2.2. Preparation of SPCIONPs

Ferric chloride hexahydrate (2 g) and ferrous sulfate heptahydrate (1.4 g) were dissolved in 5 ml of water, followed by ultrasonication until the chemicals were completely dissolved. The solution was then transferred to a three-necked flask and stirred under nitrogen. Subsequently, 20 ml of 0.1% citric acid solution was added to the solution, which was then stirred for 10 min. The three-necked flask was then placed in a water bath at 85°C. A total of 20 ml ammonium hydroxide was added with a dropping funnel; the addition was completed within 10 min, and stirring was continued for another 30 min [27,28]. The resulting suspension was centrifuged at 8500 r.p.m. for 10 min, the sediment was discarded, and the supernatant was removed to obtain the ferrofluid of citric acid-coated magnetite particles (SPIONPs). Finally, 5 ml of the ferrofluid of citric acid-coated magnetite particles was added to MES buffer solution with a pH of 5, and EDC and NHS were subsequently added under stirring to activate the carboxyl group on the surface of the ferrofluid of citric acid-coated magnetite particles, followed by the addition of 5 ml of 20 mg ml⁻¹ 50 kDa CS solution in acetic acid and stirring at 300 r.p.m. at room temperature for 6 h [29]. The liquid was collected after 48 h of dialysis with a dialysis bag (retained molecular weight over 50 000). A portion of the liquid was lyophilized and stored at 4°C for later use.