Layered double hydroxide LDH nanoparticles are developed as

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

Current therapeutics for cancer Surgery Chemotherapy Radiotherapy Immunotherapy

RNAi based cancer therapy Three strategies: sh. RNA, mi. RNA, si. RNA http: //www. alnylam. com/rnai_primer/rna-interference-pg 5. htm

Naked si. RNA • Low blood stability • Low membrane permeability • Immunogenicity

Current delivery systems for si. RNA drug Drug si. RNA-Eph. A 2 DOPC Target Eph. A 2 Administration Delivery system route Disease LNP Intravenous Advanced injection Cancers Atu 027 PKN 3 LNP Intravenous injection Advanced Solid I Tumors Silence Therapeutics Gmb. H TKM-080301 PLK 1 LNP Hepatic intraarterial administration Multiple Cancers I National Cancer Institute (NCI) ALN-VSP 02 KSP and VEGF LNP Intravenous injection Solid tumors I Alnylam Pharmaceuticals CALAA-01 RRM 2 Cyclodextrin NP Intravenous injection Cancer Solid Tumor I Calando Pharmaceuticals LODER polymer Intratumoral administration Pancreatic I Ductal Adenocarcinom a Pancreatic Cancer si. G 12 D LODER KRAS Phase I Company M. D. Anderson Cancer Center Silenseed Ltd Xu, C. F. , & Wang, J. (2015). Delivery systems for si. RNA drug development in cancer therapy. Asian Journal of Pharmaceutical Sciences, 10(1), 1 -12.

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

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

Mg 2 Al-Cl-LDH nanoparticles for si. RNA delivery High loading capacity High biocompatibility Low cytotoxicity Low cost Cl. H 2 O si. RNA

LDH nanoparticle endocytosis and cellular trafficking

What is the optimal parameters for LDH-si. RNA delivery? • Task I: Synthesis and characterisation of LDH nanoparticles • Task II: Optimisation of si. RNA delivery with LDH and si. RNA mimicking ds. DNA-cy 5 -Optimise the mixing style -Optimise the mass ratio of LDH to si. RNA -Optimise the incubation time

Task I: Synthesis of Mg 2 Al-Cl-LDH nanoparticles Mg. Cl 2 Al. Cl 3 Na. OH LDH slurry LDH suspension Step 1 Step 2 Step 3 Step 4 Mixed solution containing Mg. Cl 2 and Al. Cl 3 was quickly added into Na. OH solution, under vigorous stirring After stirring, pure LDH slurry was obtained via centrifuge and wash Resuspend LDH with deionised water Transfer LDH into autoclave, followed by hydrothermal treatment

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

Task II: Optimisation of si. RNA delivery with LDH and ds. DNA-cy 5 Different mixing styles ds. DNA-cy 5 Different LDH/ds. DNA mass ratios Different incubation time LDH FACS CNE 2(Nasopharyngeal cancer cell)

Cytotoxicity of LDH nanoparticles to nasopharyngeal cancer cells Even at the concentration of 400µg/ml, LDH did not show cytotoxicity to CNE 2 cells

Optimisation of mixing style ds. DNA-cy 5 LDH Mixing style I

Optimisation of mixing style ds. DNA-cy 5 LDH Mixing style II

Optimisation of mixing style ds. DNA-cy 5 LDH Mixing style III

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

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

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

Optimisation of incubation time After four hours, approximately 70% of NPC cells were positive. The ds. DNA concentration was 40 n. M

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

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