1. THERMORESPONSIVE POLYMER NANOPARTICLES José Paulo Farinha CQFM – Centro de Química-Física Molecular IN – Institute of Nanoscience and Nanotechnology Instituto Superior Técnico http://cqfm.ist.utl.pt Ana Tristany, “Interfaces” (www.anatristany.com)

2. Nanoparticles at CQFM Smart Polymer NPs for DNA bioassays Core-Shell Silica-Polymer NPs for Advanced Coatings Polymer NPs for Hybrid Aerogels Polymer NPs for Metal Detection and Removal Polymer - Metal and Semiconductor NPs for Photonics and Imaging Core-Shell Hybrid NPs for Photodynamic Cancer Therapy Polymer NPs as hosts for Fluorescence Sensors Magnetic Smart Polymer NPs for Antibody Separation

3. Stimuli responsive (“smart”) polymers Water soluble polymers that change size in response to a change in temperature, pH, ionic force,… High surface area of polymer nanoparticles Immobilization and separation of biomolecules, biological diagnostic tests, etc. p L p D Volume phase transition (VPT)

4. + + + + + + + + + + + + + + + + + + + + Core diameters 30 nm – 300 nm 10 4 – 10 5 charges per particle Thermoresponsive nanoparticles SEM TEM Cationic particles by two steps seeded emulsion polymerization Cationic PNIPAM shell PMMA glassy core N-isopropylacrylamide, aminoethyl methacrylate and methylene bisacrylamide cross-linker

5. Hydrodynamic diameter by dynamic light scattering + + + + + + + + + + + + + + + + + +  5 nm shell  40 nm shell Volume phase transition (VPT) Thermoresponsive nanoparticles + + + + + + + + + + + + + + + + + + + + D c =130 nm

6. + + + + + + + + + + + + + + + + + + + + dT 25 -ROX  8 nm Polythymine labeled with Rhodamine X Fluorescently labeled DNA Less polar environment Temperature increase dT 25 -ROX shows a blue shift with temperature increase (less polar environment).

7. Fluorescently labeled DNA In water Adsorbed onto particles Less polar PNIPAM shell dehydrates above the VPT. Less polar environment Temperature increase dT 25 -ROX shows a blue shift with temperature increase (less polar environment). Fluorescence spectral shift of dT 25 -ROX adsorbed into the particles

8. + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Adsorbed Fluorescence lifetime of dT 25 -ROX by single photon timing dT 25 -ROX in water 32ºC Fluorescence lifetime PNIPAM shell is more compact above the VPT higher n

9. Fluorescence Anisotropy HOOC 5 6  S 1  S 0 Steady state anisotropy II E I // II II E Photoselection using irradiation by linearly polarized light. Molecular motion leads to depolarization.

10. dT 25 -ROX adsorbed dT 25 -ROX in water Lower mobility Photoselection using irradiation by linearly polarized light. Adsorbed DNA is less mobile above the VPT Fluorescence Anisotropy Molecular motion leads to depolarization. HOOC 5 6  S 1  S 0 Steady state anisotropy

11. Polarized fluorescence decays Parallel (II) and perpendicular (  ) fluorescence decays of dT 25 -ROX The anisotropy decay goes to zero because dT 25 -ROX rotates freely in solution. In water (pH=5.5) II I //

12. Polarized fluorescence decays In water (pH=5.5)Adsorbed on nanoparticles (30ºC) Parallel (II) and perpendicular (  ) fluorescence decays of dT 25 -ROX II I // + + + + + + + + + + + + + + + + + + The anisotropy at long times (r  ) shows that the motion of dT 25 -ROX on the nanoparticles is hindered.

13. R 0 2R 0 w 10 -4 10 10 2 5 8 0 R 0 2R 0 r  = 1 nm 6 ns - 1 Rate coefficient of Energy Transfer A r  D – donor lifetime  2 – orientation factor R 0 – Förster radius Spectroscopic ruler (1 – 20 nm) Resonance Energy Transfer Förster resonance energy transfer (FRET) How are the DNA distributed at the particle shell?

14. J. Phys. Chem. B 2004, 108, 16392 J. Phys. Chem. C 2008, 112, 10591 FRET Distribution Model Systems with restricted volume and spherical symmetry Resonance Energy Transfer Distribution of labeled DNA in the particle shell Donor dT 25 -ROX dT 25 -MG Acceptor

15. T = 23 ºC Core radius R c Increasing [DNA] R H – R c  40 nm Hydrodynamic radius R H DNA at the particle-water interface J. Phys. Chem. C, 2008, 112, 10591 J. Phys. Chem. C, 2008, 112, 16331

16. T = 45 ºC Core radius R c Hydrodynamic radius R H J. Phys. Chem. C, 2008, 112, 10591 J. Phys. Chem. C, 2008, 112, 16331 R H – R c  5 nm DNA at the particle-water interface At 45ºC the shell is thinner than the DNA length.

17. ROX cGene-ROX ROX 5' TAT TAG CCC AGA GGT GAT GTC TCT C 3' - ROX DNA sequence from the human F5 gene with a 1691 G-A mutation that originates the Leiden V Factor (blood clotting). MG MG Gene-MG MG - 5' G AGA GAC ATC ACC TCT GGG CTA ATA 3' (complementary sequence ) DNA hybridization Pure and Applied Chemistry 2009, 81, 1615; ChemPhysChem 2010, 11, 1749 20ºC 40ºC Hybridization detected by FRET

18. MG ROX No difference in the fluorescence spectra before and after addition of a non ‑ complementary dT 25 -MG to nanoparticles containing cGene ‑ ROX, at 40ºC. No FRET for non-complementary DNA strands (solution or PNP) Non ‑ complementary DNA strands

19. Hybridization efficiency: 70% in the PNPs solution PNP 40ºC pH=4 Physiologic ionic strength - 0.18 M NaCl DNA hybridization 80% in solution ROXMG Nanoparticles with cGene ‑ ROX and Gene ‑ MG

20. Efficient hybridization in the particles, even at very low ionic strength solutionPNP DNA hybridization Low ionic strength - 0.001 M NaCl Hybridization efficiency: 70% in the PNPs  0% in solution 40ºC pH=4 ROXMG Nanoparticles with cGene ‑ ROX and Gene ‑ MG

21. Conclusions No need for organic cosolvents, high ionic strength, surfactants, proteins or chelating agents. High hybridization efficiency even at very low ionic strength. Below the VPT, PNIPAM is expanded and hydrated Above the VPT, the shell dehydrates and collapses on the core The adsorbed DNA is fixed on the shell with no covalent binding Selectivity: single pair mismatch Recovery of test particles and DNA Magnetic smart polymer nanoparticles Future work

22. Acknowledgments CQFM – IST Telmo Prazeres Leila Moura Prof. J. M. G. Martinho Funding FCT – POCTI/QUI/47885 FCT – PDCT/CTM/68451 Ana Tristany, “Interfaces” (www.anatristany.com) Thank you CNRS – BioMerrieux Dr. C. Pichot