Stimuli-Responsive Biodegradable Hyperbranched Polymer

Apr 4, 2016 - †Huaxi MR Research Center (HMRRC), Department of Radiology, West China Hospital and ‡Laboratory of Stem Cell Biology, State Key Labo...
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Stimuli-Responsive Biodegradable Hyperbranched Polymer− Gadolinium Conjugates as Efficient and Biocompatible Nanoscale Magnetic Resonance Imaging Contrast Agents Ling Sun,†,§ Xue Li,‡,§ Xiaoli Wei,† Qiang Luo,† Pujun Guan,† Min Wu,† Hongyan Zhu,*,‡ Kui Luo,*,† and Qiyong Gong† †

Huaxi MR Research Center (HMRRC), Department of Radiology, West China Hospital and ‡Laboratory of Stem Cell Biology, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China S Supporting Information *

ABSTRACT: The efficacy and biocompatibility of nanoscale magnetic resonance imaging (MRI) contrast agents depend on optimal molecular structures and compositions. Gadolinium [Gd(III)] based dendritic macromolecules with well-defined and tunable nanoscale sizes are excellent candidates as multivalent MRI contrast agents. Here, we propose a novel alternate preparation of biodegradable hyperbranched polymer−gadolinium conjugates via a simple strategy and report potentially efficient and biocompatible nanoscale MRI contrast agents for cancer diagnosis. The enzyme-responsive hyperbranched poly(oligo-(ethylene glycol) methacrylate)−gadolinium conjugate (HB-POEGMA-Gd) was prepared via one-step reversible addition−fragmentation chain transfer (RAFT) polymerization and Gd(III) chelating, and the cRGDyK functionalized polymer (HBPOEGMA-cRGD-Gd) was obtained via click chemistry. By using an enzyme similar to lysosomal cathepsin B, hyperbranched conjugates of high molecular weights (MW) (180 and 210 kDa) and nanoscale sizes (38 and 42 nm) were degraded into low MW (25 and 30 kDa) and smaller products (4.8 and 5.2 nm) below the renal threshold. Conjugate-based nanoscale systems had three-fold more T1 relaxivity compared to clinical agent diethylenediaminepentaacetic acid (DTPA)-Gd. Animal studies with the nanoscale system offered greater tumor accumulation and enhanced signal intensity (SI) in mouse U87 tumors of which the greatest activity was conferred by the cRGDyK moiety functionalized hyperbranched conjugate. In vitro cytotoxicity, hemocompatibility and in vivo toxicity studies confirmed no adverse events. This design strategy for multifunctional Gd(III)-labeled biodegradable dendritic macromolecules may have significant potential as future efficient, biocompatible polymeric nanoscale MRI diagnostic contrast agents for cancer. KEYWORDS: nanoscale MRI contrast agent, cancer diagnosis, biocompatibility, biodegradable, hyperbranched polymer

1. INTRODUCTION Magnetic resonance imaging (MRI), a noninvasive procedure, offers high spatial resolution and 3D images of soft tissues as well as quantitative assessments of disease pathogenesis and thus is an important tool for diagnosing cancer and monitoring therapeutic response in solid tumors.1 For tumor imaging, contrast-enhanced MRI enables visualization of margins between normal and diseased tissues using MRI contrast agents. Currently, paramagnetic Gd(III)-based complexes, such as Gd(III)-diethylenediaminepentaacetic acid ([Gd-(DTPA)]−2) (Magnevist) and Gd(III)-N,N′,N″,N′″-tetracarboxymethyl-1,4,7,10-tetraazacyclodod-ecane ([Gd-(DOTA)]−1) (Dotarem), have been used as contrast agents for brain tumor diagnosis. However, these agents are small molecules with low sensitivity, nonspecificity, and are rapidly eliminated.2 Therefore, repeated administration is required, and side effects are problematic. Additionally, accurate delineation of tumor boundaries and quantification of tumor volumes are limited.3 The emergence and development of © 2016 American Chemical Society

nanotechnology for biomedical applications and nanoscale systems may offer better contrast agents than those currently in clinical use.4 Some nanoscale systems loaded or conjugated with Gd(III) chelates enhance relaxivity and offer high accumulation in tumors via enhanced permeability and retention (EPR) effects.2 Among these nanoscale platforms, polymeric nanoscale systems may have the greatest potential for clinical use.5,6 Dendritic polymers, including dendrons, dendrimers, and hyperbranched polymers (HBPs), are novel polymer architectures with 3D dendritic architectures.7−11 Dendritic polymers as drug or imaging delivery carriers can significantly enhance diagnostic and therapeutic indices for cancer due to precise, monodispersible sizes, biocompatibility, modifiable surface Received: January 25, 2016 Accepted: April 4, 2016 Published: April 4, 2016 10499

DOI: 10.1021/acsami.6b00980 ACS Appl. Mater. Interfaces 2016, 8, 10499−10512

Research Article

ACS Applied Materials & Interfaces Scheme 1. Synthesis of Monomers MA-GFLGK-MA and MA-DOTA

Figure 1. Illustration of the preparation of HB-POEGMA-Gd and HB-POEGMA-cRGD-Gd.

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DOI: 10.1021/acsami.6b00980 ACS Appl. Mater. Interfaces 2016, 8, 10499−10512

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ACS Applied Materials & Interfaces functionality, water solubility, and multivalency.12−16 Previously, a series of peptide dendrimers/dendron-based nanoscale systems had been used for cancer diagnosis and therapy,17,18 and we noted that both tumor accumulation and circulation duration were molecular weight (MW) dependent.19 Additionally, the longitudinal relaxivity could be rapidly enhanced by increasing MW.20 For example, poly(ethylene glycol) functionalized (PEGylated) low generation dendrimer conjugated with Gd(III) chelates possessed biosafety and enhanced longitudinal relaxivity because PEG could extend serum retention time by decreasing nonspecific interactions with endogenous components and macrophages.20,21 However, PEGylated dendrimer is difficult to prepare. Specifically, functionalized dendrimers/dendrons have issues of steric hindrance in chemical reactions.22 Alternating dendrimers or dendrons, HBPs can be easily prepared in scalable quantities with diverse and variable functionalities.23 Additionally, HBPs can be optimally sized to allow passive tumor targeting via the EPR effect. Previous work indicates that nanoscale systems of 10−100 nm could efficiently and passively target tumors due to the hyper-vascularized, leaky, and compromised lymphatic drainage system of tumors.24,25 To enhance the targeting efficacy of contrast agents, nanoscale systems possessing covalently grafted receptor specific ligands can deliver MRI labels to tumors in vivo, increasing target-tobackground ratios and significantly improving imaging of pathological forms, as grafted ligands can specifically recognize biomarkers overexpressed in cancer cells or the tumor vasculature.26 Among these biomarkers, αvβ3 integrin as a receptor for extracellular matrix protein is overexpressed on activated endothelial cells of the tumor vasculature but not normal vasculature.27 A cyclic peptide, containing an arginineglycine-aspartic tripeptide sequence (cRGD), has higher binding affinity to αvβ3 integrin; thus, some multivalent cRGDcontaining MRI contrast agents have demonstrated enhanced contrast as compared to noncovalent cRGD compounds.28,29 Unfortunately, few examples of targeting HBPs MRI contrast agents have been reported for tumor diagnosis. Additionally, for hyperbranched tumor-targeting agents, it is yet to be determined whether observed results of multivalent ligand platforms are truly due to multivalent interactions or merely a size effect. In addition to relaxivity and tumor accumulation, biosafety is important for nanoscale MRI contrast agents. For polymeric nanoscale systems to have a high EPR effect, MWs must exceed 50 kDa.30,31 When MWs are optimized to 100−300 kDa, polymeric nanoscale systems remain in the circulation and accumulate into tumors.32−34 In contrast, the long circulation time of polymer-gadolinium conjugate based MRI contrast agents may be toxic due to Gd(III), which may cause nephrogenic systemic fibrosis (NSF).35,36 Previously, we designed and prepared biodegradable polymer−drug conjugates with stimuli-responsive linker (glycylphenylalanylleucylglycine tetra-peptide Gly-Phe-LeuGly, GFLG) in the main chain;33,37−39 this GFLG linker can be degraded in the presence of cathepsin B, a lysosomal cysteine protease overexpressed in many tumor and endothelial cells of the tumor vasculature.40 Polymeric conjugates with high MWs have high tumor accumulation and circulate longer but with relative safety due to biodegradable features. Thus, we sought to create biodegradable and functional hyperbranched polymer− gadolinium conjugates with tumor-targeting effects that are both efficient and safe nanoscale MRI contrast agents for cancer diagnosis.

We designed functional hyperbranched poly(oligo-(ethylene glycol) methacrylate)-gadolinium conjugates (HB-POEGMAGd and HB-POEGMA-cRGD-Gd) via reversible addition− fragmentation chain transfer (RAFT) polymerization and click chemistry and evaluated their potential efficacy and safety as nanoscale contrast agents for tumor diagnosis. Compound synthesis appears in Scheme 1 and Figure 1. Physicochemical properties of nanoscale systems, such as MW, size, zeta potential, and mass, were measured, and in vitro and in vivo MRI efficiency and biodistribution were evaluated. Additionally, in vitro and in vivo toxicity and hemocompatibility studies were conducted as overt toxicity studies and histological analysis to determine if the biodegradable hyperbranched copolymers were safe and efficient as nanoscale MRI contrast agents for tumor diaognosis.

2. EXPERIMENTAL SECTION 2.1. Materials and Methods. The 2,2′-[azobis(1methylethylidene)]bis[4,5-dihydro-1H-imidazole] dihydrochloride (VA044), 4-cyanopentanoic acid dithiobenzoate (CTA), gadolinium chloride hexahydrate (GdCl3·6H2O), and ethylene glycol methacrylate (OEGMA, Mw = 500 Da) were purchased from Sigma-Adrich. Alkynyl-cRGDyK and MA-N3 were prepared as previously described.41,42 The synthesis of monomers MA-GFLGK-MA and MA-DOTA was described in the Supporting Information. Number-average molecular weight (Mn), weight-average molecular weight (MW), and polydispersity (PDI) were measured using size-exclusion chromatography (SEC) on an Ä KTA/FPLC system (GE Healthcare) using GE Healthcare (location missing) columns. Superose 6 HR10/30 with sodium acetate buffer containing 30% acetonitrile (pH = 6.5) served as the mobile phase (flow rate: 0.4 mL/min). UV−vis spectra was measured on a Varian Cary 400 Bio UV−visible spectrophotometer. Dynamic light scattering (DLS) and zeta potential were measured by means of Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). Inductively coupled plasma mass spectrometry (ICP-MS, Elan DRC-e) was used for measuring Gd(III) in MRI contrast agents and qualitatively assessing Gd(III) taken up by different tissues in vivo. 2.2. Synthesis of Hyperbranched Poly[oligo-(ethylene glycol) methacrylate]−Gadolinium Conjugates. OEGMA (1.13 g, 2.25 mmol), MA-DOTA (1.29 g, 2.5 mmol), MAGFLGK-MA (82 mg, 125 μmol), MA-CH2CH2−N3 (154 mg, 1 mmol), CTA (14.8 mg, 53 μmol), and VA044 (5.7 mg, 17.8 μmol) were dissolved in a solution of deionized water/methanol (10 mL, 3:1). The solution was bubbled with nitrogen for 45 min at 0 °C. The solution was stirred at 45 °C for 10 h and quenched in liquid nitrogen. The copolymer was precipitated in acetone twice, isolated by centrifugation, and purified by dissolution− precipitation in methanol/acetone twice, and then the residue was fractionated/purified by SEC using Superose 6 HR10/30 (MW range for hydrophilic neutral polymers 15−300 kDa/14 mL separation volume) column on an Ä KTA FPLC system (GE Healthcare) column with sodium acetate buffer containing 30% acetonitrile (pH = 6.5) as the mobile phase. After dialysis against water in the dark and freeze-drying, the final product (hyperbranched conjugate: HB-POEGMA, 51% yield, 1.34 g) was obtained. The content of DOTA derivate was obtained via acid−base titration analysis, as previously reported.43 The HB-POEGMA conjugate (1.2 g) and GdCl3·6H2O (1.66 g, 4.5 mmol) were dissolved in 50 mL of distilled water, and then the pH of the reaction mixture was adjusted to 5.2−5.4 with 0.1 M NaOH. The reaction was stirred vigorously at room 10501

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(Siemens Sonata) at room temperature in 0.1 M PBS aqueous solutions with different Gd(III) concentrations (0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, and 0.7 mM). T1-weighted images were acquired with a spin echo (SE) sequence under the following parameters: TE = 8.7 ms; TR = 25, 30, 50, 70, 90, 110, 150, 170, 190, 210, 250, 300, 400, 600, 700, and 800 ms; Fov = 200 mm; slice thickness = 2.0 mm; matrix dimensions = 256 × 256. The r1 value was determined from the slope of 1/relaxation time (s−1) versus Gd(III) plot. After degradation of the conjugates, the relaxivity of the degraded products was measured as described in section 2.4. 2.6. In Vivo MRI Study. U87MG cells derived from human glioblastoma multiforme tumors were used for in vivo MRI. First, 6−8 weeks old female BALB/c nude mice were inoculated subcutaneously into the right lower back (1 × 107 cells suspended in 200 mL of PBS). Solid U87 tumors were allowed to reach 3−5 mm diameter, and animals were randomly divided into three groups (n = 5/group) for in vivo MRI study after treatment with DTPA-Gd, HB-POEGMA-Gd, or HB-POEGMA-cRGD-Gd. Mice were anesthetized with pentobarbital sodium salt, and a contrast-enhanced imaging study was performed on a 3.0 T imaging system (Siemens Sonata Medical System) equipped with a customized mice coil for transmission and reception of the signal using a multisection single-echo T1 weighted TSE sequence. Parameters were as follows: TR = 450 ms; TE = 11 ms; slices = 11; voxel size = 0.2 × 0.2 × 1.5 mm3; Fov = 51 mm. DTPA-Gd, HB-POEGMA-Gd, or HB-POEGMAcRGD-Gd was injected via the tail vein (0.08 mmol Gd(III)/kg mice). Images were acquired precontrast at 10 min, 30 min, 1 h, 3 h, 19 h, and 24 h postinjection. The relative enhancement of the signal-to-noise ratio (ΔSNR) was calculated using the following equation: ΔSNR = SI (tumor)/SI (water), ΔSNR = SI (bladder)/SI (water), where SI (tumor), SI (bladder), and SI (water) were the signal intensity of tumor, bladder, and water mold, respectively, within the regions of interest.44 Signal changes were evaluated semiquantitatively by plotting the ΔSNR versus time.45 A Student’s two-tailed t-test was used to calculate p-values at different time points postinjection, assuming statistical significance at p < 0.05. 2.7. Distribution of the Agents in Tissues. Mice bearing U87 tumor (n = 7) were injected with DTPA-Gd, HBPOEGMA-Gd, or HB-POEGMA-cRGD-Gd conjugates via the tail vein (0.08 mmol Gd(III)/kg) for the contrast-enhanced imaging study and then sacrificed by cervical dislocation at 24 h, 6 days, and 12 days postinjection. The heart, liver, spleen, lung, kidneys, and tumors were collected, weighed, and digested in H2O2 (1 mL) and HNO3 (3 mL), and then samples were heated at 120 °C for 2 days. Solutions were diluted with deionized water, and the Gd(III) concentration was measured using ICP-MS. Gd(III) content in each main organ/tissue was calculated as the percent of injected dose/g of organ/tissue (% ID/g) by the average value of the experimental mice. 2.8. Cytotoxicity Assay. In vitro cytotoxicity of DTPA-Gd, HB-POEGMA-Gd, and HB-POEGMA-cRGD-Gd conjugates was assayed with a cell counting kit-8 assay (CCK-8, Dojindo, Japan). U87 and LO2 cells were added to a 96-well plate (5 × 103 cells/well) in DMEM supplemented with 10% FBS. After 24 h of incubation, the medium was replaced with DMEM containing different Gd(III) concentrations (50, 100, 250, and 500 nmol/ mL) of DTPA-Gd, HB-POEGMA-Gd, and HB-POEGMAcRGD-Gd conjugate solutions. After incubation for another 24 h and 48 h, cells were washed three times with PBS to eliminate remaining polymers and other impurities. Then samples were

temperature for 24 h in the dark. After work-up, the solution was dialyzed in the dark for 2 days to remove excess Gd(III). The solution was removed by freeze-drying to yield HB-POEGMAGd as a white solid (1.23 g). ICP-MS analysis confirmed that Gd(III) composed 6.2% of the product weight. Under a nitrogen atmosphere, HB-POEGMA-Gd with azido groups (600 mg) and cRGD peptides with alkyne groups (500 mg), CuSO4·5H2O (45 mg, 0.18 mmol), and sodium ascorbate (71 mg, 0.36 mmol) were added to 20 mL of H2O (1:1, V/V). The mixture was stirred for 12 h in the dark. Then the solution was dialyzed in the dark. The solution was dialyzed with 1 mM EDTA-Na2 aqueous solution using a dialysis membrane (MW cutoff = 3500). The solution was freeze-dried to yield HBPOEGMA-cRGD-Gd as a white solid (620 g). ICP-MS analysis confirmed that Gd(III) composed 4.9% of the product weight. The peptide content of the two conjugates was analyzed via amino acid analysis with HCl hydrolyzed high-performance liquid chromatography (HPLC) methodology. Three milligrams of each sample was weighed with a readability of 0.1 mg and was added to a test tube with 5 mL of 6 M HCl. A test tube was vacuumed to 0 Pa, protected with nitrogen, and closed. The hydrolysis reaction was maintained at 110 °C for 24 h. The test tube was opened, and the sample was diluted into a final volume of 50 mL. Ten milliliters of the solution was transferred to a distillation flask in water bath at 60 °C, which was then transferred to a rotary evaporator (model RE-52AA, Shanghai, China) for evaporation. The residue was dissolved in 10 mL of HCl (0.02 M) and filtered with 0.22 μm film. One milliliter of solution containing the sample was injected into an autosampler bottle and placed in an amino acid autoanalyzer model L-8900 (Hitachi High-Technologies Corporation, Japan). The amount of each amino acid in the samples was calculated using EZChrom Elite (Hitachi High-Technologies Corporation, 2004) software, and relative amino acid content was expressed as a percentage of the total sample weight. 2.3. Size and Zeta Potential. The material was assayed with DLS and transmission electron microscopy (TEM). HBPOEGMA-Gd and HB-POEGMA-cRGD-Gd conjugates were diluted with ultrapure water to a count rate of 100−300 kcps and sonicated before measurement. Sample particle size and zeta potential were characterized using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). Each measurement was performed in triplicate, and data were processed with DTS software version 3.32. Freshly prepared conjugate solution (1 mg/mL, 10 μL) was applied onto a 150-mesh carbon-coated copper grid, and excess solution was removed with filter paper. The sample was obtained after drying at room temperature. 2.4. Biodegradability Study. The biodegradability of HBPOEGMA-Gd and HB-POEGMA-cRGD-Gd was assessed in solution with or without papain, which has similar activity to lysosomal cathepsin B. Papain in McIlvaine’s buffer (50 mM citrate/0.1 M phosphate; 2 mM EDTA, pH = 5.4) was quantified by UV detection at 480 nm. Glutathione solution in McIlvaine’s buffer (10 mM) was added to an equal volume of enzyme stock solution and preincubated for 5 min at 37 °C. Conjugates were incubated in McIlvaine’s buffer (3 mg/mL) with papain (4 mM) at 37 °C for 18 h. Subsequently, the conjugates were also incubated in PBS (pH 7.4) at 37 °C for 48 h. After incubation, samples were withdrawn and analyzed using DLS and scanning electron microscopy (SEM). 2.5. T1 Relaxivity of DTPA-Gd and Conjugates. In vitro relaxivity of HB-POEGMA-Gd and HB-POEGMA-cRGD-Gd conjugates was measured on a clinical 1.5 T MRI scanner 10502

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3, or 5 mg/mL. All samples were incubated at 37 °C for 12 h, and then the RBC suspensions were centrifuged at 1000 × g for 5 min, and supernatants were collected. Positive (100% lysis) and negative controls were produced by adding 15 μL of human blood suspension to 300 μL of distilled water and PBS, respectively. Supernatants (200 μL) were transferred into a 96well culture plate, and sample absorbance was measured using a Varioscan Flash (ThermoFisher SCIENTIFIC) microplate reader at 540 nm. Hemolytic tests were carried out in triplicate. RBC lysis was calculated as follows:46,47

incubated at 37 o C for another 2 h after the addition of 100 μL of CCK-8 solution. Absorbance was measured using a microplate reader Varioscan Flash (ThermoFisher SCIENTIFIC) at 450 nm (630 nm as a reference). Untreated control cells were considered 100% viable. 2.9. Hemolysis Tests. Blood was collected from healthy unmedicated donors into an evacuated 4.5 mL glass tube containing 3.2% sodium citrate (blood/anticoagulant ratio = 9:1). Then each contrast agent (300 μL) was added to a blood suspension (16% v/v in PBS, 15 μL) to a final concentration of 1, %hemolysis =

Mean of the test sample OD − Mean of the negative control OD × 100 Mean of the positive control OD − Mean of the negative control OD

2.10. Plasma Coagulation. Fresh blood was drawn from a healthy unmedicated donor into tubes containing 3.8% sodium citrate at ratio 9:1 (blood/anticoagulant). Platelet poor plasma (PPP) was isolated by centrifugation at 3000 × g for 15 min at room temperature and used immediately. The PPP (360 μL) was mixed with 40 μL of the conjugate solutions, and control experiments were carried out with identical volumes of PBS. To evaluate clotting time, activated partial thromboplastin time (APTT) and prothombin time (PT) were measured with an automatic coagulation analyzer (SYSMEX CA-7000) with corresponding reagents. Each experiment was repeated three times, and data were compared with controls using a Student’s two-tailed t-test assuming statistical significance at p < 0.05. 2.11. Thromboelastography (TEG). Fresh citrated whole blood was mixed with HB-POEGMA-Gd or HB-POEGMAcRGD-Gd conjugates. Typically, 900 μL of blood was mixed with 100 μL of HB-POEGMA-Gd or HB-POEGMA-cRGD-Gd for a final concentration of 0.1 and 1 mg/mL in a tube containing kaolin. Equivalent PBS was used as a control. After thorough mixing, 340 μL of the suspension was transferred into a TEG cup, and analysis was performed on Thromboelastograph Hemostasis System 5000 (TEG) (Hemoscope Corporation, city, state) after addition of 20 μL of CaCl2. 2.12. Red Blood Cell Morphologies and Aggregation. Citrated whole blood was centrifuged at 1000 × g for 5 min at room temperature and erythrocytes (RBCs) were sedimented. After plasma and the buffy coat were removed, RBCs were washed with PBS three times, and 20 μL of RBCs was added to a 100 μL PBS solution containing HB-POEGMA-Gd or HBPOEGMA-cRGD-Gd (final concentration 5 mg/mL). Mixtures were spun, and the same volume of PBS solution was used as a control. After incubation for 15 min, mixtures were washed with PBS and then fixed with 4% paraformaldehyde overnight. RBCs suspensions were dropped onto glass slides, and samples were dehydrated with 75, 85, 95, and 100% (v/v) ethanol for 10 min. Samples were placed in a static environment and dried overnight in air at room temperature. Finally, dried RBCs were coated with gold and observed with an SEM analyzer. 2.13. In Vivo Toxicity. Animal experiments were conducted in accordance to national welfare legislation and approved by the animal experiments ethical committee. Healthy and tumor-free female BALB/c mice (20 ± 2 g, 6−8 weeks-of-age) were purchased from West China Animal Culture Center of Sichuan University. Every 4 days, mice randomly divided into four groups (n = 7 per group) and were given 0.08 mmol Gd(III)/kg of DTPA-Gd, HB-POEGMA-Gd, or HB-POEGMA-cRGD-Gd three times via tail vein (200 mL injection volume), and control mice received saline. Mouse weight was measured every 2 days,

and behaviors were observed. After 9 days, all animals were allowed to recover for another 6 days and then were sacrificed. Heart, liver, spleen, lung, and kidneys were removed, washed with PBS and fixed in 4% formaldehyde, embedded in paraffin, sectioned, and then stained with hematoxylin and eosin for histological analysis. 2.14. Statistical Analysis. For all data, p < 0.05 was considered statistically significant. Data means ± SD, ANOVA, and a Student’s two-tailed t-test were used to analyze data.

3. RESULTS AND DISCUSSION 3.1. Design, Preparation, and Characterization of Hyperbranched Polymeric Conjugates. To enhance the sensitivity of polymeric MRI contrast agents, increased tumor accumulation and high longitudinal relaxivity were achieved through increasing the MW. A greater EPR was conferred by

Figure 2. SEC profiles of HB-POEGMA-Gd (A, MW 180 kDa, PDI 2.48), HB-POEGMA-cRGD-Gd (B, MW 210 kDa, PDI 2.54), and their degraded products (A, MW 25 kDa; B MW 30 kDa) after incubation of conjugates (3 mg/mL) with 4 μM papain for 12 h at 37 °C (pH = 5.4). 10503

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Figure 3. DLS studies of HB-POEGMA-Gd (A, 38 nm), HB-POEGMA-cRGD-Gd (C, 42 nm), and their metabolites (B, 4.8 nm; D, 5.2 nm) after incubation of conjugates (3 mg/mL) with 4 μM papain for 12 h at 37 °C (pH = 5.4).

branched macromolecules may have superiority as drug delivery vehicles as branched polymers overcome inherent shortcomings of linear molecules such as low nanoscale size, size distribution, and rapid renal excretion. We previously reported that PEGylated low generation dendrimers and Gd(III)-chelate labeled dendritic polymeric conjugates were safe and had enhanced longitudinal relaxivity.20 Therefore, MW, size, shape, surface functionality, and rigidity may also affect biodistribution, pharmacokinetics, biosafety, and MRI sensitivity.49 Thus, we designed hyperbranched poly(oligo-(ethylene glycol) methacrylate)−gadolinium conjugates with biodegradable GFLG linkers and branched chains via RAFT polymerization. Our synthetic approach allowed manipulation of the desirable properties mentioned previously (Figure 1). The CTA was used to synthesize polymers from monomers of OEGMA, MA-DOTA, and MA-CH2CH2−N3, and addition of a cross-linking agent led to the formation of hyperbranched polymers mixed with linear polymeric chains. Therefore, the cross-linker MA-GFLGK-MA and monomer MA-DOTA were first prepared here (Scheme 1), and successful synthesis of these was verified by 1H NMR, 13C NMR, and liquid chromatography−mass spectrometry (LC−MS) (Figures S1−S10). Because the average MW of a linear polymer chain was required to be