All the reagents were purchased from Sigma-Aldrich ACS grade and used without further purification. Technetium-99m was obtained from chromatographic 99Mo/99mTc generator “99mTc-GT-TOM” produced by Tomsk Polytechnic University (TPU)—Tomsk, Russia.

Three types of nanoparticles were selected to obtain nanocolloids labeled with 99mTc. The first type of colloids was created on the basis of metal chelates with chemically modified complexons of diethylenetriamine-pentaacetic acid (DTPA). It should be noted that the DTPA molecule itself, like its complexes with metals, is hydrophilic and does not tend to form colloidal particles. The introduction of hydrophobic fragments into its structure allowed the preparation of water-insoluble modified DTPA complexes21. The original substance of the modified DTPA (DTPAmod) was synthesized in Tomsk Polytechnic University. Preparation of colloid solution DTPAmod was produced using the following method. A sample of modified DTPA with the mass of 28 mg was quantitatively transferred to a volumetric flask of 50 ml and dissolved in 20 ml of 5% NaHCO3 solution by heating to 80 °C. After that, the volume was adjusted with the same solvent up to the mark. In order to reduce the particle size the container with suspension was heated in water to 70 °C and treated with ultrasound for 40 min, which reduced the average particle radius up to 55 nm. The general scheme for the synthesis of 99mTc-DTPAmod is shown in Fig. 2.

Figure 2

The general scheme for the synthesis of 99mTc-DTPAmod.

The second type of colloids is iron nanoparticles coated with a carbon shell of Fe@C (Fig. 3a). These particles were obtained from the Institute of Metal Physics, UrB RAS (Ekaterinburg, Russia). In order to impart lipophilic properties to iron-carbon particles and to increase their stability in solution in the form of a colloid, a technique for preliminary deposition of organic radicals, aryldiazonium tosylates (ADT), onto the surface of these particles has been developed. An effective method for the synthesis of ADT followed by their application onto the carbon surface of particles was developed at the Tomsk Polytechnic University22. The general scheme for the synthesis of Fe@C particles and their subsequent interaction with 99mTc is shown in Fig. 3b.

Figure 3

(A) Carbon encapsulated iron nanoparticle; (B) the general scheme for the synthesis of Fe@C particles.

In the third type of colloids technetium-99m was adsorbed on aluminum oxide powder. A powder of low-temperature (cubic) modification of gamma-oxide Al2O3, prepared from aluminum hydroxide powder Al(OH)3 by its calcination in a muffle furnace, was used. The substance was synthesized in Tomsk Polytechnic University.

A reducing agent—tin (II) chloride dihydrate was used in order to obtain complexes of 99mTc with colloids.

Gelatin powdered, (Ph. Eur., USP-NF) pure, pharma grade. CAS Number: 9000-70-8 was purchased from (AppliChem GmbH (Darmstadt, Germany).


Method for preparation of 99mTc labeled nanocells

The introduction of the radioactive label 99mTc into a colloidal substance was carried out by mixing of the selected substance with the reducing agent SnCl2∙2H2O (0.175–0.35 mg/ml) in different ratios and then adding a 4.0 ml of eluate of 99mTc (280–500 MBq/ml) to the mixtures. The mixtures were incubated for 30 min at a temperature of 70–80 °C. The preparation is ready for use after cooling at room temperature. The reducing agent SnCl2∙2H2O was used as a hydrochloric acid solution. The amount of 0.07 g of tin chloride (II) is added to the vial and 0.2 ml of 1 M hydrochloric acid (HCl) is then added for its preparation. After dissolution, the volume is adjusted with distilled water to 10 ml. Dissolution was carried out in an inert gas (argon) medium.

Determination of the size of 99mTc labeled colloidal particles

The determination of the size of the labeled nanocolloids was carried out by spectroscopy on a Nanophox particle size analyzer (“Sympatec GmbH”, Germany), and also by a technique, based on measuring the activity of the suspension before and after filtering it successively through filters with predetermined pore sizes: 220, 100, and 50 nm. Three samples were taken with a volume of 5 μl from each initial solution and filtrates for the subsequent measurement of their activity. Calculations of the yield of products with different particle sizes were determined according to the formulas given below:

$$C_{220} = frac{{A_{vc} – A_{1} }}{{A_{vc} }};,C_{100} = frac{{A_{1} – A_{2} }}{{A_{1} }};,C_{50} = frac{{A_{2} – A_{3} }}{{A_{2} }},$$

where Avc is the activity of the initial suspension prior to filtration; A1 is the activity measured after filtration through a 220 nm filter; A2 is the activity after filtration through 100 nm filter; A3 is the activity measured after filtration through 50 nm filter.

In parallel, determination of the radiochemical purity (RCP) of preparations by thin layer chromatography method was carried out.

Thin-layer chromatography (TLC) procedure

To determine radiochemical purity of 99mTc–nanocolloid 5 µl of prepared sample was spotted on silica-gel impregnated strip (Sorbfil, Russia), 2 × 15 cm. To determine pertechnetate content of the radiopharmaceutical sample, first strip was developed using acetone as the mobile phase (time of chromatography 10 min). In this system, pertechnetate migrated with the front of the mobile phase (Rf = 0.9). To determine the colloid content of the preparations, the second strip was developed using ethanol:water:ammonium hydroxide (2:5:5) as the mobile phase (time of chromatography 40 min). In this system, the 99mTc–nanocolloid migrated with the front of the mobile phase (Rf = 0.9)23.


The stability of 99mTc–nanocolloid was studied in vitro by mixing of 5 ml of normal serum and 0.5 ml of 99mTc–nanocolloid following by incubation at 37 °C for 8 h. At different time points (1 h, 4 h and 8 h) 0.2 ml aliquots of complex were removed and evaluated for radiochemical purity using TLC24.

Determination of the functional suitability of preparations for scintigraphic detection of SLN

A study to assess the functional suitability of new nanocolloid RPs was performed in 3 series of experiments involving 5 white Wistar male rats weighing 300–350 g. Injection of RP in a dose of 18–20 MBq was performed between the first and second fingers of the rat’s hind paw. The animals were anesthetized with ether before the subcutaneous injection and during the scintigraphic study. Since the introduction, the kinetics of radiopharmaceutical distribution throughout organs and tissues was recorded by a frame-by-frame recording for 15 min (one frame—30 s) in a 64 × 64 pixel matrix. Static scintigraphy was performed after 1, 2, 3 and 24 h in the anterior and posterior projections in a matrix of 256 × 256 with a set of 500 pulses, scintigraphy of animals was performed on an E.CAM Signature 1800 gamma camera (Siemens, Germany). The results of scintigraphic studies determined the percentage of accumulation of RP in the lymph node and the injection site. The maintenance and participation of the animals in the experiment was carried out in accordance with the rules adopted by the “European Convention for the Protection of Vertebrates Used for Experiments or Other Scientific Purposes” (Strasbourg, 1986). The experimental protocols were approved by Cancer Research Institute Biomedical Ethics Committee, Protocol number 7/15. All invasive manipulations with animals were performed using inhalation or drug anesthesia.

Statistical analysis

All mean values are expressed as %ID/g ± SD. Data were analyzed statistically using methods of general statistics with a commercially available software package “Statistics for Windows” (StatSoft Inc., Version 6.0).


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