Layered double hydroxide (LDH) nanoparticles are developed as highly efficient siRNA carriers

Yanheng Wu

Advisory Team:A/Prof. Zhi Ping (Gordon) Xu

Dr. Wenyi GuProf. Chen Chen

Surgery

Radiotherapy

Chemotherapy

Immunotherapy

Current therapeutics for cancer

http://www.alnylam.com/rnai_primer/rna-interference-pg5.htm

RNAi based cancer therapy

Three strategies: shRNA, miRNA, siRNA

Naked siRNA

• Low blood stability

• Low membrane permeability

• Immunogenicity

Drug Target Delivery systemAdministration route Disease Phase Company

siRNA-EphA2-DOPC

EphA2 LNP Intravenous injection

Advanced Cancers

I M.D. Anderson Cancer Center

Atu027 PKN3 LNP Intravenous injection

Advanced Solid Tumors

I Silence Therapeutics GmbH

TKM-080301 PLK1 LNP Hepatic intra-arterial administration

Multiple Cancers

I National Cancer Institute (NCI)

ALN-VSP02 KSP and VEGF LNP Intravenous injection

Solid tumors I Alnylam Pharmaceuticals

CALAA-01 RRM2 Cyclodextrin NP Intravenous injection

Cancer Solid Tumor

I Calando Pharmaceuticals

siG12D LODER KRAS LODER polymer Intratumoral administration

Pancreatic Ductal Adenocarcinoma Pancreatic Cancer

I Silenseed Ltd

Xu, C. F., & Wang, J. (2015). Delivery systems for siRNA drug development in cancer therapy. Asian Journal of Pharmaceutical Sciences, 10(1), 1-12.

Current delivery systems for siRNA drug

[M2+(1-x)M

3+x(OH)]An-

(x/n)·m H2O

LDH can protect polar and anionic biomolecules through surface absorbance and anion exchange

Layered double hydroxide (LDH) nanoparticles

Drug delivery Anti-cancer drugs(e.g. doxorubicin, doxifluridine,and 5-fluorouracil)

Antiinflammatory drugs (e.g. diclofenac, ibuprofen, and glucuronic acid)

Gene delivery Hepatitis B virus DNA vaccineOligonucleotide, PCR fragment

Protein Bovine serum albumin (BSA) , Ovalbumin(OVA)

siRNACl-

H2O

High loading capacity

High biocompatibility

Low cytotoxicity

Low cost

Mg2Al-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 Mg2Al-Cl-LDH nanoparticles

MgCl2 AlCl3

NaOHLDH slurry LDH suspension

Step1Mixed solution containing MgCl2 and AlCl3 was quickly added into NaOH solution, under vigorous stirring

Step2After stirring, pure LDH slurry was obtained via centrifuge and wash

Step 3Resuspend LDH with deionised water

Step 4Transfer LDH into autoclave, followed by hydrothermal treatment

Characterisation of Mg2Al-CL-LDH

Particle size distribution (A),TEM image (B), FTIR spectra (C) and XRD pattern (D) of Mg2Al-Cl-LDH nanoparticles in the suspension

A B

C D

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