1 / 21

Dr Gillian Hutcheon Reader in Biomaterials School of Pharmacy and Biomolecular Sciences

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

johnreaves
Download Presentation

Dr Gillian Hutcheon Reader in Biomaterials School of Pharmacy and Biomolecular Sciences

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

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

  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 • 4th 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 microRNA Messenger RNA miRNA binding to target mRNA blocks translation X No Protein

  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. NCMPsNanocomposite Microparticles • Need aerosolisable microparticles for pulmonary delivery (1-5mm) • 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. Poly glyceroladipate-co-pentadecalactone PGA-co-PDL Combined enzyme catalysed ring- opening and condensation polymerisation • MWt ~ 16kDa G. Hutcheon, LJMU 2005

  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. Optimisation of size and charge The size and charge of the cationic NPs was dependent on the concentration of DOTAP 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 vitrorelease 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 2 µm 10 µm Buchi Mini Spray Dryer B-290

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

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

  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 Response RT-qPCR

  19. Conclusions • NPs were within required sizerange (~ 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 (2-5mm) 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. Acknowledgements • Dr Imran Saleem • Dr Kehinde Ross • Dr Nitesh Kunda • Adel Mohamed

  21. Thank You

More Related