Gd³⁺-DTPA-DG: novel nanosized dual anticancer and molecular imaging agent

Figures & data

Figure 1 A) Chemical structure of ethylenedicysteine deoxyglucose. B) General procedure for synthesis of (a) DTPA-DA and (b) DTPA-DG.

Abbreviations: DTPA, diethylenetriamine penta-acetic acid-D-glucosamine; DTPA-DG, diethylenetriamine penta-acetic acid-D-deoxy-glucosamine.

Figure 2 Schematic illustration of performance of in vitro T1/T2 measurements using magnetic resonance imaging apparatus. For each concentration, triplicate sampling and the maximum region of interest were considered. Five concentrations of Gd³⁺-DTPA-DG (0.33, 0.17, 0.08, 0.04, and 0.02 mmol/mL) were obtained by dilution with sodium chloride 0.9%.

Abbreviation: Gd³⁺-DTPA-DG, gadopentetate dimeglumine-D-deoxy-glucosamine.

Figure 3A MTT results of 48 hours of Gd³⁺-DTPA-DG exposure to HT1080 and HT29 cell lines. The average percent of viable cells is also shown.

Abbreviation: Gd³⁺-DTPA-DG, gadopentetate dimeglumine-D-deoxy-glucosamine.

Figure 3B MTT results of 48 hours of Gd³⁺-DTPA-DG exposure to HKC-5 and HEK-293 human kidney cell lines. No significant toxic effect was observed.

Abbreviation: Gd³⁺-DTPA-DG, gadopentetate dimeglumine-D-deoxy-glucosamine.

Figure 4 Effect of Gd³⁺-DTPA-DG and DTPA on TNF-α release. As depicted, the complex increased TNF-α release significantly.

Abbreviations: DTPA, diethylenetriamine penta-acetic acid; Gd³⁺-DTPA-DG, gadopentetate dimeglumine-D-deoxy-glucosamine.

Figure 5 Effect of DTPA-DG on blood glucose level in rats. No significant changes were observed.

Abbreviation: DTPA-DG, diethylenetriamine penta-acetic acid-D-deoxy-glucosamine.

Table 1A Positive hexokinase enzyme response to Gd³⁺-DTPA-DG, glucose, and glucosamine

Glycosylated compounds at same concentrations	Hexokinase activation after three minutes of incubation (340 nm)
Glucose	+
Glucosamine	+
Gd^3+-DTPA-DG	+

Abbreviation: Gd³⁺-DTPA-DG, gadopentetate dimeglumine-D-deoxy-glucosamine.

Figure 6 Effects of the experimental complex on coagulation factors. No significant changes were observed in PT and APTT factors.

Abbreviations: PT, prothrombin time; APTT, activated partial thromboplastin time.

Figure 7 Morphological changes in HT1080 cells. A) Control group without any treatment, B) 0.125 mg/mL Gd³⁺-DTPA-DG, and C) 0.5 mg/mL Gd³⁺-DTPA-DG. The amount of HT1080 cancer cells was observed to diminish significantly only in the Gd³⁺-DTPA-DG group.

Abbreviation: Gd³⁺-DTPA-DG, gadopentetate dimeglumine-D-deoxy-glucosamine.

Table 1B T1 values (msec) derived from equations 1${\mathit{\text{Signal}}}_{SE}=S_0{e}^{-R_2·TE}$(1) and 2${\mathit{\text{Signal}}}_{SE}=S_0\hspace{0.17em}(1-e^{-TR/T_1})$(2)

TR	Concentration					Water
	1/3	1/6	1/12	1/24	1/48
60	551	358	260	206	186	116
140	1035	729	468	330	251	156
240	1385	1076	764	532	394	259
340	1542	1292	971	694	520	350
440	1628	1458	1133	835	635	434
600	1705	1581	1317	1005	794	559
800	1756	1667	1478	1197	956	692
1000	1767	1684	1544	1304	1070	793
2000	1800	1767	1739	1649	1488	1212
T1	161.2643122	253.421186	420.3446826	666.2225183	916.5902841	1367.427868
1/T1	0.006201	0.003946	0.002379	0.001501	0.001091	0.0007313
R^∧2 value	0.99	0.99	0.9875	0.9964	0.9971	0.9973

Table 1C T2 values (msec) derived from equations 1${\mathit{\text{Signal}}}_{SE}=S_0{e}^{-R_2·TE}$(1) and 2${\mathit{\text{Signal}}}_{SE}=S_0\hspace{0.17em}(1-e^{-TR/T_1})$(2)

TE	Concentration					Water
	1/3	1/6	1/12	1/24	1/48
10	1762	1602	1340	1022	800	546
25	1372	1263	1097	862	711	478
40	1200	1172	1044	835	697	463
55	1043	1018	943	775	653	456
70	962	920	911	762	641	445
1/T2	0.0099	0.00883	0.00615	0.00462	0.00352	0.00305
T2	101.010101	113.25028	162.601626	216.450216	284.09	327.87
R^∧2 value	0.96	0.97	0.92	0.88	0.91	0.8

Table 1D The average corresponding Magnevist^® T1 values as comparison standard

Magnevist 1.5 mM	T1 relaxation time
1/3	178
1/6	280
1/12	433
1/24	678
1/48	924
Water	1363

Table 1E Mean Gd³⁺-DTPA-DG T1 values (msec) from stability testing. Each sample was repeated at least three times

Gd^3+-DTPA-DG concentration	T1 relaxation time
1/3	175
1/6	275
1/12	429
1/24	677
1/48	922
Water	1363

Abbreviation: Gd³⁺-DTPA-DG, gadopentetate dimeglumine-D-deoxy-glucosamine.

Table 2 Mean in vivo T1, T2 data for Gd³⁺-DTPA-DG and Magnevist^® relaxation times at different time points and tissues are shown as the mean ± standard error of the mean for tumor-bearing nude mice (mean weight 18 ± 1.3 g, n = 3 in each group)

Tissue	Brain [Gd^3+-DTPA-DG]	Heart [Gd^3+-DTPA-DG]	Tumor [Gd^3+-DTPA-DG]	Brain [Magnevist]	Heart [Magnevist]	Tumor [Magnevist]
T1 (10 minutes)	365 ± 7	155 ± 9	145 ± 5	1333 ± 10	1123 ± 12	543 ± 8
T1 (20 minutes)	191 ± 4	87 ± 8	118 ± 8	1321 ± 11	1167 ± 10	442 ± 9
T2 (10 minutes)	189 ± 6	98 ± 10	102 ± 9	1002 ± 12	876 ± 13	398 ± 11
T2 (20 minutes)	101 ± 9	45 ± 7	89 ± 8	999 ± 14	882 ± 11	298 ± 7

Notes:

* Decimal values are not shown;

** Square brackets indicate concentrations.

Abbreviation: Gd³⁺-DTPA-DG, gadopentetate dimeglumine-D-deoxy-glucosamine.

Figure 8A T2 data based on spin echo and gradient echo protocols.

Figure 8B T1 data based on spin echo and gradient echo protocols.

Figure 9A Magnetic resonance images (T1 weighted method) prior and 20 minutes after Gd³⁺-DTPA-DG intravenous injection (A and B) in mice bearing human lung cancer (each image was performed on three mice). The tumor site was definitely enhanced. Magnetic resonance images (T1 weighted method) 20 minutes post Gd³⁺-DTPA + Gd³⁺-DTPA-DG (C) and Gd³⁺-DTPA (Magnevist^®, D) intravenous injection in mice bearing human lung cancer.

Abbreviations: Gd³⁺-DTPA-DG, gadopentetate dimeglumine-D-deoxy-glucosamine; Gd³⁺-DTPA, gadopentetate dimeglumine.

Figure 9B Illustration of the whole body magnetic resonance images prior and 20 minutes post intravenous Gd³⁺-DTPA-DG injection in mice bearing human lung cancer cells.

Abbreviation: Gd³⁺-DTPA-DG, gadopentetate dimeglumine-D-deoxy-glucosamine.

Figure 10 Illustration of the ability of Gd³⁺-DTPA-DG to penetrate the blood-brain barrier. The nude mice brain was significantly enhanced 20 min post intravenous Gd³⁺-DTPA-DG injection. As observed, Magnevist^® could not enhance the animal brain resolution. Gd³⁺-DTPA-DG seems to show the same pharmacokinetics as ¹⁸FDG in the brain as well as in the gastrointestinal tract and tumor sites.

Abbreviation: Gd³⁺-DTPA-DG, gadopentetate dimeglumine-D-deoxy-glucosamine.

Figure S1 Illustration of the original U.V spectrums of DTPA (A), D-Glucosamine (B), DTPA-DG (C), and Gd³⁺-DTPA-DG (D).

Figure S2 Size and zeta potential distribution. A) size and B) zeta potential distribution of the DTPA-DG compound according to concentration.