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Transepithelial transport of PAMAM dendrimers across isolated human intestinal tissue Dallin Hubbard, Michael Enda, Tanner Bond, Seyyed Pouya Hadipour Moghaddam, Josh Conarton, Courtney Scaife, Eric Volckmann, and Hamidreza Ghandehari Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00541 • Publication Date (Web): 28 Sep 2015 Downloaded from http://pubs.acs.org on October 6, 2015
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Molecular Pharmaceutics
Transepithelial transport of PAMAM dendrimers across isolated human intestinal tissue. Dallin Hubbard1-2, Michael Enda3, Tanner Bond4, Seyyed Pouya Hadipour Moghaddam1,5, Josh Conarton2, Courtney Scaife6, Eric Volckmann6, Hamidreza Ghandehari1,2,5 1. Utah Center for Nanomedicine, Nano Institute of Utah, University of Utah, 36 S. Wasatch Dr. Salt Lake City, UT, 84112, USA. 2. Department of Bioengineering, University of Utah, 36 S. Wasatch Dr. Salt Lake City, UT, 84112, USA. 3. Juan Diego Catholic High School, 300 E 11800 S, Draper, UT 84020, USA. 4. Department of Chemistry, Brigham Young University Idaho, Rexburg, ID, 83460, USA 5. Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, 36 S. Wasatch Dr. Salt Lake City, UT, 84112, USA. 6. Department of Surgery, University of Utah, 30 North 1900 East Salt Lake City, UT 84132, USA. KEYWORDS: poly(amido amine) dendrimers, oral drug delivery, Ussing chambers, human intestinal mucosae, transport, toxicity
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ABSTRACT
Poly(amido amine) (PAMAM) dendrimers have shown transepithelial transport across intestinal epithelial barrier in rats and across Caco-2 cell monolayers. Caco-2 models innately lack mucous barriers, and rat isolated intestinal tissue has been shown to overestimate human permeability. This study is the first report of transport of PAMAM dendrimers across isolated human intestinal epithelium. It was observed that FITC labeled G4-NH2 and G3.5-COOH PAMAM dendrimers at 1mM concentration do not have a statistically higher permeability compared to free FITC controls in isolated human jejunum and colonic tissues. Mannitol permeability was increased at 10mM concentrations of G3.5-COOH and G4-NH2 dendrimers. Significant histological changes in human colonic and jejunal tissues were observed at G3.5-COOH and G4-NH2 concentrations of 10mM implying that dose limiting toxicity may occur at similar concentrations in vivo. The permeability through human isolated intestinal tissue in this study was compared to previous rat and Caco-2 permeability data. This study implicates that PAMAM dendrimer oral drug delivery may be feasible, but may be limited to highly potent drugs.
INTRODUCTION PAMAM dendrimers are a class of polymeric nanoparticles that have shown potential in drug delivery.1–3 These constructs have a hyper-branched structure that allows for the attachment of multiple therapeutic and imaging moieties to surface groups.4 They are synthesized with repeating alternating units of ethylene diamine and methyl methacrylate.5 The surface charge of dendrimers can be controlled based on functionalization of dendrimer termini with carboxyl, amine or hydroxyl groups. The hydrophilic structure of dendrimers can be utilized to enhance the
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Molecular Pharmaceutics
solubility and intestinal permeability of drugs covalently attached or admixed with dendrimers.6,7 These properties provide rationale for the application of PAMAM dendrimers in drug delivery. Given their macromolecular nature, appreciable transport across intestinal epithelial barrier has been observed giving rationale for the development of PAMAM dendrimers as an oral drug delivery vehicle.3,8 Dosing of drugs via the oral route has many advantages over parenteral administration including patient preference, cost-effectiveness and enhanced patient quality of life.9 PAMAM dendrimers have the ability to enhance the oral permeability of drugs based on previous work in Caco-2 cell culture, isolated tissue and in vivo models.1–3,6,8,10–12 Dendrimer transport in Caco-2 monolayers has been observed to be size, charge, concentration and incubation-time dependent.10,11 The transepithelial transport of dendrimers ranging from generation 1 (G1) to generation 4.5 (G4.5) have been observed in Caco-2 studies. The apparent permeability (Papp) was observed to vary with increasing generational size in anionic G1.5-3.5 dendrimers.10,13 Rates of transport were also found to increase with increasing incubation time for positively charged G0 and G1 dendrimers in Caco-2 monolayers.10 The Papp of G3.5-FITC labeled dendrimers was found to be ~6×10-6 cm/s at 120 min incubation times and 1.0mM concentrations in Caco-2 cells.13 Concentrations of anionic G3.5–COOH terminated dendrimers greater than 1.0mM were found to be toxic via lactose dehydrogenase (LDH) release in Caco-2 cells at 150 min incubation times.14 Cationic G4-NH2 dendrimers were found to be toxic at concentrations of 0.1mM via LDH release, unless surface groups were substituted with neutral of hydrophobic moleceules.10,15 Kitchens et al. noted that 1.0mM G4-FITC dendrimers with a 1:8 loading ratio and 120 min incubation time did not induce cytotoxicity via the WST-1 assay. The Papp of this compound was ~24×10-6 cm/s.13
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Work in everted rat intestinal tissue models has found that the endocytic index of PAMAM dendrimers was higher for anionic G5.5 PAMAM dendrimers than cationic G4 dendrimers at similar concentrations and incubation times.3 G3.5-FITC labeled dendrimers were found to have a permeability higher than that of control and G4-FITC dendrimers at 1.0mM concentrations in isolated rat jejunum mounted in Ussing chambers.2 Further work in isolated tissue models using rat jejunum has shown that 1.0mM concentrations of G3.5 and G4 dendrimers caused no significant damage to isolated tissue compared to controls. Discrepancies in the toxic concentration of G4 dendrimers between Caco-2 cells and isolated rat intestinal models may be due to the lack of mucous, morphology and other qualities present in isolated tissue models. Further research in vivo has shown that PAMAM dendrimers cross the intestinal epithelium and enter the systemic blood circulation. Thiagarajan et al. observed an oral fraction absorbed of 9.4% in CD-1 mice dosed with G6.5 anionic dendrimers.1 Evidence also exists that PAMAM dendrimers have the ability to enhance the permeability of drugs mixed with or encapsulated in dendrimers. Sadekar et al. noted the ability of PAMAM dendrimers to increase the oral area under the curve (AUC) of camptothecin two fold when dendrimers were dosed at 1000 mg/kg (~7mM intestinal concentration) to CD-1 mice.6 Additional studies on the intestinal morphology after said treatment noted a lack of histopathological changes in the villi structure. The lack of morphological damage in CD-1 mice intestine at 7mM concentrations led us to evaluate similar concentrations in human tissues to assess the effects of high concentrations on epithelial morphology. While these cell culture and animal studies have established the groundwork for PAMAM dendrimer oral delivery, translation into clinical use has not yet been accomplished. Significant differences exist between human, rat and Caco-2 permeability studies including transport
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Molecular Pharmaceutics
proteins, metabolic proteins, morphological structure and lymphoid tissue.16–23 Our aim in this study is to predict the human intestinal permeability of PAMAM dendrimers based on isolated human tissue results.24 The hypothesis is that the isolated intestinal transport through human tissue will provide predictions of the applicability of PAMAM dendrimer drug delivery systems (or the lack thereof) for oral drug delivery. Additionally a basic understanding of the relationship between permeability in Caco-2, rat and human permeability experiments using PAMAM dendrimers as a test probe is to be observed. Experimental Section Materials Fluorescein isothiocyanate (FITC), fluorescein isothiocyanate dextrans, N-(3dimethylaminopropyl)-N’-ethylcarbodiimide (EDC) were obtained from Sigma Aldrich (MO, USA). 14C mannitol (0.1mCi/mL in sterile water) was obtained from American Radiolabeled Chemicals (MO, USA). PAMAM dendrimers (G3.5 and G4.0) in methanol were obtained from Dendritech Inc. (MI, USA). All other reagents were obtained from VWR. Methods FITC labeled dendrimers (G3.5 and G4) were synthesized as reported previously.13 PAMAM dendrimers that had carboxyl termini, were first modified with ethylene diamine to create sites for FITC attachment. G3.5 dendrimers were reacted with EDC and ethylene diamine in PBS pH 7.4 followed by overnight stirring and then addition of FITC (
