Curcumin-encapsulating Nanogels as an Effective Anticancer Formulation for Intracellular Uptake.

Nanoscale drug delivery systems represent an attractive strategy to improve both the efficacy and safety of anticancer drugs. In this work, we describe nanoformulation of curcumin, a most potent natural anticancer compound capable of killing cancer cells while sparing the normal tissues. Since curcumin is a natural hydrophobic polyphenol, it has a low aqueous solubility and bioavailability, which are challenging to its therapeutic efficacy. We developed and evaluated a novel colloidal nanogel carrier for encapsulation of curcumin to increase its solubility and cytotoxicity. Amphiphilic Poloxamer-cationic network in the nanogel NG127 was designed to efficiently encapsulate curcumin. Homogenous drug complexes were obtained with 20-25% content of curcumin and the particle size of ca. 150 nm. Using ImageStream multispectral imaging flow cytometry, we demonstrated that the curcumin-nanogel formulation (C-NG) was readily internalized into MDA-231 breast cancer cells. A real-time cell growth electronic sensing assay was used to measure proliferation responses of various breast cancer cells to C-NG treatments. Our results indicated that the C-NG formulation was 70-85% more effective in inhibiting growth, at concentrations lower than IC50 of free curcumin. This was also confirmed morphologically by modified acridine orange/ethidium bromide staining and fluorescent microscopy. Importantly, nanocarrier NG127 alone displayed practically no cytotoxicity. We conclude that nanogel carriers offer an innovative way to encapsulate curcumin and to obtain more effective anticancer therapeutics than curcumin alone with a potential to specific tumor targeting, such as using antibodies against surface receptors specific to breast cancer cells.

High throughput, parallel imaging and biomarker quantification of human spermatozoa by ImageStream flow cytometry.

Spermatid specific thioredoxin-3 protein (SPTRX-3) accumulates in the superfluous cytoplasm of defective human spermatozoa. Novel ImageStream technology combining flow cytometry with cell imaging was used for parallel quantification and visualization of SPTRX-3 protein in defective spermatozoa of five men from infertile couples. The majority of the SPTRX-3 containing cells were overwhelmingly spermatozoa with a variety of morphological defects, detectable in the ImageStream recorded images. Quantitative parameters of relative SPTRX-3 induced fluorescence measured by ImageStream correlated closely with conventional flow cytometric measurements of the same sample set and reflected the results of clinical semen evaluation. Image Stream quantification of SPTRX-3 combines and surpasses the informative value of both conventional flow cytometry and light microscopic semen evaluation. The observed patterns of the retention of SPTRX-3 in the sperm samples from infertility patients support the view that SPTRX3 is a biomarker of male infertility.

Dual infection with Helicobacter bilis and Helicobacter hepaticus in p-glycoprotein-deficient mdr1a-/- mice results in colitis that progresses to dysplasia.

Patients with inflammatory bowel disease (IBD) are at increased risk for developing high-grade dysplasia and colorectal cancer. Animal IBD models that develop dysplasia and neoplasia may help elucidate the link between inflammation and colorectal cancer. Mdr1a-/- mice lack the membrane efflux pump p-glycoprotein and spontaneously develop IBD that can be modulated by infection with Helicobacter sp: H. bilis accelerates development of colitis while H. hepaticus delays disease. In this study, we determined if H. hepaticus infection could prevent H. bilis-induced colitis. Unexpectedly, a proportion of dual-infected mdr1a-/- mice showed IBD with foci of low- to high-grade dysplasia. A group of dual-infected mdr1a-/- animals were maintained long term (39 weeks) by intermittent feeding of medicated wafers to model chronic and relapsing disease. These mice showed a higher frequency of high-grade crypt dysplasia, including invasive adenocarcinoma, possibly because H. hepaticus, in delaying the development of colitis, allows time for transformation of epithelial cells. Colonic epithelial preparations from co-infected mice showed increased expression of c-myc (5- to 12-fold) and interleukin-1alpha/beta (600-fold) by real-time polymerase chain reaction relative to uninfected wild-type and mdr1a-/- animals. This animal model may have particular relevance to human IBD and colorectal cancer because certain human MDR1 polymorphisms have been linked to ulcerative colitis and increased risk for colorectal cancer.

Helicobacter bilis infection accelerates and H. hepaticus infection delays the development of colitis in multiple drug resistance-deficient (mdr1a-/-) mice.

mdr1a-deficient mice lack P-glycoprotein and spontaneously develop colitis with age. Helicobacter spp. are gram-negative organisms that have been associated with colitis in certain mouse strains, but Helicobacter spp. have been excluded as contributing to the spontaneous colitis that develops in mdr1a-/- mice. We wished to determine whether infection with either H. bilis or H. hepaticus would accelerate the development of inflammatory bowel disease (IBD) in mdr1a-/- mice. We found that H. bilis infection induced diarrhea, weight loss, and IBD in mdr1a-/- mice within 6 to 17 weeks post-inoculation and before the expected onset of spontaneous IBD. Histopathology of H. bilis-induced IBD included crypt hyperplasia, inflammatory cell infiltrates, crypt abscesses, and obliteration of normal gut architecture. Reverse transcription-polymerase chain reaction and Taqman analysis from colonic tissue showed increased transcripts for interferon-gamma and interleukin-10 from H. bilis-infected colitic mdr1a-/- mice. Additionally, mesenteric lymph nodes had increased cellularity with expansion of CD4+ and CD8+ T cells and B cells and increased proliferation to soluble H. bilis antigens with elaboration of interferon-gamma, tumor necrosis factor-alpha and interleukin-10. In contrast, H. hepaticus infection of mdr1a-/- mice did not accelerate disease but rather delayed the onset of spontaneous colitis which was milder in severity. mdr1a-/- mice infected with Helicobacter spp. may provide a useful tool to explore the pathogenesis of microbial-induced IBD in a model with a presumed epithelial cell "barrier" defect.