Article pubs.acs.org/Biomac
Functional Hyperbranched Polylysine as Potential Contrast Agent Probes for Magnetic Resonance Imaging Guangyue Zu,†,‡,§ Min Liu,†,§ Kunchi Zhang,§ Shanni Hong,§ Jingjin Dong,§ Yi Cao,§ Bin Jiang,§ Liqiang Luo,‡ and Renjun Pei*,§ ‡
Department of Chemistry, College of Sciences, Shanghai University, Shanghai 200444, China Key Laboratory of Nano-Bio Interface, Division of Nanobiomedicine, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, China
§
S Supporting Information *
ABSTRACT: Researchers have never stopped questing contrast agents with high resolution and safety to overcome the drawbacks of small-molecule contrast agents in clinic. Herein, we reported the synthesis of gadolinium-based hyperbranched polylysine (HBPLL-DTPA-Gd), which was prepared by thermal polymerization of L-lysine via onestep polycondensation. After conjugating with folic acid, its potential application as MRI contrast agent was then evaluated. This contrast agent had no obvious cytotoxicity as verified by WST assay and H&E analysis. Compared to Gd(III)-diethylenetriaminepentaacetic acid (Gd-DTPA) (r1 = 4.3 mM−1 s−1), the FA-HBPLL-DTPA-Gd exhibited much higher longitudinal relaxivity value (r1 = 13.44 mM−1 s−1), up to 3 times higher than Gd-DTPA. The FA-HBPLL-DTPA-Gd showed significant signal intensity enhancement in the tumor region at various time points and provided a long time window for MR examination. The results illustrate that FA-HBPLL-DTPA-Gd will be a potential candidate for tumor-targeted MRI.
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for drug release, gene delivery, and MRI CAs.24−29 For example, poly(propyleneimine) or poly(amidoamine) dendrimers with high generations have been designed as MRI CAs, which can enhance the longitudinal relaxivities and prolong the half-life in the blood.24,25 However, the general procedure for preparation of dendrimers was usually complicated and wasted lots of time with low yield. Hyperbranched polymers then emerged as an alternative to dendrimers. Compared to dendrimers, hyperbranched polymers can be prepared through a one-step polymerization reaction with a high yield. So far, more and more hyperbranched polymers have been investigated as CAs, drug and gene deliver vesicles.30−35 Liu and co-workers prepared a Gd3+ based hyperbranched polyglycerol as a MRI CA and had longitudinal relaxivity value (r1) as high as 6.12 mM−1 s−1 (7 T, 25 °C).31 Sideratou and co-workers employed hyperbranched polyester as a biodegradable MRI CA with longitudinal relaxivity of 12.3 mM−1 s−1 (2.35 T, 25 °C).33 These results illustrated that hyperbranched Gd(III) complexes could be potential contrast agent for magnetic resonance imaging. However, the hyperbranched polylysine used as contrast agents have never been reported. In this study, the hyperbranched polylysine was prepared by thermal polymerization of L-lysine via one-step polycondensation, followed by conjugation of DTPA-Gd and targeting molecule. The properties of this targeted macromolecular contrast agent (mCA) were then evaluated both in vitro and in vivo, including relaxivity, toxicity,
INTRODUCTION In the past few decades, compared to X-ray, micro CT and ultrasound imaging techniques, magnetic resonance imaging (MRI) technique has attracted more and more attention in clinical diagnosis of cancer with high spatial resolution and noninvasive, especially used for early diagnosis of cancer.1−5 However, the routinely MRI detection cannot always distinguish the pathological tissue and normal tissue clearly in clinic. According to statistics, nearly 50% of MRI detection needs the aid of contrast agents (CAs) to improve the contrast resolution.6−8 So far, the majority of MRI CAs used in clinic is gadoliniumbased small molecules, which can shorten the relaxation time and provide a positive signal image. Over 10 million MRI scans need CAs to enhance the contrast between the pathological tissues and normal tissues every year due to the high paramagnetism and stability of Gd chelates.9−12 However, the commercial CAs usually consist of individual Gd3+ chelated with a low molecular weight acyclic or cyclic ligand. These small-molecule CAs possess low relaxivity, nonspecificity, and short blood residence time, which have been major limitations for their clinical applications.13−16 Currently, various platforms including polymers, dendrimers, liposomes, vesicles and hybrid or inorganic nanoparticles have been developed as contrast agents, and these multifunctional CAs show excellent properties.17−23 Among these carriers, dendrimer, a macromolecule with precise molecular structure, perfectly monodisperse and lots of chemical groups on the surface, can be conveniently functionalized as different platform © XXXX American Chemical Society
Received: April 28, 2016
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DOI: 10.1021/acs.biomac.6b00605 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
groups. Briefly, HBPLL-DTPA-Gd obtained above was dissolved in 10 mL sodium bicarbonate buffer, then, 5 mL DMSO solution of NHSfolate (10 mg/mL) was added to this solution and stirred for 6 h. Afterward, 1 mL of DMSO solution of FITC (1 mg/mL) was added into the above solution and stirred for another 30 min. The unreacted NHSfolate and FITC were removed through Sephadex G25 and the final products were obtained by lyophilizing. The absorbance of FA-HBPLLDTPA-Gd at 286 and 356 nm demonstrated that folic acid was successfully conjugated onto HBPLL-DTPA-Gd. In addition, the fluorescence intensity of FITC labeled FA-HBPLL-DTPA-Gd at 520 nm indicated that FITC was conjugated onto FA-HBPLL-DTPA-Gd successfully. Relaxivity Measurement. Relaxivity measurements and T1weighted MR images were performed on 0.5 T NMR-analyzer (GYPNMR-10) at 35 °C. FA-HBPLL-DTPA-Gd was diluted into different concentrations of Gd3+, and Gd-DTPA was selected as control. The T1weighted images were acquired with spin echo acquisition (TE = 8.6 ms, TR = 100 ms). The r1 value was calculated from the curve fitting of 1/T1 (s−1) versus the Gd3+ concentration (mM). Toxicity Assay. WST assay was carried out to evaluate the cytotoxicity of FA-HBPLL-DTPA-Gd against KB and 293T cells. KB cells or 293T cells were separately seeded into 96-well plates at a density of 8000 cells per well. After 24 h of incubation, the medium was replaced with 100 μL of fresh medium containing FA-HBPLL-DTPA-Gd and Gd-DTPA at various Gd3+ concentration respectively, and incubated for another 24 h. Afterward, the medium was aspirated off and replaced with 100 μL of fresh medium and 10 μL of WST solution, the cells were then incubated for another 2 h. The absorbance at 450 nm was measured with Biotek Cytation 3. The relative cell viability was calculated according to the following equation:
and MR imaging. In this way, this promising mCA, with improved efficiency, high resolution, and excellent biocompatibility, will be a potential MRI CA in clinic.
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EXPERIMENTAL SECTION
Materials. Gadolinium chloride hexahydrate (GdCl3·6H2O), folic acid, 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), fluorescein isothiocyanate (FITC), and RPMI-1640 medium without folic acid were obtained from Sigma-Aldrich. Diethylenetriaminepentacetate acid (DTPA) was obtained from Tokyo Chemical industry Co. Ltd. L-Lysine hydrochloride was obtained from J&KCHEMICA. Fetal bovine serum (FBS) was obtained from GIBCO. N,N,N,N-Tetramethylethylenediamine (TEMED) and all the other reagents were acquired from domestic suppliers and used as received. Instrument. 1H NMR spectra were recorded on a Bruker digital NMR spectrometer at 400 MHz. The Gd3+ concentration was measured by an inductively coupled plasma atomic emission spectrometer (ICPAES). WST assay was measured with Biotek Cytation 3. The longitudinal relaxation time was detected on 0.5 T NMR analyzer (GY-PNMR-10). MR imaging was performed on 1.5 T micro-MRI system. Cell Culture. Human nasopharyngeal epidermoid carcinoma cells (KB cell) were cultured in RPMI 1640 without folic acid, supplemented with 10% fetal bovine serum (FBS), 100 units/mL of streptomycin and penicillin. The cells were maintained at 37 °C and 5% CO2 in a humidified incubator. Synthesis of Hyperbranched Polylysine. Hyperbranched polylysine (HBPLL) was synthesized according to the previous literatures with a minor revision.34,36 In brief, L-lysine hydrochloride (10.958 g, 60.0 mmol) and KOH (3.030 g, 54.1 mmol) were mixed thoroughly in a mortar until a consistency pulp formed, and then this mixture was added into a 500 mL three-necked flask. After adding 50 mL of paraffin oil, the mixture was maintained at 150 °C for 48 h. The reaction should be under nitrogen atmosphere during the whole polymerization procedure which facilitate the escape of water formed in the polymerization. Then, the mixture was cooled down to room temperature. After removing the paraffin oil, the residual was then washed thrice with petroleum ether, followed by evaporating the excess petroleum ether. The obtained brownish solid product was then dissolved in methanol, and the KCl byproduct was removed through filtration. Finally, tetrahydrofuran was poured into the filtrate and yellow-brown precipitation appeared. The precipitation was collected and dissolved in deionized water. After adjusting the pH value to 5, the solution was dialysized (MWCO = 3500 Da) against deionized water and then lyophilized. A total of 2.5 g yellow-brown solid was obtained with a yield of 29%, and then the product was preserved at −20 °C. 1H NMR (400 MHz, D2O): δ 4.21 (br, COCH(R)NH), 3.81 (br, COCH(R)NH2), 3.62 (br, COCH(R)NH2), 3.21 (m, CH2NH), 2.99 (m, CH2NH2), 1.81−1.36 (br, CH2). The weight-average molecular weight of HBPLL was measured to be 9.6 × 103 with the PDI of 1.5. Synthesis of Gd-DTPA-Modified HBPLL. HBPLL (460 mg) and DTPA (3 g) were dissolved in 10 mL of deionized water. Then, the pH value of the solution was adjusted to 6 with TEMED, and 240 mg EDC was added into the mixture. After stirring for 6 h at room temperature, the crude product was purified through Sephadex G25 to remove the unreacted DTPA and then lyophilized. 1H NMR (400 MHz, D2O): δ 4.19 (br, COCH(R)NH), 3.84 (s, CH2), 3.62 (br, COCH(R)NH2), 3.21 (m, CH2NH), 3.07 (br, CH2), 2.99 (m, CH2NH2), 1.81−1.36 (br, CH2). The weight-average molecular weight of HBPLL-DTPA was measured to be 2.7 × 104 with the PDI of 3.3. The purified HBPLL-DTPA (600 mg) was dissolved in 20 mL of water. GdCl3·6H2O (600 mg) was dissolved in another 5 mL of water, then this solution was added dropwise to the solution of HBPLL-DTPA and maintained the pH at 6 with 1 M NaOH, followed by stirring at 42 °C for 6 h. Afterward, the product was purified through Sephadex G25 to remove the unreacted GdCl3 and then was lyophilized. Folic Acid and FITC Conjugation. Folic acid and FITC were conjugated to the HBPLL-DTPA-Gd through its surface residue amine
relative cell viability(%) = 100 × (ODsample − ODbackground )/(ODcontrol − ODbackground ) Hematoxylin-Eosin (HE) staining was conducted to further investigate the in vivo toxicity of FA-HBPLL-DTPA-Gd. Female athymic nude mice (4 weeks old, 20 g) were purchased from Nanjing Sikerui Biological Technology Co. LTD and acclimated for 1 week. Mice were raised with standard pellet diet and pure water. All animal experiments were performed in compliance with the relevant laws and institutional guidelines. The mice were tail-vein injected with 200 μL of FA-HBPLL-DTPA-Gd solution at the Gd3+ concentrations of 0.1 mmol/kg and 0.3 mmol/kg, respectively. Meanwhile, 200 μL of physiological saline was also tail-vein injected as the control group. After raising for 2 days, the mice were sacrificed to collect organs, including heart, liver, spleen, lung, and kidney for histology analysis. Cellular Uptake of FA-HBPLL-DTPA-Gd. KB cells were seeded into a 24-well plate with glass slices at a density of 50000 cells/well. When the cells reached 60% confluency, the first well was treated with physiological saline, the second well was treated with FITC-labeled FAHBPLL-DTPA-Gd at a Gd3+ concentration of 2 mM, KB cells in the third well were pretreated with 5 mM free folic acid, followed by the same treatment as the second well. Following incubation for 1 h, the KB cells were washed with PBS thrice. Afterward, the cells were fixed with 4% paraformaldehyde at 37 °C for 30 min, followed by washing with PBS thrice. Finally, the cell nuclei were stained with DAPI at 37 °C for 20 min. The cells were washed with PBS thrice before performing on the laser scanning confocal microscope. Tumor Implantation. Adult female nude mice were obtained from Nanjing sikerui Biological Technology Co. LTD at 4-weeks of age and acclimated for 1 week. Then, to induce a tumor, 100 μL of PBS containing 2 × 106 KB cells was injected subcutaneously into the armpit of nude mice. In Vivo MR Imaging. At about 8 days after tumor implantation, the tumor size reached around 5 mm. Then the tumor-bearing mice were anesthetized by 100 μL of 20% urethane solution through intraperitoneal injection. All the mice were prescanned and then treated with FA-HBPLL-DTPA-Gd, HBPLL-DTPA-Gd, and Gd-DTPA, respectively. In all cases, 200 μL samples in physiological saline were tail-vein injected and the Gd dose was maintained at 0.1 mmol/kg for each B
DOI: 10.1021/acs.biomac.6b00605 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules Scheme 1. Synthesis Scheme of Gd(III)-Based Hyperbranched Polylysine
sample. Finally, the mice were transferred to an animal handing system, and then inserted into the 1.5 T microimaging system (35 °C). MR images were acquired at 1, 3, 5, 8, and 24 h after injection of contrast agents. Spin echo T1-weigthed imaging was performed with the following parameters: TR/TE = 100/14.26 ms, matrix = 512 × 256 mm, FOV = 80 × 45 mm, slice thickness = 0.8 mm. Gd Retention in Mice Tissue. The Gd retention of FA-HBPLLDTPA-Gd was evaluated on female athymic nude mice. After treated with FA-HBPLL-DTPA-Gd at the Gd dose of 0.1 mmol/kg and raised for another 10 days, the mice were sacrificed to collect the major organs and tissues, including heart, lung, liver, spleen, kidney, and muscle. Then, the samples were completely digested in 5 mL of a solution of nitric acid through heating, followed by fixing to 10 mL. After centrifuging at 13000 rpm for 5 min, the Gd3+ concentration was measured by ICP-AES. Afterward, the averaged Gd content in each organ or tissue was calculated and converted to percentage of injected dose (ID) per organ/tissue. Statistical Analysis. All the data analyses were performed using OriginPro 8.5 program, and the results are presented as mean ± standard deviation (SD). p < 0.05 was considered statistically significant.
will result in gelation. In addition, we used paraffin oil as a thermally conductive solvent instead of using catalyst. After dialyzing against deionized water and filtration, the small molecule and cross-linked product were removed. Afterward, DTPA was conjugated onto the HBPLL through EDC chemistry (as shown in Scheme 1). The characteristic peaks appearing at 3.82, 3.07 ppm indicated the successful conjugation of DTPA onto HBPLL. In addition, after conjugation of DTPA, the zeta potential changed from +32 to −25.5 mV, which further confirmed that DTPA was conjugated onto HBPLL successfully. Longitudinal Relaxation Rates. Spin−lattice (T1) relaxation time was performed on 0.5 T MRI scanner through inversion−recovery method. As shown in Figure 1a, the r1 of GdDTPA calculated from the slope was 4.3 mM−1 s−1, which is in good agreement with literature.11 Meanwhile, the r1 of FAHBPLL-DTPA-Gd was found to be 13.44 mM−1 s−1, up to 3× higher than that of Gd-DTPA. In addition, T1-weighted MR images of FA-HBPLL-DTPA-Gd with various Gd3+ concentrations were performed to further confirm the results above, and Gd-DTPA and H2O were chosen as the controls. As shown in Figure 1b, compared to Gd-DTPA and H2O, FA-HBPLL-DTPAGd produced much brighter images at each Gd3+ concentration. Attaching Gd(III) chelate to macromolecule can obviously slow down the rotational motion of the complex, which can be explained through Bloembergen-Solomon-Morgan theory. The relaxivities of gadolinium chelate was closely related to two important factors: the residence lifetime of the coordinated water molecules (τM) and the rotational correlation time (τR).14 In this study, a mCA based on hyperbranched polymer was prepared. Compared to the small-molecule Gd-DTPA, the larger molecular size and numerous peripheral chemical groups on the surface not only enhance the rotational correlation time (τR), but also
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RESULTS AND DISCUSSION Synthesis and Characterization of FA-HBPLL-DTPAGd. The purpose of this study was to synthesize and characterize a targeted mCA based on hyperbranched polylysine. The thermal polymerization of L-lysine has been proven as a viable route for the preparation of hyperbranched polylysine.36 Herein, we took the one-step method to synthesize hyperbranched polylysine by thermal condensation polymerization of L-lysine. The synthesis procedure was shown in Scheme 1. During the synthesis process, there are two important factors which affect the yield of thermal polymerization. One is the amount of KOH, and the other is the reaction time and temperature. The excess KOH and the reaction time over 48 h or the reaction temperature higher than 150 °C C
DOI: 10.1021/acs.biomac.6b00605 Biomacromolecules XXXX, XXX, XXX−XXX
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cytotoxicity of FA-HBPLL-DTPA-Gd against 293T cells was also evaluated, and the viability of 293T cells remained nearly 100% even when the Gd3+ concentration went up to 5 mM (Figure S1). It seemed that FA-HBPLL-DTPA-Gd showed a slight higher toxicity against KB cells in comparison with 293T cells. The possible reason may be attributed to the receptor-mediated interaction between FA-HBPLL-DTPA-Gd and KB cells. Histological toxicity assessment was conducted to further evaluate the in vivo toxicity of this mCA. The results are shown in Figure 3. For the FA-HBPLL-DTPA-Gd treatment groups, no signs of inflammatory response were observed in the hepatocytes, no pulmonary fibrosis was observed in the lung samples, and no necrosis was found in the test samples. The kidney morphology was also hardly altered. Generally, the histological changes were negligible in all investigated organs, and nearly all of the tissues were found normal, preserving the same structures compared to the untreated mice. Cellular Uptake of FA-HBPLL-DTPA-Gd. KB cell, a wellknown cell line which overexpresses the folate recptor (FR), has been often used to evaluate the targeting capability of folic acidlabeled materials. In the top row of Figure 4, KB cells were treated with pure medium, and no fluorescent was observed. In the third row of Figure 4, KB cells were incubated with FITClabeled FA-HBPLL-DTPA-Gd, and all the KB cells exhibited a bright fluorescent signal in comparison with the cells treated with pure medium. In order to further verify whether the uptake of FA-HBPLL-DTPA-Gd was mediated through FR-dependent targeting, we used free folic acid to inhibit the uptake of FAHBPLL-DTPA-Gd, and then, fluorescence was dramatically reduced (second row of Figure 4). These results suggested that the free folic acid could specifically inhibit the combination between FR-positive cells and FA-HBPLL-DTPA-Gd, and thus reducing the fluorescence. To further confirm the targeting capability of the FA-HBPLLDTPA-Gd, in vitro MRI study was carried out for FA-HBPLLDTPA-Gd, HBPLL-DTPA-Gd, and Gd-DTPA in KB cells. Figure S2 showed the T1-weighted cellular MR images of the different samples and the Gd3+ concentration was maintained at 0.2 mM. Among all the cellular MR images, the image produced by Gd-DTPA was slightly brighter than that of untreated cells. Meanwhile, the image produced by HBPLL-DTPA-Gd was brighter than that of Gd-DTPA. However, the image produced by FA-HBPLL-DTPA-Gd was the brightest in all these images. The signal brightness produced by these contrast agents was on the order of FA-HBPLL-DTPA-Gd > HBPLL-DTPA-Gd > GdDTPA. These results further confirm that the target molecules endow the mCA with remarkable targeting specificity, which is in good agreement with the fluorescent image study. In Vivo MRI Contrast Enhancement. To further investigate whether FA-HBPLL-DTPA-Gd can enhance the sensitivity and accuracy of in vivo MRI, tumor-bearing mice were tail-vein injected with FA-HBPLL-DTPA-Gd, HBPLL-DTPAGd, and Gd-DTPA, respectively. The Gd3+ concentration for each sample was maintained at 0.1 mmol/kg, and T1-weighted MR images of mice were then acquired at various periods, including 1, 3, 5, 8, and 24 h postinjection. The results were shown in Figure 5, and the relevant signal brightness were on the order of FA-HBPLL-DTPA-Gd > HBPLL-DTPA-Gd > GdDTPA. During the whole experiment, the acquisition parameters were kept the same in order to make the images comparable. The intensity of the tumor regions in the T1-weighted images was then quantified at various time points after injection of CAs. As
Figure 1. (a) Longitudinal relaxation rate (1/T1) of FA-HBPLL-DTPAGd and Gd-DTPA, and (b) T1-weighted MR images of FA-HBPLLDTPA-Gd (A) and Gd-DTPA (B) at different Gd3+ concentrations, H2O was used as control. All these experiments were performed on a 0.5 T NMR analyzer at 35 °C.
improve the loading density of Gd-DTPA, which would increase the relaxivity. Furthermore, hyperbranched polymers own a rigid structure in comparison with the linear polymers so that they can keep Gd3+ on the surface of the materials to guarantee a high water exchange rate, which would result in high T1 relaxivity. Toxicity Assay. Although macromolecular contrast agents possess high spatial resolution and long imaging window, the toxic side effect is a key factor which limits their clinical utility. WST assay was performed with KB cells to investigate the cytotoxicity of FA-HBPLL-DTPA-Gd. The results were shown in Figure 2. The cytotoxicity of FA-HBPLL-DTPA-Gd against KB cells was found to be negligible when the Gd3+ concentration below 1.75 mM, and the viability remained 90% even when the Gd3+ concentration went up to 2.5 mM. Furthermore, the
Figure 2. WST assay of FA-HBPLL-DTPA-Gd and Gd-DTPA in KB cells. D
DOI: 10.1021/acs.biomac.6b00605 Biomacromolecules XXXX, XXX, XXX−XXX
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Figure 3. H&E-stained tissue sections from mice injected with FA-HBPLL-DTPA-Gd at different dose of Gd3+ (0.1 mmol/kg, 0.3 mmol/kg) 2 d postinjection and mice injected with physiological saline were used as control.
Figure 4. Cellular uptake of fluorescently labeled FA-HBPLL-DTPA-Gd. Top row: fluorescent images of untreated KB cells; Second row: fluorescent images of KB cells treated with FA-HBPLL-DTPA-Gd in the presence of free folic acid (5 mM); Third row: fluorescent images of KB cells treated with FA-HBPLL-DTPA-Gd.
Figure 5. MR images of tumor-bearing mice at various time points (1, 3, 5, 8, and 24 h). Mice were intravenously injected with HBPLL-DTPA-Gd (top row), FA- HBPLL-DTPA-Gd (second row), and Gd-DTPA (third row), and the yellow dot circle shows the location of tumor.
for the control group (third row of Figure 5), nearly no enhancement in the tumor region was observed in the images at all the time points. The main reason may be ascribed to the small molecular weight of Gd-DTPA, which excreted from the body rapidly and could not offer a time window long enough for the
MRI scanning. However, for the HBPLL-DTPA-Gd group (top row of Figure 5), the periphery between the tumor and normal tissue can be seen clearly, up to 1.5× to 2× enhancement. In addition, an obvious enhancement (2× enhancement) could be seen even at 24 h postinjection, which illustrated that HBPLLE
DOI: 10.1021/acs.biomac.6b00605 Biomacromolecules XXXX, XXX, XXX−XXX
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The Gd3+ retentions in major organs and tissues were investigated 10 days after injection of FA-HBPLL-DTPA-Gd, as shown in Figure 7. Nearly no Gd3+ was detected in the lung
DTPA-Gd can improve the contrast between the tumor region and normal tissues and also provide a long imaging window for MR examination compared to Gd-DTPA. Furthermore, a significant contrast enhancement in the T1-weighted images produced by FA-HBPLL-DTPA-Gd (second row of Figure 5) was clearly observed at each time points in comparison with HBPLL-DTPA-Gd group. The FA-HBPLL-DTPA-Gd can markedly increase the contrast between the tumor site and the normal tissues, up to 2.0 to 3.2× enhancement, and the consequently clear margin of solid tumor is important for anatomy diagnosis. Combining the results obtained in Figures 5 and 6, we can find that both HBPLL-DTPA-Gd and FA-HBPLL-DTPA-Gd can
Figure 7. Retention of Gd3+ in the main organs and tissues of mice 10 days after intravenous injection of FA-HBPLL-DTPA-Gd at the dose of 0.1 mmol Gd/kg.
and muscle, and the Gd % retented in the organs of heart, liver, spleen, and kidney were 0.00320%, 0.121%, 0.0315%, and 0.137%, respectively, which illustrated that little Gd3+ was retained in these organs. The data reported in our article was similar to that of small-molecule contrast agent (Gd-(DTPABMA)), while much lower than that of mCA (PAMAM-G6-(GdDO3A); liver > 5%, muscle > 3%, spleen > 1%, kidney > 1%).37,38 These results confirmed that the mCA based on hyperbranched polylysine with long imaging window and high signal enhancement can be cleared rapidly with little Gd3+ retention in the main tissues and organs.
Figure 6. Quantitative analysis of MR images. The average relative enhanced MR signal intensity of FA-HBPLL-DTPA-Gd, HBPLLDTPA-Gd, and Gd-DTPA at different time points.
produce obviously contrast images between the tumor region and the normal tissue. But, the enhancement ratios are different. The contrast images produced by FA-HBPLL-DTPA-Gd are much brighter than that of HBPLL-DTPA-Gd at each time point. Thus, it can be inferred that the folic acid truly endows the macromolecular contrast agent with positive targeting ability so that the FA-HBPLL-DTPA-Gd can accumulate more and faster in the tumor site. The signal enhancement produced by FAHBPLL-DTPA-Gd may be ascribed to several factors, such as the acquisition parameters, the dosage of CAs, the structure of macromolecular contrast agent and Gd accumulation locations. For this experiment, the dosage of CAs and the acquisition parameters were maintained the same. Thus, the molecular structure of mCA and the targeting molecule are supposed to play a dominant role in signal enhancement behaviors. Just as described above, the rigid structure and the high Gd loading density will affect the rotational correlation time and, thus, result in the increasing of relaxivities. In addition, the HBPLL-DTPAGd was labeled with folic acid, which can improve the accumulation in the positive tumors. All these factors make the FA-HBPLL-DTPA-Gd to present excellent imaging contrast. Thus, a contrast agent providing high resolution, tumor targeting specificity and long imaging window will be an excellent candidate for tumor-targeted MRI. Gd Tissue Retention. An ideal MRI CA should not only provide long imaging window for MR examination, but also be able to excrete from the body rapidly. Since the accumulated mCAs in body might be internalized by healthy cells and metabolize into toxic Gd3+, and resulted in systematic toxicity.20
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CONCLUSIONS In summary, we have successfully synthesized a hyperbranched polylysine based macromolecular contrast agent with excellent biocompatibility, which presented the improved r1 of 13.44 mM−1 s−1 in comparison with small molecule Gd-DTPA. The cellular uptake experiment confirmed that FA-HBPLL-DTPAGd was specifically mediated by the folate receptor. Finally, in vivo MRI study has shown that FA-HBPLL-DTPA-Gd possessed long imaging window and high signal enhancement than the nonspecific ones. Therefore, the FA-HBPLL-DTPA-Gd will be a potential contrast agent with high relaxivity and good biocompatibility and then provide a new platform for cancer diagnosis.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.6b00605. WST assay of FA-HBPLL-DTPA-Gd in 293T cells (Figure S1); T1 weighted images of KB cells (Figure S2; PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel.: 86-512-62872776. Author Contributions †
These authors contributed equally (G.Z. and M.L.).
F
DOI: 10.1021/acs.biomac.6b00605 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (21304106, 61571278), the CAS Hundred Talents program, and the CAS/SAFEA International Innovation Teams program.
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DOI: 10.1021/acs.biomac.6b00605 Biomacromolecules XXXX, XXX, XXX−XXX
