Introduction

Elemental radionuclides are combined with other elements to form chemical compounds, or else combined with existing pharmaceutical compounds, to form radiopharmaceuticals. The radiopharmaceutical, once administered to the patient, can localize to specific organs or cellular receptors allowing imaging the extent of a disease-process in the body, based on the cellular function and physiology, rather than relying on physical changes in the tissue anatomy. In some diseases nuclear medicine studies can identify medical problems at an earlier stage than other diagnostic tests [1, 2]. Several 99mTc-labeled compounds have been developed for imaging purposes [3–11] and some of them are routinely employed in diagnostic nuclear medicine [12–15].

Doxorubicin (DOX) is an anthracycline antibiotic with potent antineoplastic properties effective against a broad spectrum of malignancies, such as non-Hodgkin’s lymphoma, acute lymphoblastic leukemia, breast carcinoma, and several other types of cancers [16]. Doxorubicin interacts with DNA by intercalation and inhibition of macromolecular biosynthesis [17, 18]. The mechanism of cytotoxicity involves specific interaction of the planar anthracycline nucleus of DOX to the DNA double helix, resulting in prevention of further DNA replication [19, 20]. It has also been one of the most widely reported agents used in localized therapy approaches. In combination with ethiodized oil and gelatin sponge, DOX has shown response rates of up to 50% [21]. Children and adolescents are particularly susceptible to the cardiotoxic effects of anthracyclines chemotherapy and a significant portion of children treatment with DOX develop cardiomyopathy a year or more after cessation of chemotherapy [22–24].

In this paper, the direct labeling of DOX with 99mTc is described. Labeling efficiency, stability, quality control, and characterization were also studied.

Experimental

Materials and methods

Doxorubicin hydrochloride for intravenous injection was obtained from Zhejiang Hisun Pharmaceutical Co. Ltd. Karachi, Pakistan. 99mTc was obtained from a locally produced fission based Pakgen 99Mo/99mTc generator. All chemicals used were AR grade.

Synthesis of 99mTc–DOX

Optimization of labeling efficiency of DOX with 99mTc was performed by varying the amounts of DOX 100–500 μg, SnCl2·2H2O 5–20 μg and pH range 4–9. The pH was adjusted by using 0.5 M NaOH. Reaction mixture volume used in all experiments was 1.5 ± 0.2 mL. After addition of all reagents ~370 MBq Na99mTcO4 in saline was injected into the vial. All experiments were carried out at room temperature of 25 ± 2 °C and in darkness because the ligand was photosensitive.

Quality control

Radiochemical yield of 99mTc–DOX was checked by thin layer chromatographic method using Whatman No. 3 paper and ITLC-SG strips (Gelman Sciences). Free 99mTcO4 − in the preparation was determined by using Whatman No. 3 paper as the stationary phase and acetone as the mobile phase. Reduced and hydrolyzed activity was determined by using instant thin layer chromatography (ITLC-SG strips) as the stationary phase and THF as a mobile phase. Radiocolloids were also determined by passing the preparation through 0.22 μm bacteria filter (Millipore Filter Corp.). Activity remaining on the filter and in the solution was counted by a gamma-counter (Ludlum). The stability of 99mTc–DOX was checked for 5 h at room temperature. The distribution of radioactivity on chromatographic strips was measured by a 2π scanner (Berthold, Germany). Alternatively, the strips were cut into 1 cm segments and counted by a gamma-counter.

In vitro stability

Stability of the 99mTc–DOX was studied in vitro. 1.8 mL of normal human serum was mixed with 0.2 mL of 99mTc–DOX and incubated at 37 °C. 0.2 mL aliquots were withdrawn during the incubation at different time intervals up to 24 h and subjected to chromatography for determination of 99mTc–DOX, reduced/hydrolyzed 99mTc and free 99mTcO4 −.

Electrophoresis of 99mTc–DOX

The charge on 99mTc–DOX was studied by using Deluxe electrophoresis chamber (Gelman) system. The Phosphate buffer of pH 6.8 was used in this experiment. Whatman No. 1 paper of 30 cm long strip was used. A drop of 99mTc–DOX at the middle of the strip was put and electrophoresis was run for 30–60 min at a voltage of 300 V. After completion of electrophoresis, the strip was scanned by using 2π scanner to know the charge on labeled DOX.

HPLC of 99mTc–DOX

HPLC of radiotracer 99mTc–DOX was studied by using D-200 Elite HPLC system. The column of C-18 was used as stationary phase and a mixture of acetonitrile and disodium hydrogen phosphate buffer (pH adjusted up to 4–5 by using 0.5 M NaOH) ratio 45:55 (v/v %) was used as mobile phase. The flow rate was adjusted up to 1 mL/min at a wavelength of 254 nm.

Results and discussion

The DOX molecule contains (8S,10S)-10-[(3-amino-2,3,6-trideoxy-α-l-lyxo-hexopyranosyl)oxy] and 8-glycolyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy bonded with 5,12-naphthalenedione hydrochloride. The structure of DOX HCl is shown in Fig. 1. The direct method of labeling of DOX with 99mTc was exploited which is simple, rapid, efficient and does not require bifunctional chelating agents. The various chelates of 99mTc, which serve as radiopharmaceuticals are formed by interaction between specific chelating agents and a reduced form of 99mTc. In order to form bonds with technetium, the chelator must contain electron donors like nitrogen, oxygen and sulfur. Space between multiple electron donor atoms is required to allow several bonds to form with the central metal. DOX has several functional groups, such as –NH2, –OH, –O– to form bonds with 99mTc. Although the chemistry of formation and molecular structure are unknown, 99mTc–DOX is assumed to be a chelator complex with one or more DOX ligands attached to reduced 99mTc.

Fig. 1
figure 1

Structure of DOX HCl

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Labeling efficiency, radiochemical purity, and stability were determined by a combination of ascending paper chromatography and ITLC on silica gel. In paper chromatography using acetone as the solvent, free 99mTcO4 − moved towards the solvent while 99mTc–DOX and reduced/hydrolyzed 99mTc remained at the point of spotting. In ITLC-SG chromatography using THF as solvent, 99mTc–DOX and free 99mTcO4 − moved towards the solvent, while reduced/hydrolyzed 99mTc remains at the point of spotting. Radiocolloids were also determined by passing the preparation through sterile filters (0.22 μm). In this method radiocolloids were retained in the pores of filter while 99mTc–DOX and free 99mTcO4 − were passed through the filter. The results obtained from both methods were in excellent agreement. The amount of radiocolloid in the final preparations was ≤2.0%.

The effects of pH are shown in Fig. 2. At low pH (4–6) the minimum labeling efficiency is 70%, while at pH 6–7 the labeling efficiency of 99mTc–DOX is >92%. In basic media at pH 8–9 the labeling efficiency is decreased (68–53%). Hence further experiments were performed at pH 6–7.

Fig. 2
figure 2

Effect of pH on the labeling efficiency of 99mTc–DOX

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The amount of the reducing agent, SnCl2·2H2O, which gave the highest labeling efficiency was 12 μg (Fig. 3). Experiments were performed with varying amount of the ligand (DOX), and 200 μg gave the maximum labeling efficiency of >92% (Fig. 4). To avoid colloid formation, the optimum amount of reducing agent was used. The complexation of 99mTc with DOX was not rapid and maximum labeling efficiency was achieved after 15 min. The resulting complex of 99mTc–DOX was quite stable and labeling of ≥ 92% was maintained for up to 5 h (Fig. 5). The electrophoresis result indicates that the complex of 99mTc–DOX is neutral in nature (Fig. 6). HPLC results indicates that the purity of ligand was >98% (Fig. 7). The retention time of inactive and labeled DOX was nearly 10 min. Figure 8 shows that the labeling efficiency was ~95% and complex was single specie. When the preparation was incubated with normal human serum at 37 °C, insignificant increase in free pertechnetate or reduced/hydrolyzed 99mTc up to 24 h was noticed. The total impurities were ~7% (Table 1).

Fig. 3
figure 3

Effect of SnCl2·2H2O amount on the labeling efficiency of 99mTc–DOX

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Fig. 4
figure 4

Effect of ligand amount on the labeling efficiency of 99mTc–DOX

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Fig. 5
figure 5

Rate of complexation of 99mTc with DOX and stability of 99mTc–DOX

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Fig. 6
figure 6

Electrophoresis of DOX indicates its neutral nature

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Fig. 7
figure 7

HPLC analysis of Ligand shows >98% purity of ligand

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Fig. 8
figure 8

HPLC analysis of 99mTc–DOX shows single specie of complex

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Table 1 In vitro stability of 99mTc–DOX in normal human serum
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The final formulation for the radiotracer 99mTc–DOX was DOX 200 μg, SnCl2·2H2O 12 μg, pH 6–7, 99mTc 370–450 MBq, reaction mixture volume ~1.5 mL, and incubation time 15 min at room temperature.

Conclusions

Methodology for radiolabeling of DOX with 99mTc has been developed and standardized. Radiolabeling efficiency of 99mTc–DOX monitored by paper and ITLC-SG was more than 92%. The resulting complex of 99mTc–DOX was quite stable and labeling of ≥92% was maintained for up to 5 h. The complex was stable in serum. HPLC shows single specie of 99mTc–DOX. No post-labeling purification is required for further studies.