Chitosan (CS) shows in vitro and in vivo efficacy for siRNA delivery but with contradictory findings for incompletely characterized systems. For understanding which parameters produce effective delivery, a library of precisely characterized chitosans was produced at different degrees of deacetylation (DDAs) and average molecular weights (M). Encapsulation and transfection efficiencies were characterized in vitro. Formulations were selected to examine the influence of M and N:P ratio on nanoparticle uptake, metabolic activity, genotoxicity, and in vitro transfection. Hemocompatibility and in vivo biodistribution were then investigated for different M, N:P ratios, and doses. Nanoparticle uptake and gene silencing correlated with increased surface charge, which was obtained at high DDA and high M. A minimum polymer length of ∼60-70 monomers (∼10 kDa) was required for stability and knockdown. In vitro knockdown was equivalent to lipid control with no metabolic or genotoxicity. An inhibitory effect of serum on biological performance was dependent on DDA, M, and N:P. In vivo biodistribution in mice show accumulation of nanoparticles in kidney with 40-50% functional knockdown.

The selective estrogen receptor downregulator (SERD) fulvestrant can be used as second-line treatment for patients relapsing after treatment with tamoxifen, a selective estrogen receptor modulator (SERM). Unlike tamoxifen, SERDs are devoid of partial agonist activity. While the full antiestrogenicity of SERDs may result in part from their capacity to downregulate levels of estrogen receptor alpha (ER␣) through proteasome-mediated degradation, SERDs are also fully antiestrogenic in the absence of increased receptor turnover in HepG2 cells. Here we report that SERDs induce the rapid and strong SUMOylation of ER␣ in ER␣-positive and -negative cell lines, including HepG2 cells. Four sites of SUMOylation were identified by mass spectrometry analysis. In derivatives of the SERD ICI164,384, SUMOylation was dependent on the length of the side chain and correlated with full antiestrogenicity. Preventing SUMOylation by the overexpression of a SUMO-specific protease (SENP) deSUMOylase partially derepressed transcription in the presence of full antiestrogens in HepG2 cells without a corresponding increase in activity in the presence of agonists or of the SERM tamoxifen. Mutations increasing transcriptional activity in the presence of full antiestrogens reduced SUMOylation levels and suppressed stimulation by SENP1. Our results indicate that ER␣ SUMOylation contributes to full antiestrogenicity in the absence of accelerated receptor turnover. E strogens, mainly 17␤-estradiol (E2), play a crucial role in normal breast development but also contribute to mammary tumorigenesis. Antiestrogens (AEs) used for breast cancer treatment and prevention, such as tamoxifen (Tam), raloxifene (Ral), or fulvestrant (9, 17, 52, 63), block the proliferative effects of estrogens on breast epithelial and carcinoma cells by competing for estrogen receptors (ERs) (ER␣ and ER␤). Similar to other nuclear receptors, ERs activate gene transcription by binding to specific DNA sites and recruiting transcriptional coactivators in a liganddependent manner (13,40,41,70,85).AEs prevent ER activation through the induction of an altered conformation of the receptor ligand binding domain (LBD) that suppresses the recruitment of coactivators (8,66,79) and/or increases the recruitment of corepressors (23,34,45,61,77,83,94). However, selective ER modulators (SERMs) (which include Tam and Ral) have partial agonist activity in a tissue-and gene-specific manner. For example, both have estrogenic effects on bone mass (6), and Tam has estrogenic effects on the uterus (2,5,21,88,90), while Ral does not cause uterine hypertrophy (6). Tam and, to a lesser extent, Ral also have partial agonist activity in breast cancer cells in a gene-specific manner (22). The tissue-specific recruitment of coactivators and corepressors is thought to underlie selective partial agonist activity, and alterations in the expression patterns or activity of ER cofactors in breast cancer cells could contribute to the development of resistance to AE-based therapy (23,37,38,45,77,81,82).Other AEs,...

Although artificial C2-H2 zinc fingers can be designed to recognize specific DNA sequences, it remains unclear to which extent nuclear receptor C4 zinc fingers can be tailored to bind novel DNA elements. Steroid receptors bind as dimers to palindromic response elements differing in the two central base pairs of repeated motifs. Predictions based on one amino acid—one base-pair relationships may not apply to estrogen receptors (ERs), which recognize the two central base pairs of estrogen response elements (EREs) via two charged amino acids, each contacting two bases on opposite DNA strands. Mutagenesis of these residues, E203 and K210 in ERα, indicated that both contribute to ERE binding. Removal of the electric charge and steric constraints associated with K210 was required for full loss of parental DNA-binding specificity and recognition of novel sequences by E203 mutants. Although some of the new binding profiles did not match predictions, the double mutation E203R-K210A generated as predicted a mutant ER that was transcriptionally active on palindromes of PuGCTCA motifs, but not on consensus EREs. This study demonstrates the feasibility of designing C4 zinc finger mutants with novel DNA-binding specificity, but also uncovers limitations of this approach.