Improving the blood clearance time of ¹²⁵I labeled Dex-g-PMAGGCONHTyr by copolymerization

Abstract

Dextran graft copolymers, including dextran graft poly(N-methacryloylglycylglycine) copolymers conjugated with polyethylene glycol and tyrosine (Dex-g-PMAGGCONHPEG_3k-NHTyr), dextran graft poly(N-(2-hydroxypropyl) methacrylamide-co-N-methacryloylglycylglycine)-tyrosine conjugates (Dex-g-P(HPMA-co-MAGGCONHTyr)), and dextran graft poly(methacrylpolyethylene glycol-co-N-methacryloylglycylglycine)-tyrosine conjugates (Dex-g-P(MPEG-co-MAGGCONHTyr)) were synthesized for the purpose to improve the biodistribution and blood clearance time of ploy(N-methacryloylglycylglycine)-tyrosine conjugates (Dex-g-PMAGGCONHTyr). Dynamic light scattering (DLS) results indicated that no aggregation formed in 0.9% saline solution. The graft copolymers were labeled with ¹²⁵I and the labeled copolymers are stable in 0.9% saline and 1% BSA of PBS solutions. Pharmacokinetics studies showed that ¹²⁵I labeled graft copolymer Dex-g-P(HPMA-co-MAGGCONHTyr) had a longer blood clearance time than the others. Biodistribution images confirmed the preferable liver and spleen accumulation at 1 h after injection, and especially for blood tissue, the mean %ID/g value of the PHPMA-modified graft copolymer Dex-g-P(HPMA-co-MAGGCONHTyr) is 7 folds higher than that of Dex-g-PMAGGCONHTyr.

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1. Introduction

A reasonable biodistribution and longer blood clearance time are needed for an ideal tumor-targeted radiotherapy carrier besides biocompatible, biodegradable, non-toxic and non-immunogenic properties.^1–7 In particular, sufficient blood clearance time is critical for both imaging and in vivo therapy. Therefore, many efforts have been made to prolong the blood clearance time of tumor-targeted radiotherapy carriers. Among many carriers that were investigated, the presence of poly(ethylene glycol) (PEG) or poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA) were found to prolong the blood clearance time of the carriers.^6–20 PEGylated copolymers can prolong the blood clearance time, which is due to that the presence of PEG can reduce the rapid recognition of the carriers by the reticuloendothelial system.^7–12HPMA based copolymer can prolong blood clearance time and increase tumor concentration, which suggest that HPMA based copolymer drug delivery systems are attractive tools for more effectively treating various forms of advanced solid malignancy.^6,13–20 Moreover, macromolecular carriers with PEG or PHPMA parts are mainly determined by the molecular weight and the physicochemical properties of the polymer.¹⁴

Dextran is a water-soluble, biodegradable, non-immunogenic and non-toxic 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^21–23 and can be potentially used as a carrier of toxic drug or radioactive nuclides for cancer therapy.²⁴ In previous work, dextran graft ploy(N-methacryloylglycylglycine) copolymer-tyrosine conjugates (Dex-g-PMAGGCONHTyr) were synthesized and the biodistribution and blood clearance time were investigated.²⁵ However, the biodistribution and blood clearance time are still not sufficient enough for the further usages as the drug and radionuclide carriers. In order to improve the blood clearance time of Dex-g-PMAGGCONHTyr copolymers, the structure and components of the copolymers are needed to be redesigned. On the consideration of the advantages of PEG or PHPMA components on the biodistribution and blood clearance time,^6–20 PEG or PHPMA can be introduced into the Dex-g-PMAGGCONHTyr copolymers. Therefore, new dextran graft copolymers, including dextran graft poly(N-(2-hydroxypropyl)methacrylamide-co-N-methacryloylglycylglycine)-tyrosine conjugates (Dex-g-P(HPMA-co-MAGGCONHTyr)), dextran graft poly(methacrylpolyethylene glycol-co-N-methacryloylglycylglycine)-tyrosine conjugates (Dex-g-P(MPEG-co-MAGGCONHTyr)), and dextran graft poly(N-methacryloylglycylglycine) copolymers conjugated with O-(2-aminoethyl) polyethylene glycol (M_n = 3000 g mol⁻¹) and tyrosine (Dex-g-PMAGGCONHPEG_3k-NHTyr) were designed and synthesized. The effect of different side chain components on the biodistribution and the blood clearance time of ¹²⁵I labeled dextran graft copolymers were investigated. The general relationship between the physicochemical characteristics and in vivo behavior of soluble graft copolymers was discussed.

2. Experimental

2.1. Materials

Dextran (100 kDa, Fluka) was purified by previous method before use.²⁵N-methacryloylglycylglycine (MAGGCOOH)²⁶ and HPMA²⁷ were synthesized according to the literatures. Potassium persulfate (K₂S₂O₈) and sodium hydrogen sulfite (NaHSO₃) were A.R. grade and supplied by local chemical suppliers. Poly(ethylene glycol) methacrylate (M_n = 360 g mol⁻¹, Aldrich) and O-(2-Aminoethyl)polyethylene glycol (M_n = 3000 g mol⁻¹, Fluka) were used as received. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), N-(3-dimethyl aminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (≥98%, Fluka), N-hydroxysuccimide (NHS) (≥97%, Fluka), L-Tyrosine (99%, Alfa Aesar), Iodogen (Sigma, St. Louis, MO), Na¹²⁵I (Beijing Atom High Tech., China) and other chemicals and solvents were used as received. Dulbecco's Modified Eagle's Medium (DMEM) and Fetal Bovine Serum (FBS) were purchased from Gibico. MG-63 human osteosarcoma cells were provided by Graduate School of Chinese Academy of Sciences. Water with the resistivity of 18.2 mΩ cm from Milli-Q Reference Water Purification System (Millipore) was used for the reaction and purification of the graft copolymers.

2.2. Synthesis and ¹²⁵I labeling of the dextran graft copolymers

The synthesis of Dex-g-P(MPEG-co-MAGGCONHTyr), Dex-g-P(HPMA-co-MAGGCONHTyr) and Dex-g-PMAGGCONH-PEG_3k-HTyr is similar to that of Dex-g-PMAGGCONHTyr that reported in previous work.²⁵ The details of the synthesis and characterization of the PEGylated or PHPMA-modified dextran graft copolymers are given in the ESI.† The chemical structures of the synthesized dextran graft copolymers are shown in Scheme 1.

Scheme 1 The chemical structure of dextran graft copolymers.

The dextran graft copolymers were labeled with ¹²⁵I using the Iodogen method.^28,29 Briefly, 100 μg of dextran graft copolymers 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 resultant labeled copolymers were purified by a PD MiniTrapTM G-25 column (28-9180-07-07, GE Healthcare) equilibrated with phosphate buffer (0.2 M, pH 7.4) to remove unreacted radioiodide. The ¹²⁵I labeled copolymer fractuibs 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 Bruker 400 MHz Avance NMR instrument using D₂O as the solvent. Elemental analysis of the graft copolymers was performed on a Flash EA 1112 Elemental Analyzer. 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 the three graft copolymers were dissolved in water and 0.9% saline, the solutions were stirred for two 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 the solution concentrations were 0.5 mg mL⁻¹. The hydrodynamic radius (<R_h>) was obtained by fitting the correlation function with the CONTIN program.

2.4. In vitro cytotoxicity experiment

The cytotoxicity of dextran graft copolymers was evaluated by MTT assay.^30–32MG-63 human osteosarcoma cells were routinely cultured with Dulbecco's modified Eagle's medium (DMEM), supplied with 200 mM L-glutamine, 15% of heat-inactivated foetal calf serum and a mixture of antibiotic/antimycotic compounds (100 U mL⁻¹ of penicillin, 100 μg mL⁻¹ of streptomycin and 0.25 μg mL⁻¹ of amphotericin B),³³ and 10% fetal bovine serum (FBS) in a humidified incubator with 5% CO₂ at 37 °C, and plated into 96-well plates at a cell density of 2000 cells/well in 200 μL of DMEM/FBS medium/well. These cells were allowed to attach to the wells for 48 h of incubation at 37 °C in a humidified atmosphere of 5% CO₂. After that, the culture medium was removed carefully, and 200 μL of the graft copolymers' culture medium were added in each well, with concentration of copolymers ranged from 0.8 to 4 mg mL⁻¹, and incubated as the above condition for 1, 2, and 3 days, respectively. After incubation, 20 μL of the MTT solution was added to wells. After 4 h incubation, the culture medium was discarded and the precipitate of each well was dissolved by 150 μL of dimethyl sulphoxide (DMSO). The optical density (OD) was measured by the microplate reader (MULTISKANMK3, Thermo Electron Corporation) with setting absorbance wavelength at 570 nm. The cell viability (%) was calculated by comparison the OD value of treated cells to untreated cells. All experiments were tested in triplicate.

2.5. Animal studies

All the three ¹²⁵I labeled graft copolymers were purified using 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% bovine serum albumin (BSA) before purification. After loaded with radiotracer (∼100 μL), the PD MiniTrap™ G-25 column was then washed with 4 mL of PBS, the 0.5 mL between 0.6 and 1.1 mL of eluent were collected. Doses for mouse studies were prepared by dissolving the purified radiotracer in 0.9% saline to give a concentration of 100 μCi mL⁻¹ for biodistribution studies. 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.

Body distribution. For biodistribution studies, sixteen 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⁻¹. 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⁻¹). The biodistribution data and blood clearance curve were reported as an average plus the standard variation. 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.

Blood clearance time. For pharmacokinetics studied, seven BALB/c normal mice were used as one group for the blood clearance experiments 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⁻¹).

3. Results and discussion

3.1. Synthesis and ¹²⁵I labeling of the dextran graft copolymers

The synthesis of dextran graft copolymers and the following conjugation of the L-tyrosine are similar to those of Dex-g-PMAGGCONHTyr in previous work,²⁵ except that monomers or mixed monomers with different molar ratio were added for the graft reaction. The details of the synthesis of the graft copolymers and the characterization are given in the ESI.† The details of the synthesis of the dextran graft copolymers and the optimism of the graft copolymerization of each dextran graft copolymer are given in the ESI (Table S1–S3).† The reactivity ratio of the monomers in the graft copolymerization were estimated by comparing the feeding ratio of the monomers and the degree of substitution of the average monomer unit per glucose ring and the results are listed in Table S1 and S2 in the ESI.† The results show that when the feeding ratio of the monomers kept at n_[–COOH]:n_[MPEG] = 1.95 and n_[–COOH]:n_[HPMA) = 0.77, the DS_–COOH/DS_MPEG ≈ 1 and the DS_–COOH/DS_MPEG ≈ 0.8. The results suggest that the reactivity ratio of the monomers MAGGCOOH/MPEG and MAGGCOOH/HPMA is around 1 : 2 and 1 : 1, respectively. The chemical structure of the synthesized dextran graft copolymers are shown in Scheme 1.

Fig. 1 shows the ¹H NMR spectra of the dextran graft copolymers. On the ¹H NMR spectrum of Dex-g-P(MPEG-co-MAGGCONHTyr), the peak at δ = 4.12–4.28 (m, 2H) ppm comes from the methylene protons (O=C–O–CH₂–) on the PMPEG side chains (Fig. 1b). On the ¹H NMR spectrum of Dex-g-P(HPMA-co-MAGGCONHTyr) (Fig. 1c), the peak of methylene (O=C–NH–CH₂–) protons of the PHPMA side chains appears at chemical shift of δ = 3.00–3.30 (m, 2H) ppm besides the typical peaks from protons of Dex-g-PMAGCOONHTyr (Fig. 1a and 1c). On the ¹H NMR spectrum of Dex-g-PMAGGCONHPEG_3k-NHTyr, new peak appears at a chemical shift of δ = 3.75 (m, 2H) ppm, which comes from the methylene protons (–O–CH₂–CH₂–) of the conjugated –NHPEG chains (Fig. 1d). ¹H NMR results indicate the successful synthesis of the dextran graft copolymers. More details of the synthesis and characterization of the graft copolymers are provided in the ESI (Fig. S1 and S2).† Different dextran graft copolymers with similar degree of substitution of –COOH groups per glucose unit of dextran (DS_–COOH ∼ 0.55) were selected for further investigation. The details of the synthesized copolymers are summarized in Table 1.

Fig. 1 ¹HNMR spectra of dextran graft copolymers in D₂O. (a) Dex-g-PMAGGCONHTyr, (b) Dex-g-P(MPEG-co-PMAGGCONHTyr), (c) Dex-g-P(HPMA-co-PMAGGCONHTyr) and (d) Dex-g-PMAGGCONHPEG_3k-NHTyr.

Table 1 Details for the dextran copolymers that used for the mouse experiments

	Graft copolymers	DSCOOH^a	N%^b	DSPEG3k^a	DSMPEG^a	DSHPMA^a	N Tyr ^c	M w (kDa)^d
a The degree of substitution (DS) of the corresponding groups is defined as the average number of corresponding groups or polymer chains per glucose ring of dextran. b N% is the quality percent of N in the graft copolymer by element analysis. c The average amount of L-tyrosine per glucose ring of dextran. d M w is determined by ^1HNMR spectra.
I	Dex-g-PMAGGCONHTyr	0.57	6.43	—	—	—	0.014	165
II	Dex-g-PMAGGCONHPEG3k-NHTyr	0.54	5.75	0.028	—	—	0.004	213
III	Dex-g-P(MPEG-co-MAGGCONHTyr)	0.60	4.68	—	0.12	—	0.016	195
IV	Dex-g-P(HPMA-co-MAGGCONHTyr)	0.58	6.84	—	—	0.44	0.015	205

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3.2. Dextran graft copolymers in solutions

The chain conformation of the graft copolymers in physiological environment is important for application as ¹²⁵I carriers. Fig. 2 shows the hydrodynamic radius of the Dex-g-PMAGGCONHTyr, Dex-g-PMAGGCONHPEG_3k-NHTyr, Dex-g-P(MPEG-co- MAGGCONHTyr) and Dex-g-P(HPMA-co-PMAGGCONHTyr) in water and 0.9% saline solution. The results indicate that the hydrodynamic radius (R_h) of the dextran graft copolymers has a double distribution mode in water solution, which suggests the formation of aggregates in the system. A single distribution mode of the R_h of the graft copolymers can be observed in 0.9% saline solutions, which suggests that there is no aggregation was formed in the 0.9% saline solutions. The average R_h is around19–22 nm. The size and its distribution of the dextran graft copolymers synthesized in present work is similar to those of Dex-g-PMAGGCONHTyr (Fig. 2a), which indicates that the introduction of PEG or PHPMA units has no obvious effect on the solution properties of the dextran based graft copolymers.

Fig. 2 Hydrodynamic radius distribution of (a) Dex-g-PMAGGCONHTyr, (b) Dex-g-PMAGGCONHPEG_3k-NHTyr, (c) Dex-g-P(MPEG-co-PMAGGCONHTyr), and (d) Dex-g-P(HPMA-co-PMAGGCONHTyr) in 0.9% saline and water solutions. The concentration of the copolymers is kept at 0.5 mg mL⁻¹.

3.3. The labeling of three new graft copolymers with ¹²⁵I and in vitro stability

PEGylated or PHPMA-modified dextran graft copolymers were labeled with ¹²⁵I using the Iodogen method.^28,29,32 The resulting labeled copolymers were purified with a PD MiniTrap TM G-25 column. Fig. 3 shows the typical ITLC chromatograms of ¹²⁵I labeled Dex-g-P(HPMA-co-MAGGCONHTyr) using acetone as the eluent. By this method, free ¹²⁵I migrated to the solvent front while ¹²⁵I labeled copolymer remained at the origin. The radiochemical yields are above 64% and radiochemical purity (RCP) is around 100% after the purification by using PD MiniTrapTM G-25 column. The labeling of the PEGylated dextran graft copolymers, including Dex-g-P(MPEG-co-MAGGCONHTyr) and Dex-g-PMAGGCONHPEG_3k-NHTyr, were also achieved with radiochemical yield above 99% and the RCP about 100% after the purification by PD MiniTrapTM G-25 column (ESI, Fig. S3 and S4†). Fig. 4 shows the stability of the ¹²⁵I labeled dextran copolymers in 0.9% saline and 1% BSA of PBS solution. The results indicate that the ¹²⁵I labeled graft copolymers are quite stable in 0.9% saline, and the RCP remains above 99% at 36 h post-purification. The ¹²⁵I labeled PEGylated dextran copolymers have good stability in 1% BSA of PBS solution and the RCP remains above 97% at 36 h post-purification. However, ¹²⁵I labeled Dex-g-P(HPMA-co-MAGGCONHTyr) copolymer is less stable than PEGylated dextran copolymers in 1% BSA of PBS solution, and the RCP decreased to about 72% at 36 h post-purification.

Fig. 3 Representative ITLC chromatograms of ¹²⁵I labeled Dex-g-P(HPMA-co-MAGGCONHTyr) (a) original kit and (b) after purification. Free ¹²⁵I⁻ migrated to the solvent front while ¹²⁵I labeled polymer remained at the origin.

Fig. 4 Solution stability of ¹²⁵I labeled (a) Dex-g-PMAGGCONHPEG_3k-NHTyr, (b) Dex-g-P(MPEG-co-PMAGGCONHTyr), and (c) Dex-g-P(HPMA-co-MAGGCONHTyr) in 0.9% saline and 1% BSA of PBS solutions.

3.4. In vitro cytotoxicity of dextran copolymers

The cytotoxicity of the dextran graft copolymers was estimated by the viability of MG-63 human osteosarcoma cells using MTT assay and compared with that of dextran and Dex-g-PMAGGCONHTyr. 200 μL (co)polymer solutions with different concentrations were used to treat the cells in every well. The proportion of viable cells in the treated group was compared to that of the negative control. The cell viability (%) as a function of time is shown in Fig. 5. The results show that the RGR of the cells is around 100% for the cells treated with 0.8 mg mL⁻¹(co)polymer solutions for 3 days (Fig. 5a), which suggests that all the (co)polymers are non-cytotoxic at this concentration.³⁴ For the cells treated with Dex-g-P(HPMA-co-MAGGCONHTyr) at different concentrations, from 0.8 to 4 mg mL⁻¹, cell viability decreases from >90% to 70–80% (Fig. 5b), which suggests that Dex-g-P(HPMA-co-MAGGCONHTyr) copolymer only has a slight cytotoxicity at high concentration.³⁴ In the following mouse experiments, the injection concentration of the graft copolymers is about 0.25 mg mL⁻¹ in 0.9% saline solutions, which is non-cytotoxic.

Fig. 5 Cytotoxicity of MG-63 human osteosarcoma cells of dextran and its graft copolymers. (a) The cells were incubated for 1, 2, and 3 days in the presence of the dextran and its copolymers at 0.8 mg mL⁻¹ in 0.9% saline solutions and (b) the effect of copolymer concentration of Dex-g-P(HPMA-co-MAGGCONHTyr) on the cell viability of MG-63 cells. The control group corresponds to the assay just with cells in culture medium.

3.5. Biodistribution and blood clearance of the ¹²⁵I labeled dextran graft copolymers

The information of the dextran graft copolymers for mouse experiments is listed in details in Table 1. The dextran copolymers with similar molecular weight, DS_–COOH, and N_Tyr were selected for the mouse experiments, by which the influences of the molecular weight, the number of negative charges per glucose unit of dextran can be eliminated. The similar N_Tyr of the dextran graft copolymers can confirm the similar labeling efficiency of ¹²⁵I for all graft copolymers. Moreover, it is known that the blood clearance time is independent on molecular weight for the polymers with similar structure at the molecular weight higher than 70 kDa.³⁵ Therefore, copolymers with the molecular weight higher than 165 kDa were selected for the mouse experiments.

Fig. 6 shows biodistribution of the ¹²⁵I labeled dextran graft copolymers in different organs as a function of time after injection. The results indicate that, PEGylated graft copolymers have a more preferable liver and spleen accumulation than those of Dex-g-PMAGGCONHTyr and Dex-g-P(HPMA-co-MAGGCONHTyr) copolymers at 1 h after injection. For blood tissue at 1 h after intravenous injection, the mean organ uptake of ¹²⁵I labeled Dex-g-PMAGGCONHTyr is 0.97%ID/g, and the mean organ uptake of the ¹²⁵I labeled Dex-g-P(MPEG-co-MAGGCONHTyr), Dex-g-PMAGGCONHPEG_3k-NHTyr and Dex-g-P(HPMA-co- MAGGCONHTyr) are 1.22, 1.30, 7.22%ID g⁻¹, respectively. Although PEGylated dextran copolymers have the effect of biological inert and can be slow down the rapid recognition by the reticuloendothelial system,^7–12 it is obvious that blood percentage of PEGylated dextran copolymers in present work is only slightly higher than that of Dex-g-PMAGGCONHTyr. This may be due to the molecular weight of PEG on blood tissue distribution. It is known that PEG with molecular weight lower than 5000 g mol⁻¹ has no obvious effect on blood tissue distribution, whereas PEG with the molecular weight higher than 1 × 10⁴ g mol⁻¹ can significantly improve the blood percentage.⁵ On the contrast, the mean organ uptake of ¹²⁵I labeled Dex-g-P(HPMA-co-MAGGCONHTyr) is above 7 folds as that of ¹²⁵I labeled Dex-g-PMAGGCONHTyr. The results suggest that Dex-g-P(HPMA-co-MAGGCONHTyr) copolymers are the promising drug carrier for the cancer therapy.

Fig. 6 Tissue distributions of ¹²⁵I labeled dextran graft copolymers as a function of time after injection in BALB/c normal mice. (a) Dex-g-PMAGGCONHTyr, (b) Dex-g-PMAGGCONHPEG_3k-NHTyr, (c) Dex-g-P(MPEG-co-MAGGCONHTyr), and (d) Dex-g-P(HPMA-co-MAGGCONHTyr). The concentration of which was expressed as the percentage of injected dose per gram of tissue. Each data point represents an average of biodistribution data in four animals.

Fig. 7 shows the blood clearance of ¹²⁵I labeled dextran graft copolymers. It was found that PEGylated dextran graft copolymers have a rapid blood clearance, even faster than that of Dex-g-PMAGGCONHTyr. More than 90% of the PEGylated dextran graft copolymers were washed out from the blood at 20 min after the injection. In contrast, the blood clearance of Dex-g-P(HPMA-co-MAGGCONHTyr) is much slower than other dextran graft copolymers. More than 10% of the graft copolymer remains in the blood at 120 min postinjection. The striking prolonged circulation time may be attributed to the enhanced permeability and retention (EPR) effect of PHPMA component.³⁶

Fig. 7 Blood clearance of ¹²⁵I labeled dextran graft copolymers in BALB/c normal mice. Each data point represents an average of blood clearance data in seven animals.

4. Conclusion

For the purpose of improving the biodistribution and blood clearance time of ploy(N-methacryloylglycylglycine)-Tyrosine conjugates (Dex-g-PMAGGCONHTyr), PEG or PHPMA-modified Dex-g-PMAGGCONHTyr graft copolymers, including Dex-g- PMAGGCONHPEG_3k-NHTyr, Dex-g-P(HPMA-co-MAGGCONHTyr), and Dex-g-P(MPEG-co- MAGGCONHTyr) were synthesized and labeled with ¹²⁵I. The graft copolymers have a good solubility in 0.9% saline solution. The labeling yields for the ¹²⁵I labeled PEG- or PHPMA-modified graft copolymers were around 99% and 64%, respectively. All the ¹²⁵I labeled dextran graft copolymers are stable in 0.9% saline, and the RCP remains above 99% at 36 h post-purification. The mean organ uptake of ¹²⁵I labeled Dex-g-P(HPMA-co-MAGGCONHTyr) is more than 7 folds than that of ¹²⁵I labeled Dex-g-PMAGGCONHTyr. The striking prolonged clearance time shows that PHPMA-modified dextran graft copolymer Dex-g-P(HPMA-co-MAGGCONHTyr) is a potentially excellent radiotherapy carrier, which can be applied to in vivo efficient radiotherapy for tumors in the further. Tumor mouse experiments are under investigation.

Acknowledgements

The financial support of the National Natural Science Foundation of China (Grant No. 20974114, and 50821062) and the Knowledge Innovation Program of the Chinese Academy of Sciences (Grant No. KJCX2-YW-H19) is greatly appreciated.

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Footnote

† Electronic supplementary information (ESI) available: Details of the synthesis, ¹²⁵I labeling, and characterization of the graft copolymers. See DOI: 10.1039/c1py00168j

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