Biodegradable Nanoglobular Magnetic Resonance Imaging Contrast

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Biological and Medical Applications of Materials and Interfaces

Biodegradable Nano-globular Magnetic Resonance Imaging Contrast Agent Constructed with Host-guest Self-assembly for Tumor-targeted Imaging Yi Cao, Guangyue Zu, Ye Kuang, Yilin He, Zheng Mao, Min Liu, Dangsheng Xiong, and Renjun Pei ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b08021 • Publication Date (Web): 20 Jul 2018 Downloaded from http://pubs.acs.org on July 23, 2018

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ACS Applied Materials & Interfaces

Biodegradable Nano-globular Magnetic Resonance Imaging Contrast Agent Constructed with Host-guest Self-assembly for Tumor-targeted Imaging Yi Cao1,2, Guangyue Zu1, Ye Kuang1, Yilin He1, Zheng Mao1, Min Liu1, Dangsheng Xiong*,2, and Renjun Pei*,1

1

CAS Key Laboratory of Nano-Bio Interface, Division of Nanobiomedicine, Suzhou Institute

of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, China. 2

School of Materials Science and Engineering, Nanjing University of Science and

Technology, Nanjing 210094, China.

ABSTRACT: Gadolinium-based macromolecular magnetic resonance imaging (MRI) contrast agents (CAs) have attracted increasing interesting in tumor diagnosis. However, their practical application is potentially limited because the long-term retention of gadolinium ion in vivo will induce toxicity. Here, a nano-globular MRI contrast agent (CA) PAMAM-PG-g-s-s-DOTA(Gd)+FA was designed and synthesized based on the facile host-guest interaction between β-cyclodextrin (β-CD) and adamantane, which initiated the self-assembly of poly(glycerol) (PG) separately conjugated with gadolinium chelates by disulfide bonds and folic acid (FA) molecule onto the surface of poly(amidoamine) (PAMAM) dendrimer, finally realizing the biodegradability and targeting specificity. The nano-globular CA has a higher longitudinal relaxivity (r1) than commercial Gd-DTPA，showing a value of 8.39 mM-1s-1 at 0.5 T, and presents favorable biocompatibility on the observations of 1

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cytotoxicity and tissue toxicity. Furthermore, MRI on cells and tumor-bearing mice both demonstrate the obvious targeting specificity, based on which the effective contrast enhancement at tumor location was obtained. In addition, this CA exhibits the ability of cleavage to form free small-molecule gadolinium chelates, and can realize minimal gadolinium retention in main organs and tissues after tumor detection. These results suggest that the biodegradable nano-globular PAMAM-PG-g-s-s-DOTA(Gd)+FA can be a safe and efficient MRI CA for tumor diagnosis.

KEYWORDS: host-guest self-assembly, biodegradability, tumor targeting, magnetic resonance imaging, contrast agent

2


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INTRODUCTION Gadolinium-based macromolecular magnetic resonance imaging (MRI) contrast agents (CAs), constructed with the strategy of conjugating small-molecule gadolinium chelates to macromolecular carriers, have been intensively investigated in recent decades due to their favorable performance on improving longitudinal relaxivity (r1), increasing blood circulation, and efficient accumulation at pathological tissues, in contrast with small-molecule ones used in clinic.1-6 Nevertheless, these macromolecular CAs generally suffer from slow and ineffective excretion after administration, which may lead to the release and long-term deposition of gadolinium ions, and possibly cause nephrogenic systemic fibrosis (NFS) especially for patients with renal dysfunction.7-8 Thus, it is imperative to endow gadolinium-based macromolecular CAs with appropriate biodegradability to realize the rapid elimination of gadolinium chelates after MRI examination. To promote the excretion of gadolinium chelates on macromolecular CAs, one promising strategy is employing biodegradable chemical bonds to design macromolecular systems, in which acid liable bonds9-10, enzymatically degradable bonds11-12 or redox-sensitive bonds13-15 are usually incorporated to realize the disintegration of macromolecular structure. On the basis of these environmentally liable chemical bonds, the designed macromolecular CAs not only maintain the intrinsic advantages for MRI, but also possess the capability of biodegradability, inducing the facile excretion of gadolinium chelates following intravenous administration. Among the various kinds of biodegradable macromolecular CAs, those incorporated with disulfide bonds for redox-responsive biodegradability, relying on the thiol exchange of disulfide bonds with endogenous thiols, have become of increasing interest.16-18 3



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For instance, the host-guest adducts with gadolinium chelates were developed, and simultaneously the disulfide bridges were introduced between polymeric scaffold and gadolinium chelates, realizing the degradation to accelerate the elimination after administration.16 In addition, the polydisulfide composite containing gadolinium chelates was designed as a biodegradable macromolecular contrast agent (CA), which exhibited the excellent potential for imaging cardiovascular and cancer, and more importantly, showed the minimal tissue deposition of gadolinium ion.17 In this scenario, the development of gadolinium-based macromolecular CAs with biodegradability derived from disulfide bonds is potentially satisfied with the requirement of gadolinium chelate’s quick clearance from the body after finishing diagnostic function. Additionally, it should be noted that introducing the disulfide bonds between gadolinium chelates and macromolecular carrier rather than the usually incorporating disulfide bonds in the macromolecular backbone will favor the rapid excretion, since the smaller fragments containing gadolinium chelate are readily achieved after cleavage based on the former strategy13-14. Hence, a reasonable strategy for designing disulfide bond-incorporated macromolecular CAs is that gadolinium chelates are directly conjugated to the macromolecular carrier via disulfide bonds. Poly(glycerol) (PG) is a kind of polymer possessing excellent biocompatibility and potential for various chemical modification, based on which it has been widely applied in the field of biomedicine.19-23 Our previous work has reported employing PG to develop gadolinium-based macromolecular CAs, which exhibited desirable blood circulation time, extraordinary ability to enhance magnetic resonance (MR) signal at tumor location, and favorable biocompatibility in vitro and in vivo.24-26 However, there remains an imperious 4


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desire to introduce biodegradability into the PG-based CAs, facilitating the rapid clearance of gadolinium chelates, due to their relatively slow excretion inducing high gadolinium ion retention in vivo compared with small-molecule ones. In addition, supramolecular chemistry, taking the host-guest self-assembly from β-cyclodextrin/adamantane (β-CD/Ad) recognition for example, provides a convenient and powerful approach to construct nanostructured materials for biomedical application, allows for precisely controlling over the structure and properties of nanoparticles, and easily satisfies the requirement of single nanocarrier simultaneously equipped with various functions in the nano-biomedical field.27-31 Therefore, it is reasonable to utilize the host-guest self-assembly of β-CD/Ad to construct gadolinium-based macromolecular CAs with controllable structure and properties. In the field of nano-biomedicine, nano-globular macromolecules with well-defined nanosize have been designed as MRI CAs and therapeutic delivery platforms, which can realize the efficient diagnosis and treatment of tumors.2, 32-36 In compared with traditional linear macromolecules, usually demonstrating the random morphology and hydrodynamic volume that may result in inconsistent pharmacokinetics and prolonged tissue retention, nano-globular ones generally possess the controllable size and morphology, which will reduce the possibility of nonspecific interaction with healthy tissues and minimize the unpredictable accumulation.37 Take this into account, nano-globular structure is an appropriate selection for constructing gadolinium-based macromolecular CAs to effectively avoid the unexpected systemic retention of gadolinium ions. Herein, a biodegradable nano-globular MRI CA based on poly(glycerol) was rationally designed and fabricated through the host-guest recognition between β-cyclodextrin (β-CD) 5



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and adamantane (Scheme 1). Poly(amidoamine) (PAMAM) modified with β-CD (PAMAM-β-CD)

was

employed

as

a

core

of

the

nano-globular

structure.

Adamantanemethanol initiated the polymerization of poly(glycerol), which was linked with gadolinium chelates (DOTA(Gd)) via disulfide bond at the side chain, and terminally conjugated with folic acid (FA) moiety to obtain Ad-PG-g-s-s-DOTA(Gd) and Ad-PG-FA, respectively. On the basis of β-CD/Ad recognition, the functionalized poly(glycerol) (including Ad-PG-g-s-s-DOTA(Gd) and Ad-PG-FA) and PAMAM-β-CD were self-assembled to nano-globular PAMAM-PG-g-s-s-DOTA(Gd)+FA with biodegradability and targeting specificity. As far as we know, the nano-globular MRI CA based on polymers with cleavable gadolinium chelates has never been reported. With the purpose of verifying the promising application in tumor diagnosis, the comprehensive properties of biodegradable nano-globular MRI CA, including relaxivity, biocompatibility, biodegradability, targeting specificity, MRI of tumor tissue and biodistribution were systematically investigated.

6


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Scheme 1. Schematic illustration of the biodegradable nano-globular MRI CA formed through the host-guest self-assembly.

EXPERIMENTAL SECTION Materials and Methods. 1-adamantanementhanol (Ad-OH, >99%, TCI) was dried with vacuum chamber containing P2O5 at 60 oC for 24 h. Toluene (≥99.5%, Sinopharm) was treated with metallic sodium and distilled before use. Dimethyl sulfoxide (DMSO, ≥99%, Sinopharm) was treated with CaH2 and distilled under reduced pressure before use. CuBr (98%, Sigma), NaH (60% dispersion in paraffin liquid, TCI), propargyl bromide (80% in toluene, TCI), tri(2-carboxyethyl) phosphine hydrochloride (TCEP, 98%, Energy Chemical) and N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDETA, 99%, Energy Chemical) were used without any treatment. Some other reagents and solvents mentioned were purchased from Sinopharm and used as received. The reagents for cell culture were all achieved from Gibco. The detailed process of preparing PAMAM (G2.5), monoamine-modified 7



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β-cyclodextrin (β-CD-NH2) and disulfide-contained linker (PPA-cyst-CI) are presented in the Supporting Information. Azide group-modified folic acid (N3-FA) and ethoxyethyl glycidol ether (EEGE) were synthesized according to the previous methods reported by our group.24, 38 Azido group-modified gadolinium chelate (N3-DOTA(Gd)) was prepared as the reported method.39 All data of nuclear magnetic resonance (NMR) spectroscopy were measured using a Varian 400 MHz NMR spectrometer. The number-average molecular weight (Mn) was estimated on a Waters GPC system with THF as an eluent (1.0 mL/min), and the calibration curve was obtained using PEG stands. Fourier transform infrared (FTIR) spectroscopies were collected through a Thermo Fisher FTIR spectrometer. Ultraviolet-visible (UV-vis) absorption spectra were measured on a Perkin-Elmer spectrophotometer. The gadolinium concentrations were determined through a Thermo-X series 2 inductively coupled plasma mass spectrometry (ICP-MS). The measurements of zeta potential and hydrodynamic size were implemented on a Malvern Zetasizer Nano ZS. Synthesis of PAMAM-β-CD. β-CD-NH2 (6.5 g, 5.52 mmol) and PAMAM (G2.5) (345 mg, 0.057 mmol) were dissolved in 15 mL DMF, and the mixture reacted at 50 oC for 3 days. Afterward, the mixture was purified through dialysis in water for 5 days (3500 Da MWCO dialysis tube). The white powder was finally obtained after lyophilization (0.7 g, 50%). 1H NMR spectrum was used to confirm the chemical structure of PAMAM-β-CD (Figure S14). Synthesis of Ad-PEEGE. To a schlenk flask, Ad-OH (99.6 mg, 0.60 mmol), potassium (17 mg, 0.43 mmol) and 2 mL toluene were added, and the mixture reacted at 70 oC under

8


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argon atmosphere for 8 h to obtain yellow precipitation. Afterward, the temperature was increased to 95 oC, and the solvent was removed using an oil pump. After back filled with argon, 10 mL DMSO and EEGE (10.5 g, 72.0 mmol) were sequentially added, and then the reactant was thermostatted at 95 oC for 48 h. After cooling to the room temperature, 100 mL methanol and 20 g ion-exchanged resin were added to terminate the reaction. Then, the resin was removed and the solution was concentrated. The final brown viscous liquid was achieved through drying at 95 oC in vacuum for 48 h (9.9 g, 94%). The Mn of Ad-PEEGE was assessed by GPC (Mn=14304 g/mol，PDI=1.36), and 1H NMR spectrum was used to confirm the chemical structure (Figure S15). Synthesis of Ad-PG. 5 g Ad-PEEGE was dissolved in 200 mL THF, and then the resultant solution was mixed with 5 mL concentrated HCl and reacted for 2 h at room temperature. Afterward, the formed precipitation was collected and dissolved with a little amount of methanol, and then the solution was poured into excess diethyl ether for precipitation. The process of precipitation was repeated twice. The honey-like solid was finally achieved by drying at 60 oC in vacuum for 48 h (2.3 g, 90%). 1H NMR spectrum was used to verify the chemical structure of Ad-PG (Figure S16). Synthesis of Ad-PG-g-s-s-alkynyl. Ad-PG (560 mg, 0.06 mmol, OH: 7.42 mmol), PPA-cyst-CI (947 mg, 2.97 mmol) and Et3N (310 µL, 2.23 mmol) were dissolved in 15 mL DMSO, and the mixture reacted at 60 oC for 3 days. Afterward, the reaction solution was poured into excess diethyl ether and the formed precipitation was collected by centrifugation. The collected solid was dissolved in methanol and further precipitated in excess diethyl ether twice. The pale yellow product was finally achieved by drying in vacuum for 48 h (823 mg, 9



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61%), and 1H NMR spectrum was used to confirm the chemical structure (Figure S17). Synthesis of Ad-PG-g-s-s-DOTA(Gd). To a schlenk flask, Ad-PG-g-s-s-alkynyl (820 mg, 0.06 mmol) and N3-DOTA(Gd) (1.04 g, 1.63 mmol) were dissolved in 30 mL DMSO. After degassing with three freeze-pump-thaw cycles, CuBr (155 mg, 1.09 mmol) and PMDETA (225 µL, 1.09 mmol) were added under argon atmosphere. The reactant was thermostatted at 50 oC and stirred for 48 h. After that, the mixture was purified by dialysis (3500 Da MWCO) against 0.5% EDTA solution and deionized water for 2 days, respectively. The final solution was lyophilized to obtain the yellow solid (757 mg, 50%). The chemical structure of Ad-PG-g-s-s-alkynyl was verified by FTIR spectrum (Figure S18). Synthesis of Ad-PG-alkynyl. Under argon atmosphere, Ad-PEEGE (1.74 g, 0.10 mmol) and NaH (8 mg, 0.20 mmol, 60% dispersion in paraffin liquid) were dissolved in 10 mL THF and the reactant was stirred for 30 min. After that, the mixture was cooled to 0 oC, and propargyl bromide (220 µL, 2 mmol, 80% in toluene) mixed with 5 mL THF was added dropwise. After stirring at room temperature for 12 h, 80 mL ethanol and 2 mL HCl were added, and the mixture reacted at room temperature for 2 h. The obtained reaction mixture was concentrated and purified in excess diethyl ether. The purification process was repeated twice. The honey-like product was finally achieved after centrifugation and drying in vacuum for 48 h (804 mg, 90%). 1H NMR spectrum was used to confirm the chemical structure of Ad-PG-alkynyl (Figure S19). Synthesis of Ad-PG-FA. To a schlenk flask, Ad-PG-alkynyl (662 mg, 0.07 mmol) and N3-FA (77 mg, 0.15 mmol) were dissolved in 20 mL DMSO. After degassing with three

10


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freeze-pump-thaw cycles, CuBr (10.5 mg, 0.07 mmol) and PMDETA (15.3 µL, 0.07 mmol) were added under argon atmosphere. The resultant reactant was thermostatted at 50 oC and stirred for 48 h. After that, the product was purified by dialysis (3500 Da MWCO) against 0.5% EDTA solution and deionized water for 2 days, respectively. The resultant solution was lyophilized to obtain the yellow solid (270 mg, 40%). 1H NMR spectrum was used to verify the chemical structure (Figure S20). Host-guest

Self-assembly

PAMAM-PG-g-s-s-DOTA(Gd)+FA.

of

Ad-PG-g-s-s-DOTA(Gd) (510 mg, 0.02 mmol) and Ad-PG-FA (83 mg, 0.0087 mmol) were dissolved in 12 mL H2O, and then PAMAM-β-CD (39 mg, 0.0016 mmol) dissolved in 5 mL DMSO was added dropwise under vigorous stirring. The product was purified by dialysis (35 kDa MWCO) against deionized water for 3 days. The obtained solution was concentrated through

ultrafiltration for further experiments.

In addition, the control sample

PAMAM-PG-g-s-s-DOTA(Gd) without special targeting was prepared using the similar procedure, just not adding Ad-PG-FA. Measurements

of

Hydrodynamic

The

Size.

hydrodynamic

sizes

of

PAMAM-PG-g-s-s-DOTA(Gd)+FA and PAMAM-β-CD were determined with dynamic light scattering at room temperature. The samples were treated with 0.45 µm filter prior to measurements. Specially, the sample PAMAM-β-CD was dissolved in 0.5 M NaOH for the measurement. MRI

Measurements

in

Solution.

PAMAM-PG-g-s-s-DOTA(Gd)+FA

in

The

r1

physiological

and saline

T1-weighted at

varying

MRI

of

gadolinium

11



Page 12 of 41

concentrations were acquired on a 0.5 T NMR-analyzer (GY-PNMR-10). For the measurements of T1 relaxation time, the inversion recovery method (TR (repetition time)=10 s) was implemented. In the experiments of T1-weighted imaging, the images were obtained through with spin-echo sequence, and the parameters were set as follows: TR (repetition time)=100.0 ms, TE (echo time)=8.6 ms, NS (number of scans)=1. The curve-fitting of 1/T1 (s-1) versus the gadolinium concentration (mM) was implemented to calculate the r1 values. Small-molecule Gd-DTPA, as a commercially available gadolinium-based CA, was used as a control sample. In addition, the r1 value of PAMAM-PG-g-s-s-DOTA(Gd)+FA at 1.5 T was also recorded. Determination

of

Biodegradability.

The

biodegradability

of

PAMAM-PG-g-s-s-DOTA(Gd)+FA based on disulfide bonds was evaluated by monitoring changes in longitudinal relaxation time T1 and r1 through incubating with TCEP, a reducing agent for cleaving disulfide bond. Detailly, the sample PAMAM-PG-g-s-s-DOTA(Gd)+FA with a gadolinium concentration of 1 mM was incubated with 10 mM TCEP at 37 oC and the T1 values were recorded over the course of 4 h. The final r1 value was determined for comparison with the initial one. Biocompatibility Assay. PAMAM-PG-g-s-s-DOTA(Gd)+FA with a series of gadolinium concentrations were used to evaluate the cytotoxicity on KB and HUVEC cells. The cell viability was determined through the standard WST assay. Additionally, Hematoxylin-Eosin (H&E) staining was employed to investigate the tissue toxicity. The detailed procedures are shown in the Supporting Information.

12


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Cellular

MRI.

Three

samples,

Gd-DTPA,

PAMAM-PG-g-s-s-DOTA(Gd)

and

PAMAM-PG-g-s-s-DOTA(Gd)+FA, were used to observe the MRI effectiveness on the cellular level. The experimental procedures are similar to that in the previously reported literature26, and the detailed procedures are shown in the Supporting Information. MRI in Vivo. The contrast effectiveness of tumor-bearing mice respectively treated with Gd-DTPA, PAMAM-PG-g-s-s-DOTA(Gd) and PAMAM-PG-g-s-s-DOTA(Gd)+FA, were systematically investigated, and the detailed procedures are shown in the Supporting Information. Assay of Gadolinium Retention in Vivo. PAMAM-PG-g-s-s-DOTA(Gd)+FA was employed to evaluate the gadolinium retention after intravenous injection, the experimental procedures are similar to that in the previously reported literature26. The detailed procedures are shown in the Supporting Information. Statistical Analysis. The data are shown as means and standard deviations (SD) and p < 0.05 is considered statistically significant. The data analyses are based on Origin 8.5 and SigmaPlot 12.5 software.

RESULTS AND DISCUSSION Synthesis and Characterization of PAMAM-PG-g-s-s-DOTA(Gd)+FA. To endow biocompatible PG-based macromolecular MRI CA with biodegradability to reduce the retention time of gadolinium chelate in the body and further ensure the biosafety for application, the biodegradable disulfide bond was employed to conjugate gadolinium chelate to PG. Moreover, the strategy of host-guest self-assembly between β-CD and adamantane 13



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was utilized to facilitate the structural control and manufacturing procedures of CA. In addition, to realize the targeting specificity to tumor tissue, the FA moiety was introduced to the periphery of CA. Specifically, the PAMAM dendritic molecule was modified with β-CD as a cyclodextrin-containing core (PAMAM-β-CD). Furthermore, adamantanemethanol initiated the polymerization reaction to gain linear PG (Ad-PG), which was functionalized with

gadolinium

chelates

at

side

group

through

disulfide

conjugation

(Ad-PG-g-s-s-DOTA(Gd)) and modified with FA moiety at the terminal (Ad-PG-FA), respectively.

Finally,

upon

the

host-guest

self-assembly,

the

guest

molecules,

Ad-PG-g-s-s-DOTA(Gd) and Ad-PG-FA, together with the cyclodextrin-containing core PAMAM-β-CD formed a nano-globular inclusion complex. The detailed synthetic procedure for PAMAM-PG-g-s-s-DOTA(Gd)+FA is demonstrated in Figure 1. PAMAM-β-CD was prepared through the amidation reaction between PAMAM (G2.5) and β-CD-NH2. In order to make more β-CD molecule conjugate to the surface of dendritic PAMAM, an excess amount of β-CD-NH2 was added in the reaction. The chemical structure of PAMAM-β-CD was characterized by the 1H NMR spectrum (Figure S14), based on which the number of modified β-CD on each PAMAM dendrimer was calculated to be about 16 (see details in the Supporting Information). Although there are 32 active sites on the PAMAM dendrimer for conjugation, only about half of them can be utilized due to the existence of steric hindrance. For the preparation of Ad-PG-g-s-s-DOTA(Gd), potassium was used to react with adamantanemethanol and the resultant alkoxide polymerized EEGE to form Ad-PEEGE. The product was confirmed through the 1H NMR spectrum (Figure S15), and the 14


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polymerization degree was calculated to be 118 according to the integration ratio of characteristic peaks (see details in the Supporting Information). The GPC result of Ad-PEEGE demonstrates that the Mn value is 14304 g/mol with a PDI of 1.36. Then, the acetal groups were removed after adding HCl, and the chemical structure of formed Ad-PG was verified by 1H NMR spectrum (Figure S16). Furthermore, the hydroxys of Ad-PG were partially reacted with PPA-cyst-CI to obtain Ad-PG-g-s-s-alkynyl, whose side chains consisted of the disulfide bonds for biodegradation and the alkynyl groups for conjugating gadolinium chelate. The chemical structure of Ad-PG-g-s-s-alkynyl was characterized by 1H NMR spectrum (Figure S17), based on which the number of alkynyl group was calculated to be 18 (see details in the Supporting Information), and the FTIR spectrum further confirmed the successful modification, demonstrating the characteristic peak of alkynyl group at about 2100 cm-1 (Figure S18). Finally, the Ad-PG-g-s-s-DOTA(Gd) was achieved through the efficient click chemistry reaction between Ad-PG-g-s-s-alkynyl and N3-DOTA(Gd). To ensure all the alkynyl groups could be reacted, excess N3-DOTA(Gd) was added in this procedure.

Compared

with

Ad-PG-g-s-s-alkynyl,

the

FTIR

spectrum

of

Ad-PG-g-s-s-DOTA(Gd) (Figure S18) shows the disappearance of alkynyl peak, revealing the complete transformation of alkynyl group, and the number of conjugated gadolinium chelate can be considered as 18. Ad-PEEGE was further employed to synthesize Ad-PG-FA for constructing the function of targeting specificity. The terminal hydroxy group of Ad-PEEGE was modified to alkynyl group

through

the

reaction

with

propargyl

bromide,

and

then

the

resultant

Ad-PEEGE-alkynyl was treated with HCl to remove the acetal groups, obtaining the 15



Page 16 of 41

Ad-PG-alkynyl. The product was verified with 1H NMR spectrum (Figure S19). Finally, on the basis of click chemistry reaction, N3-FA was conjugated with Ad-PG-alkynyl to form Ad-PG-FA, whose chemical structure was confirmed by 1H NMR spectrum (Figure S20). Nano-globular PAMAM-PG-g-s-s-DOTA(Gd)+FA was obtained based on the host-guest self-assembly

between

PAMAM-β-CD

and

Ad-PG-g-s-s-DOTA(Gd)

together with

Ad-PG-FA. After the architecture-controllable and convenient self-assembly, the obtained nano-globular particle composed of dendritic PAMAM as a core and gadolinium chelate-modified PG as a shell, and was conjugated with targeting molecule FA at the periphery. As a macromolecular MRI CA incorporated with disulfide bonds and FA moiety, nano-globular PAMAM-PG-g-s-s-DOTA(Gd)+FA possessed the abilities of biodegradability and targeting specificity. On the one hand, to confirm the self-assembly process, the hydrodynamic sizes of PAMAM-β-CD and PAMAM-PG-g-s-s-DOTA(Gd)+FA were estimated (Figure S21), which demonstrates the size switches from 3.95 nm to 7.52 nm after the host-guest self-assembly, validating the successful conjugation originated from host-guest recognition. Moreover, the nano-globular PAMAM-PG-g-s-s-DOTA(Gd)+FA exhibits the stable hydrodynamic size over time (shown in Figure S22) and almost neutral zeta potential (shown in Figure S23). On the other hand, to verify the introduction of FA molecule on nano-globular CA, the UV-vis spectrum of PAMAM-PG-g-s-s-DOTA(Gd)+FA was recorded (Figure S24). In comparison with the control sample PAMAM-PG-g-s-s-DOTA(Gd), it is found that PAMAM-PG-g-s-s-DOTA(Gd)+FA displays the obvious characteristic peaks of FA at 280 nm and 360 nm, thus the targeting function is constructed as expectation. In addition, on the basis of gadolinium concentration determined by ICP-MS and UV-vis absorption 16


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spectra of PAMAM-PG-g-s-s-DOTA(Gd)+FA sample, the number of gadolinium chelate and FA ligand on each nano-globular MRI CA was approximately calculated to be 216 and 4, respectively (see detailed calculation in the Supporting Information). In a word, utilizing the host-guest

self-assembly

between

β-CD

and

adamantane,

the

gadolinium-based

nano-globular MRI CA with the functions of biodegradability and targeting specificity was conveniently and accurately fabricated.

17



Page 18 of 41

Figure 1. Synthetic routes of (A) PAMAM-β-CD, (B) Ad-PG-g-s-s-DOTA(Gd) and (C) Ad-PG-FA. 18


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Longitudinal

Relaxivity

and

T1-weighted

MR

Images.

The

potential

of

PAMAM-PG-g-s-s-DOTA(Gd)+FA used as a T1 MRI contrast agent was investigated on a 0.5 T NMR-analyzer. As indicated in Figure 2(a), through the linear fitting between T1 relaxation rate and gadolinium concentration, a longitudinal relaxivity r1 was calculated to be 8.39 mM-1s-1, which is obviously higher than that of commercial Gd-DTPA (4.41 mM-1s-1). The r1 value of PAMAM-PG-g-s-s-DOTA(Gd)+FA measured in a 1.5 T magnetic field is 7.97 mM-1·s-1 (shown in Figure S26), revealing a slight decrease because of the improved magnetic intensity. Moreover, the T1-weighted MR images at various gadolinium concentrations were recorded (shown in Figure 2(b)), demonstrating the improvement of signal intensity with increasing concentration, and significantly brighter images than that generated by Gd-DTPA at each concentration. All the positive results reveal the favorable potential on T1 MRI CA. In comparison with small-molecule Gd-DTPA, the improved longitudinal

relaxivity

and

strengthened

MR

signal

intensity

of

PAMAM-PG-g-s-s-DOTA(Gd)+FA derive from the increased rotational correlation time of gadolinium chelates, which is induced when gadolinium chelates are conjugated to the nano-globular molecule with large size40. Furthermore, the shell of nano-globular CA mainly consists of hydrophilic PG, in which the conjugated gadolinium chelates can access water molecules freely. As a result, during the MRI, the water-exchange of gadolinium chelates with free water molecules can be effectively ensured. As a whole, the improved rotational correlation time and hydrophilic shell structure both contribute to the favorable r1, which will be beneficial to the MRI.

19



Page 20 of 41

Figure 2. (a) Linear relationship of T1 relaxation rate (1/T1) with gadolinium concentrations for Gd-DTPA and PAMAM-PG-g-s-s-DOTA(Gd)+FA in physiological saline at 0.5 T. (b) T1-weighted MR images of Gd-DTPA and PAMAM-PG-g-s-s-DOTA(Gd)+FA with various gadolinium concentrations in physiological saline.

Biodegradability

in

Vitro.

The

disulfide

bond-based

biodegradability

of

PAMAM-PG-g-s-s-DOTA(Gd)+FA was evaluated by incubating nano-globular MRI CA in the presence of TCEP that is normally used to reduce disulfide bonds, and measuring the longitudinal relaxation time T1 as a function of time and the change of relaxivity r1 at 0.5 20


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T(shown in Figure 3). During the first 60 min of incubation, the value of T1 gradually increased, and then it stays at a relatively stable state until 240 min. Moreover, when T1 reaches the stable level, the r1 value is 4.808 mM-1s-1, showing a significant decrease compared to that in the initial state. On the basis of relaxivity theory, small-molecule gadolinium chelates conjugated to the macromolecular carrier will exhibit improvement of r1, which is contributed to the increasing rotational correlation time. Inversely, when gadolinium chelates are cleaved to form the free small molecules, the value of r1 will decrease correspondingly. For PAMAM-PG-g-s-s-DOTA(Gd)+FA, the obvious increase of T1 and decline of r1, which emerged during the incubation with a reduced agent, confirmed the feasibility of biodegradability depending on disulfide bonds.

21



Figure

3.

(a)

Variation

of

longitudinal

relaxation

Page 22 of 41

time

T1

for

PAMAM-PG-g-s-s-DOTA(Gd)+FA treated with TCEP over time. (b) Longitudinal relaxivity r1 of PAMAM-PG-g-s-s-DOTA(Gd)+FA before and after treated with TCEP.

Cytotoxicity and Tissue Toxicity. To make a systematic evaluation on the 22


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biocompatibility of PAMAM-PG-g-s-s-DOTA(Gd)+FA, the investigations about cytotoxicity and tissue toxicity were implemented. As indicated in Figure 4, both groups for HUVEC and KB cells present the negligible cytotoxicity after incubation with samples for 24 h, demonstrating the high cell viability above 90%, even when the gadolinium concentration goes up to 2.00 mM. Moreover, as shown in Figure 5, based on the comparison with blank group, the H&E-stained results of PAMAM-PG-g-s-s-DOTA(Gd)+FA indicate that the injection concentration of 0.1 and 0.3 mmol/kg both exhibit no appreciable adverse influence on the main organ tissues. All the sections in 0.1 and 0.3 mmol/kg group reveal the normal pathomorphologies, and there is no histopathological damage response in all organ tissues. Particularly, no signs of necrosis, swelling and inflammatory cell infiltration are seen from hepatocytes and other liver cells, and lungs present no pulmonary fibrosis. All the positive results about cytotoxicity and histological analysis mentioned above show that PAMAM-PG-g-s-s-DOTA(Gd)+FA, as a macromolecular MRI CA, is equipped with excellent biocompatibility for biomedical application, which can be reasonably attributed to the favorable performance of PG in biomedicine.

23



Page 24 of 41

Figure 4. Cytotoxicity of PAMAM-PG-g-s-s-DOTA(Gd)+FA with various gadolinium concentrations on HUVEC and KB cells after incubation for 24 h. Data presented as mean±SD (n=5).

24


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Figure 5. H&E-stained images (200× magnification) of main organs obtained from the blank mice, and mice intravenously injected with PAMAM-PG-g-s-s-DOTA(Gd)+FA at a gadolinium concentration of 0.1 mmol/kg and 0.3 mmol/kg, respectively. The mice were raised for 3 days after intravenous injection.

MRI of Cells in Vitro. The targeting ability of PAMAM-PG-g-s-s-DOTA(Gd)+FA, provided by the FA moiety, was evaluated through the MRI study on KB cells, which express a high level of FA receptor. Simultaneously, the groups for cells treated with non-targeted PAMAM-PG-g-s-s-DOTA(Gd) and Gd-DTPA were chosen as control. The MR images of blank cells and cells incubated with Gd-DTPA, PAMAM-PG-g-s-s-DOTA(Gd) and PAMAM-PG-g-s-s-DOTA(Gd)+FA are shown in Figure 6(a). Obviously, compared to the blank group, the groups going through incubation with CAs exhibit the brighter images. Furthermore, the brightness for the groups of Gd-DTPA and PAMAM-PG-g-s-s-DOTA(Gd) are similar, while that of PAMAM-PG-g-s-s-DOTA(Gd)+FA treated group is the highest. The signal intensity of MR images was further analyzed, and the results are indicated in Figure 6(b). Selecting the signal intensity of blank group as a baseline, the signal intensity ratio for 25



the

groups

of

Gd-DTPA,

Page 26 of 41


and

PAMAM-PG-g-s-s-DOTA(Gd)+FA are 140±5%, 142±2% and 174±4%, respectively, which further clearly indicate that the group of PAMAM-PG-g-s-s-DOTA(Gd)+FA possess the strongest

MR

signal.

On

the

basis

of

cellular

MRI,

it

is

concluded

that

PAMAM-PG-g-s-s-DOTA(Gd)+FA is capable of targeting tumor cells through FA-mediated interaction.

Figure

6.

(a)

MR

images

of

KB

cells

incubated

with

Gd-DTPA,

PAMAM-PG-g-s-s-DOTA(Gd) and PAMAM-PG-g-s-s-DOTA(Gd)+FA for 2 h, respectively. Cells without any treatment were selected as a blank group. (b) Analysis of signal intensity ratio for cellular MRI in each group. The statistically significant difference was observed on signal intensity (*p < 0.05, n=3). 26


Page 27 of 41 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


MRI

of

Tumor-bearing

Mice.

To

evaluate

the

application

of

PAMAM-PG-g-s-s-DOTA(Gd)+FA on tumor diagnosis, tumor-bearing mice were employed and the variation of MR images versus time was systematically investigated. At the same time, the small-molecule Gd-DTPA and non-targeted PAMAM-PG-g-s-s-DOTA(Gd) were also studied as control groups. The coronal MR images of mice contrasted by these CAs at different time points are demonstrated in Figure 7. For the group of Gd-DTPA, there is no obvious MR signal enhancement at the tumor location during the whole time window in the investigation. In the group of PAMAM-PG-g-s-s-DOTA(Gd), the MR signal located in tumor region presents a modest increase at the time point of 0.5 h after intravenous injection, and the contrasted effectiveness is slightly weakened in the following 1.5 h, then the lightness further decreases gradually. By contrast, PAMAM-PG-g-s-s-DOTA(Gd)+FA, equipped with targeting specificity to tumor tissue, can provide a strong contrast enhancement of tumor location at 0.5 h, and further improve the lightness at 1 h. Subsequently, the MR signal recedes slowly from the time point of 2 h. To quantitatively evaluate the change of contrast enhancement located in the tumor tissue, the values of intensity increasing rate (IIR) at different time points after intravenous injection with these CAs were also measured (shown in Figure 8). The IIR value of group contrasted by Gd-DTPA stays stable around 100%, which is due to the rapid clearance of small-molecule CAs after intravenous injection41. At the recorded time points, the vast majority of Gd-DTPA have been excreted by the kidney, which finally results in the extremely low signal intensity at tumor location. In contrast, an obvious increase of IIR value is observed in the groups of macromolecular CAs, and sufficient time window for imaging is also obtained. However, the difference between 27



Page 28 of 41

PAMAM-PG-g-s-s-DOTA(Gd) and PAMAM-PG-g-s-s-DOTA(Gd)+FA on detailed contrast effectiveness is noticeable. For the group of PAMAM-PG-g-s-s-DOTA(Gd), IIR value increases to 241±13% in 0.5 h after intravenous injection, and then gradually decreases to 139±7% at 5 h. In the group of PAMAM-PG-g-s-s-DOTA(Gd)+FA, IIR value presents a higher increase in 0.5 h, revealing an extent of 294±13%, and then exhibits a further increase to 365±28%, after that it decreases gradually to 167±5% at 5 h. The favorable performance of PAMAM-PG-g-s-s-DOTA(Gd)+FA on tumor imaging, including faster enhancement of MR signal and better contrast effectiveness, can be attributed to the targeting specificity of FA molecule. Moreover, compared with Gd-DTPA and PAMAM-PG-g-s-s-DOTA(Gd), the excellent contrast effectiveness of PAMAM-PG-g-s-s-DOTA(Gd)+FA was further verified through its efficient accumulation in the tumor at the time point of 1 h (shown in Figure S27). In addition, it is worth noting that the MR signals induced by macromolecular CAs both reduce to a relatively low level at 5 h, which may derive from the cleavage of disulfides in molecular structure causing the formation of free small-molecule gadolinium chelates with easy elimination. In the whole process of MR imaging, to confirm whether there exists the phenomenon that gadolinium chelates are excreted through renal filtration, the MR images of bladder for the group of PAMAM-PG-g-s-s-DOTA(Gd)+FA at different time points were also recorded (shown in Figure S28). Generally, the threshold of renal clearance is considered to be 30~40 kDa.42 Accordingly, the bright images at bladder after intravenous injection demonstrate the obvious excretion of small-molecule gadolinium chelates43, and indirectly indicate the existence of degradability based on disulfide bonds. Hence, the nano-globular MRI CA incorporated with FA molecule and disulfide bonds, benefiting from the targeting 28


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specificity and biodegradability, can provide excellent contrast effectiveness on tumor tissue as well as realize relatively rapid excretion of gadolinium chelates.

Figure 7. Coronal MR images of KB tumor-bearing mice before and after intravenously injected

with

Gd-DTPA,


and

PAMAM-PG-g-s-s-DOTA(Gd)+FA at a gadolinium dose of 0.1 mmol/kg. The time points after injection for investigation were 0.5, 1, 2, 3, 4, and 5 h. Red dotted circles indicate the location of tumor tissue.

29



Page 30 of 41

Figure 8. Intensity increasing rate (IIR is calculated by SIpost/SIpre, SI stands for signal intensity, IIR=100% means no signal enhancement) of MR signal located at tumor region in the

research

groups

of

Gd-DTPA,


and

PAMAM-PG-g-s-s-DOTA(Gd)+FA. The statistically significant difference was observed on signal intensity (*p < 0.05, n=3).

Biodistribution in Vivo. The contents of gadolinium ion in main organs and tissues were detected at 7 day after injection to investigate the biodistribution of CA, which might contain undegradable macromolecule and free gadolinium chelates (shown in Figure 9). Heart and lung exhibit the relatively lower content of gadolinium ion, and the values are all below 0.05 %ID/g. In the spleen, kidney and tumor, the contents are less than 0.16 %ID/g. For the liver, the content of gadolinium ion is highest, demonstrating an approximate value of 0.25 %ID/g. Compared to our previous nondegradable macromolecular CA with a similar 30


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globular structure based on PG26, whose lowest gadolinium content in the lung is around 0.5 %ID/g and highest content in the liver is about 3.8 % at 7 day of injection, the gadolinium retention from biodegradable PAMAM-PG-g-s-s-DOTA(Gd)+FA is rather lower. Moreover, the contents of gadolinium retention in main organs and tissues are at the same level with that of other reported disulfide bonds-containing mCAs17, and even lower particularly in kidney and liver. The low retention content of gadolinium in vivo is mainly attributed to the biodegradability

of

nano-globular

small-molecular-weight

fragments

macromolecular containing

CA makes

gadolinium

the

chelate.

formation As

a

of

whole,

PAMAM-PG-g-s-s-DOTA(Gd)+FA, as a biodegradable macromolecular CA, can realize the relatively fast excretion of gadolinium chelates and low retention of gadolinium ion after intravenous injection, probably making the process of tumor diagnosis safe and reliable.

31



Page 32 of 41

Figure 9. Biodistribution of gadolinium ion in main organs and tissues, including heart, liver, spleen,

lung,

kidney

and

tumor,

at

7

day

after

intravenous

injection

of

PAMAM-PG-g-s-s-DOTA(Gd)+FA with a gadolinium dose of 0.1 mmol/kg. Data presented as mean±SD (n=3).

CONCLUSIONS In summary, a nano-globular PAMAM-PG-g-s-s-DOTA(Gd)+FA with biodegradability and tumor specificity was systematically designed and prepared as macromolecular MRI CA for effective tumor diagnosis and rapid excretion. Based on the convenient host-guest self-assembly between β-CD and adamantane, PG separately conjugated with gadolinium chelates using disulfide bonds at the side chain and FA molecule at the terminal site were simultaneously linked to the surface of PAMAM dendrimer to form a nano-globular MRI CA. Compared with small-molecule Gd-DTPA, the nano-globular CA exhibits a higher r1 of 8.39 32


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mM-1s-1. Moreover, negligible toxicity was observed both on the investigations of cytotoxicity and tissue toxicity. Due to the targeting specificity, nano-globular CA can realize the favorable targeting to tumor cells and tumor tissue, and provide obvious contrast enhancement at tumor location. Additionally, since the disulfide bonds will be cleaved in reducing environment, the gadolinium chelates can be excreted by renal filtration and there is relatively low gadolinium retention in vivo. Therefore, PAMAM-PG-g-s-s-DOTA(Gd)+FA is a promising macromolecular CA for tumor diagnosis with excellent contrast effectiveness and biosafety.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Detailed experimental procedures of synthesis and characterization of PAMAM (G2.5), β-CD-NH2, and PPA-cyst-CI; 1H NMR, FTIR and UV-vis absorption spectra; hydrodynamic sizes

and

zeta

potential;

relaxivity

analysis

at 1.5

T;

structural

analysis

of

PAMAM-PG-g-s-s-DOTA(Gd)+FA; detailed procedures of cell and animal experiments; analysis of gadolinium accumulation in tumor; MR images for the bladder of mice treated with nano-globular CA.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. Tel: 86-512-62872776. 33



Page 34 of 41

*E-mail: [email protected]. Tel: 86-25-84315325. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (21575154), the National Key Research and Development program (2016YFA0101500), Natural Science Foundation of Jiangsu Province (BK20161262), National Postdoctoral Program for Innovative Talents (BX201700276), the Key Research Program of Chinese Academy of Sciences (ZDRW-ZS-2016-2) and the CAS/SAFEA International Innovation Teams program.

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Biodegradable Nanoglobular Magnetic Resonance Imaging Contrast

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