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Surface Modification of Nanoparticles Surface Modification of Nanoparticles for Biomedical Applicationsfor Biomedical Applications

Cristina Resetco

Polymer and Materials Science

University of Toronto

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Functions of Surface Ligands on Nanoparticles

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Biomedical Applications of NanoparticlesBiomedical Applications of Nanoparticles

Gold Optical absorption, stability

Thiol disulfide amine

Biomolecular recognitionsensing

CdSe quantum dots

Luminescencephoto-stability

Thiolphosphine pyridine

Imaging sensing

Fe2O3

nanoparticles

Magnetic Diolamine

MR imaging, biomolecule purification

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Phase Transfer of NanoparticlesPhase Transfer of Nanoparticles

(1) Ligand exchange (2) Additional ligand layer (3) Amphiphilic polymer

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Solubility in organic solvents and water where PEG is heavily hydrated, forming random coils

Less non-specific binding in cells by PEG-modified nanoparticles

Introduction of new functional groups on nanoparticles by bifunctional PEG

Separation by gel electrophoresis of nanoparticles with a defined number of chemical groups with PEG with molecular weight above 5000 g/mol, which forms discrete bands

Nanoparticles modified with NH2-PEG-NH2 yield nanoparticles with exactly one or two amino groups, separated by gel electrophoresis (Sperling et al. 2006).

PEG-Modified NanoparticlesPEG-Modified Nanoparticles

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Requirements for Solubilization and Bioconjugation of Nanoparticles

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 High quantum yield compared to common fluorescent dyes Broadband absorption: light that has a shorter wavelength than the emission maximum wavelength can be absorbed, peak emission wavelength is independent of excitation sourceTunable and narrow emission, dependent on composition and sizeHigh resistance to photo bleaching: inorganic particles are more photostable than organic molecules and can survive longer irradiation times Long fluorescence lifetime: fluorescent of quantum dots are 15 to 20 ns, which is higher than typical organic dye lifetimes.Improved detection sensitivity: inorganic semiconductor nanoparticles can be characterized with electron microscopes

Quantum Dot PropertiesQuantum Dot Properties

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Quantum dots conjugated with fQuantum dots conjugated with folateolate––PEGPEG––PMAMPMAM for for ttargeting argeting ttumor umor ccellsells

Folate–poly(ethylene glycol)–polyamidoamine ligands encapsulate and solubilize CdSe/ZnS quantum dots and target folate receptors in tumor cells.

Dendrimer ligands with multivalent amino groups can react with Zn2+ on the surface of CdSe/ZnS QDs based on direct ligand-exchange reactions with ODA ligands

Y. Zhao et al. Journal of Colloid and Interface Science 350 (2010) 44–50.

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More dense than linear ligands, which improves stability

More anchoring groups, which generate strong interactions between QDs and PAMAM

Terminal groups (amine, carboxyl, and hydroxyl) of polyamidoamine (PAMAM) dendrimers can be modified with different functionalities to link with various biomolecules

Poly(amidoamine) (PAMAM)Poly(amidoamine) (PAMAM) D Dendrimerendrimer LigandsLigands

Y. Zhao et al. Journal of Colloid and Interface Science 350 (2010) 44–50

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Figure 2. Phase contrast images (top row) and fluorescence image NIH-3T3 cells incubated with QDs2; (c) SKOV3 cells were incubated with QDs2

FPP-QDs specifically bind to tumor cells via the membrane expression of FA receptors on cell surface

Quantum Dots for Imaging of Tumor CellsQuantum Dots for Imaging of Tumor Cells

Y. Zhao et al. Journal of Colloid and Interface Science 350 (2010) 44–50.

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Surface Density of Ligands on NanoparticlesSurface Density of Ligands on Nanoparticles

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Monofunctionalized Nanoparticles by a Solid Phase Exchange Reaction

Bifunctional alkanethiol ligands with a carboxylic acid group are immobilized on a solid support such as polymeric Wang resin at a low density.

Exchange reaction of resin-bound thiol ligands with gold nanoparticles results in one resin-bound thiol ligand on each nanoparticle.

Cleavage from the resin yields nanoparticles with a single carboxylic acid functional group.

Schaffer, et al. Langmuir, Vol. 20, No. 19, 2004.

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Monofunctionalized Gold Nanoparticles

For solid phase exchange product there is minimal hydrogen bonding since C=O stretching vibration band appears at higher wavenumbers.

Figure 1. TEM image of gold nanoparticle dimers formed by coupling reaction of carboxylic group modified gold nanoparticles with bifunctional 1,2-ethylenediamine.

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Schaffer, et al. Langmuir, Vol. 20, No. 19, 2004.

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Bioconjugation of NanoparticlesBioconjugation of Nanoparticles

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Covalent Binding

Non-Covalent Interactions

Bifunctional linkersmercaptoacetic acid is used to link quantum dots with biomolecules

Silanizationalkosiloxane molecules form covalent Si-O-Si bonds

Electrostatic interactionshigh affinity of cationic biomolecules for negatively charged backbone of DNA

Hydrophobic forces used to link modified acrylic acid  polymer to TOPO capped quantum dots

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(1) PEG with N-hydroxysuccinimide (NHS) group at one end and an orthopyridyl disulfide (OPSS) group at the other is attached to the surface of the nanocages by breaking the disulfide bond of the OPSS group and forming a gold-thiolate bond

(2) Primary amine on antibody reacts with the NHS group of PEG molecule

Precise tuning of LSPR

Potential to trap drug molecules or enzymes in pores and release them through an externally controlled mechanism

Photothermal effect for cancer therapeutics

Gold Nanocages

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Au nanocages are synthesized by galvanic replacement reaction between Ag nanocubes and HAuCl4 in water.

Temperature-sensitive polymer based on poly(N-isopropylacrylamide) (pNIPAAm) changes conformation due to variations in temperature.

Photothermal effect induced by laser beam with a wavelength matching the absorption peak of Au nanocage, causes light to be absorbed and converted into heat

Drug release due to temperature increase that causes polymer chains to collapse exposing nanocage pores

Figure 2. TEM images of Au nanocages covered

by a pNIPAAm-co-pAAm copolymer

Figure 1. Drug release from gold nanocages

Gold nanocages covered by smart polymers for controlled release with NIR light

Yavuz, et al. Nature Materials. Vol 8, December 2009.

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Atom-transfer radical polymerization of N-isopropylacrylamide (NIPAAm) and acrylamide (Aam) initiated by a disulphide initiator forming polymer with tunable low critical solution temperature (LCST) between 32-50 C.

Polymer Synthesis by ATRPPolymer Synthesis by ATRP

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Figure 2. Cell viability for samples (C-1) cells irradiated with a pulsed near-infrared laser for 2 min without Au nanocages (C-2) cells irradiated with the laser for 2 min in the presence of Au nanocages; and (2/5 min) cells irradiated with the laser for 2 and 5 min in the presence of doxorubicin (Dox)-loaded Au nanocages.

Controlled Drug Release from NanocagesControlled Drug Release from Nanocages

Figure 1. Controlled release of alizarin dye from the Au nanocages covered by a copolymer with an LCST at 39 C Absorption spectra of alizarin-PEG released from the copolymer-covered Au nanocages

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Nanoparticles for imaging: quantum dots

Targeting agent: antibody or peptide

Cell-penetrating agent: peptide

Stimulus-sensitive element for drug release

Stabilising polymer to ensure biocompatibility: polyethylene glycol

Multifunctional NanoparticlesMultifunctional Nanoparticles

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Multifunctional Magnetic NanoparticlesMultifunctional Magnetic Nanoparticles

• Magnetic nanocrystals as ultrasensitive MR contrast agents: MnFe2O4

• Anticancer drugs as chemotherapeutic agents: doxorubicin, DOX

• Amphiphilic block copolymers as stabilizers: PLGA-PEG

• Antibodies to target cancer cells: anti-HER antibody (HER, herceptin) conjugated by carboxyl group on the surface of the MMPNs

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Yang, etal. Angew. Chem. 2007, 119, 8992 –8995.

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Targeted Drug Delivery and Inhibition of Tumor GrowthTargeted Drug Delivery and Inhibition of Tumor Growth

Human epidermal growth factor receptor (HER2) -- tumor-targeting marker for breast cancer

Fibroblast NIH3T6.7 cells -- highly express the HER2/neu cancer markers

MDA-MB-231 cells -- express low levels of the cancer markers

Figure 2. MR signal intensity and colour maps of NIH3T6.7 and MDA-MB-231 cells treated with IRR-MMPNs; black, HER-MMPNs; white.

Yang, etal. Angew. Chem. 2007, 119, 8992 –8995.

Figure 1. Multifunctional magneto-polymeric nanohybrids (MMPNs) containing manganese ferrite (MnFe2O4) nanocrystals prepared by nanoemulsion with anticancer drug (doxorubicin, DOX) and PLGA-PEG

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HER-MMPNs had the greatest tumor growth inhibition than since HER-MMPNs were target-delivered to HER2/neu receptors of NIH3T6.7 cells and DOX was released

Inhibition of Tumor Growth by Magnetic NanoparticlesInhibition of Tumor Growth by Magnetic Nanoparticles

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Nanoparticles affect biological behaviour at cellular, subcellular, protein, and gene levels by formation reactive oxygen species (ROS).

Nanoparticle ToxicityNanoparticle Toxicity

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1H NMR spectroscopy

Fourier Transform Infrared Sectroscopy (FTIR)

UV/VIS Spectrophotometry

transmission electron microscopy (TEM)

dynamic light scattering

gel electrophoresis

size exclusion chromatography

analytical ultracentrifugation

fluorescence correlation spectroscopy

Characterization of Nanoparticles and Surface LigandsCharacterization of Nanoparticles and Surface Ligands

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Acknowledgements

Professor Eugenia Kumacheva

Siyon (Lucy) ChungDr. Jemma VickeryDr. Kun LiuAriella LukashAnna LeeDan VoicuEthan TumarkinDr. Jesse GreenerJai Il ParkDr. Ziliang WuDr. Dinesh Jagadeesan