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WP 5: Target cell uptake and intracellular trafficking of siRNA/pDNA nanoparticles

WP 5: Target cell uptake and intracellular trafficking of siRNA/pDNA nanoparticles WP leader: Ghent Laboratory of General Biochemistry and Physical Pharmacy. WP5 Objectives: Contribute to understand the intracellular behaviour of siRNA/pDNA nanoparticles in cancer and endothelial cells.

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WP 5: Target cell uptake and intracellular trafficking of siRNA/pDNA nanoparticles

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  1. WP 5: Target cell uptake and intracellular trafficking of siRNA/pDNA nanoparticles WP leader: Ghent Laboratory of General Biochemistry and Physical Pharmacy

  2. WP5 Objectives: • Contribute to understand the intracellular behaviour of siRNA/pDNA nanoparticles in cancer and endothelial cells. • Identify the most critical steps in delivery of siRNA/pDNA to target cells, depending on type of nanoparticles. • Use this knowledge to design nanocarriers in WP1/WP3 for succesfull in vivo delivery of nucleic acids to cancer and endothelial cells.

  3. WP5 progress in Year 2 WP5 activities were continued as planned for year 3 of MEDITRANS

  4. WP5 activities • 5.1: Quantification of cellularuptake (months 25-42) • polyphosphazenes, PEG-PEI and cationicliposomes (UU): Stand-by • PEGylatedbiodegradabledextrannanogels (Ghent): Completed • biodegradablebranched polyesters (Marburg): Stand-by • 5.2: Quantification of endosomal escape (months 25-42) • PhotochemicalInternalisationtechnology (PCI): Ongoing • MRI imaging probes (Unito) • 5.3: Quantification of intracellulardissociation (months 25-42): Ongoing • 5.4: Quantification of cytosolicnucleic acid degradation (months 25-42)Ongoing • 5.5: Quantification of nucleic acid mobility and nuclearpDNAuptake(months25-42) • 5.6: Quantification of the biologicalactivity (months 25-42): Completed

  5. PEGylation of dextrannanogels

  6. WP5 activities • 5.1: Quantification of cellularuptake (months 25-42) • polyphosphazenes, PEG-PEI and cationicliposomes (UU): Stand-by • PEGylatedbiodegradabledextrannanogels (Ghent): Completed • biodegradablebranched polyesters (Marburg): Stand-by • 5.2: Quantification of endosomal escape (months 25-42) • PhotochemicalInternalisationtechnology (PCI): Ongoing • MRI imaging probes (Unito) • 5.3: Quantification of intracellulardissociation (months 25-42): Ongoing • 5.4: Quantification of cytosolicnucleic acid degradation (months 25-42)Ongoing • 5.5: Quantification of nucleic acid mobility and nuclearpDNAuptake(months25-42) • 5.6: Quantification of the biologicalactivity (months 25-42): Completed

  7. 5.2: Quantification of endosomal escape • PCI on PEGylatednanogels • Dual color SPT • Mathematical model ready, testing of model is ongoing • Colocalization of labelednanogels and endosomes planned • Colocalization of labeledsiRNA and endosomes planned

  8. WP5 activities • 5.1: Quantification of cellularuptake (months 25-42) • polyphosphazenes, PEG-PEI and cationicliposomes (UU): Stand-by • PEGylatedbiodegradabledextrannanogels (Ghent): Completed • biodegradablebranched polyesters (Marburg): Stand-by • 5.2: Quantification of endosomal escape (months 25-42) • PhotochemicalInternalisationtechnology (PCI): Ongoing • MRI imaging probes (Unito) • 5.3: Quantification of intracellulardissociation (months 25-42): Ongoing • 5.4: Quantification of cytosolicnucleic acid degradation (months 25-42)Ongoing • 5.5: Quantification of nucleic acid mobility and nuclearpDNAuptake(months25-42) • 5.6: Quantification of the biologicalactivity (months 25-42): Completed

  9. FluorescenceCorrelationSpectroscopy (FCS) • dual color FCS to measure stability of complexes in serum. • Stability correlates with biological effect of nanocarriers

  10. WP5 activities • 5.1: Quantification of cellularuptake (months 25-42) • polyphosphazenes, PEG-PEI and cationicliposomes (UU): Stand-by • PEGylatedbiodegradabledextrannanogels (Ghent): Completed • biodegradablebranched polyesters (Marburg): Stand-by • 5.2: Quantification of endosomal escape (months 25-42) • PhotochemicalInternalisationtechnology (PCI): Ongoing • MRI imaging probes (Unito) • 5.3: Quantification of intracellulardissociation (months 25-42): Ongoing • 5.4: Quantification of cytosolicnucleic acid degradation (months 25-42)Ongoing • 5.5: Quantification of nucleic acid mobility and nuclearpDNAuptake(months25-42) • 5.6: Quantification of the biologicalactivity (months 25-42): Completed

  11. Intracellulardegradation of siRNA: workperformedby FRET-FCS: siRNA shows long stability in intracellular environment. pDNA: currentlynogoodmethodsavailableforintracellularmeasurements of degradation and diffusion development of newmeasurementsbasedon Single ParticleTracking (SPT) INTACT pDNA: DEGRADED pDNA: colocalizedmovementno-colocalizedmovement

  12. WP5 activities • 5.1: Quantification of cellularuptake (months 25-42) • polyphosphazenes, PEG-PEI and cationicliposomes (UU): Stand-by • biodegradabledextrannanogels (Ghent): Completed • biodegradablebranched polyesters (Marburg): Stand-by • 5.2: Quantification of endosomal escape (months 25-42) • PhotochemicalInternalisationtechnology (PCI): Completedfornakednanogels • MRI imaging probes (Unito) • 5.3: Quantification of intracellulardissociation (months 25-42): Work in progress. • 5.4: Quantification of cytosolicnucleic acid degradation (months 25-42) • 5.5: Quantification of nucleic acid mobility and nuclearpDNAuptake(months25-42) • 5.6: Quantification of the biologicalactivity (months 25-42): Continued

  13. Publications: Raemdock K, Van Thienen TG, Vandenbroucke RE, et al. (2008). Dextranmicrogels for time-controlled delivery of siRNA . AdvancedFunctionalMaterials Volume: 18 Issue: 7 Pages: 993-1001 Raemdonck, K.; Demeester, J.; De Smedt, S., Advancednanogel engineering for drug delivery. Soft Matter 2009, 5, (4), 707-715. Raemdonck, K.; Naeye, B.; Buyens, K.; Vandenbroucke, R. E.; Hogset, A.; Demeester, J.; De Smedt, S. C., BiodegradableDextranNanogelsfor RNA Interference: FocusingonEndosomal Escape and IntracellularsiRNADelivery. Advanced Functional Materials 2009, 19, (9), 1406-1415. Raemdonck, K.; Vandenbroucke, R. E.; Demeester, J.; Sanders, N. N.; De Smedt, S. C., Maintaining the silence: reflections on long-term RNAi. Drug Discovery Today 2008, 13, (21-22), 917-931. Naeye, B.; Raemdonck K.; Demeester, J.; De Smedt, S.C., PEGylation of dextrannanogelsforsiRNAdelivery. Langmuir2009, Submitted. Merkel, O.M., Librizzi, D., Pfestroff, A., Schurrat, T., Buyens, K., De Smedt, S.C., Béhé, M. and Kissel, T. (2009). Influence of in vivostability of various PEI/siRNAcomplexesonpharmacokinetics and biodistribution – A correlationstudy of fluorescencefluctuationspectroscopy and nuclearimaging data. Journal of Controlled Release ,Submitted.

  14. Conference publications: Naeye B., Raemdonck K., Demeester, J., De Smedt S.C. (2008). Pegylation of biodegradable dextrannanogels for controlled siRNA release. ESF-UB conference: Nanomedicine 2008, SantFeliu de Guixols, Spain. Naeye B., Raemdonck K., Demeester, J., De Smedt S.C. (2008). Optimization of biodegradable dextrannanogels for controlled siRNA delivery.Meditrans 2nd Annual Meeting; from 26/03/2008 to 29/03/2008, Rehovot, Israel.

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