Up-Conversion Nanoparticles for Optical Molecular Imaging Karen Köhler NanoMed Berlin, March 6th, 2009

NanoMed 2009 Dr. Karen Köhler Page 2 Outline  Application of Photoluminescent Nanoparticles  Market Needs  Types of Photoluminescent Nanoparticles  Upconverting Nanophosphors  Mechanism  Synthesis  Functionalization  Coupling of Antibodies  Performance Check  Take-Home Message Introduction Main Part Summary

NanoMed 2009 Dr. Karen Köhler Page 3 Solar cells Multiplexed biolabeling Diagnostic imaging Optical fiber-based telecommunications Light-emitting displays Solid-state lasers Authentification for objects Photoluminescent Nanoparticles – What for ? Solar cells

NanoMed 2009 Dr. Karen Köhler Page 4 In-vivo In-vitro Luminescent Nanoparticles in Diagnostics Biosensors POC, Laboratory Pharmacogenomics  personalized medicine SNP-analysis DNA assays Gene expressions analysis Antibody, antigen Protein, enzyme Immuno assays Improvement of established processes Higher sensitivity: Less sample material Smaller: Small size, low-weight instruments Higher specificity: More reliable Faster: Shorter response time More information in a single test: multiplexing Introduction of new systems Lab-on-a-chip: incl. sample preparation and detection (DNA, proteins, cells) Imaging Migration in cells / organisms (e.g. tumors) Drug tracking and targeting Contrast- / imaging techniques Surveillance tasks, e.g. continuous control of glucose levels, presence of cancer cells Control over surface under various conditions (pH, temperature, salt,...) Specificity Sensitivity Clarification of tox issues  introduction of nanoprobes (blood circulation or lymphatic system) and ex-vivo detection

NanoMed 2009 Dr. Karen Köhler Page nm particle size 3.8 nm  Crystalline semiconductor nanoparticles  Materials: CdSe, CdS, InAs, InP, …  Size: 1-10 nm  just a few 1000 atoms  Confined electron motion leads to altered optical, physical and chemical properties Quantum Dots - Baydots ® 5.9 nm Supplier: Bayer Technology Services ZnS CdSe Polymer layer functional groups Evident Design +

NanoMed 2009 Dr. Karen Köhler Page 6 Phosphor Materials  Well established material  TV-screens, fluorescent lamps...)  Doped crystalline particles  Size: µm  Host: Y 2 O 3, Y 2 O 2 S, LaPO 4, ZnS,...,  Dopants: Lanthanides, transition elements (Mn, Ag, Cu,...) Novel synthesis routes for nanophosphor production had to be developed... defined by dopants not by size

NanoMed 2009 Dr. Karen Köhler Page 7 Nanophosphors CePO 4 :Tb First system:  Individual nanoparticles: ca nm (!)  Bright fluorescence (quantum yield up to 50%) Excitation < 300 nm Excitation Emission  Environmentally friendly 20 nm  Low production costs (no necessity for core-shell or size control) sensitivity ~ 1 fmol Fulfills the requirements: Dy 3+ Tb 3+ Eu 3+ Eu 2+ Source: Bayer Research Magazine (2004)  Multiplexing capability by variation of lanthanoid ions

NanoMed 2009 Dr. Karen Köhler Page 8 Upconversion-Phosphors NaYF 4 :Yb,Er NaYF 4 :Yb,Tm  Host lattice: fluorides, oxysulfides  Dopants: sensitizer (absorber) + fluorescent center (emitter)  lanthanides  Energy of two or more IR photons is transferred to one lanthanide dopant ion Excitation: IR  Emission: VIS

NanoMed 2009 Dr. Karen Köhler Page 9 Upconversion Nanophosphors Advantages of uc-nanophosphors as biolabels  Minor toxicity  Highly stable (photo, thermal, oxidation, shelf-life)  Multiplexing capability by variation of lanthanoid ions  No photodamage of biological tissues  No autofluorescence of tissue 100 nm IR NaYF 4 :Yb,Er NaYF 4 :Yb,Tm NaYF 4 :Yb, Er/Tm  Size: nm  Monodisperse  Hexagonal or cubic lattice Most important applications  Imaging  Diagnostics  Therapy S. Heer, K. Kömpe, H. Güdel, M. Haase, Adv. Mat. (2004), 16, 2102.

NanoMed 2009 Dr. Karen Köhler Page 10 In-vivo Imaging of uc-Nanophosphors High laser intensityLow laser intensity

NanoMed 2009 Dr. Karen Köhler Page 11 Targeted Nanoparticle-Bioconjugates

NanoMed 2009 Dr. Karen Köhler Page 12 Synthesis of Upconversion Nanophosphors solvent NH 4 F Na + + solvent HEEDA Solution IISolution I  uc-nanophosphors dispersed in organic solvent  particles have to be water-dispersable for bio applications HEEDA = N-(2-hydroxyethyl)ethylenediamine NaYF 4 :Yb, Er

NanoMed 2009 Dr. Karen Köhler Page 13 Surface Functionalization poly(allylamine hydrochloride) poly(acrylic acid) poly(ethylenimine) Polyanion Polycation  Electrostatic adsorption of hydrophilic polyelectrolytes on the charged particle surface  Choose appropriate polymer and molecular weight for a specific particle type  Ensure colloidal stability under physiological conditions water dispersable uc-nanophosphors functional groups for coupling of biomolecules stable under physiological conditions -COOH

NanoMed 2009 Dr. Karen Köhler Page 14 PEGylation  increase of systemic retention by PEG Polyethylene glycol -COOH -CONH -COOH H2NH2N EDC, sulfo-NHS  incomplete reaction with functional PEG leaves carboxylic groups for coupling of biomolecules Amber tutorials ¯ Nanophosphor Antibody 40 nm PEG-chain

NanoMed 2009 Dr. Karen Köhler Page 15 Biofunctionalisation – Test with Streptavidin  Coupling of streptavidin to the carboxylic groups of poly(acrylic acid) BCA-Assay Amber tutorials ¯ Nanophosphor Streptavidin (Bicinchoninic acid) Estimation: ≈ 21 nm 2 /Streptavidin (maximal loading without PEG) Protein + Cu 2+ OH - Cu + BCA absorption at 562 nm Determination of the overall protein amount

NanoMed 2009 Dr. Karen Köhler Page 16 Activity of Bound Streptavidin titration with biotin- 4-fluorescein streptavidin fluorescence quenching when bound to SA

NanoMed 2009 Dr. Karen Köhler Page 17 The Target healthy celltumor cell target

NanoMed 2009 Dr. Karen Köhler Page 18 Development of a Suitable Conjugate Selection of target structure  MUC-1 Selection of a suitable antibody for detection of the target  M12 Selection of an optimal fragment for binding / imaging  M12-scFv

NanoMed 2009 Dr. Karen Köhler Page 19 Coupling of Antibodies Conjugates EDC/s-NHS-mediated coupling COOH-functionalized nanophosphor EDC/s-NHS M12-scFv conjugate with His-tag

NanoMed 2009 Dr. Karen Köhler Page 20 Proof of Specificity with Quantum Dots - in vitro QD - M12-scFv - conjugates

NanoMed 2009 Dr. Karen Köhler Page 21 Summary  uc-nanophosphors are a new generation of photoluminescent nanoparticles emissionexcitation  high application potential in imaging, diagnostics and therapy synthesis of monodisperse particles surface functionalization coupling of antibody conjugates targeting

NanoMed 2009 Dr. Karen Köhler Page 22 Acknowledgement Stefan Barth Markus Haase Claudia Walter Helga Hummel Volker Weiler Volker Bachmann Jens Waldeck Christoph Bremer BMBF Biophotonic II LUNA Thank you for your Attention !!! Werner Hoheisel Bastian Budde Diana Landen