Developing lipid-coated calcium carbonate/phosphate hybrid nanoparticles as a gene/drug co-delivery platform for combined cancer therapy

Abstract

Cancer is one of the most life-threatening types of diseases. Various agents, such as chemical drugs, oligonucleotides, and bioactive proteins, are developed for cancer treatment. Due to the unsatisfied outcomes of treatment relying on single anticancer agent, more studies are focused on developing regimens for combination therapy. Nowadays, nanoparticle (NP) based drug carriers have shown great potential to increase the anticancer agent efficacy as they are able to provide protection during circulation and improve tumour site accumulation via targeting delivery. Moreover, a proper NP-based carrier can deliver multiple anticancer agents with the similar efficacy for combinational treatment of tumours. This Ph.D. project aims to engineer lipid-coated calcium carbonate/phosphate (LCCP) hybrid nanoparticles (NPs), with optimised cargo release property, to carry two anti-cancer agents and achieve effective cancer therapy.Calcium carbonate (CaC) and calcium phosphate (CaP) are the most commonly used inorganic materials for gene delivery. The difference between the dissolution behaviours of CaC/CaP nanoparticles suggests that the pH for the release of loaded therapeutic agents is adjustable by controlling the composition of mixed CaC/CaP nanoparticle core. The coated bilayer phospholipids endow the particles with good colloidal stability, and are able to load hydrophobic drug. Moreover, the bilayer lipids can be conjugated with target moieties for target delivery to cancer cells. This Ph.D. thesis first optimised the LCCP core composition (i.e. carbonate to phosphate molar ratio) to achieve gene release at endosomal pH using dsDNA-cy5 as an example. The obtained LCCP NPs showed good colloidal stability with the average particle size of 40 nm, high gene loading capacity (~60%), desirable gene release profile, and enhanced cellular uptake efficacy. Compared to lipid-coated calcium phosphate (LCP) NPs, LCCP NPs achieved higher sensitivity and quicker release under mild acidic pH conditions (6.0-5.5). As-prepared LCCP hybrid NPs were then used to deliver small interference RNA (siRNA) for mouse programme death ligand 1 (PD-L1) and polo-like kinase 1 (PLK1) to B16F10 melanoma cancer cells, with the delivery efficacy higher than Oligofectamine®. This tendency endows faster siRNA release during the endocytosis and quicker gene down-regulation after NP endocytosis, but not affect their long term gene silencing efficacy.Folic acid (FA) was then conjugated on the surface to enhance the delivery efficacy via interacting with the FA receptor overexpressed on cancer cells. Subsequently, the hydrophobic anticancer drug, a-tocopheryl succinate (α-TOS), was loaded in the lipid bilayer in combination with Allstar Cell Death siRNA (CD siRNA) in the core of target LCCP NPs for combined target therapy of melanoma cells. The optimised FA density was 5% of lipids on the outer layer, as determined by cellular uptake of NPs by B16F0 cells. The combination of CD siRNA and α-TOS in LCCP NPs effectively inhibited the cell growth via an additive/synergic way.To further preventing cancer metastasis, α-TOS loaded LCCP NPs (NP-TOS15) were employed to treat 4T1 tumour in combination with interferon-gamma (IFN-g). The optimised NP-TOS15 showed an α-TOS loading efficiency of ⁓60%, and enhanced the uptake by 4T1 metastatic cancer cells. The IFN-γ/NP-TOS15 treatment significantly induced 90% cell death and inhibited migration of tumour cells. Moreover, NP-TOS15 upregulated the anticancer immunity via downregulating program death ligand 1 (PD-L1) expression induced by IFN-γ, and remarkably prevented the lung metastasis (metastasis index decreased from 10800 to 500), particularly in combination with IFN-γ. Further investigation revealed that this combination therapy also modulated the CD4+ and CD8+ cytotoxic lymphocyte infiltration into the tumour tissue for tumour elimination. Taken together, the NP delivery of α-TOS in combination with IFN-γ provides an applicable strategy for cancer therapy.In summary, the LCCP NPs were well developed to be a promising platform for gene/drug co-delivery to combined cancer treatment.