Developing inorganic nanoplatforms for efficient siRNA delivery to improve cancer immunotherapy

Abstract

For many decades, cancer treatments basically include surgery, chemotherapy and radiation therapy.Over the last decade, targeted therapies using small molecules have been also included in treatmentsmany cancers. Recently, the development of immune checkpoints and adoptive cell transfer cell-based methods has largely changed the paradigm of cancer treatments, while the new methods arestill accompanied with the low responsive rate in solid tumour, high recurrence rate, serious adverseeffects and expense for patients. Thus, a more effective immunotherapy is required. One possiblesolution is to silence the inhibitory immune checkpoints (such as programmed cell death-1: PD-1 andprogrammed cell death ligand 1: PD-L1) on T cells with small interfering RNA (siRNA), whichrequires more effective and workable siRNA delivery systems.This PhD project aims to develop effective nano-carriers to deliver functional siRNA to T cells tosilence inhibitory immune checkpoints and thus enhance their killing efficacy to cancer cells. Bytaking advantages of the low toxicity, good biocompatibility, excellent colloidal stability and efficientcellular uptake, MgAl-layered double hydroxide (MgAl-LDH) and lipid-coated calcium phosphate(LCP) nanoparticles (NPs) are examined and compared as nanoplatforms for cancer immunotherapy.This project first optimised the LDH-based delivery system by examining the delivery effects ofoperation parameters, including the loading method of siRNA by LDH NPs, the LDH:siRNA massratio and incubation time. siRNA molecules directly mixed with LDH followed by a dilution withculture medium resulted in >80% cells being transfected in 4 h. MCF-7 cells took up most dsDNA orsiRNA at the LDH/gene mass ratio of 10:1-20:1. However, the most suitable LDH:siRNA mass ratiowas around 5:1 for efficiently silencing PD-L1 gene due to the control release of siRNA from LDH-siRNA complexes in cytosol.Subsequently, the PD-1 silencing in T lymphoma cell line EL4 and human tumour infiltratinglymphocytes (TILs) was examined using LDH and LCP NPs in order to compare their capability forgene silence in T cell. Under similar conditions, LCP NPs more efficiently delivered siRNA to EL4cells and silenced the PD-1 gene. The better transfection ability of LCP NPs was also confirmed inTILs to knockdown the PD-1 expression. As a proof of concept, LCP NPs were used to deliver siRNAs to silence PD-1 in PD-1 positive TILsfrom breast cancer patients and PD-L1 in breast cancer cells MCF-7, respectively. The siRNAsdelivered by LCP NPs efficiently down-regulated the target genes (PD-1 and PD-L1) in target cells(TILs and MCF-7 cells) in terms of both mRNA and protein expression levels. The PD-1 or PD-L1knockdown significantly increased the killing efficiency of TILs to MCF-7 cells. Moreover,simultaneous knockdown of both inhibitory immune checkpoints PD-1 and PD-L1 further improvedthe immune cytotoxicity of patient TILs to breast cancer cells MCF-7. This study thus implies aclinical application for breast cancer patients by silencing both PD-1 and PD-L1 usingnanotechnology.Finally, the expression of PD-L1/L2 in breast and colon cancer stem cells (CSCs) was determined.The stemness of these cancer cells was confirmed by their surface markers. We have for the first timefound that PD-L1 expression was higher in both CSCs compared to the non-CSCs by flow cytometrydata and expression of cellular PD-L1 proteins. In contrast, only trace amounts of PD-L2 weredetected in both CSCs and non-CSCs. A further investigation showed that the increase of PD-L1expression in CSCs may be mainly induced by insulin promotion of the protein production and EGFpromotion of the transport of the protein. Thus, breast and colon cancers may be sensitive to PD-1/PD-L1 immunotherapy, and warrant further investigations of CSC-targeted PD-1/PD-L1immunotherapy using the developed nanoparticles, such as LCP NPs.In conclusion, this PhD research project promises a nanoparticle-based PD-1/PD-L1-targetedimmunotherapy in the near future.