Synthesis of a gadolinium based-macrocyclic MRI contrast agent for effective cancer diagnosis

Background Gadolinium-based contrast agents are widely used as a contrast agent for magnetic resonance imaging. Since gadolinium ions are toxic, many chelators are developed to bind gadolinium ions to prevent free gadolinium-associated disease. However, many reports indicated that linear chelator-based contrast agents are associated with nephrogenic systemic fibrosis (NSF) in patients with low kidney function. Therefore, the demand for stable macrocyclic chelator-based contrast agent is now increasing. Method 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetate (DOTA) was conjugated to lactobionic acid (LBA) through DCC-NHS coupling reaction. Gd3+ (gadolinium ion) was chelated to 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetate-lactobionic acid (DOTA-LAE) and free Gd3+ was removed using a cation exchange column. In vitro cytotoxicity of contrast agent towards normal cells was measured using MTT assay. For in vivo MR imaging, contrast agents were intravenously injected to tumor-bearing mice and imaged by a MR imaging scanner. Results This new macrocyclic gadolinium-based contrast agent showed enhanced in vitro paramagnetic properties compared to Gadovist. In addition, Gd-DOTA-LAE showed a 29% increased contrast enhancement of tumor tissue compared to normal tissue within 20 min past IV injection. Conclusions We developed a new macrocyclic T1-weighted MR contrast agent. This new contrast agent offers various opportunities for cancer detection and diagnosis.


Background
Early detection of cancer is essential for treatment and the survival rate of patients [1,2]. Magnetic resonance imaging (MRI) is most frequently used imaging method for detection and diagnosis of cancer. MRI is a noninvasive method to detect soft tissue, such as organs, ligament, cartilage, and cancer regions without exposing to radiation. However, it is still hard to distinguish between tumor regions and normal region. Many contrast agents (CAs) have been developed to enhance contrast intensity and contrast effect on region of interest. Gadolinium, manganese, iron oxide, and iron platinum-based CAs are used for clinical application, because Gd 3+ -based T1 CAs have been proven its great safety in many clinical cases and Gd 3+ -based T1 CAs are the most widely applied CAs on these days. Moreover, there have been various attempts to improve the signal intensity and sensitivity of Gd 3 + -based CAs to region of interest [3][4][5][6]. Improvement of signal intensity is related to the concentration of CAs in region of interest and shortening the longitudinal relaxation time of surrounding water protons nearby Gd 3+ ions [7][8][9][10]. One of the methods is accumulation of CAs using conjugation with bioactive moieties to increase concentration of CAs in specific regions, tumor specific antibody, and stimuli-responsive polymer [11][12][13][14][15][16]. The other method is decreasing longitudinal relaxation time of surrounding water proton using CAs [17,18]. Increasing the signal intensity of the CAs is also important, but there are more concerns about stability of CAs. For a decade, there have been reports of nephrogenic systemic fibrosis (NSF) associated with the use of Gd 3+ -based CAs [19,20], especially for patients with low kidney function [21]. NSF is a rare and serious disease that causes severe fibrosis of skin and internal organs [22]. It is known that release of free Gd 3+ from unstable chelator may associated with NSF. For this reason, The European Medicine Agency (EMA) has recommended a restriction for linear chelator-based CAs to prevent against any dangers from release of Gd 3+ [23]. Therefore, there are demands for stable and safe macrocyclic chelator CAs. The purpose of this study was to design new macrocyclic gadolinium-based contrast agent for MR imaging of tumors. In this respect, we designed a hydroxyl group rich material conjugated CA for high water proton exchange to improve T1-weighted signal intensity.

Synthesis of lactobionic acid-ethylenediamine (LAE)
DOTA-lactobionic acid was synthesized using carbodiimde reactions. Briefly, 1 g of lactobionic acid (LBA) was dissolved in DMF (10 mL) and activated by DCC (1.2 mol equiv. of LBA) and NHS (1.2 mol equiv. of LBA) at room temperature for 12 h. The by-product of the reaction, dicyclohexylurea, was removed by 0.45 μm syringe filter. Ethylenediamine (10 mol equiv. of LBA) was diluted with DMF (10 mL) and added dropwise to the solution of activated LBA. The solution reacted for 24 h at room temperature and purified by precipitation in cold ether and washed three times with ether. Lactobionate-ethylendiamine (LAE) was obtained under vacuum. The product of LAE was confirmed by 300 MHz 1 H NMR spectrometer. (Bruker, Germany).

Synthesis of DOTA-LAE
Five hundred seventy-three milligrams of Tri-tert-butyl 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetate (tri BOC-DOTA) was dissolved in DMF (5 mL) and activated by DCC (1.2 mol equiv. of tri BOC-DOTA) and NHS (1.2 mol equiv. of tri BOC-DOTA) at room temperature for 12 h. The by-product of the reaction, dicyclohexylurea, was removed by 0.45 μm syringe filter. Four hundred eighty milligrams of LAE (1.2 mol equiv. of tri BOC-DOTA) was added into the solution and reacted for 24 h at room temperature. Product (tri BOC-DOTA-LAE) was precipitated by adding cold ether and kept at deep freezer for 10 mins to complete the precipitation. The tri BOC-DOTA-LAE was washed three times with ether then dried under vacuum. 0.5 g of tri BOC-DOTA-LAE was dissolved in 3 ml of 75% TFA in DCM and treated for 40 min to remove tert-butyl group and dried under reduced pressure. The final product was dissolved in distilled water (D.W) and placed in a dialysis bag (molecular weight cutoff 500 Da) against D.W for 3 days. DOTA-LAE was obtained by lyophilizing. The product of DOTA-LAE was confirmed by 300 MHz 1 H NMR spectrometer. (Bruker, Germany).

Grafting of gadolinium (III) to DOTA-LAE
Five hundred milligrams of DOTA-LAE was dissolved in D.W (10 ml) and gadolinium chloride hexahydrate (GdCl 3 •H 2 0) (1.2 mol equiv. of DOTA-LAE) was added to this solution, and pH was adjusted to 6 with 0.1 M KOH solution [24]. The solution was heated to 40°C for 24 h under stirring. Free Gd 3+ ion was removed by dialysis (molecular weight cutoff 500 Da) in D.W for 3 days. One gram of chelax®100 resin was added to the solution at pH 5 and stirred gently for 1 h, then supernatant was decanted and lyophilized.

Estimation of paramagnetic properties of Gd-DOTA-LBA
The T1 and T2 relaxation times of LBA and Gadovist were measured in test tube with various Gd 3+ concentrations. The longitudinal rate (R 1 ) and transverse rate (R 2 ) were obtained by calculating the slope of the 1/T1 and 1/T2. All studies were performed on a 4.7 T animal MRI scanner (Biospec 47/40, Bruk-er BioSpin, Ettlingen, Germany) with 72 mm coil at Korea Basic Science Institute in Ochang.

Cytotoxicity studies of Gd-DOTA-LAE
The cytotoxicities of Gd-DOTA-LAE, Gadobutrol (Gadovist), and free Gd 3+ were evaluated for 24 h using the (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) MTT assay. Chang liver cells were seeded into 96 well plates and incubated under 5% CO 2 at 37°C. Gd-DOTA-LAE, Gadovist, and free Gd 3+ in 100 μl of SF medium were added to each well in a concentration-and time-dependent manners. To measure cytotoxicities by time-dependent manners, 0.1 mM concentration of Gd-DOTA-LAE, Gadobutrol (Gadovist), and free Gd 3+ were treated at each time point. Then 10 μl of MTT solution (0.5 mg/ml) was added to each well and incubated for additional 4 h. Media containing MTT was removed and the blue formazan crystals trapped in living cells were dissolved in DMSO (100 μl). The absorbance of formazan crystal in the wells was measured using a microplate reader at 570 nm.

Removal of free gadolinium ion
One gram of DOTA-LBE was dissolved in 100 ml of D.W and 5 g of chelex ®100 resin was added into the solution to remove unreacted Gadolinium. Solution was stirred gently for 1 h and supernatant was filtered and lyophilized. Concentration of free gadolinium ion was determined using xylenol orange method [25]. The ratio of absorbance at 573 and 433 nm is proportional to the free Gd 3+ concentration. The

Statistical analysis
Data was represented as mean ± SD for all the groups. The statistical analysis was performed by Student's t-test and p < 0.01 was considered statistically significant.

Comparison of in vitro paramagnetic properties between Gd-DOTA-LAE and Gadovist
Comparison of in vitro paramagnetic properties between Gd-DOTA-LAE and Gadovist was estimated using 4.7 MR scanner. Gadovist was used for comparison study as a macrocyclic chelate-based CA. For CA, R1 and R2 relaxivities of compound are major factor to indicate the contrast efficacy of T1 and T2-weighted CAs. For T1-weighted CAs, shortening T1 relaxation time is important to increase the contrast effect. Therefore, T1-weighted CAs have high R 1 ratio and low R 2 /R 1 ratio. On the other hand, T2-weighted CAs have high R2 ratio and relatively high R2/R1 ratio compared to T1-weighted CAs. As shown in Fig. 3a, contrast intensities of phantom images were obtained at various Gd 3+ ion concentration. GD-DOTA-LAE showed similar contrast intensity with Gadovist (Fig. 3a) and Gd-DOTA-LAE showed the relatively high R 1 value and similar R 2 value compare to Gadovist (Fig. 3b & c). In addition, R 2 /R 1 ratio of Gd-DOTA-LAE (0.84) is relatively lower than that of Gadovist (0.89). These results indicate that Gd-DOTA-LAE may be used for potential CA for T1-weighted MR imaging.

Cytotoxicity studies of Gd-DOTA-LAE
Gadolinium, as a free ion, is causing serious disease known as nephrogenic systemic fibrosis (NSF) to patients with low kidney function [21]. Therefore, we evaluated the cytotoxicity of Gd-DOTA-LAE, free Gd 3+ ion and Gadovist on chang liver cell line via MTT assay. Gd-DOTA-LAE and Gadovist showed no serious toxic effect at high dose. Whereas, free Gd 3+ ion showed a serious toxic effect at high dose. In addition, Gd-DOTA-LAE and Gadovist showed no serious toxic effect in time-dependent manner. We supposed that DOTA-LAE strongly bind to Gd 3+ ions and minimizing cell interaction with Gd 3+ . Thereby DOTA-LAE did not show the cytotoxicity at high concentration or long periods of time, whereas, free Gd 3+ showed high cytotoxicity (Fig. 4).

Concentration of free gadolinium ion and bounded gadolinium
Content of free gadolinium is determined using xylenol orange method (Fig. 5a & b). The ratio of absorbance at 573 nm and 433 nm is proportional to the free Gd 3+ concentration (Fig. 5c). In 1 g of Gd-DOTA-LAE, 7 μg of free gadolinium (0.007%) contents were determined using this method (Fig. 5d). Xylenol assay and ICP-MS results indicated that most of Gd 3+ ions are tightly chelated with DOTA-LAE and free Gd 3+ ions were successfully removed by cation exchange column.

In vivo MR imaging
To evaluate the cancer diagnosis efficacy, 100 μl of Gd-DOTA-LAE (0.1 mmol/kg) and Gadovist (0.1 mmol/ kg) were injected into the lateral tail vein of tumor bearing male BALB/c mice and MR image was obtained by 4.7 T animal MR scanner (Fig. 6a). The contrast enhancement of region of interest (ROI) was calculated with the following equation. Contrast enhancement = (ROI post-injection /ROI pre-injection ) × 100. In the case of Gd-DOTA-LAE, normal region showed 20% enhanced T1 contrast within 20 min, the tumor tissue showed 53% enhanced T1 contrast effect within 15 min (Fig. 6b). In the case of Gadovist, normal region showed 20% enhanced T1 contrast within 10 min, the tumor tissue showed 42% enhanced T1 contrast effect within 15 min (Fig. 6b). The contrast enhancement of tumor tissue compared to normal tissue was calculated by following equation. Contrast enhancement efficacy = [%, (Tumor post-injection /Tumor pre-injection ) × 100-(normal tissue  (Fig. 6b). Specific accumulation of Gd-DOTA-LAE was observed in cancer region. Therefore, Gd-DOTA-LAE can be used as a CA for cancer diagnosis.

Discussion
For several decades, various attempts have been applied for accurate diagnosis of cancer. Magnetic resonance imaging is one of the most significant technologies for diagnosis. MR imaging technology helps surgeon make an accurate diagnosis and surgery. In this study, we designed DOTA conjugated lactobionic acid as a tumor diagnosis CA. Gd-DOTA-LAE and Gadovist showed 29 and 26% enhanced contrast intensity in tumor tissues compared to normal tissues within 20 min post injection, respectively. These enhanced contrast effects are explained by the water proton exchange rate. As is known to all, T1-weighted contrast effect is obtained by shortening the spin-lattice relaxation time of water protons around Gd 3+ . We used a lactobionic acid as biocompatible organic acid conjugated with DOTA. In addition, LBA has many hydroxyl groups which improve the water proton density around Gd 3+ ion by hydrogen bonding, thus it provides the enhanced water proton exchange rate. Therefore, we obtained an enhanced contrast intensity with Gd-DOTA-LAE. Furthermore, there have been reports of nephrogenic systemic fibrosis (NSF) associated with the use of linear chelator-based CA in the past decade. For this reason, there are increasing demands for macrocyclic chelator-based CAs. In this respect, Gd-DOTA-LAE could be used for T1-weighted MR CA in clinical application.

Conclusion
In conclusion, this study aims to investigate new macrocyclic chelator-based CA. This CA was synthesized DOTA with primary amine modified lactobionic acid using DCC-NHS coupling reaction. In vitro paramagnetic properties showed relatively enhanced T1 contrast effect compared to conventional macrocyclic CA.
In addition, Gd-DOTA-LAE showed 29% enhanced contrast intensity at tumor sites compared to normal tissues within 20 min post injection. These results support that Gd-DOTA-LAE can be used for clinical application for MR imaging.