*3.3. A Two-Stage Drug Release Effect of SLPs*

A few nanocarriers have been proposed capable of transporting and releasing an encapsulated drug upon activation by different stimuli. Two approaches can be adopted in designing stimuli-responsive drug nanosystems. In one approach, endogenous stimuli can be exploited for enhancing drug release. These include pH or enzymatic degradation [41]. This effect requires the selection of appropriate materials for designing the nanocarriers, which should respond to a specific endogenous stimulus releasing all of the encapsulated

drug simultaneously (the so-called "burst release"). In the second approach, physical stimuli are applied externally to targeted tissue after administration of drug-loaded specific nanocarriers. These exogenous stimuli include temperature, light, magnetic field, electric field, and ultrasound [42]. The application of these exogenous stimuli is responsible for the alteration of the structure of specifically designed nanocarriers, which leads to drug release at targeted tissues [43,44]. In some nanosystems such as liposomes [45], the drug is released in two steps. The first is upon contact with blood, releasing the drug adsorbed on their surfaces producing a "burst-release effect" that triggers a peak of drug activity at the local or systemic level that is somewhat similar to the effect seen when the free drug is administered. During the second phase, the intraparticle cargo is released.

To investigate the effect of these DOX-loaded SLPs in vitro, we used malignant melanoma cells. Fluorescence confocal microscopy imaging of the cultures revealed that the specific DOX fluorescence progressively accumulated in the cell nucleus during the first 48 h (Figure 2a, red channel), showing an efficient release of drug from the SLPs in the cells.

**Figure 2.** DOX release from DOX-loaded SLPs (SLPs-DOX) in cultured cells and in vitro. (**a**) Fluorescence confocal microscopy projection images of malignant melanoma cells treated with SLPs-DOX for 2, 16, and 48 h. The SLPs appear in the green channel, and microtubules are shown in the blue channel. The red nuclear fluorescence is indicative of the presence of released DOX. (**b**) In vitro drug release profile of the SLPs-DOX particles in physiological conditions (PBS at 37 ◦C). The results are mean ± SEM of three experimental replicas. (**c**) Scheme of treatment with SLPs-DOX against metastasis in the lungs.

Drug release from SLPs-DOX was also quantified in vitro (Figure S4). To that purpose, 0.3 mL of SLPs-DOX was resuspended in PBS at a concentration of 20 mg DOX/mL and incubated in rotation at 37 ◦C inside dialysis membranes. Figure 2b shows how DOXloaded SLPs present an initial burst-release phase, where ca. one-third of the encapsulated drug is released in the media during the first 5 h. After this initial step, a pattern of prolonged and sustained drug release was observed that lasted for more than 40 days. Drug release data were fitted to a Korsmeyer–Peppas model [46]. The release profile of

DOX can be explained by this model, with a release exponent value of 0.28 and *R* <sup>2</sup> value of 0.96. The mechanism of drug release confirmed that the SLP drug release mechanism was diffusion (Fickian model) with a slope of <0.5. These release results are consistent with other systems based on lipid matrix particles described elsewhere [47–49].
