Shiga Toxin-B Targeted Gold Nanorods for Local Photothermal Treatment in Oral Cancer Clinical Samples.

Introduction: A great challenge in nanomedicine, and more specifically in theranostics, is to improve the specificity, selectivity, and targeting of nanomaterials towards target tissues or cells. The topical use of nanomedicines as adjuvants to systemic chemotherapy can significantly improve the survival of patients affected by localized carcinomas, reducing the side effects of traditional drugs and preventing local recurrences. Methods: Here, we have used the Shiga toxin, to design a safe, high-affinity protein-ligand (ShTxB) to bind the globotriaosylceramide receptor (GB3) that is overexpressed on the surfaces of preneoplastic and malignant cancer cells in the head and neck tumors. Results: We find that ShTxB functionalized gold nanorods are efficiently retrotranslocated to the GB3-positive cell cytoplasms. After 3 minutes of laser radiation with a wavelength resonant with the AuNR longitudinal localized surface plasmon, the death of the targeted cancer cells is activated. Both preclinical murine models and patient biopsy cells show the non-cytotoxic nature of these functionalized nanoparticles before light activation and their treatment selectivity. Discussion: These results show how the use of nanomedicines directed by natural ligands can represent an effective treatment for aggressive localized cancers, such as squamous cell carcinoma of the oral cavity.

Targeting Nanomaterials to Head and Neck Cancer Cells Using a Fragment of the Shiga Toxin as a Potent Natural Ligand.

Head and Neck Cancer (HNC) is the seventh most common cancer worldwide with a 5-year survival from diagnosis of 50%. Currently, HNC is diagnosed by a physical examination followed by an histological biopsy, with surgery being the primary treatment. Here, we propose the use of targeted nanotechnology in support of existing diagnostic and therapeutic tools to prevent recurrences of tumors with poorly defined or surgically inaccessible margins. We have designed an innocuous ligand-protein, based on the receptor-binding domain of the Shiga toxin (ShTxB), that specifically drives nanoparticles to HNC cells bearing the globotriaosylceramide receptor on their surfaces. Microscopy images show how, upon binding to the receptor, the ShTxB-coated nanoparticles cause the clustering of the globotriaosylceramide receptors, the protrusion of filopodia, and rippling of the membrane, ultimately allowing the penetration of the ShTxB nanoparticles directly into the cell cytoplasm, thus triggering a biomimetic cellular response indistinguishable from that triggered by the full-length Shiga toxin. This functionalization strategy is a clear example of how some toxin fragments can be used as natural biosensors for the detection of some localized cancers and to target nanomedicines to HNC lesions.

A custom-made functionalization method to control the biological identity of nanomaterials.

Here we propose a one-step strategy to endow nanomaterials with a custom-designed bio-identity. This study designs a universal 'nanomaterial binding domain' that can be genetically attached to any protein ensuring precise and spontaneous protein orientation. We demonstrate how, despite the simplicity of the method, the bioconjugation achieved: (i) is highly efficient, even in the presence of competing proteins, (ii) is stable at extreme physiological conditions (pH ranges 5.2-9.0; NaCl concentrations 0-1 M); (iii) prevents unwanted protein biofouling days after incubation in biologically-relevant conditions; and finally, (iv) avoids nanoparticle interaction with promiscuous unspecific receptors. In summary, this protein biocoating technique, applicable to a wide array of nano-designs, integrates material science and molecular biology procedures to create hybrid nanodevices with well-defined surfaces and predictable biological behaviors, opening a chapter in precision nanodiagnostics, nanosensing or nanotherapeutic applications.

Dye-doped biodegradable nanoparticle SiO2 coating on zinc- and iron-oxide nanoparticles to improve biocompatibility and for in vivo imaging studies.

In vivo imaging and therapy represent one of the most promising areas in nanomedicine. Particularly, the identification and localization of nanomaterials within cells and tissues are key issues to understand their interaction with biological components, namely their cell internalization route, intracellular destination, therapeutic activity and possible cytotoxicity. Here, we show the development of multifunctional nanoparticles (NPs) by providing luminescent functionality to zinc and iron oxide NPs. We describe simple synthesis methods based on modified Stöber procedures to incorporate fluorescent molecules on the surface of oxide NPs. These procedures involve the successful coating of NPs with size-controlled amorphous silica (SiO2) shells incorporating standard chromophores like fluorescein, rhodamine B or rhodamine B isothiocyanate. Specifically, spherical Fe3O4 NPs with an average size of 10 nm and commercial ZnO NPs (ca. 130 nm), both coated with an amorphous SiO2 shell of ca. 15 and 24 nm thickness, respectively, are presented. The magnetic nanoparticles, with a major presence of magnetite, show negligible coercitivity. Hence, interactions (dipolar) are very weak and the cores are in the superparamagnetic regime. Spectroscopic measurements confirm the presence of fluorescent molecules within the SiO2 shell, making these hybrid NPs suitable for bioimaging. Thus, our coating procedures improve NP dispersibility in physiological media and allow the identification and localization of intracellular ZnO and Fe3O4 NPs using confocal microscopy imaging preserving the fluorescence of the NP. We demonstrate how both Fe3O4 and ZnO NPs coated with luminescent SiO2 are internalized and accumulated in the cell cytoplasm after 24 hours. Besides, the SiO2 shell provides a platform for further functionalization that enables the design of targeted therapeutic strategies. Finally, we studied the degradation of the shell in different physiological environments, pointing out that the SiO2 coating is stable enough to reach the target cells maintaining its original structure. Degradation took place only 24 hours after exposure to different media.