In vitro study of novel gadolinium-loaded liposomes guided by GBI-10 aptamer for promising tumor targeting and tumor diagnosis by magnetic resonance imaging

Abstract

Novel gadolinium-loaded liposomes guided by GBI-10 aptamer were developed and evaluated in vitro to enhance magnetic resonance imaging (MRI) diagnosis of tumor. Nontargeted gadolinium-loaded liposomes were achieved by incorporating amphipathic material, Gd (III) [N,N-bis-stearylamidomethyl-N′-amidomethyl] diethylenetriamine tetraacetic acid, into the liposome membrane using lipid film hydration method. GBI-10, as the targeting ligand, was then conjugated onto the liposome surface to get GBI-10-targeted gadolinium-loaded liposomes (GTLs). Both nontargeted gadolinium-loaded liposomes and GTLs displayed good dispersion stability, optimal size, and zeta potential for tumor targeting, as well as favorable imaging properties with enhanced relaxivity compared with a commercial MRI contrast agent (CA), gadopentetate dimeglumine. The use of GBI-10 aptamer in this liposomal system was intended to result in increased accumulation of gadolinium at the periphery of C6 glioma cells, where the targeting extracellular matrix protein tenascin-C is overexpressed. Increased cellular binding of GTLs to C6 cells was confirmed by confocal microscopy, flow cytometry, and MRI, demonstrating the promise of this novel delivery system as a carrier of MRI contrast agent for the diagnosis of tumor. These studies provide a new strategy furthering the development of nanomedicine for both diagnosis and therapy of tumor.

Keywords:

• magnetic resonance imaging
• gadolinium
• liposomes
• tenascin-C
• GBI-10 aptamer
• tumor targeting

Supplementary materials

Our research design was based on the schematic illustrations shown Figures S1–S3.

Synthesis of DTPA⋅BA

DTPA (2 g, 5 mmol), acetic anhydride (6 mL, 63.5 mmol), and pyridine (10 mL, 124.2 mmol) were placed in a 100 mL flask equipped with a condenser and a magnetic stirrer. The reaction was carried out at reflux for 4 hours. The resulting anhydride was filtered and washed thoroughly with acetic anhydride, CH₃CH₂OH, and dry diethyl ether. After drying under vacuum, the solid turned to cream-colored powder, 1.64 g. (Figure S4, compound DTPA⋅BA, yield: 90%).

¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 12.15 (s, COOH), 3.7 (s, 8H, 2× –CH₂COOCOCH₂−); 3.30 (s, 2H, –NCH₂COOH); 2.74 (t, J=6.65 Hz, 4H, 2× HOOCCH₂NCH₂CH₂–); 2.58 (t, J=6.65 Hz, 4H, 2× HOOCCH₂NCH₂CH₂–). ¹³C NMR (101 MHz, DMSO-d₆): δ (ppm) 172.95 (COOH); 166.27 (–COOCO–); 55.05 (NCH₂COOH); 53.08 (–CH₂COOCOCH₂–); 52.18 (HOOCCH₂NCH₂CH₂–); 51.19 (HOOCCH₂NCH₂CH₂–).

Synthesis of DTPA⋅BSA

A solution of octadecylamine (1.5 g, 5.57 mmol) dissolved in chloroform (300 mL) was added to a solution of DTPA BA (1 g, 0.560 mmol) previously dissolved in DMF (100 mL). The reaction was stirred at 40°C for 2 hours. The white precipitate formed was collected by filtration and washed with acetone and dried under vacuum overnight. The white solid was stirred in water at 80°C for 3 hours to dissolve any excess DTPA. The insoluble residue was filtered and dried under vacuum overnight. The white solid was then washed with boiling chloroform and stirred in chloroform for 2 hours and then filtered and dried 1 g. (Figure S4, compound DTPA⋅BSA, 40% yield).

¹H NMR (400 MHz, CF3-COOD at δ=11.50 ppm): δ (ppm) 4.38~3.90 (m, 14H, 3× HOOCCH₂N–, 2× –NH₂CONH–, 2× NCH₂CH₂N); 3.60 (s, 4H, 2× NCH₂CH₂N); 3.24~3.22 (d, 4H, J=8.0, 2× NHCH₂); 1.46 (s, 4H, 2× NHCH₂CH₂); 1.18 (s, 60H, 30× CH₂ alkyl chain); 0.73~0.75 (d, 6H, J=8.0, 2× CH₂CH₃). ¹³C NMR (101 MHz, CF₃-COOD at δ: 160.4~161.70 and 109.35~117.80 ppm): δ (ppm) 168.44 (CH₂COOH); 164.04 (NCH₂CONHCH₂CH₂); 56.4 (HOOCCH₂–); 55.0 (NCH₂ CONH–); 53.1 (NCH₂CH₂NCH₂CO– OH)CH₂CH₂N); 42.5 (NCH₂CH₂N(CH₂COOH)CH₂CH₂N); 40.44 (NHCH₂CH₂–); 30.84 (–CH₂CH₂ CH₃); 28.56 (chain CH₂’s); 25.63 (NHCH₂CH₂CH₂); 21.39 (–CH₂ CH₃); 11.74 (CH₃). HRMS (MALDI⁺) calculated for C₅₀H₉₄N₅O₈ m/z 895.7119, found 896.6882 (M+H)⁺

Synthesis of Gd⋅DTPA⋅BSA

A solution of GdCl₃⋅6H₂O (241 mg, 0.65 mmol) dissolved in water (5 mL) was added dropwise to a solution of DTPA⋅BSA (100 mg, 0.11 mmol) previously dissolved in water (100 mL). The reaction was stirred at 90°C for 3 hours (pH dropped to 3.5). The water was freeze-dried to yield a white powder 105 mg. (Figure S4, compound Gd⋅DTPA⋅BSA, 89% yield).

HRMS (MALDI⁺) calculated for C₅₀H₉₄GdN₅O₈ m/z 1,050.6343, found 1,073.5421 (M+Na)⁺, 1,089.5105 (M+K)⁺.

Synthesis of 6-(trifluoroacetamido)-1-hexanol

A solution of 1-amino-6-hexanol (2 g, 17.0 mmol) in 1-amino-6-hexanol in dichloromethane (50 mL) was slowly added to CF₃COOEt (4.2 mL, 34.9 mmol). The reaction mixture was stirred for 8 hours at room temperature. Purification was achieved by flash chromatography over silica gel using a mixture of CH₂Cl₂-MeOH (30:1) as eluent to afford a white powder 3.2 g. (Figure 1, compound 6-(trifluoroacetamido)-1-hexanol, 88% yield).

¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.36 (s, 1H, CONH); 4.33~4.30 (t, 1H, J=4, –CH₂OH); 3.38~3.33 (m, 2H, –CH₂OH); 3.18~3.13 (m, 2H, CONHCH₂–); 1.50~1.23 (m, 8H, –NHCH₂CH₂ CH₂CH₂CH₂ CH₂OH). ¹³C NMR (101 MHz, DMSO-d₆): δ (ppm) 156.7 (–CONH–); 117.89 (–CF₃); 61.06 (–CH₂OH); 39.34 (–CONHCH₂–); 32.84 (–CH₂CH₂OH); 28.72 (–CONHCH₂CH₂–); 26.52 (–CONHCH₂– CH₂CH₂–); 25.55 (–CH₂CH₂CH₂OH). HRMS (ESI⁻) calcd for C₈H₁₄F₃NO₂ m/z 213.0977, found 212.0902 (M-H)⁻.

Synthesis of spacer amino phosphoramidite

6-(trifluoroacetamido)-1-hexanol (300 mg, 1.41 mmol) and 1H-tetrazole (296 mg, 4.23 mmol) was dissolved in anhydrous CH₂Cl₂ (25 mL). The solution was cooled on an ice bath, and 2-cyanoethoxy-N′,N′,N,N-tetraisopropyl-phosphoramidite (1.42 mL, 4.23 mmol) was added under dry nitrogen. After stirring at room temperature for 2 hours, the solution was washed with 5% NaHCO₃ and brine, dried over Na₂SO₄, and concentrated by vacuum. Purification was achieved by flash chromatography over silica gel using a mixture of CH₂Cl₂-EtOAc (10:1) as eluent to afford a colorless oil 500 mg. (Figure 1, compound spacer amino phosphoramidite, 86% yield).

¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.37 (s, 1H, –CONH–); 3.75~3.51 (m, 6H, 2× –POCH₂–, 2× –CH(CH₃)₂); 3.19~3.14 (m, 2H, –NHCH₂–); 2.77~2.74 (t, 2H, J=4, CNCH₂–); 1.56~1.26 (m, 8H, –NHCH₂(CH₂)₄–); 1.18~1.12 (m, 12H, 2× –CH(CH₃)₂); ¹³C NMR (101 MHz, DMSO-d₆): δ (ppm) 156.78 (CONH–); 120.49 (NCCH₂–); 120.33 (CF₃–); 62.79 (POCH₂CH₂–); 45.44 (POCH₂CH₂CN); 40.61 (–CH(CH₃)₂); 30.22 (–CONHCH₂–); 28.57 (–POCH₂CH₂–); 26.13 (–NHCH₂CH₂–); 25.12 (–NHCH₂CH₂– CH₂–); 23.66 (–NHCH₂CH₂CH₂CH₂–); 22.49 (–CH(CH₃)₂); 11.35 (NCCH₂–). ³¹P NMR (162 MHz, DMSO-d₆): δ (ppm) 146.33.

Synthesis of amino-modified GBI-10 aptamer

Amino-modified GBI-10 aptamer (Figure 1) was synthesized on ABI 394 automated RNA/DNA synthesizer (Thermo Fisher Scientific, Waltham, MA, USA). The products were purified by C18 reverse high-performance liquid chromatography (XBridgeTM OST C18, 2.5 μm, 10 mm ×50 mm) using a linear gradient of 5%→20% eluent A in 35 minutes (Figure S5). Solutions of 0.1 M Et₃N-H₂CO₃ in water, pH =7.7, were used as eluent B, and CH₃CN was used as eluent A. The oligonucleotide solutions were desalted by Sephadex G25 column. The oligonucleotide compositions were confirmed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) (Figure S6).

Figure S1 Schematic illustration for the preparation of the liposome formulations and the targeting effect of GTLs to TN-C in tumor cells and tumor neovasculature.

Abbreviations: Gd⋅DTPA⋅BSA, Gd (III) [N,N-bis-stearylamidomethyl-N′-amidomethyl] diethylenetriamine tetraacetic acid; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; NTLs, nontargeted gadolinium-loaded liposomes; PTLS, pretargeted liposomes; EDC, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide; NHS, N-hydroxysuccinimide; GTLs, GBI-10-targeted gadolinium-loaded liposomes; CHOL, cholesterol.

Figure S2 Size distribution of NTLs and GTLs by intensity using dynamic light scattering analysis.

Abbreviations: NTLs, nontargeted gadolinium-loaded liposomes; GTLs, GBI-10-targeted gadolinium-loaded liposomes.

Figure S3 Photography of cell pellets used in the MR imaging assay.

Abbreviation: MR, magnetic resonance.

Figure S4 The synthetic routes of Gd⋅DTPA⋅BSA.

Abbreviations: DTPA⋅BSA, [N,N-Bis-stearylamidomethyl-N′-amidomethyl] diethylenetriamine tetraacetic acid; DTPA⋅BA, DTPA bis-anhydride; Gd⋅DTPA⋅BSA, Gd (III) [N,N-bis-stearylamidomethyl-N′-amidomethyl] diethylenetriamine tetraacetic acid; ODA, octadecylamine.

Figure S5 The separation results of amino-modified GBI-10 aptamer with HPLC.

Notes: Dionex UltiMate 3000 HPLC, XBridgeTM OST C18 column (2.5 μm, 10 mm ×50 mm), gradient program =5%–20% eluent A in 35 minutes (A: 0.1 M Et₃N-H₂CO₃ in water, pH =7.7; B: CH₃CN), column temperature 40°C.

Abbreviation: HPLC, high-performance liquid chromatography.

Figure S6 MALDI-TOF-MS of amino-modified GBI-10 aptamer.

Notes: Found: 10,637.3; Calcd: 10,637.93.

Abbreviations: MALDI-TOF-MS, matrix-assisted laser desorption/ionization time of flight mass spectrometry; int, intensity; Calcd, calculated.

Acknowledgments

The authors gratefully acknowledge the financial support from the Natural Science Foundation of Beijing, People’s Republic of China (Grant No 7122101), and the Ministry of Science and Technology of China (Grant No 2012CB720604). The authors thank Dr Shirley Wang of Harvard University for her kind help in preparing the manuscript.

Disclosure

The authors report no conflicts of interest in this work.