1. 5th Asia-Pacific Summit on
Cancer Therapy
Preparation of optimized lipid-coated calcium phosphate nanoparticles for enhanced siRNA delivery to breast cancer cells
Jie Tang,a Li Li,a Christopher B. Howard,a Stephen M. Mahler,a,b Leaf Huang,c Zhi Ping Xu*a
a Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, QLD 4072, Australia.
b School of Chemical Engineering, The University of Queensland, St Lucia, QLD 4072, Australia.
c Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
21st July, 2015 1

2. RNA interference (RNAi) for cancer therapy
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RNA interference (RNAi) for cancer therapy
Trehan S, Sharma G, Misra A. siRNA: Sojourn from discovery to delivery challenges and clinics[J]. Systematic Reviews in Pharmacy, 2010, 1(1): 1. 2

3. RNAi based cancer therapy drugs in clinical trials.
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RNAi for cancer therapy
RNAi based cancer therapy drugs in clinical trials.
Drug
Target
Delivery system
Administration route
Disease
Phase
Company
ClinicalTrials.gov identifier
siRNA-EphA2-DOPC
EphA2
LNP
Intravenous injection
Advanced Cancers I
M.D. Anderson Cancer Center
NCT
Atu027
PKN3
Advanced Solid Tumors
Silence Therapeutics GmbH
NCT
TKM
PLK1
Hepatic
intra-arterial administration
Multiple Cancers
National Cancer Institute (NCI)
NCT
ALN-VSP02
KSP and VEGF
Solid tumors
Alnylam Pharmaceuticals
NCT
siG12D LODER
KRAS
LODER polymer
Intratumoral administration
Pancreatic Ductal Adenocarcinoma Pancreatic Cancer
Silenseed Ltd
NCT
LNP, lipid nanoparticle; EphA2, ephrin type-A receptor 2; VEGF, vascular endothelial growth factor; PKN3, protein kinase N3; PLK1, polo-like kinase 1; KSP, kinesin spindle protein.
Xu C, Wang J. Delivery systems for siRNA drug development in cancer therapy[J]. Asian Journal of Pharmaceutical Sciences, 2015, 10(1): 1-12. 3

4. Criteria of an ideal vehicle for cancer therapy
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Criteria of an ideal vehicle for cancer therapy
evasion of the mononuclear phagocytic system (MPS) long circulation;
extravasation from the vascular
endothelial barrier;
diffuse through the extracellular
matrix;
d. be taken up into the cell;
e. escape the endosome;
f. unpackage and release the siRNA to
the RNAi machinery;
K.A. Whitehead, R. Langer, D.G. Anderson, Knocking down barriers: advances in siRNA delivery, Nat. Rev. Drug Discov. 8 (2) (2009) 129–138 4

5. Promising candidate as a nano-platform
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LCP (Lipid Calcium Phosphate) nanoparticle
1. Biocompatible and biodegradable;
2. Ca ions form complexes with nucleic acid backbone;
3. pH-sensitive: Control release in cytoplasm after endosome escape;
Promising candidate as a nano-platform
4. Colloidal stability;
Nucleic acid
PEGylation:
Long circulation in vivo;
Functional groups:
Amino or Maleimide
Protein/peptide
Functionalized with multiple ligands
Hydrophobic drug
Calcium phosphate (CaP) nanoparticle
Lipid coated calcium phosphate (LCP) nanoparticle
Li J, Yang Y, Huang L. Calcium phosphate nanoparticles with an asymmetric lipid bilayer coating for siRNA delivery to the tumor[J]. Journal of Controlled Release, 2012, 158(1): 5

6. LCP (Lipid Calcium Phosphate) nanoparticle
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LCP (Lipid Calcium Phosphate) nanoparticle
silencing of the three oncogenes
in metastatic nodules A B C
inhibition of lung metastases (~70–80%)
(0.36 mg/kg)
No observed toxicity
Yang Y, Li J, Liu F, et al. Systemic delivery of siRNA via LCP nanoparticle efficiently inhibits lung metastasis[J]. Molecular Therapy, 2012, 20(3): 6

7. Background
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Synthesis of LCP nanoparticle and optimization of key parameters in preparation process
Adjustable parameters:
1.Ca and P ion concentration;
2. Ionic strength;
3. pH value;
4. surfactants…
5. Ca/P molar ratio
CaP Cores with first layer lipid coating
film-rehydration
LCP
The outline for LCP preparation. 7

8. Synthesis parameters: Ca/P ratio influence on LCP NP ???
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LCP (Lipid Calcium Phosphate) Nanoparticle
Synthesis parameters:
Ca/P ratio influence on LCP NP ???
LCP (25~40 nm)
D. Olton et al. / Biomaterials
(2007) 1267–1279
Ca/P ratio
Particle size
Ca2+ to HxPO4x-3 ratio on
pure Nano CaP
PEG
Asymmetric
lipid bilayer
Calcium phosphate core:
siRNA (or other hydrophilic molecular)
The structure for LCP NP 8

9. Aim To elucidate the effects of the Ca/P molar ratio:
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To elucidate the effects of the Ca/P molar ratio:
the particle size and stability of synthesized LCP NPs;
the siRNA loading efficiency and protection of loaded siRNA;
the cellular uptake efficiency and cell growth inhibition of optimized LCP NP. 9

10. The effect of the Ca/P molar ratio on particle size and zeta potential
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The effect of the Ca/P molar ratio on particle size
and zeta potential
The average particle size and the zeta potential of LCPs with varying Ca/P ratios.
The data presented as average ± standard error (n = 3) 10

11. Loading siRNA/dsDNA into LCP NP
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Loading siRNA/dsDNA into LCP NP
42.4±1.2%
66.6±2.4%
39.8±1.2%
72.8±4.9%
66.6±2.4%
~35%
32.1±2.2 µg/mg
116.1±18.2 µg/mg
Then LCP NPs were used to encapsulate siRNA-mimicking Cy3-dsDNA with three different loading methods at a fixed Ca/P molar ratio of 100. As shown in Fig. 3A, Obviously, Method 3 (Ca&P) leds to the highest encapsulation efficiency, where half amount of Cy3-dsDNA was separately mixed with calcium and phosphate solution.
Next, the encapsulated efficiency and loading capacity of dsDNA were determined for the LCP NPs synthesized with method 3 at various Ca/P ratios. As shown in Fig 2B, the encapsulation efficiency of dsDNA by LCP NPs synthesized at the Ca/P ratio of 50 and 100 was much higher than that at the Ca/P ratio of 200 and 400. These data clearly indicate that the Ca/P ratio is critical in controlling the encapsulate efficiency of dsDNA by the LCP particles.
On the other hand, the loading capacity of LCP NPs increased with the Ca/P molar ratio increasing from 50 to 400. Reversely, the lower Ca/P ratio results in higher encapsulation efficiency and more LCP NPs, but the loading capacity per LCP particles is relatively low. As a trade-off, LCP NPs synthesized at Ca/P ratio of 100 seem to be optimal to load siRNA with reasonably high encapsulation efficiency (66.6±2.4%) and loading capacity (58.7±2.1 µg/mg). The loading capacity of 58 ug/mg present that in a siRNA loaded LCP NP, there is about 5.8 weight percent is siRNA.
Ca/P ratio of 100 seem to be optimal to load siRNA with reasonably high encapsulation efficiency (66.6±2.4%) and loading capacity (58.7±2.1 µg/mg).
(A). Encapsulation efficiency of dsDNA via different loading methods at the Ca/P
ratio of 100; (B). Effect of the Ca/P ratio on gene encapsulation efficiency and loading
capacity. Values presented as the mean ± S.E. from 3 independent experiments 11

12. Protection of siRNA from serum RNase degradation
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Protection of siRNA from serum RNase degradation
Optimal Ca/P ratio
After that, The ability of LCP NPs synthesized at different Ca/P ratios to protect siRNA from degradation by serum enzymes was studied by agarose gel electrophoresis. As clearly shown, 1 h incubation with serum largely degraded naked siRNA and there was no siRNA left after 2 h incubation. We also observed that siRNA encapsulated in LCP NPs prepared at the Ca/P ratio of 400 was degraded quickly and almost no siRNA was protected after 4 h incubation. Relatively, there was a large proportion of siRNA protected by LCP NPs prepared at Ca/P ratios of 50, 100 and 200 at 1 h, the protection was extended to 4 h. Interestingly, the siRNA protection by the LCP NPs prepared at Ca/P = 100 seems to be the highest.
Considering the loading efficiency, the loading capacity and the protection of loaded siRNA from enzyme degradation, we suggest that siRNA-LCP NPs prepared at Ca/P =100 are the optimised delivery system, which will be used in the subsequent testings.
Effect of the Ca/P ratio on serum stability of siRNA encapsulated by LCP NPs 12

13. Physicochemical properties of optimized LCP NPs
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Physicochemical properties of optimized LCP NPs A C
~20 nm
~40 nm
-15 mV
amorphous calcium phosphate (ACP) phase B D
(A) The particle size distribution of optimized LCP NPs (Ca/P ratio=100);
(B) TEM image of CaP cores (a), LCP NPs before (b) and after negative staining (c).
(C) XRD pattern of LCP NP; (D) FT-IR spectrum of LCP NP. 13

14. Colloidal stability of LCP NPs
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Colloidal stability of LCP NPs
Colloidal stability of LCP NPs in PBS and in medium with 10% FBS (37°C, 5% CO2) as a function of time. Data given as the mean ± SD of triplicates.
Viability of MDA-MB-468 cells in the presence of blank LCPs. 14

15. pH-controlled dissolution
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pH-sensitive
The dissolution of Ca2+
~93%
pH-controlled dissolution
pH sensitive siRNA release
&
Endosome escape
~24%
~40%
~15%
The dissolution profile of LCP NPs in aqueous solutions with different pHs. 15

16. Cellular uptake and siRNA delivery efficacy of LCP NPs
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Cellular uptake and siRNA delivery efficacy of LCP NPs
The in vitro cellular uptake efficiency of LCPs
Dose-dependent
The cellular uptake of non targeted LCP NPs was quantitatively assessed in the human breast cancer cell line MDA-MB-468, using flow cytometry analysis. 50 nM Cy3 lablled gene was used for tracking LCP NPs. As shown in Fig. 7, the cellular uptake increased gradually with the concentration of LCPs in medium, which indicates that the cellular uptake of LCP is dose-dependant. And we noticed that the uptaking amount significantly decreased when the LCPs were modified with 10% peg which indicates that pegylation could block the non-specific uptake of LCPs due to its large steric hindrance.
The effect of LCP NP dose on the cellular uptake of MDA-MB-468 cells, represented by the mean fluorescent intensity (MFI) of viable cells (A) and the percentage of positive cells (B) after incubation for 4 h. 16

17. Cellular uptake and siRNA delivery efficacy of LCP NPs
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Cellular uptake and siRNA delivery efficacy of LCP NPs
The in vitro cellular uptake efficiency of LCPs
Confocal microscopy image using fluorescence-labelled dsDNA also shows the enhanced efficiency in the cellular uptake of Cy3-dsDNA via LCP NPs (Fig. 8).
Fluorescence photographs of cultured MDA-MB-468 cells after treatment with LCP-Cy3-dsDNA NPs for 4 h. 17

18. In vitro inhibition of cancer cell growth
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In vitro inhibition of cancer cell growth
The in vitro cellular uptake efficiency of LCPs
kill~40%
Dose-dependent
~83%
In order to preliminarily test the anti-tumor effect of LCP NP loading cell death siRNA in vitro. The growth inhibition of MDA-MB-468 cell line by LCP-CD-siRNA NPs (CD-LCP NPs) was assessed using MTT assay. As shown in Fig. 9, the cell viability was CD siRNA dose dependent. As a comparison, the commercial delivery system OligofactamineTM reduced the cell viability to ~60% (P<0.001) at 80 nM, which is corresponding to that when treated with 20 nM CD siRNA loaded in LCP particles. At the CD siRNA dose of 40 and 80 nM, LCP-CD siRNA NPs were able to kill 65% and 83% MDA-MD-468 cells, with much higher inhibition than the commercial transfection reagent Oligofectamine™.
Viability of MDA-MB-468 cells in the presence of LCP-CD siRNA NPs at different concentrations. Data presented as the mean ± S.E. from 3 independent experiments. 18

19. Cell morphology after siRNA transfetion
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Cell morphology after siRNA transfetion
The in vitro cellular uptake efficiency of LCPs
blue staining
The morphology change and much less MDA-MB-468 cells in the LCP-CD siRNA-treated groups further confirmed the cell growth inhibitory effect of LCP-CD siRNA NPs. With increasing the CD siRNA concentration in LCP NPs, more cells became around with a small cell size. Particularly, LCP-CD siRNA NPs with the siRNA concentration of 40 and 80 nM induced more severe cell morphology changes than OligofectamineTM. Most cells died, floated and clustered in the complete medium at 80 nM of CD siRNA in LCP NPs. In the case of 80 nM CD siRNA in LCP NPs, there was 40 µg/mL of LCP NPs. This low dose of LCP NPs thus clearly shows that the inhibition is solely attributed to the high delivery efficacy of CD siRNA using LCP NPs.
The morphology of MDA-MB-468 cells in vitro after treatment with LCP-CD siRNA NPs. OligofectamineTM loaded with 80 nM CD siRNA was used as a positive control. 19

20. Conclusions
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Summary
The optimized LCP has a hollow spherical structure with a size of about 40 nm and possesses an asymmetric lipid bilayer at the surface which helps to well dispersed in aqueous solution;
The particle size and zeta potential were predominantly determined by the Ca/P ratio;
The loading efficiency of siRNA and the protection of the loaded siRNA from enzyme degradation were also significantly determined by the Ca/P ratio;
Optimized LCP NP system can efficiently deliver the functional CD-siRNA to MDA-MB-468 cancer cells and more effectively inhibit the cell growth in comparison with the commercial transfection agent. 20

21. Acknowledgements Advisory team in AIBN A/Prof. Zhi Ping (Gordon) Xu
A/Prof. Stephen Mahler
Dr. Christopher Howard
All of my group mates
Chinese Scholarship Council (CSC)

22. Thank you!