Cross-Linked and Biodegradable Polymeric System as a Safe

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Crosslinked and Biodegradable Polymeric System as Safe Magnetic Resonance Imaging Contrast Agent Qiang Luo, Xueyang Xiao, Xinghang Dai, Zhenyu Duan, Dayi Pan, Hongyan Zhu, Xue Li, Ling Sun, Kui Luo, and Qiyong Gong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16345 • Publication Date (Web): 20 Dec 2017 Downloaded from http://pubs.acs.org on December 21, 2017

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ACS Applied Materials & Interfaces

Crosslinked and Biodegradable Polymeric System as Safe Magnetic Resonance Imaging Contrast Agent Qiang Luo,†,1 Xueyang Xiao,†,1 Xinghang Dai,†,§ Zhenyu Duan,† Dayi Pan,† Hongyan Zhu,‡,* Xue Li, ‡ Ling Sun,† Kui Luo,†,* Qiyong Gong†

†

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

‡

Laboratory of Stem Cell Biology, State Key Laboratory of Biotherapy/Collaborative

Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China, §

West China School of Medicine, Sichuan University, Chengdu, Sichuan 610041, China.

*Corresponding authors. Prof. Zhu is to be contacted at Tel/fax: +86 28 85423503. Prof. Luo is to be contacted at Tel.: +86 28 85414308; fax: +86 28 85410653. E–mail addresses: [email protected] (Pro. Zhu); [email protected] (Pro. Luo) 1

Luo Q. and Xiao X.Y. contributed equally to this work.

1

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ABSTRACT: Owing to the low efficacy of clinically used small-molecule gadolinium (Gd)-based magnetic resonance imaging (MRI) agents, we designed and explored biodegradable macromolecular conjugates as MRI contrast agents. The linear polymeric structure and core-crosslinked formulation possessed of different characteristics and features, so we prepared and comparatively studied the two kinds of Gd-based N-(2-hydroxypropyl) methacrylamide (HPMA) polymeric systems (the corecrosslinked pHPMA-DOTA-Gd and the linear one) using the clinical agent diethylenetrianmine pentaacetic acid-Gd(III) (DTPA-Gd) as a control. This study aimed to find the optimal polymeric formulation as a biocompatible and efficient MRI contrast agent. The high molecular weight (MW, 181 kDa) and core-crosslinked copolymer was obtained via the cross-linked block linear copolymer, and could be degraded to low MW segments (29 kDa) in the presence of glutathione (GSH), and cleaned from the body. Both core-crosslinked and linear pHPMA-DOTA-Gd copolymers displayed 23-fold increased relaxivity (r1 value) than DTPA-Gd. Animal studies demonstrated that two kinds of macromolecular systems led to much longer blood circulation time, higher tumor accumulation and much higher signal intensity (SI) compared with DTPA-Gd. Additionally, the core-crosslinked pHPMA-DOTA-Gd copolymer showed superior MRI features compared to the linear and clinical ones. Finally, in vivo and in vitro toxicity studies indicated that the two macromolecular agents had great biocompatibility. Therefore, we performed preliminary but important studies on the Gd-based HPMA polymeric systems as biocompatible and efficient MRI contrast agents, and found that the biodegradable core-crosslinked pHPMA-DOTA-Gd copolymer might have greater benefits for the foreground. KEYWORDS: Biodegradable, Core-crosslinked polymer, MRI, Contrast agent, 2


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Biocompatibility

1. INTRODUCTION For clinical tumor imaging, the changed signal contrast between the diseased and normal tissues is utilized to produce clear and accurate images via MRI contrast agents. 1,2

Currently, the DTPA-Gd and other Gd-based chelate agents are most commonly used

in clinical MRI applications. However, their efficiency, sensitivity, and retention time in the circulatory system were still limited in terms of their low molecular weight (MW), short circulation time, and other related unpleasant optimized characteristics.

3

Meanwhile, to achieve better imaging results, repeated injections or high doses are required, which may cause side effects.

3,4

Spurred by recent progress in material

chemistry and polymeric nanotechnology, some of the shortcomings could be addressed by conjugating small molecular Gd-units to one macromolecular skeleton, resulting in macromolecular MRI probes.

4-6

These macromolecular agents with large

sizes accumulated greater in tumors via the enhanced permeability and retention (EPR) effect.

7

The polymeric carriers, including liposomes,

8-10

micelles,

11,12

dendritic

polymers, 13 and other polymeric systems, 14-16 have great impact on potential clinical applications in the foreseeable future. 17 Previous studies proposed that the HPMA polymer has been proved to be one of the leading candidates for the delivery of diagnostic and therapeutic agents. That is due to its biocompatibility, non-immunogenicity, neutral charge and water-soluble properties. 18-20

For these HPMA polymeric carriers with high MWs, their unique structural,

physicochemical and biological properties are more superior than low MW carriers, as a long retention time in the circulatory system and high pharmaceutical efficiency are 3



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closely associated with high MW character. 21-22 In order to further enhance the targeting efficacy of contrast agents, it is necessary to optimize the structure of carriers and regulate the macromolecular system size to meet the requirements of the EPR effect. 2325

Recently, some HPMA copolymers with high MWs had been prepared to enhance

the EPR efficacy, resulting in higher accumulation in tumors and significantly increased anticancer efficacy.26-30 Studies on the HPMA copolymer/dendrimer hybrid and linear HPMA

copolymer-based

drug

delivery

systems

demonstrated

that

the

copolymer/dendrimer hybrid held natural advantages in therapeutic indexes that might be attributed to the dendritic structure compared with linear polymers. 31 However, few studies have focused on the dendritic HPMA copolymer-based macromolecular systems for MRI-enhanced contrast agents. High relaxivity is essential to enhance the contrast between diseased and normal tissues for the contrast agents in MR images. According to the Solomon-BloembergenMorgan theory, the contrast agents with high relaxivity should obtain the following characteristics: a ligand able to combine a greater number of water molecules, an optimally short water residence time, and a slow tumbling rate.

32

Researchers tried

their best to improve the relaxivity of contrast agents via these ways even though it was extremely difficult. Previous studies have found that the relaxivity was correlated well with the MWs in a series of polymeric contrast agents, as high MW ones could slow down the tumbling time.

32,33

Previous studies on dendritic polymeric MRI contrast

agents demonstrated their high relaxivity and great potential for cancer diagnosis. However, it was not easy to synthesize those dendritic polymers due to step-by-step organic synthesis and purification, especially for the polymers with high MWs. 34,35 In addition to relaxivity, biosafety is one more primary issue for macromolecular 4


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MRI contrast agents. 36,37 Due to glomerular filtration, the upper threshold of MW is 50 kDa.

38

For the gadolinium-based MRI agents, too long of a circulation time of

conjugates might lead to nephrogenic systemic fibrosis (NSF), which might be caused by the increasing risk of toxicity of Gd(III) ions.25 Therefore, biodegradable HPMA copolymer-based delivery systems for cancer therapy and diagnosis have been designed and prepared.

14

Some researchers reported sensitive biodegradable multiblock

copolymers which had great anticancer efficacy, and could be degraded to small molecules around a specific microenvironment and finally eliminated from the body. 39 On the other hand, the dendritic polymers had shown some superiority as carriers for drug/imaging probes delivery.

39,40

Thus, we sought to design and prepare

biodegradable core-crosslinked HPMA copolymer-gadolinium conjugate by crosslinking the low MW linear copolymer with a biodegradable linker, and explore whether the macromolecular and core-crosslinked polymeric conjugate-based contrast agents could result in significantly enhanced MRI imaging efficacy as well as good biocompatibility. In this study, we designed and prepared core-crosslinked and linear pHPMA-DOTAGd as efficient MRI contrast agents via RAFT polymerization and cross-linking chemistry, as shown in Figure 1. The comparative studies, including physicochemical properties, biodegradability, in vitro and in vivo MRI imaging and biocompatibility, were carried out between the core-crosslinked copolymer and the linear one to investigate which formulation may be a superior safe and efficient MRI contrast agent. 2. MATERIALS AND METHODS 2.1. Materials and Measurements Momoners HPMA, N-[2-(2-Pyridyldithio)]ethyl methacrylamide (PTEMA), and 5



MA-DOTA were prepared as previously reported.

7, 41

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4-Cyanopentanoic acid

dithiobenzoate (CTA), trimethylolpropane tris(3-mercaptopropionate), VA044 (2,2azobis[2-(2-IMidazolin-2-yl)Propane]Dihydrochloride), 2-mercaptoethanol and V501 (Polyvinyl acetate) were purchased from Sigma-Adrich. Weight-average MW and polydispersity (PDI) of the copolymers were measured via size-exclusion chromatography (SEC) on an ÄKTA/FPLC system (GE Healthcare). Size and zeta potential were detected by Zetasizer (Malvern, Worcestershire, UK). All animal experiments were performed under the approval and guidance of the Animal Experiments Ethical Committee of West China Hospital of Sichuan University. 2.2. Synthesis of Core-crosslinked and Linear Gadolinium-based HPMA Copolymers 2.2.1. Synthesis of Linear HPMA Copolymer. HPMA (1.4 g, 9.75 mmol), 4cyanopentanoic acid dithiobenzoate (4-CTA, 12 mg, 79 µmol), MA-DOTA (3.34 g, 6.5 mmol) and VA044 (8.5 mg, 26.3 µmol) were put in a vial. The vial was closed and a solution of di-water/methanol (5: 1, 22 mL) was added. The solution was bubbled with argon at 0oC for 40 min and stirred at 44°C for 7.5 h. Then the mixture was stirred at 0oC for 5 min. The copolymer was precipitated in acetone twice. The solid was dissolved in water, after dialyzing against water for 12 h and drying by freeze dryer, the final product (pHPMA-DOTA with dithiobenzoate group) with a slight pink color (43% yield, 2.1 g; MW = 26 kDa, PDI 1.04) was obtained. 2.2.2. Synthesis of Pyridine Disulfide Modified pHPMA-DOTA Copolymer. The copolymer (pHPMA-DOTA with dithiobenzoate group) (1.2 g) and N-[2-(2pyridyldithio)]ethyl methacrylamide (PTEMA, 200 mg, 0.79 mmol) were put in a vial. Deionized water/methanol solution (3: 1, 8 mL) containing VA044 (3.2 mg, 10 µmol) 6


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was added to the vial. The solution was bubbled with argon at 0oC for 40 min and stirred at 44°C for 4 h. After quenched in liquid nitrogen and precipitated in acetone twice. The solid was dissolved in the solvents of deionized water/methanol (4: 1, 8 mL) and was dropped into the stirred acetone (200 mL). The solid was then dried, providing the product (Pyridine Disulfide Modified pHPMA-DOTA, 75% yield, 1.05 g; MW = 29 kDa, PDI 1.15). 2.2.3. Synthesis of Linear pHPMA-DOTA-Gd. The copolymer (Pyridine Disulfide Modified pHPMA-DOTA) (900 mg) and V501 (80 mg) was dissolved in 5 mL methanol. The solution was stirred at 70oC for 3 hours. The solution was cooled to room temperature, and 200 mg 2-mercaptoethanol was added. The solution was stirred over night, after dialyzing against water for 24 h and drying by freeze dryer, the final product (pHPMA-DOTA, 94% yield, 840 mg; MW = 28 kDa, PDI 1.10) was obtained. The copolymer (pHPMA-DOTA, 800 mg) and GdCl3·6H2O (928 mg, 2.5 mmol) were dissolved with 25 mL of di-water. We used 0.1 M of NaOH to adjust the pH value to 5.2-5.4. The solution with a pH of 5.2-5.4 was stirred at room temperature for 24 h. After dialyzing against water for 12 h and drying, the final product (pHPMA-DOTAGd, 810 mg; MW = 29 kDa, PDI 1.09) was obtained. The 6.5% Gd(III) loading was measured via ICP-MS analysis. 2.2.4. Synthesis of Core-crosslinked pHPMA-DOTA. Under nitrogen, the pyridine disulfide modified pHPMA-DOTA copolymer (900 mg) was dissolved in 20 mL of deionized

water/acetone

(6:

1).

While

maintaining

vigorous

stirring,

trimethylolpropane tris(3-mercaptopropionate) (20 mg) in ethyl acetate/acetone (1:1, 1 mL) was added into the solution by dropwise for 1 h and then stirred for another 6 h. The products were precipitated in acetone (200 mL) and further purified by SEC. The 7



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useful fractions were collected, dialyzed against water, and freeze-dried, resulting in product (Pyridine Disulfide Modified Core-crosslinked pHPMADOTA, 870 mg, MW = 181 kDa, PDI 1.98). The copolymer (860 mg) was reacted with V501 (>30 fold) and 2mercaptoethanol (200 mg), as preparation of pHPMA-DOTA. The solution was dialyzed against water for 24 h. After drying by freeze dryer, the final product (corecrosslinked pHPMA-DOTA, 95% yield, 820 mg; MW = 182 kDa, PDI 1.93) was obtained. 2.2.5. Synthesis of Core-crosslinked pHPMA-DOTA-Gd. The core-crosslinked copolymer (800 mg) was reacted with GdCl3·6H2O (928 mg, 2.5 mmol) to prepare gadolinium-based core-crosslinked HPMA copolymer (core-crosslinked pHPMADOTA-Gd; MW = 181 kDa, PDI 1.95), as described above. The 6.1% Gd(III) loading capacity was obtained via ICP-MS analysis. 2.3. Ex vivo and In Vivo MRI Study The relaxivity of the core-crosslinked pHPMA-DOTA-Gd and the linear one was assessed in ultrapure water by a clinical MRI scanner, as previously reported.

41

The

signal intensities were obtained by noose tool and the same size circle was applied in every measurement. Through the special computational formula and analysis method of data as previously reported, the 1/T1 value was obtained. The relaxivity value r1 was calculated by plotting the 1/T1 as a function of different Gd(III) molar concentrations. The core-crosslinked pHPMA-DOTA-Gd was incubated with GSH (10 mM) at 37 oC for 12 h, and the relaxivity of degradated products was measured. To evaluate the accumulation ability of conjugates in tumors, an in vivo MRI study was performed. Subcutaneous tumor models were built using 4T1 cells derived from mice breast 8


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carcinoma tumors. Female 4-6 week-old BALB/c mice (20 ±2 g) were purchased from Chengdu Dossy Experimental Animals Co., Ltd. At first, 4T1 cells [7×105 cells suspended in 70 μL of phosphate buffered saline (PBS)] were accurately inoculated in the right lower back of the mice. Animals were randomly divided into three groups until the volume of solid breast cancer tumors neared 15 mm3. Then, the 1.5 T or 3.0 T MRI scanner was used to obtain the contrast-enhanced images. The MRI imaging method and parameters were described in the supplementary information, as previously reported.

41,42

Animals were injected with three different agents: the core-crosslinked

pHPMA-DOTA-Gd, the linear one, and DTPA-Gd (0.08 mmol Gd(III)/kg) via the tail vein. At pre-injection, and at 10, 30, 45, 60, and 180 minutes after injection, MRI images were acquired. 2.4. In Vivo Major Organ MR imaging Three groups of (n=5 for each group) healthy female 4-6 week-old BALB/c mice (20 ± 2 g) were injected with 0.08 mmol Gd(III)/kg the core-crosslinked pHPMA-DOTAGd, the linear one and DTPA-Gd through tail-vein for in vivo major organ MR imaging. At pre-injection and 10 min, 30 min, 1.5 h, 3 h, 6 h, 9 h, and 12 h post-injection, the T1weighted MR images were obtained using the 3.0 T clinical MR system. The scanning parameters were as follows: TR = 500 ms, TE = 11 ms, slices = 15, thickness=1.5mm, and Fov = 50 mm. The intensity of major organs could be quantitatively measured by the use of noose tool in the scanning system. 2.5. Blood Circulation and Pharmacokinetics For in vivo blood circulation and pharmacokinetics, healthy female 4-6 week-old BALB/c mice (20 ±2 g) were injected with 0.08 mmol Gd(III)/kg the core-crosslinked pHPMA-DOTA-Gd, the linear one, and DTPA-Gd through tail-vein. At 5 min, 15 min, 9



30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h post-injection, 20 μL blood from fundus venous plexus was harvested. After digested by HNO3 and H2O2 solution, these ultimate samples were measured by inductively coupled plasma mass spectrometry (ICP-MS) to obtain the remaining concentration of Gd(III). The pharmacokinetic parameters were obtained by PKSolver software. 43 2.6. Biodistribution of Conjugates in Tissues Fifteen subcutaneous breast carcinoma mouse models were randomly divided into three groups, and were injected with 0.08 mmol Gd(III)/kg the core-crosslinked pHPMA-DOTA-Gd, the linear one, and DTPA-Gd through the tail-vein, and then sacrificed at 24 h post-injection to evaluate the distribution of agents in organs. The six major organs (heart, liver, spleen, lung, kidney and bladder) and tumors were separated and fixed with 4% formaldehyde. All of these organs were washed with PBS, weighed, and digested with H2O2 (1 mL) and HNO3 (3 mL). Organs were heated at 120oC until all of these samples digested completely, then they were diluted to 4 mL with deionized water. These ultimate samples were measured by inductively coupled plasma mass spectrometry (ICP-MS) to obtain the remaining agent concentration. 2.7. In Vitro Cell Viability This experiment was used to evaluate the toxicity of the two conjugates to tumor cells and normal human cells. The 4T1 (breast cancer) and LO2 (normal liver) cells were chosen to complete this progress. Cell counting kit-8 assay (CCK-8, Dojindo, Japan) was used to assay the cytotoxicity of the core-crosslinked pHPMA-DOTA-Gd, the linear one and DTPA-Gd. The 4T1 and LO2 cells were seeded on 96-well plates (5 × 103 cells/well) in DMEM supplemented with 10% FBS, and put in a carbon dioxide incubator with 5% CO2 at 37oC for 24 h. Cells were washed twice after removing the 10


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medium. The DMEM solutions with core-crosslinked pHPMA-DOTA-Gd, the linear one and DTPA-Gd were added to wells, whereas the Gd(III) concentrations were 25, 50, 100, and 200 μg/mL. The untreated cells were processed as the controls. After incubating in a carbon dioxide incubator for 24 h, cells were washed thrice with PBS, and then added 100 μL of CCK-8 into each well. 2 hours later, the absorbance of wells with cells was measured via the microplate reader Varioscan Flash (Thermo Fisher Scientific) at 450 nm to assay the cells’ viability (%). The untreated contrast cells were considered to be 100% viable. The transverse and longitudinal contrast could reflect the cytotoxicity of polymers. 2.8. Hemolysis Tests Hemolysis tests were performed to assess the influence of macromolecular systems on red blood cells (RBCs). The agents of the core-crosslinked pHPMA-DOTA-Gd and the linear one were added into a blood suspension (15 μL) from healthy donors, and the final solutions (1, 3, and 5 mg/mL) were obtained. After incubating at 37 oC for 12 h, RBCs were carefully collected via centrifuging (1000 × g, 5 min). Negative controls contained human blood suspension (15 μL) and PBS, while positive controls with distilled water were equal to 100% lysis. All samples were dropped onto a 96-well culture plate and the cells’ hemolysis (%) was measured via the microplate reader Varioscan Flash (Thermo Fisher Scientific) at 540 nm to assay. Hemolysis tests were carried out 3 times. The influence of polymers on RBC was evaluated through the transverse and longitudinal contrasts and using a previous formula.27 2.9. Plasma Coagulation Fresh blood samples from healthy donors mixed with citrate were used in this analysis. After centrifuging at room temperature (3, 000 × g, 15 min), the platelet was 11



separated, and the platelet poor plasma (PPP) were obtained. Then 40 μL solution with polymers was added into 360 μL of PPP as the final detecting samples. The equal volume of PBS was added into the PPP as the negative control. Activated partial thromboplastin time (APTT) and prothrombin time (PT) were measured to evaluate the clotting time, by means of an automatic coagulation analyzer (SYSMEX CA-7000). 2.10. Effect on RBCs’ Morphologies and Aggregation RBCs were separated from fresh healthy blood by centrifuging at room temperature (1,000 × g, 5 min) and washing 3 times with PBS solution. Then 20 μL of RBCs were added into the solution (100 μL) with core-crosslinked pHPMA-DOTA-Gd and the linear one. The final concentration of polymers was 2.5 mg/mL. The RBCs (20 μL) were added into the PBS solution (100 μL) as the control group. After washing with PBS twice, all samples were fixed with 4% paraformaldehyde for at least 1 h. Erythrocyte suspensions (10 μL) were transferred onto clean glass slides and dehydrated with gradient concentration ethanol as previously reported. Samples were dried in a static platform at room temperature. Finally, all sample photos were obtained under the SEM analyzer after coating with gold. 2.11. In Vivo Toxicity Female 4-6-week-old BALB/c mice (20 ±2 g) were randomly divided into 4 groups (n=7, each group), weighed and recorded, and then marked with fluorescence dyestuff. Three groups were given the core-crosslinked pHPMA-DOTA-Gd, the linear one and DTPA-Gd at a concentration of 0.08 mmol Gd(III)/kg. The injection was administrated every 4 days via tail vein (200 μL), 3 times totally. The other group received the same volume of saline as the control group. We observed the behavior of the mice and weighed them every 2 days. 20 days later, fresh blood was collected through the 12


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eyeballs for routine blood and biochemical examinations, then all animals were killed and primary organs were separated for histological analysis. 2.12. Statistical Analysis Data are presented as means ± SD. Student's two-tailed t test was performed to test the statistical significance. The value of p less than 0.05 or 0.01 was considered to be significant and highly significant, respectively. 3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of Gd-based HPMA Copolymers The accumulation of polymeric contrast agents in tumors is highly related to the EPR effect, which is associated with their size. Meanwhile, for MRI contrast agents, the most important parameter, “relaxivity,” could also be improved by increasing the MW. Therefore, the high MW copolymers with large size had been designed and prepared in this study. Previous studies reported that high MW copolymers have been used as drug delivery carriers for cancer therapy, 26 and biodegradable copolymers as MRI contrast agents,

45,46

which implied that our design of the biodegradable core-crosslinked and

linear polymers as contrast agents might be reasonable to some extent. Additionally, as the main chain of HPMA copolymer is not degradable, the biodegradable corecrosslinked copolymer with high MWs was designed and prepared for the good biosafety (Figure 1).

13



S

O

O

+

C

S

NH

S

O C HOO N N

CH2 O

OH

O

OH

m O

CH2 O

C

o

HN

S S

NH

N

N N

NH

C

CH2 O

C

o

CH2 O

C

n

HN

Pyridine Disulfide Modified pHPMA-DOTA HS

CH2 O

S

S

S

N

N

S

O

S

N

HN S

H COO

O CN C CH2

S

O

GdCl3· 6H2O

S

S

O

O

S

C

r HN

O

H COO

O

S

O

N

pHPMA-DOTA

O

O O

S

O

OH

SH O

HS

O

C HOO

O

O

O

NH

S

CH2

C

q

CH2 O

HN

O

CH2 O

S

C

n

HN

CN

NC

C

CH2 O

HN

C

o

CH2 O

C

n

HN

CH2 O

NH C HOO

N

S

NH

S

S

O

OH

S

Gd N N

HO

O

HN

O CN C CH2

NC

S

O

O S

S

O

O

S

C

r HN

O

O O

CH2

q

C

CH2 O

HN

O

C

p

CH2 O

S

S

O

S

O C HOO

S O

S

S

HO

HN

S

S

O

O

S

r HN

O

S

H COO

H COO

CN

O

S

N

N

O C CH2

NC

N

N

GdCl3· 6H2O

S

O

O

O

NH

S

Core-crosslinked pHPMA-DOTA O O

OH

m

HO

S

O

O

CH2 O

HN

OH

pHPMA-DOTA-Gd

C

n

HN

O

S

H COO

H COO

S

O

O

S

N N

S O

O O

O

O O

N

N

OH

V501, 70oC HS

O HO

S

O

O N

O

HN

OH

m

HO

S

Pyridine Disulfide Modified Core-crosslinked pHPMA-DOTA

OH

m

CH2 O

HN

S

O

O

S

C

p

N

O HO

O

OH

m

HO

S

O

OH

HS

HN

H COO

H COO

CN

HN

N

N

V501, 70oC

NC

N

N

2). V501, 70 C

pHPMA-DOTA with dithiobenzoate group

HO

O

O C HOO

H COO

O

OH

m

HO

o

H COO

CH2 O

HN

S

1). RAFT Polymerization

N

C

n

HN

N

C HOO

CH2 O

S N

O

H COO

CN C

S

HN

HO

CN

N

H COO

CH2 O

HN

RAFT Polymerization

N

C

n

HN

O

HO

S

CN S

HN

HN

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C

CH2

O

q

C

CH2 O

HN

S

O

C

p

CH2 O

C

n

HN

CH2 O

OH

m O

HN

HO

S

NH

S

O

O

O OH

N

O N

Gd N N

O O

O O

Core-crosslinked pHPMA-DOTA-Gd Figure 1. The scheme of the gadolinium-based HPMA conjugates, and the illustration of molecular structures.

In addition to the MW and size, the structure is also optimized to enhance the relaxivity and biocompatibility. Previous studies have reported both dendritic and linear polymeric systems were used for contrast agents or drug/gene carriers and imaging 14


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probes, and concluded that the dendritic copolymers might be superior compared with the linear ones.47,49 Therefore, core-crosslinked copolymers that bind with gadolinium chelates and biodegradable linkers were designed to enhance the blood circulation time and accumulation into tumors, relaxivity and biocompatibility, as shown in Figure 1.

A

B

C

D

E

Figure 2. 1H NMR spectrum of copolymers of pHPMA-DOTA with dithiobenzoate group (A), Pyridine Disulfide Modified pHPMA-DOTA (B), Pyridine Disulfide Modified Core-crosslinked pHPMA-DOTA (C), pHPMA-DOTA (D) and core-crosslinked pHPMA-DOTA (E).

However, due to renal filtration, the good biosafety of the HPMA polymer requires the MW is below 50 kDa. The emergence of advances in living radical polymerization has provided the chance to prepare biodegradable and high MW HPMA copolymers.2629

To balance the biosafety and efficacy, the linear copolymer (pHPMA-DOTA with

dithiobenzoate group) was firstly prepared via RAFT polymerization of monomers of HPMA and MA-DOTA, and the end was modified with the dithiobenzoate group 15



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(pHPMA-DOTA with dithiobenzoate group, see Figure 1). The presence of dithiobenzoate group was confirmed via 1H NMR (Figure 2A) owing to the peaks (δ = 7.90 ppm, 7.71 ppm and 7.53 ppm). Additionally, no peaks ranging from 5.0 ppm to 6.0 ppm was observed, indicating that the monomers have been cleared completely. Since the peaks assigned to double bond was about 5.60 (s, CH3–CH(R)–CH2 –Ha) and 5.20 (s, CH3–CH(R)–CH2–Hb). To obtain core-crosslinked polymers with high MWs and star-like structures, the copolymer with the dithiobenzoate group (pHPMA-DOTA with dithiobenzoate group) was used as the macroCTA to further mediate RAFT polymerization of PTEMA, resulting in the block and linear HPMA copolymer (Pyridine Disulfide Modified pHPMA-DOTA, see Figure 1) functionalized with pyridine disulfide groups. The obvious and broad peaks (δ = 8.40 ppm, 7.80 ppm and 7.28 ppm) in the 1H NMR spectra (Figure 2B) indicated that the pyridine disulfide groups had been introduced to the copolymer. Additionally, the MW of the copolymer was slightly increased to 29 kDa from 26 kDa. Those results indicate the formation of block polymers. The linear and pyridine

disulfide-modified

pHPMA-DOTA

polymer

was

reacted

with

trimethylolpropane tris(3-mercaptopropionate), and the reaction was carefully controlled. The successful preparation of the crosslinked copolymer was confirmed via SEC, as the MW was significantly increased to 181 kDa from the 29 kDa. Compared to the 1H NMR spectra of pHPMA-DOTA with dithiobenzoate group (Figure 2B), the peaks ranging from 7.0 to 9.0 were changed, and the peaks assigned to pyridine disulfide groups and dithiobenzoate groups were observed, as shown in Figure 2C. To avoid the possibility of toxicity mediated by dithiobenzoate group, a radical reaction of the initial copolymer with V501 was carried out, whereas excess V501 (>3016


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fold molar) was used for end-modification. The similar studies had been reported. 50 For example, the functionalized V501 agents were used to replace the end dithiobenzoate groups, resulting in total conversion.50,51 Meanwhile, the excessive and un-reacted pyridine disulfide groups were reacted with 2-mercaptoethanol to avoid the potential toxicity of pyridine disulfide groups. Since the broad peaks assigned to alcohol hydroxyl groups in the 1H NMR spectra (δ = 7.71 ppm in Figure 2D and 7.69 ppm in Figure 2E) were obviously observed. The successful preparation of different copolymers and core-crosslinked copolymer was confirmed by SEC (see Table S1), DLS and SEM studies. The hydrodynamic sizes of two conjugates (core-crosslinked pHPMA-DOTA-Gd and the linear one) were 180 nm and 6 nm (Figure 3), respectively. The significantly increased MW and hydrodynamic size suggested the formation of core-crosslinked copolymer. The size of core-crosslinked pHPMA-DOTA-Gd was also measured via SEM, and the size is about 120 nm in the dried state (Figure S1). Generally, for polymeric nanoscale systems, hydrodynamic size may be larger than that of their dried state via SEM.34 Due to the negatively charged Gd-DOTA and the neutral HPMA copolymers, the zeta potential of two gadolinium-based copolymers was -17 mV and -21 mV, respectively (Figure S2). The negative copolymers indicated their potential stability and reduced being taken up by the macrophages.

41

These results indicated that the

biodegradable core-crosslinked copolymer with high MW might enhance gadolinium accumulation into tumor tissue, while reducing potential gadolinium toxicity via fast renal excretion.

17



Figure 3. DLS studies of the core-crosslinked pHPMA-DOTA-Gd (A, 180 nm) and the linear one (B, 6 nm).

3.2. Biodegradable study of Gd-based HPMA Copolymers The cross-linked copolymer (core-crosslinked pHPMA-DOTA-Gd) was incubated with GSH medium, and was degraded to small segments (Table 1). The MW of degraded segments was 29 kDa (Figure 4). The MW of the degraded products agreed well with that of the block and linear pHPMA-DOTA-Gd. Since the linker used for cross-linking have disulfide bone, which could be degraded by the GSH that highly expressed in the tumor cells. The concentration of GSH found in extracellular (~2–10 μM) and intracellular (~2–10 mM) compartments, and in tumor tissues compared with healthy ones.52 The 29 kDa presented high molecular weight below the renal threshold of 50 kDa. The core-crosslinked pHPMA-DOTA-Gd was incubated with glutathione (10 mM), and the solution was also measured via DLS. The small segments (about 7 nm) were observed (Figure S3). The control group without GSH was also measured. However, the size was not obviously changed after incubation with 37 oC for 12 h (Figure S3B). It is notable that the size of glutathione was about 200 nm (Figure S3C). Those results also indicated the core-crosslinked polymer can be degraded at presence of glutathione. These results suggested that this biodegradable core-crosslinked copolymer with high MW could be cleaned from the body via renal excretion after ending its task as a macromolecular MRI contrast agent.

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Figure 4. The SEC profiles of core-crosslinked pHPMA-DOTA-Gd (MW 181 kDa, PDI 1.95) and their degraded products (MW 29 kDa, PDI 1.22) after incubation of copolymer (4 mg/mL) with GSH (3 mM, pH = 5.4) for 12 h at 37 °C.

Table 1. The MWs and PDI of the degraded products after incubation with the core-crosslinked pHPMA-DOTA-Gd (4 mg/mL) in buffer in the presence of GSH (3 mM, pH = 5.4) at 37 °C.

MW (kDa) PDI

0h 181 1.95

1h 151 2.26

2h 121 2.31

6h 71 1.77

8h 30 1.26

12 h 29 1.22

3.3. Ex Vivo MRI Studies To investigate the T1 contrast efficacy of the two polymeric systems as MRI contrast agents, in vitro MR imaging was performed, which can be measured by the longitudinal relaxation rate, also named r1 relaxivity. As shown in the T1-weighted MR images (Figure 5A), the core-crosslinked pHPMA-DOTA-Gd and the linear one resulted in a much brighter signal compared with the clinical DTPA-Gd under the equal Gd(III) concentration (Figure 5B). In addition, the r1 relaxivity value was calculated by plotting the 1/T1 as a function of different Gd(III) molar concentrations. The r1 value obtained from a 3.0 Tesla MRI scanner showed that the core-crosslinked pHPMA-DOTA-Gd and the linear one displayed r1 values of 10.49 and 6.42 mM-1·s-1, which was a 2-3-fold 19



increase compared to DTPA-Gd (3.38 mM-1·s-1) (Figure 5B). The 1.5 Tesla MRI results are shown in Figure S4, the core-crosslinked pHPMA-DOTA-Gd and pHPMA-DOTAGd displayed r1 values of 13.88 and 9.52 mM-1·s-1, which was also a 3-4-fold increase compared to DTPA-Gd (3.62 mM-1·s-1). The relaxivity (r1) of the degradation products was also measured by MRI scanner (Figure S5). The r1 of the degradation products is 6.9 mM-1·s-1, which was similar with that of linear pHPMA-DOTA-Gd (6.42 mM-1·s1

). The corresponding relaxivity behavior furtherly indicated the core-crosslinked

polymer is biodegradable. The relaxivity of the gadolinium-labeled polymers in 3.0 T MRI was lower than that in 1.5 T scanner. That is due to the influence of the magnetic field strength on the agents. The core-crosslinked pHPMA-DOTA-Gd copolymers resulted higher r1 values in comparison with the the linear one, which indicated that the core-crosslinked copolymers had better T1 contrast efficacy as MRI contrast agents. The results might be based on the specific structural characteristics of the corecrosslinked copolymers and the high MW. According to the Bloembergen-SolomomMorgan theory, the relaxivity of the polymeric systems could be improved by lengthening the rotational correlation time (τR) of the systems, which could be achieved by increasing the MW and molecular size. The hydrophobic and biodegradable linker that formed the high MW core-crosslinked copolymer had been recognized as playing an essential role. The macromolecules also attached to a large number of low MW DTPA-Gd moieties, and the water could effectively bind with the DTPA-Gd. Thus, the modified polymeric systems exhibited high proton relaxivity as confirmed by T1 weighted images.

53

In addition, the prepared polymeric systems were highly water-

soluble, the internal motion of the polymeric systems therefore could also influence the measured relaxivity. 20


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Figure 5. (A) T1-weighted MRI image of copolymers with gradient Gd(III) concentration; (B) The relaxivity curve of the core-crosslinked pHPMA-DOTA-Gd, the linear one and DTPA-Gd in the 3.0 T scanner.

3.4. In Vivo MRI Imaging of Tumor Based on the good T1 contrast efficacy in vitro, the in vivo MRI imaging performance was explored further to investigate the passive targeting of prepared polymeric systems as MRI contrast agents for diagnosing tumors. As shown in Figure 4, the tumors contrasted by the clinically used DTPA-Gd at 10 min post-injection showed slight brighter compared with the normal tissues or water, indicating that some DTPA-Gd molecules accumulated in the tumors. However, the signal brightness did not increase at 30 min post-injection and even began to decrease at 45 min post-injection. On the other hand, the mice injected with the polymeric systems showed a much stronger contrast enhancement at the tumor sites than the surrounding tissues at 30 min postinjection (Figure 6A), making the tumor clearly identified. At 3 h post-injection, the enhancement signal in tumors was still observed. To quantitatively analyze the contrast enhancement of agents in tumors, the relatively enhanced signal intensity (SI) was calculated via comparing the SI from the tumor regions with the water mold (Figure 6B). The core-crosslinked pHPMA-DOTA-Gd, the linear one and DTPA-Gd performed the similar relatively MRI enhancement SI (about 220%) at the 10 min post-injection. After that, the relatively enhanced SI data of the 21



core-crosslinked pHPMA-DOTA-Gd and the linear one steadily increased, and was up to about 260% at 3 h post-injection, indicating a greater accumulation in tumors for the two macromolecular contrast agents. However, for the DTPA-Gd agent, the enhancement value data fell slowly after 10 min, and recovered to the pre-injection level at 3 h post-injection. That may mean a short circulation time in the body before being excreted out of the body. Meanwhile, the core-crosslinked pHPMA-DOTA-Gd showed higher relatively enhanced MRI SI data at 10 min post-injection (Figure 6B). As previously mentioned, the EPR phenomenon of passive accumulation through the permeable neovasculature and localization in the interstitial tumor was observed for macromolecular agents in many solid tumors. The results showed that the corecrosslinked polymeric systems obtained optimal sizes to permeate the defective vasculature and resulted in an increased level of tumor accumulation compared with the linear polymeric systems. It is notable that the relatively enhanced SI data in the bladder was exactly the opposite of the tumor data (Figure S6). Data from the mice treated with the DTPA-Gd showed higher enhancement at 10 min post-injection, and then slowly increased until 3 h post-injection. However, the polymeric systems showed less enhancement compared with DTPA-Gd. That may be due to the different circulation time in the blood and the low renal excretion ratio of the polymeric systems and was deeply associated with the MW of the agents. Based on the above observations, the polymeric systems displayed strong enhancement ability, and the core-crosslinked polymer was better than the linear one.

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Figure 6. (A) MR axial images of the BALB/c mice models. (B) Relative enhancement signal intensity (SI) of copolymers in tumors in comparison with DTPA-Gd (*p < 0.05, n=5 each group).

3.5. Major Organ Imaging

The major organ regions (heart, liver, kidneys and bladder) became brighter after the injection of these contrast agents, but the variation trends of enhancement were different among these three different agents according to the quantitative measurement, as showed in Figure 7. Firstly, the relative signal enhancement of major organs increased rapidly at the first 30 minutes, especially in bladder, after the administration of polymeric macromolecular agents (the core-crosslinked pHPMA-DOTA-Gd and the linear one). After 1.5 h post-injection, the relative signal enhancement decreased slowly with time, and eventually dropped to the initial state because of the gradual elimination from the bloodstream in heart, liver and kidney. However, the signal in bladder kept high relative enhancement with time, which demonstrated the degradation and elimination in vivo and emphasized the long circulation time of these two agents again. Then, the injection of pHPMA-DOTA-Gd caused relatively mild augment of enhancement compared with the core-crosslinked polymer, suggesting better contrast efficacy and longer circulation time of the core-crosslinked polymer than the linear one. Finally, for clinically used DTPA-Gd, the duration time of enhancement in major 23



organs was limited in comparison with macromolecular agents. Especially, in bladder, the signal enhancement began to decrease at 1.5h post the injection. It was mainly due to the small MW of DTPA-Gd, which obtained shorter circulation time and could be rapidly excreted out of the body. The results demonstrated that polymeric molecular agents have excellent contrast efficacy in vivo, moreover, they could provide clear major organ MR images and could be easily excreted out of the body. On basis of current results, our future studies will focus on the more precisely diagnosis using functional Gd-based macromolecular polymeric MRI contrast agents, which is not only tumors, as well as inflammatory diseases, neurodegenerative diseases and other diseases.

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Figure 7. The T1-weighted MR images after injection and relative enhancement signal intensity in major organs of the core-crosslinked pHPMA-DOTA-Gd (A, B), the linear one (C, D) and DTPAGd (E, F).

3.6. Pharmacokinetics study

Pharmacokinetics study can further illustrate the circulation and metabolism of these materials in the body. As shown in Figure 8, the concentration of Gd(III) in blood samples was reduced with time after the administration of agents. In comparison with clinically used DTPA-Gd, these two macromolecular agents resulted longer circulation time and relatively mild reduction with time. The half-decay time (t1/2) of the corecrosslinked pHPMA-DOTA-Gd and the linear one were 521 and 421 min, much longer than DTPA-Gd (11 min). The longer circulation time might be one the basis of the larger molecular weight and size of polymeric agents, and negative charge in the surface. Additionally, compared with linear pHPMA-DOTA-Gd, the core-crosslinked one had a longer circulation time. Long half-decay time is an essential character for the application of these materials in both blood pool and tumor MR imaging, since the long circulation time permits a greater chance for materials to reach tumor tissue via EPR effect. The result suggested that these two macromolecular agents would have a better contrast efficacy in vivo, which was completely in agreement with the MR scanning results. In addition, it also proved that the core-crosslinked pHPMA-DOTA-Gd had better contrast efficacy than the linear one.

25



Figure 8. The pharmacokinetic data obtained for the core-crosslinked pHPMA-DOTA-Gd, the linear one and clinical used DTPA-Gd.

3.7. Biodistribution Analysis One more important influence factor of relatively enhanced SI data in tumors is the distribution of agents in the body. Compared with the clinical agent DTPA-Gd, the polymeric systems agents in tumors showed a much higher content of Gd(III) in tumors at 24 h post-injection (Figure 9), which indicated that polymeric systems as MRI contrast agents could obtain a higher accumulation in tumors. These results might be benefited from macromolecule property and size of polymeric systems. As previously reported in studies on copolymers, the charge and MWs played an essential role in the long circulation in the blood circulation system. Tumor growth triggered rapid angiogenesis, which resulted in extensive highly fenestrated vascular networks. The porous vascular beds of the tumors allow the proper size to selectively concentrate. These results were consistent with the findings of the in vivo imaging experiment. Compared to the pHPMA-DOTA-Gd, the core-crosslinked pHPMA-DOTA-Gd conjugate showed much higher Gd(III) contents in tumors. That may be due to the characteristics of core-crosslinked polymeric system, including high MW, larger size and negative zeta potential. However, the results of biodistribution of gadolinium indicated the potential residual gadolinium in the body. To accelerate the metabolism 26


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of the gadolinium, one of strategies is to design the backbone-degradable polymers. The relative studies are in progress, and will be reported in our further publications.

Figure 9. Quantitative analysis of Gd(III) residual in tissues and tumors at 24 h post-injection in BALB/c mice models, (n=7, per group).

3.8. Cytotoxicity Analysis To further confirm the low cytotoxicity of the polymeric system, an in vitro cytotoxicity experiment of the core-crosslinked pHPMA-DOTA-Gd, the linear one, and DTPA-Gd was precisely performed using CCK-8. In comparison with the DTPA-Gd, the polymeric systems showed higher cell viability in 4T1 cells (Figure 10A) and LO2 cells (Figure 10B) across the measured concentration range of Gd(III) (from 25 μg/mL to 200 μg/mL) incubation. According to the results, the polymers had no cytotoxicity to the 4T1 and LO2 cells, and had obvious side-effect on the proliferation of the cells, which indicated the potential good biocompatibility of the polymeric systems. The negative surface charge of these conjugates may contribute to these results. From these results, we could speculate that the two polymeric systems possessed low cytotoxicity.

27



Figure 10. Cytotoxicity of the core-crosslinked pHPMA-DOTA-Gd, the linear one and DTPA-Gd at 4T1 (A) and LO2 (B) cell lines.

3.9. Blood Compatibility Effect of Polymeric Systems on RBC Lysis. The RBCs could conjugate with specific antibodies and form an immunocomplex, which could activate the alexin and induce the lysis of RBC. For the polymeric systems, cell-polymer interactions first occur on the RBC cell membrane, which might induce the alexin activation, so it was crucial for us to detect the effect of polymeric systems on RBC lysis. In this study, prepared polymeric systems were incubated with blood, and the hemolytic activity was measured by a microplate reader. The American Society for Testing and Materials (ASTM) standard demonstrated that the accepted standard is less than 10% and the high hemocamptibility is less than 5%. Herein, there was no significant hemolysis for the polymeric systems even for 12 h incubation, whereas the concentration of the polymeric systems was 1.0, 3.0 and 5.0 mg/mL (Figure 11). The highest percentage of hemolysis was obtained below 2.0%. The low hemolysis of the core-crosslinked pHPMA-DOTAGd and the linear one may result from their suitable MWs, biodegradable features, and negative surface charge, indicating that these polymeric conjugate-based polymeric systems could be utilized as MRI contrast agents with good blood biosafety. 28


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Figure 11. RBC lysis in the presence of the core-crosslinked pHPMA-DOTA-Gd (A) and the linear one (B); Quantitative analysis of hemolysis with various concentrations of copolymers (C).

APTT and PT. For the normal organism, intrinsic/extrinsic materials could stimulate the release of blood coagulation factors and platelet aggregation, then induce coagulation. Thus, it is crucial to evaluate the effect of polymeric systems on blood coagulation properties. In this study, the APTT and PT were measured after prepared polymeric systems mixed with blood, which represented the capacity of foreign materials to cause direct blood coagulation of human erythrocytes. APTT is used to evaluate intrinsic coagulation pathways and PT is usually used to evaluate the extrinsic coagulation pathway. As shown in Figure 12, the clotting time of contrast agents had no significant change in comparison with the PBS. Thus, these results indicated that the core-crosslinked and linear polymeric systems did not obviously alter the activity of the coagulation capability of the plasma.

29



Figure 12. The effect of the core-crosslinked pHPMA-DOTA-Gd (A) and the linear one (B) on APTT and PT. And effect of the core-crosslinked pHPMA-DOTA-Gd (2.5 mg/mL, C) and the linear one (2.5 mg/mL, D) and PBS (E) on RBCs’ aggregation and morphology via SEM.

3.10. Effect on RBC Aggregation and Morphology According to previous reports, the electrostatic interaction between the negatively charged RBC surface and positively charged materials could contribute to aggregation and morphology changes of RBCs, as well as the hydrophobic interaction between hydrophobic groups of amphiphilic polymers and the lipid bilayers of the RBC membrane. Erythrocytes have the highest abundance in blood cells, with about a 4050% volume fraction in whole blood and display an atypical concave disk, which gives them a larger surface area. Based on these characteristics, the interaction between the RBCs and the agents might affect the biological functions of the RBCs, including aggregation and morphology, and further influence the function of organisms after injecting the agents via the vein. Therefore, an investigation of the effect on RBC aggregation and morphology is necessary for the biosafety of polymeric systems. RBCs 30


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incubated with various concentrations of polymeric systems still kept a smooth surface and complete cell membrane in the images ( ×2000) via SEM (Figure 12). RBC aggregation and morphology showed no significant difference in comparison with the PBS control group, indicating that the polymeric systems had no significant effects on the RBCs. Based on the above results, the polymeric systems showed the potential to be safe carriers because of the observed blood compatibility. 3.11. In Vivo Toxicity on Normal Mice The above studies investigated the toxicity of macromolecular MRI contrast agents in vitro, and the results showed good biosafety, which provides the possibility to investigate the in vivo toxicity. For the in vivo toxicity study, animal behavior and body weights were observed and studied. To evaluate the chronic toxicity of the injected agents, the blood and blood components were tested. Additionally, the organs have been harvested for histological analysis 20-days post injection.54-57 During the entire 20 days after administration, no significant signs of dehydration, locomotor impairment, muscle loss, anorexia, or other symptoms related to animal toxicity were detected in each group. The core-crosslinked pHPMA-DOTA-Gd and the linear one-injected groups did not show a significant drop in body weight during the 20 days after the injection (Figure S7). Due to the fact that the agents would circulate in the blood after administration, it is necessary to evaluate changes to blood and blood components, including red blood cells (RBCs), hemoglobin (HGB), hematocrit (HCT), platelets (PLTs), mean platelet volume (MPV), and white blood cells (WBCs). These routine blood parameters were analyzed on the 20th day after injection (Figure S8-9). No significant difference was detected in this analysis compared with the controls, and all of the results were within the normal 31



range, which demonstrated that the polymeric conjugated agents had good blood biocompatibility; this is consistent with the in vitro results. Biochemical examinations were also investigated to evaluate the liver and kidney functions. All of the biochemical parameters for the groups injected with agents were mostly within normal range and without a significant difference, which suggested the agents had no obvious damage to the liver and kidneys and had good biosafety. To further investigate the in vivo toxicity, histological analysis of five important organs was performed to evaluate the side effects of polymeric conjugated agents, including damage, inflammation, and lesions. All of the major organs were normal and no apparent histopathological abnormalities were observed, indicating no obvious in vivo signs of cell and tissue damage (Figure 13). All above, no significant toxicity to blood, tissues, and organs was observed, which may be owe to the characteristics of the polymeric conjugated agents’ molecular structures, including biodegradable features and surface negative charge.

Figure 13. Histological tests of mice administrated with saline (A), pHPMA-DOTA-Gd (B), and Core-crosslinked pHPMA-DOTA-Gd (C) at 20 days post-injection (n=7). Scale bar: 20 μm for all images.

4. CONCLUSION In summary, we reported two kinds of HPMA copolymer-gadolinium conjugates 32


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with different dimensional structures as macromolecular MRI contrast agents. Both core-crosslinked pHPMA-DOTA-Gd and pHPMA-DOTA-Gd longer blood circulation time in comparison with the clinical agent DTPA-Gd, which contributed to the higher accumulation in tumors via the EPR effect. The two agents resulted in r1 values of 10.49 and 6.42 mM-1·s-1 (per gadolinium), respectively, which was a 2-3-fold increase compared to that of DTPA-Gd. Core-crosslinked pHPMA-DOTA-Gd demonstrated better imaging results, which might have profited from the relatively higher MW compared with pHPMA-DOTA-Gd. In vivo and in vitro toxicity studies demonstrated both of the polymeric agents have good biocompatibility. The preliminary but important research piece on the safety and efficiency of core-crosslinked pHPMADOTA-Gd and pHPMA-DOTA-Gd as contrast agents found that the former might have greater benefits. Overall, we have provided an alternative strategy for the preparation of stimuli-responsive and biodegradable core-crosslinked copolymer-gadoliniumbased polymeric MRI contrast agents.

33



ASSOCIATED CONTENT Supporting Information Characterization studies and data, biodegradability study, MRI scanning parameters, Table S1, Figure S1-S9 are shown in Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors *E-mail (Zhu): [email protected] *E-mail (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, 81220108013), National Natural Science Foundation of China (51373104, 51673127, 8162103, 81361140343, 81371536) and International Science and Technology Cooperation Program of Chengdu (2016-GH0300005-HZ).

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Cross-Linked and Biodegradable Polymeric System as a Safe

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