ABSTRACT
Purpose
Poly(amidoamine) (PAMAM) dendrimers show great promise for utilization as oral drug delivery vehicles. These polymers are capable of traversing epithelial barriers, and have been shown to translocate by both transcellular and paracellular routes. While many proof-of-concept studies have shown that PAMAM dendrimers improve intestinal transport, little information exists on the mechanisms of paracellular transport, specifically dendrimer-induced tight junction modulation.
Methods
Using anionic G3.5 and cationic G4 PAMAM dendrimers with known absorption enhancers, we investigated tight junction modulation in Caco-2 monolayers by visualization and mannitol permeability and compared dendrimer-mediated tight junction modulation to that of established permeation enhancers. [14C]-Mannitol permeability in the presence and absence of phospholipase C-dependent signaling pathway inhibitors was also examined and indicated that this pathway may mediate dendrimer-induced changes in permeability.
Results
Differences between G3.5 and G4 in tight junction protein staining and permeability with inhibitors were evident, suggesting divergent mechanisms were responsible for tight junction modulation. These dissimilarities are further intimated by the intracellular calcium release caused by G4 but not G3.5. Based on our results, it is apparent that the underlying mechanisms of dendrimer permeability are complex, and the complexities are likely a result of the density and sign of the surface charges of PAMAM dendrimers.
Conclusions
The results of this study will have implications on the future use of PAMAM dendrimers for oral drug delivery.
Similar content being viewed by others
REFERENCES
Esfand R, Tomalia DA. Poly(amidoamine) (PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications. Drug Discov Today. 2001;6:427–36.
Tomalia DA, Reyna LA, Svenson S. Dendrimers as multi-purpose nanodevices for oncology drug delivery and diagnostic imaging. Biochem Soc Trans. 2007;35:61–7.
Kaminskas LM, Boyd BJ, Porter CJ. Dendrimer pharmacokinetics: the effect of size, structure and surface characteristics on ADME properties. Nanomedicine. 2011;6:1063–84.
El-Sayed M, Ginski M, Rhodes C, Ghandehari H. Transepithelial transport of poly(amidoamine) dendrimers across Caco-2 cell monolayers. J Control Release. 2002;81:355–65.
El-Sayed M, Rhodes CA, Ginski M, Ghandehari H. Transport mechanism(s) of poly (amidoamine) dendrimers across Caco-2 cell monolayers. Int J Pharm. 2003;265:151–7.
Kitchens KM, El-Sayed ME, Ghandehari H. Transepithelial and endothelial transport of poly (amidoamine) dendrimers. Adv Drug Deliv Rev. 2005;57:2163–76.
Kitchens KM, Kolhatkar RB, Swaan PW, Eddington ND, Ghandehari H. Transport of poly(amidoamine) dendrimers across Caco-2 cell monolayers: influence of size, charge and fluorescent labeling. Pharm Res. 2006;23:2818–26.
Goldberg DS, Vijayalakshmi N, Swaan PW, Ghandehari H. G3.5 PAMAM dendrimers enhance transepithelial transport of SN38 while minimizing gastrointestinal toxicity. J Control Release. 2011;150:318–25.
Kolhatkar RB, Swaan P, Ghandehari H. Potential oral delivery of 7-ethyl-10-hydroxy-camptothecin (SN-38) using poly(amidoamine) dendrimers. Pharm Res. 2008;25:1723–9.
Sadekar S, Thiagarajan G, Bartlett K, Hubbard D, Ray A, McGill LD, et al. Poly(amido amine) dendrimers as absorption enhancers for oral delivery of camptothecin. Int J Pharm. 2013;456:175–85.
Thiagarajan G, Ray A, Malugin A, Ghandehari H. PAMAM-camptothecin conjugate inhibits proliferation and induces nuclear fragmentation in colorectal carcinoma cells. Pharm Res. 2010;27:2307–16.
Banna GL, Collova E, Gebbia V, Lipari H, Giuffrida P, Cavallaro S, et al. Anticancer oral therapy: emerging related issues. Cancer Treat Rev. 2010;36:595–605.
Goldberg DS, Ghandehari H, Swaan PW. Cellular entry of G3.5 poly (amido amine) dendrimers by clathrin- and dynamin-dependent endocytosis promotes tight junctional opening in intestinal epithelia. Pharm Res. 2010;27:1547–57.
Kitchens KM, Kolhatkar RB, Swaan PW, Ghandehari H. Endocytosis inhibitors prevent poly(amidoamine) dendrimer internalization and permeability across Caco-2 cells. Mol Pharm. 2008;5:364–9.
Kitchens KM, Foraker AB, Kolhatkar RB, Swaan PW, Ghandehari H. Endocytosis and interaction of poly (amidoamine) dendrimers with Caco-2 cells. Pharm Res. 2007;24:2138–45.
Sweet DM, Kolhatkar RB, Ray A, Swaan P, Ghandehari H. Transepithelial transport of PEGylated anionic poly(amidoamine) dendrimers: implications for oral drug delivery. J Control Release. 2009;138:78–85.
Lindmark T, Schipper N, Lazorova L, de Boer AG, Artursson P. Absorption enhancement in intestinal epithelial Caco-2 monolayers by sodium caprate: assessment of molecular weight dependence and demonstration of transport routes. J Drug Target. 1998;5:215–23.
Tomita M, Hayashi M, Awazu S. Absorption-enhancing mechanism of EDTA, caprate, and decanoylcarnitine in Caco-2 cells. J Pharm Sci. 1996;85:608–11.
Tomita M, Hayashi M, Awazu S. Absorption-enhancing mechanism of sodium caprate and decanoylcarnitine in Caco-2 cells. J Pharmacol Exp Ther. 1995;272:739–43.
Berridge MJ, Dawson RM, Downes CP, Heslop JP, Irvine RF. Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides. Biochem J. 1983;212:473–82.
Streb H, Irvine RF, Berridge MJ, Schulz I. Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate. Nature. 1983;306:67–9.
Jaken S. Protein kinase C isozymes and substrates. Curr Opin Cell Biol. 1996;8:168–73.
Schulman H. The multifunctional Ca2+/calmodulin-dependent protein kinases. Curr Opin Cell Biol. 1993;5:247–53.
Turner JR, Angle JM, Black ED, Joyal JL, Sacks DB, Madara JL. PKC-dependent regulation of transepithelial resistance: roles of MLC and MLC kinase. Am J Physiol. 1999;277:C554–62.
Ma TY, Tran D, Hoa N, Nguyen D, Merryfield M, Tarnawski A. Mechanism of extracellular calcium regulation of intestinal epithelial tight junction permeability: role of cytoskeletal involvement. Microsc Res Tech. 2000;51:156–68.
Cunningham KE, Turner JR. Myosin light chain kinase: pulling the strings of epithelial tight junction function. Ann N Y Acad Sci. 2012;1258:34–42.
Ghandehari H, Smith PL, Ellens H, Yeh PY, Kopecek J. Size-dependent permeability of hydrophilic probes across rabbit colonic epithelium. J Pharmacol Exp Ther. 1997;280:747–53.
Smith RJ, Sam LM, Justen JM, Bundy GL, Bala GA, Bleasdale JE. Receptor-coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness. J Pharmacol Exp Ther. 1990;253:688–97.
Hidaka H, Sasaki Y, Tanaka T, Endo T, Ohno S, Fujii Y, et al. N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, a calmodulin antagonist, inhibits cell proliferation. Proc Natl Acad Sci U S A. 1981;78:4354–7.
Tokumitsu H, Chijiwa T, Hagiwara M, Mizutani A, Terasawa M, Hidaka H. KN-62, 1-[N, O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II. J Biol Chem. 1990;265:4315–20.
Saitoh M, Ishikawa T, Matsushima S, Naka M, Hidaka H. Selective inhibition of catalytic activity of smooth muscle myosin light chain kinase. J Biol Chem. 1987;262:7796–801.
Balda MS, Gonzalez-Mariscal L, Matter K, Cereijido M, Anderson JM. Assembly of the tight junction: the role of diacylglycerol. J Cell Biol. 1993;123:293–302.
Macia E, Ehrlich M, Massol R, Boucrot E, Brunner C, Kirchhausen T. Dynasore, a cell-permeable inhibitor of dynamin. Dev Cell. 2006;10:839–50.
Bauer H, Zweimueller-Mayer J, Steinbacher P, Lametschwandtner A, Bauer HC. The dual role of zonula occludens (ZO) proteins. J Biomed Biotechnol. 2010;2010:402593.
Farkas AE, Capaldo CT, Nusrat A. Regulation of epithelial proliferation by tight junction proteins. Ann N Y Acad Sci. 2012;1258:115–24.
Balda MS, Matter K. The tight junction protein ZO-1 and an interacting transcription factor regulate ErbB-2 expression. EMBO J. 2000;19:2024–33.
Fanning AS, Van Itallie CM, Anderson JM. Zonula occludens-1 and -2 regulate apical cell structure and the zonula adherens cytoskeleton in polarized epithelia. Mol Biol Cell. 2012;23:577–90.
Nagumo Y, Han J, Bellila A, Isoda H, Tanaka T. Cofilin mediates tight-junction opening by redistributing actin and tight-junction proteins. Biochem Biophys Res Commun. 2008;377:921–5.
DePina AS, Langford GM. Vesicle transport: the role of actin filaments and myosin motors. Microsc Res Tech. 1999;47:93–106.
Tiriveedhi V, Kitchens KM, Nevels KJ, Ghandehari H, Butko P. Kinetic analysis of the interaction between poly(amidoamine) dendrimers and model lipid membranes. Biochim Biophys Acta. 2011;1808:209–18.
Barfod ET, Moore AL, Van de Graaf BG, Lidofsky SD. Myosin light chain kinase and Src control membrane dynamics in volume recovery from cell swelling. Mol Biol Cell. 2011;22:634–50.
Albertazzi L, Serresi M, Albanese A, Beltram F. Dendrimer internalization and intracellular trafficking in living cells. Mol Pharm. 2010;7:680–8.
ACKNOWLEDGMENTS AND DISCLOSURES
Financial support was provided in part by an American Foundation for Pharmaceutical Education predoctoral fellowship to B. Avaritt. Dr. Jason Hill and Dr. Andrew Ziman provided guidance with live intracellular calcium imaging studies.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Avaritt, B.R., Swaan, P.W. Intracellular Ca2+ Release Mediates Cationic but Not Anionic Poly(amidoamine) (PAMAM) Dendrimer-Induced Tight Junction Modulation. Pharm Res 31, 2429–2438 (2014). https://doi.org/10.1007/s11095-014-1338-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11095-014-1338-y