Transcript (LDH) nanoparticles are developed

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
Current therapeutics for cancer
Surgery
Chemotherapy
Radiotherapy
Immunotherapy
RNAi based cancer therapy
Three strategies: shRNA, miRNA, siRNA
http://www.alnylam.com/rnai_primer/rna-interference-pg5.htm
Naked siRNA
•
Low blood stability
•
Low membrane permeability
•
Immunogenicity
Current delivery systems for siRNA drug
Drug
siRNA-EphA2DOPC
Target
EphA2
Administration
Delivery system route
LNP
Intravenous
injection
Atu027
PKN3
LNP
Intravenous
injection
Advanced Solid I
Tumors
Silence
Therapeutics
GmbH
TKM-080301
PLK1
LNP
Hepatic intraarterial
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
I
Ductal
Adenocarcinom
a Pancreatic
Cancer
Disease
Advanced
Cancers
Phase
I
Company
M.D. Anderson
Cancer Center
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.
Layered double hydroxide (LDH) nanoparticles
[M
2+
(1-x)M
3+
n-
x(OH)]A (x/n)·m
H2O
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)
Mg2Al-Cl-LDH nanoparticles for siRNA delivery
High loading capacity
High biocompatibility
Low cytotoxicity
Low cost
ClH2O
siRNA
LDH nanoparticle endocytosis and cellular trafficking
What is the optimal parameters for LDH-siRNA delivery?
• 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
Task I: Synthesis of Mg2Al-Cl-LDH nanoparticles
MgCl2
AlCl3
NaOH
LDH slurry
LDH suspension
Step1
Step2
Step 3
Step 4
Mixed solution
containing MgCl2
and AlCl3 was
quickly added
into NaOH
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 Mg2Al-CL-LDH
A
C
B
D
Particle size distribution (A),TEM image (B), FTIR spectra (C) and XRD
pattern (D) of Mg2Al-Cl-LDH nanoparticles in the suspension
Task II: Optimisation of siRNA delivery with LDH and dsDNA-cy5
Different mixing styles
dsDNA-cy5
Different LDH/dsDNA mass ratios
Different incubation time
LDH
FACS
CNE2(Nasopharyngeal cancer cell)
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
Optimisation of mixing style
dsDNA-cy5
LDH
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.
Optimisation of LDH:siRNA mass ratio
The optimal LDH:siRNA mass ratio for siRNA delivery is
ranging from 15:1 to 30:1
Trade-off between the loading amount and
the carrier dose
<15:1
15:1 to 30:1
>30:1
Mass ratio of LDH:dsDNA
Optimisation of incubation time
After four hours, approximately 70% of NPC cells were positive.
The dsDNA concentration was 40nM
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