Synthesis, Characterization, and In Vivo Biodistribution of 125I

ARTICLE pubs.acs.org/Biomac

Synthesis, Characterization, and In Vivo Biodistribution of 125I-Labeled Dex-g-PMAGGCONHTyr Deqian Wang,†,‡ 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 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 in pH 7.4 PBS and 0.9% saline solutions. The graft copolymers were labeled with 125I, and the labeling stability in 0.9% saline solution was investigated. Pharmacokinetics studies showed a rapid clearance of 125I-labeled graft copolymers from the blood pool. Biodistribution images confirmed the preferable liver and spleen accumulation within 1 h after injection and rapid clearance from all the organs over time. The graft copolymer with molecular weight of 9.8 kDa was eliminated from the kidney significantly faster than those with higher molecular weight. The effect of the numbers of
COOH groups on the graft copolymers on the biodistribution was also investigated. It was found that the graft copolymers with the average number of
COOH groups per glucopyranose unit (DS
COOH) of 0.57 and 0.18 are mainly distributed in liver and spleen at 1 h after injection, whereas the graft copolymer with DS
COOH of 0.07 is mainly accumulated in kidney.

1. INTRODUCTION Macromolecular-based drug carriers have attracted increasing interest because of their potential applications in cancer diagnosis, imaging, and treatment.1
14 The macromolecules are usually water-soluble, nontoxic, biocompatible, and conjugates of small-molecule drugs/radionuclide15
17 improving drug solubility, blood clearance time, and the possibility of target delivery of the drug/radionuclide payloads via passive18,19 or active targeting approach.20,21 The biodistribution and blood clearance time of such drug conjugates depends on the physicochemical characteristics of the carrier system.6,22 To achieve the suitable biodistribution and blood clearance time of the drug conjugates, we have investigated both synthetic and natural polymers as the carriers of drug delivery system. Among the synthetic polymers, N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers have the advantages of biocompatibility, nonimmunogenicity, nontoxicity, and water solubility and have been investigated extensively, for example, 99mTc-radiolabeled HPMA copolymer6 and its RGE4C conjugate,5 90Y/99mTcr 2011 American Chemical Society

radiolabeled HPMA copolymer-RGE4C conjugate,7 and 111Inradiolabeled HPMA copolymer-(RGDfK)-(CHX-A00 -DTPA) conjugates.8 Among natural polymers, dextran is a water-soluble, biodegradable, nonimmunogenic, and nontoxic glucose polymer that can be enzymatic digestion in the human body. Dextran and its derivatives have been exploited extensively in biomedical, biotechnological, and pharmaceutical fields.23 Dextran labeled with radionuclides 99mTc, 188R, 14C, and so on, are the potential biological imaging agents.24,25 The conjugation with therapeutic/imaging agents can improve the pharmacokinetics and pharmacodynamics of these agents.26
29 Meanwhile, the 125I-EGFdextran can remain cell-associated for more than 20 h, which is much longer than that of unconjugated EGF. Therefore, dextran can be potentially used as a carrier of toxic drug or radioactive Received: February 11, 2011 Revised: March 28, 2011 Published: April 06, 2011 1851

dx.doi.org/10.1021/bm200194s | Biomacromolecules 2011, 12, 1851–1859

Biomacromolecules

ARTICLE

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

nuclides for cancer therapy.30 Besides that, the pharmacokinetics of normal dextran derivatives have also been investigated, such as [99mTc] MAG3-mannosyl-dextran,31 14C-radiolabeled diethylaminoethyldextran (DEAED), and 14C-radiolabeled carboxymethyldextran (CMD).32 However, the efficient radiotherapy of HPMA-based copolymers and dextran derivatives is still limited for the lack of active carboxyl groups. Graft copolymerization may be a versatile approach for the introduction of desired active carboxyl groups for the desirable conjugation of radionuclide 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 because of its excellent solubility, biocompatibility, and containing
COOH group for further conjugating of functional groups. The effect of molecular weight and negative charge numbers on the biodistribution and the blood clearance time of 125I-radiolabeled Dex-g-PMAGGCONHTyr after intravenous injection was studied. The general relationship between the physicochemical characteristics and in vivo behavior of soluble graft copolymer was discussed.

2. EXPERIMENTAL SECTION 2.1. Materials. Dextran (6, 40, and 100 kDa) (Fluka) was purified before use. In detail, dextran was first dissolved in water and filtered to remove insoluble impurities. The solution was dialyzed (cut off molecular 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 to obtain the purified dextran. Potassium persulfate (K2S2O8) and sodium hydrogen sulfite (NaHSO3) were A.R. grade and supplied by Beijing Reagent, and N-(3-dimethyl aminopropyl)-N0 -ethylcarbodiimide hydrochloride (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 chemicals and solvents were used as received. Water with the resistivity of 18.2 mΩ 3 cm from Milli-Q Reference Water 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 the synthesis of the graft copolymers are as follows. Synthesis of Dex-g-PMAGGCOOH Copolymers (I). N-methacryloylglycylglycine (MAGGCOOH) was synthesized according to literature.33 The graft copolymerization of MAGGCOOH onto dextran was carried out using K2S2O8/NaHSO3 redox system as initiators. Dextran (250 mg) was first dissolved in 6 mL of water; then, K2S2O8 (2.7 mg, 0.01 mmol) and NaHSO3 (1.4 mg, 0.01 mmol) were added. The solution was bubbled with nitrogen at room temperature for 30 min to remove the oxygen. Then, 4 mL of MAGGCOOH aqueous solution was added slowly under a nitrogen atmosphere. The reaction mixture was transferred 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-off molecular weight of 14 kDa) and dialyzed against water (replaced every 12 h) for 48 h to remove the remained initiators and homopolymers. The resultant solutions were freeze-dried; then, the possibly remaining homopolymers were removed by Soxhlet extraction with acetone. The resultant copolymers were dried in vacuum at 60 C.34 Yield: 86%. 1852

dx.doi.org/10.1021/bm200194s |Biomacromolecules 2011, 12, 1851–1859

Biomacromolecules H 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 min for preactivation,35
37 after which L-tyrosine and Na2CO3 mixed solution (n(Tyr):n(Na2CO3) 1:9) was added dropwise. The reactant feeding ratio was n(
COOH):n(Tyr):n(EDC):n(NHS) 1:1:1:0.5. The reaction mixture was kept in water bath at 25 C and stirred for 3 h and then 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 the nonreacted L-tyrosine and catalysts. The resultant solutions were freezedried to obtain the dextran graft poly (N-methacryloylglycylglycine) copolymer-tyrosine conjugates (Dex-g-PMAGGCONHTyr). Yield: 75.6%. 1 H 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). 125 I-Labeled Dex-g-PMAGGCONHTyr (III). Dex-g-PMAGGCONHTyr was labeled at room temperature and ambient pressure with 125I using the Iodogen method as previously reported.38 Briefly, 100 μg of Dex-g-PMAGGCONHTyr and 37 MBq of Na125I in phosphate buffer solution (0.2 M, pH 7.4) were added to a glass vial coated with 20 μg Iodogen for 10 min for the 125I labeling. The molar ratio of Na125I to Ltyrosine groups is 10
120 in the feeding reaction mixture. Generally, all the L-tyrosine groups can be labeled under this labeling condition. The resultant labeled copolymer was purified by a PD MiniTrap G-25 column (28-9180-07-07, GE Healthcare) equilibrated with phosphate buffer (0.2 M, pH 7.4) to remove unreacted radioiodide. The radioactive fractions containing 125I-labeled copolymer were collected and passed through a 0.2 μm syringe filter for further in vivo experiments. 2.3. Characterization and Instruments. The 1H NMR measurements were carried out on a Bruker 400 MHz Avance NMR instrument using D2O as the solvent. In general, each proton NMR spectrum was collected by 16 scans with a relaxation time of 3 s. Bruker TOPSPIN 2.0 software was used for the integration of the NMR spectra. Elemental analysis of the graft copolymer was performed on a Flash EA 1112 elemental analyzer. The FTIR spectra (KBr) were recorded on a BrukerEquinox 55 FT-IR spectrometer. Dynamic light scattering (DLS) experiments were carried out on the ALV/SP-150 spectrometer equipped with 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 the light 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 2 days at room temperature and filtered through the Millipore Millex-FH nylon filter (0.45 μm) before DLS experiments. All measurements were carried out at the scattering angle of 90 at 25 C. All solution concentrations were 0.5 mg/mL. The hydrodynamic radius () was obtained by fitting the correlation function with the CONTIN program. 2.4. Animal Studies. All the 125I-labeled copolymer were purified using a PD MiniTrap G-25 column before mouse studies. The PD MiniTrap G-25 column was washed with 6 mL of PBS and was activated with 2 mL of 1% BSA before purification. After the PD MiniTrap G-25 column was loaded with radiotracer (∼100 μL) and was then washed with 4 mL of PBS, the 0.5 mL between 0.6 and 1.1 mL of eluent was collected. We prepared doses for mouse studies by dissolving the purified radiotracer in 0.9% saline to give a concentration of 100 μCi/ mL for biodistribution studies and 2.5 mCi/mL for imaging. Each mouse was injected with 0.1 mL of radiotracer solution (10 μCi/mouse). All animal experiments were performed in accordance with guidelines of Peking University Health Science Center Animal Care and Use Committee.

ARTICLE

1

2.5. Pharmacokinetics and Biodistribution Experiments. For pharmacokinetics studied, seven BALB/c normal mice were used as one group for the blood clearance experiment of one graft copolymer radiotracer. The 125I-labeled copolymer (10 μCi in 0.1 mL 0.9% saline) was administered intravenously to each mouse. Blood was harvested from orbital sinus at 1, 3, 5, 7, 10, 15, 20, 30, 60, 90, and 120 min postinjection (p.i.), and the radioactivity was measured using a γ-counter (Wallac 1470-002, Perkin-Elmer, Finland). The uptakes of radiotracer in blood were calculated as the percentage of the injected dose per gram of blood mass (%ID/g). For biodistribution studies, 16 BALB/c normal mice were randomly divided into four groups, each of which had four mice. The 125I-labeled copolymer (10 μCi in 0.1 mL 0.9% saline) was administered intravenously to each mouse. Mice were anesthetized with intraperitoneal injection of sodium pentobarbital at a dose of 45.0 mg/kg. Time-dependent biodistribution studies were carried out by sacrificing mice at 1, 4, 24, and 48 h postinjection. Blood, heart, liver, spleen, kidney, stomach, intestine, muscle, and bone were harvested, weighed, and measured for radioactivity in a gamma counter (Wallac 1470-002, Perkin-Elmer, Finland). The organ uptake was calculated as a percentage of injected dose per gram of wet tissue mass (%ID/g). The biodistribution data and blood clearance curve were reported as an average plus the standard variation. A comparison between two different radiotracers was also made using the one-way ANOVA test (GraphPad Prim 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 administered with 250 μCi of 125I-labeled copolymer in 0.1 mL 0.9% saline. Mice were anesthetized with intraperitoneal injection of sodium pentobarbital at a dose of 45.0 mg/kg and then were placed supine on a three-head γ-camera (GE Healthcare, Millennium VG SPECT) equipped with a parallel-hole, low-energy, and high-resolution collimator. Anterior images were acquired at 4 h post injection and stored digitally in a 128  128 matrix. The acquisition count limits were set at 200 K. The mouse was sacrificed by cervical dislocation after the completion of imaging study.

3. RESULTS AND DISCUSSION 3.1. Synthesis of the Graft Copolymers. Radicals can be resulted on the oxidation of polysaccharides on the chain,39 which will then initiate the polymerization of a vinyl monomer to yield a graft copolymer.40
43 Among various methods that can initiate the free radical copolymerization on to polysaccharides, the redox initiators are proven to be effective.43 In this work, the K2S2O8/NaHSO3 redox pair was used to initiate the graft copolymerization. The synthesis route of the graft copolymers and hereafter labeling with 125I is shown in Scheme 1. For the synthesis of Dex-g-PMAGGCOOH graft copolymers, the glucose 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 MAGGCOOH monomers to result graft copolymers. The reaction parameters 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 the selection of the optimal graft copolymerizing conditions are shown in Figure S1 of the Supporting Information. The influences of monomer concentrations on the graft ratio (G) and graft efficiency (Ge) are listed in Table 1. G and Ge were defined as G = W3/W2  100 wt % and Ge = W3/W1  100 wt %, where W1, W2, and W3 are the mass of the conversed monomer, the graft copolymer, and the side chains in the copolymer, respectively. PMAGGCOOH homopolymers are generally obtained as 1853

dx.doi.org/10.1021/bm200194s |Biomacromolecules 2011, 12, 1851–1859

Biomacromolecules

ARTICLE

Table 1. Experimental Details and Information for the Dex-gPMAGGCOOH Graft Copolymersa n(Dex)/ run n(monomer) Ge (%)b G (%)b DS
COOHb N (%)c Mw (kDa)d 1 2

1:1 1:0.33

68 62

3

1:0.14

64.5

40.5 17

0.57 0.18

6.43 2.84

165 120

8.3

0.07

1.87

108

a

Molecular weight of dextran is Mw = 100 kDa. All reactions were carried out at 30 C for 9 h with the initiator concentration c(K2S2O8) = c(NaHSO3) = 1 mmol/L. b Ge%, G%, and the degree of substitution of
COOH groups (DS
COOH) per glucose ring were calculated by 1 H NMR. c Calculated by element analysis. d Mw of the graft copolymer was calculated by 1H NMR.

Table 2. Experimental Details and Information for the Dex-gPMAGGCONHTyr Graft Copolymersa Mw,copolymer run 1

(kDa)a 9.8

DS
COOHb

N%c

Tyr (%)d

nTyre

NTyre

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

1.2

Reactions were carried out at 25 C for 3 h, the feeding ratio of n[
COOH]:n[Tyr]:n[EDC]:n[NHS] 1:1:1:0.5. The Dex-g-PMAGGCOOH copolymers with Mw of 9.8 and 65 kDa were synthesized from dextran with Mw of 6 and 40 kDa, respectively. Mw values of the graft copolymers were calculated by 1H NMR. b Average MAGGCOOH unit per glucose ring. c N % is nitrogen element content in the graft copolymers estimated by element analysis. d Molar percent of L-tyrosine to all
COOH. e nTyr and NTyr are average number of L-tyrosine per glucose ring and per copolymer chain estimated by Tyr%  DS
COOH and Tyr%  DS
COOH  DPdex, respectively. a

the byproduct in the graft copolymerization. Therefore, the resultant reaction mixture was dialyzed against pure water to remove the homopolymer and initiators, by which purified graft copolymers 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-tyrosine and the
COOH groups on the Dex-g-PMAGGCOOH graft copolymers. The amide reaction was carried out in aqueous solution at pH 10 to 11 with Dex-g-PMAGGCOOH graft copolymer 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 performed at 25 C for 3 h. To evaluate the biological properties of the graft copolymers as the drug carriers, small amounts of L-tyrosine are needed to conjugate with Dex-g-PMAGGCOOH for labeling 125I. It was found that the amount of L-tyrosine conjugated to Dex-g-PMAGGCOOH is predetermined by the molar ratio of the feeding EDC to
COOH in the reaction system (Supporting Information, Figure S2). In the present work, n(EDC):n(
COOH) 1 was used for the linkage of L-tyrosine to Dex-g-PMAGGCOOH, and the details of the resultant 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 of Dex-g-PMAGGCOOH (Figure 1b) besides the typical peaks

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.

from protons of dextran backbone at δ 3.40
4.00 and 4.95 (Figure 1a). This peak comes from hydrogen protons of the methyl (
CH3) and methylene (
C
CH2
) of the PMAGGCOOH side chains. 13C NMR data also confirms the successful synthesis of Dex-g-PMAGGCONHTyr. The peaks of PMAGGCOOH side chains appear at chemical shift of δ 177.19
180.07, 174.37
175.53, 170.59
172.36 (Supporting Information, Figure 3a). On the 1H NMR spectrum of Dex-g-PMAGGCONHTyr, 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 shown besides the typical peaks of PMAGGCOOH (Figure 1c). The characteristic peaks of carbon atoms from benzene ring appear at 1854

dx.doi.org/10.1021/bm200194s |Biomacromolecules 2011, 12, 1851–1859

Biomacromolecules

ARTICLE

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

Figure 3. Hydrodynamic radius distribution of graft copolymer Dexg-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.

chemical shift of δ 115.44, 129.03, 130.71 on the 13C NMR spectrum (Supporting Information, Figure S3a). Figure 2 shows the FTIR spectra of dextran and its graft copolymers. The absorption peaks at 1534, 1737, and 1655 cm
1 correspond to N
H stretching vibration (ν(N
H)), CdO stretching vibration (ν(CdO)) of carboxyl groups, and amide groups at 1655 cm
1, respectively (Figure 2b,c), which confirms the successful synthesis of the dextran graft copolymers. The absorption peak of the ν(CdO) at 1737 cm
1 becomes weak and also indicates that part of the carboxyl groups were conjugated with Ltyrosine (Figure 2c). Elemental analysis also shows the presence of nitrogen atoms in the graft copolymers (Tables 1 and 2), which also confirms 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
COOH groups per glucopyranose unit (DS
COOH), can be estimated by DS
COOH = A2/2A1, where A1 and A2 are the integrated areas of the protons on dextran backbone (δ 4.95) and the methylene of
C
CH2
groups of the PMAGGCOOH side chains (δ 1.62
2.10 ) on 1H NMR spectra, respectively. The molecular weight of Dex-g-PMAGGCOOH graft copolymers was calculated by Mw,Dex-g-PMAGGCOOH = Mw,Dex þ 184nDS
COOH, where Mw,Dex is the molecular weight of dextran and 184 is the molar mass of MAGGCOOH, and n is the average numbers of glucose unit per dextran chain. The

DS
COOH and the molecular weight of the Dex-g-PMAGGCOOH graft copolymers that are used for the amide formation 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 ortho position hydrogen numbers of benzene ring groups from L-tyrosine at around δ 7.10 and A1 and A3 are the integrated areas of the hydrogen of dextran backbone at δ 4.95 and the ortho-position hydrogen of benzene ring groups from L-tyrosine at around δ 7.10 on 1H NMR spectra, respectively. The details of the synthesized Dex-g-PMAGGCONHTyr for further 125I labeling are listed in Table 2. 3.2. Dex-g-PMAGGCONHTyr Copolymers in Aqueous Solutions. The chain conformation of the Dex-g-PMAGGCONHTyr in physiological environment is important for the application as 125I carrier. Figure 3 shows the hydrodynamic radius of the Dex-g-PMAGGCONHTyr in water, PBS solution at pH 7.4, and 0.9% saline solution. The results indicate that in the aqueous solution, the average hydrodynamic radius () shows a double distribution mode, which suggests the formation of aggregates in the system. This could be attributed to hydrophobic action to make Dex-g-PMAGGCONHTyr macromolecular chains partially aggregated in the aqueous solution, which due to carboxyl pKa value is lower than 6.5; most carboxyl groups have not been ionized in the aqueous solution at pH of 5 to 6, and a small amount of benzene ring groups also appeared in the side chains from L-tyrosine.44,45 In the PBS or 0.9% saline solution, the of the Dex-g-PMAGGCONHTyr shows a single distribution mode. The average is about 10 and 20 nm for Dex-g-PMAGGCONHTyr with molecular weight of 65 and 165 kDa, respectively. The results suggest that no aggregation was formed in the pH 7.4 PBS and 0.9% saline solutions of Dex-gPMAGGCONHTyr copolymers, which is due to the fact that the COOh groups alongside chains of the graft copolymers have been 1855

dx.doi.org/10.1021/bm200194s |Biomacromolecules 2011, 12, 1851–1859

Biomacromolecules shielded by the counterions to prevent the aggregation of the graft copolymers. 3.3. Labeling of Dex-g-PMAGGCONHTyr with 125I and In Vitro Stability. Dex-g-PMAGGCONHTyr was labeled with 125I by using the Iodogen method.46
48 The resultant labeled copolymers were purified by PD MiniTrapTM G-25 column. Figure 4 shows the representative ITLC chromatograms of 125Ilabeled Dex-g-PMAGGCONHTyr with the acetone as elute. By this method, free 125I migrated to the solvent front whereas 125I-

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

ARTICLE

labeled copolymer remained at the origin. The labeling yields for all 125I-labeled graft copolymers were around 56
70%, and the radiochemical purities (RCP) for 125I-labeled graft copolymers after PD MiniTrapTM G-25 column purification were higher than 99%. The solution stability of the PD MiniTrapTM G-25 column purified 125I-labeled Dex-g-PMAGGCONHTyr was monitored by ITLC for 36 h. Figure 5 shows the solution stability of the 125I-labeled Dex-g-PMAGGCONHTyr with molecular weight of 9.8, 65, and 165 kDa in 0.9% saline. The results indicate that the labeled graft copolymer is quite stable in 0.9% saline, and the RCP remains above 80% at 36 h of postpurification. 3.4. Pharmacokinetics and Biodistribution of the 125ILabeled Dex-g-PMAGGCONHTyr. Effect of Molecular Weight on Pharmacokinetics and Biodistribution. Figure 5 shows biodistribution in different organs of the 125I-labeled Dex-g-PMAGGCONHTyr with different molecular weight as a function of time after injection. The results indicate that the graft copolymers are washed out rapidly from blood circulation and distributed into different organs at 1 h. All graft copolymers are mainly distributed in liver and spleen at 1 h, which may be due to the discontinuous endothelium and basement membranes of the vasculature in the two organs, which allow easy permeation of the Dex-g-PMAGGCONHTyr carrier and initial entrapment in the extravascular space.49 Concentration of the graft copolymers then decreased rapidly from all organs over time, suggesting reduced normal organ toxicity due to a reasonably efficient clearance, for example, from liver (Figure 6d). Meanwhile, comparing the graft copolymers with different molecular weight

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 a function 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. 1856

dx.doi.org/10.1021/bm200194s |Biomacromolecules 2011, 12, 1851–1859

Biomacromolecules

ARTICLE

Figure 7. Representative scintigraphic image of the BALB/c normal mice administered with ∼250 μCi of molecular weight after injection of 4 h.

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

(9.8, 65 kDa), the graft copolymer with molecular weight of 165 kDa shows a much lower radioactivity uptake in normal organs, which will be an advantage as a carrier further conjugating with targeting moiety to do receptor-targeted tumor imaging or therapy. Figure 7 shows a typical scintigraphic image of the mouse at 4 h after administration of ∼250 μCi 125 I-labeled Dex-gPMAGGCONHTyr graft copolymer using the BALB/c nude mice. The results show that the 125I-labeled graft copolymer (9.8 kDa) excreted by both hepatobiliary system and renal system (Figure 7a). The continued renal clearance may be attributed to the fact that the molecular weight of the graft copolymer is