|Wednesday, July 20|
Detecting or inhibiting the production of reactive oxygen species for biomedical applications of gold nanorods irradiated with ultrashort laser pulses
Sarra Mitiche, CentraleSupélec - Université Paris-Saclay, France
Syrine Gueffrache, CentraleSupélec - Université Paris-Saclay, France
Sylvie Marguet, CEA, France
Jean-Frédéric Audibert, Ecole Normale Supérieure Paris-Saclay - Université Paris-Saclay, France
Robert Bernard Pansu, Ecole Normale Supérieure Paris-Saclay - Université Paris-Saclay, France
* Bruno Palpant, CentraleSupélec - Université Paris-Saclay, France
Gold nanoparticles can produce reactive oxygen species (ROS) under ultrashort pulsed light. While beneficial for photodynamic therapy, this phenomenon is prohibitive for other biomedical applications such as imaging or targeted gene or drug delivery by plasmonic photothermal conversion. ROS are usually detected by employing specific fluorescent molecules. SOSG is widely used to probe singlet oxygen (1O2). However, the chemical environment of the nanoparticles in the solution may interact with these probes and disturb the determination of ROS production. It is then needed to control the conditions in which the latter is carried out. Besides, gold nanorods (AuNRs) are an iconic choice for biomedical applications as they can exhibit an intense longitudinal plasmon mode in the transparency windows of biological tissues. A surfactant molecule, CTAB, ensures their growth and stability in solution, but it is cytotoxic. Coating AuNRs with a silica layer makes AuNRs biocompatible by removing CTAB from their surface, prevents them from shape modification under high laser intensity, and avoids the formation of a protein corona. In addition, a mesoporous silica shell can be loaded with drugs, dyes, or imaging agents. We will first investigate the effect of the CTAB-SOSG interaction for reliable 1O2 detection when produced by ultrashort laser-pulse irradiation of AuNRs, and suggest proper AuNR surface chemistry to assess the ROS production. Then, we will study the effect of coating AuNRs with silica on their ROS generation. AuNRs were synthesized using a seeded growth method in the presence of CTAB. Silica was added using the 3-mercaptopropyl trimethoxysilane precursor. ROS are produced in water by irradiating AuNRs with 400-fs laser pulses tuned to their plasmon resonance (1030 nm wavelength) and detected by fluorescence spectroscopy under 515-nm light excitation. SOSG is used to detect 1O2 while DRH probes 1O2 and •OH. Calculations combining the Boundary Element Method and Boltzmann’s equation for the hot electron dynamics in the AuNRs enable us to determine the ultrafast transient optical near-field, a key parameter to interpret ROS production properties. We first demonstrate that the SOSG emission properties and sensitivity to 1O2 strongly depend on the CTAB concentration due to CTAB micelles. With AuNRs acting as 1O2 photosensitizers, we show that 1O2 detection is not possible in CTAB, whereas a high sensitivity of SOSG for 1O2 is obtained in citrate and PEG. We then evidence that a dense silica shell added onto AuNRs inhibits the formation of 1O2 and •OH efficiently. The plasmonic field enhancement at the AuNR tips is reduced. With the multiphotonic ejection of electrons and the Dexter energy transfer to 3O2 being also blocked, the silica coating hinders all the possible pathways for ROS production. To conclude, citrate and PEG are two alternatives to CTAB for reliable use of SOSG to investigate 1O2 generation by plasmonic AuNRs. In addition, by inhibiting ROS production, silica-coated AuNRs are not suitable for use as direct photosensitizers but are safer than pure AuNRs for a range of other applications in biomedicine.
Development of DOTAREM-Gold-Complex: A new multifunctional nanotheranostic agent for Cholangiocarcinoma
* Memona Khan, CSPBAT Laboratory , France
Dotarem (DOTA) is a macrocyclic contrast agent of type T1 used in magnetic resonance imaging (MRI) 1. This contrast agent is known as a non-toxic and effective to improve the contrast of MRI images 1. The particularity of DOTA is to have gadolinium in the core and DOTA, around it to protect. Thus, gadolinium remains effective while being less toxic to the body. In this work a novel hybrid gold nanoparticle system was developed and formulated, in which the contrast agent was combined with drug on the same nanoparticles by chelation methodology (Method IN) 2. For this purpose, firstly bimetallic Au-DOTA nanoparticles were synthesized by complexation and stabilized with lactose- modified Chitosan, Chitlac (CTL) to form DOTA IN-CTL AuNPs. The choice of polymer was done because of its anti-cancer properties and because it recognizes the protein Galectin-1 (Gal-1), overly expressed on cancer cells. Physical and chemical evaluation was performed by spectroscopic analysis techniques (Raman spectroscopy, UV-visible and transmission electron microscopy TEM). The biological results on three lines: Mia PaCa-2 (human pancreatic cancer cell line), TIB-75 (Murine liver cell line) and KKU-M213 (Cholangiocarcinoma cell line) show that DOTA IN-CTL AuNPs alone are not toxic. However, when these cell lines were irradiated (with DOTA IN-CTL AuNPs) with a laser of 808 nm, a clear death cell can be observed. More, relaxivity measures by MRI, has shown effectiveness of contrast with these nanoparticles. DOTA IN-CTL AuNPs are stable under physiological conditions, are nontoxic, and are very efficient as PTT agents. The highlights such as high stability and preliminary MRI in vitro and in vivo models may be suitable for diagnosis and therapy. These hybrid gold nanoparticles open a great pathway in the field of theranostic, their application as MRI and PTT agents will bring a new approach for disease diagnosis and clinical therapies.
Gold/Polyoxometalate core/shell nanoparticles for combined chemotherapy-photothermal cancer therapy
* Anne Vallée, UVSQ, France
Despite the evolution of tumor treatments, actual cancer treatments still have many defects with a limited efficacity and side effects. It is therefore essential to find an alternative to current treatments. Gold nanoparticles (AuNPs) are amongst the state-of-the-art actors for biomedical applications because of their low toxicity and the possibility to consider combinatorial therapy to reach an optimized global therapeutic effect1. AuNPS show excellent performances in photothermal therapy (PTT) and can be easily functionalized with therapeutic agent allowing the facilitation of cellular internalization of the active agents while limiting their quantity. The combination of hyperthermia and chemotherapy has great promises to enhance the efficiency of cancer treatment due to the stronger therapeutic effect compared to a single therapy. Polyoxometalates (POMs), a class of molecular oxides composed of metals in high oxidation states, typically WVI and MoVI, known for wide range of magnetic, redox and catalytic properties, are emerging as antitumor agents 2. Additionally, POMs can be used as protecting ligands for metallic nanoparticles. In this context, the use of POMs as active agents supported by gold nanoparticles (AuNPs) is a very promising alternative to current treatments. In this study3, core/shell POMs/gold nanoparticles were designed. Polyoxomolybdate functionalized with zoledronate Mo4Zol2MnIII having shown very good antitumoral properties4 were used to decorate spherical gold nanoparticles. These new composites provided a combined antitumor activity through drug delivery and photothermal therapy. The NPs inhibited in vitro the proliferation of prostate cancer cells (PC3) in a dose-dependent manner.  Beik, J.; Khateri, M.; Khosravi, Z.; Kamrava, S. K.; Kooranifar, S.; Ghaznavi, H.; Shakeri-Zadeh, A. Gold nanoparticles in combinatorial cancer therapy strategies Coordination. Chemistry Reviews 2019,387,299-324  Bijelic, A.; Aureliano, M.; Rompel, A. Polyoxometalates as Potential Next-Generation Metallodrugs in the Combat Against Cancer. Angewandte Chemie - International Edition 2019, 58,2980-2999  Tomane, S ; Wilhelm, C ; Boujday, S. ; Fromain, A. ; Miche, A. ; Bourdreux, F. ; Dolbecq, A.; Mialane, P. and Vallée, A.* Gold/Polyoxometalate Core/Shell Nanoparticles for Combined Chemotherapy−Photothermal Cancer Therapy, ACS Applied Nano Materials 202, 4, 2339-2344.1  Boulmier, A. X. Feng, Oms,O.; Mialane, P.; Rivière, E.; Shin, C. J.; Yao, J.; Kubo, T.; Furuta, T. ; Oldfield, E.; Dolbecq, A. Anticancer Activity of Polyoxometalate-Bisphosphonate Complexes: Synthesis, Characterization, in Vitro and in Vivo Results, Inorganic Chemistry 2017, 56, 7558–7565.
High-sensitivity kinetic imaging of ultrasmall gold nanoparticles permeation across healthy and psoriatic human skin
* Mahmoud Omar, University Laval, Canada
Mariia Kiseleva, University Laval, Canada
Geneviève Rioux, University Laval, Canada
Roxane Pouliot, University Laval, Canada
Marc-André Fortin, University Laval, Canada
The diffusion of ultra-small gold-nanoparticles (GNPs) through the skin is now well documented, and formulations of GNPs are being developed as drug vectors for a variety of topical skin applications.1 In the pharmaceutical sciences, measurement of drugs and NPs permeation across the skin are often performed using diffusion-cells. These devices are made of two compartments (donor -DC; and acceptor -AC), separated by the tested membrane (e.g. skin). Tested solutions are administered in the DC, and the concentration diffusing into the AC, is measured by different techniques (e.g. UV/vis, FTIR,..etc.). Recently, the superior sensitivity of positron emission tomography (PET) over these techniques for the measurement of GNPs permeation across the skin, was reported.2 Diffusion-cells can be specifically designed for operation in PET3, thus allowing to monitor in continuous, in real-time, and at high sensitivity the concentration of drugs or NPs in the DC, AC, and potentially accumulating in the membrane. Many recent studies highlighted the advantage of treating psoriasis using ultra-small GNPs, either as a drug delivery vector or due to their intrinsic anti-inflammatory properties.4,5&6 The present study aimed at measuring at high temporal precision and molecular sensitivity, the permeation kinetics of GNPs across both healthy and psoriatic reconstructed-human skin samples. GNPs (4nm diam.-TEM; hydrodynamic-diam. 19nm diam.-DLS) were grafted with deferoxamine (DFO, chelator for Zr(IV); for PET measurements), and with the fluorescent dye Cy 5 (max.: 666nm; for histology analyses). The physicochemical properties of GNPs were characterized by FTIR, X-ray photoelectron spectroscopy (XPS), and elemental analysis. Radiolabeling was performed with 89Zr(IV) (half-life: 3.3 days; 95% radiochemical yield; Au:Zr molar ratio: 1:1.7). The permeation of GNP through healthy and psoriatic skin was measured for 20h in continuous by PET using a specially designed diffusion-cell (Figure 1).2 Permeation profiles and kinetic parameters (lag-time, influx,..etc.) were extracted. Finally, GNPs distribution inside the skin was evaluated microscopically. DFO grafting was confirmed by FTIR (hydroxamate peaks: 1629.0cm-1, 1569.0cm-1; amine peak: 3312.0cm-1) and XPS, whereas Cy5 absorption was confirmed by UV/visible (650nm). Radiolabeled GNPs were detected by the PET at a concentration as low as 6.8x10-6nM of 89Zr-DFO and 1nM of Au. The kinetic profiles measured by PET, revealed two distinct permeation phases (Figure 2). GNPs permeated faster in psoriatic skin compared with healthy skin (lag-time 2h and 4h, respectively). The PET system also allows to measure the accumulation of GNP in the skin (not possible by conventional diffusion-cell analysis). GNPs accumulate faster in psoriatic skin (>30min) than healthy skin (>5h), and the particle’s influx through psoriatic skin is double that observed for healthy skin (0.8 versus 0.4 %/h). A histological evaluation of GNPs distribution in skin samples in being conducted. The present study aimed at measuring at high temporal precision and molecular sensitivity, the permeation kinetics of GNPs across healthy and psoriatic reconstructed-human skin. The results demonstrate that PET can efficiently be used to measure the kinetic parameters of nanoparticle diffusion across the skin. Such technology could represent a powerful tool for the development of GNP-based topical formulations (creams,..etc.) in particular for the treatment of psoriasis.
In Vitro Optoporation Mediated by Gold Nanoparticles to Insert siRNA in Retinal Cells
* Isabelle Largillière, Polytechnique Montréal, Canada
Jennyfer Zapata-Farfan, Polytechnique Montréal, Canada
Przemyslaw Sapieha, University of Montreal, Canada
* Michel Meunier, Polytechnique Montréal, Canada
Age-related macular degeneration (AMD) is a retinal disease involving blindness with a prevalence of 170 million people worldwide in 2016 and is expected to increase significantly in the following years However, no cure for this disease exists yet, only treatments to slow down its progression. New techniques have been investigated to tackle this problem such as gene therapy to downregulate the vascular endothelial growth factor (VEGF) production, which is a protein at the origin of the disease. Different cargos have been studied to deliver VEGF targeting siRNA into retinal cells. We propose to use optoporation, a new technique based on the interaction between ultrafast laser irradiation and gold nanoparticles (AuNPs) to perforate the cell membrane and let the siRNA penetrate the cells. This technique has several advantages: less inflammatory issue and immune response than virus-based transfection method and double specificity thanks to the functionalization of AuNPs to aim precise cell population and the tuning of the irradiation area. It has been shown that this technique allows transfecting labeled siRNA in vitro and in vivo in rat’s retinal ganglion cells (RGC) but it has not been pushed further to check the downregulation induced by siRNA , . Using anti-CD44 functionalized 100nm AuNPs and a femtosecond laser, optoporation was performed first with Lucifer Yellow fluorophore and Cy3-siRNA. It allows us to find the optimal parameters for the optoporation of ARPE-19 cells and the laser power range to induce optoporation without decreasing the viability of the cells. Here, we prove that not only we can introduce the siRNA inside the cells but also that the siRNA is still active despite the irradiation and allows to downregulate significantly the VEGF. Reaching this step will open the way to in vivo translation of the technique on an animal model to analyze the efficacy of the treatment in a more representative environment.  A. M. Wilson et al., “in vivo laser-mediated retinal ganglion cell optoporation using K V 1.1 conjugated gold nanoparticles,” Nano Lett., 18, (2018)  C. Boutopoulos, E. Bergeron, and M. Meunier, “Cell perforation mediated by plasmonic bubbles generated by a single near infrared femtosecond laser pulse,” J. Biophotonics, 9,. 26–31 (2016)
In vivo performance of therapeutic gold nanoparticles applied as radiosensitizers in cervical cancer radiotherapy
* Mariia Kiseleva, Centre de Recherche du CHU de Québec Université Laval, Canada
Svetlana Selivanova, Centre de Recherche du CHU de Québec Université Laval, Canada
Marc-André Fortin, Centre de Recherche du CHU de Québec Université Laval, Canada
Cervical cancer is the fourth most common malignancy among women. The disease can be treated by brachytherapy (internal radiotherapy), which can be further enhanced by administration of gold nanoparticles (AuNPs) as radiosensitizers. The easy access to the cervix allows the local administration of such particles. To facilitate their administration, hydrogels are of particular interest since they are well tolerated by patients and can be 3D-printed to better conform to the irregular-shaped cervical wall. The main objective of this study was to develop and to evaluate in vivo (murine model) the performance of a 3D-printable AuNP-releasing hydrogel system (nanoparticle releasing properties, biodistribution, impact on cells) co-administered with a radioactive device. Overall, a biocompatible 3D-printable hydrogel formulation was developed for the delivery of AuNPs to the cervix. The brachytherapy inserts developed for this project were adapted to the mouse model and could prove useful for various studies for investigating the synergistic effects between radiotherapy and radiosensitizing AuNPs in cervical cancer therapy.