The characteristics, cellular uptake and intracellular trafficking of nanoparticles made of hydrophobically-modified chitosan

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Journal of Controlled Release 146 (2010) 152–159

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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l

The characteristics, cellular uptake and intracellular trafficking of nanoparticles made of hydrophobically-modified chitosan Ya-Ling Chiu a, Yi-Cheng Ho b, Yu-Ming Chen a, Shu-Fen Peng a, Cherng-Jyh Ke a, Ko-Jie Chen a, Fwu-Long Mi b, Hsing-Wen Sung a,⁎ a b

Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan, ROC Department of Biotechnology, Vanung University, Chungli, Taoyuan, Taiwan, ROC

a r t i c l e

i n f o

Article history: Received 10 March 2010 Accepted 19 May 2010 Available online 23 May 2010 Keywords: N-palmitoyl chitosan Endocytosis Internalization pathway Caveolae Molecular dynamic simulation

a b s t r a c t It has been reported that nanoparticles (NPs) prepared by hydrophobically-modified polymers could accumulate passively in the tumor tissue; however, their cellular uptake mechanism and intercellular trafficking pathway have never been understood. This study was designed to address these concerns, using NPs prepared by a hydrophobically-modified chitosan (N-palmitoyl chitosan, NPCS). Molecular dynamic simulations found that a degree of substitution (DS) of 5% of palmitoyl groups on its backbone was sufficient to allow NPCS to form NPs, due to a significant increase in the intra- and intermolecular hydrophobic interactions. With an increase of DS, there were more palmitoyl groups present on the surface of NPs which were then able to interact with the cell membranes. A greater extent of cellular uptake of NPCS NPs was observed with increasing the DS on NPCS. The internalization of NPCS NPs was clearly related with the lipid raft-mediated routes; with increasing the DS on NPCS, the caveolae-mediated endocytosis became more important. The results obtained in the intracellular trafficking study showed that NPCS NPs entered cells via caveolae and transiently localized to caveosomes before trafficking to the endosomal pathway. These results suggest that the prepared NCPS NPs may serve as a carrier for intracellular delivery of therapeutic agents. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Chitosan (CS), a natural-origin polysaccharide, is biodegradable, non-toxic and soft-tissue compatible, and thus has been used extensively in biomedical applications [1]. It is known that the pKa of CS is approximately 6.5 [1,2]. In aqueous media at pH 7.4, CS was found to form dissociated precipitates due to deprotonation of its amine groups. To enhance its intermolecular contact, we conjugated a hydrophobic palmitoyl group onto the free amine groups of CS in a previous study [3]. The synthesized N-palmitoyl CS (NPCS) in an aqueous environment was demonstrated to exhibit a rapid nanostructure transformation within a narrow pH range through a proper balance between charge repulsion and hydrophobic interaction. Subcutaneous injection of aqueous NPCS into a rat model resulted in rapid formation of a massive hydrogel at the location of injection. In dilute aqueous media, we found that NPCS polymers were able to self-assemble into nanoparticles (NPs), due to the hydrophobic interaction between their conjugated palmitoyl groups. Hydrophobically-modified polymers have been used to fabricate NPs as a drugdelivery vehicle for therapeutic applications [4–6]. Previous studies have shown that these NPs could accumulate passively in the tumor ⁎ Corresponding author. Department of Chemical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC. Tel.: +886 3 574 2504; fax: +886 3 572 6832. E-mail address: [email protected] (H.-W. Sung). 0168-3659/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2010.05.023

tissue [4,5]. The cellular uptake mechanism and intracellular fate of 5β-cholanic-acid conjugated glycol CS, a hydrophobic glycol CS (HGC), has been reported by Nam et al. [7]; interestingly, several distinct uptake pathways were identified to be involved in the internalization of HGC with a single degree of substitution (DS). Whether different DS (or hydrophobicity) could affect the endocytosis of hydrophobically-modified polymers remains to be understood. In the present study, we prepared NPs of NPCS with different DS; their cellular uptake and the potential endocytosis and intracellular trafficking pathways were investigated herein. A few synthetic methods have been developed to conjugate hydrophobic side chains on the CS backbone. Nevertheless, these synthetic methods led to either a relatively low DS of 1–5% [8–12] or caused an uncontrollable DS and produced undesired by-products [13]. In this study, we employed a method which was able to produce NPCS with relatively high and controllable DS (up to 20% DS). The synthesis was accomplished in a single-step reaction which had been used in forming amide bonds in the synthesis of peptides [3,14]. The NPs prepared by the synthesized NPCS polymers with different DS were characterized using dynamic light scattering (DLS) and molecular dynamic (MD) simulations. The cytotoxicity of NPs was evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide) assay as well as the lactate dehydrogenase (LDH) assay. The cellular internalization efficiency, potential endocytosis mechanism and intracellular trafficking pathway of test

NPs were investigated via a flow cytometer and a confocal laser scanning microscope (CLSM). 2. Materials and methods 2.1. Materials CS (viscosity 5 mPa s, 0.5% in 0.5% acetic acid at 20 °C, MW 50 kDa) with a degree of deacetylation of approximately 85% was purchased from Koyo Chemical Co. Ltd. (Tokyo, Japan). Palmitic acid Nhydroxysuccinimide ester was obtained from Sigma-Aldrich (St. Louis, MO, USA). N-hydroxysuccinimide (NHS)-functionalized cyanine 5 (Cy5-NHS) and fluorescein (fluorescein-NHS) were acquired from Amersham Biosciences (Piscataway, NJ, USA) and Thermo Scientific (Chicago, IL, USA), respectively. All other chemicals and reagents used were of analytical grade. 2.2. Synthesis of NPCS A mixture of CS (1 g) and aqueous acetic acid (50 mL, 1% w/v) was stirred for 24 h to ensure total solubility. The pH was adjusted to 6.0 by slow addition of 1N NaOH and the final volume of CS solution was 100 mL. A solution of palmitic acid N-hydroxysuccinimide ester (0.1, 0.2, 0.3 or 0.4 g) in absolute ethanol was added drop-wise to the CS solution at 98 °C and reacted for 36 h. Subsequently, the prepared solution was cooled at room temperature, added acetone and precipitated by adjusting its pH value to 9.0. The precipitate (NPCS) was then filtered, washed with an excess of acetone and air-dried. The synthesized NPCS was analyzed by the proton nuclear magnetic resonance (1H NMR) and Fourier transformed infrared (FT-IR) spectroscopy and the results were presented in our previous report [3]. Additionally, the DS on NPCS was determined by the ninhydrin assay [15] and the potassium polyvinylsulfate (PVSK) titration method [16]. 2.3. Preparation and characterization of NPCS NPs The synthesized NPCS polymers with different DS were individually dissolved in 1% aqueous acetic acid and its pH value was adjusted to 4.0 by adding a few drops of 1N NaOH under magnetic stirring to form test NPs. The size distributions and zeta potential values of the prepared NPs at pH 7.4 were investigated using DLS (Zetasizer 3000HS, Malvern Instruments Ltd., Worcestershire, UK).

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2.5. Cytotoxicity of NPCS NPs The cytotoxicity of test NPs was evaluated in vitro using the MTT and LDH assays; the group without any treatment was used as a control. HT1080 (human fibrosarcoma, ATCC CRL-121) cells were seeded in 12well plates at 1 × 105 cells/well, allowed to adhere overnight and incubated with a growth medium [DMEM with fetal bovine serum (FBS), pH 7.4] containing NPCS NPs at a concentration of 100 μg/mL. It was noted that precipitation of test NPs in the culture media was found with time, when a higher dose of test NPs was used in the study. After 24 h, test samples were aspirated and cells were incubated in a growth medium containing 1 mg/mL MTT reagent for an additional 4 h; a 1000 μL of dimethyl sulfoxide (DMSO) was added to each well to ensure solubilization of formazan crystals. Finally, the optical density readings were performed using a multiwell scanning spectrophotometer (Dynatech Laboratories, Chantilly, VA, USA) at a wavelength of 570 nm [21,22]. As an additional test, we also measured the leakage of LDH in the culture medium. After NP treatment, media were collected and centrifuged, and the activity of LDH released from the cytosol of damaged cells was assessed using the LDH Cytotoxicity Assay Kit (Sigma-Aldrich). The maximum releasable LDH activity in the cells, induced by the addition of a lysis solution, was measured and used as a 100% LDH released. The optical density at a wavelength of 490 nm was measured using the multiwell scanning spectrophotometer [22]. 2.6. Preparation of fluorescent NPCS Cy5-labeled NPCS (Cy5-NPCS) and fluorescein-labeled NPCS (fluorescein-NPCS) were synthesized as per the methods described in the literature [23]. Briefly, a solution of Cy5-NHS or fluorescein-NHS in DMSO (1 mg/mL) was prepared and added gradually into an aqueous NPCS (2 mg/mL) while stirring; the weight ratio of fluorescent dye to NPCS was kept at 1:50 (w/w). The reaction was maintained at pH 5.5 and stirred continuously for 12 h in the dark. To remove the unconjugated fluorescent dyes, the synthesized Cy5-NPCS and fluorescein-NPCS were dialyzed in the dark against deionized water and replaced on a daily basis until no fluorescence was detected in the supernatant. The resultant Cy5-NPCS and fluorescein-CS were lyophilized in a freeze dryer and used to prepare the fluorescent NPCS NPs as described above. 2.7. Internalization of NPCS NPs

2.4. Coarse-grained (CG) MD simulations MD simulations of the self-assembly of NPCS polymers with different DS into NPs in an aqueous environment was performed by a CG-MD method developed by Marrink et al. [17]. CG-MD simulations were accomplished with the program NAMD [18] using parameters adapted from the CHARMM 27 force field [19]. The NCPS polymers used in MD simulations were oligomers consisting of 20 mers. The models were minimized to remove unfavorable contacts, brought to 310 K by velocity rescaling and equilibrated for 1 ns. Before any MD trajectory was run, 40 ps of energy minimization were performed to relax the conformational and structural tensions. This minimum structure was the starting point for the MD simulations. For this purpose, the molecule was embedded into a cubic simulation box of 80 Å. A cutoff distance of 60 Å was employed for the nonbonded and electrostatic interactions. The heating process was performed from 0 to 310 K through Langevin damping with a coefficient of 10 ps− 1. A time step of 2 fs was employed for rescaling the temperature. After 20 ps heating to 310 K, equilibration trajectories of 1 ns were recorded, which provided the data for the structural and thermodynamic evaluations. The equations of motion were integrated with the Shake algorithm with a time step of 1 fs. Figures displaying atomistic pictures of molecules were generated using VMD [20].

The ability of cellular internalization of NPCS NPs was visualized and quantified by CLSM (Cy5-labeled NPCS NPs) and flow cytometry (fluorescein-labeled NPCS NPs), respectively. HT1080 cells were cultured in DMEM media supplemented with 2.2 g/L sodium bicarbonate and 10% FBS. Cells were subcultured according to the ATCC recommendations without using any antibiotics. To track the internalization of test NPs, cells were seeded in 12-well plates at 1 × 105 cells/well and incubated. Subsequently, cells were rinsed twice with serum media (DMEM without FBS, pH 7.0) and replenished with 1 mL serum-free media containing the Cy5labeled NPCS NPs at a concentration of 50 μg/mL. After incubation for 2 h, test samples were aspirated. Cells were then washed twice with the pre-warmed phosphate buffered saline (PBS) before they were fixed in 4% paraformaldehyde. Finally, the fixed cells were counterstained to visualize nuclei by propidium iodide (PI, SigmaAldrich) and examined under a CLSM (TCS SL, Leica, Germany). To quantify the cellular uptake of NPs, cells were plated in 12well plates at 2 × 105 cells/well and incubated with the fluoresceinlabeled NPCS NPs at a concentration of 10 μg/mL for 2 h. After incubation, cells were detached by 0.025% trypsin/ethylenediaminetetraacetic acid (EDTA) and transferred to microtubes. Subsequently, cells were resuspended in PBS containing 1 mM EDTA and

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Table 1 Degrees of substitution on the synthesized N-palmitoyl chitosan (NPCS) determined by the ninhydrin assay and the potassium polyvinylsulfate (PVSK) titration method (n = 5). CS: chitosan; PNS: palmitic acid N-hydroxysuccinimide ester. Samples

NPCS-5% NPCS-10% NPCS-15% NPCS-20%

Feed ratio

Degree of substitution (%)

CS (g)

PNS (g)

Ninhydrin assay

PVSK titration

1.0 1.0 1.0 1.0

0.1 0.2 0.3 0.4

2.1 ± 0.2 10.2 ± 0.1 15.1 ± 0.2 20.5 ± 0.2

5.3 ± 0.1 12.7 ± 0.1 17.0 ± 0.0 20.7 ± 0.0

Table 2 Particle sizes and zeta potential values of test nanoparticles prepared by N-palmitoyl chitosan (NPCS) with different degrees of substitution at pH 7.4 (n = 5). Nanoparticles

NCPS-5%

NPCS-10%

NCPS-15%

NPCS-20%

Particle size (nm) Zeta potential (mV)

207.1 ± 4.3 3.7 ± 0.2

190.6 ± 5.0 3.8 ± 0.1

184.0 ± 2.4 3.9 ± 0.2

168.0 ± 15.2 3.1 ± 0.5

2% FBS and fixed in 4% paraformaldehyde. Finally, the cells were analyzed by a flow cytometer equipped with a 488-nm argon laser (Beckman Coulter, Fullerton, CA, USA).

2.8. Endocytosis inhibition The effect of temperature block was studied by pre-incubating the cells at 4 °C for 1 h and then treated with the fluorescein-labeled NPCS NPs (10 μg/mL) for 2 h at 4 °C [24]. To study the effect of various inhibitors on the uptake of NPs, cells were pre-incubated with the following inhibitors individually at concentrations which were not toxic to the cells: 7 μg/mL of chlorpromazine [24–26], 500 nM wortmannin [27], 10 μg/mL of cytochalasin D [24], 3 mM methyl-βcyclodextrin (MβCD) [25,28] and 200 μM genistein [26,29]. In the study, the group without any treatment was used as a background in the flow cytometry analysis, while the groups in the presence of test NPs but without inhibitor treatment were used as controls and their fluorescence intensities were expressed as 100%. Following the preincubation for 1 h, the inhibitor solutions were removed, and the freshly prepared NPCS NPs (fluorescein-labeled) in media containing inhibitors at the same concentrations were added and further incubated for 2 h. Subsequently, the cells were washed three times with PBS, collected according to the methods described above and analyzed by flow cytometry.

2.10. Statistical analysis Comparison between two groups was analyzed by the one-tailed Student's t-test using statistical software (SPSS, Chicago, Ill, USA). Data are presented as mean ± SD. A difference of P b 0.05 was considered statistically significant.

3. Results and discussion Hydrophobically-modified polymers, which are water-soluble polymers bearing hydrophobic side chains, have found an increasing number of biological applications [30–32]. Because the contact between the hydrophobic side chains and water is energetically unfavorable, the hydrophobically-modified polymers have a strong tendency to self-assemble into aggregates on the nanometer scale in an aqueous environment [33]. In this study, we synthesized hydrophobically-modified CS (NPCS) with different DS for the preparation of NPs; their characteristics, cellular uptake and intracellular trafficking were thoroughly investigated.

3.1. Characterization of NPCS and test NPs The synthesized NPCS was analyzed by 1H NMR and FT-IR; the results, presented in our previous report [3], indicated that the palmitoyl group was successfully conjugated onto the free amine groups of CS. The DS on the synthesized NPCS polymers, determined by the ninhydrin assay and the PVSK titration method, are listed in Table 1. As shown, with increasing the amount of palmitic acid Nhydroxysuccinimide ester used during synthesis, the DS on NPCS polymers increased significantly (P b 0.05). It was noted that the reactant solution was too viscous to be manipulated with when attempting to synthesize NPCS with a DS more than 20%. In an aqueous environment at pH 7.4, CS formed dissociated precipitates because the aggregation of CS polymers occurred too rapidly and locally. In contrast, the hydrophobic palmitoyl groups were inclined to form local aggregates that acted as physical crosslinks between NPCS polymers, thus producing NPs spontaneously. With increasing the DS on NPCS polymers, the diameter of the prepared NPs decreased significantly due to a stronger hydrophobic interaction formed between the conjugated palmitoyl groups (P b 0.05), while their zeta potential values were comparable (P N 0.05, Table 2).

2.9. Intracellular trafficking To study the intracellular trafficking of test NPs, cells were treated with the Cy5-labeled NPCS NPs in the serum-free medium. After incubation at predetermined time points, cells were washed twice with the pre-warmed PBS before they were fixed in 4% paraformaldehyde. The fixed cells were investigated using the immunohistochemical stains to identify caveolae, caveosomes, Golgi apparatus, endoplasmic reticula (ER), early endosomes and lysosomes. The antibodies used in the study were polyclonal rabbit anti-caveolin-1 antibody (Cell Signaling Technology #3238, Beverly, MA, USA), mouse monoclonal anti-giantin antibody (Abcam #ab37266, Cambridge, MA, USA), mouse monoclonal anti-calnexin antibody (Abcam #ab31290), rabbit polyclonal anti-EEA1 antibody (Abcam #ab2900) and monoclonal mouse anti-LAMP2 antibody (Abcam #ab25631). The stained cells were counterstained to visualize nuclei by PI and examined using CLSM.

Fig. 1. Schematic illustrations showing the self-assembly of N-palmitoyl chitosan (NPCS) polymers into nanoparticles (NPs) via the coarse-grained (CG) molecular dynamic (MD) simulations and the potential cellular uptake mechanism and the intracellular trafficking pathway of NPCS NPs.

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Fig. 2. (a) Results of the coarse-grained molecular dynamic simulations of the self-assembly of N-palmitoyl chitosan (NCPS) with different degrees of substitution into nanoparticles (NPs) at pH 7.4; (b) schematic illustrations of the interaction of NPCS NPs with the cell membrane (lipid rafts and caveolae).

3.3. Cytotoxicity of test NPs

Fig. 3. Results of the cell viability after being treated with nanoparticles of N-palmitoyl chitosan (NPCS) with different degrees of substitution, determined by (a) the MTT assay and (b) the LDH assay (n = 5). Control: the group without any treatment; CS: the group treated with chitosan dissociated precipitates.

To examine the cytotoxicity, we incubated HT1080 cells with test NPs for 24 h; the viability of cells without coincubation with test NPs was used as a control. The mitochondrial activity of living cells was measured by the MTT assay. As shown in Fig. 3a, no significant differences in mitochondrial activity were observed between the groups treated with different test samples and the control (P N 0.05). Issues of cytotoxicity are ultimately dependent on the degree of membrane disruption [22]. The LDH assay is a means of measuring the membrane integrity as a function of the amount of cytoplasmic LDH leaked into the medium [22]. Compared with the control, the LDH leakage in the medium for the groups treated with different test NPs did not significantly increase (P N 0.05, Fig. 3b). These results indicate that there was no significant cytotoxicity observed for the NPs prepared by NPCS polymers with different DS. 3.4. Cellular uptake of test NPs

3.2. CG-MD simulations In the CG models, small groups of atoms are treated as single beads: each blue bead represents 1 unit of sugar ring on the CS backbone, while 4 red beads stand for 1 palmitoyl side chain (Fig. 1). The beads interact with each other via the screened Lennard-Jones and Coulombic potentials. This model leads to an approximately 100fold increase in speed with respect to atomistic simulations [34], therefore allowing the capture of the self-assembly process of NPCS into NPs. This methodology has recently been extended to enable simulations of the self-assembly of protein and detergent into mixed micelles [35,36]. Results of the CG-MD simulations confirmed that CS formed dissociated precipitates at pH 7.4, while a DS of 5% of palmitoyl groups on its backbone was sufficient to allow NPCS to form NPs due to a significant increase in the intra- and intermolecular hydrophobic interactions. At pH 7.4, the backbone of NPCS was deprotonated and became relatively hydrophobic; therefore, the conjugated hydrophobic palmitoyl groups had the opportunity to expose to the surface of NPs. With an increase of DS, there were more palmitoyl groups present on the surface of NPs (Fig. 2a). These surface palmitoyl groups were then able to interact with the cell membranes (Fig. 2b).

In this part of the study, Cy5-labeled NPCS polymers with different DS were synthesized and employed to prepare test NPs for the CLSM investigation. CLSM images of cells taken at 2 h after exposure to test NPs are shown in Fig. 4a. Intracellular accumulation of Cy5-labeled NPs was observed; the fluorescence intensity observed in cells increased notably with increasing the DS of NPCS used in the preparation of NPs. The percentage of cells that internalized the fluorescein-labeled NPs and their fluorescence intensity were quantified by flow cytometry and the results are shown in Fig. 4b. Among all studied groups, the group treated with CS dissociated precipitates had the lowest cellular uptake (P b 0.05, Fig. 4c and d). For the groups treated with NPs of NPCS with different DS, there were no significant differences in the percentage of cells that internalized the fluoresceinlabeled NPs (P N 0.05). However, the fluorescence intensity was significantly enhanced with increasing the DS of NPCS (P b 0.05), an indication of a greater extent of cellular uptake of test NPs. 3.5. Endocytosis pathway of test NPs Endocytic uptake is an energy-dependent mechanism; it can be strongly inhibited by lowering the temperature [24,37]. In order to

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investigate if the cellular uptake of NPs was an energy-dependent process, a cellular uptake study was carried out at 4 °C. Upon reducing the incubation temperature, the cellular uptake reduced significantly (P b 0.05, Fig. 5a) in comparison to the control (at 37 °C) but did not cease completely, indicating that NPCS NPs were taken up inside the cells via energy-dependent pathways. To elucidate their potential cellular uptake pathways, the interaction between NPCS NPs (fluorescein-labeled) and cell mem-

branes was investigated by treating cells with different chemical inhibitors of clathrin-mediated endocytosis, macropinocytosis and caveolae-mediated endocytosis and then analyzed by flow cytometry. Their counterparts in the absence of inhibitors were used as controls. Inhibition of clathrin-mediated uptake was tested by using the cationic amphiphilic drug chlorpromazine, which causes clathrin to accumulate in late endosomes, thereby inhibiting coated pit endocytosis [24–26,38]. Compared with the control, the cellular uptake of

Fig. 4. Results of CLSM observation and flow cytometry analysis of the cellular uptake of nanoparticles (NPs) of N-palmitoyl chitosan (NPCS) with different degrees of substitution. (a) Confocal images (scale bar, 20 μm) of cells after exposure to test NPs of Cy5-labeled NPCS with different degrees of substitution for 2 h; (b) intracellular fluorescence intensities of test NPs of NPCS with different degrees of substitution determined by flow cytometry; (c) percentages of cells internalized (n = 5); and (d) intracellular fluorescence intensities (n = 5). NC: negative control (the group without any treatment).

NPCS-5% NPs increased significantly in the presence of chlorpromazine (P b 0.05, Fig. 5b). Increase in cell uptake after inhibitor treatment has also been reported by other investigators [24,38,39]. It is possible that other cellular uptake pathways that are not normally involved may be up-regulated in the presence of inhibitors. In contrast, those treated with NPCS-10% and NCPS-15% NPs stayed almost the same (P N 0.05), an indication of non-clathrin-mediated endocytosis. On the other hand, the fluorescence intensity for the group treated with NPCS-20% NPs in the presence of chlorpromazine showed a 13% inhibition (P b 0.05), indicating that clathrin-mediated uptake may be involved. Wortmannin is a phosphatidyl inositol-3-phosphate inhibitor, which can inhibit macropinocytosis [27]. Cytochalasin D is known to inhibit actin polymerization and membrane ruffling, which is involved in macropinocytosis [24]. Cellular uptakes of NPCS-5% and NPCS-10% NPs were increased in the presence of either inhibitor (Fig. 5c and d), indicating that none of these two NPs appeared to be taken up by macropinocytosis. In contrast, cells treated with wortmannin or cytochalasin D caused a significant decrease in cellular uptake of NPCS-15% and NPCS-20% NPs by about 10% and 20−30%, respectively (P b 0.05), suggesting that macropinocytosis may be involved in the uptake of NPCS NPs with a higher DS. Lipid rafts are cholesterol- and sphingolipid-enriched microdomains found in cell membranes. It was reported that cholesterol and

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lipid rafts are involved in membrane trafficking [28,40]. The packing of cholesterol with the saturated acyl chains of sphingolipids is thermodynamically favored over that with unsaturated acyl chains, and cholesterol is essential to the process of raft formation. If either sphingolipid or cholesterol is depleted, the other follows and vice versa [40]. In order to examine the role of lipid rafts in the uptake of NPCS NPs, the plasma membrane cholesterol was depleted. MβCD, a water-soluble cyclic oligomer of glucopyranoside and an inhibitor of lipid rafts, acts strictly on the cell surface, selectively extracting cholesterol without being incorporated into the plasma membrane [25,28]. As compared to the controls, cells treated with MβCD significantly diminished the fluorescence intensity in all studied groups (P b 0.05, Fig. 5e), showing that the internalization of NPCS NPs was related with cholesterol and lipid rafts. Caveolae, a specialized type of lipid rafts, are flask-shaped invaginations in the plasma membrane enriched in proteins as well as cholesterol and sphingolipids [41,42]. Genistein, a tyrosine kinase inhibitor, is used to block caveolae-mediated endocytosis [26,29]. As shown in Fig. 5f, treatment with genistein resulted in a significant inhibition of uptake of NPCS NPs (P b 0.05); the inhibition in cellular uptake of NPs was more remarkable with an increase of DS on NPCS polymers. These results suggest that the internalization of NPCS NPs was clearly related with the lipid raft-mediated routes; with increasing the DS on NPCS polymers, the caveolae-mediated endocytosis became more important (Fig. 2b). Little is known about how cargo is selected for the caveolaemediated endocytosis. Surface properties of cargos play a key role in the cellular uptake and can affect the internalization process. It was reported that poly(D,L-lactide-co-glycolide) NPs with a hydrophobic surface were taken up to a greater extent via endocytosis than their counterparts with a hydrophilic surface [43]. Additionally, it is known that palmitoylated proteins, a change from the hydrophilic nature of proteins to one that is hydrophobic, are able to target to lipid rafts [44,45]. This might explain why a greater extent of cellular uptake was observed for the NPs of NPCS with a higher DS (Fig. 4d), which had more hydrophobic palmitoyl groups present on their surfaces (Fig. 2a). 3.6. Intracellular trafficking of test NPs

Fig. 5. Effects of inhibitors on the internalization of NPs of NPCS with different degrees of substitution (fluorescein-labeled). Intracellular fluorescence intensities (n = 5) after the cells were treated (a) at 4 °C or 37 °C or in the presence of (b) chlorpromazine (7 μg/mL); (c) wortmannin (500 nM); (d) cytochalasin D (10 μg/mL); (e) MβCD (methyl-βcyclodextrin, 3 mM); and (f) genistein (200 μM). Control: the groups in the presence of test NPs but without inhibitor treatment were used as controls and their fluorescence intensities were expressed as 100%.

Caveolae are characterized by the presence of a family of caveolin proteins including caveolin-1 (CAV1). CAV1 is a 21 kDa integral membrane protein that functions as a principal structural component of invaginated caveolae in most mammalian cells [41,42]. To further support the entry of NPCS NPs via the caveolae-mediated pathway, the potential co-localization of CAV1 and test NPs during and after entry into cells was investigated. In the study, caveolae were identified by immunohistochemical staining using CAV1 antibody (in green, Fig. 6). As early as 15 min after incubation, NPCS NPs (in purple) were observed in cell membranes and co-localized with CAV1 (in white). After incubation for 30 min, the internalized NPs were found associated with CAV1-positive structures (in white) near the cell periphery, whereas at a later time point (2 h after), the NPs located in peri-nuclear regions were no longer co-localized with CAV1. These results suggest that the internalized NPs transiently associate with CAV1 at cell membranes and at a peripheral CAV1positive structure coupled with caveosomes before trafficking to a CAV1 free intracellular compartment (Fig. 1). Cargo that traffics through the caveosome may be transported to the Golgi apparatus/ER [41,42] or the early endosome [41,42,46]. As shown in Fig. 7, co-localization of NPs (in purple) with either the Golgi apparatus (Gaintin, a Golgi marker, in green) or the ER (Calnexin) was not observed throughout the entire course of the study. Instead, high levels of NPs were found co-localized with the early endosomes (EEA1, in white) at 1 h after incubation; the early endosomes then matured into late endosomes or multi-vesicular bodies, and then to lysosomes (LAMP2, in white). The aforementioned results suggest that NPCS NPs

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Fig. 6. Confocal images (scale bar, 20 μm) of the intracellular trafficking of NPCS NPs at the indicated times using the immunohistochemical stains to identify caveolae and caveosomes. Area defined by a square is shown at a higher magnification in the inset.

entered cells via caveolae and transiently localized to caveosomes before trafficking to the endosomal pathway. It has been proposed that targeting NPs to the endolysosomal pathway has potential for improving drug delivery for the treatment of lysosomal storage diseases, cancer and Alzheimer's disease [47].

Acknowledgements This work was supported by a grant from the National Science Council (NSC 98-2120-M-007-007), Taiwan, Republic of China.

References 4. Conclusions In conclusion, the extent of cellular uptake of NPs was significantly enhanced with increasing the DS on NPCS. The internalization of NPCS NPs was clearly related with the lipid raft-mediated routes. With increasing the DS on NPCS polymers, the caveolae-mediated endocytosis became more important. The internalized NPs transiently associate with CAV1 at cell membranes and at a peripheral CAV1positive structure coupled with caveosomes before trafficking to the endosomal pathway.

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Fig. 7. Confocal images (scale bar, 20 μm) of the intracellular trafficking of NPCS NPs at the indicated times using the immunohistochemical stains to identify the Golgi apparatus (Giantin), endoplasmic reticula (Calnexin), early endosomes (EEA1) and lysosomes (LAMP2). Area defined by a square is shown at a higher magnification in the inset.

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