L-Serine–modified polyamidoamine dendrimer as ahighly potent renal targeting drug carrierSatoru Matsuuraa,1, Hidemasa Katsumia,1,2, Hiroe Suzukia, Natsuko Hiraia, Hidetaka Hayashia, Kazuhiro Koshinob,Takahiro Higuchib,c, Yusuke Yagid, Hiroyuki Kimurad, Toshiyasu Sakanea, and Akira Yamamotoa

aDepartment of Biopharmaceutics, Kyoto Pharmaceutical University, 607-8414 Kyoto, Japan; bDepartment of Bio-Medical Imaging, National Cerebral andCardiovascular Center Research Institute, 565-8565 Osaka, Japan; cDepartment of Nuclear Medicine, Wuerzburg University, 97080 Wuerzburg, Germany;and dDepartment of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, 607-8414 Kyoto, Japan

Edited by Robert M. Stroud, University of California, San Francisco, CA, and approved August 28, 2018 (received for review May 11, 2018)

Effective delivery of drug carriers selectively to the kidney ischallenging because of their uptake by the reticuloendothelialsystem in the liver and spleen, which limits effective treatment ofkidney diseases and results in side effects. To address this issue,we synthesized L-serine (Ser)–modified polyamidoamine den-drimer (PAMAM) as a potent renal targeting drug carrier. Approx-imately 82% of the dose was accumulated in the kidney at 3 hafter i.v. injection of 111In-labeled Ser-PAMAM in mice, while i.v.injection of 111In-labeled unmodified PAMAM, L-threonine modi-fied PAMAM, and L-tyrosine modified PAMAM resulted in kidneyaccumulations of 28%, 35%, and 31%, respectively. Single-photonemission computed tomography/computed tomography (SPECT/CT) images also indicated that 111In-labeled Ser-PAMAM specifi-cally accumulated in the kidneys. An intrakidney distribution studyshowed that fluorescein isothiocyanate-labeled Ser-PAMAM accu-mulated predominantly in renal proximal tubules. Results of a cel-lular uptake study of Ser-PAMAM in LLC-PK1 cells in the presenceof inhibitors [genistein, 5-(N-ethyl-N-isopropyl)amiloride, and lyso-zyme] revealed that caveolae-mediated endocytosis, micropinocy-tosis, and megalin were associated with the renal accumulation ofSer-PAMAM. The efficient renal distribution and angiotensin-converting enzyme (ACE) inhibition effect of captopril (CAP), anACE inhibitor, was observed after i.v. injection of the Ser-PAMAM-CAP conjugate. These findings indicate that Ser-PAMAM is a prom-ising renal targeting drug carrier for the treatment of kidney dis-eases. Thus, the results of this study demonstrate efficient renaltargeting of a drug carrier via Ser modification.

drug delivery | renal targeting | L-serine | dendrimer

The kidney plays an important role in maintaining the ho-meostasis of body fluids, and filters waste products and extra

water from the blood to produce urine (1–3). Various drugs, suchas angiotensin-converting enzyme (ACE) inhibitors, steroids,and immunosuppressive agents, have been developed for thetreatment of kidney diseases, including renal cancer, glomerulardisease, and acute and chronic renal failure. However, deliveringthese drugs selectively to the kidney is difficult, which limits ef-fective treatment of kidney diseases and results in side effects.Thus, there is an urgent need for an effective renal targetingsystem that can improve the therapeutic efficacy of drugs forkidney diseases.Of the various strategies available, conjugation of drugs with

targeting ligands via chemical modification appears to be apromising approach for renal drug targeting (4–6). However,chemically modified conjugates are generally distributed in theliver and spleen because of uptake by the reticuloendothelialsystem (7, 8). Several studies have demonstrated the successfuluse of lysozyme, a low molecular weight protein that is filtered inthe glomerulus and reabsorbed in the proximal tubules, and poly(vinylpyrrolidone-codimethyl maleic acid) (PVD) as renal tar-geting ligands (9–11). Haas et al. (9) reported that a conjugate ofnaproxen and lysozyme was taken up in proximal convolutedtubules via endocytosis. Kamada et al. (11) reported that a

conjugate of superoxide dismutase and PVD, synthesized from4 to 4′-azobis(4-cyanovaleric acid), N-vinyl-2-pyrrolidone, anddimethylmaleic anhydride via radical copolymerization, accu-mulated predominantly in the kidney after i.v. injection in mice.Although these ligands were effective for renal targeting, theimmunogenicity of lysozyme as an exogenous protein is a con-cern, and PVD exhibits size polydispersity because it is synthe-sized via a classical radical reaction; moreover, the number offunctional groups available for chemical modifications is limited.Furthermore, PVD is hardly metabolized after administration.The present study is a tissue distribution study of various types

of amino acid-modified dendrimers for kidney-targeted drugdelivery. Our aim was to develop a renal targeting system usingL-serine (Ser) modification, and to characterize the relationshipbetween the physicochemical properties and the tissue distributionof Ser-modified macromolecules, with the goal of establishing astrategy for the rational design of Ser-modified macromolecules asdrug carriers and their use as therapeutics for kidney diseases. Tothis end, we selected polyamidoamine dendrimer (PAMAM) as amacromolecule (12–14) and examined the tissue distribution ofSer-modified PAMAM (Ser-PAMAM) after i.v. injection in micein terms of PAMAM generation, physicochemical properties,and dose. Furthermore, the intrakidney distribution, delivery

Significance

Delivery of most drug carriers to the kidney is limited becauseof their uptake by the reticuloendothelial system in the liverand spleen. We have developed L-serine (Ser)–modified poly-amidoamine dendrimer (PAMAM) as a potent renal targetingdrug carrier for the treatment of kidney diseases. Pharmaco-kinetic and single-photon emission computed tomography/computed tomography studies indicated that Ser modificationresults in efficient kidney targeting of PAMAM. Ser-PAMAMaccumulated predominantly in proximal tubules, a pattern as-sociated with the pathogenesis of renal cell carcinoma andchronic renal failure. Efficient renal distribution and pharma-cologic effect of captopril was observed after i.v. injection ofthe Ser-PAMAM-captopril conjugate. Thus, our results demon-strate successful kidney targeting of a drug carrier via Sermodification.

Author contributions: H. Katsumi designed research; S.M., H. Katsumi, H.S., N.H., H.H.,K.K., T.H., Y.Y., and H. Kimura performed research; K.K., T.H., Y.Y., and H. Kimura con-tributed new reagents/analytic tools; S.M., H. Katsumi, T.S., and A.Y. analyzed data; andS.M., H. Katsumi, and A.Y. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1S.M. and H. Katsumi contributed equally to this work.2To whom correspondence should be addressed. Email: hkatsumi@mb.kyoto-phu.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1808168115/-/DCSupplemental.

Published online September 24, 2018.

www.pnas.org/cgi/doi/10.1073/pnas.1808168115 PNAS | October 9, 2018 | vol. 115 | no. 41 | 10511–10516

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route to the kidney, and mechanism of renal uptake of Ser-PAMAM were investigated after i.v. injection in mice. Finally,the tissue distribution and pharmacologic effects of captopril (CAP),an ACE inhibitor, was examined in mice after i.v. injection of aSer-PAMAM-CAP conjugate, in which multi-CAP molecules werecovalently bound to Ser-PAMAM through disulfide linkages.

ResultsTable 1 shows the physiochemical properties of PAMAM, L-tyrosine–modified PAMAM (Tyr-PAMAM), L-threonine–modified PAMAM(Thr-PAMAM), Ser-modified PAMAM (Ser-PAMAM), and CAP-conjugated Ser-PAMAM (Ser-PAMAM-CAP). For this study, weselected the second, third, and fourth generations of PAMAM(G2, G3, and G4) as bioinert dendrimer backbones (12–14). Themean diameters of PAMAM derivatives were ∼2–5 nm. PAMAMderivatives had a positive charge ranging from 2.58 to 24.77 mV,and the positive charge gradually increased in association with theirgeneration and amino groups. The number of CAP modificationson PAMAM was 5.75 in Ser-PAMAM (G3)-CAP.Fig. 1 shows the plasma concentration profiles and tissue

distribution of 111In-labeled PAMAM derivatives after i.v. in-jection in ddY mice. PAMAM (G4) and Tyr-PAMAM (G3)accumulated mainly in the liver and kidney tissues. Thr-PAMAM(G3) slowly disappeared from blood circulation, and ∼34.9% ofthe dose accumulated in the kidney within 180 min after in-jection (Fig. 1 A–C).

Ser-PAMAMs rapidly disappeared from the blood circulation,and the plasma retention of Ser-PAMAMs was inversely pro-portional to the generation of PAMAM. Approximately 47.9%,81.7%, and 47.2% of the dose was accumulated in the kidney at180 min after i.v. injection of Ser-PAMAM (G2), Ser-PAMAM(G3), and Ser-PAMAM (G4), respectively. Although Ser-PAMAMs accumulated slightly in the liver (∼4.15%), no sig-nificant radioactivity was detected in the spleen, heart, or lungs(Fig. 1 D–F).Table 2 shows the pharmacokinetics parameters of Ser-PAMAM

(G3), Thr-PAMAM (G3), Tyr-PAMAM (G3), and PAMAM (G4).The hepatic uptake clearance (CLliver) of Ser-PAMAM (G3) wasalmost equivalent to that of Thr-PAMAM and much lower thanthat of PAMAM (G4) and Tyr-PAMAM (G3). The renal uptakeclearance (CLkidney) of Ser-PAMAM (G3) was ∼4.87 mL/h, whichwas almost 79.1% of the total body clearance.Fig. 2 A and B show the in vivo and ex vivo biodistribution images

of near- infrared (NIR) fluorescence dye-labeled Ser-PAMAM[NIR-labeled Ser-PAMAM (G3) and NIR-labeled PAMAM (G4)],obtained using the IVIS imaging system (PerkinElmer) after i.v.injection in HR-1 mice. Fluorescence intensity derived from NIR-PAMAM (G4) was almost absent in vivo, with weak signals de-tected in the liver and kidney ex vivo. In contrast, high fluores-cence intensity derived from Ser-PAMAM (G3) was specificallyobserved in the kidney at 60 min after i.v. injection.Fig. 2C shows the biodistribution image of 111In-labeled Ser-

PAMAM (G3), obtained using single-photon emission computedtomography/computed tomography (SPECT/CT) after i.v. in-jection in ddY mice. Specific renal accumulation of 111In-labeledSer-PAMAM was clearly observed, although slight bladder ac-cumulation was also observed.Fig. 3 shows the microscopic images of mouse renal sections at

60 min after i.v. injection of fluorescein isothiocyanate (FITC)-labeled Ser-PAMAM (G3). As shown in Fig. 3 A and B, thefluorescence intensity of FITC-labeled Ser-PAMAM (G3) wasalmost absent in the renal medulla, whereas high fluorescenceintensity was observed in the renal cortex. Furthermore, fluo-rescence from FITC-labeled Ser-PAMAM (G3) was clearly ob-served in the proximal tubules (Fig. 3C).To elucidate the effect of glomerular filtration on the renal

distribution of Ser-PAMAM (G3), we performed a pharmaco-kinetic study of 111In-labeled Ser-PAMAM (G3) after i.v. in-jection in mice with HgCl2-induced acute renal failure (ARF).The glomerular filtration rate was significantly decreased afterHgCl2 treatment, indicating that ARF was established by thismethod (Fig. 4D). Although HgCl2-induced ARF had no sig-nificant effect on the plasma concentration profile of Ser-PAMAM (G3) (Fig. 4A), the renal accumulation and uptakeclearance of 111In-labeled Ser-PAMAM (G3) were decreased inmice with HgCl2-induced ARF (Fig. 4 B and C).Fig. 5 A and B show the time course of apical-to-basolateral

(absorptive direction) and basolateral-to-apical (secretory di-rection) transport of FITC-labeled Ser-PAMAM (G3) andPAMAM (G4) across the monolayers of LLC-PK1 cells (anepithelial cell line derived from proximal tubular cells of porcine

Table 1. Physiochemical properties of PAMAM derivatives

Compound Mean diameter, nm Mean ζ potential, mV

PAMAM (G4) 4.20 ± 0.09 4.56 ± 0.81Ser-PAMAM (G2) 2.50 ± 0.12 6.04 ± 0.31Ser-PAMAM (G3) 4.03 ± 0.29 4.76 ± 0.70Ser-PAMAM (G4) 4.39 ± 0.26 24.77 ± 0.67Thr-PAMAM (G3) 4.15 ± 0.35 2.58 ± 1.36Tyr-PAMAM (G3) 3.17 ± 0.35 5.26 ± 3.00Ser-PAMAM (G3)-CAP 4.75 ± 0.27 3.43 ± 0.61

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Fig. 1. Time courses of plasma concentration and tissue accumulation ofvarious amino acid-modified dendrimers in mice after i.v. injection at a doseof 1 mg/kg. (A) 111In-labeled PAMAM (G4). (B) Thr-PAMAM (G3). (C) Tyr-PAMAM (G3). (D) Ser-PAMAM (G2). (E) Ser- PAMAM (G3). (F) Ser-PAMAM(G4). Results are expressed as mean ± SE values for three mice. 〇, plasma;▲, liver; ■, kidney; ◇, spleen; △, heart; □, lung.

Table 2. Pharmacokinetic parameters of PAMAM derivatives

CompoundDose,mg/kg

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Clearance, mL/h

Total Liver Kidney

Ser-PAMAM (G3) 1 16.25 6.16 0.10 4.87Thr-PAMAM (G3) 1 80.98 1.23 0.05 0.55Tyr-PAMAM (G3) 1 1.17 85.25 33.44 25.19PAMAM (G4) 1 4.72 21.19 8.21 5.37

AUC, area under the plasma concentration–time curve.

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kidneys). There were no significant differences in the transportof FITC-labeled PAMAM (G4) in the two directions. In con-trast, the apical-to-basolateral (absorptive direction) transport ofFITC-labeled Ser-PAMAM (G3) was higher than the basolateral-to-apical (secretory direction) transport. These results indicatethat Ser-PAMAM (G3) was preferentially transported in theabsorptive direction.Fig. 5C shows the cellular uptake of 111In-labeled Ser-

PAMAM (G3) in the presence of cellular uptake inhibitors.Chlorpromazine (a clathrin-mediated endocytosis inhibitor) hadno significant effect on the cellular uptake of 111In- labeled Ser-PAMAM (G3). In contrast, genistein (a caveolae-mediated en-docytosis inhibitor), 5-(N-ethyl-N-isopropyl)amiloride (a macro-pinocytosis inhibitor), and lysozyme (a megalin substrate)significantly decreased the cellular uptake of 111In-labeled Ser-PAMAM (G3). In addition, cellular uptake of Ser-PAMAM(G3) was significantly inhibited in the presence of excess un-labeled Ser-PAMAM (G3).Fig. 6A shows the plasma concentration and tissue distribution

of 111In-labeled Ser-PAMAM (G3)-CAP after i.v. injection in

mice. Although the plasma retention of 111In-labeled Ser-PAMAM (G3)-CAP was slightly higher than that of 111In-labeled Ser-PAMAM (G3) (Figs. 1 and 6), 111In-labeled Ser-PAMAM (G3)-CAP accumulated predominantly in the kidney.Approximately 80.9% of the dose was accumulated in the kid-ney at 180 min after i.v. injection of 111In-labeled Ser-PAMAM(G3)-CAP.Fig. 6 B and C show the pharmacokinetics of CAP after i.v.

injection of CAP alone and Ser-PAMAM (G3)-CAP. Theplasma concentration of CAP after i.v. injection of Ser-PAMAM(G3)-CAP was slightly higher than that after injection of CAPalone (Fig. 6B). In addition, the renal accumulation of CAP afterinjection of Ser-PAMAM (G3)-CAP was greater than that afterinjection of CAP alone (Fig. 6C). After incubation of Ser-PAMAM (G3)-CAP in plasma for up to 4 h, free CAP was al-most undetectable in plasma.Fig. 6D shows renal ACE activity after i.v. injection of CAP

alone or Ser-PAMAM (G3)-CAP. CAP alone and Ser-PAMAM(G3)-CAP significantly inhibited renal ACE activity 30 at minafter i.v. injection in ddY mice. Furthermore, the decrease in the

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Fig. 2. In vivo and ex vivo imaging of NIR fluores-cence dye-labeled Ser-PAMAM (G3) and PAMAM(G4) 60 min after i.v. injection in mice. The fluores-cence intensities were measured in (A) whole miceand (B) tissues (liver, kidney, spleen, heart, and lung).(C) SPECT/CT imaging of 111In-labeled Ser-PAMAM(G3) at 180 min after i.v. injection in a mouse.

Fig. 3. Intrakidney distribution of FITC-labeled Ser-PAMAM (G3) in renaltissue sections at 60 min after i.v. injection in mice. (A) Cortex. (Scale bar:200 μm.) (B) Medulla. (Scale bar: 200 μm.) (C) Magnified image of the cortex.(Scale bar: 25 μm.) Fluorescence intensity was observed using a confocallaser-scanning microscope.

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Fig. 4. Plasma concentration, renal accumulation, renal clearance, andglomerular filtration rate (GFR) of 111In-labeled Ser-PAMAM (G3) after i.v.injection at a dose of 1 mg/kg in normal mice and mice with HgCl2-inducedacute renal failure (ARF). Time course of (A) plasma concentration and (B)renal accumulation. 〇, normal mice; ●, HgCl2-induced ARF mice. (C) Renalclearance and (D) GFR in normal mice (normal) and mice with HgCl2-inducedARF (ARF). Results are expressed as the mean ± SE for three mice.

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ACE activity persisted for a longer duration after injection ofSer-PAMAM (G3)-CAP than after injection of CAP alone.Fig. 7 shows plasma concentrations of creatinine and blood

urea nitrogen (BUN), along with histological micrographs of thekidney after i.v. injection of Ser-PAMAM (G3). Plasma con-centrations of creatinine and BUN were significantly increased

by s.c. injection of HgCl2 as a positive control (Fig. 7 A and B). Inaddition, infiltration of inflammatory cells and necrotic and/ordamaged cells was observed in the histological sections of renaltissue from ddY mice treated with HgCl2 (Fig. 7C). In contrast,Ser-PAMAM (G3) had no significant effect on creatinine andBUN levels, and histological sections of renal tissue from Ser-PAMAM (G3)–treated mice were similar to those from naïveand PBS-treated mice (Fig. 7C).

DiscussionIn the present study, biodistribution and imaging studiesrevealed that Ser-PAMAM (G3) was specifically distributed tothe kidneys after i.v. injection in mice. Thus, our results dem-onstrate effective renal targeting using Ser modification. BecauseSer is a biomolecule and a biocompatible compound, Ser mod-ification represents a safer mode of drug delivery.Fluorescent microscopic images indicated that Ser-PAMAM

(G3) was localized predominantly to the renal cortex, especiallyto the proximal tubule. The proximal tubules are involved in thepathogenesis of kidney diseases, such as chronic kidney failureand renal cell carcinoma (15–18). Thus, these findings indicatethat Ser-PAMAM (G3) represents a promising drug carrierfor the treatment of kidney diseases. Nanoparticles with adiameter <5.5 nm are efficiently filtered in the glomerulus andexcreted into urine (19). Given the mean diameter of Ser-PAMAM (G3) of ∼4 nm in the present study, we hypothesizedthat Ser-PAMAM (G3) is filtered in the glomerulus and reab-sorbed in the lumen of the proximal tubule. We demonstratedthat the renal clearance of Ser-PAMAM (G3) was proportionalto the glomerular filtration rate in the mice with HgCl2-inducedARF. These results, together with the permeability directionfindings in LLC-PK1 cells, indicate that Ser-PAMAM (G3) wasdelivered to the proximal tubule through glomerular filtration.In the present study, renal accumulation of Ser-PAMAM (G3)

was saturated at high doses (10 mg/kg; SI Appendix, Fig. S2). Inaddition, the results of the in vitro cellular uptake study in LLC-PK1 cells suggest the involvement of caveolae-mediated endo-cytosis, macropinocytosis, or megalin in the renal accumulation

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Fig. 5. Transport and cellular uptake of various amino acid-modified den-drimers in LLC-PK1 cells. Time course of (A) FITC-labeled Ser-PAMAM (G3)and (B) PAMAM (G4) transport across LLC-PK1 cell monolayers in the ab-sorptive directions and secretory directions. ■,▲, absorptive directions; □,△, secretory directions (C) Cellular uptake study of 111In-labeled Ser-PAMAM in LLC-PK1 cells in the presence of various inhibitors. a, 111In-labeled Ser-PAMAM (G3). b, 111In-labeled Ser-PAMAM (G3) + 100 μg/mLunlabeled Ser-PAMAM (G3). c, 111In-labeled Ser-PAMAM (G3) + 100 μMchlorpromazine. d, 111In-labeled Ser-PAMAM (G3) + 370 μM genistein. e,111In-labeled Ser-PAMAM (G3) + 100 μM 5-(N-ethyl-N-isopropyl)amiloride. f,111In-labeled Ser-PAMAM (G3) + 1 mM lysozyme. Results are expressed asmean ± SE for three experiments. *P < 0.05, significantly different from the111In-labeled Ser-PAMAM (G3) group a.

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Fig. 6. Plasma concentration and tissue accumula-tion of 111In-labeled Ser-PAMAM (G3)-CAP and therenal accumulation and ACE inhibition activity ofCAP alone and Ser-PAMAM (G3)-CAP. (A) Timecourses of plasma concentration and tissue accumu-lation of 111In-labeled Ser-PAMAM (G3)-CAP after i.v.injection in mice at a dose of 1 mg/kg. 〇, plasma; △,liver; ■, kidney. (B and C) Plasma concentration (B)and renal accumulation (C) of CAP after i.v. injectionof CAP alone and Ser-PAMAM (G3)-CAP in mice at adose of 2 mg CAP/kg. 〇, CAP alone; ▲, Ser-PAMAM(G3)-CAP. (D) Effect of CAP alone and Ser-PAMAM(G3)-CAP on ACE activity in kidney 30 min or 120 minafter i.v. injection in mice at a dose of 0.5 mg CAP/kg.Results are expressed as mean ± SE for three mice.*P < 0.05, significantly different from the naive group.#P < 0.05, significantly different from the CAP and Ser-PAMAM (G3)-CAP groups at the same time.

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of Ser-PAMAM (G3). Although further studies are needed toelucidate the detailed mechanism of renal accumulation, theseresults indicate that active transport mechanisms contribute tothe renal uptake of Ser-PAMAMs.Pharmacokinetic studies of Ser-PAMAMs showed the effect

of size and the degree of Ser modification on the renal targetingof Ser-PAMAMs. Although the renal clearance of Ser-PAMAMwas proportional to the degree of Ser modification on PAMAM,the renal accumulation of a midsized Ser-PAMAM molecule,Ser-PAMAM (G3), was the greatest of all the Ser-PAMAMs.This is probably because glomerular filtration of Ser-PAMAMdecreased as the size of Ser-PAMAM increased. Thus, Ser-PAMAM (G3) strikes the best balance between molecular sizeand affinity for the proximal tubules.We also examined the pharmacokinetics of Thr-PAMAM

(G3) and Tyr-PAMAM (G3) because Thr and Tyr are aminoacids with hydroxyl groups, with a similar structure to Ser. Al-though Thr-PAMAM accumulated in the kidneys, the renal ac-cumulation of Thr- PAMAM (G3) was much lower than that ofSer-PAMAM (G3) because of the greater urinary excretion (SIAppendix, Fig. S3). This is likely because, compared with Ser, Thrhas one additional methylene group in its side chain, which mightplay a key role in attaining the optimal conformation for in-creased kidney accumulation. In contrast, Tyr-PAMAM (G3)and PAMAM (G4) accumulated in both the liver and the kidney.Tyr has a phenolic hydroxyl group, and PAMAM (G4) containsseveral amino groups. These findings indicate that efficient renaltargeting requires the presence of an alcoholic hydroxyl group.The increased renal accumulation of CAP after administration

of Ser-PAMAM (G3)-CAP is consistent with the pharmacoki-netics of 111In-Ser-PAMAM (G3). These results indicate thatSer-PAMAM (G3)-CAP released negligible amounts of CAP inthe blood circulation, and CAP modification had no significanteffect on the affinity of Ser toward the kidney. Prolonged ACEinhibition with Ser-PAMAM (G3)-CAP was observed because ofthe greater renal accumulation of CAP after i.v. injection ofCAP-loaded Ser-PAMAM. Because CAP was bound to Ser-PAMAM (G3) through disulfide linkage, we hypothesize thatthe linkage was cleaved by reduced thiols, such as glutathione,

and that pharmacologically active CAP was released in the cy-toplasm after renal distribution (20–23).Our toxicity study results indicate that Ser-PAMAM (G3)

showed no acute toxicity after repetitive administration. Al-though a long-term toxicity study is needed before approval forclinical use, these results indicate that Ser-PAMAM (G3) is arelatively safe drug carrier for kidney-targeted drug delivery.In conclusion, the renal targeting of PAMAM was success-

fully achieved using Ser modification. Approximately 82% of thedose accumulated in the kidney at 3 h after i.v. injection of 111In-labeled Ser-PAMAM (G3) in mice. An intrakidney distribu-tion study showed that FITC-labeled Ser-PAMAM (G3) accu-mulated predominantly in proximal tubules. The efficient renaldistribution and ACE inhibition effect of CAP, an ACE in-hibitor, was observed after i.v. injection of a Ser-PAMAM (G3)-CAP conjugate. These results indicate that Ser modification is

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2) Ser-PAMAM (G3)

SPDP-CAP

A

B

Fig. 8. Synthesis and structures of Ser-PAMAM (G3) (A) and Ser-PAMAM(G3)-CAP (B). SPDP, N-succinimidyl 3-(2-pyridyldithio)propionate.

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promising approach for renal targeting using a macromoleculardrug carrier.

Materials and MethodsFor this study, we selected PAMAMdendrimers with an ethylenediamine core(G2, G3, or G4) (Sigma-Aldrich) as bioinert dendrimer backbones. Ser-PAMAM(G3) was synthesized by reacting PAMAM (G3) with Boc-Ser(tBu)-OH den-drimers, using the HBTU-HOBt method (Fig. 8A) (24, 25). The reaction mix-tures were incubated at room temperature until the ninhydrin test yieldednegative results on TLC. Ser-PAMAM (G3) was identified using matrix-assistedlaser desorption/ionization time-of-flight mass spectrometry (Bruker) and1H-NMR spectroscopy (Bruker) in deuterated water. The mass of PAMAM(G3) was 6960 Da, and this increased to 9684 kDa, corresponding to ∼32molecules of conjugated Ser (SI Appendix, Fig. S1A). The peaks correspondingto Ser were observed in the 1H NMR spectrum at δ 3.72–3.85 (m, 2H) of Ser-PAMAM, and the integral ratio of the methylene protons of Ser to themethylene protons (CONHCH2) of PAMAM (G3) indicates that the desiredproduct was obtained (SI Appendix, Fig. S1B) (26).

To synthesize Ser-PAMAM (G2), Ser-PAMAM (G4), Thr-PAMAM (G3), andTyr-PAMAM (G3), we reacted each generation of PAMAM with Boc-Ser(tBu)-OH, Boc-Thr(tBu)-OH, or Boc-Tyr(tBu)-OH, using the same method as for Ser-PAMAM (G3) synthesis. To synthesize CAP-conjugated Ser-PAMAM (G3) [Ser-PAMAM (G3)-CAP], we conjugated CAP with Ser-PAMAM (G3) throughdisulfide linkages (Fig. 8B). The mean diameter and ζ potential in PBS wereanalyzed using a Zetasizer Nano (Malvern Instruments) at 25 °C.

For tissue distribution studies, we radiolabeled PAMAM dendrimer de-rivatives with 111In using a bifunctional chelating agent, diethylene-triaminepentaacetic anhydride, according to the method described byHnatowich et al. (27). Each modified PAMAM dendrimer was administeredi.v. to ddY mice at a dose of 1 or 10 mg/kg. To evaluate the pharmacokinetics

of CAP, CAP solution or Ser-PAMAM (G3)-CAP solution was administered i.v.to ddY mice at a dose of 2 mg CAP/kg. We used a previously describedmethod with slight modifications to analyze CAP (28).

To elucidate the effect of glomerular filtration on the renal distribution ofSer-PAMAM (G3), we performed a pharmacokinetic study of 111In-Ser-PAMAM (G3) in mice with HgCl2-induced ARF (11). Ex vivo and in vivo tissuedistribution were evaluated using the PerkinElmer IVIS imaging system orSPECT/CT (Bioscan) to image NIR fluorescence dye-labeled or 111In-labeledSer-PAMAM (G3) after i.v. injection. Intrakidney distribution of FITC-labeledSer-PAMAM (G3) after i.v. injection was observed with a fluorescence mi-croscope. Transport and cellular uptake of Ser-PAMAM (G3) were evaluatedin LLC-PK1 cells (an epithelial cell line derived from proximal tubular cells ofporcine kidney). To evaluate the toxicity of Ser-PAMAM (G3), we measuredcreatinine and BUN levels and observed kidney sections under a microscope(KEYENCE) after i.v. injection of Ser-PAMAM (G3) (1 mg/kg) once dailyfor 5 d in ddY mice.

All animal experiments were conducted according to the principles andprocedures outlined in the National Institutes of Health’s Guide for the Careand Use of Laboratory Animals (29). The Animal Experimentation Commit-tee of the Kyoto Pharmaceutical University and the Institutional Animal CareCommittee of the National Cerebral and Cardiovascular Center approved allexperimental protocols that used animals.

The experimental procedures are described in detail in SI Appendix,Materials and Methods.

ACKNOWLEDGMENTS. We thank Dr. Shugo Yamashita (Kyoto Pharmaceu-tical University) for supporting the synthesis of PAMAM derivatives. Thiswork was supported by the Japanese Ministry of Education, Culture, Sports,Science and Technology-Supported Program for the Strategic ResearchFoundation at Private Universities.

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