Document Type

Thesis - University Access Only

Award Date

2006

Degree Name

Master of Science (MS)

Department / School

Pharmaceutical Sciences

Abstract

Polyamines (putrescine, spermidine and spermine) are abundant in all living systems and are essential for cell proliferation and carcinogenesis. Prostate cancer cell proliferation requires an increased rate of polyarnine biosynthesis and metabolism. Several studies have demonstrated that polyamines have the potential to be used as prostate cancer biomarkers. However, their major set back for in vivo application is rapid elimination from the body. To circumvent this problem, we propose to conjugate the polyarnines or its precursor to a carrier system such as a dendrimer. We hypothesize that: (a) as polyamines are taken up by carrier mediated transport across the cell membrane, conjugation of putrescine and its precursors ( arginine and ornithine) to P AMAM (polyamidoamine) dendrimer will reduce their in vivo clearance, and (b) these surface modified dendrimers are likely to be taken up by the same transport pathway as that of polyamines ( carrier mediated uptake) when administered in vivo. The primary amine groups on the surface of P AMAM dendrimers were grafted with F-moc blocked L-omithine and L-arginine. The cellular uptake study was conducted with fluorescein isothiocyanate (FITC) labeled dendrimers of different concentrations (0.1-10 μg/mL) by incubating with human prostate cancer (PC-3) and normal (CV-1) cells for 4 hrs at 37°C. The cells were observed under the laser scanning confocal microscope and by flow cytometry. Toxicity of the dendrimers to various cell lines [IPEC-J2, CV-l(monkey kidney) and Caco-2 cells] was evaluated by MTT (Methylthiazoletetrazolium) assay. The NMR and MALDI-TOF spectral analysis showed that more than 55 molecules of ornithine and arginine were coupled to a G4 NH2 terminal P AMAM dendrimer. Toxicity of the surface modified dendrimers was comparable to that of the native PAMAM dendrimers. However, above 100 μg/mL the arginine-conjugated dendrimers were significantly more toxic than P AMAM dendrimers (p< 0.05). The permeability coefficients (Papp) for the surface modified dendrimers in both Caco-2 (1.17 x 10 -6 cm/s for ornithine modified and 1.29 x 10 -6 emfs for arginine modified) and IPEC-J2 cells (0.87 x 10 -G emfs for omithine modified and 0.99 x 10 -6 emfs for arginine modified) were significantly higher than the PAMAM dendrimers (0.93 x 10 -6 cm/s for Caco-2 cells and 0.71 x 10 -6 emfs for IPEC-J2 cells). The confocal microscopy and flow cytometry studies revealed that the uptake was in the following order: PAMAMArginine> PAMAM-Omithine> PAMAM dendrimers. As the cellular uptake of polyamine-conjugated dendrimers was higher, it is likely that these polyamine-conj ugated dendrimers could serve as efficient carriers for gene delivery. A gene transfection study was performed in HEK 293 T (human embryonic kidney) cells using green fluorescent protein (GFP) plasmid as a model plasmid. The transfection efficiency of the PAMAM-arginine, PAMAM-ornithine and PAMAM were 35%, 4% and 2% respectively. In conclusion, to reduce the polyamine elimination from the body, polyamineconjugated P AMAM dendrimers were synthesized and characterized. The novel surface modified dendrimers were found to be relatively selective towards prostate cancer cells and were having high permeability coefficients than native P AMAM dendrimers. The arginine modified dendrimer showed improved gene expression in HEK 293 T cells. The results suggest that novel surface modified dendrimers have a great potential as carriers in: ( a) in the diagnosis and targeted therapy of prostate cancer and (ii) targeted gene therapy of prostate cancer.

Library of Congress Subject Headings

Polyamines in the body

Dendrimers

Prostate -- Cancer

Biochemical markers

Format

application/pdf

Number of Pages

133

Publisher

South Dakota State University

Download

Share

COinS