A double emulsification technique was used to produce the PLA/MMT/TRA NPs. Briefly, 50 mg of PLA (Sigma-Aldrich) were dissolved in 2 mL of dichloromethane (Sigma-Aldrich) and added to 200 µL of PVA (Sigma-Aldrich) solution (0.1% w/v, and 50 mg of Trastuzumab (TRA, Herceptin, Roche Laboratories). This solution was sonicated (UP100H, Hielscher, Teltow, Germany) for two cycles of 30 s at 45 W. Then, 4 mL of aqueous PVA solution (0.7% w/v) and 0.42% clay (MMT; Montmorillonite; Sigma-Aldrich) were added and sonicated (UP100H, Hielscher, Teltow, Germany) for 60 s. The residual organic solvent was evaporated (Rotavapor R114, Buchi, Postfach, Switzerland) under reduced pressure for 1 h. Finally the NPs were recovered by ultra-centrifugation (20,000 rpm) at 25 °C for 20 min (Centrifugal Beckman Coulter J 25, Brea, California, USA). Physicochemical property measurements of freshly synthesized NPs were immediately performed61 (link). Monoclonal antibody entrapment efficacy in PLA/MMT/TRA particles was measured using a BCA assay in parallel62 (link).
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