R E S EARCH ART I C L E

DRUG DEL IVERY

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An inflammation-targeting hydrogel for local drugdelivery in inflammatory bowel diseaseSufeng Zhang,1,2,3* Joerg Ermann,4,5* Marc D. Succi,1,2,5 Allen Zhou,2 Matthew J. Hamilton,5,6

Bonnie Cao,6 Joshua R. Korzenik,5,6 Jonathan N. Glickman,5,7 Praveen K. Vemula,8

Laurie H. Glimcher,9 Giovanni Traverso,1,5,10† Robert Langer,1,3,11† Jeffrey M. Karp2,5,11,12†

There is a clinical need for new, more effective treatments for chronic and debilitating inflammatory bowel disease(IBD), including Crohn’s disease and ulcerative colitis. Targeting drugs selectively to the inflamed intestine may im-prove therapeutic outcomes and minimize systemic toxicity. We report the development of an inflammation-targetinghydrogel (IT-hydrogel) that acts as a drug delivery system to the inflamed colon. Hydrogel microfibers were generatedfrom ascorbyl palmitate, an amphiphile that is generally recognized as safe (GRAS) by the U.S. Food and Drug Admin-istration. IT-hydrogel microfibers loadedwith the anti-inflammatory corticosteroid dexamethasone (Dex) were stable,released drug only upon enzymatic digestion, and demonstrated preferential adhesion to inflamed epithelial surfacesin vitro and in two mouse colitis models in vivo. Dex-loaded IT-hydrogel enemas, but not free Dex enemas,administered every other day to mice with colitis resulted in a significant reduction in inflammation and were asso-ciated with lower Dex peak serum concentrations and, thus, less systemic drug exposure. Ex vivo analysis of colontissue samples from patients with ulcerative colitis demonstrated that IT-hydrogel microfibers adhered preferentiallyto mucosa from inflamed lesions compared with histologically normal sites. The IT-hydrogel drug delivery platformrepresents a promising approach for targeted enema-based therapies in patients with colonic IBD.

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INTRODUCTION

Inflammatory bowel disease (IBD) in its twomain variants, Crohn’s dis-ease and ulcerative colitis (UC), affects about 1.4 million Americans,and its incidence is increasing around the world (1, 2). Currently avail-able therapies fail to control symptoms adequately in a significant num-ber of patients, adversely affecting quality of life (3, 4).

One approach to develop more efficacious and safer therapies couldbe inflammation-targeting drug delivery to achieve high drug concen-trations locally at the site of inflammation with minimal exposure ofhealthy or distant tissues. Enemas as a basic form of targeted drug de-livery to the inflamed colon are routinely used in mild-to-moderate co-litis (5). However, typical enema-based formulations require the patientto retain the enema for extended periods of time, which is difficult whensuffering from diarrhea and fecal urgency. The need for frequent dosingnegatively affects patient compliance (6). Furthermore, high concentra-tions of active drug may result in significant absorption and systemicside effects.

Inflammation targeting can potentially be achieved using drug de-livery systems that exploit specific features of the diseased tissue. Inflam-

1The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute ofTechnology, Cambridge, MA 02139, USA. 2Center for Regenerative Therapeutics,Biomedical Research Institute, Brigham and Women’s Hospital, Boston, MA 02115, USA.3Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge,MA 02139, USA. 4Division of Rheumatology, Immunology and Allergy, Brigham andWomen’s Hospital, Boston, MA 02115, USA. 5Harvard Medical School, Boston, MA 02115,USA. 6Division of Gastroenterology, Hepatology and Endoscopy, Brigham and Women’sHospital, Boston, MA 02115, USA. 7Miraca Life Sciences, Newton, MA 02464, USA. 8Institutefor Stem Cell Biology and Regenerative Medicine (inStem), Bangalore 560065, India. 9WeillCornell Medical College, New York, NY 10065, USA. 10Division of Gastroenterology,Massachusetts General Hospital, Boston, MA 02114, USA. 11Harvard–Massachusetts Instituteof Technology Division of Health Sciences and Technology, Cambridge, MA 02139, USA.12Harvard Stem Cell Institute, Cambridge, MA 02138, USA.*These authors contributed equally to this work.†Corresponding author. E-mail: rlanger@mit.edu (R.L.); jmkarp@partners.org (J.M.K.);ctraverso@partners.org (G.T.)

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mation of the colonicmucosa is accompanied by depletion of themucuslayer and in situ accumulation of positively charged proteins includingtransferrin (7), bactericidal/permeability-increasing protein, and anti-microbial peptides (8–11). This results in the buildup of positive chargesat the damaged epithelial surface, providing a molecular target andanchor for drug carriers with negative surface charge (12, 13). Inflam-mation is furthermore accompanied by up-regulation and release ofdegradative enzymes including esterases andmatrix metalloproteinases(MMPs) (14, 15). A drug delivery system with an overall negative sur-face charge and containing an enzyme-labile linker should thereforepreferentially adhere to inflamed mucosa and release drug in responseto enzyme activities present at the site of inflammation. Additionally,binding of the drug carrier system to the mucosa should prolong localdrug availability and permit a reduction in dosing frequency.

To identify suitable inflammation-responsive compositions, weexamined hundreds of compounds from the Generally Recognized asSafe (GRAS) list of theU.S. Food andDrugAdministration. Inflammation-responsive materials that have been described (16, 17) require an or-ganic synthesis step for the introduction ofMMP-labile linkers, whichis complex and costly. We reasoned that the use of GRAS reagents,which are generally safe for oral consumption, inexpensive, and readilyavailable in large quantities, should accelerate translation to the clinic.On the basis of our search for agents with enzyme-labile bonds thatcould be cleaved in inflammatory environments, we selected ascorbylpalmitate (AP), an amphiphile capable of self-assembly into a hydrogelin vitro (18).

We report here that a hydrogel made from AP can be used as aninflammation-targeting hydrogel (IT-hydrogel) for drug delivery inIBD (Fig. 1, A and B). IT-hydrogel microfibers encapsulate hydropho-bic drugs and, owing to their negative surface charge, preferentially ad-here to the inflamedmucosa in twomurine colitismodels,T-bet−/−Rag2−/−

ulcerative colitis (TRUC) (19) and dextran sulfate sodium (DSS)–inducedcolitis, as well as to tissue samples from patients with UC. Using the

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corticosteroid dexamethasone (Dex) as a model drug, we demonstratethat drug-loaded IT-hydrogel microfibers administered to colitic micevia enema are therapeuticallymore efficacious and result in less system-ic drug exposure than free Dex. Our study provides proof of concept forIT-hydrogel as a safe and potentially effective drug delivery platform forcolonic IBD and other inflammatory diseases.

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RESULTS

Dex is efficiently encapsulated and released fromIT-hydrogel in vitroAP consists of a partially charged hydrophilic head (ascorbic acid) and ahydrophobic tail (palmitic acid) joined by an ester bond (Fig. 1C). In adimethyl sulfoxide (DMSO)/water solvent mixture, AP assembles intoextended micellar structures and interdigitated bilayers with a hydro-phobic core and hydrophilic outer layer that form fibers at the micro-scopic level. After heating to facilitate the dissolution of AP, gelation

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occurs when the AP/solvent mixture cools to room temperature(Fig. 1D).

Here, we loaded the IT-hydrogel by adding the Dex pro-drugDex-21 palmitate (Dex-Pal) and, for imaging purposes, the fluores-cent dye DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine,4-chlorobenzenesulfonate salt) before gelation. These hydrophobiccompounds are expected to interact with the palmitic acid tails of APand integrate into the core of the hydrogel fibers. Scanning electron mi-croscopy revealed the fibrous structure of the IT-hydrogel with a fiberdiameter of 1 to 2 mm and a length of 20 to 50 mm (Fig. 1E), whichwas similar for unloaded andDex-loaded hydrogels. All IT-hydrogel for-mulations had a similar negative surface charge (Fig. 1F). For in vitro andin vivo applications, the IT-hydrogel was easily suspended in PBS,yielding a mixture of microscopic fiber particles of various sizes (Fig. 1G)that could easily be handled with pipettes and syringes.

To analyze variables affecting drug encapsulation and release,Dex-loaded IT-hydrogels were generated using two concentrations ofgelator [4 and 8% (w/v) AP] and two concentrations of Dex-Pal (Dex

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Fig. 1. IT-hydrogel targets drug release to the inflamed mucosa. (A) Alipophilic drug is loaded into IT-hydrogel during gelation. The drug inte-

degrade the gel, resulting in drug release. PMN, polymorphonuclear leuko-cytes. (C) Molecular structure of AP (top) and the assembled IT-hydrogel

grates into the hydrophobic core of the lipid bilayer. Microfibers representhigher-order assemblies of extended bilayer structures, which are sus-pended in phosphate-buffered saline (PBS) to yield amixture of microscopichydrogel fiber particles of various sizes. (B) Negatively charged IT-hydrogelmicrofibers do not adhere to the intact mucosal surface. The inflamed mu-cosa is characterized by mucus depletion, accumulation of positivelycharged proteins, and increased permeability of the epithelial cell layer. Neg-atively charged IT-hydrogel microfibers adhere to the positively chargedinflamed epithelium. Hydrolytic enzymes released by inflammatory cells

(bottom) demonstrating the alignment of the hydrophobic tails in thecenter and the hydrophilic heads on the outside of the bilayer. (D) APbefore and after gelation. (E) Environmental scanning electron microscopyimages of unloaded and Dex-loaded IT-hydrogel before suspension in PBS.(F) z Potential of IT-hydrogel (Gel) or gel loaded with Dex (Dex/gel), DiD(DiD/gel), or Dex +DiD [(Dex+DiD)/gel]. Data aremeans ± SD (n= 6pooledfrom two experiments); P = 0.1485 determined by one-way analysis of var-iance (ANOVA). (G) Polarized optical microscopy image of IT-hydrogelsuspended in PBS.

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equivalent, 5 and 10 mg/ml) on the basis of our previous experience withthe gels and drug-loading and delivery requirements, respectively. A lowergelator concentration (4%) andahigherDex-Pal concentration (10mg/ml)resulted in the best drug-loading efficiency among the formulations tested(14%, Fig. 2A). In contrast, the encapsulation efficiency was not substan-tially affected by the gelator and Dex-Pal concentrations (Fig. 2B).

Dex-loaded IT-hydrogel incubated in PBS at 37°C was stable for16 days, without any measurable Dex release (Fig. 2C). Addition of anesterase, Thermomyces lanuginosus lipase, induced a rapid and dose-dependent release of Dex (Fig. 2C and fig. S1A). The simultaneous gen-eration of free ascorbic acid and Dex (fig. S1, B and C) suggests thatesterase hydrolyzed the ester bond in AP, causing gel disassembly whilesimultaneously converting Dex-Pal to active Dex by cleaving the esterbond in the palmitated pro-drug. The Dex-Pal pro-drug could not bedetected in themobile phase at any time point. Dexwasmore efficientlyreleased from a 4% IT-hydrogel than from an 8% IT-hydrogel (fig. S1D).

Macrophages and other immune cells secrete enzymes at the site of in-flammation that would be expected to hydrolyze the IT-hydrogel (14, 15).To simulate drug release from the IT-hydrogel under inflammatoryconditions, Dex-loaded 4% IT-hydrogel microfibers were incubatedin vitro with supernatant from human or mouse macrophages culturedwith or without lipopolysaccharide (LPS). Enzymes secreted by bothmouse and human macrophages were capable of inducing drug releasefromDex-loaded IT-hydrogel (Fig. 2D).Macrophage activation by LPSenhanced the accumulation of enzymatic activity in the supernatantcompared with unstimulated cells.

For subsequent in vitro and in vivo studies, we chose to use a 4% IT-hydrogel, which was less viscous than the 8% gel (fig. S1E) and thus

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easier to pass through syringes and small-bore tubes. We found no evi-dence for cytotoxicity of IT-hydrogel preparations (3 to 8% AP, ± Dex)in two human intestinal epithelial cell lines, Caco2 and HT-29, after72 hours in vitro (fig. S2).

IT-hydrogel preferentially adheres to inflamed mucosa inmice with colitisThe negative surface charge of the IT-hydrogel (Fig. 1F) should facilitateits adhesion to the positively charged inflamed colon epithelium (7–11).To test this hypothesis, we first analyzed the adhesive properties of IT-hydrogel microfibers in vitro using synthetic surfaces. IT-hydrogelloaded with both DiD and Dex [(DiD + Dex)/gel] was incubated onpolystyrene plates coated with human recombinant transferrin (posi-tively charged) or porcinemucin protein (negatively charged), simulatinginflamed and healthy epithelium, respectively (Fig. 3A). Transferrin-coated plates retained a 7.6-fold higher fluorescence signal from the(DiD + Dex)/gel after washing compared to mucin-coated or uncoatedplates (Fig. 3A). We confirmed charge interactions as the main mecha-nismof IT-hydrogel adhesionusing chemically defined (amine- or carboxyl-modified) substrates (fig. S3A). In another control experiment, we incubated(DiD + Dex)/gel with a cationic polyallylamine solution to convert thesurface charge of themicrofibers fromnegative to positive. As predicted,this abrogated the preferential adhesion of IT-hydrogel to transferrinand enhanced adhesion to the uncoated and mucin-coated surfaces(fig. S3B).

We then examined the adhesion of IT-hydrogel to inflamed colonepithelium using two established mouse IBD models: chemically in-duced DSS colitis and the spontaneous TRUC model. The distal colon

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was removed from mice with colitis andhealthy controls, incubated with (DiD +Dex)/gel ex vivo, washed, and imagedusing an IVIS fluorescence imaging sys-tem. Colons from wild-type mice withDSS colitis and from colitic TRUC miceshowed significantly greater retention offluorescence than colons from wild-typemice without colitis and Rag2−/− controlmice, respectively (Fig. 3B). (DiD + Dex)/gel adhered to the apical surface of theinflamed colon, as shown by confocal mi-croscopy of frozen colon sections from aDSS mouse (fig. S4).

Preferential adhesion of IT-hydrogelto inflamed mucosa was further validatedin vivo. Wild-type mice with DSS colitisand untreated controls, or TRUC andRag2−/− control mice received a single ene-ma of (DiD + Dex)/gel. Animals were sac-rificed 12 hours later, the distal colon wasremoved, and fluorescence retention wasquantified. Colons from colitic mice dem-onstrated more gel adherence comparedto the respective controls in bothmodels(Fig. 3C). Administration of free DiD viaenema to mice with DSS colitis did notresult in retention of the fluorescence sig-nal when the colon was analyzed 12 hourslater (fig. S5). Together, these experiments

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Fig. 2. Dex is efficiently encapsulated into IT-hydrogel and released by esterase activity and super-natant frommacrophages. (A and B) Drug-loading and encapsulation efficiencies for 4 and 8% AP gelator

with Dex-Pal at Dex equivalent of 5 and 10 mg/ml. (C) Esterase-responsive Dex release from IT-hydrogel.Esterase (T. lanuginosus lipase, 100 U/ml) was added on day 6. (D) Dex release from IT-hydrogel uponincubation with culture supernatant from activated mouse or human macrophages for 24 hours at 37°C.The 4% IT-hydrogel in (C) and (D)was loadedwithDex-Pal (Dex equivalent, 5mg/ml). Data aremeans ± SD(n = 3, performed at least twice); P values in (D) were determined by Student’s t test with the Holm-Sidakmethod to correct for multiple comparisons.

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provide evidence that IT-hydrogel microfibers preferentially adhere tothe inflamed colon mucosa mediated by electrostatic interaction.

Drug delivery via IT-hydrogel enema improvestherapeutic efficacyWe then tested IT-hydrogel in a therapeutic setting in TRUCmice. Wedid not examine the therapeutic efficacy of Dex-loaded IT-hydrogel inthe DSS model because conflicting data about the efficacy of cortico-steroid administration on the severity of DSS colitis have been reported(20, 21). Dexwas used at 70 mg per dose on the basis of published reportsof treatment studies in rodent colitis models (22, 23). Colitic TRUCmice received an enema of Dex-Pal–loaded IT-hydrogel (Dex/gel) orwater-soluble Dex-21 phosphate in PBS (free Dex) on experimentaldays 1 and 3. Untreatedmice (Control) andmice that received gel with-out drug (Gel) served as controls. All mice were sacrificed on day 5 for

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blinded histopathological analysis of the colon by a board-certified gas-trointestinal pathologist (Fig. 4A). Disease severity was significantly re-duced in mice given Dex/gel (mean colitis score, 1.4) compared to allother experimental groups, whereas mice in the free Dex group (meancolitis score, 3.3) did not differ significantly from untreated mice (meancolitis score, 3.4) or mice that had received gel only (mean colitis score,4.1) (Fig. 4B). TRUC disease is characterized by infiltration of the colonlamina propria with neutrophils and mononuclear inflammatory cells,crypt hypertrophy, and superficial erosions (19, 24). Representativeimages (Fig. 4C) demonstrate that histological inflammation wasdiminished in the mice treated with two Dex/gel enemas, but not inmice receiving two enemas with the equivalent amount of free Dex. Co-lon weight, myeloperoxidase (MPO) activity, and expression of tumornecrosis factor (TNF) in the distal colon were evaluated as additionalparameters of disease activity in a second independent experiment

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tified using an IVIS imaging system (left). The same experimental setupcompared colitic TRUCmice and age-matched Rag2−/−micewithout colitis

uncoated, mucin-coated (simulating healthy epithelium), or transferrin-coated (simulating inflamed epithelium) surfaces at 37°C for 1 hour. Fluores-cence images obtained after rinsing (left) were quantified using ImageJsoftware (right). Data aremeans ± SD (n = 9, triplicate samples, three imagesper sample); P values were determined by one-way ANOVA with Tukeypost hoc test. aU, arbitrary unit. (B) The distal colon of wild-type (WT) micewith DSS-induced colitis and healthy controls was incubated ex vivo with(DiD+Dex)/gel at 37°C for 30min andwashed, and fluorescencewas quan-

(right). (C) WTmice with DSS-induced colitis and healthy controls receivedan enema of (DiD + Dex)/gel. The animals were sacrificed 12 hours later,and fluorescence of the distal colon was measured (left). The same exper-imental setup compared colitic TRUC and controlRag2−/−mice (right). In (B)and (C), the total fluorescence intensity was determined in a standard-sizeregion of interest (ROI) drawn around the individual colon pieces; data aremeans ± SEM (n = 5 to 7 mice per group); P values were determined byStudent’s t test.

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(Fig. 4D). We observed a reduction of all three parameters in micetreated with Dex/gel enemas compared with the other experimentalgroups; the reductions of colon weight and MPO activity were sta-tistically significant.

Although two enemas with free Dex (70 mg of Dex equivalent perdose) had no measurable effect (Fig. 4, B to D), we did observe a sig-nificant reduction in colitis severity when free Dex was administeredintraperitoneally for four consecutive days (Fig. 4, E and F), demon-strating biological activity of the compound in ourmodel. Thus, deliveringDex to colitic mice encapsulated in IT-hydrogel was significantly moreefficacious than giving the equivalent amount of free Dex via enema.

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We also performed an in vivo barrier function assay as part of theexperiment reported in Fig. 4D. Treatment with two enemas of Dex/gelhad no statistically significant beneficial effect on intestinal epithelialpermeability compared with free Dex and control groups (fig. S6),which likely reflects both incomplete resolution of inflammation onthe time scale of the experiment and the experimental noise of the assay.Longer-term and ex vivo studiesmay be needed to address this questionin the future.

To demonstrate that the therapeutic benefit of Dex/gel enemas wasnot simply an effect of administering the pro-drug Dex-Pal, coliticTRUC mice were given two enemas of Dex-Pal suspended in a 5%

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measured in a second independent experiment. Data aremeans± SD (n=10mice per group); P values were determined by one-way ANOVA with Tukey

on days 1 and 3 after overnight (O/N) fasts; animals were sacrificed for anal-ysis on day 5. (B) Histopathology scores after treatment with enemas of IT-hydrogel (Gel), free Dex, or Dex-loaded IT-hydrogel (Dex/gel, 70 mg of Dexequivalent per dose in both groups). Control mice received no treatment.Data aremeans± SD (n=10mice per group). P=0.0029by one-wayANOVA;comparison of individual groups by Tukey post hoc test. (C) Representativehematoxylin and eosin (H&E) histology images of the experimental groups in(B). (D) Colon weight, MPO activity, and TNF mRNA levels in the distal colon

post hoc test. (E) TRUCmice received four daily intraperitoneal (i.p.) injectionsof free Dex (Dex-21 phosphate, 70 mg of Dex equivalent per dose) or PBS;animals were sacrificed on day 5. (F) Histopathology scores for mice in (E)(n= 9mice per group). (G) Histopathology scores for TRUCmice treatedwithtwo enemas of Dex-Pal (70 mg of Dex equivalent per dose) in vehicle (5%ethanol + 5% Tween 80) or vehicle only (Control) (n = 8 mice per group)using the dosing regimen described in (A). Data in (F) and (G) are means ±SD; P values were determined by Student’s t test.

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ethanol/5% Tween 80 mixture on days 1 and 3, and colon histo-pathology was analyzed on day 5 (Fig. 4A). Administration of Dex-Pal without IT-hydrogel had no significant impact on disease severitycompared with vehicle controls (Fig. 4G). These results are consistentwith a model where negatively charged IT-hydrogel microfibers adhereto the inflamed colon mucosa, thereby providing a reservoir for pro-longed local drug availability and improved therapeutic efficacy.

Local drug delivery via IT-hydrogel reduces systemicdrug exposureDrug release from IT-hydrogel requires enzymatic digestion of the gel byhydrolytic enzymes such as esterases (Fig. 2C) or MMPs (18). We ana-lyzed enzyme-responsive Dex release in vivo by monitoring the serumdrug level after administration of Dex/gel to the inflamed colon. Micewith colitis received a single enema of either Dex/gel or free Dex(70 mg of Dex equivalent in both preparations), and serum Dex levelswere determined after 1, 2, 4, 6, 12, and 24 hours (Fig. 5A). Mice withDSS-induced colitis receiving free Dex had an early peak of the serumDex concentration at 1 hour (Fig. 5B); levels declined rapidly thereafterwith first-order kinetics. Inmice receivingDex/gel enemas, the peak ser-um concentration was significantly lower. The AUC as a measurementof cumulative systemic drug absorption was also significantly reducedin the Dex/gel mice compared with mice receiving free Dex (Fig. 5B).Similar pharmacokinetics was seen in TRUC mice (Fig. 5B). Thus, de-livering Dex via IT-hydrogel altered the pharmacokinetics in both DSScolitis and TRUC models, resulting in a major reduction in systemicdrug absorption.

IT-hydrogel preferentially adheres to biopsy specimens fromhuman inflamed colonTo test the adhesion of IT-hydrogel to human colonic mucosa, we ana-lyzed biopsy specimens from UC patients undergoing surveillance co-lonoscopy (n = 6; table S1) comparing inflamed mucosa with normalmucosa in the same patient. Freshly obtained biopsy specimens wereincubated ex vivo with (DiD + Dex)/gel and imaged using an IVIS flu-orescence imaging system (Fig. 6A). Specimens from inflamed sites re-tained significantly more fluorescence than did those from normal sites(Fig. 6, B andC); themean fold difference between inflamed andnormalbiopsy specimens was 5.4. The ex vivo analysis of human biopsysamples therefore demonstrates, consistent with our mouse data, thatIT-hydrogel microfibers preferentially adhere to inflamed mucosa inpatients with active UC.

DISCUSSION

Drug therapy involves the careful balancing of intended therapeuticeffects and unwanted side effects often related to activities in organsnot affected by the disease. One approach tomaximize beneficial effectsand reduce the potential for side effects is targeted drug delivery andrelease. Many oral formulations for drug delivery to the colon rely onpH-, time-, microflora-, or pressure-triggered mechanisms and targetthe intestinal region rather than the inflamed intestine (25). In contrast,inflammation-targeting drug delivery systems use specific features ofthe inflamed target organ. Poly(lactic-co-glycolic acid) nanoparticleshave been shown to accumulate in the inflamed mucosa after oral ad-ministration to rats with trinitrobenzenesulfonic acid–induced colitisowing to increased tissue permeability at the site of inflammation

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(26), and poly(thioketal) nanoparticles release drug upon degradationby reactive oxygen species present in the colon ofmicewithDSS-inducedcolitis (27). The inflamed intestine has also been targeted from the blood-stream using PEGylated poly(lactic acid) microparticles coated withantibodies against an endothelial cell adhesion molecule up-regulatedin mouse colitis models (28, 29).

Here, we demonstrate that IT-hydrogel fibers with a negativelycharged surface preferentially adhere to positively charged artificialsurfaces, to inflamed mucosa in murine colitis, and to biopsy speci-mens from inflamed colon mucosa in human UC patients. Althoughwe cannot exclude the possibility that additional factors may play arole in vivo, our in vitro data strongly suggest charge as the main fac-tor mediating adhesion of IT-hydrogel to the inflamed epithelial sur-face. This is consistent with published reports describing the use ofanionic liposomes for drug delivery to the inflamed intestine (12, 13).We were able to detect a fluorescence signal from the intestinal wall forat least 12 hours after administration of a single enema of fluorescentlylabeled IT-hydrogel to mice with colitis. This suggests that adherentIT-hydrogel microfibers generate a reservoir of encapsulated drug atthe site of inflammation, thereby prolonging local drug availability.

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received a single enema of either free Dex or Dex-loaded IT-hydrogel(containing 70 mg of Dex equivalent) at 0 hour after an overnight fast. TheserumDex concentration was determined at 1, 2, 4, 6, 12, and 24 hours afterenema, and the area under the curve (AUC) was calculated. (B) Results of thepharmacokinetic experiment described in (A) for WT mice with or withoutDSS-induced colitis and for TRUCmice comparedwith Rag2−/− controls. Dataare means ± SD (n = 7 to 10 mice per group); P values were determined byStudent’s t test.

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Drug delivery using IT-hydrogel enemas may thus allow for less fre-quent dosing in patients with IBD.

Compared with other drug delivery systems targeting the inflamedcolon mucosa, IT-hydrogel has several potential advantages. First, theIT-hydrogel described here is made from a nontoxic, GRAS compound(30), which should facilitate rapid translation into the clinic. ManyGRAS agents are relatively inexpensive and available in large quantitiesat a high grade of purity (GoodManufacturing Practice or FoodGrade).Second, the gelation process for the IT-hydrogel is simple and easy toscale up. Third, IT-hydrogel has a high drug-loading capacity, owingto the small molecular weight of the gelator and the availability of allgelator molecules to form intermolecular interactions. Fourth, drug-loaded IT-hydrogel exhibits long-term stability, which enables sus-tained drug release over several days. Presumably, the microstructureof the gel fibers protects the ester bond between ascorbic acid and pal-mitic acid from spontaneous hydrolysis by water, because water can-not efficiently penetrate into the hydrophobic fiber core to disassemblethe gel and release the drug. Drug release requires hydrolytic enzymeactivities, which are up-regulated at sites of inflammation, furtherstrengthening the inflammation-responsive aspect of this system.

IT-hydrogel microfibers made from AP are representative of a firstgeneration of inflammation-targeting drug delivery systems preparedfrom amphiphile GRAS reagents. Potential applications are not limitedto IBD but include any disease where controlled drug release at a dis-tinct location is desired.We recently reported that delivery of an immu-nosuppressive drug (tacrolimus) using an enzyme-responsive hydrogel

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prepared from another GRAS reagent (triglycerol monostearate) im-proved long-term survival of vascularized allografts in rats (31). In thatstudy, the drug-loaded hydrogel was injected subcutaneously into theallograft. Here, we used topical enzyme-responsive drug release in com-bination with inflammation-sensitive charge-based adhesion to im-prove drug delivery to the inflamed colon.

To analyze gel adhesion and drug delivery via IT-hydrogel in vivo,we used two mouse IBD models, TRUC and DSS-induced colitismodels. In both models, we found preferential adhesion of IT-hydrogelto the inflamedmucosa and reduced systemic drug absorption after en-ema administration of Dex-loaded IT-hydrogel compared with freeDex.We could demonstrate that IT-hydrogel also adhered significantlybetter to biopsy specimens from inflamedmucosal locations in patientswithUC compared with specimens from normal sites. All patients wereon anti-inflammatorymedications at the time of biopsy; our resultsmaytherefore underestimate the binding efficacy of the IT-hydrogel to theinflamed mucosa in untreated patients.

We loaded IT-hydrogel microfibers with the corticosteroid Dex toexamine the utility of this system for inflammation-targeting drug de-livery in mice in vivo. Delivering Dex encapsulated in IT-hydrogel re-duced systemic absorption compared with free Dex administered viaenema and improved its therapeutic efficacy in the TRUC model. Weanalyzed only one drug (Dex) whose anti-inflammatory properties arewell described (32, 33). However, the investigation of novel drug targetswas not the focus of our study. Rather, we consider Dex as amodel drugto demonstrate the utility of the IT-hydrogel in colonic IBD. Our ap-proach could potentially be applied to many other drugs, includingsmall-molecule inhibitors of signaling cascades (31).

We did not achieve complete resolution of colon inflammation bygiving TRUC mice two Dex/gel enemas (Fig. 4, B to D, and fig. S6).However, we chose a treatment regimen that might discern a differencein therapeutic response between Dex/gel and free Dex considering thatfree Dex effectively treats TRUC when administered in high enoughquantities (Fig. 4F). Long-term experiments with more stringent endpoints including assays of intestinal barrier function will be importantwhen testing future IT-hydrogel formulations. The use of a singlemousemodel for the analysis of therapeutic efficacy is another potentiallimitation of our study. Additional preclinical studies including largeanimal models are needed before the clinical benefits of IT-hydrogelcan be evaluated in humans.

In conclusion, we have developed a strategy for targeted drug deliv-ery to the inflamed colonicmucosa using hydrogelmicrofibers preparedfrom an amphiphilic GRAS reagent. Through attaching to the inflamedmucosa and selectively releasing drug at the site of inflammation, thissystemhas the potential to prolong local drug availability,minimize sys-temic drug absorption, reduce dosing frequency, and lower the burdenon the patient for retaining enemas after administration, all of whichshould improve compliance, reduce the risk for systemic toxicity, andmaximize therapeutic efficacy.

MATERIALS AND METHODS

Study designThe goal of this study was to engineer an inflammation-targeting drugdelivery system (IT-hydrogel) for IBD of the colon. We hypothesizedthat negatively charged IT-hydrogel microfibers should rapidly adhereto inflamed colon mucosa and selectively release their drug cargo upon

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pically normal and inflamed sites after incubation with (DiD + Dex)/gel. (B)Fluorescence intensity values for individual patient biopsy samples from his-tologically normal or inflamed locations. Values were normalized by tissuedry weight; data are means of duplicate samples. (C) Paired fluorescence in-tensity values for the patients in (B). Lines connected the paired means ofduplicate samples for individual patients (n= 6). The P valuewas determinedby paired t test.

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enzymatic digestion. After characterization of the IT-hydrogel in vitro,we examined whether drug delivery via IT-hydrogel enemas would af-fect therapeutic efficacy and systemic drug absorption in two mousemodels of IBD, DSS-induced colitis and the spontaneous TRUCmodel.Throughout this study, the anti-inflammatory drug Dex was used as amodel drug—either the hydrophobic pro-drugDex-Pal for loading intoIT-hydrogel microfibers or the water-soluble Dex-21 phosphate.Animals were randomly assigned to different treatment groups. DSS-treated animalswithoutweight losswere excluded from the study beforerandomization. For imaging experiments, 5 to 7 mice were used pergroup; for treatment and pharmacokinetic studies, we used 7 to 10miceper group on the basis of previous studies. All animals were included inthe analysis. Histopathology was analyzed by an experienced gastro-intestinal pathologist (J.N.G.) blinded to group assignment using anestablished scoring system. Adhesion of IT-hydrogel to human colonmucosa was tested using biopsy specimens from patients with UC. Pa-tients were from the Brigham and Women’s Hospital (BWH) Crohn’sand Colitis Center who had given informed consent to collect biopsyspecimens for research purposes under a protocol approved by the In-stitutional Review Board of Partners HealthCare.

MiceTRUC (T-bet−/−Rag2−/−) (19) and Rag2−/− lines on a BALB/c geneticbackground were maintained in a specific pathogen–free animal facilityat theHarvard School of PublicHealth (HSPH). Themicewere housed inmicroisolator cages with Sulfatrim [sulfamethoxazole (1 g/liter) +trimethoprim (0.2 g/liter); Hi-Tech Pharmacal] added to the drinkingwater. TRUCandRag2−/−micewereused for experiments at 8 to 10weeksof age. Adult BALB/c wild-type mice were purchased from the JacksonLaboratory. Experiments involving wild-type mice were performedeither at HSPH or at the David H. Koch Institute for Integrative CancerResearch atMassachusetts Institute of Technology (MIT). All mousestudies were performed according to institutional and National Insti-tutes of Health (NIH) guidelines for humane animal use. Experimentalprotocols were approved by the Animal Care Committees at HarvardUniversity and MIT.

Preparation of IT-hydrogelIT-hydrogel (3 to 8%, w/v) was prepared usingDMSO (Sigma-Aldrich)and H2O as a solvent pair (volume ratio, DMSO/H2O = 1:4). After dis-solving AP (Sigma-Aldrich) in DMSO, H2O was added dropwise. Thevial was heated to 60° to 80°C until AP was completely dissolved andthen allowed to cool down to room temperature (18). The formed hy-drogel was washed with PBS and centrifuged at 6000 rpm for 10 min(3×). For preparation of sterile hydrogels, all solutions were sterilizedbefore gelation using 0.20-mm syringe filters. Hydrogel morphologywas characterized by environmental scanning electron microscopy(FEI/Philips XL30 FEG, 1000×, acceleration voltage, 10.0 kV). Thewashed hydrogel pellet was suspended in PBS for polarized optical mi-croscopy imaging (Zeiss Axioplan2, 40×).

Dex encapsulationDex-Pal (Toronto ResearchChemicals Inc.) wasmixedwithAP inDMSObefore adding H2O. To quantify the drug-loading efficiency (weight ofincorporated drugdividedbyweight of drug-loaded gel) and encapsulationefficiency (weight of incorporated drug divided by weight of input drug),the hydrogel pellet was lyophilized and dissolved in DMSO for high-performance liquid chromatography (HPLC) analysis.

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Cell culturePrimary macrophages were generated by injecting wild-type BALB/cmice with 2.5ml of 3% thioglycolate (BDBiosciences) intraperitoneally.Peritoneal cells were harvested on day 3 by lavage with ice-cold PBS.Cells (1.5 × 106)were plated in 3ml of RPMI 1640 (Cellgro) supplemen-ted with 10% fetal calf serum (Atlanta Biologicals), 10 mM Hepes,1 mM sodium pyruvate, 2 mM L-glutamine, penicillin (100 U/ml),streptomycin (100 mg/ml) (all Cellgro), and 50 mM b-mercaptoethanol(Sigma-Aldrich) (RPMI-C). The cells were allowed to adhere overnight.They were then stimulated with LPS (Escherichia coli 0111:B4, Sigma-Aldrich) at 1 mg/ml. Supernatant was harvested after 24, 48, and 72 hours.THP-1 cells (American Type Culture Collection) were cultured in RPMI-C.For differentiation into macrophages, 1.25 × 106 cells were seeded onsix-well plates in 2.5 ml of RPMI-C plus 50 nM phorbol 12-myristate13-acetate 3 days before stimulation.Mediumwas changed to freshme-dium 1 day before stimulation with LPS (100 ng/ml). Supernatant washarvested after 4 and 24 hours and frozen at −80°C until analysis.

Ex vivo and in vivo gel adhesion experimentsAnimals with colitis (TRUC or DSS) and disease-free controls (Rag2−/−

or untreated wild type, respectively) were on an alfalfa-free diet (HarlanLaboratories) for 1 week before experiments. For ex vivo adhesion test-ing, the most distal 1.5 cm of colon tissue excluding the anus wasdissected. Two hundred microliters of (DiD + Dex)–loaded 4% IT-hydrogel [(DiD + Dex)/gel] was suspended in 6 ml of PBS. The colonwas inverted and incubated in 0.5 ml of gel suspension for 30 min at37°C with gentle shaking (34). After washing with PBS (3×), the colonwas opened longitudinally and imaged using an IVIS fluorescenceimager (IVIS 200, PerkinElmer) with the luminal side facing up.

For in vivo adhesion testing, animals were fasted overnight, and thefollowing morning, each mouse received an enema of 100 ml (DiD +Dex)/gel. Individual mice were anesthetized with 2.5% isoflurane, a20-gauge flexible disposable feeding needle (Braintree Scientific) wasadvanced into the rectum 3 cm past the anus, (DiD + Dex)/gel was ad-ministered, the catheter was removed, and the anus was kept closedmanually for 1 min. Animals were sacrificed after 12 hours. The distal3 cm of the colon was removed and imaged freshly without washing.The fluorescence signal intensity was quantified using Living Imagesoftware (version 4.3.1, PerkinElmer) in a standard-size ROI drawnaround individual colon pieces. Background fluorescence intensitydetermined as the average of three ROIs not containing any colon tissuewas subtracted from all specimens.

In vivo treatment of established colitisTRUC mice were randomized to one of four experimental groups:(i) control (no treatment or 100 ml of PBS enema), (ii) gel enema (4%IT-hydrogel suspended in 100 ml of PBS), (iii) free Dex enema (Dex-21phosphate in 100 ml of PBS, 70 mg of Dex equivalent), and (iv) Dex/gelenema (Dex-Pal in 4% IT-hydrogel suspended in 100 ml of PBS, 70 mgof Dex equivalent). Enemas were administered on days 1 and 3 afterfasting themice overnight as described. Duplicate aliquots of the enemasuspension or solution given to the mice in groups iii and iv were ana-lyzed by HPLC to confirm the Dex equivalent content in Dex-Pal orDex-21 phosphate.

In one control experiment (Fig. 4F), TRUCmice received four dailyconsecutive intraperitoneal injections of freeDex (70 mg ofDex equivalent)or PBS alone. In a second control experiment (Fig. 4G), TRUC micereceived enemas on days 1 and 3 of Dex-Pal (70 mg of Dex equivalent)

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dissolved in 100 ml of 5% ethanol + 5% Tween 80 (Sigma-Aldrich) orcarrier alone.

All mice were sacrificed for histopathological analysis on day 5.Colons were isolated, fixed in 4% paraformaldehyde, and embeddedin paraffin. Standard H&E-stained sections were examined and scoredby an experienced pathologist (J.N.G.) in a blinded fashion. Mono-nuclear cell infiltration, polymorphonuclear cell infiltration, epithelialhyperplasia, and epithelial injury are the four components in the scorethat were independently graded as absent (0), mild (1), moderate (2), orsevere (3), giving a total score of 0 to 12 (19, 24).

Ex vivo gel adhesion experiments with colon biopsyspecimens from patients with UCTwo biopsies from inflamed and normal mucosa as evaluated endosco-pically were taken from each patient, placed immediately in PBS, andtransported on ice to the laboratory. A third biopsy from each site wassent to the clinical pathology laboratory for routine histopathologyevaluation. The adhesion test was performed within 4 hours of thebiopsy as follows. Two hundredmicroliters of (DiD +Dex)–loaded 4%IT-hydrogel was suspended in 6 ml of PBS. Individual specimens wereincubated in 0.5 ml of the gel suspension for 30min at 37°C with gentleshaking. After washing with PBS (3×), the samples were placed on ablack plastic sheet and imaged using an IVIS fluorescence imager (IVIS200, PerkinElmer). Fluorescence signal intensity was quantified usingthe Living Image software (version 4.3.1, PerkinElmer). The specimenswere subsequently air-dried and weighed to determine the weight-normalized fluorescence signal intensity. Specimens from 11 patients(table S1) were assayed. For inclusion in the analysis (Fig. 6), we re-quired that endoscopy and histopathology reports were concordantfor either “normal” or “inflamed”mucosa; for this reason, four patientswere excluded; a fifth patient was excluded because the inflamed spec-imen was derived from the appendiceal orifice (table S1).

Statistical analysisThe two-tailed Student’s t test was used to compare differences betweentwo experimental groups, except for the study with UC patients wherethe paired t test was used. In experimentswithmultiple groups, one-wayANOVA with Tukey post hoc test was used. A value of P < 0.05 wasconsidered statistically significant. Statistical analysis and graphingwereperformed with Prism 6 (GraphPad Software).

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/7/300/300ra128/DC1MethodsFig. S1. Additional in vitro characterization of IT-hydrogel.Fig. S2. IT-hydrogel lacks in vitro cytotoxicity against human colon epithelial cells.Fig. S3. IT-hydrogel preferentially adheres to positively charged substrates.Fig. S4. Fluorescent imaging demonstrates adhesion of IT-hydrogel to the inflamed mucosa.Fig. S5. Inflamed colon mucosa retains (DiD + Dex)–loaded IT-hydrogel, but not free dye.Fig. S6. The effect of free Dex or Dex-loaded IT-hydrogel enemas on barrier function in vivo.Table S1. Clinical data of all UC patients (n = 11) recruited for this study.References (35–41)

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Acknowledgments: We thank S. Malstrom for advice on IVIS imaging setup and data analysis,W. Salmon for assistancewith polarized lightmicroscopy and the confocalmicroscopy, J. Ramirez forexpert animal care, Q. Wang for help with the rheological assay, D. Huang for help with the in vitroadhesion assay, and M. Ma and N. Bertrand for helpful discussions. Funding: S.Z. received a fellow-ship from the Natural Sciences and Engineering Research Council of Canada. P.K.V. received a Ra-malingaswami Re-entry Fellowship from the Department of Biotechnology, India. This work wassupported by Harvard Institute of Translational Immunology/Helmsley Trust Pilot Grants in Crohn’sDisease to J.E., L.H.G., and J.M.K.; NIH grant CA112663 to L.H.G.; NIH grant T32DK7191-38-S1 toG.T.; NIHgrants DE013023 and EB000244 and aMax Planck Research Award from theAlexander vonHumboldtFoundation to R.L.; and NIH grants DE023432 and AR063866 to J.M.K. Author contributions: S.Z.and J.E. designed and performed experiments, analyzed the data, and wrote themanuscript. M.D.S.andA.Z. assisted in performing experiments.M.J.H., B.C., and J.R.K. provided thebiopsy samples frompatients at the BWH Crohn’s and Colitis Center. J.N.G. analyzed histology samples. P.K.V. helpedanalyze data and edited the manuscript. L.H.G., G.T., R.L., and J.M.K. designed experiments, super-vised studies, and edited and revised themanuscript. Competing interests: L.H.G. has equity in andsits on the Board of Directors of Bristol-Myers Squibb. J.M.K., P.K.V., S.Z., and R.L. hold a patent relatedto this technology: “Nanostructured gels capable of controlled release of encapsulated agents” (U.S.Patent 20130280334A1).Data andmaterials availability:Materials are readily available andwill beprovided under the material transfer policies of the MIT and BWH.

Submitted 31 December 2014Accepted 1 July 2015Published 12 August 201510.1126/scitranslmed.aaa5657

Citation: S. Zhang, J. Ermann, M. D. Succi, A. Zhou, M. J. Hamilton, B. Cao, J. R. Korzenik,J. N. Glickman, P. K. Vemula, L. H. Glimcher, G. Traverso, R. Langer, J. M. Karp, Aninflammation-targeting hydrogel for local drug delivery in inflammatory bowel disease. Sci.Transl. Med. 7, 300ra128 (2015).

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ranslationalMedicine.org 12 August 2015 Vol 7 Issue 300 300ra128 10

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An inflammation-targeting hydrogel for local drug delivery in inflammatory bowel disease

Jonathan N. Glickman, Praveen K. Vemula, Laurie H. Glimcher, Giovanni Traverso, Robert Langer and Jeffrey M. KarpSufeng Zhang, Joerg Ermann, Marc D. Succi, Allen Zhou, Matthew J. Hamilton, Bonnie Cao, Joshua R. Korzenik,

DOI: 10.1126/scitranslmed.aaa5657, 300ra128300ra128.7Sci Transl Med

areas of epithelial inflammation in humans.regions. These findings together suggest that this new gel-based delivery system could reach and directly treat in another chemically induced mouse model of colitis, the hydrogel microfibers preferentially stuck to the inflameddid the free drug, which runs the risk of also affecting healthy tissues. In tissue samples from patients with UC and

a type of IBD. The hydrogels relieved inflammation in the animals more effectively than−−of ulcerative colitis (UC) positively charged. Dexamethasone-loaded hydrogels were administered as an enema to a genetic mouse model

self-assemble and deliver hydrophobic anti-inflammatory drugs directly to the inflamed colon surface, which is . designed a negatively charged hydrogel that couldet alget potent drugs right to the site of inflammation, Zhang

Inflammation is a driving factor of many chronic diseases, such as inflammatory bowel disease (IBD). ToCharged gels cozy up to inflamed tissues

ARTICLE TOOLS http://stm.sciencemag.org/content/7/300/300ra128

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