Minigita

TLC analysis was carried out on silica gel plates 60 F₂₅₄ (Merck), TLC Al foils (Sigma Aldrich) or Polygram SIL G/UV₂₅₄ (Macherey Nagel); radioactivity distribution was measured using a MiniGita radioTLC scanner (Raytest). An aliquot of the quenched reaction mixture (2–3 µL) was applied to a TLC plate; the plate was eluted with 4:1 hexane/EtOAc (A) or 9:1 hexane/EtOAc (B). After developing the plate was immediately covered by tape to prevent the losses of the volatile radioactive products. The R_f values of the different compounds are compiled in Table 2. The RCC (radiochemical conversion) was determined by dividing of the product peak area by the total peak area. TL-chromatograms can be found in the Supplementary Materials.

The radiochemical purity was determined via reverse-phase HPLC and ITLC. HPLC was performed on an Agilent 1260 system using a C18 column 150 × 3 mm, 3 μm (Thermo Fisher Scientific, Dreieich, Germany), and for radiochemical purity determination, a NaI (Tl) radiodetector (Gabi, Raytest, Straubenhardt, Germany) was used. The mobile phase consisted of gradient mixtures of acetonitrile and 0.1% aqueous TFA with a flow rate of 0.5 ml/min.
ITLC was performed by chromatography paper impregnated with silica-gel (ITLC-SG, Agilent Technologies) using ammonium acetate (1 M) and methanol (1 : 1) as the mobile phase. The strips were analyzed using the thin-layer chromatography scanner (miniGita, Raytest, Germany).

Radiolabeling of LEVs was achieved using [^99mTc]Tc-Annexin-V-128 (AnnV), a phosphatidylserine-targeting radiotracer for single photon emission-computed tomography (SPECT). The radiotracer was prepared by radiolabeling Annexin-V-128 lyophilizate (Advanced Accelerator Applications, Saint Genis Pouilly, France) with a fresh [^99mTc]TcO₄^- pertechnetate solution (740 MBq/100 μL) eluted from a commercial [⁹⁹Mo]Mo/[^99mTc]Tc generator (Tekcis, Curium, Paris, France). After gentle stirring, the solution was incubated for 90 min at room temperature. Radiochemical purity was assessed by instant thin layer radiochromatography using a radiochromatograph (miniGITA, Elysia-Raytest, Straubenhardt, Germany), Whatman paper (Cytiva, Marlborough, MA, USA) as stationary phase, and citric acid–citrate–dextrose solution (ACD/A solution, Fresenius-Kabi, Paris, France) as mobile phase. A radiochemical purity above 95% enabled to validate the radiolabeling. A measure of 200 MBq/100 μL of radiotracer was mixed with 15 μL of concentrated binding buffer solution (140 mmol·L⁻¹ NaCl; 25 mmol·L⁻¹ CaCl₂; 10 mmol·L⁻¹ HEPES and pH 7.4), added to a sample of 100.10⁶/50 μL LEVs, leading to a final volume of 165 µL and incubated for 20 min at room temperature.

p-SCN-Bn-DOTA (2.4 mg) was added to the Ch 18 KD suspension in carbonate buffer (pH, 9) followed by stirring with 1200 rpm at room temperature for 36 h. Purification of chitosan- p-SCN-Bn-DOTA (Ch-DOTA) conjugate was performed by 500- 1000 Da dialysis tube (spectrum labs, USA) in distilled water.
The generator was eluted with 0.5 M HCl solution. 150 µl Ch-DOTA (1.5 mg/ml) was mixed with 2 ml collected fraction (9 mCi activity) which its pH had been adjusted to 4.5 by NaOH solution 0.6 M. The mixture was incubated at 90 ^oC for 5 min. The unbounded ⁶⁸Ga was separated from solution using Amicon® centrifugal filter (10 KD cutoff).
The yield of radio labeling before and after purification was determined using thin layer chromatography (TLC) on Whatman® paper No. 3 (Sigma Aldrich, USA) and 100 mM sodium citrate solution as a mobile phase. The unbounded ⁶⁸Ga was evaluated by TLC scanner (MiniGita, Elysia-Raytest, Germany).
The radio labeled nano PECs was prepared by the method described in sec. 3.3.1 and the formulations presented in Table 1. The amount of Ch-DOTA was 45 µg/ml in each formulation which was subtracted from Ch quantity in formulation to kept Ch molarity constant.

Nanobots were labelled with [¹⁸F]F-PyTFP, on the basis of a previously established procedure with minor modifications^{33 (link)}. In brief, 200 µl of nanobot solution (1 mg ml⁻¹) was centrifuged (10 min, 13,853g), resuspended in 10 µl of PBS (1 mM, pH 8), and incubated with 4 µl of [¹⁸F]F-PyTFP in acetonitrile (about 37 MBq) for 35 min at room temperature. After incubation, the reaction mixture was diluted with water (200 µl) and purified by centrifugation (5 min, 13,853g). The resulting pellet was then rinsed three times with water before being measured in a dose calibrator (CPCRC-25R, Capintec). Radiochemical yield was calculated as the ratio between the amount of radioactivity present in the nanobots after washing and the initial amount of radioactivity. Radiochemical purity after purification was ≥99%, as determined by radio thin-layer chromatography (radio-TLC) using iTLC-SG chromatography paper (Agilent Technologies) and dichloromethane and methanol (2:1) as the stationary and mobile phases, respectively. TLC plates were analysed using a TLC reader (MiniGITA, Raytest).

Sodium [^99mTc]pertechnetate solution (Na⁺, [^99mTc]TcO₄⁻) was eluted with a physiological isotonic solution (NaCl, 0.9% m/v) from a commercial [⁹⁹Mo]Mo/[^99mTc]Tc generator (Tekcis^®, Curium, Saclay, France). After elution, radiochemical purity (RCP) was assessed using radio-thin layer chromatography using a radio-chromatograph (miniGITA^®, Elysia-Raytest, Liege, Belgium), with cellulose filter paper (Whatman #5, Cytiva, Velizy-Villacoublay, France) as the stationary phase, and acetone as the mobile phase.