Spinal Cord Repair using Polymeric Substrates

Preparation of optimized lipid-coated calcium phosphate nanoparticles for enhanced siRNA delivery to breast cancer cellsJie 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.Cancer Therapy21st July, 201515th Asia-Pacific Summit on1BackgroundAimResultsConclusionsOutlines RNA interference (RNAi) for cancer therapy

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Trehan S, Sharma G, Misra A. siRNA: Sojourn from discovery to delivery challenges and clinics[J]. Systematic Reviews in Pharmacy, 2010, 1(1): 1.SignificanceResultsFuture workBackgroundAimFuture workResultsSummaries

2BackgroundAimResultsConclusionsOutlines RNAi for cancer therapy

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Xu C, Wang J. Delivery systems for siRNA drug development in cancer therapy[J]. Asian Journal of Pharmaceutical Sciences, 2015, 10(1): 1-12.SignificanceResultsFuture workBackgroundAimFuture workResultsSummariesRNAi based cancer therapy drugs in clinical trials.

DrugTargetDelivery systemAdministration routeDiseasePhaseCompanyClinicalTrials.gov identifiersiRNA-EphA2-DOPCEphA2LNPIntravenous injectionAdvanced CancersIM.D. Anderson Cancer CenterNCT01591356Atu027PKN3LNPIntravenous injectionAdvanced Solid TumorsISilence Therapeutics GmbHNCT00938574TKM-080301PLK1LNPHepatic intra-arterial administrationMultiple CancersINational Cancer Institute (NCI)NCT01437007ALN-VSP02KSP and VEGFLNPIntravenous injectionSolid tumorsIAlnylam PharmaceuticalsNCT01158079siG12D LODERKRASLODER polymerIntratumoral administrationPancreatic Ductal Adenocarcinoma Pancreatic CancerISilenseed LtdNCT01188785LNP, 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.3BackgroundAimResultsConclusionsOutlines Criteria of an ideal vehicle for cancer therapy

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K.A. Whitehead, R. Langer, D.G. Anderson, Knocking down barriers: advances in siRNA delivery, Nat. Rev. Drug Discov. 8 (2) (2009) 129138SignificanceResultsFuture workBackgroundAimFuture workResultsSummaries

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;4Functional groups:Amino or Maleimide

AimBackgroundResultsConclusionsOutlines5Li 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): 108-114.LCP (Lipid Calcium Phosphate) nanoparticleBackgroundAimSignificanceResultsFuture workAimBackgroundResultsConclusionsFuture workCalcium phosphate (CaP) nanoparticle1. Biocompatible and biodegradable;2. Ca ions form complexes with nucleic acid backbone;3. pH-sensitive: Control release in cytoplasm after endosome escape;

Lipid coated calcium phosphate (LCP) nanoparticle4. Colloidal stability;

Nucleic acid

Protein/peptideHydrophobic drugPEGylation: Long circulation in vivo;

Functionalized with multiple ligandsBackgroundAimResultsConclusionsFuture workSummariesPromising candidate as a nano-platform5AimBackgroundResultsConclusionsOutlines6Yang Y, Li J, Liu F, et al. Systemic delivery of siRNA via LCP nanoparticle efficiently inhibits lung metastasis[J]. Molecular Therapy, 2012, 20(3): 609-615.BackgroundAimSignificanceResultsFuture workAimBackgroundResultsConclusionsFuture workBackgroundAimResultsConclusionsFuture workSummariesLCP (Lipid Calcium Phosphate) nanoparticle

inhibition of lung metastases (~7080%) (0.36 mg/kg) No observed toxicitysilencing of the three oncogenes in metastatic nodulesABC6

AimBackgroundResultsOutlines7AimSynthesis of LCP nanoparticle and optimization of key parameters in preparation processSummariesResultsAimBackgroundFuture workLCPAdjustable parameters:1.Ca and P ion concentration;2. Ionic strength;3. pH value;4. surfactants5. Ca/P molar ratioCaP Cores with first layer lipid coatingThe outline for LCP preparation.film-rehydrationAimBackgroundResultsConclusionsOutlinesBackgroundAimSignificanceResultsFuture workAimBackgroundResultsConclusionsFuture workBackgroundAimResultsConclusionsFuture workSummaries7AimBackgroundResultsOutlines8AimLCP (Lipid Calcium Phosphate) Nanoparticle SummariesResultsAimBackgroundFuture work The structure for LCP NP

PEGCalcium phosphate core: siRNA (or other hydrophilic molecular)

LCP (25~40 nm)Asymmetric lipid bilayer

D. Olton et al. / Biomaterials (2007) 12671279Ca/P ratioParticle sizeCa2+ to HxPO4x-3 ratio on pure Nano CaP AimBackgroundResultsConclusionsOutlinesBackgroundAimSignificanceResultsFuture workAimBackgroundResultsConclusionsFuture workBackgroundAimResultsConclusionsFuture workSummariesSynthesis parameters:Ca/P ratio influence on LCP NP ???8AimBackgroundResultsConclusionsFuture workAimTo 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. 9Summaries9

The effect of the Ca/P molar ratio on particle size and zeta potentialResultsAimBackgroundSignificanceFuture work10SummariesResultsAimBackgroundFuture workThe average particle size and the zeta potential of LCPs with varying Ca/P ratios. The data presented as average standard error (n = 3)10

ResultsAimBackgroundSignificanceFuture work11SummariesResultsAimBackgroundFuture workLoading siRNA/dsDNA into LCP NP(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 experiments42.41.2%66.62.4%39.81.2%72.84.9%66.62.4%~35%32.12.2 g/mg 116.118.2 g/mg Ca/P ratio of 100 seem to be optimal to load siRNA with reasonably high encapsulation efficiency (66.62.4%) and loading capacity (58.72.1 g/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 (66.62.4%) and loading capacity (58.72.1 g/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.11ResultsAimBackgroundSignificanceFuture work12SummariesResultsAimBackgroundFuture workProtection of siRNA from serum RNase degradationEffect of the Ca/P ratio on serum stability of siRNA encapsulated by LCP NPs

Optimal Ca/P ratioAfter 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. 12

ResultsAimBackgroundSignificanceFuture work13Physicochemical properties of optimized LCP NPsSummariesResultsAimBackgroundFuture work(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.

~20 nm~40 nm-15 mVamorphous calcium phosphate (ACP) phaseABCD13ResultsAimBackgroundSignificanceFuture work14SummariesResultsAimBackgroundFuture workColloidal stability of LCP NPsColloidal stability of LCP NPs in PBS and in medium with 10% FBS (37C, 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. 14ResultsAimBackgroundSignificanceFuture work15SummariesResultsAimBackgroundFuture workpH-sensitiveThe dissolution profile of LCP NPs in aqueous solutions with different pHs.

~15% ~24% ~93% The dissolution of Ca2+ pH-controlled dissolution pH sensitive siRNA release &Endosome escape

~40% 15

ResultsAimBackgroundSignificanceFuture work16ConclusionsResultsAimBackgroundFuture workDose-dependentThe in vitro cellular uptake efficiency of LCPs Cellular uptake and siRNA delivery efficacy of LCP NPsThe 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. 16ResultsAimBackgroundSignificanceFuture work17ConclusionsResultsAimBackgroundFuture workThe in vitro cellular uptake efficiency of LCPs Cellular uptake and siRNA delivery efficacy of LCP NPsFluorescence photographs of cultured MDA-MB-468 cells after treatment with LCP-Cy3-dsDNA NPs for 4 h.

17ResultsAimBackgroundSignificanceFuture work18ConclusionsResultsAimBackgroundFuture workThe in vitro cellular uptake efficiency of LCPs In vitro inhibition of cancer cell growth 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.

kill~40%Dose-dependent~83%18ResultsAimBackgroundSignificanceFuture work19ConclusionsResultsAimBackgroundFuture workThe in vitro cellular uptake efficiency of LCPsCell morphology after siRNA transfetionThe 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.

blue staining 19ResultsAimBackgroundResultsFuture work20ConclusionsSummariesAimBackgroundFuture workSummaryThe 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.

20AcknowledgementsA/Prof. Zhi Ping (Gordon) XuA/Prof. Stephen MahlerDr. Christopher Howard

All of my group mates

Chinese Scholarship Council (CSC)

Advisory team in AIBN

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Thank you!2222