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April 09, 2013

C 2013 American Chemical Society

Harnessing Nanomedicine for MucosalTheranostics;A Silver Bullet at Last?Eran Elinav†,* and Dan Peer‡,§,*

†Department of Immunology, Weizmann Institute of Sciences, Rehovot 76100, Israel, and ‡Laboratory of NanoMedicine, Department of Cell Research & Immunologyand §Center for Nanosciences and Nanotechnology, Tel Aviv University, Tel Aviv 69978, Israel

Inflammatory bowel disease (IBD) is agroup of chronic inflammatory disordersaffecting 0.5%of theWestern population,

whose incidence is steadily rising. Thepathogenesis of this common disorder re-mains unknown, but it is estimated to resultfrom loss of tolerance of the intestinal im-mune system in the presence of constantantigenic stimulus in genetically susceptibleindividuals, mediated by an immense resi-dent microbial ecosystem. The major IBDsubsets, Crohn's disease (CD) and ulcerativecolitis (UC), feature a wide variety of clinicalmanifestations. These include chronic in-flammation of the gastrointestinal tract thatvaries in uniformity (patchy disease charac-teristic in CD vs continuous disease in UC)and biogeographical distribution (diseaseisolated to the large intestine [UC] vs aninflammatory process that may involve thetract in its entirety, from mouth to anus[CD]).1,2 Inflammatory bowel disease is alsoa systemic inflammatory disease with multi-ple extra-intestinalmanifestations including,among others, the skin (erythema nodosumand pyoderma gangrenosum), eyes (uveitisand episcleritis), joints (multiple forms ofarthritis), and liver (primary sclerosingcholangitis).3 Moreover, long-standing IBDis associatedwith a significant propensity forthe development of several cancers, suchas an aggressive form of colorectal cancerand the frequently fatal cholangiocarci-noma. These risks necessitate strict patient

surveillance, which often further results inreduced quality of life and subjects patientsto heavy psychological consequences.4

While IBD has been extensively studied inthe last four decades, both in animalmodelsand in humans, treatment options remaindisappointing, nonspecific, and associatedwith multiple systemic adverse effects.Treatment options generally target themucosal and systemic inflammatory pro-cess and include immunosuppressiveand immune modulatory interventionssuch as corticosteroids, nonsteroidal anti-inflammatory drugs, antibiotics, azathiopr-ine, and methotrexate in addition to agentsthat inhibit inflammatory cytokines and ef-fector cells such as monoclonal antibodiesto tumor necrosis factor alpha (TNF-R), in-terferon gamma (IFN-γ), and novel mono-clonal antibodies against interleukin 12(IL-12) and IL-23 that are under clinicalevaluation. Additional strategies under clin-ical investigation that aim to block effectorcell recruitment into the lesions includemonoclonal antibodies to R4 and R4β7 in-tegrins and to mucosal addressin cell adhe-sion molecule 1 (MAdCAM-1) and inhibi-tors of endothelial intercellular adhesionmolecule 1 (ICAM-1) expression. All of thesemodalities feature limited efficacy and areassociated with severe infectious, meta-bolic, and neoplastic side effects that,at times, are more severe than IBD itself.5

The resultant disease course is that of

* Address correspondence toeran.elinav@weizmann.ac.il,peer@tauex.tau.ac.il.

Published online10.1021/nn400885b

ABSTRACT Inflammatory bowel disease (IBD) has been extensively studied in the last four decades both in

animal models and humans. The treatment options remain disappointing, nonspecific, and associated with multiple

systemic adverse effects. In this Perspective, we highlight issues related to emerging nanotechnologies designed

particularly for treatment and disease management of IBD and discuss potential therapeutic target options with

novel molecular imaging modalities.

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exacerbations and remissions, withsignificant morbidity, reduction inquality of life, and frequent needfor surgical intervention along thecourse of disease.

Intensive research in recent years,mostly in small animal models,has been aimed at deciphering ma-jor cellular subsets and molecularpathways that are involved in thepathogenesis of IBD. These studieselucidated an impressive and di-verse set of new therapeutic targets,including small molecules and smallinterfering RNAs, which enable pre-cise molecular manipulation of IBD-associated pathways.6 These modal-ities are highly effective in revertingintracellular processes and thus canpotentially be employed in the treat-ment of IBD, either alone or in acombination of currently used thera-pies. However, a lack of suitableplatforms for delivery to their cellulartargets greatly limits the applicabilityof thesemodalities as IBD treatments.

Potential IBD-Associated TherapeuticTargets for Nanomedicine-Based Interven-tion. Several currently recognizedmajor cellular and molecular playersin IBDmight potentially serve as use-ful therapeutic targets. The adoptiveand innate immune arms cooperatein the induction and perpetuation ofthe mucosal inflammatory processin IBD and are the major subset

targeted by currently employed ther-apeutic interventions. Adoptive cellsinvolved with IBD include multiple Tcell subsets such as TH1 (CD), TH2(UC), and TH17 (both) andpotentiallyB cells. Innate subsets involvedin the IBD inflammatory process in-clude neutrophils, natural killer cells,and members of the mononuclearphagocyte lineage includingmacro-phages and dendritic cells.7 Indeed,mononuclear phagocytes are criti-cally involved in the maintenanceof tissue integrity, as well as theinitiation and control of innate andadaptive immunity. In homeostaticconditions, subsets of lamina pro-

pria mononuclear phagocytes sam-ple the gut lumen through theintestinal epithelial tight junctions.By doing so, they coordinate andregulate the delicate balance be-tween immune tolerance to harm-less food antigens and commensalmicroorganisms and rapid initiationof the immune response to harmfulpathogens. Impaired dendritic cellfunction, due to genetic and envir-onmental causes, breaches this bal-ance and results in the developmentof IBD.8,9 Moreover, conditional ab-lation of intestinal anti-inflamma-tory lamina propria mononuclearphagocytes results in induction ofsevere autoimmune colitis, a pro-cess governed by TNF-R secretion.Altogether, it is believed that alteredbalance between these cellular sub-sets and their disregulated responsetoward components of the micro-biome leads to massive local secre-tion of cytokines such as TNF-R, IFN-γ, IL-17, and IL-1β. Indeed, poly-morphisms in the IL-23 receptorhave been associated with IBD aswell as other autoinflammatory andautoimmune disorders.10,11 More-over, immune-mediated mechan-isms of tolerance are fundamentallyimportant inpreservationofmucosaltolerance toward the “healthy” mi-crobiome. Abrogation of such toler-ance-inducing mechanisms, such asIL-10 deficiency inmice or deficiencyin IL-10 responsiveness in humans, isstrongly associated with intestinalautoinflammation.12�14

Another attractive potential targetis the intestinal epithelial layer,15�17

an extensive one-cell-thick layer thatseparates thedense luminalmicrobialecosystem from the inner sterilemilieu through a complex networkof molecular seals. In IBD, this barrierfunction is compromised, resulting inenhanced exposure of the lamina

propria compartment to intactmicrobes or microbial elements.18

Novel, nonbarrier functions shednew light at the epithelial cell beinga critical “hub” for the regulation ofthe innate immune response. Theseinclude innate sensing of pathogensor damage-associated molecular pat-terns by epithelial toll-like receptors,nod-like receptors, inflammasomes,and lectin receptors.15 Indeed, muta-tions in the nod-like receptor Nod2are strongly implicated in Crohn'sdisease susceptibility in humans.19

Other epithelial functions includecellular proliferation and anchoringof cell matrix molecules, which iscritical for mucosal repair duringchronic injury. Mice that are deficientin the mucosal repair molecule pros-taglandin E receptor 4 becomehighlysusceptible to induced colitis, whilepolymorphisms in proximity to thisgene inhumans are linked to suscept-ibility to IBD.20 Other importantepithelial functions, which may beardirect relevance to IBD, are mucusproduction by goblet cells andantimicrobial peptide secretion byPaneth cells in the small intestine.Alterations in any of these functionsmay result in enhanced microbiomeinflux into the inflamed, injured, andpermeable intestine, promoting acontinuous inflammatory state.

Other emerging therapeutic tar-gets for IBD are the extracellular ma-trix and the intestinal microbiome.Key molecules in the extracellularmatrix, including matrix metalopro-teases amongothers, are increasinglyrecognized as pivotal in switchingon the immune response, enablinginflammatory cell migration, andparticipating in the mucosal healingresponse. Specific abrogation ofspecific matrix metaloproteases wasrecently shown to ameliorate colitis

While inflammatory

bowel disease has been

extensively studied in

the last four decades,

both in animal models

and in humans,

treatment options

remain disappointing,

nonspecific, and

associated with

multiple systemic

adverse effects.

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substantially in mouse models.21

The microbiome is a highly diversemicrobial ecosystem whose intimateinteraction with the host mucosalinterphase is crucial for tolerancemaintenance and for the antipatho-genic immune response. Alterationin composition and function of theintestinal microbiome, termed dys-biosis, is considered a cornerstonefor the development of IBD.22 Whileantibiotic and probiotic treatmentshave been associated with variableclinical effects, specific targetingof “colitogenic” taxa, delivery oftherapeutics supporting tolerogenicmicrobial stains (termed prebiotics),or factors involved with host�microbiome interactions may proveefficacious as novel therapeuticmodalities.23 It is, therefore, expectedthat the next therapeutic modality intreating IBDcoulduse targetednano-medicines that deliver therapeuticcargos to bacteria or to epithelial cellsthat secrete substances that affectthe microbiome.

Cell-Specific Targeted Nanoparticlesfor IBD Treatment. Recent develop-ments in nanotechnology utilize aninterdisciplinary approach to gener-ate nanoparticles (NPs) with highspecificities toward subsets of cells,which thereby create new opportu-nities for novel targeted therapiesand specific molecular imaging thatwill aid in disease diagnosis andmanagement. These targeted NPs;also termed nanomedicines;aremade from natural biological materi-als such as lipids, sugars, proteins,or nucleic acids or from syntheticpolymers and, unlike conventionaltherapies, have the potential tooffer more effective treatment withsignificantly reduced adverse effectsthrough specific interactions withdiseased cells.24,25 These NPs enableenhanced specificity in the deliveryof large amounts of therapeuticor imaging payloads, have thecapacity to deliver multiple pay-loads simultaneously, and can incor-porate mechanisms to overcomebiological barriers in the form oftargeting agents that can directthe drug NPs into diseased cells

without collateral damage to healthytissues.

The use of NPs in IBD is still in itsinfancy. Several strategies to directNPs into the gut mucosa during IBDhave been documented, mainly forlocal (rectal) use.26�28 Local deliveryof therapeutic molecules loaded inNPs to the inflamed colon couldbe a promising strategy only forUC patients, where the disease islocated in the proximal end of thelarge intestine and the anus. A re-cent study investigated how drug-loaded polymeric NPs target the siteof inflammation and analyzed theinfluence of different colon-specificdelivery strategies.27 Three differentpolymeric NPs were formulatedusing ovalbumin as a model drug:pH-sensitive NPs were made witheudragit S100; mucoadhesive NPswere created with trimethylchitosan(TMC); and amix of polymers;poly-(lactic-co-glycolic acid) (PLGA), poly-(ethylene glycol)-PLGA (PEG-PLGA),and the diblock copolymer, PEG-poly(ε-caprolactone) (PEG-PCL);were used to obtain sustaineddrug delivery. Furthermore, ligands

targeting leukocytes (e.g., mannose)or the inflamed colon (a specificpeptide) were grafted on the PEGchain of PCL. Interaction of NPs withthe intestinal epithelium was ex-plored using Caco-2 monolayersdesigned to mimic an inflamedepithelium and then visualizedusing confocal laser microscopy.The TMC NPs had the highest ap-parent permeability for ovalbuminin the untreatedmodel. However, inthe inflamed model, there was nodifference between TMC NPs, PLGA-based NPs, and eudragit NPs. TheuptakeofNPs in the inflamedmousecolon was assessed in a horizontaldiffusion chamber. Mannose-graftedPLGA NPs showed the highest accu-mulation of ovalbumin in inflamedcolon. On the basis of these results, itwas suggested that active targetingof macrophages and dendritic cellsmay be a promising approach fortargeting the colon in IBD.27

In animal studies, luminal uptakeinto inflamedmucosal areas has beenshown to be size-dependent.29�31 Ina recent study,32 Schmidt et al. inves-tigated thepotential ofNP andmicro-particle (MP) uptake into the rectalmucosa of human IBD patients. Fluo-rescently labeled placeboNPs 250 nmin diameter and MPs 3.0 μm in di-ameter were prepared. Two hoursafter rectal application topatientswithCD or UC, confocal laser endomicro-scopy was performed to visualize theparticles in inflamed mucosal areas.In biopsies, ex vivo mucosal transportprocesses were investigated in minia-turized chambers. Overall, 33 patientswith IBD (19 patients with CD, 14patients with UC) and six healthycontrols were tested.32 A significantlyenhanced accumulation of MPs inulcerous lesions was observed(covered area = 1.28%, range0.83�3.45%, vs 0% in controls; p =0.011), while NPs were visible onlyin traces on mucosal surfaces of allpatients. This study strengthens theimportance of selective systemicdelivery of therapeutic payloads di-rectly into subsets of leukocytes andperhaps also to gut epithelial cellssince IBD is a systemic, multifactorial

Recent developments

in nanotechnology

utilize an

interdisciplinary

approach to generate

nanoparticles (NPs)

with high specificities

toward subsets of cells,

which thereby create

new opportunities for

novel targeted

therapies and specific

molecular imaging that

will aid in disease

diagnosis and

management.

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disease. To address this need, wepreviously developed a strategythat utilized a natural pathologicalprocess.33 The b7 intergrins are exclu-sively expressed on leukocytes andare dramatically upregulated duringIBD. Moreover, circulating b7-expres-sing lymphocytes home to the in-flamed gut during IBD.34�36 Wedeveloped a robust strategy to targetthese circulating lymphocytes in vivo

and showed that we can deliver ther-apeutic payloads in a b7-specificman-ner using lipid-based NPs covalentlycoated with an antibody raisedagainst the b7 integrin.33,37 This firstproof-of-principle study showed thatselectivity in vivo using NPs targetedto leukocytes is feasible.38

RNA interference (RNAi) pay-loads are currently available as IBDresearch tools and might be applic-able for clinical use. RNA interfer-ence has emerged as a powerfulstrategy for suppressing gene ex-pression, offering the potential toaccelerate in vivo drug target vali-dation dramatically as well as thepromise to create novel therapeuticapproaches if it can be effectivelyapplied in vivo.39,40 The main aim ofRNAi applied in vivo is to achievesafe, gene-specific silencing via

tissue-specific targeting using effi-cient amounts of RNAi molecules.In vivo delivery of RNAi could beaccomplished via local or systemicadministration routes, dependingon the tissues/organs that are tar-geted. Various approaches for localdelivery of RNAi molecules haverecently been applied in animalmodels. Techniques to deliver drugsinto the gastrointestinal (GI) tractcan include the provision of drugsin solution. However, such drugswill be directly affected by the pHof the stomach and are likely todegrade under acidic pH condi-tions. To circumvent degradationby stomach acidic pH or small intes-tine digestive enzymes, high drugdoses or frequent administrationare commonly used, and adverseeffects may be challenging. Enemasare often used to target drugs tothe colon, but the procedure is

cumbersome and is associated withhigh risk of local complications, in-cluding bleeding or perforation.Thus, there is an unmet need fortargeted strategies to specific areasin the GI tract, particularly the colonbut also the upper GI track such asin the case of CD patients. As theterminal end of the digestive tract,the colon is challenging to targetwith intact and quantitative amountsof drug. From oral uptake (salivaenzymes) to colon (pH 7, higherpressure), through the stomach(pH 1�3) and the small intestine(enzymatic release and pH 3�6),drugs face deleterious environ-ments.41 The fact that nucleic acidsin general are exposed to phagocy-tosis and nuclease degradation, andare membrane-impermeable, makessystemic RNAi delivery more compli-cated and challenging in general andparticularly if the target cells are notlocalized to one pathological site butare spread systemically.

To address this challenge, sys-temic delivery strategies havebeen developed to target subsetsof leukocytes. As discussed above,b7-integrin-targeted and stabilizedNPs have been used to packagedRNAi payloads and deliver theminto b7-expressing lymphocytesin vivo in an intestinal inflammationmodel. This strategy was also usedto reveal that cyclin D1 could serve asa potential novel, anti-inflammatorytarget for IBD.33 Oral delivery ofTNF-R siRNAs was also developedusing polymeric NPs and showeda proof-of-concept efficacy in thedextran sodium sulfate-induced co-lonic autoinflammation model.42,43

Although these are highly promisingexamples, many challenges still needto be addressed when using RNAito manipulate leukocytes in vivo.Among these challengesare immunetoxicity, which needs to be carefullyexamined (including complementactivation, induction of pro-inflam-matory cytokines, interferon respon-ses, and interference with naturalcoagulation cascades), and deposi-tion of the oligos and the NPs at thecellular level.

Use of Nanoparticles for Diagnosisand Follow-Up of IBD Patients. Irritablebowel disease, and in particularthat affecting the small intestine, isoften difficult to diagnose mainlybecause of difficulties accessingmany regions of the small intestine.Although innovative strategies,such as the use of capsule endo-scopy, offer novel ways of diagnos-ing and following up lesions in thesmall intestine, these approachesare costly, invasive, and may beassociated with complications suchas aspiration and capsule retention,the later more prevalent in patientswith strictures or fistulas. In addi-tion, long-term follow-up of colonicIBD is warranted in long-term pa-tients as a means of surveillancefor the aggressive and hard-to-diagnose colorectal carcinoma thatis prevalent in these patients. Eventhough periodic surveillance colo-noscopy is recommended for thesepatients, the invasive and cumber-some procedure suffers from lowcompliance rates even among at-riskpatients. Alternative approaches,such as virtual colonoscopy, necessi-tate similar preparation and bowelinflation and are often problematicin sensitivity and specificity.

Systemic molecular-based imag-ing for diagnosis and follow-upof IBD would provide more globalinformation on disease extent andseverity, while avoiding many ofthe current limitations of existingmodalities. Furthermore, such ap-proaches would be less subject tooperator-dependent variability andwould enable the kinetic assess-ment of lesions over time.

The infiltration and activation ofdendritic cells, macrophages, andT cells play key roles in the devel-opment of IBD. These cells, andother molecular markers in the pro-cess such as chemokines, cytokines,and receptors of the immunesystem, can be used as markersfor scintigraphic imaging. Currently,colonoscopy and/or small-bowelfollow-through are considered bymost to be the gold standard imag-ing techniques for the diagnosis of

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IBD. However, since the majority ofpatients need long-term follow-up, it would be ideal to rely on anoninvasive technique with goodcompliance. With the possibility ofintravascular specific contrast agentsfor blood enhancement, computedtomography (CT) scans, ultrasono-graphy, and magnetic resonanceimaging (MRI) are all able to detectincreases in biological or endo-scopic signs of disease activity(vessel dilation, wall thickening, wallstratification with thickening of thesubmucosa, wall and mesenteric hy-pervascularity, lymph node enlarge-ment, and enhancement). Nano-particles could prove highly usefulas contrast agents. Some examp-les include superparamagnetic ironoxide NPs, which have notably beenused with MRIs in diagnostic radio-logy;44 gold NPs;45,46 titanium diox-ide NPs; and gadolinium NPs.47�49

Dual-modality contrast agents,such as radiolabeled NPs, are pro-mising candidates for a numberof diagnostic applications becausethey combine the advantages oftwo different imaging modalities,namely, combining single-photonemission computed tomographyor positron emission tomographyimaging withMRI or CT.44 Molecularimaging is also becoming an impor-tant strategy in radiology. Cell-specific receptors are being labeled

using specific antibodies that areconjugated to contrast agents orradiometals. Recently, Dearling et al.

reported a nanotechnology strategyusing 64Cu-labeled anti-β7 integrinantibody to detect intestinal inflam-mation bymicroPET/CT imaging.50,51

This integrin represents a promisingtherapeutic target, as discussedabove, but could also be used forthe detection of CD and UC becauseof its specificity on a small subsetsof leukocytes and its involvementin lymphocyte recruitment to theinflamed gut and not to other princi-pal organs such as the lungs or liver.In this proof-of-concept study, theselective uptake of anti-β7 integrin64Cu-labeled antibody in the gut ofanimals with experimentally inducedcolitis suggested that integrinβ7maybe a promising target for radioimmu-nodetection of colitis, which wouldaid in the diagnosis, assessment, andtherapyguidanceof IBD (see Figure 1).Indeed, this could become a newdiseasemanagementmodality whentested clinically in humans.

FUTURE OUTLOOK

The development of nanomedi-cine for disease theranostics is pro-gressing at a fast pace and is drivenby principles from diverse fieldsof science in the postgenomic age.Nano-based theranostics are madepossibleby integrating theapplication

of molecular biomarkers across therangeof tests and interventions reach-ing from disease susceptibility testingand monitoring of clinical outcomesresulting from interventions. How-ever, the terms “nanotheranostics” or“theranostics using nanomedicine”usually reflect the ability of nanocar-riers to report on a specific pathologi-cal site or cell and to deliver drugssimultaneously directly into thesecells.52 One such strategy that mightbe relevant for IBD is tousemultistage,silicon-based NPs that can entraptherapeutic payloads with gadoli-nium-based contrast agents that en-hance T1 contrast for MRI use.53 Otherpotential options include a turn-on,near-infrared, cyanine-based probefor noninvasive intravital optical imag-ing of hydrogen peroxide.54 Thisstrategy can report on inflamed areasusing intravital optical imaging, andat the same time, H2O2 can be used asa therapeutic strategy for gut inflam-mation if small amounts are deliveredto the appropriate site in the gut.Although the number of nano-

medicines that are reaching the clinicis dramatically increasing, most ofthem thus far are cancer treat-ments.24,55 We propose that noveltheranostic nanomedicines will bedeveloped for IBD, combining noveltherapeutic and molecular imagingmodalities such as combinationsof targeted NPs that incorporate

Figure 1. Representative images of microPET/CT show the distribution of 64Cu-labeled anti-β7 monoclonal antibodies (mAb)in mice, 48 h after administration as a strategy for disease management in inflammatory bowel disease.50 The mAb raisedagainst the integrin subunit β7 shows higher uptake in the gut of mice with colitis (left) than in control mice (middle).Antibodies can also accumulate in inflamed tissue nonspecifically. The representative image on the right shows lowuptake ofnonspecific (isotype control)mAb in inflamedgut and confirms that themAbuptake in dextran sodiumsulfate-induced colitis(left) is specific (color scale = microPET image of radionuclide distribution, gray scale = microCT anatomical image).

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therapeutic payloads (such as siRNAstargeting specific genes) with imag-ing agents such as 64Cu that can

be coupled to a cell-surface-specifictargeting moiety (see Figure 2).Furthermore, individualized IBD char-acteristics such as cellular receptorexpression, pathway activation, andcytokine profile could be assessedand used to design personalizednano-based targeting vectors andpayloads. As such, the use of hostand microbiome RNA sequencing inpatients clustering to IBD subgroups,such as nonresponders versus re-sponders, may reveal new thera-peutic targets that can be down-regulated by specific siRNAs or upre-gulated by microRNAs as a means oftreatment for specific patients. Thus,

we believe that these novel strate-gies could indeed represent a long-sought new class of personalizedmedicine in IBD treatment and man-agement. This combined nanothera-nostic approach;which will includenew therapeutic payloads, possiblycoupled with and incorporated intoexisting “conventional” treatmentstrategies;combined with novelmolecular imaging approaches mayrevolutionize the management ofIBD.

Conflict of Interest: The authors de-clare no competing financial interest.

Acknowledgment. The authors wishto thank Prof. Averil Ma, Chief, Division

The development of

nanomedicine for

disease theranostics is

progressing at a fast

pace and is driven by

principles from diverse

fields of science in the

postgenomic age.

Figure 2. Schematic representation of different mechanisms by which nanoparticles (NPs) can deliver drugs and report onmucosal disease. Lipid-based NPs (as representative nanocarriers) can be employed in a variety of inflammatory andneoplastic mucosal disorders such as in the lungs, skin, and gut. Passive tissue targeting is achieved by extravasation of NPsthrough increased permeability of the vasculature and ineffective lymphatic drainage (EPR effect). Active cellular targeting(inset, left side) can be achieved by functionalizing the surfaces of NPswith ligands that promote cell-specific recognition andbinding. The NPs can release their contents in close proximity to the target cells, attach to themembrane of the cell and act asan extracellular sustained-release drug depot, or internalize into the cell. Anti-inflammatory payloads (inset, right side) suchas small molecules, RNAi, and proteins can manipulate the function of pro-inflammatory subsets of leukocytes and shift theresponse toward an anti-inflammatory response or vice versa (depending on the response). The delivery of therapeutic orimaging payloads (green star bound to the monoclonal antibodies that decorate the NPs) may be induced systemically orlocally (such as luminal delivery in the gut).

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of Gastroenterology at the University ofCalifornia, San Francisco, for his com-ments and suggestions. This work wassupported by the Kenneth Rainin Foun-dation awarded to E.E. and D.P. and by theLeonaM. andHarry B. HelmsleyNanotech-nology Research Fund awarded to D.P.

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