Gadolinium-Labeled Biodegradable Dendron–Hyaluronic Acid Hybrid

Jun 28, 2017 - Phone: +86 28 85414308. ... (MRI) contrast agents with high sensitivity and good biocompatibility are required for the diagnosis of can...
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Gadolinium-labeled Biodegradable Dendron-Hyaluronic Acid Hybrid and Its Subsequent Application as a Safe and Efficient Magnetic Resonance Imaging Contrast Agent Chunhua Guo, Ling Sun, Hao Cai, Zhenyu Duan, Shiyong Zhang, Qiyong Gong, Kui Luo, and Zhongwei Gu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06496 • Publication Date (Web): 28 Jun 2017 Downloaded from http://pubs.acs.org on July 1, 2017

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Gadolinium-labeled

Biodegradable

Dendron-Hyaluronic

Acid Hybrid and Its Subsequent Application as a Safe and Efficient Magnetic Resonance Imaging Contrast Agent Chunhua Guo,†,‡ Ling Sun,† Hao Cai,†,‡ Zhenyu Duan,†,‡ Shiyong Zhang,‡ Qiyong Gong,† Kui Luo,†,* and Zhongwei Gu†,‡



Huaxi MR Research Center (HMRRC), Department of Radiology, West China

Hospital, Sichuan University, Chengdu, Sichuan 610041, China ‡

National Engineering Research Center for Biomaterials, Sichuan University,

Chengdu 610064, China *

Corresponding author.

Prof. Luo is to be contacted at Tel.: +86 28 85414308; fax: +86 28 85410653. E–mail addresse: [email protected]

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ABSTRACT: Novel magnetic resonance imaging (MRI) contrast agents with high sensitivity and good biocompatibility are required for the diagnosis of cancer. Herein, we

prepared

and

characterized

the

gadolinium

[Gd(III)]-labeled

peptide

dendron-hyaluronic acid (HA) conjugate-based hybrid (Dendronized-HA-DOTA-Gd) by combining the advantages of hyaluronic acid and the peptide dendron. The hybrid Dendronized-HA-DOTA-Gd hybrid with 3.8% Gd(III) as a weight percentage showed a negative zeta potential (-35 mV). The in vitro degradation results indicated that the Dendronized-HA-DOTA-Gd hybrid degraded into products with low molecular weights (MW) in the presence of hyaluronidase. The Dendronized-HA-DOTA-Gd hybrid showed a 3-fold increase in longitudinal relaxivity, and much higher in vivo signal enhancement in 4T1 breast tumors of mice compared to clinical Magnevist (Gd-DTPA). The Dendronized-HA-DOTA-Gd hybrid had a higher accumulation in tumors than Gd-DTPA; it was 2~3-fold after administration. Meanwhile, the polymeric hybrid resulted in low and comparative Gd(III) residue in the body compared to Gd-DTPA. The systematic biosafety evaluations, including blood compatibility

and

toxicity

assessments,

suggested

that

the

Dendronized-HA-DOTA-Gd hybrid exhibited good biocompatibility. Thus, the gadolinium-labeled and dendronized HA hybrid shows promise as a safe and efficient macromolecular MRI contrast agent based on high sensitivity, low residue content in the body, and good biosafety. KEYWORDS: hyaluronic acid conjugate, MRI contrast agents, tumor diagnosis, biodistribution, biodegradability 2

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1. INTRODUCTION To overcome the shortcomings of current clinical MRI contrast agents, including low sensitivity and non-specificity, small Gd(III) chelates are considered to be conjugated or loaded on/in the macromolecular carriers, such as polymers, dendrimers/dendrons, liposomes and proteins.1-4 Compared to clinical agents, such as gadolinium-diethylenetriaminepentaacetic

acid

(Gd-DTPA,

Magnevist),

the

macromolecular and nanoscale Gd(III)-based MRI agents, showed high signal enhancement and enhanced accumulation in tumors via the enhanced permeability and retention (EPR) effect.5 However, the quest for novel MRI contrast agents with optimized properties has never stopped, as both high imaging efficacy and good biosafety are simultaneously required for further clinical application. Therefore, the fabrication of Gd(III)-chelate carriers and their structure optimization are essential for safe and efficient macromolecular MRI contrast agents.6 In order to enhance the sensitivity and efficacy of the agents, macromolecular Gd(III) chelates functionalized agents, as soft nanoscale systems, often have higher longitudinal relaxivities (r1) and high accumulation in tumors.5,7 Owing to the nanoscale molecular sizes, dendritic polymers, including dendron, dendrimer, and branched

polymers,

Gd(III)-chelates.8,9 properties,

have

Peptide

including

shown

the

potential

dendrimers/dendrons biodegradability,

as

carriers

possessing

good

for

some

delivery wonderful

biocompatibility,

and

immunocompatibility, have been appealing carriers for delivering therapeutic and diagnostic agents.10 Previously, we developed the novel MRI contrast agents with 3

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gadolinium-based peptide dendrimers, resulting in good biocompatibility, as the peptide branching units are biodegradable. For the dendritic MRI contrast agents, we found that the sensitivity and blood circulation time were also MW-and generation-dependent: the higher MW dendrimers resulted in higher in vitro relaxivity and longer blood circulation.11,12 However, the dendrimers/dendrons with higher generation (>fifth generation) were not easy to prepare.13,14 That was attributed to the steric hindrance to the chemical reactions. Additionally, the high-generation dendrimers were observed to have latent cytotoxicity, as the degradation was very slow.15 Recently, the “dendronized polymer” strategy, which combines the properties of dendron (third generation) and heparin, was utilized in our previous studies to construct nanoscale anticancer agents.16 The conjugate-based nanoscale vehicles exhibited increased anticancer efficacy and good biosafety, which promoted their possible use in MRI diagnosis due to the dendronized macromolecular structure and biodegradability of the natural polysaccharide and peptide dendron. In addition to heparin, hyaluronic acid (HA) is an anionic natural polysaccharide with a certain structure composed of alternating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine with β (1→4) interglycosidic linkage. HA is water soluble, nonimmunogenic, and biodegradable, which promotes it as an excellent building block for biomaterials in biomedicine.17,18 In order to broaden its functionalization and overcome its short half-life,19 the chemical modification of HA is crucial for its

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further application.20,21 Therefore, the HA-based dendronized polymers with high MWs and dendritic structures may have capacity as carriers for imaging probes. In addition to enhanced tumor-targeting efficacy and sensitivity, biocompatibility is another issue for gadolinium-based macromolecular MRI imaging probes. The prolonged gadolinium residence time in vivo may result in slow and inefficient excretion, which further increases the risk of side effects induced by the platform itself. Meanwhile, gadolinium generated from the metabolism of gadolinium chelation,22 may be responsible for nephrogenic systemic fibrosis (NSF) in patients.23 The biodegradable polymeric carriers may meet the needs of biosafety, and we previously designed and prepared stimuli-responsive biodegradable branched copolymer-gadolinium conjugates with high MWs and nanoscale sizes. Although high sensitivity and high accumulation in tumors were observed, biodegradability was slow, and gadolinium residue in the body was obvious.24 Based on the above points, we expected to construct a hybrid-based macromolecular MRI contrast agent by combining the advantages of low-generation peptide dendrons and HA. It is surmised that the macromolecular MRI contrast agent with a high MW and dendritic structure would have an increased longitudinal relaxivity value (r1) and promising higher accumulation in tumors. The biodegradability of the hybrid itself can not only minimize the side effects on the organism, but it can also alleviate the difficulty in the synthesis compared to the employment of sensitive linkers for crosslinking the non-degradable molecules.25,26

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In this study, we designed and prepared a dendronized-HA conjugate hybrid functionalized with Gd(III) chelates, and investigated its potential application as an MRI contrast agent. The HA was modified with peptide dendron (Dendron-Gd, third generation) via “click reaction,” as shown in Scheme 1. The biodegradability, longitudinal relaxivity value (r1), contrast enhancement in the breast tumor site, and biodistribution in vivo were evaluated. Its biosafety was another focus in our study, and the hybrid demonstrated good biocompatibility by systematically evaluating the blood compatibility, cytotoxicity, and in vivo toxicity, suggesting that the dendronized natural polymer hybrid may be an appealing candidate for delivery of Gd(III) chelates, and the Gd(III)-based hybrid may have potential applications as MRI probes for the diagnosis of breast cancer. 2. EXPERIMENT SECTION 2.1. Materials and Measurements. Hyaluronic acid (sodium salt; MW 10000-20000 Da) was purchased from Sunlidabio Co. (Nanjing, China). DOTA derivatives were prepared as previously reported.27 The hyaluronidase (HAase) (1148 U/mg,

bovine

testes

isolated),

2-propynylamine,

4-(4,

6-dimethoxy-1,

3,

5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), and gadolinium chloride hexahydrate (GdCl3·6H2O) were obtained from Sigma-Aldrich (USA). All of the above materials were used without further purification. 1H NMR spectroscopy data were analyzed by the Bruker Advanced Spectrometer (400 MHz). Zeta potential and dynamic light scattering (DLS) was tested on a Malvern Zetasizer Nano ZS system (Worcestershire, UK). The Gd(III) content of the MRI agents, distribution, and 6

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residual in the major tissues/organs were analyzed by the Elan DRC-e inductively coupled plasma mass spectrometry (ICP-MS, USA) method. 2.2. Preparation of the Gadolinium-labeled Peptide Dendron-Hyaluronic Acid Hybrid. The gadolinium-labeled peptide dendron (Dendron-DOTA-Gd) was synthesized according to our previous work as described in the supporting information. Hyaluronic acid sodium salt (0.4 g, 0.99 mmol) was activated by DMTMM (0.82 g, 2.97 mmol) in 200 mL of di-water for 0.5 h. After adding 2-propynylamine (0.16 g, 2.97 mmol) into the solution, the reaction was continued at room temperature for 5 h. The solvents were dialyzed against di-water for 2 days and then lyophilized, producing a white solid (HA-alkyne). The product (Dendron-Gd, 1.5 g, 0.34 mmol), HA-alkyne (0.3 g, 0.17 mmol), and sodium ascorbate (10 mol%) were put in a vial, and the vial was protected with nitrogen. Di-water (120 mL) was added under nitrogen, and copper sulfate in 5 mL of di-water (5 mol%) was added. The mixture was protected with nitrogen and stirred for 24 h at 40°C. The products of the gadolinium-labeled peptide dendron-hyaluronic acid hybrid (Dendronized-HA-DOTA-Gd) were collected via dialysis in the aqueous solution water with ethylenediaminetetra-acetic acid disodium salt (0.5 mM) for 24 h and di-water for 24 h, then freeze dried, resulting in a white powder. The content of gadolinium was determined through ICP-MS analysis, resulting in 3.8% (weight percent). 2.3.

Degradation

of

the

Dendronized-HA-DOTA-Gd

Hybrid.

The

Dendronized-HA-DOTA-Gd hybrid dissolved in PBS (5 mg/mL, pH 7.4) was 7

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incubated with hyaluronidase (150 U/mL) at 37°C on an orbital shaker. At each predetermined time point of 12 h, 24 h and 48 h, the degradation solution was heated to 100°C for 10 min to inactivate the hyaluronidase. Then the mixture was centrifuged to collect the supernate for the gel permeation chromatography (GPC) test. The hybrid treated with non-hyaluronidase in PBS under the same processes was the negative control group. 2.4. T1 Relaxivities Measurements and In Vitro MRI. The in vitro relaxivities were measured with a MRI scanner (Siemens Trio, 3T) at room temperature. In order to study the MRI phantom, the Gd-DTPA and gadolinium-based polymeric hybrid were prepared in PBS solution (0.1 M) with various Gd(III) concentrations (0.15-0.5 mM). To obtain the T1-weighted images, the parameters and sequence were set as previous reported: TE = 7 ms, TR set from 20 to 1000 ms, and matrix dimensions = 128 × 256.24 The plots of 1/relaxation time (1/T1, s-1) vs. the gadolinium concentration were used to give the relaxivity values (r1) from their plots. 2.5. Animal Model. Animal were cared for in accordance with the national welfare legislation, and experiments were approved by the Animal Experiments Ethics Committee of Sichuan University and China. Normal female BALB/c mice with ~20 were purchased from DaShuo Biotechnology Co., Ltd (Sichuan, China). About 5 × 105 4T1 breast tumor cells suspended in 50 µL of PBS solution were subcutaneously injected into the right flank. When the tumor size reached approximately 4-6 mm in diameter, the mice were used to investigate the in vivo MRI.

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2.6. In Vivo MRI of the Tumors. The 4T1 tumor-bearing mice were anesthetized by intraperitoneal injection of 1% pentobarbital sodium salt and imaged on a Siemens Trio 3T MRI system equipped with a mouse coil. Multi-section single-echo T1 weighted TSE sequence was used for the contrast-enhanced imaging study with the parameters: FOV = 51 mm, slice thickness = 1.5 mm, slices =11, TR = 450 ms, TE = 11 ms, and Flip angle = 90°. The mice were intravenously injected with Dendronized-HA-DOTA-Gd or Gd-DTPA. The dose was 0.08 mmol Gd(III)/kg mice. The images of mice were captured before and after being intravenously injected with MRI contrast agents at four different time points: 0.5 h, 1.5 h, 4 h, and up to 20 h. The signals within the regions of interest (about 0.15 cm2) were measured. The relative MRI contrast enhancement of signal-to-noise ratio defined as ∆SNR: ∆SNR = (St/Sm)/(St0/Sm0), where St/Sm was the signal ratio of tumor to the surrounding muscle in post-contrast MR images, and St0/Sm0 was that in pre-contrast images.28,29 The trends of signal changes were presented by plotting the ∆SNR vs. time. 2.7. Biodistribution and Retention of Gd(III) In Vivo. The distribution of MRI contrast agents in the major tissues/organs at different time points after the injection of MRI contrast agents was evaluated by analyzing the Gd(III) contents using ICP-MS. The mice with the 4T1 tumors were intravenously injected with Gd-DTPA or Dendronized-HA-DOTA-Gd with dose of 0.08 mmol Gd(III)/kg mice. At post-injection of 0.5 h and 20 h, the mice were killed, and the tissues/organs, including the heart, liver, lungs, kidneys, spleen, and tumors, were collected and treated for ICP-MS testing as we previously reported.24 The Gd(III) concentration of 9

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each supernatant sample was calculated via ICP-MS to calculate the Gd(III) retention in vivo as the percentage of original injection dose per gram of tissues/organs (% ID/g). In addition, the subacute Gd(III) retention of Dendronized-HA-DOTA-Gd or Gd-DTPA in vivo was studied in normal female BALB/c mice. At 15 days post-injection of the agents, the mice were killed by means of cervical dislocation, then, separated heart, liver, lungs, kidney, spleen, and muscles were collected, and treated for ICP-MS testing as we previously reported. The Gd(III) retention in those tissues was obtained according to the analytical method of Gd(III) biodistribution at 0.5 h and 20 h. 2.8. Blood Compatibility Evaluation. Blood was collected from healthy unmedicated donators and stored in sodium citrate, and the ratio of blood to -anticoagulant was 9:1. The blood compatibility measurements were performed based on previous reports.30-32 2.8.1. Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT). The effects of the agents on coagulation were tested by mixing supernatant plasma (360 µL) with Dendronized-HA-DOTA-Gd or Gd-DTPA (40 µL) for a final concentration of 10 mg/mL at 37°C. Measurements were initiated by adding coagulation reagents into the solutions and studied on the STA-R evolution automatic coagulation instrument (Stago, France, n = 3). The control group was mixed with an identical volume of PBS solution. 2.8.2.

Thromboelastography

(TEG).

The

PBS

solution

of

Dendronized-HA-DOTA-Gd or Gd-DTPA (100 µL) was added into fresh whole 10

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blood (900 µL) in a tube containing kaolin. After thoroughly mixing, the final concentrations of MRI contrast agents were 5 mg/mL and 10 mg/mL in the blood, while the blood administrated with the identical PBS solution (100 µL) was treated as the control group. We transferred 340 µL of each mixture into the TEG cup, and CaCl2 solution (20 µL, 0.2 M) was added to the agents/blood mixture to initiate the analysis. Then, the test was carried out using the thrombelastograph coagulation analyzer at 37°C. 2.8.3. Aggregation and Morphologies of Red Blood Cells. At room temperature, the red blood cells (RBCs) were precipitated from whole blood and prepared for aggregation

and

morphologies

studies,

as

previous

reported.

The

Dendronized-HA-DOTA-Gd hybrid or the Gd-DTPA was dissolved in 100 µL of PBS with final concentrations of 1 mg/mL, 5 mg/mL, and 10 mg/mL. We added 20 µL of RBCs into the solution mentioned above using PBS as the control. The mixtures were thoroughly mixed by vortexing, and incubated for 15 min. The RBCs were washed with PBS and treated with 4% paraformaldehyde, placed on glass slides, and further treated according to our previous report for scanning electron micrography (SEM) studies.33 2.8.4. Red Blood Cell Hemolysis. We mixed 1 mL of PBS solution of the Dendronized-HA-DOTA-Gd hybrid or the clinical agent Gd-DTPA with 50 µL of diluted RBCs suspension by vortexing with the final agent concentrations of 1 mg/mL, 5 mg/mL, and 10 mg/mL. RBCs incubated with the di-water and PBS were employed as positive control and negative control. Each group of the RBCs was incubated with 11

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agents in a static condition at 25°C. After 12 h, all of the samples were centrifuged at 1,000 g for 5 min and 200 µL of supernatant from each tube was added into a 96-well plate, respectively. The absorbance data at a 540 nm wavelength were tested via a microplate reader. The hemolysis percentage of RBCs was calculated as previously reported. Each sample was tested 3 times. 2.9. Cytotoxicity Assays. Mouse myoblast cell (C2C12), human embryonic kidney cell (293T), mouse embryonic fibroblasts cell (NIH/3T3), and human liver cell (L02) were cultured in 96-well plates with DMEM medium at 37°C with 5% CO2. After treating with Trypsin-EDTA (GIBCO), the digested cells were seeded (5,000 cells/well) in the 96-well plates and incubated in a humidified atmosphere for 24 h under

5%

CO2

at

37°C.

The

medium

of

DMEM

containing

the

Dendronized-HA-DOTA-Gd hybrid or the Gd-DTPA (10 - 300 nmol/mL Gd(III)) was then added into each well and the cells mixed with materials were incubated for another 24 h. After that, the medium with agents were separated and the cells were washed. The cell viabilities were obtained via CCK-8 method as previous reported.24 2.10. Histological Analysis. Normal mice were randomly divided into 3 groups. The experimental groups were administered with 200 µL of the Gd-DTPA and the gadolinium-based polymeric hybrid (0.08 mmol Gd(III)/kg) respectively via intravenous injection 3 times every 4 days. Mice administrated with physiological saline were used as the control group. The body weight of all of the mice was monitored every 2 days, and their behaviors were detected. After the final injection and recovery for 6 days, all mice were killed by cervical dislocation. The 12

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organs/tissues, including the heart, liver, spleen, lungs, and kidneys, were treated for histological analysis, as previously reported, and the blood was collected for serum biochemical analysis. 2.11. Serum Biochemical Analysis. The markers of liver and kidney function were evaluated by serum biochemical analysis. The blood from the mice that were killed and used in the analysis of Gd(III) retention was collected and the serum was obtained by centrifugation at 4°C with 3000 rpm for 10 min. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), blood urea nitrogen (BUN), and creatinine (CRE) in the serum samples were analyzed. 2.12. Statistical Analysis. Results were presented as means ± (standard deviation) SD. The levels of significantly different in all statistical analyses were set as the value of p < 0.05. Analysis of variance and t-tests was utilized for analyzing the data among groups. 3. RESULTS AND DISCUSSION 3.1. Preparation of HA Polymeric Hybrid with Gd(III) Chelates. The fabrication process of Gd(III) that chelated the functionalized and dendronized HA hybrid (Dendronized-HA-DOTA-Gd) was illustrated in Scheme 1. Considering the synthesis difficulty and the impact of the structures in their biological application, the low-generation peptide dendron (third generation) was chosen to functionalize the linear natural polymer HA, as the low-generation peptide dendron and HA were biocompatible. The dendronized polymer with Gd(III) chelates was designed to meet 13

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the biosafety and efficiency requirements of MRI due to the biodegradability and features of dendritic macromolecules. In order to construct a “dendronized polymer” structure, “click reaction” was introduced to combine the advantages of both low generation peptide dendron and HA. The tail of the flabellate peptide dendron-Gd agent was modified with one azido group, and the HA was modified with alkynyl groups. The Gd(III) was first complexed with the azido-dendron prior to the “click reaction” to avoid the latent toxicity of copper ions.8 In terms of the chemical modification of HA, the reaction was performed through carboxyl groups of HA. DMTMM was recently used to facilitate the efficient one-step condensation of small molecules and polymers, especially in the functionalization of polysaccharides.34,35 Compared to traditional activation agents, DMTMM can facilitate the reaction with high yields and relatively low cost, so we employed DMTMM as the activator to conjugate the HA and 2-Propynylamine. The excess reagents and byproducts were easily removed from the reaction via dialysis and the FPLC system. In 1H NMR spectra (Figure S1), the peak at 2.81 ppm of the proton was assigned to 2-propynylamine on the HA, with every 4 repeat glycosyl units modified with 1 alkynyl group. After the “click reaction”, the ICP-MS results demonstrated that the content of gadolinium on the Dendronized-HA-DOTA-Gd hybrid was 3.8% as a weight percentage and the final product had excellent solubility in water (˃ 100 mg/mL). GPC was employed to analyze the molecular weight distribution of the Dendronized-HA-DOTA-Gd

hybrid.

As

shown

in

Figure

S2,

the

Dendronized-HA-DOTA-Gd had a higher MW of 25200 kDa (PDI = 1.82) than that 14

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of HA, which indicated that the low-generation peptide dendron with Gd(III) chelates (Dendron-Gd) was successfully grafted onto the HA chains. The Dendronized-HA-DOTA-Gd hybrid exhibited negative zeta potential (-35 mV) (Figure S3A) that was ascribed to a bunch of hydroxyl and carboxyl groups on HA. The agent with the negative charge could avoid the drawback of rapid clearance from circulation after intravenous injection which the materials with positively charge suffered.36,37 Thus, the Dendronized-HA-DOTA-Gd hybrid with the negative charge may have a prolonged blood circulation time by reducing the interaction with serum proteins, resulting in a high accumulation into target tissue in vivo.38,39 The hydrodynamic sizes of Dendronized-HA-DOTA-Gd was 115 nm (PDI = 0.276, Figure S3B), which indicated the polymeric hybrid may be used as a nanoscale agent. 3.2. In Vitro Degradation. It has been recognized that hyaluronidase plays a major role in the biodegradation process of HA in vivo.40 In this study, the degradability of the Dendronized-HA-DOTA-Gd hybrid was estimated under the hyaluronidase treatment. Figure 1 showed the residual MW of the Dendronized-HA-DOTA-Gd hybrid after hydrolysis with hyaluronidase at different time points. Obviously, the group of hyaluronidase incubation presented faster degradable behavior than that under

the

PBS

treatment.

At

48

h,

only

24%

of

the

higher

MW

Dendronized-HA-DOTA-Gd hybrid was left, which was close to that of the Dendron-DOTA-Gd. dendrimers/dendrons

Fortunately, possess

it

has

good

been

reported

biocompatibility

that with

the

peptide

biodegradable

characteristic.41 Even though the residual fragments were the mixture of hybrid and 15

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Dendron-DOTA-Gd, it was determined that the hybrid could degrade into small molecules, which promoted its clearance out of the body and further reduced the risk from residual macromolecular contrast agents or the polymeric carriers.42,43 3.3. In Vitro MRI Relaxivity. To investigate the potential application of the prepared polymeric hybrid as an efficient MRI probe, the longitudinal relaxivity value (r1) calculated from the slopes of the plots of 1/relaxation time (s-1) against the concentrations of Gd(III) was measured on a clinical Siemens Sonata 3.0 T MR scanner at room temperature. The T1-weighted MR images were obtained (Figure 2A). The Dendronized-HA-DOTA-Gd hybrid revealed an increased signal intensity depending upon the Gd(III) concentrations, while the Gd-DTPA had no obvious signal change across the experimental concentrations (0.15-0.5 mM). Meanwhile, with the same Gd(III) concentrations, the hybrid revealed a much brighter signal intensity than that of the clinical Gd-DTPA. As the results show in Figure 2B, the r1 value of the Dendronized-HA-DOTA-Gd hybrid (7.7 mM-1 s-1) was much higher than the Gd-DTPA (2.5 mM-1 s-1). The significantly increased r1 value of the Dendronized-HA-DOTA-Gd hybrid might be mainly due to the attachment of the low MW paramagnetic gadolinium-chelates onto the dendronized-polymer-based hybrid with an increased rotational correlation time (τR).27 On the other hand, the good aqueous solubility and the homogeneous modified sites of the HA-based hybrid could evade the obstacle of water exchange between the coordinated H2O on the hybrid and bulk H2O that might happen in PEGylation,44,45 thus further increasing the r1 value of the Dendronized-HA-DOTA-Gd-based hybrid. 16

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3.4. In Vivo MRI of Tumors. The in vivo MRI experiments were performed to investigate the efficiency of the Dendronized-HA-DOTA-Gd hybrid as a cancer diagnosis MRI contrast agent on a clinical 3.0 T MR scanner. The mice bearing 4T1 breast tumors were utilized as the animal model for the administration of the Dendronized-HA-DOTA-Gd hybrid and the clinical agent Gd-DTPA. The axial MR images were obtained at various time points pre- and post-administration (Figure 3A). The mice administrated with the polymeric hybrid expressed a significantly brighter contrast enhancement at the tumor site compared to the normal tissues, which was favorable for tumor identification. Meanwhile, at each time point, signals in the tumors contrasted by the polymeric hybrid were much stronger than that contrasted by Gd-DTPA throughout the experimental period. The relative enhancements of signal-to-noise ratio (∆SNR) with time in tumors were plotted in Figure 3B to quantitatively analyze the contrast enhancement. Expectedly, at all time points, compared to the Gd-DTPA, the Dendronized-HA-DOTA-Gd hybrid generated higher ∆SNR in tumors. Actually, the ∆SNR of the Gd-DTPA fluctuated slightly around 1.0, which indicated the signal in those tumors had few changes pre- and post-administration of the Gd-DTPA. The ∆SNR in the tumors of mice injected with the Dendronized-HA-DOTA-Gd hybrid demonstrated the best result at 1.5 h (∆SNR = 1.44), and then a decline in signal intensity was observed over time. This was due to its biodegradation and the hybrid was resultantly excreted out of the body traced by the high brightness in the bladder, which further minimized the latent toxicity risk associated with gadolinium-based contrast agents. The high contrast enhancement in 17

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the tumors with the Dendronized-HA-DOTA-Gd hybrid is mainly attributed to its higher r1 value, and long blood circulation time that is favorable for its potential higher accumulation in tumors compared to the Gd-DTPA. 3.5. Biodistribution and Retention of the Agents in Vivo. The quantitative distribution and retention of the Gd(III) ions in the main organs/tissues were considered

as

important

parameters

to

evaluate

whether

the

Dendronized-HA-DOTA-Gd-based polymeric hybrid had higher tumor uptake and would be excreted out of the body. Thus, the Gd(III) distribution in major organs/tissues of the mice bearing 4T1 breast tumors were studied at 0.5 h and 20 h after being administrated with the MRI contrast agents. In Figure 4, the Gd(III) accumulation in tumors treated with the Dendronized-HA-DOTA-Gd polymeric hybrid were higher and statistically different (p < 0.05, vs. Gd-DTPA), which resulted from the prolong blood circulation time and EPR effect of the macromolecular Dendronized-HA-DOTA-Gd hybrid. Interestingly, for both of the agents at the two time points, the Gd(III) contents in the kidneys were higher than in other organs. As we know, the kidneys are responsible for the excretion of agents and the results validated that the Dendronized-HA-DOTA-Gd hybrid and its degraded products can be

cleansed

from

the

body

by

the

kidneys.

Meanwhile,

for

the

Dendronized-HA-DOTA-Gd hybrid, the Gd(III) accumulations in each experimental organs (the heart, liver, lungs, kidneys, spleen and tumors) were significantly less at 20 h compared to that at 0.5 h. The 2-fold decrease of Gd(III) accumulations from 7.3%

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(at 0.5 h) to 3.4% (at 20 h) might due to the renal excretion of Dendronized-HA-DOTA-Gd hybrid, as the hybrid is biodegradable. Generally, the high value and long-term retention of contrast agents in the body may lead to undesirable diseases induced by the Gd(III) ions, which were generated from the metastasis of the Gd(III)-based contrast agent. In order to further evaluate the subacute residue in body, the values of gadolinium in the major organs/tissues of mice injected with the polymeric hybrid were observed to have much lower gadolinium retention in the heart, liver, spleen, lung, kidney, and muscles as shown in Figure 5. In particular, the Gd(III) residue in the kidneys was 0.025%, which decreased dramatically compared to that at 20 h post-administration (2.23%). However, the total Gd(III) retention was only 0.19% at 15 days post-injection. The results were comparative with the small-molecule clinical agent Gd-DTPA; however, the too-fast cleanse of the Gd-DTPA may cause toxicity to the kidney. An optimal cleanse rate may avoid this latent side effect. This level of Gd(III) residue in vivo was much lower than our previous study in which the Gd(III) residue of copolymer-gadolinium conjugates was obvious at 12 day post-injection although the branched molecule was stimuli-responsive biodegradable.24 The low Gd(III) residue in vivo with the Dendronized-HA-DOTA-Gd hybrid was mainly due to its biodegradation and resultant excretion via the kidneys, which further minimized the potentially toxic risk from the accumulation of macromolecules in the body. 3.6.

Blood

Compatibility.

The

hemocompatibility

of

the

Dendronized-HA-DOTA-Gd and Gd-DTPA were evaluated by analyzing the blood 19

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coagulation properties, erythrocyte morphologies, aggregation and hemolysis under in vitro conditions. The effects induced by biomaterials on the blood coagulation properties are crucial for hemocompatibility evaluation.46,47 The procoagulant and anticoagulant natures of Dendronized-HA-DOTA-Gd to human plasma were evaluated by measuring the properties of APTT and PT, which both reflect the common coagulation pathway. The differences are that APTT indicates the time needed for a fibrin clot to form and characterizes the intrinsic coagulation pathway after adding the partial thromboplastin reagent and CaCl2 into the plasma, while PT represents the time needed for a fibrin clot to form and characterizes the extrinsic coagulation pathway after adding the tissue thromboplastin into the plasma.31 As presented in Figure 6, all of the values of APTT

and

PT

fell

in

the

normal

ranges,

which

indicated

that

Dendronized-HA-DOTA-Gd showed no abnormal effects on the pro- and anticoagulant natures of plasma. Thromboelastography (TEG) is an in vitro analyzer, but it has the capability of tracing the overall process of whole blood coagulation once the blood is in contact with biomaterials by replicating the in vivo conditions most accurately.48 The information that concerns the kinetics and strength of the blood clot were characterized by TEG.49 The major parameters were analyzed: R, the time for a detectable fibrin to form after initiating a run; K, the time for a substantial clot to form after starting the test, which represents the dynamics of clot formation; α angle, the

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slope of the tangent joining the point of the 2-mm split intercepting the tracing; and MA (maximum amplitude), the maximum clot strength or stiffness.50 Figure 7 displays the representative TEG results of clot formation in PBS, the Gd-DTPA, and the Dendronized-HA-DOTA-Gd hybrid, while the main parameters and data are listed in Table 1. As the results showed, for the MRI agents, all of the parameters we measured were normal values and did not result in a significant difference in blood coagulation compared with the PBS control. In other words, both the commercial agent Gd-DTPA and the novel Dendronized-HA-DOTA-Gd hybrid had no significant effects on the process of whole blood coagulation, including the initial fibrin formation time, the rate of fibrin formation, and the strength or stiffness of

the

blood

clots.

The

TEG

measurements

demonstrated

that

the

Dendronized-HA-DOTA-Gd hybrid could not cause thrombus or blood loss. Owing to their easy of handing and highest amount in blood, RBCs are employed to analyze the impact of injected agents on blood biocompatibility. Once the agents are injected into the body and blood circulation system, their interactions with RBCs are inevitable which may bring the latent side effect. The RBC membrane is a good and significative model for the study of side effects induced by foreign molecules on the mammalian cell membranes.31 Therefore, RBCs morphologies, aggregation, and hemolysis are necessary to study the hemocompatibility of foreign agents. The resulting RBCs’ morphologies and aggregations for the Dendronized-HA-DOTA-Gd hybrid and Gd-DTPA with various concentrations were displayed in Figure 8. Compared to the Gd-DTPA or the PBS control group (Figure S4), the 21

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Dendronized-HA-DOTA-Gd hybrid demonstrated RBCs monodisperse and kept their normal biconcave shape as the SEM images show. In the images, it was obviously shown that the surface of the RBCs was smooth and the cell membrane was complete. Based on the results, Dendronized-HA-DOTA-Gd had no significant side effects on the RBCs’ morphologies and aggregation. The hemolysis assay was evaluated with the percentage of hemolysis < 5% as the safe standard.51 After being incubated with the Dendronized-HA-DOTA-Gd hybrid or the Gd-DTPA for 12 h, for the RBCs, no apparent hemolytic activity was found, and no significant difference was observed compared to PBS control as shown in Figure 9A. The quantitative analysis revealed that the Dendronized-HA-DOTA-Gd hybrid with 1 mg/mL, 5 mg/mL, and 10 mg/mL induced -0.15%, 0.5%, and 0.46% lysis of RBCs, while the Gd-DTPA with the same concentrations induced -0.12%, -0.31%, and -0.43% lysis, respectively. The negative values indicated that the hemolysis percentage of the agents was low compared to the PBS control. According to previous reports, the effect of foreign agents on hemocompatibility was changed in a concentration-dependent way.31 Meanwhile, the interaction of the blood components with foreign materials is mainly due to the electrostatic attraction and/or the hydrophilic/hydrophobic interaction.32 The concentrations measured in this study were in accordance with the conventional dose used in the evaluation of agents.52 As the results showed, the Dendronized-HA-DOTA-Gd hybrid with a negative charge and hydrophilic property (even at the high concentration of 10 mg/mL), demonstrated no significant effects on the blood coagulation, erythrocyte 22

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morphologies, aggregate, and lysis, which suggested that this synthesized agent is safe to blood components. 3.7. Cytotoxicity and In Vivo Toxicity Tests. To evaluate the cytotoxic effect of the polymeric hybrid on normal cells, a CCK-8 assay was performed on C2C12, NIH/3T3, 293T, and L02 cell lines after they were incubated with the agents for 24 h in comparison to Gd-DTPA. In Figure 10, no significant cytotoxicity was observed across the measured Gd(III) concentration range for the Dendronized-HA-DOTA-Gd hybrid with the cell viability of about 100%, which suggested that the Dendronized-HA-DOTA-Gd hybrid was safe for these normal cells. As a foreign contrast agent with Gd(III) chelates for in vivo tumor diagnosis application, most of the MRI agent will reach the normal organs/tissues after being intravenously injected into body, as shown in Figure 4. The potential metabolic toxicity to normal organs/tissues may be observed due to the possibility of dissociation of Gd(III) from the carriers. Thus, we further determined whether or not the Dendronized-HA-DOTA-Gd hybrid with Gd(III) chelates could affect the health of normal mice. After being administrated with the agents, the mice's behavior and body weight shift were measured, and saline was injected as the control. Additionally, histological studies were used to assess the in vivo potential toxicity of foreign agents. Healthy mice administrated with the polymeric hybrid, Gd-DTPA, and saline (the control) did not show obvious signs of anorexia, locomotor impairment, dehydration, muscle loss, or other symptoms associated with animal toxicity throughout the 15 days evaluation 23

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period.

As

shown

in

Figure

S5,

the

mice

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injected

with

the

Dendronized-HA-DOTA-Gd hybrid showed no abnormal body weight shift and mortality compared to the controls. The steady body weight increase indicated that the Dendronized-HA-DOTA-Gd hybrid did not induce adverse effects, such as nutritional and toxicity mediated by Gd(III) to the growth of mice. Figure 11 presents the histological analysis results, which were used to indicate the side effects of the Dendronized-HA-DOTA-Gd hybrid or its degradation products to the normal organs/tissues. As the tissue slices show, the representative organs of mice administrated with Dendronized-HA-DOTA-Gd presented no damage, inflammation, or lesions from toxic exposure compared with the control groups. The clinical indexes, which were commonly used for liver function and renal function, were detected.53 As shown in Table 2, all of the data of the Dendronized-HA-DOTA-Gd hybrid did not show any significant difference compared to the saline group. It further confirmed that the Dendronized-HA-DOTA-Gd hybrid possessed good biocompatibility. The non-observed pathological abnormalities of the mice administrated with the Dendronized-HA-DOTA-Gd

hybrid

were

mainly

attributed

to

its

good

biocompatibility that was inherited from both HA and peptide dendrons with a low generation. For the Dendronized-HA-DOTA-Gd hybrid, the employment of HA modified the hybrid with a negative charge surface and aqueous solubility that were favorable for its stability and dispersity in the in vivo environment reduced the potential side effects to normal organs. Crucially, the biodegradable properties of the hybrid could promote its fast clearance via renal excretion, which was also certified in 24

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the analysis of in vitro degradation, in vivo MRI, and biodistribution. Overall, the systematic biosafety assessment in our study presented a novel hybrid with excellent biocompatibility, which suggested the Dendronized-HA-DOTA-Gd hybrid is promising for clinical applications. 4. CONCLUSION The gadolinium-labeled peptide dendron/HA hybrid was successfully designed and prepared by combining the HA-alkyne and azido-dendron-Gd via click chemistry, and its potential application as an MRI contrast agent was investigated. The hybrid exhibited

biodegradable

ability

under

the

hyaluronidase

treatment.

The

Dendronized-HA-DOTA-Gd polymeric hybrid firstly had a higher longitudinal relaxivity value (r1), which was 3-fold greater than that of Gd-DTPA. Second, the polymeric hybrid led to a much higher gadolinium accumulation in the tumors and, therefore, resulted in much stronger signal intensity and increased contrast enhancement in tumors compared to Gd-DTPA. Finally, owing to the biodegradability of the Dendronized-HA-DOTA-Gd-based hybrid, Gd(III) can be completely cleansed from the body. The systematic biosafety studies, including blood compatibility and toxicity assessments, showed the Dendronized-HA-DOTA-Gd hybrid had excellent biocompatibility, which further promoted its high potential of application in clinical cancer diagnoses. ASSOCIATED CONTENT Supporting Information

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Preparation studies and characterization data, biodegradability data, Figure S1-S5 were shown in Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *

E-mail (Prof. Luo): [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by International Science and Technology Cooperation Program of China (2015DFE52780), National Natural Science Foundation of China (51373104, 51673127, 81361140343, 81621003), Chengdu science and technology project (2016-GH03-00005-HZ) and Applied Basic Research Project of Sichuan Province (2015JY0279).

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(32) Liu, Z.; Jiao, Y.; Wang, T.; Zhang, Y.; Xue, W. Interactions Between Solubilized Polymer Molecules and Blood Components. J. Controlled Release 2012, 160, 14-24. (33) Guo, C.; Hu, J.; Kao, L.; Pan, D.; Luo, K.; Li, N.; Gu, Z. Pepetide Dendron-Functionalized Mesoporous Silica Nanoparticle Based Nanohybrid: Biocompatibility and Its Potential as Imaging Probe. ACS Biomater. Sci. Eng. 2016, 2, 860-870. (34) Kunishima, M.; Kawachi, C.; Hioki, K.; Terao, K.; Tani, S. Formation of Carboxamides by Direct Condensation of Carboxilic Acids and Amines in Alcohols Using a New Alcohol- and Water-Soluble Condensing Agent: DMT-MM. Tetrahedron 2001, 57, 1551-1558. (35) Ortega, F. R.; Ruiz, F. J. P.; Averick, S. E.; Rodríguez, G.; Aguilar, M. R.; Matyjaszewski, K.; Román, J. S. Smart Heparin-Based Bioconjugates Synthesized by a Combination of ATRP and Click Chemistry. Polym. Chem. 2013, 4, 2800-2814. (36) Malik, N.; Wiwattanapatapee, R.; Klopsch, R.; Lorenz, K.; Frey, H.; Weener, J. W.; Meijer, E. W.; Paulus, W.; Duncan, R. Dendrimers: Relationship Between Structure and Biocompatibility in Vitro, and Preliminary Studies on the Biodistribution of 125I-Labelled polyamidoamine Dendrimers in Vivo. J. Controlled Release 2000, 65, 133-148. (37) Li, S.; Zhao. Z.; Wu, W.; Ding, C.; Li, J. Dual pH-Responsive Micelles with Both Charge-Conversional Property and Hydrophobic-Hydrophilic Transition for Effective Cellular Uptake and Intracellular Drug Release. Polym. Chem. 2016, 7, 2202-2208. (38) Gao, Y.; Chen, L.; Gu, W.; Xi, Y.; Lin, L.; Li, Y. Targeted Nanoassembly Loaded with Docetaxel Improves Intracellular Drug Delivery and Efficacy in Murine Breast Cancer Model. Mol. Pharmaceutics 2008, 5, 1044-1054. (39) Aggarwal, P.; Hall, J. B.; McLeland, C. B.; Dobrovolskaia, M. A.; McNeil, S. E. Nanoparticle Interaction with Plasma Proteins as It Relates to Particle Biodistribution, Biocompatibility and Therapeutic Efficacy. Adv. Drug Delivery Rev. 2009, 61, 428-437. (40) Zhong, S.; Campoccia, D.; Doherty, P. J.; Williams, R. L.; Benedetti, L.; Williams, D. F. Biodegradation of Hyaluronic Acid Derivatives by Hyaluronidase. Biomaterials 1994, 15, 359-365. (41) Zhang, C.; Pan, D.; Luo, K.; She, W.; Guo, C.; Yang, Y.; Gu, Z. Peptide Dendrimer-Doxorubicin Conjugate-Based Nanoparticles as an Enzyme-Responsive Drug Delivery System for Cancer Therapy. Adv. Healthcare Mater. 2014, 3, 1299-1308. (42) Tang, M.; Zhou, M.; Huang, Y.; Zhong, J.; Zhou, Z.; Luo, K. Dual-Sensitive and Biodegradable Core-Cross linked HPMA Copolymer–Doxorubicin Conjugate-Based Nanoparticles for Cancer Therapy. Polym. Chem. 2017, 8, 2370-2380. (43) Duan, Z.; Zhang, Y.; Zhu, H.; Sun, L.; Cai, H.; Li, B.; Gong, Q.; Gu, Z.; Luo, K. Stimuli-Sensitive Biodegradable and Amphiphilic Block Copolymer-Gemcitabine Conjugates Self-Assemble into a Nanoscale Vehicle for Cancer Therapy. ACS Appl. Mater. Interfaces 2017, 9, 3474-3486. (44) Toth, E.; Uffelen, I. V.; Helm, L.; Merbach, A. E.; Ladd, D.; Saebo, K. B.; Kellar, K. E. Gadolinium-Based Linear Polymer with Temperature-Independent Proton Relaxivities: a Unique Interplay Between the Water Exchange and Rotational Contributions. Magn. Reson. Chem. 1998, 36, 125-134. (45) Doble, D. M. J.; Botta, M.; Wang, J.; Aime, S.; Barge, A.; Raymond, K. N. Optimization of the Relaxivity of MRI Contrast Agents: Effect of Poly(ethylene glycol) Chains on the Water-Exchange Rates of GdIII Complexes. J. Am. Chem. Soc. 2001, 123, 10758-10759. (46) Li, S.; Guo, Z.; Zhang, Y.; Xue, W.; Liu, Z. Blood Compatibility Evaluations of Fluorescent Carbon Dots. ACS Appl. Mater. Interfaces 2015, 7, 19153-19162. 29

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(47) Brockman, K. S.; Kizhakkedathu, J. N.; Santerre, J. P. Hemocompatibility Studies on a Degradable Polar Hydrophobic Ionic Polyurethane (D-PHI). Acta Biomaterialia 2017, 48, 368-377. (48) Salooja, N.; Perry, D. J. Thrombelastography. Blood Coagulation Fibrinolysis 2001, 12, 327-337. (49) Mallett, S. V.; Cox, D. J. A. Thrombelastography. Br. J. Anaesth. 1992, 69, 307-313. (50) Speranskaya, E. S.; Sevrin, C.; Saeger, S. D.; Hens, Z.; Goryacheva, I. Y.; Grandfils, C. Synthesis of Hydrophilic CuInS2/ZnS Quantum Dots with Different Polymeric Shells and Study of Their Cytotoxicity and Hemocompatibility. ACS Appl. Mater. Interfaces 2016, 8, 7613-7622. (51) Silver F. H. Biomaterials, Medical Devices and Tissue Engineering: an Integrated Approach. London: Chapman & Hall 1994, 153–192. (52) Kainthan, R. K.; Gnanamani, M.; Ganguli, M.; Ghosh, T.; Brooks, D. E.; Maiti, S.; Kizhakkedathu, J. N. Blood Compatibility of Novel Water Soluble Hyperbranched Polyglycerol-Based Multivalent Cationic Polymers and Their Interaction with DNA. Biomaterials 2006, 27, 5377-5390. (53) Pu, L.; Geng, Y.; Liu, S.; Chen, J.; Luo, K.; Wang, G.; Gu, Z. Electroneutralized Amphiphilic Triblock Copolymer with a Peptide Dendron for Efficient Muscular Gene Delivery. ACS Appl. Mater. Interfaces 2014, 6, 15344-15351.

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Captions Scheme 1. Synthesis of the Gd(III)-labeled and dendronized HA (Dendronized-HA-DOTA-Gd) hybrid.

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Table 1. Clotting kinetics values of human whole blood mixed with the clinical agent Gd-DTPA or Dendronized-HA-DOTA-Gd hybrid solutions.

R (min)

K (min)

α (deg)

MA (mm)

Normal range

5−10

1−3

53−72

50−70

PBS control

6.4

2.1

62

58.4

5 mg/mL Gd-DTPA

5.5

1.7

64.4

59.8

10 mg/mL Gd-DTPA

6.4

2.2

59.1

57

5 mg/mL Dendronized-HA-DOTA-Gd

6.1

2.2

61.2

55.8

10 mg/mL Dendronized-HA-DOTA-Gd

6.1

2.3

58.4

50.5

Table 2. Serum biochemical test of mice injected with the Dendronized-HA-DOTA-Gd hybrid, clinical agent Gd-DTPA, and saline.

ALT (U/L)

Saline

Gd-DTPA

Dendronized-HA-DOTA-Gd

48.00 ± 3.46

49.80± 4.65

51.25 ± 7.27

AST(U/L)

106.33 ± 4.63

118.50 ± 8.69

114.75 ± 7.41

ALP (U/L)

153.00 ± 9.59

154.75 ± 10.37

161.25 ± 8.46

GGT (U/L)

9.00 ± 1.00

8.75 ± 0.95

8.00 ± 1.00

BUN (mmol/L)

49.39 ± 2.47

43.24 ± 2.71

53.59 ± 3.16

CRE (µmol/L)

52.80 ± 5.54

54.33 ± 5.50

57.50 ± 6.55

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120 PBS treatment

Weight remained (%)

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100 Hyaluronidase treatment

80 60 40 20 0 0

5

10

15

20

25

30

35

40

45

50

Time (h)

Figure 1. Molecular weight remainder of the Dendronized-HA-DOTA-Gd hybrid with hyaluronidase (▲, black line, 24% remaining ) and PBS (●, red line, 57% remaining) treatment after 48 h incubation.

Figure 2. T1-weighted MRI (A), and in vitro r1 (B) of the Dendronized-HA-DOTA-Gd hybrid and Gd-DTPA in PBS at 3T.

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Figure

3.

T1-weighted

MR

imaging

in

the

tumors

of

mice

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administrated

with

the

Dendronized-HA-DOTA-Gd hybrid and Gd-DTPA (A), and the relative enhanced signal to noise ratio (∆SNR) (B) (*p < 0.05, n = 5 mice per group).

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Figure 4. Biodistribution of Gd(III) in the mice bearing 4T1 tumors at 0.5 h and 20 h post-administration of the Gd(III)-based agents. (*p < 0.05 and **p < 0.05, vs Gd-DTPA) (n = 5 mice per group, dose = 0.08 mmol Gd(III)/kg mice).

Figure 5. Retention of Gd(III) in the mice bearing 4T1 tumors at 15 days post-injection of Gd-DTPA and the Dendronized-HA-DOTA-Gd hybrid, respectively. (n = 5 mice per group, dose = 0.08 mmol Gd(III)/kg mice).

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60.00 APTT

50.00 Clotting time (sec)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

PT

40.00 30.00 20.00 10.00 0.00 PBS

Gd-DTPA

Dendronized-HA-DOTA-Gd

Figure 6. Effects of the Gd-DTPA and Dendronized-HA-DOTA-Gd hybrid on APTT and PT. (Areas between the two orange and blue dotted lines respectively represented the normal range of APTT and PT)

Figure 7. Thromboelastograph traces and results of whole blood coagulation measured by the PBS and Gd(III)-based agents.

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Figure 8. Effects of the Gd-DTPA (A) and Dendronized-HA-DOTA-Gd hybrid (B) on the aggregation and morphology of RBCs by SEM (top row: ×500, bottom row: × 2.00 k).

Figure 9. Photographs of the hemolysis of RBCs after being incubated with Gd(III)-based agents (A). Percentage of the hemolysis of RBCs incubated with the Gd(III)-based agents (B), Di-water (+) and PBS (-) were used as the positive and negative control, respectively. 37

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Gd-DTPA

Dendronized-HA-DOTA-Gd

C2C12

120

100

Cell viability (%)

Cell viability (%)

120

80 60 40 20

Dendronized-HA-DOTA-Gd NIH/3T3

100 80 60 40

0 10

120

Gd-DTPA

20

0 50 100 200 Gd concentration (nmol/mL)

Gd-DTPA

Dendronized-HA-DOTA-Gd

300

10

293 T

120

100

Cell viability (%)

Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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80 60 40 20

50 100 200 Gd concentration (nmol/mL)

Gd-DTPA

Dendronized-HA-DOTA-Gd

300

L02

100 80 60 40 20

0

0 10

50 100 200 Gd concentration (nmol/mL)

300

10

50 100 200 Gd concentration (nmol/mL)

300

Figure 10. Cell viability of Gd-DTPA and Dendronized-HA-DOTA-Gd hybrid on C2C12, NIH/3T3, 293T, and L02 cell lines.

Figure 11. Histological analysis of the organs/tissues from the normal mice administrated with the Dendronized-HA-DOTA-Gd hybrid, the clinical agent Gd-DTPA, and saline (all tissues: × 100, scale bars represent 200 µm).

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