Exploring the potential of natural extracts for pharmaceutical applications in the treatment of different diseases is an emerging field of medical research, owing to the tremendous advantages that they can offer. These include compound sustainability due to the natural origin and virtually unlimited availability. In addition, they contribute to promoting the countries in which they are extracted and manufactured. For this reason, wild active compounds derived from plants are attracting increasing interest due to their beneficial properties. Among them, Avicennia marina has been recently recognized as a potential source of natural substances with therapeutic activities for anti-cancer treatment. A. marina beneficially supplies different chemical compounds, including cyclic triterpenoids, flavonoids, iridoids, naphtaquinones, polyphenols, polysaccharides, and steroids, most of them exhibiting potent antitumor activity. The in vivo and in vitro studies on different models of solid tumors demonstrated its dose-dependent activity. Moreover, the possibility to formulate the A. marina extracted molecules in nanoparticles allowed researchers to ameliorate the therapeutic outcome of treatments exploiting improved selectivity toward cancer cells, thus reducing the side effects due to nonspecific spread.

The use of co-processed materials for Orally Disintegrating Tablets (ODT) preparation by direct compression is well consolidated. However, the evaluation of their potential for ODT preparation by 3D printing technology remains almost unexplored. The present study aimed to estimate the use of commercially available co-processed excipients, conventionally applied in compression protocols, for the preparation of ODTs with binder jetting-3D printing technology. The latter was selected among the 3D printing techniques because the deposition of multiple powder layers allows for obtaining highly porous and easily disintegrating dosage forms. The influence of some process parameters, including layer thickness, type of waveform and spread speed, on the physical and mechanical properties of the prototypes printed were evaluated. Our results suggested that binder jetting-3D printing technology could benefit from the co-processed excipients for the preparation of solid dosage forms. The process optimization conducted with the experiments reported in this work indicated that additional excipients were needed to improve the physical properties of the resulting ODTs.

The vault nanoparticle is a eukaryotic assembly consisting of 78 copies of the 99-kDa major vault protein. They generate two cup-shaped symmetrical halves, which in vivo enclose protein and RNA molecules. Overall, this assembly is mainly involved in pro-survival and cytoprotective functions. It also holds a remarkable biotechnological potential for drug/gene delivery, thanks to its huge internal cavity and the absence of toxicity/immunogenicity. The available purification protocols are complex, partly because they use higher eukaryotes as expression systems. Here, we report a simplified procedure that combines human vault expression in the yeast Komagataella phaffii, as described in a recent report, and a purification process we have developed. This consists of RNase pretreatment followed by size-exclusion chromatography, which is far simpler than any other reported to date. Protein identity and purity was confirmed by SDS-PAGE, Western blot and transmission electron microscopy. We also found that the protein displayed a significant propensity to aggregate. We thus investigated this phenomenon and the related structural changes by Fourier-transform spectroscopy and dynamic light scattering, which led us to determine the most suitable storage conditions. In particular, the addition of either trehalose or Tween-20 ensured the best preservation of the protein in native, soluble form.

Ribosome-inactivating proteins, including Saporin toxin, have found application in the search for innovative alternative cancer therapies to conventional chemo- and radiotherapy. Saporin’s main mechanism of action involves the inhibition of cytoplasmic protein synthesis. Its strong theoretical efficacy is counterbalanced by negligible cell uptake and diffusion into the cytosol. In this work, we demonstrate that by immobilizing Saporin on iron oxide nanoparticles coated with an amphiphilic polymer, which promotes nanoconjugate endosomal escape, a strong cytotoxic effect mediated by ribosomal functional inactivation can be achieved. Cancer cell death was mediated by apoptosis dependent on nanoparticle concentration but independent of surface ligand density. The cytotoxic activity of Saporin-conjugated colloidal nanoparticles proved to be selective against three different cancer cell lines in comparison with healthy fibroblasts.

Vaults are eukaryotic ribonucleoproteins consisting of 78 copies of the major vault protein (MVP), which assemble into a nanoparticle with an about 60 nm volume-based size, enclosing other proteins and RNAs. Regardless of their physiological role(s), vaults represent ideal, natural hollow nanoparticles, which are produced by the assembly of the sole MVP. Here, we have expressed in Komagataella phaffi and purified an MVP variant carrying a C-terminal Z peptide (vault-Z), which can tightly bind an antibody’s Fc portion, in view of targeted delivery. Via surface plasmon resonance analysis, we could determine a 2.5 nM affinity to the monoclonal antibody Trastuzumab (Tz)/vault-Z 1:1 interaction. Then, we characterized the in-solution interaction via co-incubation, ultracentrifugation, and analysis of the pelleted proteins. This showed virtually irreversible binding up to an at least 10:1 Tz/vault-Z ratio. As a proof of concept, we labeled the Fc portion of Tz with a fluorophore and conjugated it with the nanoparticle, along with either Tz or Cetuximab, another monoclonal antibody. Thus, we could demonstrate antibody-dependent, selective uptake by the SKBR3 and MDA-MB 231 breast cancer cell lines. These investigations provide a novel, flexible technological platform that significantly extends vault-Z’s applications, in that it can be stably conjugated with finely adjusted amounts of antibodies as well as of other molecules, such as fluorophores, cell-targeting peptides, or drugs, using the Fc portion as a scaffold.

Vaults are massive ribonucleoprotein complexes, highly conserved and abundant in eukaryotic cells, yet with unclear function. Their thin-walled barrel-shape architecture is composed of two symmetrical, antiparallel half-shells, each containing 39 copies of the major vault protein (MVP). The spacious lumen of the vault suggests a role in cellular transport. Although vaults are thought to undergo conformational changes to facilitate cargo exchange, the molecular basis for their inherent flexibility remains unknown. Here, we integrate cryogenic electron microscopy (cryo-EM) and multi-scale molecular dynamics (MD) simulations to reveal the structural determinants of the human vault particle’s flexibility. Cryo-EM identified two high-resolution alternative conformational states: a symmetric and an asymmetric structure, pointing to the vault shell’s structural plasticity. MD simulations of these conformations revealed that these structures are flexible and exhibit breathing-like motions, and porous solvent-exposed surfaces. Mutagenesis disrupting persistent MD-identified inter-half contacts reduced full MVP shell assembly, confirming the functional relevance of these flexibility determinants. Together, these findings establish the molecular basis for the human vault particle’s conformational plasticity.