Cellular Uptake Mechanism of Molecular Umbrella - Bioconjugate

Nov 17, 2009 - Molecular umbrella provided a promising avenue for the design of the intracellular delivery of hydrophilic therapeutic agents. However,...
0 downloads 0 Views 4MB Size
Download PDF
Bioconjugate Chem. 2009, 20, 2311–2316

2311

Cellular Uptake Mechanism of Molecular Umbrella Dongtao Ge,† Dewang Wu,† Zuyong Wang,† Wei Shi,*,†,‡ Ting Wu,§ Aifeng Zhang,† Shimin Hong,† Jun Wang,† Ye Zhang,§ and Lei Ren*,†,‡ Department of Biomaterials/Biomedical Engineering Research Center, College of Materials, State Key Laboratory for Physical Chemistry of Solid Surfaces, and Department of Preclinical Medicine, Medical College, Xiamen University, Xiamen 361005, China. Received July 11, 2009; Revised Manuscript Received September 23, 2009

Molecular umbrella provided a promising avenue for the design of the intracellular delivery of hydrophilic therapeutic agents. However, the limited understanding of its cellular uptake would be a roadblock to its effective application. Herein, we investigate the ability and mechanism of cellular entry of a fluorescently labeled diwalled molecular umbrella, which was synthesized from cholic acid, spermine, and 5-carboxyfluorescein, into Hela cells, with the extent of uptake analyzed by confocal fluorescence microscopy and flow cytometry. It is found that the as-synthesized diwalled molecular umbrella can greatly facilitate cellular uptake of hydrophilic agent, 5-carboxyfluorescein. In Vitro experiments with diffuse marker, endocytic marker, and inhibitors suggested that several distinct uptake pathways (e.g., passive diffuse, clathrin-mediated endocytosis, and caveolae/lipid-raft-dependent endocytosis) are involved in the internalization of diwalled molecular umbrella. These results, together with its low toxicity and good biocompatibility, thus demonstrate the suitability of molecular umbrella for application as vectors in drug delivery systems.

INTRODUCTION Cellular internalization of hydrophilic therapeutic agents such as anticancer drugs, proteins, and nucleic acids is still a challenge because the strongly hydrophobic interior of the plasma membrane inhibits the entry of these molecules (1, 2). Therefore, intracellular delivery of these pharmaceuticals relies on delivery systems that allow improved transport across the cell membrane. A novel drug delivery vector, molecular umbrella, devised by Regen’s group (3) in 1996, has been extensively studied for its potential to transport across the cell membrane. The classical molecular umbrella was composed of two facially amphiphilic units (i.e., rigid units having a hydrophobic and a hydrophilic face) and a central scaffold bearing a bioactive agent (3, 4). When the hydrophilic agent that attached to a molecular umbrella is immersed in an aqueous environment, an exposed conformation is favored such that intramolecular hydrophobic interactions are maximized and the external face of each wall is hydrated. In contrast, in a hydrophobic environment such as a lipid bilayer, the umbrella is closed on the compound in a shielded conformation so that the hydrophilic faces of the sterols now interact with the compound while the hydrophobic faces interact with the lipid tails (3-5). So far, many biologically active agents, such as glutathione (6), antisense oligonucleotides (7), and ATP (8), have been reported to be bound to the molecular umbrellas, and these agents were transported across the synthetic phospholipid bilayers by passive diffusion under the assistance of molecular umbrella. Recently, an important question for molecular umbrellas as drug carriers, i.e., whether molecular umbrellas are capable of crossing the plasma membrane of living cells, was sought by Mehiri et al. (9). Using * Corresponding authors. W. Shi and L. Ren, Department of Biomaterials/Biomedical Engineering Research Center, College of Materials,XiamenUniversity,Xiamen361005,China.Tel.:+865922188502 (W. Shi); +865922188530 (L. Ren). Fax: +865922185299. E-mail: [email protected] (W. Shi); [email protected] (L. Ren). † College of Materials. ‡ State Key Laboratory for Physical Chemistry of Solid Surfaces. § Medical College.

confocal microscopy, they found that fluorescently labeled molecular umbrellas could enter live Hela cells and the distributions of molecular umbrellas were throughout the cytoplasm and the nucleus. This finding raises the possibility that molecular umbrellas could be used as drug vectors of intracellular delivery. In order to design a new drug delivery system of a molecular umbrella for intracellular delivery more rationally, it is important to understand its cellular uptake mechanism. In general, the main routes into a cell are endocytosis, active transport, facilitated diffusion, and passive diffusion (10). Since molecular umbrella has the ability to cross liposomal membranes by passive diffusion and the cell membrane consists of phosolipid bilayer and proteins, Mehiri et al. suggested that passive diffusion should be a pathway in cellular uptake of molecular umbrella (9). However, whether there are other mechanisms that are contributing to the cellular entry of molecular umbrella is still unknown. This limited understanding of cellular uptake of the molecular umbrella has been a roadblock to its effective application. In this paper, we synthesized a fluorescently labeled diwall molecular umbrella, and evaluated the ability and mechanism of molecular umbrellas across the cell membrane. We chose cholic acid as the amphiphilic wall and spermidine as the central scaffold. The diwall molecular umbrella (N1,N3-dicholeamidospermidine) was prepared by coupling two cholic acid molecules on the spermidine. Followed by conjunction of N1,N3dicholeamidospermidine and 5-carboxyfluorescein (5-cf), 5-cf labeled molecular umbrella (5-cf-mu) was obtained. The cytotoxicity of 5-cf-mu toward Hela cells was described by MTT assays. The internalization of 5-cf-mu was tracked by confocal fluorescence microscopy (CLSM) and flow cytometry analysis (FACS). In Vitro colocation and endocytosis inhibition experiments were further used to evaluate the cellular uptake of 5-cfmu in Hela cells.

EXPERIMENTAL DETAILS Materials and Methods. Cholic acid, spermidine, and diisopropylethylamine were purchased from Acros Organic

10.1021/bc9003074  2009 American Chemical Society Published on Web 11/17/2009

2312 Bioconjugate Chem., Vol. 20, No. 12, 2009

(USA). N,N-Dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), sodium azide (NaN3), 5-carboxyfluorescein (5cf), 2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1Hbenzimidazole (Hoechst 33258), 3-hydroxy-1,2,3-benzotriazin4(3H)-one (HOObt), Nocodazole (Noc), and chlorpromazine hydrochloride (CpZ) were obtained from Sigma-Aldrich (USA). Carboxyfluorescein diacetate succinimidyl ester (CFSE), Alexa Fluor 546-transferrin, Alexa Fluor 594-Ctx B reagent, and LysoTracker Red were purchased from Molecular Probe (USA). Trypsin and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Amresco Inc. (America). Penicillin and streptomycin were purchased from Bio Basic Inc. (Canada). Fetal bovine serum (FBS) was purchased from Sangon (China). 2-deoxy-D-glucose (2-DG) and β-cyclodextrin (β-CD) were obtained from ABCR GmbH & Co. KG (Germany) and Tokyo Kasei Kogyo Co. (Japan), respectively. Hela cells were obtained from Committee on Type Culture Collection of Chinese Academy of Sciences (CTCCCAS, China). Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Invitrogen Corporation (USA). All materials used were of analytical grade and used without further purification. The glassware was thoroughly cleaned and rinsed with deionized water. HSGF 254 silica gel (150-200 µm thickness; Yantai Jiangyou Silica Gel Development Co., Shandong, China) was used for the preparative thin-layer chromatography (TLC); column chromatography employed 200-300 mesh silica gels (Sinopharm Chemical Reagent Co., Shanghai, China). Detection on TLC was made by a combination of sulfuric acid 10% in water, I2, and UV (254 and 365 nm). Melt points were recorded on an X-4 Microscopic Melting Point Apparatus (Beijing Tech Instrument Co., China). Infrared spectrum was recorded on an Avatar 360 Fourier transform infrared spectrometer (Nicolet, USA). 1H NMR was recorded on a Bruker AV-400 NMR spectrometer (Bruker, German); chemical shifts were reported in parts per million (ppm, δ). The electron spray ionization mass spectrometry (ESI-MS) was carried out on a 3200 Q TRAP instrument (Applied Biosystems, USA). Synthesis of 5-cf Labeled Molecular Umbrella. NHS Ester of Cholic Acid. To a solution of cholic acid (409 mg, 1 mmol) in dry tetrahydrofuran (8 mL) and dry acetonitrile (2 mL), NHS (127 mg, 1.1 mmol) was added. To the resulting homogeneous solution, DCC (206 mg, 1 mmol) in dry tetrahydrofuran (2 mL) was added dropwise at 10-15 °C. The mixture was stirred at 25 °C for 12 h and the precipitated N,N′dicyclohexylurea was removed by filtration. The solvent was removed under reduced pressure, and the residue was extracted with ethyl acetate (3 × 20 mL). The extract was washed successively with aqueous NaHCO3 and water. The extract was then dried over Na2SO4, and ethyl acetate was removed under reduced pressure to get crude solid. The dried white solid was purified by column chromatography [silica, ethyl acetate/ methanol (50/1, v/v)] to give the NHS ester of cholic acid as a white powder (283 mg, 60%) having Rf 0.26 [silica, ethyl acetate/methanol (50/1, v/v)] and mp 118-119 °C (lit. (11) mp 119-120 °C); IR νmax (KBr)/cm-1 3420, 1816, 1785, 1738; 1H NMR (CDCl3) δ 0.64 (s, 3 H, 18-H), 0.83 (s, 3 H, 19-H), 0.96 (d, 3 H, 21-H), 1.00-2.00 (m, 22 H, steroidal H), 2.20 (m, 2 H, 23-H), 2.78 (s, 4 H), 3.40 (m, 1 H, 3-H), 3.79 (m, 1 H, 7-H), 3.92 (m, 1 H, 12-H); ESI-MS for [C28H43NO7+Na]+ calcd 528.6, found 528.5 (100%). N1,N3-Dicholeamidospermidine. The NHS ester of cholic acid (202 mg, 0.4 mmol) was dissolved in 5 mL anhydrous N,N′dimethlformamide (DMF), and the resulting solution was added dropwise at room temperature to 1 mL of anhydrous DMF, which contained 29 mg (0.2 mmol) of spermidine and 100 µL (0.6 mmol) diisopropylethylamine. The resulting homogeneous

Ge et al.

solution was stirred for 4 h at room temperature, and the reaction mixture was poured into 50 mL of saturated aqueous solution of NaHCO3. The precipitate was collected through centrifugation and purified by column chromatography [silica, CHCl3/CH3OH/ NH4OH, 65/25/4, v/v/v] to afford 113 mg (60%) of N1,N3dicholeamidospermidine having Rf 0.63 [silica, CHCl3/CH3OH/ NH4OH, 65/25/4, v/v/v] and mp 163-165 °C; 1H NMR (CD3OD) δ 0.67 (s, 6 H), 0.88 (s, 6 H), 0.99 (d, 6 H), 1.1-2. 0 (m, 50 H), 2.23 (m, 4 H), 2.98 (m, 4H), 3.17 (m, 2 H), 3.25 (m, 4 H), 3.76 (br s, 2 H), 3.92 (br s, 2 H); ESI-MS for [C55H95O8N3+H]+ calcd 926.7, found 926.3 (100%). 5-cf Labeled Molecular Umbrella. To a solution of 5-cf (94 mg, 0.25 mmol) and HOObt (41 mg, 0.25 mmol) in 5 mL of anhydrous DMF, 52 mg (0.25 mmol) of DCC was added. The homogeneous solution was stirred at room temperature for 24 h in dark. The precipitated dicyclohexylurea was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting yellow solid was dried under reduced pressure to give the triazinyl ester (166 mg). To a stirred solution of N1,N3-dicholeamidospermidine (102 mg, 0.11 mmol) and the triazinyl ester (55 mg, 0.11 mmol) in 1.0 mL of anhydrous DMF, 62 µL (0.44 mmol) of triethylamine was added. The mixture was then stirred at room temperature for 48 h in dark. The solvent was removed under reduced pressure at 45 °C. The crude product was washed with water, then dissolved in little methanol and purified by preparative TLC [silica, CHCl3/CH3OH/NH4OH, 20/10/3 (v/v/v)] to give 35 mg (25%) of 5-cf labeled molecular umbrella (5-cf-mu) having Rf 0.36 [silica, CHCl3/CH3OH/NH4OH, 20/10/3 (v/v/v)] and 1H NMR (CD3OD) δ 8.03 (m, 1 H), 7.83 (m, 1 H), 7.34 (d, 1 H), 6.73 (s, 2 H), 6.65 (s, 2 H), 6.59 (m, 2 H), 3.98 (m, 2 H), 3.82 (s, 2 H), 3.4-3.7 (m, 8 H), 3.17 (m, 2 H), 2.2 (m, 4 H), 1.1-2.0 (m, 62 H), 0.74 (s, 6 H). ESI-MS for [C76H105N3O14+Na]+ calcd 1306.7, found 1306.6 (100%). Cells and Cell Cultures. Hela cells were cultured as exponentially growing subconfluent monolayers on 100 mm plates in DMEM supplemented with 10% (v/v) FBS, penicillin (100 units/mL), and streptomycin (100 µg/mL) in a humidified incubator containing 5% CO2 atmosphere. MTT Cell Viability Assays. The MTT assay was used to determine the toxicity of 5-cf-mu toward Hela cells. Hela cells were seeded in 96-well plate at a density of 1 × 104 cells/well and cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C. The cells were allowed to adhere to the well bottom upon overnight incubation. After that, the medium was removed and the cells were incubated with the 5-cf-mu at concentration of 25, 50, 100, and 200 µM, respectively. At the designated time (24 h), the supernatant of each well was removed and the cells were washed twice by PBS. Twenty microliters of MTT solution (5 mg/mL in PBS) and 100 µL medium were then introduced. After 4 h incubation, the formed precipitates were solubilized by 100 µL dimethyl sulfoxide (DMSO). Absorbance intensity was then measured by the microplate reader (model 680, Bio-RAD, USA) at 490 nm with a reference wavelength of 620 nm. Cell viability was expressed by the following equation: cell availability (%) )

Abssample × 100% Abscontrol

(1)

The results are given as relative value to the negative control in percent, whereas the negative control is set to be 100% viable. Flow Cytometry. To analyze the internalization of fluorochrome-labeled molecular umbrellas by FACS, exponentially growing Hela cells were dissociated with 0.25% trypsin. 6 × 105 cells were plated and cultured overnight on 60 mm dishes. The culture medium was discarded, and the cells were washed with PBS (pH 7.2). Then, PBS was discarded, and the cell

Cellular Uptake Mechanism of Molecular Umbrella

Bioconjugate Chem., Vol. 20, No. 12, 2009 2313

monolayers were incubated with different concentrations of 5-cfmu for 1 h at 37 °C to analyze dose-response of 5-cf-mu uptake. To examine the effects of time, the cells were incubated with 10 µM of 5-cf-mu at different incubation times. To investigate the cellular uptake mechanism of 5-cf-mu, the incubations of Hela cells were carried out as follows: (1) Cells were kept at 4 °C for 1 h, instead of the regular 37 °C condition. (2) For the ATP-depletion studies, the cells were preincubated in PBS supplemented with NaN3 (50 mM) and 2-DG (0.5%) for 1 h at 37 °C. (3) For cholesterol depletion assay, the cells were pretreated with β-CD (10 µM) in PBS for 1 h at 37 °C. (4) The inhibition of clathrin- and caveolae/lipid raft-mediated endocytosis were carried out by preincubating cells with CpZ (30 µM) and Noc (10 µM) in PBS for 1 h at 37 °C. Once treatments were complete, 5-cf-mu (10 µM) was added and cells were incubated for another 1 h. The cells were then resuspended in ice-cold PBS buffer solution, and the fluorescent cells with 5-cf-mu were then measured from 2 × 104 cells by using an EPICS XL flow cytometer (Beckman Coulter, USA) in the FL1 channel. Data analysis was performed with EPICS XL flow cytometer software, and the cellular uptake of 5-cf-mu was expressed by the following equation: cellular uptake (%) )

CFDi × 100% CFD37

Scheme 1. Structures of Cholic Acid and Spermidine

Scheme 2. Synthetic Route to 5-cf Labeled Molecular Umbrella

(2)

where, CFDi and CFD37 represent the mean value of cell fluorescence distribution for each incubation condition and the normal condition (37 °C), respectively. Confocal Laser Scanning Microscope. To investigate the colocalization of 5-cf-mu and LysoTracker Red, 5-cf-mu (10 µM) was added and incubated with Hela cells (5 × 105) in a 30 mm cell culture coverslip for 1 h. After washing away the extracellular 5-cf-mu with PBS, cells were then treated with LysoTracker Red for 15 min for lysosome staining. For investigation of endocytosis, Hela cells (5 × 105/30 mm cell culture coverslip) were incubated with 5-cf-mu (10 µM) in a serum-free medium, together with CFSE (10 µM), Alexa Fluor 546-transferrin (50 µg/mL), and Alexa Fluor 594-Ctx B (10 µg/ mL) for 5 h at 37 °C, respectively. Extracellularly bound 5-cfmu was removed by washing three times with PBS. After fixing with paraformaldehyde (4% in PBS) for 30 min and subsequently washing twice with PBS, the stained cells were finally observed with CLSM (Fluoview FV1000, Olympus, Japan) equipped with a Plan-Apochromat 60 × 0.7 NA lens (λex, 488 nm, and λem, 520 nm, for 5-cf-mu; λex, 577 nm, and λem, 590 nm, for LysoTracker Red; λex, 488 nm, and λem, 530 nm, for CFSE; λex, 556 nm, and λem, 573 nm, for Alexa Fluor 546; λex, 590 nm, and λem, 617 nm, for Alexa Fluor 594). Images were captured by multitracking to avoid bleedthrough between the fluorophores and further processed and analyzed with Olympus CLSM software.

first activated with NHS to afford the corresponding NHS ester of cholic acid and then coupled to both terminal amino groups of spermidine to give the diwalled molecular umbrella. 5-cf with high hydrophilicity was selected as a fluorescent label because it was difficult to transport across the biological membrane and because its green fluorescence could be easily detected (14). The carboxyl group of 5-cf was preactivated with DCC/HOObt and then coupled to the secondary amino group of spermidine on the molecular umbrella. The synthetic route of 5-cf labeled molecular umbrella was shown in Scheme 2. Cytotoxicity of 5-cf-mu. To verify that molecular umbrellas could be used for drug delivery vectors, it is important to gain information about its toxicity toward the cells. Here, the cytotoxicity of 5-cf-mu was evaluated in Hela cells by the MTT assay. The viability of Hela cells after 24 and 48 h treatment with 5-cf-mu at concentrations ranging from 50 to 200 µM was illustrated in Figure 1. 5-cf-mu exhibited low toxicity to Hela cells at concentrations as high as 200 µM following 24 h incubation. Although the concentration of 5-cf-mu from 50 to 200 µM exhibited some inhibition to cells after 48 h incubation, 5-cf-mu showed no toxicity to Hela cells at 25 µM even after 48 h. The MTT assay results thus indicated that the molecular umbrella was a good biocompatible vector.

RESULTS AND DISCUSSION Design and Synthesis of 5-cf Labeled Molecular Umbrella. Molecular umbrellas are synthesized from materials that are potentially biocompatible. Generally, cholic acid and spermidine are selected as amphiphilic wall and central scaffold, respectively (12, 13). Cholic acid, a major component of bile acids, has a sterol nucleus that presents a hydroxylated, hydrophilic face and also a face that is all-hydrocarbon and lipophilic. Spermidine, which is also found in mammalian cells, has terminal amino groups that can be used for wall attachment, and a secondary amino group can serve as a handle to bind a cargo. Their structures were shown in Scheme 1. Here, we focus on the diwalled molecular umbrella, which has amphiphilic walls for effective shielding. Cholic acid was

Figure 1. Cytotoxicity of 5-cf-mu toward Hela cells. The cell viability following 24 and 48 h exposure of Hela cells to 5-cf-mu was quantified by the colorimetric MTT assay. All samples were tested in triplicate, and results are given as the average values ( standard.

2314 Bioconjugate Chem., Vol. 20, No. 12, 2009

Ge et al.

Figure 4. Colocalization (yellow dots) with carboxyfluorescein diacetate succinimidyl esters (CFSE, red) with 5-cf-mu (green) in Hela cells.

Figure 2. (a) CLSM images for 5-cf-mu (10 µM) and 5-cf (10 µM) treated Hela cells. (b) The average fluorescence intensity of cells in (a) (n ) 6). Figure 5. Colocalization of 5-cf-mu (green) and lysosome tracker (red) in Hela cells. Overlay of the two fluorescent layers results in yellow fluorescent emission wherever overlap is complete.

Figure 3. (a) Concentration dependence by FACS analysis. Cells were exposed to 5-cf-mu at the corresponding concentration for 60 min. (b) Incubation dependence by FACS analysis. Cells were exposed to 5-cfmu (10 µM) for corresponding hours.

Cellular Uptake of 5-cf-mu. Molecular umbrellas may function as novel carriers for hydrophilic therapeutic agents which need transportation into living cells. Previously, many studies demonstrated that molecular umbrellas could transport these molecules across lipid bilayers (3-8, 15, 16). Herein, we investigated the membrane-penetrating ability of diwalled molecular umbrella into living cells. 5-cf was set as the fluorescently label molecule facilitating the detection of molecular umbrella entering into cells. As shown in Figure 2a, 5-cfmu (green) was demonstrated to enter into Hela cells by tomography of CLSM as lots of green dots distributed in the cytoplasm district. Note that 5-cf only entered into cells slightly. For quantitative analysis of internalization of 5-cf-mu and 5-cf, cells within the visual field were selected randomly and presented as the ellipse. The average cell fluorescence intensity of 5-cf-mu was about 4.8-fold higher than that of 5-cf (Figure 2b), indicating that molecular umbrellas can greatly facilitate cellular uptake of 5-cf. Moreover, the fluorescence information within cells with different depths indicated that most 5-cf-mu was distributed in the middle of Hela cells (data not shown).

The entrance of 5-cf-mu into cells was further confirmed by FACS. As shown in Figure 3, the cell fluorescence increased gradually along with the concentration and incubation time, respectively. It is thus suggested that the cellular uptake of 5-cfmu has the characteristics of both concentration- and incubation time-dependence. Internalization Pathways of 5-cf-mu. Different entry mechanisms may be preferred depending on the application, as the mode of entry affects cell-type specificity, the rate of internalization, and the fate of the compound once inside the cell. In order to design molecular umbrella-based drug delivery systems more rationally, it is important to understand the physiological processing of molecular umbrellas. Despite molecular umbrellas having been used as carriers of peptides, nucleotides, and oligonucleotides (7, 15, 16), the mechanism of transduction responsible for the entry of molecular umbrellas into cells is still poorly understood. In a pioneering work, Mehiri et al. (9) ascribed the cellular uptake of exogenously added molecular umbrellas to a diffusion pathway. One of the limitations of this hypothesis is based on the fact that the treatment of NaN3 and 2-deoxyglucose (to deplete ATP production) causes no significant reduction of molecular umbrella cellular uptake. However, some studies indicated that ATP depletion might specifically impair macropinocytosis process (17-19), but not the whole endocytic pathway, which is indeed subdivided into four categories: (1) clathrin-mediated endocytosis, (2) caveolae/lipid raft-mediated endocytosis, (3) macropinocytosis, and (4) phagocytosis (20). Interestingly, Mehiri et al. (9) also found no cellular uptake of molecular umbrellas at 4 °C, which is consistent with the endocytic pathway. Hence, one question might arise regarding whether the transport of molecular umbrellas across cell membranes could be due to the combination of diffusion and endocytosis. In the work reported herein, we sought to answer this question. To obtain direct visual evidence of the internalization diffusion pathway described by Mehiri et al. (9), we first cocultured 5-cf-mu with CFSE, which is a well-known marker

Cellular Uptake Mechanism of Molecular Umbrella

Bioconjugate Chem., Vol. 20, No. 12, 2009 2315

Figure 6. Colocalization of 5-cf-mu with Alexa Fluor 546-labeled transferrin (a) and Alexa Fluor 546-labeled Ctx B (b) in Hela cells, respectively. The emissions for 5-cf and Alexa Fluor 546 are green and red, respectively. Overlay of the two fluorescent layers results in yellow fluorescent emission wherever overlap is complete.

for diffusion (21, 22). It is indicated from Figure 4 that 5-cfmu (green) was partly colocalized with CFSE (red) presenting as yellow dots. Note that CFSE is distributed as a dispersion within both cytoplasm and nucleus, while the distribution of 5-cf-mu was an association between diffuse and puncta. The former is generally considered to be an indication of cellular entry via passive diffusion, while the latter implies that 5-cfmu might enter Hela cells by a fundamentally different pathway, most probably via endocytosis. It is noteworthy that the distribution of 5-cf-mu is not all the same as that of persulfated molecular umbrellas synthesized by Mehiri et al. (9), indicating that the framework of molecular umbrellas might affect their internalization pathway. The above finding inspires us to further investigate the internalization pathways of 5-cf-mu. Taking into account the fact that the cell membrane comprises not only lipid bilayers but also a lot of specific districts and proteins which might offer the cell membrane the active transport ability via endocytosis for carrying materials into cells, it is reasonable to suppose that molecular umbrellas could undergo endocytosis. To test this hypothesis, we determined whether 5-cf-mu internalized by the cells is indeed in endosomal compartments. LysoTracker Red was used to track endosomes in the cells because it has been extensively shown to partition to acidic compartments within a cell, e.g., lysosomes and endosomes (23). The CLSM analysis indicated part of 5-cf-mu (green) in the endosome (red), presented as dots (Figure 5). The FACS analysis further demonstrated that the uptake of 5-cfmu was considerably inhibited at 4 °C compared to 37 °C (Supporting Information). On this basis, we posit that endocytosis is one of internalization pathways for cellular uptake of 5-cf-mu. To further explore subcategories of endocytosis, we respectively performed colocalization experiments by using Alexa Fluor 594-labeled transferrin (a positive marker of clathrinmediated endocytosis) and Alexa Fluor 594-labeled Ctx B (a marker for caveolae/lipid-raft-mediated endocytosis) on Hela cells. It is observed from CLSM images (Figure 6a) that a part of 5-cf-mu (green) colocalized with Alexa Fluor 594-labeled transferrin (red) presented as yellow dots. On the other hand, 5-cf-mu (green) had much colocalization with Alexa Fluor 594labeled Ctx B (red), presented as yellow dots (Figure 6b). We also examined the effects of the endocytosis inhibitors on the uptake of 5-cf-mu into the cells by using four different types of chemical inhibitors: (1) NaN3/2-DG, which is a ATPdepleting agent, for macropinocytosis (17-19), (2) β-CD, which is a cholesterol-depleting agent, for caveolae/lipid raft-mediated endocytosis pathway (17, 20, 24), (3) Noc, which can disrupt the intracellular vesicles transport along microtubule, for ca-

Figure 7. Cellular uptake mechanism of 5-cf-mu as measured by FACS. The inhibitory effect of 4 °C, NaN3/2-DG, β-CD, or CpZ on the cellular uptake of 5-cf-mu, compared to normal cellular uptake efficiency (37 °C). Means ( SD are indicated (n ) 3).

veolae/lipid raft-mediated endocytosis pathway (25), (4) CpZ, which can interact with clathrin, for clathrin-mediated endocytosis pathway (20, 26). It is demonstrated from Figure 7 that the cellular uptake of 5-cf-mu was not inhibited by pretreating cells with NaN3/2-DG. This is consistent with Mehiri’s result (9). In contrast, it is observed that the cellular uptake level of 5-cf-mu from CpZ, β-CD, and Noc pretreated cells dramatically plunged to 72.11%, 26.59%, and 50.29%, respectively. With these results taken together, our experimental data thus suggested that the following uptake pathways are involved in the cell uptake of 5-cf-mu: (1) passive diffuse; (2) clathrin-mediated endocytosis; (3) caveolae/lipid-raft-dependent endocytosis.

CONCLUSIONS A diwalled molecular umbrella bearing 5-cf as a fluorescent label was synthesized. The molecular umbrella was prepared through coupling of cholic acid to both terminal amino groups of spermidine with the DCC and NHS. Further attachment of 5-cf to the remaining secondary amine on the molecular umbrella was performed with DCC and HOObt to obtain 5-cfmu. Little toxicity was detected even after 24 h incubation at a high concentration of 5-cf-mu by MTT assay. Confocal microscopy showed that molecular umbrellas were able to transport high hydrophilic agent 5-cf across biological membrane of Hela cells. FACS results indicated that the uptake of 5-cfmu depends on incubated time and concentration. By analyzing the mechanism of transmembrane movement, we reason that 5-cf-mu transported across the cell membrane in an association pathway between diffusion and endocytosis. All of these results indicate that the molecular umbrella is highly feasible as a smart intracellular delivery carrier for hydrophilic molecules, molecular probes, and therapeutic drugs.

2316 Bioconjugate Chem., Vol. 20, No. 12, 2009

ACKNOWLEDGMENT We are extremely grateful to the National Nature Science Foundation of China (No. 30670559, 30870648, 30870617), the National Basic Research Program of China (973 Program) (2007CB935603), and the Program for New Century Excellent Talents in Fujian Province University for supporting this research. Supporting Information Available: 1H NMR, ESI-MS, and IR spectra of 5-cf-mu, and FACS analysis of inhibited factors on the cellular uptake of 5-cf-mu. This material is available free of charge via the Internet at http://pubs.acs.org.

LITERATURE CITED (1) Alper, J. (2002) Drug delivery-breaching the membrane. Science 296, 838–839. (2) Mehiri, M., Chen, W. H., Janout, V., and Regen, S. L. (2009) Molecular umbrella transport: exceptions to the classic size/ lipophilicity rule. J. Am. Chem. Soc. 131, 1338–1339. (3) Janout, V., Lanier, M., and Regen, S. L. (1996) Molecular umbrellas. J. Am. Chem. Soc. 118, 1573–1574. (4) Janout, V., Jing, B. W., and Regen, S. L. (2002) Molecular umbrellas-assisted transport of thiolated AMP and ATP across phospholipid bilayers. Bioconjuate Chem. 13, 351–356. (5) Janout, V., and Regen, S. L. (2009) Bioconjugate-based molecular umbrellas. Bioconjugate Chem. 20, 183–192. (6) Janout, V., Zhang, L. H., Staina, I. V., DiGiorgio, C., and Regen, S. L. (2001) Molecular umbrella-assisted transport of glutathione across a phospholipid membrane. J. Am. Chem. Soc. 123, 5401–5406. (7) Janout, V., Jing, B. W., and Regen, S. L. (2005) Molecular umbrella-assisted transport of an oligonucleotide across cholesterolrich phospholipid bilayers. J. Am. Chem. Soc. 127, 15862–15870. (8) Janout, V., Jing, B. W., Staina, I. V., and Regen, S. L. (2003) Selective transport of ATP across a phospholipid bilayer by a molecular umbrella. J. Am. Chem. Soc. 125, 4436–4437. (9) Mehiri, M., Jing, B. W., Ringhoff, D., Janout, V., Cassimeris, L., and Regen, S. L. (2008) Cellular entry and nuclear targeting by a highly anionic molecular umbrella. Bioconjugate Chem. 19, 1510–1513. (10) Puckett, C. A., and Barton, J. K. (2008) Mechanism of cellular uptake of a ruthenium polypyridyl complex. Biochemistry 47, 11711–11716. (11) Okahata, Y., Ando, R., and Kunitakb, T. (1979) Catalytic hydrolysis of p-nitrophenyl esters in the presence of representative ammonium aggregates-Specific activation of a cholesteryl nucleophile bound to a dialkylammonium bilayer membrane. Bull. Chem. Soc. Jpn. 52, 3647–3653. (12) Shawaphun, S., Janout, V., and Regen, S. L. (1999) Chemical evidence for transbilayer movement of molecular umbrellas. J. Am. Chem. Soc. 121, 5860–5864.

Ge et al. (13) Debart, F., Abes, S., Deglane, G., Moulton, H. M., Clair, P., Gait, M. J., Vasseur, J. J., and Lebleu, B. (2007) Chemical modifications to improve the cellular uptake of oligonucleotides. Curr. Top. Med. Chem. 7, 727–737. (14) Onoue, S., Liu, B. S., Nemoto, Y., Hirose, M., and Yajima, T. (2006) Chemical synthesis and application of C-terminally 5-carboxyfluorescein-labelled thymopentin as a fluorescent probe for thymopoietin receptor. Anal. Sci. 22, 1531–1535. (15) Janout, V., and Regen, S. L. (2005) A needle-and-thread approach to bilayer transport: permeation of a molecular umbrella-oligonucleotide conjugate across a phospholipid membrane. J. Am. Chem. Soc. 127, 22–23. (16) Jing, B. W., Janout, V., and Regen, S. L. (2003) Fully detachable molecular umbrellas as peptide delivery agents. Bioconjugate Chem. 14, 1191–1196. (17) Khalil, I. A., Kogure, K., Akita, H., and Harashima, H. (2006) Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. Pharmacol. ReV. 58, 32–45. (18) Vazquez, E., Ferrer-Miralles, N., and Villaverde, A. (2008) Peptide-assisted traffic engineering for nonviral gene therapy. Drug DiscoVery Today 13, 1067–1074. (19) Hewlett, L. J., Prescott, A. R., and Watts, C. (1994) The coated pit and macropinocytic pathways serve distinct endosome populations. J. Cell Biol. 124, 689–703. (20) Wang, Z. Y., Zhao, Y., Ren, L., Jin, L. H., Sun, L. P., Yin, P., zhang, Y. F., and Zhang, Q. Q. (2008) Novel gelatin-siloxane nanoparticles decorated by Tat peptide as vectors for gene therapy. Nanotechnology 19, 445103. (21) Sun, L., Zhu, C. J., Tan, P., and Wong, M. C. (2004) Endothelial progenitor cells’ ‘homing’ specificity to brain tumors. Gene Ther. 11, 811–818. (22) Muranski, P., Chmielowski, B., and Ignatowicz, L. (2000) Mature CD4+ T cells perceive a positively selecting class II MHC/peptide complex in the periphery. J. Immunol. 164, 3087– 3094. (23) Huynh, K. K., Eskelinen, E. L., Scott, C. C., Malevanets, A., Saftig, P., and Grinstein, S. (2007) LAMP proteins are required for fusion of lysosomes with phagosomes. EMBO J. 26, 313– 324. (24) Wang, Y., Li, D., Fan, H., Tian, L. J., Zhong, Y. C., Zhang, Y., Zhang, Y. M., Yuan, L., Jin, C. N., Yin, C. H., and Ma, D. L. (2006) Cellular uptake of exogenous human PDCD5 protein. J. Biol. Chem. 281, 24803–24817. (25) Malorni, W., Giammarioli, A. M., Garofalo, T., and Sorice, M. (2007) Dynamics of lipid raft components during lymphocyte apoptosis: the paradigmatic role of GD3. Apoptosis 12, 941– 949. (26) Richard, J. P., Melikov, K., Brooks, H., Prevot, P., Lebleu, B., and Chernomordik, L. V. (2005) Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors. J. Biol. Chem. 280, 15300–15306. BC9003074