Montmorillonite

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 performed^{61 (link)}. Monoclonal antibody entrapment efficacy in PLA/MMT/TRA particles was measured using a BCA assay in parallel^{62 (link)}.

Acros-Organics company was the supplier of Chitosan (CS) with a molecular weight of 100,000–300,000 (Zeel West Zone 2, Janssen Pharmaceuticalan 3a, B2440, Geel, Belgium). Poly(vinyl alcohol) (PVOH) (low molecular weight i.e., 13,000–23,000, hydrolysis degree 87–89%) was purchased by SIGMA-ALDRICH (Co., 3050 Spruce Street, St. Louis, MO, USA, 314-771-5765). Nanocor Inc. was provided the powder nanoclay (2870 Forbs Avenue, Hoffman Estates, IL, USA). Montmorillonite which contained 1.53% halloysite nanotubes (Al₂Si₂O₅(OH)₄∙2H₂O, 99.5% clay, 1.68% CaO, 3.35% Fe₂O₃, 62.9% SiO₂, 19.6% Al₂O₃, and 3.05% MgO) was obtained by Sigma-Aldrich (product 685445, Sigma-Aldrich, St. Louis, MO, USA). The used Thyme Oil (TO) was produced by the Chemco company (Via Achille Grandi, 13–13/A, 42030 Vezzanosul, Crostolo, Italy).

Acros-Organics company (Zeel West Zone 2, Janssen Pharmaceuticalan 3a, B2440, Geel, Belgium) was the provider of chitosan with a molecular weight of 100,000–300,000. SIGMA-ALDRICH, Co., 3050 Spruce Street, St. Louis, MO 63103 USA 314-771-5765 was the supplier of poly(vinyl alcohol) (PVOH) with low molecular weight (13,000−23,000) and hydrolysis degree of 87−89%. The same company was also the provider of zinc acetate dihydrate (Zn(CH₃COO)₂·2H₂O), 25% ammonia solution, and montmorillonite (Mt) (code name Nanomer PGV) with a mass density of 2.6 g/cm³ and CEC value of 145 meq/100 g. The last chemical was purchased from Nanocor Inc., 2870 Forbs Avenue Hoffman Estates, IL 60192, IL, United States. Sigma-Aldrich was the supplier of the montmorillonite (product 685445, Sigma-Aldrich, St. Louis, MO, USA) with a chemical composition of 1.53% halloysite nanotubes (Al₂Si₂O₅(OH)₄·2H₂O, 99.5% clay), 1.68% CaO, 3.35% Fe₂O₃, 62.9% SiO₂, 19.6% Al₂O₃, and 3.05% MgO.