Evaluation of Poly (Amidoamine) Dendrimer Permeability and Transport Mechanisms across Caco-2 2 Cell Monolayers

Transcription

1 Evaluation of Poly (Amidoamine) Dendrimer Permeability and Transport Mechanisms across Caco-2 2 Cell Monolayers Kelly Marie Kitchens Center for Nanomedicine and Cellular Delivery Department of Pharmaceutical Sciences University of Maryland, Baltimore

2 Poly (Amidoamine) Dendrimers G1 Ethylenediamine Core Cationic Neutral Anionic PAMAM-NH 2 PAMAM-OH PAMAM-COOH G0 G2 Defined mass, size, shape and surface chemistry Potential use as oral drug delivery systems Florence (ed.) Adv Drug Del Rev, (2005)

3 AB BA AB BA Papp x 10 6 (cm/s) PAMAM-NH 2 Permeability El-Sayed et al., J Control Rel, (2002) G0 G1 G2 G3 G4 G0 G1 G2 G3 G minutes minutes AB AB BA BA Papp x 10 6 (cm/s) C-Mannitol Permeability El-Sayed et al., J Bioactive Compat, (2003) Control G0 G1 G2 G3 G Control G0 G1 G2 G3 G minutes minutes Papp x 10 6 (cm/s) AB 4.00 BA AB BA 14 C-Mannitol Permeability El-Sayed et al., J Bioactive Compat, (2003) G-0.5 G0.5 G1.5 G2.5 G3.5 G G-0.5 G0.5 G1.5 G2.5 G3.5 G minutes minutes Control EDTA Control EDTA

4 Research Objectives I. Influence of surface charge and size II. Effect of hydrophobic drug loading III. Transport mechanisms of PAMAM

5 Experimental Methodology: PAMAM Permeability PAMAM NH 3+ + Fluorescein-5-N=C=S G4NH 2 :FITC = 1:1, 1:4, 1:8 PBS,Acetone PAMAM NH-C=S NH-Fluorescein PAMAM PAMAM ph = 7.4 OH + Fluorescein-5-N=C=S 1 Mouse anti-occludin 2 Alexa Fluor 488 anti-mouse IgG PAMAM COO - + Fluorescein-5-N=C=S TEA,DMF Heat,N 2 C 2 H 8 N 2,EDC PBS,Acetone PAMAM Rhodamine Phalloidin Mannitol PAMAM O-C=S NH-Fluorescein O = CNH(CH 2 ) 2 NH-C=S NH-Fluorescein

6 Structural Features of PAMAM Dendrimers Generation Surface Group # Surface Groups M W (Da) a Label Content (mmol FITC / g dendrimer) b G2 -NH , ± G2 -OH 16 3, ± G1.5 -COOH 16 2, ± G2.5 -COOH 32 6, ± G3.5 -COOH 64 12, ± G4:FITC (1:1) -NH , ± G4:FITC (1:4) -NH , ± G4:FITC (1:8) -NH , ± a Reported by the manufacturer, Dendritech, Inc., Midland, MI b Label content is reported as mean ± SD (n = 3) Kitchens et al., Pharm Res, (2006)

7 Cell Viability via WST-1 Assay 140 HBSS G2OH 120 G2OH-FITC G1.5COOH % Viability G1.5COOH-FITC G2.5COOH G2.5COOH-FITC G3.5COOH G3.5COOH-FITC G2NH2 G2NH2-FITC G4NH2 20 G4NH2:FITC (1:1) G4NH2:FITC (1:4) mm 0.1 mm 1.0 mm G4NH2:FITC (1:8) Triton X-100 PAMAM Concentration Mean ± SD (n = 3) P < 0.05, reduction in viability Kitchens et al., Pharm Res, (2006)

8 Permeability across Caco-2 Cell Monolayers hour 2 hours Permeability x 10 6 (cm/s) 20 5 Mean ± SEM (n = 9) Toxic (WST-1 assay) 0 G2OH G1.5 G2.5 G3.5 G2NH2 G4:FITC G4:FITC G4:FITC (1:1) (1:4) (1:8) Kitchens et al., Pharm Res, (2006)

9 Mannitol Permeability in the Presence of PAMAM hour 2 hours Permeability x 10 6 (cm/s) Mean ± SEM (n = 9) Toxic (WST-1 assay) P < 0.05, increase in P app 0 Mannitol G2OH G2OH- G1.5 G1.5- G2NH2 G2NH2- G4NH2 G4:FITC FITC FITC FITC (1:8) Kitchens et al., Pharm Res, (2006)

10 Influence of PAMAM on Tight Junction Protein Occludin Mean ± SD (n = 3) P < 0.05, increase in signal Bars = 100 µm Control G2NH 2 G2OH 34.2 ± 13.4% 14.4 ± 4.0% G1.5COOH G2.5COOH G3.5COOH 60x oil objective 100 µm pinhole 0.40 µm z-step size z y x 50.8 ± 8.6% 56.6 ± 5.9% 51.9 ± 7.5% Kitchens et al., Pharm Res, (2006)

11 Influence of PAMAM on Tight Junction Protein Actin Mean ± SD (n = 3) P < 0.05, increase in signal Bars = 100 µm Control G2NH 2 G2OH 36.1 ± 3.5% 13.1 ± 6.8% G1.5COOH G2.5COOH G3.5COOH 60x oil objective 100 µm pinhole 0.40 µm z-step size z y x 54.8 ± 20.9% 44.6 ± 2.0% 63.3 ± 2.2% Kitchens et al., Pharm Res, (2006)

12 Permeability x 10 6 (cm/s) Suggested Transport Mechanisms Permeability x 10 6 (cm/sec) C 4 C 2.37 G2 Permeability 4.21 Permeability x 10 6 (cm/sec) Permeability w/palmitoyl Carnitine PC + PC min 150 min 0 G2 Mannitol G2 Mannitol 14 C-Paclitaxel Permeability G2 Permeability G2 + G Paclitaxel + Paclitaxel Permeability x 10 6 (cm/sec) Permeability x 10 6 (cm/sec) AB AB BA BA 0 AB BA El-Sayed et al., Int J Pharm, (2003)

13 Experimental Methodology: Transport Mechanisms G2-FITC & G1.5-FITC 1 - Antibodies against endocytosis markers 2 - Alexa Fluor 568 goat anti-mouse IgG LysoTracker Red Colocalization Coefficient M x = x i,coloc i x i i Merge/Colocalization Manders et al., J Microsc, (1993)

14 Colocalization with Clathrin FITC Clathrin Merge Tf Mx = 80.3 ± 3.1% G2 Mx = 74.3 ± 5.2% G1.5 Mx = 70.8 ± 3.9% 20 minutes Mean ± SEM (n = 6) Bars = 5 µm

15 Colocalization with LAMP-1 FITC LAMP1 Merge Tf Mx = 63.9 ± 7.3% G2 Mx= 37.3 ± 5.9% G1.5 Mx = 58.7 ± 2.0% 20 minutes Mean ± SEM (n = 6) Bars = 5 µm

16 Colocalization: Clathrin & LAMP-1 Clathrin LAMP-1 Tf Mx = 83.4 ± 0.2% Tf Mx = 57.0 ± 8.6% G2 Mx = 73.7 ± 3.8% G2 Mx = 59.0 ± 5.2% G1.5 Mx = 75.3 ± 11.2% G1.5 Mx = 48.9 ± 6.9% 60 minutes Mean ± SEM (n = 6) Bars = 5 µm

17 PAMAM Effect on Microvilli G2NH 2 (1 mm) G1.5COOH (1 mm) Control 12,500x Magnification Bars = 1 µm G4NH 2 (1 mm) Generation-dependent effect G3.5COOH (1mM)

18 G4NH 2 Effect on Microvilli Control G4NH 2 (0.01mM) G4NH 2 (0.1 mm) G4NH 2 (1 mm) 12,500x Magnification Bars = 1 µm Concentration-dependent effect

19 Conclusions Cationic and large generation dendrimers had greater permeability than anionic and neutral Hydrophobic drug loading reduced toxicity and enhanced permeability PAMAM dendrimers modulate tight junctions PAMAM dendrimers colocalize with endocytosis markers Cationic dendrimers cause cytotoxicity by compromising microvilli integrity Design dendrimers for optimized oral drug delivery applications

20 Acknowledgements Hamid Ghandehari, Ph.D. Peter Swaan, Ph.D. Amy Foraker Rohit Kolhatkar, Ph.D. Richard Coleman, Ph.D. Maritza Patton UMB Pharmaceutical Sciences NRSA NIH pre-doctoral fellowship F31-GM67278