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Transport of PAMAM Dendrimers Across Biological Barriers

Transport of PAMAM Dendrimers Across Biological Barriers. Ghaith Al-Jayyoussi , Will Ford & Mark Gumbleton Welsh School of Pharmacy, Cardiff University, Cardiff, CF10 3NB. Introduction. Methods.

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Transport of PAMAM Dendrimers Across Biological Barriers

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  1. Transport of PAMAM Dendrimers Across Biological Barriers Ghaith Al-Jayyoussi, Will Ford & Mark Gumbleton Welsh School of Pharmacy, Cardiff University, Cardiff, CF10 3NB Introduction Methods Anionic (-COOH surface termini) and cationic (NH2 termini) PAMAM dendrimers have been covalently coupled to the fluorescent dye, Oregon Green 488 (OG) cadaverine or its homologue succinimidyl ester, respectively. Figure (3) exhibits the fluorescent spectrum and the chemical structure of OG cadaverine. Polyamidoamine (PAMAM) dendrimers are a class of branched polymers that have the potential to serve as drug carriers. This is primarily due to their extremely low polydispersity index, the ability to precisely control their size and charge, and the multiple functional groups that they bear on their surfaces giving the ability to conjugate a wide range of therapeutic molecules[1]. The purpose of these studies is to investigate the transport of these polymers across a variety of in-vitro cell monolayers to further dissect their transport mechanisms across biological barriers. We are currently looking at a strategy for the biologically-stable conjugation between PAMAM dendrimers and therapeutic molecules to elicit intestinal- submucoual immunomodulation without allowing access to the CNS. Fluorescently labelled dendrimers were applied to the apical surface (Fig 4, A) of epithelial cell monolayers grown on semi-permeable inserts. Cumulative dendrimer transport was determined by measuring the temporal increase in fluorescence on the basolateral side(Fig 4, B) over time. Cell lines used were MDCK-I (highly restrictive), the colon cell line Caco-2 (moderately restrictive) and kidney cell line MDCK-II (less restrictive). The biocompatibility of these polymers was also examined by assessing their effect on monolayer transepithelial electrical resistance (TEER) and permeability to the paracellular probe 14C-mannitol. Fig 3. The structure and fluorescent spectrum of OG cadaverine A B www.dendritech.com Fig 2. The controlled exponential growth of a dendrimer. Biological macromolecules of similar size are as shown for comparison.[3] Fig 1. The 3D Structure of a G4 PAMAM dendrimer. Fig 4. Diffusion of a fluorescent compound across a cell monolayer grown on a semi-permeable insert Results & Discussion Figure (5A) shows the permeability coefficients of CACO-2 monolayers treated with a size-range of probes. Transport of PAMAM dendrimers was predominantly dependant on probe size (Fig 5B), implicating a restricted diffusional transport process. The extent of PAMAM 1.5 transport across MDCK monolayers (Fig 5C) was greatly reduced in the MDCK-I strain which forms more restrictive tight junctions and displays higher TER values. Incubation of monolayers with anionic PAMAM dendrimers caused neither a significant decrease in TER (Figure 6A) nor an increase in permeability to 14C-mannitol (Figure 6B). In contrast, incubation with a cationic PAMAM dendrimer of comparable size (G3) was associated with significant increases in TER and permeability to 14C mannitol. The disruptive effect of cationic PAMAM dendrimers is likely due to interaction of cationic dendrimers with anionic components of the cell membranes and tight junctions. B A C * Exponential increase in size ** A B Fig 6. (A). The effect of 0.5 mM PAMAM G3 or G3.5 on CACO-2 monolayer integrity as exhibited by monolayer permeability to 14C-Mannitol as compared to control. (B). The effect of 0.5 mM PAMAM G3 or G3.5 on CACO-2 monolayer integrity as exhibited by their effect on the trans epithelial electrical resistance (TEER) compared to control. Fig 5. (A) CACO-2Permeability coefficients for 14C-Mannitol, Oregon Green 488 Cadaverine and various PAMAM dendrimers. (B) A plot of monolayer permeability Vs. 1/3√Mwt for the range [ 14C – Mannitol(•), OG Cadaverine(•), PAMAM G1.5 (•), PAMAM G3 (•), PAMAM G3.5 (•), PAMAM G5.5 (•) ], R2 =0.9869. (C) A comparison between percentage PAMAM 1.5 absorbed across a highly restrictive endothelial cell line (MDCK I, Average TEER = 2956 ± 57 Ω.cm2) and a much less restrictive cell line from the same source (MDCK II, Average TEER = 235 ± 16 Ω.cm2) * Signifies p<0.05 ** Signifies p<0.001 Conclusions The results suggest that whilst the major pathway of dendrimer transport across biological barriers is the paracellular pathway, the extent and rate of transport is limited and their utility in delivering therapeutics across biological barriers may be much more limited than previously anticipated[2]. These studies also indicate that care must be taken when interpreting permeability data for PAMAMs when they significantly decrease the restrictiveness of the barrier properties as appears to be the case for cationic dendrimers. Preliminary biopharmaceutical studies described here support the strategy of absorption of dendrimer-drug conjugates through submucousal tissue but reduced accessibility across more restrictive biological barriers such as the blood brain barrier. 1. Tomalia, D.A., Naylor, A. M., & Goddard III, W. A., Starbustdendrimers: Molecular-level control of size, shape, surface chemistry, topology, and flexibility from atoms to macroscopic matter.AngewandteChemie - International Edition in English, 1990: 29(2), p. 138-175. 2. Kitchens, K.M., et al., Transport of poly(amidoamine) dendrimers across Caco-2 cell monolayers: Influence of size, charge and fluorescent labeling.Pharm Res, 2006. 23(12): p. 2818-26. 3. Esfand, R., et al., Poly(amidoamine) (PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications.DrugDiscov Today, 2001 Apr 1;6(8):427-436 References

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