Document Type

Dissertation - University Access Only

Award Date

2010

Degree Name

Doctor of Philosophy (PhD)

Department / School

Pharmaceutical Sciences

Abstract

Skin represents an easily accessible site for delivery of drugs. However, the delivery of drug molecules is limited by the remarkable barrier property of skin. As a result only a handful of molecules are presently delivered through skin. Several chemical and physical enhancement strategies have evolved to expand the scope of drugs delivered through skin. This dissertation focuses on exploring the use of dendrimer as a novel nanocarrier for transcutaneous delivery of small and large drug molecules. To this end, the main objective was to understand structure-permeability relationship and use this knowledge to design dendrimer based transcutaneous drug delivery systems. The first objective was to investigate the skin penetration enhancing properties of dendrimer using 5-tluorouracil (5FU) as a model hydrophilic molecule. The second objective was to study the interaction of dendrimer with skin as a function of surface charge, generation and treatment time. The dendrimer was compared with other polymeric and lipid nanocarriers. The third objective was to determine if dendrimer can be used as a charged nanocarrier for iontophoretic delivery of oligonucleotides. The fourth objective was to investigate the transcutaneous iontophoretic delivery of bcl-2 antisense oligonucleotide - dendrimer complex for skin cancer treatment. All the in-vitro skin penetration experiments were performed using excised porcine skin. Results from in-vitro skin penetration studies showed that poly (amidoamine) (PAMAM) dendrimer increased the skin penetration of 5FU by two to three fold when delivered from lipophilic vehicles. Skin pre-treatment with dendrimer enhanced the 5FU skin penetration greater than co-treatment, indicating that dendrimer enhances penetration mainly by interacting with skin. Structure-activity studies showed that skin penetration enhancement was in the following decreasing order, cationic dendrimer>neutral dendrimer>anionic dendrimer. The skin penetration enhancement of 5FU was dependent on molecular weight of dendrimer where, lower generation dendrimer caused higher permeation enhancement than higher generation dendrimers. Further, dendrimer showed a linear increase in 5FU permeation as a function of concentration (0.1 - 10 mM). Using FT-IR spectroscopy, skin resistance measurements and trans-epidermal water loss (TEWL), it was found that dendrimer increased the skin permeation of 5FU mainly by interacting with skin lipids. To understand the structure - permeability relationship, flouroisothiocynate (FITC) labeled polymers and fluorescein labeled liposome were used. Confocal laser scanning microscope (CLSM) analysis showed that the dendrimer was mainly limited to stratum corneum (SC) after 2 hrs of application. Neutral dendrimers penetrated the skin deeper compared to charged dendrimers after 12 hrs treatment. On the other hand, in presence of 0.3 mA/cm2 electric current, cationic dendrimer showed deeper skin penetration. An increase in dendrimer generation (molecular weight from 3.2 kDa to 58k.Da), caused an exponential decrease in the skin penetration in presence and absence of iontophoresis. Symmetrically branched and spherical dendrimers showed greater extent of skin penetration compared to irregularly branched, polyethylene imine (PEI-B) and linear polyethylene imine (PEI-L) in presence iontophoresis. On the other hand, cationic dendrimer and cationic liposome showed similar skin penetration. The feasibility of using dendrimer as an iontophoretic carrier was studied using bcl-2 as a model antisense oligonucleotide (ASO, 18 mer). Dendrimer formed a smaller complex with ASO (152 ± 35 nm) compared to PEI-B (173 ± 21) and liposome (205 ± 35). CLSM studies showed that after passive application both polymer and liposome complexed ASO showed similar skin penetration. Similarly, free ASO was also limited to SC. On the other hand, application of iontophoresis (0.3 mA/cm2) enhanced the skin penetration up to a depth of 100 μm for ASO-dendrimer and ASO-liposome complexes; while, ASO-PEI-B complex penetrated up to a depth of 80 μm. Colocalization studies showed that the complex was intact within the skin layers. Skin penetration studies using 32P-ASO showed similar results to that of CLSM. ASO-dendrimer and ASO-liposome complexes showed similar flux and was 2.5 times higher compared to iontophoresis of ASO-PEI-B complex and 19 fold higher than iontophoresis of free ASO. The cell-uptake of ASO-dendrimer complex was studied in human epidermoid carcinoma cells (A431 ). Cell uptake studies using FITC labeled ASO and flow cytometric analysis showed that ASO-dendrimer complex internalized into the cells within 15 minutes. On the other hand, free ASO was not taken-up by the cells even after 120 minutes treatment. Protein analysis showed that ASO-dendrimer complex decreased the bcl-2 protein levels; while, free ASO or sense oligonucleotide had no effect. In-vivo studies were performed in skin cancer mouse model where tumors were induced by 7, 12-dimethyl benzanthracene (DMBA) and 12-O-tetradecanoylphorbol 13-acetate (TPA) application. Results showed that iontophoresis of ASO-dendrimer complex significantly reduced the tumor size (by 55%) compared to control. Iontophoresis of free ASO, passive application of ASO-dendrimer complex, intradermal administration of free ASO, ASOdendrimer complex did not show significant reduction in tumor volume. Similarly, results from bcl-2 protein analysis showed that iontophoresis of ASO-dendrimer suppressed the bcl-2 protein levels by 45%. Apoptosis assay showed that only the skin samples treated with iontophoresis of ASO-dendrimer complex underwent apoptosis mediated cell death. The findings from this study demonstrate that dendrimers are promising nanocarriers for delivery of small and large molecule drugs into and across skin.

Library of Congress Subject Headings

Dendrimers in medicine

Transdermal medication

Nanomedicine

Drug delivery systems

Format

application/pdf

Number of Pages

252

Publisher

South Dakota State University

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