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Background Adaptor proteins such as growth factor receptor-bound protein-2 (Grb2) play important roles in cancer cell signaling. In the present study, we examined the biological effects of liposomal antisense oligodeoxynucleotide that blocks Grb2 expression (L-Grb2) in gynecologic cancer models. Materials and methods Murine orthotopic models of ovarian (OVCAR5 and SKOV3ip1) and uterine (Hec1a) cancer were used to study the biological effects of L-Grb2 on tumor growth. In vitro experiments (cell viability assay, Western blot analysis, siRNA transfection, and reverse phase protein array) were carried out to elucidate the mechanisms and potential predictors of tumor response to L-Grb2. Findings Treatment with L-Grb2 decreased tumor growth and metastasis in orthotopic models of ovarian cancer (OVCAR5, SKOV3ip1) by reducing angiogenesis and increasing apoptosis at a dose of 15 mg/kg with no effect on mouse body weight. Treatment with L-Grb2 and paclitaxel led to the greatest decrease in tumor weight (mean ± SEM, 0.17 g ± 0.10 g) compared with that in control mice (0.99 g ± 0.35 g). We also observed a reduction in tumor burden after treatment with L-Grb2 and the anti-VEGF antibody B-20 (86% decrease in tumor weight compared with that in controls). Ovarian cancer cells with ErbB2 amplification (OVCAR8 and SKOV3ip1) were the most sensitive to Grb2 downregulation. Reverse phase protein array analysis identified significant dysregulation of metabolites (LDHA, GAPDH, and TCA intermediates) in ovarian cancer cells after Grb2 downregulation. Interpretation L-Grb2 has therapeutic efficacy in preclinical models of ovarian and uterine cancer. These findings support further clinical development of L-Grb2.

A major obstacle in the treatment of glioblastoma (GBM) is the blood brain barrier (BBB), which restricts drug molecules in the bloodstream from reaching the tumor. The BBB may be disrupted at or near the GBM tumor core, but it is intact at the growing edge of the tumor, where invasive cells reside and can cause disease recurrence. To combat GBM more effectively, novel drug transport technology that crosses intact BBB is urgently needed. Brain microvascular endothelial cells (BMEC) found in the BBB are lined with low-density lipoprotein receptors (LDLr) and LDL-like receptor proteins (LRP), which are essential to the endocytosis/ transcytosis processes of BMEC. GBM cells are known to express higher levels of LDLr and LRP than normal brain cells. The objective of our research is to develop a novel lipid nanoparticle (NP) that targets LDLr and LRP to facilitate BMEC endocytosis/ transcytosis and GBM uptake. We focused on developing antisense oligonucleotides (oligos) as the drug cargo for our NP. Less than 20% of disease-associated genes are targetable by conventional drug molecules; however, we hypothesize that disease-associated genes, like EZH2 and ZEB1 transcriptional repressors, are amendable to antisense oligo inhibition. We engineered a novel NP which is composed of neutral lipids: phosphatidylcholine (PC), cholesterol (Chol) and a polymer called poloxamer (PXR). Toxicology studies indicated that NP composed of PC/Chol/PXR did not have adverse effects on mice. Flow cytometry and fluorescent microscopy revealed that PC/Chol/PXR NP was avidly taken up by BMEC, and that PC/Chol/PXR efficiently delivered antisense oligos to GBM cells. Additionally, intravenous injection of PC/Chol/PXR led to deposition of antisense oligos in intracranial GBM xenografts. Future studies will determine the efficacy of the innovative PC/Chol/PXR NP in suppressing target gene expression in intracranial GBM. (Supported by Florida Center for Brain Tumor Research and Accelerate Brain Cancer Cure).

While a wealth of literature for tissue-specific liposomes is emerging, optimal formulations to target the cells of the peripheral nervous system (PNS) are lacking. In this study, we asked whether a novel formulation of phospholipid-based liposomes could be optimized for preferential uptake by microvascular endothelia, peripheral neurons and Schwann cells. Here, we report a unique formulation consisting of a phospholipid, a polymer surfactant and cholesterol that result in enhanced uptake by targeted cells. Using fluorescently labeled liposomes, we followed particle internalization and trafficking through a distinct route from dextran and escape from degradative compartments, such as lysosomes. In cultures of non-myelinating Schwann cells, liposomes associate with the lipid raft marker Cholera toxin, and their internalization is inhibited by disruption of lipid rafts or actin polymerization. In contrast, pharmacological inhibition of clathrin-mediated endocytosis does not significantly impact liposome entry. To evaluate the efficacy of liposome targeting in tissues, we utilized myelinating explant cultures of dorsal root ganglia and isolated diaphragm preparations, both of which contain peripheral neurons and myelinating Schwann cells. In these models, we detected preferential liposome uptake into neurons and glial cells in comparison to surrounding muscle tissue. Furthermore, in vivo liposome administration by intramuscular or intravenous injection confirmed that the particles were delivered to myelinated peripheral nerves. Within the CNS, we detected the liposomes in choroid epithelium, but not in myelinated white matter regions or in brain parenchyma. The described nanoparticles represent a novel neurophilic delivery vehicle for targeting small therapeutic compounds, biological molecules, or imaging reagents into peripheral neurons and Schwann cells, and provide a major advancement toward developing effective therapies for peripheral neuropathies.