**4. Conclusions**

This study attempted to design and optimize RSV-loaded NLC prepared by a combination of high shear homogenization and ultrasound method. After selecting the critical process variables affecting particle size (PS) and polydispersity index (PDI), a 22 factorial design with triplicate of the central point was employed to plan and perform the experiments. Zeta potential, morphology, drug entrapment efficiency, production yield, and stability index were also measured. RSV-loaded NLC and NLC without RSV (placebo) were prepared. Optimized NLC formulation was prepared based on the predicted optimum levels of the independent variables, shear intensity, and homogenization time of the factorial design using Pareto charts and surface response charts, and on instability index. Thus, optimal parameters for NLC were obtained using shear intensity of 19,000 rpm and shear time of 6 min, producing NLC with PS around 135 nm and DPI around 0.4. Moreover, these production process parameters produced particles with high entrapment efficiency (~93%) and production yield (~65%).

**Author Contributions:** A.A.M.S., M.H.A.S., A.R.V.F., and E.M.B.S. contributed for the conceptualization, methodology, validation, formal analysis, and investigation. A.A.M.S., A.R.V.F., E.S.-L., and N.R.E.F. contributed for the Writing—Original draft preparation. A.A.M.S., M.H.A.S., and E.M.B.S. contributed for supervision, Writing—Review and editing, project administration, resources, and funding acquisition. All authors have made a substantial contribution to the work.

**Funding:** The work has received financial support from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo), Project's numbers 2017/034968 and 2014/27200-2 and from the Portuguese Science and Technology Foundation, Ministry of Science and Education (FCT/MEC) through national funds, and co-financed by FEDER, under the Partnership Agreement PT2020 for the project M-ERA-NET/0004/2015-PAIRED.

**Conflicts of Interest:** The authors declare no conflict of interest.
