**4. Discussion**

Considerable therapeutic effort in oncology has been focused on stopping cancer growth. Currently, patients with advanced metastasis have a low probability of recovery because there are no treatments to stop or prevent this process. Conventional drug systems and conventional drug carrier production methods have several limitations, such as frequent dosing, poor bioavailability, or poor patient compliance [50]. These formulations are designed in such a way that the therapeutic concentration of the drug must always be within the therapeutic window while trying to avoid the toxic effects that are produced by an overdose [51].

For this purpose, it is essential to study the drug concentration level in blood. On one hand, a high single dose of the drug could cause toxic side effects. On the other hand, lower administrated doses at different times can maintain the drug concentration in plasma, but without an efficient response from the clinical point of view. Therefore, targeted and maintained frequent drug administration with low doses could be a possible solution. In this scenario, smart delivery systems can make a significant contribution. The benefits of such delivery systems are (i) lower dosing frequency dictated by the matrix that releases the drug at a predetermined rate, (ii) higher bioavailability, (iii) improved drug stability, (iv) reduced toxic effect of the drug due to chronic and repetitive use, and (v) reduced drug loss due to continuous elimination [52]. In this context, there are numerous types of drug delivery nanocarriers but there are also many issues associated with them: toxicity, low encapsulation efficacy, lack of drug stability, inability to encapsulate more than one drug, or rapid and ineffective release.

The SLPs chosen in this work are stable, inert, and safe, and most importantly, they provide long-term retention of drugs [53], with the ability to encapsulate drugs with limited solubility. The results presented here show that the drug encapsulation efficacy was 86% with a sustained release for more than 40 days at a predetermined rate (Figure 2). This is an important advantage of these systems compared with other nanocarriers such as liposomes that, in general, cannot achieve such high encapsulation efficiency values (recently developed high-pressure processes [54] can achieve drug encapsulation above 90% but are still not common practice). Furthermore, as shown in Figure S2a, these SLPs are retained in the lungs, allowing sustained release of the drug directly in tissues with a high risk of metastasis.

In this study, we encapsulated DOX for different reasons. It is a well-known drug that is commonly used in a variety of cancers; also, its encapsulation contains its undesirable effects such as low specificity to tumor cells, toxic effect in healthy tissues, and an initial burst release followed by a strong decrease in drug concentration when administered on its own [47]. In fact, its clinical use is limited due to its cardiotoxicity and nephrotoxicity [55,56]. We demonstrate that the encapsulation of the drug in SLPs improves the antimetastatic effect of the free drug (Figure 5), improving the biocompatibility in the mice (Figures S5 and S6).

As mentioned above, stable and controlled long-term sustained drug release provides superior therapeutic outcomes than periodically supplied individual higher doses [6,7]. Intravenous administration of conventional drugs usually leads to initial high concentrations followed by a rapid concentration drop below therapeutic limits as a result of drug metabolization or elimination [22]. Thus, our system hypothesizes that the drug release occurs in two well-differentiated steps. The initial one, attributed to the first 5 h of release observed in vitro, occurs while the nanocarrier is circulating in the bloodstream. This allows the elimination of most of the circulating cancer cells. The second, a sustained-release step, takes place upon nanocarrier arrival at the target destination. This surely inhibits tissue colonization by metastatic cells. This drug release pattern would significantly reduce the initial drug dosages and decrease the adverse effects of chemotherapy. Since in the preparation of these solid lipid particles the drug is both integrated within the matrix and adsorbed on the surface of the particles, as concluded by the significant change in the ζ potential from −10 to +23 mV, our system is able to provide this unique two-step drug release effect.

It is well known that mouse models are an important tool to understand the complex mechanism involved in the metastatic process and to identify new targets or improve therapeutic approaches [57]. Previous studies have already established the antitumor efficacy of this kind of formulation in melanoma models [26,27]. Here, we have focused on the effect of SLPs-DOX on metastasis inhibition. To evaluate this antimetastatic effect in vivo, a melanoma lung metastasis model was generated according to Figure 4. This model presents the advantage of producing naked-eye-visible black spots on the lung tissue corresponding to metastatic tumor metastatic colonies.

In previous studies, SLPs have been demonstrated to be effective against metastasis using chemotherapeutic drugs such as paclitaxel [58], etoposide [59], and gallic acid ester derivatives [60], among others. Our results indicate that these particles exhibit a higher antimetastatic effect than unformulated DOX without any detectable toxicity. This antimetastatic effect may be due to the stable drug release over time from these SLPs. This rate of drug release is particularly interesting because it maintains the drug level in blood within the therapeutic window, which is crucial for primary tumor inhibition. This drug release rate in combination with local accumulation in the lungs exerts an improved

antimetastatic therapeutic output compared with that of the conventional drug; specifically, SLPs-DOX show 60% more antimetastatic effect than free DOX.

Liposomal doxorubicin formulations (Doxil®, Caelyx®, and Myocet®) are an encapsulated form of doxorubicin, with an improved pharmacokinetic profile and the ability to selectively accumulate in tumor tissue [61]. As a result, the tolerated dose of the drug can be increased, followed by a lower incidence of neutropenia and cardiotoxicity compared with treatment with free doxorubicin. However, the common adverse effect that limits the treatment dose regimen is palmoplantar erythrodysesthesia syndrome [62]. This side effect is a distinctive and relatively common toxic reaction associated with some chemotherapeutic agents. Doxorubicin, cytarabine, docetaxel, and fluorouracil are the agents most frequently implicated. This syndrome appears to be dose dependent, and its occurrence is determined by both the maximum drug concentration and the total cumulative dose. Withdrawal or reduction of the dose of the involved drug usually leads to an improvement in symptoms [63].

In summary, we have demonstrated here the ability to encapsulate DOX in SLPs and evaluated the potential of these particles to release the drug in metastatic tumors in mice. We have shown how the lipid fraction of the particles can significantly improve drug loading and thus generate formulations with improved drug loading and superior stability. We showed how these SLPs allow high retention of the drug in the matrix triggering sustained DOX release upon target tissue arrival [62]. We also demonstrated a significant reduction of the potential adverse effects of the encapsulated DOX, as is the case for the FDA-approved liposomal formulations. However, more interestingly, we demonstrated how the sustained release of the drug upon particle arrival to the target tissue results in an improved antimetastatic effect. This sustained in situ release effect allows improved local efficacy and reduced toxicity compared with that reported for liposomal formulations, where the release is faster and with a higher drug concentration, causing possible adverse effects such as palmoplantar erythrodysesthesia syndrome.

Finally, SLPs can also be prepared with encapsulated iron nanoparticles inside, which in future works will serve not only to trigger on-demand drug release but also for use in imaging techniques or hyperthermia therapy, enhancing the effect of DOX in the tumor [42].
