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Quantitative Assessment of the Intracellular Uptake of Chlorotoxin in a U87 Human Glioma Mouse Model for the Targeted Drug Delivery System
Quantitative Assessment of the Intracellular Uptake of Chlorotoxin in a U87 Human Glioma Mouse Model for the Targeted Drug Delivery System
Submitted on 09 Feb 2018

Alan D. Roberts, Joseph O. Johnson, Aleksandra Karolak, Norma Alcantar, Katarzyna A. Rejniak, Marzenna Wiranowska
Research Scholarly Concentration Program Morsani College of Medicine; Moffitt Cancer Research Center
This poster was presented at USF Health Research Day 2018
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Poster Abstract
This study quantitatively evaluates the tumor cell targeting capabilities and intracellular uptake pattern of Chlorotoxin (CTX) when delivered intratumorally (IT) and intravenously (IV) using subcutaneous (SC) U87 human glioblastoma mouse model. CTX is a scorpion derived polypeptide that selectively binds to tumor cells of neuroectodermal origin, such as glioma and a wide range of other tumor cells, but not normal cells. The quantification of CTX cellular uptake presented here will assist in the further development of our tumor targeting drug delivery system (DDS) consisting of a chemotherapeutic encapsulated in non-ionic surfactant vesicles embedded in a thermosensitive cross-linked chitosan hydrogel, which has affinity for MUC1 receptor overexpressed on many tumor cells, including low-differentiated glioma. CTX addition to the DDS will serve as a second tumor targeting molecule.
In this study, U87 human glioma cells were implanted SC in the flank of nude athymic/ncr/nus mice. When tumors reached approximately the volume of 0.25
cm3, mice were injected either IT or IV (tail vein) with 50 μl of 100 μg CTX or phosphate buffer saline (PBS) as a control. The tumors were harvested at 5 minutes post IT injection or 7 hours post IV injection, fixed, sectioned at 5 μm, and followed by either immunofluorescence or immunocytochemistry using anti- CTX primary antibody and AlexaFluor-594 conjugated secondary antibody or immunoperoxidase- AEC kit, and evaluated by confocal or scanning microscopy, respectively. CTX levels were compared between three different tumor regions (A, B, and C; A = injection site marked by methylene blue dye, C = most distant from injection site, B = equidistant between these two) after direct IT injection and compared to CTX levels in tumor tissues following IV injection. Quantitative evaluation of CTX levels were performed with ImageJ software, while further intracellular analysis of CTX presence utilized MATLAB system.
CTX was found to be evenly distributed between cells within each region post IT injection, but decreased in intensity from the injection site (A) toward the tumor edge. With region A set at 100% CTX intensity, the levels decreased to 23.7% (in region B), and 2.1% (in region C), compared to 1.8% post IV injection. Our previous in vitro studies showed CTX localization near the nucleus in U87 glioma cells but not in normal human astrocytes. Current evaluation of tumor z- sections quantified levels of CTX inside U87 glioma cells in vivo. These results confirm the specificity of CTX uptake by U87 human glioma cells in vitro and in vivo with CTX tissue levels post IV injection, which is comparable to those post IT injection in the distant C tumor region. This proves CTX is an important second tumor targeting molecule for our DDS system.Report abuse »
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