- Email: [email protected]
Transport of dendrimer nanocarriers through epithelial cells via the transcellular route
Journal of Controlled Release 97 (2004) 259 – 267 www.elsevier.com/locate/jconrel
Transport of dendrimer nanocarriers through epithelial cells via the transcellular route Rachaneekorn Jevprasesphant, Jeffrey Penny, David Attwood, Antony D’Emanuele * School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester, M13 9PL, UK Received 26 November 2003; accepted 12 March 2004 Available online 30 April 2004
Abstract The mechanism of transport of G3 PAMAM and surface-modified (with lauroyl chains) G3 PAMAM dendrimer nanocarriers across Caco-2 cell monolayers has been investigated. Flow-cytometry studies following quenching of extracellular fluorescence demonstrated the cellular internalisation of dendrimers. Optical sectioning of cells incubated with fluorescein isothiocyanate (FITC)-conjugated dendrimer and lauroyl – dendrimer using confocal laser scanning microscopy revealed colocalisation of a marker for cell nuclei (4V,6-diamidino-2-phenylindole, DAPI) and FITC fluorescence, also suggesting cellular internalisation of dendrimers. Transmission electron microscopic analyses of cells incubated with gold-labelled G3 PAMAM dendrimers confirmed endocytosis-mediated cellular internalisation when dendrimers were applied to the apical domain of Caco-2 cells. These findings are in agreement with our previous studies using Caco-2 cell monolayers that showed a significant decrease of dendrimer uptake in the presence of colchicine (endocytosis inhibitor) and when temperature was reduced from 37 to 4 jC. D 2004 Elsevier B.V. All rights reserved. Keywords: Mechanism of transepithelial transport of dendrimer nanocarriers; Transcellular and paracellular routes; Dendrimers; Drug delivery
1. Introduction Dendrimers represent a relatively new class of polymers that have found several pharmaceutical applications [1 – 3]. They have been shown to be capable of enhancing the solubility of poorly soluble drugs, enhancing the delivery of DNA and oligonucleotides, and may form a scaffold for the development of targeted delivery systems. * Corresponding author. Tel.: +44-161-275-2333; fax: +44-709203-0763. E-mail address: [email protected] (A. D’Emanuele). 0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2004.03.022
StarburstR polyamidoamine (PAMAM) dendrimers are a specific family of dendritic polymers that are based on an ethylenediamine core and an amidoamine repeat branching structure and commercially available (Dendritech) as whole-(cationic) or half (anionic)-generation polymers. Despite the growing interest in the biomedical applications of dendrimers, there is a paucity of information available on their acceptability for use in humans. There are limited and conflicting reports specifically evaluating the toxicity and biodistribution of dendrimers. Roberts et al. [4] investigated a number of biological properties of dendrimers (G3, G5 and G7
260
R. Jevprasesphant et al. / Journal of Controlled Release 97 (2004) 259–267
PAMAM) in V79 cells and Swiss-Webster mice including toxicity, immunogenicity and biodistribution. Biological complications were observed only with G7 dendrimers at high concentrations. Duncan and coworkers [5] demonstrated from a systematic study on the effect of dendrimer generation and surface functionality on biological properties that dendrimers bearing amine surface groups caused concentration dependent hemolysis and changes in red cell morphology; cationic dendrimers were, in general, found to be cytotoxic. A recent study by Fischer et al. [6] examined the effect of a range of polycations (used for biomedical applications) on cell viability and hemolysis. It was reported that PAMAM dendrimers (G3) were amongst the most biocompatible cationic polymers and did not cause blood cell hemolysis even at concentrations of up to 10 mg/ml. Compared to other polycations, dendrimers did not cause any reduction in metabolic activity in vitro up to concentrations of 10 mg/ml (after 24 h). The biological profile of a dendrimer-based delivery system (with a payload of drug and surface modifiers) is likely, however, to be very different to that of unmodified dendrimers. It has been shown [7] that DNA/PAMAM dendrimer complexes are less mytotoxic than free dendrimer, probably because the complex reduces the overall positive charge of the dendrimer. Oligonucleotide – dendrimer complexes have also been shown to be less cytotoxic than unmodified dendrimer [8]. D’Emanuele and coworkers [9,10] reported on the development of dendrimer conjugates as potential drug carrier systems and found that surface engineering (by the addition of lauroyl or PEG chains) significantly reduced the cytotoxicity of PAMAM dendrimers. Dendrimers have been shown to cross cell barriers at sufficient rates to act as potential carrier/delivery systems [11,12]. Recent work [13] has demonstrated that dendrimer nanocarriers may be used to enhance the transport of propranolol across Caco-2 cells. Propranolol is an insoluble drug and a substrate for the P-glycoprotein (P-gp) efflux transporter. When conjugated to G3 PAMAM, propranolol was shown to bypass the efflux system. Thus, dendrimer nanocarriers may be used to enhance the bioavailability of drugs that are poorly soluble and/or substrates of efflux transporters. Permeability studies using Caco-2 cell monolayers [9] revealed that cationic dendrimers (G2, G3, G4)
exhibited a significantly greater apical (A) to basal (B) apparent permeability ( Papp) than anionic dendrimers (G2.5, G3.5). Cationic dendrimers decreased the transepithelial electrical resistance (TEER) and significantly increased the permeability of [14C]mannitol. The permeation of the dendrimers was higher in the presence of ethylenediaminetetraacetic acid (EDTA), lower in the presence of colchicine (endocytosis inhibitor), and lower at 4 jC than at 37 jC. A significant reduction of cytotoxicity and enhancement of permeation was achieved by surface modification of the dendrimers with lauroyl moieties. These findings are consistent with transport involving both transcellular and paracellular pathways. El-Sayed et al. [14,15] have reported similar findings. In this study, we have carried out a detailed analysis to identify the mechanisms of transport of G3 PAMAM dendrimers and dendrimer conjugates across Caco-2 cell monolayers using flow cytometry, confocal laser scanning microscopy, and transmission electron microscopy (TEM). The purpose of this study was to understand the correlation between dendrimer characteristics and their epithelial permeability, which could aid in the future design of dendrimer nanocarriers for delivery via the gastrointestinal tract.
2. Material and methods 2.1. Materials G3 PAMAM dendrimers with ethylenediamine cores were purchased from Dendritech (Michigan, USA). Gold chloride, trypan blue, fluorescein isothiocyanate (FITC), and 4V,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich (Poole, Dorset, UK). [14C]mannitol (specific activity 50 mCi/mmol) was purchased from Amersham Pharmacia Biotech (Little Chalfont, Bucks, UK). Cell culture materials were from Gibco Life Technologies (Paisley, Scotland). Foetal bovine serum was from Labtech International (Sussex, UK). Polycarbonate cell culture inserts (TranswellR 12 mm diameter) were purchased from Corning Costar UK (High Wycombe, Bucks, UK). Glutaraldehyde and sodium cacodylate were purchased from Agar Scientific (Essex, UK). Osmium tetroxide and epoxy resin were purchased from TABBR (Aldermaston,
R. Jevprasesphant et al. / Journal of Controlled Release 97 (2004) 259–267
Berks,UK) and uranyl acetate and Reynolds lead citrate were purchased from Leica Microsystems (Milton Keynes, UK). A lauroyl –dendrimer conjugate with an average of nine lauroyl chains covalently bonded to a G3 dendrimer (G3L9) was synthesised as described previously [9]. 2.2. Cell culture Caco-2 cells (passage 41 – 63) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10 % (v/v) foetal bovine serum, 2 mM glutamine, 10 mM non-essential amino acids, 50 IU/ ml penicillin and 50 Ag/ml streptomycin, at 37 jC and in an atmosphere of 5% CO2. Growth medium was changed on alternate days. Cell number was assessed by trypan blue exclusion analyses [9]. Caco-2 cells were seeded onto polycarbonate filters at a density of 1.2 105 cells/cm2 and confluent monolayers (21 – 28 days) used for confocal microscopic and TEM studies. Monolayer integrity was determined by measurement of TEER using a voltohmmeter (EVOM, World Precision Instruments, Sarasota, FL, USA) and by assessing permeation of [14C]mannitol. Only monolayers with a TEER in the range of 800 –1000 V/cm2 were used. For flow-cytometry studies, approximately 5 105 Caco-2 cells were seeded into six-well tissue culture plates and maintained until confluent (2 days). Dendrimers were labelled with FITC using the method described previously [9]; in vitro stability tests at 37 jC in phosphate-buffered saline (PBS) pH 7.4 revealed no dissociation of FITC from the dendrimer over a period of 5 days, indicating a stable linkage of FITC to dendrimer. 2.3. Flow cytometry Trypan blue is widely used as a quencher of FITC fluorescence [16,17] and is excluded from viable cells. In the present study, it was used to distinguish between FITC-labelled dendrimer that is bound to the cell surface and dendrimer that has been internalised by Caco-2 cells. FITC-labelled G3 or FITC-labelled lauroyl-G3 dendrimer conjugates (10 AM) were incubated with confluent Caco-2 cell monolayers for 3 h. Cells were typsinised, 0.1% (w/v) trypan blue was added, and after a 10-min incubation, cells were washed and resuspended in PBS. Fluorescence was
261
measured using a flow cytometer (B.D. Biosciences FACSort, CA, USA) equipped with an argon ion laser having a single laser excitation wavelength of 488 nm, with computer-assisted data analysis (CellQuest). FITC fluorescence (emission wavelength 518 nm) was captured using the FL-1 channel (detects emitted light of wavelength 500– 600 nm). FITC fluorescence was plotted vs. number of cells, and median peak values were obtained before and after quenching. At least 10,000 cells were analysed in each sample. Each experiment was repeated four times. 2.4. Confocal laser scanning microscopy Confluent Caco-2 cell monolayers on filter inserts were incubated with FITC-labelled G3 or FITC-labelled lauroyl-G3 dendrimer conjugates 10 Am [9]. After 3 h, cells were washed with PBS and incubated for 10 min with DAPI (which intercalates into DNA and acts as a marker for cell nuclei). Time-lapse series and single images were examined and collected using confocal laser scanning microscopy (BioRad MRC1024 MP, CA, USA), with a scan head on a Nikon E800 microscope ( 40 objective). Images were analysed using Adobe Photoshop. Excitation and emission wavelengths were 364 and 461 nm, respectively, for DAPI and 488 and 518 nm for FITC. A gallery of 30 optical sections (1 Am) through the zplane was obtained and composites processed. Fluorescence signals were collected using appropriate filters allowing analysis of the different fluorophores. 2.5. Synthesis of gold – dendrimer nanocomposites for transmission electron microscopy studies Electron-dense gold was complexed to G3 and lauroyl-G3 dendrimer conjugates by a one-phase reaction procedure to enable the location of dendrimers in cells using TEM [18]. An aqueous solution of HAuCl43H2O was mixed with a methanolic solution of dendrimer at a ratio of two, four or six gold atoms per dendrimer. A further increase in the number of gold atoms per dendrimer led to precipitation of the conjugates. Nitrogen was bubbled through the solution for 2 h to produce an electrostatically bound G3 – NH3+[AuCl4 ]n complex. This was reduced by slow addition (10 ml/min) of excess 0.15 M aqueous solution of NaBH4. Upon addition of the reducing
262
R. Jevprasesphant et al. / Journal of Controlled Release 97 (2004) 259–267
agent to the dendrimer – gold conjugate, a colour change from pale yellow to deep red wine was observed, indicating the reduction of the gold anions coordinated to the dendrimer – nitrogen atoms into zero-valent gold (metallic gold) and the formation of a nanocomposite of structure G3 – NH2[Au(0)]n. The same synthetic procedure without dendrimer yielded large, insoluble gold particles. The complexes were characterised by (i) UV – visible spectroscopy, scanning wavelengths between 200 and 600 nm (Thermo Spectronic, Unicam UV 300, UK), (ii) 1H-NMR (Bruker Avance 300, Coventry, UK) and (iii) static and dynamic light scattering (Brookhaven BI 200 S combined with a Brookhaven BI 9000 AT digital correlator, NY, USA, with vertically polarised incident light of wavelength 488 nm supplied by a 2-W argon ion laser) [9]. Conjugates were designated G3Aux and G3L9Aux where x represents the number of gold atoms.
water, stained with 2% (w/v) uranyl acetate (to stain DNA and RNA) at 50jC for 50 min, washed briefly, stained with Reynolds lead citrate (membrane staining) at 25jC for 10 min and finally washed in water (LKB BROMMA 2168 Ultrastainer, Bromma, Sweden). The dried grids were visualised using a transmission electron microscope (TEM, Philips CM10, Cambridge, UK).
3. Results 3.1. Flow cytometry Flow-cytometry studies revealed high levels of fluorescence associated with cells following incuba-
2.6. Transmission electron microscopy Confluent cell monolayers were incubated with G3Au6 or G3L9Au6 dendrimer nanocomposites (10 Am). After 3 h, cells were washed twice with PBS buffer. Filters containing the cell monolayers were removed from the Transwell and fixed in a 2.5% (v/v) sodium cacodylate buffer containing 0.1 M glutaraldehyde for a minimum of 4 h, washed with 0.1 M sodium cacodylate buffer for 20 min and then post-fixed in a solution of 1% (w/v) osmium tetroxide in 0.1 M sodium cacodylate buffer for 30 min. Fixed cells were washed with sodium cacodylate buffer for 10 min and dehydrated sequentially in a graded series of aqueous ethanol solutions with ethanol contents of 50% for 10 min, 70% for 10 min, 90% for 10 min and twice with 100% for 10 min. Cells were soaked twice in propylene oxide (intermediate reagent) for 10 min and then infiltrated sequentially with epoxy resin/ propylene oxide solutions with epoxy resin contents of 50% for 20 min, 70% for 20 min, 90% for 20 min and 100% for 20 min. Filters containing cell monolayers were then embedded in freshly prepared resin moulds and polymerised at 60 jC overnight. Ultrathin sections (thickness 100– 120 nm) were cut using an Ultracut E Microtome (Reichert-Jung, Austria) and placed on 200-mesh copper grids (Agar Scientific). The grids were then washed in distilled
Fig. 1. Flow-cytometry results of FITC-labelled (A) G3 dendrimers and (B) lauroyl – G3 dendrimer conjugates (G3L9), pre- (a) and post (b) quenching with trypan blue. The marker, M1, defines the region of fluorescence exhibited by the FITC-labelled dendrimer.
R. Jevprasesphant et al. / Journal of Controlled Release 97 (2004) 259–267
tion of Caco-2 cell monolayers with FITC-labelled dendrimer or lauroyl – dendrimer. Quenching of extracellullar fluorescence by trypan blue resulted in only a small decrease in cell fluorescence intensity. The cell fluorescence intensity detected in the M1 region ( F SD) was 87 F 3% pre-quenching (Fig. 1A, curve a) and 75 F 1% post-quenching (Fig. 1A, curve b) for FITC-labelled G3 dendrimer and 95 F 3% prequenching (Fig. 1B, curve a) and 83 F 4% postquenching (Fig. 1B, curve b) for FITC-labelled lauroyl G3 dendrimer. These data indicate internal-
263
isation of FITC-labelled dendrimers into Caco-2 cells. The higher cell fluorescence intensity observed ( P < 0.05) with the FITC-labelled lauroyl – dendrimer conjugates suggests that lauroyl moieties enhance dendrimer penetration into Caco-2 cells. 3.2. Confocal laser scanning microscopy Caco-2 cells were imaged in real time using confocal laser scanning microscopy to determine the cellular disposition of G3 dendrimer and lauroyl-G3
Fig. 2. Optical sections [sections 6 (A), 8 (B), 12 (C) and 24 (D)] of Caco-2 cell monolayers incubated with lauroyl – G3 dendrimer conjugates (G3L9). The series were from the basolateral side (section 1) to apical side (section 30).
264
R. Jevprasesphant et al. / Journal of Controlled Release 97 (2004) 259–267
dendrimer during the transport process. Serial optical sections of the cells (each 1 Am in thickness) demonstrated FITC (green) fluorescence in sections between 2 and 25 Am of the apical domain of the cells, with colocalisation of DAPI (blue) and FITC fluorescence in sections between 5 and 11 Am from the basal boundary of the cells (Fig. 2). These findings are consistent with the internalisation of both FITC-labelled G3 dendrimers and lauroyl – G3 dendrimer conjugates within the cells after 3 h incubation. 3.3. Transmission electron microscopy Comparison of the UV spectrum of unconjugated gold with that of the complex between gold and dendrimers (Fig. 3) confirmed the formation of gold – dendrimer nanocomposites. The UV spectrum of unconjugated gold (HAuCl43H2O) exhibits an absorption peak at 221 nm and a shoulder at 290 nm. Reduction of the gold – dendrimer complex with NaBH4 resulted in the disappearance of the peak at 221 nm and appearance of broad peaks at 280 and 530 nm (particularly evident with increasing gold content), indicating formation of gold – dendrimer nanocomposite [19]. The UV –visible spectra of gold –dendrimer nanocomposites that had been stored in PBS buffer at 37 jC for 21 days were identical to those of freshly prepared complexes, indicating stable complexation between gold and dendrimer.
Comparison of the 1H-NMR spectra (not shown) of unmodified dendrimer and gold – dendrimer nanocomposites did not provide any conclusive evidence of complexation, possibly because the broad dendrimer peak obscured expected changes in the spectrum arising from the interaction of dendrimer with gold. The weight-average molar masses of nanocomposites measured using static light scattering techniques were 7.06, 8.01 and 9.96 103 g mol 1 for G3, G3Au6 and G3L9Au6, respectively. These values are within F 2.5% of values expected for gold – dendrimer and gold – lauroyl dendrimer conjugates having the appropriate number of complexed gold atoms (6.91, 8.09 and 9.73 103 g mol 1, respectively, as calculated using the literature value of the weight-average molar mass of the G3 PAMAM dendrimer [3]). Diameters ( F S.E.) determined from dynamic light scattering by extrapolation of diffusion data to zero concentration were 3.2 F 0.06, 4.1 F 0.02 and 4.7 F 0.02 nm for G3, G3Au6 and G3L9Au6, respectively, in agreement with sizes determined by electron microscopy. Transmission electron microscopy was used to investigate the mechanism of internalisation of gold – dendrimer nanocomposites. TEM images of Caco-2 cell monolayers revealed cells with classic columnar shape with basally located nuclei, highly differentiated microvilli and tight junction complexes,
Fig. 3. UV spectra of (o) HAuCl43H2O, (5) G3 dendrimer, (w ) G3Au2, (X) G3Au4, (+) G3Au6, (D) G3L9Au6.
R. Jevprasesphant et al. / Journal of Controlled Release 97 (2004) 259–267
265
Fig. 4. Electron micrograph showing the localisation of G3 dendrimer nanocomposites in Caco-2 cells. (A) cell surface attachment, (B) endocytotic invagination and (C) endosome-containing nanocomposites.
characteristics that are indicative of mature absorptive cells resembling the in vivo intestinal epithelium. Fig. 4 shows the attachment of nanocomposites to the apical cell surface and membrane invagination to
internalise nanocomposites into endosomes. Fig. 5 shows their internalisation and localisation in multivesicular bodies that were found throughout the cell interior. Addition of gold to dendrimers did not
Fig. 5. Electron micrograph showing the localisation of lauroyl – G3 dendrimer nanocomposites (G3L9) in Caco-2 cells. (A) endocytotic invagination and (B) multivesicular bodies.
266
R. Jevprasesphant et al. / Journal of Controlled Release 97 (2004) 259–267
significantly change their toxicity (MTT assay) [9] or permeation (apparent permeability coefficient) [9] characteristics (data not shown).
expected for a paracellular transport mechanism, we have no direct evidence for a lack of a paracellular component to dendrimer transport.
4. Discussion
5. Conclusion
Whilst there have been a significant number of recent publications examining the potential of dendrimers as vehicles for drug delivery [2,3], limited data are available relating to the mechanism(s) of dendrimer transport through cells. Recent studies suggest that dendrimer transport can occur via both paracellular and transcellular pathways [9,15]. In this study, the transport mechanisms of G3 PAMAM dendrimers and lauroyl – G3 PAMAM dendrimer conjugates across Caco-2 cell monolayers have been investigated using high-resolution imaging techniques. Flow-cytometry studies reveal a significant level of internalisation of dendrimer and dendrimer conjugate into Caco-2 cells, with low levels of dendrimer associated with the cell surface. Similar findings were obtained using confocal laser scanning microscopy, where high levels of fluorescently tagged dendrimers were observed throughout the cell interior. Detailed information from transmission electron microscopy on the mechanism of transport of dendrimer molecules tagged with gold atoms showed cellular internalisation of apically located nanocomposites by endocytosis. Early endosomes containing electron-dense nanocomposites were found immediately below the apical surface. In addition, endosomes appeared to aggregate to produce multivesicular bodies found throughout the cell, which would support the notion that nanocomposites are transported through the cell in these vesicular structures. In studies using a DNA – gold/G5 PAMAM dendrimer complex as a gene transfection agent, Bielinska et al. [20] also reported endocytosis-mediated dendrimer uptake into monkey kidney Cos-1 cells. Further evidence for an endocytotic transcellular pathway of dendrimer and dendrimer conjugates through Caco-2 cells comes from our previous studies [9] that showed a significant decrease in dendrimer uptake in the presence of colchicine (endocytosis inhibitor) and on reducing the temperature from 37 to 4 jC. Although in the present studies, no nanocomposites were found within the intercellular spaces as might be
The results of this study show that endocytosis plays an important role in the transepithelial transport of PAMAM dendrimers through cells. We have shown visual evidence of localisation of G3 dendrimers and lauroyl – G3 dendrimer conjugates in individual endocytotic vesicles at the apical domain of the cell and their association with multivesicular bodies in the cell interior. These results complement our earlier studies [9] which have demonstrated that these dendrimer and lauroyl – dendrimer conjugates are able to effectively cross Caco-2 cell monolayers (conjugated dendrimers having greater permeation coefficients than unmodified dendrimers) and demonstrate the potential of dendrimers as nanocarriers for the enhancement of bioavailability.
Acknowledgements The authors would like to thank Dr. Catherine O’Neill for the Caco-2 cells and Dr. Michael Shaw, University of Oxford, for cell biology advice. We would also like to express our gratitude to Dr. Alan Curry and Tricia Rowland for providing the transmission electron microscopy expertise, and the Government Pharmaceutical Organisation (GPO), Thailand, for its financial support.
References [1] D.A. Tomalia, Dendrimer molecules, Sci. Am. 272 (1995) 62 – 66. [2] R. Esfand, D.A. Tomalia, Poly(amidoamine) (PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications, Drug Discov. Today 6 (2001) 427 – 436. [3] A. D’Emanuele, D. Attwood, R. Abu-Rmaileh, Dendrimers, in: J. Swarbrick, J.C. Boylan (Eds.), 2nd ed., Encyclopedia of Pharmaceutical Technology, issue 3, Marcel Dekker, New York, 2003, pp. 1 – 21. [4] J.C. Roberts, M.K. Bhalgat, R.T. Zera, Preliminary biological evaluation of polyamidoamine (PAMAM) Starburst dendrimers, J. Biomed. Mater. Res. 30 (1996) 53 – 65.
R. Jevprasesphant et al. / Journal of Controlled Release 97 (2004) 259–267 [5] N. Malik, R. Wiwattanapatapee, R. Klopsch, K. Lorenz, H. Frey, J.W. Weener, E.W. Meijer, W. Paulus, R. Duncan, Dendrimers: relationship between structure and biocompatibility in vitro, and preliminary studies on the biodistribution of 125Ilabelled polyamidoamine dendrimers in vivo, J. Control. Release 65 (2000) 133 – 148. [6] D. Fischer, Y. Li, B. Ahlemeyer, J. Krieglstein, T. Kissel, In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis, Biomaterials 24 (2003) 1121 – 1131. [7] G.A. Brazeau, S. Attia, S. Poxon, J.A. Hughes, In vitro myotoxicity of selected cationic macromolecules used in non-viral gene delivery, Pharm. Res. 15 (1998) 680 – 684. [8] H. Yoo, R.L. Juliano, Enhanced delivery of antisense oligonucleotides with fluorophore-conjugated PAMAM dendrimers, Nucleic Acids Res. 28 (2000) 4225 – 4231. [9] R. Jevprasesphant, J. Penny, D. Attwood, N.B. McKeown, A. D’Emanuele, Engineering of dendrimer surfaces to enhance transepithelial transport and reduce cytotoxicity, Pharm. Res. 20 (2003) 1543 – 1550. [10] R. Jevprasesphant, J. Penny, R. Jalal, D. Attwood, N.B. McKeown, A. D’Emanuele, The influence of surface modification on the cytotoxicity of PAMAM dendrimers, Int. J. Pharm. 252 (2003) 263 – 266. [11] A.T. Florence, T. Sakthivel, I. Toth, Oral uptake and translocation of a polylysine dendrimer with a lipid surface, J. Control. Release 65 (2000) 253 – 259. [12] R. Wiwattanapatapee, B. Carreno-Gomez, N. Malik, R. Duncan, Anionic PAMAM dendrimers rapidly cross adult rat intestine in vitro: a potential oral delivery system, Pharm. Res. 17 (2000) 991 – 998.
267
[13] A. D’Emanuele, R. Jevprasesphant, J. Penny, D. Attwood, The use of a dendrimer-propranolol prodrug to bypass efflux transporters and enhance oral bioavailability, J. Control. Release 95 (2004) 447 – 453. [14] M. El-Sayed, M. Ginski, C. Rhodes, H. Ghandehari, Transepithelial transport of poly(amidoamine) dendrimers across Caco-2 cell monolayers, J. Control. Release 81 (2002) 355 – 365. [15] M. El-Sayed, C.A. Rhodes, M. Ginski, H. Ghandehari, Transport mechanism(s) of poly(amidoamine) dendrimers across Caco-2 cell monolayers, Int. J. Pharm. 265 (2003) 151 – 157. [16] E.S. Van Amersfoort, J.A.G. Van Strijp, Evaluation of a flow cytometric fluorescence quenching assay of phagocytosis of sensitized sheep erythrocytes by polymorphonuclear leukocytes, Cytometry 17 (1994) 294 – 301. [17] W. Chaka, J. Scharringa, A.F.M. Verheul, J. Verhoef, A.G. Van Strijp, I.M. Hoepelman, Quantitative analysis of phagocytosis and killing of Cryptococcus neoformans by human peripheral blood mononuclear cells by flow cytometry, Clin. Diagn. Lab. Immunol. 2 (1995) 753 – 759. [18] F. Groehn, B.J. Bauer, Y.A. Akpalu, C.L. Jackson, E.J. Amis, Dendrimer templates for the formation of gold nanoclusters, Macromolecules 33 (2000) 6042 – 6050. [19] L. Balogh, R. Valluzzi, K.S. Laverdure, S.P. Gido, G.L. Hagnauer, D.A. Tomalia, G.L. Hagnauer, Formation of silver and gold dendrimer nanocomposites, J. Nanopart. Res. 1 (1999) 353 – 368. [20] A. Bielinska, J.D. Eichman, I. Lee, J.R. Baker Jr., L. Balogh, Imaging {Au0-PAMAM} gold – dendrimer nanocomposites in cells, J. Nanopart. Res. 4 (2002) 395 – 403.
Transport of dendrimer nanocarriers through epithelial cells via the transcellular route Rachaneekorn Jevprasesphant, Jeffrey Penny, David Attwood, Antony D’Emanuele * School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester, M13 9PL, UK Received 26 November 2003; accepted 12 March 2004 Available online 30 April 2004
Abstract The mechanism of transport of G3 PAMAM and surface-modified (with lauroyl chains) G3 PAMAM dendrimer nanocarriers across Caco-2 cell monolayers has been investigated. Flow-cytometry studies following quenching of extracellular fluorescence demonstrated the cellular internalisation of dendrimers. Optical sectioning of cells incubated with fluorescein isothiocyanate (FITC)-conjugated dendrimer and lauroyl – dendrimer using confocal laser scanning microscopy revealed colocalisation of a marker for cell nuclei (4V,6-diamidino-2-phenylindole, DAPI) and FITC fluorescence, also suggesting cellular internalisation of dendrimers. Transmission electron microscopic analyses of cells incubated with gold-labelled G3 PAMAM dendrimers confirmed endocytosis-mediated cellular internalisation when dendrimers were applied to the apical domain of Caco-2 cells. These findings are in agreement with our previous studies using Caco-2 cell monolayers that showed a significant decrease of dendrimer uptake in the presence of colchicine (endocytosis inhibitor) and when temperature was reduced from 37 to 4 jC. D 2004 Elsevier B.V. All rights reserved. Keywords: Mechanism of transepithelial transport of dendrimer nanocarriers; Transcellular and paracellular routes; Dendrimers; Drug delivery
1. Introduction Dendrimers represent a relatively new class of polymers that have found several pharmaceutical applications [1 – 3]. They have been shown to be capable of enhancing the solubility of poorly soluble drugs, enhancing the delivery of DNA and oligonucleotides, and may form a scaffold for the development of targeted delivery systems. * Corresponding author. Tel.: +44-161-275-2333; fax: +44-709203-0763. E-mail address: [email protected] (A. D’Emanuele). 0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2004.03.022
StarburstR polyamidoamine (PAMAM) dendrimers are a specific family of dendritic polymers that are based on an ethylenediamine core and an amidoamine repeat branching structure and commercially available (Dendritech) as whole-(cationic) or half (anionic)-generation polymers. Despite the growing interest in the biomedical applications of dendrimers, there is a paucity of information available on their acceptability for use in humans. There are limited and conflicting reports specifically evaluating the toxicity and biodistribution of dendrimers. Roberts et al. [4] investigated a number of biological properties of dendrimers (G3, G5 and G7
260
R. Jevprasesphant et al. / Journal of Controlled Release 97 (2004) 259–267
PAMAM) in V79 cells and Swiss-Webster mice including toxicity, immunogenicity and biodistribution. Biological complications were observed only with G7 dendrimers at high concentrations. Duncan and coworkers [5] demonstrated from a systematic study on the effect of dendrimer generation and surface functionality on biological properties that dendrimers bearing amine surface groups caused concentration dependent hemolysis and changes in red cell morphology; cationic dendrimers were, in general, found to be cytotoxic. A recent study by Fischer et al. [6] examined the effect of a range of polycations (used for biomedical applications) on cell viability and hemolysis. It was reported that PAMAM dendrimers (G3) were amongst the most biocompatible cationic polymers and did not cause blood cell hemolysis even at concentrations of up to 10 mg/ml. Compared to other polycations, dendrimers did not cause any reduction in metabolic activity in vitro up to concentrations of 10 mg/ml (after 24 h). The biological profile of a dendrimer-based delivery system (with a payload of drug and surface modifiers) is likely, however, to be very different to that of unmodified dendrimers. It has been shown [7] that DNA/PAMAM dendrimer complexes are less mytotoxic than free dendrimer, probably because the complex reduces the overall positive charge of the dendrimer. Oligonucleotide – dendrimer complexes have also been shown to be less cytotoxic than unmodified dendrimer [8]. D’Emanuele and coworkers [9,10] reported on the development of dendrimer conjugates as potential drug carrier systems and found that surface engineering (by the addition of lauroyl or PEG chains) significantly reduced the cytotoxicity of PAMAM dendrimers. Dendrimers have been shown to cross cell barriers at sufficient rates to act as potential carrier/delivery systems [11,12]. Recent work [13] has demonstrated that dendrimer nanocarriers may be used to enhance the transport of propranolol across Caco-2 cells. Propranolol is an insoluble drug and a substrate for the P-glycoprotein (P-gp) efflux transporter. When conjugated to G3 PAMAM, propranolol was shown to bypass the efflux system. Thus, dendrimer nanocarriers may be used to enhance the bioavailability of drugs that are poorly soluble and/or substrates of efflux transporters. Permeability studies using Caco-2 cell monolayers [9] revealed that cationic dendrimers (G2, G3, G4)
exhibited a significantly greater apical (A) to basal (B) apparent permeability ( Papp) than anionic dendrimers (G2.5, G3.5). Cationic dendrimers decreased the transepithelial electrical resistance (TEER) and significantly increased the permeability of [14C]mannitol. The permeation of the dendrimers was higher in the presence of ethylenediaminetetraacetic acid (EDTA), lower in the presence of colchicine (endocytosis inhibitor), and lower at 4 jC than at 37 jC. A significant reduction of cytotoxicity and enhancement of permeation was achieved by surface modification of the dendrimers with lauroyl moieties. These findings are consistent with transport involving both transcellular and paracellular pathways. El-Sayed et al. [14,15] have reported similar findings. In this study, we have carried out a detailed analysis to identify the mechanisms of transport of G3 PAMAM dendrimers and dendrimer conjugates across Caco-2 cell monolayers using flow cytometry, confocal laser scanning microscopy, and transmission electron microscopy (TEM). The purpose of this study was to understand the correlation between dendrimer characteristics and their epithelial permeability, which could aid in the future design of dendrimer nanocarriers for delivery via the gastrointestinal tract.
2. Material and methods 2.1. Materials G3 PAMAM dendrimers with ethylenediamine cores were purchased from Dendritech (Michigan, USA). Gold chloride, trypan blue, fluorescein isothiocyanate (FITC), and 4V,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich (Poole, Dorset, UK). [14C]mannitol (specific activity 50 mCi/mmol) was purchased from Amersham Pharmacia Biotech (Little Chalfont, Bucks, UK). Cell culture materials were from Gibco Life Technologies (Paisley, Scotland). Foetal bovine serum was from Labtech International (Sussex, UK). Polycarbonate cell culture inserts (TranswellR 12 mm diameter) were purchased from Corning Costar UK (High Wycombe, Bucks, UK). Glutaraldehyde and sodium cacodylate were purchased from Agar Scientific (Essex, UK). Osmium tetroxide and epoxy resin were purchased from TABBR (Aldermaston,
R. Jevprasesphant et al. / Journal of Controlled Release 97 (2004) 259–267
Berks,UK) and uranyl acetate and Reynolds lead citrate were purchased from Leica Microsystems (Milton Keynes, UK). A lauroyl –dendrimer conjugate with an average of nine lauroyl chains covalently bonded to a G3 dendrimer (G3L9) was synthesised as described previously [9]. 2.2. Cell culture Caco-2 cells (passage 41 – 63) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10 % (v/v) foetal bovine serum, 2 mM glutamine, 10 mM non-essential amino acids, 50 IU/ ml penicillin and 50 Ag/ml streptomycin, at 37 jC and in an atmosphere of 5% CO2. Growth medium was changed on alternate days. Cell number was assessed by trypan blue exclusion analyses [9]. Caco-2 cells were seeded onto polycarbonate filters at a density of 1.2 105 cells/cm2 and confluent monolayers (21 – 28 days) used for confocal microscopic and TEM studies. Monolayer integrity was determined by measurement of TEER using a voltohmmeter (EVOM, World Precision Instruments, Sarasota, FL, USA) and by assessing permeation of [14C]mannitol. Only monolayers with a TEER in the range of 800 –1000 V/cm2 were used. For flow-cytometry studies, approximately 5 105 Caco-2 cells were seeded into six-well tissue culture plates and maintained until confluent (2 days). Dendrimers were labelled with FITC using the method described previously [9]; in vitro stability tests at 37 jC in phosphate-buffered saline (PBS) pH 7.4 revealed no dissociation of FITC from the dendrimer over a period of 5 days, indicating a stable linkage of FITC to dendrimer. 2.3. Flow cytometry Trypan blue is widely used as a quencher of FITC fluorescence [16,17] and is excluded from viable cells. In the present study, it was used to distinguish between FITC-labelled dendrimer that is bound to the cell surface and dendrimer that has been internalised by Caco-2 cells. FITC-labelled G3 or FITC-labelled lauroyl-G3 dendrimer conjugates (10 AM) were incubated with confluent Caco-2 cell monolayers for 3 h. Cells were typsinised, 0.1% (w/v) trypan blue was added, and after a 10-min incubation, cells were washed and resuspended in PBS. Fluorescence was
261
measured using a flow cytometer (B.D. Biosciences FACSort, CA, USA) equipped with an argon ion laser having a single laser excitation wavelength of 488 nm, with computer-assisted data analysis (CellQuest). FITC fluorescence (emission wavelength 518 nm) was captured using the FL-1 channel (detects emitted light of wavelength 500– 600 nm). FITC fluorescence was plotted vs. number of cells, and median peak values were obtained before and after quenching. At least 10,000 cells were analysed in each sample. Each experiment was repeated four times. 2.4. Confocal laser scanning microscopy Confluent Caco-2 cell monolayers on filter inserts were incubated with FITC-labelled G3 or FITC-labelled lauroyl-G3 dendrimer conjugates 10 Am [9]. After 3 h, cells were washed with PBS and incubated for 10 min with DAPI (which intercalates into DNA and acts as a marker for cell nuclei). Time-lapse series and single images were examined and collected using confocal laser scanning microscopy (BioRad MRC1024 MP, CA, USA), with a scan head on a Nikon E800 microscope ( 40 objective). Images were analysed using Adobe Photoshop. Excitation and emission wavelengths were 364 and 461 nm, respectively, for DAPI and 488 and 518 nm for FITC. A gallery of 30 optical sections (1 Am) through the zplane was obtained and composites processed. Fluorescence signals were collected using appropriate filters allowing analysis of the different fluorophores. 2.5. Synthesis of gold – dendrimer nanocomposites for transmission electron microscopy studies Electron-dense gold was complexed to G3 and lauroyl-G3 dendrimer conjugates by a one-phase reaction procedure to enable the location of dendrimers in cells using TEM [18]. An aqueous solution of HAuCl43H2O was mixed with a methanolic solution of dendrimer at a ratio of two, four or six gold atoms per dendrimer. A further increase in the number of gold atoms per dendrimer led to precipitation of the conjugates. Nitrogen was bubbled through the solution for 2 h to produce an electrostatically bound G3 – NH3+[AuCl4 ]n complex. This was reduced by slow addition (10 ml/min) of excess 0.15 M aqueous solution of NaBH4. Upon addition of the reducing
262
R. Jevprasesphant et al. / Journal of Controlled Release 97 (2004) 259–267
agent to the dendrimer – gold conjugate, a colour change from pale yellow to deep red wine was observed, indicating the reduction of the gold anions coordinated to the dendrimer – nitrogen atoms into zero-valent gold (metallic gold) and the formation of a nanocomposite of structure G3 – NH2[Au(0)]n. The same synthetic procedure without dendrimer yielded large, insoluble gold particles. The complexes were characterised by (i) UV – visible spectroscopy, scanning wavelengths between 200 and 600 nm (Thermo Spectronic, Unicam UV 300, UK), (ii) 1H-NMR (Bruker Avance 300, Coventry, UK) and (iii) static and dynamic light scattering (Brookhaven BI 200 S combined with a Brookhaven BI 9000 AT digital correlator, NY, USA, with vertically polarised incident light of wavelength 488 nm supplied by a 2-W argon ion laser) [9]. Conjugates were designated G3Aux and G3L9Aux where x represents the number of gold atoms.
water, stained with 2% (w/v) uranyl acetate (to stain DNA and RNA) at 50jC for 50 min, washed briefly, stained with Reynolds lead citrate (membrane staining) at 25jC for 10 min and finally washed in water (LKB BROMMA 2168 Ultrastainer, Bromma, Sweden). The dried grids were visualised using a transmission electron microscope (TEM, Philips CM10, Cambridge, UK).
3. Results 3.1. Flow cytometry Flow-cytometry studies revealed high levels of fluorescence associated with cells following incuba-
2.6. Transmission electron microscopy Confluent cell monolayers were incubated with G3Au6 or G3L9Au6 dendrimer nanocomposites (10 Am). After 3 h, cells were washed twice with PBS buffer. Filters containing the cell monolayers were removed from the Transwell and fixed in a 2.5% (v/v) sodium cacodylate buffer containing 0.1 M glutaraldehyde for a minimum of 4 h, washed with 0.1 M sodium cacodylate buffer for 20 min and then post-fixed in a solution of 1% (w/v) osmium tetroxide in 0.1 M sodium cacodylate buffer for 30 min. Fixed cells were washed with sodium cacodylate buffer for 10 min and dehydrated sequentially in a graded series of aqueous ethanol solutions with ethanol contents of 50% for 10 min, 70% for 10 min, 90% for 10 min and twice with 100% for 10 min. Cells were soaked twice in propylene oxide (intermediate reagent) for 10 min and then infiltrated sequentially with epoxy resin/ propylene oxide solutions with epoxy resin contents of 50% for 20 min, 70% for 20 min, 90% for 20 min and 100% for 20 min. Filters containing cell monolayers were then embedded in freshly prepared resin moulds and polymerised at 60 jC overnight. Ultrathin sections (thickness 100– 120 nm) were cut using an Ultracut E Microtome (Reichert-Jung, Austria) and placed on 200-mesh copper grids (Agar Scientific). The grids were then washed in distilled
Fig. 1. Flow-cytometry results of FITC-labelled (A) G3 dendrimers and (B) lauroyl – G3 dendrimer conjugates (G3L9), pre- (a) and post (b) quenching with trypan blue. The marker, M1, defines the region of fluorescence exhibited by the FITC-labelled dendrimer.
R. Jevprasesphant et al. / Journal of Controlled Release 97 (2004) 259–267
tion of Caco-2 cell monolayers with FITC-labelled dendrimer or lauroyl – dendrimer. Quenching of extracellullar fluorescence by trypan blue resulted in only a small decrease in cell fluorescence intensity. The cell fluorescence intensity detected in the M1 region ( F SD) was 87 F 3% pre-quenching (Fig. 1A, curve a) and 75 F 1% post-quenching (Fig. 1A, curve b) for FITC-labelled G3 dendrimer and 95 F 3% prequenching (Fig. 1B, curve a) and 83 F 4% postquenching (Fig. 1B, curve b) for FITC-labelled lauroyl G3 dendrimer. These data indicate internal-
263
isation of FITC-labelled dendrimers into Caco-2 cells. The higher cell fluorescence intensity observed ( P < 0.05) with the FITC-labelled lauroyl – dendrimer conjugates suggests that lauroyl moieties enhance dendrimer penetration into Caco-2 cells. 3.2. Confocal laser scanning microscopy Caco-2 cells were imaged in real time using confocal laser scanning microscopy to determine the cellular disposition of G3 dendrimer and lauroyl-G3
Fig. 2. Optical sections [sections 6 (A), 8 (B), 12 (C) and 24 (D)] of Caco-2 cell monolayers incubated with lauroyl – G3 dendrimer conjugates (G3L9). The series were from the basolateral side (section 1) to apical side (section 30).
264
R. Jevprasesphant et al. / Journal of Controlled Release 97 (2004) 259–267
dendrimer during the transport process. Serial optical sections of the cells (each 1 Am in thickness) demonstrated FITC (green) fluorescence in sections between 2 and 25 Am of the apical domain of the cells, with colocalisation of DAPI (blue) and FITC fluorescence in sections between 5 and 11 Am from the basal boundary of the cells (Fig. 2). These findings are consistent with the internalisation of both FITC-labelled G3 dendrimers and lauroyl – G3 dendrimer conjugates within the cells after 3 h incubation. 3.3. Transmission electron microscopy Comparison of the UV spectrum of unconjugated gold with that of the complex between gold and dendrimers (Fig. 3) confirmed the formation of gold – dendrimer nanocomposites. The UV spectrum of unconjugated gold (HAuCl43H2O) exhibits an absorption peak at 221 nm and a shoulder at 290 nm. Reduction of the gold – dendrimer complex with NaBH4 resulted in the disappearance of the peak at 221 nm and appearance of broad peaks at 280 and 530 nm (particularly evident with increasing gold content), indicating formation of gold – dendrimer nanocomposite [19]. The UV –visible spectra of gold –dendrimer nanocomposites that had been stored in PBS buffer at 37 jC for 21 days were identical to those of freshly prepared complexes, indicating stable complexation between gold and dendrimer.
Comparison of the 1H-NMR spectra (not shown) of unmodified dendrimer and gold – dendrimer nanocomposites did not provide any conclusive evidence of complexation, possibly because the broad dendrimer peak obscured expected changes in the spectrum arising from the interaction of dendrimer with gold. The weight-average molar masses of nanocomposites measured using static light scattering techniques were 7.06, 8.01 and 9.96 103 g mol 1 for G3, G3Au6 and G3L9Au6, respectively. These values are within F 2.5% of values expected for gold – dendrimer and gold – lauroyl dendrimer conjugates having the appropriate number of complexed gold atoms (6.91, 8.09 and 9.73 103 g mol 1, respectively, as calculated using the literature value of the weight-average molar mass of the G3 PAMAM dendrimer [3]). Diameters ( F S.E.) determined from dynamic light scattering by extrapolation of diffusion data to zero concentration were 3.2 F 0.06, 4.1 F 0.02 and 4.7 F 0.02 nm for G3, G3Au6 and G3L9Au6, respectively, in agreement with sizes determined by electron microscopy. Transmission electron microscopy was used to investigate the mechanism of internalisation of gold – dendrimer nanocomposites. TEM images of Caco-2 cell monolayers revealed cells with classic columnar shape with basally located nuclei, highly differentiated microvilli and tight junction complexes,
Fig. 3. UV spectra of (o) HAuCl43H2O, (5) G3 dendrimer, (w ) G3Au2, (X) G3Au4, (+) G3Au6, (D) G3L9Au6.
R. Jevprasesphant et al. / Journal of Controlled Release 97 (2004) 259–267
265
Fig. 4. Electron micrograph showing the localisation of G3 dendrimer nanocomposites in Caco-2 cells. (A) cell surface attachment, (B) endocytotic invagination and (C) endosome-containing nanocomposites.
characteristics that are indicative of mature absorptive cells resembling the in vivo intestinal epithelium. Fig. 4 shows the attachment of nanocomposites to the apical cell surface and membrane invagination to
internalise nanocomposites into endosomes. Fig. 5 shows their internalisation and localisation in multivesicular bodies that were found throughout the cell interior. Addition of gold to dendrimers did not
Fig. 5. Electron micrograph showing the localisation of lauroyl – G3 dendrimer nanocomposites (G3L9) in Caco-2 cells. (A) endocytotic invagination and (B) multivesicular bodies.
266
R. Jevprasesphant et al. / Journal of Controlled Release 97 (2004) 259–267
significantly change their toxicity (MTT assay) [9] or permeation (apparent permeability coefficient) [9] characteristics (data not shown).
expected for a paracellular transport mechanism, we have no direct evidence for a lack of a paracellular component to dendrimer transport.
4. Discussion
5. Conclusion
Whilst there have been a significant number of recent publications examining the potential of dendrimers as vehicles for drug delivery [2,3], limited data are available relating to the mechanism(s) of dendrimer transport through cells. Recent studies suggest that dendrimer transport can occur via both paracellular and transcellular pathways [9,15]. In this study, the transport mechanisms of G3 PAMAM dendrimers and lauroyl – G3 PAMAM dendrimer conjugates across Caco-2 cell monolayers have been investigated using high-resolution imaging techniques. Flow-cytometry studies reveal a significant level of internalisation of dendrimer and dendrimer conjugate into Caco-2 cells, with low levels of dendrimer associated with the cell surface. Similar findings were obtained using confocal laser scanning microscopy, where high levels of fluorescently tagged dendrimers were observed throughout the cell interior. Detailed information from transmission electron microscopy on the mechanism of transport of dendrimer molecules tagged with gold atoms showed cellular internalisation of apically located nanocomposites by endocytosis. Early endosomes containing electron-dense nanocomposites were found immediately below the apical surface. In addition, endosomes appeared to aggregate to produce multivesicular bodies found throughout the cell, which would support the notion that nanocomposites are transported through the cell in these vesicular structures. In studies using a DNA – gold/G5 PAMAM dendrimer complex as a gene transfection agent, Bielinska et al. [20] also reported endocytosis-mediated dendrimer uptake into monkey kidney Cos-1 cells. Further evidence for an endocytotic transcellular pathway of dendrimer and dendrimer conjugates through Caco-2 cells comes from our previous studies [9] that showed a significant decrease in dendrimer uptake in the presence of colchicine (endocytosis inhibitor) and on reducing the temperature from 37 to 4 jC. Although in the present studies, no nanocomposites were found within the intercellular spaces as might be
The results of this study show that endocytosis plays an important role in the transepithelial transport of PAMAM dendrimers through cells. We have shown visual evidence of localisation of G3 dendrimers and lauroyl – G3 dendrimer conjugates in individual endocytotic vesicles at the apical domain of the cell and their association with multivesicular bodies in the cell interior. These results complement our earlier studies [9] which have demonstrated that these dendrimer and lauroyl – dendrimer conjugates are able to effectively cross Caco-2 cell monolayers (conjugated dendrimers having greater permeation coefficients than unmodified dendrimers) and demonstrate the potential of dendrimers as nanocarriers for the enhancement of bioavailability.
Acknowledgements The authors would like to thank Dr. Catherine O’Neill for the Caco-2 cells and Dr. Michael Shaw, University of Oxford, for cell biology advice. We would also like to express our gratitude to Dr. Alan Curry and Tricia Rowland for providing the transmission electron microscopy expertise, and the Government Pharmaceutical Organisation (GPO), Thailand, for its financial support.
References [1] D.A. Tomalia, Dendrimer molecules, Sci. Am. 272 (1995) 62 – 66. [2] R. Esfand, D.A. Tomalia, Poly(amidoamine) (PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications, Drug Discov. Today 6 (2001) 427 – 436. [3] A. D’Emanuele, D. Attwood, R. Abu-Rmaileh, Dendrimers, in: J. Swarbrick, J.C. Boylan (Eds.), 2nd ed., Encyclopedia of Pharmaceutical Technology, issue 3, Marcel Dekker, New York, 2003, pp. 1 – 21. [4] J.C. Roberts, M.K. Bhalgat, R.T. Zera, Preliminary biological evaluation of polyamidoamine (PAMAM) Starburst dendrimers, J. Biomed. Mater. Res. 30 (1996) 53 – 65.
R. Jevprasesphant et al. / Journal of Controlled Release 97 (2004) 259–267 [5] N. Malik, R. Wiwattanapatapee, R. Klopsch, K. Lorenz, H. Frey, J.W. Weener, E.W. Meijer, W. Paulus, R. Duncan, Dendrimers: relationship between structure and biocompatibility in vitro, and preliminary studies on the biodistribution of 125Ilabelled polyamidoamine dendrimers in vivo, J. Control. Release 65 (2000) 133 – 148. [6] D. Fischer, Y. Li, B. Ahlemeyer, J. Krieglstein, T. Kissel, In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis, Biomaterials 24 (2003) 1121 – 1131. [7] G.A. Brazeau, S. Attia, S. Poxon, J.A. Hughes, In vitro myotoxicity of selected cationic macromolecules used in non-viral gene delivery, Pharm. Res. 15 (1998) 680 – 684. [8] H. Yoo, R.L. Juliano, Enhanced delivery of antisense oligonucleotides with fluorophore-conjugated PAMAM dendrimers, Nucleic Acids Res. 28 (2000) 4225 – 4231. [9] R. Jevprasesphant, J. Penny, D. Attwood, N.B. McKeown, A. D’Emanuele, Engineering of dendrimer surfaces to enhance transepithelial transport and reduce cytotoxicity, Pharm. Res. 20 (2003) 1543 – 1550. [10] R. Jevprasesphant, J. Penny, R. Jalal, D. Attwood, N.B. McKeown, A. D’Emanuele, The influence of surface modification on the cytotoxicity of PAMAM dendrimers, Int. J. Pharm. 252 (2003) 263 – 266. [11] A.T. Florence, T. Sakthivel, I. Toth, Oral uptake and translocation of a polylysine dendrimer with a lipid surface, J. Control. Release 65 (2000) 253 – 259. [12] R. Wiwattanapatapee, B. Carreno-Gomez, N. Malik, R. Duncan, Anionic PAMAM dendrimers rapidly cross adult rat intestine in vitro: a potential oral delivery system, Pharm. Res. 17 (2000) 991 – 998.
267
[13] A. D’Emanuele, R. Jevprasesphant, J. Penny, D. Attwood, The use of a dendrimer-propranolol prodrug to bypass efflux transporters and enhance oral bioavailability, J. Control. Release 95 (2004) 447 – 453. [14] M. El-Sayed, M. Ginski, C. Rhodes, H. Ghandehari, Transepithelial transport of poly(amidoamine) dendrimers across Caco-2 cell monolayers, J. Control. Release 81 (2002) 355 – 365. [15] M. El-Sayed, C.A. Rhodes, M. Ginski, H. Ghandehari, Transport mechanism(s) of poly(amidoamine) dendrimers across Caco-2 cell monolayers, Int. J. Pharm. 265 (2003) 151 – 157. [16] E.S. Van Amersfoort, J.A.G. Van Strijp, Evaluation of a flow cytometric fluorescence quenching assay of phagocytosis of sensitized sheep erythrocytes by polymorphonuclear leukocytes, Cytometry 17 (1994) 294 – 301. [17] W. Chaka, J. Scharringa, A.F.M. Verheul, J. Verhoef, A.G. Van Strijp, I.M. Hoepelman, Quantitative analysis of phagocytosis and killing of Cryptococcus neoformans by human peripheral blood mononuclear cells by flow cytometry, Clin. Diagn. Lab. Immunol. 2 (1995) 753 – 759. [18] F. Groehn, B.J. Bauer, Y.A. Akpalu, C.L. Jackson, E.J. Amis, Dendrimer templates for the formation of gold nanoclusters, Macromolecules 33 (2000) 6042 – 6050. [19] L. Balogh, R. Valluzzi, K.S. Laverdure, S.P. Gido, G.L. Hagnauer, D.A. Tomalia, G.L. Hagnauer, Formation of silver and gold dendrimer nanocomposites, J. Nanopart. Res. 1 (1999) 353 – 368. [20] A. Bielinska, J.D. Eichman, I. Lee, J.R. Baker Jr., L. Balogh, Imaging {Au0-PAMAM} gold – dendrimer nanocomposites in cells, J. Nanopart. Res. 4 (2002) 395 – 403.