1. SRI SRIDHAR, Ph.D. NCI/NSF NANOMEDICINE SCIENCE AND TECHNOLOGY ELECTRONIC MATERIALS RESEARCH INSTITUTE PHYSICS DEPARTMENT NORTHEASTERN UNIVERSITY SUPPORTED BY NSF/NCI and AFRL

2. Collaborators : Dattatri Nagesha Evin Gultepe Mansoor Amiji, Pharma Sciences Robert Campbell, Pharm Sci Vladimir Torchilin, Pharma Sciences Charles DiMarzio, ECE Latika Menon, Physics Alain Karma, Physics Don O’Malley, Biology Carol Warner, Biology Ahmed Busnaina, MIE Shashi Murthy, Chemical Engineering Laura Lewis, Chem Engg Nikos Soukos, Forsyth Dental Institute Mike Makrigiorgos & Robert Cormack, BWH-DFCI

3. NANOMEDICINE RESEARCH PLAN

4. Multi-functional Nanoplatforms PEG Spacer TAT Y pH-Sensitive Fluorescent Label Radioactive Label Antibody Endosome Buffering Agent HIV TAT Peptide pDNA or Oligonucl eotide Magnetic core Au or Fe-Au NP 1-100nm Micelles 10-50nm Liposomes 100-250 nm Polymeric NP ~ 20nm – 10  m TITANIA AND ALUMINA NANOTEMPLATES SENSING, TARGETING, DELIVERY SENSING, DRUG ELUTING PLATFORMS, SCAFFOLDS AND TEMPLATES, NEUROCHIP

5. Y Y Y Y Fe Au or Fe-Au Au + PEG Au + PEG +Fluorescent label Au + PEG +LM609 Antibody TARGETING MRI CONTRAST ENHANCEMENT MICROWAVE HYPERTHERMIA GENE DELIVERY Y Y Y Y Intracellular experiments Cellular Trafficking Au + PEG +LM609 Antibody + pDNA MULTI FUNCTIONAL NANOPARTICLES

6. Au NANOPARTICLES WITH HETERO-BIFUNCTIONAL PEG FOR BIOMEDICAL APPLICATIONS Wei Fu, Dinesh Shenoy, Curtis Crasto, Matt Bouchard, Jane Liu Mansoor Amiji, Graham Jones, Sanjeev Mukerjee, Charles di Marzio, Sri Sridhar Au Au + PEG Au + PEG +Fluorescent label Intracellular experiments Cellular Trafficking Int. J. Nanomedicine (2005), Bioorganic & Medicinal Chemistry Letters (2005)

7. Nucleus Multifunctional Nanoparticle Protein Release mRNA Early Endosome Cell Membrane Protein Expression Nuclear Uptake Endocytosis Non-Specific Uptake TAT-Mediated Uptake Receptor-Mediated Uptake Free DNA LIFE-CYCLE OF A NANOPARTICLE VECTOR Mechanisms and Pathways Binding Endocytosis Endosomal Escape Cytosolic Transport Degradation Nuclear Uptake Transfection Protein Expression Magnetic control with MagNP

8. 2-Photon Luminescence – Gold nanoparticles in Mouse Embryonic Stem Cells Int. J. of Nanomedicine (2007) Gold nanoparticles when excited by a 2-photon femto second laser source, exhibits photoluminescence Photoluminescence of 20  l gold NPs dried on glass slide Collaboration: Dr. Charles Dimarzio and Dr. Gary Laevsky (ECE) Paula Lampton, Carol Warner (Biology), Int. J. Nanomedicine (2007) Imaging without a fluorophore

9. Texas Red (568nm)Alexa 488 conj. to FNGAu (760nm) from FNG 20X lens IMAGING OF LARVAL ZEBRAFISH USING MULTI- PHOTON LUMINESCENCE OF Au NANOPARTICLES Fluor-gold nanoparticles and Texas Red Fluorophores for validation of technique (IGERT project) Collaboration with Sucharita Saha, Don O’Malley, Chuck DiMarzio New technique for studying gene delivery for spinal cord regeneration

10. BrightfieldScale Bar 100µm Two Photon λ=750nm Confocal Reflectance λ=920nm Second Harmonic λ=920nm Ag NANOPARTICLES IN ANIMAL SKIN With Yogesh Patel, Chuck DiMarzio

11. z-stack of excised skin sample recording an image every 3µm from the surface down 30µm MOVIE 15µm below glass coverslip 21µm below glass coverslip Confocal Reflectance Two-Photon Second-Harmonic

12. MAGNETIC NANOPLATFORMS A variety of applications Separation Science: cells, biomarkers, harvesting MRI contrast enhancement Localized energy delivery – hyperthermia, ablation Magnetic Manipulation- magnetic targeting, magnetic tweezers Magnetic biosensors

13. Blocking temperature (T B ) of SPIONs Superparamagnetism No hysteresis in M-H curves SuperParamagnetic Iron Oxide Nanoparticles (SPIONS)

14. Surface Modification Techniques for Magnetic Nanoparticles - - - - - - - - - - - - - - - - - - - Iron oxide nanoparticles +vely charged polyelectrolyte + +vely charged Iron oxide nanoparticles + +++ + + ++ + + a) Surface coating of magnetic NPs for direct attachment to cells Cell membrane is –vely charged and will bind to +vely charged particles through electrostatics. OH+EtO-Si-(CH 2 ) 3 -R OEt O-Si-(CH 2 ) 3 -R OH R R R R R = NH 2, SH, CHO b) Attachment of molecules for conjugation of antibodies Antibodies are conjugated through the R- functional group c) Gold coating to form core-shell morphology Iterative addition HAuCl 4 + Hydroxylamine Gold surface can be readily conjugated with various biomolecules Iron oxide NPsIron oxide-gold Core-shell NPs MAGNETIC NANOPARTICLES FOR MRI CONTRAST ENHANCEMENT

15. TEM image shows encapsulation of iron oxide NPs in the core of the micelle SPION Encapsulated PEG-PE Micelle System 2C5-Iron oxide NP-Micelles incubated with BT20 cells. Nuclei stained with Hoechst (blue) and 2C5-Iron oxide NP-Micelle stained with Rhodamine B (Red). Nanosized Cancer Cell-Targeted Polymeric Immunomicelles Loaded with Superparamagnetic Iron Oxide Nanoparticles Rishikesh M. Sawant a, Rupa R. Sawant a, Evin Gultepe b, Dattatri Nagesha b, Brigitte Papahadjopoulos-Sternberg c, Srinivas Sridhar b, Vladimir P. Torchilin a

16. MULTI-MODAL USE OF MAGNP IMAGE GUIDED MAGNETO THERAPY MRI CONTRAST ENHANCEMENT MAGNETIC TARGETING –MAGC LIPOSOMES : CAMPBELL collaboration –SPION-IMMUNOMICELLES : TORCHILIN colaboration MAGNETIC HYPERTHERMIA NOVEL NANOPARTICLE APPROACHES TO MRI NEW NANOPARTICLES, eg. Mn-Fe-O MIX T1 AND T2 AGENTS EXPLOIT NEW PULSE SEQUENCES FOR DIFFERENT TIME SCALES BOUND VS. FREE NANOPARTICLES

17. SPION-Micelles SPION SPIONs encapsulated in micelle specific anti-body attached SPION- Micelle packages intra-cellular experiments in vivo studies NMR studies Targeting MRI Contrast Agents Magnetic Hyperthermia

18. mPEG 2000 -DSPE Iron oxide NPs Lipid film hydration Sonication Encapsulation in micelles system Iron oxide NPs PEG-PE micelle T = 260K H (A/m) M(A/m) SQUID Magnetization Measurement With Sawant, Torchilin

19. TEM image shows encapsulation of iron oxide NPs in the core of the micelle SPION Encapsulated PEG-PE Micelle System 2C5-Iron oxide NP-Micelles incubated with BT20 cells. Nuclei stained with Hoechst (blue) and 2C5-Iron oxide NP-Micelle stained with Rhodamine B (Red).

20. + NH 2 Aqueous buffer pH 8-8.5, 4˚C Tumor-cell specific antinuclear antibody 2C5 was attached onto surface of SPION-micelles via para-nitrophenyl-PEG-PE mAb2C5 SPION-micelles RHODAMINE filter Bovine IgG SPION-micelles- Bare SPION-micelles HOECHST filter Rhodamine-labeled-SPION-micelles were incubated with human breast tumor MCF-7 cells in vitro. Associated fluorescence was observed using epifluorescence microscopy. Anti-body conjugation

21. Associated rhodamine fluorescence was also quantified using fluorescence spectroscopy. Fluorescence spectroscopy

22. Guided Drug Delivery Solid tumor Apply magnetic field to concentrate particles Modulate field to release drug from particles Inject NMPs IV, NMP will circulate through the blood stream Other options for targeting: 1 - Direct injection into tumor site 2 - Coating NMP with antibodies to target tumor

24. Magnetic Targeting

25. Iron oxide NPs encapsulated PEG-PE micelle system Show significant R2 enhancement Torchilin, Sawant (Pharmaceutical Sciences ), Kautz (Barnett) NMR of Iron oxide NPs PEG-PE micelle Animal MRI studies are ongoing at MGH

26. Relaxivity R2 (1/T2) signal associated with MCF-7 cells was measured using Varian INOVA 500MHz NMR Spectrometer. Tumor-cell-specific-2C5-SPION micelles interact better and thus deliver more signal to the human breast tumor MCF-7 cells compared to the “plain” or non-specific IgG-SPION micelles. R2 enhancement validated in in vitro cell studies Animal MRI studies are ongoing at MGH

27. mAb2C5-SPION-micelle SPION-micelle retains all the magnetic properties of the nanoparticles Magnetic targeting leads to 2x increase in tumor accumulation R2 relaxation significantly increased, leading to potential MRI contrast enhancement advantages

28. MAGNETIC CATIONIC LIPOSOMES FOR MRI AND HYPERTHERMIA With Robert Campbell (NU) Key advantages of MagC Targets tumor vasculature Very high cargo loading of nanocarrier Can be combined with drugs, genes, etc

29. Healthy Mouse 1hr After Injection (H2) Healthy Mouse 24hr After Injection (H1) 1hr after: the kidneys are dark because of the accumulation of iron oxide. 24hr after: kidneys are clear Healthy Mouse (H2)

30. Tumor Mouse 24hr After Injection (T1) Tumor Mouse 24hr After Injection + Magnet for 1hr (T2) Heavily T2 weighted images

31. Angiography Pre-Injection Angiography Post-Injection Arteries are invisible in post-injection images because of the circulating iron oxide.

32. Conclusion No animal death because of the injection The concentration used is enough for the contrast Immediately after injection iron-oxides circulate in the blood for at least one hour 24hr after injection the kidneys are cleared 1hr magnet placement has a positive effect on the accumulation in the tumor even after 24 hours.

33. Magnetic nanoparticles heating up due to the oscillating magnetic field. Gram-positive bacterium, E. faecalis, is exposed to ac magnetic field after incubating with nanoparticles for 5 minutes.     % killing vs exposure time MAGNETIC HYPERTHERMIA FOR CANCER AND INFECTIOUS DISEASES With Nikos Soukos, Forsyth Dental 100% kill achieved in 5-10 mins Promising results, further studies needed to assess clinical potential Potential application to Biofilm disruption Killing seems to be non-thermal Mechanism needs to be understood

34. LOCALIZED ENERGY DELIVERY EM induced drug release from liposomes With Campbell, Torchilin Cancer Hyperthermia and Thermal Ablation BIOMOLECULAR CONTROL Manipulating genetic structure by localized energy delivery EM Microbicide EM induced cell membrane disruption, eg. hemolysis

35. Cross section SEM images of Titania nanohole arrays. The average height of the film is ~ 250nm and pore diameter 25-35nm. Nanoporous Titania and Alumina Arrays Nanoassembly, Drug delivery Applications

36. Nanobead Assembly More than 85% coverage over 1cm 2 area. 50nm beads 80nm nanoholes 10V, electrophoresis plate counter electrode. Applied Physics Letters (2007)

37. Carbon Nanotube Assembly SEM micrographs of assembled SWCNTs in anodic alumina array. Applications: Biosensors High Density Memory

38. Elution from Nanotemplates www.csmc.edu Example: Drug eluting stents T. C. Woods, A. R. Marks Ann. Rev. Med., 55, 2004 Coronary artery stenosis Balloon angioplasty Stent Implantation Drug eluting stent bare stent Patent artery Restenosis

39. Elution from Nanotemplates Concentration of the drug is measured in the release medium over the time. A model drug, Doxorubicin, is loaded into alumina nanotemplates to observe release kinetics from a porous medium. excitation wavelength in-situ measurement 532 nm spectrometer

40. Anodic Alumina Nanoholes Pore diameter: 190nm Pore thickness: 10um Templates after loading with DOX. E: empty template

41. Titania Nanotubes Pore diameter: 125nm Pore thickness: 2.5um

42. Drug Elution from Nanotemplates t<10min, best fit is a power. C(t) ∝ t 1/2 ⇛ Fickian diffusion t>10min, best fit is logarithmic. C(t) ∝ log(t) ⇛ t 1/2 log(t)

43. www.igert.neu.edu A NEW INTERDISCIPLINARY IGERT PH.D. PROGRAM FUNDED BY THE NATIONAL CANCER INSTITUTE AND NATIONAL SCIENCE FOUNDATION $3.3M 2005-2010 PI & Dir: Professor Sri Sridhar Mansoor Amiji, Sanjeev Mukerjee, Mary Jo Ondrechen, Gilda Barabino, Ph.D. A NEW MODEL FOR GRADUATE EDUCATION DRIVEN BY REAL WORLD RESEARCH PROBLEMS

44. Approaches and methods  Reports  Publication s  Presentati ons  Research Integrity  Ethics of Nanomedici ne  Applications of Nanosystems in Medicine  Nanosystems at Biological Interfaces  Scientific Skills, Ethics, and Commercialization  Nanomedical Technology Seminar  Introduction to Nanomedical Technology  Nanosystems Design for Biomedical Applications  Commercialization  Product development  Patent writing  Intellectual property  Cultural diversity  International outlook NP Preparation & functionalization Electromagnetic energy delivery Magnetic Nanoparticles for sensing and bio-control Cellular trafficking and modeling Mitochondrial gene therapy Cellular Bio-sensing Industrial and international internships Multi-disciplinary courses Education and training Communicati on Skills and Professional Conduct Inter-disciplinary research groups Interdisciplinary graduate education, research, and training at the interface of nanotechnology, biotechnology and medicine Vision IGERT Nanomedicine Science and Technology

45. Timeline and Requirements OUTREACH K-12 Coordinated by Claire Duggan (www.ret.neu.edu) HOSPITAL / INDUSTRY INTERNSHIP Co-mentoring by a scientist outside NU Admission to home department Ph.D. in core discipline with specialization in Nanomedicine New Nanomedicine courses (12 Semester Hours) IGERT FUNDING

46. New Nanomedicine courses First of its kind 1.Nanomedicine Science and Technology Seminar (Jan 2006) 2.Intro to Nanomedicine S&T ( started Fall 2006) 3.Nanosystems Design for Biomedical Applications (started Spring 2007) Guest lecturers from Mass Gen, Dana Farber, Beth Israel, Advance Nanotech, …

47. IGERT NANOMEDICINE Bouve College of Health Sciences College of Arts and Sciences College of Engineering Biology Paula Lampton Advisor: Carol Warner Anthony D’Onofrio Advisor: Kim Lewis Sucharita Saha Advisor: Don O’Malley Chemistry Heather Broadkin Advisor: Mary Jo Ondrechen Adam Hendricks Advisor: Robert Hanson Tatyana Chernenko Advisor: Max Diem Electrical and Computer Engineering Yogesh Patel Advisor: Charles DiMarzio Mechanical and Industrial Engineering Robert Camp Advisor: Jeff Ruberti Chemical Engineering Savidra Lucatero Advisor: Rebecca Carrier Pharmaceutical Sciences Lilian van Vlerken Advisor: Mansoor Amiji Mattia Migliore Advisor: Barbara Waszczak Lara Jabr Advisor: Volkmar Weissig Luis Brito Advisor: Mansoor Amiji Padmaja Magadala Advisor: Mansoor Amiji

48. Lilian van Vlerken Modulation of Intracellular Ceramide using Polymeric Nanoparticles to Overcome Multidrug Resistance (MDR) of Breast Cancer Advisor: Dr. Mansoor M. Amiji Mattia M. Migliore Advisor: Dr. Barbara Waszczak Intranasal delivery of GDNF using nanoparticle technology for the treatment of Parkinson’s disease occurance of apoptosis in MDR breast cancer cells, previously resistant to cell death, with experimental therapy Substantia nigra dopamine neurons ( 40x view) of a rat that was administered cationic liposomes loaded with Alexa-488 ovalbumin intranasally and sacrificed after 24hrs IGERT NANOMEDICINE + PHARMA SCI

49. Padmaja Magadala Department: Pharmaceutical Sciences Advisor: Mansoor Amiji "HER2/neu Receptor-Targeted Gelatin-Based Nanovectors for Pancreatic Cancer Gene Therapy" C) Blank Gel NPsUnmodified Gel NPs PEG-Gel NPsPep-PEG-Gel NPs Quantitative and qualitative tranfection efficiency study of control and modified gel-NPs in panc-1 cells by FACS (A), ELISA (B) and fluorescence microscopy (C)

50. Paula Lampton Advisor: Dr.Carol Warner Department of Biology Nanoparticles for analysis of immune system molecules in mouse embryonic stem cells IGERT NANOMEDICINE + BIOLOGY Sucharita Saha Advisor: Donald O'Malley Visualization and Enhancement of Spinal Cord Regeneration using Nanoparticles Application of Nanoarray Technologies to Spinal Stimulation and Regeneration: the Nano-BMI (brain- machine interface) See poster at this meeting

51. IGERT K-12 outreach Research Experiences for Teachers (RET) Young Scholars Program Lesson Development Field Trips/Presentations Science Fair Mentoring and Support

52. www.igert.neu.edu