rXXXX American Chemical Society A dx.doi.org/10.1021/bm200194s | Biomacromolecules XXXX, XXX, 000–000

ARTICLE

pubs.acs.org/Biomac

Synthesis, Characterization, and In Vivo Biodistributionof 125I-Labeled Dex-g-PMAGGCONHTyrDeqianWang,†,‡ Jiyun Shi,§ Junjun Tan,†Xin Jin,†,‡Qinmei Li,†,‡Honglang Kang,†Ruigang Liu,*,† Bing Jia,*,§

and Yong Huang*,†,||,^

†Laboratory of Polymer Physics and Chemistry, Beijing National Laboratory of Molecular Sciences, Institute of Chemistry,Chinese Academy of Sciences, Beijing 100190, China‡Graduate University, Chinese Academy of Sciences, Beijing 100039, China§Medical Isotopes Research Center, Peking University, Beijing 100191, China

)Natural Research Center for Engineering Plastics, Technical Institute of Physics & Chemistry, Chinese Academy of Sciences,Beijing 100190, China^Laboratory of Cellulose and Lignocellulosics Chemistry, Guangzhou Institute of Chemistry, Chinese Academy of Sciences,Guangzhou, 510650, China

bS Supporting Information

1. INTRODUCTION

Macromolecular-based drug carriers have attracted increasinginterest because of their potential applications in cancer diag-nosis, imaging, and treatment.1�14 The macromolecules areusually water-soluble, nontoxic, biocompatible, and conjugatesof small-molecule drugs/radionuclide15�17 improving drug so-lubility, blood clearance time, and the possibility of targetdelivery of the drug/radionuclide payloads via passive18,19 oractive targeting approach.20,21 The biodistribution and bloodclearance time of such drug conjugates depends on the physico-chemical characteristics of the carrier system.6,22 To achieve thesuitable biodistribution and blood clearance time of the drugconjugates, we have investigated both synthetic and naturalpolymers as the carriers of drug delivery system. Amongthe synthetic polymers,N-(2-hydroxypropyl) methacrylamide(HPMA) copolymers have the advantages of biocompatibility,nonimmunogenicity, nontoxicity, and water solubility and havebeen investigated extensively, for example, 99mTc-radiolabeledHPMA copolymer6 and its RGE4C conjugate,5 90Y/99mTc-

radiolabeled HPMA copolymer-RGE4C conjugate,7 and 111In-radiolabeled HPMA copolymer-(RGDfK)-(CHX-A00-DTPA)conjugates.8

Among natural polymers, dextran is a water-soluble, biode-gradable, nonimmunogenic, and nontoxic glucose polymer thatcan be enzymatic digestion in the human body. Dextran and itsderivatives have been exploited extensively in biomedical, bio-technological, and pharmaceutical fields.23 Dextran labeled withradionuclides 99mTc, 188R, 14C, and so on, are the potentialbiological imaging agents.24,25 The conjugation with therapeu-tic/imaging agents can improve the pharmacokinetics and phar-macodynamics of these agents.26�29 Meanwhile, the 125I-EGF-dextran can remain cell-associated for more than 20 h, which ismuch longer than that of unconjugated EGF. Therefore, dextrancan be potentially used as a carrier of toxic drug or radioactive

Received: February 11, 2011Revised: March 28, 2011

ABSTRACT: Dextran graft poly (N-methacryloylglycylglycine)copolymer�tyrosine conjugates (dextran-g-PMAGGCONHTyr)were synthesized and characterized. Dynamic light scattering(DLS) results indicated that the graft copolymers are soluble inpH 7.4 PBS and 0.9% saline solutions. The graft copolymerswere labeled with 125I, and the labeling stability in 0.9% salinesolution was investigated. Pharmacokinetics studies showed arapid clearance of 125I-labeled graft copolymers from the bloodpool. Biodistribution images confirmed the preferable liverand spleen accumulation within 1 h after injection and rapidclearance from all the organs over time. The graft copolymer with molecular weight of 9.8 kDa was eliminated from the kidneysignificantly faster than those with higher molecular weight. The effect of the numbers of�COOH groups on the graft copolymerson the biodistribution was also investigated. It was found that the graft copolymers with the average number of�COOH groups perglucopyranose unit (DS�COOH) of 0.57 and 0.18 are mainly distributed in liver and spleen at 1 h after injection, whereas the graftcopolymer with DS�COOH of 0.07 is mainly accumulated in kidney.

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nuclides for cancer therapy.30 Besides that, the pharmacokineticsof normal dextran derivatives have also been investigated, such as[99mTc] MAG3-mannosyl-dextran,31 14C-radiolabeled diethyla-minoethyldextran (DEAED), and 14C-radiolabeled carboxy-methyldextran (CMD).32 However, the efficient radiotherapyof HPMA-based copolymers and dextran derivatives is stilllimited for the lack of active carboxyl groups. Graft copolymer-izationmay be a versatile approach for the introduction of desiredactive carboxyl groups for the desirable conjugation of radio-nuclide ligands and target moiety.

In this work, dextran graft poly(N-methacryloylglycylglycine)copolymer-tyrosine conjugates (Dex-g-PMAGGCONHTyr)were synthesized by radical polymerization in aqueous medium.N-methacryloylglycylglycine (MAGGCOOH) was used becauseof its excellent solubility, biocompatibility, and containing�COOH group for further conjugating of functional groups.The effect of molecular weight and negative charge numbers onthe biodistribution and the blood clearance time of 125I-radi-olabeled Dex-g-PMAGGCONHTyr after intravenous injectionwas studied. The general relationship between the physicochem-ical characteristics and in vivo behavior of soluble graft copoly-mer was discussed.

2. EXPERIMENTAL SECTION

2.1. Materials. Dextran (6, 40, and 100 kDa) (Fluka) was purifiedbefore use. In detail, dextran was first dissolved in water and filtered toremove insoluble impurities. The solution was dialyzed (cut off molec-ular weight of 3.5 kDa for dextran 6 kDa and 14 kDa for 40 and 100 kDa

dextran) against water (replaced every 12 h) for 48 h and freeze-dried toobtain the purified dextran. Potassium persulfate (K2S2O8) and sodiumhydrogen sulfite (NaHSO3) were A.R. grade and supplied by BeijingReagent, and N-(3-dimethyl aminopropyl)-N0-ethylcarbodiimide hydro-chloride (EDC) (g98%, Fluka), N-hydroxysuccimide (NHS) (g97%,Fluka), L-tyrosine (99%, Alfa Aesar), Iodogen (Sigma, St. Louis, MO),Na125I (Beijing Atom High Tech., Beijing, China), and other chemicalsand solvents were used as received. Water with the resistivity of 18.2mΩ 3 cm fromMilli-Q ReferenceWater Purification System (Millipore)was used for the reaction and purification of the graft copolymers.2.2. Synthesis of the Graft Copolymers. The synthesis route of

dextran graft copolymers is shown in Scheme 1. The details of thesynthesis of the graft copolymers are as follows.

Synthesis of Dex-g-PMAGGCOOH Copolymers (I). N-methacryloyl-glycylglycine (MAGGCOOH) was synthesized according to literature.33

The graft copolymerization of MAGGCOOH onto dextran was carriedout using K2S2O8/NaHSO3 redox system as initiators. Dextran (250mg) was first dissolved in 6 mL of water; then, K2S2O8 (2.7 mg, 0.01mmol) andNaHSO3 (1.4mg, 0.01mmol) were added. The solution wasbubbled with nitrogen at room temperature for 30 min to remove theoxygen. Then, 4 mL of MAGGCOOH aqueous solution was addedslowly under a nitrogen atmosphere. The reaction mixture was trans-ferred to a water bath set at 30 �C for 9 h for the graft copolymerization.The reaction mixture was then transferred to a dialysis bag (cut-offmolecular weight of 14 kDa) and dialyzed against water (replacedevery 12 h) for 48 h to remove the remained initiators and homo-polymers. The resultant solutions were freeze-dried; then, the possiblyremaining homopolymers were removed by Soxhlet extraction withacetone. The resultant copolymers were dried in vacuum at 60 �C.34Yield: 86%.

Scheme 1. Synthesis of Dex-g-PMAGGCONHTyr Copolymers and Labeling with 125I

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1H NMR (400 MHz, D2O, δ): 0.87�1.22 (3H, m, �CH3), 1.62�2.10 (2H,m,�C�CH2�), 3.40�4.00 (m, 9H, dextran), 4.95 (s, 1H,H1

from ring of dextran).Synthesis of Dex-g-PMAGGCONHTyr (II).Dex-g-PMAGGCOOH (50

mg) was dissolved in water to obtain a solution with pH of 3.5�4.5.Then, certain amounts of EDC/NHS were added and stirred for 10 minfor preactivation,35�37 after which L-tyrosine and Na2CO3 mixedsolution (n(Tyr):n(Na2CO3) 1:9) was added dropwise. The reactantfeeding ratio was n(�COOH):n(Tyr):n(EDC):n(NHS) 1:1:1:0.5. Thereaction mixture was kept in water bath at 25 �C and stirred for 3 h andthen transferred to a dialysis bag (cut-off molecular weight of 3.5 kDa)and dialyzed against water (replaced every 12 h) for 48 h to remove thenonreacted L-tyrosine and catalysts. The resultant solutions were freeze-dried to obtain the dextran graft poly (N-methacryloylglycylglycine)copolymer-tyrosine conjugates (Dex-g-PMAGGCONHTyr). Yield: 75.6%.

1H NMR (400 MHz, D2O, δ): 0.87�1.22 (3H, m, �CH3), 1.62�2.10 (2H, m, �C�CH2�), 2.70 (2H, s, �CH2� from L-tyrosine),3.40�4.00 (m, 9H, dextran), 4.95 (s,1H, H1 from ring of dextran), 6.70(s, 2H, m-phenyl hydrogen), 7.10 (s, 2H, o-phenyl hydrogen).

125I-Labeled Dex-g-PMAGGCONHTyr (III). Dex-g-PMAGGCONH-Tyr was labeled at room temperature and ambient pressure with 125Iusing the Iodogen method as previously reported.38 Briefly, 100 μg ofDex-g-PMAGGCONHTyr and 37 MBq of Na125I in phosphate buffersolution (0.2 M, pH 7.4) were added to a glass vial coated with 20 μgIodogen for 10 min for the 125I labeling. The molar ratio of Na125I to L-tyrosine groups is 10�120 in the feeding reaction mixture. Generally, allthe L-tyrosine groups can be labeled under this labeling condition. Theresultant labeled copolymer was purified by a PD MiniTrap G-25column (28-9180-07-07, GE Healthcare) equilibrated with phosphatebuffer (0.2M, pH 7.4) to remove unreacted radioiodide. The radioactivefractions containing 125I-labeled copolymer were collected and passedthrough a 0.2 μm syringe filter for further in vivo experiments.2.3. Characterization and Instruments. The 1H NMR mea-

surements were carried out on a Bruker 400 MHz Avance NMR instru-ment using D2O as the solvent. In general, each proton NMR spectrumwas collected by 16 scans with a relaxation time of 3 s. Bruker TOPSPIN2.0 software was used for the integration of the NMR spectra. Elementalanalysis of the graft copolymer was performed on a Flash EA 1112elemental analyzer. The FTIR spectra (KBr) were recorded on a Bruker-Equinox 55 FT-IR spectrometer. Dynamic light scattering (DLS)experiments were carried out on the ALV/SP-150 spectrometer equippedwith an ALV-5000 multi-τ digital time correlator and a solid-state laser(ADLS DPY 425II, output power ca. 400 MW at λ = 632.8 nm) as thelight source. All graft copolymers were dissolved in water, 0.9% saline,and phosphate buffer (0.1 M, pH 7.4); the solutions were stirred for 2days at room temperature and filtered through the Millipore Millex-FHnylon filter (0.45 μm) before DLS experiments. All measurements werecarried out at the scattering angle of 90� at 25 �C. All solutionconcentrations were 0.5 mg/mL. The hydrodynamic radius (<Rh>)was obtained by fitting the correlation function with the CONTINprogram.2.4. Animal Studies. All the 125I-labeled copolymer were purified

using a PD MiniTrap G-25 column before mouse studies. The PDMiniTrap G-25 column was washed with 6 mL of PBS and was activatedwith 2 mL of 1% BSA before purification. After the PD MiniTrap G-25column was loaded with radiotracer (∼100 μL) and was then washedwith 4 mL of PBS, the 0.5 mL between 0.6 and 1.1 mL of eluent wascollected. We prepared doses for mouse studies by dissolving thepurified radiotracer in 0.9% saline to give a concentration of 100 μCi/mL for biodistribution studies and 2.5mCi/mL for imaging. Eachmousewas injected with 0.1 mL of radiotracer solution (10 μCi/mouse). Allanimal experiments were performed in accordance with guidelinesof Peking University Health Science Center Animal Care and UseCommittee.

2.5. Pharmacokinetics and Biodistribution Experiments.For pharmacokinetics studied, seven BALB/c normal mice were used asone group for the blood clearance experiment of one graft copolymerradiotracer. The 125I-labeled copolymer (10 μCi in 0.1 mL 0.9% saline)was administered intravenously to each mouse. Blood was harvestedfrom orbital sinus at 1, 3, 5, 7, 10, 15, 20, 30, 60, 90, and 120 minpostinjection (p.i.), and the radioactivity was measured using aγ-counter (Wallac 1470-002, Perkin-Elmer, Finland). The uptakes ofradiotracer in blood were calculated as the percentage of the injecteddose per gram of blood mass (%ID/g). For biodistribution studies, 16BALB/c normal mice were randomly divided into four groups, each ofwhich had four mice. The 125I-labeled copolymer (10 μCi in 0.1 mL0.9% saline) was administered intravenously to each mouse. Mice wereanesthetized with intraperitoneal injection of sodium pentobarbital at adose of 45.0 mg/kg. Time-dependent biodistribution studies werecarried out by sacrificing mice at 1, 4, 24, and 48 h postinjection. Blood,heart, liver, spleen, kidney, stomach, intestine, muscle, and bone wereharvested, weighed, and measured for radioactivity in a gamma counter(Wallac 1470-002, Perkin-Elmer, Finland). The organ uptake was calcu-lated as a percentage of injected dose per gram of wet tissue mass (%ID/g).The biodistribution data and blood clearance curve were reported as anaverage plus the standard variation. A comparison between two differentradiotracers was also made using the one-way ANOVA test (GraphPadPrim 5.0, San Diego, CA). The level of significance was set at p = 0.05.2.6. Scintigraphic Imaging. Imaging studies were performed

using three BALB/c normal white mice. Each mouse was administeredwith 250 μCi of 125I-labeled copolymer in 0.1 mL 0.9% saline. Mice wereanesthetized with intraperitoneal injection of sodium pentobarbital ata dose of 45.0 mg/kg and then were placed supine on a three-headγ-camera (GE Healthcare, Millennium VG SPECT) equipped with aparallel-hole, low-energy, and high-resolution collimator. Anteriorimages were acquired at 4 h post injection and stored digitally in a128 � 128 matrix. The acquisition count limits were set at 200 K. Themouse was sacrificed by cervical dislocation after the completion ofimaging study.

3. RESULTS AND DISCUSSION

3.1. Synthesis of the Graft Copolymers. Radicals can beresulted on the oxidation of polysaccharides on the chain,39

which will then initiate the polymerization of a vinyl monomer toyield a graft copolymer.40�43 Among various methods that caninitiate the free radical copolymerization on to polysaccharides,the redox initiators are proven to be effective.43 In this work, theK2S2O8/NaHSO3 redox pair was used to initiate the graftcopolymerization. The synthesis route of the graft copolymersand hereafter labeling with 125I is shown in Scheme 1. For thesynthesis of Dex-g-PMAGGCOOH graft copolymers, the glu-cose rings of the dextran were first oxidized by the K2S2O8/NaHSO3 to result in free radicals on the dextran chain. Then,graft copolymerization took place after the addition of MAGG-COOH monomers to result graft copolymers. The reactionparameters were first optimized for the graft copolymerization.The optimal parameters are at the initiator of c(K2S2O8) =c(NaHSO3) = 1 mmol/L at 30 �C for 9 h. The details of theselection of the optimal graft copolymerizing conditions areshown in Figure S1 of the Supporting Information. The influ-ences of monomer concentrations on the graft ratio (G) and graftefficiency (Ge) are listed in Table 1. G and Ge were defined asG = W3/W2 � 100 wt % and Ge = W3/W1 � 100 wt %, whereW1, W2, and W3 are the mass of the conversed monomer, thegraft copolymer, and the side chains in the copolymer, respec-tively. PMAGGCOOH homopolymers are generally obtained as

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the byproduct in the graft copolymerization. Therefore, theresultant reaction mixture was dialyzed against pure water toremove the homopolymer and initiators, by which purified graftcopolymers can be obtained, as indicated in Experimental Section.Dex-g-PMAGGCONHTyr graft copolymers were synthesized

by the amide reaction between the �NH2 groups on L-tyrosineand the �COOH groups on the Dex-g-PMAGGCOOH graftcopolymers. The amide reaction was carried out in aqueoussolution at pH 10 to 11 with Dex-g-PMAGGCOOH graftcopolymer concentration of 4.1 mg/mL and the n(�COOH):n(Tyr):n(EDC):n(NHS) 1:1:1:0.5, and the reaction was per-formed at 25 �C for 3 h. To evaluate the biological properties ofthe graft copolymers as the drug carriers, small amounts ofL-tyrosine are needed to conjugate with Dex-g-PMAGGCOOHfor labeling 125I. It was found that the amount of L-tyrosineconjugated to Dex-g-PMAGGCOOH is predetermined by themolar ratio of the feeding EDC to �COOH in the reactionsystem (Supporting Information, Figure S2). In the presentwork, n(EDC):n(�COOH) 1 was used for the linkage ofL-tyrosine to Dex-g-PMAGGCOOH, and the details of the resul-tant graft copolymer for labeling 125I are listed in Table 2.Figure 1 shows the 1H NMR spectra of dextran and the graft

copolymers. The new peaks appear at chemical shift of δ 0.87�1.22 (m, 3H), 1.62�2.10 (m, 2H) in the 1H NMR spectrum ofDex-g-PMAGGCOOH (Figure 1b) besides the typical peaks

from protons of dextran backbone at δ 3.40�4.00 and 4.95(Figure 1a). This peak comes from hydrogen protons of themethyl (�CH3) and methylene (�C�CH2�) of the PMAGG-COOH side chains. 13C NMR data also confirms the successfulsynthesis of Dex-g-PMAGGCONHTyr. The peaks of PMAGG-COOH side chains appear at chemical shift of δ 177.19�180.07, 174.37�175.53, 170.59�172.36 (Supporting Informa-tion, Figure 3a). On the 1H NMR spectrum of Dex-g-PMAGG-CONHTyr, new peaks at δ 2.70 (s, 2H), 6.70 (s, 2H), and 7.10(s, 2H) according to the protons of L-tyrosine groups are shownbesides the typical peaks of PMAGGCOOH (Figure 1c). Thecharacteristic peaks of carbon atoms from benzene ring appear at

Table 1. Experimental Details and Information for the Dex-g-PMAGGCOOH Graft Copolymersa

run

n(Dex)/

n(monomer) Ge (%)b G (%)b DS�COOH

b N (%)c Mw (kDa)d

1 1:1 68 40.5 0.57 6.43 165

2 1:0.33 62 17 0.18 2.84 120

3 1:0.14 64.5 8.3 0.07 1.87 108aMolecular weight of dextran isMw = 100 kDa. All reactions were carriedout at 30 �C for 9 h with the initiator concentration c(K2S2O8) =c(NaHSO3) = 1 mmol/L. b Ge%, G%, and the degree of substitutionof�COOH groups (DS�COOH) per glucose ring were calculated by1H NMR. cCalculated by element analysis. dMw of the graft copolymerwas calculated by 1H NMR.

Table 2. Experimental Details and Information for the Dex-g-PMAGGCONHTyr Graft Copolymersa

run

Mw,copolymer

(kDa)a DS�COOHb N%c Tyr (%)d nTyr

e NTyre

1 9.8 0.60 7.60 15.3 0.098 3.5

2 65 0.53 6.02 6.9 0.037 9

3 165 0.57 6.43 2.6 0.014 9

4 120 0.18 2.84 2.8 0.005 3

5 108 0.07 1.87 2.8 0.002 1.2aReactions were carried out at 25 �C for 3 h, the feeding ratio ofn[�COOH]:n[Tyr]:n[EDC]:n[NHS] 1:1:1:0.5. The Dex-g-PMAGG-COOH copolymers with Mw of 9.8 and 65 kDa were synthesized fromdextran with Mw of 6 and 40 kDa, respectively. Mw values of the graftcopolymers were calculated by 1H NMR. bAverage MAGGCOOH unitper glucose ring. cN % is nitrogen element content in the graftcopolymers estimated by element analysis. dMolar percent of L-tyrosineto all �COOH. e nTyr and NTyr are average number of L-tyrosine perglucose ring and per copolymer chain estimated by Tyr% � DS�COOH

and Tyr% � DS�COOH � DPdex, respectively.

Figure 1. 1HNMR spectra of (a) dextran, (b) Dex-g-PMAGGCOOH,and (c) Dex-g-PMAGGCONHTyr in D2O at 400 MHz.

Figure 2. FTIR spectra of (a) dextran, (b) Dex-g-PMAGGCOOH, and(c) Dex-g-PMAGGCONHTyr.

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chemical shift of δ 115.44, 129.03, 130.71 on the 13C NMRspectrum (Supporting Information, Figure S3a). Figure 2 showsthe FTIR spectra of dextran and its graft copolymers. Theabsorption peaks at 1534, 1737, and 1655 cm�1 correspond toN�H stretching vibration (ν(N�H)), CdO stretching vibra-tion (ν(CdO)) of carboxyl groups, and amide groups at1655 cm�1, respectively (Figure 2b,c), which confirms thesuccessful synthesis of the dextran graft copolymers. The absorp-tion peak of the ν(CdO) at 1737 cm�1 becomes weak and alsoindicates that part of the carboxyl groups were conjugated with L-tyrosine (Figure 2c). Elemental analysis also shows the presence ofnitrogen atoms in the graft copolymers (Tables 1 and 2), which alsoconfirms the successful synthesis of the dextran graft copolymers.The content of the graft side chain PMAGGCOOH in the

graft copolymers, which was defined as the average �COOHgroups per glucopyranose unit (DS�COOH), can be estimated byDS�COOH = A2/2A1, where A1 and A2 are the integrated areasof the protons on dextran backbone (δ 4.95) and the methy-lene of�C�CH2� groups of the PMAGGCOOH side chains(δ 1.62�2.10 ) on 1H NMR spectra, respectively. Themolecular weight of Dex-g-PMAGGCOOH graft copoly-mers was calculated by Mw,Dex-g-PMAGGCOOH = Mw,Dex þ184nDS�COOH, where Mw,Dex is the molecular weight ofdextran and 184 is the molar mass of MAGGCOOH, and nis the average numbers of glucose unit per dextran chain. The

DS�COOH and the molecular weight of the Dex-g-PMAGG-COOH graft copolymers that are used for the amide forma-tion are listed in Table 1.The average numbers of L-tyrosine (NTyr) per glucopyranose

unit can be estimated by NTyr = A3/2A1, where 2 is the orthoposition hydrogen numbers of benzene ring groups fromL-tyrosine at around δ 7.10 and A1 and A3 are the integratedareas of the hydrogen of dextran backbone at δ 4.95 and theortho-position hydrogen of benzene ring groups from L-tyrosineat around δ 7.10 on 1HNMR spectra, respectively. The details ofthe synthesized Dex-g-PMAGGCONHTyr for further 125I label-ing are listed in Table 2.3.2. Dex-g-PMAGGCONHTyr Copolymers in Aqueous So-

lutions. The chain conformation of the Dex-g-PMAGGCONH-Tyr in physiological environment is important for the applicationas 125I carrier. Figure 3 shows the hydrodynamic radius of theDex-g-PMAGGCONHTyr in water, PBS solution at pH 7.4, and0.9% saline solution. The results indicate that in the aqueoussolution, the average hydrodynamic radius (<Rh>) shows adouble distribution mode, which suggests the formation ofaggregates in the system. This could be attributed to hydro-phobic action to make Dex-g-PMAGGCONHTyr macromole-cular chains partially aggregated in the aqueous solution, whichdue to carboxyl pKa value is lower than 6.5; most carboxyl groupshave not been ionized in the aqueous solution at pH of 5 to 6, anda small amount of benzene ring groups also appeared in the sidechains from L-tyrosine.44,45 In the PBS or 0.9% saline solu-tion, the <Rh> of the Dex-g-PMAGGCONHTyr shows a singledistribution mode. The average <Rh> is about 10 and 20 nm forDex-g-PMAGGCONHTyr with molecular weight of 65 and165 kDa, respectively. The results suggest that no aggregationwas formed in the pH 7.4 PBS and 0.9% saline solutions of Dex-g-PMAGGCONHTyr copolymers, which is due to the fact that theCOOh groups alongside chains of the graft copolymers have been

Figure 3. Hydrodynamic radius distribution of graft copolymer Dex-g-PMAGGCONHTyr with the molecular weight of (a) 65 and(b) 165 kDa in different media by DLS at room temperature,c = 0.5 mg/mL.

Figure 4. Representative ITLC chromatograms of 125I-labeledDex-g-PMAGGCONHTyr the original kit (a) and after purification(b). Free 125I�migrated to the solvent front whereas 125I-labeled polymerremained at the origin.

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shielded by the counterions to prevent the aggregation of thegraft copolymers.3.3. Labeling of Dex-g-PMAGGCONHTyr with 125I and In

Vitro Stability.Dex-g-PMAGGCONHTyr was labeled with 125Iby using the Iodogen method.46�48 The resultant labeledcopolymers were purified by PD MiniTrapTM G-25 column.Figure 4 shows the representative ITLC chromatograms of 125I-labeled Dex-g-PMAGGCONHTyr with the acetone as elute. Bythis method, free 125I migrated to the solvent front whereas 125I-

labeled copolymer remained at the origin. The labeling yields forall 125I-labeled graft copolymers were around 56�70%, and theradiochemical purities (RCP) for 125I-labeled graft copolymersafter PD MiniTrapTM G-25 column purification were higherthan 99%. The solution stability of the PD MiniTrapTM G-25column purified 125I-labeled Dex-g-PMAGGCONHTyr wasmonitored by ITLC for 36 h. Figure 5 shows the solutionstability of the 125I-labeled Dex-g-PMAGGCONHTyr withmolecular weight of 9.8, 65, and 165 kDa in 0.9% saline. Theresults indicate that the labeled graft copolymer is quite stablein 0.9% saline, and the RCP remains above 80% at 36 h ofpostpurification.3.4. Pharmacokinetics and Biodistribution of the 125I-

Labeled Dex-g-PMAGGCONHTyr. Effect of Molecular Weighton Pharmacokinetics and Biodistribution. Figure 5 shows biodis-tribution in different organs of the 125I-labeled Dex-g-PMAGG-CONHTyr with different molecular weight as a function of timeafter injection. The results indicate that the graft copolymers arewashed out rapidly from blood circulation and distributed intodifferent organs at 1 h. All graft copolymers are mainly distrib-uted in liver and spleen at 1 h, which may be due to the dis-continuous endothelium and basement membranes of the vas-culature in the two organs, which allow easy permeation of theDex-g-PMAGGCONHTyr carrier and initial entrapment in theextravascular space.49 Concentration of the graft copolymersthen decreased rapidly from all organs over time, suggestingreduced normal organ toxicity due to a reasonably efficientclearance, for example, from liver (Figure 6d). Meanwhile,comparing the graft copolymers with different molecular weight

Figure 5. Solution stability of 125I-labeled graft copolymers withmolecular weight of (a) 9.8, (b) 65, and (c) 165 kDa in 0.9% saline.

Figure 6. Tissue distributions of 125I-labeled Dex-g-PMAGGCONHTyr graft copolymers with molecular weight of (a) 9.8, (b) 65, and (c) 165 kDa at afunction of time after injection in BALB/c normal mice. The concentration of which was expressed as the percentage of injected dose per gram of tissue.The data points show the average of four animals.

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(9.8, 65 kDa), the graft copolymer with molecular weight of165 kDa shows a much lower radioactivity uptake in normalorgans, which will be an advantage as a carrier further conjugatingwith targeting moiety to do receptor-targeted tumor imaging ortherapy.Figure 7 shows a typical scintigraphic image of the mouse at

4 h after administration of ∼250 μCi 125I-labeled Dex-g-PMAGGCONHTyr graft copolymer using the BALB/c nudemice. The results show that the 125I-labeled graft copolymer(9.8 kDa) excreted by both hepatobiliary system and renal sys-tem (Figure 7a). The continued renal clearance may be attri-buted to the fact that the molecular weight of the graft copolymeris <20 kDa.26 For the 125I-labeled, Dex-g-PMAGGCONHTyrgraft copolymer withMw of 65 and 165 kDa, the copolymers aremainly accumulated in the liver and spleen organs. Figure 8shows the blood clearance of 125I-labeled copolymers with

different molecular weight. It shows that all graft copolymershave a rapid blood clearance and ∼90% of the graft copolymerswere washed out from the blood at 20 min after the injection; theresults may be because dextran is not biological inert and can berecognized by the reticuloendothelial system to lead to rapidclearance from blood.Effect of Negative Electronic Charge Numbers on Biodistri-

bution. For larger molecules with molecular weight above70 kDa, the effect of molecular charges on biodistribution seemsto be important. Negatively charged compounds showed higherradioactivity accumulation in the tumor than neutrally andpositively charge ones.32 The biodistribution of the graft copo-lymer with theDS�COOH of 0.07, 0.18, and 0.57 was investigated.It shows that the graft copolymer with DS�COOH of 0.57 and0.18 have preferable liver and spleen accumulation at 1 h afterinjection and have a similar lower radioactivity uptake innormal organs, whereas the graft copolymer with DS�COOH

of 0.07 is mainly accumulated in the kidney (Figure 9). It wasreported that carboxylated PVPs showed relatively high renalaccumulation. The highest renal accumulation was obtainedwith 20% carboxylated PVP; both noncarboxylated and 100%carboxylated PVP hardly accumulated in the kidneys.50 There-fore, we believe that there may be a threshold for targetingkidney accumulation, which is determined by the percent ofcarboxylated carriers, and DS�COOH of 0.07 may be thethreshold of Dex-g-PMAGGCONHTyr graft copolymers fortargeting kidney accumulation. All three Dex-g-PMAGG-CONHTyr graft copolymers show a rapid blood clearancebehavior (Figure 10).

4. CONCLUSIONS

A series of 125I-labeled Dextran-g-PMAGGCONHTyr graftcopolymers were successively synthesized. The graft copolymersare soluble in pH 7.4 PBS and 0.9% saline solutions. The labelingyields for all 125I-labeled graft copolymers were around 56�70%,and the labeled graft copolymer is quite stable in 0.9% saline, and

Figure 7. Representative scintigraphic image of the BALB/c normal mice administered with ∼250 μCi of 125I-labeled copolymers with differentmolecular weight after injection of 4 h.

Figure 8. Blood clearance of 125I-labeled Dex-g-PMAGGCONHTyrgraft copolymer in BALB/c normal mice. The data points show theaverage seven animals.

H dx.doi.org/10.1021/bm200194s |Biomacromolecules XXXX, XXX, 000–000

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the RCP remains above 80% at 36 h postpurification. The graftcopolymers have preferable liver and spleen accumulationand rapid blood clearance, whereas the graft copolymer withDS�COOH of 0.07 is mainly accumulated in kidney. The mechan-ism of kidney accumulation requires further investigation. Thiswork makes the first-step biological evaluation for 125I-labeledDex-g-PMAGGCONHTyr graft copolymers with desired�COOH numbers, which may be applied to in vivo efficientradiotherapy for tumors in the future.

’ASSOCIATED CONTENT

bS Supporting Information. The data on the selection ofthe optimal parameters of the graft copolymerization and amidereaction, 13CNMR spectra, and 1HNMR spectra with integral ofthe peaks of the graft copolymers are available. This material isavailable free of charge via the Internet at http://pubs.acs.org.

’AUTHOR INFORMATION

Corresponding Author*E-mail: rgliu@iccas.ac.cn (R.L.), jiabing@bjmu.edu.cn (B.J.),and yhuang@mail.ipc.ac.cn (Y.H.).

’ACKNOWLEDGMENT

The financial support of theNational Natural Science Founda-tion of China (grant nos. 20974114, 20774105, and 50821062) andthe Knowledge Innovation Program of the Chinese Academy ofSciences (grant no. KJCX2-YW-H19) is greatly appreciated.

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Figure 9. Tissue distributions of 125I-labeled Dex-g-PMAGGCONHTyr graft copolymer with different DP�COOH after injection of (a) 1, (b) 4, (c) 24,and (d) 48 h in BALB/c normal mice. The concentration of which was expressed as the percentage of injected dose per gram of tissue. The data pointsshow an average of four animals. Mw,Dex = 100 kDa.

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