Etodolac hydrazide and a novel series of etodolac hydrazide-hydrazones 3-15 and etodolac 4-thiazolidinones 16-26 were synthesized in this study. The structures of the new compounds were determined by spectral (FT-IR, (1)H NMR, (13)C NMR, HREI-MS) methods. Some selected compounds were determined at one dose toward the full panel of 60 human cancer cell lines by the National Cancer Institute (NCI, Bethesda, USA). 2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-yl)acetic acid[(4-chlorophenyl)methylene]hydrazide 9 demonstrated the most marked effect on the prostate cancer cell line PC-3, with 58.24% growth inhibition at 10(-5) M (10 µM). Using the MTT colorimetric method, compound 9 was evaluated in vitro against the prostate cell line PC-3 and the rat fibroblast cell line L-929, for cell viability and growth inhibition at different doses. Compound 9 exhibited anticancer activity with an IC(50) value of 54 µM (22.842 µg/mL) against the PC-3 cells and did not display any cytotoxicity toward the L-929 rat fibroblasts, compared to etodolac. In addition, this compound was evaluated for caspase-3 and Bcl-2 activation in the apoptosis pathway, which plays a key role in the treatment of cancer.

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Topical application of plasmid DNA represents an attractive route of gene delivery. Although chitosan (CS) has been widely investigated as a gene-carrier, there is very limited information about the skin application of CS-based systems for DNA. This study evaluated pDNA-loaded chitosan nanoparticles (CS-NPs) for skin gene delivery. NPs were prepared by inducing the gelation of CS upon interaction with sodium tripolyphosphate. pSV-β-Gal was used as a reporter gene. The size, surface charge, and the other in vitro characteristics of CS-NPs were examined. Primary human dermal fibroblast cells (HDF) and mouse fibroblast NIH 3T3 cell lines (ATCC CCL-92) were used for in vitro transfection studies. In in vivo study, CS-NPs were applied to the skin of baby and adult Sprague Dawley rats by spreading on the shaved area of the back of animals. During a week animals were sacrificed and skin biopsies were taken for β-Gal expression. β-galactosidase enzyme activity was determined spectrophotometrically at 420 nm. The distribution of β-galactosidase expressing cells within the skin tissue was observed by X-gal histochemical method. β-galactosidase was continuously expressed at the nanoparticle-treated skin during the 7 days. High and continuous β-Gal expressions were obtained with CS-NPs, although it was low in the first day. When a comparison was made between the data of baby and adult rats, markedly high transfection were measured in the skin samples of the baby rats. NPs protected pDNA against the enzyme and serum attacks. In conclusion, CS-NPs showed in vivo transfection potential in rats for skin gene delivery.

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Skin delivery of antisense oligonucleotides (AsODNs) has exciting potential in the treatment of skin diseases. However, the therapeutic applications of oligonucleotide-based therapies are limited by the instability of these molecules toward nucleases, short half-life in vivo, and insufficient cellular uptake. The purpose of this study was to investigate in vivo antisense effect of AsODN-loaded chitosan nanoparticles after topical application. AsODN-loaded chitosan nanoparticles were topically applied to Sprague Dawley rats (adult and baby). At 1, 3, 6, 9, and 12 days posttransfection, animals' skin samples were taken for measurement of beta-galactosidase (beta-Gal) expression and histological control. After topical application of AsODN-loaded chitosan nanoparticles in different doses, beta-Gal expression reduced significantly. Highest inhibition was observed after 6 days of transfection of nanoparticles. Free AsODNs exhibited 35% of beta-Gal inhibition on the first day. beta-Gal expression was inhibited in approximately 82-85% with transfection of nanoparticles containing 30 microg AsODNs at 6 days. The antisense effect of AsODN-loaded chitosan nanoparticle in baby skin was evaluated at 6 days: 77-86% of beta-Gal suppression was measured and differences between the doses were not significant. Thus, chitosan nanoparticles are useful carrier for delivery of AsODNs into skin cells of rats and may be used for topical application on human skin.

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VEGF is an angiogenic factor promoting the proliferation and migration of endothelial cells. Inhibition of VEGF by RNAi mechanism is one of the novel and the most important strategies in antiangiogenesis therapy. In this study, the tumor silencing efficiency of ternary complexes after addition of protamine to chitosan complexes containing VEGF targeting shRNA was investigated. Besides chitosan, protamine is an effective gene delivery material. Binary and ternary complexes consisting of chitosan, protamine, and shRNA were prepared to target VEGF, their morphology, size, and zeta potential of the complexes being measured. The average size of the complexes was between 173 and 284 nm and zeta potential was between +10 and 16 mV. In the ternary complexes, size decreased as the chitosan ratio increased; however, its molecular weight had no effect on the size of complexes. HeLa, HEK293, and MCF-7 cell lines were used for in vitro transfection. VEGF was assayed by ELISA. A higher silencing effect was obtained using ternary complexes. Transgene expression was increased by adding protamine to chitosan complexes. Gene inhibition values in cell lines followed the rank HEK293>HeLa>MCF-7. The addition of protamine to the chitosan/shRNA (VEGF) complexes increased the knockdown of VEGF genes in the cell lines, and no cytotoxicity was found after the complexes had been incorporated into the cells.