Layered double hydroxide (LDH) nanoparticles are developed as highly efficient siRNA carriers Yanheng Wu Advisory Team: A/Prof. Zhi Ping (Gordon) Xu Dr. Wenyi Gu Prof. Chen Chen

Surgery Radiotherapy Chemotherapy Immunotherapy Current therapeutics for cancer

RNAi based cancer therapy Three strategies: shRNA, miRNA, siRNA

Naked siRNA Low blood stability Low membrane permeability Immunogenicity

DrugTargetDelivery system Administration routeDiseasePhaseCompany siRNA-EphA2- DOPC EphA2LNPIntravenous injection Advanced Cancers IM.D. Anderson Cancer Center Atu027PKN3LNPIntravenous injection Advanced Solid Tumors ISilence Therapeutics GmbH TKM PLK1LNPHepatic intra- arterial administration Multiple Cancers INational Cancer Institute (NCI) ALN-VSP02KSP and VEGFLNPIntravenous injection Solid tumorsIAlnylam Pharmaceuticals CALAA-01RRM2Cyclodextrin NPIntravenous injection Cancer Solid Tumor ICalando Pharmaceuticals siG12D LODERKRASLODER polymerIntratumoral administration Pancreatic Ductal Adenocarcinom a Pancreatic Cancer ISilenseed Ltd Xu, C. F., & Wang, J. (2015). Delivery systems for siRNA drug development in cancer therapy. Asian Journal of Pharmaceutical Sciences, 10(1), Current delivery systems for siRNA drug

[M 2+ (1-x) M 3+ x (OH)]A n- (x/n) ·m H 2 O LDH can protect polar and anionic biomolecules through surface absorbance and anion exchange Layered double hydroxide (LDH) nanoparticles

Drug deliveryAnti-cancer drugs (e.g. doxorubicin, doxifluridine,and 5-fluorouracil) Antiinflammatory drugs (e.g. diclofenac, ibuprofen, and glucuronic acid) Gene deliveryHepatitis B virus DNA vaccine Oligonucleotide, PCR fragment ProteinBovine serum albumin (BSA), Ovalbumin(OVA)

siRNA Cl - H2OH2O High loading capacity High biocompatibility Low cytotoxicity Low cost Mg 2 Al-Cl-LDH nanoparticles for siRNA delivery

LDH nanoparticle endocytosis and cellular trafficking

Task I: Synthesis and characterisation of LDH nanoparticles Task II: Optimisation of siRNA delivery with LDH and siRNA mimicking dsDNA-cy5 -Optimise the mixing style -Optimise the mass ratio of LDH to siRNA -Optimise the incubation time What is the optimal parameters for LDH-siRNA delivery?

Task I: Synthesis of Mg 2 Al-Cl-LDH nanoparticles MgCl 2 AlCl 3 NaOH LDH slurry LDH suspension Step1 Mixed solution containing MgCl 2 and AlCl 3 was quickly added into NaOH solution, under vigorous stirring Step2 After stirring, pure LDH slurry was obtained via centrifuge and wash Step 3 Resuspend LDH with deionised water Step 4 Transfer LDH into autoclave, followed by hydrothermal treatment

Characterisation of Mg 2 Al-CL-LDH Particle size distribution (A),TEM image (B), FTIR spectra (C) and XRD pattern (D) of Mg 2 Al-Cl-LDH nanoparticles in the suspension AB CD

Task II: Optimisation of siRNA delivery with LDH and dsDNA-cy5 CNE2(Nasopharyngeal cancer cell) Different mixing styles Different LDH/dsDNA mass ratios Different incubation time dsDNA-cy5 LDH FACS

Cytotoxicity of LDH nanoparticles to nasopharyngeal cancer cells Even at the concentration of 400µg/ml, LDH did not show cytotoxicity to CNE2 cells

Optimisation of mixing style dsDNA-cy5 LDH Mixing style I

dsDNA-cy5 LDH Optimisation of mixing style Mixing style II

Optimisation of mixing style dsDNA-cy5 LDH Mixing style III

Optimisation of mixing style Mixing style III exhibited the highest uptake efficiency for the siRNA delivery to NPC cells.

The optimal LDH:siRNA mass ratio for siRNA delivery is ranging from 15:1 to 30:1 Optimisation of LDH:siRNA mass ratio

Mass ratio of LDH:dsDNA Trade-off between the loading amount and the carrier dose 15:1 to 30:1 <15:1 >30:1

After four hours, approximately 70% of NPC cells were positive. The dsDNA concentration was 40nM Optimisation of incubation time

Summary LDH nanoparticles are stable inorganic materials with low cytotoxicity. The optimal parameters for LDH-siRNA delivery are: -Mixing style: direct mixing of LDH and siRNA -Mass ratio of LDH:siRNA from 15:1 to 30:1 -Incubation time=4 hours

Acknowledgements Prof. Zhi Ping (Gordon) Xu Dr. Wenyi Gu Prof. Chen Chen All of my colleagues Financial support: UQ International Scholarship