Intercellular imaging by a polyarginine derived cell penetrating peptide labeled magnetic resonance contrast agent, diethylenetriamine pentaacetic acid gadolinium

Abstract

Background 

The cellular plasma membrane represents a natural barrier to many exogenous molecules including magnetic resonance (MR) contrast agent. Cell penetrating peptide (CPP) is used to internalize proteins, peptides, and radionuclide. This study was undertaken to assess the value of a new intracellular MR contrast medium, CPP labeled diethylenetriamine pentaacetic acid gadolinium (Gd-DTPA) in molecular imaging in vitro.

Methods 

Fluorescein-5-isothiocyanate (FITC) and Gd-DTPA respectively labeled with CPP (FITC-CPP, Gd-DTPA-CPP) were synthesized by the solid-phase method. Human hepatic cancer cell line-HepG₂ was respectively stained by FITC-CPP and FITC to observe the uptake and intracellular distribution. HepG₂ was respectively incubated with 100 nmol/ml Gd-DTPA-CPP for 0, 10, 30, 60 minutes, and imaged by MR for studying the relationship between the incubation time and T1WI signal. The cytotoxicity to NIH3T3 fibroblasts cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay (MTT).

Results 

The molecular weights of CPP labeled imaging agents, which were determined by MALDI mass spectrometry (FITC-CPP MW=2163.34, Gd-DTPA-CPP MW=2285.99), were similar to the calculated molecular weights. Confocal microscopy suggested HepG₂ translocated FITC-CPP in cytoplasm and nucleus independent with the incubation temperature. MR images showed HepG₂ uptaken Gd-DTPA-CPP had a higher T1 weighted imaging (T₁WI) signal, and that the T1WI signal intensity was increasing in a time-dependent manner (r=0.972, P=0.001), while the signal intensity between the cells incubated by Gd-DTPA for 60 minutes and the controlled cells was not significantly different (P=0.225). By MTT, all concentrations from 50 nmol/ml to 200 nmol/ml had no significant (F=0.006, P=1.000) effect on cell viability of mouse NIH3T3 fibroblasts, compared with the control group.

Conclusions 

The newly constructed CPP based on polyarginine can translocate cells by carrying FITC and MR contrast agent Gd-DTPA, and the intracellular concentrations are readily detectable by MR imaging, suggesting a new way for MR molecular imaging.

Chin Med J 2007;120(1):50–55

Magnetic resonance contrast agents using paramagnetic metals such as gadolinium are routinely used in magnetic resonance imaging to shorten longitudinal relaxation times (T1) of water protons and enhance image contrast. Because these contrast agents are confined to extracellular spaces, lesions can only be contrasted when there is a significant damage to extracellular spaces. In other words, the chance for diagnosis and therapy of earlier intracellular changes can be missed. Cell penetrating peptide (CPP) or membrane permeable peptide, is able to translocate into cells with many kinds of cargoes. The purpose of this study was to detect a modified CPP and assess the value of a new intracellular MR contrast agent CPP labeled diethylenetriamine pentaacetic acid gadolinium (Gd-DTPA) in molecular imaging.

METHODS

Peptide synthesis

An L-CPP, LAGRRRRRRRRRK, containing nine arginines (R), one lysine (K), one alanine(A), leucine (L) and glycine (G), was manually prepared on a peptide-synthesis column by the solid-phase peptide synthesis method. The synthesis took place on Rink resin (Novabiochem, Germany) when 9-fluorenylmethyloxycarbonyl (Fmoc)-protected amino acids were used with standard benzotriazole-1–1-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate/N-hydroxybenzotriazo le coupling chemistry. The amino acids used standard side chain protecting groups, except lysine reside containing (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) ethyl Dde Sigma-Aldrich, USA). The ε-amino group of lysine (Novabiochem) was allowed to undergo orthogonal synthesis by selective deprotection of Dde when the peptide was attached to the resin.

Synthesis of Gd-DTPA-CPP

After the synthesis and final selective deprotection of Dde were completed, diethylenetriaminepentaacetic acid anhydride (DTPA, Sigma-Aldrich) was added to react with the ε-amino group of lysine of CPP-resin by dissolving 50 mg of DTPA in 1 ml of dimethyl sulfoxide (DMSO, Sigma-Aldrich) and 4 ml of dimethylformamide (DMF, Novabiochem). The DTPAa solution reacted with 100 mg of peptide resin, which had been washed previously with N, N′-diisopropyl ethylamine (Novabiochem) and dichloromethane (Novabiochem). The coupling of DTPA was allowed to proceed with stirring overnight by nitrogen gas at room temperature. The reaction was verified by a negative ninhydrin reaction. DMF was removed, and resin was washed twice each with DMF followed by acetonitrile and then dried under argon. The peptide was cleaved from resin, and the protecting groups were removed by stirring in trifluoroacetic acid (TFA) stock solution (TFA 10 ml, phenol 0.75 g, thioanisole 0.5 ml, deionized water 0.5 ml, and ethanedithiol 0.25 ml) at room temperature for 3 hours. The cleaved and deprotected peptide was then filtered through glass wool to separate from resin, precipitated in cold mixture of ether and ethanol (V/V=1:1), and pelleted by centrifugation (10 000 r/min ×15 minutes). Finally, the supernatant was removed. The pelleted peptide was dissolved in deionized water and reacted with gadolinium oxide (Gd₂O₃ Sinopharm Chemical Reagent Co, Ltd, China) overnight at 37°C, precipitated in cold mixture of ether and ethanol (V/V=1:1), pelleted by centrifugation and lyophilized under reduced pressure (FD-1/1E VACUUM FREEZE-DRY SYS China) to obtain dried crude product. The peptide was purified by C18 column (BONDAPAKTM C18 P/N84176 5 μm water, USA) on liquid chromatography (BioCAD 700E Perfusion Chromatography Work Station USA) and identified by Voyager MALDI-TOF Mass Spectrometry (Applied Biosystems USA). The white peptide was stored at -20°C.

Synthesis of FITC-CPP

The same method described above, fluorescein-5-isothiocyanate (FITC, Sigma-Aldrich) was added in 30 mg of peptide resin suspended in 30–35 μl of anhydrous triethylamine. The ε-amino group of lysine of CPP-resin coupling was allowed to proceed with stirring for 5 hours. DMF was removed, and resin was washed twice, each with DMF followed by acetonitrile and then dried in argon. The peptide was cleaved from resin, and the protecting groups were removed by stirring in TFA stock solution (TFA 10 ml, phenol 0.75 g, thioanisole 0.5 ml, deionized water 0.5 ml, and ethanedithiol 0.25 ml) at room temperature for 3 hours. The cleaved and deprotected peptide was then filtered through glass wool to separate from the resin, precipitated in cold mixture of ether and ethanol (V/V=1:1) and pelleted by centrifugation and lyophilized to obtain dried crude product. The peptide was purified by chromatography and identified by Voyager MALDI-TOF Mass Spectrometry. The light yellow peptide was stored at -20°C.

Cell culture

Human hepatic cancer cell line HepG₂, a donation from Dr. LEI Ying-feng (Department of Microbiology of Fourth Military Medical University, Xi'an, China) and mouse fibroblast NIH3T3 cell line, a donation from Professor HE Da-lin (Department of Urology, Xi'an Jiao Tong University, Xi'an, China), were kept in Dulbecco modified Eagle's medium (Gibco, Life Technologies, USA) containing 10% heat-inactivated foetal bovine serum (FBS, Hyclone-Pierce, USA), 2 mmol/L glutamine, 1 mmol/L sodium pyruvate, 0.1 mg/L fungizone in a 60 mm² cell culture plate containing 10% FBS at 37°C in a humidified 5% CO₂ incubater. The medium was renewed 3 times per week.

Fluorescein translocating experiments

For fluorescein uptake assay, FITC-CPP and FITC 100 nm were respectively diluted in 5 ml DMEM plus 10% FBS. Then the solution was added into HepG₂ on the glass slides in 60 mm² cell culture plates. After 10-minute incubation at 4°C, room temperature, and 37°C, the solution was abandoned, and cells were washed by phosphate balanced solution (PBS, 0.1 mol/L) for 5 times and 5 minutes each time. All of the cells were then mounted and analyzed by a confocal microscope (LM200 ARCUTUROUS USA).

MR contrast media uptake experiments

For Gd-DTPA uptake assay, 1.6 μmol of Gd-DTPA-CPP and Gd-DTPA (Amersham Health Co, USA) were respectively diluted in 16 ml DMEM to a concentration of 100 nmol/ml. Five groups were enrolled for MR imaging: groups 1 to 3 (5×10⁶ cell per group) were respectively incubated by a 4 ml Gd-DTPA-CPP solution for 10, 30 and 60 minutes. Group 4 was incubated by a 4 ml 100 nmol/ml Gd-DTPA/DMEM for 90 minutes, and group 5 comprising controlled cells was only incubated by DMEM. After incubation, the solution was abandoned, and cells were washed by 0.1 mol/L PBS (5 minutes × 5 times), trypsinized, and terminated by pelleting the cells for 1000 r/min × 10 minutes. The cell pellet was suspended in 400 μl 1.0% agarose in PBS in a 500 μl-Eppendof tube. The Eppendof tube was placed in a beaker containing 100 ml 1.0% agarose in PBS and imaged with the 1.5T superconducting magnet (SIGNA EXCITE 1.5T GE) using a 3-in surface coil. The imaging protocol consisted of transverse T1 weighted fast spin-echo: TR 440 ms, TE 13 ms, field of view (FOV) 12 mm ×12 mm, slice thickness 2 mm, gap 0.2 mm, matrix 288×192, NSA 4, and flip angle 90°. Subsequently, the T1WI signal intensities in the tube (Iin) and in 1% agarose (Iout) were measured.

Cell viability

To determine whether CPP-DTPA-Gd would be more toxic to normal cells, CPP-DTPA-Gd was diluted in DMEM plus 10% FBS at the concentrations of 50 nmol/ml, 100 nmol/ml, 150 nmol/ml, and 200 nmol/ml, respectively. NIH3T3 cells (1×10³/200 μl) were planted in a 96-well cell culture plate and incubated with 50, 100, 150, 200 nmol/ml of CPP-DTPA-Gd and the controlled medium for a period of 72 hours. Four hours at the end of the incubation, 20 μl of MTT solution was added into each well containing cells, and the plate was incubated in a CO2 incubator at 37°C for 4 hours. After removal of the medium, 200 μ1 of DMSO was added to each well and pipetted up and down to dissolve crystals. The plate was placed in an incubator at 37°C for 5 minutes. The plate reader (BMG Labtechnologies, Germany) was used to measure the absorbance at A550.

Images and statistical analysis

Triple samples were analyzed to see signal characteristics and statistical significance of differences of each group. The T1WI signal intensity of the five groups (Iin) and the intensity of 1% agarose (Iout) were determined by region of interest (ROI). The data of signal intensity were expressed as mean ± standard deviation (SD). With SPSS13.0, analysis of variance (ANOVA) and LSD test were employed for multiple comparisons, and the paired samples t test was used to compare the intensity between two groups. Group differences among the groups were considered to be significant if P<0.05.

RESULTS

Purification and identification

Crude peptide was purified by the C-18 column of reversed-phase high-pressure liquid chromatography (HPLC) at a flow rate of 1 ml/min (an eluent mixture of 0.1% TFA in 5% acetonitrile/95% water and 0.1% TFA in 80% acetonitrile/15% water). The main fraction (Gd-DTPA-CPP retention time was 15.13 minutes, FITC-CPP retention time was 15.84 minutes) collected was found to be CPP-DTPA-Gd and CPP-FITC by TOF-MS (CPP-DTPA-Gd M/Z = 2285.99, calculated value = 2285.78, Fig. 1A; CPP-FITC M/Z =2 163.34, calculated value = 2163.55, Fig. 1B).

Fig. 1. A:: MS analysis of CPP-DTPA-Dd. The first agent peak showing MW=2285.99. B: MS analysis of CPPs-FITC. The first agent peak showing MW=2163.34.

In vitroimaging

FITC labeled with CPP was used in cell fluorescent uptake experiments to determine the ability of penetration and subcellular localization. By confocal microscopy, HepG2 transported FITC-CPP into the cytoplasm and nucleus in 10 minutes (Fig. 2A-2D) but no fluorescein was observed in cells incubated for 60 minutes by FITC. Signals of HepG₂ incubated with Gd-TPA or Gd-DTPA-CPP were observed on 1.5T MRI. Gd-DTPA-CPP uptaken by HepG2 in 10 minutes and had a high T1WI signal intensity (Fig. 3), and the signal intensity between groups 4 and 5 appeared to be not different. Furthermore, half-quantitative analysis of ROI showed that the T1 signal intensity was increasing in a time-dependent manner (r=0.972, P<0.001). ANOVA revealed that the signal intensities in groups 1 to 5 were significantly different (Table 1) (P<0.001), but between group 4 and the control group (group 5) not different (Table 2) (P=0.225).

Fig. 2.: HepG₂ imaged on 10 × microscopy. (A) HepG₂ incubated by FITC-CPP at 4°C. (B) at room temperature. (C) and at 37°C. (D) imaged on confocal microscope.

Fig. 3.: T1WI of MR imaging: 1, 2 and 3 respectively represent group 1 (Ii1) incubated 100 nmol/ml Gd-DTPA-CPP for 10 minutes, group 2 (Ii2) incubated 100 nmol/ml Gd-DTPA-CPP for 30 minutes and group 3 (Ii3) for 60 minutes. 4 and 5 respectively represent the group 4 (Ii4) incubated 100 nmol/ml Gd-DTPA for 60 minutes and the controlled cells (Ii5). 6 represents the background signal (Io).

Table 1: Comparison of intensity between four groups incubated with Gd-DTPA-CPP (100 nmol/ml)

Table 2: Comparison of intensity between cell incubated with Gd-DTPA and control cell

MTT assay

MTT assay demonstrated that concentrations up to 200 nmol/ml exerted no significant effect on the viability of NIH3T3 cells (Table 3) (F=0.006, P=1.000).

Table 3: NIH3T3 cell viability (A550 nm, MTT assay)

DISCUSSION

Since molecular therapies are available for a range of diseases, there is an increasing demand for imaging strategies that provide information at the cellular and molecular level with excellent specificity for a given disease. But a significant problem in molecular imaging or therapies is the inadequate delivery of medium into the cell because of the cell wall barrier. The cellular plasma membrane represents a natural barrier to many exogeneous molecules including most MRI contrast agents. To overcome this barrier for therapeutic and diagnostic drugs, a variety of strategies have been proposed to ferry agents through the cellular membrane. These strategies include microinjection or electroporation, which could damage the membrane.^1,2 The noninvasive methods involve the use of liposomes carriers,³ which could liberate the entrapped drug into the cytoplasm.

During the last decade, several proteins and peptides have been found to traverse through the cellular membrane in a so-called process “protein transduction”, delivering their cargo molecules into the cytoplasm and/or nucleus. These proteins and peptides have been used for intracellular delivery of various cargoes with molecular weights several times greater than their own. This process of protein transduction was discovered first by Green⁴ and Frankel et al⁵ independently, who found that 86-mer trans-activating transcriptional activator (TAT) from HIV-1 was efficiently taken up by various cells, when added to the surrounding medium. Subsequently, this property of translocation was found in a series of short peptides.^6–10 More precisely, their ability to translocate across the plasma membranes is confined to short sequences of less than 20 amino acids, which are highly rich in basic residues. Such sequences are called membrane permeable peptide (MPP) or protein transduction domain (PTD) or cell penetrating peptide (CPP). According to a recent classification,¹¹ CPP can be divided into three classes: protein derived CPPs such as TAT, penetratin; model peptides such as MAP, polyarginine; and designed CPPs such as MPG, transportan. Although the mechanism¹¹ of internalization of CPP has not been clarified, many molecules carried by CPP can be translocated into the cell, including protein,^12,13 antibody,¹⁴ DNA,¹⁵ lipsomes,¹⁶ nonoparticles and imaging agents,¹⁷ which provide a new way to molecular imaging and therapy.

Contrast agents using paramagnetic metals such as gadolinium are routinely used in magnetic resonance imaging to shorten longitudinal relaxation times (T1) of water protons and enhance image contrast. But these agents are confined to the extracellular space. Bhroade et al¹⁸ demonstrated that HIV-TAT could translocate 111In-tetreazcyclododecane tetraacetic acid (DOTA) and Gd-DOTA into fresh lymphocytes from rat spleen and uptake with increased incubation time and concentration. Prantner et al¹⁹ showed TAT-DOTA-Gd can cross the cell membrane into lymphocytes, and the relaxivity is better than Gd-DTPA. Invasive intercellular imaging can be achieved by CPP labeled with MR contrast agents, but MR intercellular imaging by Gd-DTPA-CPP has not been found. By MALDI-TOF mass spectrometry, Burlina et al²⁰ suggested that nine-arginine-based CPP can more effectively transport than TAT and penetratin. Hence, according to the basic sequence of nine-arginine-based CPP, a new CPP was designed and synthesized by solid-phase methods in this study for two aims, one was to determine whether the new constructed short peptide still has the ability to translocate into the cell with cargo, and the other is to observe whether the new peptide carrying Gd-DTPA can be detected by MRI. Identified by mass spectrometry, the measured molecular weight approximates the calculated value, which means a successful synthesis. By confocal microscopic imaging, modified CPP also could be transported and distributed in the cytoplasm and nucleus at 4°C, room temperature, and 37°C. By MR imaging, Gd-DTPA can be translocated into the cell by CPP, and the uptake and intercellular T1 signal increase in a time-dependent manner. The relationship between the incubation concentration and T1WI signal needs a further study.

CPP, as a carrier without carrying the targeted cargo such as antibody, should be translocated into many kinds of cells. In this study, hepatoma cell HepG2 showed significantly uptake CPP, but the difference between hepatoma cells and normal hepatic cells needs a further study. As an intercellular MR probe, CPP may be used to track stem cells. Because of intercellular penetration, the cell viability affected by the MR probe can be measured by MTT. Compared with the control group in this study, four concentrations (50 nmol/ml, 100 nmol/ml, 150 nmol/ml and 200 nmol/ml) of Gd-DTPA-CPP presented no significant effect on the cell viability of NIH3T3 cells. But the toxicity to normal human cells needs to be further studied.

Compared with D-membrane permeable peptide,²⁰ L-membrane permeable peptide can also effectively transport Gd-DTPA into the cell for intercellular imaging, and there is no toxicity to NIH3T3 cells. It is indicated that this new intercellular MR contrast agent can be modified with the appropriate targeting functionality for high targeting selectivity.

Acknowledgment:

We are grateful to the help of Dr. XU Min and ZHANG Yi-li for cell MR imaging experiments and Professor DU Yu-xue for statistical analysis.