Materials

Amriya Pharmaceutical Co. (Alexandria, Egypt) generously supplied TAM and soybean lecithin (Phospholipon 90G, 90% w/w of phosphatidyl choline). United Company for Chemical and Medical Preparations (Cairo, Egypt) supplied the 99% methyl alcohol (absolute). Stearic acid and tween 80 were purchased from EL-Gomhoria Company, Cairo, Egypt. Chloroform, Labscan Ltd, Dublin, Ireland. We bought cellulose membrane for dialysis tubing (Molecular Weight Cut Off 12,000–14,000) from Sigma Aldrich in Germany. The [99Mo]Mo/[99 mTc]Tc generator provided by the Egyptian Second Research Reactor (ETRR- 2) of the Radioisotopes Production Facility (RPF), Egyptian Atomic Energy Authority, was used to elute sodium pertechnetate (Na[99mTc]TcO4). All other chemicals and solvents were of analytical grade.

MethodsPreparation of TAM-SLNs

The SLNs were made using a modified low-temperature solidification and melt emulsion/ultrasonication process [21, 22]. Weighed 20 mg of TAM were dissolved in methanol and poured into different concentrations of lipid melt (stearic acid) at 65–70 °C to produce a uniform, transparent drug-lipid combination. A crude emulsion was produced by emulsifying the drug-lipid mixture in an aqueous solution of a surfactant (Tween 80 or lecithin), which was mechanically stirred at 6,000 rpm and heated to 10 °C above the melting point of the lipids. Then, using an ultrasonic processor, the crude emulsion was ultrasonically treated in a water bath for 10 min at 90 °C [23]. After cooling to room temperature, the SLN nanoparticles were produced and suspended in the medium. The effects of surfactant type, concentration, and lipid concentration were studied (Table 1).

Table 1 Composition of different prepared TAM-SLNs formulasCharacterization of prepared TAM-SLNs formulasDrug loading and entrapment efficiency

A reverse phase column C18 phenomax 150 × 4.6 mm with a porosity of 5 × 10–3 mm as a stationary phase was used in HPLC with a Shimadzu chromatographic system to calculate the percentage of drug loading capacity (%DL) and entrapment efficiency (%EE). For Tamoxifen citrate separation, Monteagudo et al. (2012) [24] determined that a polar mobile phase of triethylamine acetate buffer: methanol with a ratio of 24.3:75.7 v/v was ideal. The drug was detected by UV at 275 nm, and the injection volume was 50 µL with a flow rate of 1.5 mL/min. The %DL and %EE refer to the percentage of drug entrapped in SLNs according to the following equations, respectively:

$$=\frac}}\times 100$$

(1)

$$=\frac}}\times 100$$

(2)

Assessment of particle size, PDI, and zeta potential

Using a Zetasizer (Malvern NanoZS, Malvern Instruments, Malvern, UK), the dynamic light scattering method was used to quantify the mean particle size (PS) and polydispersity index (PDI) of TAM-SLNs. Disposable cells were used to average five observations at a 173° angle in order to acquire the results. Disposable simple folded capillary zeta cells were used to measure the zeta potential (ZP) of the samples. At 25 ± 2 °C, every measurement was made at least five times.

Imaging with transmission electron microscope

Using a transmission electron microscope (Thermo Scientific Apreo S, Waltham, MA, USA) at high vacuum, the size and surface characteristics of nanoparticles were investigated. For this, a Leica EMACE 600 (Leica Microsystems, Wetzlar, Germany) brand coating apparatus was used to first coat the samples with 80% gold and 20% palladium at a thickness of 7 nm. The vacuum used to prepare the coating was 5 × 10−4 mbar. The coated samples were scanned under elevated voltage settings of 5 kV and at a magnification range of × 50,000.

Selection of formula candidate for further studies

Based on the results of previously performed investigations, a certain criterion was implemented for selection of the best formula to be subjected for further testing. The formula showing the least particle size, least PDI accompanied with maximum encapsulation efficiency and Zeta potential will be considered as the best produced preparation.

Radiolabelling process and quality control test

To prepare TAM solution; 10 of tamoxifen was dissolved in 1 ml methanol: H2O (2:1) to ensure proper solubility before radiolabelling. Technetium-99m is typically obtained as sodium pertechnetate (Na99mTcO₄) by using 0.9% saline solution from a molybdenum-99 generator,in a hepta-oxidation state, which cannot label any chemical compound through direct addition. So, shortly before the labelling operation, [99mTc]Tc reduction is required to convert [99 mTc]Tc + 7 from the hepta state to a beneficial lower oxidation state, which can combine with the ligand to generate the radiopharmaceuticals [25]. This reduction process was done by using SnCl2 as a reducing agent. The reduced technetium [99mTc]Tc interacts with TAM using the direct labelling technique forming the radiolabeled complex. The radiolabelled SLNs were prepared using the radiolabelled TAM, following the same procedure described previously. In order to identify the ideal reaction conditions, the required amounts (50–5000 µg) of TAM were transferred into clean 10-mL vials which were kept under positive N2 pressure. Then, the pH was adjusted using different volumes of 0.1 M HCl, 0.1 M NaOH or buffer solutions. After that, the required amount of SnCl2·2H2O (10–200 µg) and then 100 µL of freshly eluted pertechnetate solution (195 MBq) were added to each vial at different time intervals.

Each point's experiments were carried out in triplicate, and the student's t-test was used to assess data differences. Mean ± SD is used to present the results. A significant threshold of P < 0.05 was established. For conclusive results, the radiochemical purity was assessed and optimized utilizing both thin-layer chromatography (TLC) and paper electrophoresis. Acetone was utilized as the developing solvent in thin-layer chromatography to determine the free 99mTcO4−, while ethanol, water, and ammonium hydroxide (2: 5: 1) was used as a developing solvent to calculate the (R/H)-[99mTc]NaTcO4 [26, 27].

Thus, the contribution of [99mTc]Tc-ligand can be calculated as follows:

$$\left[^}\text\right]\text-\text=100-\left(\;^}}_^}+\left(\text/\text\right)-\left[^}\text\right]}_\right)$$

(3)

The strips were dried, cut into 0.5 cm pieces, and counted in a well-type γ-scintillation counter once the mobile phase had fully developed. A Whatman paper sheet measuring 2 cm in width and 47 cm in length was used for the paper electrophoresis study. A reaction mixture containing 1–2 μL was deposited on the paper sheet 12 cm away from the cathode edge. Normal saline solution (0.9% w/v) was used as the electrolyte solution for the 1.5-h electrophoresis, which was conducted at a voltage of 300 V. Following full development, the paper was taken out, dried, and cut into 0.5 cm long strips, which were subsequently counted in a well-type γ-counter [28].

Determination of [99mTc]Tc-TAM-SLNs and [99mTc]Tc-TAM in vitro stability

For 24 h, the in vitro stability of both [99mTc]Tc-TAM-SLNs and [99mTc]Tc-TAM were assessed at room temperature (25 ± 1 °C). Additionally, they were evaluated at 37 °C with human serum (compound: serum ratio of 1:8). Samples (2 µL, n = 3) were extracted from the reaction mixture at the proper intervals (1, 2, 4, 6, and 24 h post-incubation), and were subjected to the previously described chromatographic procedure and the radiochemical purity was assayed using a well-type gamma counter (Sesa Uniscaller) [26].

In-vivo Biodistribution study

With ethical clearance number 17PA/23, all animal studies were performed in accordance with EAEA NCRRT-REC criteria, and the study was carried out in accordance with national and international laws controlling the use of radioactive material. For the 99mTc-ligand complexes'in vivo biodistribution investigations, four groups of twelve female Swiss Albino mice each were used (25–30 gm). Each animal received a 0.2 ml injection in the tail vein of a freshly produced solution comprising 200–400 KBq of [99mTc]Tc-TAM-SLNs. For the necessary amount of time, the mice were housed in metabolic cages. After administration, at appropriate time intervals of 15, 60, 120, and 240 min, mice were anesthetized and blood samples were withdrawn by cardiac puncture into pre-heparinized polypropylene tubes. Tissue samples from the main organs were isolated, washed with 0.9% saline, weighed in pre-weighed tubes, and their activity was measured in a well-type γ-scintillation counter (SR- 7) [29]. The radioactivity uptake in each tissue sample was calculated as a fraction of the injected dose per gram (ID/g). It was estimated that the weight of muscle, bone, and blood constituted 7%, 10%, and 40% of the total body weight, respectively [30].

A solid tumor was induced in Swiss Albino mice using Ehrlich ascites carcinoma cells (EAC), a model based on a mouse mammary carcinoma [31]. In female Swiss Albino mice, a line of Ehrlich ascites carcinoma (EAC) was maintained by intraperitoneal (I.P.) transplantation of 2.5 X 106 tumor cells/mouse. Aseptic needle aspiration was used to extract EAC cells. 2.5 X 106 cells were counted microscopically using a hemocytometer in 0.1 mL of the ascitic fluid after it was diluted with sterile saline. Then, 0.2 mL of the solution was injected intramuscularly in the right thigh muscle to create a solid tumor [32, 33].

Pharmacokinetic behavior

The WinNonlin program (Ver. 1.5, Scientific Consulting Inc., Cary, NC) was used to calculate the pharmacokinetics parameters of both the plain drug and the formulation using non-compartment analysis [34]. The mean amount of radioactivity uptake (%ID/g) in blood and solid tumor samples was plotted against time (h). The radioactivity uptake is a direct indication of the amount of labelled TAM (either in [99mTc]Tc-TAM solution or loaded in the [99mTc]Tc-TAM-SLNs). This relationship allows recording the maximum concentrations of their uptake (Cmax) and the time for maximum uptake (Tmax). Additionally, the area under the curve from 0 to 240 min (AUC (0–240), min %ID/g), and from 0 to infinity (AUC (0-∞), min %ID/g), were estimated. The capacity of the formula to target the tumor through the intravenous route has been determined by the drug targeting efficiency (DTE) and the relative targeting efficiency (RTE) [35]. The AUC used here is that from 0 to 240 min and from 0 to ∞ following intravenous administration for both drug and delivery system ([99mTc]Tc-TAM solution and [99mTc]Tc-TAM-SLNs) and the plain drug. DTE is defined as the average partitioning time ratio between the tumor and the blood for both the nanoformula and the plain drug and can be calculated by dividing the AUC from 0 to 240 min for the tumor and blood. The DTE and the RTE were calculated using the following formulas [36].

$$RTE\%=\frac}0-240\;}100$$

(5)