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Gadolinium-Loaded Poly(N-vinylcaprolactam) Nanogels: Synthesis, Characterization, and Application for Enhanced Tumor MR Imaging Wenjie Sun, Sabrina Thies, Jiulong Zhang, Chen Peng, Guangyu Tang, Mingwu Shen, Andrij Pich, and Xiangyang Shi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14219 • Publication Date (Web): 09 Jan 2017 Downloaded from http://pubs.acs.org on January 11, 2017

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ACS Applied Materials & Interfaces

Gadolinium-Loaded Poly(N-vinylcaprolactam) Nanogels: Synthesis, Characterization, and Application for Enhanced Tumor MR Imaging

Wenjie Suna1, Sabrina Thiesb1, Jiulong Zhangc, Chen Pengc*, Guangyu Tangc, Mingwu Shena, Andrij Pichb*, Xiangyang Shia*

a

State Key Laboratory for Modification of Chemical Fiber and Polymer Materials, College of

Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, People’s Republic of China b

DWI-Leibniz-Institute for Interactive Materials e.V., Functional and Interactive Polymers, Institute

for Technical and Macromolecular Chemistry, RWTH Aachen University, 52056 Aachen, Germany c

Department of Radiology, Shanghai Tenth People's Hospital, Tongji University School of Medicine,

Shanghai 200072, People’s Republic of China

____________________________________________________________ * Corresponding authors. E-mail: [email protected] (X. Shi), [email protected] (A. Pich), and [email protected] (C. Peng). 1

Authors contributed equally to this work.

1

ACS Paragon Plus Environment


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Abstract We report the synthesis of poly(N-vinylcaprolactam) nanogels (PVCL NGs) loaded with gadolinium (Gd) for tumor MR imaging applications. The PVCL NGs were synthesized via precipitation polymerization using the monomer N-vinylcaprolactam (VCL), the comonomer acrylic acid (AAc), and the degradable crosslinker 3,9-divinyl-2,4,8,10-tetraoxaspiro-[5,5]-undecane (VOU) in

aqueous

solution,

followed

by

covalently

binding

with

2,2',2''-(10-(4-((2-aminoethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triy l) triacetic acid (NH2-DOTA-GA)/Gd complexes. We show that the formed Gd-loaded PVCL NGs (PVCL-Gd NGs) having a size of 180.67 ± 11.04 nm are water dispersible, colloidally stable, uniform in size distribution, and non-cytotoxic in a range of the studied concentrations. The PVCL-Gd NGs also display a r1 relaxivity (6.38-7.10 mM-1s-1), which is much higher than the clinically used Gd chelates. These properties afforded the use of the PVCL-Gd NGs as an effective positive contrast agent for enhanced MR imaging of cancer cells in vitro as well as a subcutaneous tumor model in vivo. Our study suggests that the developed PVCL-Gd NGs could be applied as a promising contrast agent for T1-weighted MR imaging of diverse biosystems.

Keywords: Poly(N-vinylcaprolactam); nanogels; Gd-DOTA; MR imaging; tumors

2


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Introduction Magnetic resonance (MR) imaging is a critical clinical technology widely used for cancer diagnosis, due to its advantages of noninvasive nature, high spatial resolution, and nonionizing radiation source based on the strong magnetic fields.1 In order to improve the MR detection sensitivity and acquire high quality MR images of different diseases, contrast agents are indispensable in the clinic practice.2-5 The currently used contrast agents in MR imaging can be classified into two categories

including

gadolinium

(Gd)

chelate-based

T1-weighted

positive

agents

and

superparamagnetic iron oxide nanoparticle (NP)-based T2 negative agents.6-7 The Gd chelate-based contrast agents have gained much attention owing to their capability to positively enhance the imaging contrast.8 However, the Gd chelates used in clinic such as Magnevist® (Gd-DTPA, gadopentetate dimeglumine), Omniscan® (Gd(DTPA-BMA), gadodiamide), and Dotarem® (Gd-DOTA, gadoterate) are all low molecular weight chelates, which possess a comparatively low r1 relaxivity and short blood circulation time, thus having short imaging time. These drawbacks limit their clinical applications.1 To overcome these shortcomings, many Gd chelates were loaded onto macromolecules to improve their relaxivities and to prolong their blood circulation time.9-10 For instance, Gd chelates have been conjugated to dendrimers,11-12 polyethylenimine,13-14 polylysine,15-16 dextran,17 proteins,18 micelles,19 and nanogels (NGs).20-21 Hybrid NGs, which integrate the properties of both hydrogels and nanomaterials, are three dimensional physically or chemically crosslinked polymer particles with a unique spherical shape and a colloidal dimension.22 Due to their softness and excellent fluidity, NGs can be easily endocytosed by tumor cells, leading to the enhanced cellular uptake.23-24 By varying the monomers, crosslinkers and initiators, various functional groups can be introduced into the polymer network. This allows a further 3



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flexible functionalization of the NGs with different molecules such as dyes, drugs, metal complexes, or NPs for applications in the fields of nanomedicine, in particular imaging, controlled drug release, and hyperthermia therapy.25-32 Moreover, depending on the monomers they are made of, many smart NGs have been developed for biomedical applications, owing to their ability to swell or shrink as triggered by different external stimuli including temperature, pH, redox or ionic strength.33-34 In our previous work, N-vinylcaprolactam (VCL) has been used as a main monomer to synthesize poly(N-vinylcaprolactam) (PVCL) NGs, which are temperature sensitive and exhibit a lower critical solution temperature of about 32 oC in water.35-37 PVCL shows a great biocompatibility and is therefore suitable for in vivo medical applications.38-40 Moreover, the abundant functional groups on the surface of the PVCL NG shells may allow their modification with varying amounts of Gd chelates to overcome the drawbacks of small molecular Gd chelate-based MR contrast agents. To test our hypothesis, in this present study, PVCL NGs loaded with Gd-DOTA chelates (PVCL-Gd NGs) were prepared for MR imaging of tumors. The pure PVCL NGs with carboxylic acid groups

were

firstly

synthesized

by

the

acid

degradable

crosslinker

of

3,9-divinyl-2,4,8,10-tetraoxaspiro-[5,5]-undecane (VOU) in aqueous solution via precipitation polymerization. Briefly, the biocompatible VCL was used as the main monomer, and in addition acrylic acid (AAc) was introduced as a comonomer in the NG shell to achieve a better control over the NG diameter and to introduce carboxylic acid groups for further functionalization. Finally, the synthesized

PVCL

NGs

were

functionalized

with

NH2-DOTA(Gd)-GA

complexes

via

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) coupling reaction. The formed PVCL-Gd NGs were well characterized in terms of their structure, composition, size, morphology, stability, and MR relaxometry. Their cytocompatibility was evaluated by cytotoxicity assay and cell morphology 4


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observation. The potential of the PVCL-Gd NGs to be used as an effective positive contrast agent for T1-weighted MR imaging of cancer cells in vitro as well as a subcutaneous tumor model in vivo was finally assessed. To the best of our knowledge, no previous reports related to the use of PVCL NGs conjugated with Gd chelates for in vivo T1 MR imaging applications have been published. VOU

O

(a)

+ VCL

(b)

O

O

O

ACMA water, 70 °C, N2

O

N

O

O

= NG C

OH

(PVCL NGs)

OH

AAc

O NG C + R1 N C N R2 OH EDC

H+

H R1 N C N R2 O O C NG

NH2

HN O HO O

O

HO N

NHS

O

- EDC

O N

N

O

Gd3+ O O

N

N

O O

O O H NG C N DOTA(Gd)-GA

NH2-DOTA(Gd)-GA

O NG C O N

= NH2-DOTA(Gd)-GA

-NHS

(PVCL-Gd NGs)

O

Scheme 1. Schematic illustration of (a) the synthesis PVCL NGs via precipitation polymerization and (b) the formation PVCL-Gd NGs via EDC/NHS coupling between the carboxylic acid group on the NG surface and NH2-DOTA(Gd)-GA.

Experiment Sections Synthesis of PVCL NGs. The PVCL NGs were synthesized via a simple precipitation polymerization method (as shown in Scheme 1a). Initially VCL (1.878 g, 13.5 mmol), VOU (0.08 g, 0.377 mmol) and sodium dodecyl sulfate (SDS, 0.02 g, 0.0694 mmol) were dissolved in water (120 mL) and stirred in a double wall reactor at 70 oC for 30 min to achieve a complete homogenization. 5



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After

that

the

polymerization

was

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initiated

with

2,2-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] (ACMA, 0.07 g, 0.204 mmol, dissolved in 5 mL water) acting as a free-radical initiator. Five to ten minutes after the initiation, AAc (0.108 g, 1.5 mmol, in 25 mL water) was dropwise placed into the reaction mixture. This corresponds with a theoretical amount of 10 mol% AAc. According to the literature,36 NGs with a core-shell structure, where VCL is located in the NG core and AAc in the shell were obtained via this method. The polymerization was carried out for additional 4 h at 70 oC. After that the dispersion was dialyzed at room temperature against water for five days to remove non-reacted monomers. For dialysis, a cellulose membrane with a molecular weight cut-off (MWCO) of 12 000 to 14 000 was used and the water was changed twice a day. After freeze-drying, the PVCL NGs, a colorless solid, were obtained for further use. Reaction of PVCL NGs with NH2-DOTA(Gd)-GA Complexes. The PVCL NGs with surface carboxylic acid groups were covalently reacted with the NH2-DOTA(Gd)-GA complexes through an EDC coupling reaction (Scheme 1b). Prior to the experiment, NH2-DOTA(Gd)-GA complexes were formed by mixing equivalent amounts of Gd(III) chloride hexahydrate and the ligand NH2-DOTA-GA dissolved in water and stirring overnight at 40 oC. Typically, PVCL NGs (500 mg) were well dispersed in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (20 mL, 0.1 M, pH = 4.7, containing 0.9% NaCl). To this dispersion 0.132 g EDC (10-fold molar excess) and 0.198 g N-hydroxysuccinimide (NHS) were added and stirred well at room temperature for 20 min. After that the pH value of the dispersion was increased above 7 using phosphate buffered saline (PBS). Subsequently NH2-DOTA(Gd)-GA complexes with varying amounts were added and the mixture was vigorously stirred for 2 h at room temperature. The dispersion was transferred to a cellulose membrane with an 6


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MWCO of 12000 to 14000 and was dialyzed against PBS for three days to obtain the final Gd-loaded PVCL NGs (PVCL-Gd NGs). To make it simple, the PVCL-Gd NGs were named as PVCL-Gd NGs-0.1%, PVCL-Gd NGs-1%, and PVCL-Gd NGs-5%, respectively according to the feeding ratio.

Characterization Techniques. The final Gd ratios in the PVCL-Gd NGs were measured by inductively coupled plasma-optical emission spectroscopy (ICP-OES, Leeman Prodigy, Hudson, NH). Zeta potential and dynamic light scattering (DLS) measurements were tested on a Zetasizer NanoZS (Malvern, Worcestershire, UK). A He-Ne laser (4.0 mW, 633 nm) was used with a scattering angle of 173°. Each sample was prepared by adding a single drop of the NG dispersion to 1 mL of 10 mM PBS with a pH value of 7.4. Transmission electron microscopy (TEM) images were captured with the LibraTM 120 transmission electron microscope (Zeiss, Oberkochen, Germany) or with the SU9000 ultra-high resolution scanning electron microscope (Hitachi, Tokyo, Japan) in the scanning transmission electron microscopy (STEM) mode. Carbon coated copper grids were used to hold water suspension of the NG samples, and one drop of the NG sample (1 mg/mL) was deposited onto each grid, followed by air drying prior to measurements. The stability of DOTA-Gd complexes were evaluated through the relative Gd contents (% of original) in the supernatants of PVCL-Gd NGs with different Gd loading for different storage time after centrifugation. To analyze the dispersion stabilities of the PVCL NGs with or without Gd-loading, the sedimentation velocities were measured with an analytical centrifuge (Lumisizer®, LUM-GmbH, Berlin, Germany). The measurements were performed in aqueous dispersion in PBS. The rotation velocity was set at 2000 rpm. The incorporation of Gd within the NGs was evaluated by energy-dispersive spectroscopy (EDS), which was coupled with the SU9000 ultra-high resolution scanning electron microscope. 7



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MR Phantom and Relaxometry Measurements. The PVCL-Gd NGs (0.1, 1, and 5wt%) were dispersed into PBS, where the concentrations of Gd was quantified by ICP-OES. Subsequently, PBS solutions containing PVCL-Gd NGs with different Gd concentrations (0.05, 0.1, 0.2, 0.3, and 0.4 mM, respectively) were subjected to MR phantom and MR relaxometry studies through an NMR Analyzing and Imaging system (0.5 T, NMI20, Shanghai Niumag Corporation, Shanghai, China). The instrumental setting were similar to those reported in our previous work.41-42 For comparison, clinical Magnevist○R product was also tested under similar conditions. Cell Viability Assay and Cell Morphology Observation. HeLa cells were regularly cultivated and passaged in DMEM containing FBS (10%) and penicillin-streptomycin (1%) in a Thermo Scientific cell incubator (Waltham, MA) at 37 oC and 5% CO2. To test the cytotoxicity of NGs, the 96-well plates were seeded with HeLa cells at a density of 1 × 104 cell/well overnight. The next day, the adherent HeLa cells were incubated with 200 µL fresh DMEM containing PVCL-Gd NGs at various Gd concentrations (10, 25, 50, 75, 100, 200, and 300 µM, respectively) for another 24 h. After that, the cells were rinsed three times with PBS, and then incubated with 100 µL of DMEM without FBS but supplemented with 10% CCK-8 for 4 h. Subsequently, a ELISA reader (Multiskan MK3, Thermo scientific, Logan, UT) was employed to measure the absorbance of each well at 450 nm. The control group was the HeLa cells treated with PBS only, and for each sample 5 parallel wells were analyzed to give a mean value and standard deviation. Laser scanning confocal microscopic imaging of HeLa cells after co-cultured with the PVCL-Gd NGs was also performed to evaluate the cytotoxicity of the NGs. Confocal microscopic images were acquired using confocal laser scanning microscopy (CLSM, LSM 700, Carl Zeiss, Jena, Germany) with a 63× oil immersion objective lens. The cells were treated and samples were processed according 8


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to the literature.43 MR Imaging of Cancer Cells in Vitro. HeLa cells were seeded into culture flasks (25 cm2, 5 × 106 cells per flask) with 5 mL of DMEM containing FBS (10%) and penicillin-streptomycin (1%) and then incubated at 37 oC and 5% CO2. Through the use of the procedures reported in the literature,24, 41-42, 44

the cells were treated with PVCL-Gd NGs at different Gd concentrations for 4 h, rinsed with

PBS, trypsinized, centrifuged, and finally redispersed in 1 mL PBS (containing 0.5% agarose) prior to MR imaging. MR Imaging of a Subcutaneous Tumor Model in Vivo. All animal treatments were performed under the guidelines of the Institutional Animal Care and Use Committees (IACUC), and also in compliance with the policy of the National Ministry of Health. BALB/c male nude mice (15-20 g, four-week-old) purchased from Shanghai Slac Laboratory Animal Center (Shanghai, China) were utilized to build up a tumor model. Concretely, a suspension of HeLa cells dispersed in PBS at a density of 1 × 107 cells/mL was prepared and subcutaneously injected to the right mouse back (200 µL per mouse). When the diameter of tumor reached about 0.5 cm, the mice were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg). Then, PVCL-Gd NGs ([Gd] = 4 mM, in 100 µL PBS) were intravenously delivered via tail vein. MR scanning of the mice was carried out before injection and at 20, 40, 60, and 80 min postinjection by a clinical MR system (3.0 T, MAGNETOM VERIO, SIEMENS Medical Systems, Erlangen, Germany) with a custom-built rodent receiver coil (Chenguang Med Tech, Shanghai, China). We set the MR scanning parameters as follows: TE = 15 ms, TR = 620 ms, FOV = 8 × 10 cm, and 256 × 171 matrix. For comparison, a commercial Magnevist○R with the same Gd dose was also injected and MR scanning was performed under the same instrumental conditions. 9



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In Vivo Biodistribution of NGs and Histological Examinations. The HeLa tumor-bearing nude mice were subjected to in vivo biodistribution analysis to evaluate the metabolic behavior of the PVCL-Gd NGs. At the same MR scanning time points, the HeLa tumor-bearing mice were sacrificed after injection of the PVCL-Gd NGs ([Gd] = 4 mM, in 100 µL PBS). Then we extracted and weighted the main organs including heart, liver, spleen, lung, kidney, and tumor of the mice. These organs and tumors were cut into small pieces, treated by an aqua regia solution (2 mL, hydrochloric acid/nitric acid, v/v = 3:1) for two days, and diluted with water. Finally, the Gd content of different organs and tumors was quantified by ICP-OES measurements. The tumor-bearing mice treated with equivoluminal PBS were set as control. The long-term in vivo biodistribution analysis and hematoxylin and eosin (H&E) staining of main organs were studied to evaluate the toxicity of the PVCL-Gd NGs. For both cases, healthy male Kunming mice (20-30 g, Shanghai Slac Laboratory Animal Center) were injected with the PVCL-Gd NGs (100 µL, [Gd] = 4 mM) intravenously. For the long-term in vivo biodistribution analysis, the mice were sacrificed at 7, 14, and 30 days postinjection, respectively. The main organs including heart, liver, spleen, lung, and kidney were dissected and processed in the same manner as described above to quantify the Gd content by ICP-OES. Meanwhile, the corresponding organs were sectioned into slices with a thickness of 4 µm for H&E staining according to standard protocols.24 The histological images of these organ sections were acquired by an inverted phase contrast microscope (DM IL LED, Leica, Wetzlar, Germany). The organs from mice injected with PBS (100 µL) were used as control after the above treatments.

Results and Discussion 10


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Preparation and Characterization of PVCL-Gd NGs. PVCL NGs used in the present work were synthesized by precipitation polymerization. As shown in Table S1 (Supporting Information), the practical Gd loading percentages for different PVCL-Gd NGs were calculated to be 0.09%, 0.54%, and 2.87%, respectively based on ICP-OES measurements. The hydrodynamic sizes of the PVCL NGs and PVCL-Gd NGs with different Gd loadings were measured with DLS. As shown in Figure 1a, the pure PCVL NGs have a hydrodynamic diameter of about 140 nm, whereas the hydrodynamic diameter of the PVCL-Gd NGs tends to increase with the amount of Gd-DOTA loaded, and reaches up to 200 nm in average with a Gd-DOTA loading of 5 wt%. We assume that PVCL-Gd NGs increase in size because Gd-DOTA complexes are covalently bound to the NG shell. Nevertheless the particle size of 200 nm is still absolutely fine to find applications in vivo. The PDIs of the NGs, which are illustrated in Figure S1 (Supporting Information), show that all samples are quite monodisperse. With a higher amount of Gd-DOTA modified, the NG PDI has a tendency to increase. This can be explained to be due to the functionalization of the NGs with the Gd-DOTA complexes. Figure 1b shows the zeta potential measurements of PVCL NGs and PVCL-Gd NGs in PBS. The zeta potentials of the NGs are in the range between -20 mV and -30 mV without any particular trend. These negative zeta potentials can be explained with the fact that the NGs contain carboxylic acid groups on their surface. The NG dispersions with zeta potentials in this range are considered to be colloidally stable due to the combination of steric (dangling chains) and electrostatic (charges) stabilization mechanisms. To confirm the good colloidal stability of NGs, the sedimentation velocities of PVCL NGs and PVCL-Gd NGs were measured at pH 7.4 and 20 oC. Pure PVCL NGs have a sedimentation velocity of 2.5 µm/s, which indicates a very good dispersion stability (Figure 1b). The diagram shows that the 11



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dispersion stability slightly decreases with the increase of Gd-DOTA loading up to sedimentation velocities of about 8 µm/s, which can be explained by the increase of the NG size shown in Figure 1a. Sedimentation velocity values less than 10 µm/s indicate very good colloidal stability of colloidal systems, which additionally confirms the efficient stabilization of NGs in aqueous solutions.45-46

Figure 1. (a) Hydrodynamic diameter and (b) sedimentation velocities and zeta potential measurements of PVCL NGs and PVCL-Gd NGs loaded with varying amounts of Gd-DOTA (10 mM PBS, pH 7.4, T = 20 oC). TEM images of PVCL NGs (c) and PVCL-Gd NGs-0.1% (d).

To analyze the nature of the PVCL-Gd NGs further, TEM images of pure PVCL NGs and PVCL-Gd NGs were captured (Figure 1c, 1d, and Figure S2, Supporting Information). The size distributions of pure PVCL NGs and PVCL-Gd NGs are displayed in Figure S3 (Supporting 12


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Information). As expected, the gels show no significant difference in size, size distribution and morphology before and after the Gd loading. The average diameter (dTEM) is 180.67 ± 11.04 nm. In comparison to the results of the hydrodynamic diameter from the DLS measurements, the diameter from the TEM measurements (dTEM) indicates a slightly spreading of the NGs on the TEM grid, likely due to the electron beam interaction with the samples. All samples show a high monodispersity. Due to the comonomer acrylic acid (AAc), the PVCL NGs show a good contrast in the TEM images and the morphology of all gels is highly homogeneous. The loading of Gd within the NGs was qualitatively confirmed by energy-dispersive spectroscopy (EDS), as shown in Figure S4a (Supporting Information). There is the characteristic signal at 6.056 keV for the Lα of Gd, proving the successful modification of Gd-DOTA. In the following layered TEM image (Figure S4b, Supporting Information), it becomes obvious that the Gd-DOTA complex is mainly located in the area of the NGs, which again proves that the covalent bounding was successful. The image was captured in the TEM mode. Free Gd can probably be explained with an image shift during the measurements. Overall, our study suggests that the synthesized PVCL NGs with or without Gd loading are colloidally stable. The stability of the Gd-DOTA complexes after loaded onto the NGs was further examined by ICP-OES after the NGs were exposed to PBS at 37 oC for 1, 3, and 7 days. As shown in Figure S5 (Supporting Information), the relative Gd contents (% of original) in the supernatants of PVCL-Gd NGs with different Gd loadings after centrifugation are all less than 0.1%, suggesting that the immobilized DOTA-Gd chelates have a minimum Gd leakage. MR Relaxometry. The r1 relaxivity is an important index to evaluate the imaging performance of the T1 MR contrast agents. Therefore, the T1 relaxation times of the PVCL-Gd NGs with different Gd loadings at different Gd concentrations were measured. After linear fitting, as shown in Figure 2a, 13



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there is no significant change of the r1 relaxivities of PVCL-Gd NGs with different Gd loadings (7.07, 7.10, and 6.38 mM-1 s-1, respectively), implying that the T1 MR relaxometry of the PVCL-Gd NGs is not significantly impacted by Gd loading degrees. The average value of r1 relaxivities of PVCL-Gd NGs with different Gd loadings was calculated to be 6.85 mM-1 s-1, much higher than that of clinical product of Magnevist○R (4.56 mM-1 s-1). The higher r1 relaxivities of PVCL-Gd NGs may be ascribed to the fact that the proton relaxation process is significantly affected by the structure of PVCL NGs. After the DOTA-Gd complexes are modified onto the surface of PVCL NGs, the molecular dimension of the complexes increase. Hence the rotational motion of the complexes is slowed down, resulting in increased r1 relaxivities.1, 47 It can also be observed that the PVCL-Gd NGs could enhance the MR signal intensity with the increasing of Gd concentration (Figure 2b), with an increasing tendency better than DTPA(Gd) chelates (Magnevist○R ). These results demonstrate that the synthesized PVCL-Gd NGs hold a promising potential to be applied as positive contrast agents for MR imaging.

Figure 2. Linear fitting of 1/T1 (a) and the T1-weighted MR images (b) of the PVCL-Gd NGs with different Gd loadings and clinical Magnevist○R at various Gd concentrations.

14


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Cytotoxicity Assay and Cell Morphology Observation. It is crucial to investigate the cytotoxicity of the PVCL-Gd NGs prior to their in vitro and in vivo imaging applications. CCK-8 assay of cell viability was performed (Figure 3). Clearly, HeLa cells after co-cultured with the PVCL-Gd NGs with various Gd loadings display a cell viability of 70% in a range of studied Gd concentrations (10-300 µM), suggesting their good cytocompatibility. However, the viability of cells treated with the PVCL-Gd NGs-5% was lower than that of the other groups, which may be due to their relatively high Gd content in a single NG. Taking into account of the results of stability, r1 relaxivity, and cell viability assay data, the PVCL-Gd NGs-0.1% were chosen for further characterizations. PVCL-Gd NGs 0.1% PVCL-Gd NGs 1% PVCL-Gd NGs 5%

125

Cell viability (%)

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100 75 50 25 0

PBS 10 25 50 75 100 200 300

Gd concentration (µM) Figure 3. CCK-8 assay of the viability of HeLa cells after treated with the PVCL-Gd NGs with different Gd loadings at different Gd concentrations for 24 h.

HeLa cells treated with the PVCL-Gd NGs-0.1% at various Gd concentrations were also observed by CLSM after 4’ 6-Diamidino-2-phenylindole (DAPI) staining of cell nucleus (Figure S6, Supporting Information). The cytoplasm and cell nucleus of HeLa cells could be observed clearly. There were no 15



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any significant morphological changes in terms of cytoplasm and cell nucleus of HeLa cells treated with the PVCL-Gd NGs-0.1% in the studied Gd concentration range, when compared to the control group treated with PBS. The results of cell morphology observation are in good accordance with the CCK-8 assay data, confirming that the PVCL-Gd NGs-0.1% possess good cytocompatibility in the given Gd concentration range.

Figure 4. In vitro T1-weighted MR images (a) and MR SNR (b) of HeLa cells treated with the PVCL-Gd NGs-0.1% at different Gd concentrations for 4 h.

MR Imaging of Cancer Cells In Vitro. Based on the high r1 relaxivity and good cytocompatibility of the PVCL-Gd NGs-0.1%, we next used them as a positive contrast agent of MR imaging of cancer cells in vitro (Figure 4). As shown in Figure 4a, the color of the T1-weighted MR images gradually changes from green to red, meaning that the MR signal intensity of HeLa cells treated with the PVCL-Gd NGs-0.1% increases with the Gd concentration. Moreover, the signal to 16


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noise ratio (SNR) of these T1-weighted MR images was quantitatively analyzed (Figure 4b). The MR SNR results also quantitatively demonstrates the ability of the NGs to increase the MR signal intensity of cells with the Gd concentration. Specifically, at the Gd concentration of 0.3 mM, the MR SNR of HeLa cells could reach a value of 33.6, about 2-fold higher than that of the control group. These data demonstrate that the formed PVCL-Gd NGs-0.1% hold a promising potential to be used as a positive contrast agent for MR imaging of cancer cells in vitro.

Figure 5. In vivo T1-weighted MR images of the nude mice bearing subcutaneous HeLa tumors before and at different time points postinjection of the PVCL-Gd NGs-0.1% (a) and the clinical agent Magnevist○ (b) ([Gd] = 4 mM, in 100 µL PBS) and tumor MR SNR (c) at different time points R

postinjection of the above materials.

MR Imaging of a Subcutaneous Tumor Model In Vivo. The feasibility to use the PVCL-Gd NGs-0.1% as a positive T1-weighted contrast agent for tumor MR imaging in vivo was next investigated. Figure 5a shows that the MR signal intensity of the tumor region increases firstly and then decreases with the time postinjection. In particular, the strongest MR signal intensity was 17



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observed at 20 min postinjection. Later on, the MR signal intensity in the tumor area is recovered to some extent at 80 min postinjection. For comparison, the tumor MR images after injection of Magnevist○R were also recorded (Figure 5b). Clearly, the MR intensity change follows the same trend. The MR signal intensity change can be further quantitatively confirmed by plotting the tumor MR SNR as a function of time postinjection (Figure 5c). Specifically, the tumor MR SNR was increased from 13.72 to 26.57 at 20 min postinjection of the PVCL-Gd NGs-0.1%. However, the tumor MR SNR was just increased from 13.68 to 17.35 after 20 min postinjection of Magnevist○R . Obviously, the tumor MR SNR after injection of the PVCL-Gd NGs-0.1% was significantly higher than that after injection of Magnevist○R especially at the time points of 20, 40 and 60 min (p < 0.001). Overall, the above data suggest that the PVCL-Gd NGs-0.1% are able to be employed as a positive contrast agent for enhanced tumor MR imaging, possibly by means of the passive enhanced permeability and retention (EPR) effect. In Vivo Biodistribution and Histological Examinations. In order to investigate the in vivo metabolic behavior of the PVCL-Gd NGs-0.1% in mice after intravenous injection, the biodistribution of Gd element in different organs including the heart, liver, spleen, lung, kidney, and tumor was quantitatively analyzed by ICP-OES at 20, 40, 60, and 80 min postinjection (Figure S7, Supporting Information). Generally, the nanomaterials could be delivered to different organs by the blood circulation after intravenous injection. The liver, spleen, and lung of the mice treated with the PVCL-Gd NGs displayed much higher Gd content than that of the mice treated with PBS. This may be because the three organs are the macrophage-rich organs, which could clear the nanomaterials.48-49 Moreover, the Gd content in tumor sites starts to decrease at 40 min postinjection, in accordance with the above tumor MR imaging data. In addition, the percentage injected dose per gram of tissue (%ID/g) 18


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at 20 min postinjection was calculated to evaluate the tumor uptake efficiency through EPR effect. The value of %ID/g was calculated to be 1.11 ± 0.32. The %ID/g could be improved by surface modification of the NGs with targeting ligands to render the particles to have targeting specificity in our future efforts. Control 7 days 14 days 30 days

40 30

Gd (µg/g)

20 10

y id ne K

Lu ng

en Sp le

Li ve r

ea rt

0

H

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Figure 6. Biodistribution of Gd in the major organs of the healthy mice including the heart, liver, spleen, lung, and kidney at 7, 14, and 30 days post intravenous injection of the PVCL-Gd NGs-0.1% ([Gd] = 4 mM, in 100 µL PBS, for each mouse). In addition, the long-term in vivo biodistribution studies and H&E staining of different organs after intravenous injection of the PVCL-Gd NGs-0.1% were evaluated in healthy mice to further investigate the metabolic activity and the side effect of the NGs to the major organs. As shown in Figure 6, the Gd element was mostly accumulated in liver and spleen (the reticuloendothelial system (RES) rich organs) after 14 days, and then slowly excreted after 30 days. This means that the injected PVCL-Gd NGs are able to be finally metabolized and removed away from the animal body. It should be noted that the metabolic behavior of the NGs is different in different time periods. After a short injection time, due to the rich macrophages in lung, part of NGs were accumulated in lung through

19



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pulmonic circulation (Figure S7). After a longer circulation time, the NGs were accumulated at the other macrophage-rich organs such as liver and spleen and further be cleared out of the body (Figure 6). H&E staining (Figure 7) data reveal that no appreciable morphological changes, no inflammatory infiltrate, and no necrosis can be found in the major organs after injection of the PVCL-Gd NGs-0.1% for 7, 14, and 30 days compared to the control group. In other words, the PVCL-Gd NGs-0.1% do not exert any possible in vivo toxicity to the mice during 30 days. Take into account of all the in vivo data, we can safely draw a conclusion that the developed PVCL-Gd NGs-0.1% are biocompatible to mice, and can be metabolized and finally cleared away from the mice body, which is crucial for translational medicine applications.

Figure 7. Histological examinations of different organs from healthy mice at 7, 14, and 30 days 20


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postinjection of the PVCL-Gd NGs-0.1% ([Gd] = 4 mM, in 100 µL PBS, for each mouse).

Conclusion In this present work, uniform PVCL-Gd NGs were synthesized by precipitation polymerization of the monomer VCL and the comonomer AAc in aqueous solution, followed by covalently binding with Gd-DOTA complex. The synthesized PVCL-Gd NGs with an average diameter (dTEM) of about 180.67 ± 11.04 nm are water dispersible, colloidally stable, and non-cytotoxic in a range of the studied concentrations. The r1 relaxivities of the PVCL-Gd NGs was measured to be 6.38-7.10 mM-1 s-1, which are higher than that of the clinical Gd chelates. These properties render the use of the NGs as a positive contrast agent for MR imaging of cancer cells in vitro and a subcutaneous tumor model in vivo. The residual carboxyl groups derived from acrylic acid shell of the PVCL NGs may offer the potential to build up multifunctional NGs by further modification with different targeting ligands, imaging molecules or drugs for theranostics of different biosystems.

Acknowledgements This research is financially supported by the Sino-German Center for Research Promotion (GZ899), the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning, and the Fundamental Research Funds for the Central Universities (M. Shen and W. Sun). A. Pich thanks VW Stiftung and DFG SFB 985 “Functional Microgels and Microgel Systems” for support of this work.

Supporting Information

21



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Additional experimental details and data of PDI, TEM, size distribution, EDS, stability assessment of PVCL-Gd NGs, confocal microscopy images, and in vivo biodistribution of the NGs. This material is available free of charge via the Internet at http://pubs.acs.org.

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