Dendritic pro-drug for local and selective treatment of locally advanced breast cancer

Abstract

Event Abstract Back to Event Dendritic pro-drug for local and selective treatment of locally advanced breast cancer Nuria Oliva1, Mariana Atilano1, 2*, João Conde1, 3*, Elazer R. Edelman1, 4* and Natalie Artzi1, 5* 1 MIT, IMES, United States 2 IQS, Chemical Engineering, Spain 3 Queen Mary University of London, School of Engineering and Materials Science, United Kingdom 4 BWH, Harvard Medical School, Cardiovascular Division, United States 5 BWH, Harvard Medical School, Department of Medicine, United States Introduction: Systemic neoadjuvant therapy has been established as the preferred therapeutic approach for locally advanced breast cancer, downstaging the disease and preventing mastectomy. However, complications of systemic chemotherapy are devastating. Local therapy would prevent high concentrations of circulating drug and reduce off-target tissue retention. Yet, the means to attain ideal release kinetics and selective uptake remain elusive. We have developed a novel class of biocompatible and biodegradable adhesive materials based on dendrimers and dextrans[1] that can coat the tumor and locally release drugs in a controlled manner. In this work, I have developed and optimized a dendritic pro-drug capable of discerning between healthy and cancer cells. It selectively enters EGFR-overexpressing breast cancer cells through receptor-mediated endocytosis (RME) and releases doxorubicin inside the cells. They will be added to our adhesive hydrogel for local and sustained delivery. Materials and Methods: PAMAM dendrimer generation 5 (Dendritech) was conjugated to EGF-mimicking peptides[2] (Biopolymer Lab, MIT) and also to doxorubicin (Cayman) through a pH-sensitive linker[3]. Cancer cells (MDA-MB-468, ATCC) and healthy mammary epithelial cells (HMEpC, ATCC) were cultured in their recommended media. Cells were treated with 10 uM tagged-dendrimer solutions and uptake was assessed by FACS. Doxorubicin release from the dendrimer was monitored through UV-VIS spectroscopy in PBS (pH 7.4) and acetate buffer (pH 5.5). Cancer and healthy cells were incubated with 10 uM dendritic pro-drug for 48 hours to study cytotoxicity. Results and Discussion: Fluorescence microscopy showed indiscriminate uptake of naked dendrimer independent of cell type (Fig. 1a-b), while dendrimer-peptide uptake was higher in EGFR+ cancer cells than in EGFR- healthy cells (Fig. 1d-e). Blocking of the receptor using an antibody caused abrogation of dendrimer-peptide uptake, but not naked dendrimer (Fig 1c and f). These results were corroborated by FACS (not shown). Taken together, these data prove that our dendrimer conjugates are being uptaken by RME through EGFR, as opposed to diffusion-driven uptake observed for naked dendrimer. Doxorubicin was conjugated to dendrimer-peptide through a cis-aconityl pH-sensitive linker to form the dendritic pro-drug. Incubation of the dendritic pro-drug in PBS (pH 7.4) showed no statistically significant doxorubicin release over 12 hours, while 45% of the drug was released in acetate buffer (pH 5.5) in the first 3 hours (not shown), thus corroborating pH-triggered release. The dendritic pro-drug showed 86% cytotoxicity after 48 hours in cancer cells, while no toxicity was observed in healthy cells (Fig. 1g-k). Conclusions: We have demonstrated that we can successfully develop a dendritic pro-drug that selectively treats EGFR-overexpressing tumors while minimizing side effects in healthy cells surrounding the tumor. The dendritic pro-drug will be incorporated to our adhesive hydrogel and release kinetics and in vivo efficacy will be assessed. Generalization of this platform with peptides targeting other commonly overexpressed growth factor receptors in cancer (FGF2R, VEGFR or PDGFR) will expand the targeting capabilities of our delivery system. We aim to develop a delivery platform capable of treating tumors in a local and selective manner. Marie Curie International Outgoing Fellowship and Funding (FP7-PEOPLE-2013-IOF, Project 626386); Dr. Dong Soo Yun for cryo-TEM assistance at the Peterson Nanotechnology Materials Core Facility; KI MIT Biopolymers Lab; Dr. Glenn Paradis for FACS assistance with Cancer Center Support (FACS core); KI Genomics Core/ MIT BioMicro Center; Timothy E. Cheng for assistance with computation of methods