While it has become common practice for dendrimers to deliver imaging and therapeutic agents there are few reported examples of cellular internalization of dendrimers. Moreover, targeting of dendrimers to the mitochondria in cells has not yet been reported. Previously, we have delivered small molecule imaging agents into glioma and breast cancer cells by targeting the translocator protein (TSPO; formerly known as the peripheral benzodiazepine receptor or PBR) with a family high affinity conjugable ligands. The 18 kiloDalton (kDa.) multimeric TSPO is expressed in steroid-producing cells, primarily on the outer mitochondrial membrane. This protein is a prime candidate for molecular targeting because tumors and other disease related cells contain high densities of TSPO. Here we present the synthesis, characterization and cellular internalization into C6 rat glioma cells of a TSPO targeted dendrimer imaging agent.

Chemotherapeutics such as doxorubicin (DOX) and paclitaxel (PXL) have dose limiting systemic toxicities including cardiotoxicity and peripheral neuropathy. Delivery strategies to minimize these undesirable effects are needed and could improve efficacy, while reducing patient morbidity. Here DOX and PXL were conjugated to a nanodendron (ND) through an MMP9-cleavable peptide linker, producing two new therapies, ND2DOX and ND2PXL designed to improve delivery specificity to the tumor microenvironment and reduce systemic toxicity. Comparative cytotoxicity assays were performed between intact ND-drug conjugates and the MMP9 released drug in cell lines with and without MMP9 expression. While ND2DOX was found to loose cytotoxicity due to the modification of DOX for conjugation to the ND; ND2PXL was determined to have the desired properties for a prodrug delivery system. ND2PXL was found to be cytotoxic in MMP9-expressing mouse mammary carcinoma (R221-Aluc) (53%) and human breast carcinoma (MDA-MB-231) (66%) at a concentration of 50 nM (in PXL) after 48 hours. Treating ND2PXL with MMP9 prior to the cytotoxicity assay resulted in a faster response; however, both cleaved and intact versions of the drug reached the same efficacy as the unmodified drug by 96 hours in the R221A-luc and MDA-MB-231 cell lines. Further studies in modified Lewis lung carcinoma cells that either do (LLCMMP9) or do not express (LLCRSV) MMP9 demonstrate the selectivity of ND2PXL for MMP9. LLCMMP9 cells were only 20% viable after 48 hours of treatment while LLCRSV were not affected. Inclusion of an MMP inhibitor, GM6001, when treating the LLCMMP9 cells with ND2PXL eliminated the response of the MMP9 expressing cells (LLCMMP9). The data presented here suggests that these NDs, specifically ND2PXL, are non-toxic until activated by MMP9, a protease common in the microenvironment of tumors, indicating that incorporation of chemotherapeutic or cytostatic agents onto the ND platform have potential for tumor-targeted efficacy with reduced in vivo systemic toxicities.

Advances in probes for cellular imaging have driven discoveries in biology and medicine. Primarily, antibodies and small molecules have been made for contrast enhancement of specific proteins. The development of new dendrimer-based tools offers opportunities to tune cellular internalization and targeting, image multiple modalities in the same molecule and explore therapeutics. The translocator protein (TSPO) offers an ideal target to develop dendrimer tools because it is well characterized and implicated in a number of disease states. The TSPO-targeted dendrimers reported here, primarily ClPhIQ-PAMAM-Gd-Liss, are cell membrane permeable nanoparticles that enable labeling of TSPO and provide contrast in fluorescence, electron microscopy and magnetic resonance imaging. The molecular binding affinity for TSPO was found to be 0.51 μM, 3 times greater than the monomeric agents previously demonstrated in our laboratory. The relaxivity per Gd 3+ of the ClPhIQ 23-PAMAM-Gd 18 dendrimer was 7.7 and 8.0 mM −1 s −1 for r 1 and r 2 respectively, approximately double that of the clinically used monomeric Gd 3+ chelates. In vitro studies confirmed molecular selectively for labeling TSPO in the mitochondria of C6 rat glioma and MDA-MB-231 cell lines. Fluorescence co-registration with Mitotracker Green® and increased contrast of osmium-staining in electron microscopy confirmed mitochondrial labeling of these TSPO-targeted agents. Taken collectively these experiments demonstrate the versatility of conjugation of our PAMAM dendrimeric chemistry to allow multimodality agents to be prepared. These agents target organelles and use complementary imaging modalities in vitro, potentially allowing disease mechanism studies with high sensitivity and high resolution techniques.

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Mannose-TEMPO functionalized G4-PAMAM dendrimers with increasing mannose loadings have been synthesized and characterized by MALDI-TOF MS and EPR spectroscopy. Analysis of linebroadening effects in the EPR spectra of these dendrimers allowed us to determine the relative presentation of mannose and TEMPO on the dendrimer surface. Hemagglutination assays and affinity chromatography/EPR experiments to assess the activity of the mannose-TEMPO dendrimers with Concanavalin A are presented.