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Volume 5, Issue 1

Inter-PEN QuarterlyInter-PEN QuarterlyNewsletterNewsletter

Principal Investigators for the new PEN Contracts 2010 - 2015

Principal Investigator of the Administrative Center

Gang Bao Ralph Weissleder Zahi A. Fayad Robert S. Langer Michael J. Welch Karen L. Wooley

Emory UniversityGeorgia Tech

UC - Davis

Mass. Gen. Hosp.Brigham & Women’s Hosp.

Harvard UniversityMass. Instit. Tech.

Mass. Instit. Tech.Brigham & Women’s Hosp.

Columbia UniversityNew York University

Mount Sinai - New York

Broad Institute

Washington University

UT - Southwestern Med Center

Texas A&MUC - Berkeley

UC - Santa Barbara

The goal of NHLBI Programs of Excellence in Nanotechnology is to develop nanotechnology – based tools for the diagnosis and treatment of heart, lung, and blood diseases, and to move the translation of these technologies towards clinical application. The program will bring together multi-disciplinary teams from the biological, physical, and clinical sciences for the focused development and testing of nanoscale devices or devices with nanoscale components, and apply them to cardiovascular, hematopoietic, and pulmonary diseases. The program will also develop investigators with the interdisciplinary skills to apply nanotechnology to heart, lung, and blood disease problems.

Our Mission

NHLBI Program Offi cial

Denis B. Buxton Robert J. GroplerNHLBI Washington University

Administer Multi-Center ProgramRobert J. Gropler, M.D., of Washington University School of Medicine, was selected to serve as the Prinicipal Investigator of the NHLBI-PEN Administrative Center. Dr. Gropler will be responsible for the oversight of the administration, coordination and implementation of the Administrative Center PEN (ACPEN). The ACPEN will produce a comprehensive website for the general public, and a secure site for the Inter-PEN community. The secure site will facilitate information sharing of protocols, reagents, outcomes and products, between the PENs. Finally, the ACPEN will provide an infrastructure that ensures opportunities for collaborations and interdisciplinary interactions. The ACPEN will work with Denis B. Buxton, Ph.D., the NHLBI Program Offi cial, as needed.

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A Word from our Program Coordinator

Congratulations to the four new PEN Contracts! We welcome back three former PENs, and introduce one new PEN. The returning PENs are: Gang Bao’s of The Georgia Institute

of Technology, Emory University, and a new addition to their team: The University of California-Davis; Ralph Weissleder’s PEN with contributions from Massachusetts General Hospital, Brigham and Women’s Hospital, The Broad Institute, Harvard Medical School, and Massachusetts Institute of Technology; Michael J. Welch will lead the Washington University PEN, along with Co-PI, Karen L. Wooley, of Texas A&M University, and continued input from the University of California-Berkeley, University of California-Santa Barbara, and the University of Texas Southwestern Medical Center at Dallas.

We are pleased to welcome the new PEN led by Zahi A. Fayad, of Mount Sinai of New York, and co-lead by Robert S. Langer of the Mas-sachusetts Institute of Technology. Additional members of their team include participants from Brigham and Women’s Hospital, Columbia University, New York University. To learn more about these two new Principal Investigators, see the “Spotlight On” segment on pages 14 and 15.

Robert J. Gropler, of the Washington University School of Medicine, was named the Principal Investigator of the

In this issue . . .

PEN Administrative Center (ACPEN) for the new NHLBI-PEN Contracts. To learn more, see “Introducing” on page 3.

On January 7, 2011, the Principal Investigators from the new PEN Contracts held their fi rst Executive Committee teleconference meet-ing with the NHLBI Program Offi cial, Denis Buxton. These meet-ings will to continue to be held on the fi rst Friday of each quarter.

The date for the upcoming 5th Annual Inter-PEN Meeting (all four PENs) has been set for November 4-5, 2011. The meet-ing will be held at Washington University School of Medicine, in St. Louis, Missouri. For more information, please turn to the back cover of this issue for hotel and meeting room logistics.

We appreciate the National Heart Lung and Blood Institute’s contin-ued support and funding. We look forward to continued collabora-tions among the PENs.

Eileen A. Cler, B.S.Program Coordinator for the NHLBI-PEN Contractsclere@mir.wustl.edu

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PAGE 13

MEET OUR PENRalph WeisslederMass Gen Hosp/Harvard

HOT EMERGINGBREAKTHROUGHSUnpublished data

RESEARCH

PAGE 4

Mark GoodmanEmory University

AWARDSMichael J. WelchDistinguished Investigator PAGE 8

SPOTLIGHT

PAGE 15

Mount Sinai, New YorkPrincipal InvestigatorZahi A. FayadPAGE 14

Mass. Instit. Tech (MIT)Co-Principal InvestigatorRobert S. Langer

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R o b e r t J . G r o p l e r , M . D . , W a s h i n g t o n U n i v e r s i t y S c h o o l o f M e d i c i n e

INTRODUCING ...

Dr. Robert J. Gropler of Washington University School of Medicine was named the Principal Investigator of the PEN Administrative Center. This is a new position created for the 2010-2015 PEN Contracts. Some of the responsibilites of the Administrative Center include:

The coordination of the annual Inter-PEN Meeting, conducting the quarterly Executive Committee meetings with the Program Offi cial and Principal Investigators, production of the NHLBI Inter-PEN Quarterly Inter-PEN Newsletter, developing and maintaining a common website for the four PENs, maintaining and distributing a PEN Personnel Directory, posting a list of PEN-funded publications on the public website, and also providing a secure area where protocols, outcomes, reagents and products created by the four PENs may be shared among the Inter-PEN Community.

Dr. Gropler is the Mallinckrodt Institute of Radiology (MIR) Interim Director of Radiological Sciences. The Division of Radiological Sciences is composed of 6 laboratories (Biomedical MR, Cardiovascular, Molecular, Neuroimaging, Optical and Radiological Chemistry). The division consists of 118 faculty members and 314 staff. Also part of the division responsible are two facilities: the Cyclotron facility which consists of 3 cyclotrons producing both clinical and research products and the Small Animal MR facility. For the past 15 years Dr. Gropler has been the Lab Chief of the Cardiovascular Imaging Laboratory at the Mallinckrodt Institute of Radiology which is currently composed of 25 faculty and staff and is a basic science and translational research laboratory. In addition for the past 4 years, Dr. Gropler has been the Principal Investigator of the program project grant, PO1-HL13581; “Cyclotron Produced Isotopes in Biology and Medicine”. As a result, Dr. Gropler has extensive experience in managing large and diverse research agendas of similar scope to this Administrative Core.

Dr. Gropler will provide oversight of the administration, coordination and implementation of the Administrative Center PEN (ACPEN). Dr. Gropler has been an investigator on the current PEN grant since its inception fi ve years ago. His role in this grant has been to provide cardiologic input into the selection of molecular targets, the design of experiments and the analysis of data. His role in this previous proposal gives him the expertise in nanotechnologies to be Principal Investigator on this Administrative Center.

PRINCIPAL INVESTIGATOR OF ADMINISTRATIVE CENTERPRINCIPAL INVESTIGATOR OF ADMINISTRATIVE CENTER

2011 - April 1, July 1 and October 7

Quarterly Teleconference Calls(Denis Buxton and Principal Investigators only)

Upcoming Executive Committee Meetings

Karen M. Kharasch Eileen A. Cler

Dr. Gropler will be assisted by Karen M. Kharasch, B.S., Business Director, and Eileen A. Cler, B.S., Program Coordinator, both of Washington University School of Medicine. If you have questions regarding the PEN Administrative Center, please contact Eileen A. Cler at clere@mir.wustl.edu.

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C e n t e r f o r T r a n s l a t i o n a l C a r d i o v a s c u l a r N a n o m e d i c i n eGEORGIA TECH, EMORY, & UC-DAVIS

Small Radiolabeled-Molecule Drug Discovery

Mark M. Goodmanmgoodma@emory.edu, gang.bao@bme.gatech.edu

Cardiovascular disease (CVD) is the leading cause of death for both men and women in the United States. In 2006, 831,000 Americans died from CVD. One in fi ve Americans has been

estimated to have some form of cardiovascular disease; the estimated economic cost for all cardiovascular diseases in the United States for 2010 will exceed $500 billion. There is clearly an unmet clinical need in diagnosing and treating atherosclerosis and myocardial infarction (MI). Atherosclerosis is an infl ammatory disease that is character-ized by punctuated exacerbations of the infl ammatory process which creates instability in some vascular atherosclerotic lesions causing them to rupture, often with serious or fatal consequences. The stan-dard clinical approaches rely on the assessment of the hemodynamic restrictions of lesion on coronary blood fl ow. These are typically de-tected by nuclear imaging, angiography or ultrasound. None of these technologies provide insights into the state of infl ammatory activity in the vessel wall. Furthermore, treatments are directed toward modify-ing risk factors including lipid levels, blood pressure levels and lifestyle changes. Arguably, no available therapies are attacking the infl amma-tory process directly. Thus, there is a great unmet need to develop in-novative diagnostic modalities that inform the activity of the infl am-matory disease and to guide evaluation of therapy

The goal of the Radiopharmaceutical Drug Discovery Lab (RDDL), Department of Radiology at Emory University School of Medicine led by Dr. Mark M. Goodman, is to de-velop radiopharmaceuticals for the study and management of treatment of cardiovascular disease, cancer, cocaine addiction, depression, anxiety, suicide, dementia and psychomotor dis-orders. To accomplish this goal, the RDDL is focused on PET and SPECT radiotracer development of imaging agents with special emphasis on fl uorine-18, carbon-11, iodine-123 and Cu-64. The RDDL developed and translated [123I]BMIPP, [18F]FACBC, [18F]FACPC, [18F]FECNT, [123I]MMG-142/IPT, [11C]HOMADAM, and [123I]mZIENT from the bench to bed-side for, heart disorders, cancer, Parkinson’s Disease, cocaine addiction and mood disorders, respectively. Hallmarks of the RDDL are the methylbranched fatty acid [

123I] BMIPP which

has been commercially introduced in Japan as Cardiodine for monitoring fatty acid metabolism in heart disorders and the translation of the fi rst reported synthetic amino alicyclic acid radiolabeled with the PET radioelement fl uorine-18, anti-3-fl uoro-1-aminocyclobutane-1-carboxylic acid ([

18F]FACBC ),

for imaging both intracranial tumors and prostate cancer in patients. To carry out the projects in the GaTech-Emory-UC Davis PEN, RDDL in collaboration with the laboratories of Dr.

Gang Bao and Dr. Niren Murthy will develop PET labeled MR nanoparticles and Hoechst-based agents (DNA binding dyes) to visualize vulnerable plaques.

The major distinguishing features of novel imaging tech-nologies developed by the RDDL are the combined use of [18F] label-ing, non-metabolized amino acids, and PET and SPECT imaging. The radiolabel [18F] provides signifi cant logistical and economic advan-tages over [11C] (t1/2=20 min.). The longer half-life of [18F] (t1/2=110 min.) allows off-site distribution and multiple doses from a single of radiotracer. The use of non-metabolized amino acids provides the opportunity for wider application as an imaging agent for certain systemic solid tumors that do not image well with [18F]FDG PET. These non-metabolized amino acids move across tumor capillaries by carrier-mediated transport involving either the “L” large-neutral amino acid or “A” alanine amino acid transport systems to image brain and systemic tumors in vivo based upon elevated expression of amino acid transporters in tumors compared to normal tissue. Fi-nally, the “functional” imaging capabilities of PET and SPECT have specifi c advantages over CT and/or MRI imaging in particular clinical situations. While CT and MRI provide exquisite anatomical and mor-phologic detail, they do not always provide information that yields a defi nitive diagnosis. PET and SPECT are alternative imaging modali-ties that complement CT and MRI by providing the opportunity to assess physiological and biochemical activity in normal tissues, and to directly compare functional activity in diseased tissue.

Figure 1. PET and MR images of a patient with glioblastoma multiforme. Contrast-enhanced T1-weighted MR images taken before biopsy show a lesion in the left frontal lobe (top row). [18F]2-FDG PET images taken 8 wks after biopsy (middle row). PET images using [18F]FACBC taken 1 week after surgery show residual tumor with a suggestion of midline invasion along the corpus callosum (bottom row). Shoup, T. M.; Olson, J.; Hoff man, J. M.; Votaw, J.; Eshima, D.; Eshima, L.; Camp, V. M.; Stabin, M.; Votaw, D.; Goodman, M. M. (1999). Synthesis and Evaluation of [18F]1-amino-fl uorocyclobutane-1-carboxylic Acid to Image Brain Tumors. J. Nucl. Med., 40, 331-338.

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About the Author

PEN

Mark M. Goodman, Ph.D.Endowed Chair in Imaging Sciences

Director, PET Core Facility & PET ChemistryDirector, Radiopharmaceutical Discovery Lab

Professor of Radiology, Psychiatry, Hematology & Oncology

Emory University School of Medicine

The RDDL has multiple ongoing imaging projects involving the novel [18F] radiolabeled amino acid technology, with the goal of translating this basic imaging research to patients in the clinic. A re-cent and exciting success story involves the translation of [18F]FACBC from the lab into a diagnostic tool for the management and treatment of brain and prostate cancer. The RDDL began development of the novel, [18F]-labeled cyclobutyl nonmetabolized amino acid radiotracer technology in 1994. A patent was submitted on this technology in 1995, and the fi rst in human study on a glioblastoma multiforme patient was published in 1999 (Figure 1), demonstrating a 6-fold enhancement of radiolabel uptake in the tumor, compared to normal brain tissue. In addition, the technology was commercially licensed which provided funding to study anti-[18F]FACBC in cohorts of glioma (n=10) and prostate (n=15) cancer patients. In 2006, twelve years following the initial concept, Dr. Goodman and colleagues were awarded a DCIDE application for toxicity testing, followed by an IND. In 2007, the RDDL reported the fi rst use of synthetic amino alicyclic acid radiolabeled with the PET radioelement fl uorine-18, anti-3-fl uoro-1-aminocyclobu-tane-1-carboxylic acid ([18F]FACBC), for imaging prostate cancer in patients.

“This project has been funded in whole or in part with Federal funds from the National Heart, Lung, and Blood Institute, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN26820100004xC”.

Acknowledgement Text

Did you know?

for the new PEN Contracts is:

“The goal of the Radiophar-maceutical Drug Discovery Lab ... is to develop radiophar-maceuticals for the study and management of treatment of cardiovascular disease, cancer, cocaine addiction, depression, anxiety, suicide, dementia and psychomotor disorders. “

With “x” being your PEN’s unique number

SKILLSDevelopment

As part of the MSSM-MIT Skills Development Core, Drs. Kevin D. Costa, Willem Mulder, and David Cormode have created a new course entitled “Foundations of Nanomedicine”. This course will be offered for the fi rst time in Spring 2011 as a web-accessible graduate course at Mount Sinai. The course will cover the synthesis and characterization of multifunctional nanoparticles, nanomaterial applications in imaging, use of innovative tissue engineering platforms, and development of nanomaterials as novel therapeutics. Dr. Martin Schwarz at MSSM is Director of the Skills Development Core.

SEMINARA collaboration among PENs

Niren Murthy, Ph.D., Senior Investigator on the Georgia Institute of Technology PEN, gave a presentation on “New Materials for Drug Delivery and Molecular Imaging” at the Washington University School of Medicine, in St. Louis, MO. Fifty-eight people attended the meeting locally, while faculty, students and postdocs from TAMU, UCB, UCSB and UTSW connected via Cisco WebEx conferencing software.

March 3, 2011

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M G H , B W H , B I , H A R V A R D , M I TT r a n s l a t i o n a l P r o g r a m o f E x c e l l e n c e i n N a n o t e c h n o l o g y

Detection of Macrophages in Aortic Aneurysms by Nanoparticle Positron

Emission Tomography Computed Tomography1

Jason R. McCarthy and Ralph Weisslederweissleder@helix.mgh.harvard.edu,jason_mccarthy@hms.harvard.edu

Aortic aneurysms (AA) occur in up to 5% of the elderly population, with 50% of the larger aneurysms rupturing, resulting in signifi cant mortality.2 Patients with aneurysms

undergo routine anatomic imaging in order to monitor size and progression of the disease, with surgery being one option to preempt rupture. One of the main issues with this strategy is that risk assessment using current imaging strategies is prone to failure, as smaller aneurysms may rupture between imaging sessions, while some large lesions that undergo surgical intervention may have been relatively stable. Thus, there is a need to develop strategies for more accurate risk prediction.

Recent evidence points to the role of innate immune cells, namely the monocyte and macrophage, in the progression of AA. These cells infi ltrate the vessel wall and degrade the extracellular matrix via the action of proteases, such as matrix metalloproteinases (MMPs). These immune cells also release cytokines, further promoting the infl ammatory process. Thus, Nahrendorf at al. hypothesized that nanoparticles targeted to macrophages could be used to detect infl ammation in AAs.1 To accomplish this, the authors synthesized 18F-labeled magnetofl uorescent crosslinked dextran coated iron oxide nanoparticles (CLIO), which has previously shown applicability in the

imaging of infl ammatory macrophages in a number of other diseases.

In order to induce AA in mice, the researchers utilized a model based upon atherosclerotic apolipoprotein E defi cient mice on high cholesterol diet. The mice were further implanted subcutaneously with minipumps for the administration of angiotensin II (AT-II) for 7 or 28 days, depending

upon the cohort. Additionally, one cohort received a splenectomy, as the splenic monocyte reservoir are released into the blood pool on AT-II administration, and can contribute up to 50% of the myeloid cells in infl amed tissue. Initial micro-CT imaging allowed the researchers to examine incidence, location, and dimension of AAs, as compared to wild-type control mice, with aneurysms commonly observed in the ascending aorta and in the abdominal aorta. Importantly, 6 mice from the cohort that received 28-days of AT-II died, with autopsy confi rming aneurysm rupture.

PET-CT imaging was next utilized to image nanoagent accumulation in AA (Figure 1). As compared to atherosclerotic apoE-/- mice not receiving AT-II or wild type control mice, the AT-II treated mice demonstrated 2 and 3-fold higher PET signal from the aneurysms, respectively. These fi nding were further validated ex vivousing scintillation counting and autoradiography, which additionally

Figure 1. PET-CT imaging in mice with AAs. A, Representative PET-CT

images. Yellow arrows and dotted lines outline the aneurysmatic aorta. MIP indicates maximum intensity projection. * indicates liver signal. B, In vivo PET signal derived from the aneurysmatic vessel wall at the site of the aneurysm (left) wild-type controls (middle), and apoE-/- mice that did not receive AT-II (right bar). Data are presented as mean ± SEM, *P<0.01. C, A correlation of the PET signal with aortic diameter (measured by CT) after AT-II delivery in apoE -/- mice.

“One of the main issues with this strategy is that risk assessment using current imaging strategies is prone to failure, as smaller aneurysms may rupture between imaging sessions, while some large lesions that undergo surgical intervention may have been relatively stable. “

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Jason R. McCarthy, Ph.D. Senior Investigator

Center for Systems BiologyMassachusetts General Hospital

Harvard Medical School

Ralph Weissleder, M.D., Ph.D. Principal Investigator

Director, Center for Systems BiologyMassachusetts General Hospital

Professor of Radiology and Systems BiologyHarvard Medical School

PEN

About the Authors

demonstrated co-localization via fl uorescence refl ectance imaging. Cell specifi c uptake of the nanoagent was examined by microscopy and fl ow cytometry. Comparison of fl uorescence microscopy images of the fl uorescent nanoparticles with immunohistochemical staining for macrophages (MAC-3) in adjacent aortic section revealed that the agent accumulated co-localized with macrophage rich regions of the aneurysm. For fl ow cytometry, aneurysmic sections of the aorta were digested and labeled with a cocktail of antibodies to distinguish the relevant cell populations, and identify those that contain the fl uorescently labeled nanoparticle. While there was minimal uptake of the agent by neutrophils and lymphocytes, this experiment revealed that greater than 90% of the PET signal in aneurysms can be attributed to macrophages.

As the authors overall goal was the development of an imaging strategy that could detect infl ammatory aneurysms before they rupture, they investigated the ability to be able to detect infl ammation in younger, evolving aneurysms. To do this they treated the mice with AT-II for only 7 days and imaged nanoparticle localization. They found that, even at the earlier time point, the PET signal was already signifi cantly elevated, which correlated well with the number of monocytes and macrophages, as determined by fl ow cytometry. They next investigated the blunting of the infl ammatory process by removing the spleen. As splenic monocytes give rise to macrophages, the researchers hypothesized that removing the spleen would decrease

Would you like to learn more about the four PEN contracts?

Visit our website at www.nhlbi-pen.net to read about:

• Aims of each PEN Contract (2010-2015)• Research slides from past Inter-PEN meetings• Past newsletter issues• Seminar Series• Initial PEN Grant information (2005-2010)

Figure 2. Histology from an aneurysmatic aorta after 28 days of

AT-II administration. A, A fl uorescence image showing nanoparticle accumulation in the aneurysm wall (arrows). B, Hematoxylin/eosin (H&E) histology. C and H, Immunohistochemistry indicating the presence of macrophages (arrows). D, E, and I, Fluorescence images depicting nanoparticle distribution (arrows) in an adjacent section. Although there was some accumulation in macrophages in luminal atherosclerotic plaque, robust signal was located in adventitial macrophages next to degraded media (asterisks in H to J). F, G, and J, Autofl uorescence images of the aorta.

the number of macrophages in the aneurysm, thereby decreasing the PET signal. As expected, the PET signal decreased by 3-4 fold.

In this study, the authors conclude that the targeting of macrophages with imaging may offer early insight into the course of AA, as an approach that targets a key component of the biology underlying aneurysm rupture may help to more accurately determine individual risk as well as population-based risk factors.

References1. Nahrendorf, M.; Keliher, E.; Marinelli, B.; et al. “Detection of

Macrophages in Aortic Aneurysms by Nanoparticle Positron Emission Tomography-Computed Tomography”, Arterioscler Thromb Vasc Biol, 2011.

2. Hirsch, A.T.; Haskal, Z.J.; Hertzer, N.R.; et al. “ACC/AHA 2005 Practice Guidelines for the management of patients with peripheral arterial disease.” Circulation, 2006, 113, e463-654.

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Recognition (honors, appointments, awards) received by members of the four PENsAWARDS

MGH, BWH, Broad, Harvard, MIT

WUSTL, TAMU, UCB, UCSB, UTSWBy Eileen A. Cler, B.S.

Emory, Georgia Tech, UCD

Jason R. McCarthy, Ph.D., was promoted to Assistant Professor in Radiology at Harvard Medical School and Massachusetts General Hospital.

Craig J. Hawker, Ph.D. of the University of California, Santa Barbara, was elected a Fellow of the Royal Society (2010), received the Macro Group UK International Medal for Outstanding Achievement (2010) and received the Arthur C. Cope Scholar, from the American Chemical Society (2011).

Yongjian Liu, Ph.D. won the 3rd place at the Young Investigator at North American So-ciety For Cardiovascular Imaging (NASCI) Meeting in October 2010.

Michael J. Welch, Ph.D. received the Dis-tinguished Investigator Award from Wash-ington University School of Medicine.

Michael E. Davis, Ph.D., was elected a Fellow of the American Heart Association at the AHA.

Gang Bao, Ph.D., was recently named the Director of Nanomedicine Research Institute at Georgia Tech.

Shuming Nie, Ph.D., was honored at the Emory University Annual MilliPub Club Reception in October 2010, for his scholarly publications that have each received more than 1000 citations. Dr. Nie has published 4 original research papers that have each

been cited over 1000 times. Overall, Dr. Nie’s work has received more than 16,000 citations, making him one of the most cited authors at Emory and Georgia Tech.

Don P. Giddens, Ph.D., was elected a Fellow in American Association for the Advance-ment of Science (AAAS); the incoming pres-ident of the American Society for Engineer-ing Education; also the incoming chair of the Bioengineering Section of the National Academy of Engineering (NAE).

Hakho Lee, Ph.D., was promoted to Assistant Professor in Radiology at Harvard Medical School and Massachusetts General Hospital.

Mt. Sinai, MIT, BWH, Columbia, NYU

Dr. Giddens was also elected a Fellow in American Association for the Advancement of Science (AAAS), and he will be a Distin-guished Visiting Fellow at Imperial College, London, in Septem-ber – October 2011.

David Cormode, Ph.D., a Post-Doctoral Fellow at the Mount Sinai School of Medi-cine published an article “Atherosclerotic Plaque Composition: Analysis with Multi-color CT and Targeted Gold Nanoparticles” that was named the 2010 Philips CT NetFo-rum Publication of the Year.

Kevin D. Costa, Ph.D., of MSSM was recently awarded an NIH S10 Shared Instrument Grant entitled “Integrated AFM and Real-time Confocal Microscope Core” to establish a state-of-the-art atomic force microscope facility for nano-scale biomedical research applications.

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Emerging Breakthroughs

Meet our PEN

Principal Investigator Program Offi cialRalph Weissleder Denis Buxton

Brigham and Women’s HospitalBroad Institute

Harvard University School of MedicineMassachusetts General Hospital

Massachusetts Institute of Technology

Scott A.

Hakho Lee

Matthias

RalphJason R.

Center for Systems Biology

Stanley ShawPeter Libby

Investigators and PostdocsInvestigators and Postdocs

Mikael J.

T r a n s l a t i o n a l P r o g r a m o f E x c e l l e n c e i n N a n o t e c h n o l o g y

Monty Liong

Carlos Tassa

Virna

Yali Li Arezou

Edmund

Thibaut Quillard Koichi Shimizu

Guobin Shan

Jae Hoon Chung

S. Sibel ErdemMcCarthy

NahrendorfHilderbrand

Weissleder Ghazani

Pittet Cortez-Retamozo

Keliher

By Eileen A. Cler, B.S.

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WUSTL, TAMU, UCB, UCSB , & UTSWI n t e g r a t e d N a n o s y s t e m s f o r D i a g n o s i s a n d T h e r a p y

Multi-functional Trackable Dendritic Scaffolds and Delivery Agents

Roey J. Amir, Lorenzo Albertazzi, Jenny Willis, Anzar Khan, Taegon Kang and Craig J. Hawker*

hawker@mrl.ucsb.edu, welchm@mir.wustl.edu

Dendrimers are very attractive candidates for biological ap-plications as carriers for diagnostic probes and/or drugs due to their globular shape, tunable structure, mono-

dispersity and plurality of functional end groups.[1] Up to date, a number of architectures and related structures aimed at design-ing dendritic nano-capsules that can carry bioactive molecules to their target cells or tissue have been reported in the literature.[2]

The two major approaches of loading dendritic carriers with drug or dye molecules are: encapsulation[3] and loading of the dendrimer surface.[4] While the encapsulation of drugs or dyes inside the in-ner cavities of the dendrimer seems to be very promising, in most cases few guest molecules were encapsulated even with dendrimers of high generations. Moreover, the non-covalent nature of encap-sulation makes it challenging to control the stability of the loaded carrier and the release of the payload. An alternative approach is to use the multiple end groups at the dendrimer surface to carry the cargo molecules. However, loading large amounts of hydropho-bic drugs or dyes alters the dendrimer surface properties and may decrease its solubility and biocompatibility. Partial loading of the surface groups offers a solution to this problem, but it results in loss of control over the exact amount of loaded molecules and in many cases only low amounts can be loaded without signifi cantly changing the surface properties.

In order to maintain both high loading and mono-dis-persity, an alternative design is to covalently attach the cargo mol-ecules to the interior of the dendrimer. This strategy overcomes the challenges associated with surface functionalization, allowing high and reproducible loading without signifi cantly altering the surface prop-erties of the dendritic scaffold. To illus-trate the power of this novel strategy, we developed an accelerated synthesis of orthogonal surface and internally func-tionalized dendrimers and used them as multi-functional dendritic scaffolds (Figure 1). As model delivery and di-agnostic units, two different dyes were conjugated to the dendrimer: multiple coumarin units (blue in fi gure) were loaded internally through a cleavable

linker and a single Alexa647 dye (red in Figure 1) was conjugated to the surface through a stable amide bond. This dual labelling allows the dendritic scaffold and the model payload to be individually tracked in living cells at the same time. An additional facet of the platform design is the pres-ence of numerous protonated amino groups at the chain ends of the dendritic fragments (yellow in Figure 1), which are designed to induce cell internalization through endocytosis and while cat-ionic materials may be toxic, they serve as a useful model system. Once inside the cell, hydrolytic enzymes would cleave the internal coumarin units from the scaffold, resulting in release of the model coumarin payload and appearance of fl uorescence.

We used an alternating sequence of ‘amine-epoxy’ and ‘thiol-yne’ coupling reactions to synthesize a fourth generation dendrimer (Figure 2a) in only four steps as both reactions allowed the formation of a new generation at each synthetic step (an AB2/CD2 approach). The ‘amine-epoxy’ reactions yielded orthogonal hydroxyl groups that would be used to bind the cargo molecules. To serve as a soluble support and to the enhance water solubil-ity of the dendritic carrier, a 10kD bis-amine polyethylene glycol (PEG) linear polymer was used as a core. The fl uorescence spec-tra of free and dendrimer-bonded coumarin showed that the dyes are quenched inside the dendritic carrier (Figure 2b), due to the high local concentration and proximity of dyes in the interior of the dendrimer. To demonstrate that the covalently attached dyes inside the dendrimer are accessible to enzymes, we incubated the loaded dendrimers with Porcine Liver Esterase (PLE). The increase of fl uorescence, observed only in the presence of PLE (Figure 2c), implies that PLE can access and cleave the dyes, while no release is observed for the control solutions. The dequenching of the couma-rin dye after release is of crucial importance for the cell studies as it allows highlighting the kinetic and the localization of the cleavage of the ester bond inside living cells.

Figure 1: Schematic representation of the dendritic carrier. Upon enzymatic cleavage, the covalently attached “blue” dyes are release from the carrier. The non-cleavable “red” dye allows monitoring the dendrimer itself.

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Having demonstrated the increase in fl uorescence on pay-load release, a series of model compounds were prepared for in vi-tro experiments and to examine the internalization of these hybrid structures into living cells. For tracking of the dendritic scaffold, an amino functionalized 4th generation dendrimer was conjugated with an average of one FITC dye to yield a labelled 4th genera-tion dendrimer ([G-4]-FITC). Incubation of the labeled dendrimer with living B16 mouse melanoma cells showed membrane binding and internalization (Figure 3a). Following these results, Alexa647 was coupled with the dendrimer to yield the labelled derivative, [G-4]-(coumarin)20-Alexa, having an average of one Alexa dye at the chain ends, which allows monitoring of the dendritic scaffold, and ~20 coumarin units (corresponds to ~ 25 wt%) attached to the internal dendrimer framework. Confocal microscopy on living B16 mouse melanoma cells revealed that upon membrane binding and internalization of our carrier intracellular enzymes cleaved the dye-dendrimer bonds releasing the payload (coumarin) in the cyto-plasm (Figure 3b). To the best of our knowledge this is the fi rst time that a dendritic carrier and the release of its payload are monitored

Figure 2: a) Schematic representation of the dendritic carrier; (b) Fluorescence spectra of free coumarin and [G-4]-(Coumarin)20; (c) Fluorescence intensity of [G-4]-(coumarin)20 at 480 nm as a function of time in the presence of esterase at pH 7.4 and in the absence of esterase at pH = 5.0 and 7.4.

simultaneously inside living cells. This data is critical for the design of dendritic carriers for the delivery of diagnostic probes and drugs, as it sheds light on the intracellular fate of the loaded molecules and the corresponding carrier.

References1. Tomalia, D.A., Reyna, L.A.; Svenson, S. “Dendrimers as multi-

purpose nanodevices for oncology drug delivery and diagnos-tic imaging”, Biochem. Soc. T., 2007, 35, 61-67.

2. Lee, C.C.; MacKay, J.A.; Fréchet, J.M.J.; Szoka, F.C. “Design-ing dendrimers for biological applications”, Nat. Biotechnol., 2005, 23, 1517-1526.

3. D’Emanuele, A. ; Attwood, D. “Dendrimer-drug interactions”, Adv. Drug Deliv. Rev., 2005, 57, 2147-2162.

4. Almutairi, A. ; Guillaudeu, S.; Berezin, M.; Achilefu, S.; Fréchet, J.M.J. “Biodegradable pH-Sensing Dendritic Nano-probes for Near-Infrared Fluorescence Lifetime and Intensity Imaging”, J. Am. Chem. Soc., 2008, 130, 444-445.

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Craig J. Hawker, Ph.D.Senior Investigator

Director, Materials Research LaboratoryDepartment of Chemistry

University of California, Santa Barbara

Roey J. Amir, Ph.D.Postdoctoral Research Associate

Materials Research LaboratoryUniversity of California, Santa Barbara

Lorenzo AlbertazziNEST, Ph.D. Candidate

NEST, Scuola Normale Superiore and CNR-INFM and IIT@NEST, Center for

Nanotechnology Innovation, Pisa, ItalyFormer visiting student at the

Materials Research Laboratory, UCSB

Taegon KangPh.D. Candidate

Materials Research LaboratoryUniversity of California, Santa Barbara

Anzar Khan, Ph.D.FormeSenior scientist

Department of Materials, Institute of Polymers ETH-Zurich

Zurich, Switzerland

Jenny WillisMiddle School Science Teacher, Ventura, CA

Former visiting teacher as part of the Research Experience for Teachers Program

Materials Research Laboratory, UCSB

About the Authors

Figure 3: Subcellular confocal images of B-16 cells treated with (a) dendrimer [G4]-FITC (green) and (b) dendrimer G4-(coumarin)20-Alexa (coumarin – blue and Alexa – red) at 8 hours.

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Exciting new ideas and/or research in the fi eld of nanotechnology on unpublished data

Late breaking news in the fi eld of Nanotechnology

Dr. Michael E. Davis’ laboratory at Emory University School of Medicine has recently developed modifi ed nanoparticles that signifi cantly increase uptake by non-phagocytic cells. Using carbohydrate chemistry, we are able to demonstrate improved delivery of agents to cardiomyocytes and cardiac progenitor cells. This may improve cardiac function by directly introducing factors that enhance contractile function and survival directly to myocytes, as well as allowing complements to existing cell transplantation strategies. Current work in the laboratory is moving toward cell-specifi c targeting and temporal control of drug delivery including intravenous infarct targeting of compounds Gang Bao

Ralph Weissleder

Karen L. Wooley

By Eileen A. Cler, B.S.

Dr. Peter Libby of Brigham and Women’s Hospital and Dr. Jason R. McCarthy of Massachusetts General Hospital have developed stimuli-actuated delivery vehicles for the prevention of transplant rejection. These nanoscaffolds respond to proteases present in the rejecting heart, liberating an immunosuppressive drug. In vitro and in vivo examina-tion of nanoagent effi cacy are currently underway.

Jason R. McCarthy

PeterLibby

Michael E. Davis

Dr. James C. Sacchettini’s group at Texas A&M University has performed HTS to identify compounds that re-sensitize resistant Pseudomonas aeruginosa to the aminoglycoside antibiotic, tobramycin. Nanoparticles will be dual-loaded with tobramycin and the presumed inhibitor of aminoglycoside resistance determinants to test for effi cacy against tobramycin resistant strains. Dr. Carolyn L. Cannon’s group at the University of Texas, Southwestern Medical Center at Dallas, has completed transcriptional profi ling of silver-treated P. aeruginosa to identify silver-

Carolyn L. Cannon

James C. Sacchettini

Michael J. Welch

Robert S. Langer

Zahi A. Fayad

Dr. Ira Tabas’ laboratory at Columbia University College of Physicians and Surgeons has conducted a pilot experiment using LXR activator-containing NPs to address the hypothesis that LXR activation of macrophages that have undergone proteolytic clevage of the efferocytosis receptor MerTK would show faster recovery of the receptor due to enhancement of receptor induction by LXR. The preliminary data support this hypothesis, pending further testing with a series of further optimized LXR activator-containing and control NPs. Under the direction of Dr. Omid C. Farokhzad, the team at Brigham and Women’s Hospital has successfully developed PLGA-PEG nanoparticles encapsulating the LXR agonist (GW3965) and IL-10 protein. For the LXR encapsulated nanoparticles we obtained good encapsulation effi ciencies, drug loading and particle sizes of below 150 nm. We are currently optimizing various parameters for IL-10 encapsulating nanoparticles and also integrating microfl uidic systems into the project to improve particle size and encapsulation effi ciency.

Ira Tabas

Hot Upcoming TopicsHot Upcoming Topics

Omir C. Farokhzad

Emerging BreakthroughsEmerging Breakthroughs

resistance determinants. The Sacchettini group will perform HTS of P. aeruginosa strains that over-express the identifi ed resistance determinants, grown in the presence of silver, to fi nd compounds that will sensitize the bacteria to killing by nanoparticles co-loaded with silver and the “hits,” presumed inhibitors of silver resistance determinants.

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P E N S P O T L I G H T O N :

Zahi S. Fayad, Ph.D., FAHA, FACC, serves as professor of Radiology and Medicine (Cardiology) at the Mount Sinai School of Medicine. Dr. Fayad trained at the Johns Hopkins University and at the University of Pennsylvania. From 1996 to 1997 he was junior faculty in the Department of Radiology

and the University of Pennsylvania. In 1997 he joined the faculty at Mount Sinai School of Medicine.

Dr. Fayad is the Interim Director of the Translational and Molecular Imaging Institute; Director and Founder of the Eva and Morris Feld Imaging Science Laboratories; and Director of Cardiovascular Imaging Research at the Mount Sinai School of Medicine and Mount Sinai Medical Center. He is also the Principal Investigator of the Imaging Core of the Mount Sinai National Institute of Health (NIH)/Clinical and Translational Science Awards (CTSA).

Dr. Fayad will participate in the teaching, outreach and training as part of the Skills Development Program, and also work closely with Dr. Martin Schwarz on the Skills Development program organization and implementation. He has made many contributions to the clinical and preclinical research cardiovascular sciences (MRI, multimodality imaging, and molecular imaging) through his seminal work on atherosclerosis imaging. Dr. Fayad is one of the world leaders in the development and use of multimodality cardiovascular imaging including, Cardiovascular Magnetic Resonance (MR), computed tomography (CT), and positron emission tomography (PET), as well as molecular imaging and nanomedicine to study and treat cardiovascular disease. His focus in the past 12 years at Mount Sinai has been on the noninvasive assessment and understanding of atherosclerosis (Sanz and Fayad Nature 2008; 451:953-957). He holds 8 US and Worldwide patents in the fi eld of imaging. He has authored more than 250 peer-reviewed publications, 50 book chapters, and over 400 meeting presentations. He is currently the principal investigator of four federal grants funded by the NIH’s National Heart, Lung and Blood Institute (NHLBI) and National Institute of Biomedical Imaging and Bioengineering (NIBIB).

Dr. Fayad is past-deputy Editor of Magnetic Resonance in Medicine (MRM), past-president of the Society of Atherosclerosis and Prevention (SAIP), fellow of the American Heart Association (AHA) where he served on the National Research Committee and on the Council on Cardiovascular Radiology and Intervention (CVRI). He is also a fellow of the American College of Cardiology (ACC), where he served on the Cardiovascular Collaborative Imaging (CCI) Committee. He is a member of the NIH’s NHLBI Cardiovascular Strategic Planning Working Group on Vascular Disease and Hypertension. He is a member of the Foundation of the NIH (FNIH) Biomarkers Consortium.

Dr. Fayad is on the editorial boards of Arteriosclerosis Thrombosis and Vascular Biology (ATVB), Journal of Cardiovascular Magnetic Resonance (JCMR), Nature Reviews Cardiology, Atherosclerosis, and Journal of the American College of Cardiology Imaging (JACC Imaging). He often serves as guest editor for several journals in the fi elds of imaging, vascular biology, cardiology and radiology. He participates regularly to the AHA/ACC writing groups. He is a member of the Medical Imaging (MEDI) study section and ad-hoc member on numerous other study sections including those from NIH and the National Academy of Science. He is a member of the New York University Program in Computational Biology. He is a past member of the board of trustees of the Society of Cardiovascular Magnetic Resonance (SCMR) and he is past member of the Scientifi c Program Committee of the International Society of Magnetic Resonance in Medicine (ISMRM). He also serves on the boards of several national and international scientifi c boards, committees and foundations.

In 2007 he was given the John Paul II Medal from Krakow, Poland, in recognition for the potential of his work on humankind. As a teacher and mentor, Dr. Fayad has trained over 30 postdoctoral fellows, clinical fellows and students. His trainees have received major awards, fellowships, and positions in academia and industry. In 2008 he received the Outstanding Teacher Award from the International Society of Magnetic Resonance in Medicine (ISMRM) for his teaching on cardiovascular imaging and molecular imaging. Recently, in 2009 he was awarded the title of Honorary Professor in Nanomedicine at Aarhus University in Denmark.

Z a h i A . F a y a d , P h . D . , P r i n c i p a l I n v e s t i g a t o r , M t . S i n a i

The “Spotlight” rotates between the PENs. If someone on your PEN has made a signifi cant contriubtion to your PEN’s success and you would like to recognize their work, please send a brief biography to Eileen A. Cler at clere@mir.wustl.edu.

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p

W E L C O M E N E W P I sR o b e r t S . L a n g e r , S c . D . , C o - P r i n c i p a l I n v e s t i g a t o r , M I T

Robert S. Langer, Sc.D., is the David H. Koch Institute Professor (there are 14 Institute Professors at Massachusetts Institute of Technology). He received his Bachelor’s Degree from Cornell University in 1970 and his Sc.D. from the Massachusetts Institute of Technology in 1974, both in Chemical

Engineering.

Dr. Langer has written approximately 1,050 articles. He also has approximately 750 issued and pending patents worldwide. Dr. Langer’s patents have been licensed or sublicensed to over 220 pharmaceutical, chemical, biotechnology and medical device companies. He is the most cited engineer in history. He served as a member of the United States Food and Drug Administration’s SCIENCE Board, the FDA’s highest advisory board, from 1995 – 2002 and as its Chairman from 1999-2002.

Dr. Langer has received over 170 major awards including the 2006 United States National Medal of Science; the Charles Stark Draper Prize, considered the equivalent of the Nobel Prize for engineers and the 2008 Millennium Prize, the world’s largest technology prize. He is the also the only engineer to receive the Gairdner Foundation International Award; 72 recipients of this award have subsequently received a Nobel Prize. Among numerous other awards Langer has received are the Dickson Prize for Science (2002), Heinz Award for Technology, Economy and Employment (2003), the Harvey Prize (2003), the John Fritz Award (2003) (given previously to inventors such as Thomas Edison and Orville Wright), the General Motors Kettering Prize for Cancer Research (2004), the Dan David Prize in Materials Science (2005), the Albany Medical Center Prize in Medicine and Biomedical Research (2005), the largest prize in the U.S. for medical research, induction into the National Inventors Hall of Fame (2006), the Max Planck Research Award (2008) and the Prince of Asturias Award for Technical and Scientifi c Research (2008). In 1998, he received the Lemelson-MIT prize, the world’s largest prize for invention for being “one of history’s most prolifi c inventors in medicine.” In 1989 Dr. Langer was elected to the Institute of Medicine of the National Academy of Sciences, and in 1992 he was elected to both the National Academy of Engineering and to the National Academy of Sciences. He is one of very few people ever elected to all three United States National Academies and the youngest in history (at age 43) to receive this distinction. Forbes Magazine (1999) and Bio World (1990) have named Dr. Langer as one of the 25 most important individuals in biotechnology in the world. Discover Magazine (2002) named him as one of the 20 most important people in this area. Forbes Magazine (2002) selected Dr. Langer as one of the 15 innovators world-wide who will reinvent our future. Time Magazine and CNN (2001) named Dr. Langer as one of the 100 most important people in America and one of the 18 top people in science or medicine in America (America’s Best). Parade Magazine (2004) selected Dr. Langer as one of 6 “Heroes whose research may save your life.” Dr. Langer has received honorary doctorates from Harvard University, the Mt. Sinai School of Medicine, Yale University, the ETH (Switzerland), the Technion (Israel), the Hebrew University of Jerusalem (Israel), the Universite Catholique de Louvain (Belgium), the University of Liverpool (England), the University of Nottingham (England), Albany Medical College, the Pennsylvania State University, Northwestern University, Uppsala University (Sweden) and the University of California, San Francisco Medal.

Inna Gurewitz, M.P.H.Administrator

Mt. Sinai/MIT/BWH/Columbia/NYU PENDepartment of Radiology

Mount Sinai School of Medicineinna.gurewitz@mssm.edu

PEN

“Spotlights” compiled by

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N o v e m b e r 4 - 5 , 2 0 1 1 - W a s h i n g t o n U n i v e r s i t y S c h o o l o f M e d i c i n e

5th Annual Inter-PEN Meeting

NHLBI Inter-PEN Quarterlyhttp://www.nhlbi-pen.net/default.php?pag=news

Program OfficialDenis B. Buxton, Ph.D.National Heart, Lung, and Blood InstituteEmail: buxtond@nhlbi.nih.govhttp://www.nhlbi.nih.gov

Principal Investigator of the PENAdministrative CenterRobert J. Gropler, M.D.Principal Investigator of Administrative CenterWashington University School of MedicineDepartment of Radiology

Production, Design, Editor, PhotographerEileen A. Cler, B.S.Program Coordinator for NHLBI-PEN ContractsWashington University School of MedicineEmail: clere@mir.wustl.edu

Eileen A. Cler, B.S.Program Coordinator for the PEN ContractsWashington University School of MedicineDepartment of Radiology(314) 747-0386clere@mir.wustl.edu

The meeting will be held at:

Washington University School of Medicine Farrell Learning and Teaching Center Connor Auditorium 520 S. Euclid Avenue St. Louis, MO 63110

Hotel accommodations will be at:

Parkway Hotel 4550 Forest Park Avenue St. Louis, Missouri 63108 (314) 256-7700 http://www.theparkwayhotel.com/

Principal Investigator, Michael J. Welch, and Co-Principal Investigator, Karen L. Wooley, will host the 5th Annual Inter-PEN meeting on Novem-ber 4-5, 2011, at Washington University School of Medicine. This will be

the fi rst Annual Inter-PEN Meeting for the new PEN Contracts. All PEN partici-pants are welcome to attend.

The hotel is less than one block from the meeting location. Both the hotel and meeting location may be accessed by taking the Metrolink train from the airport to the Central West End station. The senior in-vestigators will stay on the Executive Level (top three fl oors) of the Parkway Hotel, with the students and postdocs on the Lower Level.

If you have any questions, please contact Eileen A. Cler at clere@mir.wustl.edu.

Coming soon ...

The new Inter-PEN Administrative Center website will be avail-able in May 2011.

The website will use the same URL as the Inter-PEN website for the Initial PENs. www.nhlbi-pen.net