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.

Artificial and Naturally Derived Phospholipidic Bilayers as Smart Coatings of Solid-State Nanoparticles: Current Works and Perspectives in Cancer Therapy.

Recent advances in nanomedicine toward cancer treatment have considered exploiting liposomes and extracellular vesicles as effective cargos to deliver therapeutic agents to tumor cells. Meanwhile, solid-state nanoparticles are continuing to attract interest for their great medical potential thanks to their countless properties and possible applications. However, possible drawbacks arising from the use of nanoparticles in nanomedicine, such as the nonspecific uptake of these materials in healthy organs, their aggregation in biological environments and their possible immunogenicity, must be taken into account. Considering these limitations and the intrinsic capability of phospholipidic bilayers to act as a biocompatible shield, their exploitation for effectively encasing solid-state nanoparticles seems a promising strategy to broaden the frontiers of cancer nanomedicine, also providing the possibility to engineer the lipid bilayers to further enhance the therapeutic potential of such nanotools. This work aims to give a comprehensive overview of the latest developments in the use of artificial liposomes and naturally derived extracellular vesicles for the coating of solid-state nanoparticles for cancer treatment, starting from in vitro works until the up-to-date advances and current limitations of these nanopharmaceutics in clinical applications, passing through in vivo and 3D cultures studies.

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.

Nanotechnological engineering of extracellular vesicles for the development of actively targeted hybrid nanodevices.

BACKGROUND: We propose an efficient method to modify B-cell derived EVs by loading them with a nanotherapeutic stimuli-responsive cargo and equipping them with antibodies for efficient targeting of lymphoma cells. RESULTS: The post-isolation engineering of the EVs is accomplished by a freeze-thaw method to load therapeutically-active zinc oxide nanocrystals (ZnO NCs), obtaining the so-called TrojanNanoHorse (TNH) to recall the biomimetism and cytotoxic potential of this novel nanoconstruct. TNHs are further modified at their surface with anti-CD20 monoclonal antibodies (TNHCD20) achieving specific targeting against lymphoid cancer cell line. The in vitro characterization is carried out on CD20+ lymphoid Daudi cell line, CD20-negative cancerous myeloid cells (HL60) and the healthy counterpart (B lymphocytes). The TNH shows nanosized structure, high colloidal stability, even over time, and good hemocompatibility. The in vitro characterization shows the high biocompatibility, targeting specificity and cytotoxic capability. Importantly, the selectivity of TNHCD20 demonstrates significantly higher interaction towards the target lymphoid Daudi cell line compared to the CD20-negative cancerous myeloid cells (HL60) and the healthy counterpart (lymphocytes). An enhanced cytotoxicity directed against Daudi cancer cells is demonstrated after the TNHCD20 activation with high-energy ultrasound shock-waves (SW). CONCLUSION: This work demonstrates the efficient re-engineering of EVs, derived from healthy cells, with inorganic nanoparticles and monoclonal antibodies. The obtained hybrid nanoconstructs can be on-demand activated by an external stimulation, here acoustic pressure waves, to exploit a cytotoxic effect conveyed by the ZnO NCs cargo against selected cancer cells.

Insight into Sonoluminescence Augmented by ZnO-Functionalized Nanoparticles.

Recent advances in optical imaging techniques rely on the use of nanosized contrast agents for in vitro and in vivo applications. We report on an imaging method based on the inertial cavitation of ultrasound-irradiated water solutions that lead to sonoluminescence (SL), here, newly proposed in combination with semiconductor nanoparticles, in particular, aminopropyl-functionalized zinc oxide nanocrystals. The obtained measurements confirm the ability of such nanocrystals to increase the sonoluminescence emission, together with the ability to modify the SL spectrum when compared to the pure water behavior. In particular, it is shown that the UV component of SL is absorbed by the semiconductor behavior that is also confirmed in different biologically relevant media. Finally, optical images of nanocrystal-assisted SL are acquired for the first time, in particular, in biological buffers, revealing that at low ultrasound intensities, SL is measurable only when the nanocrystals are present in solution. All of these results witness the role of amine-functionalized zinc oxide nanocrystals for sonoluminescence emission, which makes them very good candidates as efficient nanocontrast agents for SL imaging for biological and biomedical applications.

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.

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.

Gold Nanoparticles Radio-Sensitize and Reduce Cell Survival in Lewis Lung Carcinoma.

It has been suggested that particle size plays an important role in determining the genotoxicity of gold nanoparticles (GNPs). The purpose of this study was to compare the potential radio-sensitization effects of two different sized GNPs (3.9 and 37.4 nm) fabricated and examined in vitro in Lewis lung carcinoma (LLC) as a model of non-small cell lung cancer through use of comet and clonogenic assays. After treatment with 2Gy X-ray irradiation, both particle sizes demonstrated increased DNA damage when compared to treatment with particles only and radiation alone. This radio-sensitization was further translated into a reduction in cell survival demonstrated by clonogenicity. This work indicates that GNPs of both sizes induce DNA damage in LLC cells at the tested concentrations, whereas the 37.4 nm particle size treatment group demonstrated greater significance in vitro. The presented data aids in the evaluation of the radiobiological response of Lewis lung carcinoma cells treated with gold nanoparticles.

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.

Polydopamine Nanoparticles as an Organic and Biodegradable Multitasking Tool for Neuroprotection and Remote Neuronal Stimulation.

Oxidative stress represents a common issue in most neurological diseases, causing severe impairments of neuronal cell physiological activity that ultimately lead to neuron loss of function and cellular death. In this work, lipid-coated polydopamine nanoparticles (L-PDNPs) are proposed both as antioxidant and neuroprotective agents, and as a photothermal conversion platform able to stimulate neuronal activity. L-PDNPs showed the ability to counteract reactive oxygen species (ROS) accumulation in differentiated SH-SY5Y, prevented mitochondrial ROS-induced dysfunctions and stimulated neurite outgrowth. Moreover, for the first time in the literature, the photothermal conversion capacity of L-PDNPs was used to increase the intracellular temperature of neuron-like cells through near-infrared (NIR) laser stimulation, and this phenomenon was thoroughly investigated using a fluorescent temperature-sensitive dye and modeled from a mathematical point of view. It was also demonstrated that the increment in temperature caused by the NIR stimulation of L-PDNPs was able to produce a Ca2+ influx in differentiated SH-SY5Y, being, to the best of our knowledge, the first example of organic nanostructures used in such an approach. This work could pave the way to new and exciting applications of polydopamine-based and of other NIR-responsive antioxidant nanomaterials in neuronal research.

Engineered Extracellular Vesicles as a Reliable Tool in Cancer Nanomedicine.

Fast diagnosis and more efficient therapies for cancer surely represent one of the huge tasks for the worldwide researchers' and clinicians' community. In the last two decades, our understanding of the biology and molecular pathology of cancer mechanisms, coupled with the continuous development of the material science and technological compounds, have successfully improved nanomedicine applications in oncology. This review argues on nanomedicine application of engineered extracellular vesicles (EVs) in oncology. All the most innovative processes of EVs engineering are discussed together with the related degree of applicability for each one of them in cancer nanomedicines.

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.