Newborn bovine serum

Vero cells were purchased from the UK Health Service (Biological Collection). Vero cells were cultured in Minimum Essential Medium (MEM) (Gibco, New York, NY, USA) supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere with 5% CO₂, and passaged when the cells were cultured to 90% density. Trypsin is an example of an animal-based enzyme, while TrypLE is an example of an animal-origin-free enzyme. The 0.25% EDTA-Trypsin was provided by vaccine room six of the Beijing Institute of Biological Products Co., Ltd. (Beijing, China). TrypLE was 1× and purchased from Gibco (Brooklyn, NY, USA), and contains 200 mg/L KCl, 200 mg/L KH₂PO₄, 8000 mg/L NaCl, 2160 mg/L Na₂HPO₄-7H₂O, 457.6 mg/L EDTA, and 100 mg/L Phenol Red. Non-enzymatic cell lysate was purchased from Sciencell Company (Carlsbad, CA, USA), and the product number was 0123. Vero cell growth medium containing 10% bovine serum was provided by vaccine room six of the Beijing Institute of Biological Products Co., Ltd. Newborn bovine serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. (Zhejiang, China).

The size, polydispersity (PDI), and zeta potential of SHK@HA-MPDA nanoparticles were measured by a Zeta Sizer Nanoparticle Analyzer (Malvern Panalytical, Malvern, UK). The morphological observation of SHK@HA-MPDA was carried out by transmission electron microscopy (TEM). The drug encapsulation efficiency (EE%) and drug-loading capacity (DL%) of SHK@HA-MPDA were determined by high-performance liquid chromatography (HPLC) (1260 Infinity, Agilent Technologies, Santa Clara, USA) equipped with Agilent-C18 (5 μm, 250 mm × 4.6 mm). The chromatographic conditions for SHK determination were listed as follows: mobile phase composed of methanol (90%) and phosphoric acid (0.01%) aqueous solution, flow rate of 1 mL/min, and detection wavelength of 516 nm. The in vitro drug release was evaluated by a dialysis method using a dialysis membrane (MWCO 8,000–14,000 Da) in PBS (pH 5.0) containing 0.5% (w/v) SDS with 150 rpm/min shaking at 37 °C. The released drugs were quantified by HPLC as described above. The serum stability of SHK@HA-MPDA was evaluated by suspending them in PBS containing 10% new-born bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd, Hangzhou, China) with a general shaking at 150 rpm at 37 °C. The size of particles at a specified time was measured by a Zeta Sizer Nanoparticle Analyzer.