Differential effects on membrane permeability and viability of human keratinocyte cells undergoing very low intensity megasonic fields

Among different therapeutic applications of Ultrasound (US), transient membrane sonoporation (SP) - a temporary, non-lethal porosity, mechanically induced in cell membranes through US exposure - represents a compelling opportunity towards an efficient and safe drug delivery. Nevertheless, progresses in this field have been limited by an insufficient understanding of the potential cytotoxic effects of US related to the failure of the cellular repair and to the possible activation of inflammatory pathway. In this framework we studied the in vitro effects of very low-intensity US on a human keratinocyte cell line, which represents an ideal model system of skin protective barrier cells which are the first to be involved during medical US treatments. Bioeffects linked to US application at 1 MHz varying the exposure parameters were investigated by fluorescence microscopy and fluorescence activated cell sorting. Our results indicate that keratinocytes undergoing low US doses can uptake drug model molecules with size and efficiency which depend on exposure parameters. According to sub-cavitation SP models, we have identified the range of doses triggering transient membrane SP, actually with negligible biological damage. By increasing US doses we observed a reduced cells viability and an inflammatory gene overexpression enlightening novel healthy relevant strategies

In vitro analysis of the mechanical and biological effects induced by the ultrasound-cell interaction

The exposition to megasonic fields can induce several biological effects with potential damage for human health. The more significant ones involve membrane poration, with the consequent permeabilization of cells to exogenous particles and the triggering of inflammatory and cytotoxic processes. We developed an experimental setup with the purpose to perform an in vitro systematic analysis of the biological effects related to the ultrasound-cell interaction. Our study involved two using different cell models, human keratinocyte (HaCaT) and skin melanoma (SK-Mel28). The ultrasound-induced biological effects have been investigated at two frequencies (0.5 MHz and 1 MHz) as a function of different exposure parameters

Effect of 1-MHz ultrasound on the proinflammatory interleukin-6 secretion in human keratinocytes

Keratinocytes, the main cell type of the skin, are one of the most exposed cells to environmental factors, providing a first defence barrier for the host and actively participating in immune response. In fact, keratinocytes express pattern recognition receptors that interact with pathogen associated molecular patterns and damage associated molecular patterns, leading to the production of cytokines and chemokines, including interleukin (IL)-6. Herein, we investigated whether mechanical energy transported by low intensity ultrasound (US) could generate a mechanical stress able to induce the release of inflammatory cytokine such IL-6 in the human keratinocyte cell line, HaCaT. The extensive clinical application of US in both diagnosis and therapy suggests the need to better understand the related biological effects. Our results point out that US promotes the overexpression and secretion of IL-6, associated with the activation of nuclear factor-kappa B (NF-kappa B). Furthermore, we observed a reduced cell viability dependent on exposure parameters together with alterations in membrane permeability, paving the way for further investigating the molecular mechanisms related to US exposure

PLGA based particles as “drug reservoir” for antitumor drug delivery: characterization and cytotoxicity studies

Doxorubicin (DOX) is commonly used to treat several tumor types, but its severe side effects, primarily cardiotoxicity, represent a major limitation for its use in clinical settings. In this study we developed and characterized biodegradable and stable poly(D,L-lactic-co-glycolic) acid (PLGA) submicrocarriers employing an osmosis-based patented methodology, which allowed to optimize the drug loading efficiency up to 99%. Proceeding from this, we evaluated on MCF-7, a human breast cancer cell line, the ability of PLGA to promote the internalization of DOX and to improve its cytotoxicity in vitro. We found that the in vitro uptake efficiency is dramatically increased when DOX is loaded within PLGA colloidal carriers, which adhere to the cell membrane behaving as an efficient drug reservoir. In fact, the particles provide a diffusion-driven, sustained release of DOX across the cell membrane, resulting in high drug concentration. Accordingly, the cytotoxic analysis clearly showed that DOX-loaded PLGA exhibit a lower 50% inhibitory concentration than free DOX. The decay time of cell viability was successfully compared with DOX diffusion time constant from PLGA. The overall in vitro results highlight the potential of DOX-loaded PLGA particles to be employed as vectors with improved antitumor efficacy

SERS-based diagnostics: selective targeting of different human cancer cells using functionalized gold nanoparticles

In the last few decades, the development of novel spectroscopic techniques, often combining a very high sensitivity with huge spatial resolution, allowed the possibility of investigating down-scaled phenomena, as for example biochemical and biophysical processes in living systems. Surface Enhanced Raman Spectroscopy (SERS) is one of the most established techniques in this framework [1]. SERS is based on the plasmonic resonance of metal nanostructures: the collective electronic excitation at the metal surface can indeed be excited by light and give rise to the localization of strong electromagnetic fields close to the nanoparticle surface, which can be used for spectroscopy. In the past years, SERS has allowed to reach not only the threshold of single molecule vibrational spectroscopy [2], but also the implementation of devices in the field of biosensing, capable of detecting specific biomolecules at very low concentration by means of Raman spectroscopy [3]. Moreover, the implementation of SERS-labelled nanomaterials, such as functionalized metallic nanoparticles, paved the way for the application of these systems in the emerging field of nanomedicine [4]. Much interest has lately risen, indeed, around the concept of “theranostics”, i.e. combining diagnostics with therapy, the latter to perform selectively on cancer cells without damaging the healthy tissue [4]. Plasmonics-based theranostics is often designed combining SERS and photothermal bleaching. One of the open problems in biomedicine is the early detection of cancer, i.e. the capability of reveal the presence of the disease when it is not yet advanced. Addressing this problem, we designed a biocompatible system based on gold nanoparticles functionalized with the Raman active bifunctional linker 4-aminothiophenol and further conjugated with folic acid, a biomolecule with an essential role in cell reproduction. Our system can be considered a nanobiovector, as it is capable of targeting a specific kind of cell and locate on the folate receptors, on the cell membrane [5]. Folic acid receptors are generally overexpressed in many types of cancer cells, as these reproduce more frequently then ordinary ones [6]. The presence of folate receptors on the membrane strongly depends on the physiology of the cell line considered. In this presentation, we will show that the high specificity of our system allowed us not only to target cancer cells, but also to be able to distinguish different cell lines based on their level of expression of folate receptors [5]

Antifolate SERS-active nanovectors: Quantitative drug nanostructuring and selective cell targeting for effective theranostics

One of the frontiers of nanomedicine is the rational design of theranostic nanovectors. These are nanosized materials combining diagnostic and therapeutic capabilities, i.e. capable of tracking cancer cells and tissues in complex environments, and of selectively acting against them. We herein report on the preparation and application of antifolate plasmonic nanovectors, made of functionalized gold nanoparticles conjugated with the folic acid competitors aminopterin and methotrexate. Due to the overexpression of folate binding proteins on many types of cancer cells, these nanosystems can be exploited for selective cancer cell targeting. The strong surface enhanced Raman scattering (SERS) signature of these nanovectors acts as a diagnostic tool, not only for tracing their presence in biological samples, but also, through a careful spectral analysis, to precisely quantify the amount of drug loaded on a single nanoparticle, and therefore delivered to the cells. Meanwhile, the therapeutic action is implemented based on the strong toxicity of antifolate drugs. Remarkably, supplying the drug in the nanostructured form, rather than as a free molecule, enhances its specific toxicity. The selectivity of the antifolate nanovectors can be optimized by the design of a hybrid folate/antifolate coloaded nanovector for the specific targeting of folate receptor α, which is overexpressed on numerous cancer cell types

Surface enhanced Raman microimaging allows for screening single cells with different folate binding capabilities

In the last few decades, the development of novel spectroscopic techniques, often combining a very high sensitivity with huge spatial resolution, allowed the possibility of investigating down-scaled phenomena, as for example biochemical and biophysical processes in living systems. Surface Enhanced Raman Spectroscopy (SERS) is one of the most established techniques in this framework [1]. SERS is based on the plasmonic resonance of metal nanostructures: the collective electronic excitation at the metal surface can indeed be excited by light and give rise to the localization of strong electromagnetic fields close to the nanoparticle surface, which can be used for spectroscopy. In the past years, SERS has allowed to reach not only the threshold of single molecule vibrational spectroscopy [2], but also the implementation of devices in the field of biosensing, capable of detecting specific biomolecules at very low concentration by means of Raman spectroscopy [3]. Moreover, the implementation of SERS-labelled nanomaterials, such as functionalized metallic nanoparticles, paved the way for the application of these systems in the emerging field of nanomedicine [4]. Much interest has lately risen, indeed, around the concept of “theranostics”, i.e. combining diagnostics with therapy, the latter to perform selectively on cancer cells without damaging the healthy tissue [4]. Plasmonics-based theranostics is often designed combining SERS and photothermal bleaching. One of the open problems in biomedicine is the early detection of cancer, i.e. the capability of reveal the presence of the disease when it is not yet advanced. Addressing this problem, we designed a biocompatible system based on gold nanoparticles functionalized with the Raman active bifunctional linker 4-aminothiophenol and further conjugated with folic acid, a biomolecule with an essential role in cell reproduction. Our system can be considered a nanobiovector, as it is capable of targeting a specific kind of cell and locate on the folate receptors, on the cell membrane [5]. Folic acid receptors are generally overexpressed in many types of cancer cells, as these reproduce more frequently then ordinary ones [6]. The presence of folate receptors on the membrane strongly depends on the physiology of the cell line considered. In this presentation, we will show that the high specificity of our system allowed us not only to target cancer cells, but also to be able to distinguish different cell lines based on their level of expression of folate receptors [5]