Our study investigated the manufacturing of lipid-based nanotherapeutics with stealth properties for siRNA delivery by employing a diffusion-driven microfluidic process in one or two-steps strategies to produce siRNA-loaded lipid nanocarriers and lipoplexes, respectively. In the one-step synthesis, siRNA in the aqueous phase is introduced from one inlet, while phospholipids dispersed in anhydrous ethanol are introduced from other inlets, generating the lipid nanocarriers. In the two-steps strategies, the pre-formed liposomes are complexed with siRNA. The process configuration with an aqueous diffusion barrier exerts a significant effect on the nanoaggregates synthesis. Dynamic light scattering data showed that lipid nanocarriers had a bigger particle diameter (298 ± 24 nm) and surface charge (43 ± 6 mV) compared to lipoplexes (194 ± 7 nm and 37.0 ± 0.4 mV). Moreover, DSPE-PEG(2000) was included in the formulation to synthesize lipid-based nanotherapeutics containing siRNA with stealth characteristics. The inclusion of PEG-lipid resulted in an increase in the surface charge of lipoplexes (from 33.7 ± 4.4–54.3 ± 1.6 mV), while a significant decrease was observed in the surface charge of lipid nanocarriers (30.3 ± 8.7 mV). The different structural assemblies were identified for lipoplex and lipid nanocarriers using Synchrotron SAXS. Lipid nanocarriers present a lower amount of multilayers than lipoplexes. Lipid-PEG insertion significantly influenced lipid nanocarriers’ characteristics, drastically decreasing the number of multilayers. This effect was not observed in lipoplexes. The association between process configuration, lipid composition, and its effect on the characteristics of lipid-based vector systems can generate fundamental insights, contributing to gene-based nanotherapeutics development.