Cellular Uptake Mechanism of an Inorganic Nanovehicle and Its Drug

Oct 21, 2006 - Biomacromolecules 0 (proofing), .... João Tedim , Mário Ferreira , Rosa Freitas , Amadeu M. V. M. Soares , Susana Loureiro .... Iván...
2 downloads 0 Views 594KB Size
Download PDF
Bioconjugate Chem. 2006, 17, 1411−1417

1411

Cellular Uptake Mechanism of an Inorganic Nanovehicle and Its Drug Conjugates: Enhanced Efficacy Due To Clathrin-Mediated Endocytosis Jae-Min Oh,§ Soo-Jin Choi,§ Sang-Tae Kim,§ and Jin-Ho Choy* Center for Intelligent Nano-Bio Materials, Division of Nanoscience and Department of Chemistry, Ewha Womans University, Seoul 120-750, Korea. Received May 23, 2006; Revised Manuscript Received September 4, 2006

We present the mechanism for the cellular uptake of layered double hydroxide (LDH) nanoparticles that are internalized into MNNG/HOS cells principally via clathrin-mediated endocytosis. The intracellular LDHs are highly colocalized with not only typical endocytic proteins, such as clathrin heavy chain, dynamin, and eps15, but also transferrin, a marker of the clathrin-mediated process, suggesting their specific internalization pathway. LDHs loaded with an anticancer drug (MTX-LDH) were also prepared to confirm the efficacy of LDHs as drug delivery systems. The cellular uptake of MTX was higher in MTX-LDH-treated cells than in MTX-treated cells, giving a lower IC50 value for MTX-LDH than for MTX only. The inhibition of the cell cycle was greater for MTX-LDH than for MTX only. This result clearly shows that the internalization of LDH nanoparticles via clathrin-mediated endocytosis may allow the efficient delivery of MTX-LDH in cells and thus enhance drug efficacy.

INTRODUCTION Nanomaterials such as nanoparticles, nanohybrids, or nanocomposites, as well as nanoporous materials, have been widely studied in the view point of physics, chemistry, biosciences, and materials engineering (1-6). Among these nanomaterials, the use of nanoparticles as nanomedicines (7) for alternative drug delivery vehicles (8-12) or molecular diagnostics (1315) has attracted much interest. Designing nanomedicines with enhanced and intentional functionalities required an understanding of the intracellular trafficking pathway of nanoparticles so that their fate in the cell can be predicted. Recently, it has been reported that certain nanoparticles, such as iron oxide and silica, as well as carbon nanotubes, are internalized in cells via the endocytic pathway (11, 16-21). Endocytosis is a conserved process in eukaryotes by which extracellular components are taken up into cells by invagination of the plasma membrane to form vesicles that enclose these materials. In the present study, we have used layered double hydroxide (LDH) to investigate the cellular interaction and uptake of nanoparticles, since LDH is considered as a candidate for drug delivery nanovehicles (4, 11, 12). LDHs are positively charged metal hydroxide layers in which solvated exchangeable anions are stabilized to compensate for the positive layer charges (22). Due to their hydrolysis behavior in acidic media and their anionic exchange capacity, LDHs are already used as antiacid and antipepsin agents (23). More recently, we have successfully demonstrated that the positively or neutrally charged LDHs which encapsulate anionic molecules are easily attached to the surface of a negatively charged plasma membrane, and in such a way their internalization into cells could be facilitated. Thus, LDHs may prove useful as gene or drug delivery vectors (11, 12).

EXPERIMENTAL PROCEDURES Preparation of LDHs. All reagents, Mg(NO3)2‚6H2O, Al(NO3)3‚9H2O, NaOH, NaHCO3, methotrexate (MTX), and * Corresponding author. E-mail: [email protected], Tel.: +8202-3277-4305, Fax.: +82-02-3277-4340. § Equally contributed to the work reported.

fluorescein 5′-isothiocyanate (FITC) were purchased from Aldrich Co. Ltd. An LDH with the formula Mg0.68Al0.32(OH)2(CO3)0.16‚0.1H2O was prepared by coprecipitation at room temperature by titration to pH ∼ 9.5 with NaOH/NaHCO3 solution (0.5 M/0.5 M). The obtained mixed solution was then hydrothermally treated at 100 °C for 24 h to give even particle size distribution (CO3-LDH), as described previously (24). LDH loaded with the anticancer drug MTX (MTX-LDH), and FITC-loaded LDH (FITC-LDH) were also prepared by respectively coprecipitation and anion exchange, as previously reported (12). Powdered MTX was dissolved in decarbonated water, and 0.5 M NaOH solution was added carefully to give a 0.032 M solution of MTX at pH 7. A mixed solution of Mg(NO3)2‚6H2O (0.032 M) and Al(NO3)3‚9H2O (0.016 M) was then added. The pH of the mixture was adjusted to 9.5 by NaOH titration. For FITC-LDH, a 0.032 M solution of FITC was prepared with decarbonated water and NaOH titration. LDH particles (Mg0.68Al0.32(OH)2(NO3)0.32‚0.1H2O) dispersed in decarbonated water were then added to the FITC solution. Both the MTX and FITC mixtures were left to stand for 48 h at room temperature. Each LDH sample (CO3-LDH, MTX-LDH, and FITC-LDH) was thoroughly washed with decarbonated water and dried in vacuum to give a powder, which was then redispersed in decarbonated water for the next step. The prepared LDHs were analyzed by powder X-ray diffraction (PXRD; Phillips PW3710 diffractometer with Ni-filtered Cu KR radiation (λ ) 1.5418 Å)) and by scanning electron microscopy (SEM; HITACHI S-4300). Immunofluorescence/Confocal Microscopy. MNNG/HOS cells were seeded on 12-well slides (5 × 103 cells/well) and incubated with 50 µg/mL FITC-LDH for 10 min, 1, 2, or 3 h. The effects of a membrane entry inhibitor were examined by incubating the cells with 10 µg/mL chlorpromazine for 1 h at 37 °C followed by treatment with FITC-LDH as described above. The cells were washed several times with ice-cold PBS, fixed by incubation with freshly made 3% formaldehyde (containing 1.5% methanol) in PBS (pH 7.4) for 15 min, and permeabilized by incubation with 0.1% Triton X-100 in PBS for 10 min. The cells were incubated with primary antibodies against mouse clathrin, caveolin-1, eps15, dynamin, and Trans-

10.1021/bc0601323 CCC: $33.50 © 2006 American Chemical Society Published on Web 10/21/2006

1412 Bioconjugate Chem., Vol. 17, No. 6, 2006

Oh et al.

Figure 1. (A) Powder X-ray diffraction (PXRD) patterns, (B) schematic diagram of the structure, and (C) SEM images of three different LDH particles. (a) CO3-LDH (b) MTX-LDH, and (c) FITC-LDH. (Scale bars in part C represent 100 nm).

Figure 2. (A) Fluorescence microscopic images of FITC-LDH-treated MNNG/HOS cells depending on incubation time. (B) Immunofluorescence microscopic images showing the colocalization of clathrin and LDH in MNNG/HOS cells. (C) Confocal microscopy: colocalization of FITCLDH and clathrin in MNNG/HOS cells. Localization of (a) nucleus, (b) clathrin, and (c) FITC-LDH, the merged image (d) in MNNG/HOS cells. Cells were incubated with FITC-LDH for 2 h, treated with clathrin antibodies, and stained by TR and DAPI. Scale bar represents 10 µm. (D) Magnified images of the white boxes in part C are also given. The images identify areas showing colocalization of FITC-LDH and clathrin, as shown merged in yellow.

ferrin (Tf) for 1 h, washed, and then incubated with secondary antibodies conjugated to Texas Red (TR : Molecular Probe) or Alexa fluor 594 (Molecular Probe). The cells were then stained by incubation with 2 µg/mL DAPI dye for 10 min at room temperature in the dark. The cells were washed and visualized by fluorescence microscopy using an Axioplan Zeiss microscope and photographed with a digital camera (CCD). For laser scanning confocal microscopy (LSCM), the cells were

visualized using a Zeiss LSM 510 confocal microscope (Carl Zeiss Inc., Germany) equipped with Argon (488 nm) and HeNe (543 nm) lasers for fluorescence. Image acquisition and analysis was done using LSM 510 software. Each experiment was repeated three times on separate days. MTX Quantification by HPLC. Cells (1 × 106) were seeded on a 60 mm culture dish and treated with 50 µM/mL MTX or the equivalent amount of MTX-LDH with respect to MTX

Clathrin-Mediated Endocytosis of Inorganic Nanoparticles

Bioconjugate Chem., Vol. 17, No. 6, 2006 1413

Table 1. Chemical Formula and Colloidal Properties of Each LDH Sample (MTX: C20H20N8O52-, FITC: C21H10O5SN2-) sample

chemical formula

particle size (nm)a

CO3-LDH MTX-LDH FITC-LDH

Mg0.68Al0.32(OH)2(CO3)0.16‚0.1H2O Mg0.68Al0.32(OH)2(MTX)0.16‚0.1H2O Mg0.68Al0.32(OH)2(FITC)0.16‚0.1H2O

136 ( 29 127 ( 25 147 ( 31

zeta potential (mV) ∼+20 ∼+5.6 ∼-4.9

a Each sample was dispersed in deionized water, and the suspension was dropped on the flat Si-wafer. After evaporating water in the oven, particles on the Si-wafer were measured by scanning electron microscopy. Fifty particles were randomly selected from SEM image, and the diameters of particles were measured. Zeta potential was measured at pH 7.

Figure 3. Colocalization of FITC-LDH with clathrin-mediated endocytic proteins and markers. FITC-LDH-treated MNNG/HOS cells were fixed and processed for immunofluorescence microscopy. (A) Fluorescence microscopic images of the cells treated with clathrin, eps15, and dynamin antibodies and then stained with secondary antibodies conjugated to Alexa Fluor 594, showing the same localization pattern. (B) Colocalization of FITC-LDH and Alexa Fluor 594-labeled Tf, a well-known marker of clathrin-mediated endocytosis. The localization of LDH and Tf clearly overlaps. Scale bars, 20 µm. (C) The effect of clathrin inhibition on the entry of LDHs in MNNG/HOS cells. The cells initially incubated with a clathrin inhibitor, CPZ, showed a considerable reduction in LDH particle uptake.

concentration. After 0.5, 1, 2, 4, 8, and 24 h, the cells were washed twice with PBS, harvested by trypsin-EDTA treatment, and cell pellets were collected by centrifugation. After the pellets were washed twice with PBS, cell lysis buffer (20 mM TrisHCl, pH 7.5, 1% Triton X-100, 150 mM NaCl, and 1 mM EDTA) was added and the pellets were incubated on ice for 20 min followed by ultrasonication for 20 s (Sonics & Materials Inc.) The crude cell lysates were then centrifuged at 10 000g for 5 min. Two volumes of acetonitrile were added to the cell supernatants and mixed vigorously to precipitate proteins. After centrifugation at 10 000g for 3 min, four volumes of chloroform were added to the supernatant to remove lipids. The supernatants were then collected by centrifugation at 2000g for 10 min and analyzed by high performance liquid chromatography (HPLC). MTX was quantified by HPLC using an Agilent 1100 series HPLC system on a C18 column (Nucleosil, 250 × 4.6 mm, Alltech). The mobile phase was 0.05 M KH2PO4, 10% acetonitrile, pH 6.6, and flow rate was 1 mL/min. Column temperature was maintained at a constant 40 °C and MTX was detected by UV absorption at 304 nm. Each experiment was repeated three times on separate days. In Vitro Cell Viability/Cytotoxicity Studies. Cells were seeded onto 96-well plates at a density of 5 × 103 cells/well and incubated overnight at 37 °C in a 5% CO2 atmosphere. The medium in the wells was then replaced with the fresh medium containing nanoparticles (CO3-LDH or MTX-LDH) in a

concentration range of 0 to 2 mg/mL. After 72 h, cell viability and cytotoxicity were monitored by counting viable (trypan blue excluding) cells in a hemacytometer. Flow Cytometric Analysis of the Cell Cycle. Cells (1 × 106) were treated with indicated concentrations of drugs for 20 h. They were then harvested by trypsin treatment, washed with PBS, and fixed by incubation with cold 70% ethanol on ice for 1 h. The fixed cells were washed with PBS and incubated with PI (40 µg/mL PI, 320 µg/mL RNase-A in PBS) at 4 °C for 30 min. Flow cytometric measurement was done on a flow cytometer (FACS Calibur, Becton Dickinson BD). For each analysis, 20 000 events were monitored.

RESULTS AND DISCUSSION We used three different kinds of LDHs in this study. Pristine LDH with a formula of Mg0.68Al0.32(OH)2(CO3)0.16‚0.1H2O (CO3-LDH) and fluorescein isothiocyanate (FITC)-loaded LDH (FITC-LDH) were used to check, by electron and fluorescence microscopy, the cellular uptake pathway of the drug delivery vector, LDH. LDH loaded with the anticancer drug methotrexate (MTX) (MTX-LDH) was used to confirm the increased efficacy of drug delivery by LDH encapsulation. The powder X-ray diffraction patterns showed that each LDH had a typical LDH structure (Figure 1A), and the d-spacing of each LDH determined was 7.6, 20.8, and 18.8 Å, respectively, upon

1414 Bioconjugate Chem., Vol. 17, No. 6, 2006

Figure 4. Cellular accumulation of free MTX molecules in MNNG/ HOS cells treated with either MTX (O) or MTX-LDH (b), as quantified by HPLC analysis (see Experimental Procedures). (A). Cell viability/cytotoxicity of MNNG/HOS cells treated with LDH (2), MTX (O), and MTX-LDH (b), as monitored by trypan blue exclusion, with respect to drug concentration (see Experimental Procedures) (B).

molecular size of interlayer anions such as CO32-, MTX, and FITC (Figure 1B), as previously reported (11, 12, 24). The SEM results (Figure 1C) showed that all the LDH particles have characteristic hexagonal plate like morphology with uniform size. When dispersed in water (neutral pH), the particle size of approximately 150 nm was determined with narrow distribution as shown in Table 1, and the zeta potential of each colloidal LDH was positive or neutral. The LDH particle should have positive layer charge due to the isomorphous substitution of Mg2+ ions by Al3+ ions. This is the reason why negatively charged organic molecules such as MTX and FITC could be intercalated into the interlayer space of LDH. In this way, charge neutralization of the reaction could be satisfied (Table 1). There are several possible endocytic pathways for internalizing LDH nanoparticles, such as clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrin-caveolae-independent endocytosis. We determined the specific endocytic pathway used by the LDH nanoparticles for membrane entry by immunofluorescence and confocal microscopic studies. We used tumorogenic osteosarcoma MNNG/HOS cells treated with the fluorescent FITC-LDH to measure the intracellular fluorescence intensity with respect to time. The fluorescence microscopic

Oh et al.

images (Figure 2A) showed an increasingly strong fluorescent signal inside the cell as the incubation time increased to 2 h. For the immunofluorescence study, the cells were treated with FITC-LDH for 2 h, during which the maximum fluorescence intensity was attained, and stained with either anti-clathrin antibody or anti-caveolin-1 antibody both conjugated to TR. The immunofluorescence microscopic images showed that both the clathrin staining red fluorescence (TR) and the LDH green one (FITC) overlapped each other perfectly. Though the red immunofluorescent caveolin-1 was observed but faintly, no clear merged image could be seen when treated with the LDH fluorescence as shown in Figure 2B. MNNG/HOS cells treated with FITC-LDH and the anti-clathrin antibody showed that, as expected, FITC-LDHs were mainly located in the cytosol, with a perinuclear distribution and was highly colocalized with clathrin (Figure 2C). The magnified image (Figure 2D) revealed that the green FITC fluorescence (LDH) and the red fluorescent clathrin staining overlapped well to produce a yellow fluorescence. This result clearly suggests that clathrin-mediated endocytosis is the principal mechanism for the cellular internalization of LDH nanoparticles. Caveolae-mediated endocytosis does not appear to be responsible for LDH uptake. We confirmed the clathrin-mediated cellular uptake of LDH nanoparticles by determining the involvement of certain accessory proteins related to clathrin-mediated endocytosis. Thus, we investigated whether FITC-LDH colocalized with typical endocytic proteins such as dynamin and eps15. Dynamin regulates endocytic vesicle formation in different endocytic pathways (25), and eps15 is involved in regulating clathrincoated pit assembly (26). We incubated MNNG/HOS cells with FITC-LDH and stained them with antibodies against clathrin, dynamin, and eps15, with the red Alexa Fluor 594 conjugated to the secondary antibodies as the probe (Figure 3A). We found that FITC-LDH was well colocalized with clathrin, dynamin, and eps15. eps15 plays a critical role only in clathrin-mediated endocytosis by associating with the plasma membrane adaptor, the AP-2 protein (26), which is essential for receptor-mediated endocytosis. As a positive control, we also compared the internalization of FITC-LDH with that of Tf, which is wellknown to undergo the clathrin-mediated endocytosis through the transferrin receptor (27). Intracellular FITC-LDH was highly colocalized with the Tf labeled with Alexa Fluor 594 (Figure 3B). This is consistent with the results seen for clathrin, dynamin, and eps15, confirming the clathrin-mediated internalization of LDH nanoparticles. We confirmed LDH entry via clathrin-coated vesicles biochemically by studying the effect of chlorpromazine (CPZ), a clathrin inhibitor, on LDH entry. CPZ is a cationic amphiphilic drug that prevents clathrin-mediated endocytosis by disrupting the assembly of the clathrin adaptor protein at the cell surface (28). Thus, we incubated MNNG/HOS cells with CPZ before observing the cellular uptake of FITC-LDH. Unsurprisingly, clathrin inhibition gave rise to a considerable reduction of the uptake of the LDH particles (Figure 3C). Altogether, it is concluded that LDH nanoparticles are transported into MNNG/ HOS cells principally via clathrin-mediated endocytosis. The targeted entry of nanoparticles into cells is important in nanomedicines, as their specific intracellular delivery can considerably reduce drug toxicity and increase therapeutic effects. Drugs conjugated with a ligand that can be efficiently and specifically recognized by receptor-mediated endocytosis have been already used to deliver drugs into cells; for example, transferrin/transferrin receptor-mediated drug delivery (29, 30). Thus, our results showing that LDH nanoparticles primarily select the clathrin-mediated process such as the transferrin ligand/receptor interaction for cellular entry suggest that they may prove promising as nanomedicines and drug delivery

Clathrin-Mediated Endocytosis of Inorganic Nanoparticles

Bioconjugate Chem., Vol. 17, No. 6, 2006 1415

Figure 5. G1 phase arrest of MNNG/HOS cells exposed to either MTX or MTX-LDH at different concentrations for 20 h. The cell cycle was studied by FACS analysis of PI-stained cells. DNA histograms are shown, with the x-axis representing the DNA content and the y-axis representing the cell number. Note that 10 and 320 µg/mL MTX are equivalent to 22 and 704 µg/mL of MTX-LDH, respectively, based on MTX content in MTX-LDH.

nanovehicles. Their intracellular trafficking mechanism may also be predicted, being transported to endosomes, and subsequently to the Golgi complex or/and lysosomes after uptake (31). From our results, we would expect an enhanced internalization of the drug in vitro due to their being encapsulated in the LDH nanoparticles. Thus, we investigated the applicability of LDH nanoparticles as drug delivery nanovectors for anticancer chemotherapy using a well-known anticancer drug, MTX, a folate antagonist. MTX binds competitively and reversibly to dihydrofolate reductase and inhibits important biochemical pathways, including the syntheses of thymidine, purine, DNA, RNA, and proteins (32). MTX is used in patients with certain forms of leukemia, and also in patients with various malignant diseases, such as lymphoma, choriocarcinoma, osteosarcoma, and uterus, breast, lung, head, neck, and ovarian cancers. However, high doses of MTX are required because of the antifolate properties of MTX, which is one of the major disadvantages of MTX medication (33). Thus, new chemotherapeutic approaches are urgently needed to limit its toxic effects. We prepared MTX-LDH and quantified the level of intracellular “free MTX in the cells” by HPLC to determine its accumulation in cells (Figure 4A). The concentration of MTX within MTX-LDH-treated cells was considerably higher than in the cells treated with MTX only at all incubation times. This result strongly suggests that in an osteosarcoma cell culture line,

the clathrin-mediated endocytosis of LDH nanoparticles enhances the internalization of conjugated drug molecules. As shown in Figure 4B, MTX-LDH was more toxic than MTX only as demonstrated that the IC50 value of MTX-LDH in the cells was about 2.5 lower than that of MTX only which means that MTX-LDH penetrates the cell membrane more effectively than MTX only, resulting in a better efficacy. This result indicates that the drug efficacy of MTX-LDH significantly increased over MTX only, resulting from effective internalization of MTX-LDH. Furthermore, LDH alone does not influence the cell viability at levels up to 500 µg/mL (Figure 4 B). We also investigated the effect of MTX and MTX-LDH on cell cycle distribution to confirm further the efficacy of MTXLDH. The cell cycle consists of interphases (G1, S, and G2) and mitosis (M). During the G1 period, cells increase in size, produce RNA, and synthesize proteins for DNA formation. During the S phase, DNA replication occurs and the cells continue to grow, producing new proteins at the G2 phase. Nuclear and cytoplasmic division occurs at the M stage. We used fluorescence-activated cell sorting (FACS) analysis to study propidium iodide (PI)-stained cells treated with either MTX or MTX-LDH (Figure 5). Over 20 h, MTX and MTX-LDH treatments resulted in an accumulation of cells in the G1 phase and a decrease in the number of cells in the S and G2 phases versus control cells. This is probably due to inhibition of DNA synthesis and blockage at the G1/S boundary. However, the

1416 Bioconjugate Chem., Vol. 17, No. 6, 2006

inhibition of G1/S transition was more evident in MTX-LDHtreated cells than in MTX-treated cells at the same concentration (63.87% versus 75.09% at 10 µM/mL) and was considerably increased at higher concentrations of MTX-LDH. This suggests that using LDH as drug delivery vector effectively enhances MTX efficacy, consistent with above results (Figure 4A,B). LDH alone did not affect the cell cycle at concentrations between 1.5 and 384 µg/mL. The G1 checkpoint stops replication of cells with DNA damage and is involved the development of cancer. Proteins involved in the G1 checkpoint, such as p53, a tumor suppressor protein, and cyclin-dependent kinases may be targets for cancer therapeutics (34). We have shown using immunofluorescence and confocal microscopy that LDH nanoparticles are internalized in MNNG/ HOS cells via clathrin-mediated endocytosis. FITC-LDHs were highly colocalized with clathrin and Tf, and also clathrinmediated endocytosis substrates, such as dynamin and eps15. We also studied the efficacy of MTX-LDH as a potential drug delivery nanovector. The cellular uptake of MTX-LDH was found to be considerably higher than that for MTX only, resulting in lower IC50 values and an increased inhibition of the G1 cell cycle. LDH nanoparticles can stabilize MTX during cell permeation and intracellular trafficking by encapsulation and can release it in the cytoplasm in a stable and constant manner due to its anion exchange capacity in acidic pH. The most common endocytic pathway, the clathrin-coated pit process, for LDH uptake may contribute to the efficient delivery of MTX-LDH in the cells and thus enhance drug efficacy. These results allow us to propose new directions in designing target-specific nanomedicine. Our research, particularly in designing drug delivery nanovehicles based on metal hydroxide nanoparticles, may provide a new strategy based on a better understanding of their cellular uptake pathways and intracellular delivery mechanism.

ACKNOWLEDGMENT We thank Dong-Ok Kim for her help in supporting cell preparations. This work was financially supported by the SRC program of MOST/KOSEF through the Center for Intelligent Nano-Bio Materials at Ewha Womans University (grant: R112005-008-00000-0), and partly by MOCIE through the National R&D project for Nano Science and Technology (grant: 20050980-1 and 2005-0546-1). Supporting Information Available: Supplementary data. This material is available free of charge via the Internet at http:// pubs.acs.org.

LITERATURE CITED (1) Choy, J. H., Kwon, S. J., and Park, G. S. (1998) High-T-c superconductors in the two-dimensional limit: [(Py-CnH2n + 1)(2)HgI4]-Bi2Sr2Cam-1CumOy (m ) 1 and 2). Science 280, 15891592. (2) Choy, J. H., Jang, E. S., Won, J. H., Chung, J. H., Jang, D. J., and Kim, Y. W. (2003) Soft solution route to directionally grown ZnO nanorod arrays on Si wafer; room-temperature ultraviolet laser. AdV. Mater. 15, 1911-+. (3) Usuki, A., Hasegawa, N., Kadoura, H., and Okamoto, T. (2001) Three-dimensional observation of structure and morphology in nylon6/ clay nanocomposite. Nano Lett. 1, 271-272. (4) Choy, J. H., Oh, J. M., Park, M., Sohn, K. M., and Kim, J. W. (2004) Inorganic-biomolecular hybrid nanomaterials as a genetic molecular code system. AdV. Mater. 16, 1181-+. (5) Maxwell, D. J., Taylor, J. R., and Nie, S. M. (2002) Self-assembled nanoparticle probes for recognition and detection of biomolecules. J. Am. Chem. Soc. 124, 9606-9612.

Oh et al. (6) Choy, J. H., Kwak, S. Y., Park, J. S., Jeong, Y. J., and Portier, J. (1999) Intercalative nanohybrids of nucleoside monophosphates and DNA in layered metal hydroxide. J. Am. Chem. Soc. 121, 13991400. (7) Moghimi, S. M., Hunter, A. C., and Murray, J. C. (2005) Nanomedicine: current status and future prospects. FASEB J. 19, 311-330. (8) Zhao, W., Gu, J., Zhang, L., Chen, H., and Shi, J. (2005) Fabrication of Uniform Magnetic Nanocomposite Spheres with a Magnetic Core/ Mesoporous Silica Shell Structure. J. Am. Chem. Soc. 127, 89168917. (9) Andersson, J., Rosenholm, J., Areva, S., and Linden, M. (2004) Influences of material characteristics on ibuprofen drug loading and release profiles from ordered micro- and mesoporous silica matrices. Chem. Mater. 16, 4160-4167. (10) Lai, C. Y., Trewyn, B. G., Jeftinija, D. M., Jeftinija, K., Xu, S., Jeftinija, S., and Lin, V. S. Y. (2003) A mesoporous silica nanosphere-based carrier system with chemically removable CdS nanoparticle caps for stimuli-responsive controlled release of neurotransmitters and drug molecules. J. Am. Chem. Soc. 125, 44514459. (11) Choy, J. H., Jung, J. S., Oh, J. M., Park, M., Jeong, J., Kang, Y. K., and Han, O. J. (2004) Layered double hydroxide as an efficient drug reservoir for folate derivatives. Biomaterials 25, 3059-3064. (12) Choy, J. H., Kwak, S. Y., Jeong, Y. J., and Park, J. S. (2000) Inorganic layered double hydroxides as nonviral vectors. Angew. Chem., Int. Ed. 39, 4042-4045. (13) Portet, D., Denizot, B., Rump, E., Lejeune, J. J., and Jallet, P. (2001) Nonpolymeric coatings of iron oxide colloids for biological use as magnetic resonance imaging contrast agents. J. Colloid Interface Sci. 238, 37-42. (14) Wunderbaldinger, P., Josephson, L., and Weissleder, R. (2002) Tat peptide directs enhanced clearance and hepatic permeability of magnetic nanoparticles. Bioconjugate Chem. 13, 264-268. (15) Won, J., Kim, M., Yi, Y. W., Kim, Y. H., Jung, N., and Kim, T. K. (2005) A magnetic nanoprobe technology for detecting molecular interactions in live cells. Science. 309, 121-5. (16) Oh, J. M., Park, M., Kim, S. T., Jung, J. Y., Kang, Y. K., Choy, J. H. (2006) Efficient delivery of anticancer drug MTX through MTX-LDH nanohybrid system. J. Phys. Chem. Solids. 67, 10241027. (17) Berry, C. C. (2005) Possible exploitation of magnetic nanoparticlecell interaction for biomedical applications. J. Mater. Chem. 15, 543-547. (18) Xing, X., He, X., Peng, J., Wang, K., and Tan, W. (2005) Uptake of silica-coated nanoparticles by HeLa cells. J. Nanosci. Nanotechnol. 5, 1688-93. (19) Kam, N. W. S., and Dai, H. J. (2005) Carbon nanotubes as intracellular protein transporters: Generality and biological functionality. J. Am. Chem. Soc. 127, 6021-6026. (20) Choy, J. H., Kwak, S. Y., Park, J. S., and Jeong, Y. J. (2001) Cellular uptake behavior of [gamma-P-32] labeled ATP-LDH nanohybrids. J. Mater. Chem. 11, 1671-1674. (21) Kam, N. W., Liu, Z., and Dai, H. (2006) Carbon nanotubes as intracellular transporters for proteins and DNA: an investigation of the uptake mechanism and pathway. Angew. Chem., Int. Ed. 45, 577-81. (22) Vaccari, A. (1998) Preparation and catalytic properties of cationic and anionic clays. Catal. Today 41, 53-71. (23) Tarnawski, A., Pai, R., Itani, R., and Wyle, F. A. (1999) The antacid Talcid adsorbs and neutralizes all proteins secreted by H. pylori including VacA cytotoxin: A new mechanism for its ulcerhealing action? Digestion 60, 449-455. (24) Oh, J. M., Hwang, S. H., and Choy, J. H. (2002) The effect of synthetic conditions on tailoring the size of hydrotalcite particles. Solid State Ionics 151, 285-291. (25) Sever, S., Damke, H., and Schmid, S. L. (2000) Garrotes, springs, ratchets, and whips: putting dynamin models to the test. Traffic 1, 385-92. (26) Benmerah, A., Bayrou, M., Cerf-Bensussan, N., and DautryVarsat, A. (1999) Inhibition of clathrin-coated pit assembly by an Eps15 mutant. J. Cell Sci. 112 (Pt 9), 1303-11. (27) Singh, M. (1999) Transferrin As A targeting ligand for liposomes and anticancer drugs. Curr. Pharm. Des. 5, 443-51.

Bioconjugate Chem., Vol. 17, No. 6, 2006 1417

Clathrin-Mediated Endocytosis of Inorganic Nanoparticles (28) Kirchhausen, T. (2000) Three ways to make a vesicle. Nat. ReV. Mol. Cell. Biol. 1, 187-98. (29) Li, H., and Qian, Z. M. (2002) Transferrin/transferrin receptormediated drug delivery. Med. Res. ReV. 22, 225-50. (30) Widera, A., Norouziyan, F., and Shen, W. C. (2003) Mechanisms of TfR-mediated transcytosis and sorting in epithelial cells and applications toward drug delivery. AdV. Drug DeliVery ReV. 55, 1439-66. (31) Medina-Kauwe, L. K., Xie, J., and Hamm-Alvarez, S. (2005)

Intracellular trafficking of nonviral vectors. Gene Ther. 12, 173451. (32) McGuire, J. J. (2003) Anticancer antifolates: current status and future directions. Curr. Pharm. Des. 9, 2593-613. (33) Ackland, S. P., and Schilsky, R. L. (1987) High-dose methotrexate: a critical reappraisal. J. Clin. Oncol. 5, 2017-31. (34) Shapiro, G. I., and Harper, J. W. (1999) Anticancer drug targets: cell cycle and checkpoint control. J. Clin. InVest. 104, 1645-53. BC0601323