*4.1. Anticancer Peptides*

Typically, anticancer e ffects of peptides occur through two possible mechanisms. One of these arises when the peptide is able to disrupt the normal function of the membrane, while the other occurs when it triggers pro-apoptotic pathways. The membrane-disrupting mechanism operates when the peptides are enriched with cationic amino acids: lysine (Lys, K; amine), arginine (Arg, R; guanidine), and histidine (Hys, H; imidazole). Although this mechanism is not fully clear, these peptides demonstrated a certain membrane-lytic e ffect similar to other cationic species [134]. The first evidence on the topic was observed with TAT-targeted nanosystems [135], as well with other cell-membrane-penetrating peptides [136].

Regarding the use of cationic peptides as therapeutic agents, it is highly desirable to hide their cationic character to prevent premature damages such as hemolysis and embolisms during tra fficking. Apart from pore loading, which preserves cargoes from undesired interactions, another interesting possibility arises from the use of additional negative components to balance the resulting surface charge. One elegant strategy, reported by Zhang and coworkers, employed citraconic anhydride to transform an octa-lysine into a negatively charged peptide able to balance the cationic K8(RGD)2 functionalized MSNs [111]. The resulting outermost layer was able to accomplish three tasks: target integrin receptors through the RDG moiety, show a highly biocompatible neutral charge, and carry a detachable but toxic K8 sequence. Unfortunately, the reported studies mainly focused on assessing the e ffect of carried DOX than on studying the e ffect of the peptide release, overlooking any potential anticancer e ffect. In a later contribution by these authors, their system was evolved by including a triphenyl phosphonium group into the KLA peptide sequence (KLAKLAKKLAKLAK) in order to target the mitochondrion. Mitochondria appeared as a key target in cancer treatment because of their roles in regulating cell apoptosis and metabolism [137]. The delivery of antitumoral therapeutic molecules to mitochondria may improve their therapeutic e fficacy while avoiding resistance pathways. Furthermore, stimulation of apoptosis mediated by mitochondria could also improve the e fficacy of cancer therapy. The resulting organelle-targeted compound was bound to MSNs through disulfide bonds, known to be broken intracellularly by glutathione. Finally, to prevent undesired damages, the authors created a PEGylated anionic polymer shell aimed at increasing biocompatibility and reducing the leakage of topotecan [112]. In this system, the non-loaded model was able to exert a significant pro-apoptotic even at low concentrations, proving the potential of peptides in anticancer therapies; however, as expected, the highest antiproliferative e ffect occurred when topotecan was loaded within the mesopores.

Encouraged by these results, this group also addressed the synthesis of a multiple pro-apoptotic nanosystem: delivery of DOX, in combination with the delivery of two therapeutic peptides with glutathione-mediated cleavage [113]. In this model, the employed peptides contained di fferent membrane-disrupting sequences, together with two di fferent targeting elements: one of them specific to mitochondrion (C-GKGG-DKLAKLAKKLAKLAK) and the other specific to membranes (C-GRKKRRQRRRPPQ-RGDS). Additionally, to increase the potential cellular damage, the mesopores were loaded with DOX while the peptides were bonded to particles through disulfide bonds, in order to induce oxidative stress upon glutathione depletion. As result, the complete system showed high efficiency against HeLa and COS7 cells, although this e ffect decreased significantly in the absence of the drug, which highlights the limited e ffect of membrane-lytic peptides. More recently, this group also employed the glutathione-mediated disulfide cleavage to prepare a drug delivery system for DOX and a membrane-targeted therapeutic peptide rich in tryptophan ((RGDWWW)2KC) [114]. In this case, this peptide was designed to have a DNA-intercalant e ffect due to the high concentration of indoles. This postulate seems to be justified based on the toxicity obtained for such a nanosystem; however, as expected, the best e ffect was obtained when DOX was co-delivered. More recently, Feng's group also employed this coating approach to co-deliver DOX and the anticancer peptide KLA. In this model, the final capping was done with a bovine serum albumin (BSA) corona [115], which permitted achieving a double e ffect: creating a di ffusion barrier for both therapeutic agents, and enabling a

protease/glutathione-mediated intracellular release. An interesting aspect of the system is that BSA was employed in its wild-type form, which might trigger an additional cellular response when in combination with the remaining multi-apoptotic e ffects.

Regarding the delivery of peptides that trigger pro-apoptotic routes, the reported examples focused on threading such peptides into the mesopores. For example, Martínez-Máñez's group reported the use of ε-polylysine as a coating layer to prevent the leakage of the pro-apoptotic C9h (YVETLDDIFEQWAHSEDLK) peptide [116]. In this model, the polylysine coating had multiple roles, as it favored cellular uptake due to its cationic character, while maintaining the C9h peptide within the mesopores until proteases cleaved this protective layer. The in vitro testing of this model proved that the encapsulated peptide showed a better therapeutic profile that its free form. However, this e ffect reached a maximum plateau over which higher dosages did not augmen<sup>t</sup> apoptosis. This points out once again the limited anticancer e ffect of peptides, which demand additional chemotherapeutic agents to obtain satisfactory results. In another similar example employing pore modifications, Cao et al. reported the delivery of a di fferent proapoptotic peptide. In this model, they employed large-pore MSNs to deliver a bifunctional RGD-containing Hylin a1 peptide (IFGAILPLALGALKNLIK) able to target and kill cancerous cells [117]. To accommodate the peptide, the authors functionalized the internal facets of mesopores to favor threading and enable a pH-dependent release. In vitro studies of this system showed that encapsulation drastically reduced the hemolytic rate shown by the free peptide without a ffecting the potent cytotoxic e ffect against HeLa and Hep2 cells in vitro. Published in vivo studies with this system showed a clear tumor growth arrest in murine models, although, unfortunately, complete tumor remission could not be achieved.

As previously outlined, the anticancer e ffect of peptides and proteins is relatively low; thus, their use is generally limited to therapeutic adjuvants of more active species. However, the overall therapeutic effect may be increased if higher doses can be delivered; in this case, high-loading carriers gain interest. Among silica-based carriers, HMSNs are the most suitable candidates, since they theoretically allow loading in larger quantities than their porous analogues. The diverging aspects of MSNs vs. HMSNs in peptide delivery were studied by Rahmani et al., who focused on pepstatin A, a cathepsin D inhibitor peptide [118]. Surprisingly, the authors found two unexpected behaviors: HMSNs loaded less peptide than typical large-pore MSNs, but their e ffect was higher. Therein, the authors justified such behavior based on the release patterns observed from HMSNs, which provided a more sustained release (longer therapeutic e ffect), in comparison to regular pore-expended MSNs, which showed a burst-like release.

Another promising anticancer peptide is NuBCP9 (FSRSLHSLL), which is able to bind the Bcl-2 protein, highly overexpressed in cancer cells, turning a cell protector into an apoptosis inductor [138]. Along this line, Wu et al. reported folate-targeted, large-pore MSNs able to deliver this NuBCP9 peptide into HeLa tumors in zebrafish [119]. The resulting ca. 35-nm-width MSNs with pores in the range of 20 nm showed e ffective internalization into folate-positive HeLa cells, reaching up to 70% reduction of viability when loaded with the peptide. On the other hand, these nanoparticles showed fantastic biocompatibility, as they permitted obtaining fish survival above 80% in concentrations up to 200 μg/mL. In addition to this targeted example, this peptide was also employed in combination with a typical chemotherapeutic. In this contribution, the authors employed ca. 30-nm MSNs with large pores to load the peptide and an outer coating of a fifth-generation polyamidoamine (PAMAM) dendrimer able to load DOX within the structure [120]. As a result, the nanosystem was able to co-deliver both pro-apoptotic species to several cancer cell lines, achieving almost complete cell destruction in concentrations up to 1 μg/mL. To validate the potential of such a combination, the authors tested the efficiency of their nanosystem against resistant cell lines, obtaining outstanding results except for the case of the DOX-resistant MCF-7 line. Nevertheless, these results must be carefully accounted for, as the outermost PAMAM cationic coating may produce a decrease in overall biocompatibility.
