Surface Modification of PAMAM Dendrimers Modulates the

Bioconjugate Chem. 2009, 20, 693–701

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Surface Modification of PAMAM Dendrimers Modulates the Mechanism of Cellular Internalization Angkana Saovapakhiran,† Antony D’Emanuele,‡ David Attwood,† and Jeffrey Penny*,† School of Pharmacy and Pharmaceutical Sciences, The University of Manchester, Oxford Road, Manchester M13 9PT, and School of Pharmacy and Pharmaceutical Sciences, University of Central Lancashire, Preston, Lancashire PR1 2HE, United Kingdom. Received June 13, 2008; Revised Manuscript Received January 27, 2009

The aim of this study was to investigate the influence of dendrimer surface properties on cellular internalization and intracellular trafficking in the human colon adenocarcinoma HT-29 cell line. Third-generation (G3) polyamidoamine (PAMAM) dendrimers were modified to contain either two lauroyl chains (G3L2), two propranolol molecules (G3P2), or two lauroyl and two propranolol molecules (G3L2P2) at the dendrimer surface. Surfacemodified and unmodified dendrimers were labeled with fluorescein isothiocyanate (FITC) at an average molar ratio of 1:1. The mechanisms of cellular internalization and intracellular trafficking of dendrimers were analyzed by confocal laser scanning microscopy and flow cytometry. The internalization of G3 and G3P2 dendrimers involved both caveolae-dependent endocytosis and macropinocytosis pathways; internalization of G3L2P2 dendrimer appeared to involve caveolae-dependent, and possibly clathrin-dependent, endocytosis pathways; and internalization of G3L2 dendrimer occurred via caveolae-dependent, clathrin-dependent, and macropinocytosis pathways. Subcellular colocalization data indicated that unmodified and all surface-modified G3 PAMAM dendrimers were internalized and trafficked to endosomes and lysosomes. It is therefore apparent that the initial mode of dendrimer internalization into HT-29 cells is influenced by the surface properties of G3 PAMAM dendrimer.

INTRODUCTION Due to their highly branched nature and the presence of multivalent surface groups that can be conjugated with drugs, polyamidoamine (PAMAM) dendrimers offer significant potential as versatile carriers for drug delivery (1). Our previous studies have established that the conjugation of propranolol, a P-glycoprotein substrate with poor bioavailability, to cationic PAMAM dendrimer increased drug solubility and enhanced the permeation of propranolol across Caco-2 cell monolayers (2). Numerous studies have reported that surface modification of dendrimers can cause significant changes in their biological properties, including cytoxicity and permeation through cell monolayers (2-6). Addition of lauroyl moieties to cationic PAMAM dendrimers has been reported to increase transcellular apical-to-basal transport of both dendrimer and dendrimer-drug conjugates in Caco-2 cells (2-4, 7, 8). Addition of FITC onto cationic G4 PAMAM dendrimer at a ratio of 8:1 resulted in a decrease in dendrimer cytotoxicity and significant increase in permeation through Caco-2 cell monolayers (5). A study of the influence of surface acetylation of cationic G2 and G4 PAMAM dendrimer on the viability of Caco-2 cells revealed that acetylation resulted in a significant decrease in cytotoxicity. Cytotoxicity decreased as the number of surface acetyl groups increased while the permeability across cell monolayers was maintained (6). These findings reveal that PAMAM dendrimers offer potential as nanoparticulate drug delivery systems. However, although surface modification is able to enhance transcellular dendrimer permeation, little is known of the precise influence of surface modification on the mechanism(s) of dendrimer cellular internalization and intracellular trafficking. * Corresponding author. E-mail: [email protected]; Telephone: +44 (0)161 275 8344; Fax: 0161 275 8349. † The University of Manchester. ‡ University of Central Lancashire.

PAMAM dendrimers are able to traverse cell monolayers via paracellular and transcellular pathways (2, 4, 5, 9, 10), and the endocytosis pathway is believed to be an important route for dendrimer internalization. Kitchens et al. (11, 12) reported that uptake of cationic and anionic (G2, G4, and G1.5) PAMAM dendrimers into Caco-2 cells was via clathrin-dependent endocytosis, whereas cationic G4 PAMAM dendrimer were predominately internalized via a cholesterol-dependent route in B16F10 melanoma cells (13). In this study, we investigated the effect of conjugating lauroyl and/or propranolol groups onto the surface of G3 PAMAM dendrimers on the mechanism of cellular internalization and subsequent intracellular trafficking in HT-29 cells, which are capable of clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis (14-19). G3 PAMAM dendrimers were employed since they are less cytotoxic than both G4 and G5 PAMAM dendrimers (3, 4) and were chosen in preference to G2 dendrimers, because the latter possess significantly fewer surface groups available for attachment of surface moieties. In addition, G2 and anionic dendrimers, e.g., G2.5, G3.5, demonstrate less apparent permeation through cell monolayers than G3 dendrimer (4). A better understanding of the influence of the surface properties of dendrimers on their internalization mechanisms is important in the development of dendrimer-mediated drug delivery systems.

EXPERIMENTAL PROCEDURES Materials. G3 PAMAM dendrimer was purchased from Dendritech Inc. (Michigan, USA). Sephadex G-25, fluorescein isothiocyanate (FITC), chlorpromazine hydrochloride, filipin III, 5-(N-ethyl-N-isopropyl) amiloride, nystatin, trypan blue, formaldehyde, and bovine serum albumin were purchased from Sigma-Aldrich Co. Ltd. (Gillingham, Dorset, UK). Saponin was obtained from Fluka (Steinheim, Germany). Cell culture materials, Lysotracker Red DND-99, Alexa Fluor 555 rabbit antigoat

10.1021/bc8002343 CCC: $40.75  2009 American Chemical Society Published on Web 03/09/2009

694 Bioconjugate Chem., Vol. 20, No. 4, 2009

IgG (heavy and light chains), Alexa Fluor 555 goat antirabbit IgG (heavy and light chains), and Alexa Fluor 555 conjugated transferrin (from human serum) were obtained from Invitrogen (Paisley, UK). Caveolin-1 (N-20) rabbit polyclonal IgG and tetramethylrhodamine-5-isothiocyanate (TRITC)-clathrin HC (C20) goat polyclonal IgG were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). Cytotoxicity detection kit (lactate dehydrogenase, LDH) was purchased from Roche Applied Science (Mannheim, Germany). All other chemicals were purchased from BDH Laboratory Supplier (Poole, Dorset, UK). Synthesis of Surface-Modified G3 PAMAM Dendrimers. The synthesis of surface-modified PAMAM dendrimers containing two lauroyl chains (G3L2) was performed using 4-nitrophenyl chloroformate as a linker (7). Lauroyl alcohol, 200 mg (1 mmol), was dissolved in 5 mL of anhydrous tetrahydrofuran (THF), and triethylamine (TEA) 0.3 mL (2.1 mmol) was added. 4-Nitrophenylchloroformate, 417 mg (2 mmol), was dissolved in 1.5 mL of anhydrous THF, added dropwise to the mixture, and stirred for 24 h under nitrogen at room temperature. Following evaporation of solvent, the residue was dissolved in hexane/diethylacetate (85:15), filtered (Whatman No. 1), and purified by silica gel column chromatography using a hexane/ diethylacetate (85:15) mobile phase. The mobile phase was applied with a flow rate of 120 mL per h, and 5 mL fractions were collected and analyzed by TLC (using the same mobile phase) to identify the purified compound. Purified lauroyl 4-(nitrophenyl carbonate) was characterized by 1H NMR as described previously (CDCl3): 0.88 (3H, t, J ) 6.8, CH3), 1.27-1.42 (18H, m, (CH2)8), 1.76 (2H, pentet, J ) 7.0, CH2), 4.29 (2H, t, J ) 6.6, CH2COO), 7.32-7.39 (2H, m, Ar-H2 + Ar-H6), 8.27-8.30 (2H, m, Ar-H3 + Ar-H5) (7). G3 PAMAM dendrimer, 0.21 mg (0.03 mmol), was dissolved in 20 mL of anhydrous N,N-dimethylformamide (DMF). Lauroyl (4-nitrophenyl carbonate), 0.06 mmol (2 lauroyl molecules/1 G3 PAMAM dendrimer), was dissolved in 5 mL of anhydrous DMF and added dropwise to the reaction mixture (Figure 1). The reaction was carried out, with stirring, at room temperature for 5 days. Lauroyl-G3 PAMAM conjugate (G3L2) (Figure 1-(1)) was purified by chromatography using Sephadex LH20 with a methanol/water (4:1) mobile phase applied at a flow rate of 90 mL per h. 1.5 mL samples were collected and absorbance measured at 230 nm. The ratio of lauroyl to G3 PAMAM dendrimer was verified by using 1H NMR. The yield of G3L2 was 66.23% (w/w). 1H NMR (d4-MeOD): 0.89 (6H, CH3), 1.29 (36H, (CH2)9), 1.43 (4H, CH2), 2.38 (120H, CH2CdONH), 2.58 (120H, CH2NH + CH2N), 2.80 (124H, NCH2), 3.31 (120H, OdCNHCH2), 4.00 (4H, CH2COO). Synthesis of G3 PAMAM dendrimer conjugates containing either two propranolol molecules (G3P2) (Figure 1-(2)) or two propranolol molecules and two lauroyl molecules (G3L2P2) (Figure 1-(3)) employed chloroacetyl chloride as a linker (2). Propranolol base, 7.6 mmol, was dissolved in 20 mL of anhydrous dichloromethane (DCM) and TEA, 22.8 mmol, was added. Chloroacetyl chloride, 7.6 mmol, was added slowly and the reaction stirred for 1 h on ice. The dark brown solution obtained was sequentially washed once with equal volumes of saturated solutions of sodium bicarbonate and then sodium chloride. The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The crude residue was dissolved in mobile phase (hexane/diethylacetate (1:1)) and purified by silica gel column chromatography. The mobile phase was applied with a flow rate of 120 mL per h and 5 mL fractions collected and analyzed by TLC to identify fractions containing purified propranolol-N-chloroacetyl. Characterization of propranolol-N-chloroacetyl was by 1H NMR and 13C NMR. The yield of propranolol-N-chloroacetyl was 70.05% (w/w). 1H NMR

Saovapakhiran et al.

Figure 1. Synthetic scheme for surface modification of G3 PAMAM dendrimers.

(CDCl3): 1.30 (6H, dd J ) 26.06 Hz, J ) 6.71 Hz, (CH3)CH), 3.66 (1H, dd, J ) 14.6 Hz, J ) 1.9 Hz (CH3)CH), 3.72 (1H, dd, J ) 14.6 Hz, J ) 7.8 Hz (CH3)CH), 4.09 (1H, m, CHN), 4.17 (2H, s, CH2Cl), 4.21 (2H, m, CH2O), 4.26 (1H, m, CHO), 6.84 (1H, d, J ) 7.47 Hz, 1H, Ar-H2), 7.45 (4H, m, Ar-H3 + Ar-H4 + Ar-H7 + Ar-H8), 7.79 (1H, m, Ar-H6), 8.20 (1H, m, Ar-H9). 13C NMR: 21.0 (2 × CH3), 41.0 (CH2Cl), 46.1 (CH2NCH), 49.8 ((CH3)2CHN), 69.4 (CHOH), 71.3 (CHCH2O), 104.4 (CH, Ar), 120.3 (CH, Ar), 121.1 (CH, Ar), 124.8 (CH, Ar), 125.5 (CH, Ar), 126.0 (CH, Ar), 126.1 (C, Ar), 127.6 (CH, Ar), 134.0 (C, Ar), 153.5 (C, Ar), 168.9 (ONCCH2). In order to conjugate propranolol to dendrimer, either G3 PAMAM dendrimer, 0.21 mg (0.03 mmol), or G3L2 PAMAM dendrimer, 0.22 mg (0.03 mmol), was dissolved in 15-20 mL of anhydrous DMF. Propranolol-N-chloroacetyl, 0.06 mmol (1 propranolol molecules/1 G3 PAMAM dendrimer or 1 G3L2 PAMAM dendrimer), was dissolved in 5 mL of anhydrous DMF, added dropwise to the mixture, and stirred at room temperature for 12 h. Propranolol-G3 PAMAM conjugate (G3P2) was purified by column chromatography using Sephadex LH20 with methanol/water (4:1) mobile phase. The latter was applied at a flow rate of 90 mL per h. A series of 1.5 mL samples were collected and absorbance measured at 230 nm. The number of propranolol molecules attached was confirmed by using 1H NMR. The yields of G3P2 and G3L2P2 were 31.01% and 54.37% (w/w), respectively. G3P2 (d4-MeOD): 1.286-1.308 (12H, (CH3)2CH), 2.372 (120H, CH2CdONH), 2.583 (120H, CH2NH + CH2N), 2.789 (124H, NCH2), 3.272-3.346 (120H, OdCNHCH2), 3.593-4.695 (16H, CH2NCH + CHN + CH2O +HNCH2CdON+CHO),6.878-7.046(2H,Ar-H2),7.376-7.483 (8H, Ar-H3 + Ar-H4 + Ar-H7 + Ar-H8), 7.803-7.814 (2H,

Mechanisms of Dendrimer Internalization

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Figure 2. 1H NMR spectra (270 MHz) of (a) G3, (b) G3P2, (c) G3L2, and (d) G3L2P2 PAMAM dendrimers (d4-MeOD).

Ar-H6), 8.293 (2H, Ar-H9). G3L2P2 (d4-MeOD): 0.89 (6H, CH3), 1.23 (48H, (CH2)9 + (CH3)2CH), 1.54-1.67 (4H, CH2), 2.46-2.53 (120H, CH2CdONH), 2.60 (120H, CH2NH + CH2N), 2.87 (124H, NCH2), 3.05-3.06 (120H, OdCNHCH2), 3.36-3.53 (16H, CH2NCH + CHN + CH2O + NHCH2CdON + CHO), 3.94-4.19 (4H, CH2COO), 7.43-7.58 (8H, Ar-H3 + Ar-H4 + Ar-H7 + Ar-H8), 7.85 (2H, Ar-H6), 8.23 (2H, Ar-H9). Preparation and Characterization of FITC-Labeled G3 PAMAM Dendrimers. G3 PAMAM dendrimer and surfacemodified dendrimers were labeled with FITC via a thiourea bond between the isothiocyanate group of FITC and an amine group of PAMAM dendrimer, as described previously (4). Briefly, 15 mL of a methanolic solution of FITC containing 0.036 mmol was slowly added to 0.030 mmol of G3 PAMAM dendrimer (a molar ratio 1.2:1 of PAMAM dendrimer to FITC) in 15 mL PBS and stirred at room temperature for 24 h in the dark. FITClabeled G3 PAMAM dendrimer was purified by size exclusion chromatography on a Sephadex G-25 column (mobile phase: phosphate buffered saline (PBS)). One milliliter fractions were collected and those containing FITC-dendrimer conjugate identified using TLC (mobile phase: chloroform/methanol/ ammonia, 5:4:1). Fractions containing FITC-dendrimer were pooled and dialyzed against distilled water for 24 h to remove salts. Manipulations involving FITC-labeled dendrimers were carried out in a low-light environment. The ratio of FITC to dendrimer was confirmed using 1H NMR. Analysis of size distribution of FITC-labeled G3 dendrimer and surface-modified dendrimers was performed using dynamic light scattering (Zetasizer Nano, Malvern Instruments, UK) (4, 20). Samples were dissolved in PBS (10 mg/mL) and filtered through a Nalgene filter (0.2 µm pore size) into the scattering cell. All measurements were carried out at 37 °C. Diameters are represented as mean ( standard deviation of triplicate measurements. Cell Culture. HT-29 cells (passage number 36-67) were maintained in Dulbecco’s Modified Eagles Medium (DMEM) containing 10% (v/v) fetal calf serum, 2 mM L-glutamine, 10 mM nonessential amino acids, 50 IU/mL penicillin, and 50 µg/ mL streptomycin at 37 °C in an atmosphere of 5% CO2. LDH Assay. Approximately 1 × 104 HT-29 cells/well were cultured in 96-well plates overnight. The cells were treated with

chlorpromazine (5-50 µM), filipin (0.25-5 µg/mL), nystatin (5-50 µM), and EIPA (50-250 µM) for 4 h. To measure dendrimer cytotoxicity, 100 µL of supernatant was transferred into an optically clear 96-well plate. The LDH reaction mixture was freshly prepared according to the manufacturer’s instruction (Roche Diagnostics), 100 µL added to each well, and the plate incubated for 30 min at 25 °C. The absorbances at 450 and 650 nm were measured using a multiplate reader (MRX, Dynatech Laboratories, Guernsey, UK). Determination of the Effect of Endocytosis Inhibitors on Uptake of G3 PAMAM Dendrimers. Chlorpromazine (inhibitor of clathrin-dependent endocytosis) (21, 22), nystatin and filipin (inhibitors of caveolae-dependent endocytosis) (23-25), and ethyl isopropyl amiloride (inhibitor of macropinocytosis) (26, 27) were dissolved in Opti-MEM I medium containing 12.5 mM HEPES, pH 7.4. Approximately 1 × 106 HT-29 cells/well were seeded in 6-well plates and incubated at 37 °C in an atmosphere of 5% CO2 until cells reached 80% confluency. Cells were washed twice with PBS and pretreated with Opti-MEM I medium with 12.5 mM HEPES pH 7.4 containing inhibitor (at the concentrations indicated in Figure 4) for 30 min at 37 °C in an atmosphere of 5% CO2. FITC-labeled G3 PAMAM dendrimer and FITC-labeled surface-modified PAMAM dendrimers were added at a final concentration of 2.5 µM and cells incubated for 1 h. Following a 1 h incubation, cells were washed three times with ice-cold PBS and incubated with FITC-quench solution (PBS containing 0.4% w/v trypan blue) for 10 min to quench extracellular fluorescence. Cells were washed three times with ice-cold PBS and treated with 0.25% (v/v) trypsin-EDTA solution for 5 min at 37 °C. The cell suspension was centrifuged at 12 000 g for 3 min, the pellet resuspended in PBS, and intracellular fluorescence analyzed immediately by flow cytometry (Dako Cytomation, Glostrup, Denmark). A flow rate of less than 200 events/s and an excitation wavelength of 488 nm (530/ 40 bandpass filter) were employed. Subcellular Localization of FITC-Labeled G3 PAMAM Dendrimers. HT-29 cells were seeded onto collagen-coated coverslips and grown to 70% confluency. DMEM growth medium was replaced with Opti-MEM I medium with 12.5 mM HEPES, pH 7.4, containing 2.5 µM FITC-labeled G3 PAMAM dendrimer or FITC-labeled surface-modified PAMAM den-



Figure 3. 1H NMR spectra (270 MHz) of (a) FITC-labeled G3 PAMAM dendrimer and size distribution of (b) FITC-labeled G3, (c) FITC-labeled G3P2, (d) FITC-labeled G3L2, and (e) FITC-labeled G3L2P2 PAMAM dendrimers.

drimers. In immunofluorescence studies, cells were incubated for 1.5 h with dendrimer. In endosome localization studies, cells were incubated for 1 h, while in lysosome localization studies, cells were incubated for 12 h (G3), 3 h (G3P2), 6 h (G3L2), or 12 h (G3L2P2). Growth medium was removed, and cells were washed three times with ice-cold PBS. For detection of clathrin and caveolin-1, HT-29 cells were fixed with 4% (v/v) formaldehyde in PBS for 10 min. Free aldehyde groups were quenched by incubating with 50 mM NH4Cl for 10 min, and cells were then permeabilized by treatment with 0.1% (w/v) saponin in PBS. After three washes with PBS, permeabilized cells were incubated with PBS containing 5% (w/v) BSA to reduce nonspecific antibody binding and then incubated with either anticlathrin heavy-chain goat polyclonal IgG or anticaveolin-1 rabbit polyclonal IgG (1: 100) for 2 h. Cells were washed three times with PBS and incubated with either Alexa Fluor 555 rabbit antigoat IgG or Alexa Fluor 555 goat antirabbit IgG (1:100) for 1 h. After thorough washing with PBS, coverslips were mounted onto microscope slides using mounting medium. Cell-associated fluorescence was analyzed by confocal laser scanning microscope. Transferrin was used as a positive control of receptormediated endocytosis and trafficking into endosomes. Cells were incubated with Alexa Fluor 555-transferrin (50 µg/mL) in OptiMEM I medium at 37 °C in an atmosphere of 5% CO2 for either 10 min (G3P2, G3L2, G3L2P2) or 60 min (G3). Cells were washed with ice-cold PBS containing 0.5% w/v BSA and then with ice-cold PBS. Cells were fixed immediately with 4% w/v formaldehyde in PBS and treated with 50 mM NH4Cl to quench residual formaldehyde fluorescence. Following further washing with PBS, coverslips were mounted onto microscope slides using mounting medium. Alexa Fluor 555-transferrin was visualized using confocal laser scanning microscopy. For detection of lysosomes, HT-29 cells were incubated in Opti-MEM I medium containing Lysotracker Red DND-99 for 45 min at 37 °C. Cells were washed three times with PBS prior to confocal laser scanning microscopy. Confocal Laser Scanning Microscopic Analysis and Image Processing. Cells were examined with a Zeiss LSM 510 Meta ConfoCor 2 using 63× oil immersion objective. FITC-

labeled G3 PAMAM dendrimer or FITC-surface-modified PAMAM dendrimers were analyzed using an argon laser (excitation 488 nm; emission 505-550 nm). Lysotracker Red DND-99 or Alexa Fluor 555 labeled antibodies and Alexa Fluor 555-transferrin were analyzed using a helium/neon laser (excitation 543 nm; emission 560 nm). Images and a gallery of 30 optical sections (0.5 µm) through the z plane were collected and processed using Combi LSM-FCS and LSM Image Browser v4.0 software (Germany). Colocalization studies were carried out on three discrete regions of the microscope slide (with between 3 and 6 individual cells analyzed per slide). Data were analyzed using ImageJ v 1.410 (National Institutes of Health, USA). Channel overlap was determined by calculation of Pearson’s coefficient and the true extent of three-dimensional colocalization defined by the Overlap Coefficient. Statistical Analysis. Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Dunnett’s test (SPSS for Windows version 10.0). Probability values of P < 0.05 were considered to be significant.

RESULTS Conjugation of Dendrimers with Lauroyl and Propranolol Molecules. 1H NMR studies revealed that, on average, G3 PAMAM dendrimer (Figure 2a) was successfully conjugated with two propranolol molecules (Figure 2b), with two lauroyl chains (Figure 2c), and with both two lauroyl chains and two propranolol molecules (Figure 2d). The successful conjugation of lauroyl chains to the G3 PAMAM dendrimer is indicated by the additional peaks at the chemical shift 0.89-1.43 ppm, which confirmed the attachment of lauroyl chain onto G3 PAMAM dendrimer (Figure 2b). The number of lipid chains was calculated by comparing the peak integrations of the chemical shift at 1.29 ppm of lauroyl and at 2.38 ppm of PAMAM dendrimer (Figure 2b). Attachment of propranolol molecules to dendrimer is indicated by the additional peaks at the chemical shifts 1.29-1.31 ppm, 3.59-4.70 ppm, and 6.88-8.29 ppm. The integration of the peak between 7.43 and 7.49 ppm of propranolol and 2.38 ppm of PAMAM dendrimer revealed that two propranolol molecules were attached to G3 PAMAM



Figure 4. Effect of endocytosis and macropinocytosis inhibitors chlorpromazine 50 µM (CMZ), filipin 1 µg/mL (F), nystatin 50 µM (NYS), 5-(Nethyl-N-isopropyl) amiloride 150 µM (EIPA), and low-temperature (4 °C) treatment on dendrimer internalization into HT-29 cells. Dendrimer accumulation in untreated cells was set at 100%. Data are given as mean ( SD of triplicate experiments. Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Dunnett’s test (* p < 0.05, ** p < 0.01, *** p < 0.001).

dendrimer (Figure 2c). However, there was an overlap between propranolol molecules and lauroyl moieties at a chemical shift of 1.23 ppm (Figure 2d). Therefore, the integration of peaks at chemical shift 0.89 of lauroyl moieties, 2.39-2.43 ppm of G3 dendrimer, and 6.90-7.01 ppm of propranolol were used to establish the presence of two lauroyl moieties and two propranolol molecules on the G3 dendrimer (Figure 2d). Synthesis and Characterization of FITC-Labeled G3 PAMAM Dendrimers. 1H NMR analyses indicated parent G3 PAMAM dendrimer (Figure 3a) and surface-modified dendrimers (data not shown) were successfully labeled with FITC at an average ratio of 1:1. Size distribution curves from dynamic light scattering for FITC-labeled G3 (Figure 3b), -G3P2 (Figure 3c), -G3L2 (Figure 3d), and -G3L2P2 (Figure 3e) PAMAM dendrimers yielded mean diameters of 5.18 ( 0.24, 17.00 ( 2.28, 11.81 ( 1.50, and 10.73 ( 0.01 nm, respectively. The data show that addition of the FITC label to the G3 dendrimer caused an increase of hydrodynamic diameter of approximately 1-2 nm. Addition of the lauroyl and/or propranolol groups to G3 PAMAM dendrimers resulted in an increase in hydrodynamic diameter of between 5.6 and 11.8 nm indicating successful attachment of moieties, with possible dimer and trimer formation due to chain entanglement (G3L2) and π-stacking of aromatic rings (G3P2). We also noted that the hydrodynamic diameter of G3L2P2 appeared to be less than that of G3P2, possibly a consequence of differing extents of dendrimer aggregation. Effect of Dendrimers on Cell Viability. Studies were initially carried out to measure the viability of HT-29 cells exposed to dendrimer (2.5 µM). Viability of cells incubated with G3 was set at 100%, with viabilities of 100 ( 4.65%, 100 ( 4.65%, and 87.90 ( 2.60% observed for cells incubated with G3L2, G3P2, and G3L2P2, respectively. Effect of Surface Modification on the Rate of Internalization of Dendrimers. The effect of surface modification on the rate of intracellular internalization of dendrimer into HT-29 cells was analyzed by flow cytometry. The rate of internalization of unmodified parent G3 dendrimer was 118.64 ( 0.22 absorbance

units/hour (au/h). Addition of, on average, two lauroyl molecules to the parent dendrimer, to yield G3L2, significantly (P < 0.05) increased the rate of internalization to 235.74 ( 11.47 au/h. In contrast, the rate of uptake of parent dendrimer modified to possess, on average, two propranolol molecules (G3P2) at 49.10 ( 4.38 au/h, was significantly (P < 0.001) less than the uptake rate of G3 dendrimer. Addition of, on average, two lauroyl and two propranolol molecules to the parent dendrimer (G3L2P2) had no significant effect on the rate of internalization of modified dendrimer, 144.68 ( 6.64 au/h, compared to unmodified G3. Effect of Endocytosis Inhibitors on the Internalization of Dendrimers. Studies were carried out using endocytosis inhibitors in order to examine the possible mechanisms of dendrimer internalization in HT-29 cells. Initial measurement of the cytotoxicity of endocytosis inhibitors showed that exposure of cells to chlorpromazine (50 µM) (21, 28), flipin (1 µg/mL) (12), nystatin (50 µM) (28), and EIPA (150 µM) (26) resulted in cell viabilities of 74.22 ( 7.38% (compared to untreated cells at 100% viability), 78.23 ( 5.20%, 81.11 ( 6.01%, and 98.31 ( 0.61%, respectively. Confocal analyses of cells treated with endocytosis inhibitors at these concentrations revealed that cells possessed a healthy morphology, and subsequent inhibition studies were carried out using these inhibitor concentrations. Flow cytometry studies were carried out to investigate the effects of endocytosis inhibitors on dendrimer internalization. Treatment of HT-29 cells with chlorpromazine (50 µM), an inhibitor of clathrin-dependent endocytosis, had no significant effect on the intracellular accumulation of FITC-labeled unmodified G3 dendrimer (Figure 4a), G3P2 (Figure 4b), or G3L2P2 (Figure 4d) but significantly reduced intracellular accumulation of lauroyl-modified (G3L2) dendrimer (Figure 4c). Treatment of cells with filipin (1 µg/mL) and nystatin (50 µM) (inhibitors of caveolae-dependent endocytosis) and incubation at reduced temperature (4 °C) significantly reduced intracellular accumulation of FITC-labeled unmodified G3 dendrimer and all of the dendrimer conjugates. EIPA (150 µM) (inhibitor of



Figure 5. Subcellular localization of FITC-labeled G3 PAMAM dendrimers in HT-29 cells. HT-29 cells were incubated with the G3 dendrimers then either subjected to indirect immunostaining for caveolin-1 or clathrin or incubated with Alexa Fluor 555-transferrin or Lysotracker Red DND. Arrows indicate regions of colocalization of FITC-labeled dendrimers and endosome or lysosome marker.

macropinocytosis) significantly reduced intracellular accumulation of unmodified parent G3, G3P2, and G3L2 but had no significant effect on G3L2P2. These findings suggest that cellular internalization of unmodified G3 dendrimer and G3P2 dendrimer conjugate occurs via both caveolae-dependent and macropinocytosis pathways. G3L2 dendrimer conjugate is internalized via clathrin-dependent, caveolae-dependent, and macropinocytosis pathways. However, internalization of G3L2P2 dendrimer conjugate appears to involve a caveolae-dependent endocytosis pathway. Subcellular Localization of FITC Labeled-Unmodified and Surface-Modified G3 PAMAM Dendrimer in HT-29 Cells. To clarify further both the mechanism of internalization and the intracellular trafficking routes of dendrimers in HT-29 cells, subcellular colocalization of G3 PAMAM dendrimer and key endocytosis markers was investigated using confocal laser scanning microscopy. Clathrin was used as a marker of clathrindependent endocytosis and caveolin-1 as a marker of cavelaedependent endocytosis. Transferrin undergoes receptor-mediated endocytosis and trafficks via the endosome pathway; therefore, Alexa Fluor 555-transferrin was used as an endosome marker. Lysotracker Red DND-99 was employed to identify lysosomes. Unmodified FITC-labeled G3 dendrimer was localized both in the plasma membrane and intracellularly (Figure 5a,b,c,d, green). Indirect immunostaining of these cells with Alexa Fluor 555-caveolin-1 antibody showed caveolin-1 to be located predominantly in the plasma membrane, with some cytoplasmic localization (Figure 5a, red). Combining FITC and Alexa Fluor fluorescence revealed colocalization of G3 PAMAM dendrimer and caveolin-1 within the plasma membrane of HT-29 cells (Figure 5a, yellow). Confocal analyses of HT-29 cells indirectly immunostained with anticlathrin antibody showed fluorescence

to be predominantly located at the plasma membrane (Figure 5b, red). However, negligible colocalization between FITClabeled G3 dendrimer and clathrin were observed (Figure 5b). Alexa Fluor 555-transferrin was located both within the plasma membrane and intracellularly (Figure 5c, red). Areas of colocalization between G3 dendrimer and transferrin were observed both intracellularly and within the plasma membrane (Figure 5c, yellow). When HT-29 cells were incubated with FITClabeled G3 dendrimer, sectioning in the z-plane indicated that fluorescence was associated with intracellular vesicle-like structures. Lysotracker Red DND was localized intracellularly (Figure 5d, red). Combining FITC and Alexa Fluor fluorescence of HT-29 cells incubated with FITC-labeled unmodified G3 dendrimer and Lysotracker Red DND-99 clearly revealed overlapping fluorescence of dendrimer and lysosome probe (Figure 5d, yellow). The confocal images of HT-29 cells incubated with FITClabeled G3P2 dendrimer revealed that fluorescence was associated both with the plasma membrane and with intracellular vesicle-like structures (Figure 5e, f, g, h, green), as was observed for FITC-labeled unmodified G3 dendrimer. FITC-labeled G3P2 dendrimer was found to colocalize with caveolin-1 (Figure 5e, yellow). There was negligible colocalization between this surface-modified G3 dendrimer and clathrin (Figure 5f), but colocalization with Alexa Fluor 555-transferrin (Figure 5g, yellow) and with Lysotracker Red DND (Figure 5h, yellow) was observed. FITC-labeled G3L2 dendrimer localized at the plasma membrane, with some intracellular fluorescence observed (Figure 5i, j, k, l, green). This modified dendrimer colocalized with caveolin-1 (Figure 5i, yellow) and with clathrin (Figure 5j, yellow). These subcellular colocalization results are in


agreement with our finding that nystatin, filipin, and chlorpromazine reduce intracellular internalization (Figure 4c). FITClabeled G3L2 dendrimer colocalized with Alexa Fluor 555-transferrin (Figure 5k, yellow) and was also observed to colocalize with Lysotracker Red DND (Figure 5l, yellow). Incubation of HT-29 cells with FITC-labeled G3L2P2 dendrimer revealed FITC-associated fluorescence at the plasma membrane (Figure 5m, n, o, p, green). This modified dendrimer colocalized with caveolin-1 (Figure 5m, yellow) and with clathrin (Figure 5n yellow). Our studies revealed that FITClabeled G3L2P2 dendrimer also colocalized with Alexa Fluor 555-transferrin (Figure 5o, yellow) and Lysotracker Red DND (Figure 5p, yellow). All dendrimers and dendrimer conjugates studied are trafficked to lysosomes. In order to determine the effect of surface modification on the extent of dendrimer accumulation in lysosomes, semiquantitative analysis was carried out using ImageJ to obtain the overlap coefficient, which defines the extent of true 3-dimensional colocalization. The level of colocalization of G3 dendrimer with the lysosomal marker Lysotracker Red DND (overlap coefficient 0.8 ( 0.05) was significantly (P < 0.0001) higher than the colocalization levels observed with Lysotracker Red DND and G3P2 (0.5 ( 0.04), G3L2 (0.4 ( 0.07), or G2L2P2 (0.6 ( 0.08), suggesting that surface modification influences the extent of dendrimer trafficking to lysosomes.

DISCUSSION Dendrimers are an attractive nanoparticulate drug delivery system which have been shown to significantly enhance delivery of several drugs such as propranolol, naproxen, terfenadine, 5-aminosalicylic acid, 5-fluorouracil, cisplatin, and doxorubicin (2, 7, 8, 29-32). However, little is known about the precise cellular mechanisms of dendrimer uptake and how modification of the dendrimer surface affect the internalization mechanism(s). Cellular dendrimer internalization has been reported to involve both caveolae- and clathrin-mediated endocytosis (11-13, 33). In the HT-29 human intestinal adenocarcinoma cell line, confocal laser scanning microscopy revealed that caveolae and clathrin-1 were expressed in a manner consistent with the distribution patterns reported previously in HT-29 cells (19). All dendrimers studied colocalized with caveolin-1, as has been observed with the dendrimer-DNA complex in EAhy 926 cells (33), while G3L2 and G3L2P2 were found to colocalize with clathrin-1, consistent with our flow cytometry findings. The dendrimers used in the present study are cationic in nature, and several studies suggest that clathrin-dependent endocytosis may represent one of the most important pathways of internalization for several cationic lipoplexes in COS7, A549, and HeLa cells (34-36). Subcellular colocalization studies revealed that all dendrimers colocalized with transferrin, consistent with studies that report that both FITC-labeled cationic and anionic PAMAM dendrimers (G2 and G1.5) colocalized with early endosomal antigen-1 (EEA-1) in Caco-2 cells (11) and with studies showing goldlabeled G3 PAMAM dendrimer localized into endosomes in Caco-2 cells (37). To establish the effects of surface modification on the rate of intracellular internalization of dendrimers into HT-29 cells, flow cytometry studies were carried out. Associated with surface modification is a change in the size of dendrimer nanoparticles. These studies revealed no correlation between dendrimer size and either the rate or internalization mechanism of dendrimer uptake into HT-29 cells. These findings can be explained by the fact that linked with a change in size is a concomitant change in physicochemical characteristics, including charge and hy-


drophobicity, which have important roles in influencing interaction of nanoparticles with the cell surface. Surface modification by the addition of, on average, two lauroyl molecules to G3 dendrimer significantly increased the rate of internalization. In addition, these studies revealed that G3 is taken up by both caveolae-mediated endocytosis and macropinocytosis. These results are in agreement with a reduction in cell-associated Orgeon green labeled G4 PAMAM dendrimer fluorescence observed in B16F10 murine melanoma cells in the presence of methyl-β-cyclodextrin (13) and the significant inhibition of uptake of second-generation dendrimer modified with 18 aminolaevulinic acid uptake in the presence of EIPA in PAM 212 cells (27). However, modification of G3 to G3L2 resulted in dendrimer internalization via clathrinmediated endocytosis, as well as via caveolae-mediated endocytosis and macropinocytosis. These findings are consistent with those reported in Caco-2 cells in which the apparent permeability of G3 dendrimer modified with nine lauroyl moieties was reduced by inhibitors of clathrin-mediated endocytosis and caveolae-mediated endocytosis (Rachaneekorn Jevprasaphant, Antony D’Emanuele, David Attwood, Jeffrey Penny, unpublished results). Thus, modification of parent dendrimer with lauroyl moieties appears to offer an additional internalization pathway for G3 dendrimer uptake into HT-29 cells, and it is feasible that cellular uptake via an additional pathway has the potential to increase the rate of nanoparticle delivery. The rate of intracellular internalization of G3 dendrimer possessing, on average, two propranolol molecules into HT-29 cells was significantly lower than that of unmodified G3 dendrimer. Furthermore, the rate-enhancing effect of lauroyl moieties on dendrimer uptake appeared to be negated in dendrimer possessing both lauroyl and propranolol moieties (G3L2P2). Multiple pathways are involved in the uptake of dendrimer and modified dendrimers into HT-29 cells, and the precise mechanism by which propranolol reduces the rate of dendrimer internalization is unknown. The plasma membrane is an extremely heterogeneous environment, possessing microenvironments with lipid, protein, and receptor profiles that differ significantly compared to the bulk of the plasma membrane. It is likely that the rate of dendrimer internalization is influenced by the physicochemical characteristics of the nanoparticles and the differential abilities of dendrimers to interact with the heterogeneous domains and microenvironments of the plasma membrane. All dendrimers and dendrimer conjugates studied are trafficked into lysosomes, acidic organelles rich in hydrolytic enzymes. These findings are consistent with colocalization of FITC-labeled G1.5 and G2 PAMAM dendrimers with lysosomeassociated membrane protein (LAMP-1) in Caco-2 cells (11). It is important to determine the relationship between surface modification and the extent of lysosome accumulation, since exposure to a hydrolytic environment may impact on the efficacy of dendrimer-mediated drug delivery. Semiquantitative colocalization studies revealed addition of lauroyl moieties to G3, to yield G3L2, resulting in significantly less modified dendrimer being trafficked into lysosomes. Similarly, semiquantitative colocalization studies revealed that, as with G3L2, significantly less G3P2 and G3L2P2 were associated with lysosomes compared to unmodified G3. It therefore appears that surface modification of G3 dendrimer influences not only the rate of intracellular accumulation in HT-29 cells, but also the extent of dendrimer accumulation within lysosomes.

CONCLUSION Studies to elucidate the mechanisms and pathways of dendrimer internalization and trafficking are important, as they may


help define how surface modification may enhance cellular uptake of nanoparticulate dendrimer-drug complexes and potentially increase drug delivery across physiological cell barriers. The present study provides evidence that the surface properties of dendrimers influence the initial mode of dendrimer internalization in HT-29 cells. Modification of parent G3 dendrimer with lauroyl moieties increases the rate of intracellular uptake, a finding that could be exploited to increase the rate of oral drug delivery. In addition, modification of the nanoparticles with lauroyl moieties appears to reduce the extent of lysosomal accumulation, suggesting potentially less exposure of the dendrimer conjugate to the highly acidic lysosomal environment and hydrolytic enzymes, a potentially important issue when considering delivery of acid-labile drugs or drug conjugates. Thus, dendrimer modification with lauroyl chains is attractive, since it enhances the internalization kinetics of modified dendrimer into human intestinal epithelial cells and may have the potential for improving drug delivery via the oral route.

ACKNOWLEDGMENT The authors would like to thank the Ministry of Science and Technology in Thailand for its financial support and Lesley Wright for secretarial assistance.

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