The standard CNPs (SCNPs) were prepared using the Bottom-up method. Briefly, 5.33 mg/mL of MO (Danisco A/S, Denmark) were dissolved in ethanol and dispersed in an aqueous phase at a ratio of 1:3 v/v.38 (link) Dextran (Pharmacosmos, Denmark) and Kolliphor® P407 (BASF, Germany) (4.32 mg/mL and 173 μg/mL, respectively) were previously dissolved in MilliQ water. The resulting formulation was mixed for 15 minutes by moderate magnetic stirring.
For the preparation of the cationic and chitosan-coated cubosomal nanoparticles liquid formulations, several proportions of lipid mixtures were prepared by the thin lipid film hydration method to evaluate their influence on the physicochemical properties of the resulting cubosomes. Briefly, the lipid mixtures were composed of 70:25:5, 60:30:10, 50:35:15 and 40:40:20%MOL of MO, CHO (Sigma-Aldrich, USA) and charged lipids (DOTAP or DOPS, cationic and anionic lipids, respectively) (Lipoid GmbH, Germany). The lipids were introduced into a round-bottomed opaque flask and dissolved by a mixture of organic solvents (dichloromethane:methanol 50:50 v/v). Except for the blank formulations, the PP (Biochem Partner, China) was previously dissolved in the organic solvent-lipid mixture.
The organic solvents were removed within 20 minutes using a Rotavapor R-205 (Büchi Labortechnik, Flawil, Switzerland) at 60°C to produce a thin lipid film at the internal surface of the round-bottom flask. The pressure was set at 250 mmHg for 10 minutes and 150 mmHg for the next 10 minutes.
Finally, the Bottom-up method was used to generate the CNPs. This process relies on the lipid film dissolution by ethanol and, then, by the precipitation of the cubosomes by adding of an excess of an aqueous phase. In this work, Kolliphor® P407 (10% w/w of lipid mixture) was dissolved in MilliQ water to stabilize the colloidal system.
A further coating step of the anionic CNPs with chitosan followed this procedure to produce the chitosan-coated CNPs (ChCNPs) liquid formulation, this procedure was followed by a further step of coating the anionic CNPs with chitosan low molecular weight (50–190 kDa, 75–85% deacetylated, Sigma-Aldrich, USA). A solution of 10% w/v of chitosan was prepared in 1% v/v acetic acid solution. Then, this solution was added drop by drop under vigorous magnetic stirring for 10 minutes in the previously formed anionic liquid formulation to obtain a final product with 0.1% w/v of chitosan. So, the anionic CNPs were positively charged thanks to the chitosan coating.
For the preparation of the cationic and chitosan-coated cubosomal nanoparticles liquid formulations, several proportions of lipid mixtures were prepared by the thin lipid film hydration method to evaluate their influence on the physicochemical properties of the resulting cubosomes. Briefly, the lipid mixtures were composed of 70:25:5, 60:30:10, 50:35:15 and 40:40:20%MOL of MO, CHO (Sigma-Aldrich, USA) and charged lipids (DOTAP or DOPS, cationic and anionic lipids, respectively) (Lipoid GmbH, Germany). The lipids were introduced into a round-bottomed opaque flask and dissolved by a mixture of organic solvents (dichloromethane:methanol 50:50 v/v). Except for the blank formulations, the PP (Biochem Partner, China) was previously dissolved in the organic solvent-lipid mixture.
The organic solvents were removed within 20 minutes using a Rotavapor R-205 (Büchi Labortechnik, Flawil, Switzerland) at 60°C to produce a thin lipid film at the internal surface of the round-bottom flask. The pressure was set at 250 mmHg for 10 minutes and 150 mmHg for the next 10 minutes.
Finally, the Bottom-up method was used to generate the CNPs. This process relies on the lipid film dissolution by ethanol and, then, by the precipitation of the cubosomes by adding of an excess of an aqueous phase. In this work, Kolliphor® P407 (10% w/w of lipid mixture) was dissolved in MilliQ water to stabilize the colloidal system.
A further coating step of the anionic CNPs with chitosan followed this procedure to produce the chitosan-coated CNPs (ChCNPs) liquid formulation, this procedure was followed by a further step of coating the anionic CNPs with chitosan low molecular weight (50–190 kDa, 75–85% deacetylated, Sigma-Aldrich, USA). A solution of 10% w/v of chitosan was prepared in 1% v/v acetic acid solution. Then, this solution was added drop by drop under vigorous magnetic stirring for 10 minutes in the previously formed anionic liquid formulation to obtain a final product with 0.1% w/v of chitosan. So, the anionic CNPs were positively charged thanks to the chitosan coating.
