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Profiling gene expression in Hydra vulgaris following Gold Nanoparticle-mediated hyperthermia

Periodic Reporting for period 1 - HyHeat (Profiling gene expression in Hydra vulgaris following Gold Nanoparticle-mediated hyperthermia)

Reporting period: 2015-09-01 to 2017-08-31

Hyperthermia (HT) is currently used as a non-invasive technique for cancer therapy, whereby biological tissues are exposed to higher than normal temperatures, for selective ablation of tumoral cells. Heating treatments can be applied using external heating sources such as ultrasounds, however, heating only malignant cells selectively is difficult to obtain, and therefore the translation of this modality to the clinic is still challenging. Recently, there has been a growing interest in the use of gold nanoparticles (AuNPs) to selectively generate heat in a spatiotemporal fashion, which is known as photothermal therapy (PTT). The rationale for such therapy is based on the fact that metallic NPs can be synthetized to absorb incident light coming from a laser outside the body and generate heat only in the tissues where NPs are allocated. Moreover, AuNPs are highly biocompatible and can be finely tuned to absorb light in the near-infrared (NIR) spectral range, known as the “biological window” (690-1100 nm). In this range tissue is maximally transparent, and therefore, the light itself would not damage the tissue.
While significant efforts have been made to develop suitable NPs with appropriate heating capabilities, to date, the molecular mechanisms underlying the in vivo cellular responses to heat stress remain unclear.
Therefore, the overall aim of the HyHeat project is to use an invertebrate model (Hydra vulgaris) to screen the heating capabilities of different AuNPs in vivo, with the grand aim of understanding the cellular responses to heat stress and therefore taking the first steps towards improving nanoparticle mediated HT efficacy for therapeutic purposes. A simple invertebrate organism have been used, in line with European strategies aimed to reduce vertebrate experimentation. During the project, we have synthesized different types of AuNPs and fully characterized them. The toxicity in the animals have been assessed using different techniques, before proceeding to laser irradiation. Lastly, gene expression profiling after laser irradiation in Hydra has been performed. It has been possible to characterize the heating capabilities of the AuNPs in vivo and select deregulated genes upon irradiation.
Two types of gold nanoparticles with localized surface plasmon resonance band in the near infrared have been synthesized. The as-synthesized AuNPs were stabilized using a bifunctional poly(ethylene glycol)(PEG). After being synthetized, these NPs have been fully characterized, by means of Transmision Electron Microscopy (TEM), UV-Vis, inductively coupled plasma mass sprectrometry (ICP-MS) and thermogravimetric analysis(TGA). To improve the cellular uptake and to be able to track the AuNPs once in the animals, further functionalizaition of the surface has been carried out using glucose and a commercial dye, i.e. Tamra.
Both types of AuNPs have been incubated with Hydra to test the toxicity in adult animals, using different assays, namely alterations in the morphology of the animals, in the regeneration rates or the reproduction processes. To study the biodistribution of the nanoparticles, fluorescence microscopy, TEM and ICP have been used.
Laser irradiation conditions were tuned to obtain a mild and sublethal hyperthermia treatment. For measuring the modulation of the genes upon laser irradiation, qRT-PCR was performed. Cell necrosis and viability were also evaluated using DAPI and propidium iodide staining. After the molecular analysis we have been able to classify the AuNP heating capabilities in vivo and to identify the genes that are deregulated upon laser irradiation. In mice, it has been shown that AuNPs are not toxic either at short or long term, which is a good point for the design of future PTT experiments.
The main results of the project have been disseminated using different channels depending on the target audience. In a scientific context, one manuscript have been published, and three are being prepared or under review. The results have been presented in four scientific conferences and invited talks at different Research Institutes. Outreach activities for general public have been performed for elementary and secondary schools, as well as publishing an article in a non-scientific journal.
The objectives proposed in the project have been successfully accomplished. By determining the gene expression profile after laser irradiation, it has been shown which AuNPs are better nanoheaters in vivo. This can help to take the first steps towards improving PTT efficacy for clinical purposes. Also, the main genes deregulated after irradiating the animals have been assessed, both in mild and sublethal conditions. Lastly, it has been shown that Hydra could serve as an excellent model to reduce vertebrate experimentation, which is in line with guidelines of ECVAM for the development of new testing species. Gaining understanding in fundamental areas such as PTT will help to enhance the excellence and competitiveness in the Nanotechnology field. Project results will be useful for the community working with gold nanoparticles and PTT, as it enhances the understanding of the molecular mechanisms underlying the cellular responses to heat stress caused by PTT. Also, people working with vertebrates could be attracted to start working with invertebrate models due to ethical and monetary restrictions.
Overview of HyHeat project