ESTROGEN ANCHORED NANOMICELLES FOR COMBINATIONAL DRUG DELIVERY WITH ENHANCED THERAPEUTIC EFFICACY: A PROTEOMICS GUIDED INVESTIGATION FOR MULTIFUNCTIONAL NANOTHERAPEUTICS

The employment of the stable isotope labeling with amino acids in cell culture (SILAC) based proteomic analysis as a guidance tool for investigation of cellular response of a multifunctional nanodelivery system has been described for cancer therapy. The multifunctional nanodelivery system in this study was based on an estrogen anchored multimodal nanomicelle (NPG). The key components of the multifunctional nanomicelle consist: β-cyclodextrin conjugated estrone (CDE1), an escort molecule; polymeric BH3 mimetic, a proapoptotic BcL-2 inhibitor; and the mitotic catastrophe agent paclitaxel. They were assembled into the nanomicelle by multiple weak interactions including hydrophobic/hydrophilic interaction and host-guest recognition. The resulting nanomicelle exhibited unimodal morphology with average size of 170 nm with sustained release. It was found that the nanoparticle exhibits excellent anti-tumor activities for the treatment of breast adenocarcinoma with exceptional targeting efficacy both in MCF-7 tumor bearing mice. NPG as a new drug delivery system demonstrated several merits such as the increased drug uptake in breast tumor tissue, low toxicity, potent tumor growth retardation and metastasis inhibition, as well as potential clinical practicality without compromising liver, kidney and immune function and ameliorating the conventional chemotherapeutics induced phlebitis in breast tumor bearing nude mice model. The systematic SILAC based proteomics study and the subsequent validation revealed that the synergistic induction of mitotic catastrophe through enhanced G2/M phase arrest and PI3K/Akt/mTOR mediated autophagy, account for the exceedingly potent anti-tumor activity of this convergent nanomicelle. Additionally, the verification of the top upregulated gene from the proteomics profiling revealed that the overexpression of zinc finger protein 350 (ZNF350/ZBRK1) is associated with the enhanced antitumor effect induced by NPG.KEYWORDS:  Drug targeting, Estrogen Receptor, Proteomics, Paclitaxel, Gossypol, SynergismÂ

Synthesis and biological evaluation of novel folic acid receptor-targeted, β-cyclodextrin-based drug complexes for cancer treatment

Drug targeting is an active area of research and nano-scaled drug delivery systems hold tremendous potential for the treatment of neoplasms. In this study, a novel cyclodextrin (CD)-based nanoparticle drug delivery system has been assembled and characterized for the therapy of folate receptor-positive [FR(+)] cancer. Water-soluble folic acid (FA)-conjugated CD carriers (FACDs) were successfully synthesized and their structures were confirmed by 1D/2D nuclear magnetic resonance (NMR), matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS), high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR), and circular dichroism. Drug complexes of adamatane (Ada) and cytotoxic doxorubicin (Dox) with FACD were readily obtained by mixed solvent precipitation. The average size of FACD-Ada-Dox was 1.5-2.5 nm. The host-guest association constant Ka was 1,639 M-1 as determined by induced circular dichroism and the hydrophilicity of the FACDs was greatly enhanced compared to unmodified CD. Cellular uptake and FR binding competitive experiments demonstrated an efficient and preferentially targeted delivery of Dox into FR-positive tumor cells and a sustained drug release profile was seen in vitro. The delivery of Dox into FR(+) cancer cells via endocytosis was observed by confocal microscopy and drug uptake of the targeted nanoparticles was 8-fold greater than that of non-targeted drug complexes. Our docking results suggest that FA, FACD and FACD-Ada-Dox could bind human hedgehog interacting protein that contains a FR domain. Mouse cardiomyocytes as well as fibroblast treated with FACD-Ada-Dox had significantly lower levels of reactive oxygen species, with increased content of glutathione and glutathione peroxidase activity, indicating a reduced potential for Dox-induced cardiotoxicity. These results indicate that the targeted drug complex possesses high drug association and sustained drug release properties with good biocompatibility and physiological stability. The novel FA-conjugated β-CD based drug complex might be promising as an anti-tumor treatment for FR(+) cancer

Liver X Receptor Agonist Therapy Prevents Diffuse Alveolar Hemorrhage in Murine Lupus by Repolarizing Macrophages

The generation of CD138+ phagocytic macrophages with an alternative (M2) phenotype that clear apoptotic cells from tissues is defective in lupus. Liver X receptor-alpha (LXRα) is an oxysterol-regulated transcription factor that promotes reverse cholesterol transport and alternative (M2) macrophage activation. Conversely, hypoxia-inducible factor 1-α (HIF1α) promotes classical (M1) macrophage activation. The objective of this study was to see if lupus can be treated by enhancing the generation of M2-like macrophages using LXR agonists. Peritoneal macrophages from pristane-treated mice had an M1 phenotype, high HIFα-regulated phosphofructokinase and TNFα expression (quantitative PCR, flow cytometry), and low expression of the LXRα-regulated gene ATP binding cassette subfamily A member 1 (Abca1) and Il10 vs. mice treated with mineral oil, a control inflammatory oil that does not cause lupus. Glycolytic metabolism (extracellular flux assays) and Hif1a expression were higher in pristane-treated mice (M1-like) whereas oxidative metabolism and LXRα expression were higher in mineral oil-treated mice (M2-like). Similarly, lupus patients’ monocytes exhibited low LXRα/ABCA1 and high HIF1α vs. controls. The LXR agonist T0901317 inhibited type I interferon and increased ABCA1 in lupus patients’ monocytes and in murine peritoneal macrophages. In vivo, T0901317 induced M2-like macrophage polarization and protected mice from diffuse alveolar hemorrhage (DAH), an often fatal complication of lupus. We conclude that end-organ damage (DAH) in murine lupus can be prevented using an LXR agonist to correct a macrophage differentiation abnormality characteristic of lupus. LXR agonists also decrease inflammatory cytokine production by human lupus monocytes, suggesting that these agents may be have a role in the pharmacotherapy of lupus

Controllable drug uptake and nongenomic response through estrogen-anchored cyclodextrin drug complex

Breast cancer is a leading killer of women worldwide. Cyclodextrin-based estrogen receptor-targeting drug-delivery systems represent a promising direction in cancer therapy but have rarely been investigated. To seek new targeting therapies for membrane estrogen receptor-positive breast cancer, an estrogen-anchored cyclodextrin encapsulating a doxorubicin derivative Ada-DOX (CDE1-Ada-DOX) has been synthesized and evaluated in human breast cancer MCF-7 cells. First, we synthesized estrone-conjugated cyclodextrin (CDE1), which formed the complex CDE1-Ada-DOX via molecular recognition with the derivative adamantane-doxorubicin (Ada-DOX) (Kd =1,617 M(-1)). The structure of the targeting vector CDE1 was fully characterized using (1)H- and (13)C-nuclear magnetic resonance, mass spectrometry, and electron microscopy. CDE1-Ada-DOX showed two-phase drug-release kinetics with much slower release than Ada-DOX. The fluorescence polarization analysis reveals that CDE1-Ada-DOX binds to recombinant human estrogen receptor α fragments with a Kd of 0.027 µM. Competition assay of the drug complex with estrogen ligands demonstrated that estrone and tamoxifen competed with CDE1-Ada-DOX for membrane estrogen receptor binding in MCF-7 cells. Intermolecular self-assembly of CDE1 molecules were observed, showing tail-in-bucket and wire-like structures confirmed by transmission electronic microscopy. CDE1-Ada-DOX had an unexpected lower drug uptake (when the host-guest ratio was >1) than non-targeting drugs in MCF-7 cells due to ensconced ligands in cyclodextrins cavities resulting from the intermolecular self-assembly. The uptake of CDE1-Ada-DOX was significantly increased when the host-guest ratio was adjusted to be less than half at the concentration of CDE1 over 5 µM due to the release of the estrone residues. CDE1 elicited rapid activation of mitogen-activated protein kinases (p44/42 MAPK, Erk1/2) in minutes through phosphorylation of Thr202/Tyr204 in MCF-7 cells. These results demonstrate a targeted therapeutics delivery of CDE1-Ada-DOX to breast cancer cells in a controlled manner and that the drug vector CDE1 can potentially be employed as a molecular tool to differentiate nongenomic from genomic mechanism

The TEM (a) and AFM (b) images of FACD-Ada-Dox molecules.

<p>The TEM (a) and AFM (b) images of FACD-Ada-Dox molecules.</p

Determination of intraceullar ROS, GPx and GSH levels in mouse 3T3 cells.

<p>Plot a shows the level of intracellular ROS in 3T3 cells treated with Dox, Ada-Dox or FACD-Ada-Dox at 5.0 µM over 60 min at 37°C in the culture media. Cells were treated with CM-H₂DCFDA. Plot b displays the activity of GPx in 3T3 cells in the presence of Dox, Ada-Dox or FACD-Ada-Dox at 5.0 µM. Plot c shows the GSH concentrations (expressed as nmol/mg protein) in 3T3 cells in the presence of Dox, Ada-Dox or FACD-Ada-Dox at 5.0 µM. Values are the mean ± SD of three different homogenates of cells analyzed in triplicate. ^*<i>P</i><0.05; ^**<i>P</i><0.01; and ^***<i>P</i><0.001.</p