To surface adsorb NP +/- chitosan with PspA4Pro, 1 mL of 1 mg/mL PspA4Pro was added to the 3 mL of 25 mg NP suspension (produced in 2.2 and 2.3) and mixed with a magnetic stirrer at room temperature for 1 h. The NP suspension was ultracentrifuged at 80,000 × g in a Beckman Coulter type 70.1 rotor. The adsorption of PspA4Pro on the NPs was quantified using a previously developed high performance liquid chromatography (HPLC) method in our group (Kunda et al., 2015). The adsorption was calculated by the difference between the initial amount of PspA4Pro and the amount detected in the supernatant

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