Poly(glycerol) Used for Constructing Mixed Polymeric Micelles as T1

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Poly (glycerol) used for constructing Mixed Polymeric Micelles as T1 MRI Contrast Agent for Tumor-targeted Imaging Yi Cao, Min Liu, Kunchi Zhang, Guangyue Zu, Ye Kuang, Xiaoyan Tong, Dangsheng Xiong, and Renjun Pei Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01437 • Publication Date (Web): 21 Nov 2016 Downloaded from http://pubs.acs.org on November 26, 2016

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Poly (glycerol) used for constructing Mixed Polymeric Micelles as T1 MRI Contrast Agent for Tumor-targeted Imaging Yi Cao†,‡,※, Min Liu†,※, Kunchi Zhang†, Guangyue Zu†, Ye Kuang†, Xiaoyan Tong†, Dangsheng Xiong*,‡, and Renjun Pei*,† †

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

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

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

Technology, Nanjing 210094, China.

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ACS Paragon Plus Environment

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ABSTRACT There was much interest in the development of nanoscale delivery vehicles based on polymeric micelles to realize the diagnostic and therapeutic applications in biomedicine. Here, with the purpose of constructing a micellar magnetic resonance imaging (MRI) contrast agent (CA) with well biocompatibility and targeting specificity, two types of amphiphilic diblock polymers, mPEG-PG(DOTA(Gd))-b-PCL and FA-PEG-b-PCL, were synthesized to form mixed micelles by co-assembly. The nanostructure of the resulting micellar system consisted of poly (caprolactone) (PCL) as core, and poly (glycerol) (PG) and poly (ethylene glycol) (PEG) as shell, simultaneously modified with DOTA(Gd) chelates and folic acid (FA), affording functions of MRI contrast enhancement and tumor targeting. The mixed micelles in aqueous solution presented a hydrodynamic diameter of about 85 nm. Additionally, this mixed micelles exhibited higher r1 relaxivity (14.01 mM-1·S-1) compared with commercial Magnevist® (3.95 mM-1·S-1), and showed negligible cytotoxicity estimated by WST assay. In vitro and in vivo MRI experiments revealed excellent targeting specificity to tumor cells and tissue. Furthermore, considerably enhanced signal intensity and prominent positive contrast effect were achieved at tumor region after tumor-bearing mice were intravenously injected with the mixed micelles. These preliminary results indicated the potential of the mixed micelle as T1 MRI CA for tumor-targeted imaging.

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INTRODUCTION Polymeric micelles, one class of promising nanotechnology-based delivery vehicles, have been widely investigated as carriers for effective diagnostic and therapeutic modalities.1-6 Especially, several ones of excellent potentiality have been evaluated in clinical trials applied for anti-cancer drugs.7-9 The core-shell architecture of polymeric micelles, usually assembles from amphiphilic block copolymers above critical micelle concentration (CMC) in water, providing the advantages of reducing the sides effects stemming from the incorporated drugs, prolonging the circulation time in blood and allowing the easy introduction of targeting moieties to the surface.10-12 Some of these properties are appealing for fabricating macromolecular magnetic resonance imaging (MRI) contrast agents (CAs) based on gadolinium for tumor imaging. Micellar contrast agents with strong increase of relaxivity and long-circulating properties were easily gained through grafting gadolinium chelates to the hydrophilic layer of polymer micelles.13-14 Additionally, enhanced tumor accumulation of micellar contrast agents was achieved when they were imparted specific targeting by conveniently conjugating the targeting ligand onto the surface.15 Obviously, polymeric micelle-based carries modified with gadolinium chelates have a potential as T1 MRI CAs for tumor imaging.

Mixed polymeric micelles, as a special class of polymeric micelles, have also attracted intensive interest in the field of medical imaging and drug delivery.16-21 Contrast to traditional polymeric micelles, they are usually assembled from two or more kinds of block polymers. Normally, various mixed micelles were designed with the purpose of endowing them with special properties, such as controlled drug release,18 dependable stability19 and tumor 3


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targeting20-21. Typically, Liu and coworkers reported a mixed micelle which was co-assembled from two types of amphiphilic diblock copolymers, respectively modified with gadolinium chelates and folic acid (FA), together with chemotherapeutic drugs as an integrated multifunctional platform of targeting specificity.21 Although some efforts have been made, mixed polymeric micelles as gadolinium-based CAs have been far less explored.

Poly (ethylene glycol) (PEG) is widely considered as an excellent polymer to construct the hydrophilic shell of polymeric micelles, since it is well known to be highly hydrated, well biocompatible, and capable of forming “stealth effect” when circulating in blood.22-24 Simultaneously, linear Poly (glycerol) (lin-PG), structurally similar to PEG, is increasingly recognized as an alternative to conventional PEG in recent years.25 In contrast to PEG, multihydroxy-functional lin-PG exhibits a similar or even slightly superior biocompatibility profile.26-28 Moreover, the characteristic of multihydroxy provides abundant groups for chemical modification, which is favorable for the design of multi-functional nanocarriers.29 Especially for preparing gadolinium-based CAs of macromolecule, lin-PG could offer not only many active sites for conjugating gadolinium chelates but also hydrated microenvironment for better realizing effective water-exchange. Thus, it is reasonable to speculate that taking lin-PG as the hydrophilic block to build polymeric micelle for gadolinium-based CAs would be a good selection. However, according to our knowledge, there is no such work which has been published.

Herein, amphiphilic polymer (mPEG-PG(DOTA(Gd))-b-PCL) consisted of lin-PG and poly (ε-caprolacton) (PCL) was prepared and gadolinium chelate was conjugated to the side 4


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chain of lin-PG. At the same time, another bi-block polymer FA-PEG-b-PCL, terminally modified with folic acid, was synthesized to co-assemble with mPEG-PG(DOTA(Gd))-b-PCL to form mixed polymeric micelles, which were designed as gadolinium-based contrast agent (CA) for tumor-targeted imaging. Furthermore, preliminary evaluations of the imaging efficacy of this mixed micelle-based gadolinium CA at cellular and animal levels were performed by in vitro MR imaging of KB cells and in vivo study on xenograft nude mice model.

EXPERIMENTAL SECTION Materials and Measurements. N,N,N′,N′′,N′′-Pentamethyldiethylenetriamine (PMDETA, 99%, Sigma-Aldrich), folic acid (FA, >97%, Sigma-Aldrich), sodium ascorbate (reagent grade,

Sigma-Aldrich),

CuBr

(98%,

Sigma-Aldrich),4-Nitrophenyl

chloroformate

(p-NPC, >98%, TCI) andanhydrouspyridine (>99%, J&K) were used as received. Poly (ethylene glycol) methyl ether (mPEG, average Mn=350, Sigma-Aldrich) was dried by azeotropic distillation from anhydrous toluene. ε-caprolactone (ε-CL, >99%, TCI) was stored over CaH2 and purified by vacuum distillation. Stannous octoate (Sn(Oct)2, 97%, Energy Chemical) was distilled in vacuum before use. Poly (ethylene glycol) alkynyl ether (alkynyl-PEG-OH, Mn=2000 g/mol, Sinopeg Biotech Co, Ltd) was dried in vacuum for 48h.Cell culture reagents including RPMI 1640folic acid free media and fetal bovine serum (FBS) were acquired from Gbico. Potassium, NH4HCO3, CuSO4·5H2O, toluene, hydrochloric acid (HCl), tetrahydrofuran (THF), dichloromethane (DCM) and dimethyl sulfoxide (DMSO) in analytical grade were all obtained from Sinopharm Chemical Reagent Co, Ltd and used without further purifications. The synthetic process of azido-folic acid (N3-FA) and 5


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alkynyl-DOTA(Gd) are shown in Supporting Information. Ethoxyethyl Glycidol Ether (EEGE) and 1-azido-3-aminopropane were synthesized according to the reported methods.30-31

1

H NMR spectroscopy data were obtained from a Varian 400 MHz spectrometer and the

chemical shifts were reported in ppm on the δ scale. The molecular weight of mPEG-PEEGE, mPEG-PG-b-PCL and alkynyl-PEG-b-PCL were estimated by a Waters GPC system with THF (1.0 mL/min), DMF (0.6 mL/min) and THF (1.0 mL/min) as eluent, respectively, and calibration curves were generated through PEG stands. Inductively coupled plasma atomic emission spectrometer (ICP-OES, Angilent) was used to identify the gadolinium concentration of samples. FTIR spectroscopies were collected using a Thermo Fisher FTIR spectrometer and the samples were pelletized with KBr before measurement. The morphologies and size distribution of mixed polymeric micelle were observed by transmission electron microscope (TEM, Hitachi) and dynamic light scattering (DLS, Malvern), respectively.

Synthesis of mPEG-PEEGE. This polymer was synthesized by a modified method used in the published literature.32 Briefly, to a Schlenk flask, mPEG (700 mg, 2 mmol) and potassium (39 mg, 1 mmol) were added under argon atmosphere. The mixture reacted under vacuum at 60 oC for 0.5 h to obtain 50% deprotonation of mPEG. After back-filled with argon, the resulting potassium alkoxide was heated to 95 oC, and then EEGE (13.14 g, 90 mmol) was added in one portion with a syringe. The reaction was cooled to room temperature and terminated by excess methanol together with acid ion-exchange resin after 48 h. 6


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Afterward, the solid was removed by filtration and the resulting solution was concentrated under reduced pressure. The final honey-like product was obtained by vacuum drying at 80 o

C for 48h (11.2 g, 79%). The monomer conversion was determined by 1H NMR (82%

monomer conversion), and the molecular weights were assessed by GPC (Mn=3.14×103, PDI=1.47) and 1H NMR spectrum (Figure S7 in Supporting Information).

Synthesis of mPEG-PG-b-PCL. To a schlenk flask, mPEG-PEEGE (2.87 g, 0.5 mmol), ε-CL (3.99 g, 35 mmol) and Sn(Oct)2 (2.8 mL, 0.1 M solution in toluene) were dissolved in 30 mL toluene under argon atmosphere, which was heated to 100 oC and reacted for 48 h. After cooled to room temperature, the solvent was removed under reduce pressure and the resulting mixture was redissolved in 150 mL THF. Afterward, the solution was added 2 mL HCl and stirred at room temperature for 2 h. Then, the mixture was concentrated using rotary evaporator and precipitated in excess cold ethyl ether. The procedure of precipitation was repeated twice. The final brown solid was collected by filtration and dried under vacuum for 24 h (3.8 g, 55%). The monomer conversion was determined by 1H NMR (43% monomer conversion), and the molecular weights were assessed by GPC (Mn=1.34×104, PDI=1.29) and 1

H NMR spectrum (Figure S8 in Supporting Information).

Synthesis of mPEG-PG(NPC)-b-PCL. mPEG-PG-b-PCL (1.3 g, 0.2 mmol, 7.4 mmol hydroxy), pyridine (202 µL, 2.5 mmol) and 40 ml DCM were stirred to form a homogenous solution, which was left in the ice bath to cool to about 4 oC. p-NPC (447 mg, 2.2 mmol) dissolved in 10 mL DCM was dropwise added to the above solution. After the addition, the resulting mixture was left at room temperature and reacted for 48 h. Then, the reaction 7


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solution was concentrated and purified by precipitation in excess ethyl ether. This procedure was repeated twice. The final product was collected by centrifugation and dried under vacuum for 48 h (555 mg, 32%). The chemical structure was confirmed by 1H NMR spectrum (Figure S9in Supporting Information).

Synthesis of mPEG-PG(N3)-b-PCL. mPEG-PG(NPC)-b-PCL obtained in above step was dissolved in 15 mL DMSO and 1-azido-3-aminopropane (666 mg, 6.6 mmol) mixed with 10 mL DMSO was added dropwise. Afterward, the mixture reacted at room temperature for 48 h. The product was purified by precipitation in ethyl ether three times and dried in vacuum for 48 h (382 mg, 69%). The chemical structure of mPEG-PG(N3)-b-PCL was confirmed by 1H NMR spectrum (Figure S10 in Supporting Information) and FT-IR spectrum (Figure S11 in Supporting Information).

Synthesis of mPEG-PG(DOTA(Gd))-b-PCL. mPEG-PG(N3)-b-PCL (215 mg, 0.03 mmol, N3: 0.15 mmol) and alkynyl-DOTA(Gd) (179 mg, 0.3 mmol) were dissolved in 10 mL DMSO under argon atmosphere. After that, PMDETA (32 µL, 0.15 mmol) and CuBr (22 mg, 0.15 mmol) were added, and the reaction mixture reacted at 50 oC for 48 h. The resulting mixture was dialyzed against 0.5% EDTA solution and water, respectively. Finally, through lyophilization, faint yellow solid was obtained (240 mg, 79%). FT-IR spectrum was carried out to confirm the chemical structure of mPEG-PG(DOTA(Gd))-b-PCL (Figure S11 in Supporting Information).

Synthesis of alkynyl-PEG-b-PCL. To a schlenk flask, alkynyl-PEG-OH (200 mg, 0.1 mmol), ε-CL (380 mg, 3.3 mmol) and Sn(Oct)2 (260 µL, 0.1M solution in toluene) were 8


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dissolved in 10 mL toluene under argon atmosphere. The resulting mixture was left at 100 oC and reacted for 24 h. Afterward, the reaction solution was added to excess ethyl ether to form white precipitation. This procedure was repeated twice. The final product was obtained through centrifugation and dried under vacuum for 48 h (374 mg, 64%). The monomer conversion was determined by 1H NMR (81% monomer conversion), and the molecular weights were assessed by GPC (Mn=5.86×103, PDI=1.25) and 1H NMR spectrum (Figure S12 in Supporting Information).

Synthesis of FA-PEG-b-PCL. Alkynyl-PEG-b-PCL (254 mg, 0.05 mmol) and N3-FA (39 mg, 0.075 mmol) were dissolved in 15 mL NH4HCO3 solution (10 mM), which was degassed by three freeze-pump-thaw cycles on a schlenk line. Then, sodium ascorbate (7.4 mg, 0.0375 mmol) and CuSO4·5H2O (3.7 mg, 0.015 mmol) were introduced under argon. After reacting at room temperature for 24 h, the resulting mixture was mixed with 80 mL saturated brine and extracted with DCM (20 mL×3). The combined organic phase was concentrated by rotary evaporation and precipitated in excess ethyl ether to form yellow solid, which was collected by centrifugation and dried under vacuum for 24 h (150 mg, 49%). The chemical structure of FA-PEG-b-PCL was confirmed by

1

H NMR spectrum (Figure S13 in Supporting

Information)

Fabrication of Mixed Polymeric Micelles. Mixed micelles co-assembled from mPEG-PG(DOTA(Gd))-b-PCL and FA-PEG-b-PCL were fabricated through common solvent approach. Briefly, mPEG-PG(DOTA(Gd))-b-PCL (8 mg) and FA-PEG-b-PCL (2 mg) were dissolved in 5 mL DMSO, and then slowly added to 20 mL H2O under stirring. Afterward, 9


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the mixture solution was dialyzed against water using 3500 MWCO dialysis tube for 24h. The final colloidal dispersion was concentrated by ultra-filtration for further experiments. Simultaneously, single micelles, just without FA modified block polymer, were prepared using a similar approach as the control group.

Cell Culture and Animal Tumor Model. Human oral epidermoid carcinoma cells (KB cells), obtained from Cell Bank of Chinese Academy of Sciences (Shanghai, China), were cultured in RPMI 1640 folic acid free media with 10% FBS, supplemented with 100 units/mL of streptomycin and penicillin, and maintained at 37 oC in a humidified atmosphere of 5% CO2.

Female athymic nude mice (4 weeks old) were obtained from Nanjing Sikerui Biological Technology Co. Ltd. and acclimated under pathogen-free conditions for 2 weeks before use. All animal experiments were conducted in accordance with the relevant laws and institutional guidelines following the approval of the Ethics Committee of Chinese Academy of Sciences. To induce a tumor, 2×106 KB cells were suspended in 100 µL of PBS and injected subcutaneously into the armpit of nude mice. When the tumor size reached about 5 mm in diameter after about 10 days, the nude mice were used for MR imaging study.

In vitro Cytotoxicity Assay.WST assay was used to evaluate the cytotoxicity of mixed polymeric micelles against KB cells. Firstly, KB cells were seeded with a density of 8000 cells/well in 96-well plate. Then, when cells achieved 60-70% confluence after 24 h of incubation, the medium was replaced with fresh complete medium containing samples with various concentrations (mg/mL), and incubated for further 24 h. Finally, the cell viability was 10


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assessed by adding 10 µL WST solution and incubating for another 2 h. The absorbance at 450 nm was measured with a Perkin-Elmer microplate reader. The cell viability was determined by the standard WST assay protocols.

In vitro MRI Research. For the measurement of T1 relaxivity (r1) and T1-weighted MR images, 200 µL micellar solution and Magnevist® with different gadolinium concentration were transferred to tubes, and the data were acquired with spin-echo acquisition on 0.5 T NMR-analyzer (GY-PNMR-10). The parameters were set as follows: TR (repetition time) = 100.0 ms, TE (echo time) = 8.6 ms, NS (number of scans) = 1. The r1 values were calculated from the slope of curve-fitting result of 1/T1 (s-1) versus the gadolinium concentration (mM). Additionally, the measurement in a magnetic field of 1.5 T (most commonly used in MRI scanners) was also carried out.

For the cellular MRI, KB cells were seeded into culture dishes with 10 mL culture medium. When the cells reached 80% confluence, the medium was replaced with fresh medium containing different samples (micelles and mixed micelles at the same gadolinium concentration of 0.1mM). Meanwhile, another dish only replaced with fresh medium was selected as control. After 2 h of incubation, the medium was removed and washed with PBS for 3 times. Then, cells were harvested by treatment with trypsin and centrifugation. The resulting cells were transferred to 200 µL Eppendorf tubes, and then centrifuged at 1000 rpm to obtain a compact pellet at the bottom of tube and directly measured for MRI.

In vivo animal MRI study. The T1 weighted MR images of animals were acquired on a 1.5 T scanner at designated time and the detailed imaging parameters were set as follows: 11


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Number excitation = 1; TR/TE = 100/16.8 ms; Matrix = 512×256; Slice thickness = 0.3 mm; 128 slices and no gap between slices. The tumor-bearing mice were firstly anesthetized by intraperitoneal injection of 20% urethane solution at a dose of 5 mL/kg, followed by collecting the coronal MR images of the tumor-bearing mice as the data of pre-injection. Afterward, the mice were treated with 200 µL sample in physiological saline (0.03 mmol/kg based on gadolinium concentration) through tail intravenous injection and coronal images were obtained at special time intervals (9 h, 24 h, 48 h). Overall, two groups of MR imaging experiment, including micelles and mixed micelles, were carried out to evaluate the tumor-targeting ability of mixed micelles.

StatisticAnalysis. Statistical analysis was performed using a Student’s t-test. The data were described with means and standard deviations (SD) and p < 0.05 was considered as statistically significant. All data analyses were performed using Origin 8.5 and SigmaPlot 12.5 software.

RESULTS and DISCUSSION Synthesis of mPEG-PG(DOTA(Gd))-b-PCL and FA-PEG-b-PCL. Based on the design of biocompatible carrier, PCL, PEG and lin-PG were selected to fabricate the main structure of amphiphilic diblock polymers, and then gadolinium chelate and FA were introduced by “click chemistry” to realize the MR imaging and tumor specificity. The detailed synthetic routes of mPEG-PG(DOTA(Gd))-b-PCL and FA-PEG-b-PCL were shown in Figure 1.

The terminal hydroxyl of mPEG-OH was reacted with potassium to obtain alkoxide, which initiated the polymerization of EEGE. The chemical structure was verified via 1H NMR 12


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(Figure S7), and the degree of polymerization of EEGE was calculated to be 37 (see details in Supporting Information). GPC analysis revealed a number-average molecular weight (Mn) of 3

3.14×10 g/mol (Mw/Mn=1.47). The obtained mPEG-PEEGE was further employed as an initiator for the ring-opening polymerization (ROP) of ε-CL, and the resulting copolymer was treated with HCl to cleave the acetal group to achieve mPEG-PG-b-PCL. The chemical structure was characterized by1H NMR (Figure S8), and signal integration ratio of characteristic peaks indicated that the degree of polymerization was 30 (see Supporting Information for details). The Mn of mPEG-PG-b-PCL estimated by GPC to be 1.34×104 g/mol (Mw/Mn=1.29).

The hydroxyl in mPEG-PG-b-PCL was then partially modified with azido group using p-NPC

mediatedhydroxyl

amine

coupling

reaction.

The

chemical

structures

of

mPEG-PG(NPC)-b-PCL and mPEG-PG(N3)-b-PCL were confirmed with 1H NMR (Figure S9 and Figure S10). The result of mPEG-PG(NPC)-b-PCL revealed that about 5 hydroxyl groups were reacted with p-NPC (see detailed calculation in Supporting Information). After reacting with 1-azido-3-aminopropane, the disappearance of characteristic peaks (7.5 ppm and 8.3 ppm) of nitrobenzene inmPEG-PG(N3)-b-PCLindicated the complete reaction, thus the number of azido group could be considered as 5 in this diblock copolymer. Furthermore, FTIR absorption was carried out to estimate the mPEG-PG(N3)-b-PCL and the result obviously showed the characteristic peak of azido group at 2100 cm-1 (Figure S11), which further confirmed the successful modification.

Conjugation of gadolinium chelates was realized through copper (Ⅰ) catalyzed 13


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alkyne-azide click reaction, which had been widely applied in various functionalized polymers because of its high reaction efficiency.33 In the presence of CuBr-PMDETA catalyst, mPEG-PG(N3)-b-PCL was reacted with excess alkynyl-DOTA(Gd) to ensure the complete reaction of azido groups. The disappearance of characteristic peak (2100 cm-1) of azido groups in the FTIR spectrum(Figure S11) for mPEG-PG(DOTA(Gd))-b-PCL verified the quantitative transformation. Therefore, it was reasonable to consider that there were five DOTA(Gd) chelates on mPEG-PG(DOTA(Gd))-b-PCL diblock copolymer.

Another amphiphilic copolymer (FA-PEG-b-PCL) was synthesized by a combination of ROP and “click chemistry”. Alkynyl-PEG was selected as an initiator for polymerization of ε-CL. The chemical structure of the obtained product was characterized by 1H NMR (Figure S12), and the polymerization degree was calculated to be 27(see details in Supporting Information), which was similar to that of PCL block in mPEG-PG(DOTA(Gd))-b-PCL, providing

benefit

for

the

formation

of

co-assembly.

GPC

characterization

of

alkynyl-PEG-b-PCL revealed a Mn of 5.86×103 g/mol (Mw/Mn of 1.25). Finally, FA moiety was successfully introduced to the terminal by “click chemistry”, which was confirmed through the characteristic peaks of FA between 9.0 ppm to 6.5 ppm detected in 1H NMR spectrum of FA-PEG-b-PCL (Figure S13).

14


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Figure 1.Synthetic routes of mPEG-PG(DOTA(Gd))-b-PCL (A) and FA-PEG-b-PCL(B).

Preparation and Characterization of Mixed Polymeric Micelles. The mixed polymeric micelles were prepared through the co-assembling from two kinds of diblock copolymers, mPEG-PG(DOTA(Gd))-b-PCL and FA-PEG-b-PCL, in an aqueous solution at a weight ratio of 4:1, affording the core-shell structure as sketchily described in Figure 2(a). The core was formed from the aggregation of PCL block, and the shell was consisted of PG and PEG. Simultaneously, DOTA(Gd) chelates were carried in the shell, ensuring the sufficient contact 15


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with water molecules, and FA moieties were distributed on the outer layer of the micelle to realize the tumor-targeted function. The characterization of TEM (Figure 2(c)) showed that the mixed polymeric micelles were monodispersed with a uniform morphology. The particle size of mixed polymeric micelles was analyzed with DLS and the result was presented in Figure 2(b). The mean hydrodynamic diameter was approximately 85 nm (PDI=0.27), which was little higher than that determined through TEM arising from the swelling of micelles in aqueous solution.

Figure 2.Schematic of core-shell structure (a), DLS measurement (b) and TEM image (c) of mixed polymeric micelles co-assembled from mPEG-PG(DOTA(Gd))-b-PCL and FA-PEG-b-PCL.

Characterization of Longitudinal Relaxivity (r1) and In Vitro MRI of Mixed Micelles. 16


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To explore the potential of mixed micelles as T1 MRI contrast agent, longitudinal relaxivity (r1), reflecting the ability to shorten the T1 longitudinal relaxation time of water protons, was measuredon a 0.5 T MRI scanner at 35 oC. At the same time, commercial CA (Magnevist®) was also evaluated as a control. The final values were calculated from the slope of curves in Figure3(a). The r1 value of mixed micelles was 14.01 mM-1·S-1 for per Gd, which was about 3.5 times as high as commercial Magnevist® (3.95 mM-1·S-1). The r1 in the higher magnetic field (1.5 T) showed a value of 13.49 mM-1·S-1 for per Gd, indicating a slight decrease due to the improvement of magnetic intensity34. Further research on T1-weighted images (Figure3(b)) revealed that mixed micellespresented significant brighter images than Magnevist® at the same gadolinium concentration. These results suggested that mixed micelles had higher imaging

enhancement

on

MR

than

Magnevist®.

According

to

the

Bloembergen-Solomon-Morgan theory, the higher relaxivity of mixed micelle was attributed to the sufficient exchange of water protons with gadolinium and the increase of rotational correlation time (τR).35-37 Generally, the shell structure of micelles is formed from the hydrophilic block of amphiphilic polymers after self-assembly in water, which could ensure the exchange of water protons with gadolinium when gadolinium chelates are modified in the shell of micelles.14, 21 In this study, the sufficient exchange of water protons with gadolinium also could be ensured because of the well-known hydrophilicity of poly (glycerol), constituting the shell of mixed micelles. Moreover, when small-molecule DOTA(Gd) chelates were modified to the shell of micelles with relatively large size, the value of τR was obviously increased. Similar to the published results of some mCAs38-39, the value of r1 was obviously improved. Overall, the results of mixed micelles in MRI characterization indicated the good 17


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potential for applications as T1 MRI CA.

Figure 3. Relaxivity measurements (a) and in vitro T1-weighted imaging of various Gd concentrations (0.08mM, 0.14mM, 0.20mM and 0.26 mM) (b) for mixed polymeric micelles and Magnevist® in pathological saline. The measurements were performed on a 0.5 T MRI scanner at 35 oC.

In vitro Toxicity Assay. To better understand the performance of mixed micelles on toxicity, KB cells were employed to evaluate the cytotoxicity of mixed micelles with various concentrations using the standard WST assay. At the same time, Magnevist®, an MRI CA used in clinic, was selected as a control. As shown in Figure 4, similar to Magnevist®, mixed micelles presented negligible cytotoxicity, even at the gadolinium concentration up to 1.6 mM (the approximate concentration of mixed micelles was 4 mg/mL).This favorable result may attribute to the well-recognized biocompatibility of lin-PG, PEG and PCL, which constituted the main parts of mixed micelles. Therefore, it was concluded that mixed micelles used as MRI CA possessed excellent biocompatibility.

18


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Figure 4. In vitro cytotoxicity of mixed micelles and Magnevist® determined by WST assay against KB cells.

Cellular MR Imaging. The targeting efficiency of mixed micelles co-assembled from mPEG-PG(DOTA(Gd))-b-PCL and FA-PEG-b-PCL was estimated by MR imaging of KB cells in vitro. KB cells were incubated with pathological saline (blank), Magnevist®, micelles (assembled from mPEG-PG(DOTA(Gd))-b-PCL, without FA) and mixed micelles, respectively, and the T1-weighted MR images of treated cells were measured (shown in Figure 5(a)). In comparison with cells incubated with pathological saline, cells treated with samples without targeting ligands (Magnevist® and micelles) presented no or slight improvement of brightness. However, the cells treated with mixed micelles exhibited obviously more brightness, which could attribute to the affinity between KB cells (expressing high FA receptor) and mixed micelles mediated by FA. Moreover, the intensity of signal was measured to quantitatively analyze the difference of MR signals (shown in Figure 5(b)). 19


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When the blank group was set as a baseline, the signal intensity of Magnevist®-treated and micelles-treated cells were nearly not improved and slightly improved to about 118±6%, respectively, whereas, that of cells incubated with mixed micelles was up to 162±7%. Obviously, the targeting ability of mixed micelles was verified through the results obtained above.

Figure 5. MR images (a) and signal intensity ratio (b) of KB cells treated pathological saline, Magnevist®, micelles and mixed micelles. Cells were incubated with samples at a Gd concentration of 0.1 mM for 2 h, and for the blank sample, the same volume of pathological saline was added. Signal intensity showed a statistically significant difference (*p < 0.05, n=3). 20


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In Vivo MR Imaging on Tumor-bearing Mice. The performance of mixed micelles used as CA for imaging of tumor-bearing mice was evaluated at a 1.5 T MRI scanner. Micelles, only self-assembled from the block polymer of mPEG-PG(DOTA(Gd))-b-PCL, was selected as a control to estimate the targeting specificity of mixed micelles in vivo. Simultaneously, the commercial Magnevist®, as a small-molecule MRI CA, was also evaluated as a comparison. Samples were intravenously injected at a gadolinium dose of 0.03 mmol/kg, and coronal T1-weighted MR images of pre-injection and at various time points after injection were acquired (shown in Figure 6). After the injection of samples, the MR signal intensity of tumor contrasted by Magnevist® and micelles presented no significant change, however, that induced by mixed micelles exhibited obvious signal enhancement. Careful observation of tumor suffered from mixed micelles showed that the boundary of tumor could be preliminarily confirmed after 9 h, and then the tumor tissue could be clearly detected after 24 h. Finally, the signal intensity relatively reduced at the time point of 48 h. All the results revealed that mixed micelles possessed the favorable ability for enhancing MR imaging at tumor location.

21


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®

Figure 6. Coronal MR images of KB tumor-bearing mice injected with Magnevist , micelles and mixed micelles at Gd dose of 0.03mmol/kg. The images of pre-injection were also acquired to estimate increasing rate. The red dotted circle shows the location of tumor.

To better understand the change of MR signals, intensity increasing rate (IIR, based on gray intensity estimated by ImageJ Software) of signals on tumor location was quantitatively analyzed and the results were shown in Figure 7. On the whole, the tumor treated with Magnevist® and micelles showed lower IIR than that contrasted with mixed micelles at each time point after injection, and the values of IRR were kept stable around 100%. In details, the difference of contrast effect induced by Magnevist®, micelles and mixed micelles was also apparent. For the sample of Magnevist®, the value of IIR maintained around 100% at each time point observed. For the sample of micelles, the value of IIR presented nearly no change (about 100%) in 9 h after injection, and then it gradually increased to 120±6% in 24 h, after that, the value decreased to 110±3% in 48 h. The change of MR signals in whole procedure was inconspicuous. For the sample of mixed micelles, the value of IIR ascended to 180±27% 22


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in 9 h after injection, and the signal intensity was further improved to around 310±28% in 24 h. Even after 48 h, the IIR was kept about 210±31%. According to the published results40-42, the high IIR value of 310±28% is sufficient to detect the location of tumor tissues. In contrast to the Magnevist® and micelles, the mixed micelles exhibited excellent contrast effect, which was ascribed to not only the long circulation time in blood of micellar carriers but also the targeting ability of FA moiety. In conclusion, mixed micellesco-assembled from mPEG-PG(DOTA(Gd))-b-PCL and FA-PEG-b-PCL, as T1 CA, were suitable for tumor-targeted MR imaging.

Figure 7. Intensity increasing rate of MR signal for KB tumor bearing mice (n=3) injected with ®

Magnevist , micelles and mixed micelles at Gd dose of 0.03mmol/kg. Intensity increasing rate showed a statistically significant difference (*p < 0.05, n=3).

CONCLUSION 23


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In this study, systematic synthesis of two types of biocompatible diblock polymers, mPEG-PG(DOTA(Gd))-b-PCL and FA-PEG-b-PCL, was demonstrated, and then mixed micelles were successfully prepared through the co-assembly of them, affording monodisperse nanoparticles used as T1 CA for MR imaging. The potentiality of this micellar CA was preliminarily explored in vivo and in vitro. The characterization of relaxivity indicated much higher r1 value of 14.01 mM-1·S-1 compared to commercial Magnevist®, and the cytotoxicity assay in vitro revealed the excellent biocompatibility. Tumor-targeted ability of the mixed micelles was validated through MR imaging of cell and tumor-beared mice. MR imaging in vivo also presented significant contrast effect of tumor location. This newly developed micellar system of targeting specificity could be used as T1 CA for the MR imaging of tumor tissue.

ASSOCIATED CONTENT Supporting Information Synthetic procedure and 1H NMR characterization of alkynyl-DOTA(Gd); synthetic method and1H NMR characterization ofN3-FA; 1H NMR spectra and analysis of mPEG-PEEGE, mPEG-PG-b-PCL, mPEG-PG(NPC)-b-PCL, mPEG-PG(N3)-b-PCL, alkynyl-PEG-b-PCL, FA-PEG-b-PCL;

FTIR

absorption

spectroscopiesof

mPEG-PG(N3)-b-PCL

and

mPEG-PG(DOTA(Gd))-b-PCL. This material is available free of charge via the Internet at http://pubs.asc.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], Tel: 86-512-62872776 (R.P.) 24


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*E-mail: [email protected], Tel: 86-25-84315325 (D.X.) Author Contributions ※ These authors have contributed equally. Notes These authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the Natural Science Foundation of China (21575154, 21304106), the Science and Technology Foundation of Suzhou (SYG201432), the CAS/SAFEA International Innovation Teams program and the 2016 Foundation for Imported Leading Talent Teams in Universities and Colleges of Anhui Province.

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Graphical Abstract


Poly(glycerol) Used for Constructing Mixed Polymeric Micelles as T1

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