Abstract P1-01-13: Triple negative breast cancer targeting paramagnetic nanoparticle for non-invasive tumor imaging
Authors: Meser M Ali, Nadimpalli RS Varma, Branislava Janic, Li Zhang, James R Ewing
Journal: Cancer Research (2015)

Abstract Triple-negative breast cancer (TNBC) refers to a subtype of breast cancer that is negative for estrogen receptors (ER) and/or progesterone receptors (PR) , and lacks HER2 overexpression. Therefore, this subtype of breast cancer lacks the benefits of specific therapies which target these receptors. TNBC has been characterized by an acidic extracellular environment. In human aggressive breast tumors, pHe has been measured by microelectrode and the values are in good agreement with the values observed in animal systems i.e. that the pHe is significantly acidic in the range from 6.2 to 7.0. However, tumor intracellular pH is either neutral or alkaline. Interestingly, a similar pH gradient is not observed in normal tissues. Therefore, this acidic extracellular pH (pHe) within tumor tissues can be exploited for targeted delivery of drugs and imaging agents. Recently, A pH low insertion peptide (pHLIP) derived from the protein bacteriorhodopsin has been found to target tumor acidic pH. The peptide inserts across cell membranes as an α-helix when extracellular pH (pHe) is acidic, but does not form the helix at normal or alkaline pH. Since aggressive TNBC has an acidic environment, the pHLIP will insert into the cancer cell membrane, but the pHLIP will not insert into the cell membranes of normal tissues, providing excellent specificity for targeting TNBC. Here, we demonstrate that pHLIP-tagged nanoparticles bind to and are internalized by TNBC cells in vitro. Systemic delivery of the Gd-G5-pHLIP leads to accumulation of the nanoparticles in a flank mouse model of TNBC tumor that are detected by optical and MR imaging. We have synthesized pH-responsive MRI nanoprobe, phosphonate G5-(GdDOTA-4AmP) by following our published synthetic method. The MW of the conjugated G5 dendrimer was estimated at 79,082 g/mole by maldi-tof analysis. This corresponds to a G5-dendrime with an average of 44 chelated Gd3+ ions per dendrimer. Gd44-G5 dendrimer was reacted with heterobifunctional cross-linker (sulfo-SMCC) to form reactive maleimides and then maleimide- Gd44-G5 dendrimer was coupled with C-terminus cysteine group of biotinylated Bt-pHLIP (AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG-pegBiotin). The HABA assay with biotin and avidin revealed that on average 3.1 molecules of biotin are present in Gd44-G5-Bt-pHLIP dendrimer. Finally, Rhodamine dye was conjugated to amines surface of preloaded Gd44-G5-Bt-pHLIP3 in order to achieve final conjugate Rhodamine-Gd44-G5-Bt-pHLIP3. To study pH-dependent translocation of molecules across the cell membrane, we have added Rho-Gd-G5-Bt-pHLIP to the cells and incubated for 3 h at pH 7.4 and 6.5. The cellular uptake of Rho-Gd-G5-pHLIP was significantly higher at pH 6.5. When Rho-Gd-G5 was used, the cellular uptake was considerable lower at both, pH 6.5 and 7.4. Hence, we have shown the ability of pHLIP peptide for intracellular delivery of Gd-G5 nanoparticles in vitro at pH 6.5 but the same ability is attenuated significantly at neutral pH. We have created a mouse model of TNBC MDA-MB-231 tumor. The pharmacokinetics of Gd44-G5-pHLIP was visualized in the MDA-MB-231 tumor over the course of 105 min post-contrast administration. Citation Format: Meser M Ali, Nadimpalli RS Varma, Branislava Janic, Li Zhang, James R Ewing. Triple negative breast cancer targeting paramagnetic nanoparticle for non-invasive tumor imaging [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-01-13.