Phospholipid-modified PEI-based nanocarriers for in vivo siRNA therapeutics against multidrug-resistant tumors.

Multidrug resistance (MDR) mediated by P-glycoprotein overexpression in solid tumors is a major factor in the failure of many forms of chemotherapy. Here we evaluated phospholipid-modified, low-molecular-weight polyethylenimine (DOPE-PEI) nanocarriers for intravenous delivery of anti-P-pg siRNA to tumors with the final goal of modulating MDR in breast cancer. First, we studied the biodistribution of DOPE-PEI nanocarriers and the effect of PEG coating in a subcutaneous breast tumor model. Four hours postinjection, PEGylated carriers showed an 8% injected dose (ID) accumulation in solid tumor via the enhanced permeability and retention effect and 22% ID in serum due to a prolonged, PEG-mediated circulation. Second, we established the therapeutic efficacy and safety of DOPE-PEI/siRNA-mediated P-gp downregulation in combination with doxorubicin (Dox) chemotherapy in MCF-7/MDR xenografts. Weekly injection of siRNA nanopreparations and Dox for up to 5 weeks sensitized the tumors to otherwise non-effective doses of Dox and decreased the tumor volume by threefold vs controls. This therapeutic improvement in response to Dox was attributed to the significant, sequence-specific P-gp downregulation in excised tumors mediated by the DOPE-PEI formulations

Dynamic Analysis of Human Natural Killer Cell Response at Single-Cell Resolution in B-Cell Non-Hodgkin Lymphoma

Natural killer (NK) cells are phenotypically and functionally diverse lymphocytes that recognize and kill cancer cells. The susceptibility of target cancer cells to NK cell-mediated cytotoxicity depends on the strength and balance of regulatory (activating/inhibitory) ligands expressed on target cell surface. We performed gene expression arrays to determine patterns of NK cell ligands associated with B-cell non-Hodgkin lymphoma (b-NHL). Microarray analyses revealed significant upregulation of a multitude of NK-activating and costimulatory ligands across varied b-NHL cell lines and primary lymphoma cells, including ULBP1, CD72, CD48, and SLAMF6. To correlate genetic signatures with functional anti-lymphoma activity, we developed a dynamic and quantitative cytotoxicity assay in an integrated microfluidic droplet generation and docking array. Individual NK cells and target lymphoma cells were co-encapsulated in picoliter-volume droplets to facilitate monitoring of transient cellular interactions and NK cell effector outcomes at single-cell level. We identified significant variability in NK-lymphoma cell contact duration, frequency, and subsequent cytolysis. Death of lymphoma cells undergoing single contact with NK cells occurred faster than cells that made multiple short contacts. NK cells also killed target cells in droplets via contact-independent mechanisms that partially relied on calcium-dependent processes and perforin secretion, but not on cytokines (interferon-γ or tumor necrosis factor-α). We extended this technique to characterize functional heterogeneity in cytolysis of primary cells from b-NHL patients. Tumor cells from two diffuse large B-cell lymphoma patients showed similar contact durations with NK cells; primary Burkitt lymphoma cells made longer contacts and were lysed at later times. We also tested the cytotoxic efficacy of NK-92, a continuously growing NK cell line being investigated as an antitumor therapy, using our droplet-based bioassay. NK-92 cells were found to be more efficient in killing b-NHL cells compared with primary NK cells, requiring shorter contacts for faster killing activity. Taken together, our combined genetic and microfluidic analysis demonstrate b-NHL cell sensitivity to NK cell-based cytotoxicity, which was associated with significant heterogeneity in the dynamic interaction at single-cell level