A comparative analysis of low intensity ultrasound effects on living cells: from simulation to experiments.

Ultrasounds are already broadly exploited in clinical diagnostics and are now becoming a powerful and not harmful tool in antitumoral therapies, as they are able to produce damages towards cancer cells, thank to inertial cavitation and temperature increase. The use of US alone or combined to molecular compounds, microbubbles or solid-state nanoparticles is the focus of current research and clinical trials, like thermoablation, drug sonoporation or sonodynamic therapies. In the present work, we discuss on the non-thermal effects of ultrasound and the conditions which enable oxygen radical production and which role they can have in provoking the death of different cancer cell lines. In this perspective, we set a mathematical model to predict the pressure spatial distribution in a defined water sample volume and thus obtain a map of acoustic pressures and acoustic intensities of the applied ultrasound at different input powers. We then validate and verify these numerical results with direct acoustic measurements and by detecting the production of reactive oxygen species (ROS) by means of sonochemiluminescence (SCL) and electron paramagnetic resonance (EPR) spectroscopy, applied to the same water sample volume and using the same US input parameters adopted in the simulation. Finally, the various US conditions are applied to two different set of cancer cell lines, a cervical adenocarcinoma and a hematological cancer, Burkitt's lymphoma. We hypothesize how the ROS generation can influence the recorded cell death. In a second set of experiments, the role of semiconductor metal oxide nanocrystals, i.e. zinc oxide, is also evaluated by adding them to the water and biological systems. In particular, the role of ZnO in enhancing the ROS production is verified. Furthermore, the interplay among US and ZnO nanocrystals is evaluated in provoking cancer cell death at specific conditions. This study demonstrates a useful correlation between numerical simulation and experimental acoustic validation as well as with ROS measurement at both qualitative and quantitative levels during US irradiation of simple water solution. It further tries to translate the obtained results to justify one of the possible mechanisms responsible of cancer cell death. It thus aims to pave the way for the use of US in cancer therapy and a better understanding on the non-thermal effect that a specific set of US parameters can have on cancer cells cultured in vitro.

Nanoparticle-assisted ultrasound: A special focus on sonodynamic therapy against cancer.

At present, ultrasound radiation is broadly employed in medicine for both diagnostic and therapeutic purposes at various frequencies and intensities. In this review article, we focus on therapeutically-active nanoparticles (NPs) when stimulated by ultrasound. We first introduce the different ultrasound-based therapies with special attention to the techniques involved in the oncological field, then we summarize the different NPs used, ranging from soft materials, like liposomes or micro/nano-bubbles, to metal and metal oxide NPs. We therefore focus on the sonodynamic therapy and on the possible working mechanisms under debate of NPs-assisted sonodynamic treatments. We support the idea that various, complex and synergistics physical-chemical processes take place during acoustic cavitation and NP activation. Different mechanisms are therefore responsible for the final cancer cell death and strongly depends not only on the type and structure of NPs or nanocarriers, but also on the way they interact with the ultrasonic pressure waves. We conclude with a brief overview of the clinical applications of the various ultrasound therapies and the related use of NPs-assisted ultrasound in clinics, showing that this very innovative and promising approach is however still at its infancy in the clinical cancer treatment.

Anti-CD38 targeted nanotrojan horses stimulated by acoustic waves as therapeutic nanotools selectively against Burkitt's lymphoma cells.

The horizon of nanomedicine research is moving toward the design of therapeutic tools able to be completely safe per se, and simultaneously be capable of becoming toxic when externally activated by stimuli of different nature. Among all the stimuli, ultrasounds come to the fore as an innovative approach to produce cytotoxicity on demand in presence of NPs, without invasiveness, with high biosafety and low cost. In this context, zinc oxide nanoparticles (NPs) are among the most promising metal oxide materials for theranostic application due to their optical and semi-conductor properties, high surface reactivity, and their response to ultrasound irradiation. Here, ZnO nanocrystals constitute the stimuli-responsive core with a customized biomimicking lipidic shielding, resembling the composition of natural extracellular vesicles. This core-shell hybrid structure provides high bio- and hemocompatibility towards healthy cells and is here proofed for the treatment of Burkitt's Lymphoma. This is a very common haematological tumor, typically found in children, for which consolidated therapies are so far the combination of chemo-therapy drugs and targeted immunotherapy. In this work, the proposed safe-by-design antiCD38-targeted hybrid nanosystem exhibits an efficient selectivity toward cancerous cells, and an on-demand activation, leading to a significant killing efficacy due to the synergistic interaction between US and targeted hybrid NPs. Interestingly, this innovative treatment does not significantly affect healthy B lymphocytes nor a negative control cancer cell line, a CD38- acute myeloid leukemia, being thus highly specific and targeted. Different characterization and analyses confirmed indeed the effective formation of targeted hybrid ZnO NPs, their cellular internalization and the damages produced in Burkitt's Lymphoma cells only with respect to the other cell lines. The presented work holds promises for future clinical applications, as well as translation to other tumor types.

Role of Diacylglycerol Kinases in Acute Myeloid Leukemia.

Diacylglycerol kinases (DGKs) play dual roles in cell transformation and immunosurveillance. According to cancer expression databases, acute myeloid leukemia (AML) exhibits significant overexpression of multiple DGK isoforms, including DGKA, DGKD and DGKG, without a precise correlation with specific AML subtypes. In the TGCA database, high DGKA expression negatively correlates with survival, while high DGKG expression is associated with a more favorable prognosis. DGKA and DGKG also feature different patterns of co-expressed genes. Conversely, the BeatAML and TARGET databases show that high DGKH expression is correlated with shorter survival. To assess the suitability of DGKs as therapeutic targets, we treated HL-60 and HEL cells with DGK inhibitors and compared cell growth and survival with those of untransformed lymphocytes. We observed a specific sensitivity to R59022 and R59949, two poorly selective inhibitors, which promoted cytotoxicity and cell accumulation in the S phase in both cell lines. Conversely, the DGKA-specific inhibitors CU-3 and AMB639752 showed poor efficacy. These findings underscore the pivotal and isoform-specific involvement of DGKs in AML, offering a promising pathway for the identification of potential therapeutic targets. Notably, the DGKA and DGKH isoforms emerge as relevant players in AML pathogenesis, albeit DGKA inhibition alone seems insufficient to impair AML cell viability.

Wiskott-Aldrich syndrome protein interacts and inhibits diacylglycerol kinase alpha promoting IL-2 induction.

Background: Phosphorylation of diacylglycerol by diacylglycerol-kinases represents a major inhibitory event constraining T cell activation upon antigen engagement. Efficient TCR signalling requires the inhibition of the alpha isoform of diacylglycerol kinase, DGKα, by an unidentified signalling pathway triggered by the protein adaptor SAP. We previously demonstrated that, in SAP absence, excessive DGKα activity makes the T cells resistant to restimulation-induced cell death (RICD), an apoptotic program counteracting excessive T cell clonal expansion. Results: Herein, we report that the Wiskott-Aldrich syndrome protein (WASp) inhibits DGKα through a specific interaction of the DGKα recoverin homology domain with the WH1 domain of WASp. Indeed, WASp is necessary and sufficient for DGKα inhibition, and this WASp function is independent of ARP2/3 activity. The adaptor protein NCK-1 and the small G protein CDC42 connect WASp-mediated DGKα inhibition to SAP and the TCR signalosome. In primary human T cells, this new signalling pathway is necessary for a full response in terms of IL-2 production, while minimally affecting TCR signalling and restimulation-induced cell death. Conversely, in T cells made resistant to RICD by SAP silencing, the enhanced DAG signalling due to DGKα inhibition is sufficient to restore apoptosis sensitivity. Conclusion: We discover a novel signalling pathway where, upon strong TCR activation, the complex between WASp and DGKα blocks DGKα activity, allowing a full cytokine response.

Synergistic Phenomena between Iron-Doped ZnO Nanoparticles and Shock Waves Exploited against Pancreatic Cancer Cells.

We propose the use of iron-doped zinc oxide nanoparticles (Fe:ZnO NPs) showing theranostic capabilities and being synergistically active against pancreatic ductal adenocarcinoma once combined with mechanical pressure waves, such as shock waves. Fe:ZnO NPs are synthesized by employing oleic acid as a capping agent and are functionalized with amino-propyl groups. We first report their superior characteristics with respect to undoped ZnO NPs in terms of magnetic properties, colloidal stability, cytocompatibility, and internalization into BxPC-3 pancreatic cancer cells in vitro. These Fe:ZnO NPs are also cytocompatible toward normal pancreatic cells. We then perform a synergistic cell treatment with both shock waves and Fe:ZnO NPs once internalized into cells. We also evaluate the contribution to the synergistic activity of the NPs located in the extracellular space. Results show that both NPs and shock waves, when administered separately, are safe to cells, while their combination provokes an enhanced cell death after 24 h. Various mechanisms are then considered, such as dissolution of NPs, production of free radicals, and cell membrane disruption or permeation. It is understood so far that iron-doped ZnO NPs can degrade intracellularly into zinc cations, while the use of shock waves produce cell membrane permeabilization and possible rupture. In contrast, the production of reactive oxygen species is here ruled out. The provoked cell death can be recognized in both apoptotic and necrotic events. The proposed work is thus a first proof-of-concept study enabling promising future applications to deep-seated tumors such as pancreatic cancer, which is still an unmet clinical need with a tremendous death rate.

Extracellular Vesicles Tropism: A Comparative Study between Passive Innate Tropism and the Active Engineered Targeting Capability of Lymphocyte-Derived EVs.

Cellular communications take place thanks to a well-connected network of chemical-physical signals, biomolecules, growth factors, and vesicular messengers that travel inside or between cells. A deep knowledge of the extracellular vesicle (EV) system allows for a better understanding of the whole series of phenomena responsible for cell proliferation and death. To this purpose, here, a thorough immuno-phenotypic characterization of B-cell EV membranes is presented. Furthermore, the cellular membrane of B lymphocytes, Burkitt lymphoma, and human myeloid leukemic cells were characterized through cytofluorimetry assays and fluorescent microscopy analysis. Through cytotoxicity and internalization tests, the tropism of B lymphocyte-derived EVs was investigated toward the parental cell line and two different cancer cell lines. In this study, an innate capability of passive targeting of the native EVs was distinguished from the active targeting capability of monoclonal antibody-engineered EVs, able to selectively drive the vesicles, enhancing their internalization into the target cancer cells. In particular, the specific targeting ability of anti-CD20 engineered EVs towards Daudi cells, highly expressing CD20 marker on their cell membrane, was proved, while almost no internalization events were observed in HL60 cells, since they did not express an appreciable amount of the CD20 marker on their plasma membranes.

Iron-Doped ZnO Nanoparticles as Multifunctional Nanoplatforms for Theranostics.

Zinc oxide nanoparticles (ZnO NPs) are currently among the most promising nanomaterials for theranostics. However, they suffer from some drawbacks that could prevent their application in nanomedicine as theranostic agents. The doping of ZnO NPs can be effectively exploited to enhance the already-existing ZnO properties and introduce completely new functionalities in the doped material. Herein, we propose a novel synthetic approach for iron-doped ZnO (Fe:ZnO) NPs as a multifunctional theranostic nanoplatform aimed at cancer cell treatment. Pure ZnO and Fe:ZnO NPs, with two different levels of iron doping, were synthesized by a rapid wet-chemical method and analyzed in terms of morphology, crystal structure and chemical composition. Interestingly, Fe:ZnO NPs featured bioimaging potentialities thanks to superior optical properties and novel magnetic responsiveness. Moreover, iron doping provides a way to enhance the electromechanical behavior of the NPs, which are then expected to show enhanced therapeutic functionalities. Finally, the intrinsic therapeutic potentialities of the NPs were tested in terms of cytotoxicity and cellular uptake with both healthy B lymphocytes and cancerous Burkitt's lymphoma cells. Furthermore, their biocompatibility was tested with a pancreatic ductal adenocarcinoma cell line (BxPC-3), where the novel properties of the proposed iron-doped ZnO NPs can be potentially exploited for theranostics.

Focalization Performance Study of a Novel Bulk Acoustic Wave Device.

This work illustrates focalization performances of a silicon-based bulk acoustic wave device applied for the separation of specimens owing to micrometric dimensions. Samples are separated in the microfluidic channel by the presence of an acoustic field, which focalizes particles or cells according to their mechanical properties compared to the surrounded medium ones. Design and fabrication processes are reported, followed by focalization performance tests conducted either with synthetic particles or cells. High focalization performances occurred at different microparticle concentrations. In addition, preliminary tests carried out with HL-60 cells highlighted an optimal separation performance at a high flow rate and when cells are mixed with micro and nanoparticles without affecting device focalization capabilities. These encouraging results showed how this bulk acoustic wave device could be exploited to develop a diagnostic tool for early diagnosis or some specific target therapies by separating different kinds of cells or biomarkers possessing different mechanical properties such as shapes, sizes and densities.

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.

Remotely Activated Nanoparticles for Anticancer Therapy.

Cancer has nowadays become one of the leading causes of death worldwide. Conventional anticancer approaches are associated with different limitations. Therefore, innovative methodologies are being investigated, and several researchers propose the use of remotely activated nanoparticles to trigger cancer cell death. The idea is to conjugate two different components, i.e., an external physical input and nanoparticles. Both are given in a harmless dose that once combined together act synergistically to therapeutically treat the cell or tissue of interest, thus also limiting the negative outcomes for the surrounding tissues. Tuning both the properties of the nanomaterial and the involved triggering stimulus, it is possible furthermore to achieve not only a therapeutic effect, but also a powerful platform for imaging at the same time, obtaining a nano-theranostic application. In the present review, we highlight the role of nanoparticles as therapeutic or theranostic tools, thus excluding the cases where a molecular drug is activated. We thus present many examples where the highly cytotoxic power only derives from the active interaction between different physical inputs and nanoparticles. We perform a special focus on mechanical waves responding nanoparticles, in which remotely activated nanoparticles directly become therapeutic agents without the need of the administration of chemotherapeutics or sonosensitizing drugs.

Zinc Oxide Nanocrystals and High-Energy Shock Waves: A New Synergy for the Treatment of Cancer Cells.

In the last years, different nanotools have been developed to fight cancer cells. They could be administered alone, exploiting their intrinsic toxicity, or remotely activated to achieve cell death. In the latter case, ultrasound (US) has been recently proposed to stimulate some nanomaterials because of the US outstanding property of deep tissue penetration and the possibility of focusing. In this study, for the first time, we report on the highly efficient killing capability of amino-propyl functionalized ZnO nanocrystals (ZnO NCs) in synergy with high-energy ultrasound shock waves (SW) for the treatment of cancer cells. The cytotoxicity and internalization of ZnO NCs were evaluated in cervical adenocarcinoma KB cells, as well as the safety of the SW treatment alone. Then, the remarkably high cytotoxic combination of ZnO NCs and SW was demonstrated, comparing the effect of multiple (3 times/day) SW treatments toward a single one, highlighting that multiple treatments are necessary to achieve efficient cell death. At last, preliminary tests to understand the mechanism of the observed synergistic effect were carried out, correlating the nanomaterial surface chemistry to the specific type of stimulus used. The obtained results can thus pave the way for a novel nanomedicine treatment, based on the synergistic effect of nanocrystals combined with highly intense mechanical pressure waves, offering high efficiency, deep and focused tissue penetration, and a reduction of side effects on healthy cells.

The Synergistic Effect of Nanocrystals Combined With Ultrasound in the Generation of Reactive Oxygen Species for Biomedical Applications.

Reactive oxygen species (ROS) effects on living cells and tissues is multifaceted and their level or dose can considerably affect cell proliferation and viability. It is therefore necessary understand their role also designing ways able to regulate their amount inside cells, i.e., using engineered nanomaterials with either antioxidant properties or, for cancer therapy applications, capable to induce oxidative stress and cell death, through tunable ROS production. In this paper, we report on the use of single-crystalline zinc oxide (ZnO) round-shaped nanoparticles, yet ZnO nanocrystals (NCs) functionalized with amino-propyl groups (ZnO-NH2 NCs), combined with pulsed ultrasound (US). We show the synergistic effects produced by NC-assisted US which are able to produce different amount of ROS, as a result of inertial cavitation under the pulsed US exposure. Using Passive Cavitation Detection (PCD) and Electron Paramagnetic Resonance (EPR) spectroscopy, we systematically study which are the key parameters, monitoring, and influencing the amount of generated ROS measuring their concentration in water media and comparing all the results with pure water batches. We thus propose a ROS generation mechanism based on the selective application of US to the ZnO nanocrystals in water solutions. Ultrasound B-mode imaging is also applied, proving in respect to pure water, the enhanced ecographic signal generation of the aqueous solution containing ZnO-NH2 NCs when exposed to pulsed ultrasound. Furthermore, to evaluate the applicability of ZnO-NH2 NCs in the biomedical field, the ROS generation is studied by interposing different tissue mimicking materials, like phantoms and ex vivo tissues, between the US transducer and the sample well. As a whole, we clearly proof the enhanced capability to produce ROS and to control their amount when using ZnO-NH2 NCs in combination with pulsed ultrasound anticipating their applicability in the fields of biology and health care.

A Microwave-Assisted Synthesis of Zinc Oxide Nanocrystals Finely Tuned for Biological Applications.

Herein we report a novel, easy, fast and reliable microwave-assisted synthesis procedure for the preparation of colloidal zinc oxide nanocrystals (ZnO NCs) optimized for biological applications. ZnO NCs are also prepared by a conventional solvo-thermal approach and the properties of the two families of NCs are compared and discussed. All of the NCs are fully characterized in terms of morphological analysis, crystalline structure, chemical composition and optical properties, both as pristine nanomaterials or after amino-propyl group functionalization. Compared to the conventional approach, the novel microwave-derived ZnO NCs demonstrate outstanding colloidal stability in ethanol and water with long shelf-life. Furthermore, together with their more uniform size, shape and chemical surface properties, this long-term colloidal stability also contributes to the highly reproducible data in terms of biocompatibility. Actually, a significantly different biological behavior of the microwave-synthesized ZnO NCs is reported with respect to NCs prepared by the conventional synthesis procedure. In particular, consistent cytotoxicity and highly reproducible cell uptake toward KB cancer cells are measured with the use of microwave-synthesized ZnO NCs, in contrast to the non-reproducible and scattered data obtained with the conventionally-synthesized ones. Thus, we demonstrate how the synthetic route and, as a consequence, the control over all the nanomaterial properties are prominent points to be considered when dealing with the biological world for the achievement of reproducible and reliable results, and how the use of commercially-available and under-characterized nanomaterials should be discouraged in this view.

ZnO nanocrystals shuttled by extracellular vesicles as effective Trojan nano-horses against cancer cells.

Aim: The effective application of nanoparticles in cancer theranostics is jeopardized by their aggregation in biological media, rapid degradation and clearance. The design of biomimetic nanoconstructs with enhanced colloidal stability and non-immunogenicity is therefore essential. We propose naturally stable cell-derived extracellular vesicles to encapsulate zinc oxide (ZnO) nanocrystals as efficacious nanodrugs, to obtain highly biomimetic and stable Trojan nano-horses (TNHs). Materials & methods: Coupling efficiency, biostability, cellular cytotoxicity and internalization were tested. Results:In vitro studies showed a high internalization of TNHs into cancer cells and efficient cytotoxic activity thanks to ZnO intracellular release. Conclusion: TNHs represent an efficient biomimetic platform for future nanotheranostic applications, with biomimetic extracellular vesicle-lipid envelope, facilitated ZnO cellular uptake and potential therapeutic implications.

Enhanced selective sonosensitizing efficacy of ultrasound-based anticancer treatment by targeted gold nanoparticles.

AIM: This study investigates cancer targeted gold nanoparticles as ultrasound sensitizers for the treatment of cancer. METHODS: The ultrasound sensitizer activity of folate-PEG decorated gold nanoparticles (FA-PEG-GNP) has been studied on human cancer cell lines that overexpress folate receptors (KB and HCT-116) and another that does not (MCF7), at two ultrasound energy densities (8 × 10-6 J cm-2 and 8 × 10-5 J cm-2, for 5 min at 1.866 MHz). RESULTS: FA-PEG-GNP selectively targeted KB and HCT-116 cells and a remarkable reduction in cancer cell growth was observed upon ultrasound exposure, along with significant reactive oxygen species generation and increase in necrotic cells. CONCLUSION: The combined use of targeting capacity and the ultrasound sensitizing effect, make FA-PEG-GNP promising candidates for the site-specific cancer treatment.