ABSTRACT
Systemic off-target toxicities, including neurotoxicity, are prevalent side effects in cancer patients treated with a number of otherwise highly efficacious anticancer drugs. In the current study, we have: (1) developed a new analytical metric for the in vivo preclinical assessment of systemic toxicities/neurotoxicity of new drugs and delivery systems; and (2) evaluated, in mice, the in vivo efficacy and toxicity of a versatile and modular NanoDendron (ND) drug delivery and imaging platform that we recently developed. Our paclitaxel-carrying ND prodrug, ND(PXL), is activated following proteolytic cleavage by MMP9, resulting in localized cytotoxic chemotherapy. Using click chemistry, we combined ND(PXL) with a traceable beacon, ND(PB), yielding ND(PXL)-ND(PB) that functions as a theranostic compound. In vivo fluorescence FRET imaging of this theranostic platform was used to confirm localized delivery to tumors and to assess the efficiency of drug delivery to tumors, achieving 25-30% activation in the tumors of an immunocompetent mouse model of breast cancer. In this model, ND-drug exhibited anti-tumor efficacy comparable to nab-paclitaxel, a clinical formulation. In addition, we combined neurobehavioral metrics of nociception and sensorimotor performance of individual mice to develop a novel composite toxicity score that reveals and quantifies peripheral neurotoxicity, a debilitating long-term systemic toxicity of paclitaxel therapy. Importantly, mice treated with nab-paclitaxel developed changes in behavioral metrics with significantly higher toxicity scores indicative of peripheral neuropathy, while mice treated with ND(PXL) showed no significant changes in behavioral responses or toxicity score. Our ND formulation was designed to be readily adaptable to incorporate different drugs, imaging modalities and/or targeting motifs. This formulation has significant potential for preclinical and clinical tools across multiple disease states. The studies presented here report a novel toxicity score for assessing peripheral neuropathy and demonstrate that our targeted, theranostic NDs are safe and effective, providing localized tumor delivery of a chemotherapeutic and with reduced common neurotoxic side-effects.
Subject(s)
Antineoplastic Agents, Phytogenic/therapeutic use , Breast Neoplasms/drug therapy , Drug Delivery Systems , Mammary Neoplasms, Experimental/drug therapy , Paclitaxel/therapeutic use , Prodrugs/therapeutic use , Animals , Antineoplastic Agents, Phytogenic/administration & dosage , Antineoplastic Agents, Phytogenic/adverse effects , Heterografts , Matrix Metalloproteinase 9/metabolism , Mice , Motor Activity/drug effects , Nociception/drug effects , Paclitaxel/administration & dosage , Paclitaxel/adverse effects , Prodrugs/administration & dosage , Prodrugs/adverse effectsABSTRACT
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 Gd3+ of the ClPhIQ23-PAMAM-Gd18 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 Gd3+ 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 multi-modality 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.
ABSTRACT
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, ND2(DOX) and ND2(PXL), 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 ND2(DOX) was found to lose cytotoxicity due to the modification of DOX for conjugation to the ND; ND2(PXL) was determined to have the desired properties for a prodrug delivery system. ND2(PXL) was found to be cytotoxic in MMP9-expressing mouse mammary carcinoma (R221A-luc) (53%) and human breast carcinoma (MDA-MB-231) (66%) at a concentration of 50 nM (in PXL) after 48 h. Treating ND2(PXL) 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 h in the R221A-luc and MDA-MB-231 cell lines. Further studies in modified Lewis lung carcinoma cells that either do (LLC(MMP9)) or do not (LLC(RSV)) express MMP9 demonstrate the selectivity of ND2(PXL) for MMP9. LLC(MMP9) cells were only 20% viable after 48 h of treatment, while LLC(RSV) were not affected. Inclusion of an MMP inhibitor, GM6001, when treating the LLC(MMP9) cells with ND2(PXL) eliminated the response of the MMP9 expressing cells (LLC(MMP9)). The data presented here suggests that these NDs, specifically ND2(PXL), are nontoxic 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.
Subject(s)
Drug Delivery Systems , Matrix Metalloproteinase 9/metabolism , Animals , Cell Line, Tumor , Dipeptides/pharmacology , Doxorubicin/administration & dosage , Humans , Matrix Metalloproteinase Inhibitors/pharmacology , Paclitaxel/administration & dosage , Prodrugs/administration & dosage , RatsABSTRACT
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 of high-affinity conjugable ligands. The 18 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.
Subject(s)
Dendrimers/pharmacokinetics , Diagnostic Imaging/methods , Drug Carriers/chemistry , Drug Delivery Systems/methods , Receptors, GABA/analysis , Animals , Cell Membrane Permeability , Dendrimers/therapeutic use , Glioma/diagnosis , Glioma/pathology , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Rats , Receptors, GABA/metabolismABSTRACT
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.