MAGNETIC NANOCOMPOSITE PARTICLES FOR BIO-APPLICATIONS Ana Luísa Daniel da Silva ana.luisa@ua.pt

Nanoparticles vs Nanobiosystems 1 10 100 nm hemoglobina ribossoma The relative particle size between nanoparticles and nanobiosystems

Bio-applications of magnetic nanoparticles Magnetite Fe3O4 Maghemite g-Fe2O3 Magnetic Separation Protein purification Cell isolation Biocatalysis .... Endorem®, Resovist® Diagnosis MRI (contrast enhancement agents) Biosensors .... Chem Comm 12 (2008) 1203 Drug Delivery Magnetic drug targeting Controlled drug release (magnetic heating) .... Therapeutics Magnetic Hyperthermia Cancer therapy .... Adv. Mater. (2003) 15, 1729

Multifunctional Magnetic Nanoparticles Combine various components Multiple bio-applications Sun et al., Adv Drug Delivery Rev 60 (2008) 1252 Gao et al., Acc Chem Res 42 (2009) 1097

Superparamagnetism H=0 : negligible remanent magnetization superparamagnetic Iron Oxide NPs: Ø< 15 nm H=0 : negligible remanent magnetization minimal NPs agglomeration Biomedical applications Hysteresis loop (M vs H): ferrimagnetic superparamagnetic

Research activities - Inorganic functional nanomaterials and organic-inorganic hybrids (Research Group 1) - Nanostructured Materials Magnetic NPs (Iron oxides,…) Metallic NPs Semiconducting NPs Nanocomposites biopolymers synthetic polymers …. Bio-applications Heterogeneous catalysis Paints and coatings …. Synthesis Materials characterization Surface modification Interface phenomena ….

Aim of the work Magnetic Nanocomposites based on Thermosensitive Hydrogels (Drug delivery applications) Why to use thermoreversible gels? T Controlled drug release upon local heating induced by external stimuli Biofunctionalization, magnetic driving, MRI..

Aim of the work Magnetic Nanocomposites based on Thermosensitive Hydrogels (Drug delivery applications) - Synthesis of magnetic bio-nanocomposites Magnetic properties Stability to oxidation Rheological properties - Synthesis of composite nanoparticles - Biofunctionalization - In vitro release studies

In-situ synthesis of magnetic bio-nanocomposites Co-precipitation within carrageenan biopolymer Fe2+ + 2 Fe3+ + 8 OH- Fe3O4 + 4H2O Prevention of the agglomeration Control of the particle size ~ 8 -15 nm (superparamagnetism) > Gel strength A.L. Daniel-da-Silva et al., Biomacromolecules 8 (2007) 2350

Particle size & Gel strength In-situ synthesis of magnetic bio-nanocomposites Chemical stability to oxidation ? k- 0.5% k- 2.0% i- 2.0% l- 2.0% k- 2.0%* Fe3O4 g-Fe2O3 Magnetite oxidation Diffusion based mechanism Chemical Stability Balance between Particle size & Gel strength A.L. Daniel-da-Silva et al., Biomacromolecules 8 (2007) 2350

Rheological studies in bio-nanocomposites k-carrageenan /Fe3O4 Sol Gel k-1.0wt% Sol Gel Tgel In-situ & Ex-situ synthesis G’ (k-car+Fe3O4+ K+) > G’ (k-car + K+) Ex-situ synthesis G’ (k-car + Fe3O4) < G’ (k-car) Reinforcement of gel Combined effect Fe3O4 & K+ ions A.L. Daniel-da-Silva et al., J. Colloid Interf Sci 324 (2008) 205

increases [K+] within “polymer cages” electrostatic repulsions Rheological studies in bio-nanocomposites Reinforcement of gel Combined effect Fe3O4 & K+ ions KOH Stronger gels Fe3O4 K+ increases [K+] within “polymer cages” z (Fe3O4) < 0 electrostatic repulsions magnetite / -OSO3- Weaker gels (as for Carr/SiO2 NPs) - Model system k-carrageenan /SiO2 NPs A.L. Daniel-da-Silva et al., J. Colloid Interf Sci 320 (2008) 571 A.L. Daniel-da-Silva et al., J. Colloid Interf Sci 324 (2008) 205

Biofunctionalized magnetic hydrogel nanospheres (k-carrageenan/Fe3O4) NP size control W=[H2O]/[surfactant] A.L. Daniel-da-Silva et al., Nanotechnology 20 (2009) 355602

Biofunctionalized magnetic hydrogel nanospheres NP Surface carboxymethylation followed by bovine IgG antibody conjugation (Carbodiimide-mediated reaction) T 37-45ºC DSC Superparamagnetic properties (MRI, magnetic targeting) Biofunctionalization (local targeting) Thermosensitive (Thermally controlled drug release) A.L. Daniel-da-Silva et al., Nanotechnology 20 (2009) 355602

In vitro release profiles ONGOING WORK k-carrageenan discs k-carrageenan discs (bulk) Methylene Blue Parameters of AzM encapsulation in nanospheres k-carrageenan nanospheres EE(%) LC(%) k-car1 19±8. 5 0.18±0.0 7 k-car4 42±2. 8 0.36±0.0 4

In vitro release profiles ONGOING WORK k-carrageenan/Fe3O4 nanocomposite discs 37ºC

Main Conclusions - Magnetic nanocomposites were prepared via in situ co-precipitation of Fe3O4 NPs. Carrageenan plays a relevant role in the control of size and chemical stability of NPs. The resulting bio-nanocomposites are thermoreversible hydrogels with superparamagnetic properties. Successful preparation of k-carrageenan/Fe3O4 bio-nanocomposite particles showing NP size <100 nm Superparamagnetic properties Thermosensitivity in T range acceptable for living cells Ability for coupling to active biomolecules. Envisaged applications include local targeted and thermally controlled drug delivery. In vitro release tests regarding their behavior as carrier for controlled drug release are in progress. Evidences of possible reaction to external stimuli (radiation).

Perspectives of Future Work - In vitro release tests upon magnetic stimuli - Evaluation of toxicity - Extension of this work to other systems (in progress) -magnetic bionanocomposites with other nanofillers (e.g. MWCNTs) -other thermosensitive hydrogels

Acknowledgements Ana Estrada (UA) Ana M. Gil (UA) António J. Guiomar (UC) Benilde F.O. Costa (UC) Brian Goodfellow (UA) Ernest Mendoza (ICN-Barcelona) Joana Moreira (UA) José A. Lopes-da-Silva (UA) Luciana Ferreira (UA) Nuno João Silva (UA) Sara Fateixa (UA) Tito Trindade (UA) SFRH/BPD/39105/2007 FEDER (PTDC/QUI/67712/2006) NANO/NMed-SD/0140/2007

Nanocomposite Particles for Bio-Applications Materials and Bio-Interfaces edited by Tito Trindade & Ana Luísa Daniel-da-Silva (University of Aveiro, Portugal) One of the first books on bio-applications of nanocomposite particles. Includes contributions from specialists with international published research in their field of expertise. Illustrated with excellent figures and containing a very good survey of references to accompany each chapter. For more information, please email sales@panstanford.com or visit www.panstanford.com Sep 2010, 400 pages (approx.) 978-981-4267-78-6 (Hardback)