GRP75-driven, cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticles underlies distinct gene therapy effect in ovarian cancer

Abstract

Practice of tumor-targeted suicide gene therapy is hampered by unsafe and low efficient delivery of plasmid DNA (pDNA). Using HIV-Tat-derived peptide (Tat) to non-covalently form Tat/pDNA complexes advances the delivery performance. However, this innovative approach is still limited by intracellular delivery efficiency and cell-cycle status. In this study, Tat/pDNA complexes were further condensed into smaller, nontoxic nanoparticles by Ca2+ addition. Formulated Tat/pDNA-Ca2+ nanoparticles mainly use macropinocytosis for intercellular delivery, and their macropinocytic uptake was persisted in mitosis (M-) phase and highly activated in DNA synthesis (S-) phase of cell-cycle. Over-expression or phosphorylation of a mitochondrial chaperone, 75-kDa glucose-regulated protein (GRP75), promoted monopolar spindle kinase 1 (MPS1)-controlled centrosome duplication and cell-cycle progress, but also driven cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticles. Further in vivo molecular imaging based on DF (Fluc-eGFP)-TF (RFP-Rluc-HSV-ttk) system showed that Tat/pDNA-Ca2+ nanoparticles exhibited highly suicide gene therapy efficiency in mouse model xenografted with human ovarian cancer. Furthermore, arresting cell-cycle at S-phase markedly enhanced delivery performance of Tat/pDNA-Ca2+ nanoparticles, whereas targeting GRP75 reduced their macropinocytic delivery. More importantly, in vivo targeting GRP75 combined with cell-cycle or macropinocytosis inhibitors exhibited distinct suicide gene therapy efficiency. In summary, our data highlight that mitochondrial chaperone GRP75 moonlights as a biphasic driver underlying cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticles in ovarian cancer.

Hypoxia‐activated ADCC‐enhanced humanized anti‐CD147 antibody for liver cancer imaging and targeted therapy with improved selectivity

“…Fresh surgical samples from liver cancer patients were collected from clinic. Immunohistochemistry (IHC) staining was performed as previously described 26,32,34,58,64,65 . Briefly, after deparaffinization, rehydration, and Ag retrieval with 10 mM sodium citrate buffer (pH 6.0) at 120°C, the slides were treated with hydrogen peroxide, blocked with goat serum, and overnight incubated with primary Abs at 4°C.…”

“…Immunohistochemistry (IHC) staining was performed as previously described. 26,32,34,58,64,65 Briefly, after deparaffinization, rehydration, and Ag retrieval with 10 mM sodium citrate buffer (pH 6.0) at 120 • C, the slides were treated with hydrogen peroxide, blocked with goat serum, and overnight incubated with primary Abs at 4 • C. Goat anti-human IgG-HRP was used as a secondary Ab. IHC staining was performed with an Envision two-step system.…”

Construction and Application of a Technical Platform for Determining Cell Cycle- and Autophagy-Associated Cellular Uptake of Lipid-Based Nanoparticles

“…For instance, the methods used or available in literatures could not differentiate between G2-phase and M-phase cells in physiological state [13][14][15][16][17][18][19][20] . Additionally, cell dissociation agent such as trypsin 13,[15][16][17][18][19] , exogenous stimuli (e.g., serum deprivation) 16,18 and "cell cycle blockers" (e.g., nocodazole) 13,15,[18][19][20] for artificial synchronization of cell cycle, inevitably disturbed the cellular physiology and subsequent nanoparticle uptake. Moreover, evaluation of nanoparticle uptake with confocal laser scanning microscopy (CLSM) was limited at a two-dimensional (2D) level, in which spatial cellular architecture and heterogeneous intracellular distribution of nanoparticles were both ignored 13,17,19,20 .…”

“…Nevertheless, so far only three literatures explored the impact of cell cycle on the uptake of inorganic nanoparticles including CdTe QDs 13,14 and ZnO 15 . Regarding organic nanoparticles, five literatures respectively investigated the cell cycle-associated uptake of carboxylated polystyrene nanoparticles (PS-CCOH), FITC-dextran fibroin-encapsulated microspheres, cRGD-targeting matrix metalloproteinase-sensitive nanoparticles (FITC@NPs-cRGD), folate modified poly (L-amino acid) micelles and Tat/pGL3-Ca 2+ nanoparticles [16][17][18][19][20] . It is noteworthy that several methodological limitations may interfere with the accuracy of data and result in the discrepant findings previously reported.…”

“…It is noteworthy that several methodological limitations may interfere with the accuracy of data and result in the discrepant findings previously reported. For instance, the methods used or available in literatures could not differentiate between G2-phase and M-phase cells in physiological state [13][14][15][16][17][18][19][20] . Additionally, cell dissociation agent such as trypsin 13,[15][16][17][18][19] , exogenous stimuli (e.g., serum deprivation) 16,18 and "cell cycle blockers" (e.g., nocodazole) 13,15,[18][19][20] for artificial synchronization of cell cycle, inevitably disturbed the cellular physiology and subsequent nanoparticle uptake.…”