Gene electrotransfer of IL-2 and IL-12 plasmids effectively eradicated murine B16.F10 melanoma

Gene therapy has become an important approach for treating cancer, and electroporation represents a technology for introducing therapeutic genes into a cell. An example of cancer gene therapy relying on gene electrotransfer is the use of immunomodulatory cytokines, such as interleukin 2 (IL-2) and 12 (IL-12), which directly stimulate immune cells at the tumour site. The aim of our study was to determine the effects of gene electrotransfer with two plasmids encoding IL-2 and IL-12 in vitro and in vivo. Two different pulse protocols, known as EP1 (600 V/cm, 5 ms, 1 Hz, 8 pulses) and EP2 (1300 V/cm, 100 %s, 1 Hz, 8 pulses), were assessed in vitro for application in subsequent in vivo experiments. In the in vivo experiment, gene electrotransfer of pIL-2 and pIL-12 using the EP1 protocol was performed in B16.F10 murine melanoma. Combined treatment of tumours using pIL2 and pIL12 induced significant tumour growth delay and 71% complete tumour regression. Furthermore, in tumours coexpressing IL-2 and IL-12, increased accumulation of dendritic cells and M1 macrophages was obtained along with the activation of proinflammatory signals, resulting in CD4 + and CD8 + T-lymphocyte recruitment and immune memory development in the mice. In conclusion, we demonstrated high antitumour efficacy of combined IL-2 and IL-12 gene electrotransfer protocols in low-immunogenicity murine B16.F10 melanoma

Geisha art in the Jan Colton Collection [019]

Photo of Geishas at a ceremonial tea in a Geisha house, from a Meiji period album in the Jan Colton Collection, San Diego, CaliforniaTea Cer. Geisha House Colton Coll. S.D. Meiji Per. Album Print Japan

Single Cell Electrical Characterization Techniques

International audienceIn this chapter the possibility to electrically characterize the electroporation of single cells is discussed. During the electroporation process, the application of pulsed electric field (Transmembrane Voltage Induced by Applied Electric Fields) leads to the partial permeabilization of the cell membrane, opening access to the cytoplasmic compartment for drug delivery, for instance. The electric impedance of the cell is modified by such a treatment. Impedance measurement can thus be employed to characterize the permeabilization effect, on a given angular frequencies range, which leads to an impedance spectrum. The impedance spectrum measurement can be achieved even at the level of single cells. In that case, the distance between the detection microelectrodes and the cell must be considered in order to achieve a satisfactory sensitivity. In addition, when considering the impedance measurement of cells flowing through a microfluidic channel, fast detection is also required, as the cell is present between the measurement electrodes only for a short while. A specific strategy must then be employed to achieve such goal. In this case, instead of screening the whole frequencies spectrum, a few set of frequencies should be selected and mixed to be simultaneously injected prior to synchronous detection. Another way to assess the single cell impedance is the use of indirect methods based on the mechanical behavior of cells that polarize under the application of a stationary or propagative electrical field. In particular, electrorotation experiment provides the electrical characterization of the cell, which is dependent of its level of permeabilization, after the application of electrical field pulses. When such approach is used, the vicinity of electrodes is less critical as the main requirement lays on the homogeneity of the rotating electrical field. Finally, the possibility to focus the electroporation at the subcellular level is discussed in the chapter. Indeed, the use of micro- or nanotechnology permits to localize spatially the electroporation on a tiny portion of the membrane of single cells. Adherent cells can be locally electroporated, using nanosized electrodes, subsequently used to monitor the intracellular potential

PD1 blockade potentiates the therapeutic efficacy of photothermally-activated and MRI-guided low temperature-sensitive magnetoliposomes

This study investigates the effect of PD1 blockade on the therapeutic efficacy of novel doxorubicin-loaded temperature-sensitive liposomes. Herein, we report photothermally-activated, low temperature-sensitive magnetoliposomes (mLTSL) for efficient drug delivery and magnetic resonance imaging (MRI). The mLTSL were prepared by embedding small nitrodopamine palmitate (NDPM)-coated iron oxide nanoparticles (IO NPs) in the lipid bilayer of low temperature-sensitive liposomes (LTSL), using lipid film hydration and extrusion. Doxorubicin (DOX)-loaded mLTSL were characterized using dynamic light scattering, differential scanning calorimetry, electron microscopy, spectrofluorimetry, and atomic absorption spectroscopy. Photothermal experiments using 808 nm laser irradiation were conducted. In vitro photothermal DOX release studies and cytotoxicity was assessed using flow cytometry and resazurin viability assay, respectively. In vivo DOX release and tumor accumulation of mLTSL(DOX) were assessed using fluorescence and MR imaging, respectively. Finally, the therapeutic efficacy of PD1 blockade in combination with photothermally-activated mLTSL(DOX) in CT26-tumor model was evaluated by monitoring tumor growth, cytokine release and immune cell infiltration in the tumor tissue. Interestingly, efficient photothermal heating was obtained by varying the IO NPs content and the laser power, where on-demand burst DOX release was achievable in vitro and in vivo. Moreover, our mLTSL exhibited promising MR imaging properties with high transverse r 2 relaxivity (333 mM −1 s −1), resulting in superior MR imaging in vivo. Furthermore, mLTSL(DOX) therapeutic efficacy was potentiated in combination with anti-PD1 mAb, resulting in a significant reduction in CT26 tumor growth via immune cell activation. Our study highlights the potential of combining PD1 blockade with mLTSL(DOX), where the latter could facilitate chemo/photothermal therapy and MRI-guided drug delivery

Design of a cyclotide antagonist of neuropilin-1 and-2 that potently inhibits endothelial cell migration

Neuropilin-1 and -2 are critical regulators of angiogenesis, lymphangiogenesis, and cell survival as receptors for multiple growth factors. Disulfide-rich peptides that antagonize the growth factor receptors neuropilin-1 and neuropilin-2 were developed using bacterial display libraries. Peptide ligands specific for the VEGFA binding site on neuropilin-1 were identified by screening a library of disulfide-rich peptides derived from the thermostable, protease-resistant cyclotide kalata B1. First generation ligands were subjected to one cycle of affinity maturation to yield acyclic peptides with affinities of 40-60 nM and slow dissociation rate constants (similar to 1 X 10(-3) s(-1)). Peptides exhibited equivalent affinities for human and mouse neuropilin-1 and cross-reacted with human neuropilin-2 with lower affinity. A C-to-N cyclized variant (cyclotide) of one neuropilin ligand retained high affinity, exhibited increased protease resistance, and conferred improved potency for inhibiting endothelial cell migration in vitro (EC50 approximate to 00 nM). These results demonstrate that potent, target-specific cyclotides can be created by evolutionary design and that backbone cyclization can confer improved pharmacological properties