated 4 was noticeably localized on the cell membrane. We found that branched alkyl-chain-tethered 3 was the most efficient in permeating the cell membrane barrier. In the M phase, these molecules were all localized on the plasma membrane without rapid internalization. Our findings offer a

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Supporting Information Available: Materials and methods. This material is available free of charge via the Internet at http://pubs.acs.org.

DOI: 10.1021/la101039w

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