Introduction

There is great potential interest in developing new ligands capable of sufficiently selective accumulation in tumors to permit external scintiscanning for tumor diagnosis or delivery of therapeutic radionuclides for tumor-targeted therapy. As one class of derivatives that have special biological functions in the human body’s metabolism, porphyrin has received considerable attention due to its specifically distinguish cancer cells from the normal one [1–5].

Porphyrins are essential for many vital and biological functions of normal metabolism of living organisms, such as oxygen transport and storage, photosynthesis, etc. Due to their formation of highly stable and kinetically inert complexes with many metals, they find numerous medical applications [6].

Porphyrin accumulates and causes cellular damage on light excitation, so that it has been used in both detection and treatment of certain malignant tumors by combining porphyrin drugs with light activation [7–9]. The photodynamic therapy (PDT) technique for cancer uses the interaction of a sensitizer and light to destroy cancerous cells and tumors [10].

Several researchers have utilized various porphyrin derivatives and metalloporphyrins for identifying and delineating malignant tissues [11–14]. A wide variety of porphyrin derivatives with various types of peripheral moieties were radiolabeled with several medically important radionuclides to develope a useful porphyrin-based tumor specific agent [6, 15–21].

99mTc complexes are routinely used in the diagnosis of many cancer diseases involving various organs due to 99mTc radionuclide has favorable nuclear properties (140 keV, gamma emitters; t 1/2 = 6:01 h), low cost and readily availability [22, 23].

Phytochlorin[21H,23H-Porphine-7-propanoicacid,3-carboxy-5-(carboxymethyl)13-ethenyl-18-ethyl-7,8-dihydro-2,8,12,17-tetramethyl-,(7S,8S), Fig. 1] is a water soluble porphyrin derivative.

Fig. 1.
figure 1

21H,23H-Porphine-7-propanoicacid,3-carboxy-5-(carboxymethyl)-13-ethenyl-18-ethyl-7,8-dihydro-2,8,12,17-tetramethyl-,(7S,8S)

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The aim of this study is to use 99mTc for labeling phytochlorin, to evaluate its radiochemical and biological characteristics, and finally to predict the molecular structure of the 99mTc-phytochlorin complex.

Experimental

Chemicals

Phytochlorin was purchased from Aldrich-Sigma Chemical Company. All other chemicals were purchased from Merck and they were of the highest purity grade.

Radioactive material

99mTc was eluted as 99mTcO4 − from 99Mo to 99mTc generator, Elutec Brussels, Belgium.

Methods

Method of labeling

Accurately weighted 100 mg phytochlorin was transferred to an evacuated penicillin vial. N2-purged stannous chloride aqueous solution (containing exactly 75 μg SnCl2·2H2O) was added and the pH of the mixture was adjusted to 10 then the volume of the mixture was adjusted to 1 mL by N2-purged distilled water. One mL of freshly eluted 99mTcO4 − (400 MBq) was added to the above reaction mixture. The reaction mixture was then vigorously shaken and allowed to react at room temperature (25 °C) for sufficient time (30 min) to complete the reaction.

Analysis of 99mTc-phytochlorin complex

The radiochemical purity of 99mTc-phytochlorin was performed by thin layer chromatographic method using strips of silica gel impregnated glass fiber sheets (ITLC-SG). Free 99mTcO4 − in the preparation was determined using acetone as the mobile phase. Reduced hydrolyzed technetium was determined by using ethanol: water: ammonium hydroxide mixture (2:5:1) as the mobile phase. It was further confirmed by a Shimadzu HPLC system, which consists of pumps LC-9A, UV spectrophotometric detector operated at a 220 nm (SPD-6A), and rheodyne injection valve. Chromatographic analysis of 99mTc-phytochlorin was performed by injection of 10 μL of the reaction mixture at the optimum conditions into a reversed-phase column, (Waters, Symmetry C18; 5 μm, 4.6 × 150 mm2) preceded by a guard column (Waters, Symmetry C18; 5 μm) and eluted with mobile phase consisting of acetonitrile and 0.01 M potassium dihydrogen phosphate/diethylamine (60:40:0.2 v/v) [41]. The mobile phase was filtered and degassed prior to use and the flow rate was 0.5 mL/min.

Computational method

The optimized molecular structures of the compound of interest and corresponding energies were calculated by using GAUSSIAN 09 program package [24] using density functional theory with Becke3–Lee–Yang–Parr (B3LYP) [25–27] combined with three different basis sets without any constraint on the geometries. The standard 3-21G basis set was used for carbon and hydrogen atoms and the standard 6-31G basis set was used for oxygen and nitrogen atoms. However, the last basis set was LANL2MB and it was used for technetium atom. The LANL2MB basis set treated electrons near the nuclei via effective core potentials (ECPs) and it also includes some relativistic effect, which are essential for heavy elements like technetium [28–30]. Gaussview program [31] has been considered to get visual the optimized structures. It is worthy to mention that, for the compound of interest, all the optimized structures were found to be true minima, i.e. no imaginary frequency modes were obtained.

Induction of tumor in mice

The parent tumor line (Ehrlich Ascites Carcinoma) was withdrawn from 7 days old donor female Swiss Albino mice and diluted with sterile physiological saline solution to give 12.5 × 106 cells/mL. Exactly 0.2 mL solution was injected intramuscularly in the right thigh to produce a solid tumor. The animals were maintained till the tumor development was apparent (10–15 day).

Biodistribution study in mice

In vivo biodistribution studies were carried out in groups of five female Albino mice where each animal was injected in the tail vein with 0.2 mL solution containing 5–10 kBq of 99mTc-phytochlorin. The mice were put in metabolic cages for the required time. The mice were sacrificed by cervical dislocation in groups at various time intervals after injection and the organs or tissues of interest were removed, weighted and counted. To correct for physical decay and to calculate uptake of the radiolabel compound in each tissue sample as a fraction of the injected dose, aliquots of the injected dose were counted simultaneously. The results were expressed as percentage injected dose per gram of tissue or organ (% ID/g).

Results and discussion

The radiochemical yield of 99mTc-phytochlorin was determined by using thin layer chromatography (TLC-SG) as well as HPLC. Acetone was used with TLC-SG strip to calculate the percentage of free 99mTcO4 − which moved with the solvent front (R f = 1.0) leaving 99mTc-phytochlorin and colloid at the origin. Ethanol:water:ammonium hydroxide mixture (2:5:1) was used to check the amount of reduced hydrolyzed technetium (colloid) which remained at the origin (R f = 0) while 99mTcO4 − and 99mTc-phytochlorin migrated with the solvent front (R f = 1). The radiochemical purity was determined by subtracting the sum of the % of colloid and free pertechnetate from 100 %. The radiochemical yield is the mean value of three experiments.

In case of HPLC, an HPLC radiochromatogram is presented in Fig. 2. It exhibited two peaks, one at fraction number 6 corresponding to 99mTcO −4 , while the second peak was collected at fraction number 17.3 corresponding to 99mTc-phytochlorin, which was found to coincide with the UV signal. Nearly, 97 % of the injected activity in the HPLC was recovered as collected activity.

Fig. 2
figure 2

HPLC radiochromatogram of 99mTc-phytochlorin complex

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Molecular geometry

It is impossible to determine the exact structure and composition of the reaction product because the amount of 99mTc eluted from 99Mo/99mTc generator is very tiny ca. 10−9 mol/L [32]. Consequently, we decided to use theoretical background and methods of molecular modeling to predict and design the reaction product. Plus, in order to carry out labeling of the chelating agent with 99mTc, tin chloride was added to reduce the oxidation state of technetium from IIV to V, IV or III. However, technetium oxidation states in the chelate systems were investigated in presence of SnCl2 and it was concluded and proved that Tc(V) and Tc(IV) were the most stable oxidation states [33, 34]. Moreover, pH of the reaction mixture plays an essential role in the labeling process and in our case it is found that pH 10 is the optimum pH for the labeling process. It is known also that stability of complexes containing chelate rings is more than that of complexes containing none or fewer chelate rings due to the chelate effect [35]. According to this discussion, one can consequently expect the following: Technetium will prefer to coordinate to the four nitrogen atoms of the core of the porphyrine molecule (H2L) due to the chelate effect; the coordinated technetium atom will complete its coordination sphere with OH− or O2− species since the optimum pH for the labeling reaction is an alkaline pH; and finally the oxidation state of the coordinated technetium will be +4 or +5. Accordingly, the geometry of the possible complexes {[LTcO]+, 1; [LTcO], 2; [LTcOH]2+, 3; [LTcOH]+, 4; [(L)OTcO]−, 5; [(L)OTcO]2−, 6; [(L)OTcOH], 7; [(L)OTcOH]−, 8; [(L)HOTcOH]+, 9; and [(L)HOTcOH], 10} was optimized, in a singlet and doublet state for Tc(V) and Tc(IV) complexes, respectively, by the DFT method with the B3LYP function. The fully optimized geometries of the suggested complexes are shown in Fig. 3. The energy, and dipole moment (μ), for the geometrically optimized complexes are presented in Table 1. It is clear that complex 10 has the lowest energy (−2210.75113006 a.u.) and hence this is the most stable structure for the product of the labeling process. The optimized geometrical structure and the atomic numbering of [(L)HOTcOH], 10, complex is shown in Fig. 4. The structure of complex 10 presents nearly linear HO–Tc–OH unit with an angle of 179.27° and a coplanar Tc(N1N2N3N4) unit. Some selected parameters of the geometrically optimized structure of complex 10 are presented in Table 2. The calculated bond lengths and angles for Tc–O and Tc–N are in quite good agreement with the values reported for other oxotechnetium and oxorhenium complexes of related structure [36–38].

Fig. 3
figure 3

The geometrically optimized structures of complexes 1–10

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Table 1 Energy and dipole moment for the geometrically optimized structures of possible products
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Fig. 4
figure 4

DFT optimized structure of [(L)HOTcOH], 10, with numbering of atoms

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Table 2 Some structure parameters of the optimized geometry of [(L)HOTcOH], 10
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The labeling conditions for 99mTc-phytochlorin were studied in detail as follow

Figure 5 shows the effect of phytochlorin amount on labeling yield of 99mTc-phytochlorin complex. At low phytochlorin concentration (25 mg) the labeling yield was small and equal to 33.2 % where this low labeling yield was due to phytochlorin concentration being insufficient to form the complex with all of the reduced 99mTc. Increasing the amount of phytochlorin led to higher labeling yield and the maximum yield (98.4 ± 0.6 %) was achieved at 100 mg. By increasing the phytochlorin amount over the optimum value, the labeling yield slightly decreased again till reached 92.5 % at 125 mg phytochlorin.

Fig. 5
figure 5

Effect of phytochlorin amount on percent labeling yield of 99mTc-phytochlorin

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As shown in Fig. 6, if too much SnCl2·2H2O content was used, >75 μg, the labeling yield was low (83.5 % at 125 μg SnCl2·2H2O) and the main impurity was reduced hydrolyzed technetium (12.3 % at 125 μg SnCl2·2H2O). This may be due to the fact that most of the ligand molecules are consumed in the formation of complexes, so the pertechnetate is reduced to insoluble technetium (IV) TcO2·xH2O in the absence of ligand [39] or due to the fact that the excess amount of stannous chloride leads to the formation of stannous hydroxide colloid Sn(OH) −3 in basic medium [40].

Fig. 6
figure 6

Effect of SnCl2·2H2O on percent labeling yield of 99mTc-phytochlorin

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The optimum labeling yield was obtained at 75 μg SnCl2·2H2O at which a maximum labeling yield was 98.4 ± 0.6 %. Below 75 μg SnCl2·2H2O, stannous chloride is insufficient for complete reduction of pertechnetate to form 99mTc-phytochlorin complex, this is an explanation of the presence of high quantity of free pertchnetate (59.8 % at 25 μg SnCl2·2H2O).

Figure 7 shows that, the labeling yield of 99mTc-phytochlorin is dependent on the pH of the reaction mixture in the range from 8 up to 12. The maximum labeling yield of 99mTc-phytochlorin complex (98.4 ± 0.6 %) was obtained at pH 10. The yield was decreased with decreasing the pH of the reaction mixture with an increase of the 99mTcO −4 (19.3 % at pH 8), above pH 10 the yield decreased again.

Fig. 7
figure 7

Effect of pH of the reaction mixture on percent labeling yield of 99mTc-phytochlorin

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The stability of 99mTc-complex was studied in order to determine the suitable time for injection to avoid the formation of the undesired products that result from the radiolysis of the labeled compound. These undesired radioactive products may be accumulated in non-target organs. Figure 8 shows the effect of reaction time on percent labeling yield of 99mTc-phytochlorin where at 15 min, the labeling yield of 99mTc-phytochlorin complex was low and maximized (98.4 ± 0.6 %) at 30 min then the labeling yield remained stable up to 8 h.

Fig. 8
figure 8

Radiochemical stability of 99mTc-phytochlorin complex

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Biodistribution of 99mTc-phytochlorin complex

Biodistribution of 99mTc-phytochlorin in solid tumor bearing mice is shown in Table (3). Biodistribution assay was done at time intervals 20, 60, 90 and 120 min p.i. and the results were expressed as % injected dose per gram organ or fluid (%ID/g organ). The body clearance of 99mTc-phytochlorin is mainly through the urinary pathway and the activity is not accumulated in specific organ other than the solid tumor tissue. The selectivity of 99mTc-phytochlorin was evaluated by the target/non-target (T/NT) ratio between tumor muscle (right mouse thigh muscle) and normal muscle (left mouse thigh muscle). Figure 9 shows T/NT ratio of 99mTc-phytochlorin in solid tumor bearing mice which clarify that 99mTc-phytochlorin is highly selective to the tumor cells with an accumulation ratio 3.65 at 90 min post injection. T/NT ratio of 99mTc-phytochlorin is higher than that of many other radiopharmaceuticals such as 99mTc-DETA, 99mTc-TETA, 99mTc-TEPA, [99mTc(CO)3(IDA–PEG3–CB)]−,99mTc-nitride-pyrazolo[1,5-a]pyrimidine and 99mTc-HL-91 and 99mTc-2-methoxy-isobutyl-isonitrile [41–45]. This preclinical study suggests that 99mTc-phytochlorin is a selective agent for solid tumor imaging and can be used as a potential solid tumor imaging radiotracer.

Table 3 In vivo biodistribution study of 99mTc-phytochlorin in solid tumor bearing Albino mice at different time intervals post injection, (% ID/g organ ± SE, n = 3)
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Fig. 9
figure 9

T/NT ratio of 99mTc-phytochlorin in solid tumor bearing mice

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Conclusion

99mTc-phytochlorin can easily be labeled with high labeling yield of 98.4 ± 0.6 % by direct labeling technique using SnCl2·2H2O as a reducing agent. The molecular modeling study of the labeled compound showed that the structure of 99mTc-phytochlorin complex has nearly linear HO–Tc–OH unit with an angle of 179.27° and a coplanar Tc(N1N2N3N4) unit. Moreover, the biodistribution studies of 99mTc-phytochlorin revealed that 99mTc-phytochlorin has high T/NT ratio and is rapidly cleared out from body organs and excreted via the urinary pathway. These findings, combined with the advantage of the high labeling yield and stability of 99mTc-phytochlorin complex, are promising enough to encourage clinical investigation of this new 99mTc agent as a selective potential radiopharmaceutical for solid tumor imaging.