Cellular and molecular responses of human white blood cells to microbubble-mediated ultrasound

Abstract

Microbubble-mediated ultrasound (MB-US) has received much attention as a promising strategy to enhance drug/gene delivery. The induction of membrane perforation, also referred to as sonoporation, has been considered one of the key advantages of MB-US exposure, as it may potentially facilitate the permeabilization of the extracellular molecules. It has been demonstrated in vitro that MB-US sonoporation could effectively promote the efficiency of the drug/gene delivery. To establish therapeutic utility, the context of in vivo MB-US application needs to be carefully considered. One of the important regards is the white blood cell (WBC) involved in the in vivo MB-US exposure. As the microbubbles need to be injected intravenously and circulate in the vasculatures, the circulatory WBCs were promptly exposed to microbubbles and involved in the MB-US exposure, which could be a critical safety issue in the realization of sonoporation-based therapeutics. WBCs play a principle role in the human immune system and the bioeffects of MB-US exposure on WBCs may elicit profound downstream responses in vivo. However, the involvement of WBCs also offers a reasonable possibility for the MB-US exposure to be applied to the drug delivery for leukemia treatment. This thesis therefore aimed to clarify the downstream responses of human WBCs and the leukemia cells to the MB-US exposure. Methodologies were designed to tackle the hinder parts in establishing precise measurements of the characteristics of heterogeneous WBCs after MB-US exposure. To gain comparative information for the three subsets of WBCs, the subsets were identified and individual analysis were conducted for each subset. To determine the exact responses of the heterogeneous populations of leukemia cells, meticulously-controlled cell sorting was employed to extract each population prior to the specified downstream analyses. RNA-seq was carried out for different leukemia cells to seek a comprehensive perspective of the molecular signaling triggered by MB-US exposure. In this thesis, distinct responses were found among different subsets both in normal and cancerous WBCs. Firstly, in terms of the immediate responses, molt-4 T lymphoblast leukemia cells were more resistant to MB-US exposure than HL-60 promyelocytic leukemia cells based on the high viability and low sonoporation efficiency. Among normal WBC subsets, lymphocytes showed the highest survival with significant sonoporation efficiency, while granulocytes exhibited both lowest viability and sonoporation efficiency. Secondly, both types of leukemia cells were found to preserve more cell viability after MB-US exposure than their corresponding normal subsets. Thirdly, heterogeneous long-term responses were observed between sonoporated and unsonoporated cells. The sonoporated cells bore the chronic stresses and were unable to proliferate within 24h after exposure, while the bioeffects of MB-US on the unsonoporated cells were likely to be transient. Lastly, gene responses to MB-US exposure between two leukemia cell lines were different. Differentially expressed genes of molt-4 cells were dramatically more than HL-60 cells after MB-US exposure. T-cell receptor signaling pathway and antigen processing and presentation pathway were activated for Molt-4 and HL-60 cells respectively, which indicated the immune functions might be involved in response to sonoporation.