Targeted Delivery of Nanomedicine

WP 5: Target cell uptake and intracellular trafficking of

siRNA/pDNA nanoparticles

WP leader: Ghent

Laboratory of General Biochemistry and Physical Pharmacy

Targeted Delivery of Nanomedicine

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.

Targeted Delivery of Nanomedicine

WP5 progress in Year 2

WP5 activities were continued as planned for year 3 of MEDITRANS

Deliverable Due date Date delivered/ expectedNo. Title

D61.1 Different nanoparticle carriers (nanogels, cationic polymers and liposomes) evaluated in vitro for efficiency of endosomal escape. Enhancing effect of PCI (Task 5.1, Months 24, 36, Responsibility: PCI)

Month 24 Month 25

D62.1 (a) Documented information on the extent of cellular uptake of dextran nanogels (“naked” i.e. not yet coated) by cancer and endothelial cells and on whether the uptake should be enhanced by the use of target moieties. (b) An answer to the question whether dextran nanogels sufficiently spontaneously escape from the endosomes or whether this should be enhanced by an appropriate coating (see WP1). (c) A view on whether PCI enhances the endosomal escape of the nanogels. (d) A view on the degradation of nanogels in the cytosol and whether intact siRNA is released (Task 5.1, Months 24, 36, Responsibility: GHENT)

Month 24 Month 24

Targeted Delivery of Nanomedicine

DeliverableDue date Date delivered/

expectedNo. Title

D61.2Different nanoparticle carriers (nanogels, cationic polymers and liposomes) evaluated in vitro for efficiency of endosomal escape. Enhancing effect of PCI (Task 5.1, Months 24, 36, Responsibility: PCI)

Month 36 Month 36

D62.2

(a) Documented information on the extent of cellular uptake of dextran nanogels (“PEGylated”) by cancer and endothelial cells and on whether the uptake should be enhanced by the use of target moieties. (b) An answer to the question whether dextran nanogels sufficiently spontaneously escape from the endosomes or whether this should be enhanced by an appropriate coating (see WP1). (c) A view on whether PCI enhances the endosomal escape of the nanogels.

Month 36 Month 36

D89Documented information on the intracellular uptake and processing of branched polyester-based nanoparticles in endothelial and cancer cells

Month 36

D90 MRI imaging method to monitor endosomal escape Month 36

Targeted Delivery of Nanomedicine

WP5 activities

5.1: Quantification of cellular uptake (months 25-42)•polyphosphazenes, PEG-PEI and cationic liposomes (UU): Stand-by•PEGylated biodegradable dextran nanogels (Ghent): Completed•biodegradable branched polyesters (Marburg): Stand-by

5.2: Quantification of endosomal escape (months 25-42)•Photochemical Internalisation technology (PCI): Ongoing•MRI imaging probes (Unito)

5.3: Quantification of intracellular dissociation (months 25-42): Ongoing

5.4: Quantification of cytosolic nucleic acid degradation (months 25-42) Ongoing

5.5: Quantification of nucleic acid mobility and nuclear pDNA uptake (months 25-42)

5.6: Quantification of the biological activity (months 25-42): Completed

Targeted Delivery of Nanomedicine

PEGylation of dextran nanogels

Targeted Delivery of Nanomedicine

Targeted Delivery of Nanomedicine

Targeted Delivery of Nanomedicine

WP5 activities

5.1: Quantification of cellular uptake (months 25-42)•polyphosphazenes, PEG-PEI and cationic liposomes (UU): Stand-by•PEGylated biodegradable dextran nanogels (Ghent): Completed•biodegradable branched polyesters (Marburg): Stand-by

5.2: Quantification of endosomal escape (months 25-42)•Photochemical Internalisation technology (PCI): Ongoing•MRI imaging probes (Unito)

5.3: Quantification of intracellular dissociation (months 25-42): Ongoing

5.4: Quantification of cytosolic nucleic acid degradation (months 25-42) Ongoing

5.5: Quantification of nucleic acid mobility and nuclear pDNA uptake (months 25-42)

5.6: Quantification of the biological activity (months 25-42): Completed

Targeted Delivery of Nanomedicine

Dual color SPT• Mathematical model ready, testing of model is ongoing• Colocalization of labeled nanogels and endosomes planned• Colocalization of labeled siRNA and endosomes planned

5.2: Quantification of endosomal escape

PCI on PEGylated nanogels

Targeted Delivery of Nanomedicine

WP5 activities

5.1: Quantification of cellular uptake (months 25-42)•polyphosphazenes, PEG-PEI and cationic liposomes (UU): Stand-by•PEGylated biodegradable dextran nanogels (Ghent): Completed•biodegradable branched polyesters (Marburg): Stand-by

5.2: Quantification of endosomal escape (months 25-42)•Photochemical Internalisation technology (PCI): Ongoing•MRI imaging probes (Unito)

5.3: Quantification of intracellular dissociation (months 25-42): Ongoing

5.4: Quantification of cytosolic nucleic acid degradation (months 25-42) Ongoing

5.5: Quantification of nucleic acid mobility and nuclear pDNA uptake (months 25-42)

5.6: Quantification of the biological activity (months 25-42): Completed

Targeted Delivery of Nanomedicine

dual color FCS to measure stability of complexes in serum.

Stability correlates with biological effect of nanocarriers

Fluorescence Correlation Spectroscopy (FCS)

Targeted Delivery of Nanomedicine

WP5 activities

5.1: Quantification of cellular uptake (months 25-42)•polyphosphazenes, PEG-PEI and cationic liposomes (UU): Stand-by•PEGylated biodegradable dextran nanogels (Ghent): Completed•biodegradable branched polyesters (Marburg): Stand-by

5.2: Quantification of endosomal escape (months 25-42)•Photochemical Internalisation technology (PCI): Ongoing•MRI imaging probes (Unito)

5.3: Quantification of intracellular dissociation (months 25-42): Ongoing

5.4: Quantification of cytosolic nucleic acid degradation (months 25-42) Ongoing

5.5: Quantification of nucleic acid mobility and nuclear pDNA uptake (months 25-42)

5.6: Quantification of the biological activity (months 25-42): Completed

Targeted Delivery of Nanomedicine

Intracellular degradation of siRNA: work performed by FRET-FCS: siRNA shows long stability in intracellular environment.

pDNA: currently no good methods available for intracellular measurements of degradation and diffusion development of new measurements based on Single Particle Tracking (SPT)

INTACT pDNA: DEGRADED pDNA: colocalized movement no-colocalized movement

Targeted Delivery of Nanomedicine

WP5 activities

5.1: Quantification of cellular uptake (months 25-42)•polyphosphazenes, PEG-PEI and cationic liposomes (UU): Stand-by•biodegradable dextran nanogels (Ghent): Completed•biodegradable branched polyesters (Marburg): Stand-by

5.2: Quantification of endosomal escape (months 25-42)•Photochemical Internalisation technology (PCI): Completed for naked nanogels•MRI imaging probes (Unito)

5.3: Quantification of intracellular dissociation (months 25-42): Work in progress.

5.4: Quantification of cytosolic nucleic acid degradation (months 25-42) 5.5: Quantification of nucleic acid mobility and nuclear pDNA uptake (months 25-42)

5.6: Quantification of the biological activity (months 25-42): Continued

Targeted Delivery of Nanomedicine

Targeted Delivery of Nanomedicine

Publications:Raemdock K, Van Thienen TG, Vandenbroucke RE, et al. (2008). Dextran microgels for time-controlled delivery of siRNA . Advanced Functional Materials Volume: 18 Issue: 7 Pages: 993-1001 Raemdonck, K.; Demeester, J.; De Smedt, S., Advanced nanogel 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., Biodegradable Dextran Nanogels for RNA Interference: Focusing on Endosomal Escape and Intracellular siRNA Delivery. 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 dextran nanogels for siRNA delivery.Langmuir 2009, 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 vivo stability of various PEI/siRNA complexes on pharmacokinetics and biodistribution – A correlation study of fluorescence fluctuation spectroscopy and nuclear imaging data. Journal of Controlled Release ,Submitted .

Targeted Delivery of Nanomedicine

Conference publications:Naeye B., Raemdonck K., Demeester, J., De Smedt S.C. (2008). Pegylation of biodegradable dextran nanogels for controlled siRNA release. ESF-UB conference: Nanomedicine 2008, Sant Feliu de Guixols, Spain.

Naeye B., Raemdonck K., Demeester, J., De Smedt S.C. (2008). Optimization of biodegradable dextran nanogels for controlled siRNA delivery. Meditrans 2nd Annual Meeting; from 26/03/2008 to 29/03/2008, Rehovot, Israel.