Ilia Rivkin

Paclitaxel-clusters coated with hyaluronan as selective tumor-targeted nanovectors

Paclitaxel (PTX) is a widely used anti-tumor agent in the treatment of solid tumors. Lack of selective strategies to target PTX into tumor cells together with poor solubility necessitating detergent, are severe clinical limitations. To address these hurdles, we devised a strategy that utilized PTX insolubility, mixing it with lipids that self-assemble into nanoparticle-like clusters. These clusters were then coated with hyaluronan, a glycosaminoglycan (GAG), and termed PTX-GAGs. These particles, delivered PTX selectively into tumor cells in a CD44-dependent manner. Injected systemically to mice bearing solid tumors, the PTX-GAGs showed high safety profile and tumor accumulation. Tumor progression was exponential upon treatment with free PTX or PTX in albumin nanoparticles (the FDA-approved Taxol and Abraxane, respectively). Under the same conditions, PTX-GAGs induced tumor arrest and were as potent as a 4-fold higher Taxol dose. Our findings suggest GAGs merit further investigation as vehicles for taxanes, and may be applicable as carriers in other therapeutic settings.

Three-dimensional characterization of drug-encapsulating particles using STEM detector in FEG-SEM

New drug-encapsulating particles were investigated using bright field (BF) scanning transmission electron microscopy (STEM) in a field emission gun (FEG) scanning electron microscope (SEM). Thickness characterization was done based on measuring the effective cross-section for interaction in our sample-detector configuration using calibration particles. A simplified analytical model, taking account of BF-STEM contrast and effective cross-section for interaction, was utilized for transforming projected two-dimensional BF-STEM images into three-dimensional thickness images. The three-dimensional characterization is demonstrated on a new family of biological materials composed of submicron to micron drug-free and drug-encapsulating particles. The importance of using BF-STEM in SEM, relative to other electron microscopy methods, is discussed as well as the lateral and depth resolution.

A color discriminating broad range cell staining technology for early detection of cell transformation.

Background: Advanced diagnostic tools stand today at the heart of successful cancer treatment. CellDetect® is a new histochemical staining technology that enables color discrimination between normal cells and a wide variety of neoplastic tissues. Using this technology, normal cells are colored blue/green, while neoplastic cells color red. This tinctorial difference coincides with clear morphological visualization properties, mainly in tissue samples. Here we show that the CellDetect® technology can be deployed to distinguish normal cells from transformed cells and most significantly detect cells in their early pre-cancerous transformed state. Materials and Methods: In tissue culture, we studied the ability of the CellDetect® technology to color discriminate foci in a number of two stage transformation systems as well as in a well defined cellular model for cervical cancer development, using HPV16 transformed keratinocytes. Results: In all these cellular systems, the CellDetect® technology was able to sensitively show that all transformed cells, including pre-cancerous HPV 16 transformed cells, are colored red, whereas normal cells are colored blue/green. The staining technology was able to pick up: (i) early transformation events in the form of small type 1 foci (non-invasive, not piled up small, with parallel alignment of cells), and (ii) early HPV16 transformed cells, even prior to their ability to form colonies in soft agar. The study shows the utility of the CellDetect® technology in early detection of transformation events.

Novel Histochemical Stain for Tinctorial Detection of Cancer and Neoplastic Cells

Abstract Zetiq has introduced a histochemical stain that claims to tinctorially identify cancer and neoplastic cells. Because of the potential importance of such a capability, we undertook to investigate Zetiqs CellDetect® staining technology as applied to cultured cell lines as well as an initial sample of clinical cases. This goal was pursued by investigating four types of comparisons: 1) cancer cell lines before and after differentiation; 2) cervical squamous-cell carcinoma (SCC) biopsies to non-neoplastic squamous epithelium; 3) SCCs to neoplastic, nonmalignant squamous epithelium; and 4) neo-plastic squamous cells to non-neoplastic squamous cells in cytological preparations. The clinical material was also stained with hematoxylin and eosin (biopsies) or Pap (cytologies) for diagnostic purposes. We found that all CellDetect®-stained cells exhibited one of the two tinctorial outcomes. Cell lines with malignant phenotype uniformly had red/purple cytoplasm, whereas the differentiated phenotype changed the color to blue/green. Moreover, these two color values are sufficiently distinct that they can be readily distinguished quantitatively with an ELISA reader when applied to cells in tissue culture. Biopsies of SCC and non-neoplastic tissues exhibit the same two color values: cancers had red/purple cytoplasm, whereas non-neoplastic tissues were green/blue stained. In contrast, neoplastic, nonmalignant tissues (dysplasias) stained red/purple, similar to SCCs. Cytological preparations gave similar results: neoplastic cells stained red/purple, whereas non-neoplastic cells exhibited green/blue cytoplasm. The study demonstrated that the staining was rapid and reproducible. Conclusion: The CellDetect® stain allows detecting cancer and neoplastic cells tinctorially based on a rapid, reproducible histochemical process.

Screening for cervical neoplasia: A community‐based trial comparing Pap staining, human papilloma virus testing, and the new bi‐functional celldetect® stain

Although cytological screening for cervical neoplasia has lowered mortality rates, current screening methods are plagued by sub‐optimal sensitivity and/or specificity. The purpose of this study was to compare the performance of the new CellDetect® staining technology as a potential screening tool. This initial, non‐blinded study, utilized samples are taken at a community‐based clinic. The diagnostic results using CellDetect® were compared with the performance of Pap staining and human papilloma virus (HPV) testing on the same material, as well as the follow‐up biopsies. These data were statistically analyzed in terms of sensitivity, specificity, predictive value (N.P.V and P.P.V), and inter‐observer agreement. Bi‐functional CellDetect® staining revealed morphological details and tinctorial properties that permitted recognition of neoplasia even at low magnification. Performance‐wise, CellDetect® demonstrated non‐inferiority for all statistical parameters to both Pap and HPV tests. Importantly, superior sensitivity compared with Pap staining was observed, as well as higher specificity than HPV testing with near equivalent sensitivity. We conclude that CellDetect® is a promising approach to early detection of cervical cancer because of its bi‐functional capabilities that afford high sensitivity and specificity. The data suggest that this new methodology warrants further and more extensive clinical evaluation. Diagn. Cytopathol. 2012.

Novel dual-function CellDetect® staining technology: wedding morphology and tinctorial discrimination to detect cervical neoplasia

BackgroundA persistent goal of oncologic histochemistry is to microscopically identify neoplasia tinctorially. Consequently, the newly developed CellDetect® staining technology, that appears to exhibit this property, warrants clinical evaluation. The objective of this study was to compare the diagnostic results using CellDetect® to the outcomes of standard microscopic examination based on hematoxylin and eosin (H&E) staining for the recognition of different squamous epithelial phenotypes of the uterine cervix.MethodsPairs of adjacent sections were made from 60 cervical biopsy cases that were diagnosed originally as either normal or neoplastic (CIN, SCC). One section of the pair was stained for H&E; the second section, with CellDetect®. Based on the examination of these pairs by two experienced pathologists, we investigated the following issues:(1) diagnostic agreement between the pathologists on each pair; (2) agreement between H&E and CellDetect® for each pair (3) tinctorial characteristics in micro-regions (n = 130) evaluated as either normal, reactive or neoplastic.ResultsQualitatively, CellDetect®-stained preparations displayed cyto-morphological detail comparable to H&E images. Tinctorially, non-neoplastic cells appeared green/blue when stained withCellDetect®, contrasting with cytologically neoplastic foci, where cells of every grade were red/magenta in color. Due to these tinctorial characteristics, even small foci of neoplasia could be readily distinguished that were inconspicuous on H&E at low magnification. In some instances, this prompted re-examination of the H&E and revision of the diagnosis. Quantitatively, we found that despite diagnostic variation between pathologists, in about 3% of the cases, each pathologist made the same diagnosis regardless of whether CellDetect® or H&E was used, i.e. there was 100% self-agreement for each pathologist between stains. Particularly noteworthy was the finding of a 0% false negative rate, coupled with a 10-15% false positive rate. Regarding specificity, the performance in reactive squamous processes was similar to that observed for morphologically normal squamous epithelium.ConclusionsIn this first order assessment of clinical applicability, CellDetect® staining technology was at least comparable to results using H&E, and perhaps surperior. CellDetect® provided a uniquely useful tinctorial clue for the detection of neoplasia, which exhibited an impressive 0% false negative rate. A more extensive, blinded study is needed to confirm these promising findings.

Cancer cell sensitization and improved treatment efficacy by combined sodium butyrate and paclitaxel formulations is cancer-type specific.

We queried whether cancer treatment by combinations of paclitaxel and butyrate - free or formulated in drug delivery systems - can improve therapeutic responses compared to each drug alone. Combination treatments were conducted with HT-29 and HeLa cells, as representatives of differentiation-induced and cell-death-induced cancer lines, respectively. Pre-treatment of the HT-29 cells with butyrate (at doses inducing differentiation), followed by butyrate+paclitaxel generated changes in cell cycle profile, increased the level of dead cells beyond that of each drug alone, and allowed reduction in paclitaxel doses. A similar combination treatment of HeLa cells was detrimental, indicating that whether the combination is beneficial or not is cancer-type specific. We hypothesize that while butyrate-treated HT-29 cells became sensitive to paclitaxel-induced Fas-mediated apoptosis, butyrate-adapted HeLa cells became apoptosis-resistant. We next tested the same drug combination on HT-29 cells, but each drug in a specific tumor-targeted carrier. The combination of drug carriers outperformed an equidose combination of the free drugs, showing potential to achieve high therapeutic responses (even in drug-resistant cells) at significantly lower and detergent-free paclitaxel doses, which should allow for reduction in adverse effects and risks of toxicity.

Treatment of respiratory damage in mice by aerosols of drug-encapsulating targeted lipid-based particles.

ABSTRACT The purpose of this study was to develop a treatment for respiratory damage caused by exposure to toxic industrial chemicals (TICs), including mass casualty events, by aerosols of dexamethasone and/or N‐acetyl cysteine formulated in targeted lipid‐based particles. Good encapsulation, performance as slow‐release drug depots, conservation of matter, and retention of biological activity were obtained for the three drug‐carrier formulations, pre‐ and post‐aerosolization. Weight changes over a 2 week period were applied, deliberately, as a non‐invasive clinical parameter. Control mice gained weight continuously, whereas a non‐lethal 30 minute exposure of mice to 300 ppm Cl2 in air showed a two‐trend response. Weight loss over the first two days, reversing thereafter to weight gain, but at a rate and level significantly slower and smaller than those of the control mice, indicating the chlorine damage was long‐term. The weight changes of Cl2‐exposed mice given the inhalational treatments also showed the two‐trend response, but the weight gain rates and levels were similar to those of the control mice, reaching the weight‐gain range of the control mice. Following this proof of concept, studies are now extended to include additional TICs, and biochemical markers of injury and recovery.