fall 2009 nhlbi inter-pen quarterly newsletter · 2009-11-04 · a. three branches of the lca...

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NewsletterNH

LBI

Volume 3, Issue 4

NewsletterInter-PEN QuarterlyInter-PEN Quarterly

4th Annual Inter-PEN Meeting

Boston, MassachusettsOctober 16-17, 2009

Harvard Medical SchoolMassachusetts General Hospital

EILEEN A

. CLER

Hosted by:

Supported by:The National Institutes of Health

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A Word from our Program Coordinator

MEET OUR PEN

PAGE 14

PEN SPOTLIGHT ON

HOT EMERGINGBREAKTHROUGHS

AWARDS

PAGE 8

PAGE 8

PAGE 9

Emory/Georgia TechGang Bao

Eileen A. Cler, B.S.Program Coordinator for the PEN Grants

Jean M. J. FréchetNagoya Gold Medal

Unpublished data

Stanley Y. ShawMGH-Harvard

We had an excellent turnout for the Fourth Annual Inter-PEN Meeting on October 16-17, 2009, in Boston, Massa-chusetts. Seventy-two people registered for the conference

and twenty-eight students, postdocs and investigators displayed post-ers with highlights of their recent research data and fi ndings.

Special thanks to our host, Principal Investigator - Ralph Weissleder,and his staff at the Massachusetts General Hospital and Harvard Uni-versity for their efforts in conducting the meeting. The accommoda-tions for the meeting room and the hotel were well chosen.

Over the past four and a half years, each of the PENs has strived to fi nd solutions to the medical challenges regarding various diseases affect-ing the heart, lung and blood. Many discoveries have been made, both in the research area, and also in the bringing together of people in the clinical and research disciplines. While initially there were times when it was a challenge to understand how others presented their re-sults, over time we’ve come to understand more completely how to communicate and resolve concerns.

A signifi cant amount of effort was involved in applying for the renewal of the PENs. It’s our hope that the renewal will allow us to continue our research. We have been fortunate to have the strong leadership and guidance of our NHLBI Program Offi cial, Denis Buxton.

For my part, I would like to say that it has been a pleasure working with each and every one of you during the past three years. I’ve es-pecially enjoyed producing and editing this newsletter, maintaining the Inter-PEN website, and photographing the annual meetings. In fact, I had to chuckle to myself this year when I overheard someone comment during the lineup for this year’s group photo on the steps of the Bulfi nch Building at Massachusetts General Hospital, “... Alright everybody, you know the drill, ... just do what she says and we’ll be done in no time”.

APPS FOR Ac-DEX

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Jessica L. CohenUC - Berkeley

4th INTER-PEN

PAGE 16

Ralph WeisslederMass Gen Hosp/Harvard

In this issue . . .

PEN

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N a n o t e c h n o l o g y : D e t e c t i o n & A n a l y s i s o f P l a q u e F o r m a t i o nEMORY UNIVERSITY, GEORGIA TECH

Partial Carotid Ligation is a Model of Acutely Induced Disturbed Flow,

Leading to Rapid Endothelial Dysfunction and Atherosclerosis

Hanjoong Jo, Douglas Nam, Amir Rezvan, Chih-Wen Ni, David G. Harrison, Don P. Giddens

[email protected]

Atherosclerosis is closely associated with disturbed fl ow char-acterized by low and oscillatory shear stress, but studies di-rectly linking dis-

scribed a method of isolating endothelial RNA in suffi cient quantity from mouse carotid ar-teries. Using this model and method, we have shown that partial liga-tion causes upregulation of pro-atherogenic genes, downregulation of anti-atherogenic genes, endothelial dysfunction, and rapid atheroscle-rosis in 2 wks in a p47phox-dependent manner and advanced lesions (numerous and pronounced intraplaque neovascularization) by 4 wk.

In the partial carotid ligation, three of the four caudal branches of the left common carotid artery (LCA) are ligated using sutures (Fig. 1A), resulting in substantial fl ow reduction and diastolic fl ow reversal in LCA as shown by ultrasound (Fig. 1B). We used com-

putational fl uid dynamics

Figure 1. Partial ligation of left common carotid artery (LCA) causes low and oscillatory fl ow. A. Three branches of the LCA [external carotid artery (ECA), inter-nal carotid artery (ICA), and occipital artery (OA)] were ligated in the LCA, while leaving the superior thyroid artery (STA) open. B. Ultrasound showing fl ow velocity profi les and revealing that partial ligation induces fl ow reversal (indicated by arrows) in LCA during diastole.

A. B.

“ One of key advantages of this mouse model is

that signifi cant atheroma develops rapidly and

specifi cally along the entire length of LCA, but not

in the ontralateral common carotid artery within

two weeks upon partial ligation with high-fat diet. “

turbed fl ow to atherogenesis is lacking. The major reason for this has been a lack of an animal model in which dis-turbed fl ow can be acutely induced and cause athero-sclerosis. In our recent pa-per published in American Journal of Physiology- Heart and Circulatory Physiology (297:1535-1543, 2009), we

modeling (CFD) to calculate shear stress values at the ves-sel walls and showed that time-averaged wall shear stress was reduced from ~110 dyn/cm2 preligation to ~30 dyn/cm2 postligation. We then used this model as an in vivo model for acutely induced disturbed fl ow in a region previously exposed

characterized partial carotid ligation as a model of disturbed fl ow with characteristics of low and oscillatory wall shear stress. We also de-

to unidirectional laminar fl ow. We used a novel technique for RNA isolation to collect RNA from the intima and show that specifi c genes

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Figure 3. Partial ligation and high-fat diet rapidly induce atherosclerosis in LCA of ApoE KO mice. ApoE KO mice were partially ligated and fed the high-fat diet for 1–4 wk. A. Oil Red O staining of frozen sections. B. Paraffi n sections obtained from LCA and RCA (4 weeks post ligation) were stained with pentachrome. Note needle-shapedcholesterol clefts (*) and intraplaque neovessels (arrows) containing red blood cells. Shown are representative images of at least n = 6.

B.

Figure 2. A. Partial ligation results in decreases in kruppel-like factor 2 (KLF2) and endothelial nitric oxide synthase (eNOS) while increasing bone

morphogenic protein 4 (BMP4), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). Intimal RNA from

sham and partially ligated C57BL/6 mice were collected from LCA and RCA, respectively, 2 days after surgery and analyzed by qPCR using 18S as

an internal control. Data are shown as a ratio of mRNA expressed in LCA over RCA of sham and partially ligated mice. Means SE±are shown, n =3 sham 5 ligated. B. Partial ligation induces endothelial dysfunction. Arterial rings were obtained from LCA and RCA that were partially ligated as in Fig. 1 and fed high-fat diet for 2 and 7 days in ApoE KO mice. Rings preconstricted with PGF2α were dilated with increasing concentrations of acetylcholine (A) or sodium nitroprusside (SNP; B) for endothelial-independent relaxation. Shown are means±SE, n = 2 for 2 days, and 6 for 7 days.

B.A.

such as ICAM1, VCAM1 and BMP4 are up-regulated in response to disturbed fl ow and others such as eNOS and KLF2 are down-regulat-ed (Fig 2A). Using ApoE KO mice put on high fat diet as a model of hypercholesterolemia, we demonstrate that inducing disturbed fl ow in the left carotid will cause endothelial dysfunction by 7 days (Fig 2B), and accelerated atherosclerosis with signifi cant atheroma forma-tion by 2 weeks (Fig 3A) which leads to complex lesion formation by 4 weeks post ligation (Fig 3B).

In summary, we found that partial ligation of the carotid artery results in acutely induced disturbed fl ow (low and oscillatory shear stress), resulting in upregulation of some proatherogenic genes

and downregulation of some anti-atherogenic genes (in 2 days), en-dothelial dysfunction (in 1 week), accelerated atherosclerosis (in 2 weeks) and advanced lesion development (in 4 weeks) in a hyperlip-idemic mouse model. It also allows for easy and rapid intimal RNA isolation which could be used for genome-wide studies to determine molecular mechanisms underlying fl ow-dependent regulation of vas-cular biology and disease. One of key advantages of this mouse model is that signifi cant atheroma develops rapidly and specifi cally along the entire length of LCA, but not in the contralateral common carotid artery within two weeks upon partial ligation with high-fat diet. By comparison, typical mouse models of atherosclerosis in ApoE KO or LDLR KO requires more than two to three months. This rapid in vivo

A.

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References1. Nam, D.; Ni, C.W.; Rezvan, A.; Suo, J.; Budzyn, K.; Llanos, A.;

Harrison, D.; Giddens, D.; and Jo, H. “Partial Carotid Ligation is a Model of Acutely Induced Disturbed Flow, Leading to Rap-id Endothelial Dysfunction and Atherosclerosis”, Am J Physiol Heart Circ Physio, 297:1535-1543, 2009.

2. Chang, K.; Weiss, D.; Suo, J.; Vega, J.D.; Giddens, D.; Taylor, W.R.; Jo, H. “Bone Morphogenic Protein Antagonists Are Coexpressed With Bone Morphogenic Protein 4 in Endothelial Cells Exposed to Unstable Flow In Vitro in Mouse Aortas and in Human Coronary Arteries: Role of Bone Morphogenic Pro-tein Antagonists in Infl ammation and Atherosclerosis”, Circu-lation, 116, 1258-1266, 2007.

Gang Bao, Ph.D. Principal Investigator

Distinguished ProfessorThe Wallace H. Coulter Department of

Biomedical EngineeringEmory University

The Georgia Institute of Technology

About the Authors

Hanjoong Jo, Ph.D. Senior Investigator

The Wallace H. Coulter Department of Biomedical Engineering

Emory UniversityThe Georgia Institute of Technology

Department of MedicineEmory University

Douglas NamCardiology Fellow


Amir RezvanCardiology Fellow


Chih-Wen NiPh.D. Candidate



David G. Harrison Senior Investigator

Department of CardiologyEmory University

Don P. Giddens, Ph.D.Senior Investigator



PEN

atheroma model will be extremely useful not only to obtain molecular insights into atherogenic mechanisms, but also to test various thera-peutic and diagnostic interventions targeting endothelial dysfunction and atherosclerosis, saving tremendous time, effort and cost.

“... we found that partial ligation of

the carotid artery results in acutely

induced disturbed fl ow (low and os-

cillatory shear stress), resulting in

upregulation of some proatherogenic

genes and downregulation of some

anti-atherogenic genes (in 2 days),

endothelial dysfunction (in 1 week),

accelerated atherosclerosis (in 2

weeks) and advanced lesion develop-

ment (in 4 weeks) in a hyperlipidemic

mouse model.”

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B I , B W H , M G H , 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

Oxazine Conjugated Nanoparticle Detects In Vivo Hypochlorous Acid and

Peroxynitrite Generation1

By Jason McCarthy and Ralph [email protected],[email protected]

In a number of infl ammatory diseases, such as Alzheimer’s disease or atherosclerosis, reactive oxygen or reactive nitrogen species (ROS/RNS) are considered causal to or an exacerbating factor

in their pathogenesis. The main sources of these species are heavy-metal catalyzed oxidation reactions or enzymatically via myeloper-oxidase (MPO). MPO mediates the production of hypochlorous acid (HOCl) from the chloride ion (Cl-) and hydrogen peroxide (H

2O

2), and

can oxidize a number of small molecule substrates. The promiscuity of the MPO active site and the generation of hydroxyl radicals results in secondary oxidation products, including chloramines, tyrosyl radi-cals, and nitrogen dioxide. These reactive byproducts modify proteins, generate DNA adducts, and alter lipid structure, resulting in the in-hibition of various protein functions. MPO has also been identifi ed as a biomarker of myocardial infarction and coronary artery disease. While a number of strategies have been developed to image oxidative stress in vivo, these probes have often lacked the appropriate phar-macokinetics, suitable emission wavelengths to overcome tissue auto-fl uorescence, and adequate specifi city for relevant ROS. To this end, we have developed a novel optical sensor for monitoring ROS/RNS generation, and demonstrated its applicability in vivo in a murine model of myocardial infarction. The probe, based upon the HOCl/per-oxynitrite (ONOO-)-mediated release of a masked oxazine fl uorophore, was synthesized by the reac-tion scheme depicted in fi gure 1. The free dye was initially reacted with glutaric anhydride and tri-ethylamine in dichlorobenzene to yield a conju-gatable, fl uorogenic oxazine. This molecule was then conjugated to crosslinked dextran-coated iron oxide (CLIO) nanoparticles, which had pre-viously been modifi ed with AlexaFluor 488, to yield ~400 activatable oxazines per nanoparticle. Activation of the ROS/RNS sensor by HOCl gen-erated from the MPO/H

2O

2/Cl- system results in

release of the oxazine dye from the nanoparticle scaffold and restoration of its fl uorescent proper-ties (Figure 1d), resulting in greater than a 500-fold increase in fl uorescence. The specifi city of

Figure 1. Synthesis of the ROS/RNS sensor. (a, b) Reaction of oxazine with glutaric anhydride to generate the quenched ROS responsive intermediate with conjugatable handle for attachment to the dextran shell of the iron oxide nanoparticles. Nanoparticles are dual labeled with Alexa Fluor 488 to monitor particle location. (c, d) Relative fl uorescence signal for each of the reactants and products. (e, f ) Blood half-life determination for the free oxazine dye and the ROS/RNS sensor.

the probe was tested against a panel of ROS, and demonstrated activation by only HOCl and ONOO-. In addition, the probe activation was shown to be linear over a range of micromolar concentra-tions of NaOCl. The in vitro activity of the sensor was tested in CD11b-enriched leukocytes isolated from whole mouse blood. These neutrophils con-tain MPO, and are known to generate HOCl and ONOO-. Activation of the probe was assayed by fl ow cytometry after incubation with the neutrophils, and demonstrated a marked increase in fl uorescence in the allophycocyanine (APC) channel, which corresponds to the oxa-zine dye fl uorescence. When the cells were pretreated with an MPO inhibitor, this resulted in a 95% decrease in activation, indicating that HOCl generation is dominant with respect to ONOO-. The ROS/RNS sensor was fi nally tested in vivo in a murine model of myocardial in-farction. For all experiments, the agents (sensor or quenched small molecule probe) were injected intravenously into the tail vein of the mice 12-14 h after MI and imaged 24 h later when neutrophil recruit-ment to the infarct is high. Macroscopic analysis by fl uorescence re-fl ectance imaging of coronal sections of the infarcted mouse hearts indicated that the ROS/RNS sensor localized to the infarcted zone, whereas the fl uorescence from the small molecule probe had washed out of the infarct and into the myocardium. Histological examination of the hearts further demonstrated the colocalization of the probe with neutrophils. While this probe has demonstrated utility in MI, we are currently examining its usefulness in other diseases and conditions,

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Figure 2. Ex vivo comparison of ROS/RNS sensor or free dye injected

mouse tissue and fl uorescence signal associated with oxazine release,

accumulation, and probe washout kinetics. Fluorescence refl ectance imaging (FRI) of control (a-c), ROS/RNS sensor (d-f), and Intermediate I (g, h) injected myocardial rings are shown. White light images of TTC- stained heart slices are shown (a, d, and g) with infarcted region highlighted by the dashed line throughout. Accumulation of the activated oxazine probe is detectable in the Cy5 channel (b, e, and h), while fl uorescence associated with the covalent attached AF488-labeled nanoparticle (c and f).

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

Nanotechnology: Detection & Analysis of Plaque

FormationEmory UniversityGeorgia Institute of Technology

P.I. - Gang Bao, Ph.D. Director , Emory-GT, Program of Excellence in Nanotechnology CoE Distinguished Professor The Wallace H. Coulter Department of Biomedical Engineering Georgia Institute of Technology Emory University

Nanotherapy for Vulnerable PlaqueThe Burnham InstituteUniversity of California Santa BarbaraThe Scripps Institute

P.I. - Jeffrey W. Smith, Ph.D. Professor Director, Center on Proteolytic Pathways Director, Program of Excellence in Nanotechnology Burnham Institute for Medical Research

Translational Program of Excellence in NanotechnologyHarvard UniversityMassachusetts General HospitalMassachusetts Institute of TechnologyBrigham and Women’s Hospital

P.I. - Ralph Weissleder, M.D., Ph.D. Director, Center for Systems Biology Massachusetts General Hospital Professor of Radiology and Systems Biology Harvard Medical School

Integrated Nanosystems for Diagnosis and TherapyTexas A&M UniversityWashington University in Saint LouisUniversity of California Santa BarbaraUniversity of California BerkeleyUniversity of Texas, Southwestern Medical Center at Dallas

P.I. - Karen L. Wooley, Ph.D. W. T. Doherty-Welch Chair Professor of Chemistry Texas A&M University

The Four PENs andtheir Principal Investigators

About the Authors

such as atherosclerosis, cancer, metastasis, and organ rejection, as each possesses an infl ammatory component.

References1. Panizzi, P.; Nahrendorf;, M.; Wildgruber, M.; et al. “Oxazine Conjugated Nanoparticle Detects In Vivo Hypochlorous Acid and Per-oxynitrite Generation.” J Am Chem Soc. In Press

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P E N S P O T L I G H T O N :S t a n l e y Y . S h a w , M . D . , P h . D . , I n v e s t i g a t o r a t M G H / H a r v a r d

Stanley Y. Shaw is a Principal Investigator and Co-Director of Chemical Biology in the Massachusetts General Hospital Center for Systems Biology, with a joint appointment in the Cardiovascular Research Center. His re-search uses systematic chemical biology, molecular imaging, and genomics approaches to identify novel human

phenotypes for cardiovascular and metabolic disease. A major interest of his group is the synthesis, characterization and screening of targeted nanoparticles for molecular imaging of specifi c cell-types of high biological importance for cardiovascular disease. A complementary effort is the discovery of novel cell-based phenotypes from human subjects, including from the Framingham Heart Study. “Our hope and expectation is that molecular imaging can one day con-tribute to systematic phenotyping of mouse models and human subjects. By revealing how drugs and mutations affect biological pathways in vivo, nanoparticle-based imaging agents can help build a systems-based, holistic understanding of cardiovascular disease.”

Dr. Shaw is a board-certifi ed cardiologist and attending physician in the Massachusetts General Hospital Heart Center. Before that, he also served as Chief Resident of the Medical Service at Massachusetts General Hospital.

He received his A.B. summa cum laude from Harvard College before completing his combined M.D. and Ph.D. studies

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

AWARDS

Harvard, MGH, MIT, BWHUCB, UCSB, WUSTL

By Eileen A. Cler, B.S.

Emory, Georgia TechDr. Nie's former postdoctoral fellow Dr.

Hongwei Duan has received a faculty

position as Nanyang Assistant Professor

in Singapore. He started his faculty

appointment on September 1, 2009.

Dr. Ralph Weissleder has been elected as a

new member of the U.S. National Academies

Institute of Medicine (IOM). Election to

the IOM is considered one of the highest

honors in the fi elds of health and medicine

and recognizes individuals who have

demonstrated outstanding professional

achievement and commitment to service.

Jean M. J. Fréchet, Ph.D., will receive the

Nagoya Gold Medal for organic chemistry

at a special symposium in Nagoya Japan on

November 16, 2009.

PEN

Stanley Y. Shaw, M.D., Ph.D.Senior Investigator

Massachusetts General [email protected]

If someone on your PEN has received an Award that you’d

like to mention in an upcoming newsletter, please send a

description of the award to Eileen A. Cler at [email protected].

at Harvard; his Ph.D. in Biophysics studied DNA topology under the supervision of James Wang. He completed his internship, residency, and cardiology fellowship at Massa-chusetts General Hospital; and worked with Mark Fish-man and Stuart Schreiber as a Howard Hughes Medical Institute physician post-doctoral fellow.

For more information about Stanley Y. Shaw’s re-search, visit the Center Systems Biology web site at: http://csb.mgh.harvard.edu/shaw.

About the Author

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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

Sheng Tong, Sijian Hou and Gang Bao - “In vivo drug delivery using magnetic nanoparticle carriers” - We have been developing SPIO-based nanocarriers to deliver small hydrophobic drug molecules in vivo for treating cardiovascular diseases. This approach has the advantage of combining targeting, imaging and delivery, and using external magnetic fi eld to guide and retain drug-loaded SPIOs. Gang Bao

RalphWeissleder

Karen L. Wooley

By Eileen A. Cler, B.S.

Craig J. Hawker

Craig J. Hawker and his group at UCSB are synthesizing comb copolymers with CANF peptide and 64Cu for the targeted imaging of cardiovascular disease using PET imaging. Pamela K. Woodard and colleagues at Washington University are currently evaluating some of these materials in their animal models. Pamela K. Woodard

Dr. Hakho Lee and his group, in collaboration with researchers at the Harvard School of Public Health, are screening a number of po-tential ligands for their magnetic nanoparticles in order to further improve upon the sensitivity and the specifi city of their NMR-Fil-ter system for the detection of Mycobacterium tuberculosis (MTB) This system is already capable of detecting 20 bacteria in a 1 mL sample of sputum within 30 min. The on-going research focuses on enabling the broad detection of different MTB strains in clinical settings.

Hakho Lee

Sheng Tong Sijan Hou

Emerging BreakthroughsEmerging Breakthroughs

Hot Upcoming TopicsHot Upcoming Topics

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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

Figure 1. Overview of Ac-DEX as a material for the formation of acid-sensitive particles.

Acetalated Dextran (Ac-DEX): A Biocompatible, Tunable, and Degradable

Material for Therapeutic Applications

Jessica L. Cohen, Joel A. Cohen, Kyle E. Broaders, Tristan T. Beaudette, Eric M. Bachelder, and Jean M. J. Fréchet*

[email protected]

Microparticles made from biodegradable polymers such as poly(lactic-co-glycolic acid) (PLGA) have been extensively investigated as vehicles for biomedical applications, includ-

ing gene delivery1 and chemotherapy.2 In these examples, the encap-sulated cargo is typically released over the course of several months via surface erosion and the slow degradation of the polymer.3 For many drug delivery applications, it is desirable to release therapeu-tic agents under mildly acidic conditions, as may be found in sites of infl ammation, lysosomal compartments, or in tumor tissue.4,5 Acid-sensitive liposomes, micelles, and hydrogels have been widely ex-plored, but few easily prepared polymeric materials exist that combine acid-sensitivity and biodegradability. Poly(β-amino esters), which are protonated and thus become soluble at lower pH, constitute one such material. However, these polymers become polycationic under acidic conditions and must be blended with biocompatible polyesters to re-duce their toxicity.6 We sought to create a system with the fl exibility and biocompatibility of polyester materials, but with the additional benefi t of a change in rate of payload release sensitive to physiologi-cally relevant acidic conditions.

We recently developed a biocompati-ble and pH-sensitive polymer based on modifi ed dextran which may offer advantages over polyester-based materials such as PLGA (Figure 1).7-9 Dextran (an FDA approved and clinically used biopolymer) was rendered insoluble in water by modifi cation of its hydroxyl groups through reaction with 2-methoxypropene under acid catalysis (Figure 2). The high density of pendant acetals makes the new “acetalated-dextran” (Ac-DEX) soluble in organic solvents such as dichloromethane, ethyl acetate, or acetone. Masking the hy-droxyl groups of dextran as acetals not only provides a hydrophobic material that is easily processable using various emulsion techniques,

Figure 2. Synthesis of Ac-DEX. Single-step procedure for modifi cation of water-soluble dextran to organic soluble Ac-DEX.

it also provides a trigger for introducing pH-sensitivity. Under mildly acidic aqueous conditions, the pendant acetal groups are expected to hydrolyze, thus unmasking the parent hydroxyl groups of dextran, re-sulting in the solubilization of the polymer in aqueous conditions.

Ac-DEX is a tunable and modular particle system that can be used in the delivery of various biological cargoes. We have been able to prepare particles encapsulating either hydrophobic small molecules

or hydrophilic macromolecules using single or double emulsion techniques respectively. The particle size can be easily tuned from 175 nm up to 10 μm by varying the processing condi-tions used during particle for-mation. In addition, we have shown that the rate of nanopar-ticle degradation can be easily varied by controlling the type of acetals (i.e., cyclic vs. acy-clic) formed on the pendant hy-droxyl groups of dextran.8 Dur-ing the synthesis of Ac-DEX, acyclic acetals form quickly, whereas more stable cyclic ac-etals become prevalent after

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some equilibration period. Given that cyclic acetals are more stable than their acyclic counterparts, the half-life of hydrolysis of Ac-DEX can be tuned by controlling the amount of each type of acetal present. This is particularly evident at pH 5 (Figure 3), where altering the ac-etal ratio enables tuning of degradation rates on the order of minutes to days, while the particles remain stable at pH 7.4. We have shown that this tunability can affect both the magnitude and nature of bio-logical processing of compounds encapsulated within these particles.8

Additionally, we have developed a facile and chemoselective method for modifying the surface of Ac-DEX particles with targeting groups or imaging agents. Based on prior examples of oxime forma-tion with complex polysaccharides,10-12 we hypothesized that carbohy-drate reducing chain ends present at the surface of the particles could be exploited to form stable oxime conjugates with alkoxyamine-bear-ing molecules. Incubation of Ac-DEX particles with alkoxyamine-functionalized dye led to both time and concentration dependent la-beling of the particles. The mild reaction conditions and functional group tolerance of this ligation strategy enables the particles to be similarly modifi ed with a wide range of complex bioactive molecules.

To assess the feasibility of using functionalized particles for the delivery of biologically relevant payloads, we performed proof-of-concept in vitro transfection experiments with plasmid-loaded Ac-DEX particles. Blending PLGA with poly(β-amino ester) polymers has been shown to enhance transfection effi ciency in phagocytic cell lines, presumably due to a proton-sponge effect.13 We hypothesized that functionalizing similarly prepared Ac-DEX particles with CPPs would enhance the uptake of these particles by non-phagocytic cells and lead to effi cient transfection. Ac-DEX particles (both fast- and slow-degrading) blended with 10% (w/w) poly(β-amino ester) poly-mer, and encapsulating plasmid DNA encoding fi refl y luciferase as

Figure 4. In vitro transfection of HeLa cells with plasmid-loaded

microparticles. Relative light units from the luminometer were converted into the mass of luciferase present using a recombinant standard, then normalized to the total mass of cellular protein determined from a fl uorescamine assay. Data represent the mean ± standard deviation of triplicate measurements.

a reporter, were prepared and functionalized with CPPs using the oxime chemistry described above. After a 2 day incubation, HeLa cells treated with fast-degrading CPP-modifi ed Ac-DEX particles demonstrated a signifi cant increase in expression of the luciferase reporter compared to cells incubated with unmodifi ed particles or slow-degrading Ac-DEX particles (Figure 4), which strongly suggests effi cient particle uptake and delivery of the encapsulated DNA. In conclusion, Ac-DEX-based particles represent a novel class of materi-als that have the potential to be used in various biomedical applica-tions due to their biocompatibility, biodegradability, and tunability.

Figure 3. (a) Composition of acetals modifying dextran over the course of the reaction. Acyclic acetals dominate the acetal population early in the reaction, but are replaced by cyclic acetals as the reaction continues. (b) Degradation of Ac-DEX over the course of 48 h in pH 5 acetate buff er. The degradation rate of particles depends on acetal modifi cation.

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PEN

About the Authors

Jean M. J. Fréchet, Ph.D.Senior Investigator

Professor of ChemistryUniversity of California, Berkeley

Jessica L. CohenPh.D. Candidate in the Fréchet Laboratory

Department of ChemistryUniversity of California, Berkeley

Joel A. Cohen, Ph.D.Postdoc in the Fréchet Laboratory


Kyle E. Broaders

,Ph.D. Candidate in the Fréchet LaboratoryDepartment of Chemistry

University of California, Berkeley

Tristan T. BeaudettePh.D. Candidate in the Fréchet Laboratory


Eric M. Bachelder, Ph.D.Postdoc in the Fréchet Laboratory


Want to learn more about the four PENs?Visit our website at www.nhlbi-pen.net to read about:

• Aims of each PEN Grant• Research slides from past Inter-PEN meetings• Past newsletter issues• Joint Seminar Series with the Siteman Center

of Cancer Nanotechnology Excellence

Next Meeting - December 4

Program of Excellence in Nanotechnology (PEN) Executive Committee, (from left to right): Gang Bao (Emory/Georgia Tech), Karen L. Wooley (WUSTL/TAMU/UCB/UCSB/UTSW), Ralph Weissleder (Broad/BWH/Harvard/MGH/MIT), Jeffrey W. Smith (Burnham/Scripps/UCSB) and Denis Buxton (NHLBI)

Upcoming Executive Committee Meetings(Denis Buxton and Principal Investigators only)

References 1. Gvili, K.; Benny, O.; Danino, D.; Machluf, M. Biopolymers,

2007, 85, 379-391.2. Sengupta, S.; Eavarone, D.; Capila, I.; Zhao, G. L.; Watson, N.;

Kiziltepe, T.; Sasisekharan, R. Nature, 2005, 436, 568-572.3. Matsumoto, A.; Matsukawa, Y.; Suzuki, T.; Yoshino, H. J. Con-

trolled Release, 2005, 106, 172-180.4. Sun-Wada, G. H.; Wada, Y.; Futai, M. Cell Struct. Funct. 2003,

28, 455-463.5. Helmlinger, G.; Sckell, A.; Dellian, M.; Forbes, N. S.; Jain, R. K.

Clin. Cancer Res., 2002, 8, 1284-1291.6. Little, S. R.; Lynn, D. M.; Puram, S. V.; Langer, R. J. Controlled

Release, 2005, 107, 449-462.7. Bachelder, E. M.; Beaudette, T. T.; Broaders, K. E.; Dashe, J.;

Fréchet, J. M. J. J. Am. Chem. Soc., 2008, 130, 10494-10495. PMCID: PMC2673804.

8. Broaders, K. E.; Cohen, J. A.; Beaudette, T. T.; Bachelder, E. M.; Fréchet, J. M. J. Proc. Nat. Acad. Sci. U.S.A., 2009, 106, 5497-5502. PMCID: PMC2666992.

9. Beaudette, T. T.; Cohen, J. A.; Bachelder, E. M.; Broaders, K. E.; Cohen, J. L.; Engleman, E. G.; Fréchet, J. M. J. J. Am. Chem. Soc., 2009, 131, 10360-10361. NIHMSID: NIHMS131727.

10. Cervigni, S. E.; Dumy, P.; Mutter, M. Angew. Chem. Int. Ed.,1996, 35, 1230-1232.

11. Hatanaka, Y.; Kempin, U.; Jong-Jip, P. J. Org. Chem. 2000, 65, 5639-5643.

12. Zhao, Y. M.; Kent, S. B. H.; Chait, B. T. Proc. Nat. Acad. Sci. U.S.A., 1997, 94, 1629-1633.

13. Little, S. R.; Lynn, D. M.; Ge, Q.; Anderson, D. G.; Puram, S. V.; Chen, J. Z.; Eisen, H. N.; Langer, R. Proc. Nat. Acad. Sci. U.S.A., 2004, 101, 9534-9539.

Author Jessica L. Cohen, at the 4th Annual Inter-PEN Meeting as she talks to Jered B. Haun of Massachusetts General Hospital about his poster on “Click Labeling of Intracellular Cancer Markers Using Dual Fluorescent-Magnetic Nanoparticles”.

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Ritu ShresthaPh.D. Candidate in the Wooley Lab

Washington UniversityDepartment of Chemistry

Eileen A. Cler, B.S.Photographer and PEN Coordinator

Washington University in Saint LouisDepartment of Chemistry

Carolyn J. Anderson, Ph.D.SKILLS Component Leader

Professor in Radiology, Biochemistry and Molecular Biophysics

Washington University School of MedicineDepartment of Radiology

Michael J. Welch, Ph.D. Senior Investigator


Charles R. Glaus, Ph.D.Postdoc in the Welch Laboratory


Postdoc Charles R. Glaus (WUSM – Welch Lab) and Ph.D. Candidate Ritu Shrestha (WUSTL – Wooley Lab) perform a radiolabeling experiment with newly-synthesized SCK nanoparticles. Interdisciplinary collaborations such as the one between polymer chemists from the Wooley Lab and radiochemists from the Welch Lab encourage the sharing of expertise. Having input from both teams improves nanoparticle development and experimental design. Ultimately, nanoparticles with desirable biodistribution, high degree of radiolabeling, and strong affi nity for their biological target are selected for in vivo imaging studies.

Karen L. Wooley, Ph.D.Principal InvestigatorTexas A&M University

Department of Chemistry

Participants

CROSS‐TRAINING“Ultimately, nanoparticles with desirable biodistribution, high degree of radiolabeling, and strong affi nity for their biological target are selected for in vivo imaging studies.”

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Principal Investigator Program Offi cialGang Bao Denis Buxton

Our Students, Postdocs and Staff ScientistsOur Students, Postdocs and Staff Scientists


p

Wayne Alexander No students currently

Jin Suo

Xiaoping HuNo students currently

Gang Bao

Kathy K. Griendling No students currently

David G. Harrison Heinrich Lob

Michael E. DavisInthirai Somasuntharam

Allison M. DennisSijian Hou

Nazanin Masoodzadehgan

Qingfen PanWong Jong Rhee

Sheng TongAmy H. Tang*

*Senior Research Project Coordinator

Zilan (Annie) Zheng

Don P. Giddens

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About the AuthorEileen A. Cler, B.S.

Photography and Graphic DesignProgram Coordinator for the PENs


Hanjoong JoHaiwei QiuDong Ju Son

Meredith B. Baker

Niren Murthy

Dongmei (May) Wang

Shuming NieMin Kuang

Michael C. ManciniAndrew Smith

Ximei Qian

Greg M. BoothIan C. Campbell

Michelle A. Consolini (not pictured)

Sarah F. KnightNatalia Landazuri

Nick Willett

Ji Woong Han

Kousik KunduSungmun Lee

Jay C. Sy

Charles D. Searles

W. Robert Taylor

Young-Sup Yoon

John Phan

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44444444444444444444444444th Annual Inter-PEN Meeting

Boston, MassachusettsOctober 16-17, 2009

Harvard Medical SchoolMassachusetts General Hospital

Inter-PEN Meeting attendees gather on the steps of the historical Bulfi nch Building of Massachusetts General Hospital, where “Ether Day” is celebrated each year. Th e Ether Dome here is where anesthesia was discovered on October 16, 1846.

Hosted by: Poster Winners - Monty Liong (MGH/Harvard), Ritu Shrestha (WUSTL), Jay C. Sy (Georgia Tech) and Natalie A. Forbes (UC-Santa Barbara)

Supported by:The National Institutes of Health

EILEEN A

. CLER

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(Left to right) Amy H. Tang of Emory/Geor-gia Tech with Event Organizers, Dianne M. Moschella of MGH/Harvard and Eileen A. Cler of Washington University in St. Louis. (Mikhail) Misha Pivovarov of MGH/Harvard (2nd from right) assisted with IT.

About the AuthorEileen A. Cler, B.S.

Photography and Graphic DesignProgram Coordinator for the PENs


Senior Investigator, Charles D. Searles (left), of Emory University talks with poster winner and Ph.D. Candidate, Jay C. Sy, of Georgia Tech about Jay’s poster on “New Strategies for Treating Cardiac Dysfunction”.

(Left to right) Neal K. Devaraj, Hahko Lee, and Huilin Shao of Massachusetts General Hospi-tal discuss Lee’s and Shao’s poster “Miniatur-ized NMR Chip for Highly Sensitive Medical Diagnosis”.

Poster winner and Ph.D. Candidate, Ritu Shres-tha, of Washington University in St. Louis (from Karen L. Wooley’s Lab) presents her poster to Jayeeta Bhaumik of the Massachusetts General Hospital. The poster was titled “Shell Cross-linked Nanoparticles with PNAs as Imaging agents for iNOS” and was a collaboration with fellow Washington University Ph.D. Candidate, Yuefei Shen (from John-Stephen A. Taylor’s Lab).

Postdoctoral fellow, Kousik Kundu,of The Georgia Institute of Tech-nology (from Niren Murthy’s Lab) presents his poster on “Hydrocya-nines/Deuterocyanines: A New Family of Probes for Imaging Radi-cal Oxidants In Vitro and In Vivo”.

Senior Investigator, Jason R. McCarthy (left), of Massa-chusetts General Hospital and NHLBI Program Offi cial, Denis B. Buxton, pre-view the Inter-PEN Meeting agenda.

Poster winner and Ph.D. Candidate, Natalie A. Forbes(from Joseph A. Zasadzin-ski’s lab) of the University of California at Santa Barbara (UCSB) presents her poster to fellow UCSB Ph.D. Candi-date, Matthew J. Black.

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Massachusetts General Hospital, Boston, Massachusetts October 16-17, 2009

4th ANNUAL INTER-PEN MEETING

Dianne M. Moschella Administrative Director Center for Systems Biology Mass. Gen. Hosp. Harvard Medical School (617) 643-3220 [email protected]

Good luck to everyone in the Renewal. We expect the research awards

to be announced in January. The Administrative Core selection will be

announced in February.

Thank you to our Inter-PEN Meeting host, Principal Investigator, Ralph Weissleder, and his staff members,

Dianne M. Moschella, Jason R. McCarthy, and Serena D. Laft, of the Center for Systems Biology at the Massachusetts General Hospital.

Summary slides from the Boston meeting will soon be posted on the Inter-PEN website at www.nhlbi-pen.net.

Serena D. Laft Assistant to Dr. Weissleder Administrative Manager Center for Systems Biology Harvard Medical School Mass. Gen. Hosp. (617) 726-7870 [email protected]

Eileen A. Cler Program Coordinator for the PEN Grants Washington University in Saint Louis (314) 935-7482 [email protected]

Renewal

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

Program OfficialDenis B. BuxtonNational Heart, Lung, and Blood InstituteEmail: [email protected]://www.nhlbi.nih.gov

Principal Investigators Gang BaoEmory UniversityGeorgia Institute of TechnologyEmail: [email protected]://pen.bme.gatech.edu/

Jeffrey W. SmithThe Burnham InstituteThe Scripps Research InstituteUniversity of California, Santa BarbaraEmail: [email protected]://www.pennvp.org

Ralph WeisslederHarvard UniversityMassachusetts General HospitalMassachusetts Institute of TechnologyBrigham and Women’s HospitalEmail: [email protected]://csb.mgh.harvard.edu/

Karen L. WooleyTexas A&M UniversityEmail: [email protected]

Production Designer, Editor, PhotographerEileen A. ClerWeb Designer and DeveloperProgram Coordinator for NHLBI-PEN GrantsWashington University in Saint LouisEmail: [email protected]

Poster SessionBoston, MA

PEN Executive Committee, (from left to right): Principal Investigators - Gang Bao, Karen L. Wooley, Ralph Weissleder, Jeffrey W. Smith and Program Offi cial, Denis Buxton.

Inter-PEN Event Organizers:

fall 2009 nhlbi inter-pen quarterly newsletter · 2009-11-04 · a. three branches of the lca...

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