How does the cell overcome LCP nanoparticle-induced calcium toxicity?

Abstract

To address the question of how cells respond to the possible Ca(2+) toxicity caused by the release of Ca(2+) into the cytoplasm by LCP nanoparticles, a series of in vitro and in vivo studies using Ca(2+) pump inhibitors were conducted. The results indicated that two major Ca(2+) pumps on the plasma membrane and the mitochondrial membrane, respectively, were able to rapidly respond to the elevated cytosolic Ca(2+) concentration and prevent Ca(2+)-induced apoptosis or necrosis. However, exposure to specific inhibitors of calcium pumps would cause LCP-treated H460 cells to undergo necrosis both in vitro and in vivo. These results demonstrated that the Ca(2+) delivered by LCP was not toxic to cells when the cells contain functional Ca(2+) pumps.

Figure 1. Calcium pumps are important for Ca²⁺ toxicity management

Shown here are stills taken from the recorded video of H460 cells at the indicated time points for four different treatment groups. Red color indicates low intracellular Ca²⁺ concentration under physiological conditions. Green color indicates elevated intracellular Ca²⁺ concentration. Phase contrast images taken at 8 min are included to show round, swollen cells.

Figure 2. Tumor necrosis in vivo which has been induced by LCP with Ca²⁺ pump inhibitors

H460 subcutaneous tumor sections with H&E staining showing that LCP toxicity was only observed in the presence of Ca²⁺ pump inhibitors. (A) Control H460 tumor without any treatment. (B) Tumor that has received an IT injection of two Ca²⁺ pump inhibitors. (C) Tumor that has received an IV injection of LCP. (D) Tumor that has received an IT injection of two Ca²⁺ pump inhibitors and an IV injection of LCP. (E) Tumor that has received an IT injection of two Ca²⁺ pump inhibitors and an IT injection of LCP. (F) Tumor that has received an IT injection of two Ca²⁺ pump inhibitors and an IT injection of LCP without PEGylation. Necrotic lesions were recognized as the “ghost” cells morphology on the H&E-stained sections. Blue and black arrows indicate necrotic and severely necrotic regions, respectively.

Figure 3. Schematic illustration of the Ca²⁺ removing mechanism after LCP dosing

The elevated cytosolic Ca²⁺ delivered by LCP was rapidly removed by the PMCA on the plasma membrane and the MCU on the mitochondria. Mitochondrial calcium-uptake 1 (MICU1) is a calcium sensor that regulates the Ca²⁺-influx capacity of MCU.