layered double hydroxide (ldh) nanoparticles are developed as highly efficient sirna carriers...
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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
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