Development and biostability evaluation of hybrid nanoconstructs for cancer therapy based on zinc oxide nanocrystals

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The shielding effect of phospholipidic bilayers on zinc oxide nanocrystals for biomedical applications

Zinc oxide nanocrystals (ZnO NCs), thanks to their unique properties, are receiving much attention for their use in nanomedicine, in particular for therapy against cancer [1]. To be efficiently employed as diagnostic and therapeutic (yet theranostic) tools [2], highly dispersed, stable and non-toxic nanoparticles are required. In the case of ZnO NCs, there is still a lack of knowledge about cytotoxicity mechanisms and stability in the biological context, as well as immunological response and haemocompatible features. Most of these above-mentioned behaviours strongly depends on physico-chemical and surface properties of the nanoparticles. We thus propose a novel approach to stabilize the ZnO NCs in various biological media, focusing on NC aggregation and biodegradation as a function of the surface functionalization. We synthesized bare ZnO NCs, amino-propyl functionalized ones, and lipid bilayer-shielded NCs, and we characterized their morphological, chemical and physical properties. The stability behavior of the three different samples was evaluated, comparing their biodegradation profiles in different media, i.e. organic solvents, water, and different simulated and biological fluids. The studies aim to investigate how the particle surface functionalizations, and thus chemistry and charge, could influence their hydrodynamic size, zeta potential and consequent aggregation and degradation in the different solvents. We demonstrated that bare and amino-functionalized ZnO NCs strongly and rapidly aggregate when suspended in both simulated and biological media. Long-term biodegradation analysis showed small dissolution into potentially cytotoxic Zn-cations, also slightly affecting their crystalline structure. In contrast, high colloidal stability and integrity was retained for lipid-shielded ZnO NCs in all media, rendering them the ideal candidates for further theranostic applications [3]. [1] P. Zhu, Z. Weng, X. Li, X. Liu, S. Wu Adv. Mater. Interfaces 3 (2016) 1500494. [2] E. Lim, T. Kim, S. Paik, S. Haam, Y. Huh, and K. Lee, Chem. Rev. 115 (2015) 327−394. [3] B. Dumontel, M. Canta, H. Engelke, A. Chiodoni, L. Racca, A. Ancona, T. Limongi, G. Canavese and V. Cauda, J. Mater. Chem. B, under review The support from ERC Starting Grant – Project N. 678151 “Trojananohorse” and Compagnia di Sanpaolo are gratefully acknowledged

Acoustic waves and smart biomimetic nanoparticles: combination treatment from 2 to 3D colorectal cancer models

Colorectal Cancer (CRC) is the second leading cause of death among tumors worldwide. Conventional treatments are often accompanied by emerging immunotherapies, trying to reduce the burden of advanced and metastatic stages. Recently, nanomedicine therapies have been under intensive research to offer new perspectives to patients. Motivated by this rationale, this work proposes the formulation of advanced biomimetic and targeted nanoparticles (NPs) enabling a stimuli responsive and localized therapy, triggered by the safe use of acoustic shockwaves, a deep penetrating tissue stimulation. Iron-doped zinc oxide nanocrystals were synthetized and enveloped in a biomimetic lipid bilayer shell, conjugating a peptide (YSA) as selective targeting toward CRC cells. Comparative studies, performed both in 2D monolayer and 3D spheroids of CRC models, between non-targeted (L-ZnO) and targeted (YSA-L-ZnO) nanoparticles demonstrated the superior capability of targeted nanosystems to dock and be internalized by CRC cells. The YSA-L-ZnO are proven to be highly biocompatible and hemocompatible, and capable of inducing selective damage, once activated by safe shockwaves. This mechanism is able to synergistically ablate tumor cells in both 2D and 3D models, proofing the concept of an innovative stimuli responsive nanomedicine with a targeted and biomimetic strategy to offer future options for cancer fight

Biomimetic Non-Immunogenic Nanoassembly for the Antitumor Therapy

Nanoassembly (1) for inducing apoptosis in cancer cells comprising: a core (2) comprising at least a nanoparticle of a nano structured and semiconductor metal oxide, said nanoparticle being monocrystalline or polycrystalline; a shell (3) formed by a double phospholipid layer and proteins derived from an extracellular biovesicole chosen between an exosome, an ectosome, a connectosome, an oncosome and an apoptotic body, and an oncosome, said core (2) being enclosed inside said shell (3); and a plurality of targeting molecules (4, 4', 4") of said cancer cells, preferably monoclonal antibodies (4, 4', 4"), said molecules (4, 4', 4") being anchored to the external surface of said biovesicole

Lipid-coated zinc oxide nanocrystals as innovative ROS-generators for photodynamic therapy

Photodynamic Therapy (PDT) is a medical treatment that combines the administration of a nontoxic drug, called photosensitizer (PS), with light irradiation of the targeted region. It has been proposed as a new cancer therapy, promising better selectivity and fewer side-effects compared to traditional chemo- and radio-therapies. PSs indeed can accumulate specifically within the region of interest so that when the light is directly focused only in that region the therapeutic effect is highly localized. Traditional PSs, like chlorins and porphyrins, suffer from several drawbacks such as aggregation in biological media and poor biocompatibility. Thus, the development of innovative photosensitizers able to overcome these issues is crucial to the therapeutic action of PDT. Among the others, nanostructured Zinc Oxide (ZnO) has been recently proposed as new therapeutic agent and PS thanks to its semiconducting properties, biocompatible features, and ease of functionalization [1]. Nevertheless, further efforts are needed in order to improve its colloidal stability in biological media and to unravel the effective therapeutic mechanism. Here, we propose the synthesis and characterization of lipid-coated ZnO nanoparticles as new photosensitizer for cancer PDT [2]. First, by Dynamic Light Scattering (DLS) experiments, we show that the lipid-coating increases the colloidal stability of the ZnO NPs in Phosphate buffered saline (PBS). Then, using Electron Paramagnetic Resonance (EPR) coupled with the spin-trapping technique, we demonstrate and characterize the ability of bare and lipid-coated ZnO NPs to generate Reactive Oxygen Species (ROS) in water only when remotely actuated via light irradiation. Interestingly, our results aware that the surface chemistry of the NPs greatly influence the type of photo-generated ROS. Finally, we show that our NPs are effectively internalized inside human epithelial carcinoma cells (HeLa) via a lysosomal pathway and that they are able to generate ROS inside cancer cells. [1] B. Dumontel, M. Canta, H. Engelke, A. Chiodoni, L. Racca, A. Ancona, T. Limongi, G. Canavese and V. Cauda, ‎J. Mater. Chem. B. under revision. [2] A. Ancona, H. Engelke, N. Garino, B. Dumontel, W.Fazzini and V. Cauda, to be submitted. The support from ERC Starting Grant – Project N. 678151 “Trojananohorse” is gratefully acknowledged

BIOMIMETIC NON - IMMUNOGENIC NANOASSEMBLY FOR THE ANTITUMOR THERAPY

Nanoassembly ( 1 ) for inducing apoptosis in cancer cells comprising : a core ( 2 ) comprising at least a nanoparticle of a nano structured and semiconductor metal oxide , said nanoparticle being monocrystalline or polycrystalline ; a shell ( 3 ) formed by a double phospholipid layer and proteins derived from an extracellular biovesicole chosen between an exosome , an ectosome , a connectosome , an oncosome and an apoptotic body , and an oncosome , said core ( 2 ) being enclosed inside said shell ( 3 ) ; and a plurality of targeting molecules ( 4 , 4 ' , 4 " ) of said cancer cells , preferably mono clonal antibodies ( 4 , 4 ' , 4 " ) , said molecules ( 4 , 4 , 4 " ) being anchored to the external surface of said biovesicole

Enhanced Biostability and Cellular Uptake of Zinc Oxide Nanocrystals Shielded with Phospholipid Bilayer

The widespread use of ZnO nanomaterials for biomedical applications, including therapeutic drug delivery or stimuli-responsive activation, as well as imaging, imposes a careful control over the colloidal stability and long-term behaviour of ZnO in biological media. Moreover, the effect of ZnO nanostructures on living cells, in particular cancer cells, is still under debate. This paper discusses the role of surface chemistry and charge of zinc oxide nanocrystals, of around 15 nm in size, which influence their behaviour in biological fluids and effect on cancer cells. In particular, we address this problem by modifying the surface of pristine ZnO nanocrystals (NCs), rich of hydroxyl groups, with positively charged amino-propyl chains or, more innovatively, by self-assembling a double-lipidic membrane, shielding the ZnO NCs. Our findings show that the prolonged immersion in simulated human plasma and in the cell culture medium leads to highly colloidally dispersed ZnO NCs only when coated by the lipidic bilayer. In contrast, the pristine and amine-functionalized NCs form huge aggregates after already one hour of immersion. Partial dissolution of these two samples into potentially cytotoxic Zn2+ cations takes place, together with the precipitation of phosphate and carbonate salts on the NCs’ surface. When exposed to living HeLa cancer cells, higher amounts of lipid-shielded ZnO NCs are internalized with respect to the other samples, thus showing a reduced cytotoxicity, based on the same amount of internalized NCs. These results pave the way for the development of novel theranostic platforms based on ZnO NCs. The new formulation of ZnO shielded with a lipid-bilayer will prevent strong aggregation and premature degradation into toxic by-products, and promote a highly efficient cell uptake for further therapeutic or diagnostic functions

Smart Shockwave Responsive Titania-Based Nanoparticles for Cancer Treatment

Nanomedicine is an emerging treatment approach for many cancers, characterized by having high sensitivity and selectivity for tumor cells and minimal toxic effects induced by the conventional chemotherapeutics. In these context, smart nanoparticles (NPs) are getting increasingly relevant in the development of new therapies. NPs with specific chemical composition and/or structure and being stimuli-responsive to magnetic, light or ultrasound waves are new promising tools. In the present work, amorphous-titania propyl-amine functionalized (a-TiO2-NH2) NPs, coated with bovine serum albumin (BSA), are stimulated with high energy shock waves to induce cytotoxic effects in cancer cells. First, a new method to coat a-TiO2-NH2 NPs with BSA (a-TiO2-NH2/BSA) was proposed, allowing for a high dispersion and colloidal stability in a cell culture media. The a-TiO2-NH2/BSA NPs showed no cancer cell cytotoxicity. In a second step, the use of shock waves to stimulate a-TiO2-NH2/BSA NPs, was evaluated and optimized. A systematic study was performed in in vitro cell culture aiming to impair the cancer cell viability: NP concentrations, time steps and single versus multiple shock waves treatments were studied. The obtained results highlighted the relevance of NPs design and administration time point with respect to the shock wave treatment and allow to hypothesize mechanical damages to cells