1. Dr Gillian Hutcheon Reader in Biomaterials School of Pharmacy and Biomolecular Sciences Liverpool John Moores University Polymeric nanoparticles for the pulmonary delivery of miRNA to treat Chronic Obstructive Pulmonary Disease (COPD)

2. Research Aims Develop polymeric nanoparticles (NPs) for the pulmonary delivery of miRNA to treat COPD Optimise LJMU technology platform: Nanocomposite microparticles (NCMPs) for the pulmonary delivery of biopharmaceuticals using dry powder inhalation for local and systemic drug delivery.

3. COPD Heterogeneous inflammatory disease 4 th leading cause of death worldwide Current treatment for symptoms –Bronchodilators –Corticosteroids –Antibiotics –Oxygen Need to treat progression Locally via pulmonary route

4. Micro RNA Modulation of miRNA expression and function is a strategy for therapeutic intervention. In COPD fibroblasts cytokine-dependent induction of miR-146a is impaired increasing inflammation Naked miRNA cannot cross anionic cell membrane X No Protein microRNA Messenger RNA miRNA binding to target mRNA blocks translation

5. Nanoparticles (NPs) Formulation of miR-146a oligonucleotide with polymeric NPs to treat COPD by compensating for impaired miR-146a levels. Adsorption prevents loss of miRNA function

6. NCMPs Nanocomposite Microparticles Need aerosolisable microparticles for pulmonary delivery (1-5  m) Spray dry NPs with leucine to form NCMP Applications: local and systemic –Lung disease –Vaccine delivery –Gene delivery –Cancer therapy

7. Delivery System Cell uptake

8. Research plan Polymer synthesis Particle preparation Macromolecule adsorption Spray drying Aerosolisation In vitro assays

9. G. Hutcheon, LJMU 2005 Combined enzyme catalysed ring- opening and condensation polymerisation MWt ~ 16kDa Poly glyceroladipate-co-pentadecalactone PGA-co-PDL

10. Preparation of Nanoparticles Single emulsion solvent evaporation – DOTAP (dioleoyltrimethylammoniumpropane) at concentrations ranging from 0-20 (%w/v) used to produce cationic NPs [ Zetasizer – NP size –Charge

11. The size and charge of the cationic NPs was dependent on the concentration of DOTAP Optimisation of size and charge NP size NP surface charge

12. miRNA Adsorption Fluorescently-labelled miRNA was adsorbed onto the 15% DOTAP NPs at room temp Maximum adsorption of 40 μg/ml miRNA was 3.625±0.035 μg per mg NP after 24 h with no significant change after 2 h In vitro release in PBS, 77% after 24 h

13. Spray Drying Nanocomposite Microparticles (NCMPs) prepared using L-Leucine as a carrier PGA-co-PDL/L-leu NCMPs (1:1.5) Currently optimised using BSA as a model Yield: 50.01 ± 0.3% SEM Buchi Mini Spray Dryer B-290 10 µm 2 µm 20/06/2013

14. 25/03/2013 In vitro Aerosolisation studies Next Generation Impactor (NGI). Plates coated with 1% (tween 80: acetone) solution. Samples drawn at a flow rate of 60 L/min for 4 s BSA analysed using HPLC Fine Particle Fraction (FPF) 60.92 ± 3.25% Mass Median Aerodynamic Diameter (MMAD) 1.57 ± 0.09 µm

15. NP Toxicity Over 65% of A549 and Calu-3 cells remained viable following 24 h exposure to DOTAP NPs at 1.25 mg/ml Local concentration in vivo will be much less

16. Intracellular Uptake A) Nucleus stained with DAPI (B) NPs stained with Nile Red (C) merged image after 1hr Fluorescence microscopy Confocal microscopy ABCABC

17. miRNA-NPs Uptake A) Nucleus stained by DAPI B) FAM labelled miRNA-NPs C) Merged image after 1h Confocal microscopy Fluorescence microscopy ABCABC

18. miR-146a Functionality Activity of miR-146a against targeted gene, interleukin-1 receptor-associated kinase (IRAK1) Dose dependant effect on target gene repression compared with untreated cells RT-qPCR Response

19. Conclusions NPs were within required size range (~ 200nm) for cellular uptake and ZP was positive (15% DOTAP ) Fluorescently-labelled miRNA was adsorbed onto cationic NPs and ~ 77 % was released in PBS after 24 h. BSA-NCMP (2-5  m) prepared and aerosolisaton tested – FPF 60.92 ± 3.25%, MMAD 1.57 ± 0.09 µm miRNA-NPs were distributed in cytoplasm and at periphery of nucleus region in A549 cells miRNA-NPs had dose depended effect on target gene repression and response reduction compared with untreated cells

20. –Dr Imran Saleem –Dr Kehinde Ross –Dr Nitesh Kunda –Adel Mohamed Acknowledgements

21. Thank You