Smart drug delivery systems for siRNA therapeutics in prostate cancer immunotherapy
University College Cork
Prostate cancer (PCa) as an immunosuppressive cancer remains a serious condition threatening the health of men due to the complicated nature of the tumour microenvironment (TME). Conventional treatments of PCa face challenges including poor prognosis and tumour resistance, therefore new therapeutic strategies are urgently needed. Tumour-associated macrophages (TAMs) are a potential therapeutic target in cancer immunotherapy. Colony stimulating factor-1/colony stimulating factor-1 receptor (CSF-1/CSF-1R) pathway plays a crucial role in the polarization of the immunosuppressive TAMs, M2 macrophages. Downregulation of CSF-1R expression by small interfering RNA (siRNA), a double-stranded non-coding RNA, is known to reprogram the immunosuppressive TAMs, M2 macrophages, to the immunostimulatory phenotype, M1 macrophages. Sialic acid (SA) is a ligand for Siglec-1 (CD169) which is overexpressed on M2 macrophages with little expression in other phenotypes. M2 macrophage-targeting peptide (M2pep) is an artificially designed peptide and could specifically target M2 macrophages, the sequence of which was selected by subtractive phage biopanning. Beta-cyclodextrins (CDs) are cyclic oligosaccharides derived from starch, consisting of at 7 glucose subunits linked by α-1,4 glycosidic bonds, the primary and secondary sides of the ring structure are hydrophilic and the cavity is hydrophobic. Cationic CDs have been used for delivery of therapeutic nucleic acids including siRNA as non-viral vectors. Therefore, in this thesis, 2 different CD-based nanoparticles (NPs) with PEGylated SA or PEGylated M2pep as the ligand was designed and characterized for the delivery of CSF-1R to M2 macrophages in both human and mouse cells to study the macrophage reprogramming efficacy. In the meanwhile, a Transwell model was built for co-culture of M2 macrophages and PCa cells to test the anti-PCa effectiveness in vitro. In vitro results show that the sizes of three NPs were around 250 nm, and the surface charges were 11 ~ 29 mV, the poly dispersity index (PDI) was lower than 0.30. All CD-based formulations were stable in both salt- and serum-containing media, and did not present any cytotoxicity in either human or mouse macrophages. Both two NPs achieved M2 macrophage reprogramming, and post-transfected cells expressed M1 macrophage markers. The co-culture results show that the formulations caused the apoptosis of PCa cells due to the M2 macrophage reprogramming. In the in vitro studies, the CD.siRNA.DSPE-PEG-M2pep formulation presented the best CSF-1R gene silencing efficacy, superior M2 macrophage reprogramming and anti-tumour efficacy. Therefore, the CD.siRNA.DSPE-PEG-M2pep formulation was selected for assessment in vivo. A PCa-bearing mouse model was built in C57 BL/6 mice by subcutaneous injection of PCa cells and used to study the targeting and anti-PCa efficacy of CD.siRNA.DSPE-PEG-M2pep in vivo. The NPs increased M2 macrophage targeting in vivo, promoting the release of M1 factors and simultaneously downregulating the levels of M2 factors through TAM reprogramming. The subsequent remodelling of the TME resulted in a reduction in tumour growth in the PCa mouse model, mainly mediated through the recruitment of cytotoxic T cells. In summary, the CD-based targeted siRNA NPs successfully reprogrammed the immunosuppressive M2 macrophages thus promoting PCa immunotherapy and providing an alternative strategy for PCa treatment worthy of further investigation.
Modified cyclodextrins , RNA interference , Sialic acid , M2pep , Tumor-associated macrophages , Prostate cancer , Immunotherapy
Sun, Y. 2023. Smart drug delivery systems for siRNA therapeutics in prostate cancer immunotherapy. PhD Thesis, University College Cork.